[The angiographic picture of arteriovenous fistulae (author's transl)]. The angiographic appearances and the significance of various angiographic signs are discussed in relation to 12 arteriovenous fistulae demonstrated angiographically in 11 patients. Dilatation of the supplying artery is an indication of the size of the shunt and is particularly prominent in the presence of multiple, congenital microfistulae. The technique developed in our clinic for investigating suspected or known A-V fistulae is described. This technique simplifies the differential diagnosis between the Klippel-Trénaunay syndrome and the prognostically less favourable Weber syndrome.


[Polyneuropathies as a diagnostic and therapeutic task]. Despite their variety the syndromes of peripheral neuropathy are variations of just one subject. The most different lesions might be their cause. Therefore, one should not be satisfied with the diagnosis peripheral neuropathy, but should always try to clarify the etiology. Form and extension of the disturbances give essential hints. As far as possible, therapy should be directed against the respective causes. For several types of neuropathy prevention is possible. Symptomatic treatment is also important and in many cases even essential for survival.


[Chemodectoma in the carotid triangle region]. The only treatment of the extensive and infiltrative carotid body tumors is radical resection. Exact preoperative diagnosis in unknown tumors of the neck avoid hazardous complications during operative resection. This report contains diagnosis and definition of these rare tumors. Various operative procedures of tumor-resection are discussed. Carotid angiographies and operative photographs from three cases demonstrate the extent and close relation of the carotid artery.


[Prevention and therapy of viral diseases with special reference to interferon]. Human leukocyte interferon (HLI) was used for treatment of human diploid fibroblasts before and after infection with vaccinia virus, herpes simplex virus type 1 (HSV 1), herpes simplex virus type 2 (HSV 2), and varicella zoster virus (VZV). Vero cells were infected with Medical Lake macaque herpes virus (MLMV), and treated with HLI in the same way. In all of these systems HLI exhibited an antiviral effect when administered before infection, and this effect could be increased by additional HLI treatment after infection. In vivo studies with HLI treatment were performed in monkeys experimentally infected with vaccinia virus, HSV 1, and MLMV. Vaccinia and herpes keratitis were prevented by local, prophylactic administration of HLI. Generalized infections with vaccinia virus and MLMV in monkeys immunosuppressed by antilymphocyte globulin, were significantly modified by either prophylactic or therapeutic systemic treatment with HLI.


Localization of chymotrypsin-like cationic protein, collagenase and elastase in azurophil granules of human neutrophilic polymorphonuclear leukocytes. The subcellular localization of granulocyte collagenase, elastase and chymotrypsin-like cationic protein was determined using velocity centrifugation of cytoplasmic granules of human polymorphonuclear leukocytes. The proteases were assayed by immunochemical and enzymatic methods. Measurements of lactoferrin and myeloperoxidase distinguish exactly between constituents of specific and azurophil granules. Collagenase, elastase and chymotrypsin-like cationic proteins showed an almost identical sharp and unimodal distribution. They co-sedimented with myeloperoxidase demonstrating that these enzymes are localized exclusively in the azurophil granules.


Patients released after Wyatt: where did they go? The author investigated the posthospital outcome of a random sample of 228 patients released from Bryce Hospital between June 1972 and June 1973. Approximately 72% of the ex-patients participating in the follow-up study had been released from the hospital to a household containing a member of the family. At the time of the follow-up interviews, conducted between May 1975 and June 1976, approximately 63% of the ex-patients were found to be living in non-institutional settings. The study also found that a majority of the patients had not been readmitted to a state psychiatric facility since their release.


Myofibroblasts and related cells in malignant fibrous and fibrohistiocytic tumors. Myofibroblasts were detected by electron microscopy in five of five cases of fibrosarcoma and in five of six cases of malignant fibrous histiocytoma. In some areas myofibroblasts constituted up to 75 per cent of the tumor cells. Most myofibroblasts contained only sheaves of myofilaments along the margins of the cells, but some cells contained larger bundles of myofilaments and very closely resembled smooth muscle cells. An additional related type of cell was seen in several cases; it was large and possessed abundant eosinophilic cytoplasm, resembling a rhabdomyoblast at the light microscopic level. By electron microscopy this type of cell was seen to contain plentiful rough endoplasmic reticulum and large aggregates of fine filaments with rare dense bodies. These findings suggest that fibrosarcomas and malignant fibrous histiocytomas contain cells showing a spectrum of differentiation from fibrocytic to myogenic and that at the ultrastructural level the distinction between fibroblast and smooth muscle tumors may be blurred.


Morphologic aspects of nuclear bodies in a case of bronchiolar-alveolar carcinoma of the lung. The ultrastructural features of a bronchiolar-alveolar carcinoma of the lung are described wherein the presence of nuclear bodies in many of the tumor cells were noted. Although these bodies are not viral elements, it is surmised that they may be virus associated and consequently possibly related to the etiology of this tumor. A review of the ultrastructural findings in the pulmonary adenomatosis of sheep is presented, and these are compared with those in human disease. Experimental evidence of an infectious agent responsible for human lung carcinomas is discussed, and possible avenues for future investigation are delineated.


Benign hepatic lesions and orally administered contraceptives. A report of seven cases and a critical analysis of the literature. A consecutive series of 12 benign hepatic lesions in women consisted of six cases of focal nodular hyperplasia and six cases of liver cell adenoma. Five of the six women with liver cell adenoma and two of the six with focal nodular hyperplasia had taken orally active contraceptive hormones. These few cases reflect a similar impression gained from a critical analysis of the literature, namely, that focal nodular hyperplasia may be unrelated to the oral administration of contraceptive hormones, whereas the increase in liver cell adenoma reported in recent years is probably related to such therapy. Two women with liver cell adenomas were asymptomatic six and four years after incomplete resection of the tumor. These are the longest intervals thus far reported for uncomplicated survival in incompletely resected liver cell adenoma.


Cytomegalovirus oophoritis: ovarian cortical necrosis. Three of eight female patients with cytomegalovirus disease had evidence of ovarian infection at autopsy. All three patients with ovarian lesions were postmenopausal; the remaining five were premenopausal. The lesions, which may be bilateral and may occur without evidence of cytomegalovirus infection elsewhere, are distinctive macroscopically, pathognomonic microscopically, and characterized by acute focal ovarian cortical necrosis with numerous cytomegalic cells and a variable but usually severe inflammatory reaction. It is suggested that reduced ovarian cortical vascular perfusion reactivates a latent infection in cortical stromal cells. This elicits inflammatory necrosis and the characteristic morphological lesions. This previously unreported lesion is not likely to have clinical importance.


Red cell adenylate kinase phenotypes in the affective disorders. The red cell adenylate kinase (AK) phenotype was determined by starch gel electrophoresis in 96 adult Caucasian subjects with affective disorders (24 with bipolar illness and 72 with unipolar illness). The phenotype frequencies and the gene frequencies of the bipolar group closely resembled that of the control subjects (180 subjects drawn from the population of a large institution for the mentally retarded), the unipolar group however, showed a significant increase in the frequency of the AK2 allele. The significance of these results have been discussed in relation to the known genetic and biochemical findings in the affective disorders. It is suggested that the mechanism involved may be a reduction of the enzyme activity in the tissues of subjects with the AK 2:1 phenotype. This may present a selective disadvantage in the form of a decrease in control of energy metabolism in general, and control of adenine nucleotide levels in nervous tissue in particular.


Isolation of cell lines from embryos of the cockroach, Blattella germanica. Cell lines were isolated from three stages of embryos of Blattella germanica dissociated with trypsin. The lines have been subcultured 50 to 134 times in 3 years. Line UM-BGE-1 was isolated from germ band embryos at stages of segmentation and limb-bud formation (5 days old). Line UM-BGE-2 was derived from embryos at dorsal closure (7 days old). Line UM-BGE-4 arose from embryos in the germ band and dorsal closure stages (5 and 7 days old): these cells colonize as hollow spheres or vesicles. Line UM-BGE-5, isolated during organogenesis (10 days old), developed into two distinct sublines. Subline alpha is composed of round cells that do not attach to the flask. Subline beta grows as an attached monolayer; the cells can be removed with a saline solution containing 20 mM disodium dihydrogen Versenate. Most of the cells of these lines have the diploid chromosome number (23 or 24) excepting line UM-BGE-1 in which the tetraploid number predominates


Cyclic AMP and serum arrest of the mitotic activity of human diploid fibroblasts. Mitotic activity in confluent cultures of human diploid fibroblasts was arrested by the reduction of the serum concentration of the incubation medium to 0.5% or by the addition of 0.5 mM 6-N, 2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate (db cAMP). Under either of these conditions, cultures maintained a constant cell number for 14 days; cultures continuously exposed to medium containing 10% serum doubled their cell number during this 14-day period. The protein cotent per cell decreased by 20% when cells were maintained with 0.5% serum whereas that of cells exposed to db cAMP remained constant. Ultrastructural studies revealed that cells exposed to db cAMP exhibited a morphology typical of cells cultures with 10% serum alone, whereas cells incubated with 0.5% serum showed the ultrastructural changes in mitochondria, endoplasmic reticulum and Golgi complex previously identified with low-serum arrest. Cellular adenosine 3':5'-cyclic monophosphate (cAMP) levels remained constant during the 7-day growth period in which confluency was attained, as well as during the 14-day arrested period with 0.5% serum. These results indicated that the mitotic inhibition induced by reducing the serum concentration of the incubation medium was not mediated by increased intracellular levels of cAMP and differed from that induced by the addition of exogenous db cAMP.


Cholera toxin, ganglioside receptors and the immune response. Cholera toxin activates plasma membrane adenylate cyclase in all mammalian cell types. The structure-function relationship of the toxin has recently been clarified, and the cell membrane receptor identified. This information has made cholera toxin the "agent of choice" for studies in many biological systems of the possible regulatory role of adenylate cyclase/cyclic AMP. This article describes briefly our current knowledge about the toxin and its receptor. It then reviews recent research which has revealed that cholera toxin has strong modulating influences on the proliferation of normal and malignant lympocytes as well as on the initiation and expression of immune responses. The toxin has been found to inhibit DNA synthesis of B and T lymphocytes in vitro without inducing cell death and also to inhibit seems to decrease antibody secretion from plasma cells in vitro, and might also interfere with the release of other soluble immunological mediator subtances. In vivo cholera toxin induces a transient involution of the spleen and a more prolonged lymphocyte depletion of the thymus in mice; these effects appear to be mediated through the adrenal glands. The toxin inhibitors allograft rejection, and either stimulates or suppresses antibody formation depending on the timing of the toxin in relation to the antigen administration. It increases the capacity of the spleen cells to induce graft-vs-host reactions and the "allogeneic effect" on antibody production. An inhibitory effect on a normal suppressor population among the spleen cells has been identified. The findings illustrate the complex effects induced on the immune system by the probably most discriminative investigative tool available for stimulation of the adenylate cyclase/cyclic AMP system.


Role of macrophages in hepatitis induced by Herpes simplex virus types 1 and 2 in mice. A marked difference was found between herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in the induction of hepatic necrotic lesions in mice inoculated intraperitoneally. Although HSV-2 produced many large, progressive liver lesions in 4-week-old BALB/c mice, HSV-1 only occasionally induced a few, self-limiting foci, which eventually healed. This was reflected in the isolation of HSV from the liver and spleen, two organs that are rich in macrophages. Although HSV-1 could be only temporarily isolated, HSV-2 was found in the two organs until the mice died. On the other hand, no such difference was found in the isolation of virus from the brain, which contains no macrophages, and the mice eventually died from encephalitis. This difference in hepatic involvement caused by the two virus types was found to parallel a marked difference in the restriction of HSV-1 and HSV-2 replication by macrophages as measured by an infectious center assay in vitro. HSV-2 produced 17 times as many infectious centers in infected peritoneal macrophage cultures as did HSV-1. Furthermore, the HSV-2 plaques in the cell overlay were large and increasing in size, whereas the HSV-1 plaques were small and showed regression on prolonged incubation. It was shown that this diversity was unique to the macrophage population and not caused by differences in the uptake of virus by macrophages. This model involving two closely related virus types shows the importance of tissue macrophages in the primary host defense against virus infections.


Mechanisms of resistant of herpesviruses: comparison of the effectiveness of different cell types in mediating antibody-dependent cell-mediated cytotoxicity. Enriched populations of polymorphonuclear leukocytes (PMN) and macrophages obtained from the mammary gland and of granulocytes (PBG) and lymphocytes (PBL) prepared from peripheral blood of the same animal were compared for their ability to mediate antibody-dependent cell cytotoxocity against antibody-sensitized infectious bovine rhinotracheitis virus-infected target cells (IBR-GBK) and antibody-sensitized chicken erythrocyte targest (CRBC). The order of effectiveness was PMN leads to macrophages leads to PBG leads to PBL. The reason why PBG (86% PMN) were less than 50% as active as mammary PMN (99% PMN) was explored and discussed. The findings that PMN were more effective on a cell-to-cell basis, required less antiserum to sensitize for cytotoxicity, and destroyed IBR-GBK cells faster and more completely than other cell types could mean that PMN may be the cell type most importnat in causing early recovery from herpesvirus infections.


Effect of water temperature on a herpesvirus infection of sea turtles. The role of water temperature in the induction and maintenance of a dermal herpesvirus infection (gray-patch disease) of young, green sea turtles (Chelonia mydas) was studied under carefully controlled experimental conditions, in which the influence of other recognized stress factors was negligible. A nimals that were subjected to a gradual temperature increase from 25 to 30 degrees C, with subsequent maintenance at 30 degrees C, and those that were abruptly shifted from water at 25 degrees C to water at 30 degrees C showed a significantly shorter period before the onset of clinical signs and an increase in the severity of the lesions when compared with control animals. Animals that were subjected to a gradual increase in water temperature from 25 to 30 degrees C and a subsequent decrease to 25 degrees C, where they were maintained, had a period before onset of clinical signs and severity closer to that of control animals. Our findings indicate that both the induction of clinical gray-patch disease and the severity of the lesions are affected by water temperature and suggest that one possible means of control of this herpesvirus infection under intensive aquaculture conditions might be water temperature manipulation.


Capsular polysaccharide of Clostridium perfringens Hobbs 9. Several closely related capsular polysaccharides were isolated from a strain of Clostridium perfringens Hobbs 9 type A by extraction of encapsulated cells with cold 0.85% NaCl. The soluble polymers were precipitated with alcohol and purified by (NH4)2SO4 fractionation, enzymatic digestion with papain and ribonuclease, and chromatography on diethylaminoethyl-Sephadex A25. The polysaccharides were composed mainly of glucose, galactose, and galactosamine. The major fraction contained these constituents (representing 77% of the dry weight) in a molar ratio of 1:1.6:1.1. All of the fractions contained phosphate and peptide material that was not removed during purification. The polysaccharides were closely related but not identical as indicated by double-diffusion-in-gel experiments. Immunoelectrophoresis in agarose demonstrated that the polysaccharides had identical mobilities and that no resolution into additional fractions occurred. The immunological activity of all the purified polysaccharides was destroyed by periodate oxidation but was unaffected by protease.


Reliable in vivo model for latent herpes simplex virus reactivation with peripheral virus shedding. A long-sought laboratory model, a reliable system for in vivo reactivization of latent herpes simplex infection with peripheral shedding of active virus, is present. Results demonstrate that surgical stimulation of latently infected rabbit trigeminal ganglia induces peripheral release of infectious herpes simplex virus. Virus could be cultured from the tear film in 83% of the stimulated animals' eyes within 48 h.


Antigenic relationships among several simian varicella-like viruses and varicella-zoster virus. Cross-neutralization and complement fixation tests demonstrated the immunological identity of the Delta herpesvirus, the 592S virus, the Liverpool vervet monkey virus, the herpesvirus of patas monkeys, and the Medical Lake macaque virus. These viruses were isolated from diverse outbreaks of varicella-like disease in simians and from various simian species. All of the simian viruses were shown to be related to human varicella-zoster (V-Z) virus, as evidenced by the fact that immunization of monkeys with each of the simian viruses elicited the production of both neutralizing and complement-fixing antibodies to V-Z virus. However, cross-complement fixation tests indicated that the simian viruses are not so closely related to V-Z virus as they are to one another. Varicella or zoster infections in humans produced neutralizing and complement-fixing antibody responses to each of the simian viruses; the responses were more marked in zoster infections than in varicella infections but, in most patients, antibody levels produced to the simian viruses were much lower than those to the homologous V-Z virus.


Complement-fixing reactivity of Varicella-Zoster virus subunit antigens with sera from homotypic infections and heterotypic Herpes simplex virus infections. Various subunit antigens of varicella-zoster (V-Z) virus were examined for complement-fixing (CF) activity with sera from homotypic infections and from herpes simplex virus (HSV) infections in which a CF antibody titer rise was demonstrated with crude V-Z antigen. The subunit antigens included nucleocapsids, envelopes, a soluble antigen produced from infected culture fluids by sucrose density gradient centrifugation, a soluble antigen produced by reducing the volume of clarified infected culture fluids, a soluble antigen derived from infected cell lysates, a "viral" antigen consisting largely of enveloped particles with a few nucleocapsids, and a cell membrane-associated antigen. None was more suitable than crude V-Z antigen for serodifferentiation of V-Z virus and HSV infections. The envelope antigen, cell membrane antigen, and the soluble antigen prepared by density gradient centrifugation showed little reactivity with sera from varicella and HSV infections, but gave high antibody titers with sera from zoster infections, suggesting that a secondary V-Z virus infection is required to produce an antibody response to these subunit antigens. Patients with varicella and zoster infections and the selected patients with HSV infections all showed significant CF antibody responses to the other V-Z subunit antigens.


Solid-phase radioimmunoassay of human immunoglobulin M and immunoglobulin G antibodies against herpes simplex virus type 1 capsid, envelope, and excreted antigens. A solid-phase radioimmunoassay developed in our laboratory for detection of human viral immunoglobulin M (IgM) and IgG antibodies was applied to demonstrate human class-specific antibody response against capsid, envelope, and excreted antigens of herpes simplex virus type 1. In primary infections, a clear IgM and IgG antibody response was found predominantly against the envelope components, whereas the IgM and IgG antibodies to the capsid antigen appeared more slowly. Increasing IgG antibody titers to the excreted antigen were also found in primary infections, though appearing more slowly than antibodies to the other subunit antigens. The antibody response against capsid and envelope antigens was not type specific, whereas in primary infections IgG class antibodies against the excreted antigen showed distinct type specificity. In recurrent infections, no significant level of IgM class antibodies was demonstrated, but in the patients with a severe secondary herpes simplex virus infection a definite IgM class antibody response was found against the envelope antigen. In addition, during severe secondary infections the antibody response against the excreted antigen was enhanced. The host IgG antibody response in recurrent infections was directed against the envelope and excreted antigens, whereas the level of the capsid antibodies was relatively stable.


Contrasting effects of immunosuppression on Theiler's virus infection in mice. In the present study, cyclophosphamide and rabbit anti-mouse thymocyte serum were used to immunosuppress SJL/J mice infected with Theiler's mouse encephalomyelitis virus (TMEV) in order to delineate the potential mechanism(s) of virus-induced cellular injury in this infection. Whereas both immunosuppressive agents produced a significant increase in mortality, this treatment had differing effects on the pathological involvement of gray and white-matter structures in the central nervous system. The central nervous system of immunosuppressed TMEV-infected mice had increased microglial cell proliferation and neuronal necrosis, longer maintenance of high virus levels and spread of virus antigen to involve the neocortex and hippocampal complex. These observations indicate that TMEV causes a cytolotic infection of neurons and possibly other cells in gray matter. In contrast, immunosuppression produced a dramatic reduction in mononuclear inflammatory cells in the leptomeninges and spinal cord white matter of infected mice and prevented demyelination. Further, virus antigen was not detected in the leptomeninges and white matter of immunosuppressed and infected mice. These findings suggest that demyelination of TMEV infection is immune mediated.


Effect of Clostridium perfrongens enterotoxin in mitochondrial respiration. Clostridium perfringens enterotoxin caused a 26 to 41% reduction in the rate of oxygen consumption by rat liver mitochondria when various tricarboxylic acid cycle intermediates were used as substrate. However, P/O ratios were unaltered.


Recent advances in pulmonary gas exchange. Abnormal gas exchange frequently accompanies general anesthesia, and the mechanisms are probably variable and multiple. This chapter has focused on recent advances in both experimental and theoretical approaches to gas exchange in general, with emphasis on the potentially important areas for the anesthesiologist wishing to study gas during anesthesia. Experimentally it is clear that if complex multifactorial mechanisms of abnormal gas exchange are to be described, methods must developed that give significantly more information than is available by standard techniques. Even while such methods are beginning to appear, it is probable that even the most sophisticated of approaches cannot give all of the fine detail one might seek, and that critical evaluation of just what information can be obtained is of great importance. Notwithstanding the practical limitations of such methods, much can be gained by taking advantage of digital computer models of the lung. In only ten years since such models were first introduced, the understanding of complex interactions in pulmonary gas exchange has certainly exceeded the sum of previous knowledge in this area and has allowed clear-cut conclusions of fundamental importance to be reached.


Detection by indirect immunofluorescence of Fc receptors in cells acutely infected with Herpes simplex virus. In attempting to use the indirect fluorescent antibody test (IFA) to measure antibodies to herpes simplex virus (HSV), we found that all human sera gave a positive reaction with Chang liver cells infected with type 1 (HSV). All sera gave equivalent titers of 320-640 for acetone-fixed cells and about 40 for live cells (membrane fluorescence) in the presence of fluorescein-labeled antisera to human Ig; none of the sera reacted with uninfected cells. The fluorescence seen in fixed cells was primarily cytoplasmic; some cells showed a diffuse fluorescence, obscuring the demarcation between the nucleus and cytoplasm. Purified IgG from antibody-negative human sera and a purified Fc fragment of IgG were positive both for cytoplasmic and membrane fluorescence, whereas F(ab')2, IgM and IgA were unreactive. The reaction was also seen when an antiserum conjugate specific for the Fc fragment of IgG was used. The reactive IgG was present in freshly prepared plasma and serum; it could not be removed from serum either by ultracentrifugation or by serial absorption with HSV-infected cells. These findings suggest that the nonspecificity of the IFA results from the formation of low-avidity bonds between the large mass of native serum IgG and an Fc receptor on the plasma membrane and in the cytoplasm of cells infected with HSV. The results also suggest that extreme caution be exercised in attempting to use the IFA in the serodiagnosis of infections with HSV and perhaps the other human herpes-viruses.


Hypolipidemic effect of tibric acid. A comparison with clofibrate and placebo in type IV hyperlipoproteinemia. The effects of tibric acid, clofibrate, and placebo were compared in type IV hyperlipidemic patients for a 6-month period. The patients were divided into two pathological level groups according to their baseline triglyceride levels. Compared to the placebo, both tibric acid and clofibrate reduced the mean serum triglyceride concentration in the high pathological level group; however, clofibrate was also effective in the low pathological level group. The effects on total cholesterol were less pronounced with the two drugs. No effect was observed on esterified cholesterol, phospholipids, free fatty acids, and fasting blood sugar. After a 6-week follow-up period under placebo, no rebound of the  triglyceride and cholesterol levels could be observed after discontinuation of the two active drugs. The other biochemical changes observed after each active treatment are discussed in relation to the different degree of activity of each active drug and to the different baseline levels of triglycerides.


Free-radical formation in gamma-irradiated oriented DNA containing electron-affinic radiosensitizers. Electron paramagnetic resonance (e.p.r.) was used to study the free radicals induced by gamma-irradiation at 77 K in oriented DNA with incorporated electronaffinic radiosensitizing compounds (4-nitroacetophenone, metronidazole, and Ro-07-0582). The observed e.p.r. spectra were compared with those obtained from pure oriented DNA, which had previously been analysed in detail and found to consist mainly of two components, arising from anion redicals on thymine, and cation radicals on guanine. The major spectral changes caused by the radiosensitizers could be explained as a considerable increase in the formation of cationic free radicals on guanine. There were also indications of the formation of anion radicals on the radiosensitizer molecules. No hydrogen-addition free radicals on thymine were observed when the radiosensitized samples were annealed, in contrast to the pure DNA samples.


Attenuation of Japanese encephalitis virus by brain passage of guinea pigs. JE virus was difficult to keep alive in guinea pig brain beyond 3 days and the virus was lost after 3 days. Ten cross passages from guinea pig to mice attenuated JE virus (GP10) and the result indicated that 3 passages could induce illness in guinea pig (GP4). GP4 and GP10 did not induce viremia to mice. 0.1 ml of 1:100,000 and 1:10,000 mouse suspension of GP4 inoculated into the author subcutaneously did not cause any uncomfortable symptom.


Ultrastructure of human prostatic epithelium. Secretion granules or virus particles? The objective of this report is to demonstrate that only slight and superficial similarities exist between true virus particles and secretion granules in human prostatic epithelium. In spite of this, secretion granules have sometimes been called virus or virus-like particles. With the current interest in the possible role of viruses in the etiology of some human cancers, many investigations are in progress in search of human tumor viruses. The presence of RNA virus particles in normal as well as tumor tissues derived from animals and man, further complicates the picture. Also, in view of the fact that recent studies indicate an association between herpes simplex type 2 virus and cervical cancer, the possibility of the involvement of the same virus, which has a venereal mode of transmission, in prostatic and other genitourinary cancers in the human male has been considered. Investigators with peripheral interests in such studies are cautioned against making quick decisions on the nature of the "virus-like particles" often observed in secretory cells because these particles often happen to be cell organelles.


Purification of Sendai virions with glutaraldehyde-treated red blood cells. The use of glutaraldehyde (GA)-treated chicken red blood cells (RBC) for purification of Sendai virions by adsorption and elution was found to be advantageous over the use of untreated RBC. Biologic and enzymatic activities and structural components of the purified virions were compared with those of the original virions. The purified virions lost glucosamine-labeled non-proteinous materials. These materials did not relate to the expression of infectivity or cell fusion activity of Sendai virus. No evidence of disruption of Sendai virions during interaction with GA-RBC was observed. The use of GA-RBC provides a new method to prepare an unadsorbed virus fraction with a minimum contamination of hemagglutinins. The unadsorbed fraction consisted of Sendai virions with weak hemagglutinating activity and smaller amounts of the gp1(HN) polypeptide and with negligible hemolytic and cell fusion activities. The origin of these particles is discussed.


Psychiatric consultation services in the large general hospital: a review and a new report. Studies are reviewed documenting increased interest in psychiatric consultation services in the general hospital, and disparities between the need for and the availability/utilization of consultation services are examined. The emergence of the eclectrically trained psychiatrist as the most acceptable consultant in the general hospital is discussed. An exploratory attiduinal questionnaire, developed and circulated to a sample of attending physicians in a midwestern university teaching hospital, yielded the following data. Thirty-nine per cent (N=96) of the 244 physicians who received the questionnaire responded. Ninety-nine per cent of the respondents agreed that readily available psychiatric consultation services should be fully operative in all large general hospitals; 88% agreed that psychiatric consultations in the general hospital must be performed by a physician with special training in  psychological medicine; 50% indicated a preference for a biologically-oriented psychiatrist, and 26% preferred a family practitioner or internist who had subsequently become a psychiatrist. The majority of respondents indicated that between July 1974 and July 1975, 1-5% of their private hospitalized patients received inhospital psychiatric consultation. Eighty-six per cent of the respondents felt the patients should be billed directly at a rate equal to other medical/surgical consultations. These data are correlated with issues involved in the utilization of psychiatric consultation in the general hospital.


Comparative analysis of polypeptides induced by type 1 and type 2 strains of herpes simplex virus. The polypeptides synthesized in HEp-2 cells infected with 7 type 1 and 8 type 2 strains of herpes simplex virus were examined by PAGE. The polypeptides produced were consistent for each type and clearly different from those produced by infection with the other type.


DNA-binding proteins in mouse cells infected with polyoma virus. DNA-binding proteins from uninfected or polyoma virus-infected mouse cells (3T6) were isolated and compared by DNA-cellulose chromatography and SDS-PAGE. During the early phase of infection, few but significant and reproducible differences were observed. In the 0.4 M NaCl eluate, four major new polypeptides were detected in infected cells, with approximate molecular weights of 160 K, 130 K, 86 K and 81 K, the last two being compatible with previous estimates for polyoma T antigen molecular weight.


Structural polypeptides of Sendai virus: analysis by tryptic peptide mapping. Tryptic peptide patterns identified Sendai virus polypeptides polypeptides HN, HN, P,F0, NP, and M as unique gene products, and confirmed the precursor-product relationship of polypeptides F0 and F. By the same criterion, the major nucleocapsid polypeptides, P and NP, in virions were derived from the same genes as their counterparts in nucleocapsids obtained from infected cells.


Effect of virus infection and cycloheximide treatment on the labeling of cellular phospholipids with 32 P. The total amount and the relative proportions of labeled phospholipids were studied in chorioallantoic membrane cells (CAM), chick embryo cells (CEC) and L cells which were either infected with Newcastle disease virus (NDV), fowl plague virus (FPV) and mouse encephalomyocarditis (EMC) virus or treated with cycloheximide. In productively infected or nonproductively infected cells, the total amount of labeled phospholipids was not altered, but the relative proportions of individual phospholipids were modified. These modifications were related to the presence of viral proteins. Blocking of protein synthesis for 6 h with 7 microng cycloheximide/ml did not modify the total amount of labeled phospholipids in CAM and CEC. Less label was incorporated into cyclohexidime-treated L cells than in controls. The relative proportions of the labeled individual phospholipids were modified in all three cell types and these modifications were different from those in virus-infected cells.


The effect of phosphonoacetic acid on the in vitro replication of varicella-zoster virus. Phosphonoacetic acid (PAA) was found to inhibit in vitro varicella-zoster virus (VZV) replication. At 100 microng/ml, PAA blocked the development and spread of VZV cytopathology in both growing and contact-inhibited cultures. Formation of virus plaques by infected cells and cell-free virus was effectively blocked at concentrations as low as 25 microng/ml. The development of nuclear and cytoplasmic virus antigens was also inhibited by PAA. Inhibition by PAA suggests that VZV DNA replication, like that of other herpes-viruses, involves a virus-specific DNA polymerase.


Isolation of Aleutian disease virus of mink in cell culture. Aleutian disease virus, the causative agent of a persistent infection in mink, was isolated in a continuous line of feline renal cells when the cultures were maintained at reduced temperature (31.8 degrees). After serial in vitro passage of the virus at this temperature it had an optimum replication temperature of 37 degrees. An immunofluorescence focus assay was found to be suitable for virus quantitation. The cultured virus reproduced Aleutian disease in mink, and the virus could be reisolated from the mink 10--180 days after inoculation. The properties of the virus suggest that it is a member of the parvovirus group.


Morphological transformation of rat embryonic fibroblasts by abortive herpes simplex virus infection: increased transformation rate correlated to a defective viral genotype. Rat embryo fibroblasts were abortively infected with various stocks of herpes simplex virus type 1 strain ANG at 42 degrees. In uninfected controls all of the cells died during an 8-day incubation period at the elevated temperature, whereas varying numbers of cells in the infected cultures survived and formed colonies during subsequent incubation for 3--4 weeks at 37 degrees. All of the survivors appeared to be morphologically transformed. Two types of survivors, epithelial- and spindle-like cells, which occurred at a ratio of approximately 1:1 in all assays, could be distinguished. The observed survival rates increased from about 1 x 10-7 to 3 x 10-5, corresponding to increasing fractions (0--50%) of a defective genotype present in the infecting virus stocks. The individual survival rates do not depend exclusively on the quantity of defective virions. The existence of different subtypes of defective genomes as a further parameter is discussed.


A sensitive method for the detection of herpes simplex virus type 2 specific thymidine kinase. Thymidine kinase (TK) activity of uninfected and in vitro herpes simplex virus type 2 (HSV-2) infected human fetal lung cells was analyzed. Polyacrylamide gel electrophoresis was carried out, and subsequent incubation of equally divided gel slices in the presence of normal or anti-HSV-2 specific TK serum, together with different substrates, was performed. Small amounts of virus-specific enzyme could be detected using this combination of electrophoresis and antibodies and replacing ATP by CTP in the substrate medium.


Ultrastructural cytochemistry of the secretory granules of the hamster submandibular gland. The ultrastructure and cytochemistry of the secretory granules of the male hamster submandibular salivary gland were studied; After fixation in glutaraldehyde followed by osmium tetroxide the granules exhibit a characteristic bipartite substructure, with an electron lucid crescenteric rim and a more dense central core. A differentiation into two regions of the granules could also be visualized in specimens primarily fixed in Millonig's osmium tetroxide or in potassium permanganate. The electron lucid peripheral portion of the membrane bounded secretory granules further displays a strong positive reaction after staining of ultrathin sections with the periodic acid-chromic acid-(PA-CrA)-silver technique. The strong periodate reactivity of the rim relative to the core, suggests a difference in mucin composition of the two granule regions. With the PA-CrA-silver straining technique a positive reaction was also observed within the Golgi apparatus of the acinar cells.


Cellular distribution of sarcoplasmic calcium-binding proteins by immunofluorescence. Specific antibodies against carp paravalbumin, crayfish calcium binding protein and crayfish arginine kinase were used for indirect immunofluorescence localization of the respective proteins. Simultaneous staining of the same muscle sections with human serum containing anti-actin autoantibodies served as a probe to identify the isotropic band. Parvalbumin appears to be evenly distributed in carp white muscle. The crayfish calcium binding protein however shows a distinct localization, in the isotropic band, coincident with the actin staining. Arginine kinase, which has the same molecular weight and is extractible in the same way as the calcium binding protein does not show this distinct localization, but is evenly present in crayfish tail muscle, similarly to parvalbumin. The possible meaning of the different distribution of the two calcium binding proteins is discussed.


[Synovial sarcoma of the neck (author's transl)]. Two cases of synovial sarcoma of the neck are reported. As it is already known from comparable literature, the extraoridinary localisation and the unusual histological pattern showing sarcomatous and synovial elements may cause major difficulties in diagnosis. In both cases the treatment consisted of radical surgical removal.


Nine simultaneous primary tumors in a Boxer dog. An aged Boxer dog had 9 distinct primary tumors: chemodectoma, osteosarcoma, bronchiolo-alveolar adenocarcinoma, interstitial cell tumor, seminoma, basal cell tumor, fibropapilloma, adrenal cortical adenoma, and pancreatic adenoma. From this report, as well as from other studies, it is clear that Boxers have special susceptibilities to a variety of tumors. Analysis of clinical data on canine tumors indicated that the risk of Boxers for multiple tumors is only slightly higher than all tumors, indicating little or no specific predisposition for multiple tumors. In Boxers, however, certain tumor types occurred more frequently as multiple primary tumors than would be expected by chance.


An estimation of the fibre type compostion of eleven skeletal muscles from New Zealand White rabbits between weaning and early maturity. Fibre types in 11 skeletal muscles from New Zealand White rabbits were differentiated on the basis of histochemical staining reactions for Ca2+-adenosine triphosphatase (Ca2+ATPase) at pH 9-4, cytochrome oxidase, succinate dehydrogenase and L-glycerol-3-phosphate:menadione oxidoreductase activities. Using these enzyme reactions it was convenient to divide muscle fibres into three main categories in 'white' muscles and two in 'red' muscles. Between weaning and early maturity most muscles showed little change in fibre type composition, particularly when Ca2+-ATPase activity was used as the criterion. Many muscles showed an uneven distribution of fibre types in transverse sections; this was particularly so in the cases of longissimus, semitendinosus, soleus and semimembranosus proprius. The methods successful in resolving fibre types in mature muscles were not so capable of resolving fibre types in neonatal muscles.


Sensitive silica gel-packed flowcell for fluorometric detection of aflatoxins by high pressure liquid chromatography. Aflatoxins B1, B2, G1, and G2 were quantitatively detected by high pressure liquid chromatography on a 5 micronm Lichrosorb column, using a Lichrosorb-packed flowcell in the fluorometric detector. The relationship between peak height and the amount injected was linear only up to about 2 ng but showed a linear loglog relationship. Methods for constructing and packing the flowcell are given. A guard column and venting valve were used to minimize deterioration of the analytical column and the adsorbent-packed flowcell. The method was applied to a peanut butter extract, although with the cleanup procedure used, the life expectancy of the flowcell is limited.


Detection and semiquantitative estimation of thyroxine and diiodothyronine in liothyronine sodium. A method is presented for the detection and semiquantitative estimation of thyroxine and diiodothyronine in liothyronine sodium. Thyroxine is detected by thin layer chromatography, using silica gel H plates developed in butanol-acetone-ammonia (30 + 55 + 15) and sprayed with a 2,7-dichlorofluorescein solution, and estimated by comparison with standard spots. For quantitative results, a larger amount of sample is applied as a streak on a fluorescent silica gel H plate. After a 5 hr development, the band is scraped off and extracted in a small volume of 0.1N NaOH; the extract is centrifuged, transferred to a 2 cm microcell, and examined spectrophotometrically. The diiodothyronine content of liothyronine sodium is estimated by liquid chromatography on a Bondapak C18 column with 0.01N sodium perchloratebutanol-acetonitrile (1000 +62 + 188) as the eluting solvent.


Transcription of the hut operons of Salmonella typhimurium. We have measured, by ribonucleic acid-deoxyribonucleic acid hybrid formation, the amounts of hut-specific ribonucleic acid contained in extracts of various mutant strains of Salmonella typhimurium. Our data are consistent with a model in which regulation of Hut enzyme production occurs at the level of transcription and support earlier genetic evidence indicating that all of the hut genes are transcribed in the clockwise direction on the S. typhimurium chromosome. These results also suggest that promoter sites of the two hut operons may differ in their ability to initiate transcription.


Resolution and purification of three periplasmic phosphatases of Salmonella typhimurium. A survey of Salmonella typhimurium enzymes possessing phosphatase or phosphodiesterase activity was made using several different growth conditions. These studies revealed the presence of three major enzymes, all of which were subsequently purified: a cyclic 2' ,3'-nucleotide phosphodiesterase (EC 3.1.4.d), an acid hexose phosphatase (EC 3.1.3.2), and a nonspecific acid phosphatase (EC 3.1.3.2). A fourth enzyme hydrolyzed bis-(p-nitrophenyl)phosphate but none of the other substrates tested. No evidence was found for the existence of an alkaline phosphatase (EC 3.1.3.1) or a specific 5'-nucleotidase (EC 3.1.3.5) in S. typhimurium LT2. All three phosphatases could be measured efficiently in intact cells, which suggested a periplasmic location; however, they were not readily released by osmotic shock procedures. The nonspecific acid phosphatase, which was purified to apparent homogeneity, yielded a single polypeptide band on both sodium dodecyl sulfate and acidic urea gel electrophoretic systems.


Regulation of two phosphatases and a cyclic phosphodiesterase of Salmonella typhimurium. The regulation of three Salmonella typhimurium phosphatases in reponse to different nutritional limitations has been studied. Two enzymes, an acid hexose phosphatase (EC 3.1.3.2) and a cyclic phosphodiesterase (EC 3.1.4.d), appear to be regulated by the cyclic adenosine 3' ,5'-monophosphate (AMP) catabolite repression system. Levels of these enzymes increased in cells grown on poor carbon sources but not in cells grown on poor nitrogen or phosphorus sources. Mutants lacking adenyl cyclase did not produce elevated levels of these enzymes in response to carbon limitation unless cyclic AMP was supplied. Mutants lacking the cyclic AMP receptor protein did not produce elevated levels of these enzymes in response to carbon limitation regardless of the presence of cyclic AMP. Since no specific induction of either enzyme could be demonstrated, these enzymes appear to be controlled solely by the cyclic AMP system. Nonspecific acid phsphatase activity (EC 3.1.3.2) increased in response to carbon, nitrogen, phosphorus, or sulfur limitation. The extent of the increase depended on growth rate, with slower growth rates favoring greater increases, and on the type of limitation. Limitation for either carbon or phosphorus resulted in maximum increases, whereas severe limitation of Mg2+ caused only a slight increase. The increase in nonspecific acid phosphatase during carbon limitation was apparently not mediated by the catabolite repression system since mutants lacking adenyl cyclase or the cyclic AMP receptor protein still produced elevated levels of this enzyme during carbon starvation. Nor did the increase during phosphorus limitation appear to be mediated by the alkaline phosphatase regulatory system. A strain of Salmonella bearing a chromosomal mutation, which caused constitutive production of alkaline phosphatase (introduced by an episome from Escherichia coli), did not have constitutive levels of nonspecific acid phosphatase.


Inactivation of fructose diphosphatase by sucrose in yeast. In Saccharomyces carlsbergensis, the addition of sucrose to the medium led to the inactivation of fructose diphosphatase. Sucrose itself probably triggered this process.


Mutational analysis of serine-glycine biosynthesis in Rhodopseudomonas capsulata. Rhodopseudomonas capsulata possesses the enzymes of both the "phosphorylated" and the "non-phosphorylated" pathways of serine biosynthesis. Certain mutants with lesions in the phosphorylated pathway are serine-glycine auxotrophs, though they still produce enzymes of the non-phosphorylated sequence. These results indicate that the phosphorylated pathway is essential for the synthesis of serine and glycine in R. capsulata under the condtions tested.


Specific thiamine monophosphate phosphohydrolase in Micrococcus denitrificans. A phosphohydrolase specific for thiamine monophosphate was isolated from Micrococcus denitrificans, partially purified approximately 100-fold, and separated from acid phosphatase.


Biochemical characterization of an Escherichia coli hisT strain. An Escherichia coli hisT strain was characterized biochemically and shown to contain altered transfer ribonucleic acid and to be altered in the regulation of amino acid biosynthesis.


Differential binding of cyclic adenosine 3' ,5'-monophosphate to the cyclic adenosine 3' ,5'-monophosphate receptor protein in Escherichia coli. Binding of cyclic adenosine 3' ,5'-monophosphate (cAMP) by the cAMP receptor protein in crude cell-free extracts of Escherichia coli was characterized. When cell were grown in glucose, binding was inhibited 50% relative to extracts from cells grown with succinate as carbon source . This inhibition could be relieved by dialysis.


Purification and characterization of catalytic subunit of skeletal muscle adenosine 3':5'-monophosphate-dependent protein kinase. The catalytic subunit of rabbit skeletal muscle cyclic adenosine 3':5'-monophosphate-dependent protein kinase has been isolated in pure form. It has a molecular weight of 41,300, as determined by sedimentation equilibrium, which is in good agreement with the value of 41,000 determined by electrophoresis in the presence of sodium dodecyl sulfate. Sedimentation velocity determinations indicate that the subunit has an S20,w value of 3.12 which is essentially independent of protein concentration. These experiments are interpreted as indicating that the catalytic subunit dissociated from the holoenzyme exists as a monomer in solution. The least abundant amino acid is half-cystine, which was calculated to be present at 2.8 mol/mol of protein. The sulfhydryl reagents, N-ethylmaleimide, p-chloromercuribenzoic acid, and 5,5'-dithiobis(2-nitrobenzoic acid) inhibit the enzymatic activity of the subunit; inhibition by the two latter compounds can be reversed by 2-mercaptoethanol. Binding of 1 mol of N-ethylmaleimide/mol of protein results in almost complete inhibition. The isolated catalytic subunit contains 2.2 mol of tightly bound phosphate/mol of protein. Identification of either O-phosphoserine or O-phosphothreonine after partial acid hydrolysis indicates that at least part of the endogeneous phosphate exists as the phospho ester of one of these amino acids. Two peaks of catalytic activity corresponding to isoelectric points of pH 7.4 and 8.5 were identified by isoelectric focusing. Both forms utilize the same substrates and have similar sedimentation constants.


Intracellular utilization of superoxide anion by indoleamine 2,3-dioxygenase of rabbit enterocytes. The participation of superoxide anion (O2-) in the intracellular indoleamine 2,3-dioxygenase activity was studied using the dispersed cell suspension of the rabbit small intestine. The dioxygenase activity was assayed by measuring [14C]formate released from DL-[ring-2-14C]tryptophan. The addition of diethyldiethiocarbamate, a superoxide dismutase inhibitor, markedly accelerated the intracellular dioxygenase activity while the superoxide dismutase activity decreased concomitantly. Furthermore, substrates of xanthine oxidase such as inosine, adenosine, and hypoxanthine also increased the dioxygenase activity in the cells, particularly in the presence of methylene blue. This increase was completely abolished by the addition of allopurinol, a specific inhibitor of xanthine oxidase. These results, taken together, indicate that the intracellular accumulation of O2- results in acceleration of the in situ dioxygenase activity, and that indoleamine 2,3-dioxygenase utilizes O2- in the isolated intestinal cells.


Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone. Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.


Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes. The dehydrogenation reaction of cholest-7-en-3beta-ol (I) to cholesta-5,7-dien-3beta-ol (II) in the presence of NADH was studied in rat liver microsomes and in microsomal acetone powder preparations, using [3alpha-3H]cholest-7-en-3beta-ol. It was found that the reaction was inhibited by menadione, adenosine diphosphate, potassium ferricyanide, and cytochrome c while p-cresol had no effect. These results indicated the participation of a microsomal electron transport system in the dehydrogenation of cholest-7-en-3beta-ol. The conversion of cholest-7-en-3beta-ol to cholesta-5,7-dien-3beta-ol was also observed in the absence of NADH when ascorbic acid was included in the incubation mixture. However, the ascorbic acid-catalyzed dehydrogenation was not inhibited by potassium ferricyanide. Immunological evidence that microsomal cytochrome b5 is involved in the dehydrogenation of (I) to (II) was obtained. Antibodies specific for rat liver microsomal cytochrome b5 were elicited in rabbits. The anticytochrome b5 immunoglobulin fraction inhibited rat liver microsomal NADH-cytochrome c reductase but not NADPH-cytochrome c reductase. Also, the extent of reduction of cytochrome b5 was not affected by the antibodies. The conversion of (I) to (II) by rat liver microsomes was inhibited (73%) by anticytochrome b5 immunoglobulin at a ratio of microsomal protein:immunoglobulin of 1:5.6. These results are consistent with the participation of microsomal cytochrome b5 in the introduction of the C-5 double bond in cholesterol biosynthesis. A close analogy of the microsomal dehydrogenation of fatty acids and of cholest-7-en-3beta-ol is apparent and this suggests a possible similarity in the mechanisms of the two reactions.


Purification of phosphoprotein phosphatase from bovine cardiac muscle that catalyzes dephosphorylation of cyclic AMP-binding protein component of protein kinase. A phosphoprotein phosphatase that catalyzes the dephosphorylation of cyclic adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase from bovine cardiac muscle has been purified to homogeneity by a modification of the procedure of Brandt et al. (Brandt, H., Capulong, Z.L., and Lee, E. Y. C. (1975) J. Biol. Chem. 250, 8038-8044). Treatment of the enzyme preparation with ethanol during the early stages of purification results in activation concomitant with reduction in molecular weight to 30,000. The purified activated enzyme has a Km for phospho-protein kinase in the presence or absence of 1.2 mM Mn2+ of 5 and 22 micronM, respectively. Phosphatase activity on phospho-protein kinase but not on other phosphoprotein substrates was cAMP-dependent. This selective activation by cAMP reflects the preference of the phosphatase for the free, phosphorylated cAMP-binding protein rather than the phosphoholoenzyme.


Glucuronosyl-N-acetylglucosaminyl pyrophosphoryldolichol. Formation in SV40-transformed human lung fibroblasts and biosynthesis in rat lung microsomal preparations. Incubation of SV40-transformed human lung fibroblasts with [3H]glucosamine for 1 h. followed by chloroform:methanol extraction and thin layer chromatographic analysis, revealed the presence of a major radioactive lipid that was isolated and characterized as GIcUA-(1 leads to 4)-GlcNAc-P-P-dolichol. An identical lipid was formed in smaller quantities under similar incubation conditions in several fibroblastic lines, HeLa cells, and in mouse L cells. Rat lung microsomal preparations catalyze the synthesis of the disaccharide lipid in the following sequence of reactions: UDP-[3H]GlcNAc + dolichol-P leads to [3H]GlcNAc-P-P-dolichol (1) [3H]GlcNAc-P-P-dolichol + UDP-[14C]GlcUA leads to [14C]GlcUA-[3H]GlcNAc-P-P-dolichol (2) The double-labeled lipid was identical to the lipid isolated from SV40-transformed fibroblasts with regard to its behavior on thin layer and silicic acid chromatography. Further, the double-labeled disaccharide released from the lipid by mild acid hydrolysis was identical to GlcUA-(1 leads to 4)-GlcNAc in its chromatographic and electrophoretic behavior and in its composition. The occurrence of a polyprenol derivative of GlcUA-(1 leads to 4)-GlcNAc suggests a possible role for this lipid in the biosynthesis of the repeating disaccharide units of proteoglycans, such as heparin.


Ionic inhibition of catalytic phosphorylation of histone by bovine brain protein kinase. The effects of various ions commonly found in protein kinase assays upon the rate of histone phosphorylation catalyzed by the highly purified bovine brain enzyme, protein kinase I, have been investigated. Sodium, potassium, and magnesium were found to inhibit histone phosphorylation by protein kinase I  in a similar manner. The degree of inhibition by any of these cations was demonstrated to be directly proportional to the square root of the ionic strength of the assay medium. The relationship between the ionic strength of the assay medium and the rate of histone phosphorylation catalyzed by protein kinase I was employed to correct the rate of histone phosphorylation at various magnesium acetate concentrations to a standard ionic strength. When this was done an analysis of the previously postulated rate law for histone phosphorylation c atalyzed by protein kinase I gave a binding constant for the magnesium-ATP complex which was in agreement with that expected for this complex on  the basis of various binding constants available in the literature. These results demonstrate that it is unnecessary to postulate a specific ion inhibition process for protein kinase I by the ions employed in this study. They also support the reasonable assumption that magnesium ion binds to ATP at or prior to the rate-determining step in histone phosphorylation catalyzed by protein kinase I. The expression developed in this paper for the effect of ionic strength upon protein kinase I activity can now be used to correct activity measurements made under various assay conditions to a standard assay state, allowing facile comparisons of kinetic data. It should be possible to develop similar expressions for other protein kinases and substrates to permit useful interpretation of kinetic data.


Ca2+-dependent effect of ATP on spin-labeled sarcoplasmic reticulum. Vesicular fragments of sarcoplasmic reticulum (SR) were labeled with the --SH-directed spin label 2,2,6,6-tetra-methyl,4-amino(N-iodoacetamide). Colorimetric titrations of the remaining --SH residues and determinations of unbound spin label indicated that primarily 3 residues/enzyme molecule were labeled under saturating conditions. This labeling was accompanied by minimal losses in activity, providing precautions were taken to prevent sulfhydryl oxidation during the labeling process.  Additions of ATP produced a new "highly constrained" component in the ESR spectrum of the labeled SR, an effect not noted in previous studies. It is demonstrated that the changes produced by ATP are reversible, and require both substrate binding and Ca2+ binding. However, hydrolysis of the substrate is not required. It is further demonstrated that the labeled residue(s) responsible for the spectral change is not in the immediate vicinity of the ATP binding site. It is apparent that the observed spectral change is related to a conformational effect of ATP and Ca2+ on the ATPase protein, which is associated with a large free energy change occurring on binding. It is also suggested that the conformational effect extends to a significant distance from the nucleotide binding site and may be a precursory step to Ca2+ translocation.


Phospholipid synthesis in isolated fat cells. Studies of microsomal diacylglycerol cholinephosphotransferase and diacylglycerol ethanolaminephosphotransferase activities. Diacylglycerol cholinephosphotransferase (EC 2.7.8.2) and diacylglycerol ethanolaminephosphotransferase (EC 2.7.8.1) activities were investigated in microsomes from isolated rat fat cells. Assays based on the conversion of CDP-[14C]choline of CDP-[14C]ethanolamine to phosphatidylcholine or phosphatidylethanolamine utilized ethanol-dispersed diacylglycerols and 1 to 5 microng of protein. Cholinephosphotransferase and ethanolaminephosphotransferase activities had similar dependences on MgCl2 and pH, and were inhibited similarly by CaCl2, organic solvents, Triton X-100, Tween 20, and dithiothreitol. Ethylene glycol bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid stimulated both activities similarly. With 1,2-dioleoyl-sn-glycerol, the cholinephosphotransferase activity had an apparent Km for CDP-choline of 23.9 micronM and a V max of 8.54 nmol/min/mg. CDP-ethanolamine and CDP were competitive inhibitors of the cholinephosphotransferase activity (apparent Kl values of 227 micronM and 360 micronM, respectively). With 1,2-dioleoyl-sn-glycerol, the ethanolaminephosphotransferase activity had an apparent Km of 18.3 micronM for CDP-ethanolamine and a V max of 1.14 nmol/min/mg. CDP-choline appeared to be a noncompetitive inhibitor of the ethanolaminephosphotransferase activity (apparent Kl of 1620 micronM). Inhibition of the ethanolaminephosphotransferase activity by CDP appeared to be of a mixed type. The dependences on diacylglycerols containing fatty acids 6 to 18 carbons in length were investigated...


Preliminary results using superfractionation in the treatment of glioblastoma multiforme. Preliminary results of a phase I--phase II trial of the use of superfractionation radiation treatment in the treatment of Glioblastoma Multiforme are presented with the rationale for this form of treatment. A comparison is made with an historic control group from the same institution, and a significant improvement in survival is noted in the experimental group over this control group.


Putrescine transport is greatly increased in human fibroblasts initiated to proliferate. Putrescine (diaminobutane) was previously found to stimulate proliferation of human fibroblasts in tissue culture, and a growth factor produced by these cells was identified as putrescine. In the present paper putrescine transport is studied. The rate of putrescine transport was dependent on temperature, and most of the labeled putrescine was retained by the cells after washing with excess unlabeled putrescine. The concentration of radioactivity after a [14 C]putrescine pulse was 85 times higher in the cells than in the medium, and over 95% of the radioactivity in the cells was as unchanged putrescine. Butanol treatment removed 70% of the radioactivity from the cells. The calculated Km was about the same for rapidly growing and for starved cultures, while Vmax was higher for the former than for the latter cultures. Putrescine transport was inhibited to varying degrees by other polyamines, but not by amino acids or divalent cations. Stimulation of cell proliferation by serum was followed by an 18-100-fold increase in the rate of putrescine transport, which was not inhibitable with cyclic AMP, dibutyryl cyclic AMP, or prostaglandin E1. Removal of serum resulted in a rapid decrease in the rate of putrescine transport. Insulin in low serum medium and trypsin in the absence of serum also accelerated putrescine transport. Moreover, the rate of putrescine transport was dependent on cell density. It was faster in sparsely populated than in densely populated cultures. SV40-transformed human fibroblasts responded to addition and removal of serum in the same way as the untransformed parent cell line.


Characterization of surface glycoproteins of mouse lymphoid cells. We have labeled exposed surface glycoproteins of mouse lymphoid cells by the galactose oxidase-tritated sodium borohydride technique. The labeled glyco-proteins were separated by polyacrylamide slab gel electrophoresis and visualized by autoradiography (fluorography). The major thymocyte surface proteins have molecular weights of 170,000 and 125,000. Thymocytes from TL antigen-positive mouse strains showed an additional band with a molecular weight of 27,000. Highly purified T lymphocytes contain two major surface glycoproteins with molecular weights of 180,000 and 125,000. Purified B lymphocytes have one major surface glycoprotein with a molecular weight of 210,000. When T lymphocytes are stimulated in vitro by concanavalin A or phytohemag-glutinin, the major proteins characteristic of T cells are relatively weakly labeled, but new components of lower molecular weights appear on the cell surface. A similar change is seen in B lymphocytes stimulated by Escherichia coli lipopolysaccharide. T lymphoblasts isolated from mixed lymphocyte cultures show a slightly different surface glycoprotein pattern. A polypeptide with a molecular weight of 57,000, which was labeled without enzymatic treatment by tritiated sodium borohydride alone, is strongly labeled in proliferating cells.


Identification and transmembranous localization of active cytochrome oxidase in reconstituted membranes of purified phospholipids by electron microscopy. Cytochrome oxidase vesicles with high oxidase activity and respiratory control ratio (greater than 3.5) were characterized by the freeze-etch technique for electron microscopy. By the use of this technique, cytochrome oxidase is shown to be an inner membrane particle. By locating cross-fractured vesicles in the same preparation, cytochrome oxidase particles are shown to extend across the phospholipid bilayer membranes. When cytochrome oxidase is added to preformed liposomes respiratory control is not observed, but high oxidase activity is maintained. In this preparation the cytochrome oxidase particles are located on the outer vesicle membrane surface. These observations provide direct evidence that cytochrome oxidase is found in a transmembranous position in closed, activecytochrome oxidase vesicles having respiratory control.


Nuclear mutations affecting mitochondrial structure and function in Chlamydomonas. Wild type cells of the green alga Chlamydomonas reinhardtii can grow in the in the dark by taking up and respiring exogenously supplied acetate. Obligate photoautotrophic (dark dier, dk) mutants of this alga have been selected which grow at near wild type rates in the light, but rapidly die when transferred to darkness because of defects in mitochondrial structure and function. In crosses of the dk mutants to wild type, the majority of the mutants are inherited in a mendelian fashion, although two have been isolated which are inherited in a clearly nonmendelian fashion. Nine mendelian dk mutants have been analyzed in detail, and belong to eight different complementation groups representing eight gene loci. These mutants have been tentatively grouped into three classes on the basis of the pleiotropic nature of their phenotypic defects. Mutants in Class I have gross alterations in the ultrastructure of their mitochondrial inner membranes together with deficiencies in cytochrome oxidase and antimycin/rotenone-sensitive NADH-cytochrome c reductase activities. Mutants in Class II have a variety of less severe alterations in mitochondrial ultrastructure and deficiencies in cytochrome oxidase activity. Mutants in Class III have normal or near normal mitochondrial ultrastructure and reduced cytochrome oxidase activity. Eight of the nine mutants show corresponding reductions in cyanide-sensitive respiration.


Lipogenesis and the synthesis and secretion of very low density lipoprotein by avian liver cells in nonproliferating monolayer culture. Hormonal effects. The nonproliferating chicken liver cell culture system described yields cell monolayers with morphological and lipogenic properties characteristic of the physiological-nutritional state of donor animals. Synthesis and secretion of fatty acid, cholesterol, and very low density lipoprotein (VLDL) occur at in vivo rates and respond to hormones and agents which affect these processes in vivo. Cells derived from fed chickens maintain high rates of synthesis of fatty acid and cholesterol for several days if insulin is present in the medium. High rates of fatty acid synthesis are correlated with the appearance of membrane-enclosed triglyceride-rich vesicles in the cytoplasm; deletion of insulin causes a decrease (T1/2 = 22 h) in fatty acid synthetic activity. Addition of glucagon or cyclic AMP (cAMP) causes an immediate cessation of fatty acid synthesis and blocks the appearance of the triglyceride-rich vesicles. Fatty acid synthesis in liver cells prepared from fasted chickens is less than 5% that of cells from fed animals. After 2-3 days in culture with serum-free medium containing insulin +/- triiodothyronine, fatty acid synthesis is restored to normal; glucagon or dibutyryl cAMP blocks this recovery. Liver cells derived from estradiol-treated chickens synthesize and secrete VLDL for at least 48 h in culture. Electron micrographs of these cells reveal more extensive development of the rough endoplasmic reticulum and Golgi complex compared to cells from untreated chickens. Whereas [3H]leucine incorporation into total protein is unaffected by estrogen treatment, [3H]leucine incorporation into cellular and secreted immunoprecipitable VLDL is markedly increased indicating specific activation of VLDL apopeptide synthesis; 8-10% of the labeled protein synthesized and secreted is VLDL. Dodecyl sulfate-acrylamide gel electrophoresis of immunoprecipitated 3H-VLDL reveals three major apopepetides of 300,000, 11,000, and 8,000 daltons corresponding to those of purified chicken VLDL.


Estradiol-induced redistribution of lysosomal proteins in rat preputial gland. Evidence from immunologic probes. The influence of estrogen on the subcellular localization and distribution of lysosomal components of preputial gland was investigated in the ovariectomized rat. Antisera of high titer and specificity toward high-density lysosomal lipoproteins of this organ were raised in rabbits. The immunologic effectiveness of the IgG fraction so obtained was confirmed by microcomplement fixation, immunodiffusion, and immunoelectrophoresis. By both direct and indirect immunofluorescence techniques, cryostat sections of preputial gland from the control animals exhibited pinpoint cytoplasmic fluorescence, of dimensions corresponding to those of lysosomes. In contrast, specific immunoreactive material in corresponding target cells from animals receiving 0.1 microng of estradiol-17 beta/100 g body wt only 2 min earlier was distributed more homogeneously, indicating release of antigen from the membrane-bounded organelles. Moreover, specific immunofluorescence became evident at cell surfaces and in peri- and supranuclear localization, sites essentially negative in the controls. These effects were intensified at 15 min, as well as by maximal physiologic dose (0.5 microng/100 g body wt) of hormone. The relatively less active epimer, estradiol-17 alpha, exhibited only very limited effectiveness by some of these criteria. These observations, taken together with independent biochemical and ultrastructural evidence, lead to the conclusion that structural labilization of lysosomal constituents and their translocation to the nuclear compartment are early correlates of estrogen action.


Isolation and characterization of the urothelial lumenal plasma membrane. The lumenal plasma membrane has been isolated from transitional epithelial cells (urothelium) lining the urinary bladder in sheep by a modified technique involving treatment with hypotonic thioglycolate. The isolated membranes, like those in situ, are distinguished morphologically by arrays of hexagonal particles (in plague regions) separated by smooth interplaque regions. These plaque regions, specifically, can be isolated from the lumenal plasma membrane. Of the proteins constituting the lumenal plasma membrane, five were found to characterize the plaque regions and, in particular, the 33,000-dalton species appears to be most heavily concentrated in the sodium dodecyl sulfate-polyacrylamide gel pattern of the isolated plaque regions. Lipid analyses showed that there are approximately 0.93 mg of phospholipid and 0.27 mg of cholesterol for each milligram of protein, giving a value of 55% lipids and 45% proteins for the composition of the lumenal plasma membrane. The total sialic acid content was measured to be approximately 0.038 micronmol/mg protein for the plasma membrane. Several plasma membrane marker enzymes were found to be associated with the lumenal plasma membrane fraction, but only the 5'-nucleotidase activity was found to be further enriched in the plaque region fraction. Amino acid analysis of the intrinsic proteins of the plaques indicated a polarity index of 45%.


Growth and biochemical characteristics of a detachment variant of CHO cells. A variant subline of Chinese hamster cells (line CHO) was isolated that had increased resistance to detachment from the substratum. Comparisons between parental and variant cells of the complex carbohydrates liberated during trypsin detachment showed that the variant cells synthesized little or no hyaluronic acid. These cells also had reduced amounts of other complex carbohydrates in the cell periphery. However, parental and variant cells did not differ in morphology, growth control, or cyclic AMP concentration. Profound changes in the physical nature of the cell periphery, in themselves, evidently are insufficient to cause changes in many aspects of cell behavior.


Comparative studies of glucose-fed and glucose-starved hamster cell cultures: responses in galactose metabolism. The metabolic flow of trace amounts of D-[14C]-galactose was followed in cultures of transformed and untransformed hamster cells over a period ranging from five minutes to two hours. The results of chromatographic and enzymatic analyses of the soluble pools are described. Non-glycolytic cells(previously deprived of sugar periods of up to 24 hours) convert D-galactose to galactose-1-phosphate and uridine diphosphoglucuronic acid in 10 to 20 minutes. In the same short assay time, glycolytic cells which have been maintained for 24 hours in media containing glucose or galactose convert D-galactose to uridine diphsphogalactose and uridine diphosphoglucose (ratio 1.4:1). Long term diprivation of sugar also results in 3- to 4-fold increases in the uptake of galactose. In addition, the incorporation of galactose label into chloroformethanol soluble material appears to be influenced by the culture conditions of the untransformed cells while incorporation in the transformed cells appears unaffected. When cycloheximide is included in the maintenance medium for extended periods, the non-glycolytic cells also show increases in galactose uptake rates but the glucose-fed, glycolytic cells llose uptake ability. UDPhexose is the main galactose metabolic peak in the soluble pools of the cycloheximide-treated, glycolytic and the cycloheximide-treated, non-glycolytic cells. The results of these experiments suggests that uptake of galactose and its subsequent metabolism are under separate control.


The relative amounts of the cytoplasmic RNA species in normal, transformed and senescent cultured cell lines. We have examined the relative quantities of 18S and 28S rRNA, 4S RNA and poly (A) + mRNA in the following cultured cells: the mouse fibroblast lines 3T3 and 3T6 in the resting (contact inhibited) and growing (sparse) states, 3T3 clones transformed with SV40 (SV3T3) and with both SV40 and polyoma SV-Py 3T3), hamster lung fibrobalsts (v79), human cervical carcinoma cells (HeLa), and human diploid fibroblasts at early and late passage. The relative quantities of the RNA species were determined by labeling the cells to equilibrium with 32PO4 and measuring the amount of label in each RNA species. The ratio of mRNA to rRNA varied form 1.1% to 2.7% in the different cell lines, the more rapidly growing cell lines usually giving a higher ratio. In cells experiencing growth limitation either by contact inhibition or due to senescence, the ratio of mRNA to rRNA was about 30% lower than in the corresponding cells in the growing state. In most cell lines the ratio of 4S RNA to 18S rRNA was between 0.8 and 1.2, but in seescent fibroblasts, this ratio increased to greater than 1.7. Senescent fibroblasts also contained much more total RNA per unit of DNA than the same cells at early passage or than 3T6 or 3T3 cells.


Surface exposure of glycosaminoglycans in resting, growing and virus transformed 3T3 cells. Glycosaminoglycans (GAG's) were released by trypsin from the surface of cultured mouse cells (3T3) in two different growing states: during log-growth phase and during resting due to serum starvation. Doubly labelled molecules from resting cells were compared with those from growing as well as from trnsformed cells. Reproducible differences in the elution pattern during ion exchange chromatography and in susceptibility to specific hydrolytic enzymes have been demonstrated: the GAGs pattern of growing normal cells is similar to the pattern of the cells transformed by either Polyoma or SV-40 viruses and very different from the pattern of resting cells. Growing and transformed 3T3 show a relatively low amount of trypsin removable heparan sulphate (HS) and a relatively high amount of hyaluronic acid (HA) while resting cells exhibit an opposite ratio between the two GAG'S. The lowering of HS and the increase of HA in the cell coat is therefore suspected to be more dependent upon growth than upon transformation.


Cell cycle changes in transformed cells growing under serum-free conditions. Polyoma transformed hamster cells (PyBHK) and SV40 transformed mouse cells (SV3T3) were transferred in culture using crystalline trypsin followed by neutralisation with soybean trypsin inhibitor. Such cells were able to proliferate freely in defined medium without any serum supplement and without any intervening period of adaptation. However, growth rates were reduced under serum-free conditions. Re-establishment of rapid growth rates could be achieved by addition of serum, with the rate attained being proportional to the serum concentration. Irrespective of the prevailing rates of growth, percentages of cells synthesising DNA were the same. However, the rate at which DNA was being synthesised was found to change proportionately with the changes in overall growth rate.


Quantitative recovery of free and esterified fatty acids from thin-layer plates coated with silica gel. A method is described for the recovery of fatty acids from silica gel on thin-layer plates. The method is simple and rapid and depends on the conversion of silica gel to potassium silicate, using potassium hydroxide, followed by acidification in such a way that the silicic acid is kept in solution. Fatty acids are then extracted by shaking with solvent. Fatty acid recovery from free fatty acid, phospholipid, glyceride and cholesterol ester zones is more than 90% efficient. There is no oxidation or isomerization of unsaturated fatty acids.


Dry column chromatography of phospholipids. Separation of common phospholipids can be effected by dry column chromatography on silica gel. The method involves packing the column with dry gel and developing it in solvent mixtures used for thin-layer chromatography of the same lipids. Solvent is allowed to migrate only to the end of the column; access to the bands of separated material is obtained by using columns with a removable glass front. RF values of lipids on development columns and those on thin-layer plates are nearly identical when the column is packed with thin-layer chromatography gel. Such columns, however, develop very slowly. Columns packed with fine silica gel designed for elution column chromatography develop very rapidly and yield separations that are still quite comparable to those obtainable from thin-layer plates. Such columns are convenient for the purification of phospholipids in amounts of 10 mg to about 10 g. Column design and construction are described in detail.


Sequence analysis of synthetic oligonucleotides by high-performance liquid anion-exchange chromatography. A simple procedure for the sequential anlysis of small oligonucleotides is reported. The method is based on the simultaneous identification and quantitation of monomers released by venom phosphodiesterase digestion of oligonucleotides using high-performance anion-exchange chromatography on Permaphase AAX at room temperature and by applying isocratic elution conditions. In this way, the correct sequence of five oligomers, e.g., r-ACCUCC, r-CUGUU, r-AGGA, d-ATTACC and d-GGTAAT, could easily be established unambiguously.


[Determination of indolalkylamines after selective derivatisation (author's transl)]. The 1N-TMS-omegan-TFA-derivatives of indolalkylamines are suitable for estimation by combined gas chromatography and mass spectrometry for two reasons, viz. (1) Under electron impact charge stabilisation at the indole nucleus is favoured, so structure-specific ions form the base peak. For unsubstituted indoles m/e values of 202 are found and for hydroxyl-substituted indoles m/e values of 290. (2) The calibration curves of the derivatives are linear, even in the femtomole range. The 1N-TMS-omegan-derivatives can easily be obtained by trimethylsilylation with N-methyl-N-trimethylsilyltrifluoroacetamide containing catalytic amounts of N-trimethylsilylimidazole followed by acylation with N-methyl bis(trifluoroacetamide). The serotonin concentrations in different rat tissues are determined by this method of selective omegaN-trifluoroacylation-1N-trimethylsilylation and compared with the values obtained by pertrimethylsilylation.


Preliminary study of the use of dansyl chloride to determine cyclic-3',5'-AMP in tissues. A highly sensitive method for the determination of cyclic-3',5'-AMP has been developed which involves the reaction of the substance with dansyl chloride and the subsequent separation of the dansyl-cyclic-3',5'-AMP derivative by thin-layer chromatography. Experiments with standard solutions of 3H-cyclic-3',5'-AMP have shown that there is a direct relationship between the amount of dansyl-3H-cyclic-3',5'-AMP recovered and that dansylated. The procedure is exceedingly sensitive, allowing milligram quantities of material to be analysed for its endogeneous cyclic-3',5'-AMP content. With the use of 14C-adenine as substrate, this method permits the separation of 14C-cyclic-3',5'-AMP formed from the substrate and other 14C-containing compounds, thus allowing the turn-over of cyclic-3'-5'-AMP to be studied. The usefullness of the method is demonstrated by analysing the turn-over and endogenous content of cyclic-3'-5'-AMP in rat nervous tissue.


Identification of a receptor for somatomedin-like polypeptides in human fibroblasts. We have demonstrated a specific receptor for somatomedin-like growth polypeptides in human fibroblasts in culture using the closely related polypeptide, multiplication stimulating activity (MSA), as the radioligand. Polypeptides purified from human plasma, somatomedin A and acid soluble nonsuppressible insulin-like activity (NSILA-s), competed potently for 125I-MSA binding, as did unlabeled MSA. Although insulin and proinsulin also strongly inhibited MSA binding, the properties of the growth peptide receptor differed from those of the human fibroblasts insulin receptor. Somatomedin A, NSILA-s, MSA, insulin and proinsulin all stimulated the incorporation of [3H]thymidine into DNA in human fibroblasts. We propose that these polypeptides induce DNA synthesis through their interaction with the growth peptide receptor.


Reactivity of non-primate growth hormones and prolactins with human growth hormone receptors on cultured human lymphocytes. Ovine placental lactogen is as reactive as human growth hormone with the human growth hormone receptor of cultured human (IM-9) lymphocytes, which confirms the findings of Carr and Friesen with receptors of human liver. We now also show that bovine and ovine growth hormones and ovine prolactin have reactivity for the human growth hormone receptor on IM-9 lymphocytes that is of the same order of magnitude (0.03%) as that previously reported for human placental lactogen. The binding studies predict that these non-primate hormones will have biological effects on skeletal growth in primates, either as agonists or antagonists. Previous studies have shown that when IM-9 lymphocytes are exposed to human growth hormone for 18 h at 37 C, there is a time and concentration dependent loss of human growth hormone receptors, and the magnitude of the loss of receptors after preincubation for 18 h at 37 C is greater than the average occupancy of receptors under steady state conditions for 90 min at 30 C. In the present study we show that human and ovine placental lactogens, ovine prolactin, and bovine and ovine growth hormones also produce this effect on the human growth hormone receptor. Since the cellular process by which a hormone induces loss of its own receptors appears to require binding of the hormone to its receptor as well as one or more subsequent steps in hormone action, it is likely that all of the preparations that induce receptor loss will be shown to have some agonist activity of human growth hormone in promoting skeletal growth in primates. Further, these studies extend the interrelationships between primate and non-primate pituitary and placental hormones from what has been suggested previously from biological and structural studies.


MSH/ACTH 4-10 influences behavioral and physiological measures of attention. Eleven healthy male volunteers were infused with MSH/ACTH 4-10 and a control solution in a completely crossed and balanced procedure. Perception, attention and memory were tested as were heart rate and cephalic pulse amplitude in response to changes in the environment. Treatment of subjects with MSH/ACTH 4-10 raised their perceptual threshold for detection of simple stimuli and facilitated perceptual integration of patterned information. Administration of the heptapeptide improved the subjects' ability to discriminate tests of relevant from irrelevant information and augmented the heart-rate deceleratory response during presentation of novel stimuli. It was speculated that MSH/ACTH 4-10 has a predominant influence on attentional processes and that it may be uniquely coded for attentional/perceptual functioning.


HCG binding and stimulation of testosterone biosynthesis in the human fetal testis. The role of hCG in the regulation of testicular steroid production in human fetuses from 14 to 20 weeks gestational age was studied. Saturable binding of 125I-hCG to testicular homogenates was demonstrated, and physiologic concentrations of hCG were able to stimulate testosterone formation in testicular minces without the addition of exogenous precursors. In five fetses of 16-20 weeks gestational age, the capacity to bind hCG varied from 25.6 to 42.2 pg/mg wet tissue. The association constant of binding was 1.07+/-0.12 X 10(10) M-1. Testicular minces from six other fetuses (gestational age 14-19 weeks) were incubated in the presence of concentrations of 0, 0.5, 5 or 50 ng/ml NIH-hCG (1 mg=10,000 IU), which are within the physiologic range. Preincubation of 30 min in excess buffer was necessary to observe clear differences in testosterone production rates between controls and hCG stimulated testicular tissues. The greatest increase in testosterone production occurred when the hCG concentration was increased from 0.5 to 5 ng/ml. Little additional stimulation was observed at a concentration of 50 ng/ml. Maximal production rates of up to 12 ng/mg tissue/h were seen. It is concluded that human fetal testes bind hCG, and that physiologic levels of hCG stimulate fetal testicular testosterone formation in vitro at this stage of gestation.


Inactivation of classical and alternative pathway-activated bactericidal activity of human serum by sodium polyanetholsulfonate. Sodium polyanetholsulfonate (SPS) at a final concentration of at least 250 microng/ml (0.025%) was required for inhibition of the bactericidal activity of 80% (vol/vol) of fresh human serum against "promptly serum-sensitive" strains of Serratia marcescens and control strain Escherichia coli C, i.e., for inhibition of the classical pathway of complement activation. In contrast, SPS at 125 microng/ml (0.0125%) was sufficient for neutralization of the bactericidal activity of 80% (vol/vol) fresh human serum against "delayed serum-sensitive" strains of S. marcescens known to activate the alternative pathway of human complement. Addition of up to 500 microng of SPS per ml to 80% (vol/vol) fresh human serum failed to neutralize transferrin-mediated, "late" bacteriostasis against control strain E. coli C, an effect that was demonstrable only after prolonged, i.e., overnight, incubation of the test strain. However, this late inhibitory effect against E. coli C was not observed in SPS-treated 20% (vol/vol) fresh human serum or in 10 or 20% (vol/vol) conventionally heat-inactivated human serum. Immunoelectrophoretic examination disclosed that SPS did not precipitate transferrin from either fresh or heat-inactivated human serum. Thus, SPS, at 250 microng/ml, was demonstrated to be sufficient for the inhibition of both classical and alternative complement pathway-activated bactericidal activity of 80% (vol/vol) human serum. However, SPS at a concentration of 500 microng/ml failed to antagonize one antimicrobial system of 80% (vol/vol) human serum, namely transferrin-mediated bacteriostasis.


Infectious causes of equine respiratory disease on Ontario standardbred racetracks. Upper respiratory disease has been a serious problem in Standardbred horses on racetracks in Ontario, with outbreaks occurring once or twice annually in late winter and early spring seasons. To determine the causes of these epidemics, a 3-year investigation was carried out in which nasal swabs and serum samples were obtained at intervals from apparently healthy horses and from horses suffering from upper respiratory disease. The nasal swabs were used to isolate bacteria and viruses. The serum samples were examined for the presence and level of antibodies to equine influenza viruses and equine herpesvirus 1. None of the bacteria isolated were associated with the outbreaks of disease. Equine herpesvirus 2 was isolated 72 times from both diseased and apparently healthy horses. Equine herpesvirus 1 was isolated 10 times from horses with respiratory disease, both during and between epidemics. Influenza equine/1 virus was isolated seven times and influenza equine/2 was isolated once during severe outbreaks of upper respiratory disease. Serological evidence confirmed that influenza viruses were the causes of the major epidemics, with the equine/1 strain being involved most often.


Correlation of cytopathic effect, fluorescent-antibody microneutralization, and plaque reduction test results for determining avian infectious bronchitis virus antibodies. A microneutralization fluorescent-antibody (MFA) test was effective in determining the level of antibodies to avian infectious bronchitis virus. A comparison of the MFA test with the cytopathic effect microneutralization (MNT) test and 50% plaque reduction (PR) test resulted in positive correlations that were significant (P less than 0.001). The PR test was more sensitive than either the MFA or the MNT test, but there was no significant difference between the sensitivities of the MFA and MNT tests. The MFA test has advantages over the PR test in the capacity to test large numbers of sera in a shorter period of time. The MFA test also can be completed in one-half the time required for the MNT test.


Purification of infectious feline leukemia virus from large volumes of tissue culture fluids. Feline leukemia virus from tissue culture fluids was concentrated 100 times by continuous-flow molecular filtration. Virus concentrates retained 100% of their original infectivity. Further purification was accomplished by a single sucrose density gradient centrifugation.


Microcalorimetry as a tool for evaluation of blood culture media. Evaluation of optimal compositions of blood culture media has called for extensive and laborious work in comparative studies of large series of clincal specimens. Bacterial growth is accompanied by heat production, and calorimetry provides an analytical tool for its detection and quantification. A twin microcalorimeter of the heat conduction type was used to register heat effects in experimentally infected blood cultures. When studying Escherichia coli and Staphylococcus aureus, larger heat effects were produced with 0.05% sodium polyanetholsulfonate than with 600 IU of heparin per ml, which was also the case when using 10% sucrose. The addition of IsoVitaleX (BBL) increased the heat effects produced by the two species mentioned, whereas it had the opposite effect in cultures of Neisseria meningitidis. The present study indicates that microcalorimetry is a valuable and time-saving tool for the evaluation of optimal compositions of bacterial culture media.


Effect of dilution on recovery of bacteria from blood. The multiplication rate of bacteria in undiluted blood containing sodium polyanethol sulfonate was compared with growth rates obtained in dilutions of blood ranging from 1:2 to 1:8. Although all organisms tested grew in the undiluted blood, increased growth rates were seen in the 1:2 dilution. Further dilution resulted in growth rates equivalent to that obtained with the 1:2 dilution. In view of these results, we question the present recommendations that blood be diluted 1:10 or 1:20.


Comparative complement fixation and serum neutralization antibody titers to herpes simplex virus type 1 and Herpesvirus simiae in Macaca mulatta and humans. The serological relationship of herpes simplex type 1 virus and Herpesvirus simiae was studied. Antibody titers to these viruses were determined in 163 Macaca mulatta sera and 67 human sera by serum neutralization (SN) and complement fixation (CF) tests. Both groups of sera were also tested by CF with envelope and capsid antigens of herpes simplex type 1. By SN, the majority of the monkeys and all of the humans had a higher titer to herpes simplex type 1 than to H. simiae. By CF, with crude antigens the titers in the monkey sera were greater to H. simiae than to herpes simplex type 1, although four sera had equal titers to both antigens; the titers in the human sera were conversely higher with the herpes simplex type 1 antigen, except for four sera which had equal titers to both antigens. The capsid CF antigen of herpes simplex type 1 was reactive with the human sera but virtually nonreactive with the monkey sera; the envelope CF antigen of herpes simplex type 1 was reactive with both monkey and human sera but was somewhat less reactive than the crude herpes simplex type 1 CF antigen. In addition, serum samples from a patient recently infected with H. simiae were examined by CF and SN for antibody to both herpes simplex type 1 and H. simiae viruses. The serological profile indicated a positive correlation with the infecting virus. Although the SN titers did not conclusively reflect an infection with H. simiae, the CF titers were higher to H. simiae than to herpes simplex in later sera and thus appeared to be compatible with H. simiae infection.