Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells. Properties of the purified polymerase. Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells, partially purified by chromatography on DNA-agarose, was obtained as a more than 80% homogeneous preparation by isoelectric focusing in a sucrose gradient. The polymerase activity was shown to be associated with the major protein in the preparation. Results obtained by electrophoresis in the presence of sodium dodecyl-sulfate indicated that poly(ADP-ribose) polymerase consists of a polypeptide chain with a molecular weight of 130 000. Ultracentrifugation at non-denaturating conditions indicated that the active enzyme may be an oligomeric form of this polypeptide chain. The isoelectric point of the polymerase was 9.40. The effects of various additions to the assay mixture on the synthesis of poly(ADP-ribose) as well as some kinetic data, are given. It is shown that poly(ADP-ribose) is a highly efficient inhibitor of its own synthesis, and results are presented which suggest that the well-known stimulatory effect of DNA on the synthesis is due to reduction of this inhibitory effect of the product.


The spin-state transition of the hemochrome non-equilibrium conformation in partially reduced human methemoglobin. A pulse-radiolysis study of aqueous-methanol solutions of methemoglobin. The effect of external parameters on the relaxation process of the hemochrome-type non-equilibrium conformation in partially reduced methemoglobin has been investigated. The relaxation of the intermediate ferrous low-spin state to the high-spin equilibrium conformation of hemoglobin appears to be facilitated particularly by protons and phosphate ions. In addition to studying the spin-state transition in aquomethemoglobin we have also studied it in complexes of the heme group in methemoglobin with fluoride, azide and cyanide anions.


Metabolism of apolipoproteins A-I and A-II and its influence on the high density lipoprotein subfraction distribution in males and females. Rate zonal ultracentrifugation of plasma samples from ten healthy age-matched volunteers (five males, five females) indicated that the high density lipoprotein subfraction ratio (HDL2:HDL3) in females was significantly higher than in males. The cause of this phenomenon was investigated by simultaneous examination of the metabolism of the major HDL apoproteins (apoA-I and apoA-II) in both groups. The results show that there is no significant sex-related difference in the plasma pool size, fractional catabolic rate, or synthetic rate of either apoprotein. We conclude that the increased HDL2:HDL3 ratio in femalse versus males does not derive from measurable differences in the metabolic handling of either apoprotein.


Variations in apolipoproteins B and A1 during the course of myocardial infarction. The plasma apolipoproteins B and A1, and plasma lipids and lipoproteins, were studied in fifteen patients with acute myocardial infarction. In the days immediately after acute infarction there was a decrease in total cholesterol, low density lipoprotein-cholesterol, total apolipoprotein-B, low density lipoprotein apolipoprotein-B and high density lipoprotein apolipoprotein A1. High density lipoprotein-cholesterol remained unchanged. In the same period the total triglycerides, very low density lipoprotein-protein, very low density lipoprotein-cholesterol, very low density lipoprotein apolipoprotein-B and very low density lipoprotein apolipoprotein A1 were increased. A reduction of the apolipoprotein ratio CII/CIII occurred after the acute phase. After 25--30 days all these values regained their baseline values.


High density lipoprotein infusion and partial plasma exchange in Tangier disease. High density lipoprotein (HDL) infusion and partial plasma exchange were undertaken in two patients homozygous for Tangier disease. Serum samples and ultracentrifugally isolated serum fractions were analysed over a period of 7 days post infusion by agarose electrophoresis, two-dimensional immunoelectrophoresis (employing antibodies to HDL, HDL3, Apoprotein A-I, and Apoprotein A-II), Apoprotein A radioimmunoassay, and analytical polyacrylamide electrophoresis. The following observations were made: (a) immediately after HDL substitution the broad-beta band, normally visible upon agarose electrophoresis of Tangier plasma, resolved into a distinct beta and pre-beta band; (b)as HDL was catabolized, an abnormal alpha-migrating lipoprotein was generated which contained Apoprotein A-II as protein constituent; and (c) there was a proferential loss of Apoprotein A-I from HDL and the plasma compartment in the course of HDL catabolism. The results suggest that the defect in Tangier disease resides with enhanced catabolism or defective synthesis of Apoprotein A-I.


Serum lipoprotiens in four European communities: a quantitative comparison. Serum lipid and lipoprotein concentrations have been measured in 985 men and women sampled from communities in London, Naples, Uppsala and Geneva. This was done to define normal ranges, detect inter-population differences, and relate such differences to levels of lipids in the major lipoprotein classes of plasma. Cholesterol and triglyceride concentrations showed substantial differences between the four populations; levels of these lipids showed parallel trends, which were attributable to differences in low density lipoprotein and very low density lipoprotein concentrations. By contrast, high density lipoprotein cholesterol concentrations showed little or no interpopulation differences. Geographical differences in serum triglyceride and cholesterol levels were not correlated with variation in the prevalence of obesity.


The effects of alpha and beta adrenergic agents on spleen cell antigen binding in four amphibian species. Adults of two urodele amphibian species (Triturus cristatus carnifex and Cynops hongkongensis) and two anuran species (Rana temporaria and Xenopus laevis laevis) were immunized with a 25% suspension of sheep or horse erythrocytes. After eight or 14 days, splenic lymphocytes were removed, and their specific red cell-binding capacities tested by immunocytoadherence. Antigen-binding cells were classified as high-dose nonsecretory (S-) or secretory (S+), according to whether they bound a single layer or several layers or erythrocytes. The stimulation of both alpha and beta adrenoreceptors reduced the numbers of S+ rosettes formed by Triturus and Cynops lymphocytes, whereas a beta agonist increased and an alpha agonist decreased S+ rosette formation by Rana and Xenopus splenic lymphocytes. These effects were blocked by alpha and beta adrenoreceptor antagonists. Low-dose immunization of Xenopus with a 0.0025% suspension of sheep erythrocytes gave a minimal number of S+ rosettes two and eight days after immunization, and beta adrenoreceptor stimulation had no effect on antigen binding. These results are discussed in terms of the distribution of alpha and beta adrenoreceptors in amphibians and possible relationships between S+ and high-dose S- antigen-binding cells, and support the view that functional lymphocyte heterogeneity exists in these lower vertebrates.


Activation of the alternative complement pathway by human B cell lymphoma lines is associated with Epstein-Barr virus transformation of the cells. Activation of the alternative complement pathway by human B cell lymphoma lines is correlated with the presence of Epstein Barr virus (EBV) in the cell genome. EBV-negative B cell lymphoma lines produce little activation of the alternative pathway as measured either by C3 deposition on the cell surface or C3 conversion and consumption of alternative pathway activity in the supernatant serum. By contrast, EBV-positive sublines derived by in vitro EBV conversion of EBV-negative parental lines produce considerable activation of the alternative pathway. This membrane-associated complement-activating mechanism reflects an EBV-induced membrane change in these cells and may provide a mechanism whereby EBV-transformed cells are controlled in vivo.


Caffeine elicited withdrawal signs in morphine-dependent rhesus monkeys. In the dose range of 4.0--32.0 mg/kg s.c., caffeine produced most of the signs which are commonly seen after the administration of naloxone (0.05 mg/kg s.c.) to morphine-dependent monkeys. The signs designated as lying on side or abdomen, avoiding contact, vocalizing, crawling or rolling, restlessness or pacing, tremors, retching, vomiting, coughing, vocalizing when abdomen palpated, rigid abdomen and salivation were noted. A randomized and blind experimental design, which included vehicle and positive (naloxone) controls was used. The significance of the differences between total scores for the whole syndrome was tested by the Mann-Whitney U-test. In preliminary studies in naive monkeys, caffeine was found to elicit some withdrawal signs but the results were equivocal. Na benzoate also elicited some withdrawal signs in morphine-dependent monkeys at 32.0 mg/kg s.c., but few signs were seen in naive monkeys. Caffeine was found to be approximately 10X more active than Na benzoate in inhibiting cAMP phosphodiesterase activity in a neuroblastoma cell whole homogenate assay. These results are consistent with the observations of Collier and Francis that morphine abstinence in rodents is associated with increased brain levels of cAMP.


Distinct dopaminergic systems in ACTH-induced grooming. Excessive grooming behavior induced in rats by intraventricular injection of ACTH1-24 was inhibited by selective pharmacological manipulation of two functionally distinct types of dopaminergic terminals (DAe and DAi) in the neostriatum or the nucleus accumbens. It is concluded that when ACTH1-24 produces the behavioral response it modulates the activity of both the DAe and the DAi systems.


The role of protein synthesis in polymorphonuclear leukocyte phagocytosis II. Polymorphonuclear leukocytes collected from healthy human volunteers were treated with puromycin which inhibited protein synthesis in the cell. The neutrophils exhibited a marked decrease in phagocytosis after 1 h. The myeloperoxidase mediated conversion of iodide to a protein-bound form was also decreased. Hexosemonophosphate shunt activity remained similar for both puromycin treated PMN and control cells at 1 h. The results suggest that new protein synthesis is necessary to maintain PMN function at full capacity.


Mobilization of CFU-C by exercise and ACTH induced stress in man. This study was designed to investigate physiological factors that affect blood CFU-C numbers. Exercise was found to produce a fourfold rise in blood colony forming cells, and a twofold rise in cluster forming cells accompanied by a lymphocytosis. ACTH-induced stress caused a twofold rise in blood colony and cluster forming cells accompanied by a neutrophil leucocytosis. The results suggest that blood CFU-C exchange with a large marginal pool as well as a bone marrown pool. The procedures described could be exploited in the use of leukapheresed nucleated cells as a source of stem cells for bone marrow transplantation.


The effect of peripheral nerve cross-union on connections of single Ia fibers to motoneurons. The distribution of Ia terminals to alpha-motoneurons has been investigated under conditions where a) the Ia fiber innervates a foreign muscle and b) the motor axon innervates a foreign muscle. This distribution has been assessed by examining which motoneurons develop an EPSP in response to stimulation of a single Ia afferent fiber. Cross-union of the medial gastrocnemius and lateral gastrocnemius-soleus nerves has been performed in 5--8 day old kittens and the effect of this procedure on Ia connections to alpha-motoneurons has been investigated in the adult about one year later. No rearrangement of synaptic connections has been observed under these conditions. The connections from self-unioned Ia afferents and to self-unioned motoneurons are also normal. The individual EPSP's in these motoneurons with regenerated axons have normal rise times. The findings of normal Ia connections as well as normal EPSP rise times in alpha-motoneurons with regenerated motor axons indicates reversibility of the changes seen in axotomized motoneurons.


Study of antiblastomogenic action of epidermal chalones. I. The effect of epidermal chalones on some transplantable mouse tumours. The effect of chalone-containing ethanol extract of rat skin (CCE) on the growth in mice of transplanted uterine cervix and skin carcinomas, hepatoma-22a, sarcoma-180 and leukemia L-1210 was studied. When CCE is added to the suspension of tumour cells (10 mg CCE/100 mg tumour tissue/ml saline) the most obvious retention of tumour growth is observed on squamous-cell carcinoma of uterine cervix (72.6%; p less than 0.01). The effect of CCE on the growth of other transplanted tumours, including the skin carcinoma, is not significant. As compared to the uterine cervix carcinoma, the skin carcinoma lacked its primary squamous-cell structure during the tumour progression. The possibility of applicative use of chalones for cancer control is discussed.


Initiation of sperm motility in the mammalian epididymis. Spermatozoa acquire the capacity for motility as they traverse the mammalian epididymis. The biochemical basis for this induction of motility is still largely unknown. Current theories are discussed and recent studies from the authors' laboratory are described which indicate that two separate processes are involved and that these act synergistically. These processes are an increase in the intrasperm content of cyclic AMP during epididymal transit combined with the binding of a specific forward-motility protein. A second increase in cyclic AMP and stimulation by calcium ion is likely involved in the expression of the acquired potential for motility at the time of ejaculation.


[Role of the paradoxical stage in the organization of the sleep--wakefulness cycle in rats]. In chronic experiments on rats, using the Jouvet technique, paradoxical sleep deprivation entailed disturbances in both the sleep cycle and the mechanism for regulation of the circadian sleep--wakefulness cycle. The sleep cycle disturbances involved, in the first place, a considerable reduction of the paradoxical sleep, diminishing of the profound slow-wave sleep, and an increase in duration of the light slow-wave sleep. These shifts were clear on the 1st day of the deprivation and considerably reduced on the 2nd day due to an increasing rate of alternation of the sleep--wakefulness cycle's phases. The data obtained suggest a certain commonness of the mechanisms for control of the slow-wave and the paradoxical phases of sleep, as well as the great compensatory abilities of the organism at the paradoxical sleep deprivation.


[Deprivation of "rapid" sleep by stimulation of the reticular formation of rats]. Rats were repeatedly arused from REM sleep by the stimulation via implanted midbrain reticular electrodes. The 4-day seprivation reduced REM sleep by 50% whereas the slow-wave sleep was only reduced by 10%. There was no evidence of a disruption in the animal behaviour during the deprivation and no changes of emotional responsiveness in the "open-field" at the end of the deprivation. REM deprivation produced a slight but statistically significant (P = 0.025) adrenal weightlos. Thymus weight was unchanged and no stomach ulcers developed. Therefore the REM deprivation as such did not appear to produce stress in rats.


[Metabolic structure of the restitution process following physical loads of a different character]. The result of biochemical and mathematical analysis of restitution processes after various exercises determined the phases of these processes and the interrelationship between metabolic structure of restitution and the character of exercise. Metabolic structure of restitution has some common traits in recovery period after non-fatiguing exercises and exercises causing a rapidly developing fatigue. In contrast, sharp changes of this structure occur after exercises causing a slowly developing exhaustion.


[Effects of several estrogenic compounds on serum corticosteroid-binding globulin (CBG) and the estrogen thresholds for increasing CBG (author's transl)]. The effects of several estrogen substances--alone, or in combination with norgestrel--on serum CBG in humans are reported in this paper. CBG was measured by a modified charcoal adsorption technique. Daily oral doses of Premarin, from 0.3 to 2.5mg, were given to healthy postmenopausal women for 14 days. Dose-dependent increases in CBG-BC were observed. Ovral, which contains ethinylestradiol and norgestrel, also induced significant increases in the levels of CBG-BC. Similar results were seen with Lo Ovral, but it had less influence than Ovral. When given alone, daily for 21 days, norgestrel had no effect on CBG-BC. The maximum rise of serum CBG-BC was found after 10 days of the daily doses of Ovral, and then the levels remained quite constant during the period of treatment. Exogenous estrogens of other types also caused increases in CBG-BC. The response of CBG to exogenous estrogens is of use in comparing the estrogen potencies of the compounds. CBG-BC levels remained constant throughout the menstrual cycle in five normal women despite fluctuating levels of estradiol which reached peaks of 190 approximately 430 pg/ml. In pregnant women and in HMG-treated women, however, serum levels of CBG-BC increased beginning when estradiol levels were greater than 1 ng/ml.


So-called multicentric pigmented Bowen's disease. Report of a case and a possible etiologic role of human papilloma virus. Multicentric pigmented Bowen's disease (MPBD) is a bowenoid atypia in the genitocrural region with peculiar clinical appearances. A 36-year-old Japanese female patient showed a variety of lesions. Clinically the lesions on the external genitalia consisted of brown-black papillomatous eruptions, black discrete or confluent papules, and whitish macerated papules. Histologically only black papules showed bowenoid atypia, but whitish papules also showed transient bowenoid atypia. Electron microscopically, in all three kinds of the lesions, spherical particles with a diameter of about 50 nm were scattered or gathered together in the nuclei of the keratinocytes beneath the horny layer. These particles were morphologically similar to human papilloma virus. Based upon clinical, histologic and electron microscopic observations, MPBD may be regarded as a new entity, and a term such as multicentric pigmented viral papulosis may be rather preferable than MPBD.


Distribution of adenylate cyclase among membrane fractions of rat liver. Adenylate cyclase activity was detected in plasma membranes, Golgi apparatus, and endoplasmic reticulum from rat liver. Adenylate cyclase activities of purified membranes were determined biochemically by two methods. In one, the synthesis of radioactive cyclic AMP from ATalpha32P was monitored. In the other, the synthesis of cyclic AMP was quantitiated using a protein which specifically binds cyclic AMP. The enzyme activity was responsive to activation by both glucagon and sodium fluoride although differences in degree of activation were noted comparing plasma membrane, Golgi apparatus, and endoplasmic reticulum. Cytochemical studies, using both whole tissue and purified cell fractions and conducted in parallel, confirmed the biochemical results. Deposition of lead phosphate, enhanced by glucagon and NaF with samples incubated with appropriate substrates, was not restricted to plasma membranes of hepatocytes but was present in intracellular membranes as well. Adenylate cyclase of rat hepatocytes appears more widely distributed among internal membranes than previously recognized.


Transformation of skin fibroblast cells of a cystinotic patient by simian virus 40: evidence for an establishment of a permanent cell clone which retains the original metabolism defect. Human skin fibroblast cells derived from a juvenile patient with nephropathic cystinosis were transformed by simian virus 40. Transformed cell clones were isolated and established in tissue culture. In comparison to the parental cystinotic cells, the newly isolated, transformed cell clones had a higher plating efficiency, a modal chromosome number of 68, grew in soft agar, and showed a nuclear immunofluorescence typical for SV 40-specific tumor (T) antigen. The content of intracellular, unbound cystine in the transformed cell clone was of the same level (6.1 nmol 1/2 cystine/mg protein) as in the parental cystinotic cells (7.4 nmol). Control cells (SV 80 and WI-38) contained normal levels of cystine (0.31 and 0.47 nmol 1/2 cystine/mg protein). The growth characteristics make the transformed cystinotic cell clone suitable for large scale preparation of cellular constituents, i.e. lysosomes which seem to be affected in cystinotic patients.


Polygraphic studies of kitten development: respiratory rate and variability during sleep-waking states. The developmental course of respiration rate and variability during sleep states and waking was measured in chronically prepared kittens. Kittens had higher respiration rates during active sleep (AS) as compared to quiet sleep (QS) at all ages, with rates declining developmentally in both sleep states. Compared to waking, respiration rate and variability were decreased during sleep. The decrease was greatest in the youngest animals and during QS. Minute-to-minute respiratory variability declined from 10 to 40 days of age for each state, whereas breath-to-breath variability declined during the same period only during QS. Respiratory variability was higher in AS than QS in older kittens. Heart rate and variability were found to be correlated with respiratory parameters only during QS. These observations support the hypothesis that the control and development of respiration during sleep is achieved by different processes in QS and AS.


Effect of N-(3,5-dichlorophenyl)succinimide on the histological pattern and incidence of kidney tumors induced by streptozotocin rats. The effect of the nephrotoxic substance, N-(3,5-dichlorophenyl)succinimide, on the histological pattern and incidence of kidney tumors induced by streptozotocin in rats was studied. In groups administered streptozotocin alone or in combination with nicotinamide, the histological pattern and the incidence of kidney tumors in rats were similar; renal cell tumors developed in 1 of 9 rats (11.1%) and in 2 of 17 rats (11.8%), respectively. However, post-treatment with N-(3,5-dichlorophenyl)succinimide increased the induction of epithelial tumors by streptozotocin, and embryonal cell tumors were also induced nearly as frequently. Administration of nicotinamide alone did not result in the development of kidney tumors.


Application of the transfection technique for segregation of avian tumor viruses. The possibility of clong avian tumor virus (ATV) by the transfection technique was studied. The attempts required efficient facilitator for the uptake of exogenous cellular DNA by chick embryo cells (CEC) to increase transfection efficiency. The calcium method was more efficient than the DEAE-dextran method for the uptake of tritium-labeled DNA, and the calcium method was used in further segregation studies of ATV by transfection of cellular DNA from ATV-infected cells. The test viruses included the BAI-A strain of avian myeloblastosis virus (a mixture of subgroup A and B viruses) and the B77 strain of avian sarcoma virus (a mixture of transforming and transformation-defective viruses). Analysis of the recovered progeny viruses after transfection revealed that they were indeed segregates of the parent viruses.


Inhibition of macrophage-mediated cytolysis by lipoproteins from cell-free tumorous ascites. Cell-free ascites from syngeneic MM46 tumor-bearing C3H/He mice inhibited the antibody-dependent macrophage-mediated tumor lysis in vitro and the inhibitiory activity increased with the period of tumor bearing. Thus, the inhibitory activity was demonstrated at the effector cell level. Three fractions of lipoprotein and a protein-rich fraction were prepared from the cell-free ascites by sequential flotation. Among these, the very low density (p less than 1.006) and low density (1.006 less than p less than 1.063) lipoprotein fractions inhibited tumor lysis mediated by activated macrophages and syngeneic antitumor antibody, whereas the high density (1.063 less than p less than 1.21) lipoprotein and the protein-rich infranatant fractions did not. This inhibitory activity at the effector cell level may be due to functional depression of macrophages by lipoprotein fractions.


Electron microscopic observation of scirrhous carcinoma of the stomach. Electron microscopic findings obtained from seven scirrhous carcinomas of the stomach were analyzed dividing each carcinoma into superficial, deep, and peripheral parts. Proliferation of collagen fibrils, disappearance of plasma membranes, and extracellular release of cell organellae of cancer cells were considered to be peculiar findings to this type of cancer. These phenomena were observed frequently at the deep part of the cancer, but rarely at the peripheral and superficial parts. Replacement of damaged cells by the proliferated collagen fibrils at the central area of the cancer and disconnection of cancer cells from scirrhous lesion at the peripheral part were considered to contribute to the biological behaviour of this carcinoma.


Deletions of the iso-1-cytochrome c and adjacent genes of yeast: discovery of the OSM1 gene controlling osmotic sensitivity. Some of the deletions in the yeast Saccharomyces cerevisiae that encompass the CYC1 gene, which determines iso-1-cytochrome c, extend into the OSM1 gene, causing inhibition of growth on hypertonic media, and into the RAD7 gene, causing sensitivity to UV light. Two deletions (cyc1--363 and cyc1--367) encompass only the CYC1 gene, two deletions (cyc1--366 and cyc1--368) encompass the CYC1 and OSM1 genes, three deletions (cyc1--1, cyc1--364 and cyc1--365) encompass the CYC1, OSM1 and RAD7 genes, while none of the deletions extend into the closely linked SUP4 gene.


[Analysis of mutation frequency following continuous and fractionated application of ethyleneimine and ethyl methanesulfonate to male sex cells of Drosophila melanogaster]. The increase in the frequency of recessive lethal sex-linked mutations induced by fractionated effect of ethylene imine (EI) an ethylmethanesulphonate (EMS) on mature sperm of Drosophila melanogaster was observed and compared uith prolonged treatment (8h) and with the additive effect. This effect of dose fractionation was observed in the case of the treatment of sperms in male gonads and in female spermathecas. The increase of the mutation frequency was noted by brood-pattern method after fractionated treatment of spermatocytes and spermatogonia only with EMS. This increase was not observed under the effect of EI on spermatocytes and spermatogonia because of the high sierilization activity of EI. Possible mechanisms of the effects observed are discussed.


[Results of various operations for syndactylia (follow-up studies and statistical evaluation of 101 primary operations)]. 101 cases of syndactyly in 34 male and 14 female infants who were aged three months to seven years at the time of their initial surgery were reviewed one to 9.5 years thereafter. The web space was formed by a palmar and a dorsal flap; in 50% of all commissures the result was found satisfactory in the follow-up series. Straight incision - performed in two thirds of our cases - caused scar contractures and deformities of several fingers. In contrary, zig-zag or wavy incisions showed good results. Using almost exclusively full thickness skin grafts from the arm, the donor site looked poor because of non-cosmetic scares and keloids. After surgery two thirds of all patients used a special splint for finger abduction for an average of about one year; neither an amelioration of insufficient primary surgery was achieved nor reappearance of syndactylism could be avoided by this device. The mobility of joints was partially decreased regardless of the pre-operative condition. Combined functions in grasping, however, were not restricted to a considerable degree. There was no difference between cutaneous and osseous syndactylism with respect to the abduction ability. Even slight impairment of wound healing caused reappearance of syndactylism; reseparation of fingers was necessary 32 times during a ten year period of follow-up. Scar contractures - especially after necrosis of skin grafts and deep infections - required early correction, while slowly developing secondary webs had to be separated some years later. Disturbance of wound healing must, therefore, be treated adequately, or better should be avoided by precise surgery and careful management of postoperative dressing.


[The motor response potential after distal and proximal stimulation: studies in healthy volunteers and in polyneuropathies (author's transl)]. Muscle responses evoked distally and proximally were recorded in median and in peroneal nerves from controls and patients with polyneuropathies of different origin. The following parameters were studied in the individual muscle response: 1. the amplitude of the potential, measured peak to peak, 2. the amplitude of the negative phase, 3. the total duration of the potential, 4. the duration of the negative phase, 5. the area covered by the negative phase. Results of the proximal measurements were divided by those of the distal records. Distal motor latencies and nerve conduction velocities were also determined. In controls a loss of amplitude of 10-15% and an equal reduction of the area covered by the negative phase was noted in proximally evoked responses and the duration of the negative phase was increased by 2-7% when compared with distal measurements. In some polyneuropathies these parameters showed significantly pathological changes, although distal motor latency, amplitude and nerve conduction velocity were still within the normal range. Comparison of distally and proximally evoked muscle potentials may thus lead to further information about the mode of conduction in altered motor nerves.


Effect of concurrent cytomegaloviral infection and undernutrition on the growth and immune response of mice. Mice were inoculated with murine cytomegalovirus (MCMV) passaged in cell cultures within 24 h of birth and subsequently fed an adequate or a low-protein diet after weaning; increases in body weight, the blastogenic response of lymphocytes to T and B cell mitogens, and the capability to produce antibodies to T or B cell-dependent antigens were observed for the first 28 to 42 days of life. Neonatal infection resulted in immunosuppression and retarded physical growth through the first 4 weeks of life. After weaning (3 weeks), the effect of MCMV infection of well-nourished mice paralleled the effect of undernutrition in uninfected animals. Undernutrition and MCMV infection functioned synergistically.


Antibodies in urine of chimpanzees with chronic adenoviral viruria. Many chimpanzees have naturally occurring chronic intermittent viruria with an adenovirus of a new type called Pan 11. Small amounts of neutralizing antibodies to Pan 11 adenovirus were found in the urine of chimpanzees. Urinary antibodies to adenovirus were mainly of the immunoglobulin G (IgG) class with some IgA antibodies also present. There was no neutralizing activity in urine against another adenovirus, Pan 9, which has been isolated from lymph nodes, but not from urine, of chimpanzees; however, sera of all chimpanzees had neutralizing antibodies to Pan 9 virus, some with titers similar to those of antibodies against Pan 11 virus. Antibodies reacting with simian cytomegalovirus by indirect immunofluorescence were found in sera of all chimpanzees tested and in two of six urines. There was no correlation between levels of antiviral IgG antibodies in serum and urine by immunofluorescence. These findings suggest that both IgG and IgA antibodies may be locally produced in response to viral infection of the urinary tract in primates.


Suppression and enhancement of mitogen response in chickens infected with Marek's disease virus and the herpesvirus of turkeys. The kinetics of phytohemagglutinin (PHA) response of peripheral blood lymphocytes from chickens infected with oncogenic Marek's disease (MD) virus (MDV) or nononcogenic herpesvirus of turkeys (HVT) was studied with a whole blood microassay. At about 7 days after inoculation, a depression in PHA response was observed in MDV-inoculated resistant line N or susceptible line 7(2) chickens and in HVT-inoculated line 7(2) chickens. All chickens initially regained their PHA responsiveness. Susceptible chickens that died of MD or developed MD lymphoma in later stages of virus infection showed a second severe depression in PHA response. No depression was observed in HVT-vaccinated chickens when challenged with MDV. The PHA response of MDV-inoculated chickens that survived MD, HVT-inoculated chickens, and HVT-vaccinated MDV-challenged chickens showed evidence of enhancement. The depression of PHA response was studied and was attributed to the suppressive effect of macrophages on T-cell response, a finding consistent with our previous studies on MDV suppression of PHA response.


Studies on gonococcus infection. XV. Identification of surface proteins of Neisseria gonorrhoeae correlated with leukocyte association. Neisseria gonorrhoeae which exhibit high levels of leukocyte association have a surface protein which is considerably diminished in isogenic gonococci which exhibit low levels of leukocyte association (LA). The LA protein exhibits strain variation in molecular weight and immunogenicity. Membranes derived from LA+ and LA- organisms show quantitative differences in their adsorption to leukocytes; these differences are analogous to those found for the intact organisms regarding their association with leukocytes.


Vascular permeability changes in the central nervous system of rats with hyperacute experimental allergic encephalomyelitis induced with the aid of a substance from Bordetella pertussis. Development of hyperacute experimental allergic encephalomyelitis in Lewis rats after intraperitoneal administration of a mixture of guinea pig spinal cord emulsion and pertussigen from Bordetella pertussis was accompanied by an increase in vascular permeability in the central nervous system. The increased permeability was most striking in the spinal cord and seemed to be associated with the ascending development of paralysis. Rats that had completely recovered from paralysis did not have any increased permeability in the central nervous system. Rats which developed paralysis after inoculation with either guinea pig spinal cord emulsion alone or with complete Freund adjuvant had only a small degree, if any, of increased permeability in the vascular system of the central nervous system.


Immunofluorescence of hepatitis A virus antigen in chimpanzees. Chimpanzee liver biopsies and necropsy tissues were examined by immunofluorescence for hepatitis A virus antigen. Results further indicate that the liver may be the sole site of replication for the virus.


Antiviral activity in interferon-treated bovine tracheal organ cultures. The effect of bovine interferon on the replication of infectious bovine rhinotracheitis virus and vesicular stomatitis virus in bovine tracheal organ cultures was studied. After treatment of tracheal organ cultures with interferon, inhibition of infectious bovine rhinotracheitis virus and vesicular stomatitis virus replication was observed. This tracheal organ system may be useful in determining the in vivo response to interferon for viral infections of the bovine respiratory tract.


Some properties of beta-toxin produced by Clostridium perfringens type C. Purified beta-toxin from Clostridium perfringens type C was found to be a single polypeptide chain protein with a molecular weight of approximately 30,000. The toxin was heat labile, with 75% of its activity being inactivated by incubation at 50 degrees C for 5 min. Biological activity of the purified toxin was completely destroyed on exposure to trypsin for 30 min at 37 degrees C. The 50% lethal dose for mice was 1.87 microgram of purified toxin.


Herpes simplex virus type 2 infection of neonatal mice. We have demonstrated that newborn mice can be infected orally with herpes simplex virus type 2. Transplacental and transvaginal transmission of herpes simplex virus type 2 did not occur in our mice. Failure to produce natural transvaginal infection in mice may be explained by the relatively short period of exposure to the virus during parturition. These experiments demonstrate that high oral doses of herpes simplex virus type 2 can produce disseminate herpes neonatal infection in mice and therefore may be useful in future investigations into the mechanism and the treatment of this disease.


E.s.r. and spin-trapping studies of the reactions of hydrated electrons with dipeptides. The reactions of hydrated electrons (eaq-) with 55 dipeptides and 25 acetyl and formyl amino acids have been studied by e.s.r. and spin-trapping techniques. Gamma-radiolysis of deaerated aqueous solutions was used to generate eaq-, and sodium formate or t-BuOH was added to scavenge the OH radicals. t-Nitrosobutane was employed as the spin-trapping reagent. The radical,--CO---NH--, which is the initial product of the reactions of eaq- with dipeptides, was observed only for val-gly, val-ala, val-leu and ile-ala. For most of the dipeptides this radical converts to the primary deamination radical, CHR'-CONH-CHR-COO-, where R and R' are the side-chains of the common amino acids. In many cases a radical of the type CHR-COO-, formed by secondary deamination, was also observed. Only secondary deamination reactions were observed for dipeptides containing beta-alanine as the amino terminal residue and for acetyl and formyl amino acids. The secondary deamination reactions of eaq- with dipeptides, acetyl and formyl amino acids in aqueous solutions have not been observed previously. This type of reaction is of interest since it brings about main-chain scission in polypeptides and proteins.


Free radicals in U.V.-irradiated aqueous solutions of substituted amides: an e.s.r. and spin-trapping study. The radicals produced by reactions of hydroxyl radicals with alkyl substituted ureas and amides in aqueous solutions have been investigated. Hydroxyl radicals were produced by U.V. photolysis of H2O2 and the short-lived amide and urea radicals were spin-trapped by t-nitrosobutane and identified by e.s.r. For all N-alkyl derivatives of urea and acetamide, and for N,N-dimethyl propionamide and N,N-diethyl formamide, only radicals centred on N-alkyl groups were detected. Radicals situated only on alkyl groups attached to the carbonyl carbon were observed for dimethyl acetamide, trimethyl acetamide and butyramide. However, for N,N-dimethyl butyramide, N, N-diethyl butyramide, N-methyl propionamide and N, N-diethyl propionamide, free radicals were formed which were localized on the alkyl group attached to the amide carbon as well as those attached to nitrogen. The hydrogen atom bound to the carbonyl carbon was abstracted in N-ethyl formamide. Acyl radicals formed by C-N scission due to direct U.V. photolysis of N, N-dimethyl butyramide and N,N-dimethyl propionamide were also detected.


E.s.r. study of the post-radiolysis growth of spin-trapped radicals in gamma-irradiated aqueous solutions of thymine. The post-irradiation growth of the spin-adduct nitroxide radical produced by the addition of the thymine--OD radical to t-nitrosobutane (tNB) in gamma-irradiated, de-aerated D2O solutions was investigated by e.s.r. The thymine--OD radical was formed by the addition of an OD radical to the C(5) position of thymine. Growth reached a greater maximum value and was more rapid with increasing dose. At a fixed dose, growth was also greater and more rapid if oxygen was present after gamma-radiolysis. The addition of a second radical to the spin-adduct nitroxide during radiolysis to give a diamagnetic intermediate, which can regenerate the spin-adduct radical during storage in air-free and in air-saturated solutions at room temperature, was inferred to be responsible for post-irradiation growth. U.V. photolysis at 260-280 nm of a solution containing the diamagnetic intermediate rapidly regenerates the spin-adduct nitroxide. The longer lifetime of the diamagnetic intermediate in oxygen-free solutions may be relevant to an understanding of the anoxic sensitization by nitroxides in cellular systems.


Immunoenzymehistochemical demonstration of the binding of low density lipoproteins to cultured human fibroblasts. Using as indirect cytochemical immunoperoxidase technique, we were able to demonstrate the binding of low density lipoprotein to cultured human fibroblasts. With this technique, fibroblasts from a patient suffering from homozygous hyperlipoproteinaemia type IIa did not show this binding. The method described here allows study of the localization of unmodified low density lipoproteins binding to cultured fibroblasts.


ACTH-like immunoreactivity in the gastrin cell. Independent changes in gastrin and ACTH-like immunoreactivity during ontogeny. Rat antral gastrin cells have been shown to contain ACTH-like immunoreactivity. Studies on the ontogeny of the antral gastrin cells reveal that these cells start to store gastrin before they contain detectable quantities of ACTH-like immunoreactivity. At no stage studied were duodenal gastrin cells found to contain ACTH-like peptides. The data indicate that the G cells synthetizes and/or releases the two hormonal peptides independently.


Catalase positive particles from pig lung. Biochemical preparations and morphological studies. In pig lung tissue catalase positive particles (CPs) are abundant especially in type II pneumocytes and in Clara cells. In both cell types they occur as circular, oval or elongated membrane profiles surrounding a moderately electron dense matrix lacking a crystalline core. In Clara cells and in part of type II pneumocytes they are located as individual particles without any evident morphological relation to other cell organelles. In part of type II pneumocytes 5-8 particles are forming a group and their close relation to agranular endoplasmic reticulum cisterns is evident. The particles can be purified from lung homogenates by fractionated pelleting and subsequent rate sedimentation in a sucrose gradient using a zonal rotor. The catalase rich fraction bands in the middle of the gradient whereas cytochrome oxidase and part of the acid phosphatase sediments at its heavy end. A second part of acid phosphatase stays at the light end of the gradient and--according to morphological control--seems to correspond to lamellar bodies of the type II pneumocytes. The purified catalase positive particles do not contain hydroxyacid and D-aminoacid oxidases thought to be characteristic H2O2 producing enzymes of peroxisomal systems. The buoyant density of the particles (d = 1.195 g/cm3) is lower than that of liver peroxisomes. Cytochemical controls of the peroxisomal pellets exhibit the particles partly uniformly filled with reaction product, partly irregularly stained.


[Demonstration of an alkali PAS-effect using periodic acid at low concentration (author's transl)]. It has been shown that an isolated KOH PAS-effect of epithelial mucosubstances in the colonic mucosae of the rat can be demonstrated also without the conventionally used preliminary oxidation/reduction step. The method is based on the use of strongly diluted periodic acid after previous treatment of tissue sections with ethanolic KOH. As the positive reacting material has been proven to be sensitive against treatment with neuraminidase the modified KOH PAS-reaction should be related to the presence of acylated sialic acid residues in mucosubstances of the colonic mucosae of the rat.


Organ culture of adult mouse intestine IV. Stimulation of glucose-6-phosphatase in vitro. Explants of adult mouse jejunum have been maintained in organ culture with or without fructose added to the medium in order to stimulate the intestinal glucose-6-phosphatase (G-6-Pase). When the fructose is added, at the beginning of the culture, a three-fold increase of G-6-Pase in measured during the first 24 h. If the fructose is added after 24 h of culture, no significant increase of the G-6-Pase is registered in comparison with the controls. Proteins, DNA content and dissacharidase activities are not modified during the culture. Alkaline phosphatase activity presents a twofold increase in the controls and stimulated explants. The ultrastructural localization of the G-6-Pase is not altered during the culture.


Improved method for the histochemical demonstration of glucose-6-phosphatase activity. Methodological studies on the histochemical technique for the demonstration of G6Pase activity showed that the occurrence of common artifacts: morphological destruction, extracellular precipitation of reaction product and nuclear staining are dependent on the concentration of lead nitrate, buffer and substrate. By studying the effects of systematic variation of the incubation media on the histochemical reaction optimal concentrations of either of these components were determined. An improved medium containing 3.6 mM lead nitrate, 40 mM tris-maleate buffer, pH 6.5, 10 mM G6P and 300 mM sucrose was used for the study of G6Pase distribution patterns in liver acini of juvenile and adult rats of both sexes and in those of starved adult female rats. The results obtained indicate sex dependent differences in the functional organization of the liver acinus and furthermore demonstrate the rapid functional adaptability of liver parenchyma to changes of the nutritional situation.


[Distribution of enzymes in the stereocilia of the ductus epididymis of rats (author's transl)]. Using unfixed or cryostate sections from aldehyde fixed epididymides of adult rats gamma-glutamyl naphthylamides are hydrolysed in the stereocilia of the principal cells in zone 1A-4B and glycyl-prolyl naphthylamides in those of zone 1B-4A (nomenclature of Reid and Cleland, 1957); this is not the case when other amino acid and peptide naphthylamides are employed. Adenosine triphosphate (ATP) is metabolized by the stereocilia in zone 1a, 2, 3 and 4A and adenosine monophosphate (AMP) in the stereocilia of zone 3-6. With thiamine pyrophosphate (TPP) especially the Golgi apparatus of the principal cells in zone 1B and C, 2 and 3 reacts. In addition ATP, AMP and TPP stain the stereocilia of single epithelial cells outside the zones where all stereocilia react. The AMP pattern allows a further subdivision of zone 3 into 3 A and B. Substrates for the demonstration of alpha- and beta-D-glycosidases, non-specific esterases and alkaline phosphatases are either not hydrolyzed by the stereocilia or the rate of hydrolysis is low.


Enterotoxemia in two foals.

Enterotoxemia in two foals. Two Quarter Horse foals from different premises died from enterotoxemia. Clostridium perfringens toxins alpha and beta were demonstrated in the foal's intestines by mouse protection tests. Clostridium perfringens type C was isolated from the intestines of each foal. Histologic examination revealed hemorrhage, necrosis, and massive numbers of C perfringens.


Focal loss of pigment in the Belgian Tervuren dog. Hypopigmentation most commonly affecting the face and mouth of the Belgian Tervuren dog was characterized by an absence of melanocytes in the epidermis. Pigment loss usually occurred during young adulthood, and although there was partial repigmentation in some dogs, complete repigmentation did not occur. Treatment with vitamin and mineral supplements was unrewarding. The condition appeared to have some degree of heritability and to be similar to vitiligo in man.


Plasma dopamine-beta-hydroxylase in childhood psychosis. A previous report suggested that plasma dopamine-beta-hydroxylase (DBH) is elevated in childhood autism. We measured plasma DBH in 15 Children with functional psychosis and in 10 psychotic children with known organic etiology. DBH activity was significantly elevated in the children with functional psychoses and showed a significant correlation with age that is not reported for this age range in normals. It is possible that children with functional psychoses show an abnormal continuation of the rise in plasma DBH activity characteristic of infancy. No differences between children with functional psychoses and children with organic psychoses were found for red blood cell catechol-O-methyl-transferase, plasma monoamine oxidase, or plasma cyclic AMP.


Effects of antipain (a protease inhibitor) on respiration, viability, and excision of pyrimidine dimers in UV-irradiated Escherichia coli cells. The protease inhibitor antipain increases the effectiveness of UV irradiation on cessation of respiration and cell killing in Escherichia coli B/r cultures without affecting excision of pyrimidine dimers. The actions are similar to those caused by cyclic AMP in irradiated cultures.


Control of biodegradative threonine dehydratase inducibility by cyclic AMP in energy-restricted Escherichia coli. To explain the requirement for anaerobic conditions in the induction of biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, measurements of cyclic AMP (cAMP) were made during aerobic and anaerobic growth and upon an aerobic-to-anaerobic transition. Internal cAMP levels were similar (5 to 10 muM) throughout exponential growth, whether aerobic or anaerobic, but only during anaerobiosis was threonine dehydratase synthesized. When an exponentially growing aerobic culture was made anaerobic, a sharp increase in internal cAMP was noted, reaching 300 muM within 10 min and declining thereafter to normal anaerobic levels. Threonine dehydratase synthesis was detected immediately after the attainment of peak cAMP levels and continued for several generations. A similar pattern but with less accumulation of cAMP and less threonine dehydratase production was also noted upon treatment of an aerobically growing culture with KCN. Pyruvate addition at the time of anaerobic shock severely affected both cAMP accumulation and threonine dehydratase synthesis; however, externally added cAMP could partially counter the pyruvate effect on enzyme synthesis. The conclusion was reached that conditions which resulted in a temporary energy deficit brought about the major accumulation of cAMP, and this elevated level served as a signal for initiation of threonine dehydratase synthesis to supply energy by the nonoxidative degradation of threonine.


Effect of membrane-associated f1 bacteriophage coat protein upon the activity of Escherichia coli phosphatidylserine synthetase. The effects of insertion of the major coat protein of f1 bacteriophage into Escherichia coli membranes were investigated under conditions allowing in vivo analysis of phosphatidylserine synthesis. An E. coli strain possessing a temperature-sensitive phosphatidylserine decarboxylase was utilized under conditions in which the decarboxylase activity was reduced but nonlethal. The presence of the coat protein in the host membranes inhibits the activity of the phosphatidylserine synthetase and perhaps affects the activity of the phosphatidylserine decarboxylase.


Induction of cyclic AMP phosphodiesterase in Blastocladiella emersonii and its relation to cyclic AMP metabolism. Extracts of vegetative cells of Blastocladiella emersonii contain 5% or less of the cyclic AMP phosphodiesterase activity in zoospore extracts. This difference in activity could be accounted for entirely by an increase in the differential rate of phosphodiesterase synthesis during sporulation, beginning after a lag period of about 60 min and extending for at least an additional 90 min into the 4-h sporulation process. To examine the relation between enzyme synthesis and cyclic nucleotide metabolicm, we determined the substrate specificity of phosphodiesterase synthesized during sporulation and partially purified from zoospores. Zoospore extracts contain two components, separable by gel filtration chromatography, with cyclic AMP phosphodiesterase activity. The larger component accounts for 20% of the total activity and the smaller component for 80%. Both components show essentially an absolute substrate specificity for cyclic AMP among several cyclic purine and cyclic pyrimidine nucleotides tested. Nevertheless, we found no change in the total cyclic AMP content of sporulating cells before, during, or after enzyme activity increased. We speculate that some other component of cyclic AMP metabolism or function limits the rate of cyclic AMP hydrolysis in sporulating cells.


Cyclic AMP binding proteins and cyclic AMP-dependent protein kinase from Blastocladiella emersonii. The stoichiometry of cyclic AMP binding protein to cyclic AMP in sporulating cells of Blastocladiella emersonii and the resistance of protein-bound cyclic AMP to enzyme-catalyzed hydrolysis suggest that the distribution of cyclic AMP between free and protein-bound pools is an important factor in cyclic AMP metabolism. Most but not all of the cyclic AMP binding protein in sporulating cells is associated with a cyclic AMP-dependent protein kinase.


Cystine reductase in the dimorphic fungus Histoplasma capsulatum. Organo-sulfur compounds favor the transition of mycelia of Histoplasma capsulatum to the yeast form (6, 8). Investigation of the role of cystine in the transition revealed that the two phases concentrated this amino acid at comparable rates and that mutants defective in the uptake of cystine were still able to undergo the transition normally. Uptake of cystine is therefore probably not a requirement for transition to or maintenance of the yeast phase. Both phases contained a reduced nicotinamide adenine dinucleotide phosphate-dependent glutathione reductase; but a reduced nicotinamide adenine dinucleotide-dependent cystine reductase was detectable only in the yeast phase. The cystine reductase appeared early in the transition of mycelium to yeast. Treatment of mycelia with p-chloromercuriphenylsulfonic acid, which prevented the transition to yeast, had no effect on cystine uptake but strongly inhibited the cystine reductase. These results suggest that cystine reductase may provide reduced sulfhydryl groups involved in the transition of mycelium to yeast.


Saturation transfer electron spin resonance study on the rotational diffusion of calcium- and magnesium-dependent adenosine triphosphatase in sarcoplasmic reticulum membranes. The technique of saturation transfer electron spin resonance has been applied to study the rotational diffusion of spin-labeled Ca2+, Mg2+-dependent ATPase molecules in the membranes of sarcoplasmic reticulum vesicles. Comparison of the present data with those for spin-labeled hemoglobin undergoing isotropic rotation leads to a value of 2 X 10(-4) s for the apparent rotational correlation time at 20 degrees C for the membrane-bound protein. Consideration of the anisotropy of the Brownian rotation of the membrane-bound ATPase suggests that the true correlation time for the expected axial rotation may be somewhat smaller than the apparent value. An Arrhenius plot of the rotational motion shows a break, which is interpreted as indicating the occurrence of a conformational change of the ATPase molecule at about 15 degrees C.


Comparison of calcium-activated, cyclic nucleotide-independent protein kinase and adenosine 3':5'-monophosphate-dependent protein kinase as regards the ability to stimulate glycogen breakdown in vitro. A cyclic nucleotide-independent protein kinase, which was produced from its proenzyme upon limited proteolysis by a Ca2+-dependent protease (Takai, Y., Yamamoto, M., Inoue, M., Kishimoto, A., & Nishizuka , Y. (1977) Biochem. Biophys. Res. Commun. 77, 542-550), showed an ability to phosphorylate not only muscle glycogen phosphorylase kinase but also glycogen synthase, resulting in activation and inactivation of the respective enzymes, although the protein kinase was less active than adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase toward glycogen synthase. Available evidence indicates that this new protein kinase shows pleiotropic functions apparently similar to those described for cyclic AMP-dependent protein kinase. Nevertheless, these protein kinases were clearly distinguishable from each other in their response to cyclic nucleotides and susceptibility to protein inhibitor.


Carbamyl phosphate synthetase of Escherichia coli uses the same diastereomer of adenosine-5'-[2-thiotriphosphate] at both ATP sites. Carbamyl phosphate synthetase from Escherichia coli has been shown to use only the A isomer of adenosine-5'-[2-thiotriphosphate] in both the ATPase reaction (MgATP HCO3- leads to MgADP + Pi) and the carbamyl phosphate synthesis reaction (2MgATP + HCO3- + L-glutamine leads to 2MgADP + Pi + carbamyl-P + L-glutamate). The B isomer was less than 5% as reactive. In the reverse reaction, only the A isomer of adenosine-5'-[2-thiotriphosphate] is synthesized from adenosine-5'-[2-thiodiphosphate] and carbamyl-P as determined by 31P NMR and a coupled enzymatic assay with Cd2+- hexokinase. It is therefore proposed that carbamyl phosphate synthetase uses the same diastereomer of MgATP at both ATP sites.


Nonspecific effect of m7GMP on protein-RNA interactions. Cap analogs m7GMP and m7GDP inhibit binding of eukaryotic initiation factors to reovirus capped mRNA but also inhibit complex formation involving uncapped mRNA or 18 S rRNA. Furthermore, Escherichia coli DNA-dependent RNA polymerase binds 18 S rRNA and this interaction is also blocked by m7GMP. These results indicate that inhibition by cap analogs is not a stringent test for putative cap-specific binding between proteins and mRNA.


A novel electron paramagnetic resonance signal of "oxygenated" cytochrome c oxidase. It had been observed previously that a pair of transient EPR resonances (g = 1.78 and 1.69) appears within less than 5 ms on reoxidation of reduced cytochrome c oxidase by O2. Since the location of other lines that are part of the same signal was not known, the quantity of the paramagnetic species involved, and thus the significance of the observed resonances, remained questionable. We have now found a broad resonance at g = 5 which is obviously associated with those at g = 1.78 and 1.69. The width of the signal (approximately 250 mT) at the observed intensity suggests that it represents a significant fraction of one of the components of the enzyme. The signal disappears within less than 5 ms on addition of cyanide or sulfide but only within several hundred milliseconds after addition of ferrocytochrome c. This behavior suggests that it originates from the a3 component of the enzyme. It is suggested that the species represented in the signal is either identical with or part of what has been named collectively the "oxygenated" form and recently described "activated" forms of the enzyme. On reoxidation of reduced oxidase with oxygen enriched 90% in 17O, no change of signal shape was seen.


A rapid method for the purification of extrachromosomal DNA from eukaryotic cells. A simple and efficient procedure to purify the low molecular weight extrachromosomal DNA from eukaryotic cells is described. Gentle lysis of cells with urea and sodium dodecyl sulfate in 0.24 M phosphate buffer (pH 6.8) is followed by the removal of high molecular weight bulk DNA by centrifugation. Protein and RNA are removed from the supernatant by hydroxyapatite chromatography in urea/phosphate buffer. Urea is then removed with 0.15 M phosphate buffer and the extrachromosomal DNA, virtually free from protein and RNA,  is finally eluted in 0.5 M phosphate buffer. The procedure allows the recovery of about 99% simian virus 40 (SV40) DNA from infected monkey kidney cells in the extrachromosomal fraction. In normal mouse, monkey, andhuman cells, approximately 1% of total cell DNA appears to be extrachromosomal.


A K+ transport ATPase in Escherichia coli. A K+ -stimulated ATPase in membranes of Escherichia coli has been identified as an activity of the Kdp system, and ATP-driven K+ transport system. Three characteristics support association of the ATPase with the Kdp system: (i) ATPase and Kdp transport are both repressed by growth in media containing high concentrations of K+; (ii) the ATPase and Kdp system accept only K+ as substrate, neither requires Na+ nor accepts Rb+ as a substrate; (iii) the affinity of the ATPase and that of th Kdp system for K+ is similar and is altered by mutations in the structural genes of the Kdp system. Discovery of an ATPase associated with a bacterial transport system suggests functional similarities with the ATP-driven transport systems of animal cells.


Bovine sperm forward motility protein. Partial purification and characterization. A protein, present in bovine seminal plasma, initiates forward motility in immature, immotile caput spermatozoa that have been incubated with a cyclic AMP phosphodiesterase inhibitor. An improved motility assay was developed to study this process and the protein involved. This forward motility protein exhibits multiple forms when fractionated on the basis of charge or molecular weight. Molecular sieving in urea or sodium dodecyl sulfate and dithiothreitol results in a single peak of activity which will re-form the larger aggregates in the absence of these agents. The molecular weight of this monomeric motility protein, as estimated from molecular sieving under these dissociating conditions, is 37,500. The forward motility protein can be partially purified by heat treatment, gell chromatography in urea, and affinity chromatography on concanavalin A/agarose. Enzymatic treatments further suggest a glycoprotein nature, i.e. treatment with beta-galactosidase, neuraminidase, alpha-mannosidase, or galactose oxidase reduces its activity by 50%; treatment with trypsin completely abolishes forward motility protein activity. On the basis of concurrent studies on the activity, properties, and distribution of forward motility protein in bovine body fluids, it is suggested that this protein is involved in the development of the capacity for motility as sperm traverse the epididymis.


Binding of divalent cation to phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase. In order to study the action of the divalent cation which is essential for phosphorylation of sodium- and potassium-transport adenosine triphosphatase, magnesium ion, the normal ligand, was replaced with calcium ion, which had properties diffeerent from those of Mg2+, Mn2+, Fe2+, Co2+, Ni2+, or Zn2+. Phosphorylation of the enzyme from ATP at pH 7.4 in the presence of Na+ and Ca2+ yielded a Ca.phosphoenzyme (60% of the maximal level) with a normal rate of dephosphorylation following a chase with unlabeled Ca.ATP (PK = 0.092S-1 at 0 degrees C). In contrast, after a chase by a chelator, namely ethylenediaminetetraacetic acid, 1,2-cyclohexylenedinitrilotetraacetic acid, or ethylene glycol bis-(beta-aminoethyl ether)N,N'-tetraacetic acid, dephosphorylation slowed within 5 s and half of the initial phosphoenzyme remained with a stability about 5-fold greater than normal. Three states of the phosphoenzyme were distinguished according to their relative sensitivity to ADP or to K+ added during a chase. Normally prepared Mg.phosphoenzyme was sensitive to K+ but not to ADP; Ca.phosphoenzyme was sensitive either to ADP or to K+; and the stabilized phosphoenzyme prepared from Ca.phosphoenzyme by addition of a chelator was sensitive neither to ADP nor to K+ nor to both together. Addition of Ca2+ to the stabilized phosphoenzyme restored the reactivity to that of Ca.phosphoenzyme. Addition of Mg2+ to the stabilized phosphoenzyme changed the reactivity to that of Mg.phosphoenzyme. Therefore, this unreactive, stabilized state of the phosphoenzyme appeared to be a divalent cation-free phosphoenzyme. With respect to sensitivity to ouabain, Ca.phosphoenzyme was as sensitive as Mg.phosphoenzyme but calcium-free phosphoenzyme was much less sensitive. It was concluded that the divalent cation required for phosphorylation normally remains tightly bound to the phosphoenzyme and is required for normal reactivity. Calcium ion was almost unique in dissociating relatively easily from the phosphoenzyme. Strontium ion appeared to act similarly to Ca2+.


The tyrosine free radical in ribonucleotide reductase from Escherichia coli. One of the two nonidentical subunits of ribonucleotide reductase from Escherichia coli, protein B2, contains an organic free radical required for enzyme activity. Earlier isotope subtitution experiments (Sjöberg, B.-M., Reichard, P. Gräslund, A., and Ehrenberg, A. (1977) J. Biol. Chem. 252, 536-541) demonstrated that the radical was localized to a tyrosine residue of the enzyme and suggested that the spin density of the radical was centered at the methylene carbon of tyrosine. However, additional isotope substitution experiments now show that the spin density of the radical must be delocalized over the aromatic ring of the tyrosine residue.


Effect of the lipid environment on protein motion and enzymatic activity of sarcoplasmic reticulum calcium ATPase. In order to investigate the roles of the physical states of phospholipid and protein in the enzymatic behavior of the Ca2+ -ATPase from sarcoplasmic reticulum, we have modified the lipid phase of the enzyme, observed the effects on the enzymatic activity at low temperatures, and correlated these effects with spectroscopic measurements of the rotational motions of both the lipid and protein components. Replacement of the native lipids with dipalmitoyl phosphatidylcholine inhibits ATPase activity and decreases both lipid fluidity, as monitored by EPR spectroscopy on a stearic acid spin label, and protein rotational mobility, as monitored by saturation transfer EPR spectroscopy on the covalently spin-labeled enzyme. Solubilization of the lipid-replaced enzyme with Triton X-100 reverses all three of these effects. Ten millimolar CaCl2 added either to the enzyme associated with the endogenous lipids or to the Triton X-100 soulbilized enzyme inhibits both ATPase activity and protein rotational mobility but has no detectable effect on the lipid mobility. These results are consistent with the proposal that both lipid fluidity and protein rotational mobility are essential for enzymatic activity.


The role of ATP and divalent cations in the regulation of a cardiac phosphorylase phosphatase (phosphoprotein phosphatase) of Mr = 35,000. The effects of ATP and divalent cations on a divalent cation-independent phosphorylase phosphatase of Mr = 35,000 (phosphatase S) purified from canine cardiac muscle have been studied. The enzyme can be rapidly inactivated by ATP or other nucleoside di- and triphosphates and PPi, but not by AMP, adenosine, adenine, Pi, EDTA, ethylene glycol bis(beta-aminoethyl ether)N,N' -tetraacetic acid, 1,10-phenanthroline, or 8-hydroxyquinoline. After removing the inactivating agent, such as ATP or PPi, by gel filtraiton followed by exhaustive dialysis, the inactivated enzyme (apophosphatase S) can be reactivated by preincubating with Mn2+ or Co2+, but not with Mg2+, Ca2+, Ni2+, Zn2+, Fe2+, Cu2+, Ba2+, Hg2+, Pb2+, or Cd2+. The Mn2+ -reactivated enzyme, which is less active than the Co2+ -reactivated enzyme, can be again inactivated by preincubating with ATP. The present findings indicate that phosphatase S contains a tightly bound divalent cation, probably Mn2+, in the active site. ATP and PPi, due to their structural similarity to the phosphoprotein substrate and their ability to chelate metal ions, can readily enter the active site to remove the divalent cation(s) essential for the catalytic function. The present findings also indicate that phosphatase S, a common catalytic subunit of several larger molecular forms of nospecific phosphoprotein phosphatase in cardiac muscle, can exist in two interconvertible forms, a metallized form (active) and a demetallized form (inactive). ATP and metal ions may regulate this class of isozymes by mediating the interconversions.


Cross-linking studies of the self-association properties of apo-A-I and apo-A-II from human high density lipoprotein. The state of self-association of the apoprotein components of human high density lipoprotein have been studied by use of the cross-linking reagent dimethyl-suberimidate. Analusis of the cross-linked products was carried out by soduim dodecyl sulfate-gel electrophoresis and by agarose column chromatography in 6 M guanidine hydrochloride. Apo-A-I was found to exist as a monomer at low concentration, but associates to tetrameric and pentameric forms at concentrations of 0.5 mg/ml or higher. The self-association was found to be ionic strength-dependent, with association promoted by the presence of salt. Apo-A-II was also found to associate, but the major oligomeric form observed was dimeric (Mr = 34,000), and the association was less dependent on ionic strength than for apo-A-I. Cross-linking in the presence of various concentrations of guanidine hydrochloride showed that apo-A-II self-association persisted at higher concentrations of the denaturant than for apo-A-I. Studies of the effect of temperature demonstrated that the self-association of both proteins was diminished at temperatures above 30 degrees C. Recombination of apo-A-II with phospholipid resulted in the formation of particles which yielded primarily trimers upon cross-linking. This suggests that phospholipid binding causes major reorganization of the self-associated forms of apo-A-II.


Derivatized silica spheres as immunospecific markers for high resolution labeling in electron microscopy. For high resolution labeling of influenza virus cell surface antigens on HeLa cells, an immunospecific marker is used with silica sphere cores of 13--14 nm average diameter. These markers are formed using commercially available silica sphere sols. Two other size ranges are available, 7--8 nm and 22--25 nm. The steps for chemical derivatization are described in detail. Amino and aldehyde functions are covalently introduced onto the sphere surface. Sols of these derivatized silica spheres (DSS) are physicochemically stable and therefore usable for years. Coupling of IgG to DSS followed by permeation chromatography on controlled pore glass results in size-defined immunospecific silica sphere markers (DSS-markers). Saturation labeling of cell surface antigens on HeLa cells on cover slips is obtained with the final sphere concentration of 10(14) DSS-marker/cm3 within 20 min. With usual protective conditions, the marker stability and labeling ability are preserved for months. The visibility and the fine structure of the DSS-marker on cell surfaces are shown by using transmission electron microscopy (TEM) with stereo replicas and ultrathin sections.


Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient. A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.


Role of Ca++ in virus-induced membrane fusion. Ca++ accumulation and ultrastructural changes induced by Sendai virus in chicken erythrocytes. Some of the ultrastructural (freeze-etching technique), morphological, and biochemical effects of Sendai virus interaction with chicken erythrocytes have been studied under fusogenic (in the presence of CaCl2) and nonfusogenic (in the presence of ethyleneglycol-bis-N,N'-tetraacetic acid, [EGTA]) conditions. The following phenomena occur, irrespective of the presence of CaCl2 or EGTA: (a) binding of iodinated virus particles to chicken erythrocytes at 4 degrees C and their partial release from the cells at 37 degrees C; (b) gradual incorporation of the viral envelope and viral M-protein into plasma membrane, as visualized in the protoplasmic and exoplasmic fracture (P and E, respectively) faces of the membrane; and (c) virus-dependent transient clustering of intramembrane particles at 4 degrees C, which is reversible after transferring the cells back to 37 degrees C. The following virus-induced phenomena occur only in the presence of CaCl2: (a) rounding of cells followed by their fusion; (b) transient decrease in the density of intramembrane particles; and (c) the virus induces uptake of 45CaCl2 by chicken erythrocytes. The uptake is specific as it is inhibited by LaCl3, and no accumulation of [14C]glucose-1-phosphate ([14C]G-1-P) could be observed under the 45 CaCl2 uptake conditions. The data show that fusion of virus with plasma membrane is a Ca++-independent process and, as such, it should be distinguished from the virus-induced membrane-membrane and cell fusion processes. The latter is absolutely dependent on the rise of intracellular Ca++, as reflected by the fact that Ca++-induced rounding of chicken erythrocytes always precedes fusion (Volsky, D. and A. Loyter. 1977.Biochim. Biophys. Acta 471:253--259).


Effects of two blood culture anticoagulants on growth of Neisseria meningitidis. Tests of 25 strains of Neisseria meningitidis for sensitivity to sodium polyanethol sulfonate (SPS) showed that the sensitivity of strains varied with both inoculum size and SPS concentration. In Trypticase soy broth (TSB), 2 out of 13 strains were sensitive to 0.05% SPS, whereas 8 out of 13 strains were sensitive to the same concentration of SPS in brain heart infusion (BHI). In artificial blood cultures with six strains of meningococci, the addition of 10% defibrinated blood was found to eliminate the sensitivity of all six strains to SPS in BHI, but not of the two strains in TSB. Addition of 1.2% gelatin to artificial blood cultures eliminated the inhibitory effect of 0.05% SPS, whereas the addition of 1% yeast extract to blood cultures containing 0.025% or 0.05% SPS enhanced the inhibitory effect of this anticoagulant. None of the 13 strains tested was inhibited by 0.05% sodium amylosulfate in TSB or BHI alone or in artificial blood cultures with these media.


Detection of Clostridium perfringens enterotoxin in human fecal samples and anti-enterotoxin in sera. By using counterimmunoelectrophoresis (CIEP), Clostridium perfringens enterotoxin was successfully demonstrated in fecal samples collected within 1 day of attack from sick individuals involved in a bacteriologically and epidemiologically proven outbreak of C. perfringens food poisoning. In contrast, enterotoxin was not demonstrable in fecal samples of apparently healthy individuals both at high- and low-risk exposure to the organism and enterotoxin or in fecal samples collected 4 to 5 days after a food poisoning outbreak. A 100% prevalence of C. perfringens anti-enterotoxin in sera of human volunteers at high- as well as low-risk exposure to the organism and enterotoxin was recorded with CIEP.


Comparison of WI-38, MRC-5, and IMR-90 cell strains for isolation of viruses from clinical specimens. With the diminishing supply of the human fetal lung WI-38 cell strain, a replacement for viral isolation is needed. Two candidates are the human fetal lung strains MRC-5 and IMR-90. A comparison of WI-38, MRC-5, and IMR-90 was performed to evaluate efficiency and speed of viral isolation, clarity of cytophatic effect, and ease of growing the cells. The inocula were clinical specimens rather than tissue culture-adapted isolates. Frozen samples of 46 specimens that had previously yielded an isolate on WI-38 were thawed and inoculated onto WI-38, MRC-5, and IMR-90 cells. In addition, 95 freshly taken clinical specimens uf undetermined infectivity were inoculated onto the cell strains. Viral recovery rates were similar on all three strains, as were the appearance and speed of onset of the cytophatic effect. MRC-5 and WI-38 cells remained healthy until generation 36, whereas IMR-90 cells went into crisis by generation 20. The longer life span of the MRC-5 cells makes them more suitable than IMR-90 cells to replace the WI-38 strain for routine use in viral diagnosis.


Incidence and significance of hepatitis B e antigen and antibody in postnecrotic cirrhosis and primary hepatocellular carcinoma. A study of the serological markers of Hepatitis B virus (HBV) including e antigen (HBe Ag) and antibody against HBe Ag (anti-HBe) was performed in Senegalese patients suffering from cirrhosis and primary hepatocellular carcinoma, and in a control group (blood donors). It was not possible to diagnose additional HBV infections in primary hepatocellular carcinoma patients using HBe Ag or anti-HBe Ab alone as serological markers. The lower prevalence of HBe Ag among primary hepatocellular carcinoma patients as compared with cirrhotic patients suggests that active replication of HBV becomes increasingly defective during the course of the malignant process.


The regulation of skeletal muscle alanine and glutamine formation and release in experimental chronic uremia in the rat: subsensitivity of adenylate cyclase and amino acid release to epinephrine and serotonin. The mechanism of the increased alanine and glutamine formation and release from skeletal muscle in experimental uremia was investigated using epitrochlearis preparations from control and chronically uremic rats. In uremic muscle, insensitivity to epinephrine or serotonin suppression of alanine and glutamine release was observed. With control muscles, 1 nm or greater, epinephrine inhibited alanine and glutamine release, whereas with uremic muscles, epinephrine concentrations <1 muM did not alter amino acid release. Decreased alanine and glutamine release with 1 nM serotonin was observed in control muscles, but no inhibition was observed with concentrations <1 muM in uremic muscle. Muscle amino acid levels were the same in control and uremic muscles in the presence or absence of epinephrine or serotonin. The reutilization of released alanine by protein synthesis or oxidation to CO(2) was not differentially affected by epinephrine in uremic muscles as compared with control muscle. Dibutyryl-cAMP inhibited amino acid release equally in uremic and control muscles. Epinephrine or serotonin increased cAMP levels two- to four-fold or more in control than in uremic muscle. Basal- and fluoride-stimulated adenylate cyclase activities were equal in uremic and control muscle homogenates and in membrane fractions, but 10 muM epinephrine-stimulated adenylate cyclase was reduced 30-60% with uremia. At any concentration of epinephrine (0.001-100 muM), the stimulation of membrane adenylate cyclase activity was one- to twofold greater with control membranes than with uremic muscle membranes. With either control or uremic muscle, peak adenylate cyclase activity was observed at 1 muM epinephrine. These data indicate that skeletal muscle in chronic uremia acquires an insensitivity to the metabolic action of epinephrine or serotonin. This insensitivity may be attributable in part to the diminished increments in muscle cAMP levels produced by adrenergic and serotonergic agonists. The decreased cAMP levels may derive in turn from a decreased activity or subsensitization of the agonist-stimulated adenylate cyclase in uremic muscle.


Urinary excretion of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in malignancy. The urinary excretion of cyclic adenosine 3',5'-monophosphate (cAMP), corrected for urinary creatinine, was determined in 177 patients with primary or metastatic tumours and in 149 normal subjects. In 26 patients with malignancy and in 10 control subjects the excretion of cyclic guanosine 3',5'-monophosphate (cGMP) was also evaluated. The urinary cAMP/Cr ratio in human neoplasms of epithelial origin was often significantly lower than normal, irrespective of the extension of malignancy. Surgical resection of the tumour, radiotherapy, or theophylline treatment increased urinary excretion of the nucleotide. In patients with malignancy, intravenous infusion of glucagon failed to produce the degree of elevation of plasma cAMP seen in normal subjects. Urines from patients with malignant neoplasms had low values of cAMP/Cr ratio with increased values of cGMP/Cr ratio. These findings could be the result of systemic alteration in synthesis or breakdown of the nucleotides.


Localization of receptors for the dipsogenic action of angiotensin II in the subfornical organ of rat. The proposal of the subfornical organ (SFO) as a site of receptors for drinking induced by angiotensin II (AII) was investigated with several mutually confirmatory experiments. Intracranial injections of physiological doses of AII elicited drinking if and only if applied directly to the SFO (Experiment I). Ablation of the SFO selectively (Experiment 2) and permanently (Experiment 4) eliminated drinking elicited by physiological doses of intravenously infused AII. Animals in which SFO had been ablated responded normally to cellular dehydration but reduced responding to the extracellular thirsts of beta-adrenergic activation and hyperoncotic colloid dialysis (Experiment 3). Infusion of saralasin, an AII antagonist, directly into the SFO selectively and reversibly antagonized intravenous AII drinking (Experiment 5). The hypothesis that the SFO contains dipsogenic receptors for circulating AII is strongly supported.


Adenosine triphosphatases of rat pancreatic islets: comparison with those of rat kidney. Electrolyte fluxes are fundamental to normal endocrine pancreatic function. Adenosine triphosphatases (ATPases) are enzyme systems believed to modulate electrolyte movements across membranes in a number of cell types. This study was undertaken to measure cation-dependent ATPases of rat pancreatic islets. In addition, we compared effects of substances which influence endocrine pancreatic function upon ATPases in homogenates of islets and kidney, the latter being a tissue which would not be expected to have a stimulus-secretion response to substances which activate islets. Both tissues were generally similar with respect to apparent Michaelis constant (ATP) of Na(+)K(+)ATPase, Mg(++)ATPase, and Ca(++)ATPase. In islets and kidney, Na(+)K(+)ATPase specific activity was increased when the Na:K ratio was lowered from 250:1 (175:0.7 mM) to 5:1 (100:20 mM). Inhibition of Na(+)K(+)ATPase at either Na:K ratio by ouabain, an activator of secretion, and enhancement of the high-ratio Na(+)K(+)ATPase by diphenylhydantoin, an islet secretory inhibitor, were also common to both tissues. Because both inhibition and enhancement of Na(+)K(+)ATPase could be studied at the high Na:K ratio, we examined the effect of regulators of secretion upon the activity of this enzyme. Like ouabain, substances which induce or support islet secretion, glucose 16 mM or 3.3 mM, arginine 14.2 mM (with 3.3 mM glucose), or Ca(++) 1 mM, inhibited high-ratio islet Na(+)K(+)ATPase. Like diphenylhydantoin, the inhibitors of insulin secretion, diazoxide 0.22 mM, or NH(4)Cl 16 mM, enhanced this islet ATPase. Neither valine, which is non-secretogenic, nor arginine without glucose, which is a weak secretagogue, had any effect upon islet Na(+)K(+)ATPase. We examined the effect of these substances upon other cation-dependent islet ATPases. Ca(++) inhibited Mg(++)ATPase, and glucose inhibited Ca(++)ATPase. Leucine, 22.9 mM, which induces insulin secretion in the absence of glucose, suppressed islet Ca(++)ATPase and had no effect upon high-ratio Na(+)K(+)ATPase. In contrast to the observations in the islets, most substances which influence islet function had no effect on kidney ATPases, or effects which were different from those seen in islets. Except for ouabain, none of these substances influenced the three kidney ATPases in a manner similar to that seen with islets. These findings support the hypothesis that cation-dependent ATPases are involved in specificity of islet response to substances which influence endocrine pancreatic activity.


Receptors for luteinizing hormone-releasing hormone. Antisera to luteinizing hormone-releasing hormone (LH-RH) confer on Araldite sections of occasional rat pituitaries moderate immunocytochemical staining to the large secretory granules of gonadotrophs. Treatment of the sections with LH-RH before anti-LH-RH yields strong staining in all animals, irrespective of presence or absence of staining without pretreatment. This enhancement of staining is specific for LH-RH and is a high affinity, saturable reaction. Staining with or without LH-RH pretreatment is absent when anti-LH-RH absorbed with insolubilized LH-RH is used. Staining is inhibited by carboxyterminally-deficient LH-RH, unaffected by aminoterminally deficient LH-RH.


Adrenal luteinizing hormone releasing hormone receptors. Paraffin sections of mouse adrenals processed with antiserum to luteinizing hormone-releasing hormone (LHRH) in the unlabeled antibody enzyme method reveal moderate staining in the cytoplasm of cells of zona fasciculata and reticularis. The stain is intensified upon pretreatment of sections with LHRH. Pretreated sections processed with solid phase immunoabsorbed LHRH are unstained. Analogues of LHRH deficient in the C-terminal glycine amide inhibit staining, while analogues deficient in the N-terminal pyroglutamic acid have no effect. It is concluded that the adrenal contains receptors for a ligand resembling LHRH in receptor and immunoreactivity. The possibility is considered that the ligand may be an inhibitor of pineal origin.


Microneutralization tests for serological typing and subtyping of foot-and-mouth disease virus strains. A microneutralization test for serotyping of FMD viruses is described. It is based on earlier observations by Booth, Rweyemamu & Pay (1978) that dose-response relationships in quantal microneutralizations often deviated from linearity. The typing test described therefore utilizes undiluted virus preparations. In about 90% of samples a positive typing was obtained in contrast with about 50% for the complement fixation test. The test was also found to be susceptible to minimal quantities of heterotypic viral contamination. For strain differentiation the microneutralization test was carried out as a checkerboard test. When compared with the complement fixation test it was found to be more specific. The necessity to utilize virus-neutralization test systems for comparing FMD virus strains particularly for the purpose of vaccine selection is emphasized. The two dimensional microneutralization test has been applied to a study of comparing FMDV vaccine strains for Europe, South America, the Middle East and East Africa.


Conversion of Bordetella pertussis to Bordetella parapertussis. The epidemiological and drug susceptibility data on whooping cough suggested a possibility that Bordetella pertussis converts in some way to Bordetella parapertussis. To prove this, B. pertussis strain 75 was treated with N-methyl-N'-nitro-N-nitrosoguanidine and a mutant resistant to staphcillin v and eight mutants resistant to trimethoprim were isolated. The staphcillin V-resistant mutant of B. pertussis agreed with all of the criteria of B. parapertussis and the trimethoprim-resistant mutants also agreed with many of these criteria. Thus, a hypothesis is presented that B. parapertussis is a mutant of B. pertussis which appeared in nature probably by a selective pressure of antibiotics.


Serum antibody to poliovirus in patients in a mental deficiency hospital, with particular reference to Down's syndrome. Neutralization tests for poliovirus antibodies were carried out on 74 patients in an adult mental deficiency hospital: 37 patients with Down's syndrome and 37 non-Down's mental defectives. The distribution of antibody titres to poliovirus types 1, 2 and 3 did not differ significantly between the two groups. Most patients had antibody to at least one poliovirus type but less than a third had antibodies at a titre of 1/8 or greater to all three types. The low level of poliovirus immunity in this population may be of epidemiological importance.


Detection of airborne polyoma virus.

Detection of airborne polyoma virus. Polyoma virus was recovered from the air of an animal laboratory housing mice infected with the virus. Air samples were obtained by means of a high volume air sampler and further concentrated by high speed centrifugation. Total concentration of the air samples was 7.5 x 10(7). Assay for polyoma virus was by mouse antibody production tests. Airborne polyoma virus was detected in four of six samples.


Sodium polyanethole sulfonate: a new macrophage-dependent polymyxin-inhibitable, polyclonal B cell activator. The anionic detergent sodium polyanethole sulfonate was found to activate mouse B lymphocytes to increased cell division and formation of plaque-forming cells, possibly by an indirect mechanism. An increased cell division was also obtained in guinea pig and in rabbit lymphooid cell cultures. A strong similarity was found when comparing the dextran sulfate- and the sodium polyanethole sulfonate-sensitive cell population. Thus, the responding mouse cells resided in spleen and lymph node and also in fetal liver and bone marrow. The sensitive lymphocytes were dependent on the presence of macrophages or macrophage-replacing factors, since depletion of phagocytic cells resulted in loss of response. Addition of polymyxin B inhibited the stimulation, possibly through a direct interaction with the polyanion. In potency sodium polyanethole sulfonate was found to surpass dextran sulfate, making this benzene sulfonate a useful tool when studying lymphocyte responses.


Intracellular control of human neutrophil secretion. I. C5a-induced stimulus-specific desensitization and the effects of cytochalasin B. Human neutrophils released the granule constituents myeloperoxidase and lysozyme, but not the cytoplasmic enzyme lactic dehydrogenase, when pretreated with cytochalasin B and stimulated with purified human C5a. Prior exposure to C5a before the cytochalasin B, however, abrogated the subsequent secretory process. Interaction of neutrophils with C5a was shown to result in a concentration-dependent rapid desensitization that could not be overcome by later addition of cytochalasin B or of cytochalasin B and C5a. The effect was relatively stimulus specific in that neutrophils desensitized in this manner could be induced to release granule enzymes by casein or by complement-coated zymosan particles. Cytochalasin B effects on neutrophils appear to mimic those of surface binding of soluble stimuli such as C5a and immune complexes. It is suggested that desensitization in concert with surface stimulation may represent an important intracellular mechanism for limiting neutrophil secretion.


Potentiation of lymphocyte activation by colchicine. Proliferation of human lymphocytes induced by IO4- is potentiated by 30 min exposure to colchicine (10(-6)M), whereas the response to Con A is inhibited. Treatment with colchicine before or after IO4- modification has similar enhancing effects. Lumicolchicine does not alter proliferative responses. In addition to the proliferation of IO4--oxidized cells, irradiated IO4- modified lymphocytes induce proliferation when mixed with untreated lymphocytes. Enhancement occurs in both these conditions only when IO4--modified cells are treated with colchicine. Preliminary data indicate that proliferation in mixed lymphocyte cultures is also potentiated when either stimulating or responding cells are pretreated with colchicine. These findings suggest a selective stimulatory effect of colchicine on lymphocyte responses induced by cell-cell contact. Agents that modify microtubular assemblies might regulate the induction of immune responses that involve cellular interactions.


Enhanced chemotactic and phagocytic activities of leukocytes in psoriasis vulgaris. Leukocytes derived from the peripheral blood of psoriatic patients demonstrated an enhanced chemotactic response compared with leukocytes from healthy subjects. No significant difference was detected between the chemotactic response of leukocytes from patients with minimal or no skin involvement and those from patients with extensive lesions. Psoriatic leukocytes also had a significantly higher capacity to engulf 125I labeled Shigella flexneri than control leukocytes. It is postulated that a decrease in the cyclic AMP/cyclic GMP ratio in psoriatic leukocytes, similar to the imbalance of these 2 cyclic nucleotides found in the lesional epidermis of psoriasis, might be the cause of their enhanced chemotactic and phagocytic activities.


Effects of ascorbic acid deficiency on adrenal mitochondrial hydroxylations in guinea pigs. The effect of ascorbic acid deficiency on adrenal hydroxylation of cholesterol and deoxycorticosterone in guinea pigs was studied by using mitochondria and isolated cytochrome P-450 fractions. The effects obtained were compared with the effects of long-term treatment with ACTH. Advanced scurvy as well as treatment with ACTH resulted in an increase in the weight of the adrenals, the total amount of cytochrome P-450, the cholesterol side-chain cleavage activity, the cortisol level in plasma, and the excretion of unconjugated cortisol in urine. Total 11beta- and 18-hydroxylation of deoxycorticosterone were not stimulated or were stimulated only to a small extent. It is suggested that the major effects observed in advanced scurvy are due to ACTH, the level of which was significantly increased, most probably as a consequence of the stress. In animals kept on a scorbutogenic diet for 2-4 weeks or, with a small dose of ascorbate added, for several weeks, changes were observed that could not be fully explained as effects of ACTH on normal adrenals. Although the plasma levels of ACTH and cortisol were increased only to a small extent and excretion of unconjugated cortisol in urine was unaffected, there was a significant increase in the total capacity of adrenal mitochondria to hydroxylate exogenous cholesterol. It is concluded that the level of ascorbate in the adrenals might be of some importance for the capacity to convert cholesterol into pregnenolone. The normal feed-back regulation is, however, intact in moderate ascorbate deficiency and the plasma level of cortisol is kept within normal limits.


Cyclic AMP metabolism in adipose tissue of exercise-trained rats. Cyclic AMP metabolism in epididymal adipose tissue of exercise-trained rats was examined to determine if training induced changes in cyclic AMP production or inactivation. Beginning at 7 weeks of age, male rats were physically trained by 12 weeks of treadmill running. Pair-fed control rats remained sedentary in their cages for the duration of the experiment. Tissue levels of cyclic AMP were measured in epididymal adipose tissue slices incubated with norepinephrine. Adenyl cyclase was assayed in adipocyte ghost cell prepartions and low-Km phosphodiesterase was assayed in homogenates of adipose tissue. In response to norepinephrine stimulation, tissue cyclic AMP levels were reduced in trained compared to untrained rats. Training increased the ratio of activity of phosphodiesterase relative to adenyl cyclase. The results of this study indicate that cyclic AMP production in response to norepinephrine stimulation is not increased by training and may even be reduced, implying that adipose tissue cyclic AMP levels may be under a greater degree of control in trained rats. Modulation of adipose tissue cyclic AMP levels may function to regulate more closely the duration of lipolysis in exercise-trained rats.


Cyclic AMP-sensitive activation of hepatic sterol synthesis and 3-hydroxy-3-methylglutaryl coenzyme A reductase. We previously showed that preincubation of a 10,000 g supernatant (S(10)) from rat liver for 20 min at 37 degrees C dramatically increased the subsequent incorporation of [(14)C]acetate into sterols. No activation was seen with [(14)C]mevalonate as substrate. In the present studies we have examined the effect of preincubation on HMG CoA reductase. When microsomes were isolated from S(10) by calcium precipitation, preincubation of S(10) increased the specific activity of HMG CoA reductase threefold. No activation of HMG CoA reductase was observed in microsomes isolated by ultracentrifugation. Activation was cyclic AMP-sensitive. When cyclic AMP (0.001-1.0 mM) and MgATP (1 mM) were present during the preincubation period, there was little or no activation of HMG CoA reductase activity or of sterol synthesis from acetate. MgATP alone did not prevent activation. Neither cyclic AMP nor MgATP was inhibitory when present only during the assay of sterol synthesis. We propose that the in vitro activation represents the reversal of a physiologic cyclic AMP-mediated mechanism for the control of hepatic HMG CoA reductase. That a phosphoprotein phosphatase may catalyze the activation was supported by the observation that sodium fluoride, an inhibitor of phosphoprotein phosphatases, inhibited the activation. These results suggest that hormone-induced changes in the cellular level of cyclic AMP may regulate the activity of HMG CoA reductase and the rate of hepatic cholesterol synthesis.