[Directed chemotherapy of neoplastic diseases. Significance of the purine-de novo synthesis and cyclic AMP in normal and leukemic leukocytes]. Studies on purine de novo-synthesis and cyclic AMP in normal and pathologic leukocytes. The destruction of neoplastic cells by cytostatic agents depends on the existence of metabolic reactions within the cells which can be specifically blocked by these agents. Concerning the action of purine antagonists there is, as yet, no clear evidence whether de novo synthesis of purines takes place in normal as well as in leukemic leukocytes. Therefore studies were performed to evaluate the capacity of these cells to incorporate the purine precursor, glycine, into their acid-soluble adenine nucleotides. The results demonstrate that "resting" human lymphocytes are not capable of building purines de novo. However, stimulation of the cells with a mitogen (Phytohemagglutinin) leads to the induction of de novo purine synthesis. Leukemic cells do not show uniform behaviour. In order to get more information about the mechanisms by which synthesis of purines as well as the morphologic transformation of lymphocytes may be induced, the metabolism of cyclic AMP was investigated. According to our results the nucleotide does not play an essential role in initiating the transformation process, but it seems to have regulating function in the further development of cell transformation. The studies furthermore indicate an important role of cyclic AMP during proliferation of leukemic cells.


Interactions of glucagon and related peptides with chicken adipose tissue. The effect of glucagon, Vasoactive Intestinal Polypeptide (VIP), secretin and gut glucagon on the cyclic adenosine 3'5' monophosphate (cAMP) level, and on the specific binding of these 125I-peptides to the adipocyte plasma membrane was measured in chicken adipocytes and compared to the results obtained in rat adipocytes. The displacement of 125I-glucagon from its specific sites was observed with about the same concentration of unlabeled hormone in fat cell plasma membranes of both species. However, the rise in cAMP induced by glucagon was much higher in chicken than in rat adipocytes. In chicken fat cells unlike rat fat cells, the cAMP accumulation elicited by glucagon was maintained during at least 60 min even in the absence of theophylline. Theophylline at 1-10 mM potentiated the glucagon-stimulated cAMP levels in rat fat cells, but had only a slight effect, if any, in chicken adiposyces. Porcine VIP, secretin or gut glucagon exerted no detectable action on the cAMP level of chicken adipocytes. The lack of cAMP accumulation was in good agreement with the absence of binding of 125I-VIP and 125I-secretin by chicken plasma membranes. These findings suggest that: 1) the difference of glucagon effect in rat and chicken fat cells results from variations in the rate of degradation of cAMP rather than from differences in the specific binding of glucagon between the two species; 2) the use of chicken fat cells is suitable to discriminate between glucagon and structurally related peptides from mammals.


Influence of the assay conditions on the lipolytic potency in vitro of different adrenocorticotrophins. The lipolytic activity of ACTH-(1-39) and ACTH-(1-24) in vitro has been compared on a molar basis in assays performed with both adipose-tissue slices and isolated fat cells. In the tissue-slice assay ACTH-(1-24) displayed 29% of the potency of ACTH-(1-39), whereas in the isolated-cell assay, ACTH-(1-24) was 3.9 times more potent than the longer peptide. This discrepancy seems to be due to the fact that ACTH-peptides are inactivated in the adipose-tissue-slice assay, but remain stable in the isolated-fat-cell assay. ACTH-(1-24) is inactivated more rapidly than ACTH-(1-39). Pre-incubation and washing of the tissue slices removes the inactivating principle, which most likely consists of peptidases, and their responsiveness is increased, so that both ACTH-peptides display similar lipolytic activity to that seen in the isolated-cell system. Comparison of the responsiveness of isolated fat cells and isolated adrenal cells indicates that the affinity of ACTH for the receptor of adrenal cells is approximately 100 times greater than its affinity for the fat-cell receptor.


Isolated corticotrophin-deficiency found through alcohol-induced hypoglycemic coma. A case of hypoglycemic coma after alcohol ingestion was observed in a chronic alcoholic. Upon close examination isolated corticotrophin-deficiency was found. It is suggested that ethanol-induced hypoglycemia may be consistent with dysfunction of mitochondria in hepatic cells and that there may be disorder of the hypothalamus in the chronic drinker.


Urinary cyclic 3',5'-adenosine monophosphate (cAMP) in diabetic subjects with and without symptomatic hypoglycemia and in normal subjects with insulin-induced hypoglycemia. Urinary cAMP was investigated in diabetic subjects. It was found normal in patients on oral antidiabetics and on insulin in good or poor control. Patients with insulin-induced symptomatic hypoglycemia, however, excreted increased amounts of cAMP in the urine. To demonstrate the significance of hypoglycemia we induced hypoglycemia by i.v.-injection of 0.15 U insulin/kg body weight in six normal subjects. An increase of urinary cAMP was found even during the first 15 minutes with maximal hypoglycemia. Determination of urinary cAMP seems to be helpful in the diagnosis of unrecognized hypoglycemia and counterregulation.


Long-term treatment of acromegaly with bromocryptine: postprandial HGH levels and response to TRH and glucose administration. Fourteen patients with acromegaly were treated with bromocryptine (CB 154, Sandoz), 4 X 2.5 mg, for periods of up to eleven months. One patient did not tolerate the drug, ten of the remaining thirteen experienced considerable clinical improvement. There was a dose-dependent suppression of plasma growth hormone levels, but growth hormone response to TRH injection and to glucose administration was still present during therapy although reduced. TSH response to TRH was not significantly altered. The suppressive power of bromocryptine on growth hormone appears to be related to the mechanism by which TRH stimulates growth hormone secretion in acromegaly, but long-term administration of this drug may be successful in spite of an absent response to TRH in some cases. Bromocryptine appears to be a safe and effective drug for the treatment of acromegaly.


Effect of an oral contraceptive on the lysine-8-vasopressin test in the diagnostic evaluation of pituitary function. A comparative study was performed on the plasma 11-hydroxycorticosteroids (11-OHCS) responses to lysine-8-vasopressin (LVP) before and during ingestion of an oral contraceptive wieh oestrogenic activity. A total of 19 healthy women with a normal menstrual cycle and normal plasma 11-OHCS content in blood samples at 8 a.m. and 4 p.m. were included in the study. The results show that the mean response of plasma 11-OHCS to the administration of LVP was equal in magnitude before and during ingestion of the contraceptive, although the baseline value of 11-OHCS doubled as a result of the treatment. On the other hand, during contraceptive treatment the increase of plasma 11-OHCS concentration after the administration of LVP was somewhat slower, reaching its peak later than before treatment. The noticeable variation in individual responses of plasma 11-OHCS to LVP increased during the treatment. Three subjects showed weak responses to LVP, although the adrenocortical responses to exogenous ACTH were quite normal. The inidvidual responses to LVP before and during ingestion of an oral contraceptive with oestrogenic activity are discussed.


Studies in vivo on the effects of parathyroid hormone upon kidney cyclic adenosine 3',5'-monophosphate content using rapid tissue fixation by microwave irradiation. Microwave irradiation is shown to be a useful method for simultaneously killing chicks and fixing tissues. Renal adenylate cyclase and phosphodiesterase activities were rapidly abolished by microwaving. The increase in chick kidney cyclic adenosine 3',5'-monophosphate (cyclic AMP) content produced by intravenous bovine parathyroid hormone (PTH) injection was much greater in microwaved birds than in those killed by cervical dislocation with subsequent tissue fixation in liquid nitrogen. After PTH injection there was a prolonged elevation of renal cyclic AMP content. At the time of maximum response (2 minutes), log. dose-response curves were linear in the dose range 0.1-10 U. The responses to three different bovine PTH preparations were indistinguishable. Arginine vasopressin, arginine vasotocin, salmon calcitonin and prostaglandin E1 did not affect kidney cyclic AMP content within 2 minutes. Because of its specificity and precision, the method is of use for the in vivo bioassay of PTH. Injection of CaCl2 (20 mumoles) 1 minute before, or conjointly with, bovine PTH inhibited the subsequent increase in kidney cyclic AMP content. The synthetic bovine PTH peptide fragments BPTH (1-34) and BPTH (2-34) both increased chick kidney cyclic AMP content. The use of such fragments allows investigation of the structural requirements of PTH for interaction with the systems regulating cyclic AMP metabolism in the kidney in vivo.


Purification and characterization of N-acetylneuraminate lyase from Clostridium perfringens. Clostridium perfringens cells were cultivated on a large scale using an automatic system. 2) N-Acetylneuraminate lyase, which is a cytosolic enzyme, was liberated from the bacteria by cell lysis using lysozyme in hypotonic solution. The enzyme was purified 770-fold by precepitation with ammonium sulfate, filtration on Sephadex A-50 and final preparative electrophoresis in a 7.5% polyacrylamide gel. Yield: 12 mg from 1 kg wet cell paste; specific activity: 167 nkat/mg protein. 3) The enzyme preparation appeared homogeneous in analytical disc electrophoresis, in gel electrophroesis in 0.1% sodium dodecylsulfate or 8m urea and in immunoelectrophoresis. Contaminating enzyme activities were not detected. 4) The isoelectric point of pH 4.7 was found for the enzyme. At 278 nm a molar extinction coefficient of 6.4 x 10(4)M-1 Xcm-1 was determined. The enzyme exhibited a Km value for N-acetylneuraminic acid of 2.8mM at its pH optimum of pH 7.2. The pH dependence of the Km value gives evidence that an ionizing guoup in the active center of the enzyme with  a pKe value of 6.4 may be involved in the catalytic reaction. Pyruvate inhibited the cleavage reaction of N-acetylneuraminic acid competitively; Ki = 2.9mM. 5) An average molecular weight of 99200 was determined for the native enzyme using different methods. After denaturation in sokium dodecylsulfate or urea, a mean molecular weight of only 50000 could be demonstrated, indicating the existence of two enzyme subunits. The lyase molecule was shown by electron microscopy, using a negative staining technique, to consist of two hemispherical parts. 6) Two active sites per native enzyme molecule, probably corresponding to one active site per subunit, were found by incubation of the enzyme with radioactive pyruvate followed by borohydride reduction. The results obtained from chemical modification of the lyase with 5-diazonium-1H-tetrazole and iodocaetamide under various conditionsare interpreted as evidence for the presence of two reactive histidine residues in the enzyme molecule. It is probable that one residue per subunit forms the nucleophilic group participating in enzyme catalysis. A model suggesting the mechanism of reversible cleavage of N-acylneuraminic acids by the lyase is presented.


[Coenzym properties of some Ade-1- and Ade-N6-substituted NAD derivatives (author's transl)]. By reaction of NAD with different oxiranes or with aziridine, derivatives of the coenzyme are obtained with substituents in position 1 or on the amino group in position 6 of the adenine ring. While the Ade-1-substituted derivatives show high Km values with different dehydrogenases and are reduced only very slowly by these enzymes, the coenzyme derivatives substituted at the amino group behave very similarly to NAD. Correlations were found between coenzyme efficiency of the compounds and the lipophilic character of their substituents. The results can be interpreted from the structure of the active site of the dehydrogenases investigated.


Placental enzyme polymorphisms in Canadian populations. Phenotype distributions and allele frequencies of adenylate kinase and esterase D were determined for four Canadian populations. In two population samples from south-western Ontario, allele frequencies at both loci were similar to those of European populations. In two northern, indigenous populations, the allele AK2 was not detected. There was variation at the EsD locus with EsD2 having a frequency of 0.176 in an Indian population, and 0.156 in an Eskimo population.


A sensitive and reproducible assay for the quantitation of erythrophagocytosis and its correlation with reduction in tritiated thymidine uptake in a tumor target cell system modified by immunoenhancing or immunosuppressive agents. A new and sensitive assay procedure for studying erythrophagocytosis is described. The assay technique permits quantitation of the in vivo and in vitro effects of chemicals, hormones, and cell, or microbrial products, on the level of phagocytic activation of glass-adherent cells. The effect of intraperitoneal injection of BCG, Zymosan, Vitamin A, B. pertussis, cortisone, estrone, and thioglycollate on phagocytic activation of peritoneal exudate cells harvested from two days up to 28 days following drug injection was examined by this assay. Erythrophagocytosis was compared to the effect of "activated" spleen cells on tritiated thymidine uptake of a tumor target cell suspension.


Genetic role of rat macrophage cytotoxicity against tumor. Macrophages from the Lewis (Le) rat strain are significantly more cytotoxic to a Moloney sarcoma tumor both in vivo and in vitro, than are macrophages from the Brown Norway (BN) strain. Activity of macrophages from (Le x BN)F1 rats that are histocompatible with the Moloney sarcoma tumor is directed toward tumor and/or virus-associated antigens and is expressed as a dominant genetic trait. Experiments with backcross rats suggest that the genetic factors are unrelated to the major histocompatibility locus (AgB) of the rats. BN microphages, although not active against tumor and/or viral antigens, can become cytotoxic to cells displaying Le alloantigens.


Interactions of murine leukemia virus (MuLV) with isolated lymphocytes. I. Virus replication in lymphocytes infected with Friend virus and cultures in diffusion chambers in vivo. A new technique for infection of mature lymphocytes with murine leukemia virus (Friend) MuLV-F) is described. Spleen cells for normal, non-infected donors were placed into diffusion chambers (constructed with 0.22 mum por size Millipore filters) which were then implanted into the peritoneal cavities of normal syngeneic recipient mice. The cells were infected with an injection of MuLV-F into the peritoneal cavity and, in some instances, also by placing virus into the chambers. Cells were recovered by treating the chamber content with elastase and collagenase. The infection was determined in two ways: (1) cells with replicating MuLV were enumerated as infection centers (IC) on S+L- indicator cells; and (2) virus-related cell membrane antigen (MA) was detected by immunofluorescence. Cells recovered from chambers after 2-3 weeks of culture represented about 10% of the original inoculum; viability was approximately 90%. The number of IC in MuLV-F-infected chambers was about 10 times higher than obtained by infection and cultivation of spleen cells in vitro. The kinetics of IC and MA in chamber-cultured. MuLV-F-infected spleen cells was similar to that in the spleen of infected mice during the first 10 days after infection. Later on, the process of infection within the chambers slowed down, reaching about 50% MA-positive and about 10% IC-positive cells, whereas the number of both IC- and MA-positive cells in the spleen reached 80% or more. The infection of splenic lymphocytes in diffusion chambers occurred equally well when chambers were implanted into: (1) syngeneic, virus susceptible hosts; (2) syngeneic, lethally irradiated hosts; and (3) allogeneic, virus-resistant hosts, suggesting that the process within the chamber is independent of MuLV replication in the tissues of the chamber-bearing mouse. The diffusion chamber technique seems to provide an environment in which various types of isolated lymphocytes of different mouse strains can interact with MuLV almost as efficiently as in vivo.


Detection of anti-tumor antibody in virally induced tumors and its relationship to tumor growth. The humoral antibody response to virally induced tumors insyngeneic hosts has been studied. The tumors include an SV40 tumor SVT2, the Friend virus-induced leukemias FBL-3 and FLC; and Moloney sarcoma virus-induced tumors. It was found that antitumor antibodies could be detected by the isotopic antiglobulin technique in these tumor systems at a relatively early stage of tumor growth. The kinetics of the antibody response in relation to the status of tumor growth varied between different tumors. In geneumor growth than in the regressors of tumor-free hosts. Reinoculation of tumor cells or recurrence of tumor growth produced elevation of antibody levels (secondary response). The specificity of the antibody reactions also varied in different tumor systems: some antibodies were truly tumor-specific and thus might produce a biological effect on in vivo tumor immunity, whereas others were not. These studies indicated that a sensitive antibody assay could be used for early detection of tumor growth. However, its usefulness in evaluation of the status of tumor growth should be carefully studied in each tumor system.


Augmented immunogenicity of tumor cell membranes produced by surface budding viruses: parameters of optimal immunization. Membranes prepared from tumor cells infected with surface budding viruses are much more immunogenic than membranes from uninfected tumor cells. Factors affecting immunization with membranes from virus-infected tumor cells were studied. Preparations made with influenza virus were clearly superior to those prepared with vesicular stomatitis virus (VSV). Membranes infected with VSV were maximally immunogenic at a dose equivalent to a 10% cell pack whereas influenza-virus-infected membranes were immunogenic at 1/100th of this dose. Subcutaneous inoculation was better than other routes of administration. Maximum protection against challenge with viable tumor cells was afforded by two inoculations of VSV-infected membranes spaced 3 days apart or a single inoculation with influenza-virus-infected membranes. Administration of membranes in complete Freund's adjuvant either had no effect of induced a slight degree of tumor enhancement. Immunization with influenza-virus-infected membranes significantly reduced tumor size and incidence even at a challenge dose of tumor cells which was 50 times the LD100.


Inflammatory cells in solid murine neoplasms. I. Tumor disaggregation and identification of constituent inflammatory cells. Mechanical and enzymatic methods of disaggregating tumors were studied with the goals of (1) minimizing cell losses while (2) maintaining functional and surface membrane markers needed to objectively identify inflammatory cells (IC)1 in resultant suspensions. Application of the principles and methods described makes accurate estimation of the percentage of each IC type present in neoplasms possible for the first time. Compared to purely mechanical means of disaggregating tumors, all enzyme mixtures tested markedly increased yields of viable cells/g neoplasm. Best results were obtained with a combination of collagenase and a protease of broader substrate range (alpha chymotrypsin, papain, pronase or trypsin). The combination of enzymes that gave the highest yields with the least effect on inflammatory cell markers was trypsin, collagenase and DNAse (TCD). Because mechanical injury appeared to be the greatest single cause of cell loss (the enzymes themselves had little direct effect), potential sources were identified and either eliminated or minimized. With TCD, depending on the tumor system, cell recovery (measured as DNA recovered in cell suspensions) was as high as 50% and yields were as much as 6.9 X 10(8) viable cells/g tumor. Complete disaggregation was not required to obtain representative IC populations from tumor fragments. Neutrophils, eosinophils and mast cells from disaggregated neoplasms were counted in Giemsa stained cytocentrifuge preparations based on their unique morphologic appearances. Macrophages were identified by their capacity to phagocytose zymosan, a function which proved highly resistant to the effect of enzymes. Flourescent microscopic identification of brain associated thymus antigen (BATA) allowed quantification of T lymphocytes, since this marker was virtually unchanged by enzyme exposure. Surface immunoglobulin (Ig) was stripped from B lymphocytes most rapidly by pronase and chymotrypsin, slowly by trypsin and papain, and not at all by collagenase. Ig positive cells therefore could be quantified in suspensions generated by collagenase or very short (20 min) exposure of fragments to trypsin.


Increased uptake of iodide by hormone-responsive compared to hormone-independent mammary tumors in GR mice. The uptake of 125-iodide by transplanted hormone-responsive (HR) or hormone-independent (HI) mammary tumors, normal mammary tissue and skeletal muscle was compared in GR mice. The uptake of 125-iodide by HR mammary tumors in mice treated with progesterone and oestrone (P+O) was about 20 times greater than the uptake of 125-iodide by HI mammary tumors in mice not treated with P+O. This difference in uptake of 125-iodide by HR and HI mammary tumors could not be attributed to the difference in the hormonal status of the mice since uptake of 125-iodide was also low in HI tumors in mice receiving P+O treatment. The uptake of 125-iodide by HR mammary tumors was greatly reduced by the simultaneous injection of either an excess of non-radioactive iodide or of perchlorate. Uptake of 125-iodide by normal mammary tissue and skeletal muscle was similar was similar in all groups of tumor bearing mice, and was not influenced by the presence of an excess of non-radioactive iodide or of perchlorate. The possible clinical significance of these findings is discussed.


A comparison of interhemispheric transmission times as measured by verbal and manual reaction time. Interhemispheric transmission times (IHTTs) were calculated for 33 right-handed subjects. Verbal and manual response times (RTs) were recorded to visual stimuli exposed in either the left or right visual fields (LF, RF) for 50 msec. Each subject received 15 stimuli (dots) in the LF (1.5 degrees to the left of fixation), 15 stimuli in the RF, and 15 blank trials in both the verbal and manual response condition. IHTTs were calculated for each subject by subtracting the mean RF RT (I.E., uncrossed path) from the means LF RT (i.e., crossed path). Results indicated a nonsignificant IHTT obtained under the manual response condition of 5 msec and a highly significant IHTT obtained under the verbal response condition of 20 msec. While no significant correlation was found between IHTTs obtained under both response conditions, high correlations were found between component RT measures (i.e., manual RF RT, manual LF RT, verbal RF RT, and verbal LF RT).  Thus, IHTT is independent of simple RT.


The biochemistry of sugars.

The biochemistry of sugars. In animal cells the utilization of sugars involves one or more of the three major pathways known to occur in these cells. The glycolytic (Embden-Meyerhof) pathway is quantitatively the most important, and this pathway, coupled to the citric acid cycle, serves as a major source of energy. Glycolysis and the citric acid cycle also provide most of the precursors for the synthesis of proteins, nucleic acids and lipids. The pentose phosphate, or hexose monophosphate oxidation, pathway is a major source of NADPH required for the conversion of carbohydrate to the more reduced lipids and proteins, and also furnishes the ribose and deoxyribose moieties of nucleotides and nucleic acids. The functions of the third pathway, the uronic acid pathway, are less well defined. It is the source of glucuronides for mucopolysaccharides and for detoxification mechanisms, and also in most animals, is the pathway for the synthesis of ascorbic acid. The uronic acid pathway may also function as a point of entry for glucuronides, pentoses, and pentitols.


Fluorescence and neutralizing antibodies to herpes simplex virus in the cerebrospinal fluid of patients with central nervous system diseases. Herpes simplex virus antibodies were studied in 73 patients with various diseases of the central nervous system. In 29 patients, there were rising titers of fluorescence and neutralizing antibodies to herpes simplex virus in the cerebrospinal fluid (CSF). The CSF antibodies were predominantly IgG. In one patient, IgM was found during the fourth, but not during the eighth week of illness. The CSF antibody titers did not correlate with the serum antibody titers or with the severity of the neurologic defect. No fluorescence antibodies were found in the CSF of 52 of 53 control patients. The presence of CSF fluorescence antibodies may be of considerable value in the diagnosis of herpes simplex encephalitis. However, the absence of these antibodies in the early stage of the disease does not exclude the possibility of infection with herpes simplex virus.


[Current aspects of the pathogenesis of skin manifestations in psoriasis vulgaris]. This review deals with newer aspects of the pathogenesis of skin lesions in psoriasis vulgaris. Genetic determinants and mechanisms of provocation of psoriasis are discussed. The pathobiology of the psoriatic lesions is reviewed from a more functional point of view. The disturbances of epidermal cell proliferation and epidermal cell differentiation in the psoriatic lesions are discussed. Newer findings about chalones and the disturbances of the c-AMP/c-GMP-system are reviewed. The changes of capillaries and the inflammatory reaction in the psoriatic lesion, the morphology of the initial psoriatic lesion and newer immunological findings in patients with psoriasis are reviewed. A working hypothesis of the pathogenesis of the psoriatic lesion is given.


Radionuclide imaging of intestinal infarction in dogs. Intestinal infarction was produced in 10 dogs by means of microsphere embolization. Intravenous and intra-arterial injections of  99mTc-pyrophosphate were then administered and in vivo and in vitro scintigrams obtained. Positive in vitro images of the infarctions were seen for all the dogs and positive in vivo radionuclide accumulation was seen for all the dogs which had intra-arterial injections. Only 1 of the 4 dogs with intravenous injection showed positive in vivo imaging of the infarction.


Correlative morphological and biochemical study of the effects of isoprenaline on the organelle and membrane content of the rabbit parotid gland. A quantitative study has been made on the enzymic, chemical and ultrastructural changes that occur in the parotid glands of rabbits as a result of Isoprenaline-induced secretion. Emphasis has been placed on correlating changes in organelle and membrane content which are evident 2 hr after Isoprenaline administration and which have been measured stereologically with the levels of appropriate enzymic or chemical markers, taking into account the contribution made by both the acinar and duct tissue. Lower protein, alpha-amylase and beta-glycerophosphatase levels correlated with reductions in zymogen granule and lysosome volume with whilst plasmalemmal and Golgi membrane areas and their marker enzyme concentrations remained unchanged. However, declines in alkaline phosphatase and succinate dehydrogenase activity (illustrated histochemically), and p-nitrophenyl phosphatase activity at pH 4-5 in the presence of tartrate occurred without any detectable decrease in membrane area. Conversely, an increase in rough endoplasmic reticulum area was measured stereologically but no increases in chemical markers were detected. The extent of correlation of the data is discussed in the context of the mechanism of secretion and the action of Isoprenaline.


Adenosine triphosphate hydrolysis in rat dental tissues. A histochemical study to differentiate the enzymes involved. The purpose of this study was to try to differentiate histochemically between the various enzymes which may catalyze the hydrolysis of ATP in developing rat dental tissues. Freeze cut and freeze dried sections of molar and incisor teeth were incubated in lead capture-based media at pH 5.0, 7.2 or 9.4 with one of the following substrates: beta-glycerophosphate, AMP, ADP, ATP, AMP-PNP and tetrasodium pyrophosphate. To establish the enzymatic nature of the hydrolysis parallel sections were incubated after prior fixation in either formaldehyde or glutaraldehyde. By comparing the enzymatic stainings obtained with the various substrates and at the different pH:s, it was concluded that ATP can be visibly hydrolyzed in rat dental tissues by alkaline phosphatase (stratum intermedium, apical part of maturation ameloblasts, basal part of all ameloblasts, odontoblasts and subodontoblastic layer), specific ATPase (apical and basal parts of secretory ameloblasts) and ATP pyrophosphatase and/or adenylate cyclase (stratum intermedium, odontoblasts). Acid phosphatase, specific ADPase, 5'-nucleotidase, inorganic pyrophosphatase, 3':5'-cyclic-AMP-phosphodiesterase and adenylate kinase on the other hand, seem not to be engaged in the ATP hydrolysis to such a degree as to complicate the interpretation of the histochemical staining. The alkaline phosphatase part of the ATP hydrolysis appeared to be rather insensitive to aldehyde fixation, while the hydrolysis effected by specific ATPase and ATP pyrophosphatase and/or adenylate cyclase was extinguished after fixation with formaldehyde for 4 h or glutaraldehyde for 10 min.


The demonstration of acid phosphatase in cultured 3T3 mouse cells. Acid phosphatase has been demonstrated ultrastructurally in 3T3 and SV40-3T3 mouse cells using sodium beta-glycerophosphate and p-nitrophenyl phosphate as substrate. The former substrate only demonstrates the enzyme in lysosomes and elements of the Golgi apparatus while the latter demonstrates it in the cisternae of the endoplasmic reticulum and in the cell surface as well as at lysosomal sites. The significance of surface acid phosphatase activity is discussed in terms of sublethal autolysis.


[Childhood facial paralysis due to tumours (author's transl)]. Facial nerve palsy due to malignant tumours is uncommon in childhood and adolescence. If present the malignancy may be primary or metastatic, and usually brain tumours, leukaemias, or lymphosarcomas are found. Rhabdomyosarcomas, parotid malignancies, Ewing's sarcomas, Wilms' tumours and neuroblastomas are rare. The nerve lesion may be due to direct tumour infiltration, external pressure on the nerve and secondary ischaemia. In disseminated malignancy the lesion is usually due to extending intraneural infiltration and all parts of the nerve may be affected. If facial nerve palsy coexists the prognosis in childhood malignancies is extremely poor.


Evidences for complex formation between polymyxin B and lipopolysaccharides from Serratia marcescens. In vitro and in vivo complex formations of polymyxin B and lipopolysaccharides (LPS) from resistant and sensitive cells of Serratia marcescens were studied by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and electron microscopy. In vitro treatment of LPS from resistant cells with polymyxin B gave two populations of spherical complexes of differnt molecular weights as determined electrophoretically. Similar treatment of LPS from sensitive cells resulted in dissociation of the LPS-protein and subsequent complexing with the LPS moiety into stable spheres. In vivo treatment of resistant cells with polymyxin B resulted in LPS-polymyxin B complexes which were comparatively smaller and existed in two morphological forms; spheres and linear ribbons. LPS from the sensitive cells were degraded extensively into small rods and an amorphous mass by the in vivo polymyxin B treatment. In both systems, the electrophoretic results consistently matched the electron microscopic evidences for complex formation of LPS with polymyxin B. It is suggested that the disruptive effects of polymyxin B on LPS in the outer membrane of S. marcescens may be the explanation for the change in permeability barrier in the resistant cells and disorganization of the outer membrane and subsequent death in the sensitive cells. Furthermore, the ability of the LPS to complex with the polymyxin B molecules in resistant cells may be the basis of their resistance to the antibiotic.


Effect of increased gas density on pulmonary gas exchange in man. Pulmonary gas exchange was measured in seven resting supine subjects breathing air or a dense gas mixture containing 21% O2 in sulfur hexafluoride (SF6). The mean value of the alveolar-arterial oxygen difference (AaDO2) decreased from 12.4 on air to 7.0 on SF6 (P less than 0.01), and increased again to 13.4 when air breathing resumed (P less than 0.01). No differences occurred between gas mixtures for O2 consumption, respiratory quotient, minute ventilation, breathing frequency, heart rate, or blood pressure, and the improved oxygen transfer could not be attributed to changes in cardiac output or mixed venous oxygen content in the one subject in which they were measured. These results are best explained by an altered distribution of ventilation during dense gas breathing, so that the ventilation-perfusion ratio (VA/Q) variance was reduced. Of several considered mechanisms, we favor one in which SF6 promotes cardiogenic gas mixing between peripheral parallel units having different alveolar gas concentrations. This mechanism allows for observed increases in arterial carbon dioxide tension and dead space-to-tidal volume ratio during dense gas breathing, and suggests that intraregional VA/Q variance accounts for at least one-half of the resting AaDO2 in healthy supine young men.


Manganese requirement of phosphoglycerate phosphomutase and its consequences for growth and sporulation of Bacillus subtilis. In the absence of manganese, rapidly metabolizable carbohydrates such as glucose or glycerol are not completely metabolized by Bacillus subtilis growing in a nutrient sporulation medium: 3-phosphoglyceric acid (3PGA) accumulates inside the cells, growth stops at a low cell titer, and normal sporulation remains suppressed (no prespore septa). Upon the addition of manganese, 3PGA disappears, growth resumes, and normal sporulation takes place. These effects results from a specific manganese requirement of phosphoglycerate phosphomutase which catalyzes the interconversion of 3PGA and 2-phosphoglyceric acid (2PGA). Other metal ions cannot replace manganese, for which the enzyme has an apparent Km of 0.22 mM.


Arginine, a growth-limiting factor for Eubacterium lentum. Eubacterium lentum is a gram-positive, asaccharolytic, obligately anaerobic bacillus, which grows to a low turbidity (absorbancy at 650 nm = 0.05 to 0.1) in peptone-based medium. The addition of substrate amounts of arginine or citrulline dramatically increased growth (absorbancy at 650 nm =1.4). The presence of an arginine dihydrolase pathway was confirmed by measurement of the necessary enzymes and demonstration of the intermediate compounds. The production of adenosine 5'-triphosphate from the arginine dihydrolase pathway appeared to be the sole source of energy for growth of this organism. Each of 11 strains showed definite growth stimulation. Ten of the 11 strains had cytochromes. Growth stimulation with arginine and the presence of cytochromes offer two new positive criteria for the identification of E. lentum.


Soluble adenylate cyclase from the culture medium of Bordetella pertussis: purification and characterization. Culture medium of exponentially growing Bordetella pertussis (strain 114) contains significant quantities of soluble (100,000 X g for 1 h) adenylate cyclase. The enzyme was purified by chromatography on diethylaminoethyl-cellulose and Sephadex G-200. The purest material yielded a single band on sodium dodecyl sulfate-disc gel electrophoresis. It is heat labile, has a temperature optimum of 30 degrees C, a pH optimum of pH 7 to 8, and a Km for adenosine 5'-triphosphate of 0.4 mM, and requires Mg2+ for maximum activity. The molecular weight, by sodium dodecyl sulfate-disc gel electrophoresis and sucrose density gradient, is approximately 70,000. The enzyme is markedly inhibited by fluoride and weakly inhibited by monovalent salts, but its activity is not altered by alpha-keto acids of nonsubstrate nucleoside triphosphates. Thus, but its presence in the culture supernatant, its smaller molecular weight, and its insensitivity to alpha-keto acids and nucleotides, this enzyme differs from the bacterial adenylate cyclases previously described.


Use of polymyxin B, levallorphan, and tetracaine to isolate novel envelope mutants of Escherichia coli. Mutants of Escherichia coli were isolated by their resistance to the bacteriocidal effects of the membrane-active drugs polymyxin B, levallorphan, and tetracaine. The mutants were examined for additional changes in cellular physiology evoked by the lesions; many polymyxin-resistant strains had a concomitant increased sensitivity to anionic detergents, and several strains of each type had concomitant alterations in generation time and morphology. Mutants of each class (polymyxin resistant, tetracaine resistant, and levallorphan resistant) were transduced into recipient strains. The levallorphan resistance site (lev) was located at approximately 9 min on the E. coli chromosome. Polymyxin (pmx) and tetracaine (tec) resistance loci were also transduced. The lev and tec strains had a slight prolongation of generation time, in contrast with their isogenic wild-type strains. The tec transductant produced long filaments in the absence of tetracaine and had an altered colonial morphology, it reverted at high frequency, with the morphological abnormalities reverting along with the tetracaine resistance. The pmx transductant had an increased sensitivity to levallorphan and to anionic detergents. In contrast, both lev and tec mutants were more resistant to acriflavine than was the wild type or the pmx transductant. The pmx, lev, and tec loci differed in sensitivity to mitomycin C; the lev strain was more resistant, the tec strain was more sensitive, and the pmx strain was much more sensitive than the wild type. There was no difference in sensitivity to several other dyes and detergents, colicins, or T bacteriophage between the transductant and isogenic wild-type strains. Thus, lev, tec, and pmx loci confer more subtle alterations in the permeability barrier than do lipopolysaccharide-deficient mutants previously studied.


Electron microscope study of sporulation and parasporal crystal formation in Bacillus thuringiensis. A comprehensive ultrastructural analysis of sporulation and parasporal crystal development is described for Bacillus thuringiensis. The insecticidal crystal of B. thuringiensis is initiated at the start of engulfment and is nearly complete by the time the exosporium forms. The crystal and a heretofore unobserved ovoid inclusion develop without any clear association with the forespore septum, exosporium, or mesosomes. These observations contradict previous hypotheses that the crystal is synthesized on the forespore membrane, exosporium, or mesosomes. Formation of forespore septa involves densely staining, double-membrane-bound, vesicular mesosomes that have a bridged appearance. Forespore engulfment is subpolar and also involves mesosomes. Upon completion of engulfment and the following cytoplasmic changes occur: decrease in electron density of the incipient forespore membrane; loss of bridged appearance of incipient forespore membrane; change in stainability of incipient forespore, forespore, and mother cell cytoplasms; and alteration in staining quality of plasma membrane. These changes are involved in the conversion of the incipient forespore into a forespore and reflect "commitment" to sporulation.


Genetic control of the glp system in Bacillus subtilis. In pleiotropic negative glycerol utilization mutants (GlpPI mutants) of Bacillus subitilis, glycerol kinase and sn-glycerol 3-phosphate (G3P) dehydrogenase are noninducible. GlpPI mutants also fail to take up exogenous [14C]G3P. To study the regulation of the glp system in B. subtilis phenotypically, Glp+ revertants were isolated from GlpPI mutants. Four classes of revertants were identified: phenotypically, wild type; R1 type, which contains an informational suppressor, R2 type, which produced G3P dehydrogenase constitutively; and R3 type, with a temperature-sensitive Glp phenotype producing G3P dehydrogenase constitutively at permissive temperature (32 degrees C). The properties of the revertants indicate that the glpPI locus codes for a protein with a positive regulatory function.


Effect of chloramphenicol on the electron systems in Ustilago cynodontis. The mycelial cells of Ustilago cynodontis possess at least two electron transport systems: a cyanide-sensitive cytochrome pathway, which represents the major route for electron transport, and an alternative cyanide-insensitive pathway, inhibited by salicylhydroxamic acid. In the presence of chloramphenicol in the culture medium, mycelial cells respire only by the alternatuve chain. The stable induced yeast-like cells, obtained by prolonged chloramphenicol treatment of the mycelial cells, respire as the untreated mycelial cells; this result indicates that the phenotypic change induced by chloramphenicol is not related to a modification of the respiratory system.


Membrane-bound respiratory of Spirillum itersonii. The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.


Effect of polymyxin on the ultrastructure of the outer membrane of wild-type and polymyxin-resistant strain of Salmonella. The effect of polymyxin on two sets of Salmonella mutants was studied by thin-section and scanning electron microscopy. Polymyxin (in increasing concentrations, starting just below bactericidal effect) caused the appearance of the previously described rodlike projections on the cell surface of wild-type (smooth, polymyxin-sensitive) bacteria. These projections seemed to involve the outer membrane of the cell wall. In rough mutants, which are deficient in lipopolysaccharide, the projections were much smaller and flat. Higher concentrations of polymyxin were required to produce morphological effects in polyxmin-resistant mutants of both smooth and rough forms. Furthermore, in these mutants polymyxin caused vesicle-like bulging of the total outer membrane quite different in appearance from the rodlike projections of the wild type.


Growth inhibition of Proteus mirabilis by cyclic adenosine 3'-5'-monophosphate. Growth of Proteus mirabilis on a synthetic agar medium containing either glycerol, galactose, or trehalose as the sole source is inhibited by 5 mM cyclic adenosine 3',5'-monophosphate (cAMP). Inhibition on an agar medium is evident as loss of viability, but in broth cAMP only slightly inhibits growth rate. Inhibition is associated with the accumulation of methylglyoxal in the medium. A nonswarming mutant of P. mirabilis is not inhibited by cAMP on either of the three carbon sources, but it is sensitive to exogenous methylglyoxal.


New radioisotopic assays of argininosuccinate synthetase and argininosuccinase. Methods were developed for the radioisotopic assay of argininosuccinate synthetase [L-citrulline: L-aspartate ligase (AMP-forming), EC 6.3.4.5] and argininosuccinase [L-argininosuccinate arginine-lyase, EC 4.3.2.1]. The assay of argininosuccinate synthetase was based on the separation of [14C]argininosuccinate formed from aspartate and [carbamoyl-14C]citrulline in the presence of ATP from the substrate citrulline. For this, the product was converted to its anhydride form by boiling for 30 min at pH 2.0 followed by application on a column of Dowex 50W (pyridine form). Argininosuccinic anhydride was eluted with 0.3 M pyridine acetate buffer, pH 4.25, while citrulline was eluted with 0.1 M pyridine acetate buffer, pH 3.80. The assay of argininosuccinase was based on the separation of [14C]argininosuccinic acid formed from arginine and [U-14C]fumaric acid from the substrate fumarate on a column of Dowex 50W(H+ form). The argininosuccinic acid was adsorbed on the column and eluted with 1 M pyridine solution, while fumarate was not adsorbed. The distributions of these two enzymes in various organs and cell fractions were reinvestigated using these methods.


Purification and characterization of human leukocyte interferon components. Human leukocyte interferon, prepurified either by acid ethanol extraction or by affinity chromatography with antibodies, was further purified by gel filtration in the presence of sodium dodecyl sulfate. Interferon was eluted from gel filtration columns as an apparently homogeneous entity with a molecular weight of 26,600, resulting in an up to 50-fold additional purification during a single step. The antiviral activity could be further resolved into two components by hydroxylapatite adsorption chromatography. The isolated components (A and B) were distinguishable by isoelectric focusing and polyacrylamide gel electrophoresis. The apparent molecular weights were 20,000 to 16,000 and 16,000, respectively. No differences were detected in their susceptibility toward reduction of disulfide bonds by beta-mercaptoethanol. Both could be obtained on a preparative scale with minimal losses in biological activity.


Hypoxanthine transport by cultured Chinese hamster lung fibroblasts. The uptake of hypoxanthine by Chinese hamster lung fibroblasts grown in tissue culture was studied in wild type clones and 8-azaguanine-resistant mutant clones devoid of hypoxanthine-guanine phosphoribosyltransferase. Wild type fibroblasts rapidly accumulate [3H]hypoxanthine from the medium and over 80% of the intracellular radioactivity is found in acid-soluble nucleotides. The phosphoribosyltransferase-deficient clones accumulate much lower levels of hypoxanthine and over 85% of the intracellular 3H label is associated with chemically unaltered hypoxanthine. The internal level of hypoxanthine in the mutant clones rapidly approaches but does not exceed that present in the medium. Wild type and phosphoribosyltransferase-deficient cells take up hypoxanthine at almost identical initial rates at external hypoxanthine levels from 2 to 300 muM. Analysis of these data reveals two transport systems that obey the Michaelis-Menten relationship. These differ markedly in affinity, yielding average Km values of 20 and 600 muM for both cell types. Hypoxanthine transport by both low and high affinity transport systems is blocked by p-chloromercuriphenylsulfonate and N-ethylmaleimide. Counter-transport of hypoxanthine was demonstrated in phosphoribosyltransferase-deficient fibroblasts. It is concluded that hypoxanthine is transported into Chinese hamster cells by means of carrier-mediated processes (facilitated diffusion) that operate independently of phosphoribosylation.


Pulmonary angiotensin-converting enzyme. Interspecies homology and inhibition by heterologous antibody in vivo. Goat antibody against pure rabbit pulmonary angiotensin-converting enzyme was used to probe homology of converting enzymes from other species. Immunologically cross-reactive material was found in detergent-solubilized extracts of lung particles from rat, guinea pig, and dog by double immunodiffusion, radioimmunoassay, and inhibition of enzyme activity. No homology was demonstrable with bovine, frog, or chicken lung extracts. Antibodies from different individual goats yielded comparable estimates of homology by immunodiffusion and radioimmunoassay. In contrast, they varied greatly in extent and specificity of their inhibitory action on heterologous enzyme activity. The vasopressor effect of angiotensin I and the vasodepressor effect of bradykinin were diminished and potentiated, respectively, in rats treated with anti-rabbit enzyme antibody. A smaller but significant immune-dependent inhibition of the vasopressor response to angiotensin II was also observed.


Specific herpes simplex virus-induced incorporation of 5-iodo-5'-amino-2',5'-dideoxyuridine into deoxyribonucleic acid. 5-Iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd) is a novel thymidine analog which inhibits herpes simplex virus, type 1 (HS-1 virus) replication in the absence of detectable host toxicity. When murine, simian, or human cells in culture are treated with [125I]AIdUrd for up to 24 hours essentially none of the nucleoside becomes cell-associated. In contrast, upon HS-1 virus infection significant radiolabel is detected in both nucleotide pools and in DNA. The major acid-soluble metabolite has been shown by enzymic and chromatographic analysis to be the 5'-triphosphate of AIdUrd. DNA from HS-1 virus-infected Vero cells labeled with [14C]thymidine, 5-[125I]iodo-2'-deoxyuridine (IdUrd), or [125I]AIdUrd was isolated by buoyant density centrifugation and subjected to digestion by pancreatic DNase I, spleen DNase II, micrococcal nuclease, spleen, and venom phosphodiesterases. Analysis of the digestion products clearly indicate that AIdUrd is incorporated internally into the DNA structure. DNA containing AIdUrd therefore contains phosphoramidate (P-N) bonds, known to be extremely acid-labile. The selective HS-1 virus-induced phosphorylation of AIdUrd and its subsequent incorporation into DNA may account for the unique biological activity of the AIdUrd nucleoside.


Structural polypeptides of mammalian type C RNA viruses. Isolation and immunologic characterization of a low molecular weight polypeptide, p10. Low molecular weight polypeptides of several mammalian type C RNA tumor viruses were purified by sequential ion exchange chromatography and molecular sizing techniques. These included a polypeptide with a molecular weight of 10,000 to 11,000, p 10, from two type C viruses of mouse origin. Rauscher- and Moloney-murine leukemia virus (MuL virus), and from an infectious type C virus isolate of the woolly monkey. The p12 structural polypeptides of these viruses as well as Rauscher-MuL virus p15 were also purified. By using radioimmunoassays developed for each polypeptide, it was possible to demonstrate that all three low molecular weight polypeptides, p15, p12, and p10, were immunologically unique. Among type C viral structural polypeptides, p10 has been least well characterized immunologically. The results of the present study indicate that p10 is virus-coded and possesses strong group-specific antigenic determinants. By use of appropriate immunoassays, broadly reactive interspecies determinants shared by mammalian type C virus isolates of murine, feline, and primate origin, were also demonstrated. The interspecies antigenic determinants of p10 were shown to be as broadly cross-reactive as those exhibited by the major type C virus structural polypeptide, p30.


Microsomal membrane permeability and the hepatic glucose-6-phosphatase system. Interactions of the system with D-mannose 6-phosphate and D-mannose. We have proposed that glucose-6-phosphatase (EC 3.1.3.9) is a two-component system consisting of (a) a glucose-6-P-specific transporter which mediates the movement of the hexose phosphate from the cytosol to the lumen of the endoplasmic reticulum (or cisternae of the isolated microsomal vesicle), and (b) a nonspecific phosphohydrolase-phosphotransferase localized on the luminal surface of the membrane (Arion, W.J., Wallin, B.K., Lange, A.J., and Ballas, L.M. (1975) Mol. Cell. Biochem. 6, 75-83). Additional support for this model has been obtained by studying the interactions of D-mannose-6-P and D-mannose with the enzyme of untreated (i.e. intact) and taurocholate-disrupted microsomes. An exact correspondence was shown between the mannose-6-P phosphohydrolase activity at low substrate concentrations and the permeability of the microsomal membrane to EDTA. The state of intactness of the membrane influenced the kinetics of mannose inhibition of glucose-6-P hydrolysis; uncompetitive and noncompetitive inhibitions were observed for intact and disrupted microsomes, respectively. The apparent Km for glucose-6-P was smaller with intact preparations at mannose concentrations above 0.3 M. Mannose significantly inhibited total glucose-6-P utilization by intact microsomes, whereas D-glucose had a stimulatory effect. Both hexoses markedly enhanced the rate of glucose-6-P utilization by disrupted microsomes. The actions of mannose on the glucose-6-phosphatase of intact microsomes fully support the postulated transport model. They are predictable consequences of the synthesis and accumulation of mannose-6-P in the cisternae of microsomal vesicles which possess a nonspecific, multifunctional enzyme on the inner surface and a limiting membrane permeable to D-glucose, D-mannose, glucose-6-P, but impermeable to mannose-6-P. The latency of the mannose-6-P phosphohydrolase activity is proposed as a reliable, quantitative index of microsomal membrane integrity. The inherent limitations of the use of EDTA permeability for this purpose are discussed.


Rat high density lipoprotein subfraction (HDL3) uptake and catabolism by isolated rat liver parenchymal cells. Rat liver parenchymal cell binding, uptake, and proteolytic degradation of rat 125I-labeled high density lipoprotein (HDL) subfraction, HDL3 (1.10 less than d less than 1.210 g/ml), in which apo-A-I is the major polypeptide, were investigated. Structural and metabolic integrity of the isolated cells was verified by trypan blue exclusion, low lactic dehydrogenase leakage, expected morphology, and gluconeogenesis from lactate and pyruvate. 125I-labeled HDL3 was incubated with 10 X 10(6) cells at 37 degrees and 4 degrees in albumin and Krebs-Henseleit bicarbonate buffer, pH 7.4. Binding and uptake were determined by radioactivity in washed cells. Proteolytic degradation was determined by trichloroacetic acid-soluble radioactivity in the incubation medium. At 37 degrees, maximum HDL3 binding (Bmax) and uptake occurred at 30 min with a Bmax of 31 ng/mg dry weight of cells. The apparent dissociation constant of the HDL3 receptor system (Kd) was 60 X 10(-8) M, based on Mr = 28,000 of apo-A-I, the predominant rat HDL3 protein. Proteolytic degradation showed a 15-min lag and then constant proteolysis. After 2 hours 5.8% of incubated 125I-labeled HDL3 was degraded. Sixty per cent of cell radioactivity at 37 degrees was trypsin-releasable. At 37 degrees, 125I-labeled HDL3 was incubated with cells in the presence of varying concentrations of native (cold) HDL3, very low density lipoproteins, and low density lipoproteins. Incubation with native HDL3 resulted in greatest inhibition of 125I-labeled HDL3 binding, uptake, and proteolytic degradation. When 125I-labeled HDL3 was preincubated with increasing amounts of HDL3 antiserum, binding and uptake by cells were decreased to complete inhibition. Cell binding, uptake, and proteolytic degradation of 125I-labeled HDL3 were markedly diminished at 4 degrees. Less than 1 mM chloroquine enhanced 125I-labeled HDL3 proteolysis but at 5 mM or greater, chloroquine inhibited proteolysis with 125I-labeled HDL3 accumulation in cells. L-[U-14C]Lysine-labeled HDL3 was bound, taken up, and degraded by cells as effectively as 125I-labeled HDL3. These data suggest that liver cell binding, uptake, and proteolytic degradation of rat HDL3 are actively performed and linked in the sequence:binding, then uptake, and finally proteolytic degradation. Furthermore, there may be a specific HDL3 (lipoprotein A) receptor of recognition site(s) on the plasma membrane. Finally, our data further support our previous reports of the important role of liver lysosomes in proteolytic degradation of HDL3.


Cyclic adenosine 3':5'-monophosphate phosphodiesterase. Distinct forms in human lymphocytes and monocytes. Adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase activity of normal human peripheral blood leukocyte suspensions containing 90% lymphocytes and 10% monocytes showed anomalous kinetic behavior indicative of multiple enzyme forms. Kinetic analyses of purified lymphocyte (99%) or monocyte preparations (95%) indicated that only one type of phosphodiesterase was present in each cell type. None of the preparations contained any detectable guanosine 3':5'-monophosphate (cyclic GMP) hydrolytic activity. The lymphocyte enzyme had an apparent Km congruent to 0.4 muM for cyclic AMP and Vmax congruent to 0.5 picomoles/min/10(6) cells. These kinetic parameters were confirmed by several cell purification techniques used alone and sequentially. Sedimentation velocity analyses indicated that the higher Km monocyte enzyme had a molecular weight near 45,000 and that the lower Km lymphocyte enzyme most likely had a molecular weight near 98,000. A variety of procedures led to a loss of the higher molecular weight, high affinity enzyme leaving only the enzyme of 45,000 daltons with a much lower substrate affinity. A long term, stable human lymphoblastoid cell line had cyclic AMP phosphodiesterase activity that was similar to the lymphocyte enzyme by both physical and kinetic criteria. Lymphocyte cyclic AMP phosphodiesterase appears to be a soluble enzyme whose pH and temperature optima and cationic requirements are similar to those of other mammalian phosphodiesterases. The distinct cyclic AMP phosphodiesterase forms of these cells may possibly represent the basic, active subunit of mammalian cyclic nucleotide phosphodiesterases. We hypothesize that the extremely high affinity cyclic AMP phosphodiesterase of normal lymphocytes plays an important role in the regulation of normal function in these cells, and also in the rapid proliferative responses characteristic of the stimulated lymphocyte.


Preparation and characterization of hormone-sensitive, resealed erythrocyte ghosts. A method for preparing resealed turkey erythrocyte ghosts is described which utilizes hypotonic lysis and resealing following restoration of isotonicity. The resealed ghosts are isolated above 55% sucrose. The resealed ghosts are shown to be capable of maintaining high intracellular K+ concentrations in the presence of a low K+ extracellular environment. When ATP and an ATP-regenerating system are included during the resealing stage, (R)-(-)-epinephrine- and NaF-stimulated cyclic AMP accumulation, which is linear for 20 min, can be demonstrated. The concentration of (R)-(-)-epinephrine producing a half-maximal response in resealed ghosts is 1.0 +/- 0.4 X 10(-6) M. This is the same as that for (R)-(-)-epinephrine in the intact erythrocyte. The resealed ghosts are impermeable to Ca2+, but Ca2+ inhibition of cyclic AMP accumulation is noted if the divalent cation ionophore. A-23187, is present or if Ca2+ is included during the resealing stage.


Gonadotropin binding factor(s). Extraction of high affinity gonadotropin binding sites from rat testis and partial characterization of their interaction with human follitropin, lutropin, and choriogonadotropin. Factor(s) that bind gonadotropins have been extracted from rat testis by 30% ethanol (v/v) in water and their interaction with human lutropin (hLH) and human follitropin (hFSH) have been investigated by a new assay using dextran-coated charcoal. These studies reveal that: 1. Maximal binding of gonadotropin with soluble factors was observed over a broad range of pH from 6.0 to 8.0 with a relative decline in binding at extremes of pH. The binding was independent of the ionic strength of the buffer and reached equilibrium within 5 min at 4 degrees, 27 degrees, and 37 degrees. 2. The soluble factors have marked thermostability, a point of distinction from detergent-solubilized receptors. 3. The equilibrium dissociation constant (Kd) of 125I-hFSH binding to the soluble factor was 6.0 +/- 0.58 X 10(-10) M, consistent with the values obtained from the membrane binding studies. Similarly, the Kd value for 125I-hLH to the soluble factor(s) was 3.33 +/- 0.3 X 10(-9) M, comparable to the values obtained from the membrane binding studies. Hill plots demonstrated a lack of a cooperative relationship with an apparent Hill coefficient of 1.071 for hLH and 0.909 for hFSH. Furthermore, two classes of binding sites for 125I-human choriogonadotropin (hCG) were clearly discernible by both Lineweaver-Burk and Hill plots with an equilibrium dissociation constant of 2.4 +/- 0.5 X 10(-11) M and 1.35 +/- 1.2 X 10(-9) M. The apparent Hill coefficient of interaction of 125I-hCG with the soluble factors was found to be 0.923 for high affinity and 1.09 for low affinity binding sites. 4. The binding of 125I-hLH and 125I-hFSH with respect to concentrations of soluble factor(s) was found to be a saturable process, yielding an expected 4.4-fold higher Kd for hLH (294 +/- 13.8 mug/ml) compared to hFSH (66.6 +/- 4 mug/ml). These findings are comparable with the equilibrium dissociation constants, thus confirming a 5-fold higher affinity of hFSH as compared to hLH for the soluble factors, i.e. the ratio of 3.0 X 10(-9) M to 6.0 X 10(-10) M versus the ratio of 294 mug/ml to 66.6 mug/ml. 5. The hormone specificity of the interaction has been studied by using radiolabeled hFSH, hLH, hCG, prolactin, growth hormone, and bovine serum albumin. The binding of FSH at low factor concentrations was found to be 5- to 10-fold greater than prolactin, growth hormone, and albumin. 6. The soluble factors are found in higher concentration in testis compared to liver, kidney, and blood. 7. The effect of ethanol upon solubilization of the factor(s) has been investigated. The factor(s) can be extracted with buffer or water alone. However, 10 to 25% of ethanol (v/v) facilitates the process of solubilization. The treatment with 70% ethanol (v/v) or more did not extract any factor activity from testes. The factor(s) were insoluble in petroleum ether, chloroform, absolute ethanol, methanol, or lipid solvent. 8. Finally the effect of soluble factors on classical membrane binding was investigated...


Metabolism and excretion of exogenous adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate. Studies in the isolated perfused rat kidney and in the intact rat. Isolated rat kidneys were perfused with a recirculating medium containing exogenous adenosine 3':5'-monophosphate (cyclic AMP) or guanosine 3':5'-monophosphate (cyclic GMP) at an initial concentration of 0.1 mM. Both cyclic nucleotides were rapidly removed from the perfusate. Urinary excretion accounted for about 20% and 40% of the respective cyclic AMP and cyclic GMP lost from the perfusate. The metabolism of the cyclic nucleotides was studied by 14C-labeled cyclic nucleotides in the perfusate. During 60 min, 30% of added cyclic [14C]AMP was metabolized to renal [14C]adenine nucleotides (ATP, ADP, and AMP) and 30% to perfusate [14C]uric acid. Similarly, 20% of cyclic[14C]GMP was metabolized to renal [14C]guanine nucleotides (GTP, GDP, and GMP) and 30% to perfusate [14C]uric acid. Urine contained principally unchanged 14C-labeled cyclic nucleotide. Addition of 0.1 mM cyclic AMP to the perfusate elevated the renal ATP and ADP contents 2-fold. Addition of 0.1 mM of either cyclic AMP or cyclic GMP to the perfusate also elevated the renal production of uric acid 2- to 3-fold. The production and distribution of metabolites of exogenous cyclic nucleotides were also studied in the intact rat. Within 60 min after injection, 3.3 mumol of either 14C-labeled cyclic AMP or cyclic GMP was cleared from the plasma. Kidney cortex and liver were the principal tissues for 14C accumulation. Urinary excretion accounted for about 20 and 45% of the cyclic [14C]AMP and cyclic [14C]GMP lost from the plasma, respectively. The 14C found in the kidney and liver was present almost entirely as the respective purine mono-, di-, and trinucleotides. The other principal metabolite was [14C]allantoin, found in the urine and, to a lesser extent, the liver. The urine contained mostly unchanged 14C-labeled cyclic nucleotide. Unlike the findings with the perfused kidney, [14C]uric acid was not a significant metabolite of the 14C-labeled cyclic nucleotides in these in vivo experiments.


Purification and characterization of the carrier protein for juvenile hormone from the hemolymph of the tobacco hornworm Manduca sexta Johannson (Lepidoptera: Sphingidae). The larval hemolymph of the tobacco hornworm, Manduca sexta, contains a carrier protein that binds specifically and with high affinity the juvenile hormone, an important regulator of insect development. This protein serves to transport the hormone and to protect it from the action of degradative enzymes during early larval stages. Using hemolymph from the last larval stage, we have isolated a pure carrier protein using acetone precipitation, gel filtration, ion exchange chromatography, and preparative isoelectric focusing. Gel filtration, polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and equilibrium ultracentrifugation established that the carrier protein is a single chain polypeptide of approximately 28,000 daltons. The amino acid composition is unexceptional, and no evidence for hexosamine has been obtained. An ion exchange filter disc assay method was used to determine the formation of the complex between the carrier protein and isotopically labeled juvenile hormone. With this technique it was shown that each carrier protein binds one hormone molecule with a dissociation constant of 4.4 +/- 0.2 X 10(-7) M at 0 degrees.


Incorporation of D-[3-3H, U-14C] glucose into glycerolipid via acyl dihydroxyacetone phosphate untransformed and viral-transformed BHK-21-c13 fibroblasts. Untransformed BHK-21-c13 fibroblasts as well as 4 polyoma-transformed strains were incubated with D-[U-14C,3-3H]glucose. This substrate generates intracellular labeled glycerol, and also [4-3H]NADPH via the phosphogluconate oxidative pathway. The latter selectively transfers hydrogen to C-2 of glycerol in glycerolipid via the acyl dihydroxyacetone phosphate pathway. After incubation, the distribution of radioactivity and the ratios of 3H/14C at the three positions of recovered glycerol were determined in sn-glycerol 3-phosphate, saponifiable glycerolipids, alkyl ether glycerolipids, and plasmalogens. In each of the cell types examined, 3H in the sn-1 position of glycerol in the recovered ether-containing glycerolipids was negligible, yet this position contained most of the recovered 3H in sn-glycerol 3-phosphate and saponifiable glycerolipids. The 3H/14C ratio in position 2 of glycerol, measured at various incubation times, was from 5- to 200-fold greater in the saponifiable glycerolipids than in free sn-glycerol 3-phosphate. The ratio in position 2 of ether-containing glycerolipids was the same or greater than that in the saponifiable glycerolipids in all of the cell types employed. A similar pattern in the 3H/14C ratio was observed when BHK-21-c13 cells were incubated with D-[U-14C,1-3H]glucose. These observations demonstrate significant participation of the acyl dihydroxyacetone phosphate pathway in glycerolipid synthesis in BHK cells.


Progesterone-binding components of chick oviduct. In vitro effects of purified hormone-receptor complexes on the initiation of RNA synthesis in chromatin. We have investigated the manner by which progesterone receptors act to induce initiation of RNA synthesis in a cell-free system derived from chick oviduct. A method utilizing rifampicin enabled us to measure the formation of binary initiation complexes between RNA polymerase and chick oviduct chromatin (Tsai, M.-J., Schwartz, R.J., Tsai S.Y., and O'Malley, B.W. (1975) J.Biol. Chem. 250, 5165-5174) and allowed for the quantitative assessment of RNA chain initiation sites, RNA chain propagation rates, and RNA chain size under conditions which prevent secondary chain reinitiations. We have measured the available initiation sites for transcription in oviduct chromatin prepared from chicks withdrawn from all hormone and then restimulated with a secondary injection of progesterone. Within 1/2 hour after administration of progesterone, the number of initiation sites increased from 8,700 sites/pg of chromatin DNA for the control to 15,500 sites. After 1 hour, the concentration of RNA polymerase needed to saturate chromatin binding sites was increased 60% in comparison to control values, while the number of initiation sites increased 160%. This rapid increment in transcriptional activity preceded temporally the induction of synthesis of ovalbumin mRNA. To test directly the effect of progesterone receptor on transcription, in vitro, a reconstituted cell-free system was employed which contained purified cytoplasmic progesterone-receptor complexes, Escherichia coli RNA polymerase, and chromatin prepared from hormonally withdrawn chick oviducts. Purified progesterone-receptor complex stimulated transcription of oviduct chromatin in vitro by promoting an increase of 3,000 to 5,000 additional sites for RNA chain initiation. These data showed that progesterone receptor can directly increase the number of RNA polymerase binding and initiation sites in the chromatin template in the absence of a detectable change in either the rate of RNA chain propagation or the size of the RNA product. The kinetics of progesterone-receptor stimulation of RNA synthesis in chromatin revealed a t1/2 of 15 min for this effect to occur. This value was identical with the optimal time required for binding of receptor to chromatin. The concentration of receptor required for half-maximal stimulation of RNA chain initiation was approximately 5 x 10(-9) M. This value agreed closely with our previously reported estimates of the affinity (Kd approximately 5 x 10(-9)M) of the progesterone-receptor complex for oviduct chromatin. The stimulatory effect of purified progesterone receptor appeared to be relatively specific for oviduct chromatin in comparison to nontarget tissue chromatins or chick DNA. The data presented here show that steroid hormone-receptor complex can directly regulate gene transcription in vitro in a manner which mimics the events observed in vivo in target cells.


Progesterone-binding components of chick oviduct. In vitro effect of receptor subunits on gene transcription. We have studied chick oviduct progesterone receptor subunits and their effect on RNA transcription in oviduct chromatin. Initiation of RNA chain synthesis was measured by the rifampicin-challenge assay. Progesterone receptor subunits A and B were purified 200-fold by sequential chromatography on phosphocellulose, DEAE-cellulose, and hydroxylapatite. The subunits could be recombined to form the 6S receptor A-B dimer initially present in chick oviduct homogenates. The intact 6 S receptor dimer preparation increased transcription of oviduct chromatin by 40 to 60%. This increase corresponds to the creation of 5,800 new initiation sites per haploid cellular equivalent of DNA as chromatin. The increased chromatin transcription which resulted was half-maximal at a receptor concentration of about 5 x 10(-9) M. Preparations from other tissues which were devoid of progesterone-binding activity or oviduct preparations lacking progesterone did not stimulate rifampicin-resistant RNA synthesis. The oviduct receptor fraction contained no detectable glucocorticoid, androgen, or estrogen receptors. When isolated B subunits alone were incubated with chromatin, no transcriptional increase was detectable. Isolated A subunits increased chromatin transcription, but only at concentration approximately 10-fold higher than required of intact dimer. These observations suggest that the 6 S receptor dimer is the functional species in chromatin transcription in vivo; two hormones are required per dimer, one on each subunit. Since subunit A binds DNA and has stimulatory activity , a dimer dissociation at acceptor sites is postulated, liberating A subunits which stimulate RNA chain initiation sites.


Human plasma lipoproteins as accelerators of prothrombin activation. The activation rate of bovine prothrombin by Factor Xa and Ca2+ has long been known to be greatly enhanced by addition of phospholipid. Upon substitution of human plasma lipoproteins for phospholipid (cephalin) in this activation system, only very low density lipoprotein enhances prothrombin activation. Low density lipoprotein and high density lipoprotein have no stimulatory effect on prothrombin activation. On the other hand, the sonicated lipid extracts from very low, low, and high density lipoproteins all can substitute for phospholipid in potentiating prothrombin activation. The efficiency of each lipid extract, in this regard, depends upon its source of extraction, and is greatest for the lipid extract of very low density lipoprotein.


Corticosteroid-binding proteins in human colostrum and milk and rat milk. A corticosteroid-binding protein was detected in the whey of human colostrum and milk which resembles serum corticosteroid-binding globulin in certain respects: the equilibrium association constants for cortisol and progesterone binding and the apparent molecular size, as determined by Sephadex G-200 chromatography, were similar, and cortisol andd progesterone competed strongly for binding to the same site in each instance. Dexamethasone-binding activity could not be detected. The concentration of corticosteroid-binding protein in the colostrum obtained before parturition is about 0.1 muM; the concentration declines rapidly after parturition to about 0.01 muM. A corticosteroid-binding protein was found, also, in the whey of mature rat milk at levels of about 0.3 muM. This protein resembles rat serum corticosteroid-binding globulin: the equilibrium association constants for cortisol, corticosterone, and progesterone binding, and the apparent molecular size, as determined by Sephadex G-200 chromatography, were similar; the elution behavior of the respective proteins on anion exchange chromatography with DEAE-Sephadex A-50 was similar, also. Identity of the corticosteroid-binding proteins in whey with corticosteroid-binding globulin in serum is not presumed, however. Rat and human whey exhibited very little testosterone- or 17 beta-estradiol-binding activity. It is suggested the corticosteroid-binding proteins may play a significant physiological role in regulating the concentration of the bound and unbound forms of progesterone and cortisol in the fluids bathing the epithelial cells lining the mammary ducts and acini.


In vitro activation of rat liver phenylalanine hydroxylase by phosphorylation. Essentially pure phenylalanine hydroxylase from rat liver can be activated between 2.5- and 3.0-fold by treatment with Mg2+, ATP, protein kinase, and cyclic AMP. The activation is seen when the hydroxylase is assayed in the presence of tetrahydrobiopterin, but not in the presence of 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine. In the presence of [gamma-32P]ATP, activation is accompanied by incorporation of 32P into the protein to the extent of 0.7 mol/mol of hydroxylase subunit (Mr = 50,000). Cehmical analysis of the untreated enzyme shows that it already contains about 0.3 mol of Pi/mol of hydroxylase. These results suggest that the activity of the hydroxylase may be regulated by phosphorylation.


Phosphorylation of rat liver ribonucleic acid polymerase I by nuclear protein kinases. Phosphorylation of rat liver RNA polymerase I occurred when intact rat liver nuclei were incubated with [gamma32P]ATP and N6,O2' dibutyryl cyclic 3':5'-AMP. In addition, partially purified RNA polymerase I could be phosphorylated in vitro by an endogenous protein kinase. Phosphorylation by either method was followed by extensive purification of the enzyme. This revealed that 32P remained bound to the enzyme throughout purification. Analysis of the homogeneous labeled protein by polyacrylamide gel electrophoresis under nondenaturing conditions followed by autoradiography revealed that only one of the two forms of RNA polymerase I in rat liver nuclei was phosphorylated. RNA polymerase II was not phosphorylated in intact nuclei. Polyacrylamide gel electrophoresis of the phosphorylated RNA polymerase I in the presence of 0.1% sodium dodecyl sulfate followed by autoradiography demonstrated that the 32P was located primarily on enzyme subunits SA1, SA3, and SA5-SA6. High voltage paper electrophoresis of a partial acid hydrolysate of phosphorylated RNA polymerase I revealed that both serine and threonine residues were phosphroylated. N6,O2'-Dibutyryl cyclic 3':5'-AMP stimulated endogenous RNA polymerase I activity and endogenous nuclear protein phosphorylation in intact nuclei. These results suggest that phosphorylation of RNA polymerase I by nuclear protein kinases may play a role in the control of transcription in mammalian cells.


Treatment of fractures of the femoral neck by replacement with the Thompson prosthesis. The case histories of 361 subcapital fractures of the femoral neck in 354 patients treated by primary replacement with the cemented Thompson prosthesis were reviewed, and 156 survivors were seen at follow-up. The average age was eighty-one years. Three hundred and thirty fractures occurred in women and thirty-one in men. The antero-lateral approach to the hip was used at all operations because it allows immediate and unrestricted mobilisation after operation. It is considered that this method is the best treatment in the rehabilitation of elderly patients after high femoral neck fractures, because of its low morbidity and high success rate in returning the geriatric orthopaedic patient to independence. The results after an average of just over three years were available for 161 hips in 156 patients studied at follow-up. Of these, 132 (82 per cent) were satisfactory. Most of the unsatisfactory results were in patients under seventy-five years of age. Out of the whole group of 361 hips treated the important early complications were wound infections in seventeen hips 4-7 per cent), all of which resolved, and dislocation in seven hips (2 per cent). Forty-six patients (12-9 per cent) died during the first four weeks after operation, their average age being eighty-five. One hundred and seventy-one patients (47-4 per cent) were discharged from hospital within under four weeks of the operation. Acetabular erosion and loosening are shown to be the important later complications. Three patients had late sepsis.


Ecto-ganglioside-sialidase activity of herpes simplex virus-transformed hamster embryo fibroblasts. Cellular location of ganglioside-sialidase activity was determined in confluent hamster embryo fibroblasts transformed with herpes simplex virus type 2. Approximately equal specific activities of ganglioside-sialidase activity were found to be associated with the crude lysosomal and crude plasma membrane fractions isolated from whole cell homogenates. Whole transformed cells hydrolyzed exogenous ganglioside substrate, suggesting a partial location of the cellular sialidase on the outer surface of the plasma membrane of these cells. Intact cells were treated with the diazonium salt of sulfanilic acid, a nonpenetrating reagent inhibitory to ecto-enzymes (DePierre, J.W., and M. L. Karnovsky. 1974. J. Biol. Chem. 249:7111-7120). Cytoplasmic lactate dehydrogenase activity was not inhibited by this treatment, and mitochondrial succinate dehydrogenase activity was inhibited only 10%, indicating that intracellular enzymes were not affected. 5'-Nucleotidase activity was diminished 90%, and sialidase very rapidly lost 40% of its exogenously directed activity. These results show that, in herpes simplex virus-transformed fibroblasts, ganglioside-sialidase is both a lysosomal and a plasma membrane enzyme. The plasma membrane sialidase is capable of acting on endogenous plasma membrane sialolipids and also functions in the cultured transformed cell as an ecto-enzyme which can attack exogenous substrates.


Cell to substratum adhesion-promoting activity released by normal and virus-transformed cells in culture. It is demonstrated here that cultured fibroblasts release into their medium a nondialyzable, protease-sensitive factor(s) capable of promoting the adhesion and spreading of virus-transformed rat fibroblasts on a plastic substratum. A relatively sensitive biological assay is described for quantitation of the adhesion-promoting factor (APF) activity in serum-free, conditioned medium harvested from the cultures. Evidence is presented which indicates that the primary mode of action of the APF is by binding to and modifying the properties of the substratum. Conditioned media harvested after 24 h of incubation in similarly populated cultures of normal fibroblasts of diverse animal species exhibited similar levels of APF activity. However, conditioned media obtained from Rous sarcoma virus (Prague strain)-transformed and avian sarcoma virus B77-transformed rat fibroblasts exhibited three- to sixfold lower levels of APF activity than media conditioned in parallel cultures of heterologous or homologous normal fibroblasts. Cultivation of B77 virus-transformed rat cells in the presence of dibutyryl cyclic AMP and theophylline led to as much as a sevenfold increase in the level of APF activity appearing in the culture medium, with a concomitant increase in the adhesiveness of the cells to the culture substratum. The results support the role of extracellular macromolecules in cell to substratum adhesion. It is postulated that the reduced adhesiveness of transformed cells to a substratum may be at least partially owing to a deficiency in the production and/or release of APF-like macromolecules.


Fibrils attached to the nuclear pore prevent egress of SV40 particles from the infected nucleus. SV40 particles can apparently enter the nucleus intact. However, they do not leave the nucleus despite the high concentration present during the productive phase. We found structural evidence that SV40 virus is prevented from approaching the most likely site of exit, the nuclear pore complex. From these images, it is concluded that the fibrils attached to the nuclear pore complex prevent egress of SV40 particles from the infected nucleus.


Cytotoxicity for tumor cells of cationic proteins from human neutrophil granules. Cationic proteins purified from human polymorphonuclear leukocyte granules exert a cytotoxic effect on mammalian tumor cells. This effect is time and concentration dependent, is inhibited by the anionic agent heparin, and is enhanced by preheating the cationic proteins. Other strongly basic proteins (histone, protamine) also exhibited cytotoxic activity. Myeloperoxidase isolated from human leukocytes is cytotoxic when combined with H2O2 and chloride. Under these conditions, the potency of the myeloperoxidase-mediated system is greater than that of the cationic proteins.


Stimulation by N6,O2'-dibutyrul andenosine 3',5'-cyclic monophosphate of RNA and DNA synthesis and of cell proliferation of rat hepatocytes in primary tissue culture. The effects of DBcAMP in doses from 1.5 x 10(-8) to 1.5 x 10(-3) M on the compartmental apparent surface area (ASA) and (5(-3H)uridine radioactivity concentration (URC), (methyl-3H)thymidine labelling index per 1 hour ([Me-3H]Tdr LI/h) and per cent mitotic index (MI%) and colchicine metaphase index (CMI%) of young rat differentiated hepatocytes in primary tissue culture were investigated by morphometric and radioautographie methods. In these cells DBcAMP was found to elicit: (1) progressive increments in the ASA of nucleoli, karyoplasm and cytoplasm; (2) peak increases in nucleolar URC at 1.5 x 10(-8) and 10(-5) M, but a slight decrease at 1.5 x 10(-3) M; (3) singificant increments in karyoplasmic and total nuclear URC at all doses, except at 1.5 x 10(-6) and 10(-4) M, when such parameters remained at control levels; (4) steady and progressive increases in cytoplasmic and total cell URC values; (5) marked increments in (Me-3H)Tdr LI/h, MI% and CMI% up to the dose of 1.5 x 10(-4) M, but at 1.5 x 10(-3) M these parameters were found to be either much less enhanced or to approach closely to control values. cAMP in doses from 1.5 x 10(-8) to 10(-4) M also markedly incremented the in vitro hepatocyte CMI%, while having a lesser stimulatory effect at 1.5 x 10(-3)M. Finally of the various possible metabolites of DBcAMP administered at 1.5 x 10(-8) M to liver cultures, N6- and O2'-MBcAMP and, again, cAMP significantly increased the CMI%, of cultured hepatocytes, whereas 5'-AMP, adenosine and allantoin had no significant effect and Na-butyrate slightly decreased it. The present observations strengthen the hypothesis that cAMP and its butyrated derivatives, by possibly amplifying the template activity of the liver chromatin, accelerate the flow of differentiated primary young rat hepatocytes into the various stages of the mitotic cell cycle.


Detection of sodium alkanesulphonates and alkylbenzenesulphonates by polyamide thin-layer chromatography. A thin-layer chromatographic method was investigated in which two series of sodium alkanesulphonates (C4-C18) and alkylbenzenesulphonates (C0-C14) were separated. All of the compounds tested were clearly separated on polyamide layers with aqueous ammonia-pyridine and aqueous ammonia-pyridine-methanol systems and detected with high sensitivity by spraying with a pynacryptol yellow reagent and then observing under UV light (253.6nm).


Separation of drug substances by modern liquid chromatography on silver impregnated silica gels. The use of silver halide impregnated supports for high-pressure liquid chromatographic (HPLC) and thin-layer chromatographic (TLC) analysis of drug substances has been studied. Successful separations of xanthines and mixtures containing barbiturates, xanthines, ergot alkaloids, and tropane alkaloids have been achieved with isocratic conditions or by using simple gradients. The Ag-supports show a similar behavior as many chemically bonded stationary phases, such as modification fo adsorption sites on silica gel to give lower retention but better specificity; rapid reconditioning in connection with gradient elution (3-5 minutes). Reasonable cost, simplicity of preparation and usage, and good chemical and physical stability render the silver halide phases applicable in routine analysis and quite competitive with many commercially available chemically bonded stationary phases.


A method for the gas chromatographic determination of vitamin D and related structures. A procedure is described for the separation of Vitamin D and related compounds by gas-liquid chromatography using flame ionization detection. The method involves a two-step conversion: isomerization to the corresponding (all trans) isotachysterol(s) followed by methyl ether derivatization of the alcohol group(s). The procedure provides a means for identification as well as a possible basis for quantitation of Vitamin D and analogs.


Estrone and estradiol in patients with cirrhosis of the liver: effects of ACTH and dexamethasone. To study the influence of the adrenal gland on plasma estrogen levels in male patients with hepatic cirrhosis, estrone and estradiol were measured under a variety of experimental conditions. Compared to controls, estradiol levels were moderately elevated by 26% (P is less than 0.05) in patients with hepatic cirrhosis (28.5 +/- 5.4 vs. 36.0 +/- 4.7 pg/ml plasma; n: 12), whereas estrone levels exhibited a two- to threefold increase under basal conditions (32.5 +/- 5.6 vs. 67.8 +/- 20.8 pg/ml; P is less than 0.01). ACTH application resulted in a striking increase in plasma estrone levels in both patients with hepatic cirrhosis and in normal subjects (61.8 +/- 27.5 vs. 27.3 +/- 7.8 pg/ml). During stimulation with ACTH, estradiol levels showed no significant changes. After suppression of the adrenal gland by dexamethasone administered for 5 days, plasma concentrations of estrone and estradiol were found to be reduced. The absolute decrease of estrone was significantly greater in patients with hepatic cirrhosis than in healthy male subjects (35.5 +/- 12.6 vs. 21.3 +/- 6.0 pg/ml; P is less than 0.05; n: 8). Estrogen values, however, were still high in patients with hepatic cirrhosis after 5 days of dexamethasone administration (37.1 +/- 17.6 pg estrone/ml and 23.9 +/- 3.6 pg estradiol/ml plasma). It is suggested that elevated plasma values of estrogens in this disorder may be derived predominantly from adrenal production. Peripheral conversion of androgens to estrone rather than to estradiol appears to be more effective in sustaining plasma levels of estrogens in patients with hepatic cirrhosis.


Effects of estrogens on adrenal 3 beta-hydroxysteroid dehydrogenase in ovariectomized women. The acute and chronic effects of estradiol (E2) on the serum levels of four delta5,3-beta hydroxysteroids and their four delta4, 3-keto products were studied in four ovariectomized women with and without adrenal stimulation by ACTH. Six hour infusions of saline and of synthetic 1-24 ACTH were administered and later repeated with a two hour infusion of E2 50 mug/h. The patients were then given 50 mug of ethinyl estradiol (EE2) p.o. for 4 to 6 weeks and the control and ACTH infusions were again repeated. Levels of pregnenolone3 (Pe), 17alpha-hydroxypregnenolone (17 Pe), progesterone (Po), 17alpha-hydroxyprogesterone (17 Po), dehydroepiandrosterone (DHEA), androstenedione (Adione), androstenediol (Adiol), and testosterone (T), as well as cortisol and DHEA-sulfate were measured by radioimmunoassay on serum samples taken at 1200 and 1300 h. There was no significant effect of E2 or EE2 in the doses administered with or without exogenous ACTH on 3 betaOHSD activity as reflected in absolute steroid levels or in the ratio of concentrations of each delta5:delta4 steroid pair. During the 4th and 5th hour of ACTH infusion, the plasma level of 17 Pe (mean 22.5-fold stimulation) was most elevated, followed by 17 Po (12.5-fold), Pe (10-fold), cortisol (5.9-fold) and Po (4.5-fold), with smaller increases for the other steroids. These results, as well as the pattern of change in plasma levels in one of the subjects in whom fifteen minute samples were measured, provide further evidence suggesting that the major pathway for cortisol biosynthesis in vivo proceeds from Pe via 17 Pe, and not via Po.


ACTH and cortisol responses to glucagon stimulation. The effect of the intramuscular injection of various doses of glucagon in 15 healthy subjects was studied. Significant elevations of plasma ACTH, and cortisol were found to occur 3 h after the administration of 4 mg of crystalline glucagon. Mean levels in 7 subjects were for ACTH 44 +/- 30 (SD) pg/ml, and for cortisol 14 +/- 6 (SD) mug/100 ml at the beginning of the test, and rose to 109 +/- 48 (SD) pg/ml and to 23 +/- 5 (SD) mug/100 ml respectively following glucagon. The peak response of ACTH and cortisol was preceded by a significant rise of plasma insulin, by a fall of the blood glucose, which was initially increased by the administration of glucagon, and by the symptoms of nausea and sweating. This study demonstrates that the intramuscluar administration of glucagon (4 mg) provids a potent stimulus to ACTH and cortisol secretion in healthy subjects.


Stability of pseudorabies virus during freeze-drying and storage: effect of suspending media. The effect of suspending media on the stability of pseudorabies virus upon freeze-drying and subsequent storage was studied. A variety of media was tested, including: sodium glutamate; sucrose; lactose; lactalbumin hydrolysate; peptone; a combination of sucrose, dextran, and glutamate; and various combinations of sucrose, glutamate, and potassium phosphates. Suspending media containing glutamate, either alone or in combination with sucrose and either dextran or phosphates, afforded the greatest degree of protection during the freeze-drying process and upon storage. Some possible functions of these additives in preventing injury to the virus during freezing and drying have been suggested.


Recovery of applied human leukocyte interferon from the nasal mucosa of chimpanzees and humans. Intranasally applied human leukocyte interferon was recovered locally in 9 of 10 chimpanzees and 3 of 3 human volunteers tested. A 5- to 50-fold reduction in interferon was observed over 60 min.


Continuing search for the etiology of cat scratch disease. Fifty cat scratch disease patients were tested for antibodies against candidate etiological agents, with inconclusive results. Future research should emphasize new viral isolation methods to discover the elusive agent.


Plaque reduction neutralization test for human cytomegalovirus based upon enhanced uptake of neutral red by virus-infected cells. Foci of cells infected with human cytomegalovirus were noted to stain more intensely than uninfected cells with neutral red, and this provided the basis for development of a plaque assay and plaque reduction neutralization test for cytomegalovirus. Plaques demonstrable by neutral red staining could be counted at 8 days after infection; thus, results could be obtained earlier than for plaque assay systems based upon the viral cytopathic effect, a fewer manipulations were required for staining cell monolayers to demonstrate plaques. Certain variables affecting plaque size and numbers and antibody titers were defined. Addition of fresh guinea pig complement to the reaction mixtures markedly enhanced cytomegalovirus-neutralizing antibody titers of hyperimmune animal sera, but titers of human sera were enhanced only two-or fourfold.


Genetic Transformation as a tool for detection of Neisseria gonorrhoeae. A rapid method for the detection of Neisseria gonorrhoeae, making use of the ability of deoxyribonucleic acid samples from clinically isolated strains of this organism to transform nutritional mutants of a particular strain of N. gonorrhoeae, has been described. In addition to using isolated cultures, transforming deoxyribonucleic acid can be obtained directly from the material that adheres to swabs of the cervix or the urethra. The time interval for transfer of swabs to the diagnostic laboratory is not a significant factor. It is not necessary to use pure cultures on primary isolation plates to obtain definitive results. Nongonorrhoeae  neisserias, as well as a large variety of commonly encountered unrelated bacteria, do not react or interfere in the transformation assay when using one of the mutant strains under a standardized set of conditions. The entire assay can be completed in less than 24 h. It has also been shown that type T4 cells of the strain of N. gonorrhoeae employed in the present study are competent for genetic transformation, although type T4 cells are transformed at a significantly lower frequency than are type T2 cells of the same strain.


Male pseudohermaphroditism presumably due to target organ unresponsiveness to androgens. Deficient 5alpha-dihydrotestosterone binding in cultured skin fibroblasts. Maximum specific 5alpha-dihydrotestosterone (DHT) binding activity (Bmax) had been measured in intact confluent monolayers representing fibroblast strains derived form nongenital and genital (labium majus) skin of normal individuals and of 11 patients fulfilling the clinicogenetic criteria of complete testicular feminization (TF). Nine labium majus strains from adult females had a mean Bmax value three times greater than that of seven nongenital strains from adult females (33 vs. 11 fmol/mg cell protein). The Bmax results for 13 adult nongenital strains varied from 5.6 to 23.3 fmol/mg protein; the values for males and females had very similar means and ranges. The variation could not be correlated with the chronologic age of adult skin explant donors or with the in vitro age (mean population doubling level) of the cultures assayed. The Bmax activities of three nongenital strains from normal infants  (two male, one female) did not exceed 5 fmol/mg protein. Seven of eight nongenital TF strains had Bmax values below 2 fmol/mg protein; the value for the eighth coincided with the lower limit of normal adults. The lower limit of DHT binding in normal labium majus strains was 15 fmol/mg protein. Three of five labial strains from patients with TF had Bmax values close to zero; the other two fell between 10 and 15 fmol/mg protein. It is apparant that labial skin fibroblast strains from clinically homogeneous patients with TF had highly variable degrees of DHT binding deficiency, and that they permit a more reliable diagnosis of severe and intermediate degrees of DHT binding deficiency than do strains of nongenital skin fibroblasts.