beta-Endorphin and naloxone in psychiatric patients: clinical and biological effects. The authors conducted single- and double-blind studies of the responses of 7 chronic male schizophrenic patients to 10 mg of naloxone. BPRS ratings were made before and 6 hours after the injection; ACTH blood levels were determined before and 1 1/2 and 6 hours after injection. Statistically significant improvement of psychotic behavior occurred after 6 hours. The greatest improvement occurred in the patient who showed the most pronounced diurnal variation of ACTH levels, and there was no improvement in the patient who had no diurnal changes. Prolactin plasma levels following endorphin injections were apparently dose-dependent and peaked at approximately 30 minutes. The mean half-life of elimination of exogenous beta-endorphin was between 12 and 35 minutes. The authors theorize that positive and negative behavioral responses to naloxone depend--as possibly do many placebo responses in general--on the relative stress produced by experimental or therapeutic interventions.


Research and development of naltrexone: a new narcotic antagonist. The author reviews the history of federally supported research in the field of narcotic antagonist therapy, focusing on social, political, and governmental issues. He describes the criteria for establishing an optimum narcotic antagonist, the legal guidelines for drug development, and the currently available narcotic antagonists. Research supported by the National Institute on Drug Abuse has indicated that naltrexone is the most promising drug in this category. The author discusses the safety and clinic use of naltrexone, the status of the NIDA naltrexone program, and plans for future development of the drug.


Alterations in hepatocytes of mice fed a gallstone-inducing diet: occurrence of nuclear and cytoplasmic lipids. Mice fed a high cholesterol-cholic acid (lithogenic) diet for one year develop fatty livers in addition to gallstones. Light and electron micrographs demonstrate large amounts of lipids in liver parenchymal cells, often to the exclusion of most other cytoplasmic organelles. In addition, some hepatocytes exhibit nuclear lipid pseudo- and true inclusions. Other prominent features of hepatocytes after lithogenic diet include segregation of nucleolar granular and fibrillar material. Accumulation of considerable collagen in extracellular spaces is also noted. Observations suggest changes induced by the cholesterol diet are comparable to cytologic alterations seen in spontaneous and drug induced hepatic tumors, as well as to more general "fatty metamorphosis" of the liver.


Polymorphism of 14C vitamin D3 binding protein in cattle and water buffalo serum. Cattle and water buffalo sera labelled with vitamin D3[14C] (300 and 480 individual samples respectively) were subjected to starch gel electrophoresis followed by autoradiography in an attempt to identify a possible polymorphism of the proteins capable of binding this vitamin. Three phenotypes controlled by two codominant autosomal alleles were identified in cattle while in water buffalo six phenotypes controlled by three codominant autosomal alleles were observed.


Increased proportion of active sheep erythrocyte rosette-forming lymphocytes and plasma rosette enhancement in sarcoidosis. We examined blood lymphocyte subpopulations in 20 patients with sarcoidosis, 37 patients with other diseases, and 51 normal subjects. The B-lymphocytes were identified by the presence of surface immunoglobulin or B-lymphocyte-associated antigen. Lymphocytes were also centrifuged with sheep erythrocytes for 5 min at room temperature at 200 g, and rosette formation was assayed immediately (active E-rosette-forming T-lymphocytes) or after 60-min incubation at 4 degrees C (total T-lymphocytes). The B-lymphocytes counts did not differ among the groups. The proportions of total E-rosette-forming T-lymphocytes and active E-rosette-forming T-lymphocytes were increased in the sarcoid patients, whereas absolute counts of both types of E-rosette-forming T-lymphocytes were not different from control counts. Active E-rosette-forming T-lymphocytes showed an inverse correlation with serum concentration of angiotensin-1-converting enzyme, a probable indicator of the disease activity. Incubation of normal lymphocytes with sarcoid plasma increased the proportion of active E-rosette-forming T-lymphocytes. This plasma rosette enhancement was correlated with the number of active E-rosette-forming T-lymphocytes in the blood from which the plasma was separated. These results suggest that a factor in sarcoid plasma affects the number of active E-rosette-forming T-lymphocytes and that high numbers of these cells are associated with disease stability.


Impairment of isoproterenol, H2 histamine, and prostaglandin E1 response of human granulocytes after incubation in vitro with live influenza vaccines. The release of the lysosomal enzyme beta-glucuronidase from granulocytes follows incubation in vitro with complement-activated zymosan particles. Release of beta-glucuronidase is inhibited by isoproterenol, histamine, and prostaglandin E1. This in vitro model was used to study the effect of incubating a live, bivalent (A + B) influenza vaccine on the granulocyte response to the agonists described. After incubation in vitro with the live, bivalent influenza vaccine, there was a significantly impaired granulocyte reponse to all 3 agonists. The change in the response of polymorphonuclear leukocytes to isoproterenol was similar to an impairment in beta-adrenergic response found during respiratory infections in vivo. The viral-induced changes in the granulocyte response to isoproterenol may reflect similar alteration in other tissues, such as variable control of the airways and provide one explanation for the occurrence of airway dysfunction during respiratory infections.


Hormone-mediated watery diarrhea in a family with multiple endocrine neoplasms. A father and son each presented with severe watery diarrhea. The son was found to have a pancreatic islet-cell tumor associated with the pancreatic cholera syndrome, as well as a parathyroid adenoma. The father was found to have multiple islet-cell adenomas and the Zollinger-Ellison syndrome. Pancreatic tumor tissue from each patient contained detectable gastrin and vasoactive intestinal peptide; however, a much higher gastrin concentration was found in the tumor tissue from the father and a much higher vasoactive intestinal peptide content in the tumor tissue from the son. Thus, watery diarrhea may be mediated by different hormones in families having multiple endocrine neoplasia; the precise cause of the diarrheal syndrome should be defined to ensure the proper therapy.


[Nutritional and biological experiences on low-erucic acid rapeseed oil "Janpol". Studies on rats after ingestion of "Janpol" oil and other edible fats]. Comprehensive investigations were carried out for establishing the biological and nutritional value of low erucic-acid rapeseed oil from a variety of rape called Janpol selected in Poland. The pathophysiological effects of Janpol rapeseed oil were observed after giving it as the only source of fat in the diet or added in different proportions to other edible fats. In all cases the total amount of fat in the diet was 20 p. 100 kcal. The investigations were carried out on 78 young male Wistar rats aged 25 days at the beginning of the experiment. The rats were divided into 7 groups and they were given diets containing: 1) soybean oil; 2) mixed fats; 3) rapeseed oil of high erucic-acid content; 4) mixed fats containing 25 p. 100 of Janpol rapeseed oil; 5) mixed fats with 50 p. 100 of Janpol rapeseed oil; 6) mixed fats with 75 p. 100 of Janpol rapeseed oil; 7) Janpol rapeseed oil only. The experiment lasted 3 months. After its completion the rats were decapitated after 18 hours of starvation. The investigation s included : determination of weight gain, determination of the weight of selected organs (liver-lungs, heart, kidneys, testes, spleen), determination of alkaline phosphatase and pseudocholinesterase activity in the serum, determination of triglycerides and cholesterol in the serum, tests for adrenocortical function, histo-chemical investigations of the liver (alkaline and acid phosphatase, adenosine triphosphatase, fatty infiltration of the liver), macroscopic and microscopic anatomopathological examinations. The authors found the Janpol rapeseed oil caused less pronounced changes in the determined indices of the biological and nutritional evaluation as compared with high-erucic-acid rapeseed oil. Janpol repeseed oil given to experimental animals mixed with other fats in proportions of 25 p. 100 and 50 p. 100 of all fats in the diet, that is 5 p. 100 and 10 p. 100 kcal in the diet derived from Janpol oil gave in most determinations of the investigated parameters results very similar to those observed in animals receiving soybean oil. The results of these investigations show that Janpol rapesed oil can be used for nutrition of man in amounts not exceeding 10 p. 100 of the total caloric content of food.


Effect of phenytoin on bone and vitamin D metabolism. Calcium and vitamin D metabolism were evaluated in 5 adult epileptic patients before and during treatment with phenytoin. Significant decreases occurred in serum concentrations of calcium, albumin, and 25-hydroxy-cholecalciferol. The decreases in serum calcium paralleled those in serum albumin. Significant increases occurred in serum alkaline phosphatase and 1 alpha, 25-dihydroxycholecalciferol, in urinary hydroxyproline, and in the fractional gastrointestinal absorption of calcium. Urinary cyclic adenosine monophosphate and serum parathyroid hormone did not change. The results suggest that the bone disease resulting from phenytoin therapy may be associated with a deficiency of 25-hydroxycholecalciferol and not of 1 alpha, 25-dihydroxycholecalciferol, and that reduced gastrointestinal absorption of calcium or changes in parathyroid function may not be necessary for the development of bone disease.


[New amino acid from the antibiotic, ristomycin A]. A new amino acid E was isolated from a mixture of the products of the reductive hydrolysis of ristomycin A 57% HJ in the presence of red phosphorus. Its characterization was performed. The new amino acid was formed as a result of reductive dehydration of the respective beta-oxyamino acid present in the native antibiotic and being completely destroyed during general acid or alkaline hydrolysis.


Experimental viral infections of the inner ear. II. Simian virus 40 induced tumors of the temporal bone. Pathological and virological studies were performed on temporal bones of 23 hamsters which developed tumors subsequent to neonatal inoculation of simian virus 40 (SV40). Four to five months after viral inoculation, 22 hamsters developed undifferentiated sarcomas in the subcutaneous space adjacent to the temporal bone. Nine tumors invaded the temporal bone, occassionally extending to the subarachnoid space but not to the inner ear. Choroid plexus papillomas developed in four animals, with one tumor demonstrating invasion of the cochlear aqueduct, internal auditory canal, and cochlear modiolus. Cells grown from a sarcoma and a choroid plexus papilloma contained tumor antigen and established that the tumors were SV40 virus induced.


[Experimental study of the action of the RNAse from Actinomyces rimosus on the variolovaccine virus]. The effect of Actinomyces rimosus RNAase on the variolovaccine virus was studied. The inhibitory effect of the Actinomyces rimosus RNAase on the variolovaccine virus reproduction in the tissue culture cells was shown. In the experiments with the use of chick embryons and rabbits this effect was less pronounced. A 10 time increase in the infection multiplicity both in the tissue culture and the chick embryons had no noticeable effect on the level of the virus inhibition. It was supposed that the inhibitory effect of the RNAase on the variolovaccine was associated with impairment of the cell metabolism.


[Effect of dibasol and ascorbic acid on the antiviral activity of human interferon in cell culture]. The antiviral effect of human lymphocytic interferon was studied in the primary culture of human embryonic fibroblasts in the presence of dibazol and ascorbic acid. It was found that dibazol and ascorbic acid in concentrations of 5 and 10 gamma/ml respectively were capable of increasing the antiviral effect of human interferon in homological cells. The assays of 13 lots of interferon showed that its average titer in the experiments with ascorbic acid was 2.5 times higher than that in the control. The assays of 21 lots of interferon showed that its average titer in the experiments with dibazol was 3 times higher than that in the control. It is suggested that an increase in the protective properties of interferon in the presence of dibazol and ascorbic acid is connected with their capacity for stimulating the intracellular production of DNA and protein. The data obtained indicate that dibazol and ascorbic acid may be recommended in the complex of therapy and prophylaxis of antiviral infections.


[Antiviral activity of interferon and its inducers in human lymphoblastoid and somatic cells]. The antiviral effect of interferon inductors, such as poly-I--poly-C, phage f2 RNA replicative form and low molecular inductor GSN and their influence on cellular DNA synthesis were studied in the cultures of lymphoblastoid (inplanting lines Raji Namalva) and somatic human cells. The Semliki forest virus used as the test organism multiplicated well in cells Raji accumulating up to 9 lg BOU/ml. The two-strand RNA was less active in the lymphoid cells than in the somatic ones. GSN was 10 times more active and less toxic in cells Raji as compared to the fibroblasts. The lymphoblastoid interferon had higher antiviral activity as compared to the fibroblast interferon in the system of Raji--Semliki forest virus than in the system of the human embryon fibroblast--Venezuela Horse Encephalytic Virus. Romantadin actively inhibited (100 times) production of the alfavirus in both the somatic and lymphoblastoid cells.


[Determination of a dimethylaniline mixture in the intermediate products for the production of semisynthetic penicillins]. A gas-chromatographic method for determination fo the content of dimethylaniline (DMA) in 6-aminopenicillanic acid, a semi-product for production of semi-synthetic antibiotics, was elaborated. The chloroform extracts of DMA from the alkaline solutions of the preparation were analysed in a gas chromatograph with a flame-ionization detector on a stainless steel column 2 m long and the inner diameter of 2 mm filled with 10 per cent OV-17 on chromosorb W-HMDS at a temperature of 112 degrees C. The minimum detectable amount of DMA was 5.10(-9) g. The assay error was +/- 5.35 per cent. The method may be used for the assay of other semi-synthetic antibiotics.


Localization of viral DNA-replication in sections of human warts by nucleic acid hybridization with complementary RNA of human papilloma virus Type 1. Using complementary RNA of human papilloma virus type 1 (HPV1) and in situ hybridization techniques the localization of viral DNA replication was studied in sections of 38 human virus acanthomata from 31 different patients. In five cases significant labeling was detected by autoradiography. Labeling started always in the first or the second suprabasal cell layer and was strongly limited to the nuclei. A remarkable early beginning of the vacuolated process seemed to be correlated with the visible DNA replication. No labeling could be detected in the basal cell layer. This suggests that these cells contain at the most only a small number of viral genomes. Our findings represent only the situation in human warts definitely caused by HPV 1. We are not able to say, whether our negative hybridization results in the remaining warts indicate either the scarcity of viral genomes within these sections or their infection by a different agent.


[Gonadoblastoma and overgrowing dysgerminoma in Turner mosaicism [45, XO/46, Xi (Xq)] (author's transl)]. It is well established that the Y-chromosome is associated with germ cell tumor development. There is a considerable tumour risk in XY- and XY/XO-gonadal dysgenesis. In the absence of Y-chromosome germ cell tumours are extremely rare. The history of a patient with 45 XO/46 Xi (Xq)-karyotype is presented, who had a gonadoblastoma with overgrowing dysgerminoma. According to basal body temperature recordings, this patient ovulated up to the age of 22 years. After this cyclical ovarian function was exhausted; histologically no primordial follicles could be detected. Gonadotropin as well as prolactin binding sites in the tumours could not be demonstrated, suggesting hormone independency and complete malignant transformation of the tumor. In general the clinician should be aware of a possible germ cell tumour development in the absence of a Y-chromosome. However as far as the clinical management of patients with dysgenetic gonads is concerned, prophylactic gonadectomy is only indicated in the presence of a Y-chromosome.


Intranuclear inclusion bodies in epithelial cells of human vas deferens. Four different types of inclusion bodies were seen within the nucleus of the principal cells in segments of vas deferens from 40 fertile males: (1) electron-dense homogeneous bodies of various sizes (0.1--1.8 microns) and shapes, (2) granular bodies approximately 0.06--0.12 microns, (3) lipid inclusion bodies, and (4) less electron-dense, filamentous bodies. They were either membrane-bound or free within the nucleoplasm. Some membranous material was scattered within the nucleoplasm. The internal structure of nuclei containing these bodies differed from cell to cell. The nucleoplasm was composed either of dense granular elements or fine filamentous components. The aggregation and density of the ground nuclear material increased with increasing numbers of inclusion bodies. Very dense nuclei were in cells with typical features of degeneration. Frequently, extruded parts of cells or entire cells were seen within the lumen of the vas deferens.


Metastasis-induced acute pancreatitis in small cell bronchogenic carcinoma. Despite frequent metastatic involvement of the pancreas at postmortem examination in patients with small cell lung cancer, clinically observed pancreatitis due to metastatic pancreatic tumor rarely has been reported. This communication describes three cases of clinical acute pancreatitis occurring in a consecutive series of 40 patients with oat cell lung cancer. This complication may appear either as the initial manifestation of the neoplasm or during a recrudescent phase of the malignant growth. The diagnosis should be suspected in the presence of the clinical, laboratory, and radiologic features of acute pancreatitis in patients with known small cell carcinoma of the lung, especially if there is evidence of progression of the neoplastic disease elsewhere and no response to conservative medical management. Aggressive treatment with polychemotherapy can produce rapid clinical improvement and useful prolongation of survival.


[Complex bone abnormalities with fatal outcome: a new familial syndrome]. A new lethal disorder affecting two brothers is described. The features were curvature of the long bones, multiple fractures, dysmorphic facies, syndactyly and absence of ossification in the cranial vault. The condition could be inherited as an autosomal recessive or sex linked condition.


BLV-LB virus-specific sequences in cattle with spontaneous and experimentally induced leukemia. Poly(A)-containing RNAs were extracted directly from the culture fluid of BLV-producing continuous and short-term lymphocyte cultures. The abovesaid poly(A)-RNAs were used as a template to synthesize complementary [3H]DNA (BLV cDNA). BLV cDNA possessed a high degree of homology to poly(A)-RNA isolated directly from the blood plasma of a leukemic animal. In contrast of leukemic cattle BLV-related sequences in white blood cells of normal animals were absent. Leukemic cells of cattle with both, spontaneous and experimentally induced leukemia contained bovine leukemia virus sequences in their genomes.


[The effect of ethylnitrosourea in the activity of the NAD-linked glycero-3-phosphate dehydrogenase in the Vth nerve of developing rats and its relation to myelination and tumour induction (author's transl)]. The activity of NAD-linked L-alpha-glycero-3-phosphate dehydrogenase (E.C. 1.1.1.8.) in the Vth nerve from albino rats treated with a single subcutaneous injection of ethylnitrosourea in the first day of life and from controls was determined biochemically in 10 different age groups ranging from 7 to 70 days. The specific activities of the controls rise rapidly from the 7--35 days of life and remain there after almost at the same level. The activity of the ENU treated animals is significantly lower than that of the controls and reaches only at the age of 63 days the values of the 35 days old untreated animals. About 26% of the activity values of the treated animals are below the lower confidence limit of the controls.


Ultrastructural changes in rat liver cells induced by Proteus mirabilis endotoxin. Changes in individual organella of rat hepatocytes developing under the effect of Proteus mirabilis endotoxin (Pme) were observed under electron microscope. The effect of drugs which diminish liver cell injury by Pme (polymyxin, cholestyramine and hydrocortisone) on the changes was studied.


Serological identification of viral and virus-related antigens on DBA/2 mouse leukemia lymphocytes. Allogenic, semisyngeneic and syngeneic sera of animals immunized with ML-positive leukemia L1210 cells, besides anti-ML antibodies, contain antibodies which react with Gross cellular surface antigen. ML antigen and Gross cellular surface antigen were shown by the immunoferritin test in electron microscopy, and by the blocking test, to be situated on different parts of the cell surface. No budding viral particles were found on the areas occupied by these antigens. By distinguishing the ML antigen identified on the surface of leukemia L1210 cells from the Gross cellular antigen, it was shown that the MTV present in leukemias of DBA/2 mice has no leukemogenic properties. Demonstration of core and envelope antigens of the MTV and Gross MuLV, besides C particles and intracytoplasmatic A particles, which are precursors of B particles, is proof of existence of genomes of both viruses in leukemia L1210 cells. The ability of leukemia L1210 cells to absorb activity from the anti-ML sera and reaction between anti-ML sera and isolated B particles of the MTV in immunoprecipitation, indicate probable existence of an antigenic component of the MTV within the ML antigen.


The influence of immunization of Syrian hamsters with tumor cells treated with glutaraldehyde on transplantation immunity and the cytotoxic effect of lymphocytes on polyoma tumor cells. The influence of glutaraldehyde on polyoma tumor cells was studied in syngeneic Syrian hamsters, which were inoculated with glutarldhehyde-treated cells. Sera from carriers of the tumor, immunized hamsters and healthy animals inhibited in vitro cytotoxicity of sensitized lymphocytes (spleen cells) for tumor cells. If healthy animals were inoculated subcutaneously with spleen cells from immunized hamsters together with tumor cells, tumor growth was inhibited (in Winn's test) compared with animals not so inoculated and recieving only tumor cells. Cells taking part in immune responses to hamster membrane antigens were demonstrated in vitro by the microcytotoxic test. Lymphocytes from hamsters immunized with polyoma tumor cells fixed with glutaraldehyde against polyoma tumor cells were cytotoxic for target cells provided the ratio of attacking to target cells was 50:1 or higher.


Rat interferons. VI. Heterologous interferon in the treatment of viral infections. In previous studies in vitro we demonstrated the protection of mouse cells against viral infections by rat interferon. In continuation, the present paper reports results of a study designed to confirm this heterospecific activity of rat interferon in vivo. Experiments were carried out with mice of the inbred 129/AoBoy strain, inoculated intranasally or intraperitoneally with EMC-strain Col MM, VSV and influenza A 055/74 viruses, and treated with mouse or rat interferon administered by the same routes as infection. Both interferons exhibited protective action. The therapeutic effect of the mouse and rat interferons was dependent on the infecting dose of the viruses. Rat interferon proved more effective in the treatment of mice infected intraperitoneally than mice infected intranasally.


Pathogenesis of chronic myocarditis in mice infected with coxsackie B3 viruses. Infection of 300 mice of the Swiss race with Coxsackie B3 viruses gave rise to chronic myocarditis and endocarditis. The virus was cultured from the heart muscle 9 days post infection. Between days 18 and 108 post infection, virtually all mice showed evidence of an active inflammatory process in the myocardium, and in one half there was proliferation of endothelial cells, and infitration and fibrosis in the endocardium. Immunomorphologic studies demonstrated the precence of antiheart antibodies in the blood serum, and Coxsackie B3 antigen and immunoglobulin deposits in the myocardium and endocardium. Highest levels of antivirus antibodies were observed 18 days post infection.


Trials of treating myocarditis in mice infected with coxsackie B3 viruses. On the assumption of an immunologic background of chronic myocarditis after infection with Coxsackie B3 viruses, mice were treated with prednisolone or cyclophosphamide, starting 18 days after infection, for periods of three months. The favorable therapeutic effect indicates that chronic myocarditis is an immunologic disease.


[Metastasis of breast carcinoma to the pituitary. Report of a case]. The case of a 65 old woman who had a rapidly progressive neurologic symtomatology is reported. The autopsy disclosed a carcinoma of the breast with metastases in brain, cerebellum, brainstem, pituitary, thyroid gland and the adrenals. The frequency of the pituitary metastases is discussed, especially in cases of breast cancer. The macro and microscopic aspects of the metastatic pituitary gland are studied.


CSF viral antibodies. Evaluation in amyotrophic lateral sclerosis and late-onset postpoliomyelitis progressive muscular atrophy. Serum and CSF from 48 patients with amyotrophic lateral sclerosis and six patients with late-onset postpoliomyelitis progressive muscular atrophy were investigated for the presence of antibody to poliovirus types 1, 2, and 3, coxsackie viruses B3 and B4, influenza A, measles, rubella, mumps, herpes simplex types 1 and 2, cytomegalovirus, varicella-zoster, and Toxoplasma gondii. These results were compared with those from 53 control patients with neuromuscular disease matched for age, sex, race, and poliovirus vaccine exposure. There was no difference either in distribution of serum or CSF antibody titers or the geometric-mean antibody titers. There was no evidence suggesting the presence of locally produced specific viral antibody within the CNS to any of the agents studied. In particular, there was no serological evidence to suggest an association between persistent infection with any poliovirus type and amyotrophic lateral sclerosis or late-onset postpoliomyelitis progressive muscular atrophy.


Combined myelopathy and radiculoneuropathy with malignant lymphoproliferative disease. Clinicopathological findings for two similar patients in whom myelopathy and radiculoneuropathy developed in association with malignant lymphoma were reviewed. This pattern of neurological disease has not been reported in association with the malignant lymphoproliferative disorders, and it provides ground for speculation concerning lymphocyte function and viral infection in cases of neurological disease in association with remote neoplasm.


[Cyclic GMP in the eye and its correlation with externally administered insulin]. Experiments carried on Albino rabbits for detecting cyclic GMP in the eye tissues and to observe whether cGMP is influenced through local administration of insulin to the eye, externally. Whereas, for instance, intraveinous administration of insulin does not influence, in any way, concentration of cGMP -- after 3' to 10' -- in the humour aqueous, the lens, the vitreous body and the blood serum, following administration of insulin by means of retrobulbar injection, for the same time and on the same tissues, the values of cGMP appear increased. On the other hand, results yielded by our experiments allow us to estimate that environment preparing cGMP in the eye is the retina and the choroid.


Oculodentodigital dysplasia. Four new reports and a literature review. Four new patients with oculodentodigital dysplasia (ODD) have been examined. The salient and fairly constant features of ODD appear to be (1) unique facial appearance, (2) microcornea with other inconstant ocular findings, (3) syndactyly of the hands with additional characteristic phalangeal aberrations, (4) diffuse skeletal dysplasia, (5) enamel dysplasia, and (6) trichosis. Echographic studies indicate that ODD globes have microcornea with otherwise normal dimensions. An increased number of vessels crossing the optic discs was observed in three patients from one family. The distance between the inner canthi and the medial orbital walls in three patients we studied suggests that previous reports of hypertelorism may have been illusions resulting from microcornea, small palpebral fissures, and variably present epicanthus.


Melanotic neuroectodermal tumor of infancy. An ophthalmic appearance. Fullness developed in the left side of a 5-month-old male infant's face in the region of the zygoma. An incisional biopsy specimen showed the mass to be a melanotic neuroectodermal tumor, and radical excision was performed. There has been no recurrence of the tumor one year later. Tumors of this type occur in the face, particularly in the maxilla, and have only rarely been reported around the orbit.


Herpetic whitlow and heratitis.

Herpetic whitlow and heratitis. Two patients had herpes simplex infection of the fingertips (herpetic whitlow) associated with herpetic keratitis. Both patients were health-care professionals who probably acquired their disease from patients. Herpetic whitlow can be distinguished from a paronychia by the lack of a tense pulp space, formation of vesicles, and serous (rather than purulent) drainage. The fingertip inflammation resolves spontaneously, leaving normal healthy skin, in about two weeks.


Mucolipidosis IV. Histopathology of conjunctiva, cornea, and skin. The condition of a 4-year-old white girl of Ashkenazi Jewish parents was diagnosed as mucolipidosis IV on the basis of marked corneal clouding and severe psychomotor retardation, in the absence of facial-skeletal dysplasia or abnormal mucopolysacchariduria. The results of histochemical and ultrastructural studies of conjunctiva, skin, and corneal epithelium confirmed the combined storage of acid mucopolysaccharide and complex lipid substances. An unusual histopathologic feature of mucolipidosis IV is the predisposition of extreme storage involvement of corneal epithelial cells with relative sparing of the keratocytes, which is a finding of potential therapeutic implication. In addition, application of electron microscopic study of cultured amniotic cells and conjunctival biopsy specimens to assess for the parents the mother's subsequent pregnancy additional emphasizes the value of ultrastructural studies in the diagnosis of lysosomal storage disease.


Cryosurgical treatment of glomus juglare tumor. A 35 year-old Japanese female complained of a right-sided pulsation tinnitis, hearing disturbance, and facial weakness. Extensive radiographic studies including angiograms and retrograde juglar venography provided a diagnosis and localization of a tumor. Radical mastoidectomy was performed and a red grape-like glomus juglare tumor along the facial nerve was extirpated as there was a profuse hemorrhage from the tumor mass. Cryosurgery was then performed. Complete surgical removal is possible when the tumor is small, however, when the tumor involves the middle ear and mastoid area, complete extirpation cannot always be done. Radical mastoidectomy plus cryosurgery appears to be the most feasible management in the surgical treatment of glomus juglare tumor.


[A case of multiple chemodectoma: clinical and electron microscopical studies]. We observed electronmicroscopically and clinically a case of multiple chemodectoma, which has occurred from glomus caroticum sinistra and glomus jugularis sinistra of a 18 year old boy. The diagnosis was especially made by an angiography of the left common carotid artery and by a retrograde venography of the left internal jugular vein. Electronmicroscopically two kinds of tumor cells in this chemodectoma were identified. These cytoplasmas showed cored neurosecretory granules measured 125 to 250 A in diameter markedly increasing in number. Moreover in the island of the tumor cells of the carotid body tumor we happened to observe the nerve ending.


A comparative study of the age-related patterns of decay of some nucleoside monophosphate kinases in human red cells. The nucleoside monophosphate kinases, adenylate kinase (AK), guanylate kinase (GUK), and uridine monophosphate kinase (UMPK), were studied electrophoretically and quantitatively in density gradient fractions of human red cells from normal adults which contain red cells of differing mean age. The enzymes were found to differ both in their rates and patterns of decay and in secondary isozyme formation during the life of the red cell in the circulation. AK showed no appreciable enzyme decay and slight genetation of secondary isozymes; UMPK showed a rapid monophasic decline and no secondary isozyme formation; GUK showed intermediate overall loss of activity with a biphasic pattern of decay and marked secondary isozyme formation. A comparative study of the two common phenotypes of UMPK (UMPK 1 and UMPK 2-1) and of AK (AK 1 and AK 2-1) was made. The UMPK 2 isozyme showed a more rapid decay than the UMPK 1 isozyme, whereas no  difference was observed between the AK 1 and AK 2 isozymes.


The amino acid sequence of cytochrome c' from the purple sulphur bacterium Chromatium vinosum. An amino acid sequence is proposed for the cytochrome c' from the photosynthetic purple sulphur bacterium Chromatium vinosum strain D. It is single polypeptide chain of 131 residues, with haem-attachment cysteine residues at positions 121 and 124. The results discredit an earlier report [Dus, Bartsch & Kamen (1962) J. Biol. Chem 237, 3083--3093] of a di-haem peptide sequence from this protein. The sequence belongs to the same class as the published Alcaligenes and Rhodospirillum rubrum cytochrome c' squences, but the resemblance is not close. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50,093 (15 pp.) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1978) 169, 5.


A steady-state-kinetic model for formaldehyde dehydrogenase from human liver. A mechanism involving NAD+ and the hemimercaptal adduct of glutathione and formaldehyde as substrates and free glutathione as an allosteric activator of the enzyme. The steady-state kinetics of formaldehyde dehydrogenase from human liver have been explored. Non-linearities were obtained in v-versus-v[S] plots. It was necessary and sufficient to consider two reactants of the equilibrium mixture of formaldehyde, glutathione and their hemimercaptal adduct for a complete description of the kinetics. A random sequential reaction scheme is proposed in which adduct and beta-NAD+ are the substrates. In addition, glutathione can bind to an allosteric regulatory site and only the glutathione-containing enzyme is considered productive. Various alternative reaction models were examined but no simple alterative was superior to the model chosen. The discrimination was largely based on results of non-linear regression analysis. Several S-substituted glutathione derivatives were tested as activators or inhibitors of the enzyme, but all were without effect. Thio-NAD+, nicotinamide--hypoxanthine dinucleotide and 3-acetylpyridine-adenine dinucleotide could substitute for beta-NAD+ as the nucleotide substrate. alpha-NAD+ and ADP-ribose were competitive inhibitors with respect to beta-NAD+ and non-competitive with glutathione and the adduct. When used simultaneously, the inhibitors were linear competitive versus each other, indicating a single nucleotide-binding site or, if more than one, non-co-operative binding sites.


Characterization of the second prosthetic group of the flavoenzyme NADH-acceptor reductase (component C) of the methane mono-oxygenase from Methylococcus capsulatus (Bath). 1. A new two-step purification is described that routinely yields 100mg quantities of component C for biochemical studies. 2. Chemical analyses show component C purified by this procedure to contain 2 g-atoms of iron, 2 mol of acid-labile sulphide (S) and 1 mol of FAD per mol of protein. 3. The Fe-S core of component C was extruded by treating the protein with p-methoxybenzenethiol in hexamethyl phosphoramide/50mM-Tris/HCl buffer, pH 8.5 (4:1, v/v), under anaerobic conditions. The spectral properties of the extruded core suggest that component C contains 1 mol of [2Fe-2S(S-Cys)4] centre per mol of protein. 4. E.p.r. spectroscopy confirms the presence of a Fe-S centre in component C. 5. Component C catalyses the reduction by NADH of ferricyanide, 2,6-dichlorophenol-indophenol or horse heart cytochrome c, with specific activities of 50--230 units/mg of protein. 6. The optimum pH for the NADH-acceptor reductase activity is 8.5--9.0, and the apparent Km values for NADH and NADPH are 0.05mM and 15.5mM respectively. 7. Unlike methane mono-oxygenase activity, NADH-acceptor reductase activity of component C is not inhibited by 8-hydroxyquinoline or by acetylene.


Analysis of histones from the yeast Saccharomyces carlsbergensis. Basic chromosomal proteins were isolated from the chromatin of the yeast Saccharomyces carlsbergensis by extraction with H2SO4 and were purified by ion-exchange chromatography. Electrophoresis of the purified fraction on acetic acid/urea gels revealed the presence of four main components. These four proteins were identified as histones H2A, H2B, H3 and H4 on the basis of their amino acid composition, molecular weight and solubility properties, all of which are very similar to the corresponding properties of the various histone proteins from other eukaryotic organisms. A fifth basic protein could be isolated from yeast chromatin by extraction with HClO4. The available evidence indicates this protein to be an H1-type histone. Yeast thus appears to contain a complete set of histone proteins which are strongly homologous to the histones occurring in higher eukaryotes.


Nucleotide metabolism by microsomal UDP-glucuronyltransferase and nucleoside diphosphatase as determine by 31P nuclear-magnetic-resonance spectroscopy. 31P n.m.r. spectroscopy was used to study the nucleotide kinetics of UDP-glucuronyltransferase and associated reactions in the liver microsomal fraction. The effects of Mg2+ and EDTA on these reactions were investigated qualitatively. It was found that the rabbit microsomal fraction has no nucleoside pyrophosphatase activity, that UDP was immediately hydrolysed and that it was released from the microsomal surface. Reverse glucuronyltransferase could be demonstrated. The results are discussed with reference to functional coupling of UDP-glucuronyltransferase to other enzymes and the effects of Mg2+ and EDTA on the system.


Compound C2, a product of the reaction of oxygen and the mixed-valence state of cytochrome oxidase. Optical evidence for a type-I copper. Compound C2 is a product of the reaction of O2 and the mixed-valence state of cytochrome oxidase. The mixed-valence state of membrane-bound cytochrome oxidase is obtained at -24 degrees C, by using either ferricyanide or yeast peroxidase complex ES as oxidants, and the configurations of oxidized haem a and its associated copper (a3+Cua2+) and of reduced haem a3 and its associated copper (ac3+.CO.Cua3+) are obtained. The mixed-valence-state cytochrome oxidase mixed with O2 at -24 degrees C and flash-photolysed at -60 to -100 degrees C reacts with O2 and initially forms an oxy compound (A2) similar to that formed from the fully reduced state (A1). Thereafter the course of the reaction differs from that obtained in the fully reduced state, and absorbance increases are observed at 740--750 nm and 609 nm and a decrease at 444 nm, with no increase in absorbance at 655 nm. One possible attribution of the absorbance increases is to charge-transfer interaction between the iron of haem a3 and the copper associated with haem a3, Cua3(2+), having properties of a type-I 'blue' copper. A possible attribution of the decrease in absorbance at 444 nm is to liganding of a3(2+). A related explanation is that the 609 nm absorbance involves a charge-transfer interaction of both iron and copper as a mixed-valence binuclear complex, Cua3, having properties of a non-blue copper. Intermediates in addition to Compound C2 are not yet identifiable by chemical or spectroscopic tests. The kinetic and equilibrium properties of Compound C2 are described.


The mechanism of adduct formation between NAD+ and pyruvate bound to pig heart lactate dehydrogenase. 1. The rate of adduct formation between NAD+ and enol-pyruvate at the active site of lactate dehydrogenase is determined by the rate of enolization of pyruvate in solution. 2. The proportion of enol-pyruvate solutions is less than 0.01%. 3. The overall dissociation constant of adduct formation is less than 5 X 10(-8) M for pig heart lactate dehydrogenase at pH 7.0. 4. The unusual kinetics for adduct formation previously observed in the case of rabbit muscle lactate dehydrogenase [Griffin & Criddle (1970) Biochemistry 9, 1195--1205] may be attributed to the concentration of enol-pyruvate in solution being considerably less than the concentration of enzyme.


Structure and metabolism of sulphated glycosaminoglycans in cultures of human fibroblasts. Structural characteristics of co-polymeric galactosaminoglycans in sequential extracts of fibroblasts during pulse-chase experiments. 1. Human embryonic lung and skin fibroblasts were allowed to incorporate 32SO42- or 35SO42- and D-[1-3H]glucosamine. After removal of the medium the monolayer was subjected to sequential extractions by using EDTA, brief trypsin digestion, extraction with dithiothreitol ofllowed by freeze--thawing and extraction with trichloroacetic acid. The heparan sulphate and galactosaminoglycan contents of the various extracts were estimated after deaminative cleavage of the former component. Heparan sulphate was the major component of the trypsin digest, whereas galactosaminoglycans were the dominant component of other fractions. 2. Galactosaminoglycans of the various fractions were subjected to chemical (periodate oxidation/alkaline elimination) and enzymic (chondroitinase-AC and -ABC, as well as testicular hyaluronidase) degradations. Galactosaminoglycans from the insoluble cell fraction and the dithiothreitol extract contained larger amounts of L-iduronic acid than did those of other fractions. 3. Pulse-chase experiments were performed with and without replating of the cells at the start of the chase period. Radioactive glycans were isolated from the various extracts during the chase period. The half-lives of glycans of the insoluble cell fraction and the dithioreitol extract were shorter (5--8h) than were those of the trypsin digest and the EDTA extract (22h and 11h respectively). After replating of the cells in chase medium, radioactive cell-associated glycans were secreted from the cells and could be recovered in the trypsin digest, the EDTA extract and the medium. Furthermore, 35S/3H ratios of glycans from all these fractions decreased during the chase period. The following conclusions were reached. The insoluble cell fraction contains the synthesis pool and some structural material, whereas the soluble cell fraction is the storage and degradation pool. The dithiothreitol extract appears to contain the immediate precursors of secreted material. The trypsin-released glycans comprise structural components as well as material destined for pinocytosis or secretion into the medium. The EDTA extract is considered to consist of glycans en route to the medium. 4. The two presumptive precursor pools were preferentially depleted of L-iduronic acid-rich galactosaminoglycans during the chase. Glycans recovered from the trypsin digest, the EDTA extract and the medium during the chase contained larger amounts of periodate-resistant uronic acid residues (D-glucuronic acid and/or L-iduronic acid O-sulphate) than did their precursors. It is proposed that polymer-level modifications of secreted glycans are partly responsible for the results.


Turnover of prolyl hydroxylase tetramers and the monomer-size protein in chick-embryo cartilaginous bone and lung in vivo. The turnover of prolyl hydroxylase and an immunoreactive protein that corresponds in size to the smaller subunit of the enzyme was studied in vivo after injection of [(3)H]leucine into 11-day chick embryos. The specific radioactivity and total radioactivity of the monomer-size protein were much higher than those of the enzyme tetramers in the cartilaginous bone at 3h and 12h after the radioisotope injection, indicating that the monomer-size protein represents precursors rather than degradation products of the enzyme tetramers. Between 24 and 144h after the injection the specific radioactivity and total radioactivity of the two forms of the enzyme protein showed essentially identical decay rates, the observed specific radioactivity of the monomer-size protein being about 120-130% and total radioactivity about 80% of that of the enzyme tetramers. The true half-life, when corrected for dilution caused by tissue growth and re-utilization of the [(3)H]leucine, was 37.9h for the monomer-size protein and 39.0h for the tetramers. The results obtained in the lung were less reliable owing to high blank radioactivity values in the immunoprecipitation, but even so some definite differences were found between this tissue and the cartilaginous bone. The specific radioactivity of both forms of the enzyme protein at 24h was only about 20-25% of that in the cartilaginous bone. The total radioactivity of the monomer-size protein in the lung remained about 5 times that of the enzyme tetramers, whereas it was only about 0.8 times that of the tetramers in the cartilaginous bone. As in the cartilaginous bone, the decay rates of both forms of the enzyme protein were essentially identical in the lung, with a true half-life of about 46h. The results suggest that the rate of prolyl hydroxylase synthesis is slower in the lung than in the cartilaginous bone, whereas the degradation rates are fairly similar in these two tissues. The data further suggest that, in the lung at least, a large part of the monomer-size protein became degraded without being converted into enzyme tetramers.


The microbial metabolism of C1 compounds. Oxidative phosphorylation in membrane preparations of Pseudomonas AM1. A method is described for preparation of membrane vesicles (diameter 80nm) capable of respiration-linked ATP synthesis. Vesicles prepared from succinate-grown bacteria oxidized NADH, succinate and ascorbate plus NNN'N'-tetramethylphenylenediamine; vesicles prepared from methanol-grown bacteria also oxidized methanol and formaldehyde, but they were otherwise identical. The uncoupling agent carbonyl cyanide chlorophenylhydrazone and the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide both inhibited ATP synthesis, whereas they had no effect on the rate of respiration. Rotenone inhibited ATP synthesis and respiration with NADH as substrate; antimycin A inhibited with succinate as substrate, and cyanide inhibited with all substrates. P/O ratios were usually 0.7-1.3 with NADH, 0.6-1.0 with succinate and 0.2-0.6 with reduced NNN'N'-tetramethylphenylenediamine or methanol as respiratory substrate. When 2,6-dichlorophenol-indophenol was used as an alternative electron acceptor to O(2) (NADH as donor) the P/2e ratio was 1.65. Although these P/O ratios are minimum values, because they do not take into account unknown amounts of uncoupled O(2) consumption, they are consistent with previous proposals [O'Keeffe & Anthony (1978) Biochem, J.170, 561-567] based on measurements of proton translocation in whole cells. The results also confirm that methanol dehydrogenase and cytochromes c and a/a(3) are arranged so that the first step in methanol oxidation is coupled to synthesis of ATP.


The permeability of rat liver lysosomes to sugars. Evidence for carrier-mediated facilitated diffusion. 1. By the osmotic-protection method, the penetration of sugars through the rat liver lysosomal membranes was studied with a view of determining whether sugar uptake was by facilitated diffusion. 2. The following criteria for this type of transport were established: sugar specificity, the order of uptake being 2-deoxy-D-glucose less than D-glucose less than D-mannose less than D-galactose less than D-ribose less than 2-deoxy-D-ribose; stereospecificity, the uptake of L-glucose and L-ribose being 50% slower than their D-stereoisomers; inhibition by 1 MM-phlorrhizin and 1 M-cytochalastin B; competition between sugars for uptake, and a Q10 (rate difference over a 10 degrees C temperature range) for uptake of approx. 2.8. 3. It is proposed that sugar uptake into lysosomes from rat liver is by facilitated diffusion.


Interaction between insulin-storage granules and F-actin in vitro. Possible interactions between polymerized (F-) actin and insulin-storage granules from rat islets of Langerhans were examined in vitro by comparing the sedimentation of the granules in the presence of various actin concentrations. Actin in the concentration range 0.1--0.5 mg/ml produced a retardation in granule-sedimentation rates consistent with binding of the granules to the actin filaments. The interaction was increased by addition of ATP (2mM), but was decreased by CaCl2 (0.1 mM). Binding of granules to actin was unaffected by cyclic AMP or by preincubation of the granules with phospholipase C. Specificity of the interaction was confirmed by the use of depolymerized (G-) actin and of myosin to provide a solution of comparable viscosity; neither of these caused any alteration of granule sedimentation. Possible implications of this interaction of insulin-storage granules with actin for the mechanism of insulin secretion are briefly discussed.


Stimulation of glucose transport in rat adipocytes by insulin, adenosine, nicotinic acid and hydrogen peroxide. Role of adenosine 3':5'-cyclic monophosphate. Glucose transport into adipocytes of the rat was measured by monitoring the conversion of [1-(14)C]glucose into (14)CO(2). Glucose transport was made rate-limiting by increasing the flux through the pentose phosphate pathway with phenazine methosulphate, an agent that rapidly reoxidizes NADPH. Under these conditions, the observed rate of glucose disappearance from the incubation medium was about 20% higher than the rate of conversion of the C-1 of glucose into (14)CO(2). Apparent rates of glucose transport were significantly increased by insulin, H(2)O(2), adenosine and nicotinic acid. Stimulation of the apparent rate of glucose transport by insulin was dependent on adipocyte concentration, the hormone being most effective at relatively high cell concentrations. Adenosine and nicotinic acid further enhanced the maximum stimulation of glucose transport by insulin. Potentiation of insulin action by adenosine was more pronounced at lower cell concentrations. At relatively high cell concentrations the stimulatory action of insulin was markedly decreased by adenosine deaminase. Stimulation of apparent rates of glucose transport by the compounds noted above were antagonized by agents that increased intracellular cyclic AMP concentrations (theophylline and isoprenaline) and by dibutyryl cyclic AMP. Intracellular concentrations of cyclic AMP were significantly lowered when adipocytes were incubated with insulin, H(2)O(2), adenosine or nicotinic acid. These effects were observed under basal conditions or when intracellular cyclic AMP concentrations were elevated by theophylline or isoprenaline. On the basis of the above data, we suggest that insulin, H(2)O(2), adenosine and nicotinic acid may all stimulate glucose transport in rat adipocytes by lowering the intracellular cyclic AMP concentration. These data therefore support the hypothesis that cyclic AMP inhibits glucose transport in rat adipocytes.


The effects of lipid phase transitions on the interaction of mitochondrial NADH--ubiquinone oxidoreductase with ubiquinol--cytochrome c oxidoreductase. 1. The endogenous phosphatidylcholine and phosphatidylethanolamine of Complexes I and III from bovine heart mitochondria may be completely replaced with 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine with at least partial retention of activity. 2. The lipid-replaced enzymes associate in 1:1 molar ratio to give a Complex I--III unit catalysing NADH-cytochrome c oxidoreductase activity. 3. On increasing the concentration of ubiquinone-10 and the synthetic phospholipid, the lipid-replaced Complexes appear to operate independently of each other as in the natural membrane. Thus the lipid-replaced enzymes associate in exactly the same ways as the enzymes containing natural phospholipids. 4. Arrhenius plots of NADH--cytochrome c oxidoreductase activity reconstituted from lipid-replaced Complexes I and III exhibit changes in slope at 24 degrees C. When the concentrations of phospholipid and ubiquinone-10 are increased, the Arrhenius plots show discontinuities at 24 degrees C as well as changes in slope. 5. The kinetics of cytochrome b reduction by NADH were measured in mixtures containing 2 mol of Complex III/mol of Complex I. When the enzymes contained natural phospholipids. the reduction kinetics were biphasic. When the enzymes had been supplemented with further phospholipid and ubiquinone-10 the kinetics were monophasic. When lipid-replaced enzymes were supplemented with 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine and ubiquinone-10, reduction of cytochrome b was monophasic above the phase-transition temperature of the lipid but biphasic below it. 6. These findings are interpreted in terms of the model for the interaction of Complexes in the natural membrane proposed by Heron, Ragan & Trum-power [(1978) Biochem. J. 174, 791--800].


Induction of N-acetylglucosamine-catabolic pathway in spheroplasts of Candida albicans. Synthesis of N-acetylglucosamine-catabolic enzymes, namely permease (high-affinity uptake system), kinase and deaminase was studied in the spheroplasts of the yeast Candida albicans. The presence of N-acetylglucosamine as inducer is essential for the induced synthesis of these enzymes in the spheroplasts, which were active for at least 8--9 h. However, some of the newly synthesized kinase and deaminase leaked out from the spheroplasts into the medium during induction. Experiments with inhibitors of RNA and protein synthesis indicate that the appearance of new enzyme activities is dependent on concomitant new protein synthesis and the inducer operates at a transcriptional level. However, inhibitors of DNA synthesis, e.g. mitomycin-C and hydroxyurea, had no effect on the synthesis of these enzymes.


The metabolic conversion of very-low-density lipoprotein into low-density lipoprotein by the extrahepatic tissues of the rat. 1. The work reported was designed to provide quantitative information about the capacity of the extrahepatic tissues of the rat to degrade injected VLD lipoproteins (very-low-density lipoproteins, d less than 1.006) to LD lipoproteins (low-density lipoproteins, d 1.006--1.063) and to study the fate of the different VLD-lipoprotein apoproteins during the degradative process. 2. Rat liver VLD lipoproteins, radioactively labelled in their protein moieties, were produced by the perfusion of the organ and were either injected into the circulation of the supradiaphragmatic rats or incubated in rat plasma at 37 degrees C. At a time (75 min) when approx. 90% of the triacylglycerol of the VLD lipoproteins had been hydrolysed the supradiaphragmatic rats were bled and VLD lipoproteins, LD lipoproteins and HD lipoproteins (high-density lipoproteins, d 1.063--1.21) were separated from their plasma and from the plasma incubated in vitro. The apoproteins of each of the lipoprotein classes were resolved by gel-filtration chromatography into three main fractions, designated peaks I, II and III. 3. Incubation of the liver VLD lipoproteins in plasma in vitro led to the transfer of about 30% of the total protein radioactivity to the HD lipoproteins. The transfer mainly involved the peak-II (arginine-rich and/or apo A-I) and peak-III (apo C) proteins. There was also a small transfer of radioactivity (about 5% of the total) to the LD lipoproteins. 4. Injection of the liver VLD lipoproteins into the circulation of the supradiaphragmatic rat resulted in the transfer of about 15% of the total VLD-lipoprotein radioactivity to the LD lipoproteins. The transfer involved mainly the peak-I (apo B) proteins and accounted for about 20% of the total apo B protein radioactivity of the injected VLD lipoproteins. When the endogenous plasma VLD lipoprotein was taken into account the transfer of apo B protein was about 35%. 5. The transfer of peak-II protein radioactivity from the VLD to the HD lipoproteins was greater in the plasma of the supradiaphragmatic rat than in the incubated plasma suggesting that there was a net transfer of peak-II apoproteins during the VLD lipoprotein degradation. The transfer of peak-III protein radioactivity was not greater in the plasma of the supradiaphragmatic rat, but there was a loss of this radioactivity from the circulation.


Localization of oxidized nocotinamide--adenine dinucleotide glycohydrolase in the mouse liver nuclear envelope. NAD+ glycohydrolase activity located in the nuclear envelope was maximally solubilized by treatment with 0.1--0.2% Triton X-100. The residual activity largely represents the chromatin-associated NAD+ glycohydrolase. Under these conditions the phospholipids were extensively solubilized (over 90%) while leaving the nuclei physically stable, although the nuclear membranes were removed, as shown by electron microscopy. After Triton X-100 treatment, deoxyribonuclease I did not significantly affect the residual NAD+ glycohydrolase activity, although the DNA was completely broken down. This enzyme activity can be released from the nuclear pellet by incubation with phospholipase C. For comparative studies, the glucose 6-phosphatase activity, known to be present in the nuclear envelope, was investigated. Treatment with 0.01% Triton X-100 released 10--20% of the phospholipids, but without solubilizing either glucose 6-phosphatase or NAD+ glycohydrolase. Higher Triton X-100 concentrations (0.1--1.0%) inhibited glucose 6-phosphatase, but not NAD+ glycohydrolase activity. NAD+ glycohydrolase is apparently present in a latent form in the nuclear envelope. Glucose 6-phosphatase, However, shows no such latency.


Fatty acid stimulation of membrane phosphatidylinositol hydrolysis by brain phosphatidylinositol phosphodiesterase. The hydrolysis of membrane-bound phosphatidylinositol in rat liver microsomal fraction by the soluble phosphatidylinositol phosphodiesterase from rat brain was markedly stimulated by oleic acid or arachidonic acid. The stimulation did not require added calcium, although it was abolished by EDTA. Lysophosphatidylcholine also totally suppressed the stimulation. A possible role for the fatty acid content of a membrane in controlling phosphatidylinositol turnover is suggested.


Central nervous system lupus erythematosus: measurement of cerebrospinal fluid cyclic GMP and other clinical markers of disease activity. Cyclic-GMP (C-GMP), a normal constituent of the central nervous system, was found to be present in increased amounts in the cerebrospinal fluid (CSF) of systemic lupus erythematosus (SLE) patients with active neurologic disease. Twenty-four CSF samples from 17 patients with SLE were evaluated for C-GMP concentration by radioimmunoassay. This study extends our initial observations and examines three groups of SLE patients based on their clinical status at the time of each lumbar puncture: those with active neurologic and psychologic abnormalities (group I), active neurologic abnormalities (group II), and psychologic abnormalities without active neurologic involvement (group III). Groups I and II had mean CSF C-GMP values of 3.1 nM +/- 0.64 (SE) and 4.1 nM +/- 0.10 respectively, which were both significantly higher than the mean for group III (1.2 nM +/- 0.43) (P less than 0.05). Other CSF findings did not display this close correlation with activity of neurologic disease. In 4 SLE patients, significantly higher levels of CSF C-GMP were found on serial sampling during times when neurologic abnormalities were active. Thus elevated CSF C-GMP concentration may be a marker of active neurologic disease in SLE.


[The influence of clanobutin on bile secretion in the isolated perfused rat liver (author's transl)]. Isolated perfused rat liver was used to characterize the choleretic action of 4-[4-chloro-N-(4-methoxyphenyl)-benzamido]-butyric acid (clanobutin). The dose-dependent increase of the bile flow was associated with an increase of the Na+ secretion. In in vitro studies the drug inhibited the Na+-K+-ATPase of the sinusoidal membrane more than that of the canalicular membrane in a dose-dependent manner. The following hypothesis was proposed to explain the choleretic action of clanobutin. The intracellular Na+ concentration increased due to the inhibition of the sinusoidal Na+-K+-ATPase. This is then compensated by transporting the extra Na+ into the bile. However, it cannot be ruled out that Na+ is transported as counterion during hepatobiliary transport of clanobutin or else its metabolites. The effect of the drug seems to be non-specific since other enzymes of the plasma membrane were also inhibited.


The action of silybin on the mouse liver in alpha-amanitine poisoning. Histochemical and histoenzymological studies were carried out on liver slices from mice which had received alpha-amanitine two days previously, some of which had been treated with silybin, and from control mice. The toxin produced certain changes in the activity of the enzymes involved in different metabolic processes, and in the amounts of lipids and nucleic acids. Treatment with silybin, given 60 min before administering alpha-amanitine or 10 min later alike, prevents the appearance of these changes and gives results comparable to those in the control animals.


A new and rapid system for the determination of enzymatic activity of bacterial collagenases. Collagenase activity is assayed using N-CBZ-Gly-Pro-Gly-Pro-Ala as substrate in an autoanalyzer II system. The liberation of the tripeptide is monitored after the reaction with trinitrobenzenesulfonic acid. The new system shows a considerably improved precision when compared to existing methods and allows about ten times as many samples to be analyzed per day. This is of importance for laboratories where large amounts of activity determinations have to be performed.


Different appearance of hepatic collagenase and lysosomal enzymes in recovery of experimental hepatic fibrosis. 1. Both activities of hepatic collagenase and lysosomal enzymes (acid phosphatase, beta-glucuronidase and N-acetyl-beta-D-glucosaminidase) have been observed in the recovery from experimental hepatic fibrosis in rats treated with carbon tetrachloride for 6 to 20 weeks, and compared with the disappearance of newly formed collagen fibers in the recovery process. 2. In the process of experimental hepatic fibrosis, collagenase activity reached maximum on sethe accumulation of collagen fibers in reversible hepatic fibrosis, but decreased to the same level as that of non-treated rat liver in cirrhotic stage. In the reocvery from reversible hepatic fibrosis, collagenase activity reached maximum on second day after the discontinuation of carbon tetrachloride, and decreased to the same extent of that of non-treated rat liver on seventh day. 3. Lysosomal enzyme activity was parallel to the activity of hepatic collagenase and to the accumulation of collagen fibers in the process of hepatic fibrosis. In the recovery stage, lysosomal enzyme activity in mesenchymal cells within the septa increased markedly on second day after the discontinuation of toxic agent but turned to the same level of that of non-treated rat liver seven days later, which was consistent with the appearance and disappearance of collagenase activity. On the other hand the appearance of lysosomal enzymes activities in Kupffer cells and hepatocytes was different from that of collagenase activity. That is lysosomal enzyme activity in Kupffer cells decreased in early days but increased five days later, and the enzyme activity in hepatocytes markedly decreased but gradually recovered to normal level seven days later. 4. The appearance of collagenase was observed at the beginning of the recovery stage. It indicates that mammalian collagenase initiates the collagen degradation and lysosomal enzymes might have a role in the subsequent degradation of collagen.


Effect of starvation, alloxan diabetes and adrenalectomy on Na+ K+-ATPase of the mucosa of the small intestine of rat. The dietary stress conditions such as starvation influenced Na+K+-ATPase activity which increased steadily above normal fed levels between the starvation periods of 24--48 hr. Also, an increased enzyme level was observed in alloxan diabetic rats and administration of insulin to diabetic rats led to a tendency towards a lowering of Na+K+-ATPase. Adrenalectomy brought about a lowering of Na+K+-ATPase activity from those of normals while the administration of hydrocortisone induced an enhancement. The results indicate that both starvation and diabetic conditions might cause a stress-like activation of adrenal cortex resulting in increased levels of glucocorticoids which in turn activate the intestinal Na+K+-ATPase activity.


The mechanism of photodynamic inactivation of human cells in vitro in the presence of haematoporphyrin. The photosensitizing effect of haematoporphyrin (HP) on human cells of the established line NHIK 3025 has been studied. Fluorescence measurements show that HP is bound to these cells. Serum proteins also bind HP, and the presence of 10% human serum during incubation with HP (3 X 10(-4)M) reduces the cellular uptake of HP by 75% or more. The photosensitized inactivation is enhanced when the cells are suspended in D2O-buffer during irradiation. This indicates that singlet oxygen is involved in the inactivation. Two findings indicate that the photoinduced damage is repairable: firstly, the fraction of cells surviving a given light dose decreases with decreasing irradiation temperature, and secondly, the survival curves have a shoulder at low exposures of light.


Retrovirus-like particles in EBV-negative Burkitt's lymphoma cell line but not in EBV-DNA-positive lines from patients with ataxia telangiectasia and Down's syndrome. Retrovirus-like particles can be recovered by arginine deprivation from the BJAB-1 Epstein-Barr virus (EBV) negative cell line derived from an African patient with typical Burkitt's lymphoma. These particles resemble retroviruses in their morphology and in their physicochemical properties. Particles with a similar morphology were obtained from derivative cell lines established by infecting BJAB-1 cells with EBV. On the other hand, retrovirus-like particles could not be induced in EBV-DNA-positive lymphoblastoid cell lines derived from non-leukaemic patients with ataxia telangiectasia and Down's syndrome and from a patient with infectious mononucleosis.


Antibody-induced changes in levels of cyclic adenosine monophosphate in leukaemic lymphocytes. When L2C leukaemic B lymphocytes from guinea-pigs were incubated in vitro with antibody directed to their surface immunoglobulin (Ig), a rapid rise in intracellular adenosine 3':5'-phosphate (cyclic adenosine monophosphate, cAMP) was observed. Estimation of cAMP was by a protein-binding assay using bovine adrenal protein kinase. Increases up to 30-fold occurred within 30 seconds of incubation at 37 degrees C, to be succeeded by a fall which reached the basal level between 5 and 7 min. The response was proportional to the amount of antibody present. Cross-linking of surface Ig by the antibody was necessary, bivalent (Fab'gamma)2 from the antibody gave a rise in cAMP similar to that given by the parent molecule, whereas monomeric Fab'gamma was ineffective unless it was subsequently cross-linked by anti-antibody. The rise was too rapid to have required capping of the surface Ig for its induction. Not all perturbations of the plasma membrane by antibody induce such a surge in cAMP, since anti-beta2 microglobulin, also reacting with the lymphocyte surface, failed to alter cAMP concentration. The results emphasize that immunotherapy can be influenced by antibody altering the metabolic activity of target cells, quite apart from activation of immunological cytotoxic pathways.


Effect of mouse hepatitis virus infection on iron retention in the mouse liver. Increased iron uptake and iron-induced ferritin synthesis has been demonstrated in experimentally virus-infected cell cultures. However, this has not been observed in the intact animal. The results reported in this paper indicate that higher deposition of injected radioiron occurs in the livers of mice infected with MHV-3 virus compared with livers from uninfected animals. Administration of iron at different time intervals indicated that iron uptake correlates well with the degree of tissue injury in the livers of infected animals.


Cellular heterogeneity in an ethylnitrosourea-induced glioma: malignancy, karyology and other properties of tumour cell types. Two types of tumour cell have been obtained from a glioma transplacentally induced by ethylnitrosourea in a BD-IX rat. These were distinguished in culture by their different morphologies, responses to dibutyryl cyclic adenosine monophosphate, inducibility of glycerol phosphate dehydrogenase and growth in soft agar. They had different karyotypes, with distinctive numbers and arrangements of chromosomes. One cell type had an apparently normal diploid set of 42 whilst the other had 43 chromosomes. An additional chromosome No 4 was identified in the latter by Giemsa banding. Translocations and other abnormalities involving this chromosome were consistently observed. Both cell types produced malignant, astrocytic tumours when injected into newborn syngeneic rats, but with different latent periods and morphological features.


Mechanism of Lactobacillus leichmannii ribonucleotide reductase studied with Coalpha-[alpha-(Aden-9-yl)]-Cobeta-adenosylcobamide (Pseudocoenzyme B12) as coenzyme. Coalpha-[alpha-(Aden-9-yl)]-Cobeta-adenosylcobamide (pseudocoenzyme B12) purified from Clostridium tetanomorphum has been reacted with ribonucleotide reductase purified from Lactobacillus leichmannii under various conditions, and the properties of the products obtained have been compared by electron paramagnetic resonance (EPR) with those previously reported for products formed from the normal coenzyme (adenosylcobalamin). The rapidly formed intermediate and the slowly formed "doublet" species from the pseudocoenzyme have EPR spectra identical with those formed from the normal coenzyme. This and other considerations make it less likely that the unusual magnetic properties of the rapidly formed intermediate are due to strongly distorted octahedral symmetry about Co(II) as previously postulated. Instead it is probable that the EPR spectrum is due to interaction of the radical pair by both exchange coupling and magnetic dipole--dipole coupling. Although Coalpha-[alpha-(aden-9-YL)]cob(II)amide in solution does not show superhyperfine splitting in the EPR spectrum because of its base-off configuration, the cob(II)amide formed by degradation of the pseudocoenzyme within the catalytic site of the enzyme did show triplets due to a nitrogen axially coordinated to cobalt. This suggests that binding of the cob(II)amide to the reductase catalytic site causes a shift to the base-on form.


Membrane glycopeptides from virus-transformed hamster fibroblasts and the normal counterpart. Comparisons of membrane glycopeptides from baby hamster kidney fibroblasts (BHK21/C13) and a clone transformed by Rous sarcoma virus (C13/B4) were made by using cells metabolically labeled with radioactive D-glucose and L-fucose. Most of the glycopeptides were metabolically labeled with both the general and the specific glycoprotein precursors. The glycopeptides obtained from the cell surface by controlled trypsinization were representative of the surface membrane as shown by comparing them with those of purified membrane preparations. The trypsin-removable glycopeptides from both cell types were further processed and examined by successive chromatography on Sephadex G-50 and DEAE-cellulose. The chromatographic distribution patterns showed that each cell type had glycopeptides of similar characteristics, although the proportions of the glycopeptides differed dramatically between the two cell types. After transformation there was an increase in the larger, more highly charged glycopeptides. This was verified by the increased sialic acid content in these glycopeptides. Some of the glycopeptides were homogeneous after the size and charge separations, since a variety of procedures did not separate them further. The apparent homogeneity and reasonably few species obtained may be due to the methods of isolation, with the procedures selecting particular glycopeptides from the external portion of the membrane. These results corroborate the concept and show for the first time that virus transformation is accompanied by an increase in certain species of glycopeptides rather than de novo synthesis.


Partial structure of a membrane glycopeptide from virus-transformed hamster cells. The predominant surface glycopeptide from a clone of baby hamster kidney cells transformed by Rous sarcoma virus (C13/B4), metabolically labeled with L-[14C]fucose, has been characterized for the first time. This glycopeptide represents 19% of the total radioactivity removed by trypsin from the cell surface of the transformed fibroblasts and is more abundant in the transformed cells than in the normal counterpart. Purification of the glycopeptide after digestion with Pronase was by successive chromatography on DEAE-cellulose and Sephadex G-50. The monosaccharide content of the glycopeptide was 42, 127, 138, 114, and 243 nmol of fucose, sialic acid, galatose, mannose, and glucosamine, respectively. A partial structure of the glycopeptide was proposed from the results of sequential enzymatic degradation coupled with gas-liquid chromatographic analysis of the resultant monosaccharides. All of the enzymes used were purified and pretested on natural substrates and found to remove terminal monosaccharides of the correct configuration, quantitatively. The purification and properties of an alpha-L-fucosidase from rat testes were described. All of the radioactivity in the glycopeptide, recovered as fucose, was present at the core and was removed by treatment with this alpha-L-fucosidase. The proposed structure is a triantennary, completely sialylated, complex glycopeptide containing a core region of beta-D-mannose, beta-D-N-acetylglucosamine, and alpha-L-fucose.


Ubiquinone in Rhodopseudomonas sphaeroides. Some thermodynamic properties. In Rhodopseudomonas sphaeroides chromatophores there are 25 +/- 3 ubiquinone (Q) molecules/reaction center protein. They comprise several thermodynamically and functionally different ubiquinone complements. There are approx. 19 ubiquinones (Em7 = 90 mV) in the main ubiquinone complement which, within experimental resolution, appears thermodynamically homogenous and follows the redox reaction Q + 2e + 2H+ in equilibrium with QH2 from pH 5--9. A method which takes advantage of the 2H+ bound/molecule of Q reduced is described for measuring the time course of light-activated reaction center-driven reduction and oxidation of the 19 Q complement. No stable semiquinones were detected in the constitutents of the 19 Q complement. There are approx. 6 ubiquinones of lower Em which are currently unaccounted for, although one or possibly two of these can be assigned to the quinones of the reaction center protein. The remainder may be associated with the NADH-ubiquinone oxidoreductase.


Studies on the molecular organization of cytochromes P-450 and b5 in the microsomal membrane. 1. The relative orientations of the heme groups of cytochromes P-450 and b5 in the microsomal membrane have been studied by the technique of electron paramagnetic resonance. The results show that the heme plane of cytochrome P-450 lies in the same plane as the membrane surface, whereas the cytochrome b5 heme plane has a random orientation. 2. No significant broadening or change in relaxation properties of the gz component of low spin cytochrome P-450 occurred when cytochrome b5 was reduced by redox poising. It is concluded that there is little or no paramagnetic coupling between the heme groups of the two species. 3. The results favor a model in which no tight complex between cytochromes P-450 and b5 is present, the species being independent and interacting only by random molecular collisions or via other intermediate species.


The multiplicity and stoichiometry of the prosthetic groups in QH2: cytochrome c oxidoreductase as studied by EPR. 1. The EPR signal in the g = 2 region of the reduced QH2: cytochrome c oxidoreductase as present in submitochondrial particles and the isolated enzyme is an overlap of two signals in a 1 : 1 weighted ratio. Both signals are due to [2Fe-2S]+1 centers. 2. From the signal intensity it is computed that the concentration of each Fe-S center is half that of cytochrome c1. 3. The line shape of one of the Fe-S centers, defined as center 1, is reversibly dependent on the redox state of the b-c1 complex. The change of the line shape cannot be correlated with changes of the redox state of any of the cytochromes in QH2: cytochrome c oxidoreductase. 4. Lie the optical spectrum, the EPR spectrum of the cytochromes is composed of the absorption of at least three different b cytochromes and cytochrome c1. 5. The molar ratio of the prosthetic groups was found to be c1 : b-562 : b-566 : b-558 : center 1 : center 2 = 2 : 2 : 1 : 1 : 1 : 1. The consequences of this stoichiometry are discussed in relation to the basic enzymic unit of QH2 : cytochrome c oxidoreductase.


Intensity of highly anisotropic low-spin heme EPR signals. A semi-empirical formula has been derived to calculate the concentration of low-spin heme compounds that are highly anisotropic, i.e. 3 less than gz less than 4, and where information only on the gz absorption is available.


Studies on antiviral glycosides. II. Mode of action for virucidal effects on Sendai virus. Treatment of Sendai virus with p-(sec-butyl)-phenyl-6-chloro-6-deoxy-beta-D-glucopyranoside, followed by freezing and thawing resulted in a loss of hemolytic and cell fusion activities as well as infectivity without affecting hemagglutinating and neuraminidase activities. The anti-hemolytic activity of this compound was reversed by the addition of phosphatidyl choline to the virus samples. p-Azidophenyl-6-chloro-6-deoxy-beta-D[3H]glucopyranoside was successfully used for photoaffinity labeling of a specific virion site, and we confirmed the affected site of the glucoside to be the lipid components in the viral envelopes.


Effects of microtubule-disruptive and membrane-stabilizing agents on low density lipoprotein metabolism by cultured human fibroblasts. The cellular mechanisms involved in the uptake and metabolism of low density lipoprotein (LDL) by cultured normal human fibroblasts have been investigated with the aid of drugs known to disrupt cytoplasmic microtubules or to inhibit membrane fusion. Two drugs which disrupt microtubules by differing mechanisms, colchicine and vinblastine, each reduced the high affinity surface binding of 125I-labelled LDL by fibroblasts. Associated reductions of the endocytosis and degradation of the lipoprotein could be attributed almost entirely to this effect. In contrast, lumicolchicine, an analogue of colchicine without microtubule-disruptive activity, had little or no effect on 125I-labelled LDL metabolism. Each of two groups of membrane-stabilizing agents, the phenothiazines and the tertiary amine local anaesthetics, directly inhibited both the internalization of 125I-labelled LDL following high affinity binding to cell surface receptors and the catabolism of the lipoprotein subsequent to endocytosis, supporting previous morphological evidence for the importance of membrane fusion in these processes.


Isolation of luminal and antiluminal membranes from dog kidney cortex. Luminal (brush border) and antiluminal (basal-lateral) membranes were isolated from canine renal cortex. The enzyme marker for luminal membrane, alkaline phosphatase was enhanced 19-fold and the antiluminal enzyme marker, (Na+ + K+)-ATPase, was enhanced 22-fold in their respective membrane preparation, while the amount of cross contamination was minimal. Contamination of these preparations by enzyme markers for lysosomes, endoplasmic reticulum and mitochondria was also low. Routinely, more than 50 mg membrane protein was isolated for each membrane. Electron micrographs showed that the membranes were uniform in size, appearance, and vesicular in nature. An examination of the orientation of these membranes showed that 76.5% of the antiluminal membranes and 86% of the luminal membranes were right-side out.


Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells. A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 +/- 1.14 . 10(5) and (2.2 +/- 1.2) . 10(5) spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethylmaleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.


Monitoring the location profile of fluorophores in phosphatidylcholine bilayers by the use or paramagnetic quenching. Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short range interaction we developed a spectroscopic method to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase. This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.


Mass isolation of cell surface membrane fragments from pigeon heart. Cell surface membrane fragments were isolated and purified by successive rate zonal and isopycnic centrifugation of calcium oxalate-loaded pigeon heart microsomes in sucrose density gradients. The most highly purified cell membrane fraction sediments at a buoyant density of 1.105 g/ml. Some of the membrane pieces are present as open fragments and leaky vesicles, while others form tightly sealed vesicles of both inside-in and inside-out membrane orientation. The pigeon heart cell membrane preparation exhibits high (Na+ + K+ + Mg2+)-ATPase and adenylate cyclase activities. Additional activity of these enzymes is uncovered by sodium dodecyl sulfate and alamethicin, respectively. Electron microscopic inspection of the cell surface membrane preparation revealed (a) a predominance of thick-walled vesicles with smooth surfaces on negative staining and (b) binding of concanavalin A to the bulk of isolated membrane pieces following their incubation with the lectin.


Protein kinases of Dictyostelium discoideum, strain AX-2. In cell homogenates of Dictyostelium discoideum, strain AX-2, four major soluble protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) and one membrane-associated protein kinase activity were identified. The enzymes showed high affinity for casein. One of the enzymes was purified by affinity chromatography on casein-coated Sepharose. The soluble high molecular weight enzymes phosphorylated histones, whereas the low molecular weight enzymes did not. The same protein kinase species were present in vegetative and aggregation-competent cells. Their specific activity, however, changed during the development to aggregation competence. None of the enzymes was stimulated by cyclic AMP or cyclic GMP, regardless of their origin from vegetative or aggregation-competent cells.


Cyclic AMP-dependent and -independent protein kinases of the water mold, Blastocladiella emersonii. Protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) and cyclic adenosine 3',5'-monophosphate binding activities have been identified in zoospore extracts of the water mold Blastocladiella emersonii. More than 75% of these activities is found in the soluble fraction. Soluble protein kinase activity is resolved in three peaks(I, II and III) by DEAE-cellulose chromatography. Peak I is casein dependent and insensitive to cyclic AMP. Peak II is histone dependent and cyclic AMP independent; this enzyme is inhibited by the heat-stable inhibitor from bovine muscle. Peak III utilizes histone as substrate and is activated by cyclic AMP.


Mode of action of bacterial collagenase on a synthetic substrate, (Pro-Pro-Gly)5. Clostridial collagenase (EC 3.4.24.3) catalyzes the hydrolysis of (Pro-Pro-Gly)5 at minimum of three different rates, producing Pro-Pro, Gly-Pro-Pro and Gly-Pro-Pro-Gly, and various intermediate peptides. The intermediate and final products were separated by cation-exchange column chromatogrphy and identified, and their rates of formation were measured. Pro-Pro was released most rapidly with formation of the tridecapeptide. After the initial release of the N-terminal Pro-Pro, hexa- and heptapeptides were formed in larger amounts than tri-, tetra-, nona- and decapeptides from the tridecapeptide. The rates of disappearance of the intermediates decreased in the order trideca- greater than deca- and nona- greater than heptapeptide. The results indicate that the enzyme hydrolyzes inner linkages of the tridecapeptide having N- and C-terminal Gly residues, forming large peptides, preferentially to outer linkages, forming the tri- and tetrapeptides.


Purification and properties of the membrane-bound hydrogenase from Proteus mirabilis. The cytoplasmic membrane-bound hydrogenase of the facultative anaerobe, Proteus mirabilis, has been solubilized and purified to homogeneity. The purified enzyme exhibited a maximal specific activity of about 780 mumol H2 oxidized/min per mg protein (benzyl viologen reduction). The hydrogenase has a molecular weight of 205 000 and is composed of two subunits with a molecular weight of 63 000 and two of 33 000. The absorption spectrum of the enzyme was characteristic of non-heme iron proteins. The millimolar extinction coefficients at 400 and 280 nm are 106 and 390, respectively. The hydrogenase has about 24 iron atoms and 24 acid-labile sulfide atoms/molecule. Amino acid analyses revealed the presence of 39 half-cystine residues/molecule and a preponderance of acidic amino acids. The hydrogenase in its oxidized form exhibits an EPR signal of the HiPIP-type with g values at 2.025 and 2.018. Upon reduction with either dithionite or H2 the signal disappears; no other signals were detectable.


The mechanism of electron transfer in laccase-catalysed reactions. 1. The reaction of the electron acceptors in Rhus vernicifera laccase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) have been studied with stopped-flow and rapid-freeze EPR techniques. The studies have been directed mainly towards elucidation of the role of the type 2Cu2+ as a possible pH-sensitve regulator of electron transfer. 2. Anaerobic reduction experiments with Rhus laccase indicate that the type 1 and 2 sites contribute one electron each to the reduction of the two-electron-accepting type 3 site. There is also evidence that the reduction of the type 1 Cu2+ triggers the reduction of the type 2 Cu2+. 3. Only at pH values at which the reduction of the two-electron acceptor is limited by a slow intramolecular reaction can an OH- be displaced from the type 2 Cu2+ by the inhibitor F-. 4. A model describing the role of the electron-accepting sites in catalysis is formulated.


The binding of spin-labeled derivatives of NAD+ and its structural components to pig skeletal muscle lactate dehydrogenase. The binding of spin-labeled derivatives of NAD+ and its structural components to pig skeletal muscle lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) is described. In contrast to results previously obtained with the heart muscle isozyme (Wenzel, H.R., Pfleiderer, G., Trommer, W.E., Paschenda, K. and Redhardt, A. (1976) Biochim. Biophys. Acta 452,292--301), no significant increase is observed in the binding constant of N6-SL-ADP as compared N6-SL-AMP. This different behavior can be explained by the substitution of glutamine-31 for alanine in the muscle isozyme, which has been proposed to account for the the tighter binding of NADH to the heart type. In both isozymes the binding of the binding of the spin-labeled coenzyme itself is weaker than found for its structural components.


Isolation and characterization of yeast nicotinamide adenine dinucleotide kinase. NAD+ kinase (ATP:NAD+ 2'-phosphotransferase, EC 2.7.1.23) from yeast has been purified utilizing ion-exchange and NAD+-agarose affinity chromatography to give a 2100-fold purification. The apparent homogeneity of the enzyme preparation was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analytical ultracentrifugation. The enzyme has a subunit molecular weight of 31,000, and a native molecular weight of 124,000, and is, thus, probably a tetramer. The single form of the enzyme has an apparent isoelectric point of 5.85. Initial velocity studies in the forward direction with both substrates gave intersecting Lineweaver-Burk plots, and this suggests a sequential mechanism in which both substrates are bound before products are released. Replots of these data were linear and gave Km values for NAD+ and ATP of 0.68 mM and 2.3 mM, respectively.


Facilitation of ouabain binding to (Na+ + K+)-ATPase by vanadate at in vivo concentrations. In the presence of Mg2+ vanadate was shown to facilitate ouabain binding to (Na+ + K+)-ATPase in much the same way as Pi does. Thus the hypothesis that vanadate interacts with the phosphate site of the enzyme seems to be supported by ouabain binding experiments. At given ouabain concentrations maximum binding is achieved at microM concentrations of vanadate whereas mM concentrations of Pi are needed. Na+ as well as K+ counteract ouabain binding but some cardiac glycoside binding is still possible at in vivo concentrations of these cations. A minor contamination of the enzyme preparations with vanadate could explain the in vitro binding of ouabain that can be obtained with Mg2+ and in the absence of Pi.


Validation of a procedure for exogenous isotopic labeling of lipoprotein triglyceride with radioactive triolein. A procedure has been developed for the exogenous isotopic labeling of triglyceride-rich lipoproteins (chylomicrons and very low density lipoproteins) using high specific activity radioactive triglyceride in the presence of aqueous dimethyl sulfoxide. The labeled product lipoproteins showed unchanged chemical and physical properties. When the particles had also been labeled biologically by incorporation of unesterified fatty acids into the triglycerides of lipoproteins secreted by liver or intestine, both endogenous and exogenous labels were removed at the same rates in the isolated perfused heart and liver or in intact or functionally hepatectomized rats. These experiments additonally indicated that the triglyceride fatty acid composition of chylomicrons and very low density lipoproteins was unchanged during triglyceride depletion in the peripheral tissues. Using such labeled lipoproteins it has been shown that uptake of remnant lipoprotein cholesteryl ester and triglyceride by the liver is simultaneous. The labeling procedure described should prove suitable for kinetic studies of the disposition of the various lipoprotein non-polar ('core') lipids.


Structure of membrane lipids and physico-biochemical properties of the plasma membrane from Thermoplasma acidophilum, adapted to growth at 37 degrees C. Thermoplasma acidophilum, a mycoplasma-like organism, grows optimally at 56 degrees C and pH2. The low temperature extreme of growth is 37 degrees C. The plasma membrane of cells grown at 37 degrees C was isolated and characterized physicobiochemically. Membrane lipids which comprise 25% of the membrane dry weight consist mainly of two repetitively methyl-branched C40 side chains that were ether-linked to two glycerol molecules. The lipid structures were elucidated by combined gas chromatography-mass spectroscopy, direct probe mass spectroscopy and 13C NMR. 37 degrees C-grown cells contained lipids with 42% more pentane cyclization than the 56 degrees C-grown cells. In 37 degrees C-grown cells, phospholipid and serine content decreased by about 10% each, carbohydrate content increased by 5%. EPR studies demonstrated an increase in membrane lipid fluidity of 37 degrees C-grown cells with an upper transition temperature at 35 degrees C which was shifted down by 10 degrees C compared with cells grown at 56 degrees C. Membrane-bound ATPase activities also indicated similar changes upon adaptation. There is a close correlation between membrane fluidity and physiological functioning of this membrane-bound enzyme.


Transformation of 4-androsten-3,17-dione by growing cultures and cell extracts of Clostridium paraputrificum. Growing cultures of Clostridium paraputrificum transformed 4-androsten-3,17-dione to 3 alpha-hydroxy-5 beta-androstan-17-one in a sequential manner with 5 beta-androstan-3,17-dione as an intermediate. The addition of 1.5 mM menadione to log-phase cultures which had formed 5 beta-androstan-3,17-dione resulted in a partial reoxidation of this steroid to 4-androsten-3,17-dione. However, this treatment also resulted in transient inhibition of culture growth. Resumption of growth was accompanied by complete reduction of 4-androsten-3,17-dione to 5 beta-androstan-3,17-dione. Cell extracts of C. paraputrificum were capable of carrying out these reductive transformations in the absence of added cofactors. However, Sephadex G-25 treated extracts required NADH or NADPH for these reactions. A flavin nucleotide, either FAD (plus NADH or NADPH) or FMN (plus NADH) was highly stimulatory for 4-androsten-3,17-dione reduction to 5 beta-androstan-3,17-dione. NADH was the preferred reduced pyridine nucleotide for reduction of the C4-C5 double bond, while time-course measurements suggested that NADPH was the preferred donor for reduction of the 3-keto group.