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Placenta-bound immunoglobulins.

Low pH eluates were prepared from trophoblasts derived from 8 term human placentas. A qualitative analysis for immunoglobulins revealed the presence of IgG, IgA, and IgM in these eluates. IgC-rich fractions were obtained by DEAE-cellulose chromatography of ammonium sulfate-concentrated eluates. These fractions were able to neutralize, in vitro, the catalytic activity of reverse transcriptases (RT) from several retroviruses. RT from baboon endogenous virus (BEV) seemed to be more susceptible to the neutralizing activity of some eluates. This was in contrast to RT from feline leukemia virus (FeLV) which were neutralized by eluates of leukocytes from chronic myelogenous leukemia. In contrast to previous and present results with purified IgG from leukemic leukocytes, the purified IgG from placenta eluates was incapable of RT neutralization. However, such purified IgG fractions inhibited mixed lymphocyte reactions.

Effects of bacterial endotoxin and corticosteroids on plasma concentrations of alpha 2 macroglobulin, haptoglobin and fibrinogen in rats.

Bacterial endotoxin injected into rats resulted in increased plasma concentration of alpha 2 macroglobulin, haptoglobin and fibrinogen. Cortisone acetate injected i.m. by itself was sufficient to increase the plasma concentration of haptoglobin by 54% and to a lesser extent the concentrations of the other two proteins. When cortisone acetate and/or cortisol succinate were injected simultaneously with varying doses of endotoxin, the effects of the corticosteroid differed for each plasma protein. Doubtless because of the effect of cortisone by itself the slope of the dose-response relationship for haptoglobin was greatly reduced. In contrast to this the slope for alpha 2 macroglobulin was reduced and that for fibrinogen was unaffected. These findings suggest that, if effects due to endogenous corticosteroids are to be avoided, increases in plasma fibrinogen will serve best as indicators of stimulation of the acute-phase response. Since, however, the relative increase of alpha 2 macroglobulin due to the lowest dose of endotoxin was much greater than that of fibrinogen, increases in concentration of the former protein represent the most sensitive indication of the acute-phase response. Consideration of the responses in individual rats has made possible division into those with more or less than average increases for all 3 plasma proteins and those showing irregular responses. Especially in the group which had received the lowest dose of endotoxin, a much larger number than would be expected on a random basis was found to respond regularly with either more or less than average increases for all 3 proteins.

Rapid removal to the liver of intravenously injected lipoprotein lipase.

Lipoprotein lipase was purified from bovine milk and labeled with 125I. After intravenous injection to rats the labeled lipase rapidly disappeared from the blood. The initial half-life was about 1 min and more than 70% of the radioactivity was found in the liver at 10 min. 30 min after the injection about 10% of the injected radioactivity was present in acid-soluble form in blood, indicating that the enzyme had been rapidly degraded. Injection of asialofetuin, ribonuclease B or mannan in amounts known to block the hepatic receptors for glycoproteins with exposed galactose, N-acetylglucosamine or mannose residues did not retard the removal of the lipoprotein lipase. Thus, some other, as yet undefined, receptor is implicated. Lipoprotein lipase is known to bind to heparin and some related polysacchrides. Heparin injected before the enzyme delayed its removal and heparin injected after the enzyme caused an immediate increase in blood radioactivity, signifying return from tissues to blood of labeled enzyme. Lipoprotein lipase is present at the endothelium in several extrahepatic tissues and is rapidly turned over. Its presence in blood in appreciable amounts would cause a derangement of lipid transport. The efficient hepatic removal of the enzyme may thus serve an important physiological purpose in keeping the blood levels of this enzyme low.

Place of immunological examinations in extrinsic asthma.

The provocation test is - in spite of its drawbacks, especially the risk for the patient - the most reliable way to identify causative agents in extrinsic asthma. The immunological genesis of the obstruction provoked must be proved with adequate methods however. RAST (Radio-allergo-sorbent-test) and histamine liberation are valuable in vitro techniques for the identification of atopic sensitizations. Their disadvantages - the expensive equipment and the time-consuming procedure respectively - limit their widespread use. A carefully performed and evaluated prick test possesses a comparable specificity with acceptable risk. Less satisfying is the situation in nonimmediate asthma. Precipitations tests are at present most valuable. Main indications for immunological methods in extrinsic asthma are: - the identification of causative allergens and the kind of underlying immune reactions; - the control and - in the future - the individualization of hyposensitization procedure. The future use of allergen extracts containing a minimum of non-specific irritants is extremely important.

Antiviral agents: action and clinical use.

The development of antiviral agents has been hindered by a variety of problems. There are fundamental biological differences between viruses and other infectious agents. Viruses are strictly dependent on cellular metabolic processes and possess very limited intrinsic enzyme systems and building blocks which may serve as targets for drugs. Antiviral drugs must also possess the ability to enter the host cell. Viral replication consists of a series of events, each of which can be interfered with, leading to interruption of the viral replication cycle. Currently, the major antiviral agents in therapeutic use are amantadine, idoxuridine and vidarabine. Methisazone and isoprinosine are also used in some areas. Immunoglobulins have some antiviral activity. Immune serum globulin and high titred hepatitis B immune globulin have both been used in prophylaxis of viral hepatitis. However, studies in this area have not been well controlled and results in some areas are conflicting. Interferon appears to be the most exciting antiviral agent yet discovered. However, its potential is limited by its availability, which remains dependent on biological method. Significant progress has been made recently, though, which may lead to the chemical synthesis of interferon and thus to an antiviral agent active against many viruses.

[Simplification of the Papanicolaou stain which is easily reproducable (author's transl)].

A simple and well reproducable modification of the Papanicolaou stain is reported. The most important modification is the replacement of the natural stain hematoxyline by the qualitively excellent synthetic stain thionin which is immediately added to the alcoholic fixation solution for a fixation stain. The high labor intense alternation between alcohol water and alcohol is thus eliminated. The quality of the nuclear staining is very good. The fixation staining with thionin without counter-staining of the cytoplasma may be used for a rapid thionin stain which gives sufficient cytoplasmatic staining by the thionin for a rapid cytological smear for cancer. The counter-stain of the cytoplasma was also simplified. The over-all staining of this simplification of the pap smear is comparable to that of the original papanicolaou stain. This modification of the papanicolaou stain fulfills requirements which a stain in competition with the original papanicolaou stain should have. 1. The modification is qualitively equal to the original method, 2. the staining method is simpler, 3. the staining method is well reproducable, 4. the staining method is more economical.

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, II. Characterization of a second inhibitory inactive domain by amino acid sequence determination.

A short digestion with excess of trypsin releases an inhibitor with an apparent molecular weight of 14,000 from both the inter-alpha-trypsin inhibitor and the ITI-related acid-stable inhibitor. The amino acid sequence of this inhibitor was determined. The inhibitor is composed of two covalently linked homologous Kunitz-type domains. One domain has antitryptic activity, as reported. This paper characterizes the second, inactive domain as also of the Kunitz type.

Immunochemical study on basement membrane (type IV) collagens.

Basement membrane (type IV) collagens were extracted from a mouse tumour with acetic acid and from human placenta after limited enzymatic digestion. Antisera were produced against both collagens in rabbits and guinea-pigs and examined by various assays. These antisera were found to be specific for basement membrane collagen and showed little or no cross-reactions with the interstitial collagens, types I, II and III or with human placenta collagen consisting of alpha A and alpha B chains. Varying degrees of cross-reaction were observed between antisera to human and mouse type IV collagen. Immunochemical analyses demonstrated the presence of three distinct determinants in the tumour type IV collagen. Rabbit antisera against this antigen reacted with either collagenase-resistant segments or with a collagenous, disulphide-bonded segment (P3). Guinea-pig antisera recognized primarily antigenic determinants in the P3 segment. Antisera to placenta type IV collagen reacted with another collagenous, pepsin fragment (P1) which lacks disulphide bonds. These antisera showed complete cross-reaction with collagenous alpha 1 (IV) chains prepared from pepsin-digests of human placenta and bovine lens capsule.

E-rosette formation in Graves' ophthalmopathy.

We investigated the lymphocyte characteristics of 77 Graves' disease patients with and without infiltrative ophthalmopathy. Thirteen patients with infiltrative ophthalmopathy without prior antithyroid therapy and 20 euthyroid patients with progressive ophthalmopathy demonstrated decreased percentages of active and total erythrocyte rosette-forming lymphocytes compared to thyrotoxic patients without eye disease and to a control population (p less than 0.001). There was no significant difference in rosette-forming cells between untreated thyrotoxic and treated euthyroid patients with ophthalmopathy. No lymphocytotoxic antibodies or rosette inhibitory factor was present in the sera of patients with infiltrative ophthalmopathy. Untreated and treated patients with lid retraction and mild proptosis without extraocular muscle disease had decreased active rosette-forming cells (p less than 0.001) but normal total rosette-forming cells. Five patients with infiltrative ophthalmopathy who failed to improve with systemic corticosteroids demonstrated elevated active but normal total rosette-forming cells. Differences in rosette formation between ophthalmic and nonophthalmic Graves' disease may represent an associated cell-mediated abnormality that may explain why control of the thyrotoxic state need not correlate with the ophthalmic manifestations of the disorder.

Regulation of surface topography of mouse peritoneal cells. Formation of microvilli and vesiculated pits on omental mesothelial cells by serum and other proteins.

The mesothelial cells of the mouse omentum provide an in vivo model for the study of the mobilization of labile microvilli on the cell surface. These mesothelial cells are sparsely covered with microvilli and large pits 150--400 nm in diameter, termed vesiculated pits. On the unstimulated cell, the microvilli average 44/100 microns2 and pits, 30/100 microns 2 of surface and they are rapidly induced to increase in number by the intraperitoneal injection of isologous mouse serum. After 2 min, microvilli increase threefold, continue to sevenfold at 30 min, and decrease to fourfold at 90 min. Vesiculated pits increased with similar kinetics. Bovine serum albumin and gamma globulin also stimulate the microvilli and pits to form, but the response is a slow, gradual rise to five- or sixfold the normal value at 90 min. Evidence indicates that multiple factors, possibly including insulin and immunoglobulins, are involved in the effect of serum. The close physical and temporal relationship between microvilli and pits suggests that a correlation exists in their mobilization by the cell and it is hypothesized that microvilli function in the regulation of the cortical microfilament network in effecting this mobilization.

Study on the significance of bronchial hyperreactivity in the bronchus obstruction after inhalation of cat dander allergen.

The role of bronchial hyperreactivity in the process that leads to bronchial obstruction after inhalation of an allergen was investigated. In 30 asthmatic children selected because of a positive skin test to cat dander allergen, we measured the histamine threshold, the reaction after allergen inhalation, the allergen-specific IgE concentration in serum, the lowest allergen concentration to which the intracutaneous skin test was positive (skin titer), and the histamine release of leukocytes after challenge with allergen. These variables were correlated with each other. The highest correlation was found between the inhalation reaction and the combination of the histamine threshold and either the allergen-specific IgE or the skin titer. Inhalation was only positive with a decreased histamine threshold (less than or equal to 8 mg/ml). With a low histamine threshold, a positive reaction to inhalation is likely to occur at an allergen-specific IgE concentration of > or = 2 U/ml or at a skin titer of < or = 2.5 x 10(-1) micrograms/ml.

Nonspecific light loss and intrinsic DNA variation problems associated with feulgen DNA cytophotometry.

Nonspecific light loss by the cell-wall-plus-cytoplasm (CWC) can cause a 50% increase in Feulgen absorption units in peanut root-tip nuclei as determined by scanning at 450 nm, whereas this phenomenon is not evident with chicken erythrocytes. A two wavelength scanning method of subtracting nonspecific 450 nm absorption from 550 nm Feulgen absorption values eliminated the nonspecific light loss in CWC, However, the two wavelength scanning method is time consuming and somewhat impractical with a regular scanning microdensitometer such as Vickers M85. Elimination of the problem of nonspecific light loss is suggested by careful determination of background setting with the spot position close to the nucleus in CWC. The accuracy of the CWC background setting method was further tested by comparison with subtraction method. The use of plant nucleis as an internal standard in plant DNA measurements was also evaluated. Significant variation among the replicate slides due to the variation in pine nuclear DNA amounts was observed and plant nuclei generally are not reliable internal standards. Mature chicken erythrocytes are recommended as an internal standard because the cell type and metabolic state is known.

Hapten-specific T-cell responses to 4-hydroxy-3-nitrophenyl acetyl. II. Demonstration of idiotypic determinants on suppressor T cells.

The ability of NP-coupled syngeneic spleen cells to induce antigen-specific T-suppressor cells capable of binding to NP-BSA-coated Petri dishes and mediating transfer of specific suppressive activity to NP was demonstrated. Furthermore, in strains of mice bearing the Ig-1b allotype, including SJL, and in (non-Ig-1b x Ig-1b)F1 hybrids, the NP-specific suppressor cells also interferes with expression of immunity after priming with NIP-BGG. Anti-NPb anti-idiotype antiserum plus complement treatment effectively abrogated the ability to transfer suppression. Formal genetic mapping of the fine specificity of cross-reactivity with Ig-1 allotypic congenic mice implies that expression of this trait is linked to the Ig-1b heavy chain linkage group. The sensitivity of NP-suppressor cells of appropriate strains to anti-idiotype treatment was also consistent with the formal mapping data. These experiments suggest that there are shared V-region structures on antibody and T cells that are crucial in the suppression pathway for the same antigen.

Macrophage oxygen-dependent antimicrobial activity. I. Susceptibility of Toxoplasma gondii to oxygen intermediates.

A sensitive method for evaluating extracellular parasite viability was used to determine the in vitro susceptibility of virulent Toxoplasma gondii to selected oxygen intermediates. By acridine orange fluorescent staining criteria, toxoplasmas were resistant to up to either 10(-3) M reagent H2O2 or H2O2 generated by glucose-glucose oxidase. In keeping with a lack of sensitivity to H2O2, toxoplasmas contained endogenous catalase (5.7 x 10(-4) Baudhuin units/10(6) organisms). The addition of a peroxidase and halide, however, markedly accelerated killing and lowered the H2O2 requirement by 1,000-fold. In contrast, toxoplasmas were promptly killed after exposure to products generated by xanthine (1.5 x 10(-4) M) and xanthine oxidase (50 micrograms). The inhibition of this system's microbicidal activity by scavengers of O2- (superoxide dismutase) and H2O2 (catalase) indicated that although neither O2- nor H2O2 were toxoplasmacidal, their interaction was required for parasite killing. Quenching OH. and 1O2, presumed products of O2--H2O2 interaction, by mannitol, benzoate, diazabicyclooctane, and histidine, also inhibited toxoplasma killing by xanthine-xanthine oxidase. These findings suggested that O2- and H2O2 functioned in precursor roles and that OH. and 1O2 were toxoplasmacidal. The capacity of normal peritoneal macrophages to pinocytose an oxygen intermediate scavenger, soluble catalase, was also demonstrated. Appreciable extraphagosomal concentrations of catalase were achieved by exposing macrophages to 1 mg/ml of the enzyme for 3 h. Maintenance of high intracellular levels required constant exposure because interiorized catalase was rapidly degraded.

Correlation between the capacity of bacterial lipopolysaccharide to supress experimental allergic encephalomyelitis and its mitogenic activity for lymph node cells in guinea pigs.

Protection of guinea pigs from experimental allergic encephalomyelitis (EAE) was attempted using bacterial lipopolysaccharide (LPS) from 4 sources. The ability of these LPS to induce DNA synthesis in guinea pig lymph node (LN) cells in vitro was also investigated. It was found that there existed a good correlation between the capacity of LPS to suppress EAE and their degree of mitogenic activities for LN cells. LPS from Escherichia coli 0111:B4 (Ec-LPS), which was most effective in suppressing EAE and also best inducer of DNA synthesis in LN cells, enhanced the proliferation of cells forming antibody to myelin basic protein (BP) in the regional LN. These results, in addition to the previous report, suggested that at the inductive phase the proliferation of B lymphocytes or their products, antibodies to BP, could inhibit formation of T lymphocytes sensitized to BP, resulting in suppression of EAE. Lipid A but not PS fraction of Ec-LPS showed a protective activity against EAE and a mitogenic activity for LN cells although less so than whole LPS. In addition, Lipid A appeared to exert its mitogenic effect mainly on B rather than on T lymphocytes.

Isoelectric focusing and crossed immunoelectrofocusing of CSF immunoglobulins in MS.

The 3 main Ig classes and the presence of free light chains were studied by isoelectric focusing and crossed immunoelectrofocusing in 100 CSF samples from patients with clinically definitie or probable MS. Minute quantities of IgM and free light-chain (mostly lambda) components were found in 2 out of 11 and 5 out of 14 samples respectively. IgG and IgA were detected in all samples examined for these proteins and were found in the pI-ranges of 5.3-9.8 and 4.6-6.4 pH-units respectively. The gammaglobulin abnormalities found on isoelectric focusing were identified as microheterogeneous, oligoclonal IgG with predominantly kappa light-chain determinants. The IgG immunoprecipitates differed from those of normal subjects and the major abnormal components most frequently exhibited pI-values greater than 8 pH-units. The IgA immunoprecipitates had 2-4 main components with some tendency to discontinuous subfractionation. This Ig class, however, did not exhibit the marked tendency to oligoclonal distribution found for IgG.

Recurrent transient global amnesia in a case with cerebrovascular lesions and livedo reticularis (Sneddon Syndrome).

Eight attacks of transient global amnesia were observed in a female patient who suffered from livedo reticularis and a series of other neurological symptoms, which were transient in most stances. The neurological deficits include focal epileptic attacks, unilateral loss of vision, paresis of left arm and/or leg and dysarthria. The first amnestic attack was seen at the age of 19. The episodes lasted from a few to 3 days. The intervals between the amnestic episodes varied between a few days and 11 years. The livedo reticularis became more obvious during each neurological episode and was less pronounced during the time of remission. A benign type of essential hypertension and parproteinemia (gamma-M) was found. The investigations failed to show any evidence of essential thrombocythemia, polyarteriitis nodosa, lupus erythematodes and other immune complex diseases. The underlaying disease remained unclear.

Isotachophoresis in capillary tubes of CSF proteins -- especially gammaglobulins.

Isotachophoresis in polyacrylamide gel tubes (PAG-ITP) and in capillary tubes (Tachophor, LKB) have previously been found by the authors, to be very promising high-separation methods for CSF and serum proteins, especially regarding the diagnosis of MS. PAG-ITP methods for analytical and preparative use have been described by the authors elsewhere, while in this paper proper cationic systems for ITP in capillary tubes for studying gammaglobulins in microliter amounts of CSF and serum are described, i.e. the albumin injection-clog problem is avoided and the preparation time can be forced. By using microdialysis of the CSF samples for desalting, with a technique easy to perform and with high reproducibility, microliter amounts of native CSF can be performed in less than half an hour. The method seems to be even more applicable for clinical and scientific use if the capillary isotachophoretic apparatus is connected to a synchronized equipment (LKB Tachophrac) with a cellulosa acetate strip onto which the separated fractions are ejected for further analysis by immunological tests. The analytical systems used have been especially directed to gammaglobulins in CSF and serum regarding further studies on demyelinating and infectious disorders of the nervous system.

Isolation and characterization of surface antigens from Schistosoma mansoni. II. Antigenicity of radiolabeled proteins from adult worms.

Adult Schistosoma mansoni were radiolabeled in vitro with 125I Bolton-Hunter reagent. Surface membrane antigens were solubilized with non-ionic detergent, then reacted with infection or normal serum. The antigen-antibody complexes were then precipitated with staphylococcal protein A immunoadsorbent, eluted with urea and SDS, and fractionated by SDS-PAGE. The results indicated the presence of 6 to 8 tegument antigens, depending on the type of antisera used. Human antisera to S. japonicum and S. haematobium reacted with some but not all of the antigens identified with human S. mansoni infection serum; this implies the presence of species-specific tegument antigens. The molecular weights of the radiolabeled antigens ranged from 10,000 to 100,000. A large (greater than 100,000) molecular weight glycoprotein and an uncharacterized lipid fraction appeared to be precipitated nonspecifically. Immunoprecipitation methods with anti-mouse IgG and anti-mouse whole serum failed to detect the presence of hostlike antigens in the labeled extracts. Several of the labeled proteins from S. mansoni were found to react with serum from patients infected with either S. haematobium or with S. japonicum.

Synthesis and antitumor activity of 5-azacytosine arabinoside.

5-Azacytosine arabinoside (ara-AC) can be considered a combination of structural elements derived from the antitumor nucleosides cytosine arabinoside (ara-C) and 5-azacytidine (5-AC). The synthesis of ara-AC, for which standard methods were inadequate, was accomplished using the stable dihydro derivative as a synthetic intermediate. A novel dehydrogenation of the latter through the application of a trimethylsilylation-oxidation procedure gave ara-AC in good yield. Using murine L1210 leukemia as a test system, ara-AC was evaluated for antitumor properties in parallel determinations with 5-AC and ara-C. Although higher dose levels were necessary, ara-AC demonstrated a reproducibly greater efficacy in the L1210 system (% ILS = 144-148) than that shown by 5-AC (% ILS = 126-124) or ara-C (% ILS=127-121 ). Moreover, initial data suggest that ara-AC exhibits less host toxicity than either 5-AC or ARA-C. Although ara-AC can equally be considered an analogue of either 5-AC or ara-C, preliminary results indicate that ara-AC is chemically similar to 5-AC but biologically more closely related to ara-C.

Preparation of syngeneic tumor regressor serum reactive with the unique determinants of the Abelson murine leukemia virus-encoded P120 protein at the cell surface.

Antisera reactive with the Abelson murine leukemia virus (A-MuLV)-specified P120 (anti-AbT sera) were produced in C57L/J mice. Of many strains tested, only C57L/J reproducibly rejected syngenic A-MuLV-induced tumor cells; after multiple immunizations their sera would immunoprecipitate both P120 and Moloney-MuLV (M-MuLV) proteins. Using labeled A-MuLV-induced nonproducer cells, only P120 could be detected by anti-AbT sera, suggesting that it may be the only A-MuLV-specified protein. Reactivity of anti-AbT sera with P120 was not blocked by M-MuLV virion proteins, implying that the sera recognize a portion of P120 that is not homologous to any M-MuLV product. Anti-AbT sera stained the surface of live, A-MuLV-transformed nonproducer cells in a two-stage immunofluorescence assay, and such staining was not blocked by M-MuLV protein. Also, intact A-MuLV-transformed cells absorbed much of the reactivity of certain anti-AbT sera for P120. Thus a portion of P120 appears to be exposed on the surface of transformed cells. P120 lacks detectable carbohydrate, is not affected by endoglycosidase H, and cannot be labeled by lactoperoxidase-catalyzed iodination. Thus P120 is an unusual surface protein.

Search for specificity in natural cell-mediated cytotoxicity.

Differences in antigenicity between the human osteosarcoma cell line TE 85/B and its feline sarcoma virus-infected subline NIH E1041 were detected by competitive inhibition of natural cell-mediated cytotoxicity (NCMC). Whether the differences could be attributed to the viral infection was investigated by absorption and elution studies of antibodies that determine the specificity of NCMC against the cell lines. Antibodies from the serum of healthy individuals were first absorbed onto target cells against which they were to be tested and then eluted to provide antibodies putatively specific for the target cells. Trypsin-treated effector cells were restored with the absorbed serum or eluted antibodies and tested against TE 85/B and its intentionally infected sublines. The differences observed previously between TE 85/B and NIH E1041 were extended to the detection of small differences in antigenicity among all sublines. Separately maintained sublines from the same culture became antigenically different with continuous passage. The causes for these specific changes were unknown, but a role for the control of these antigens by NCMC was suggested. Differences in antigenicity between virus-infected sublines cultured separately need not be related to the virus infection.

Reliability of assessment of alcohol intake based on personal interviews in a liver clinic.

In 37 patients with alcoholic liver disease urinary alcohol was measured daily for up to 6 months. Every week the patients were asked about their drinking during the past week. Those who convinced the physicians of their abstinence were recorded as not drinking. Patients with alcohol in their urines convincingly denied alcohol intake 52% of the times that they were questioned. 25% of them denied drinking every time. Only 17% of all patients admitted it at all times. Patients who always admitted to drinking had an average urinary alcohol value of 1420 +/- 66 mg/l, compared to 81 +/- 5 mg/l in those who denied drinking every time. Those who admitted drinking intermittently had significantly higher urinary alcohol values (1001 +/- 57 mg/l) when admitting than when denying (538 +/- mg/l). The personal interview should not be used to separate populations of abstainers and non-abstainers in the follow-up of alcoholic patients. On the other hand, deniers appear to consume less alcohol than those who admit their drinking.

Uptake and retrograde axonal transport of horseradish peroxidase in normal and axotomized motor neurons during postnatal development.

The axonal uptake and somatopetal transport of horseradish peroxidase (HRP) was studied during early postnatal development of facial neurons in mice and rats. HRP injected systemically or locally into the muscles of the vibrissae, diffused into the region of the immature neuromuscular junction and was incorporated into vesicles in the axon terminals on the first and third postnatal days, at a time when synaptic vesicles were already present. HRP later was found in the nerve cell bodies of the facial nucleus in the brain stem indicating a somatopetal transport of the tracer in axons. The response of facial neurons to nerve transection changed from rapid neuronal death to prolonged survival between the 6th and 10th postnatal day. HRP was transferred to nerve cell bodies after topical application to the proximal stump of transected facial nerves in rats 3 days-of-age. In the perikaryon it was localized to vesicles and vacuoles with no signs of leakage into the cytoplasm. In the light of our findings different hypotheses for the mechanism of the neuronal death in the immature animals are discussed.

Feulgen cytophotometry of pine nuclei. II. Effect of pectinase used in cell separation.

Pectinase used for cell separation prior to cytophotometry contains a DNase that is able to penetrate the cells of pine root tips and attack nuclear DNA. When pine root tips were exposed to 1% pectinase (pH 6.0), there was a decrease in nuclear DNA content at every sample point and a sharp drop between 16 and 20 hr. The effect of the DNase was eliminated by preparing the enzyme solution in 0.01 M sodium citrate or 0.001 M EDTA. It is suggested that heat denaturation of the DNase should also be effective and might be used in combination with the magnesium chelators.

The zinc iodide-osmium (ZIO) reaction in the central nervous system: localization and relations to functional activity.

The fine structural characteristics of ZIO reaction was studied in the cerebral and cerebellar cortex and olfactory bulb of the rat and in synaptosomes prepared from rat cerebellar cortex. It was concluded that: 1. Organelles of different nerve cell types exhibit different ZIO reactions provided that the impregnation was carried out under standardized conditions. 2. 6...10 times more synaptic vesicles were stained by ZIO in the inhibitory terminals than in the excitatory ones. 3. ZIO positivity was found in all types of synaptosomes prepared from cerebral cortex. Following electrical or chemical (KCl) depolarization there was a decrease in the number of ZIO positive synaptic vesicles, which decrease was directly proportional to the parameters of stimulation. 4. By x-ray microanalysis Os, Zn and Ca were consistently detected in the ZIO precipitates. Iodine, however, could not always be found. After stimulation the presence of Ca was observed even in those synaptosomes in which the ZIO reaction product was absent. 5. On the basis of the staining characteristics the reaction, under standard conditions, can reflect certain functional states of the nerve terminals.

Immunotherapy in spring-time hay fever. A clinical and immunological study comparing two different treatment extract compositions.

In a study of the efficacy of two different treatment schedules for perennial immunotherapy, 47 adult patients with spring-time hay fever due to allergy against birch and other deciduous trees were randomly assigned to three treatment groups: one group received birch, alder and hazel allergen in Allpyral, another group received the same Allpyral mixture and in addition all relevant tree pollens in aqueous extract and a control group received no injections. For determination of antibody titres the radioallergosorbent test (RAST) and the ammonium sulphate precipitation (ASP) technique were used. Cellular responsiveness was studied by measuring birch pollen (BP) induced leucocyte histamine release in peripheral blood. The clinical and immunological response was similar in the two treated groups. Treated patients had less symptoms and a lower consumption of antihistamine tablets during the pollen season than the control group. Non-IgE BP antibodies and IgE antibodies recorded with the ASP technique increased after immunotherapy while RAST values did not change significantly. A decrease of RAST values from postseasonal values during the first year to preseasonal values in the following year was seen in all patient groups but was less pronounced in treated than in untreated patients. The decrease was more pronounced in patients with high RAST values of postseasonal sera than in patients with low RAST values. Cellular reactivity increased slightly during the first phase of therapy but returned to the pre-treatment level later. Clinical improvement was positively correlated to the percentage increase of non-IgE antibody titre and to the pre-treatment non-IgE/IgE antibody ratio. Patients with high preseasonal RAST titres or high cellular sensitivity tended to have more severe symptoms during the pollen season. It is concluded that a mixture of birch, alder and hazel is sufficient for immunotherapy in spring-term hay fever. It is obvious that changes of a single immunological variable do not account for the therapeutic results in immunotherapy.

Chronic progressive myelopathy: investigation with CSF electrophoresis, evoked potentials, and CT scan.

Chronic progressive myelopathy (CPM) is a difficult clinical problem. Many patients who present with CPM turn out to have a spinal form of multiple sclerosis (MS), but until there is clear lesion dissemination, a definite clinical diagnosis cannot be made. We have looked for MS-related abnormalities in 72 patients with CPM. The mean age of onset was 42 years, mean duration was ten years, and mean Kurtzke disability rating was 4.5. Studies performed were cerebrospinal fluid electrophoresis for oligoclonal banding, pattern-reversal visual evoked responses, blink reflex latencies, and computerized axial tomography. Oligoclonal banding was found in 32 patients (44%), patterned visual evoked responses were abnormal in 32 (44%), and blink latencies were abnormal in 40 (56%). A least one of these studies was abnormal in 61 patients (85%) and at least two in 48 (66%). The CT scan was abnormal in 38 )53%), 36 with atrophy and 3 with low-density or enhancing lesions. These results suggest that at least 44% of patients with CPM may have MS that could be diagnosed by oligoclonal bands. Other physiological tests suggesting diffuse or disseminated disease bring the total to 85%. Only autopsy follow-up will tell us the exact diagnostic accuracy of these studies in this complex syndrome.

Relatedness among coagulase-negative staphylococci: deoxyribonucleic acid reassociation and comparative immunological studies.

DNA-DNA-homology values were determined under restrictive to relaxed reassociation conditions with type strains and some additional strains of coagulase-negative staphylococci belonging to ten different species. The immunological relationship of the catalases present in the type strains of these species was also determined by applying double immunodiffusion and microcomplement fixation. The results of these studies support the previous proposal to subdivide the coagulase-negative staphylococci into at least ten separate species. However, it is evident that some of the species are more closely realted than others and can form species groups. According to the results presented in this study, the coagulase-negative staphylococci can be combined into five species groups: The Staphylococcus saprophyticus group is composed of S. saprophyticus, S. xylosus and S. cohnii. The S. epidermidis group comprises S. epidermis, S. capitis and S. warneri. The S. hominis group which exhibits a significant relationship to S. epidermidis includes S. hominis and S. haemolyticus. The species group S. sciuri consists of S. sciuri ssp. sciuri and S. sciuri ssp. lentus and the species group S. simulans is presently represented by the corresponding single species.

Separation of human leukocyte interferon components by concanavalin A-agarose affinity chromatography and their characterization.

Human leukocyte interferon (HL-IF), produced by mixed leukocytes infected with Newcastle disease virus, was resolved into three distinct fractions when chromatographed on concanavalin A-agarose. The major portion (70--75%) of interferon appeared in the breakthrough (BT fraction). The bound interferon (25--30%) was displaced from the column as two peaks: the first was eluted with 0.01 M methyl alpha-D-mannoside, yielding 15-20% of the interferon activity (alpha-MM fraction), and the second by including ethylene glycol (70%) in the eluant, yielding the remaining 5--15% of the interferon (EG fraction). No interferon was retained when HL-IF produced in the presence of glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) was chromatographed on concanavalin A-agarose, suggesting that the fraction of interferon retained by this lectin is glycosylated. The three fractions of interferon (BT, alpha-MM, and EG) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cross-species antiviral activity, and neutralization by specific antisera. The BT fraction contains exclusively the 16 000 molecular weight component of human leukocyte interferon. The majority of the alpha-MM fraction (90%) is the 21 000 molecular weight component. However, the EG fraction contains the 16 000 and 21 000--23 000 molecular weight components in essentially equal proportions. On the basis of cross-species antiviral activity and neutralization by specific antisera, the BT and alpha-MM fractions are leukocyte-type interferon and the EG fraction seems to be primarily of fibroblast type.

Effects of adenosine and adenine nucleotides on synaptic transmission in the cerebral cortex.

Adenosine and the adenine nucleotides have a potent depressant action on cerebral cortical neurons, including identified corticospinal cells. Other purine and pyrimidine nucleotides were either weakly depressant (inosine and guanosine derivatives) or largely inactive (xanthine, cytidine, thymidine, uridine derivatives). The 5'-triphosphates and to a lesser extent the 5'-diphosphates of all the purine and pyrimidines tested had excitant actions on cortical neurons. Adenosine transport blockers and deaminase inhibitors depressed the firing of cortical neurons and potentiated the depressant actions of adenosine and the adenine nucleotides. Methylxanthines (theophylline, caffeine, and isobutylmethylxanthine) antagonized the depressant effects of adenosine and the adenine nucleotides and enhanced the spontaneous firing rate of cerebral cortical neurons. Intracellular recordings showed that adenosine 5'-monophosphate hyperpolarizes cerebral cortical neurons and suppresses spontaneous and evoked excitatory postsynaptic potentials in the absence of any pronounced alterations in membrane resistance or of the threshold for action potential generation. It is suggested that adenosine depresses spontaneous and evoked activity by inhibiting the release of transmitter from presynaptic nerve terminals. Furthermore, the depressant effects of potentiators and excitant effects of antagonists of adenosine on neuronal firing are consistent with the hypothesis that cortical neurons are subject to control by endogenously released purines.

The biologic basis for combined modality treatment of cancer.

Strategies related to cancer should be aimed at optimization of local and regional treatment. Delineation of subsets of patients with high risk for recurrence would have a significant impact, upon which such adjunctive systems of therapeutic management should be evaluated. In disseminated disease, or where the probability of dissemination is high, evaluation of any realistic potential combination of treatment for tumor control should be pursued. It is necessary to delineate optimal palliative therapy where tumor control is not possible, but also to test new drugs, immunotherapeutic tools and combination regimens in all such situations where the disseminated compartment of the disease is of high probability. Only under such circumstances will the most practical and important optimization of treatment programs be achieved. The development of the above basic attributes for each tumor type needs to be studied so that protocols can be developed with a clear understanding of the nature of the disease, condition to be studied, and the general therapeutic strategies to be followed.

Site specificity of bleomycin-mediated single-strand scissions and alkali-labile damage in duplex DNA.

Form II PM2 DNA, which contained bleomycin-mediated single-strand breaks, was purified and treated with the extracellular endonuclease from Alteromonas BAL 31. This enzyme cleaves the phosphodiester backbone opposite a single-strand break to yield a double-strand break. The locations of these double-strand breaks were determined relative to the cleavage sites produced by the restriction enzyme HindIII. The experimental procedure was as follows. Form I PM2 DNA was treated with bleomycin to produce alkali-labile bonds. These were hydrolyzed by alkali treatment and the DNA, now containing single-strand breaks, was purified and treated with the BAL 31 enzyme and the HindIII enzyme to determine the positions of the original alkali-labile bonds. It was found that the single-strand breaks and alkali-labile bonds were introduced at preferred sites on the PM2 genome, since electrophoretic analyses of the DNA after the HindIII digestion revealed DNA bands of discrete sizes. The molecular weights of the DNA fragments produced by these treatments indicate that single-strand breaks and alkali-labile bonds occur at the same sites as those previously determined for direct double-strand scissions introduced by bleomycin at neutral pH. Some of the specific sites of double-strand scissions mediated by bleomycin at neutral pH (Lloyd et al., 1978b) are also shown here to be relatively more reactive than other sites when the DNA contains superhelical turns.

Inhibition of antigen-induced lymph node cell proliferation by murine amniotic fluid and its components.

Murine amniotic fluid (MAF), alpha-foeto-protein (AFP) and MAF depleted of AFP by affinity chromatography (MAF-AFP) inhibited the T-cell dependent in vitro proliferative responses of lymph node cells sensitized to a variety of soluble antigens. Variable degrees of inhibition were observed with the different antigens used in the assay. In general, the higher the proliferative response induced by a particular antigen, the less it was inhibited by the three inhibitors. Enhancement of proliferation was not infrequently observed at lower concentrations followed by a dose-dependent inhibition as the concentration of the inhibitor was increased. Usually the order of inhibition was MAF greater than MAF-AFP greater than or equal to AFP although variations in inhibitory potency were noted between different preparations of AFP and MAF-AFP. The existence of inhibitors in preparations of MAF depleted of AFP raised the question as to whether MAF contains single or multiple inhibitory factors. The most facile explanation is that two inhbitors exist; AFP and the as yet uncharacterized non-AFP suppressor present in MAF-AFP.

Kinetics of formation of metallic silver and binding of silver ions by tissue components.

The effect of time on the formation of metallic silver by tissue reducing groups follows a curve which can be divided into three main parts. In the first, which may last for several hours, the reaction is very slow, and only an undetectably small amount of metallic silver is produced. In the second period the speed of the reaction first increases in a progressive manner and then begins to decrease gradually; during the third period the speed approaches zero asymptotically. Binding of the silver ions by the tissue commences initially at its fastest rate; the level then decreases steadily to zero within about a quarter of an hour. There is no direct relationship between the amount of silver ion bound to the tissue and the formation of metallic silver. The latter cannot take place by way of direct (non-catalysed) reaction. The following mechanism is proposed for the process: Transfer of electrons from the reducing molecules to the silver ions is mediated at first by certain tissue sites (catalytic points) and then also by the steadily increasing total surface area of the metallic silver grains (autocatalysis). On the basis of this mechanism, several anomalies of both the argentaffin and argyrophil reactions are explained.

Quantitative succinate-dehydrogenase histochemistry. III. Variations in histochemical succinatedehydrogenase activity in different cross-sections of the same muscle fibre.

The variation in histochemical SDH-activity at different levels in the same muscle fibre was determined in muscle fibre cross-sections both by visual classification and quantitative determination of the formazan-deposits. This work resulted in a confirmation of the earlier micro-biochemical studies of Spamer AND Pette (1977, 1979) and Lowrey et al. (1978) that the activity of enzymes of the citric acid cycle is not homogeneously distributed in a muscle fibre over its entire length. In addition it is shown that the observed variations in histochemical SDH-activity strongly interfere with the visual muscle fibre typing. Some of the possible causes for these variations in histochemical SDH-activity (section-thickness, presence of the motor-endplate) and the implications of these findings for the relation between histochemical characteristics and functional properties of the muscle fibres are briefly discussed.

Testing the properties of an experimental batch of zoster immune gammaglobulin (ZIG).

The methods of indirct haemagglutination (IH) and precipitation in gel (ID) were employed to test the level of varicella-zoster (VZ) antibodies in an experimental batch of zoster gammaglobulin (ZIG). The titre of indirect haemagglutinating antibodies in ZIG was about 64 times higher than in the ordinary batches of normal immunoglobulin and about 8 times higher in comparison with the level of the initial plasma pool. In the reaction of precipitation in gel, ZIG produced 5 to 6 zones. In comparison with the initial pool of convalescent plasma, ZIG also showed an 8-fold concentration of precipitating antibodies. ZIG was administered preventively to 6 children with risk diagnoses. None of the children fell ill with varicella. According to the results of subsequent serological examination in the reactions of indirect haemagglutination and radioimmunologic analysis, only 3 children were definitely susceptible to VZ infection. In two other children (very low antibody titres) the risk could not be excluded. No substantial increase in the levels of IH and RIA antibodies was observed in the 4 children under serological observation in a period of 6 months following the administration of ZIG. ZIG was administered therapeutically to four children with varicella. The effect of ZIG therapy was very suggestive, especially in two newborn infants lacking maternal antibodies, where the dose of ZIG per 1 kg body weigt was unusually high.

Bidirectional axonal transport of free glycine in identified neurons R3--R14 of Aplysia.

The axonal transport of 3H-amino acids was studied in the axons of identified neurons R3--R14 in the parietovisceral ganglion (PVG) of the mollusc Aplysia. The PVG was incubated (3--24 hr) in media containing physiological concentrations of single 3H-amino acids while the isolated nerve was superfused with plain or chemically altered media. The nerve was then sliced into sequential segments for biochemical analyses or fixed for autoradiography. 3H-glucine was transported at 70 mm/day in 6X greater quantities than other amino acids which were transported at less than 40 mm/day. In the 3H-glycine experiments, greater than 80% of the label transported into the nerve remained as free glycine, comigrating with glycine in thin-layer chromatographs. In autoradiographs of sections 4 mm from the ganglion-nerve barrier, greater than 50% of the silver grains were over R3--R14 axons which occupy less than 10% of the nerve cross-sectional area. EM autoradiographs confirmed that grains were within R3--R14 and not in surrounding glia. The selective transport of glycine was inhibited by Hg2+, by vinblastine and Nocodazole, and by low Ca2+ media. Autoradiographs of vinblastine-treated nerves showed a drastic reduction in label over R3--R14 and other axons. Label was also transported retrogradely; this transport rate was similar to the orthograde rate, but 5--10 times less label moved retrogradely. Autoradiographs showed that the retrograde label was localized to R3--R14 axons. This report clearly demonstrates the rapid, selective, and bidirectional transport of a free amino acid and provides further evidence that glycine may be used as a neurochemical messenter by neurons R3--R14.

Detection of virus-associated antigen in serum and liver of patients with non-A non-B hepatitis.

In a search for serological markers of non-A non-B(NANB) hepatitis, sera from repeatedly transfused and convalescent patients were assayed by immunodiffusion against sera from 12 patients with early acute NANB hepatitis. A new antigen/antibody system distinct from HBsAg was demonstrated in 8 cases. To assess the specificity of the test, serial sera from 17 patients with acute hepatitis of known aetiology (10 due to hepatitis-B virus, 4 to hepatitis-A virus, 3 to drugs) were tested twice a month, together with sera from 14 NANB patients obtained during a prospective post-transfusion study. NANB antigen (Ag) was detected in at least one sample from 12 of the 14 NANB patients (86%) but in none of the other groups. NANB Ag appeared after or just before elevation of transaminase levels and was cleared before they fell to normal. 4 of 5 patients who showed seroconversion to NANB antibody (Ab) had transient hepatitis. In contrast, the alanine adminotransferase value returned to normal in only 1 of the 5 with persistent NANB antigenaemia during 6 months' follow-up. NANB Ag was also demonstrated by immunodiffusion in liver extracts from patients with chronic NANB hepatitis with antigenaemia. Fluorescein-isothiocyanate-labelled gammaglobulins with strong NANB Ab activity revealed specific nuclear fluorescence in foci of hepatocytes on cryostat sectons of these livers but in none of 6 control human livers. The results suggest that the antigen and antibody are specifically linked to NANB hepatitis of long incubation period.

[Combination of adriamycin and cyclophosphamide (alone or with other substances) in the treatment of breast cancer].

This paper reviews clinical trials at the University of Arizona Cancer Center which were designed to improve the outcome in breast cancer by utilizing the combination of adriamycin and cyclophosphamide (A--C) alone or with the addition of other agents or modalities. Our initial trial in advanced breast cancer with A--C produced an overall objective response rate of 78% in 51 patients with advanced breast cancer without prior chemotherapy. The median duration of disease control was 12 months. Subsequent studies showed that the addition of either vincristine or the androgen, calusterone, effectively doubled the remission duration and prolonged survival. In our surgical adjuvant trial with 6 months of treatment with A--C there has been only a 9% relapse rate in stage II patients thus far, with a median follow-up of close to 2 years. A subset of stage II patients who received regional radiotherapy along with A--C have not yet shown added benefit compared to the use of A--C alone. Since 1975, stage I patients have been treated with an abbreviated treatment schedule (3 courses of A--C over 9 weeks). While there have not yet been relapses in this category, much longer periods of follow-up will be required. The use of A--C (plus other drugs) has clearly provided excellent palliation and improved survival in patients with advanced or recurrent breast cancer; in our opinion it should be used as initial cytotoxic chemotherapy. The brief intensive program of A--C as a surgical adjuvant also shows considerable promise for erradicating occult micrometastases in both pre- and postmenopausal women.

Myxobacterial hemagglutinin: a development-specific lectin of Myxococcus xanthus.

Fruiting body formation in the bacterium Myxococcus xanthus consists of a temporal sequence of cellular aggregation and sporulation. During the period of cellular aggregation, a major new development-specific protein that has lectin-like activity is synthesized. This protein, called myxobacterial hemagglutinin (MBHA), was able to agglutinate sheep or guinea pig erythrocytes but not horse, ox, chicken, or human erythrocytes. MBHA was undetectable in extracts of vegetative cells, cells starved in liquid buffer, or in glycerol-induced cells. However, cells starved on a fruiting medium produced large amounts of MBHA (about 5% of protein synthesis), starting at about 6-8 hr of development. The protein accumulated in the soluble fraction of cells, reaching a peak of 1-2% of total protein at about the time when aggregation was completed. At later times the amount of MBHA present in the soluble fraction declined although synthesis continued. The hemagglutinating activity of MBHA could not be inhibited with simple sugars or aminosugars but could be inhibited with fetuin, a fetal calf serum glycoprotein. The O-glycosidically linked trisaccharide glycopeptide of fetuin was shown to be inhibitory by itself. The penultimate galactose of this glycopeptide was directly implicated in the inhibitory activity, because the inhibition by asialofetuin was reduced to 1/60th by periodate oxidation and to 1/15th after beta-galactosidase treatment. MBHA is an abundant biochemical marker of development in M. xanthus. The fact that it is a lectin suggests that it may play a role in cell-cell recognition or agglutination.

Structural characterization of the murine fourth component of complement and sex-limited protein and their precursors: evidence for two loci in the S region of the H-2 complex.

The S region of the murine major histocompatibility complex controls the expression of two related, serum substance-positive proteins; one (C4) has functional complement activity, whereas the other, the sex-limited protein (Slp), is hemolytically nonfunctional. The structural relationships of these molecules to each other and to their putative intracellular precursors have been examined. Radiolabeled intracellular C4 and Slp precursors were isolated from lysates of cultured peritoneal cells. The C4 and Slp precursors and their processed subunits were purified by immunoprecipitation and sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Antigenically distinct precursors for C4 and Slp were demonstrated by sequential immunoprecipitation experiments in which anti-Slp-reactive molecules were precleared by exhaustive immunoprecipitation and residual C4 molecules were precipitated by antibody to serum substance. Both molecules had apparent molecular weights of 185,000. Their molecular identities as precursors of the mature C4 and Slp proteins were established in pulse-chase studies and by comparisons of their tryptic peptide profiles with those of isolated subunits from the processed proteins. When isolated alpha- or beta-subunits from C4 and Slp proteins were compared by peptide mapping, it was possible to detect multiple distinct and multiple shared peptides. This evidence indicates that the C4 and Slp proteins derive from distinct precursor polypeptides and suggests that the primary structures of the C4 and Slp alpha- and beta-subunits are different. These results support the postulate that the S region contains two discrete structural loci that specify discrete C4 and Slp proteins.

Serum beta2-microglobulin in liver disease.

The concentration of beta 2-microglobulin in serum was determined in seventy-one patients with various liver disorders. Elevated values were found in most patients with chronic active or chronic persistent hepatitis and in over 80% of patients with alcohol-induced liver cirrhosis. In contrast, patients with alcohol-induced fatty liver, the serum beta 2-microglobulin concentrations were mostly within the normal range. Significant correlation (P less than 0.001) was noted between the elimination rate of galactose from blood and the serum beta 2-microglobulin concentration in patients with alcoholic liver damage but not in patients with chronic hepatitis. The reasons for the increased S-beta 2-microglobulin concentrations in liver diseases are unknown. Several explanations including a release of beta 2-microglobulin from necrotic liver cells or an increased synthesis of beta 2-microglobulin consequent to inflammation in the liver are possible. Alternatively, raised beta 2-microglobulin levels may reflect the hepatic synthesis during reparative growth.

Pneumococcal infections and the possible need for a vaccine.

Antibiotics and chemotherapeutics have been available for about 40 years, but still bacterial infections constitute some of the greatest problems in medicine. Pneumococci causing pneumonia, bacteremia, meningitis and otitis are a leading cause of illness and death. The exact incidence and lethality of pneumococcal infections is not known, however, since they are not reportable diseases in most countries and since microbiological diagnosis is difficult. In the latest years some significant progresses have been made for the diagnosis of infections caused by pneumococci, especially pneumonia. This is for example the counterimmunoelectrophoresis (CIE) for antigen determination, the transtracheal aspiration (TTA) for direct bacterial cultivation from trachea, and serological assays like radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) for pneumococcal capsular antibody determination. These techniques have further emphasized the significance of pneumococcal infections. The recent finding of pneumococci resistant to penicillin and some other antibiotics also emphasizes the need for immunological prophylaxis. In recent years a vaccine consisting of the purified, most common pneumococcal polysaccharides has been introduced. It has been shown to protect against pneumonia, pneumococcal infections in splenectomized individuals and people with spherocytosis and probably partly against otitis media. Indications for the vaccine are suggested.

[Soluble Babesia ovis antigen].

Dissolved antigen of plasma and of erythrocytic cytoplasma was obtained from 4 splenectomized sheep experimentally infected by Babesia ovis. Blood was taken in the stage of acute parasitemia with 23--25% parasite erythrocytes. The antigen was isolated by precipitating the plasma and the erythrocytic cytoplasma three times with an ammonium sulfate solution saturated 50 and 75%. The fraction obtained following dialysis was concentrated after McErlean's method. The specificity and activity of the isolated soluble antigen were proven by RAGP against serums of 6 weaned lamb groups: I group -- with 4 cm3 plasma, II group -- with 8 cm3 plasma, III group -- with 2 cm3 erythrocytic cytoplasma, IV group -- with live vaccine, V group -- with normal plasma and VI group -- normal weaned lambs. The same groups were used to assess the immunogenic properties of the dissolved antigen. Following provocation with a virulent B. ovis strain the lambs of group II proved as resistant as these immunized with live vaccine. The result was confirmed under production conditions -- not one of the 360 lambs immunized with 5 and 10 cm3 plasma became infected, while of the 250 control lambs 30 were infected. The conclusion is drawn that plasma in the stage of acute parasitemia can be used for immunoprophylaxis, but more supplementary studies are needed.

[X-ray diagnosis and surgical treatment of brain tumors in young children].

Among 319 patients under the age of three years with tumors of the brain verified by means of contrast X-ray examination, during operation, or on autopsy, 215 had undergone surgery (166 with tumor of the posterior cranial fossa and 84 with tumor of supratentotial localization). From analysis of 176 cases in which X-ray contrast methods of examination were applied it was established that the method is safe and yields sufficient information for making the topic diagnosis of the tumor, determining its blood supply, its relation to the system of the cerebrospinal fluid and for judging the degree of the concomitant internal hydrocephalus and occlusion of the cerebrospinal fluid passages. In addition, these methods help to solve problems of the techniques of surgical intervention or the contraindications for operations. Analysis of the results of 250 operations shows that when the child's condition and the localization of the tumor permit, the surgeon should strive to perform single-stage subtotal or total removal of the tumor because this does not increase postoperative mortality.

Treatment of generalized scleroderma: updated results.

Long-term treatment of patients with generalized progressive scleroderma by means of inhibitors of connective-tissue biosynthesis brings about total or subtotal regression of dermal sclerosis in 40.8%, partial regression in 33.1%, arrest of progression without regression in 14.8%, while in 11.3% it had no effect whatsoever. The drugs used were D-penicillamine, benzyl-penicillin-diethyl-aminoethylesterhydro-iodide, glutamine, hydralazine, chlorpromazine, L-dopa, diphenylhydantoin, and corticosteroids. Disease activity before, during and after treatment was indicated by the urinary fractions of high-molecular hydroxyproline and hydroxylysine containing peptides and of uronic acid, break-down products of collagen and acid glycosaminoglycans of connective-tissue ground substance. The prospects were better for young patients than for old, for those with a short history than for the longstanding disease cases, and for those having a large total dose than for those who had less. If left untreated, scleroderma progresses inexorably.

Argentophil neuronal perikarya and neurofibrils induced by postmortem trauma and hypertonic perfusates.

Argentophil neuronal perikarya and perikaryal neurofibrils similar to those illustrated in Ramón y Cajal's classical studies have in the present investigation been found to be manifestations of the chromophil neuron. Conclusive evidence of such association was obtained by silver impregnation with the Bodian technique of sections previously stained with cresyl violet. Regardless of the fixative used, silver-impregnated neurofibrils were evident when (1) normal tissues were fixed by immersion or unsuccessfully fixed by perfusion, (2) normal tissues were exposed and touched after death but before perfusion with the fixative, or (3) flow of perfusates was compromised by the effect of an experimental procedure, as well as when (4) a hypertonic saline solution was used in the first perfusate. These cytologic peculiarities were still discernible after 24 h of postmortem autolysis following a delay in removal of the brain or in immersion of the exposed brain in the fixative. After immersion fixation, argentophilia and chromophilia occurred ubiquitously in the brain of the newborn guinea pig; however, argentophil neurofibrils were noted in the absence of chromophil neurons in the brain stem of the newborn rat, rabbit and cat. After fixation by perfusion, perikaryal neurofibrils were not impregnated in either newborn or old animals or in animals with facial nerve transection. Affinity for Congo red or birefringency, exhibited by neurons with marked neurofilbrillary changes in human senile brain atrophy, were absent in the present material. On the basis of the current light-microscopic observations, it is concluded that argentophilia of neuronal perikarya and perikaryal neurofibrils is another manifestation of the chromophil neuron induced by postmortem trauma and of the ocellate neuron elicited by perfusion with hypertonic saline.

Formation of granulation tissue in subcutaneously implanted sponges in rats. A comparison between granulation tissue developed in viscose cellulose sponges (Visella) and in polyvinyl alcohol sponges (Ivalon).

A comparison was made between the granulation tissue formation in two different synthetic sponge types. Visella and Ivalon, of different sizes. The granulation tissue formed in the two sponge types did not differ qualitatively, and had the character of wound tissue and inflammatory tissue in man. The rate of tissue formation in the Visella sponges was faster and the tissue was more homogenous than in the Ivalon sponges. Fourteen-day-old Visella implants of either size contained more granulation tissue than Ivalon sponges, probably owing to the smaller pore size of the former material. This may also account for the more frequent occurrence of giant cells in the Visella implants. In contrast to the Visella sponges, the trabeculae of the Ivalon polymer showed calcification and positive staining properties with histological staining procedures, and deformation was frequent among the Ivalon implants. Thin sponges of either type closed in about 21 days, thick ones after about 42 days of implantation. Calculated per 2 cm3 of implant, thin sponges produced more tissue after 14 days of implantation than thick noes. It is concluded that the Visella sponge type is best suitable for this experimental model of inflammation.

Effect of azacytidine on Simian Virus 40 nucleoprotein complexes.

Simian virus 40 nucleoprotein complexes synthesized in the presence of 5-azacytidine showed small differences in sedimentation rate on neutral sucrose and buoyant density in metrizamide and cesium chloride. Simian virus 40 deoxyribonucleic acid (DNA) I, isolated from the nucleoprotein complexes of drug-treated cultures, was found to band at a higher buoyant density and therefore had a decreased ability to bind ethidium bromide. The data indicated that these molecules were deficient in superhelical turns. Treatment with 5-azacytidine was shown to inhibit protein synthesis, which preceded the inhibition of DNA synthesis. Under the same conditions, protein synthesis was inhibited to a greater degree and occurred much faster than inhibition of DNA syntheses. Upon removal of the drug, resumption of protein and DNA synthesis occurred slowly. It is concluded that the inhibition of simian virus 40 DNA synthesis and the conformational alterations in DNA I isolated from nucleoprotein complexes result from the inhibition of protein synthesis.

Partial purification of tumour-specific transplantation antigens from methylcholanthrene-induced murine sarcomas by immobilized lectins.

Plasma membranes isolated from two immunogenic, non-cross-protecting, MC sarcomas were shown to retain the specific rejection antigens of whole cells as well as serologically detected H-2 antigens. Solubilization of the membranes with sodium deoxycholate gave quantitative release of H-2 and retained the rejection specificity of the tumour from which it was derived. Polyacrylamide-gel electrophoresis (PAGE) showed no extensive degradation of membrane components during solubilization. The solubilized TSTAs were further characterized and purified on columns of 4 different lectins immobilized on sepharose beads. TSTA from both tumours bound to WGA but not to Con A, LCH or RCA columns. Specific activity was retained after elution from the WGA column. Serologically detectable H-2 bound to the Con A and LCH columns only. Clear separation of H-2 from TSTA activity was thus obtained. Furthermore the WGA-binding material represents a source for further purification of TSTA molecules in order to explore the basis for their diversity.

Regulation of internal solute concentrations of marine Vibrio alginolyticus in response to external NaCl concentration.

Slightly halophilic marine Vibrio alginolyticus grown in the range of NaCl from 0.2 to 1.5 M maintained the total internal solute concentration always higher than the external medium by about 0.25 osM. The concentrations of macromolecules such as DNA, RNA, and protein were little affected by the increase in medium NaCl. The internal K+ concentration was kept to about 400 mM in the range of medium NaCl from 0.4 to 0.8 M; it rose to 510 mM when the bacterium was grown in 1.5 M NaCl, indicating that K+ increased only slightly in response to the large increase in medium NaCl. Thus, in contrast to the case of nonhalophilic and extremely halophilic bacteria, K+ was unlikely to act as a major component to regulate the internal solute concentration of marine V. alginolyticus. The internal Na+ and Cl- concentrations were maintained always lower than those in the growth medium, but they increased in response to the increase in medium NaCl. The concentration of internal Na+ was close to that of K+ at the concentration of medium NaCl that supports the optimal growth of this organism. The total amino acid content of V. alginolyticus increased from 76 to 413 mM by the increase in medium NaCl from 0.2 to 1.5 M. The concentrations of glutamic acid and prolined were 254 and 72 mM, respectively, when grown in 1.5 M NaCl. These results indicated that Na+, Cl- and amino acids, especially glutamic acid and proline, contributed to the regulation of internal solute concentration of V. alginolyticus in response to the increased external NaCl.

Arthropod-borne encephalitides in the Americas.

The arthropod-borne encephalitides are an important cause of equine and human morbidity in the Americas. Between 1975 and 1978, 6970 human cases of arboviral encephalitis were reported in the United States of America; however, this represents only a fraction of the true incidence. St Louis encephalitis (4824 cases), California encephalitis (1035 cases), and western equine encephalitis (WEE, 947 cases) accounted for 98.5% of all reported infections. Approximately 1000-4000 cases of equine encephalitis occur annually in the United States, the majority due to WEE. In tropical America, important outbreaks of Venezuelan, eastern, and western equine encephalitis, and of Rocio encephalitis have occurred.In this article, epidemiological aspects of arboviral encephalitis outbreaks occurring within the past 5 years are reviewed. In addition, summaries of current research activities on the ecology and epidemiology of St Louis, western equine, Venezuelan equine, Rocio, and California encephalitis viruses are presented, and the problem of control of these infections is discussed.

Effects of gibberellic acid and of tunicamycin on glycosyl-transferase activities and on alpha-amylase secretion in barley.

A crude membrane fraction was prepared from isolated aleurone layers, the secretory tissue of barley grains. The layers were pre-incubated in the presence or absence of the phytohormone gibberellic acid. The membranes catalyzed the transfer of [14C]mannose from GDP-[14C]mannose and of N-[14C]acetylglucosamine from UDP-N-[14C]acetylglucosamine to endogenous and exogenous dolichyl monophosphate. When gibberellic acid was present during the pretreatment the activity of the transferases was increased by a factor of two to three. A significantly increased activity was observable within four hours after the addition of gibberellic acid, whereas the gibberellic-acid-induced secretion of the glycoprotein alpha-amylase started only after 12 h. Tunicamycin inhibited the secretion of alpha-amylase by 60 to 80%. Intracellularly, however, no alpha-amylase was found to accumulate. On the other hand, tunicamycin did not inhibit the rate of total protein synthesis by more than 10%. The possibility is discussed that the synthesis of the protein portion of glycoproteins is specifically inhibited, when glycosylation is prevented.

Substance P infusion into substantia nigra of the rat: behavioural analysis and involvement of striatal dopamine.

The purpose of the present study was to characterize the nature of the behavioural response to substance P (SP) infusion into the substantia nigra and to evaluate this response in 6-hydroxydopamine (6-OHDA) caudate lesioned rats. The effects of SP infusions (3 microgram in 1 microliter bilaterally) were assessed in an open field. Two groups of rats were used: one with 6-OHDA lesions in the caudate nucleus, and one with sham lesions. In sham rats, the first infusion produced a strong increase in sterotyped rearing and sniffing, with no concurrent enhancement of locomotion. With three subsequent infusions (made at interval of two days) the rearing response disappeared and a tendency to groom emerged. All SP-induced behavioural stimulation was blocked in the caudate-lesioned rats; an effect of the lesion itself was reduced rearing. These results suggest that the response to SP infusion is mediated through the nigro-striatal dopamine system. The behavioural profiles which emerge after SP infusion into the substantia nigra and ventral tegmental area are compared. In general, the behavioural studies of SP effects support the concept that the A9 and A10 dopamine systems can be behaviourally differentiated.

Identification and characterization of trypsin, chymotrypsin and elastase inhibitors in porcine serum.

Characterization of the trypsin-, chymotrypsin- and elastase-inhibiting properties of porcine serum was carried out by gel filtration on Ultrogel, AcA 44, and agarose gel electrophoresis with subsequent processing for protease-inhibiting activity. Moreover, by allowing the fractions obtained from gel filtration to react with antibodies to porcine serum protease inhibitors, the specific inhibiting properties of these inhibitor molecules were identified. At least six protease inhibitors were identified and partially characterized in porcine serum. Two alpha 2 -macroglobulins (alpha 2 Mf and alpha 2 Ms), homologues to human alpha 2 -macroglobulin, with slightly different electrophoretic mobilities, were both found to exhibit trypsin, chymotrypsin and elastase inhibiting activity. Alpha 1 -Protease inhibitor (Mr 51 000), a homologue to human alpha 1 -protease inhibitor (alpha 1 -antitrypsin), also showed trypsin-, chymotrypsin- and elastase-inhibiting properties. Inter-alpha-trypsin inhibitor (Mr 162 000 and 129000), a porcine serum counterpart to human inter-alpha-trypsin inhibitor, showed trypsin- and chymo-trypsin-inhibiting properties. In addition, a specific trypsin inhibitor, alpha 2 -antigrypsin (Mr 58 000), and a specific elastase inhibitor, beta-elastase inhibitor, were characterized in porcine serum, and these seem to have no counterparts in human serum.

Alpha-fetoprotein and gamma-glutamyltranspeptidase in mice. Effect of Raf gene.

The regulation of the high physiological level of serum alpha-fetoprotein (AFP) in BALB/c/J mice was studied. Serum AFP concentration in 7- to 12-week-old BALB/c/J mice varied from 460 ng/ml up to 2,790 ng/ml and in other strains tested from 10 ng/ml to 400 ng/ml. In contrast to the difference in serum AFP level, activity of the fetal enzyme gamma-glutamyltranspeptidase (GGT) in the liver of BALB/c/J mice was similar to that found in other mice. In all newborn mice, the initially high GGT activity decreased almost 100-fold during the first 10 days. The serum AFP concentration stayed relatively stable during this period. AFP decreased to the adult level between the 10th and 30th days. Partial hepatectomy and administration of carbon tetrachloride increased the serum AFP level in all mice. The elevation of AFP was 10 times higher in BALB/c/J mice than in mice with low basal AFP level. A slightly elevated GGT activity in the liver was observed in only 10 out of 40 mice after carbon tetrachloride treatment, but none of the mice after partial hepatectomy showed an elevation. These results suggest that the gene ("Raf" gene) controlling the reduced decrease of serum AFP level in young BALB/c/J mice enhances the "turning on" of production of AFP in the regenerating liver. This gene does not, however, control the expression of the other carcinofetal liver marker, GGT.

Isolation of a SV40-like Papovavirus from a human glioblastoma.

A human glioblastoma multiforme (M27) tested in early cell cultures by indirect immunofluorescence staining showed SV40-related tumor (T)-antigen, 95% of the cells being positive. SV40-related viral capsid (V)-antigen was absent in all cells tested. Experiments to rescue this virus were performed by fusing M27 cells with CV-I monkey cells, which were permissive for SV40, using polyethylene glycol (PEG) as fusion factor. We succeeded in isolating virus particles SV40-GBM which electron microscopy showed to correspond in size and morphology to papovaviruses. Serological tests (hemagglutination, neutralization, fluorescent antibody) revealed that the virus is indistinguishable from SV40. Despite this apparent antigenic identity SV40-GBM differs slightly from SV40 wild type. This virus can propagate and produce CPE in both CV-I cells and primary fetal human kidney cells. Furthermore digestion of SV40-GBM DNA with the HindII/III restriction endonucleases revealed minor differences compared with the SV40 DNA. Therefore the virus SV40-GBM obtained from glioblastoma cells seems to be closely related to the SV40-PML viruses described earlier.

Orcein, collastin and pseudo-elastica: a re-investigation of Unna's concepts.

Orcein has been recommended for identification of elastin. Since other traditional elastica stains proved to be unspecific, it was deemed of interest to determine the selectivity of orcein and to review pertinent literature. Orcein was employed as a textile dye in ancient Egypt and was used for dyeing of wool and silk until the early 20th century. It was introduced into histological technic in 1878 as a stain for cytoplasm. Unna recommended it for demonstration of elastic tissue in 1890 and retracted claims for its specifity in 1894 because orcein colored also certain collagen fibers. Unna suggested the term collastin for collagen fibers which share the affinity of elastin for acid orcein. Other orcein solutions were used as selective stains for collagen. In histochemical studies, the staining properties of resorcin-fuchsin and orcein were very similar; elastin and various collagen fibers were strongly colored. Unna's collastin is apparently identical with the pseudo-elastica described in sections stained with resorcin-fuchsin. Both dyes react with meshworks of fine fibers, embryonic, experimentally or pathologically altered collagens. It is suggested to use the term collastin, instead of pseudo-elastica, for collagenous fibers which bind the traditional elastica stains.

Studies of metallobleomycins by electronic spectroscopy, electron spin resonance spectroscopy, and potentiometric titration.

The 1:1 bleomycin-A2-Cu(II) complex shows an absorption maximum at 595 nm (epsilon 120), circular dichroism extrema at 555 nm (delta epsilon + 1.21) and 665 nm (-0.61), and electron spin resonance (ESR) signal with g = 2.211, g = 2.055, and A = 178 x 10(-4) cm-1. The formation constant (log K = 12.630) and deprotonation constant (PKc = 3.585) of the 1:1 bleomycin-Cu(II) complex were determined by computer analysis of potentiometric data. The results of potentiometric titration also indicate that the stability of bleomycin-metal complexes is in the order Fe(II) less than Co(II) less than Ni(II) less than Cu(II) greater than Zn(II) and that these divalent metal complexes have a similar coordination environment. The bleomycin-Cu(II) complex has substantially a square-pyramidal configuration in which the secondary amine nitrogen, pyrimidine(N-1) ring nitrogen, deprotonated peptide nitrogen of histidine residue, and histidine imidazole(N-1) nitrogen coordinate to Cu(II) as planar ligand donors, and the alpha-amino nitrogen as axial donor. The specific Cu(II)-binding site of bleomycin has been compared with that of human serum albumin.

Analysis of hepatitis B surface antigen components solubilized with Triton X-100.

Three glycoproteins of intact hepatitis B surface antigen (HBsAg) with mol. wt. of 32 000, 30 000 and 28 000 respectively were identified by reaction with 125I-concanavalin A (Con A) after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen was effectively disrupted with Triton X-100 to produce a structure with a sedimentation coefficient of 3.9S. Affinity chromatography of disrupted HBsAg using concanavalin A-Sepharose 4B (Con A-Sepharose) resulted in two fractions. The first contained material which did not bind to the lectin and consisted of a single polypeptide of mol. wt. 64 000. Further studies revealed this component to be serologically identical to serum albumin and to lack any affinity for antibody to HBsAg. A comparison of the tryptic peptide map of this polypeptide with that of purified serum albumin demonstrated identical amino-acid sequences. The second fraction contained material which bound to Con A and contained two polypeptides with mol. wt. of 28000 and 23000 respectively. HBsAg reactivity was associated with this fraction. This procedure allows the prepartion of HBsAg sub-units in milligram quantities for further immunological studies.

Treatment of myasthenia gravis. Report on 139 patients.

In the treatment of myasthenia gravis (MG) considerable progress has recently been achieved. Our experience is based on the observation of 139 patients with an average follow-up of 3 years and 4 months. A treatment plan and results are presented. Indications for thymectomy: all cases of MG in adult life, apart from ocular myasthenia without radiological thymoma and without electrophysiological and pharmacological signs of generalization; before puberty only cases with radiological thymoma and severely incapacitating or life-threatening signs. Median sternotomy is preferable for thymoma, the transcervical approach with a sternal split for non-neoplastic thymus. Mediastinal radiotherapy is indicated after removal of an invasive or adhesive thymoma. Indications for corticosteroids: 1) before thymectomy: respiratory weakness; 2) soon after thymectomy: life-threatening signs; 3) later after thymectomy: incapacitating or life-threatening signs; 4) as an alternative to thymectomy: when surgery cannot be performed or it is not indicated. Oral Prednisone was nearly always preferred: alternate-day high single dose (75 to 115 mg) has given good results in most cases even if in some cases a small dose was required in the "off day"; inversely a lower alternate-day or daily dose was often sufficient. Long-term results: following this schedule for adult patients good results were scored in 67% of thymomas, in 94% of hyperplasias, and in 62% of unthymectomized patients: in prepuberal life the few cases of severe MG have all shown a favorable evolution.

Pattern reversal evoked cortical responses in normals. A study of different methods of stimulation and potential reproducibility.

Using a commercially available television set for stimulation of 78 healthy subjects, the upper limit of normal for the latency of the major positive wave (p 100) of checkerboard pattern reversal evoked potentials is practically the same as that obtained by employing slide projector combined with a rotating mirror. Potentials evoked by a small pattern, for purely foveal stimulation, were often difficult to evaluate exactly. Fixation of a large pattern at the upper border of the stimulus field brought no advantages as compared to the usual fixation in the center. Continuous prolonged recordings, with successive averaging of 64 pattern reversals, confirmed statements that only the major positive wave and its latency are constantly reproducible without being influenced by fatigue or inattention. Repeated examinations in the same subjects at intervals of 2 weeks showed a considerable spread of the latencies from one session to the other but the absolute values always ranged within the normal. More or less large latency differences (up to 12ms with large pattern stimulation) were found in every subject at least once. A longer latency found at a control examination, or a newly appearing latency difference, therefore do not prove a fresh optic nerve lesion as long as the absolute values are still within the limits of normal. From repeated examinations it could also be seen that foveal stimulation with a small pattern is not suitable for routine examinations because of high variation in the results.

Amylase secretion in the rabbit parotid gland when stimulating the sympathetic nerves during parasympathetic activity.

1. In anaesthetized rabbits amylase secretion from the parotid gland was investigated. Secretion was evoked by sympathetic nerve stimulation, either alone or superimposed on a parasympathetic background secretion, imitating the resting secretion present in the waking animal.2. Sympathetic nerve stimulation at frequencies below 1 Hz was alone subthreshold for fluid secretion, but could greatly increase the amounts of amylase present in fluid secretion produced by parasympathetic nerve stimulation. The amylase output due to sympathetic nerve stimulation alone at 10 Hz did not exceed that seen in response to a stimulation at 1 Hz superimposed on parasympathetic activity.3. The amylase output in response to superimposed sympathetic stimulation was not influenced by the rate of fluid secretion, which was altered by stimulating the parasympathetic nerves at different frequencies.4. Sympathetically-evoked amylase secretion was abolished after beta(1)-block. The amylase secretion remaining on parasympathetic activation was sparse.5. It is concluded that secretion of amylase in response to sympathetic nerve stimulation requires the presence of a parasympathetic fluid secretion to be washed along the glandular ducts. Parasympathetic activity may also augment the sympathetic effect on amylase secretion.

Staining of DNA-phosphate groups with a mixture of azure A and acridine yellow.

This paper deals with staining of DNA-phosphate groups with a mixture of an equal parts of aqueous solution of azure A and acridine yellow in a 1:1 proportion and also embodies a study of the absorption properties of the stained nuclei. It also embodies results of sequential staining of nuclei stained first with azure A followed by staining with acridine yellow and vice versa, after extraction of RNA with cold phosphoric acid. The results indicate that the absorption peaks of nuclei differ from those of nuclei stained for DNA-aldehyde molecules with azure A-SO2 or acridine yellow-SO2. The in vitro absorption characteristics of an aqueous solution of azure A and those of an aqueous solution of acridine yellow are also presented herein. The conclusion obtained from this study is that all the phosphate groups of DNA do not take part in the staining process when staining is carried out with azure A or acridine yellow alone after after RNA has been extracted. This is because the nuclei stained with these dyes sequentially show the presence of acridine yellow-DNA and azure A-DNA complex.

Longitudinal contraction of isolated guinea-pig ileum induced by rapid cooling.

1. Rapid change of bath temperature from 37 degrees C to 27 degrees C and vice versa caused longitudinal contraction of the isolated guinea-pig ileum. 2. Tetrodotoxin, tropicamide, noradrenaline, isoprenaline, morphine, and the met-enkephalin analogue FK 33-824 depressed the responses or accelerated the fade of the contraction induced by rapid cooling when added after the response had reached its maximum. 3. Hexamethonium had no influence on the responses. 4. Physostigmine potentiated all responses and reversed the fade of contraction induced by rapid cooling when added after this contraction had reached its maximum. 5. The effects of rapid cooling or warming were not altered in preparations made tachyphylactic to substance P; the response to rapid warming, but not cooling, was partially inhibited under tachyphylaxis to 5-hydroxytryptamine. 6. Antazoline, phentolamine, naloxone, and indomethacin did not block the responses. 7. Capsaicin firt potentiated and subsequently depressed the responses to both rapid cooling and warming. 8. The results indicate that rapid change of bath temperature induces longitudinal contraction by excitation of postganglionic cholinergic fibres.

Observations on fast axoplasmic transport in peripheral nerve following repetitive electrical stimulation.

In a series of 13 cats the effect of electrical stimulation of peripheral nerve on the mechanism of fast axoplasmic transport was studied. Electrical stimulation was used for varying time periods at parameters reported in the range of those used to produce electroanalgesia in man. Our results indicate that at these parameters, electrical stimulation produced no effect on this important aspect of nerve function, and, therefore, our work lends support to the safety of these devices in pain states.

T helper lymphocytes recognize the VL domain of the isologous mouse myeloma protein 315.

The localization of a major determinant on an isologous myeloma protein (M315) which stimulates BALB/c helper T cells was investigated. Augmentation of the adoptive secondary antibody response to NIP-M315 and the idiotype of M315 (Id315) was used as an indicator of helper effects. Spleen cells primed with the light chain of M315 (L315) and its V-domain (VL315) were highly efficient helpers; priming with the fragment containing the two V-domains of M315 (FV315) induced a somewhat weaker helper effect than L315 or VL315. The helper effect was abolished or markedly reduced by treating the primed cells with rabbit anti-brain theta + C. Cells primed with the heavy chain of M315 (H315) effected weak but significant help. The V-domain of H315 (VH315) was incapable of eliciting cells with detectable helper effect. The data indicate that the VL315 embodies a major determinant for T helper lymphocytes. This determinant is expressed on the free VL315 as well as on the complete M315. In contrast, previous studies have shown that BALB/c antibodies produced against Id315 recognize antigenic sites that are only displayed on associated (VL315 + VH315) domains.

The association of pyloric antral tissue levels of 5-hydroxytryptamine and restraint stress ulceration in the rat.

To determine the possible role of 5-hydroxytryptamine (5-HT) in the formation of restraint ulcerations, we investigated the relationship among mucosal tissue levels of 5-HT, vagotomy, and restraint ulcerations. Three murine experimental groups and one control group were used to compare the ulcerogenic effects of restraint with and without prior vagotomy. Ulcers were graded as to frequency, size, and hemorrhage on a 0-4+ scale. 5HT was measured in the gastric mucosa of each animal. Restraint caused significant ulcerations. The mean tissue level of 5-HT was significantly higher in the group with marked ulcerations (p less than 0.001). Our data suggest an increased cellular production of 5-HT during the development of the restraint ulcers. Vagotomy protected the animals against restraint ulcerations without depleting the baseline levels of 5-HT. Previous experiments have shown that urine levels of 5-hydroxyindoleacetic acid (5-HIAA) increase during the restraint period. Thus, increased tissue and urine levels of 5-HT appear to coincide with the development of restraint stress ulcers in the rat. Accordingly, if the coincidence occurs in humans, the measurement of urinary levels of 5-HIAA may be an indirect means of determining those patients at high risk for the development of stress ulcers.

[Viral acanthomas and specialized forms of keratinosome "membrane coating granules" (author's transl)].

In the case of viral acanthomas, the stratum spinosum and granulosum presents ballooned cells which contain all transitional stages from multivesicular bodies (MVB) to keratinosomes. A particularity in condylomata acuminata are the "wagon-wheel" bodies. These structures are typical for the non keratinazed squamous epithelium. The participation of intercellular extruded "wagon-wheel" bodies, MVB and atypical keratinosomes on an irregular baso-apical diffusion-barrier in the epidermis of cases with viral acanthomas has been discussed. On the basis of the relation seen between MVB and the Golgi-apparatus, their transition to partially atypical keratinosomes in cases of viral acanthomas and their "expulsion" into the intercellular space could indicate that in keratinozytes the enzymatically regulated feed-back between the cellular surface and the capability to synthesize is changed by viral agents. The interference appears to manifest itself in the Golgi-apparatus and also appears to be "specified" by the terrain present.

A comparative study in Porcellio laevis (Lat.) and Armadillidium vulgare (Lat.): response to temperature, photoperiod and oxygen concentration.

Relationship of body weight, imposed fasting, temperature, light intensity, and oxygen concentration to oxygen consumption in Porcellio laevis and Armadillidium vulgare has been investigated in a series of laboratory experiments. It was observed that (1) the metabolic response in the two species to temperature change was a uniform increase of oxygen consumption with increasing temperature from 15 degrees C to 60 degrees C. Beyond 30 degrees C, the oxygen consumption in each species fell, and the thermal death point was reached at about 40 degrees C. (2) The response to decreasing oxygen concentrations was a corresponding decrease in oxygen consumption. Armadillidium vulgare was a partial regulator while Porcellio laevis was able to conform its internal state to the changing oxygen levels. (3) In each species there was a decrease in metabolic rate with increasing body weight. (4) On the basis of their general activity level and oxygen consumption rate, Porcellio appeared to be a nocturnal species, while Armadillidium had a day active metabolism.

Repetitive DNA and Feulgen hydrolysis kinetics in three species of Bufo.

Two North American species of the genus Bufo (Bufo cognatus and Bufo boreas, 2n = 22) and one African species (Bufo regularis, 2n = 20) were analyzed with respect to their repetitive DNA fractions and the behaviour of their chromatin to the acid hydrolysis at different times. The mean melting point of the total isolated DNA decreased from 89 degrees C to 87 degrees C with a genome size increase from 4.4 to 7.5 pg. The differences in genome size can only partly be explained on the basis of repetitive DNA fractions (renaturing up to Cot 10 in 0.12 M phosphate buffer). Several fractions in this repetitive range behave independently in the three species and the spectrum of repetitive fractions in the African Bufo regularis differs distinctly from those of the American toads. When fixed chromatin of these species in histochemical preparations is hydrolyzed with 5N HCl during the Feulgen reaction, the kinetics of depurination are equal in all species, while hydrolytic DNA breakdown proceeds distinctly more slowly in Bufo reularis as compared to the other species.

The chemical heterogeneity of human hemoglobin F. Direct evidence for the existence of three types of gamma chain, the G gamma I, A gamma I, and A gamma T chains.

Direct evidence is presented for the existence of three types of gamma chain of human hemoglobin F. A modification of a CM-cellulose chromatographic method has allowed the incomplete separation of these gamma chains while high pressure liquid chromatography and fingerprint analyses of tryptic peptides of zones of the isolated gamma chains, and amino acid analyses of isolated peptides were used to identify the chains. These studies have shown that the presence of a glycyl residue in position 136 (G gamma chain) is directly related to that of an isoleucyl residue in position 75 (I gamma chain), thus indicating the existence of an G gamma I chain, and that the presence of an alanyl residue in position 136 (A gamma chain) can be related to that of an isoleucyl residue in position 75, thus suggesting the existence of an A gamma I chain. When the isoleucyl residue at positive 75 is replaced by a threonyl residue, invariably it is related to the alanyl substitution at position 136 (A gamma T chain). These data support indirect evidence from case analyses and family studies which were published before, and indicate that the T gamma chain is an allele of the A gamma which should be renamed the A gamma T chain.

Common leukemia-associated antigen of DBA/2 mouse leukemia detected by tumor rejection and complement-dependent cytotoxicity assays.

A cytotoxic antibody for L1210 leukemia cells was found in the (C57BL/6 x DBA/2)F1 (BDF1) mice immunized with L1210 leukemia cells infected with ts mutant of HVJ (HVJ-pi) and challenged several times with uninfected L1210 leukemia cells. These immune mice fell into two categories; high and low responders regarding the titer of cytotoxic antibody produced. The antigen defined by this cytotoxic antibody was present on leukemia cells originating in DBA/2 mice but not on leukemia induced by passage-A Gross virus or spontaneous mammary tumors. This serological cross-reactivity among L1210, P388, and L5178Y leukemia cells has been substantially confirmed by the observation of cross protection against challenge with DBA/2 leukemia cells in immune BDF1 mice. These findings strongly suggested the presence of a common DBA/2 leukemia-associated antigen different from known cell-surface antigens of murine leukemia. The results obtained in the present work also demonstrated the great efficacy of non-cytopathic, viable HVJ-pi-injected tumor cells as an immunogen for inducing tumor immunity.

A serum-free medium for the growth of muscle cells in culture.

Rates of cell proliferation essentially equal to those in 10% serum were obtained when Yaffe's L6 myoblasts were incubated in Ham's F-12 medium containing 10(-5) M fetuin, 10(-6) M insulin, and 10(-7) M dexamethasone; we have designated this mixture muscle medium-1 (MM-1). Addition of other growth factors and hormones in various combinations did not increase the proliferation of myoblasts above the rate in MM-1, and neither fetuin nor insulin could be replaced by other growth factors. All glucocorticoids tested (but no other steroid hormones) were active. Fetuins prepared by the rather different procedures of Pedersen, Deutsch, and Spiro were all active, and the active material was heat labile and nondialyzable; this is the first cell culture system in which highly purified Spiro fetuin has been found active. Primary rat myoblasts proliferated more rapidly that fibroblasts in parallel cultures when incubated in MM-1. This simple medium, composed of relatively inexpensive and readily available components, should be useful for the study of muscle cell growth and differentiation.

[Identification of nerve cells in the visual cortex of the rat using Nissl and Golgi-Kopsch methods].

The neurons of the visual cortex of the albino rat were studied using both the Nissl- and Golgi-Kopsch methods. In Nissl preparations we can distinguish between a group of neurons rich in cytoplasm, a group of neurons poor in cytoplasm and an intermediate group. In the Golgi preparations the neurons can be subdivided according to the shape of their cell bodies, dendrites and axons. Spiny cells with long axonal main trunks are pyramidal cells, multiangular cells and stellate cells of layer IV. Cells with spineless dendrites and short axonal arborization are basket cells, neuroglioform cells and small double bouquet cells. Due to its spines and the short axonal arborization, the coarse fusiform cell (Martinotti cell) is an intermediate type. We assume cells having long axons and dendritic spines are category I neurons and cells having short axons and no or a few spines are category II neurons (according to SZENTAGOTHAI 1973). On the basis of homological criterions and taxonomically relevant features references for identifying the cell group rich in cytoplasm and category I neurons, on the one hand, and the cell group poor in cytoplasm and category II neurons, on the other hand, were found. The group of cells rich in cytoplasm is related to pyramidal cells, multiangular cells and stellate cells of lamina IV. The group of cells poor in cytoplasm is discussed as corresponding to cells of lamina I, round or oval forms as basket cells and neuroglioform cells, fusiform cells as double bouquet cells. The intermediate cell form in the Nissl preparations is according to the Martinotti cell in Golgi material. These findings allow quantitative studies about particular cell populations and can, completed with electron microscopical date, instruct computer models to simulate the complicate neuronal network of the visual cortex.

Effects of intra-arterial bradykinin and substance P on isolated, blood-perfused small intestine of the rat.

Experiments were conducted on rat isolated, small intestine perfused at a fixed flow rate through the superior mesenteric artery with arterial blood from a donor rat, to determine the responses of the ileum to different peptides. Drugs were closely injected into the superior mesenteric artery. Single injections of bradykinin produced monophasic fast contractions of the ileum preceded by an initial fall and a subsequent rise of tone. The fast contraction was abolished by tetrodotoxin (TTX), morphine and hexamethonium (C6), but was resistant to blockade by atropine or mepyramine. Changes in ileal tone induced by bradykinin remained evident even in the presence of these blocking agents, thereby suggesting a direct action on smooth muscle fibers of the ileum. The fast contraction in response to substance P was not influenced by either TTX, morphine, C6, atropine or mepyramine. The present results indicate that bradykinin induces the fast contraction of the ileum by excitation of myenteric neuronal elements involving cholinergic interneurons, while substance P produces the contraction by a direct stimulation of smooth muscle fibers of the ileal region.

Chlorinated pesticide residues in chicken egg.

Two hundred and twenty-one representative samples of chicken eggs of native and commercial strains of Gallus domesticus were collected during January 1975 to August 1977 throughout the Tehran area and the Northern province of Mazandarane. The samples were analyzed for chlorinated pesticide residues by gas-liquid chromatography. The insecticides [1,1,1-Trichloro-2,2-bis (P-Chlorophenyl)ethanel] (DDT), [1,1-Dichloro-2,2-bis (P-Chlorophenyl) ethane] (TDE), [1,1-Dichloro-2,2-bis (P-Chlorophenyl)ethylene] (DDE), isomers of benzene hexachloride (BHC), Aldrin/Dieldrin, Heptachlor/Heptachlor epoxide, Dieldrin, and endrin were detected in varying concentration in the eggs. The eggs of native bred chickens, mainly as a result of their food sources, showed greater concentration of all pesticides except BHC isomers than those of commercial types. Though concentration of DDT compounds in 22% of native bred and 5% of commercial eggs exceeded WHO tolerance limit, the mean concentrations of pesticides residues were not exceeding these limits. There was no correlation between concentrations of pesticides and egg shell thinning.

Circulating immune complexes, free antigen and alpha 1-antitrypsin in levels in sarcoidosis patients.

Consecutive serum samples from 26 sarcoidosis patients were examined for circulating immune complex (IC) activity. Fifteen (58%) gave IC-positive reactions in a complement consumption (CC) test and a significant difference as regards anticomplementary was observed when comparing patients in clinical stages 2 and 1 respectively (P less than 0.01) The serum alpha 1-antitrypsin levels were not correlated (r=0.36) to the results of the CC-assay. The duration of IC-occurrence was studied, by two IC-assays, over 1 1/2 to 2 years. The majority of the positive reactions were registered in patients in clinical stage 2. Isolated IC was used for immunization of rabbits. Absorbed immune sera produced a single precipitate of postalbumin mobility with seven out of 36 sarcoidosis sera. In two cases a tailing of the recipitate suggested that the antigen was in complex formation. Immunoelectrophoresis indicated identity between the antigen detected in different sarcoidosis sera. No precipitates were observed using 130 sera from other patients, and screening of 100 blood donors revealed one positive reaction. The antigen, which eluted in the first protein peak on Sephadex G-200, has not been identified serologically.

Studies on fibronectin in the skin. III. Indirect immunofluorescence studies in lichen planus.

Fibronectin is a glycoprotein which is responsible for a varity of functions in the human organism, such as mediation of contact between cells and between cells and fibres, opsonic qualities, interaction in the stabilization of fibrin, etc. Fibronectin is an important constituent of the ground substance having a special affinity to collagen. In indirect immunofluorescence studies its presence has been abundantly demonstrated in normal human skin, in collagen-rich structures such as the basement membranes, the papillary and reticular dermis, and in the vascular and neural structures, demonstrable by its characteristic staining patterns. Fibronectin is not found in the epidermis. In lichen planus, the distribution in unaffected skin is identical with that in normal skin, whereas in affected skin, changes in the pattern of fibronectin are found. The basement membrane zone becomes broader and hazy, later undergoing disintegration and destruction, concomitant with swelling and homogenization of the reticular distribution of fibronectin in the papillary dermis. Globular structures containing fibronectin are found in the basement membrane area, together with an intensified immunofluorescence in the vascular system. Fibronectin has certain adhesional properties and changes in the distribution of this glycoprotein may result in loss of tissue stability. The pathophysiological significance of the changes of fibronectin in lichen planus is, however, difficult to evaluate at present.

Cerebrospinal fluid proteins electrophoresis without prior concentration.

There is a need for new methods to study CSF proteins. Kerenyi et al., 1973 and Verheecke, 1975 have already used this method and discussed its advantages. The electrophoresis is carried out according to the method of Wieme with as supporting medium agar. The volume applied varies between 5 to 15 microliters according to the total protein content and the width of the slit. Care must be taken to work at all times with bidistilled water and pro analysis reagents. After drying, the electrophoretic plates are reduced in a 10% solution of thiodiglycol, dried again, then immersed in 2% potassium hexacyanoferrate, washed thoroughly, then revealed with a silver nitrate solution (Kerenyi et al., 1973; Verheecke P., 1975) and left in 1% acetic acid. This technique is of major value as: 1. It avoids artefacts due to concentration and loss of proteins; 2. It works with very small amounts of fluid; 3. Where the CSF is silent for antibodies restricted heterogeneity using the classical methods, it reveals marked IgG fractionation.

[Effect of pyridoxine on histamine liberation and degranulation of rat mast cells].

Pyridoxine, one of the B vitamins, has been shown to be useful in the treatment of childhood bronchial asthma by Collip et al. (1975). A double-blind study with 76 asthmatic children followed for five months indicated significant improvement in asthma following pyridoxine therapy (200 mg daily) and a reduction in dosage of bronchodilators and cortisone. Other reports have shown that nicotinamide, another B vitamin shows inhibitory activity in rat mast cell degranulation and histamine release (Bekier et al. 1974, Wiczolkowska and Maslinski, 1975, 1976). These results induced us to investigate if pyridoxine, like nicotinamide or disodium cromoglycate, exhibits pharmacological inhibitory activity in rat mast cell degranulation and histamine release induced by antigen or other non-immunological stimulants. We found that pyridoxine at concentrations of 10 (-3) M, or greater significantly inhibited rat mast cell degranulation and histamine release induced by phospholipase A, compound 48/80, antigen (egg albumin) or a mixture of dextran and phosphatidyl serine, respectively. In these experimental models, pyridoxine shows a pharmacological profile similar to nicotinamide and disodium cromoglycate, although weaker than the latter. In spite of this, the lack of toxicity of this vitamin at relatively high doses (1 or 1.5 g), the possibility that other mechanisms of action may be involved, such as the improvement in tryptophan metabolism reported by Collip following pyridoxine therapy, suggest that this vitamine merits additional research.

Phosphorescence of protein-bound eosin and erythrosin. A possible probe for measurements of slow rotational mobility.

We used a pulsed dye laser working at 540 nm to excite triplet-state formation of eosin and erythrosin, either bound or unbound to bovine serum albumin, in aqueous solution anaerobically at pH 8 and 20-22 degrees C. Delayed emission from radiative transitions of the triplet state was readily detectable, both as delayed fluorescence and as red phosphorescence. Detection of the triplet state by measurement of phosphorescence at 645 nm upwards was at least 100-fold more sensitive than by absorbance measurements of ground-state depletion at 500 nm. When immobilized in poly(methyl methacrylate), the phosphorescence of eosin and erythrosin was polarized with an anisotropy parameter [Jablonski (1961) Z. Naturforsch. A16, 1-4] of about 0.25. The phosphorescence of erythrosin is sufficiently intense to be distinguishable from the long-wavelength end of fluorescence under conditions of continuous rather than pulsed excitation. Our observations suggest that phosphorescence depolarization of eosin or erythrosin probes could be used as a highly sensitive method of measuring rotational relaxation times in region from 10(-5) to 10(-3) s, such as those of the uniaxial rotation of membrane proteins.

The immature intestine: implications for nutrition of the neonate.

The survival and prognosis of the prematurely born human infant are dependent on a successful transition from the intrauterine to the extrauterine environment. This is largely a consequence of the maturation of sufficient gastrointestinal function to provide adequate nutrition. However, the gastrointestinal tract of the premature infant, and to some extent, of the full-term infant, may be unprepared to provide the requisite absorptive function. Data presented in this symposium emphasize the dissociations in the development of human gastrointestinal function. Morphological maturation is completed early in gestation while glucose absorption increases with gestational age. Sucrase and maltase activities appear early; lactase activity begins to increase at 30 weeks and increases steadily to term. The latter pattern is accompanied by increased production of cortisol and thyroid in the fetus. The intraluminal phase of fat digestion is immature even in the full-term neonate. Both pancreatic secretory function and bile salt metabolism mature postnatally. Despite this relative immaturity, breast milk fat is absorbed with great efficiency by the term infant, and breast milk provides other important influences on intestinal development: mitogenic factor, immunological support, control of intestinal flora. The goals of nutrition support of the premature infant have been to maintain intrauterine growth standards; yet premature infants receiving pooled breast milk from mothers at 40 weeks or more may be given too little protein for their needs. Human milk from mothers of premature infants may be a more appropriate nutrient source. Supplements with higher contents of amino acids may lead to amino acid imbalance or hyperammonaemia. Additional stresses and requirements are imposed by illness or congenital anomalies. While we must apply current research findings to clinical care, we must also extend our knowledge of extrauterine human development. The ultimate measure of success in this field will be the physical and neurological capacities of infants followed prospectively.

Rapid dextran infusion in essential hypertension.

Hemodynamic parameters were studied before and after rapid dextran infusion in 34 men including 17 patients with sustained essential hypertension and 17 normotensive controls. In both groups of patients, dextran infusion induced a significant increase (p less than 0.001) in central venous pressure (CVP), cardiac output (CO), and stroke volume. The percent change in stroke volume was significantly higher in hypertensives (p less than 0.001) than in controls. Three indices of volume expansion were calculated: 1) the ratio between the change in CO and the change in volume, which was significantly higher in hypertensives (p less than 0.025), 2) the ratio between the change in CO and the change in CVP, which was similar in both groups, and 3) the ratio between the change in volume and the change in CVP, which was significantly reduced in hypertensives (p less than 0.001). In the overall population, the latter ratio was negatively correlated with the change in CO (or in stroke volume) induced by expansion ( r = -0.75). The results provided evidence that: 1) the slope of the relationship between CO and blood volume was steeper in hypertensives than in normotensives, and 2) the steeper slope was due to a reduction in the effective compliance of the vascular bed, causing a greater elevation in CO per unit rise in volume.

Phosphonoformate inhibits reverse transcriptase.

The new antiviral substance phosphonoformate (PFA) has been tested in a cell-free system for its effect on reverse transcriptases from an avian retrovirus (avian myeloblastosis virus, AMV) and from mammalian retroviruses (Rauscher leukaemia virus, RMuLV; bovine leukaemia virus; baboon endogenous virus; simian sarcoma virus; visna virus). The observed inhibitory effect of PFA has been compared with that of a structurally related substance, phosphonoacetate (PAA). Phosphonoformate, at a concentration of 100 microM, reduced the activities of all the above mentioned polymerases by 90% when (rA)n.(dT)10 was used as a template/primer. The dose-response curves for AMV and RMuLV polymerases primed with (rA)n.(dT)10 showed PFA to be a 1000-fold more active than PAA; the RMuLV polymerase activity was reduced to 50% after incubation with 0.7 microM-PFA and 0.7 mM-PAA, respectively. There was no difference in PFA inhibition of virus-associated and purified reverse transcriptase activity. Results with various synthetic templates showed that both the RNA- and the DNA-dependent polymerase activities of reverse transcriptase were inhibited by PFA. The endogenous polymerase activity of AMV was inhibited to 50% at 100 microM-PFA, while PAA had no effect. The PFA inhibition was dependent on whether Mg2+ or Mn2+ was used as divalent cation in the assay. Phosphonoformate arrested DNA synthesis immediately after being added to the assay system. The mechanism of inhibition of the AMV polymerase was non-competitive with respect to substrate and template and the apparent inhibition constants were 16 microM and 9 microM, respectively.

Comparison of the antibodies elicited by the individual structural polypeptides of foot-and mouth disease and polio viruses.

Antibody produced against preparations of VP1, one of the four structural polypeptides of foot-and-mouth disease virus, neutralized the virus and reacted with both full and empty particles in radioimmunoassays (RIA). Antiserum against VP2 reacted with artificial empty particles of the virus but not with full particles. In contrast, none of the individual polypeptides of poliovirus produced antisera which neutralized the virus nor reacted with it in RIA. However, antisera produced with VP1 and VP2 reacted with artificial empty particles in RIA.

Hepatitis B viral DNA molecules have cohesive ends.

Hepatitis B virus DNA made fully double stranded by a virion DNA polymerase reaction could be converted from circular to linear molecules by heating in 10 mM NaCl at 77 degrees C or in 100 mM NaCl at 90 degrees C for 15 min. Heat-generated linear hepatitis B virus DNA was reannealed to circular molecules by incubating in higher salt concentrations. The identity of the molecular forms was established by their electrophoretic mobility and appearance in electron micrographs. Recircularization was blocked by reacting linear molecules with nuclease S1 or avian myeloblastosis virus reverse transcriptase. These results suggest that the heated linear DNA had single-stranded ends with complementary nucleotide sequences. It also suggests that a discontinuity or nick is present in each strand of the circular DNA molecule after the single-stranded region is made double stranded by the virion DNA polymerase reaction. The difference in contour length by electron microscopy of circular and linear molecules spread under aqueous conditions suggested that the discontinuities in the two strands were about 270 base pairs apart. The amount of nucleotide incorporated into the ends of heat-generated linear hepatitis B virus DNA by reverse transcriptase suggested that the single-stranded ends were about 305 bases in length. This fully double-stranded linear DNA was cleaved with EcoRI or HpaI restriction endonuclease. The sum of the two fragments generated by each totaled 3,510 base pairs, 310 base pairs greater than the contour length of circular hepatitis B virus DNA which represents a third estimate of the distance between the discontinuities in the two DNA strands of circular DNA. Restriction endonuclease cleavage also indicated that the ends of heated linear DNA which correspond to the discontinuities in the two strands of the circular DNA are at unique sites in the DNA with respect to the restriction sites.

Changes in cardiac norepinephrine in spontaneously hypertensive and stroke-prone rats.

The norepinephrine (NE) concentration of cardiac ventricles was determined by radioenzymatic assay in normotensive Wistar Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and stroke-prone rats (SPR) at 3-6, 14-19, and over 31 weeks of age. There was no difference between strains prior to hypertension, but a progressive decrease in cardiac NE concentration occurred in SHR and particularly in SPR relative to WKY after hypertension was established. This decrease was not due to cardiac hypertrophy. The cardiac neuronal NE storage capacity in rats over 31 weeks of age was analyzed by determining the maximum concentration of NE obtained in a cardiac microsomal fraction, after saturation in vivo with exogenous NE. The results indicated that, after a long period of hypertension, there was a reduction in cardiac NE storage capacity resulting from a loss either of sympathetic nerve endings or of storage vesicles. Moreover, in addition to this reduction in the total size of the cardiac NE store, there was an independent reduction in the degree of filling of this store in both SHR and SPR. This could reflect an increased turnover of cardiac NE in chronically hypertensive SHR and SPR.

Effects of phenytoin on refractoriness and conduction in the human heart.

Using His bundle electrograms and the atrial (A2) and ventricular extrastimulus (V2) techniques, anterograde and retrograde refractory period studies were performed (in 9 and 12 patients, respectively) before and 10 min after intravenous infusion of phenytoin (DPH; mean plasma level, 17.3 micrograms/ml). DPH had no effect on the duration of the QRS complex or the H-V interval of the sinus beats; it had variable but insignificant effects on the sinus rates and the atrial, A-V nodal, and ventricular muscle refractoriness. With the use of the A2 technique, the effective refractory period (ERP) of the His-Purkinje system (HPS) could not be determined in any patient; the relative refractory period (RRP) of the HPS could be determined in 2/9 patients and shortened in both patients after DPH. With the use of the V2 technique, retrograde functional refractory period (FRP) and RRP of the HPS could be determined in all 12 patients and the retrograde ERP of the HPS in 7/12; DPH significantly shortened all these parameters (p less than 0.001, less than 0.001, and less than 0.005, respectively). Functional refractory period of the ventriculo-atrial conduction system (VACS) could be determined in 11/12 patients during control studies (the remaining one patient had complete ventriculo-atrial block). DPH significantly shortened the FRP of the VACS in those (4) patients (Group I) in whom it was determined primarily by the HPS (p less than 0.025), and had variable but insignificant effects on FRP of the VACS in the other seven patients (Group II) in whom it was determined almost exclusively by the A-V node. DPH significantly decreased the retrograde HPS conduction times of the premature impulses (V2H2 intervals) for the same coupling (V1V2) intervals (p less than 0.001). It is concluded that, in the human heart, DPH exerts its most important effects on the HPS where it significantly decreases refractoriness and enhances conduction of the premature impulses. This study also demonstrates that the V2 technique is far superior to the A2 technique for evaluating the effects of drugs on refractoriness and conduction in the HPS.

Sodium intake and plasma angiotensin level as modulators of adrenal and uterine angiotensin II receptors in the rat.

Angiotensin II receptors from rat adrenal cortex and myometrium were studied with the use of tritiated angiotensin under conditions where the sensitivity of the target organs for angiotensin II is modified. Sodium status was found to modulate the number of angiotensin receptors both in adrenal gland and uterus. In both target tissues low Na+ diet increases the number of receptors, while a high Na+ diet results in an increase in uterine receptors without modifying adrenal cortical receptors. However, a more markedly positive sodium balance, such as that observed in deoxycorticosterone acetate (DOCA) hypertension and in one-kidney Goldblatt hypertension, resulted in a reduction of the adrenocortical angiotensin II binding capacity. The endogenous angiotensin II level may also regulate the number of receptor sites as demonstrated by an increased number of receptors after suppression of circulating angiotensin II. It is proposed that the number of angiotensin II receptors is determined by the combined influences of sodium status and angiotensin II concentration. Some changes in the sensitivity of the target organ can be secondary to variations in the number of angiotensin receptors. However, others cannot be so explained and stem, therefore, from events occurring beyond the hormone-receptor interaction.

Effect of adrenergic receptor blockade on the responses to isometric handgrip: studies in normal and hypertensive subjects.

Heart rate and blood pressure (BP) responses to a standardized 3 min isometric handgrip (IHG) test were measured in 32 normotensive men and compared with those found in 35 age-matched, drug-free men with established essential hypertension (BP range: 140--170/90--110). IHG testing in the normal subjects induced significant increases in heart rate (mean +/- SE: 7.6 +/- 3.3 beats/min), systolic blood pressure (19.4 +/- 5.3 mm Hg), and diastolic blood pressure (15.6 +/- 4.6 mm Hg). Although beginning from higher resting levels the hypertensive patients had similar degrees of increase in all three parameters. After chronic treatment with propranolol, the heart rate increase with IHG was suppressed in both study groups, but blood pressure responses differed, with a diminished pressor response to IHG seen in normal subjects and augmented pressor effects in the hypertensive group. Intravenous administration of phentolamine and propranolol completely abolished the pressor effects of IHG. These observations suggest that the autonomic control mechanisms mediating the responses to isometric exercise function similarly in drug-free normal and hypertensive patients and that the responses to IHG, mediated largely by endogenous catecholamine release, can be prevented by peripheral sympathetic receptor blockade.

Electrophysiological actions of lorcainide in patients with cardiac disease.

Lorcainide is a new antiarrhythmic agent which effectively suppresses ventricular premature contractions. Its electrophysiological actions were studied in 15 cardiac patients with and without cardiac conduction abnormalities. In a dose of 2 mg/kg body weight, lorcainide decreased the sinus cycle length (SCL) and increased the corrected sinus node recovery time. The intra-atrial, atrioventricular (AV), and intraventricular conduction times were prolonged. A third-degree AV block developed in 2 patients with pre-existing conduction abnormalities. QRS widening showed that intramyocardial conduction was also affected. The effective refractory period of the atrioventricular node (ERP-AVN) was shortened, whereas the effect on the functional refractory period of the AVN was variable. The decrease in SCL and ERP-AVN may be due to a possible anticholinergic effect of the drug. The accessory pathway was blocked in 3 patients with a Wolff-Parkinson-White syndrome. The ERP of the ventricle was slightly prolonged. The electrophysiological profile of the drug, i.e., slowing of conduction velocity throughout the heart combined with shortening of SCL and ERP-AVN, differs from other antiarrhythmic agents.

Interactions between clonidine and alpha-adrenoceptor blocking drugs on the tachycardic response to stimulation of the cardiac nerve in dogs.

In pentobarbital-treated dogs clonidine (10 micrograms/kg) reduced the increase in heart rate caused by electrical stimulation of the cardiac nerve (1-10 Hz). We studied the actions of six alpha-adrenoceptor blocking drugs. Yohimbine (0.3 mg/kg) and phentolamine (1 mg/kg) potentiated the effects of nerve stimulation and antagonized the inhibitory effects of clonidine. Piperoxan (1 mg/kg) increased the response to nerve stimulation but antagonized the effects of clonidine only at the lowest frequency of stimulation. Thymoxamine (1 mg/kg) and prazosin at high doses (1 mg/kg) also antagonized the effects of clonidine but failed to increase the positive chronotropic response to stimulation of the cardiac nerve. AR-C239, a new and potent alpha-adrenoceptor blocking agent, changed neither the response to nerve stimulation nor the inhibitory effect of clonidine. The effects of all these drugs were observed at doses which reduced or reversed the pressor response to adrenaline. Therefore, our results afford further evidence for a dissimilarity between postsynaptic and presynaptic alpha-adrenoceptors in the dog. In addition, they show that the failure of an alpha-adrenoceptor blocking compound to increase the response to nerve stimulation does not necessarily indicate a lack of presynaptic alpha-adrenoceptor blockade.

Correlation between pharmacokinetics and intropic and electrophysiologic responses to digitoxin in the intact dog.

We attempted to correlate inotropic and eletrophysilogic digitoxin effects with serum digitoxin concentrations during 8 hr after a single dose. Eight pentobarbital-anesthetized Labrador dogs were given 2.0 mg digitoxin intravenously. Left heart catheterization and His bundle registration were performed. acing and programed electrical stimulation were used to determine heart rate-independant changes in dP/dt, intra-atrial, atrioventricular (AV), His-Purkinje, and intraventricular conduction velocity. The effective (A-ERP) and functional (A-FRP) refractory periods of the atrium and the functional nodal refractory period (AV-FRP) were measured. Serum digitoxin concentrations were determined by radioimmunoassay. Median serum digitoxin half-time was 5.7 hr. The dP/dt increased after digitoxin, with maximum values after 2 hr. Digitoxin concentration correlated with the inotropic response after 4 hr. Heart rate fell significantly within 2 min and remained below control values for the 8 hr observation period concimitant with an increase in AV-nodal conduction time and AV-ERP increased significantly in the late elimination phase, while A-FRP increased slightly initially and remained high. No consistent correlation was found between the electrophysilogic variables and serum concentrations. We conclude that the two main effects of digitoxin, the inotropic and electrophysiologic, are dissociated in the elimination phase after a single dose.

Action of substance P on neurotico-hypertensive rats.

The action of an eledoisin-hexapeptide analogue (EH) upon learning and memorising processes of 48 male Wistar laboratory rats aged between 5 and 6 months was studied and is reported in this paper. The animals suffered from neurogenic hypertension which had been experimentally induced by applying emotional stress. A comparison between the action of EH (Lys-Phe-Ile-Gly-Leu-MetNH2) and that of Substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH2) on conditioned-reflex learning in the intact rat had been reported by the authors in one of their previous papers [7]. The following results were obtained with regard to EH and its action upon rats with neurogenic hypertension. The learning process was favoured, as it had been by 2 or 3 weeks of exercise. Defective learning and memorizing process as well as impaired behavioural patterns, interpreted as neurotic phenomena, were normalized by doses of 250 microgram/kg and 500 microgram/kg. Blood pressures were reduced, depending on dosage. The action of the EH analogue used on the central nervous system was stronger than that on blood pressure. Discontinuance of peptide application was followed by the phenomenon of "state-dependent learning". The results are likely to suggest possible involvement of such peptide sequences in the regulation of processes which are relevant to the whole. That effect is of particular interest, as Substance P is under discussion as a transmitter or modulator in mammals.

Affinity of intramitochondrial granules for ruthenium red accompanying induced cell death in chick embryos.

To test the hypothesis that ruthenium red binding of intramitochondrial granules might reflect an altered or pathological state of membranes associated with degeneration, embryos were treated with 6-AN to induce cell death in cartilaginous skeletons of chick embryos. Cervical cartilage from normal, 6-AN-treated and nicotinamide-alleviated 6-AN embryos was examined ultrastructurally for presence of IM RR-positive granules. Mitochondria of normal cervical chondroblasts which undergo normal phenotypic expression acquire RR-positive granules, although few mature cells are observed in young embryos. Necrotic chondroblasts, chondroblasts in various stages of degeneration, and proliferating chondrogenic cells of 6-AN-treated embryos all demonstrated induced RR-positive IM granules. Foci of degenerating chondroblasts, with mitochondria demonstrating RR granules, were observed infrequently in teratogen-alleviated tissue. The cytological features induced by 6-AN confirm its lethal effect and the degenerative effect on membranes presumably "unmasks" mitochondrial Ca-affinity sites which then become RR-positive. Cytochemical observations correspond with the biochemical and structural changes induced by 6-AN and confirm the hypothesis that RR-positive sites are the result of pathological changes.

Microfluorometric quantitation of size, deoxyribonucleic acid and viral antigenic determinants in cells productively infected with HSV-2.

The interaction of herpes simplex virus type 2 (HSV-2) with the eukaryotic human cell line (HEp-2) was investigated by flow microfluorometric analysis. The three parameters that were quantitated include cell size, deoxyribonucleic acid content and the expression of virus-specific antigens. Productively infected cells are always smaller than uninfected ones, and they resolve into two populations with respect to viral antigenic content. Consistent with viral replication, one of these two populations, displaying a higher viral antigenic content, shows morphologic features characteristic of cell degeneration. On the other hand, the second population, with a relatively lower content of viral antigens, displays morphologic features consistent with increased cell growth. Indeed, microfluorometric measurements of the HSV-2-infected cells with respect to DNA content and expression of viral antigenic determinants resolves four cell populations. Of them, one displays the lowest level of viral antigens with the highest (equivalent to 8N) DNA content, features consistent with transformation. The results demonstrate the great potential of this technique for the detection, separation and quantitation of statistically significant cell populations not otherwise identified and for the analysis of virus-host cell interactions resulting in different pathologic outcomes.

Immune retention: immunological requirements for maintaining an easily degradable antigen in vivo.

Radioactive human serum albumin (125I-HSA) was injected into the hind foot pads of unimmunized mice, actively immunized mice and mice passively immunized with mouse or rabbit anti-HSA serum. Eleven days later the unimmunized mice had cleared most of the 125I-HSA. In contrast, a high concentration of 125I-HSA was retained in the feet and draining lymph nodes and, to a lesser extent, in the spleen of the actively or passively immunized mice. Although immune retention required specific antibody, it appeared to be independent of T-cells or T-cell factors, since passively immunized nude mice retained antigen as well as actively or passively immunized normal mice. Depletion of the complement system with cobra venom factor (CVF) increased antigen retention in the feet but decreased retention in the spleen. Treatment with CVF did not decrease antigen retention in lymph nodes of actively immunized mice. Such treatment did, however, decrease retention in lymph nodes of passively immunized mice although not to the same extent as in the spleen. Retention of antigen in the feet was not only complement-independent but was also Fc independent, since F(ab')2 fragments of IgG could mediate immune retention. Antigen dose response studies indicated that immune retention in lymph nodes occurred optimally with minute amounts of antigen, whereas optimal retention in the feet required much higher concentrations of antigen. Foot pad injections of non-radiolabelled HSA eliminated 60% of the radioactivity retained in the foot pads of immunized mice. In contrast, non-radioactive egg albumin (EA) had almost no effect on retained HSA. However, if the mice were immunized to both EA and HSA, an injection of EA would displace a significant amount of retained HSA. Complexes of one specificity can apparently displace some retained antigen of a differing specificity.

Chlormadinone acetate.

This monograph on chlormadinone acetate (CA) includes chemical and physical data (synonyms and trade names), structural and molecular formulae and molecular weight of CA, chemical and physical properties of CA, and the production, use, occurrence, and analysis of CA. Production of CA, which is not known to occur naturally, occurs by treatment of 17-acetoxyprogesterone with ethyl orthoformate in the presence of an acid catalyst to produce the 3-enol ether of the corresponding 3,5-dione. Typical analytical procedures for determining CA as a bulk chemical are summarized tabularly. CA, before suspension from commercial use, was marketed as an ingredient in sequential oral contraceptives. It is approved as an estrus regulator in cattle feed. Biological data relevant to the evaluation of carcinogenic risk to humans are presented in brief. Experimental work with dogs, mice, and rats using oral administration has been done. When CA is combined with mestranol and given to mice, it increases the incidence of pituitary tumors in both sexes; combined with ethinylestradiol it increased the incidence of mammary tumors. When CA was given alone to dogs, mammary tumors developed. No human case reports or epidemiological studies on CA alone are available. Hence, it was concluded that there is limited evidence for the carcinogenicity of CA in dogs. In humans, oral contraceptives containing estrogens in combination with progestins have been related causally to increased incidence of benign liver adenomas and decreased incidence of benign breast disease, and so CA is implicated in this respect.