Wound infection during the Yom Kippur war: observations concerning antibiotic prophylaxis and therapy. Eighty-eight episodes of wound associated infection were identified among 624 consecutively admitted battlefield casualties. Ninety-one per cent of infections occurred during the administration of antibiotic therapy or prophylaxis and 65% were associated with the use of multiple antibacterial agents. Gram negative bacillary and mixed microbial infection predominated and were found to increase in relative incidence after the second day of hospitalization. Appropriate therapy, based on disc sensitivity testing, was administered in only 33% of infectious episodes. The practice of antibiotic wound prophylaxis may contribute to the incidence and nature of infection in battlefield wounds. Problems unique to the handling of battlefield wounded are discussed in comparing the present data with those of other war associated and civilian studies.


Flavoxate, a potent phosphodiesterase inhibitor. The c-AMP phosphodiesterase inhibiting properties of flavoxate and of its main metabolite i.e. 3-methylflavone-8-carboxylic acid (MFCA), were assayed in vitro and compared to those of theophylline. Flavoxate and MFCA are competitive phosphodiesterase inhibitors, and are 21 and respectively 5 times more potent than theophylline. The smooth muscle relaxing activity of flavoxate possibly relies on this enzymatic mechanism.


High synovial fluid white blood cell counts in pseudogout; Possible confusion with septic arthritis. During a 12-month period, we have treated five patients with acute inflammatory arthritis and synovial fluid leukocyte counts of 65,000 to 100,00/cu mm with 93% to 100% polymorphonuclear cells, calcium pyrophosphate dihydrate crystals in the synovial fluid, and negative cultures. Four of these five were origingally treated for septic joints. We would like to emphasize this relatively uncommon, but important manifestation of pseudogout.


Changes in phosphatase activity associated with cell wall defects in Chlamydomonas reinhardi. Experiments were performed to isolate mutants lacking alkaline phosphatase in Chlamydomonas reinhardi. Mutants with null enzyme activity were obtained. A cytological study of these mutants however revealed cell wall defects, suggesting that the loss of phosphatase activity in these strains is not due to the inactivation of the corresponding phosphatase structural gene but rather to the leakage of this enzyme as a consequence of the cell wall abnormality. Incidentally, this finding provides the basis of a convenient method for selecting easily cell wall mutants of Chlamydomonas.


On the structure of the peptidoglycan of cell walls from Myxobacter AL-1 (myxobacterales). Basically the peptidoglycan of Myxobater AL-1 consists of alternating beta-1,4-linked N-acetylglucosamic-N-acetylmuramic acid chains. After splitting the aminosugar backbone with a specific algal enzyme three subunits arise: a monomer, a dimer and a timer. Investigation of the monomer with specific enzymes and comparison of the degradation products to standards derived from other bacterial peptidoglycans suggest the following structure of the monomer peptide: L-alanyl-D-glutamic-L-meso-diaminopimelic-D-alanine. A D-alanyl-D-meso-diaminopimelic acid bond is the bridgebond between the peptides of the subunits.


The effect of soluble antigens from polyoma tumor and liver cells on stimulation of lymph nodes measured by incorporation of 14C-leucine. The effect of solube antigens from polyoma tumor cells, liver antigens and PHA on lymph node cells from tumor bearers and on normal lymph node cells in CBA mice was studied. The stimulation index was calculated on the basis of incorporation of 14C-leucine in short-term cultures. The stimulation index was lower in lymphocytes derived from tumor bearers than those from normal donors. The blastoid responses to polyoma-associated antigens and PHA were similar, in contrast to liver-associated antigens. It was concluded that stimulation of liver antigens may be connected with different chemical structure, mainly of carbohydrate components.


Training examiners to administer a quantitative neurological examination for a multicenter clinical trial. A battery of quantitative neurological tests was used in a multicenter clinical trial to compare ACTH with placebo for the treatment of patients with multiple sclerosis in acute exacerbation. Since the battery of tests was administered in ten centers by physical therapists, it was critical that the examiners were well trained and remained so. Results of experiments showed that physical therapists could be successfully trained to administer the tests in a uniform fashion and remained trained for three years, the duration of the clinical trial. The fact that physical therapists are not taught tell-tale side effects of a treatment, and hence do not perceive these as do the physician examiners, introduces another level of objectivity into neurological clinical trials.


Rhinovirus type 14 RNA polymerase complexes. Rhinovirus type 14 RNA-dependent RNA polymerase complexes were isolated from microsomal and soluble fraction of infected KB cells. Maximum activities were measured at at 6 and 7 hours post inoculation (p.i.) for microsomal and soluble polymerases, respectively. Both polymerase activities are considerably reduced by 8 to 9 hours, p.i., and interval in which the in vivo rate of synthesis of viral RNA is maximal. In vitro RNA products of RNA polymerases in both fractions consist of ribonuclease-sensitive and ribonuclease-resistant RNA of heterogeneous sizes. Detergent treatment of the microsomal RNA polymerase(s) did not affect the total amount of RNA synthesized, the proportion of ribonuclease-sensitive RNA synthesized or the size of the RNA products. The data suggest that RV14RNA polymerase complexes are intially associated with membranes but are then irreversibly released into the soluble phase of the cytoplasm; possible explanations for this phenomena are discussed.


Evidence of chronic persistent infections with polyomaviruses (BK type) in renal transplant recipients. Ten renal transplant recipients showing a significant increase in human polyomavirus antibodies, indicative of an acute infection, were followed up serologically over periods ranging from two months to more than two years. Fifty-four serum specimens were available for the study and they were tested by both haemagglutination-inhibition and complement-fixation. Polyomavirus antigens were prepared from the BK and SV40-like strains of polyomaviruses, and from the SV40 virus. One strain of polyomavirus, related to the BK strain was isolated from the urine of one of these patients. Two other BK strains were recovered from the urine and kidney, respectively, of transplant recipients not included in this study. Sera of these two patients were not obtained until the transplantation was made; they were already highly positive for polyomavirus antibodies, precluding the demonstration of an increase in antibody titer. Serologic results have shown that HAI antibodies persist at high titers throughout the observation period. This persistence ranged from two to four months (four cases), seven to eleven months (three cases) and thirteen to twenty months (three cases). In none of the cases could a decrease of high titer be demonstrated. Moreover, density gradient studies have shown that specific IgM antibodies also tend to persist over many months. Similar serologic results were obtained in complement-fixation tests with a BK antigen. Titers were at least 1 in 30 in the study group, but were not observed among healthy blood donors. All sera were uniformly negative for SV40 and SV40-like antigens. One polyomavirus isolation was successful from urine obtained six months after initial serologic evidence for a polyomavirus infection. The other two viruses were isolated from materials taken four and seven months after first detection of polyomavirus antibodies at high titer. Both serologic evidence and viral isolations seem to indicate that polyomaviruses (BK type) might cause a chronic infection in humans.


Binary ethylenimine as an inactivant for foot-and-mouth disease virus and its application for vaccine production. Foot-and-mouth disease virus was inactivated with binary ethylenimine formed apart from or directly in the virus suspension by the cyclization of 2-bromoethylamine hydrobromide or 2-chloroethylamine hydrochloride under alkaline conditions. The inactivation rates with binary ethylenimine prepared apart from the virus suspension in dilute sodium hydroxide with either 2-bromoethylamine hydrobromide or 2-chlorethylamine hydrochloride were higher than with pure ethylenimine. When binary ethylenime was prepared directly in the virus suspension only 2-bromoethylamine hydrobromide gave acceptable inactivation rates. The reduced inactivation rates for binary ethylenimine directly prepared in the virus suspension are due to the different cyclization rates of 2-bromoethylamine hydrobromide and 2-chloroethylamine hydrochloride and to the interference of bicarbonate in the cyclization reaction. The complement fixing antigen of foot-and-mouth disease virus was not affected by binary ethylenimine inactivation. Vaccines prepared with foot-and-mouth disease virus inactivated by binary ethylenimine were comparable in their immunogenicity to vaccines prepared with ethylenimine or N-acetylethylenimine used as inactivants. Application of binary ethylenimine in the preparation of foot-and-mouth disease vaccines considerably reduces the potential danger associated with handling pure ethylenimine and other aziridines.


The enhancing effect of copper, nickel, and cobalt ions on plaque formation by Semliki Forest virus (SFV) in chicken embryo fibroblasts. In cultured chicken embryo fibroblasts treated with copper-(copper cation 10-minus 3.5-10-minus 5 M), nickel and cobalt salts an enhancement of the plating efficiency of Semliki Forest virus could be observed. This augmented plaque formation was most probably due to a higher adsorption rate of virions to the cell surface under the influence of the transition metal ions. The plating efficiency of West Nile virus in chicken-embryo fibroblasts and, to a lesser degree, of poliovius type 1 and 2 in KB-cells was also enhanced by copper sulfate.


Virulence of temperature-sensitive mutants of foot-and-mouth disease virus. A number of temperature-sensitive mutants isolated from two strains of foot-and-mouth disease virus were examined for their virulence in suckling mice. The majority of the mutants were found to be less virulent than the parent virus strains, ranging from slight to total attenuation, but two mutants retained parental levels of virulence. There was no correlation between mutant cut-off temperatures and virulence, or the revertant content of mutant preparations and virulence. It was not always possible to regain parental levels of virulence by isolating phenotypic revertants or wild-type recombinants from genetic crosses, but recombinants were significantly more virulent than either of the two input viruses in a genetic cross.


A bovine adenovirus type 3: isolation, characterization, and experimental infection in calves. A virus was isolated from a group of feedlot cattle with acute respiratory disease that was characterized by physicochemical methods as an adenovirus and identified serologically as a bovine adenovirus type 3. Intratracheal inoculation of three 4 month old colostrum deprived calves resulted in pyrexia, hyperpnea, dyspnea, anorexia and in one animal a mild diarrhea.


Experimental studies concerning the possibility of a latent carrier state in bovine herpes mammillitis (BHM). Adult bovines, young calves and young rats were infected with bovine herpes mammillitis (BHM) virus providing a group of BHM-recovered animals in which to investigate a possible latent carrier state. The progression of the acute infection and serological response was essentially similar to that previously reported. The effect of natural (calving, littering) and artificial (corticosteroid treatment) stress on these animals following recovery from infection was investigated, but reactivation of BHM was never detected. Long term observation of cocultures of BHM virus infected-and-recovered bovine teat dermis with bovine embryo cells failed to reveal any evidence for the induction of BHM virus replication even in those cocultures treated with mitomycin C or bromodeoxyuridine. Similarly, cocultures utilizing lumbar dorsal root ganglia from the same animals were also negative with respect to the induction of virus replication. Experiments using cytosine arabinoside indicated that an artificial in vitro latent carrier state of BHM virus could be maintained for 6 days with subsequent sporadic virus reactivation, but an attempt to maintain such a state for 12 days was unsuccessful, with no subsequent virus reactivation.


Human and primate poxviruses: I. Growth characteristics of cytolytic and tumor variants. The dual potential of poxviruses to be cytolytic and tumorgenic has been extended. Yaba monkey tumor virus formed foci on monkey and human embryonic cells. Cytolytic or plaque-forming virus was isolated from Yaba tumor homogenates by selective sucrose centrifugation and passage through monkey or human embryonic cells. Monkey pox virus (MPV) was cytolytic for monkey cells or human embryonic cells, but upon passage onto young monolayers of human carcinoma cells, HeLa, produced a restricted cytopathic effect on first transfer, foci on second transfer and cytolysis after the fourth transfer. If HeLa monolayers were compact, the rapid growth precluded overt expression of cytolytic MPV. Electron micrographs of cytolytic Yaba indicated that Yaba development was similar to vaccinia and MPV but not totally organized. Growth curves of vaccinia, MPV and cytolytic Yaba were essentially identical in monkey and human embryonic cell lines.


Exacerbation and reactivation of Herpesvirus hominis infection in mice by cyclophosphamide. Cyclophosphamide has been shown to potentiate the growth of Herpesvirus hominis in the skin of Webster Schneider mice. Higher skin yields of virus were obtained, marked dissemination of virus to the brain was produced and mortality in mice was increased. Serum neutralizing antibody titres were suppressed. The system may serve as a good model for the study of specific antiherpes drugs.


Studies of Marek's disease herpesvirus and turkey herpesvirus specific common antigen which stimulates the production of neutralizing antibodies. An agar-gel precipitation antigen prepared from the skin (feather-Ag) of chicken infected with JM strain of Marek's disease herpesvirus (MDHV) and cell extracts of cultures infected with either Type 2 plaque producing agent (PPA) of MDHV or turkey herpesvirus (HVT) (Type 2-Ag, HVT-Ag) had 1 precipitation line in common. Hyperimmune sera to the common antigen (common-Ag) neutralized to a greater extent by homologous antiserum. Absorption of HVT antiserum or hyperimmune serum to Type 2 PPA with common-Ag reduced their neutralizing activity. This reduction was almost complete with homologous antigen but less complete with heterologous antigen. The location of antigen in Type 2 PPA and HVT infected cells was determined using hyperimmune serum to the common-Ag in fluorescent antibody tests. Antigen mainly occurred in the cytoplasm of cells corresponding to the rounded refractile cells in the plaques, whereas unfixed cells showed antigen on the cytoplasmic membrane. The common-Ag associated with MDHV or HVT infections might be a virus structural component which is associated with the envelope.


Sporadic bovine meningo-encephalitis-isolation of a paramyxovirus. Isolation of a viral agent (107) directly from brain explants of a 15-month-old heifer with symptoms of a sporadic encephalomyelitis is described. The virus shares properties with the paramyxovirus family. It grows in a variety of cell cultures from different species, and induces nuclear and cytoplasmic inclusion bodies in infected cells. Nucleocapsids measuring 17 nm in diameter were found in the nucleus and cytoplasm of these cells when studied electron microscopically, thus indicating a close relationship of the agent to the measles-distemper-rinderpest group. No infectious virus was released from infected cells, although alignment of nucleocapsids was observed beneath the cell membrane, and no hemagglutinating activity could be detected with the methods employed. The 107 agent was compared serologically with parainfluenza viruses type 1, 2 and 3, simian virus 5, mumps and Newcastle disease virus (NDV), two bovine respiratory syncytial viruses and measles/subacute sclerosing panencephalitis, distemper and rinderpest viruses, always using 107 virus infected CV1 cells and antiserum of the different viruses in indirect FA tests. Positive FA reactions were observed only with two sera obtained from SSPE patients with high antibody titer to SSPE virus, and with one rabbit-anti-rinderpest serum. The titers of these sera to 107 virus, however, were significantly lower than those against homologous viruses. Five out of 9 sera from randomly selected healthy cattle showed antibody titers between 1:10 and 1:80 to 107 virus in FA tests. The significance of these results is discussed with respect to the epidemiology of SSPE in children and its possible implication with rinderpest in Europe.


The effect of arginine deprivation on the cytopathogenic effect and replication of human cytomegalovirus. Arginine is necessary for the development of the cytopathogenic effect of human cytomegalovirus in human embryonic fibroblasts. It is also required, though in greater concentrations, for the production of infective virions, the requirement being at an early stage of replication. Inhibitor studies suggested that this block in replication caused by arginine deficiency was prior to the formation of viral DNA. Withdrawal of arginine from the medium 24 or 48 hours after infection resulted in a decline in virus production indicating that the continued presence of the amino acid is necessary for constant virus production. Infected cultures deprived of arginine could be stimulated to produce cytopathic effects and infective virions by replacement of the amino acid even eight days after inoculation, demonstrating that the information for cytomegalovirus replication remains intact within the cell. This establishment of latency in vitro may be related to the ability of the virus to establish a similar state in vivo.


Early cytoplasmic vacuolization of African green monkey kidney cells by SV40. As early as 3--4 hours after infection with SV40 at a high input multiplicity, African green monkey (Cercopithecus aethiops) kidney (AGMK) cells developed cytoplasmic vacuolization. At 10--20 hours after infection, the vacuolization reached its maximal level, then disappeared and SV40 specific cytopathic change followed. This vacuolization developed before the synthesis of the specific T and V antigens. This early cytoplasmic vacuolization (ECV) was prevented by preincubating the virus with specific antiserum, or by heating the virus with MgCl2. The ECV could be induced by UV-irradiated SV40. Addition of metabolic inhibitors had no effect on the induction of the ECV. These results suggest that the capacity to induce the ECV resides in a structural component(s) of SV40 virion and the vacuolization is not associated with the replication of SV40.


Identification of strains of herpes simplex virus by comparison of the density of their DNA using the preparative ultracentrifuge. The buoyant densities of the DNA of herpes simplex virus type 1, type 2 and Pseudorabies virus, as determined in the analytical ultracentrifuge, are 1.725, 1.727 and 1.731 correlating with G+C contents of 67, 69 and 73 per cent respectively. The density differences for the DNA's of type 1 and type 2 herpes simplex viruses have been confirmed in experiments with isotopically labelled DNA from four type 1 and six type 2 strains by preparative CsCl gradient ultracentrifugation. The DNA of all the type 2 strains was denser than that of any of the type 1 strains examined. Despite these differences in DNA base composition of type 1 and type 2 strains, nearest neighbour analysis of their DNA's disclosed no obvious differences in doublet pattern or general design.


[Microadenomas and hypersecreting pituitary adenomas]. The early radiographic changes of the sella turcica in the cases of hypersecreting pituitary adenomas (Cushing disease, acromegaly, amenorrhea-galactorrhea) are reported and discussed. The finding of the double contour of the sellar floor and the small crescentic bulging of its antero-inferior wall, with the aid of the tomographic cuts of the sella, has permited the diagnosis of the microadenoma'a localization. With this it si possible the achievement of a selective excision of the lesion in the initial stage, when the tumor has not caused yet irreversible damage to the inside normal pituitary tissue. This surgery is performed in the precocious stage with the primary goal to preserve the normal gland, without hurt it to avoid postoperative hipopituitarism. A anatomo-radiologic classification of the alterations sella turcica in 4 grades is made, which is of great utility for the surgical prognostic, since we have observed clinical and biological cure over 90% in the grade 1, 75% in the grade 2, 50% in the grade 3 and up to 10% in the grade 4 in the first surgery.


[The cell-independent influence on calcification in living bone and in vitro (author's transl)]. While there is no doubt that collagen is an important extra-cellular factor in the calcification of bone tissues, the exact nature of the process remains unclear. It has been explained in turn by the nucleation effect of corresponding lattice distances of apatite and collagen, that is, an oriented crystal overgrowth of the two substances which decreases the energy of nucleus formation, or by conformity between fibril-bundles and calciumphosphate crystal, or by the binding of phosphate to anionic positions of the collagen, as well as by the bone-forming effect of a non-collagen protein which is, however, separable from collagen. Because of their high viscosity proteo-polysaccharides inhibit crystal formation. Alkaline soluble proteo-pholysaccharides, however, appear to promote calcification in vitro and also in tissue of living bone. Lathyrogene and penicillamine impair the cross-linking in the bone collagen; this probably explains their disturbing effect on ossification. Diphosphonates would, like anorganic pyrophosphate, inhibit both the formation and dissolution of apatite crystal. Our research shows, however, that methanie-bis-phosphonate clearly promotes calcification, in vitro and in vivo. In vitro, and probably also in vivo, magnesium inhibits the formation of apatite crystal nucleus. Our research indicates moreover that magnesium also slows down the reduction of calcified bone tissue on account of its inhibiting effect on parathormone emission.


Effects of chlorpromazine and diazepam on neuronal activities of the lateral vestibular nucleus in cats. The effects of chlorpromazine and diazepam on vestibular neuronal activity were studied in gallamine immobilized cats. Chlorpromazine depressed all the P, N1 and N2-waves of field potentials in the lateral vestibular nucleus evoked by electrical stimulation of the vestibular nerve. Diazepam also reduced the amplitude of postsynaptic components (N1 and N2-waves), while the presynaptic one (P-wave) was slightly increased. Chlorpromazine suppressed the spontaneous as well as the increased vestibular neuronal firing following stimulation of the vestibular nerve. Diazepam enhanced the increased vestibular neuronal firing excited by the nerve stimulation and had no effect on the spontaneous unitary discharges.


Tuberous xanthoma in homozygous type II hyperlipoproteinemia. A histologic, histochemical, and electron microscopical study. Histologic, histochemical, and ultrastructural studies of a tuberous xanthoma from a patient with homozygous type II hyperlipoproteinemia showed that all of the lipid was within histiocytic foam cells; no lipid was identified in interstitial regions or in blood vessels. Primitive mesenchymal cells, elongated perivascular and fibroblast-like cells, and lysosome-filled macrophages also were present within the xanthoma, indicating possible stages in the evolution of dermal mesenchymal cells into mature, cholesterol-rich foam cells. Morphologically, the lipid was in four different forms: large droplets, which were the dominant form, and membrane-bound crystals, concentric lamellar bodies, and ceroid. The paucity of membrane-bound lipid forms, relative to the abundant free lipid droplets, indicated that lysosomal digestion was a minor metabolic pathway for the intracellular metabolism of lipid in the xanthoma. Thus, nonlysosomal lipid storage in foam cells is a characteristic tissue response to the underlying metabolic defect in type II hyperlipoproteinemia.


Human model for studying seed-soil factors in blood-borne metastasis. The relationship between tumor "seed" and tissue "soil" in blood-borne metastasis is poorly understood. Therefore, an explanatory human model is proposed. It is based on the hypothesis that the spread of lung cancer cells to the adrenal vein provides in man not only a variable attribute, lung cancer seed, but also a constant attribute, adrenal venous soil, as in a scientific experiment. Out of 100 cases of lung cancer, four histologic types proved their invasive potentiality in 45 subjects by colonizing the adrenal parenchyma proper and yet differed appreciably in their ability to grow in the adrenal venous blood. Hence, in all probability, the systematic study of venous blood will help in the discovery of the potentialities and limitations of human blood in tumor metastasis.


Fine structure and origin of amyloid deposits in pituitary adenoma. A pituitary adenoma with amyloid deposits was studied by light and electron microscopy. The amyloid material was radially oriented, deposited in the extracellular space, and often intermingled with small vesicles. It was surrounded by histiocytes with deep cytoplasmic invaginations. The histiocytes contained large autophagic vacuoles in which amyloid was also present. These features are similar to other forms of amyloidosis and suggest a histiocytic origin of the amyloid.


Spironolactone bodies in an adrenal adenoma. Spironolactone bodies were observed in an adrenal cortical adenoma that was removed from a patient with primary hyperaldosteronism (Conn syndrome) treated preoperatively with spironolactone. The electron microscopical evaluation of this adrenal cortical adenoma shows origin of spironolactone bodies from whorls of endoplasmic reticulum in cells with the cytoplasmic features of those from the zona glomerulosa of the adrenal gland. There was no evidence that the bodies were derived from mitochondria, which confirms recent ultrastructural findings in patients treated with spironolactone. These bodies have been described in the adrenal cortex only in patients who have received spironolactone, and the pharmacologic specificity of the bodies strongly suggests a direct mode of action by spironolactone on aldosterone production by cells of the adrenal zona glomerulosa.


EMC virus and cultured human fetal pancreatic cells. Ultrastructural observations. Tissue cultures of pancreatic acinar cells of seven human fetuses were infected with either the M or the E variant of the encephalomyocarditis (EMC) virus. Between the third and fifth days after infection, severe ultrastructural damage was noted. Margination, condensation, and fragmentation of the nuclear chromatin and the appearance of numerous cytoplasmic vesicles filled with finely granular material were the major changes observed. Viral particles were not identified. One hundred plaque-forming units (PFU) were sufficient to induce these alterations with the M variant while 10,000 PFU were needed to obtain the same results with the EMC-E virus. The described lesions are qualitatively similar to those induced in vivo in experimental CD-1 mice infected with the virus.


Thyroid peroxidase deficiency in Batten-Spielmeyer-Vogt disease. Thyroid tissue from two patients with Batten-Spielmeyer-Vogt disease (BSV) was studied for peroxidase enzyme activity and for morphological abnormalities by light and electron microscopy. Diagnosis was confirmed by the demonstration of intracytoplasmic material identical to that described in other reported cases of BSV. There was a substantial decrease in peroxidase activity in the thyroid tissue from both patients. An abnormally low level of peroxidase activity had previously been demonstrated in the white blood cells of these patients. Thyroid biopsy offers obvious advantages over brain biopsy, provides adequate tissue for enzyme analysis, and allows the demonstration of the intracytoplasmic structures characteristic of BSV.


Characteristics of the sleep of men in simulated space flights. A study of the sleep of men in simulated space flight has shown that, during different effects (diminished motor activity, noise, rotation), monotony and time of the exposure is the general pathogenetic factor producing sleep disturbances. The monotony factor was responsible for typical changes in sleep function (difficulty involved in falling asleep, disturbed continuity of sleep as a process, decline in the depth, etc.), whose degree was related not only to the professional importance of the experiment but also to the individual intolerance to the main exposure. In these cases, the action of somnogenic mechanisms developed against the background of the action of analyzers (vestibular, accoustic, proprioceptive) that were under load and whose excitation during hypokinesia, noise, and rotation, especially in poorly adapted people, led to cumulation and was expressed in the phenomenon of spontaneous awakening. In addition to that, the genesis of dyssomnia, particularly during bedrest and water immersion, included hemodynamic disturbances induced by blood redistribution due to the recumbent position and monotonous motor activity. The psychosomatic feelings (congestion, edema, irresistible desire to stand up and stretch) brought about emotional strain which was later enhanced by the duration of the exposure.


Development of a live attenuated varicella vaccine. The Oka strain of varicella virus, isolated in our labolatory, was serially cultivated in guinea-pig embryo cultures (GPEC), and a considerable amount of cell-free virus was obtained from infected cell. GPEC passage virus at the 6th passage level was used in a small scale field trial. Susceptible children of 1 to 10 years old were injected subcutaneously with 100 to 1,000 PFU of virus. No clinical reactions due to the vaccination were observed in any children, and a high rate of antibody response was obtained with viral doses of more than 200 PFU. Attenuated virus obtained by passage in GPEC was propagated in human diploid (WI-38) cells, and it was also effective in inducing an immune response without clinical reactions. The results show that the Oka strain of varicella virus passaged in GPEC and human diploid (WI-38) cells may be used safely and effectively as a live attenuated vaccine.


Application of a live attenuated varicella vaccine to hospitalized children and its protective effect on spread of varicella infection. Twenty-three hospitalized children with no history of varicella or no detectable complement fixing (CF) antibody, were vaccinated with a live attenuated varicella vaccine (Oka strain) immediately after the occurrence of a case of varicella in a children's ward of hospital. These children suffered from the nephrotic syndrome, nephritis, purulent meningitis, hepatitis etc., and 12 of them were receiving steroid therapy. An antibody response was noticed in all the vaccinated children, with mild fever in 6 and a mild rash in 2 of 6. It was uncertain whether these reactions were due to vaccinatin or to naturally acquired infection modified by vaccination. No other clinical reactions or abnormalities of the blood or urine were detected. Thus the spread of varicella infection was prevented, with the exception of one severe case in an unvaccinated patient. In another trial, 16 children with renal diseases were also vaccinated. All the children showed an immune response with no clinical reactions and no abnormalities in blood and urine examinations. Thus live varicella vaccine (Oka strain) can be used safely and effectively for hospitalized children, and its effectiveness in preventing spread of varicella infection was confirmed.


Multiple cyclic nucleotide phosphodiesterase activities from rat tissues and occurrence of a calcium-plus-magnesium-ion-dependent phosphodiesterase and its protein activator. 1. Supernatant fluids from rat cerebral cortex, cerebellum, kidney, heart and liver contained more phosphodiesterase activity hydrolysing cyclic GMP than that hydrolysing cyclic AMP when assayed with sub-saturating concentrations of substrate. 2. These activities were resolved into several fractions by Sephadex G-200 gel filtration; no two tissues had similar activity profiles. 3. With every tissue examined, a fraction (fraction II) with a molecular weight of about 150,000 was obtained which hydrolysed cyclic GMP preferentially at sub-saturating substrate concentrations in the presence of micromolar concentration of Ca2+, millimolar concentration of Mg2+ and a protein activator. 4. The activity of fraction II accounted for about 60 percent in liver, more than 80 percent in heart and cerebellum, and almost 100 percent in cerebral cortex of the total activity for cyclic GMP hydrolysis, calculated from the activity profiles. 5. Km values of fraction II samples from kidney, heart and liver for cyclic GMP were 1.3, 1.7 and 5 muM respectively. 6. 3-Isobutyl-1-methylxanthine inhibited hydrolysis of cyclic GMP by fraction II with an I50 value of 3muM for heart and liver and 50 muM for cerebrum. 7. The activator protein, with an estimated molecular weight of about 30,000 was isolated from all the tissues listed in 1.8. The concentrations of activator protein and of the isolated enzyme, fraction II, did not correspond exactly.


Inhibition by ricin of protein synthesis in vitro. Inhibition of the binding of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 to ribosomes. The binding of EF2 (elongation factor 2) and of ADP-ribosyl-EF 2 to rat liver ribosomes is inhibited by ricin. This result suggests that the native enzyme and its ADP-ribose derivative have the same or closely related binding sites on the ribosome. The inhibition by ricin of the binding of EF 2 to ribosomes is consistent with the previous observation that ricin affects EF 2-catalysed translocation during polypeptide chain elongation.


Purification and some properties of a soluble benzene-oxidizing system from a strain of Pseudomonas. 1. A soluble enzyme system which oxidizes benzene to cis-1,2-dihydroxycyclohexa-3,5-diene (cis-benzene glycol) was obtained from a species of Pseudomonas grown on benzene as the major carbon source. 2. The system was shown to consist of three protein components. Two of these were non-haem-iron proteins of molecular weight approx. 21,000 and approx. 186,000 and the other was a flavoprotein of molecular weight approx. 60,000. 3. Fe2+ and NADH were essential cofactors for benzene oxidation.


Incorporation of isotopic carbon into cerebral glycogen from non-glucose substrates. 1. Measurable incorporation of radioactive carbon from [U-14C]pyruvate, [U-14C]-glutamate and [14C]bicarbonate into the glycogen synthesized by brain slices in vitro was demonstrated. 2. The fructose diphosphatase activity of guinea-pig brain was determined and found to be about 0.03 mumol of substrate degraded/min per g of fresh tissue. 3. The specific radioactivity of the glucose carbon from glycogen relative to that of the precursor added to the incubation medium gave approximate values of 0.195 for glucose, 0.006 for pyruvate, 0.039 for glutamate and 0.001 for bicarbonate.


Role of glycerol 3-phosphate dehydrogenase in glyceride metabolism. Effect of diet on enzyme activities in chicken liver. 1. The metabolic role of hepatic NAD-linked glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) was investigated vis-a-vis glyceride synthesis, glyceride degradation and the maintainence of the NAD redox state. 2. Five-week-old chickens were placed on five dietary regimes: a control group, a group on an increased-carbohydrate-lowered-fat diet, a group on a high-fat-lowered-carbohydrate diet, a starved group and a starved-refed group. In each group the specific activity (mumol/min per g wet wt. of tissue) of hepatic glycerol 3-phosphate dehydrogenase was compared with the activities of the beta-oxoacyl-(acyl-carrier protein) reductase component of fatty acid synthetase, glycerol kinase (EC 2.7.1.30) and lactate dehydrogenase (EC 1.1.1.27). 3. During starvation, the activities of glycerol 3-phosphate dehydrogenase, glycerol kinase and lactate dehydrogenase rose significantly. After re-feeding these activities returned to near normal. All three activities rose slightly on the high-fat diet. Lactate dehydrogenase activity rose slightly, whereas those of the other two enzymes fell slightly on the increased-carbohydrate-lowered-fat diet. 4. The activity of the beta-oxoacyl-(acyl-carrier protein) reductase component of fatty acid synthetase, a lipid-synthesizing enzyme, contrasted strikingly with the other three enzyme activities. Its activity was slightly elevated on the increased-carbohydrate diet and significantly diminished on the high-fat diet and during starvation. 5. The changes in activity of the chicken liver isoenzyme of glycerol 3-phosphate dehydrogenase in response to dietary stresses suggest that the enzyme has an important metabolic role other than or in addition to glyceride biosynthesis.


Electron-paramagnetic-resonance spectroscopy studies of iron-sulphur centres of submitochondrial particles from iron- and sulphur-deficient. Candida utilis. 1. Measurements were made at 12 degrees K of the electron-paramagnetic-resonance (e.p.r.) spectra of submitochondrial particles from Candida utilis cells grown under conditions that alter the amount of the mitochondrial NADH dehydrogenase (EC 1.6.99.3). 2. Iron-limited growth decreases the extent of iron-sulphur e.p.r. signals to undetectable values that are less than 1 percent of those normally found with glycerol-limited growth. 3. Small but significant signals attributable to the NADH dehydrogenase were detected in submitochondrial particles from sulphate-limited cells. 4. Measurements made on submitochondrial particles prepared from these and other phenotypically modified cells lead us to conclude that the presence of low-temperature e.p.r.-detectable iron-sulphur centres attributable to the NADH dehydrogenase are necessary but not sufficient for the coupling of ATP synthesis to the NADH dehydrogenase reaction in the mitochondrial membrane of C. utilis. 6. The amplitude of the g=2.01 signal observed in non-reduced submitochondrial particles is approximately tenfold diminished by iron limitation but not significantly altered by sulphate limitation.


Relationship between enhanced turnover of phosphatidylinositol and lymphocyte activation by mitogens. 1. Various lectins [phaseolus vulgaris phytohaemagglutinin, Glycine max (soy-bean) agglutinin, Triticum vulgaris (wheat-germ) agglutinin and Axinella polyploides agglutinin] and antibodies to pig Ig (immunoglobulin) that are found by pig lymphocytes were assessed in terms of their capacities to stimulate lymphocyte transformation and to enhance phosphatidylinositol turnover. Transformation was measured after 45h of culture by incorporation of [6-(3)H]thymidine into DNA, whereas phosphatidylinositol metabolism was assessed after 1h of cultuis and G. max agglutinins and rabbit antibodies to pig Ig) increased phosphatidylinositol turnover, but non-transforming agents (T. vulgaris and A. polyploides agglutinins and Fab fragments of rabbit antibodies to pig Ig) failed to induce any significant enhancement. Subsequent cross-linkage of the bound, non-transforming Fab fragments with a goat antiserum to rabbit Ig stimulated transformation and phosphatidylinositol turnover. 3. Each transforming agent gave characteristic optimal dose responses that were similar for both phosphatidylinositol turnover and transformation. 4. The results indicate that activation of T- and B-lymphocytes is accompanied by enhanced phosphatidylinositol turnover and that in the case of B-cells this enhancement depends on the cross-linkage of surface receptors. They are consistent with the proposal that turnover represents an essential early step in the transformation process.


Lack of relationship between activity of chromatin-bound proteinase and cell growth rates. Proteinase associated with chromatin isolated from liver and hepatoma of rat is stimulated by salt, and, of the histone fractions, lysine-rich (F1) histone is preferentially degraded by this enzyme at an ionic strength comparable with that found in the nucleus. However, there appears to be no strict relationship between the activity of the proteinase and growth rates of hepatomas. Chromatin isolated from a fast-growing tumour, Ehrlich ascites carcinoma, shows no apparent proteinase activity in the presence of salt.


The properties of hydrogen peroxide production under hyperoxic and hypoxic conditions of perfused rat liver. The properties of H2O2 production in the "haemoglobin-free", "non-circulatory" perfused liver of rats were examined. The H2O2 production with 1 mM-lactate and 0.15 mM-pyruvate was 82nmol/min per g of liver or 333nmol/min per 100g body wt. in the liver of fed rats at 30 degrees C. This rate decreased to almost half in the livers of starved and phenobarbital-pretreated rats. When H2O2 production was stimulated by urate infusion, almost all of the H2O2 produced by the uricase reaction was decomposed by the catalase reaction. During the demethylation reaction of aminopyrine, no change in H2O2 production was detected by the present method; thus microsomal H2O2 production observed in isolated subcellular fractions appeared not to contribute significantly to the H2O2 production in the whole organ. Whereas the rate of the glycolate-dependent H2O2 production was halved at an intracellular O2 concentration that caused a 10 percent increase in the reduction state of cytochrome c, the half-maximal rate of H2O2 production with lactate and pyruvate was observed at an O2 concentration that caused a 40 percent increase in the reduction state of cytochrome c in the liver. No further increase in the rates of H2O2 production was obtained by increasing O2 pressure up to 5 times 10(5) Pa. The rate of ethanol oxidation through the catalase "peroxidatic" reaction varied, depending on the substrate availability. The maximal capability of this pathway in ethanol oxidation reached approx. 1.5 mumol/min per g of liver, when a mixture of urate, glycollate and octanoate was infused to enhance H2O2 production.


The selectivity and stoicheiometry of membrane binding sites for polyribosomes, ribosomes and ribosomal subunits in vitro. Differences in the binding sites for polyribosomes, template-depleted ribosomes and large ribosomal subunits were found in microsomal derivatives of the rough endoplasmic reticulum. 1. The stoicheiometry of polyribosome and ribosome interaction in vitro with membranes was shown to be influenced by the relative concentration of interactants and the duration of their mixing. Large ribosomal subunits required a more prolonged mixing schedule to achieve saturation of membranes than did polyribosomes. 2. By using a procedure which minimized the effects on binidng by the stoicheiometric variables, competition between populations of polyribosomes, ribosomes and subunits for membrane sites showed that subunits, and to a lesser extent ribosomes, failed to block polyribosome attachment. 3. Polyribosomes isolated from liver, kidney and hepatoma 5123C entirely bound to a common membrane site, but some polyribosomes from myeloma MOPC-21 bound to other sites, perhaps influenced by their unique nascent proteins. 4. Subunit-binding sites appear on rough membranes only after endogenous polyribosomes have been removed, but no evidence that resulting changes in surface constituents are responsible was found. Large-subunit binding was largely abolished by lowering MgC12 concentration of 0.1 mM, whereas under the same conditions polyribosome binding was undiminished. 5. The large-subunit site appears to be distinct from the polyribosome site not only in the restriction of its affinity for particles but also spatially, to the extent that bound subunits do not hinder access of polyribosomes to their sites.


The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The oxidized and reduced nicotinamide-adenine dinucleotide content of flight muscle and isolated mitochondria, the adenosine triphosphate and adenosine diphosphate content of mitochondria, and the energy status of the mitochondria during controlled respiration. 1. A study is presented of the mitochondrial NADH content during controlled (state 4) and active (state 3) pyruvate oxidation by blowfly flight-muscle mitochondria. The results confirm and extend those of an earlier study (Hansford, 1972), which indicated an increased reduction in state 3. Nicotinamide nucleotide is normally highly oxidized during state 4; however, there can be substantial reduction in the presence of carnitine or high concentrations of proline, or on lengthy incubation in the presence of either of the systems used to generate intramitochondrial tricarboxylate-cycle intermediate. 2. Omission of phosphate leads to substantial reduction and this can be reversed by adding phosphate or acetate. 3. Estimations of NAD-+ and NADH in fly thoraces show a marked increase in NADH on flight, tending to corroborate the results of mitochondrial experiments and testifying to the importance of dehydrogenase activation in this tissue. 4. Determination of intramitochondrial adenine nucleotides reveals a total of 4-5 nmol/mg of protein, and an ADP content of less than 0.1 nmol/mg during state 4 oxidation of pyruvate and proline. ATP content is found to increase slowly during state 4 and this is attributed to the net phosphorylation of AMP. 5. The uncoupling agent carbonyl cyanide p=trifluoromethoxyphenylhydrazone leads to hydrolysis of some, but not all, of the mitochondrial ATP. Studies of mitochondrial ATPase (adenosine triphosphatase), measured by external pH change, show that it is inactive unless the mitochondria are allowed to respire for several minutes in state 4 in the presence of phosphate before the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is suggested that phosphate uptake is essential for maximal ATPase activity. 6. Studies of the fluorescence of the fluorochrome 8-anilino-1-naphthalensulphonic acid suggest that the energy status of the mitochondrion is high during state 4-pyruvate oxidattion, and decrease slightly in state 3. The implications of these findings are discussed.


The influence of environmental agents on prostaglandin biosynthesis and metabolism in the lung. Inhibition of lung 15-hydroxyprostaglandin dehydrogenase by exposure of guinea pigs to 100 per cent oxygen at atmospheric pressure. Enzymes in the 100 000g supernatant fraction of guinea-pig lungs, in the presence of NAD-+, converted PGF-2 alpha (prostaglanding F-2 alpha) into a less-polar compound. The u.v. spectrum of this metabolite showed a strong absorption band at 230 nm, which is characteristic of a carbonyl group in conjugation with a double bond. Reduction of this metabolite with NaBH4 resulted in a compound that behaved like PGF2 ALPHA on t.l.c. and g.l.c. From this evidence we concluded that PGF2alpha is metabolized in vitro to 15-oxo-PGF2 alpha by the NAD-+-dependent prostaglandin dehydrogenase system of guinea-pig lung. The effect of exposure of the animal to SO-2 and O2 on the rate of prostaglanding biosynthesis and catabolism by lung fractions in vitro was studied. Exposure of guinea pigs to 500 p.m. of SO2 for 5h or to 50p.p.m for 9 days (6h/day) did not alter the production or degradation of prostaglandings by lung fractions in vitro. In contrast, exposure of guinea pigs to 100% O2 for 48 h inhibited the rate of prostaglanding metabolism in vitro by 60-70% without significantly altering the rate of biosynthesis by lung fractions. Inhibition of prostaglandin dehydrogenase activity in vitro by lung fractions after exposure of the animal to O2 was dependent on the duration of exposure. Gluthathione S-aryltransferase and catechol O-methyltransferase activites of guinea-pig lung 100 000g supernatant were unaltered by exposure of the animal to O2. Thus it appears that inhibition of pulmonary prostaglandin dehydrogenase by exposure of the animal to O2 is not the result of a general toxic response. It was postulated that the inhibition of prostaglanding dehydrogenase may occur after exposure of the animal to other oxidant gases.


Properties of phosphoenolpyruvate carboxykinase (guanosine triphosphate) synthesized in hepatoma cells in the presence of amino acid analogues. Phosphoenolpyruvate carboxykinase (GTP) was induced by a combination of dibutyryl cyclic AMP, theophyline and dexamethasone in Reuber H35 hepatoma cells under conditions where an amino acid in the medium was replaced by an appropriate analogue. 2. With canavanine replacing arginine or with 5-fluorotryptophan or 6-fluorotryptophan replacing tryptophan the induced enzyme had a lower catalytic activity-relative to antibody reactivity. 3. These aberrant enzyme molecules were heat-labile in vitro. 4. Measurements of enzyme degradation in vivo indicated that the canavanine-containing enzyme and the 6-fluorotryptophan-containing enzyme were degraded more rapidly than the enzyme containing all natural amino acids.


Increased degradation rates of protein synthesized in hepatoma cells in the presence of amino acid analogues. 1. Reuber H35 hepatoma cells incorporate the arginine analogue canavanine into cell protein when arginine is omitted from the incubation medium. 2. By labelling arginine-containing proteins with (14-C)leucine and then canavanine-containing proteins with (3-H)leucine in the same cells, it is possible to measure the degradation of both types of protein during a subsequent 'chase' period. With this technique it has been shown that canavanine-containing proteins are degraded at a rate severalfold greater than normal proteins. Comparable results were found when 6-fluorotryptophan was used as an analogue to tryptophan. 3. Control experiments in which the labelling order was reversed or where the animo acid and its analogue were incubated in separate cell cultures support the conclusion that abberrant proteins are rapidly degraded in vivo.


Dynamics of oestrogen-receptor distribution between the cytosol and nuclear fractions of immature rat uterus after oestradiol administration. 1. The nuclear-myofibrilar (800g pellet) fraction of the uterus from immature (22-23 days old) rats not exposed to oestrogen exhibits saturable binding of oestradiol. The nuclear binding capacity represents approximately 10% of that of the cytosol fraction (approx. 3.5 fmol/mug of DNA). The predominant part (0.3.5 fmol/mug of DNA) of the nuclear binind sites are present in the residual pellet after extraction with 0.5 M-KC1. 2. By using an exchange technique in vitro, determinations of the nuclear binding sites have been carried out after administration of 1 mug of oestradiol in vivo. Within 0.5h after the  hormone injection, the concentration of nuclear bindng sites increased to approx. 0.4 fmol/mug of DNA in the 0.5 M-KC1-extractable fraction, and to approx. 1.2 fmol/mug of DNA in the residual fraction. Meanwhile the cytosol oestrogen-receptor concentration decreased to approx. 10% of its initial value. In the following period from 0.5 h after the oestradiol injection onwards, the concentration of nuclear oestrogen receptors decreased with halflife values of approx. 140 and 200 min for the KC1(0.5 M)-extractable and residual form respectively. At the same time, the cytosol receptor concentration increased to reach approx. 50% of the initial value by the 6h. This increase could not be blocked by cycloheximide. The initial concentration of cytosol receptor was restored approx. 11h after the injection and the increase during the 6-11h period was sensitive to cycloheximide inhibition, suggesting protein-synthesis-dependence of the process. 3. With the (more) physiological dose of oestradiol (0.1 mug), the decrease the cytosol receptor was only 50% by 4h and this was followed by a period (up to 12h after injection) during which the initial concentration was restored. During this period the increase of the receptor can be blocked by cycloheximide.


The properties and extracellular location of 5'-nucleotidase of the rat fat-cell plasma membrane. 1. A phosphohydrolase specific for 5'-nucleotides was characterized by using a particulate fraction from isolated fat-cells. 2. The activity of intact cells towards 5'-AMP was studied. 3. The activity in either situation had the same KM for AMP (45 muM) and was inhibited by low concentrations of ATP (less than 50 muM), but less potently by the ATP analogues AMP-P(CH2)P(adenylyl (beta gamma-methylene)diphosphonate) and AMP-P)NH)P (adenylylimidodiphosphate). 4. Homogenization of intact fat-cells caused no increase in activity and at least 85% of the activity was recovered in the particulate preparation. 5. The preparation of fat-cells used in this work was not freely permeable to AMP. 6. The ability of intact fat-cells to hydrolyse AMP implies that 5'-nucleotidase is an ectoenzyme in fat-cells. 7. Concentrations of ATP 100 times lower than intracellular concentrations inhibit the enzyme when added extracellularly to intact fat-cells, implying that this effect is also medicated at the extracellular face of the membrane. 8. Antibodies raised to whole liver cells and whole fat-cells inhibit 5'-nucleotidase in intact cells. 9. Incubation of intact fat-cells with adrenaline (1 mug/ml) or insulin (50 mui.u./ml) failed to alter the KM or Vmax. of the enzyme.


Metabolic interactions of glucose, acetoacetate and adrenaline in rat submaxillary gland in vitro. 1. The metabolic interactions between glucose, acetoacetate and adrenaline were studied in submaxillary-gland slices. 2. Acetoacetate (2.5 mM) inhibited glucose removal by 22% and entry of glucose carbon into the tricarboxylic acid cycle by 54%. 3. Acetoacetate caused an increase in (glucose 6-phosphate) together with an increase in (citrate), a finding that suggests that the phosphofructokinase step might be inhibited by the elevated (citrate). Support for this suggestion was obtained in experiments in which fluoracetate was used to elevate (citrate). 4. A further site of action of acetoacetate at the pyruvate dehydrogenase step was suggested by an increase in the lactate+pyruvate pool, and the finding that pyruvate removal and (3-14C)pyruvate oxidation were inhibited by acetoacetate. 5. Adrenaline, a stimulator of secretion by this tissue, increased glucose removal by 25%. Adrenaline increased glucose removal to the same extent when acetoacetate was also present in the incubation medium. In both cases the increase was accompanied by a fall in (glucose 6-phosphate). 6. Adrenaline also overcame the inhibition of pyruvate removal caused by acetoacetate. 7. The tissue (ATP) decreased by about 50% on addition of adrenaline, and a similar fall was observed in vivo after adrenergic stimulation by isoproterenol. 8. Omission of Ca-2+ from the medium prevented the fall in (glucose 6-phosphate) and (ATP) caused by adrenaline, although adrenaline was still able to stimulate glucose removal. The inhibitory effect of acetoacetate on gluocse removal was reversed by adrenaline, but there was no stimulation above the control rates. Inhibition of pyruvate removal by acetoacetate was not overcome by adrenaline in the absence of Ca-2+. 9. Dibutyryl cyclic AMP had no effect on glucose removal or on (ATP). 10. Possible mechanisms by which adrenaline can bring about its metabolic effects are discussed.


Differential effects of lipid depletion on membrane sodium-plus-potassium ion-dependent adenosine triphosphatase and potassium ion-dependent phosphatase. The phospholipid-dependence of the (Na-++K-+)-dependent ATPase (adenosine triphosphatase) (EC 3.6.1.3) and associated K-+-dependent phosphatase activity (EC 3.6.1.7) have been compared. Unlike the (Na-++K-+)-dependent ATPase activities, the K-+-dependent phosphatase activities of a number of different preparations were not closely correlated with their total phospholipid contents. After partial lipid depletion with a single extraction in Lubrol W the residual ATPase and phosphatase activities were correlated, but their magnitudes were quite different: on average only about 5% of the former remained compared with 50% of the latter. A similar differential effect on these activities was found after extraction with deoxycholate. In contrast with the ATPase, consistent restoration of the phosphatase activity of Lubrol-extracted enzymes by added exogenous phospholipids was not observed. We conclude that, although the K-+-dependent phosphatase may be lipid-dependent, the lipid requirement must be different from that of the complete ATPase system, and this difference should help investigations of their relationship.


[Anti-inflammatory activity of a new quinoid polyradical (FL-70)]. The anti-inflammatory activity of FL 70, a derivative of 2,5-dihydroxy-benzoic acid, was examined in a number of conventional experimental models. In addition, FL-70 was tested for its inhibitory action on enzymes. The results were as follows: 1. The induction of a local inflammatory reaction and the subsequent i.v. injection of trypan blue showed that FL 70 reduces the capillary permeability. 2, FL-70 significantly suppresses exudation in the formalin-induced peritonitis of the rat. 3. A slight inhibition of an edema in the footpad of the rat induced by formalin-dextran was not shown to be statistically significant. 4. Local swelling could be markedly inhibited in the turpentine-oil induced inflammatory reaction of the rabbit. 5. Exudation and formation of granulomatous tissue was inhibited in Selye's granuloma. 6. FL-70 markedly inhibited the local inflammatory reaction accompanying the cutaneous reaction in experimental vaccinia infection of the rabbit skin. The size of the infiltration after intracutaneous infection of the virus was not reduced. 7. FL-70 could not prevent the onset of clinical signs, if administered in experimental allergic encephalitis. 8. The activity of acid phosphatase was inhibited by FL-70. Alcaline phosphatase, cholinesterase, leucin aminopeptidase, glucose-6- phosphatase-dehydrogenase (G-6-PDH), trypsin and chymotrypsin were unaffe-ted. FL-70 inhibits the following, G-6-PDH activated reduction process: glucose-6-phosphate (see article).


Snythesis and antiprotozoal activity of nitro derivatives of 2, 2'-biimidazole. We synthesized a series of 2,2'-biimidazoles with different substituents and screened them against Entamoeba histolytica, Trichomonas vaginalis and Giardia muris. A 5-nitro group is necessary for the inclusion of activity and an amino group exercises favourable effect on in vivi activity.


A search for microtubular inclusions in endothelial cells in a variety of skin diseases. An electron microscopic study of a variety of skin diseases has demonstrated microtubular 'inclusions' in vascular endothelium in bullous pemphigoid for the first time. The 'inclusions' are similar to those originally reported in lupus erythematosus by Fresco (1968) and presently being detected in an increasing number and variety of pathological processes.


Micro-determination of cyclic AMP levels in human epidermis, dermis and haif follicles. In order to study the biological and possible pathological roles of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in the skin, it is mandatory to measure cyclic AMP in 50-100 mug of microdissected epidermis, dermis or appendages. In the present study, we offer a method of extracting cyclic AMP from less than 100 mug of tissue, removing contaminating nucleotides and scaling down Gilman's method to fit the analysis of small amounts of tissue. Cyclic AMP levels in the dermis, epidermis, and hair follicles (bulbs) were approximately 1, 2 and 3-5 pmols/mug dry weight tissue respectively. This procedure is applicable to the measurement of cyclic AMP levels in limited foci of healthy or diseased skin.


Cyclic AMP in skin: effects of acute ischaemia. The cyclic adenosine 3',5'-monophosphate (cyclic AMP) content of pig skin was measured several seconds to minutes after removal of the skin from the body. It increased very rapidly, reached a maximum by 2 min after removal (4 times higher than the initial level), then decreased very slowly. Propranolol injected into the animal before or added after the removal of the skin did not suppress this phenomenon. The practical significance of this finding (increase of cyclic AMP level in skin after ischaemia) is obvious-in order to measure cyclic AMP level in vivo, the sample must be frozen immediately to avoid an 'artificial' increase in cyclic AMP.


Further studies on adenyl cyclase in psoriasis. Slices of human skin obtained with a keratome were pre-incubated with [3H]adenine to label the ATP pool from which cyclic AMP was subsequently formed. The accumulation of radioactive cyclic AMP was measured as an index of adenyl cyclase activity. The data showed that both the ability to incorporate [3H] into ATP and adenyl cyclase activity were significantly lower in psoriatic plaques than in uninvolved skin of the psoriatic patients, or in normal skin of control subjects. The response of adenyl cyclase to the stimulation of 3.3 muM adrenaline was less than five fold in psoriatic plaques as compared to twelve to thirty-two fold in the uninvolved skin. The response to the stimulation of prostaglandin E2 (5 mug/ml) showed no significant difference between the plaque and normal skin. The adenyl cyclase activity in uninvolved skin of psoriatic patients appeared normal. Propranolol  (10 muM) blocked the stimulatory effect of adrenaline but not that of PGE2 in normal skin. These results suggest that the adenyl cyclase system of the skin has different regulatory sites for adrenaline and PGE2 and that the enzyme is defective in the epidermis of the psoriatic plaque, especially at the adrenaline regulatory site.


Effect of dietary copper deficiency. in the rat on fatty acid composition of adipose tissue and desaturase activity of liver microsomes. 1. Male rats were maintained from wearing to between 4 and 16 weeks of age on a semisynthetic diet which was deficient in copper. 2. Methyl esters of fatty acids from adipose tissue of the rats were analysed by gas-liquid chromatography and the desaturase activity of liver microsomes, with [1-14C]=stearic acid as the substrate, was determined. Liver and plasma Cu concentration, cytohrome c oxidas (EC 1.9.3.I) activity and caeruloplasmin activity were determined as indices of Cu status. 3. Cu deficiency was associated with decreased mono-unsaturated:saturated ratios for C16 and C18 fatty acids from subcutaneous adipose tissue and decreased desaturase activity for liver microsomes. When Cu-deficient rats were given free access to the Cu-adequate diet or were injected intraperitoneally with an aqueous solution of CuSO4, that is, when the animals were related with Cu, the indices of Cu status, and desaturase activity for liver microsomes returned to values found in control animals. 4. When Cu or a Cu-chelator (Neocuproine) was added to microsomes, there was no effect on the activity of the desaturase enzyme system; the stability of the desaturase was not affected by Cu. 5. These results are indicative of an involvement of Cu in the desaturase reaction. It is suggested that the site of this involvement could be the terminal component of the microsomal electron transport chain.


The action of colicin E2 on supercoiled lambda DNA.II. Experiments in vitro. An in vitro system has been developed to test whether colicin E2 possesses DNase activity. Purified colicin E2 preparations introduced one single-strand scission in supercoiled lambda phage DNA. Glycerol gradient fractionation of colicin E2 supports the association of in vitro action with in vivo cell-killing activity. Colicin E2 preparations also attacked superhelical SV40 DNA yielding open circles and fragments and single-stranded fd DNA molecules causing one or more endonucleolytic breaks. The possible role of contaminating nucleases in the activity of colicin E2 preparations is discussed.


Spin-label studies of tropomyosin.

Spin-label studies of tropomyosin. Studies are reported on nitroxide spin-labeled tropomyosin. The labels attach to sulfhydryl groups and to amino groups. The amino spins are highly mobile, the sulfhydryl much less so. Spin count studies show an average of approximately 0.5 labeled sulfhydryl/tropomyosin molecule and only approximately 0.15 labeled amino group/molecule. The spectra are used tostudy the denaturation of tropomyosin by guanidine hydrochloride. The information obtained reveals the course of denaturation at sites near the sulfhydryl group. It is found that these sites are more susceptible to guanidine than the bulk of the molecule; denaturation at the sulfhydryl sites is complete by 1.5 M guanidine, whereas optical studies indicate the molecule as a whole is not completely denatured until the concentration reaches 3.5 M. Spectra are also shown of tropomyosin fibers oriented variously with respect to the applied magnetic field. Strong orientation effects are seen and these indicate that the sulfhydryl-attached spins (but not the amino-attached spins) have a definite orientation in the fiber. Interpretation of the spectra reveals that the normal to the nitroxide plane is inclined to the fiber axis at an angle of 50 degrees. Circular dichroism studies in the tyrosine region also reveal drastic changes with guanidine denaturation, confirming the idea that denaturation produces pronounced increase in mobility at the beta carbon (as in the sulfhydryl casey). A strong negative band existing only in helical tropomyosin at pH's where the tyrosines are uncharged appears to be due to interaction of tyrosines with the helical backbone, whereas the appearance of a strong positive CD band at 250 nm at high pH (approximately11) seems to be ascribable to interaction between the charged phenolic groups and the dissymmetric backbone alpha-carbon atom.


Correlation between quaternary structure and ligand dissociation kinetics for fully liganded hemoglobin. The quaternary structures of fully liganded adult hemoglobin and hemoglobin Kansas (alpha2beta2 102 Asn-thr) bound by carbon monoxide or nitric oxide were spectroscopically characterized using high-resolution nuclear magnetic resonance (NMR) and ultraviolet circular dichroism (CD). The spectral markers used for the quarternary transition were the line in the NMR spectrum in H2O-14 ppm downfield from 2,2-dimethyl-2-silapentane-5-sulfonate and the negative peak at 285 nm in the ultraviolet CD spectrum. In the nitrosyl derivatives, these two structural markers were compared with the electron paramagnetic resonance (EPR) spectrum at room temperature for the purpose of correlating structural changes in the protein with changes at the heme...


Mapping of the SV40 specific sequences transcribed in vitro from chromatin of SV40 transformed cells. Chromatin was isolated from SV40 transformed mouse cells (SV3T3) and transcribed with Escherichia coli RNA polymerase. The SV40 specific transcripts were analyzed by annealing the RNA to the minus strands of purified fragments of SV40 DNA produced by cleavage of the DNA with a restriction enzyme isolated from Hemophilus aegyptius. Quantitation of the frequency of transcription from the region (fragments A and D) was transcribed five to ten times more frequently than the remaining regions. These results are in good agreement with the transcription pattern observed in the transformed cell. In contrast, transcription of purified SV3T3 DNA by E. coli polymerase produced roughly equal frequencies of transcription from all regions of the integrated SV40 DNA. Comparison of the results with the known distribution of initiation sites for E. coli RNA polymerase on linear SV40 DNA indicates that the major initiation site is relatively unavailable in SV3T3 chromatin whereas other sites are available. This restriction is not observed when purified SV3T3 DNA is used as a template and must therefore result from the association of protein or other macromolecules with the DNA of the chromatin.


Initial membrane reaction in the biosynthesis of peptidoglycan. Spin-labeled intermediates as receptors for vancomycin and ristocetin. Phospho-N-acetylmuramyl-pentapeptide translocase (UDP-MurNAc-Ala-DGlu-Lys-DAla-DAla:undecaprenyl phosphate, phospho-MurNAc-pentapeptide transferase) catalyzes the initial membrane reaction in the biosynthesis of peptidoglycan. The spin-labeled nucleotide, UDP-MurNAc-Ala-DGlu-Lys (Nepsilon-2,2,5,5-tetramethyl-N-oxyl-pyrroline-3-carbonyl)-DAla-DAla, was used as a substrate by this enzyme for the synthesis of membrane-associated undecaprenyl-diphosphate-MurNAc-Ala-DGlu-Lys(Nepsilon-Tempyo)-DAla-DAla. The spin-labeled substrate and product complex with the antibiotics vancomycin and ristocetin. The association constants for the spin-labeled nucleotide are 6.2 times 10(5) and 6.2 times 10(4) M-1 for vancomycin and ristocetin, respectively. The association constants for the spin-labeled lipid intermediate are 3.0 times 10(4) and 2.1 times 10(4) M-1 for vancomycin and ristocetin, respectively. These results indicate that the acyl-DAla termini of membranes-associated spin-labeled undecaprenyl-diphosphate-MurNAc-pentapeptide are accessible to vancomycin and ristocetin and that the association constants are smaller than those determined for the corresponding antibiotic spin-labeled UDP-MurNAc-pentapeptide complexes.


A link between rhodopsin and disc membrane cyclic nucleotide phosphodiesterase. Action spectrum and sensitivity to illumination. Frog (Rana pipiens) rod outer segment disc membranes contain guanosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.1.c) which, in the presence of ATP, is stimulated 5- to 20-fold by illumination. The effectiveness of monochromatic light of different wavelengths in activating phosphodiesterase was examined. The action spectrum has a maximum of 500 nm, and the entire spectrum from 350 to 800 nm closely matches the absorption spectrum of rhodopsin, which is apparently the pigment which mediates the effects of light on phosphodiesterase activity. trans-Retinal alone does not mimic light. Half-maximal activation of the phosphodiesterase occurs with a light exposure which bleaches 1/2000 of the rhodopsins. Half-maximal activation can also be achieved by mixing 1 part of illuminated disc membranes in which the rhodopsin is bleached with 99 parts of unilluminated membranes. Regeneration of bleached rhodopsin by addition of 11-cis-retinal is illuminated disc membranes reverses the ability of these membranes to activate phosphodiesterase in unilluminated membranes. If the rhodopsin regenerated by 11-cis-retinal is illuminated again, it regains the ability to activate phosphodiesterase. These studies show that the levels of cyclic nucleotides in vetebrate rod outer segments are regulated by minute amounts of light and clearly indicate that rhodopsin is the photopigment whose state of illumination is closely linked to the enzymatic activity of disc membrane phosphodiesterase.


Regulation of Membrane Flexibility in Human Erythrocytes. We have used spin-labels to detect prostaglandin E induced changes in erythrocyte membranes. The observed changes in spin-label resonance spectra can be mimicked in erythrocyte ghosts by loading them with cAMP or cGMP. These changes can also be observed by adding either of these cyclic nucleotides to intact cells. This entry of cyclic nucleotides into intact cells is blocked by an inhibitor of the anion channel. We suggest that the observed changes in paramagnetic resonance spectra are due to changes in lipid "fluidity" that are brought about by changes in the biochemical state of membrane-associated proteins (such as spectrin) and in the direct or indirect biophysical interactions of these proteins with membrane lipids.


Swine lipoproteins and atherosclerosis. Changes in the plasma lipoproteins and apoproteins induced by cholesterol feeding. Cholesterol feeding in miniature swine resulted in a hypercholesterolemia with a distinctive hyperlipoproteinemia and the subsequent development of atherosclerosis. Alterations in the type and distribution of plasma lipoproteins induced by cholesterol feeding were as follows: (a) the occurrence of beta-migrating lipoproteins (B-VLDL) as well as very low density lipoproteins in the d less than 1.006 ultracentrifugal fraction; (b) an increased prominence of the intermediate lipoproteins (d = 1.006-1.02); (c) an increased prominence of low density lipoproteins; and (d) the occurrence of a distinctive lipoprotein with alpha mobility which was referred to as HDLc (cholesterol induced). Characterization of the various plasma lipoproteins included chemical composition, size by electron microscopy, and apoprotein content. The B-VLDL resembled the beta-migrating lipoproteins of human Type III hyperlipoproteinemia and contained a prominent protein equivalent to the arginine-rich apoprotein in addition to the B apoprotein, apo-A-I, and the fast-migrating apoproteins (apo-C). The HDLc were rich in cholesterol, ranged in size from 100 to 240 A in diameter, and contained the arginine-rich apoprotein and apo-A0I but lacked the B apoprotein. The arginine-rich apoproteins isolated from B-VLDL and HDLc by gel chromatography were similar in amino acid analyses, with glutamic acid as their amino-terminal residue. The occurrence of a spectrum of cholesterol-rich lipoproteins which contained the arginine-rich apoprotein with the occurrence of accelerated atherosclerosis suggested an interesting, although speculative, association.


13-C nuclear magnetic resonance studies on the lipid organization in enveloped virions (vesicular stomatitis virus). 13-C nuclear magnetic resonance (NMR) studies are described regarding the lipid organization in the envelope of the vesicular stomatitis virion. The fatty acid chains (oleic acid) and the choline moiety of the 3-sn-phosphatidylcholine and spingomyelin have been labeled specifically with 13-C by growing the virions in prelabeled host cells (BHK 21 cells). The results suggest that 130C NMR spectroscopy is a very feasible method for the study of natural membranes provided the isotope is highly enriched in specific positions and incorporated biochemically. Spin-lattice relaxation (T1) measurements of particular C atoms have been carried out with whole virions, with virions deprived of their surface projections by trypsinization but unaltered in their shape and size, and with liposomes prepared from the total lipid mixture of the envelope in order to get insight into the molecular structure of this model membrane. The mobility of the central part of 11-13-C-labeled oleic acid incorporated into the ester and amide lipids and the choline group of 3-sn-phosphatidylcholine and sphingomyelin is very restricted as indicated by their short T1 times. It is concluded from the data presented here that the high cholesterol content (cholesterol/P: 0.7) of the envelope lipid phase is responsible for the rather rigidly packed envelope structure. The mode and extent of the interactions between lipids and glycoprotein surface projections are subjects for further study.


The amino acid sequence of chick skin collagen alpha1-CB7. The amino acid sequence of chick skin collagen alpha1-CB7, the 268 CNBr peptide from the helical portion of the alpha1 chain, has been determined by automatic and manual degradation of tryptic and chymotryptic peptides, and of the COOH-terminal fragment produced by cleavage with animal collagenase. The resulting sequence shows 94% identity with that of the corresponding peptide from calf skin collage (Fietzek, P. P., Rexrodt, F. W., Hopper, K. E., and Kühn, K. (1973), Eur. J. Biochem. 38, 396). The bond cleaved by animal collagenase has been identified as Gly-Ile at residues 221-222 of alpha1-CB7.


Adrenocorticotropic hormone stimulation of adrenal RNA polymerase I and III activities. Nucleotide incorporation into internal positions and 3' chain termini. In the presence of 50 mM (NH4)2SO4 and low concentrations of alpha-amanitin (7.7 mug/ml), adrenal nuclei synthesize predominately rRNA as characterized by size and base composition. Approximately 10% of the RNA synthesized under these conditions sediments at 4-5 S; this RNA synthesizing activity is inhibited by high concentrations of alpha-amanitin (231 mug/ml) indicating the presence of RNA polymerase III activity. ACTH administration to guinea pigs results in a twofold increase in adrenal nuclear RNA polymerase I and III activities at 14 hr of hormone treatment. Analysis of the amount of radiolabeled nucleoside triphosphate incorporated in vitro into 3' chain termini and into internal nucleotide positions has been utilized to measure the number of RNA chains and the average chain length synthesized in vitro. Incorporation into 3' chain termini is not changed by ACTH; incorporation into internal nucleotides is doubled in parallel with the increase in RNA polymerase I activity. These results are not due to an altered Km of RNA polymerase I for the four nucleoside triphosphates, nor to differential R Nase or phosphatase activity. These studies suggest that the regulation of RNA polymerase I by ACTH is accomplished in part through an increase in the rate of RNA chain elongation.


Synthetic position 5 analogs of adrenocorticotropin fragments and their in vitro lipolytic activity. Twenty-three analogs of the ACTH-(4-10)-heptapeptide sequence, which forms the "active core" of adrenocorticotropin (ACTH) and related hormones, have been synthesized by the solid-phase method. These analogs all contain structural modifications at or near the 5-glutamic acid residue of ACTH. The peptides were purified to electrophoretic and chromatographic homogeneity. The peptides were assayed for lipolytic activity in an isolated cell system derived from rabbit adipose tissue. In this system, it was determined that residue 5 plays a very important "spacer" role in the peptide, but that this spacer function is not very dependent on the nature of the side chain of the position 5 amino acid. It was found, however, that a number of analogs containing basic residues (arginine or lysine) in position 3 and/or position 5 of ACTH-(3-10) and ACTH-(4-10) fragments have 5 to 10 times the activity of the respective parent peptides. The presence of a latent anionic locus in the rabbit fat-cell receptor for ACTH is suggested by this study.


Effect of the human plasma apolipoproteins and phosphatidylcholine acyl donor on the activity of lecithin: cholesterol acyltransferase. The human plasma apoproteins apoA-I and apoC-I enhanced the activity of partially purified lecithin: cholesterol acyltransferase five to tenfold with chemically defined phosphatidylcholine:cholesterol single bilayer vesicles as substrates. By contrast, apoproteins apoA-II, apoC-II, and apoC-III did not give any enhancement of enzyme activity. The activation by apoA-I and apoC-I differed, depending upon the nature of the hydrocarbon chains of phosphatidylcholine acyl donor. ApoA-I was most effective with a phosphatidylcholine containing an unsaturated fatty acyl chain. ApoC-I activated LCAT to the same extent with both saturated and unsaturated phosphatidylcholine substrates. Two of the four peptides obtained by cyanogen bromide cleavage of apoA-I retained some ability to activate LCAT. The efficacy of each of these peptides was approximately 25% that of the whole protein. Cyanogen bromide fragments of apoC-I were inactive. The apoproteins from HDL, HDL2, and HDL3, at low protein concentrations, were equally effective as activators of LCATand less effective than apoA-I. Higher concentrations of apoHDL, apoHDL2, and apoHDL3 inhibited LCAT activity. ApoC and apoA-II were both found to inhibit the activation of LCAT by apoA-I. The inhibition of LCAT by higher concentrations of apoHDL was not correlated with the aopA-II and apoC content.


Synthesis of a new phosphatidylserine spin-label and calcium-induced lateral phase separation in phosphatidylserine-phosphatidylcholine membranes. A new phosphatidylserine spin label with nitroxide stearate attached at the 2 position has been synthesized by the reaction of spin-labeled CDP-diglyceride with L-serine under the catalytic action of phosphatidylserine synthetase. Some structural properties of pure phosphatidylserine (PS) and binary PS-phosphatidylcholine (PC) membranes were studied with the spin label. PS membrane became solidified on lowering solution pH, 50% solidification being attained at pH 3.5. The membrane was also solidified by addition of Ca-2+. The effect of Ba-2+,Sr-2+, and Mg-2+ was smaller than that of Ca-2+. The calcium-induced lateral phase separation in the binary membrane was studied from the side of the calcium-receiving lipid. The results confirmed and extended our previous conclusion drawn with PC spin label. The phase diagram of the binary membrane in the presence of Ca-2+ was determined. Not all PS molecules were aggregated to form the solid patches but some remained dissolved in the fluid PC matrix. The fluid PS fraction was larger for the membranes containing more PC. The membrane with 10% PS still had a significant fraction of solid phase. The rate of calcium-induced aggregation was greatly dependent on the PS content. The aggregation was almost complete within 5 min in the membrane containing 67% PS, while it was still proceeding after several hours in the membrane with 20% PS. The rate-limiting step was suggested to be in the formation of "stable" nuclei consisting of larger aggregates. The possible biological significance of the ionotropic phase separation was discussed whereby a transient density fluctuation was emphasized.


Kinetics of gonadotropin binding by receptors of the rat testis. Analysis by a nonlinear curve-fitting method. The kinetics of the reaction between human chorionic gonadotropin (hCG) and specific gonadotropin receptors in the rat testis were determined at 24 and 37 degrees, over a wide range of hormone concentrations. Hormone concentrations were corrected for the binding activity of the (-125I)hCG tracer preparations. Analysis of the experimental data was performed with an interactive nonlinear curve fitting program, based upon the second-order chemical kinetic differential equation. The mean values for the association rate constant (k1) were 4.7 x 10-7 M-1 min-1 at 24 degrees, and 11.0 x 10-7 M-1 min-1 at 37 degrees. At both temperatures, the values of kl were independent of hormone concentration. Initial dissociation rates were consistent with first order kinetics, with dissociation rate constant (k2) of 1.7 x 10 minus -3 and 4.6 x 10 minus -3 min minus -1 at 24 and 37 degrees, respectively. When studied over longer periods at 24 degrees, the dissociation process appeared to be multiexponential. The kinetics of degradation of (-125I)hCG and receptors were determined at both temperatures, and a mathematical model was developed by modification of the second-order chemical kinetic differential equation to take these factors into account. The application of such a model to hCG kinetic binding data demonstrated that reactant degradation had little significant effect on the derivation of the association rate constant (k1), but caused significant overestimation of the dissociation rate constant (k2) values derived from association experiments. The model was also applied by computer simulation to a theoretical analysis of the effects of degradation of free hormone and receptor sites upon kinetic and steadystate binding data. By this method, the initial velocities of hormone binding were shown to be less affected by degradation than the steady-state levels of hormone-receptor complex. Also, reactant degradation in simulated steady-state experiments caused an underestimate of the apparent equilibrium association constant, but had relatively less effect on the determination of binding site concentration.


Demonstration and partial characterization of cytosol receptors for testosterone. Androgen uptake was investigated in several peripheral organs after administration of (1,2,6,7 minus -3H)testosterone to castrated male rats. The animals were killed after 30 min, the organs were taken out, and the radioactivity was determined after tissue combustion. A relatively high accumulation of androgen was found in pancreas, adrenals, spleen, thigh muscle, kidneys, and liver in addition to the classical androgen target organs coagulation glands, seminal vesicles, prostate, preputial glands, and harderian glands. In a second serier of experiments, nuclear and cytosol fractions were prepared from prostate, seminal vesicles, coagulation glands, preputial glands, spleen, submaxillary glands, kidneys, and pancreas from castrated male rats give (1,2,6,7 minus -3H)testosterone, and these fractions were then characterized by thin-layer and radio-gas chromatography with respect to their patterns of labeled steroids. Only prostate and seminal vesicles were found to contain significant amounts of nuclear 5alpha-(-3H)dihydrotestosterone. The major nuclear androgen was (-3H)testosterone that was the only detectable labeled steroid in coagulation glands, preputial glands, and spleen and that constituted 70% or more of the nuclear radioactivity in seminal vesicles, submaxillary glands, kidneys, and pancreas. These results indicate that testosterone itself may be the predominant active androgen principle in vivo in most androgen target organs and that conversion to 5alpha-dihydrotestosterone is generally not a prerequisite for androgen activity. Using an ultrasensitive micromodification of isoelectric focusing (cf. M. Katsumata and A. S. Goldman (1974), Biochem. Biophys. Acta 359, 112. It was possible to show that cytosol from kidney; submaxillary gland, thigh muscle, and levator ani muscle and nuclei from kidney and submaxillary gland contained androgen-binding proteins with pI's in the region 4.6-5.1 ("4.6 minus 5.1 Complex"). This complex also formed in vitro after incubation of (1,2,6,7 minus -3H)testosterone with cytosol from kidney and submaxillary gland. (1,2,6,7 minus -3H)Testosterone was bound with high affinity to receptor proteins in cytosol from both kidney, submaxillary gland, and thigh muscle with dissociation constants of 5.0 x 10 minus -12 M (kidney), 3.3 x 10 mi;nus -11 M and 4.1 x 10 minus -10 M (two types of binding sites, submaxillary gland), 2.4 x 10 minus -12 M (thigh muscle) and 1.9 x 10 minus -12 M (levator ani muscle). The number of binding sites was in all cases between 1 and 20 fmol/mg of protein. On the basis of these results the hypothesis is presented that a common class of testosterone receptors is present in most organs and that these receptors can be detected both in vivo and in vitro provided methods sensitive enough are utilized.


Fucosyl-glycoprotein and precursor polls in HeLa cells. An enzymatic-radioactive isotope method has been developed for the direct quantitation of L-fucose in amounts as low at 0.5 plus or minus 0.05 nmol. Fucose kinase is used to transfer [32-P]phosphate from ATP to [3-H]fucose. The labeled enzymatic products are then separated electrophoretically and the amount and specific activity of the fucose are determined from the known specific activity of the phosphate donor. This assay has been used to measure the GDP-L-fucose and macromolecualar fucose in HeLa cells after extraction and purification of the sugar. It has been determined there are 0.5 nmol of GDP-L-fucose in 10-7 cells with a nine- to tenfold dilution of specific activity in converting L-[3-H] fucose to GDP-L-[3-H]fucose. After 2 to 3 days of labeling, the GDP-L-[3-H]fucose pool is essentially at equilibrium with the macromolecular pool, and hence it can be concluded that the dilution of label is due to a nine- to tenfold contribution to GDP-L-fucose from an endogenous source, as compared to exogenously supplied fucose. The fucosyl-glycoprotein pool has been shown to be much larger containing 6 to 8 nmol of fucose in 10-7 cells. It has further been shown that GDP-fucose is the only soluble fucose intermediate present in significant amount.


Molecular weights of estrogen and androgen binding proteins in the liver of Xenopus laevis. [3-H]Estradiol-17beta and [3-H]dihydrotestosterone binding proteins in the cytosol fraction of liver from both male and female Xenopus laevis were characterized by electrophoresis on polyacrylamide gels. These binding proteins, which were indistinguishable based upon their mobilities on gels of different acrylamide concentrations, migrated as single components with a molecular weight of 2.0 x 10-4. Separation of native or sodium dodecyl sulfate denatured specific estrogen-binding components on dodecyl sulfate free acrylamide gels gave similar results, i.e., a single species of molecular weight 2.0-2.5 x 10-4. The same molecular weight also was obtained when cytosol was prepared in the presence of either diisopropyl fluorophosphate or phenylmethanesulfonyl fluoride, protease inhibitors. Evidence that the liver components binding either [3-H]estradiol-17-beta or [3-H]dihydrotestosterone were not plasma contaminants was provided by the observation that the plasma sex-steroid binding globulin of Xenopus had a different mobility when separated by polyacrylamide gel electrophoresis.


Magnetic resonance and kinetic studies of pyruvate, phosphate dikinase. Interaction of oxalate with the phosphorylated form of the enzyme. Pyruvate, orthophosphate dikinase (EC 2.7.9.1) carries out its catalytic function in three successive partial reactions, the final step being the reaction of pyruvate with a stable phosphoenzyme intermediate to give phosphoenolpyruvate and free enzyme (Evans, H.J., and Wood, H. G. (1968), Proc. Natl. Acad. Sci. U.S.A. 61, 1448). Interactions of oxalate, a structural analog of enolpyruvate, with the phosphorylated form of the enzyme have been investigated by kinetic inhibition measurements and by magnetic resonance studies of manganous ion complexes with the enzyme. Oxalate inhibits the reaction catalyzed by pyruvate, phosphate dikinase, and the inhibition is linearly competitive with respect to pyruvate. The inhibitor constant for oxalate of 25 mu-M is fourfold lower than the Michaelis constant for pyruvate. The enhancement in the longitudinal relaxation rate of water protons (PRR) which occurs upon binding of Mn(II) to the enzyme has been used to monitor binding of oxalate to Mn(II)-enzyme complexes. PRR titrations indicate that the dissociation constant of oxalate from the Mn(II) complex of the free form of the enzyme is an order of magnitude weaker than the kinetically determined Ki. On the other hand, titrations of solutions which contain the phosphorylated form of the enzyme reveal a much stronger binding of oxalate. Moreover, the strength of oxalate binding to the phosphorylated enzyme is a function both of the species and of the concentration of monovalent cations in the solution. In the presence of Tl+, which has the most favorable activator constant for the final partial reaction, the dissociation constant for oxalate from its complex with the phosphorylated enzyme is less than 1 mu-M. Electron paramagnetic resonance (EPR) spectra for the enzyme-bound Mn(II) are sensitive to structural perturbations which occur upon binding of substrates or of oxalate to the enzyme. The EPR spectrum for the Mn(II)-phosphoenzyme-oxalate species is distinguished from spectra for other complexes of the enzyme by unusually narrow line widths and consequent resolution of fine structure from electronic quadrupole splitting. The narrow lines in the EPR spectrum are indicative of a rigid, pseudocrystalline environment for the bound Mn(II). The magnitude and frequency dependence of the PRR for the Mn(II)-phosphoenzyme-oxalate complex indicate that if any water molecules are bound to the Mn(II), their exchange with the bulk water is severely retarded. The kinetic and magnetic resonance studies support the hypothesis that oxalate mimics the reactive intermediate, enolpyruvate, in a complex with the phosphorylated enzyme which may resemble the structure of the transition state of the final partial reaction.


A study of protein-sodium dodecyl sulfate complexes by transient electric birefringence. The method of transient electric birefringence has been applied to study the conformation of protein-sodium dodecyl sulfate complexes. A model of a deformable prolate ellipsoid has been proposed for the protein-dodecyl sulfate complex. This model is compared to the models proposed by J. A. Reynolds and C. Tanford (1970), J. Biol. Chem. 245, 5161) and K. Shirahama, K. Tsujii, and T. Takagi (1974, J. Biochem. 75, 309). Differences between these latter two models are resolved by the model presented here. In addition, it has been demonstrated that protein molecular weights may be obtained from the slow relaxation time for transient electric birefringence of protein-dodecyl sulfate complexes.


Characterization of procollagen-derived peptides unique to the precursor molecule. A disulfide-bonded fragment with a molecular weight of about 100,000 was identified in the medium of cultured chick cranial bone and its derivation from procollagen was established by immunological criteria. The molecular weight of the fragment was reduced to 33,000 after cleavage of disulfide bonds, indicating a triple-stranded structure. The amino acid composition of the fragment lacked hydroxyproline and hydroxylysine and differed markedly from that of collagen in other respects. A similar but somewhat larger fragment was isolated after bacterial collagenase digestion of chick bone procollagen purified by chromatography on DEAE-cellulose. The characterization and comparison of these fragments further define the nature of the additional regions in procollagen and, when combined with information derived from studies of acid-extracted and dermatosparactic procollagens, support a mechanism for the conversion of procollagen to collagen which involves more than one proteolytic step.


Conformational changes in the progesterone binding globulin-progesterone complex. An improved purification procedure for the progesterone-binding globulin (PBG) of the pregnant guinea pig has been developed utilizing sulfopropyl Sephadex, a strong cation exchanger, in the first step. The method exploits the low pI (2.8) and favorable acid stability of the glycoprotein. Subsequent chromatographies on DEAE-cellulose and Sephadex G-200 afford a highly purified PBG that exhibits the previously observed polydispersity (R.M. Burton et al. (1974), Biochemistry 13, 3554-3561). Circular dichroism, optical rotatory dispersion, and difference uv spectra all indicate the purified protein to undergo a conformational transition upon forming a complex with a steroid ligand. The CD and ORD spectra cannot be interpreted in terms of tertiary structure probably due to carbohydrate contributions. However, the difference spectra indicate strong perturbation of both a tryptophan residue and the steroid chromophore in the complex.


Actions of insulin, epinephrine, and dibutyryl cyclic adenosine 5'-monophosphate on fat cell protein phosphorylations. Cyclic adenosine 5'-monophosphate dependent and independent mechanisms. Endogenous and hormone-induced protein (polypeptide) phosphorylations were studied in isolated rat fat cells, in fat pads, and in subcellular fractions obtained from fat tissue under different physiological conditions. Insulin (25-100 muU/ml) increased the incorporation of 32P into two proteins: insulin-phosphorylated proteins (IPP 140 and IPP 50; similar to 140,000 and 50,000 daltons, respectively). Epinephrine (10(-7)-10(-6) M) increased the incorporation of 32P into another protein: epinephrine-phosphorylated protein (EPP 60-65; similar to 60,000-65,000 daltons). Endogenous IPP 140 phosphorylation in fat cells obtained from fasted and refed rats was similar to that of insulin in normal cells. Studies of insulin and epinephrine interactions showed that insulin increased IPP 140 phosphorylation even in the presence of epinephrine or lithium (25 mM times 10(-3) M). dibutyryl cyclic AMP (5 times 10(-4) M) markedly stimulated EPP 60-65 phosphorylation, but neither epinephrine (10(-7)-10(-6) M) nor dibutyryl cyclic AMP reproduced insulin's phosphorylation of APP 140. Lithium inhibited both endogenous and epinephrine-stimulate EPP 60-65 phosphorylation, but did not inhibit that induced by dibutyryl cyclic AMP. These findings suggest that insulin stimulated a specific, cyclic AMP independent protein kinase for IPP 140 phosphorylation. Cell-free extracts from insulin-treated fat tissue catalyzed the specific transfer of 32P from ATP to IPP 140 more rapidly than control extracts. No differences in the total receptor protein or total protein kinase activity using [gamma(-32P]ATP were noted between insulin-treated and control preparations. IPP 140 may be either (a) an insulin-sensitive protein kinase (phosphotransferase) or (b) a protein whose function is regulated by an insulin-sensitive protein kinase or phosphatase.