Immunologic dysfunction among PBB-exposed Michigan dairy farmers. In 1973 inadvertent contamination occurred in a special farm feed supplement for lactating cows. Polybrominated biphenyls (PBBs) were used in place of magnesium oxide resulting in serious harm to farm animals, including cattle, chickens, geese, ducks. Farm families, accustomed to eating their own products, were most heavily exposed. To further study the impact of PBBs, 45 adult Michigan farm residents who were originally examined in a clinical field survey were further studied with respect to their immunologic status. For comparison, 46 dairy farm residents in Wisconsin, who had not eaten PBB-contaminated food, were examined, as were 79 healthy subjects in New York City. Abnormalities in the Michigan group included significant decrease in absolute numbers and percentages of T and B-lymphocytes and increased number of lymphocytes with no detectable surface markers ("null cells"). Significant reduction of in vitro immune function was noted in 35--40% of the Michigan farm residents who had eaten food containing PBB. Despite the absence of any apparent numerical reduction, both T and B lymphocyte subpopulations of peripheral blood lymphocytes showed evidence of functional defect. Ten of the 45 Michigan farmers studied showed impaired PHA-induced blastogeneic response, due to the decreased number and percent of T-cells in the PBLs. The decreased immune function detected among the PBB-exposed farm residents tended to affect families as a unit and was independent of exposed individuals' age or sex, speaking against the possibility of genetic predisposition.


Familial porphyria cutanea tarda in a patient with retinitis pigmentosa. The patient in this report had typical retinitis pigmentosa which apparently was of recessive inheritance. In addition, she had an exacerbation of porphyria cutanea tarda (PCT) while taking oral contraceptives. In view of the lack of evidence for hereditary transmission of PCT it is also of interest that the patient's mother had an exacerbation of PCT while taking estrogenic medication.


An overview of pituitary tumors.

An overview of pituitary tumors. Tumors arising in and around the hypophyseal fossa can cause symptoms by compression of surrounding structures or, in the case of adenomas arising from the adenohypophysis, by hypersecretion of hormones. Until recently, adenomas of the hypophysis have been classified on the basis of light microscopy into chromophobe, eosinophilic and basophilic. Presently available methods of histochemistry, immunocytology, electron microscopy and hormone assays make available a biological classification of these adenomas into two groups: (I) adenomas without secretory activity and (II) adenomas with secretory activity. Amongst the latter are included somatotroph adenomas, prolactin cell adenomas, melanocorticotroph adenomas and thyrotroph adenomas. Many of the large group of tumors formerly called "chromophobe" can now be reclassified amongst the secretory adenomas.


A mutant of Saccharomyces cerevisiae lacking catabolic NAD-specific glutamate dehydrogenase. Growth characteristics of the mutant and regulation of enzyme synthesis in the wild-type strain. NAD-specific glutamate dehydrogenase (GDH-B) was induced in a wild-type strain derived of alpha-sigma 1278b by alpha-amino acids, the nitrogen of which according to known degradative pathways is transferred to 2-oxoglutarate. A recessive mutant (gdhB) devoid of GDH-B activity grew more slowly than the wild type if one of these amino acids was the sole source of nitrogen. Addition of ammonium chloride, glutamine, asparagine or serine to growth media with inducing alpha-amino acids as the main nitrogen source increased the growth rate of the gdhB mutant to the wild-type level and repressed GDH-B synthesis in the wild type. Arginine, urea and allantoin similarly increased the growth rate of the gdhB mutant and repressed GDH-B synthesis in the presence of glutamate, but not in the presence of aspartate, alanine or proline as the main nitrogen source. These observations are consistent with the view that GDH-B in vivo deaminates glutamate. Ammonium ions are required for the biosynthesis of glutamine, asparagine, arginine, histidine and purine and pyrimidine bases. Aspartate and alanine apparently are more potent inducers of GDH-B than glutamate. Anabolic NADP-specific glutamate dehydrogenase (GDH-A) can not fulfil the function of GDH-B in the gdhB mutant. This is concluded from the equal growth rates in glutamate, aspartate and proline media as observed with a gdhB mutant and with a gdhA, gdhB double mutant in which both glutamate dehydrogenases area lacking. The double mutant showed an anomalous growth behaviour, growth rates on several nitrogen sources being unexpectedly low.


Degradation of poliovirus by adsorption on inorganic surfaces. Alteration of the specific infectivity of 3H-labeled ribonucleic acid and 14C-protein labeled poliovirus type 1 by adsorption on inorganic surfaces is investigated by application of kinetic theory to data obtained from sequential extractions of adsorbed virus. Some surfaces, e.g., SiO2, appear to have no significant effect. On the other hand, CuO substantially decreases the specific infectivity of adsorbed preparations. Differences in kinetic plots between 3H-labeled ribonucleic acid and 14C-labeled protein suggest that the inactivation observed involves physical disruption of virions. Van der Waals interactions between solid surfaces and virus are suspected to induce spontaneous virion disassembly. Surface catalyzed disassembly in aquatic and soil environments is implicated as an important mechanism controlling enterovirus dissemination. Methods developed here to evaluate complete recovery of adsorbed virus have potenital application to other degradation studied and problems concerning virus recovery from adsorbents used in virus concentrators.


Human enteroviruses in oysters and their overlying waters. The presence of enteroviruses in oysters and oyster-harvesting waters of the Texas Gulf coast was monitored over a period of 10 months. Viruses were detected in water and oyster samples obtained from areas both open and closed to shellfish harvesting. Viruses were detected periodically in waters that met current bacteriological standards for shellfish harvesting. No significant statistical relationship was demonstrated between virus concentration in oysters and the bacteriological and physiochemical quality of water and shellfish. Viruses in water were, however, moderately correlated with total coliforms in water and oysters and with fecal coliforms in oysters. Total coliforms in water were realted to total coliforms in sediment were related only to total coliforms in sediment. Among the physiochemical characteristics of water, turbidity was related statistically to the organic matter content of water and to fecal coliforms in water. There was a marked effect of rainfall on the bacteriological quality of water. Of a total of 44 water samples, 26 yielded virus in concentrations from 4 to 167 plaque-forming units per 100-gallon (ca. 378.5-liter) sample. Of a total of 40 pools of 10 to 12 oysters each, virus was found in 14 pools at a concentration of 6 to 224 plaque-forming units per 100 g of oyster meat. On five occasions, virus was found in water samples when no virus could be detected in oysters harvested from the same sites. This study indicates that current bacteriological standards for determining the safety of shellfish and shellfish-growing waters do no reflect the occurrence of enteroviruses.


Survival of bacteria in carcasses.

Survival of bacteria in carcasses. Bacteria injected into the bloodstream of guinea pigs shortly before death decreased in number in carcass tissues for about 1 h after death. If initial bacterial numbers were sufficiently low, all bacteria were eliminated, and carcass tissues were sterile 24 h after death. Carcass tissue sterility was maintained with an initial density of Clostridium perfringens or Salmonella typhimurium of 20 cells per g or with an initial density of the other species examined of several hundred cells per gram. With larger numbers of strict and facultative anaerobes, growth commenced after 3 h in carcasses incubated at 30 degrees C. Spores of C. perfringens were killed over the same period as vegetative cells, but growth did not commence until 8 h after death. Bactericidal activity in carcass tissues must therefore be taken into account in evaluating the significance of reports of deep-tissue contamination of carcasses from meat animals.


Microscopically controlled surgery for extramammary Paget's disease. In five cases of extramammary Paget's disease (EMPD), the neoplasms were removed under complete microscopical control by means of fixed-tissue chemosurgical technique in one case and fresh-tissue technique in four cases. In each lesion there were histologically involved areas extending several centimeters into clinically normal-appearing skin. All patients have been free of the disease for periods from four months to nine years. Other authors using conventional surgery have reported a recurrence rate of 44%. Microscopically controlled surgery offers the most reliable method of removing all of the neoplastic tissue EMPD and preserves as much normal tissue as possible.


Seroepidemiology of cytomegalovirus infections during the first years of life in urban communities. Using indirect haemagglutination assay, combined with a collection of blood samples on blotting cards, seroepidemiological surveys of cytomegalovirus infections during early infancy have been done in different populations (French and immigrant) in urban areas. The comparison of CMV antibody status of mothers and their children at 10 months and at 2 years of age enables possible factors of viral transmission to be defined. During the first year of life, seropositive mothers were the only source of infection and they remained the main source during the second year. Socioeconomic class and educational level are determinant factors in the incidence of viral transmission.


The spread of Coxsackie B1 infection.

The spread of Coxsackie B1 infection. During June and July 1977 5 junior boys in a boarding school for 800 pupils became ill with a mild infection caused by Coxsackie B1. The school had been taking part in a vaccine trial, and paired blood samples had been taken from new entrants in October 1976 and October 1977. 18% of the boys susceptible to the infection developed antibodies. The results suggest that dormitories are more important than the day-to-day contacts in the spread of infection. The advisability of nursing children with known or suspected enterovirus infections in open wards in hospital is questioned.


The surgical aspects of insulinomas.

The surgical aspects of insulinomas. The clinical diagnosis of insulinoma rests on the demonstration of Whipple's triad (symptoms of hypoglycimia, low circulating glucose and prompt relief of symptoms after glucose administration). Biochemically, the association of an increased value of immunoreactive insulin with a low glucose value is diagnostic of insulin-mediated hypoglycemia. Angiographic localization of these tumors is accomplished in more than 90% of cases. The pathologic changes are usually due to a single adenoma, for which surgical enucleation is the procedure of choice. Malignancy and persistent hypoglycemia occur in slightly less than 10% of cases and can be fairly successfully managed by diazoxide and streptozotocin.


Bronchioloalveolar cell carcinoma of the lung: a clinicopathological study. Thirty-nine patients with bronchioloalveolar carcinoma were reviewed. The primary lung tumor from 27 patients was examined and divided by histological criteria into three categories. The type 1 pattern was associated with a mean survival of 4.7 years. A mean survival of 3.8 years was attained in patients with type 2. Patients with the type 3 pattern had an average survival of 1.4 years. There was a statistically significant difference in survival when types 1 and 2 together were compared with type 3 (rho less than 0.05). Another statistically significant finding was a mean survival of 5.2 years in patients with negative lymph nodes after surgical resection and a 2.2 year mean survival in patients with positive nodes. The tumor histology of bronchioloalveolar carcinoma should be examined carefully to obtain helpful information in predicting survival. We recommend that these tumors be classified as well-differentiated or poorly differentiated bronchioloalveolar carcinoma.


Subacute cadmium intoxication in jewelry workers: an evaluation of diagnostic procedures. An outbreak of cadmium intoxication in a jewelry factory provided an opportunity for evaluating the usefulness of diagnostic procedures used to evaluate human cadmium toxicity. Blood cadmium levels in workers exposed to cadmium were higher (.93 micrograms/100ml vs .38 micrograms/100 ml) than in unexposed workers. A dose-response relationship was noted between blood cadmium level and symptom prevalence in four symptoms (dyspnea, chest pain, dysuria, and dizziness). Segmental hair analysis revealed highest cadmium levels (up to 19 micrograms/gm) in segments formed prior to cadmium exposure, suggesting that extrinsic contamination was the primary source of cadmium in the hair. beta2-microglobulin levels were within normal limits. No significant renal or pulmonary dysfunction was noted. Symptoms ceased after a cadmium-containing brazing alloy used in jewelry production was replaced, yet urine cadmium levels remained persistently elevated in four workers. Blood cadmium determinations were found to be useful in evaluating symptoms potentially related to cadmium intoxication.


[Stress and immunity in cattle].

[Stress and immunity in cattle]. Stress of variable length may be caused in calf by parenteral administration of supercortisol and application of acute thermal load. Hormonal or thermal stress may have negative impact on postvaccinal immunity to paratyphoid (Salmonella dublin 81), as may be interpreted with reference to the O-agglutination titre of blood serum. The extent to which immunity is suppressed by stress was found to depend on the moment of stress application, duration of stress, and age of the calf.


Crystal-like deposits of amyloid in pancreatic islet cell tumors. Numerous stellate crystal-like deposits of amyloid were observed in four human pancreatic islet cell tumors. Three of the tumors were associated with hypoglycemia or hyperinsulinemia, and the remaining one produced gastrin. Histochemical and ultrastructural studies confirmed the existence of amyloid in the deposits and also suggested the presence of adsorbed or incorporated mucopolysaccharides. Stellate amyloid deposits have previously been described in experimental systemic murine amyloidosis. The relationships of these stellate deposits to other forms of amyloid depostion are unknown.


Lesions induced by encephalomyocarditis virus and coxsackievirus B in newborn mice. Electron microscopic studies of various tissues from virus-infected newborn mice disclosed a consistent form of viral cytonecrotic ultrastructural lesion in divergent types of cells (myocardial, valvular fibroblastic, aortic, exocrine and endocrine pancreatic, and hepatic cells) due to three different picornaviruses (coxsackieviruses B4 and B1 and encephalomyocarditis virus). The lesion is characterized by numerous membrane-vesicle complexes, margination of nuclear chromatin or nuclear pyknosis, dilation of rough endoplasmic reticulum, inflammation, and, at times, mitochondrial swelling. It seems apparent that the finding of this ultrastructural type of cytonecrosis is characteristic of a picornaviral infection regardless of the tissue involved and the offending member of the picornavirus group.


Localized cytomegalovirus encephalitis contiguous to metastatic nasopharyngeal carcinoma. We report a case of focal infection of the brain with cytomegalovirus (CMV) in a 14-year-old boy with metastatic lymphoepithelioma (nasopharyngeal carcinoma). Cytomegalovirus-bearing subependymal glia were only seen in the fourth ventricle, in close proximity to tumor cells, these were associated with an intense inflammatory cell exudate. The latter was due to enterococcal meningitis. There was no evidence of systemic CMV infection, and the typical encephalitis with glial nodules seen in acquired forms of the disease was lacking. We postulate that the infection gained access to the brain either with the tumor cells, which happened to carry CMV genome, or with virus-carrying polymorphonuclear leukocytes migrating to the bacterial meningitis. It is also possible that the proliferating subependymal glia were unduly susceptible to CMV when the host defense mechanisms were compromised.


Dementia-Parkinsonism syndrome with numerous Lewy bodies and senile plaques in cerebral cortex. Five cases of age range 62-72 years with progressive dementia and muscular rigidity are reported and discussed from the clinicopathologic point of view. The neuropathology of these cases was characterized by the widespread occurrence of Lewy bodies (LB) in the CNS as well as the presence of senile changes. The presence of numerous LB in the cerebral cortex and basal ganglia was especially characteristic, although their distribution pattern in the diencephalon and brain stem was identical to that found in paralysis agitans. On the other hand, the presence of senile changes in the cerebral cortex was almost identical to the neuropathology of senile dementia or Alzheimer's disease. Nosologically, the present cases may represent either a combination of atypical paralysis agitans with senile dementia and Alzheimer's disease, or a new disease.


Ductal carcinoma of the pancreas. Rationales for total pancreatectomy. In order to evaluate total pancreatectomy as a surgical procedure for ductal carcinoma of the pancreas, a histopathological analysis was made on 18 resected specimens with special regard to the pattern of cancer growth in the pancreatic tissue. In five of them there was no lymphatic involvement or extrapancreatic invasion, but cancer extended continuously to the tail along with the pancreatic ducts and reached the end of the ducts in three cases. All 11 patients treated with Whipple's procedure died of recurrence, while of four total pancreatectomized patients, one with continuously invasive cancer to the end of the pancreatic duct has been living more than eight years postoperatively. We believe that total pancreatectomy for this type of "intraductal spreading cancer" without invasion beyond the pancreas is indicated as a radical procedure.


Cytomegalovirus infection of the colon associated with ulcerative colitis. We report a 12th case in which infection of the colon with cytomegalovirus was demonstrated in association with ulcerative collitis. Rectal biopsy has proven to be reliable in identifying cells with cytomegaloviral inclusion bodies if careful histologic evaluation is performed. Thus far, prognosis has been serious when cytomegalovirus of the colon has been documented in ulcerative colitis. Nine of 12 patients have required a colectomy, and only seven of 12 have survived.


Variation in susceptibility of HeLa cell lines to coxsackievirus A9. In the course of serial passages for several years a line of uncloned HeLa cells (A line) showed a gradual decrease in plaquing efficiency by coxsackievirus A9 (CA9 virus), while subcultures prepared from the same line kept frozen at an early passage level (A original line) did not show any change. However, it was observed later that the plaque-forming ability of the A original line (A orig. line) also decreased after serial passages as was observed with the A line. Comparing the characteristics of the same cell line at two different passage levels, it was found that the efficiency of adsorption of virus to cells was nearly the same, while virus yield at 8 hours after infection was different. The activity of alkaline phosphatase of cells was also different between these two passage levels, suggesting that a high enzymatic activity is associated with the susceptibility of cell cultures to CA9 virus. Magnesium chloride at 25 mM enhanced plaque formation by CA9 virus in highly passaged less susceptible cell cultures, and a possible role of the chemical as a stabilizer of alkaline phosphatase was discussed.


Fluorescence polarization of DPH-labeled cells adsorbing viruses and its diagnostic potential. Mammalian or avian cells were labeled with a fluorescent probe DPH (1,6-diphenyl-1,3,5-hexatriene). Within a few minutes after adsorption of various naked and enveloped viruses, the degree of fluorescence polarization (P) of the DPH embedded in the adsorbing cells as measured at 37 degrees C, was reduced, a finding indicating a decrease in the microviscosity of the lipids in the cell membrane. This change of fluidity was proportional to the concentration of the adsorbing virus and could be abolished or inhibited by homologous specific antiviral sera, but not by heterologous sera. Potential use of fluorescence polarization tests is described for titration of virus concentration, as well as for serological identification of a virus.


Detection of Lucké herpes virus antigens in infected frog pronephric cells. A frog pronephric cell line, infected with herpes virus derived from Lucké renal carcinomas of Rana pipiens was examined for the presence of Lucké herpes virus antigens. Non-infected pronephric cells were controls. Antiserum to purified Lucké tumor herpes virus was applied in blind tests to the normal and virus infected cells. Both cytoplasmic and nuclear fluorescence was found in the herpes virus infected cells after indirect immunofluorescence with the antiserum. Infected cells cultivated at the optimum growth temperature of 25 degrees C or maintained at 9 degrees C, a temperature inducive to herpes virus replication, showed equivalent fluorescence reactions. No fluorescence was found in the normal pronephric cell line. Examination of parallel herpes infected cells showed cytopathic effect in monolayers by light microscopy, and nuclear or cytoplasmic immunofluorescence. Electron microscopic examination revealed proviral elements in nuclei and sparsely scattered herpes virus coincident with cytoplasmic fluorescence.


Monkey interferon: activity on human cells and chromatographic properties. Brief report. Monkey interferon (MKIF) produced in monkey BSC-1 cells infected with Newcastle disease virus showed antiviral activity on human foreskin fibroblasts and RD114cells--a human line transformed by feline sarcoma virus. The titer of the monkey interferon in human cells was 10--30 fold greater than that found in several normal monkey (BSC-1, CV-1 or SV 40 transformed (C2, C6, T-22) monkey cell lines tested. Ten to fifteen-fold purification of MKIF without loss of activity could be achieved by chromatography on Phenyl-Sepharose CL-4B. Antiviral activity of MKIF was fully resistant to treatment with 1 per cent sodium dodecyl sulfate (SDS).


Human cytomegalovirus induced changes in rabbit cells. Brief report. After infection with human cytomegalovirus, rabbit lung fibroblasts showed, during the four week period of the experiment, cytopathic changes and virus-specific antigens demonstrable by fluorescent antibody. Infectious virus could be recovered from the infected cells by co-cultivation with human lung fibroblasts.


Guinea pig cytomegalovirus: transplacental transmission. Brief report. A well characterized strain of guinea pig cytomegalovirus (GPCMV) was used to infect pregnant guinea pigs during various periods of pregnancy. Transplacental transmission of virus with invasion of the fetus was observed, even in some mothers with preinoculation evidence of GPCMV antibody. Fetal infection occurred during the middle third of pregnancy and GPCMV was isolated from many fetal tissues although histologic evidence of infection was not noted. During the last third, abortion of the pregnancy occurred in some animals. This report demonstrates that GPCMV may invade the fetus producing a sublethal, possibly mild infection which may be very similar to the usual type of CMV infection observed in the human newborn.


Antiviral activity of virus-like particles from Lentinus edodes (Shiitake). Brief report. Interferon (IF) induction and antiviral activity by purified spherical (S) or filamentous (F) virus-like particles and S-derived RNA was studied. A single administration of S particles prior to virus challenge reduced significantly the mortality of mice infected with western equine encephalitis (WEE) virus.


Asymptomatic infection of mouse hepatitis virus in the rat. Brief report. After intranasal inoculation of suckling rats mouse hepatitis virus multiplied mostly in the nasal epithelium; though there were no symptoms, antibodies were produced. Antibodies were also demonstrated in adult rats. These findings suggest that the rat may be a natural host for the virus.


Detection of three polypeptides in preparations of bovine viral diarrhoea virus. Radiolabelled bovine viral diarrhoea/mucosal disease virus (BVDV) strains NADL and Oregon C24V were purified by different steps. Following immunoprecipitation, electrophoresis in SDS-polyacrylamide gels revealed three BVDV structural polypeptides with molecular weights of 57 (VP1), 44 (VP2), and 34 (VP3) kd. The two larger BVDV polypeptides VP1 and VP2 were found to be glycosylated (gp57, gp44). The data obtained on BVDV structural proteins demonstrate common features with hog cholera virus and indicate a common grouping with the family Togaviridae.


Antibody-dependent cell-mediated cytotoxicity (ADCC) in Aujeszky's disease. Antibody-dependent cell-mediated cytotoxicity (ADCC) was studied using as targets 51Cr-labelled Vero cells infected with the Bartha strain of Aujeszky's disease virus (ADV). Using hyperimmune anti-ADV serum to sensitize the targets, porcine leukocytes from dextran-sedimented blood were found to be efficient effector cells yielding maximal 51Cr release by 16 hours. Whilst complement-dependent cytotoxic antibody could be demonstrated no enhancement of ADCC by complement was found. The sera of pigs vaccinated i.m. with Bartha virus were titrated in ADCC using leukocytes as effector cells and the results compared with those obtained by virus neutralization. ADCC proved to be a much more sensitive technique and might, therefore, provide the basis for a reliable diagnostic test. Partially purified lymphocytes and polymorphonuclear leukocytes from blood and peritoneal exudates, and macrophages from exudates were found to mediate ADCC with hyperimmune serum, but differences were observed in the efficiency and timing of their cytotoxic effects.


Viral nucleic acid synthesis in HSV infected neural cells. HSV-1 replication and synthesis of viral DNA and RNA have been examined in gliomas of human (COX) and rat origin (C6) and in mouse neuroblastomas (D2). COX cells fully support HSV-1 replication and show patterns of viral DNA and RNA synthesis similar to those seen in continuous line cells. HSV-1 also grows to high titers in D2 cells but without concomitant high levels of viral DNA and RNA synthesis in the infected cells. Finally, HSV-1 established a persistent infection in C6 cells. Viral mRNA and DNA synthesis could not be detected in these cultures. At cycles of approximately 15--20 days, the persistently infected cultures exhibited massive CPE and relatively high production of infectious HSV.


[Lysosomal-cation test and the prospects of its use in pathomorphological and laboratory diagnostic practice]. A cytochemical lysosome-cation test is proposed as suitable for use in diagnostic laboratories. The test is based on the detection of cationic protein and determinations of the fate of phagocytized and extracellular bacteria in inflammation foci. A method for simultaneous cytochemical detection of myeloperoxidase and cationic proteins in neutrophilic and eosinophilic granulocytes was developed. Using this method, a previously unknown variety of lysosomal granules devoid of peroxidase was detected in neutrophilic promyelocytes. The lysosomal cation test was shown to be useful for improved morphological diagnosis of leukemias, for the determination of the fate of phagocytized and non-phagocytized bacteria in inflammation foci, and of the level of nonspecific and infection resistance of the host.


Adenoviral meningoencephalitis in a patient with lead toxicity. Adenovirus type 12 was recovered from the CSF of a 36-year-old woman with adenoviral meningoencephalitis and lead toxicity. The serum level of lead was 199 micrograms/dL and the CSF level was 7 micrograms/dL. After therapy with edetate disodium calcium (Calcium Disodium Versenate), she had an uneventful recovery. The possibility of exacerbation of lead poisoning with encephalopathy due to adenovirus type 12 meningoencephalitis is raised.


Viral hepatitis and intrahepatic cholestasis of pregnancy. Specific serological tests which are now available for the diagnosis of infection with hepatitis A and hepatitis B viruses, were used to distinguish viral hepatitis from other causes of liver disease in pregnancy. Forty-nine (51.6%) of 95 pregnant patients had viral hepatitis, 24 hepatitis A and 25 hepatitis B. The majority of patients (26 of 28) presenting before 22 weeks gestation had viral hepatitis. By contrast, 23 (34.3%) of the remaining 67 patients presenting with abnormal liver function tests at greater than or equal to 22 weeks gestation, had viral hepatitis; the majority of the remainder had features compatible with intrahepatic cholestasis of pregnancy. Although there were significant clinical and biochemical differences between the two groups, accurate diagnosis was only possible by using specific virological tests.


High density and low density lipoproteins in chronic renal failure before and after renal transplantation. The hyperlipoproteinaemia accompanying chronic renal failure and renal transplantation has been re-examined in terms of the apoprotein (apo A and apo B) moieties of the high density (HDL) and low density (LDL) lipoproteins. Hypertriglyceridaemia is prevalent in chronic uraemia and is not corrected by haemodialysis or renal transplantation. Hypercholesterolaemia is characteristic only of renal allograft recipients. Apo A levels are reduced in undialyzed uraemic and remain low during haemodialysis, as well as in patients with renal allografts of less than six months duration. In renal transplant recipients of greater than one year duration, apo A levels are normalized. The normalization of apo A level is related to the stability of graft function, as determined by sequential analysis of 15 renal transplant recipients. Apo B levels are normal transplant recipients. Apo B levels are normal in uraemic and dialyzed patients, but are elevated in long term transplant recipients. Unlike apo A levels, this alteration does not appear to reflect the restoration of kidney function.


Establishment of rat pancreatic endocrine cell lines by infection with simian virus 40. The feasibility of infection and transformation by SV40 (simian virus 40) of primary cell cultures derived from newborn-rat pancreas was investigated. As judged by the presence of intranuclear SV40 T-antigen, exposure to the virus resulted specifically in infection and transformation of epithelioid (predominantly endocrine) cells. The transformed cells were subcultured (more than 64 passages) and cloned. Culture medium and acid/ethanol extracts of the cells did not contain detectable amounts of immunoreactive insulin after the third subculture. However, inoculation of such SV40-transformed pancreatic cells into immunodeficient rats results in tumours in which insulin production was partially restored through the passage in vivo, since the tumour cells contained and synthesized small amounts of immunoreactive insulin which co-migrated with an insulin marker on gel chromatography. Interestingly, the transformed cells maintained under tissue-culture conditions produced a protein immunologically related to insulin, soluble in aqueous buffer but insoluble in acid/ethanol. This 3000-dalton protein is too large to be a translation product of the rat preproinsulin 9S mRNA. SV40-transformed pancreatic cells might prove useful in the investigation of the factors controlling and maintaining insulin biosynthesis.


The turnover of cytochrome c in different skeletal-muscle fibre types of the rat. The turnover of cytochrome c was determined in the three skeletal-muscle fibre types of adult male rats by a kinetic analysis that followed the time course of cytochrome c content change. Confirming evidence was obtained with double-labelling studies using delta-aminolaevulinate. Cytochrome c turnover was most rapid in the low-oxidative fast-twitch white fibre [t1/2 (half-life) about 4 days], slowest in the high-oxidative fast-twitch red fibre (t1/2 9-10 days) and relatively rapid in the high-oxidative slow-twitch red fibre (t1/2 5-6 days). Thus cytochrome c turnover does not strictly conform to either the appearance (i.e. red or white) or the contractile characteristics (i.e. fast or slow) of the muscle fibres. The synthesis rates needed to maintain the corresponding cytochrome c concentrations, however, were similarly high in the two mitochondria-rich red fibre types. These data illustrate that both the synthesis and degradation processes are important in establishing the cytochrome c concentrations that distinguish the different skeletal-muscle fibre types.


Clearance of calcium pyrophosphate dihydrate crystals in vivo. I. Studies using 169Yb labeled triclinic crystals. Synthetic triclinic calcium pyrophosphate dihydrate crystals were uniformly trace-labeled with Ytterbium-169 (169Yb), a pure gamma-emitting isotope with a halflife of 31 days. The solubility of the labeled crystals was similar to that of cold synthetic crystals. The clearance rate of labeled sterile crystals, sieved to obtain the desired size, was determined after injection of microgram quantities into 4 arthritic huuman and 3 normal adult rabbit joints and corrected by the observed rate of clearance of free 169Yb. The derived rate constants were then used to calculate the time required for half of the injected dose of CPPD to be cleared from the joint. Crystal clearance was found in all instances. Crystal removal from normal rabbit joints was much more rapid than from the much larger human arthritic joints and was inversely proportional to the size of the crystals injected.


Chemotactic factor-induced generation of superoxide radicals by human neutrophils: effect of metabolic inhibitors and antiinflammatory drugs. Human peripheral neutrophils generated superoxide radicals as assessed by ferricytochrome C reduction in response to activation by the synthetic chemotactic factor, N-formyl-methionyl-leucyl-phenylalanine. Superoxide generation was inhibited by 2-deoxy-D-glucose (ID50 4 X 10(-5)M), 2-iodoacetate (ID50 5 X 10(-5)M), and N-ethyl-maleimide (ID50 5 X 10(-6)M), suggesting a dependence on anaerobic glycolysis and sulfhydryl groups. Ouabain, microtubule-disrupting agents, inhibitors of respiration, oxidative phosphorylation, and protein and nucleic acid synthesis were without appreciable effects. Indomethacin (ID50 1 X 10(-4)M), ibuprofen (ID50 9 X 10(-4)M, and phenylbutazone (ID50 1 X 10(-5)M) all caused dose-dependent inhibition of superoxide generation at concentrations approximating those plasma and tissue levels obtained in human beings at therapeutic doses. Acetylsalicylic acid (125-500) microgram/ml) and aurothioglucose (10(-3)-10(-6)M) were without appreciable effects. Superoxide generation was inhibited only by relatively high concentrations of hydrocortisone (ID50 greater than 10(-3)M). Because superoxide radicals have been implicated in the pathogenesis of tissue injury in several forms of inflammation and arthritis in vivo, these studies suggest that the production of a potential cytotoxic factor may be subject to pharmacologic manipulation and that at least some of the antiphlogistic effects of the nonsteroidal antiinflammatory agents may be mediated through effects on superoxide production.


Plasma lipoproteins and coronary arteriography in subjects in the program on the surgical control of the hyperlipidemias. Preliminary report. Coronary arteriographic findings, plasma lipid and lipoprotein levels, and cigarette smoking history are reported for the first 101 male post myocardial infarction survivors who have been entered into the POSCH clinical trial. Estimates of the extent of stenosis in the major coronary arteries were made using 4 models ranging from a simple determination of the number of the 3 major vessels having significant (i.e. 50% or greater stenosis) disease to more complex methods of determining overall extent of disease in 14 major segments of the coronary arteries. Age was shown to be an important factor in the extent of vessel disease. When controlling for age, plasma cholesterol and LDL-cholesterol levels were shown to be related to the extent of disease, especially in Type II hyperlipoproteinemia subjects. Multiple linear regression analysis demonstrated that age and LDL-cholesterol had positive associations and HDL-cholesterol had an inverse association with the coronary artery  disease indices. In this comparatively "healthy" subgroup of the overall population of first MI survivors the major CHD risk factors are limited to plasma lipids and cigarette smoking.  This preliminary report of 10% of the recruitment objective of the project supports the currently held views of the lipid--atherosclerosis hypothesis regarding the effects of age-total plasma cholesterol, LDL--cholesterol, and HDL--cholesterol on the extent of coronary atherosclerotic plaques, as determined by coronary arteriography.


Lipid--lipoprotein composition in hypercholesterolemic children and their parents. In 13 hypercholesterolemic children, re-screened for serum cholesterol after a 1-year interval, hypercholesterolemia was confirmed in only 61.5% of the cases. A tentative explanation seems to be the statistical principle of regression towards the mean. The lipid--lipoprotein analysis showed that serum and LDL cholesterol concentrations in the 13 hypercholesterolemic children and their parents were significantly higher compared to controls (children and parents). At re-screening, hyper-LDL cholesterolemia was present in only 8 of the 13 children (61.5%); 4 cases exhibited hyper-HDL cholesterolemia (30.7%). The high prevalence of the parents repeating the lipoprotein abnormality and the electrophoretic pattern found in the propositi (children) confirms the familial aggregation of the hypercholesterolemic states (hyper-LDL and hyper-HDL cholesterolemia). In conclusion the results of our study stress the importance of determining the lipid--lipoprotein composition, rather then merely evaluating total serum cholesterol in order to make a correct diagnosis of the hypercholesterolemic state. It should also be emphasized that the lipoprotein disturbances and their familial aggregation may be detected early in childhood, suggesting that the familial screening for risk factors of atherosclerosis should be done at pediatric age.


Influence of oral polyunsaturated and saturated phospholipid treatment on the lipid composition and fatty acid profile of chimpanzee lipoproteins. The influence of treatment with polyunsaturated lecithin (EPL) and with saturated lecithin on the lipoprotein composition and fatty acid profile was investigated in 4 male chimpanzees. The animals were successively given 3 isocaloric diets containing the same amount of fat with a degree of saturation varying from 1 in the control diet to 0.2 in the diet enriched with polyunsaturated lecithin, to 4 in the diet enrich with saturated lecithin. The VLDL, LDL and HDL3 fractions were isolated by ultracentrifugal flotation; changes in their lipid and fatty acid composition were followed and their microviscosity was measured. The treatment with polyunsaturated lecithin increases the cholesterol esters and lysolecithin content in HDL3, presumably via activation of the enzyme LCAT. These modified HDL particles have a more fluid surface and a denser core and are susceptible to act as better cholesterol carriers. A complementary effect of this treatment is a decrease of the plasma triglycerides and VLDL concentration, an increase in the unsaturation ratio of the triglycerides which might take place via activation of triglyceride lipase. The saturated lecithin treatment increases the plasma VLDL and LDL concentrations and the triglyceride levels and increases mostly the saturation ratio of the cholesterol esters. These effects are likely to enhance the progression of atherosclerosis.


Accumulation and loss of cholesterol esters in monkey arterial smooth muscle cells exposed to normal and hyperlipemic serum lipoproteins. The effects of high low and very low density lipoprotein fractions from normal or hyperlipemic rhesus monkey serum on the accumulation or removal of cholesterol esters from rhesus monkey smooth muscle cells in tissue culture were determined. Serum or serum lipoproteins were labeled with [14C] free cholesterol and adjusted to the same free cholesterol level in the incubation medium. Of the two normal lipoproteins examined, the LDL fraction caused more esterification than the HDL. Cells incubated in hyperlipemic serum showed a 2-fold stimulation in esterification as compared to cells in normal serum. This was contributed by hyperlipemic VLDL and LDL and led to a concomitant increase in cellular cholesterol ester content. Both hyperlipemic LDL and HDL stimulated esterification when compared to their normal counterparts. Cholesterol ester removal was examined by incubating the serum or lipoprotein fractions with cells enriched in cholesterol ester through a prior exposure to hyperlipemic serum. The cells incubated in normal or hyperlipemic HDL or lipoprotein-deficient serum had the lowest cholesterol ester content. Thus, the lipoprotein fractions which caused the lowest levels of cholesterol esterification were also the most efficient in the removal of cellular cholesterol esters.


Decreased high density lipoprotein-cholesterol levels in male patients with transient ischemic attacks. Plasma lipid and lipoproteins levels were determined in a continuous series of 50 patients (36 males and 14 females), mean age around 50 years, with a clinical diagnosis of transient ischemic attacks (TIAs). TIA was defined as a sudden episode of focal cerebrovascular insufficiency, with complete resolution of the symptoms within 24 h. TIAs are considered an important prognostic symptom for ischemic cerebrovascular diseases, being manifest in approximately 45% of the patients later undergoing a complete stroke. Plasma total cholesterol levels did not differ in these patients, when compared with a similar series of patients of the same age and sex, free of cerebrovascular lesions. A slight elevation of mean triglyceride levels was detected in the patients of both sexes, as well as higher incidence of type IV hyperlipoproteinemia. The most significant finding, however, observed only in male TIA patients, was that of significantly reduced high density lipoprotein (HDL)-cholesterol levels. This reduction (-19.7% compared to the control group) is similar to that recently reported for patients with clear-cut ischemic cerebrovascular disease. The detection of decreased HDL-cholesterol levels in male TIA patients may be of considerable significance for a prognostic evaluation of this biochemical parameter.


Body composition in hypopituitary dwarfs before and during human growth hormone therapy. The clinical characteristics and body composition of eight hypopituitary dwarfs (10.2-21.6 yr) were analyzed before and after 6 and 12 mo of growth hormone therapy. 2 IU 3 times/wk. Before treatment, growth rate was 1.8 +/- 0.7 cm/yr, height age was 2.0-12.8 yr less, and bone age 2.0-11.1 yr less than chronologic age. Total body water (TBW), lean body mass (LBM), extracellular water (ECW), and intracellular water (ICW) were below normal for chronologic age, but normal for height. Muscle mass (MM) was below normal for age and height. During HGH therapy, growth rate was 7.1 +/- 1.6 cm/yr in the first 6 mo and 7.8 +/- 1.4 cm/yr during the next 6 mo; the ratio of change in height age to change in chronologic age was greater than or equal to 1.0 in all patients and the ratio of change in bone age to change in height age was 1.2 in one patient and less than or equal to 1.0 in the others. TBW, LBM, ECW, and ICW increased according to height increments; however, MM increased at a faster rate than expected from the height gains. Also, a relative or absolute loss of total body fat was recorded during the first 6 mo of therapy. It is suggested (1) that among the body composition parameters studied, muscle mass is the tissue most closely reflecting the lack of HGH and also its therapeutic benefits and (2) evaluation of body composition in hypopituitary dwarfs in response to HGH therapy shows striking changes not reflected by the determination of stature or weight alone.


Radiotherapy alone or with chemotherapy in the treatment of small-cell carcinoma of the lung. Medical Research Council Lung Cancer Working Party. This report gives the complete findings at one year of a study comparing radiotherapy (Rt) with radiotherapy followed by 3-drug chemotherapy (RtC3) in the treatment of histologically proven small-cell carcinoma of the lung of limited extent. Over the 12-month period there was a significantly increased survival for the RtC3 patients (P = 0.002) and at 12 months 18% of the 121 Rt but 34% of the 115 RtC3 patients were alive (P = 0.009). The median survival for the Rt series was 25 weeks and for the RtC3 series 43 weeks. There was evidence of recurrence of the primary cancer in 32 (32%) of the 99 Rt and 20 (26%) of the 76 RtC3 patients who died. Distant metastases appeared earlier and were more frequent in the Rt series (P less than 0.0001) and over the 12-month period 79% of the Rt and 57% of the RtC3 patients developed distant metastases (P less than 0.0005). At 12 months only 8% of the Rt but 26% of the RtC3 patients were alive and free of metastases. Adverse reactions occurred much more frequently in the RtC3 series; 32% of the Rt series as against 83% of the RtC3 series had reactions, the most common being nausea and vomiting (13% Rt, 71% RtC3) and the most serious being marrow depression (23% Rt, 54% RtC3). No important differences were found among the survivors in the 2 series at 3, 6 or 12 months, in general condition, physical activity or respiratory function. It is concluded that radiotherapy plus chemotherapy was superior to radiotherapy alone, although chemotherapy did not protect patients from recurrence of primary growth.


The nature of the copper atoms of cytochrome c oxidase as studied by optical and x-ray absorption edge spectroscopy. X-ray absorption edge spectroscopy has been used to study the copper of 1--2 mM cytochrome c oxidase in the resting oxidized, mixed-valence, and fully reduced states. A comparison was made of this protein with copper complexes and with natural and artificial copper proteins. Spectra were obtained with synchrotron radiation from the SPEAR storage ring using highly sensitive fluorescence detectors. Temperatures of -80 to -120 degrees C were employed further to improve the stability of the samples and to avoid the possibility of either auto- or photon-induced reduction of the materials, which might have occurred in previous studies. In order to characterize the valence states of the Cu and Fe components, the samples were monitored by infrared and visible spectroscopy before and after irradiation by the X-ray beam. The combination of the optical and X-ray absorption techniques has afforded a deconvolution of the four species of copper in the various states of cytochrome c oxidase and the tentative assignment of Cu alpha, the copper redox coupled to the heme alpha of cytochrome alpha, as a highly covalent type of copper and Cu alpha 3, the copper of cytochrome alpha 3, as a more ionic 'blue' type I copper. The implications of these findings upon the mechanism of action of cytochrome oxidase are briefly outlined.


Cyclic AMP induces the synthesis of developmentally regulated plasma membrane proteins in Dictyostelium. Dictyostelium discoideum slugs (pseudoplasmodia) were disaggregated and the resynthesis of developmentally regulated plasma membrane proteins examined. The synthesis of the majority of these proteins was inhibited when cells were overlaid with Cellophane and maintained as a monolayer. However, cell contact and movement did occur under the Cellophane. The inhibition of differentiation may result from the inability of the cells to organize specifically into multicellular aggregates. The addition of cyclic AMP (1--5 mM) induced the synthesis of certain developmentally regulated plasma membrane proteins in cells overlaid with Cellophane. Hence, this confirms other work showing that cyclic AMP is required for at least some post-aggregative gene expression. Specific cell organisation and interactions are apparently required for an increase in or maintenance of intracellular cyclic AMP levels.


The decreased membrane fluidity of in vivo aged, human erythrocytes. A spin label study. The decreased membrane fluidity of the in vivo aged, human erythrocytes is found, by monitoring the electron paramagnetic resonance (EPR) spectra of fatty acid spin labels incorporated into the membrane. In addition, the decreased cell sizes and the decreased cholesterol and phospholipids contents, without significant changes of the quantity of the membrane proteins, also the decrease of ATP and 2,3-diphosphoglycerate and the increase of ADP and AMP, in the aged cells, were observed. Further the functional impairments of the aged cells, i.e. the increased oxygen affinity and the decreased deformability, were shown. On the basis of these quantitative data, the alteration of the protein-lipid organization, due to decreased lipid/protein ratio, the modified protein-lipid interaction and/or the influences of the diminished ATP content, is suggested to contribute towards the decreased membrane fluidity of the in vivo aged erythrocytes.


ESR determination of membrane order parameter in yeast sterol mutants. ESR investigations designed to determine membrane order parameter in sterol mutants of Saccharomyces cerevisiae were conducted using the membrane probe, 5-doxyl stearic acid. These mutants are blocked in the ergosterol biosynthetic pathway and thus do not synthesize ergosterol, the end product sterol. They do not require exogenous ergosterol for growth and, therefore, incorporate ergosterol biosynthetic intermediates in their membrane. Increasing order parameter is reflective of an increase in membrane rigidity. Single mutants involving B-ring delta 8 leads to delta 7 isomerization (erg 2) and C-24 methylation (erg 6) showed greater membrane rigidity than wild-type during exponential growth. A double mutant containing both lesions (erg 6/2) showed an even greater degree of membrane rigidity. During stationary phase the order of decreasing membrane rigidity was erg 6 greater than erg 6/2 greater than erg 2 = wild-type. The increased membrane order parameter was attributed to the presence of substituted sterols rather than increased sterol content or altered fatty acid synthesis.


Infinite cis influx of cyclic AMP into human erythrocyte ghosts. Infinite cis uptake of cyclic AMP into red blood cell ghosts has been measured. The Koiic is calculated from two different integrated rate equations that are applicable when the substrate concentration is unsufficient to cause volume changes. Values of 0.69 mM and 0.66 mM are obtained for the infinite cis Km at 30 degrees C using these procedures. These values are only slightly higher than that predicted from zero trans net flux experiments. Lowering the temperature reduces Koiic from 0.69 mM at 30 degrees C to 0.478 mM at 20 degrees C, 0.108 mM at 10 degrees C and 0.072 mM at 4 degrees C (Q10 = 2.4). The Q10 for activation of influx permeability of 10(-5) M cyclic AMP is 1.55.


Sidedness of the ATP-Na+-K+ interactions with the Na+ pump in squid axons. Using dialysed squid axons we have been able to control internal and external ionic compositions under conditions in which most of the Na+ efflux goes through the Na+ pump. We found that (i) internal K+ had a strong inhibitory effect on Na+ efflux; this effect was antagonized by ATP, with low affinity, and by internal Na+, (ii) a reduction in ATP levels from 3 mM to 50 microM greatly increased the apparent affinity for external K+, but reduced its effectiveness compared with other monovalent cations, as an activator of Na+ efflux, and (iii) the relative effectiveness of different K+ congeners as external activator of the Na+ efflux, though affected by the ATP concentration, was not affected by the Na+/K+ ratio inside the cells. These results are consistent with the idea that the same conformation of the (Na+ + K+)-ATPase can be reached by interaction with external K+ after phosphorylation and with internal K+ before rephosphorylation. They also stress a nonphosphorylating regulatory role of ATP.


Effects of drugs on pigeon erythrocyte membrane and asymmetric control or adenylate cyclase by the lipid bilayer. In pigeon erythrocyte membrane, the beta-adrenergic receptor and the enzyme adenylate cyclase can be uncoupled in two different ways depending on the type of drug used. Cationic drugs: chlorpromazine, methochlorpromazine, tetracaine, n-octylamine and a neutral alcohol, octanol, abolished alprenolol receptor binding ability and in the same range of concentration of the drug, sensitized adenylate cyclase to fluoride or Gpp(NH)p stimulation. Anionic drugs: di- and trinitro-phenols, indomethacin and octanoic acid did not affect the total number of beta-adrenergic receptor sites and, with the exception of trinitrophenol, did not change the association constant for alprenolol but they abolished the stimulation of adenylate cyclase by isoproterenol, fluoride or Gpp(NH)p. These modifications of the adenylate cyclase system occurred in a range of drug concentration where cell shape and protection against hemolysis were also affected. As chemical composition varies widely from one drug to another, it is suggested that these effects are largely nonspecific and mediated by the lipid bilayer. They are probably related to a preferential sidedness of action of the drugs in the lipid bilayer, displaying the role of an asymmetric control of the adenylate cyclase system in the membrane by the two halves of this bilayer.


Accessibility of sialo components in a murine tumor cell to extracellular N-acetylneuraminate glycohydrolase (sialidase). Lipid-bound sialic acid in the murine melanoma cell is not totally inaccessible to an exogenous macromolecular probe, as formerly believed. Roughly 30% of the dialic acid bound to lipid, and an equal proportion of the sialic acid bound to protein is cleaved by the action of Clostridium perfringens N-acetylneuraminate glycohydrolase (neuraminidase, sialidase) when the purified enzyme is added to the suspenion medium of intact murine melanoma cells freshly derived from the tumor. Cleavage of lipid-bound sialic acid is indifferent to the presence of Ca (2+) in the medium. However, maximum release from protein requires a physiological concentration of this divalent cation. Variation in ionic strength has no effect on release of sialic acid. These findings show that restricted portion of the bound sialic acid may be released from the intact murine melanama cell by the extracellularly supplied enzyme acting topographically.


Interaction of polyene antibiotics with sterols in phosphatidylcholine bilayer membranes as studied by spin probes. Interaction of filipin and amphotericin B with sterols in phosphatidylcholine membranes has been studied using various spin probes; epiandrosterone, cholestanone, phosphatidylcholine with 12-nitroxide or 5-nitroxide stearate attached to 2 position and also with tempocholine at the head group. Filipin caused increase in the fluidity of cholesterol-containing phosphatidylcholine membranes near the center, while it rather decreased the fluidity near the polar surface. On the other hand, amphotericin B did not apparently affect the fluidity. In the electron spin resonance spectrum of steriod spin probes in the antibiotic-containing membranes, both bound and free signals were observed and the association constant was calculated from the siganal intensity. In the binding of steriods with filipin, both 3 and 17 positions were involved, while the 17 positions was less involved in the binding with amphotericin B. Phase change in the host membrane markedly affected the interaction of filipin with epiandrosterone probe. The bound fraction jumped from 0.4 to 0.8 on going to the crystalline state and increased further with decrease in temperature. The overall splitting of the bound signal also increased on lowering the temperature below phase transition. This change was attributed to aggregate formation of filipin-steriod complexes in the crystalline state. On the other hand, effect of phase transition was much smaller on the interaction of amphotericin B with the steriod probe.


Alteration of human erythrocyte plasma membranes by perfringolysin O as revealed by freeze-fracture electron microscopy. Studies on Clostridium perfringens exotoxins V. When human erythrocyte membranes were treated with perfringolysin O (Clostridium perfringens theta-toxin) and examined by electron microscopy after freeze-fracture, two ultrastructural alterations were observed in fracture faces of membrane. (1) A random aggregation of intramembranous particles was seen in the fracture face of the protoplasmic half (PF face) of all membranes treated with the toxin, even if at a low concentration (40 hemolytic units/ml). On the other hand, the aggregation in the fracture face of the exoplasmic half (EF face) was observed only in membranes treated with a high concentration (3300 hemolytic units/ml) for 2 h. (2) Round protrusions and "cavities" with 30 nm in diameter were visible in EF and PF faces of membranes treated with a high concentration, respectively. These structures were always protruded toward cytoplasmic side, but did not appear to form holes through the membrane. Ring and arc shaped structures with a dark center of 26 nm and a distinct border of 5 nm in width were observed when the toxin alone was negatively stained at a very high concentration (170,000 hemolytic units/ml). These structures were also produced in the presence of cholesterol even if the toxin concentration was low.


Characterization of a proteolipid complex of aminoacyl-tRNA synthetases and transfer RNA from rat liver. A high molecular weight complex containing aminoacyl-tRNA synthetases, peptidyl acetyltransferase, lipids and tRNA has been isolated from the 250,000 x g postmitochondrial supernatant from rat liver cells. Aminoacyl-tRNA synthetase activity directed towards arginine, aspartate, glutamine, glutamate, glycine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, and tyrosine is present. An endogenous pool of aminoacyladenylates is indicated by an ATP-32PPi exchange catalyzed by the native complex, which shows a dramatic increase after addition of ATP. Lysine is the only amino acid which greatly increases the exchange rate catalyzed by the native complex in vitro, whereas components of the denatured complex activate all the 13 amino acids in the presence of ATP. Six of the eight lipid fractions were glycolipids; cholesterol and cholesterol esters were absent. The extracted RNA has many characteristics of tRNA. These findings provide evidence for the organization of aminoacyl-tRNA synthetases in a complex with peptidyl acetyltransferase that also contains lipids and tRNA and that can be readily isolated from the cytosol of rat liver cells.


Removal of T4 endonuclease V-sensitive sites from SV40 DNA after exposure to ultraviolet light. The induction and removal of sites sensitive to T4 endonuclease V from ultraviolet-irradiated Simian Virus 40 (SV40) Form I (supercoiled) DNA during a lytic infection of monkey CV-1 cells was investigated by agarose tube gel electrophoresis. Endonuclease-sensitive sites were induced at a rate of 0.049 sites/SV40 genome per J/m2, or 1.4 sites/1 . 10(8) daltons of DNA per J/m2. This value is similar to the yield of endonuclease-sensitive sites and pyrimidine dimers in uninfected host CV-1 cell DNA. Removal of endonuclease-sensitive sites was dose dependent and non-linear for at least 24 h after irradiation. These results suggest that SV40 DNA is subject to the excision repair mechanisms of the host cell, and that the excision of pyrimidine dimers may be one of the biochemical events underlying host cell reactivation.


Purification of mitochondrial NADH dehydrogenase from Drosophila hydei and comparison with the 'heat-shock' polypeptides. Mitochondrial NADH dehydrogenase (NADH:(acceptor) oxidoreductase, EC .6.99.3) from either Drosophila hydei larvae or embryos has been purified 150- and 120-fold, respectively. The purified enzyme appeared homogeneous and showed a molecular weight of 57 000. The molecular weight of the nondenatured enzyme was 79 000. On isoelectro-focussing of the preparation, two fractions were observed, a major one with an isoelectric point of 6.2 and a minor fraction with an isoelectric point of 4.9. Straight-line kinetics in Lineweaver-Burk plots were observed for the purified enzyme with a Km of 0.040 mM. The Km was not changed during the purification procedure, suggesting that the enzyme was not denatured or inactivated. The pH optimum of the purified enzyme was 5.6. The molecular weight of the purified mitochondrial NADH dehydrogenase does not correspond to that of one of the 'heat-shock' polypeptides.


The nature of the substrate inhibition in lactate dehydrogenases as studied by a spin-labeled derivative of NAD. The formation of the ternary complex of lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) from pig heart and skeletal muscle with the adduct of pyruvate to NAD", spin-labeled at N6 was studied by ultraviolet spectroscopy and ESR techniques. According to ultraviolet measurements we found identical binding characteristics for the natural coenzyme and its spin-labeled analog. The rate by which the ESR signal of free spin-labeled NAD+ decreased upon addition of pyruvate to the binary complexes was substantially different in the two isozymes. With the heart type an initial drop followed by a further linear decrease, zero order in the enzyme and coenzyme concentration was observed. In case of the skeletal muscle isozyme no immediate reaction and a first order process occurred. The initial reaction can be attributed to a non-covalent enzyme/spin-labeled NAD+/pyruvate complex with a dissociation constant for pyruvate of 11 +/- 1 mM, thus explaining the well-known substrate inhibition in the heart isozyme above 2 mM pyruvate. The further reaction is then determined by the buffer dependent enolization of pyruvate. In the muscle isozyme formation of the covalent adduct is not assisted by prior binding of pyruvate in a non-covalent ternary complex and therefore the rate depends on the binary complex concentration.


Mechanism of inhibition of malate dehydrogenase by thyroxine derivatives and reactivation by antibodies: homogeneous enzyme immunoassay for thryroxine. Pig heart mitochondrial malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) is about 90% inhibited upon labelling an average of two amino groups per subunit with an active ester of thyroxine. Inhibition is probably associated primarily with thyroxine binding to one specific group which is normally unreactive but becomes activated upon noncovalent binding of thyroxine derivatives to the enzyme. Enzyme inhibition is due to a decrease in the rate of association of NAD. Antibodies to thyroxine induce a slow conformational change with partial reversal of inhibition of more heavily labelled conjugates. The antibody-induced activation is not cooperative and does not require bivalent association of the antibody. Activation can be blocked by the presence of free thyroxine and is the basis for a clinically useful assay for serum thyroxine.


Hydrogenase activity in the dry state: isotope exchange and reversible oxidoreduction of cytochrome c3. Hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) catalyzes three types of reactions, i.e., (1) conversion between hydrogen modifications, para-H2 and ortho-H2, (2) exchange reaction between hydrogen isotopes, and (3) reversible oxidoreduction of an electron carrier with H2 and protons. We observed that purified desulfovibrio hydrogenase in the dry state could catalyze not only the conversion and exchange reactions (Yagi, T., Tsuda, M., Mori, Y. and Inokuchi, H. (1969) J. Am. Chem. Soc. 91, 2801) but also the reversible oxidoreduction of the electron carrier, cytochrome c3 with H2. The rate of the conversion was in the range from 0.1 to 0.65 mol H2 converted per mol hydrogenase per s, and the ratio of the conversion rate to the exchange rate was near 5. The rate of oxidoreduction of cytochrome c3 in the dry state was 0.015 mol H2 taken up in the forward reaction and 0.003 mol H2 released in the reverse reaction per mol hydrogenase per s. The process of these reactions could be explained by the observations that the hydrogenase molecule in the dry state has protons which are directly exchangeable with H2 during catalytic process. The reversible oxidoreduction of cytochrome c3 is also explained by inter- and intramolecular electron transfer among cytochrome c3 molecules.


Ethanediol monoester hydrolysis by monoacylglycerol lipase of rat liver microsomes. The hydrolysis of long-chain monoester of ethanediol by rat,liver subcellular fractions was investigated in order to define the carboxylic acid ester hydrolase involved and to localize the enzymic activity. We found that with 1-O-hexadecanoyl [U-14C]ethanediol as substrate, hydrolytic activity was foremost associated with the rough microsomal fraction. The pH optimum occurred at 8.5. The apparent Km and V values were 6.5 . 10(-4) M and 13 mumol/h per mg microsomal protein, respectively. Enzymic activity was inhibited by p-chloromercuribenzoate and by diisopropylfluorophosphate, whereas NaF was less effective and CaCl2 did not affect apparent activity. Amongst a number of carboxylic acid esters tested as substrate, only long-chain 1-acyl and 2-acyl glycerols inhibited acyl diol hydrolysis competitively (Ki approximately 0.9 mM). It was concluded that long-chain monoesters of ethanediol are hydrolyzed by the monoacyl glycerol lipase system associated with the rat liver microsomal fraction. Because diol monoester is also utilized by the cholinephosphotransferase system of liver to form highly lytic acyl diol phosphocholines, efficient diol monoester hydrolysis by monoglyceride lipase may be a significant step in regulating acyl diol phosphocholine levels in biological systems.


Purification of human plasma lecithin:cholesterol acyltransferase and its specificity towards the acyl acceptor. A simple and convenient method for the purification of human plasma lecithin-cholesterol acyltransferase was developed. The method involves the adsorption of the enzyme from diluted human plasma on DEAE-Sephadex, treatment with 1-butanol in the presence of (NH4)2SO4, DEAE-Sephadex chromatography, treatment with dextran sulfate in the presence of Ca2+, and hydroxyapatite chromatography. The enzyme purified showed a single main band by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In addition, the enzyme obtained was stable for more than four weeks, when it was kept at 4 degrees C under N2 in a buffer of low ionic strength. The purified enzyme was used to study its specificity toward the acyl acceptor. This specificity was found to be broad in that not only sterols but also long chain primary alcohols exhibited considerable acceptor activity. Furthermore, in agreement with our previous observations with crude enzyme (Piran, U. and Nishida, T. (1976) J. Biochem. (Tokyo) 80, 887-889), the purified enzyme was found to be capable of hydrolyzing the ester linkage at the carbon-2 position of phosphatidylcholine. The transesterification, as well as the hydrolytic reaction, required the presence of the cofactor polypeptide, apolipoprotein A-I.


Preparative isoelectric focussing of apolipoproteins C and E from human very low density lipoproteins. The application of isoelectric focussing on a gel-stabilized layer for the separation of the Tris-urea-soluble apolipoproteins of very low density lipoproteins has been described. This method in one step, allows the separation of most apolipoproteins, which were then analyzed and characterized. Apolipoproteins CII and CI were isolated as single protein bands with apparent pI of 5.0 and 6.5, respectively. Apolipoprotein CII was biologically active and could activate lipoprotein lipase. Apolipoprotein CIII was separated into several protein bands with pI ranging from 4.7 to 5.1 as a function of their number of sialic acid residues. Apolipoprotein E was isolated and characterized into five polymorphic bands with pI of 5.7, 5.8, 5.9, 6.0, and 6.2, respectively.


The demonstration of very low density lipoprotein in the basal lamina of the granulosa layer in the hen's ovarian follicle. The isolated basal lamina from the granulosa layer in ovarian follicles of the domestic fowl contains an abundance of spherical particles with a modal cross-sectional diameter of 25-30 nm. The lipid in this basal lamina is predominantly triacylglycerol and its total fatty acid composition resembles that of plasma very low density lipoprotein (VLDL). Immunodiffusion studies and immunoelectrophoresis indicated that this basal lamina contains diffusible antigen identifiable with plasma VLDL. Perfusion with an alkaline buffer displaced the particles from the basal lamina and subsequent perfusion with plasma VLDL in an acidic buffer resulted in the reappearance of particles of similar size and form. Alternatively, when the perfused basal lamina was subsequently perfused with VLDL-free serum, few particulate structures were observed. Measurements of total and VLDL triacylglycerol together with electron microscope studies of the untreated and of the perfused basal lamina provided further evidence for the identification of the majority of particles with plasma VLDL. Other particulate lipoprotein is most probably plasma low density lipoprotein (LDL). These studies demonstrated that this basal lamina is permeable to the circulating VLDL of the laying fowl.


Association of lysolecithin acyltransferase with the high density lipoproteins and its activation by the low density lipoproteins in normal human plasma. The lysolecithin acyltransferase of human plasma is shown to be associated with the high-density lipoprotein fraction. Although the low density lipoproteins do not have intrinsic enzyme activity, their presence activated the enzyme 3--7-fold. This activation is not affected by heat-treatment of the low density lipoproteins, but is abolished by the addition of heparin.


Reduction of ferricytochrome c, methemoglobin and metmyoglobin by hydroxyl and alcohol radicals. We have studied the reaction of ferricytochrome c, methemoglobin and metmyoglobin with OH and alcohol radicals (methanol, ethanol, ethylene glycol and glycerol). These radicals can be divided into three groups: 1. The OH radicals which reduce the ferricytochrome c with a yield of (30 +/- 10)% and methemoglobin with a yield of (40 +/- 10)%. They do not reduce metmyoglobin. The reduction is not a normal bimolecular reaction but is most probably an intramolecular electron transfer of a protein radical. 2. Methanol and ethanol radicals which reduce all three hemoproteins with a yield of (100 +/- 5)%. This reduction is a normal bimolecular reaction. 3. Glycerol radicals which do not reduce the ferrihemoproteins under our experimental conditions. Ethylene glycol radicals do not reduce ferricytochrome c and metmyoglobin but they do reduce methemoglobin with a yield of (30 +/- 10)%.


A novel method for converting apolipoprotein B, the major protein moiety of human plasma low density lipoproteins, into a water-soluble protein. We have developed a new technique to solubilize apolipoprotein B (ApoB) in aqueous solutions. The procedure involves stirring ApoB in 6 M guanidine/20 mM NH4Cl/NH4OH in the presence of cupric ammonia complexes at pH 9.7 for 20 h in N2, and then removing these reagents by a series of dialysis in N2. The resulting Cu(NH3)4(2)+-treated (Cu2+-treated) ApoB is soluble in aqueous buffers of pH above 8.3 or below 3. Parallel experiments carried out on control proteins, human albumin, alpha-lactalbumin, and insulin, indicated no change in molecular weight and no creation of a new NH2-terminal amino acid after Cu2+-treatment. By Edman degradation, the Cu2+-treated ApoB showed no detectable NH2-terminal amino acid. These results showed that the mechanism of Cu2+-solubilization of ApoB was not due to the cleavage of peptide bonds. Electrophoresis on urea-polyacrylamide gel, Cu2+-treated ApoB showed the same number of bands as the non-treated ApoB in the separating gels (7%) near the cathode, suggesting the heterogeneity of ApoB. In SDS-polyacrylamide gel (10%), the reduced and Cu2+-treated ApoB migrated with the similar mobilities to the monomer or dimer of human albumin. Antibodies raised against Cu2+-treated ApoB gave at least two immunoprecipitin lines against the Cu2+-treated ApoB as well as the non-treated guanidine-HCl-soluble ApoB, suggesting the presence of non-identical subunits.


Receptor binding and conformational properties of bovine and ovine prolactins after chemical modification of the two tryptophan residues. The two tryptophan residues in ovine and bovine prolactins were modified by reaction with 2-nitrophenylsulfenyl chloride in 75% formic acid. These derivatives exhibited an important loss of receptor affinity (less than 1% of the native hormones) to a rabbit mammary gland preparation. To a lesser degree, they also lost their binding affinity to specific guinea pig antibodies as detected by radioimmunoassay. The chemical modifications induced a change in the folding of the polypeptide chain, which in itself could be partly or totally responsible for the loss of biological or binding activities. This conformational change has been analyzed by circular dichroism and by prediction of secondary structures from the amino acid sequence using the method of Garnier et al. (Garnier, J., Osguthorpe, D.J. and Robson, B. (1978) J. Mol. Biol. 120, 97--120). The comparison of predicted prolactin and somatotropin structures revealed almost identical alpha-helix, turns and coil regions with an overall content of 67% alpha-helix, 5% beta-sheet, 17% turn and 11% of aperiodic structures. These values were close to those obtained from circular dichroism. The conformational change of the chemically modified hormones as compared to native folding, can be described as a partial loss of alpha-helical structure and an increase in beta-sheet content.


Multiple forms of an extracellular cyclic-AMP phosphodiesterase from Dictyostelium discoideum. The extracellular cyclic-AMP phosphodiesterase of a mutant of Dictyostelium discoideum which accumulates this enzyme was found to exist in multiple forms. Using the isoelectric focusing technique the phosphodiesterase activity was distributed into three peaks with isoelectric points of 4.6, 6.5 and 8.3, designated as p4, p6 and p8. Gel filtration and sucrose gradient analysis showed that the p4 activity consisted of two forms of different sedimentation coefficients. At high enzyme concentrations, the heavy form was favored. Dilution of enzyme activity shifted the equilibrium toward the light form. Direct analysis by sucrose gradient sedimentation of all isoelectric forms demonstrated that besides p4, p6 activity also existed as a mixture of the heavy (9.7 S) and the light (5.4 S) components. In contrast, the p8 activity displayed only the light form. The heterogeneity of the p4 and p6 isoelectric forms was also observed by polyacrylamide gel electrophoresis. A procedure for a partial purification of the extracellular enzyme to about 70-fold is presented.


Collagenase, procollagenase and bone resorption. Effects of heparin, parathyroid hormone and calcitonin. 1. The addition of heparin to the culture fluid of mouse tibiae or calvaria did not cause any significant resorption of bone collagen or mineral. However, heparin (or analogue sulfated polyanions), enhanced greatly the amount of latent, trypsin-activatable collagenase (i.e. procollagenase) released by the bones in the medium without influencing that of directly active collagenase which was always very low. Heparin appeared to act by increasing the production of the enzyme which is immediately excreted. Procollagenase and collagenase are not stored in bone tissue, even under conditions where it is in active resorption. 2. Parathyroid hormone induced in the explants a resorption of both mineral and collagen that was inhibited by calcitonin. These hormones, however, had no influence on the release of procollagenase or collagenase either in the presence or in the absence of heparin. 3. Once activated, bone collagenase digested the collagen of the bone explants, and more extensively after their demineralization. Thus the latent collagenase that accumulates around non-resorbing bones has to be considered as a precursor, (and not as a residue), of active enzyme. 4. Active collagenase added to incipient cultures of bones disappeared with a half-life of 24 h. The lost enzyme could, however, not be reactivated by trypsin and thus was not transformed into latent procollagenase.


Gonadal receptors. I. Evidence for irreversibility in the binding of human chorionic gonadotropin and human luteinizing hormone. The binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors of rat testes has generally been assumed to follow an equilibrium model similar to that proposed for many enzyme systems. Our work shows that equilibrium dissociation constant (Kd) and number of hormone binding sites (Bmax) are highly sensitive to changes in hormone and/or receptor concentration and to treatment received by tissue or receptor preparation prior to the assay. The results of binding assays obtained using receptor preparation pretreated with hormone (labeled as well as unlabeled) indicated that the binding reaction between hormone and receptor was irreversible and that pretreatment of the tissue with hormone greatly alters the number of high affinity gonadotropin binding sites in the testicular homogenate. Data from studies involving increasing receptor concentrations revealed that increasing the mass of particulate receptors in the binding assays leads to higher Kd as well as Bmax values. These findings are incompatible with a binding model based upon occupancy of receptor sites and the state of equilibrium implied. The incompatibilities are analyzed and an alternate model advanced (Bhalla, V.K., Trowbridge, C.G., Chen, C.J.H., Lindeman, J.G. and Rojas, F.J. (1979) Biochim. Biophys. Acta 584, 436--453).


Gonadal receptors. II. Effects of time and reaction volume upon the binding of human chorionic gonadotropin and human luteinizing hormone to particulate receptors. The effect of reaction volume upon the binding of gonadotropins by particulate receptors was studied. Two experimental approaches were used: one involved increasing the reaction volume of the binding assay (i.e. diluting the hormone and receptor concentrations and will be referred to as buffer coincubation studies) and the other involved incubating the testicular homogenate in various buffer volumes prior to the binding assay (buffer preincubation stidies). The results showed that the number of hormone binding sites inferred from Scatchard analysis was inversely related to the reaction volume in the coincubation as well as in the preincubation studies. Time-dependent dissociation of receptors from the intact testis was demonstrated by perifusion studies and the loss of receptors from intact testis correlated with the appearance of soluble factors (Bhalla, V.K., Haskell, J., Grier, H. and Mahesh, V.B. (1976) J. Biol. Chem. 251, 4947--4957) in the eluate obtained. The results obtained along with those presented in the preceding manuscript (Chen, C.J.H., Lindeman, J.G., Trowbridge, C.G. and Bhalla, V.K. (1979) Biochim. Biophys. Acta 284, 407--435) question the validity of the rapid equilibrium model which assumes reversible hormone occupancy of a fixed number of receptor sites. An alternate binding model is proposed herein and its implications are discussed.


Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea. Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly puridied nuclei exhibited specific binding with 125I-labeled human choriogonadotropin, [3H]prostaglandin E1 and [3H]prostaglandin F2 alpha. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E1) and 8.9% (prostaglandin F2 alpha) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F2 alpha binding site and 5'-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21--26% (prostaglandins) of original specific binding despite virtual disappearance of 5'-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound 125I-labeled human choriogonadotropin and [3H]prostaglandin F2 alpha to the same extent or significantly more ([3H]prostaglandin E1, P less than 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.


Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes. Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24 degrees C. Within 20 min most of the cell lines destroyed 20% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37 degrees C. Bmax and KD for the different lymphocytes was 5.3--19 . 10(11) M and 1.8--18,5 . 10(11) M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.


Regulation of hydrogen utilisation in Rhizobium japonicum by cyclic AMP. Utilisation (uptake) of hydrogen gas by whole cells of Rhizobium japonicum was found to be influenced by the carbon source(s) present in the growth medium, with activity being highest in a medium containing sugars. Tricarboxylic acid cycle intermediates, such as malate, significantly reduced H2 utilisation. No reduction in the hydrogenase activity is observed when the enzyme is assayed directly by the tritium exchange method, indicating that the decrease in hydrogen uptake activity is not due to repression of hydrogenase biosynthesis. Cyclic AMP was found to alleviate the inhibition of H2 uptake by malate, and this requires new protein synthesis. Addition of chloramphenicol or rifampicin simultaneously with cyclic AMP eliminated the stimulation of H2 uptake in the malate medium. These results show that in R. japonicum cyclic AMP plays a major role in the regulation of H2 metabolism.


Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscles cells in vivo and in vitro. The effect of medium Ca2+ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures. There is an easily identifiable class of muscle protein which includes the Ca2+-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomysin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca2+ concentration, 0.05--0.3 mM, where fusion was prevented. Similar medium Ca2+ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as 'calcium-modulated proteins'. The increased Ca2+ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca2+ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca2+ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates. The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca2+ concentration between 0.06 and 1.8 mM. It is proposed that increase in cytoplasmic free Ca2+ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.


Distribution and properties of protein kinase and protein phosphatase activities in synaptosomal plasma membranes and synaptic junctions. Some characteristics of the protein kinase activity associated with a synaptosomal plasma membrane (synaptic membrane) fraction and a synaptic junction fraction have been compared. Autoradiography of the phosphorylated fractions separated on sodium dodecyl sulfate polyacrylamide gels showed that cyclic AMP stimulates the phosphorylation of five polypeptides in synaptic membranes, whereas no cyclic AMP dependency could be detected in synaptic junctions. Kinetic studies demonstrated that synaptic junctions contain a high Km and a low Km protein kinase activity while only the high Km activity could be detected in synaptic membranes. The intrinsic ATPase activity of synaptic membranes was shown to strongly interfere with measurements of protein kinase activity. Cyclic AMP binding experiments revealed a 2.6-fold enrichment of cyclic AMP binding capacity in synaptic junctions as compared to synaptic membranes. Protein phosphatase activity was not detected in synaptic junctions but was associated with synaptic membranes, where cyclic AMP was shown to either stimulate or inhibit the dephosphorylation of different polypeptides.


Characterization of the cyclic 3',5'-nucleotide phosphodiesterase actitivty associated with synaptosomal plasma membranes and synaptic junctions. Some characteristics of the cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) activity associated with the synaptosomal plasma membrane (synaptic membrane) and the synaptic junction fractions of rat brain are reported. Kinetic analysis revealed that only one type of phosphodiesterase activity, with a Km of 2.10 19(-4) M for cyclic AMP, is associated with both fractions. The specific activities of the phosphodiesterase in synaptic membranes and synaptic junctions have been estimated at 23.4 nmol/min per mg protein and 22.5 nmol/min per mg protein, respectively. The synaptic junction-associated activity undergoes a 30% stimulation by Ca2+ while no Ca2+ sensitivity of the synaptic membrane-associated activity could be detected. Cytochemical studies performed on the synaptic membrane fraction demonstrated a predominant localization of phosphodiesterase activity over postsynaptic densities, while dense deposits were sometimes observed over the synaptic cleft region.


Different effects of concanavalin A and its succinylated derivative on superoxide release in peritoneal macrophages. The effects of tetravalent conconavalin A and its succinylated derivative on the intracellular production of superoxide anion (O-2) and its release into cell exterior of peritoneal macrophages were observed. Both tetravalent concanavalin A and its succinylated derivative induced marked enhancement of intracellular reduction of nitroblue tetrazolium, which could be inhibited by alpha-methyl-D-glucoside. The extent of activation of nitroblue tetrazolium reduction induced by both types of the lectin paralleled the activation ratio of oxygen consumption. There was little difference in the extent of intracellular O-2 production induced by two types of the lectin. Nitroblue tetrazolium reduction was not affected significantly by pretreatment with colchicine, rotenone or malonate, inhibitors of the cytoskeletal system and of the electron transport system. In contrast, tetravalent concanavalin A induced a higher rate of superoxide release than did succinylated divalent concanavalin A, which lacks the cross-linking activity of surface glycoproteins. These results indicate that superoxide production following oxygen consumption and superoxide release into cell exterior are controlled independently by a separate membrane mechanism and that superoxide production system is not essentially dependent on the involvement of the cytoskeletal system.


The effect of calcium on somatostatin inhibition of insulin release and cyclic AMP production in mouse pancreatic islets. The effect of somatostatin on glucose-induced insulin secretion and cyclic AMP accumulation in isolated islets from obese, hyperglycemic ob/ob mice was studied in a microperifusion system. The normal biphasic pattern of insulin release as well as the inhibitory pattern of insulin release produced by somatostatin (0.5--1 microgram/ml) was matched by similar changes in the intracellular concentration of cyclic AMP. When islets were stimulated by glucose (3 mg/ml) plus 3-isobutyl-1-methylxanthine (0.1 mM), somatostatin (0.5 microgram/ml) failed to inhibit insulin secretion or cyclic AMP formation in the second phase whereas in the first phase both parameters were significantly reduced by somatostatin (0.5 microgram/ml). In batch-type incubations it was shown that addition of excess calcium (to 6 mM) reversed this inhibition. In the second phase calcium potentiated the (glucose + 3-isobutyl-1-methylxanthine)-stimulated insulin secretion without affecting the cyclic AMP production. This potentiation was inhibited by somatostatin (0.1 microgram/ml). Somatostatin (1 microgram/ml) inhibited adenylate cyclase activity in islet homogenates. No effect of somatostatin on islet glucose utilization could be demonstrated. The results indicate a dual action of somatostatin in the inhibition of insulin release, one involving the islet adenylate cyclase and one affecting the islet uptake of calcium.


Action of cholera toxin on dispersed acini from guinea pig pancreas. In dispersed acini from guinea pig pancreas cholera toxin bound reversibly to specific membrane binding sites to increase cellular cyclic AMP and amylase secretion. Cholera toxin did not alter outflux of 45Ca or cellular cyclic AMP. Binding of 125I-labeled cholera toxin could be detected within 5 min; however, cholera toxin did not increase cyclic AMP or amylase release until after 40 min of incubation. There was a close correlation between the dose vs. response curve for inhibition of binding of 125I-labeled cholera toxin by native toxin and the action of native toxin on cellular cyclic AMP. With different concentrations of cholera toxin, maximal stimulation of amylase release occurred when the increase in cellular cyclic AMP was approximately 35% of maximal. Cholera toxin did not alter the increase in 45Ca outflux or cellular cyclic GMP caused by cholecystokinin or carbachol but significantly augmented the increase in cellular cyclic AMP caused by secretin or vasoactive intestinal peptide. The increase in amylase secretion caused by cholera toxin plus secretin or vasoactive intestinal peptide was the same as that with cholera toxin alone. On the other hand, the increase in amylase secretion caused by cholera toxin plus cholecystokinin or carbachol was significantly greater than the sum of the increases caused by each agent alone.


Dissociation by cooling of hormone and cholera toxin activation of adenylate cyclase in intact cells. Cholera toxin, through adenylate cyclase activation reproduced cyclic AMP-mediated effects of thyroid-stimulating hormone (TSH) in dog thyroid slices, i.e. protein iodination, [1-14C]glucose-oxidation and hormone secretion. Iodide and carbamylcholine decreased the cyclic AMP accumulation induced by cholera toxin as well as by TSH, which supports the hypothesis of an action of these agents beyond the steps of hormone-receptor and receptor-adenylate cyclase interaction. Cooling to 20 degrees C did not impair the TSH induced cyclic AMP accumulation in thyroid slices, but completely suppressed the cholera toxin effect. This observation has been extended to other hormones and target tissues, such as the parathyroid hormone (PTH) (kidney cortex), adrenocorticotropic hormone (ACTH) (adrenal cortex) and luteinizing hormone (LH) (ovary systems). As in thyroid, cooling dissociated the cholera toxin and hormonal effects on cyclic AMP accumulation. In homogenate, cooling decreased cyclic AMP generation in the presence of cholera toxin but at 20 degrees C and 16 degrees C a cholera toxin stimulation was still observed. These results bear strongly against the hypothesis that the glycoprotein hormones TSH and LH acetivate adenylate cyclase by a mechanism identical to cholera toxin.


The influence of fluoro-phenyl-alanine on the synthesis of simian virus 40 DNA and T antigens. Treatment of Simian Virus 40 (SV40) infected monkey cells with fluorophenylalanine (FPA) resulted in increased uptake of thymidine by the cells, and progressive inhibition of both viral and cellular DNA synthesis. Viral DNA synthesis was more sensitive to inhibition by FPA than cell DNA synthesis. Synthesis of SV40 T antigens was however unaffected by FPA, as judged from immunofluorescence assays. The M.W. of the major polypetides immunoprecipitated from cell extracts by antibodies from tumor bearing hamster sera was similarly unaffected. It is suggested that T antigen synthesized in the presence of FPA is non functional.


A theory on the control of arbitrary movements. A theory dealing with the control of human, arbitrary movements is proposed. A schema is set up to suggest how the relevant information flows and what kind of operations affect it. A number of successive steps are distinguished in the production of a movement. It is assumed that the intended movement is carried out in the imagination, and that this imaginary movement is composed of a spatial trajectory and an intensity course, which are considered to be independent features of the intended movement. The spatial trajectory will be encoded in a special coding, which is related to the lengths of the muscles that effect the movement. From this special coding of the intended movement static and dynamic control signals can be derived. Because afferent and efferent signals are encoded in the same way in this schema, the evaluation and correction of the performed movement is quite simple. The higher levels in the control schema may function in an abstract way, i.e. the signals at these levels are barely concerned with details of the peripheral motor system. This abstract functioning of the higher levels is based on the numerous feedback mechanisms involved at all levels of control and in the peripheral motor system. Nevertheless, it is possible to incorporate specific peripheral properties in the generation of the control signals. The assumptions in this theory will be discussed and aspects of the proposed control schema will be compared with general control principles.