Studies on the association of the Epstein-Barr virus genome with chromosomes in human (Burkitt)/mouse hybrid cells. We have used somatic-cell hybrids of mouse fibroblast and Burkitt's lymphoblastoid tumour cells to continue our investigation of the association between Epstein-Barr virus (EBV) genome and human chromosomes. A mouse/Burkitt hybrid cell, designated CL1D/HR-1, was cloned in soft agar. Each of 10 clones was assayed for the spontaneous expression of EBV-associated nuclear antigen (EBNA), early antigen (EA) and virus capsid antigen (VCA). Six of 10 clones were EBNA-positive but negative for EA and VCA even after treatment with iododeoxyuridine. One clone, designated M44, contained approximately 90% EBNA-positive cells and 0.3--0.5 EBV genome equivalents per cell. Thirty subclones of clone M44 were obtained and analysed for EBV DNA, EBNA and human chromosomes. Four subclones (three EBNA-positive and one EBNA-negative) were studied in detail. Data obtained thus far indicate that none of the four subclones of clone M44 studied contained any intact human chromosome. Isozyme analysis of these subclones indicated that all four subclones, regardless of the status of the EBV genome, synthesized nucleoside phosphorylase, an enzyme which has been linked to human chromosome number 14.


Characterization of the replicative structures of the DNA of a herpesvirus (pseudorabies). The replication of PRV DNA occurs in two phases, early and late. During the early stages of infection newly synthesized DNA is associated with molecules sedimenting with an S-value up to two-fold greater than that of mature viral DNA. These molecules represent unit-size linear or circular molecules, as well as small concatemers in the process of replication. Initiation of replication occurs at a site situated 20 micron from one of the ends as well as at or near the end of the molecules. At later times, newly synthesized DNA is associated with large, "tangled" concatemers containing single-stranded segments of DNA. Our results indicate that at least some of the single-stranded DNA may be produced during the extraction procedure. Analysis of the large, "tangled" concatemers with restriction enzymes shows that they consist of linear arrays of viral DNA molecules.


The DNA of serially passaged herpes simplex virus: organization, origin, and homology to viral RNA. High-density DNA prepared from serially passaged herpes simplex virus contains three major classes of modified viral DNA molecules. The altered DNA molecules are composed of multiple repetitions of sequences derived from the right-hand side of the S region of the parental plaque-purified viral DNA. The repeat units contained in the three types of high-density DNA share most of their DNA sequences but differ with respect to a small region derived from the unique sequences of the S component of HSV-1 DNA. Hybridization of the defective DNA to HSV-infected cell RNA shows that the high-density DNA contains sequences complementary to both early and late viral transcripts.


Characterization of human varicella-zoster virus DNA. The DNA of varicella-zoster virus (VZV) has been characterized by sucrose gradient sedimentation, restriction enzyme cleavage with either EcoRI or HindIII site-specific endonucleases, and by isopycnic banding in caesium chloride. Comparisons of the DNAs from different clinical isolates have been made. DNAs from VZVs isolated from either varicella or herpes zoster are indistinguishable on the basis of size and restriction enzyme cleavage pattern. The buoyant density in caesium chloride of the DNA of VZV isolated from varicella was reproducibly slightly lower than that of the DNA of VZV isolated from herpes zoster.


Characterization of human cytomegalovirus DNA: infectivity and molecular weight. Human cytomegalovirus DNA was isolated from purified virions and further purified by sucrose density-gradient centrifugation. The viral DNA molecules were studied by electron microscopy and found to be linear and to have a length of 76.22 +/- 3.80 microM, corresponding to a molecular weight of 147.13 +/- 6.18 x 10(6). The DNA was infectious when tested in human embryonic lung cells using the DEAE-dextran and the calcium-phosphate techniques. The density in caesium chloride was 1.717 g/cm3.


Inhibition of the multiplication of herpes simplex virus by aliphatic nitrosamines. Non-toxic doses of dimethylnitrosamine and diethylnitrosamine inhibit the multiplication of herpes simplex virus type 1 and type 2 in HEp-2, Wi-38 and primary HEK cells. These results are somewhat unexpected, since both HEp-2 and Wi-38 cells lack detectable microsomal enzyme activity. The possible significance of the preliminary results is discussed.


Potential for immunization against herpesvirus infections with plasma membrane vesicles. Virus-free, membrane antigen (MA)-positive plasma membrane vesicles produced from herpesvirus saimiri-infected owl monkey cells were used as immunogens in cotton-top marmosets. Neutralizing antibodies as well as antibodies to MA and HVS-induced LA were induced following immunization of marmosets with these vesicles. Preliminary findings demonstrated that immunized marmosets were significantly less susceptible to tumour induction by HVS than control animals.


Herpes simplex virus vaccine: protection from stomatitis, ganglionitis, encephalitis and latency. A mouse model system was developed for studying the pathogenesis of oral infection with herpes simplex virus type 1 and the protection offered by prior immunization with a nucleic acid-free vaccine. Of non-immunized mice, 95-100% developed ulcerative lesions 3-5 days following application of virus to abraded oral epithelial surfaces. Infection of the ipsilateral sensory (trigeminal) ganglion and the cerebellum occurred by day 2 and sequentially progressed to the contralateral ganglion by day 4 and to the cerebrum by day 5. Prior immunization of mice with an inactivated virus vaccine, and most importantly, with a vaccine free of nucleic acid, protected mice from subsequent oral virus infection. Protection was demonstrated by: (i) reduction in the incidence and severity of primary oral lesions; (ii) a decrease in the number of mice with acute ganglionic infection or dying of encephalitis; and (iii) a reduction in the incidence of latent trigeminal ganglionic infection.


A live varicella vaccine used for children in hospitals. A live varicella vaccine was developed and the following results obtained: 1. The vaccine was safely and effectively used for children in hospital with various underlying diseases; some of the children had been receiving immunosuppressive therapy. 2. Spread of varicella could be prevented by immediate inoculation of the vaccine in hospital or at home when vaccine was given to susceptible children within 72 hours after exposure. 3. Persistence of neutralizing antibody and prevention of natural varicella have been confirmed in a two-year follow-up study of the vaccine recipients. 4. No contact infection from vaccine recipients to susceptible children in closed institutions was detected (Asano et al., 1976). 5. Varicella skin antigen was prepared and was found useful for clinical purposes, e.g., by providing a convenient means of determining the susceptibility of individuals to clinical varicella.


Non-protective variants of Marek's disease vaccine viruses. Two variant Marek's disease-vaccine viruses failed to induce protective immune responses in chickens and replicated poorly in vivo. One of these, MDV/200D, was defective for growth at 41 degrees C, and clones from this stock had 38 degrees C/41 degrees C plating ratios as high as 10(4). The basis for the poor in vivo replicative ability was not determined.


Interferon in the replication of herpes simplex virus in normal and pathological nerve cells. Previous results obtained in experimental and clinical trials have demonstrated that topical combined thrapy with human interferon (HI) and human colostral secretory immunoglobulin A (S-IgA) is effective against herpetic corneal infection. This therapy prevented encephalitis in rabbits but could not completely prevent recurrences either in rabbits or in patients. A number of in vitro studies were designed to elucidate the role of these factors in herpes simplex virus replication in the nervous system, with the following results: (1) HSV latency in trigeminal ganglia (TG) explanted from rabbits with experimental herpetic keratitis, topically treated with HI or HI/S-IgA: HSV was recovered in 30% TG after 15-19 days co-cultivation on RK-13 cells. (2) HSV replication in nervous ganglia and nerve of newborn rabbits in organ culture; influence of HI or HI plus IgG: a restrictive HSV productive infection was demonstrated in this system, although yields were always higher in nerve cultures. We were unable to demonstrate a direct effect of HI on HSV-1 replication. When explants were treated with HI and IgG before and after infection for 48 hours a delay in the expression of HSV-1 was detected by co-cultivation. (3) Replication of HSV-1 and HSV-2 in a C1300 murine neuroblastoma clone (NB41A3): both HSV types replicated with titres of 10(3.4) for HSV-1 AND 10(4.8) for HSV-2 at 48 hours p.i.; CPE was more marked for HSV-2 at 24 hours. HSV-specific antigens were demonstrated by the immunoperoxidase technique.


Effect of exogenous interferon on neonatal infection with murine cytomegalovirus. One dose of interferon given before inoculation of MCMV to the neonatal mouse resulted in less virus in the submaxillary glands and reduced tissue damage due to MCMV infection, probably by modulating the immune system.


Alterations in biological properties of different lines of cytomegalorivus-transformed human embryo lung cells following in vitro cultivation. Diverse alterations in biological properties of CMV-transformed cell lines were observed during prolonged in vitro cultivation. In the CMV-Mj-HEL-2 parent line there was a gradual decrease in the number of cells expressing CMV-related antigens; at the same time, an increase in oncogenicity was observed. One tumour line, designated CMV-Mj-HEL-2,T-1, however, retained the original ratio of cells expressing CMV-related antigens for over 100 in vitro passages. The cells lost their original moderate oncogenicity during this period. A later increase in the ratio of cells without CMV antigenic markers was accompanied by the return of moderate tumorigenicity and karyotypic changes. Both cell lines were studied to determine sensitivity to superinfection with herpesviruses, induction of immune response in nude mice, and release of infectious virus.


Induction of cellular DNA synthesis by defective human cytomegalovirus. The results presented in this paper show that virus stocks enriched for defective particles are more effective in stimulating cellular DNA synthesis than are stocks relatively free of defective particles. Several lines of evidence support this conclusion: (1) Low levels of UV irradiation enhance the ability of standard virus stocks to stimulate cellular DNA synthesis. (2) Three different stocks derived from individual plaques (which presumably contain very few defective particles) and passaged only twice at low multiplicity, were found to be poor inducers of cellular DNA synthesis. (3) Stocks of virions obtained after serial undiluted passage which were shown to be enriched for defective particles were quite effective in stimulating cellular DNA synthesis.


Pronephric tumour cell lines from herpesvirus-transformed cells. Pronephric tumor cell lines have been established from tumours induced after inoculation of embryos with herpesvirus cultivated in vitro. Neoplastic properties of the lines are characterized.


Inhibition of Epstein-Barr virus transformation: evidence for a block between EBNA production and cell proliferation. Non-adherent lymphocyte populations prepared from donors with previous EBV infection transformed in the usual way in response to EBV. However, transformation was completely inhibited in the presence of adult but not fetal human fibroblasts. Such inhibition was associated with lymphocytes from donors with prior EBV infection, but not from those without, and there appears to be an immunological basis for inhibition. Inhibition was associated with production of EBNA and was reversible. Addition of adult fibroblasts to non-adherent lymphocytes up to 2-4 days after infection with EBV also resulted in inhibition of transformation. These results are interpreted as indicating that EBV transformation involves two recognizable phases, with an immunological block between EBNA production and cell proliferation.


Suppression of in vitro Epstein-Barr virus infection: a new role for adult human T-lymphocytes. In vitro EBV infection of lymphocytes from different sources indicated that B-cells of adult peripheral or fetal cord blood transform with equal efficiency whether assayed by DNA synthesis induction or immortalization. Unfractionated adult lymphocytes transformed much less efficiently than those from fetal cord blood. This effect was due to the suppression of B-cell proliferation by adult T-lymphocytes.


Transfection studies in vitro and in vivo with isolated Marek's disease virus DNA. For tranfection studies in vitro and in vivo, infectious Marek's disease virus (MDV) DNA was isolated from purified virus particles. Its sedimentation coefficient in neutral sucrose gradients was found to be 53 +/- 2S and the buoyant density in caesium chloride corresponded to 1.707 g/cm3. The virus DNA was also analysed by using the restriction endonucleases EcoRI and HindIII. High molecular-weight MDV DNA precipitated by the calcium phosphate technique was shown to be infectious in cultivated chicken embryo fibroblasts and newly hatched antibody-free chickens. Three out of eight chickens which each received between 0.5 and 2 micrograms MDV DNA by the intraabdominal route produced precipitating A-antigens in the feather follicles and/or lymphoroliferative tumours in the visceral organs. Infectious MDV was also detected in spleen and blood lymphocytes. Transformation experiemnts in vitro performed with isolated thymus cells, however, have so far produced negative results. The induction of Marek's disease and tumours by purified MDV DNA also indicates that the presence of contaminating avian oncornaviruses in the inoculum is not a necessary condition for the initiation of an MDV infection.


Expression of Marek's disease virus in producer and non-producer lymphoblastoid cell lines. Continuous in vitro propagation of a producer Marek's disease (MD) lymphoblastoid cell line reduced the number of cells producing virus antigens and the inducibility of these antigens to 5-iododeoxyuridine (IUDR) but did not change the oncogenicity of the cells for chickens. In contrast, propagation of a non-producer MD cell line did not change the non-productivity of the cell line, but decreased the transplantability of the cells. The MD lines were free of replicating avian leukosis sarcoma viruses (LSV) and were susceptible to infection with LSV of subgroup A.


Surface glycoprotein patterns of human haematopoietic cell lines. Human EBV-positive and EBV-negative haematopoietic cell lines were shown to have different and easily distinguishable surface glycoprotein patterns as demonstrated using surface labelling by the galactose oxidase-tritiated sodium borohydride method.


Antibody-dependent autologous lymphocyte cytotoxicity against cells freshly transformed by Epstein-Barr virus. Cytotoxicity against lymphocytes freshly transformed by EBV was studied in an autologous system by trypan blue exclusion of EBNA-positive cells and 51Cr-release assay. When EBV-tranformed lymphocytes were incubated with EBV-positive serum and autologous unfractionated lymphocytes or K-cells from sero-positive healthy donors, the transformed cells were significantly damaged. The cytotoxicity against newly produced EBNA-positive lymphocytes was also remarkable when sero-positive healthy donor lymphocytes were exposed to EBV, immediately followed by incubation with positive serum. In contrast, EBV-transformed cells incubated with autologous lymphocytes or T-cells alone were not significantly affected. EBV-positive serum alone was also not cytotoxic, regardless of inactivation. These results strongly suggest that antibody-dependent cellular cytotoxicity (ADCC) plays an important role in immunological defence against EBV oncogenesis in healthy humans. However, ADCC was not seen when immunosuppressed patients were examined.


Detection of Epstein-Barr virus-coded antigens in lymphocytes isolated from defined patient samples. A preliminary report on the use of specific rabbit antisera raised to Epstein-Barr virus-coded antigens (EBNA and EA) for detection of these antigens in vivo is presented. Human lymphocytes were isolated on isokinetic gradients and the C3 receptor-bearing B-lymphocyte subpopulation was isolated, providing an enriched source of EBV-infected lymphocytes. Such technology was employed to establish the status of the EBV host-cell complex in recurrent exudative tonsillitis (RET), infectious mononucleosis (IM), and Hodgkin's and non-Hodgkin's lymphoma patients. Only EBNA was detected in the lymphocytes from the tonsils of RET patients and the peripheral blood of IM patients. However, the spleen and lymph-nodes of patients with lymphomas had lymphocytes synthesizing EBNA and EA.


Replication of Epstein-Barr virus DNA in epithelial cells in vivo. Epstein-Barr virus (EBV)-specific complementary RNA (cRNA) was hybridized in situ to oropharyngeal epithelial cells taken from patients with infectious mononucleosis. Cells from patients shedding virus in the throat hybridized signifnicant quantities of cRNA, whereas cells from EBV-negative sources did not. The degree of hybridization indicated a large EBV genome number per infected epithelial cell and suggested that these cells were the source of virus found in the throat. This finding may explain the presence of the EBV genome in the malignant epithelial cells of nasopharyngeal carcinoma.


Lymphocyte transformation and interferon production as measures of cellular immunity in lymphoma patients. Our studies of in vitro cellular immune responses to herpesviruses have shown that, before treatment, lymphoma patients have decreased transformation to varicella-zoster virus antigen compared to herpes simplex virus and cytomegalovirus antigens. Interferon production to varicella-zoster virus and cytomegalovirus antigens is also decreased. During the first six months of treatment, decreased lymphocyte transformation and interferon production to all herpesviral antigens was observed. Among patients in long-term remission, recovery from the suppression associated with treatment was noted except that interferon production to varicella-zoster virus antigen remained decreased.


Sequential changes in cell-mediated immune responses to herpes simplex virus following primary herpetic infection in man. During primary herpes simlex virus infections, sensitization to the virus was demonstrated at the time of presentation by lymphocyte blastogenesis. Recovery from infection coincided with development of the macrophage migration inhibition response, and it is suggested that macrophage activation by products of stimulated lymphocytes contributes to control of the infection.


Immune responses to vaginal or systemic infection of mice with herpes simplex virus type 2. Relationships among cell-mediated immune (CMI) responses, neutralizing antibody, and macrophages in host resistance to vaginal or systemic infection with HSV-2 were defined in BALB/c mice. The importance of T-lymphocytes in resistance to either systemic or vaginal infection was demonstrated by increased susceptibility of mice depleted of T-lymphocytes. A role for non-specific resistance against the systemic, but not vaginal, infection was demonstrated by increased susceptibility of mice depleted of macrophages. The temporal relationships among the serum antibody response, and the delayed-type hypersensitivity (DTH) and splenic proliferative CMI responses were defined in individual animals. Vaginal infection was associated exclusively with relatively transient CMI responses which appeared during the acute infection. After systemic infection, CMI responses appeared during the acute infection. After systemic infection, CMI responses appeared during the acute infection and persisted for approximately five weeks, while a humoral response appeared in surviving animals and persisted for at least four months. The results suggest that CMI is the predominant host response to vaginal HSV-2 infection, while both CMI and macrophage-mediated antiviral activity may be involved in recovery from primary systemic infection with HSV-2.


Herpes simplex virus latency in patients with multiple sclerosis, lymphoma and normal humans. Herpes simplex virus (HSV) was isolated from the trigeminal ganglia (TG) of 12 cadavers (10 traumatic deaths, one lymphoma and one multiple sclerosis). The cadaver with multiple sclerosis showed large bilateral trigeminal nerve root entry zone areas of demyelination. It is hypothesized that HSV is capable of migrating to the trigeminal nerve root entry zone and initiating demyelinating disease.


Immunosuppression reactivates and disseminates latent murine cytomegalovirus. When searched for by standard virological methods, murine cytomegalovirus (MCMV) becomes undetectable by four months after inoculation of mice. However, a two-week regimen of immunosuppression with antilymphocyte serum and corticosteroid results in reactivation and dissemination of the virus in virtually all animals. This system should be useful in defining the pathogenesis of generalized cytomegalic disease resulting from reactivation of latent virus in immunocompromized individuals.


Differences in susceptibility to herpes simplex virus infection of inbred strains of mice. Marked differences were observed between the susceptibility of adult C57BL/6 and DBA/2 mice to infection with HSV-1. These differences were seen after various infection schedules but were most marked after i.p. infection. A significant difference between the LD50 after i.p. infection was also observed between LPS-resistant C3H/HeJ and the closely related LPS-sensitive C3HeB/FeJ mice. Injection of LPS significantly increased the LD50 after i.p. infection with HSV in C57BL/6 or C3H/HeJ mice but not in C3HeB/FeJ mice. In vitro pretreatment with LPS was necessary to demonstrate replication of HSV in mouse spleen-cell cultures. Such replication could be demonstrated in cultures of DBA/2 and C3HeB/FeJ but not in C57BL/6 or C3H/HeJ mice.


Vaccination against diseases associated with herpesvirus infections in animals: a review. An account is presented of the development and use of herpesvirus vaccines in domestic animals, with particular reference to those viruses causing cytolytic rather than oncogenic infections. The chief infections covered are Aujeszky's disease (AD or pseudorabies), infectious bovine rhinotracheitis (IBR) and equine rhinopneumonitis (equine abortion; EHV-1). Others mentioned are feline viral rhinotracheitis and malignant catarrhal fever of cattle. Both live-modified and inactivated vaccines are widely used or under development for ADV, IBR and EHV-1. Live vaccines are generally regarded as successful in some circumstances but have major drawbacks, both in regard to safety or immunogenicity for the individual animal or to efforts to control and eliminate the viruses from whole populations. Difficulties remian in the preparation and use of inactivated vaccines, which still suffer from many of the epidemiological objections raised against modified live vaccines.


The interaction between herpesvirus and oncornavirus of guineapigs: in vitro and in vivo studies. In vitro, cultured guineapig doubly infected with both guineapig herpesvirus (GPHLV) and guineapig oncornavirus (GPOV) revealed pseudotype virus particles with oncornavirus morphology coated with GPHLV antigen as detected by immunoferritin electron microscopy. In vivo, infection of Hartley guineapigs with both GPHLV and GPOV induced hyperplasia in lymph-nodes in a higher percentage of animals than in those that received only one virus type. In addition, experimental GPHLV infection of strain 2 guineapigs caused some delay in the development of L2C leukaemia in inoculated animals.


Effect of ara-T on the replication of herpes simplex virus, varicella-zoster virus and cytomegalovirus. The thymidine analogue, 1-beta-arabinofuranosylthymine (ara-T), has previously been found to selectively inhibit herpes simplex virus (HSV) replication. At a relatively non-toxic concentration (50 microgram/ml), ara-T reduced HSV yields by a factor of 10,000-100,000. Ara-T was also effective in inhibiting the replication of variecella-zoster virus (VZV) in vitro in human embryo fibroblasts, completely preventing VZV-specific cytopathic effects (CPE). Ara-T reduced the cell-free virus and plaque-forming cell (PFC) yields of VZV as well as of the simian varicella-like virus, Delta herpesvirus. In contrast to HSV and VZV, cytomegalovirus (CMV) replication was relatively resistant to ara-T. Neither CPE nor the incorporation of 3H-thymidine into acid-insoluble material in CMV-infected cells was markedly affected. Interpretation of these results with regard to virus-induced deoxypyrimidine kinase is discussed.


Effects of theophylline on capillary tube leucocyte migration. Theophylline stimulates the capillary tube migration of human peripheral blood mixed leucocytes. Minor stimulation of polymorph migration is produced directly by theophylline and dibutyryl cyclic AMP, but polymorph migration is markedly stimulated by mononuclear leucocyte culture supernatants to which theophylline has been added. These results suggest that polymorph migration is stimulated when intracellular cyclic AMP increases, and that mononuclear leucocytes produce a potential migration stimulator whose activity is enhanced by theophylline.


Rhesus monkey (macaca mulatta) allotypes. Identification of three low density lipoprotein allotypes controlled by independent genes. Three rhesus monkey allotypes are described, which are located on distinct low density lipoprotein molecules. These three markers have been detected using double immunodiffusion in agar. They are inherited in a simple Mendelian manner and controlled by three independent genes.


Further studies on the adjuvant effect of silica on IgE antibody production in mice. The effects of amorphous silica (Aerosil) and of aluminium hydroxide on anti-DNP IgE antibody production were studied in Swiss mice preimmunized with Ascaris protein and then challenged 7 days later with DNP-ASC. Aerosil exerted an adjuvant effect especially when it was injected together with the DNP-ASC conjugate. Al(OH)3 had a weaker adjuvant effect than that produced by Aerosil and then only when it was injected with the DNP-ASC conjugate. Al(OH)3 given with Ascaris extract 7 days before complete antigen had an inhibitory effect on the anti-DNP IgE antibody production.


Detection of a microsomal antigen and its antibody in human liver diseases. Sera of 173 patients with various forms of liver disease along with serum precipitates produced by polyethylene glycol were screened for the presence of a microsomal antigen referred to as ubiquitous tissue antigen (UTA) and its antibody by double diffusion precipitation in agarose gel. UTA was detected in 7 or 26 patients with chronic active hepatitis, 1 of 5 with alcoholic hepatitis, 2 of 14 with alcoholic cirrhosis and 18 of 98 with hepatoma. Antibodies to UTA were found only in 2 patients with chronic active hepatitis, 1 with alcoholic cirrhosis and 1 with hepatoma. No UTA or its antibody were noted in sera of 5 patients with alcoholic fatty liver, 10 patients with hepatitis B, and 15 asymptomatic carriers of HBsAg. Positivity for the UTA or its antibody was restricted to severe, chronic cases irrespective of diagnosis, indicating that persistent tissue destruction might be necessary for antigen release or antibody formation.


Fluorescence study of thymus lymphocytes of X-irradiated mice and their progeny. The mechanisms of changes in the ultra-violet fluorescence (U.V.F.) intensity of mouse thymus lymphocytes 24 hours and 30 days after whole-body X-irradiation have been studied. The thymus lymphocytes of the first generation offspring (F1) from X-irradiated males and unirradiated females were also investigated. At 24 hours after irradiation the U.V.F. intensity decreased for small doses (50 and 65 rad) and increased for doses of more than 100 rad. The changes in U.V.F. intensity were related to a size-independent mechanism. It was found that the U.V.F. increase for doses of 100-700 rad was not connected with the appearance of non-viable (eosin test) cells. The changes in U.V.F. intensity and cellular composition of the thymus were the same 30 days after irradiation and for F1 mice. The increase in U.V.F. intensity was about 14% and did not depend on dose between 50 and 500 rad. About one-half of this increase was connected with an increase in the proportion of medium and large lymphocytes in the thymus. The rest of the effect was related to a size-independent mechanism.


The formation and reaction of methyl radicals produced by direct photolysis of aqueous solutions of N-acetyl substituted aliphatic amino acids at 77K. Photolysis of amino acids, peptides and their derivatives leads to the formation of free radicals in these substances. The electron-spin-resonance spectra obtained directly after irradiation at 77 K are not very well resolved.  They are recognizable as the superposition of the spectra of different types of photoproduced radicals. CH3 radicals are formed by U.V. irradiation if methyl groups are present in the molecule. These radicals are easily detectable because of their four line spectrum. In this paper the formation of methyl radicals and their reaction with undamaged molecules of N-acetyl-substituted amino acids in investigated. The number of CH3 radicals present after a 30 min U.V. irradiation is higher if preceding U.V. irradiations and heat treatments are performed. The overall concentration of radicals is reduced only partially during this heat-treatment, while the CH3 radicals decay completely. Other experiments show that the rate of and the yield of CH3 radicals by U.V. irradiation increase with the dose of a preceding gamma-irradiation. The results suggest that there are substances present which are responsible for the higher production rate of methyl radicals after a preceding treatment. It is assumed that radicals are precursors of the fast-formed CH3 radicals


Naturally occurring vitamin D3 in fish products analysed by HPLC, using vitamin D2 as an international standard. A chemical method for the analysis of naturally occurring vitamin D is proposed. The unsaponifiable matter of oils and tissues is prepared, cholesterol is partly removed by double precipitation at low temperature in methanol. The vitamin D fraction is collected on an adsorption column by high performance liquid chromatography. The fraction is further purified and the vitamins D2 and D3 are separated on a partition column (reverse phase) by HPLC. Recovery was 89 to 93%, standard deviation 3%. The only vitamin D analogue found in fish oils, livers and fillets, was cholecalciferol (D3). Hence, ergocalciferol (D2) could be used as an internal standard. The provitamins ergosterol and 7-dehydrocholesterol, as well as the previtamins, were separated from the vitamin D-fraction on the adsorption column. Results in the range 0.050 to 134 microgram D3 per gram (2 to 5360 I.U. per gram) are given. One cod liver oil was analysed in a rat bioassay, giving supporting results.


Induction of the FAD synthetase system in rat liver by phenobarbital administration. FMN and FAD synthesizing activities in rat liver were investigated when drug metabolizing enzymes were induced by phenobarbital. The FAD content of the liver mitochondrial and microsomal fractions were increased by either one shot administration or three successive daily administrations of phenobarbital. Similarly the flavokinase and FAD pyrophosphorylase activities also increased in the mitochondrial, microsomal and cytosol fractions. The authors have verified that activation of the FAD synthesizing system is induced by phenobarbital and suggest that the FMN and FAD contents are regulated not only by variations of flavin enzyme apoprotein, but also by changes in flavokinase and FAD pyrophosphorylase activities.


Collagenase from corneal cell cultures and its modulation by phagocytosis. The uptake of latex by fibroblasts in confluent primary culture results in the secretion of collagenase at a linear rate for a prolonged period. Phagocytosis might therefore constitute an important level of collagenase regulation in corneal ulceration. The collagenase in cell cultures is present in a latent form (40,000 MW) like that obtained from organ cultures of ulcerating corneas and can be activated proteolytically. Production of the latent collagenase in cell culture depends upon the presence of serum and diminishes greatly when serum is removed from the medium. Collagenase activity can be demonstrated after the latent collagenase has been separated from serum antiproteases in the media. Alternatively, careful titration of the crude media with trypsin to saturate serum antiproteases, to release collagenase from the complex with alpha 2-macroglobulin, and to activate latent collagenase also results in measurable collagenase activity. The collagenase that is secreted cleaves fibrillar type I collagen and cleaves soluble type I collagen into the typical 3/4 and 1/4 length fragments, as demonstrated by SDS-gel electrophoresis and electron microscopy.


Electron paramagnetic resonance study of iron oxalate in calcium oxalate renal stones. Weak electron paramagnetic resonance (EPR) signals from a number of calcium oxalate renal stones are attributed to an iron oxalate component. The g-value of the resonance is 2.0036 and its width is approximately 9 gauss. The EPR resonance from stones has the same characteristics as resonances from iron introduced into calcium oxalate and oxalic acid as an impurity. A sharp increase in EPR signal when calcium oxalate renal stones are exposed to intense light is attributable to the reduction of Fe3+ and the formation of the oxalate radical ion (C2O4)-.


Quantitative analysis of alpha and beta adrenergic receptor densities in the lower urinary tract of the dog and the rabbit. Using specific radioligand receptor binding assays, we have, for the first time, quantitated both alpha- and beta-adrenergic receptors in the musculature of the lower ureter, bladder body, posterior bladder base, and proximal urethra of the dog, and in the bladder base and body of the rabbit. For the beta receptor assay 3H-dihydroalprenolol was used as the ligand, and, for the alpha receptor assay, 3H-dihydroergocryptine was used. In the dog, there was a significantly greater density of beta receptors in the bladder body than in either the ureter or the posterior bladder base. The urethra had an intermediate density. The bladder base and the proximal urethra had a significantly greater density of alpha receptors than either the ureter or the bladder dome. The dissociation constant of beta receptors for DHA was approximately 9 nM for all tissues studied, and that of alpha receptors for DHE approximately 7 nM. A similar distribution of alpha-and beta adrenergic receptors was seen in the rabbit bladder.


Etiology of infantile enteritis in South Africa. Infantile enteritis constitutes a major health problem in developing countries. Several investigations into the etiology of this condition among various South African populations have been undertaken during the past few years. Recent studies of black urban infants have revealed that salmonellae, shigellae, enterotoxigenic Enterobacteriaceae, and rotaviruses play a relatively minor role in infantile enteritis. On the other hand, all studies, including a number performed several years ago, have demonstrated that Escherichia coli strains belonging to traditional enteropathogenic serotypes are more frequently recovered from patients with diarrhea than from matched control subjects. These bacteria are particularly prominent during the summer months at the height of the annual gastroenteritis epidemic. Recent studies have indicated the importance of Campylobacter fetus in infantile enteritis, especially in children younger than nine months. The data point to a complex multiple etiology of infantile enteritis in South Africa, which will require clarification before specific preventive measures can be instituted.


Transitory hypoadrenalism due to long-term treatment with antiovulatory compounds. A considerable number of women receiving antiovulatory compounds or estrogens complain of weakness and fatigability, suggesting a state of clinical hypoadrenalism. For this reason, levels of plasma ACTH and plasma cortisol were determined in 25 women with such complaints both during treatment and at various intervals after cessation of this treatment. The results obtained showed that there was a significant inhibition of ACTH secretion during long-term treatment with antiovulatory compounds or estrogens, and in half of the cases, there was a delay in normalization of the pituitary-adrenal axis following interruption of the drug, supporting a state of transitory hypoadrenalism.


[Cutaneous cysts and cystic skin tumors]. The nomenclature and pathogenesis of cutaneous cysts is discussed along the lines of their pathological properties. On the basis of histological and experimental evidence it is concluded that most cysts represent benign neoplasms derived from pluripotential cells.


[Clear-cell hidradenoma of an unusual location]. A case of clear cell hidradenoma, which develops from the eccrine glands of the skin, is reported. Specially the histological characteristics, the histogenesis and the unusual localisation of this tumor are described.


[Multilocular extramammary Paget's disease]. Paget cells were found histologically in a patient with erythemato-erosive lesions on the genitoperineale areas. These cells were also observed in the right axillary skin within a tiny depigmented spot and even in the left axillary skin which was macroscopically almost intact. There were no signs of metastatic or deep-seated adnexal adenocarcinoma. Treatment consisted of radical operation of the pathological lesions with skin grafting and removal of one testicle. After the operation the x-ray irradiation was performed.


[Extramammary Paget's disease].

[Extramammary Paget's disease]. A case of extensive extramammary Paget's disease in the scrotum and penis is demonstrated. The disease started a few years before as eczema-like lesions. No underlying carcinoma was found. The surgical excision resulted in a good cosmetic effect.


Isolation of picornavirus from feces and semen from an infertile bull. Virus was isolated from semen and fecal samples from a bull with orchitis, testicular degeneration, aspermatogenesis, and loss of libido. Both isolates were classified as picornavirus, bovine enterovirus serotype I, on the basis of physical, chemical, and serologic characteristics. Veterinary practitioners that may suspect viral infection as a cause of bovine infertility should submit both semen and fecal samples for virus isolation and identification.


Serologic survey for equine infectious anemia virus in Louisiana. In 1975, a survey was conducted in East Baton Rouge Parish, Louisiana, to determine the prevalence of equine infectious anemia. Using the agar gel immunodiffusion test, 94 of 1,398 horses (6.7%) were found to be infected. Infection rates were especially high in areas where clinical cases of equine infectious anemia had been diagnosed. Clinical signs compatible with the disease were noted in 1 of the 94 seropositive horses. The sample set of 1,398 horses represented 22% of the census population obtained during the 1971 Venezuelan equine encephalomyelitis vaccination campaign.


Group aftercare for chronic schizophrenia. This study evaluated the effectiveness of group follow-up in the aftercare of chronic schizophrenia. Fifteen outpatient schizophrenics were reviewed to compare their rehospitalization rates and percentage of time spent in the community during a 2 year period of group aftercare with the 2 year per-group period. Rehospitalization rates were 0.2 admissions/patient year during the group therapy period versus 1.1 admissions/patient year during the pre-group period ( less than .001). Attendance compliance rates during the group therapy were 97%. The results suggest that for these patients, group therapy was an effective method of maintaining schizophrenics in the community.


Spin label studies on the interactions of bovine adrenodoxin with NADPH-adrenodoxin reductase and with cytochrome P-450scc. Adrenodoxin of bovine adrenocortical mitochondria was spin-labeled with two different spin-labeling reagents, N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)imidazole (I) and N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (II), without major loss of its activity for electron transport from NADPH to cytochrome c. The EPR spectrum of adrenodoxin spin-labeled with either of the reagents showed a pattern typical of a moderately immobilized spin label. When adrenodoxin was treated with (I), approximately two amino acid residues per molecule were spin-labeled, whereas a single residue was labeled by (II). While assition of NADPH to adrenodoxin spin-labeled with (I) did not diminish the EPR signal intensity, addition of the reductant to the labeled adrenodoxin in the presence of adrenodoxin reductase caused slow reduction of the spin label, the rate of which was dependent on the aerobicity. Addition of adrenodoxin reductase to adrenodoxin spin-labeled with (I) or (II) resulted in the appearance of a more immobilized component in the EPR spectrum. The ratio of the more immobilized component to the less immobilized component was saturated at a molar ratio of one to one. Addition of cytochrome P-450scc to adrenodoxin labeled with (I) had similar effects on the EPR spectrum.


The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. I. Effect of calcium-dependent regulator protein on the calcium sensitivity of microtubule assembly. We examined the effect of porcine brain Ca2+-dependent regulator (CDR) protein on microtubule (MT) assembly from microtubular proteins isolated from porcine brain by temperature-dependent cycles of assembly-disassembly. CDR exhibited a potent inhibitory effect on MT assembly in the presence of Ca2+, whereas it had little or no effect on the extent of MT assembly in the absence of Ca2+. The increase in KCl concentration greatly potentiated the Ca2+-dependent inhibitory effect of CDR. The effect of CDR was reversible in a Ca2+ concentration-dependent manner, and the extent of inhibition by CDR at a fixed concentration of free Ca2+ was roughly proportional to the concentration of CDR. Moreover, the Ca2+ concentration required for the half-maximal inhibition of MT assembly from a fixed concentration of purified microtubular proteins (PMP) decreased with increasing CDR concentration. On the basis of these results, together with data on the Ca2+-dependent association of CDR and tubulin (J. Biochem., accompanying paper), we propose the following model; Ca2+ + CDR in equilibrium Ca2+-CDR Ca2+-CDR + tubulin in equilibrium Ca2+-CDR-tubulin (nonpolymerizable).


The interactions between calcium-dependent regulator protein of cyclic nucleotide phosphodiesterase and microtubule proteins. II. Association of calcium-dependent regulator protein with tubulin dimer. The Ca2+-dependent regulator protein (CDR) of cyclic nucleotide phosphodiesterase (PDE) was reported to be a Ca2+-dependent regulator of microtubule (MT) assembly in the preceding paper. In this paper, the binding of Ca2+-CDR complex to tubulin dimer was investigated in order to elucidate the Ca2+-dependent inhibitory action of CDR on MT assembly. Purified microtubular proteins (PMPs) isolated from porcine brain did not affect the ability of CDR to activate Ca2+-activatable PDE, and did not include any inhibitory protein of Ca2+-activatable PDE. The binding of CDR to the tubulin dimer was observed on Sephadex G-200 gel filtration and ammonium sulfate fractionation in a Ca2+-dependent manner. CDR did not bind to microtubule associated proteins. We now assume that Ca2+-dependent inhibition of MT assembly by CDR is due to the binding of CDR to tubulin dimer in a Ca2+-dependent manner.


Use of recombinant plasmids to characterize collagen RNAs in normal and transformed chick embryo fibroblasts. Two recombinant plasmids containing chick collagen DNA sequences have been used to characterize messenger RNAs for pro-alpha1 (type I) and pro-alpha2 collagen. Poly(A)-containing RNA from chick embryo calvaria and long bones, tissues which are very active in collagen synthesis, were electrophoresed on agarose gels containing methylmercuric hydroxide and transferred to diazobenzyloxymethyl paper; these covalently bound RNAs were hybridized to 32P-labeled pro-alpha1 or pro-alpha2 collagen DNA sequences derived from the recombinant plasmids. The pro-alpha1 collagen probe identified two RNAs, a major species of 5000 bases and a minor species of 7100 bases; the pro-alpha2 collagen probe hybridized to a major species very similar in size to the pro-alpha1 mRNA, about 5200 bases, and a minor species of 5700 bases. It is possible that the 7100 and 5700 base RNAs represent precursors of pro-alpha1 and pro-alpha2 collagen mRNA, respectively. When similar hybridization experiments were performed with RNA from chick embryo fibroblasts, both the pro-alpha1 and pro-alpha2 collagen mRNAs were observed, as well as their corresponding larger species. With RNAs from fibroblasts transformed by Rous sarcoma virus, however, the levels of all RNA species which hybridized with the pro-alpha1 and pro-alpha2 collagen DNA probes were significantly reduced.


Declining procollagen mRNA sequences in chick embryo fibroblasts infected with rous sarcoma virus. Correlation with procollagen synthesis. Chick cells infected with Rous sarcoma virus are characterized by a wide variety of changes known collectively as transformation. Among these are decreases in the level of procollagen biosynthesis and in the level of procollagen mRNA. In this communication, we examine the time course of the decrease in procollagen biosynthesis, as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and collagenase assay, and compare it with the decrease in procollagen mRNA sequences measured by hybridization to a complementary DNA. Procollagen biosynthesis and procollagen mRNA sequences decrease simultaneously after infection. Even the initial decrease in procollagen biosynthesis, therefore, is due to a decline in the level of procollagen mRNA.


Effects of spermine on activity and stability of calcium-dependent guanosine 3',5'-monophosphate phosphodiesterase. Spermine in micromolar concentrations decreased the basal activity of a guanosine 3',5'-monophosphate (cGMP) phosphodiesterase from bovine brain but had no effect in the presence of Ca2+ plus the calcium-dependent regulatory protein (CDR) which increased the activity of the enzyme 4- to 6-fold. Similar effects of spermine were observed on the enzyme at several stages of purification. Spermidine and putrescine were also inhibitory but higher concentrations were required. In the absence of Ca2+ and CDR, the enzyme exhibited two apparent Km values for cGMP (2.5 and 20 microM) which were unaltered by spermine. In the presence of Ca2+ and CDR (when spermine had no effect on activity), a single Km (3.5 microM) was observed. Enzyme purified by chromatography on CDR-Sepharose was rapidly inactivated during incubation at 30 degrees C in 5 mM potassium phosphate buffer (pH 7.0) with EDTA and ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA). Spermine (20 microM) partially stabilized enzyme activity under these conditions, although it was somewhat less effective than 2 mM MgCl2. The inhibitory effects of spermine (or other polyamines) on basal phosphodiesterase activity, which can be overcome by Ca2+ and CDR, could be important in the regulation of cellular cyclic nucleotide content.


Thyroglobulin interactions with thyroid plasma membranes. The existence of specific receptors and their potential role. Thyroglobulin binds to isolated thyroid plasma membrane preparations. Binding is pH- and temperature-dependent with 10-fold better binding at pH 5.0 and 37 degrees C than at 0 degrees C and pH 6.0 through pH 7.5. Binding is, however, maximal in 90 min at all pH values and temperatures examined. Although salts can inhibit or enhance thyroglobulin binding depending on the temperature or pH, conditions approaching those of the physiological state are not inhibitory; physiological conditions do inhibit thyrotropin binding to the same membrane preparations. 125I-Labeled thyroglobulin binding is poorly reversed by unlabeled thyroglobulin at all pH values and temperatures studied; excess unlabeled thyroglobulin can, however, readily prevent binding. At pH values greater than 6.0 and at 0 degrees C, the iodine content of thyroglobulin can affect binding, and the 27 S thyroid iodoprotein is relatively ineffective in preventing the binding of the 19 S species. At pH 5.0 and 37 degrees C, there is no difference in binding of highly and less iodinated thyroglobulin, and the 27 S thyroglobulin iodoprotein is effective in preventing 19 S thyroglobulin binding. The complex nature of these results is interpreted in the light of additional data which show (i) that the thyroid membrane recognizes asialothyroglobulin and (ii) that at pH 5.0 and 37 degrees C a membrane-associated neuraminidase is activated which removes sialic acid from thyroglobulin. Vibrio cholerae neuraminidase can substitute for the endogenous neuraminidase. The receptor on thyroid membranes for asialothyroglobulin is similar to the asialoglycoprotein receptor on liver membranes (Morell, A.G., Gregoriadis, G., Scheinberg, I.H., Hickman, J., and Ashwell, G. (1971) J. Biol. Chem. 246, 1461-1467) in that sialic acid on the receptor is critical for receptor expression. It is distinct from the liver asialoglycoprotein receptor in its binding specificity and in its sensitivity to different bacterial and mammalian neuraminidase preparations. Relationships between thyroglobulin and thyrotropin receptors on thyroid membranes are explored, and the functional role of the thyroglobulin receptor is discussed.


The activity of 2,4,5-triamino-6-hydroxypyrimidine in the phenylalanine hydroxylase system. The pyrimidine moiety of a pterin, 2,4,5-triamino-6-hydroxypyrimidine, has been found to be active in the phenylalanine-hydroxylating system. The phenylalanine-dependent, phenylalanine hydroxylase-catalyzed reaction in the presence of the pyrimidine is largely, but not completely, uncoupled; the ratio of DPNH oxidized to tyrosine formed is about 20 to 1. In addition to the pyrimidine having activity with phenylalanine hydroxylase, a product of the pyrimidine is also a substrate for dihydropteridine reductase. The activity of the pyrimidine with the hydroxylase indicates that neither carbon atoms 6 or 7 of the pterin ring is involved in activation of oxygen during the hydroxylase-catalyzed reaction.


Interaction of cytochrome c, ferrous ion, and phosphate. Electron transfer within a stoichiometric complex. The rate and extent of electron transfer from ferrous ion to ferricytochrome c are enhanced by the presence of inorganic orthophosphate at concentrations comparable to those of reductant and oxidant. Evidence, obtained by the method of continuous variations, shows that the electron transfer occurs within a stoichiometric complex composed of cytochrome c, ferrous ion, and phosphate in molar proportions of about 1:1:1. The incorporation of the anion into this complex appears to result in a modulation of the extent and rate of cytochrome c reduction. The rate of electron transfer obeys a first order rate law, characterized by an apparent first order rate constant of 1.4 min-1. The complex has kinetic significance only; equilibrium dialysis, gel filtration, and sedimentation velocity experiments yielded no evidence for stable binding of phosphate and iron, or of aggregation, on a significant scale. The extent of reduction is limited (for reasons not yet known) to about one-half of the available cytochrome molecules. Reduction in excess of 50% can be achieved only when both, ferrous ion and phosphate, are present in excess of the cytochrome concentration. Kinetic data indicate that reduction to extents over and below 50% occurs by different mechanisms.


Purification and properties of fructose-1,6-bisphosphatase of Bacillus subtilis. Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrase, EC 3.1.3.11) of Bacillus subtilis is a constitutive enzyme that was purified 1000-fold (30% yield) to 80% purity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis where it exhibits a band corresponding to 72,000 daltons. It sediments at 15 S in sucrose density gradients indicating a molecular weight of 380,000, but apparently is very asymmetric. Its activity is irreversibly inactivated in the absence of Mn2+. The enzyme specifically catalyzes dephosphorylation of D-fructose 1,6-bisphosphate with a pH optimum of 8.0. It has 40 to 60% of full activity in the absence of P-enolpyruvate; 20 microM P-enolpyruvate activates it maximally. High concentrations of monovalent cations also activate, NH4+ being most effective. Inhibitors fall into two groups. 1) Nucleoside monophosphates, phosphorylated coenzymes, and polynucleotides inhibit competitively with P-enolpyruvate (AMP (Ki = 2 microM) and dAMP are most effective). 2) The inhibition by nucleoside di- and triphosphates, PPi, and highly phosphorylated nucleotides (guanosine 5'-triphosphate 3'-diphosphate (pppGpp) and adenosine 5'-triphosphate 3'-diphosphate are most effective) is not competed by P-enolpyruvate but is partially overcome by fructose 1,6-bisphosphate (2 microM). Therefore, highly phosphorylated nucleotides (pppGpp and others), produced in over 0.2 mM concentrations upon step down from fast to slow growth rates (Gallant, J., and Lazzarini, R.A. (1976) in Protein Synthesis (McConkey, E.H., ed) Vol. 2, pp. 309-349, Marcel Dekker, Inc., New York), can reduce the conversion rate of fructose 1,6-bisphosphate to fructose 6-phosphate during gluconeogenesis. Comparing glycolytic growth on D-glucose and gluconeogenic growth on L-malate, the intracellular concentrations of fructose 1,6-bisphosphate differ but are both above the Km (13 microM) of the enzyme, those of AMP are similar, whereas those of P-enolpyruvate (0.18 mM versus 1.3 mM) indicate that the enzyme has only 40% of its full activity during glycolysis; nucleotides other than AMP may inhibit additionally. Thus, the futile cycle of fructose 1,6-bisphosphate synthesis and degradation during glycolysis is partially avoided, but the cells are poised for rapid adaptation upon change to gluconeogenic growth conditions.


Vesicular stomatitis virus infection reduces the number of active DNA-dependent RNA polymerases in myeloma cells. Infection of mouse myeloma (MPC-11) cells with vesicular stomatitis virus resulted in rapid loss in activity of cellular RNA polymerases associated with nuclear chromatin. No RNA polymerase inhibitor could be detected in extracts of infected cell nuclei. Reconstitution experiments with solubilized RNA polymerases dissociated from chromatin of infected and uninfected cells demonstrated that vesicular stomatitis viral infection did not affect the ability of the polymerases to function on endogenous or exogenous templates; nor did infection alter the template capability of the chromatin. Measurement of the number of actively growing RNA chains revealed that infected cell nuclei contained fewer active polymerase units; however, the rates of RNA chain elongation were the same in nuclei from infected and uninfected cells. Quantitation of the number of polymerase units active in nuclear chromatin revealed that the alpha-amantin-sensitive polymerase II was more severely reduced by viral infection than were polymerases I and III.


Hormone-stimulated phosphorylation of liver phosphofructokinase in vivo. The effect of glucagon on the phosphorylation and the enzymic activity of phosphofructokinase in rat liver in vivo was investigated. Glucagon stimulated the phosphorylation of liver phosphofructokinase approximately 3- to 5-fold and increased cAMP levels 5-fold and blood glucose levels 2-fold over the values obtained for control animals. The specific radioactivity of ATP isolated from liver was the same in both control and hormone-treated animals. During the purification of the 32P-labeled enzyme from both animals, no difference was observed in the total or specific enzyme activities of the enzymes from the various fractions. Thus, phosphofructokinase appears to be phosphorylated in vivo by a cyclic AMP-dependent protein kinase. Although phosphorylation does not affect the maximum catalytic activity of the enzyme, it does render the enzyme significantly more sensitive to ATP inhibition. Thus, at a given concentration of ATP, the phosphorylated phosphofructokinase exhibits considerably lower activity than the unphosphorylated enzyme. The possible relationship between our observations and glucagon-mediated control of glycolysis is discussed.


Processing and function of undermethylated chicken embryo fibroblast mRNA. Cycloleucine (1-aminocyclopentane-1-carboxylic acid) is a potent inhibitor of RNA methylation in B77 sarcoma virus-infected chicken embryo fibroblasts. Under conditions where 40 mM cycloleucine is present, internal N-6-methyladenosine and 5'-terminal cap 2'-O-ribose methylations of poly(A)+ RNA are inhibited greater than 90%. The methylation of the 5'-terminal 7-methylguanosine, however, does not appear to be significantly affected. The poly(A)+ RNA synthesized in cycloleucine-treated cells is transported from the nucleus to the cytoplasm and associates with polyribosomes at rates comparable to poly(A)+ RNA in untreated cells. On the other hand, the transport and utilization of newly synthesized ribosomal RNA in cycloleucine-treated cells is impaired, and the accumulation of mature 18 S and 28 S rRNA is reduced.


A protein activator for the adenylate cyclase of Bordetella pertussis. The activity of Bordetella pertussis extracytoplasmic adenylate cyclase is 100-fold higher in organisms grown on blood agar than in those grown in synthetic medium. This increase in activity is due to in vivo activation of the enzyme by a factor present in erythrocytes. Activation also occurs in killed or disrupted organisms. The activator can be separated from heme proteins and has been purified approximately 100-fold from erythrocytes, yielding material of approximately 105,000 daltons. It is sensitive to trypsin and alpha-chymotrypsin and exhibits considerable heat stability. Activation of cyclase in intact B. pertussis organisms exhibits a lag of 3 to 4 min and is not reversed by washing. Response to the activator decreases with increasing purification of the adenylate cyclase and is absent in the pure enzyme. The activation does not appear to be proteolytic and does not appear to change access to the substrate, ATP. The activator has no effect on a number of eukaryotic cyclases. We conclude that this is a new type of activation and that the activator differs from all those previously described.


Testicular steroidogenesis after human chorionic gonadotropin desensitization in rats. When a single injection of 500 I.U. of human chorionic gonadotropin (hCG) is given to rats there is an initial acute rise of plasma testosterone and of testicular content for both cyclic AMP and testosterone. This response correlates with an increase in both lyase and 17 alpha-hydroxylase activities. Thereafter both plasma and testicular testosterone decline and do not increase after a second injection of hCG. During this period of desensitization, isolated Leydig cells were insensitive to the steroidogenic stimulatory effect of both hCG and dibutyryl cyclic AMP. The post-cyclic AMP block is not due to an alteration of the cyclic AMP-dependent protein kinase but it is correlated with a decrease in both lyase and 17 alpha-hydroxylase activities of the Leydig cell's microsomes. This decrease is not caused by the absence of the recently described cytosol activator of this enzyme because its addition did not restore the enzymatic activity. Within 60 to 96 h after hCG injection there was a spontaneous increase of both plasma and testicular testosterone and this parallels the recovery of lyase and 17 alpha-hydroxylase activities. These results suggest that both enzymatic activities are regulated, directly or indirectly, by hCG, and that this is partly responsible for the hCG-induced steroidogenic refractoriness of Leydig cells.


An adenosine 3':5'-monophosphate-dependent protein kinase from the human erythrocyte membrane. Purification and characterization. An adenosine 3':5'-monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) has been isolated from the human erythrocyte memebrane and the phosphotransferase activity exhibited by this enzyme has been purified 800-fold. In concentrated solutions, the membrane-derived protein kinase undergoes aggregation with a concomitant loss in observed phosphotransferase activity. This loss of activity can be restored by means of inducing deaggregation. The phosphotransferase activity of the protein kinase is virtually obliterated in the presence of high (300 mM) concentrations of sodium chloride. This effect is also reversible. The pH optimum for the phosphotransferase reaction that is catalyzed by the membrane-derived protein kinase is approximately 8. Micromolar concentrations of cAMP are optimal with respect to promoting the phosphotransferase reaction. Initial velocity and product inhibition studies were conducted on the cAMP-independent protein kinase derived from the cAMP-dependent enzyme. These studies indicate that the phosphotransferase reaction proceeds by a sequential kinetic mechanism.


Total synthesis of a tyrosine suppressor transfer RNA gene. XIII. Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence -1 to -29 in the promoter region. Chemical syntheses of two tridecanucleotides, d(G-C-A-T-C-A-T-A-T-C-A-A-A) and d(G-C-G-T-C-A-T-T-T-G-A-T-A), and three undecanucleotides, d(G-G-A-A-G-C-G-G-G-G-C), d(T-G-A-T-G-C-G-C-C-C-C), and d(T-G-A-C-G-C-G-C-C-G-C), are described. These deoxyribo-oligonucleotide segments together represent the DNA duplex corresponding to the previously determined nucleotide sequence -1 to -29 of the promoter region of the tyrosine tRNA gene (Sekiya, T., van Ormondt, H., and Khorana, H.G. (1975) J. Biol. Chem. 250, 1087-1098). Chemical syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains. The desired condensation products were isolated by solvent extraction methods in the case of di- and trincleotides and by anion exchange chromatography in the case of longer chains. All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling at the 5'-ends with a [32P]phosphate group.


Total synthesis of a tyrosine suppressor transfer RNA gene. XVI. Enzymatic joinings to form the total 207-base pair-long DNA. The total synthesis of a 207-base pair-long DNA, which is biologically functional as a tyrosine suppressor transfer RNA gene, has been completed. The synthesis involved the enzymatic joining of the previously synthesized duplexes. Thus, the duplex corresponding to the promoter region [P] (Sekiya, T., Brown, E.L., Ramamoorthy, B., Fritz, H.-J., Gait, M.J., Lees, R.G., Ryan, M.J., Khorana, H.G., and Norris, K.E. (1979) J. Biol. Chem. 254, 5781-5786) was jointed to Duplex [I] (Caruthers, M.H., Kleppe, R., Kleppe, K., and Khorana, H.G. (1976) J. Biol Chem. 251, 658-666) to form [P + I]. Separatively, Duplex [III + IV + Vb] was prepared from the previously described Duplexes [III], [IV], and [Vb]. (Loewen, P.C., Miller, R.C., Panet, A., Sekiya, T., and Khorana, H.G. (1976) J. Biol. Chem. 251, 642-650; Sekiya, T., Besmer, P., Takeya, T., and Khorana, H.G. 1976) J. Biol. Chem. 251, 634-641; Ramamoorthy, B., Lees, R.G., Kleid, D., and Khorana, H.G., (1976) J. Biol Chem. 251, 676-694). The product [P + I], was joined to Duplex [II] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H.G. (1976) J. Biol. Chem. 251, 651-657) and then to [III + IV + Vb] without isolation of the intermediates. In all the above joinings, the duplexes carried 32P-labeled phosphate groups at the appropriate 5'-ends. The total DNA and the intermediate duplexes were all characterized by their relative mobilities in electrophoresis on polyacrylamide gel slabs, by nearest neighbor analysis, and by degradation to 5'-nucleotides of radioactively labeled joined products. Two succeeding papers describe the transcription in vitro and the suppressor activity in vivo, of the synthetic gene now described.


The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. I. The tryptic peptides from the maleylated protein. Six tryptic peptides ranging in size from 3 to 126 residues were isolated from maleylated Fragment A of diphtheria toxin after tryptic hydrolysis. These peptides accounted for all 193 residues found by amino acid analysis. After demaleylation, the six peptides were purified by chromatography on Sephadex G-50, coupled with paper chromatography and electrophoresis, and were analyzed by various methods. The compositions and properties of the peptides are reported. Almost 70% of the residues were positioned within these peptides.


The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. II. The cyanogen bromide peptides. Cyanogen bromide cleavage of Fragment A from diphtheria toxin at the four methionines present in each molecule resulted in five major peptides which were isolated and studied by sequence methods. These five peptides of 4, 11, 14, 63, and 101 residues account for all 193 residues in Fragment A and provide overlaps for the tryptic peptides from the maleylated protein. Two additional peptides were isolated and shown to be shorter forms (8 and 10 residues) of the COOH-terminal cyanogen bromide peptide (11 residues).


The amino acid sequence of fragment A, an enzymically active fragment of diphtheria toxin. III. The chymotryptic peptides, the peptides derived by cleavage at tryptophan residues, and the complete sequence of the protein. Fragment A (21,145 daltons in its longest known form) may be derived from diphtheria toxin (60,000 daltons) by mild tryptic digestion and reduction. Purified Fragment A consists of a mixture of 3 molecules of 190, 192, and 193 residues; the first 190 residues are in common and correspond to the NH2-terminal region the toxin. All three species of Fragment A are active in catalyzing ADP ribosylation of elongation factor 2, an essential component of protein synthesis. This reaction inactivates the factor and is responsible for the toxin's action in inhibiting protein synthesis in animal cells. It is believed that Fragment A or similar enzymically active fragments released into the cytosol of toxin-treated cells mediate this inhibition. The complete amino acid sequence of Fragment A has been determined from 32 chymotryptic peptides, three peptides derived by chemical cleavage of Fragment A at its 2 tryptophan residues, five cyanogen bromide peptides, and six tryptic peptides from the maleylated protein.


Enzymic activity of cholera toxin. II. Relationships to proteolytic processing, disulfide bond reduction, and subunit composition. Cholera toxin containing intact A chain (Mr = 29,000) was isolated, and its enzymic properties were characterized. The "unnicked" form of the toxin, produced by a protease-deficient, hypertoxinogenic mutant of Vibrio cholerae 569B, had greatly reduced activity in catalyzing the NAD+-glycohydrolase and ADP-ribosyltransferase reactions as compared to the naturally nicked form commonly isolated. In the latter, the intact A chain has been cleaved by bacterial proteases to yield disulfide-linked A1 and A2 chains (Mr = 23,000 and 6,000, respectively). Digestion of unnicked toxin with trypsin or elastase yielded a nicked form similar to or identical with the naturally nicked toxin, but chymotryptic digestion did not. Disulfide bond reduction was necessary for expression of enzymic activity by naturally nicked or trypsin-nicked toxin, or the A1A2 protomer. Fractionation of thiol-treated, nicked cholera toxin by ion exchange, molecular exclusion, or affinity chromatography gave results suggesting that the reduced toxin displays enzymic activity while remaining structurally intact.


Structure of the lipopolysaccharide from an Escherichia coli heptose-less mutant. I. Chemical degradations and identification of products. The structure of lipopolysaccharide from a heptose-less mutant of Escherichia coli K-12 has been investigated. Lipopolysaccharide isolated from 32P-labeled cells was treated with mild alkali to yield two separable components: [OH-LPS]-I (approximately 70%) and [OH-LPS]-II (approximately 30%). Mild acidic treatment of [OH-LPS]-I gave mainly a product which was identified as (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine 1-phosphate (Compound I). Further acidic hydrolysis of both [OH-LPS]-I and [OH-LPS]-II yielded as the main product (4-O-phosphoryl-N-beta-hydroxymyristyl-D-glucosaminyl)-beta(1 leads to 6)-N-beta-hydroxymyristyl-D-glucosamine (Compound II). The structures of the above products were deduced by a combination of compositional analyses, sensitivity to phosphomonoesterase, rates of hydrolysis of the phosphate groups and alkali-catalyzed beta elimination of the phosphate residues following appropriate oxidation of hydroxyl groups. These studies together with work reported in the accompanying papers have led to the identification of two species of lipopolysaccharide in the E. coli strain both of which contain a single glucosamine dissacharide unit but differ in having monosubstituted phosphate or pyrophosphate groups at the glycosidic position. Each species of lipopolysaccharide also appeared to be heterogeneous with respect to the number of esterified fatty acyl groups.


Cytochrome c peroxidase. Interconversion of chemically and enzymatically reactive and unreactive forms of the ferric protein. Ferric yeast cytochrome c peroxidase in the presence of different anions may assume a number of forms which differ in optical spectra and chemical properties. In solutions whose only anion is acetate, two spectral forms are present together in an equilibrium. Each of these spectral species is believed to bear bound acetate anion. A form characterized by an intense absorption maximum at 620 nm is unreactive enzymatically and does not react with hydrogen peroxide or with dithionite. A form characterized by a less intense absorption near 645 nm is enzymatically and chemically reactive. Increasing temperature and increasing pH displace the equilibrium toward the 645 nm form. Increasing cytochrome c peroxidase concentration favors the 620 nm form. In kinetic experiments in which the 645 nm form is removed by rapid reaction with H2O2 or dithionite, the 620 nm form is converted in a first order reaction (k = 0.36 s-1, 15 degrees C) to the 645 nm form. In solutions whose sole anion is phosphate a 645 nm form is the only demonstrable spectral species. The enzymatic activity and rates of chemical reaction of 645 nm spectral forms occurring in acetate and in phosphate buffers are the same.


Two molecular forms of (Na+ + K+)-stimulated ATPase in brain. Separation, and difference in affinity for strophanthidin. The brain contains two distinct molecular forms of the (Na,K)-ATPase (sodium and potassium ion-stimulated adenosine triphosphatase). They can be resolved by gel electrophoresis in sodium dodecyl sulfate, and can be identified by sodium-dependent, potassium-sensitive phosphorylation by [gamma-32P]ATP. They are present in the brain of every animal species examined, while only one molecular form is detected in the other organs examined. They are located in different kinds of cells within the brain, and can be physically separated while fully active by gentle tissue fractionation procedures. One is the only (Na,K)-ATPase of brain non-neuronal cells (astrocytes), while the other is the only (Na,K)-ATPase of axolemma (plasma membrane of myelinated axons). They differ in at least one kinetic parameter: the affinity for the specific inhibitor strophanthidin. They have similar one-dimensional peptide maps, but differ in their sensitivity to digestion by trypsin and in the number or reactivity of sulfhydryl groups. It is anticipated that they will be found to play functionally different roles in the complex ion transport mechanisms of the brain.


The role of oxygen in the biosynthesis of cytochrome c oxidase of yeast mitochondria. The formation of cytochrome c oxidase in yeast is dependent on oxygen. In order to examine the oxygen-dependent formation of the active enzyme, the effect of oxygen on the synthesis and the assembly of cytochrome c oxidase subunits was studied. Pulse-labeling experiments revealed that oxygen has no significant immediate effect on the synthesis of the three mitochondrially made subunits I to III; however, its presence causes subunits I and II to form a complex with the cytoplasmically made subunits VI and VII. This "assembly-inducing" effect can be demonstrated with intact yeast cells as well as with isolated mitochondria. It is independent of cytoplasmic or mitochondrial protein synthesis. After anaerobic growth for 10 or more generations, the intracellular concentrations of individual cytochrome c oxidase subunits drop 10- to 100-fold. Most of these residual subunits are not assembled within a functional cytochrome c oxidase molecule.


Sequence of two phosphorylated sites in the catalytic subunit of bovine cardiac muscle adenosine 3':5'-monophosphate-dependent protein kinase. The sequences of two phosphopeptides isolated from the catalytic subunit of bovine cardiac muscle cAMP-dependent protein kinase (type II) and from two of its cyanogen bromide fragments, have been determined. One phosphorylation site is a threonyl residue located approximately 180 residues from the blocked NH2 terminus. Its sequence is: -Gly-Arg-Thr-Trp-Thr(P)-Leu-Cys- and includes one of the three sulfhydryl groups present in the molecule. The second phosphorylated site within the sequence: -Val-Ser(P)-Ile-Asn- is located towards the carboxyl end of the protein where the other 2 cysteinyl residues also reside. The finding that phosphorylation of the catalytic subunit occurs on two discrete sites rather than at random suggests that it might be of physiological importance, e.g. in the regulation of enzyme activity.


Synthesis of a new 8-spin-labeled analog of adenosine 5'-phosphate and its interaction with AMP nucleosidase. The synthesis of a new 8-spin-labeled analog of AMP, 8-[[[(2,2,5,5-tetramethyl-1-oxy-3-pyrrolidinyl)carbamoyl]methyl]thio]adenosine 5'-phosphate (8-slAMP), is described. The procedure is facile and results in high yields. 8-slAMP is a competitive inhibitor of AMP nucleosidase with a Ki of 19 microM as compared to a Km of 100 microM for AMP. The analog is not a substrate for the enzyme and does not displace MgATP2- from the allosteric sites under the usual assay conditions. The EPR spectrum of the bound spin probe reveals a highly immobilized nitroxide group. Binding studies with 8-slAMP at 8 degrees C indicate three independent binding sites (Kd = 1.4 microM) per molecule of enzyme (Mr = 320,000). These properties make 8-slAMP a good spin probe for AMP nucleosidase. The analog may also be useful for other proteins known or suspected of binding AMP analogs in a syn conformation.


NAD-dependent ADP-ribosylation of arginine and proteins by Escherichia coli heat-labile enterotoxin. Escherichia coli heat-labile enterotoxin (labile toxin, LT) catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide and the ADP-ribosylation of arginine (Moss, J., and Richardson, S.H. (1978) J. Clin. Invest. 62, 281-285). Analysis of the product of the ADP-ribosylation of arginine by nuclear magnetic resonance spectroscopy indicated that the reaction was stereospecific and resulted in the formation of alpha-ADP-ribosyl-L-arginine. This reaction product rapidly anomerized to yield a mixture of the alpha and beta forms. In the presence of [adenine-U-14C]NAD, E. coli enterotoxin catalyzed the transfer of the radiolabel to proteins; the ADP-ribosylation of proteins was inhibited by arginine methyl ester, an alternative substrate. Digestion of the 14C-protein with snake venom phosphodiesterase released predominantly 5'-AMP. No product was obtained with a mobility similar to that of 2'-(5''-phosphoribosyl)-5'-AMP. This result is consistent with the covalent attachment by the enterotoxin of ADP-ribose rather than poly(ADP-ribose) to protein. Thus, LT is catalytically equivalent to choleragen, an enterotoxin of Vibrio cholerae, and activates adenylate cyclase through a similar stereospecific ADP-ribosylation reaction.


Total assignments, including four aromatic residues, and sequence confirmation of the decapeptide tyrocidine A using difference double resonance. Qualitative nuclear overhauser effect criteria for beta turn and antiparallel beta-pleated sheet conformations. The complete assignments of all the proton magnetic resonance signals from each NH-CalphaH-CbetaH2 moiety in a complex peptide containing several residues of the same type has not yet been achieved without specific or stereospecific isotopic enrichment. We report the sequencing and proton magnetic resonance spectral assignments, including those of 4 aromatic residues, of tyrocidine A, an analog of the decapeptide gramicidin S. Two complementary methods, proton-proton nuclear Overhauser enhancements and scalar decoupling, evaluated by two distinct forms of difference double resonance, were used. All chemical shifts, scalar coupling constants, and [1H:1H] nuclear Overhauser enhancements for the backbone protons are reported. The [1H:1H] nuclear Overhauser enhancements are consistent with tyrocidine A possessing a beta-I turn/beta-II' turn/antiparallel beta-pleated sheet conformation. In addition to the previously proposed nuclear Overhauser enhancement criteria for beta turns and antiparallel beta sheets, another criterion for identifying the antiparallel beta sheet is demonstrated; namely, the nuclear Overhauser enhancement between 2 CalphaH protons of the central resisdues, in this case the Phe7CalphaH and Orn2CalphaH.


Effect of plasma membranes on solute transport in 3T3 cells. Sparse cultures of Swiss 3T3 cells are arrested early in the G1 phase of growth by the addition of a plasma membrane fraction obtained from confluent 3T3 cells. We have examined whether the changes in solute transport which are usually associated with cessation of growth at confluency also take place when cell growth is arrested by the addition of plasma membranes. We find that the rate of uptake of alpha-aminoisobutyric acid and uridine is decreased after the addition of plasma membranes to 3T3 cells, but the rate of uptake of 2-deoxyglucose and phosphate is not. We conclude from these observations that uptake of uridine and alpha-aminoisobutyric acid are related to contact inhibition of growth, while the decline in the rate of uptake of 2-deoxyglucose and phosphate observed at high cell density must be due to changes other than cell to cell contact.


T4 gene 32 protein trypsin-generated fragments. Fluorescence measurement of DNA-binding parameters. The intrinsic fluorescence of the T4 helix-destabilizing protein specified by gene 32 (32P) is not altered by the proteolytic removal of either the 6200-dalton COOH-terminal "A" region (32P*-A) or both the A and the 2300-dalton NH2-terminal "B" region (32P*-(A + B)). The intrinsic fluorescence of 32P, 32P*-A, and 32P*-(A + B) is decreased 23% by the addition of d(pT)8 and 34% by the addition of poly(dT). Saturation binding curves of the percentage of change in protein fluorescence as a function of nucleotide concentration show that the intact 32P as well as the two proteolysis-generated fragments all have association constants of approximately 10(6) M-1 for d(pT)8. This demonstrates that the DNA binding site is not contained within either the A or B regions of 32P. Both 32P and 32P*-A bind cooperatively to poly(dT) as evidenced by a 400- to 1000-fold increase in association constant for poly(dT) compared to d(pT)8. Since within the limits of our measurements 32P and 32P*-A bind equally well to poly(dT) (Kassoc approximately 5 . 10(8) M-1), the enhanced helix-destabilizing properties previously reported for 32P*-A cannot be accounted for by a significant increase in binding affinity of 32P*-A for single-stranded DNA. The binding constant for the 32P*-(A + B):poly(dT) complex is only 3-fold higher than that for the 32P*-(A + B):d(pT)8 complex, which confirms our proposal that the B region is essential for cooperative 32P:32P protein interactions.


Brain pyridoxal kinase. Purification, substrate specificities, and sensitized photodestruction of an essential histidine. Pyridoxal kinase has been purified 2,000-fold from pig brain. The enzyme preparation migrates as a single protein and activity band on analytical gel electrophoresis. Pyridoxal kinase, 60,000 molecular weight, catalyzes the phosphorylation of pyridoxal (Km = 2.5 x 10(-5) M) and pyridoxine (Km = 1.7 x 10(-5) M). Pyridoxamine is not a substrate of the purified kinase. Irradiation of the kinase in the presence of riboflavin leads to irreversible loss of catalytic activity. Riboflavin binds to the kinase with a KD = 5 microM as shown by fluorometric titrations. Singlet excited oxygen, generated by energy transfer from the lowest triplet of riboflavin to oxygen, acts as the oxidizing agent of approximately one histidine residue per mol of enzyme. The amino acid residues tyrosine, tryptophan, and cysteine are not photooxidized by the sensitizer bound to the enzyme. It is postulated that histidine is involved in the binding of the substrate ATP to the catalytic site of pyridoxal kinase.


Adeno-associated virus DNA replication complexes in herpes simplex virus or adenovirus-infected cells. A complex which is active in in vitro synthesis of adeno-associated virus (AAV) DNA was solubilized from Vero cells that were co-infected with AAV and either adenovirus (Ad5) or a herpes simplex virus type 1 (HSV-1) as the helper virus. The complexes from the Ad5 and HSV-1-infected cells sedimented at 23 S and 28 S, respectively. The optimal conditions for in vitro DNA synthesis for the two types of complex using the endogenous AAV template and the endogenous DNA polymerase, differed with respect to the effect of KCl and K2SO4 concentration. In addition the complex from HSV-1-infected cells, but not that from Ad5-infected cells, was inhibited by phosphonoacetic acid. Thus, the two complexes appear to contain different DNA polymerase activities. This was verified by phosphocellulose chromatography of the DNA polymerases solubilized from the isolated complexes. The major activity in the complex from HSV-1 infected cells was the HSV-induced DNA polymerase with lesser amounts of cellular DNA polymerase alpha and gamma or both. The complex from the Ad5-infected cells contained mainly a cellular DNA polymerase gamma.


Acetate kinase from Veillonella alcalescens. Regulation by succinate and substrates. The kinetic properties of acetate kinase from Veillonella alcalescens were investigated. In the presence of high concentrations of nucleotide both forward and reverse reactions were observed. In the presence of succinate the degree of cooperativity between subunits of the homodimer decreased, i.e. the Hill coefficient, n, decreased from 2.5 to 1.4 for acetyl phosphate in the presence of succinate. At low substrate concentrations hyperbolic kinetic data were observed with succinate. We have proposed a modified version of the concerted symmetry model to describe the kinetics observed with this enzyme. The primary differentiating feature of the proposed model is the requirement for activator ligand binding for catalysis. In the absence of succinate, the substrate (acetate or acetyl phosphate) also functions as an activating ligand.


Purification and partial characterization of a nonprimate growth hormone receptor. Pregnant rabbit liver membranes have been shown to possess two types of receptors by displacement analysis, a growth hormone (GH) receptor which binds bovine growth hormone with an affinity constant (KA) of 3 x 10(9) M-1 and ovine prolactin with a KA of 3 x 10(8) M-1, and a prolactin (Prl)-specific receptor which binds ovine prolactin with a KA of 5 x 10(9) M-1. The prolactin-specific receptor when solubilized with Triton exhibits a 4-fold increase in the its KA while the KA of the growth hormone receptor decreases slightly to 2 x 10(9) M-1 after solubilization. The 10-fold difference in affinity which results has been exploited to facilitate the separation of these two receptors by differential affinity chromatography on human growth hormone (hGH) affinity gels. The growth hormone receptor is eluted from the gel with 4 M urea while 5 M MgCl2 is required to elute the prolactin receptor. Conditions of affinity chromatography have been optimized, and further purification of the GH receptor by preparative isoelectric focusing and Sepharose 6B gel filtration resulted in a more than 8000-fold purification of the receptor. This material had a Stokes radius of 62 A, consistent with a molecular weight of 300,000 and gave one main band (75,000 to 80,000) and two minor bands on sodium dodecyl sulfate (SDS) polyacrylamide gels, which could be interpreted as indicating a tetrameric receptor. The GH receptor was shown to be a sialoglycoprotein (or closely associated with sialoglycoprotein) by analytical isoelectric focusing with an isoelectric point of 4.6. Specificity studies with the highly purified receptor confirmed the initial hypothesis that this receptor is capable of binding bovine growth hormone (bGH) with high affinity and ovine prolactin (oPrl) with low affinity, in contrast to the prolactin-specific receptor.


Leydig cell hypoplasia: a cause of male pseudohermaphroditism. A patient with male pseudohermaphroditism is presented whose testicular biopsy showed marked paucity of the Leydig cell populations to which is attributed testosterone deficiency and impaired spermatogenesis.


Selective hypopituitarism with severe hyponatremia and secondary hyporeninism. A female patient presenting clinically a severe hyponatremia was found to have a selective hypopituitarism with predominant ACTH and partial FSH, LH, and GH deficiency as well as a suppression of plasma renin activity and aldosterone. The adrenal cortex responded well in cortisol increase to ACTH infusion and in plasma aldosterone increase to angiotensin II infusion. The patient had pressor hyperreactivity to angiotensin II. The hyponatremia was caused by a negative sodium balance induced by excessive urinary loss which remained unaffected by mineralocorticoid treatment. Substitution doses of cortisol, however, corrected the disturbance with an increase in plasma renin activity and improvement in the sodium balance. The data are interpreted as indicating a direct or indirect regulatory (permissive?) effect of low doses of cortisol on plasma renin activity correcting the underlying disturbance--the secondary hyporeninism.


Suppression of plasma catecholamines and flushing by clonidine in man. Administration of the anti-hypertensive agent clonidine as a single (0.5 mg) oral dose or as multiple doses (0.2-0.4 mg/day for 4 days) markedly reduced plasma catecholamines (decrement = 81 +/- 3% and 68 +/- 5%, respectively; X +/- SE, % of basal; both P less than 0.001) in normal male volunteers. Five patients with various metabolic disorders showed similar responses. The absolute decrements in plasma catecholamines correlated significantly with basal catecholamine levels (P less than 0.001). Clonidine-induced decrements in mean arterial blood pressure correlated significantly with decrements in plasma catecholamines (P less than 0.001). The clonidine effect upon catecholamine levels was reversed by phentolamine (clonidine = -68 +/- 5%; clonidine with phentolamine = -1 +/- 16%). The decrements in catecholamines induced by clonidine in normal subjects were associated with increased sensitivity to the pressor effect of infusion of exogenous norepinephrine. In an analogous fashion flushing associated with endogenous adrenergic discharge was blocked by clonidine, whereas that due to exogenous catecholamines was intensified. These data are compatible with data in experimental animals suggesting that clonidine acts at least in part by interaction with a central alpha adrenergic receptor.


DDAVP (1-desamino-8-D-arginine-vasopressin) treatment of central diabetes insipidus--mechanism of prolonged antidiuresis. DDAVP, 1-desamino-8-D-arginine-vasopressin, is a synthetic analog of arginine vasopressin which produces prolonged antidiuresis after intranasal administration to patients with complete central diabetes insipidus. We have studied the mechanism of the prolonged antidiuretic effect by specific radioimmunossay of DDAVP in plasma of patients and by in vitro studies on the adenylate cyclase-cylic AMP system of the rat outer renal medulla. When DDAVP was administredd to patients, all responded, but the duration of response among patients varied from 5-21 h. The peak level of DDAVP in plasma was achieved up to 4 h after administration indicating a slow absorption from the nasal mucosa. The disappearance time of DDAVP from plasma correlated significantly with the duration of antidiuresis, P less than 0.001. On a molar basis DDAVP was 3-fold greater than AVP in its stimulation of outer medullary adenylate cyclase activity and 10-fold greater than AVP in its stimulation of cyclic AMP content. The prolonged antidiuresis of intranasally administered DDAVP is due to slow absorption, presistence in plasma, and enchanced effect on the kidney.


Prolonged infusions of Ile5-angiotensin-II in sodium replete and deplete man: effects on aldosterone, ACTH, cortisol, blood pressure, and electrolyte balance. Angiotensin II (Ile5) was infused for 72 h into 4 sodium replete (3 ng/kg/min) and 8 sodium deplete (3 or 6 ng/kg/min) healthy young men after appropriate control periods, and the effects on aldosterone secretion, plasma cortisol, ACTH, renin activity, plasma and urinary electrolytes, and blood pressure were assessed. Sustained contrived elevation of plasma angiotensin II levels in sodium replete subjects to the range of moderate sodium depletion led to a sustained increase in plasma and urinary aldosterone levels, which further and significantly increased between the 1st and 2nd days of angiotensin II infusion, when gross sodium retention during infusion was prevented. This additional increase may be explained as the expression of a "trophic" effect of angiotension II on the zona glomerulosa. In the sodium deplete state, the absolute increment of aldosterone secretion for a given elevation of angiotensin II levels diring infusion was larger than in sodium replete subjects. This confirms the conclusions from previous short-term angiotensin II infusion experiments that sodium deficiency sensitizes the zona glomerulosa against angiotensin II. The "trophic" effect of angiotensin II on the adrenal gland seems to be one mechanism by which the sensitization is brought about, but insufficient for its full explanation. Since plasma ACTH and cortisol, plasma sodium and potassium concentrations, and potassium blance did not change significantly across sodium depletion or angiotensin II infusion, the mechanism of sensitization awaits its full elucidation. The effect of angiotensin II on blood pressure was blunted by soidum depletion. The opposite shifts in sensitivity against angiotensin II of the zona glomerulosa and of resistance blood vessels with changes in the sodium state seem to be an effective and important means in the regulation of body sodium.


Effect of human chorionic gonadotropin on human thyroid tissue in vitro. Thyroid stimulating substances other than TSH have been found in certain disease states associated with hyperthyroidism. The thyroid stimulator associated with the thyrotoxicosis of trophoblastic disease is uncertain; however, recent evidence suggests a role for hCG. To explore the thyroid stimulating properties of hCG further, we examined the ability of hCG to displace [1252]TSH from receptors on human thyroid membrane and to generate cyclic-AMP (c-AMP) from human thyroid slices. Human chorionic gonadotropin at a concentration of 40 IU/ml displaced labeled TSH from human thyroid membranes and, at a concentration of 69 IU/ml, hCG caused the generation of c-AMP in thyroid slices. These results suggest that hCG can bind to the TSH receptor on thyroid cells and can stimulate them to produce c-AMP at concentrations of hCG within the range that is found in trophoblastic disease.


Effects of the intravenous administration of calcium on nephrogenous cyclic AMP: use as a parathyroid suppression test. The question of parathyroid autonomy in primary hyperparathyroidism has been the subject of conflicting immunoassay data. We studied the effects of calcium infusion (12 mg/kg/3h) and calcium injection (3 mg/kg/10 min) on peripheral plasma parathyroid hormone (iPTH) determined with a multivalent antiserum and on the excretion of nephrogenous cyclic AMP in normal subjects and in 7 patients with primary hyperparathyroidism who displayed only mild, intermittent hypercalcemia. In control subjects, calcium administration resulted in small (13-20%) reductions in iPTH, whereas some 4/5 (77-81%) of the nephrogenous cyclic AMP was rapidly and uniformly suppressed. In the patients with primary hyperparathyroidism, both analyses revealed a lack of absolute parathyroid autonomy in response to calcium, with overlapping iPTH responses between a majority of the patients and the control group. In contrast, the nephrogenous cyclic AMP responses provided a clear separation of the 2 groups after both calcium infusion and calcium injection (mean values for both studies, patients: 2.93 nmol/100 ml GF vs. normal sugjects: 0.38 nmol/100 ml GF), and measurements of total cyclic AMP excretion also clearly distinguished the 2 groups. When a sensitive antiserum with predominantly carboxy-terminal reactivity was employed, the iPTH responses to calcium injection provided an improved separation of patients and normal subjects. The data suggest that 1) although parathyroid autonomy is not, in general, a feature of primary hyperparathyroidism, abnormal parathyroid suppressibility is easily demonstrated even in patients with a subtle form of the disorder; 2) the determination of nephrogenous cyclic AMP provides an optimal method for assessing rapid changes in parathyroid function; and 3) the interpretation of iPTH results from such studies is dependent on a number of technological features of the assay employed.


Thyrotoxicosis: comparison of effects of thyroid ablation and beta-adrenergic blockade on metabolic rate and ventilatory control. To elucidate the role of the beta-sympathetic system in thyrotoxicosis (THY), we examined cardiac sensitivity to infused beta-agonist and compared the effect of beta-blockade with that of resolution of the hyperthyroid state. Beta-sympathetic (beta-SYM) sensitivity was measured as the heart rate response to isoproterenol in THY patients and in normal subjects. The patients with THY showed both lower threshold (P less than 0.05) and increased slope (P less than 0.05) of the heart rate-isoproterenol response, suggesting beta-SYM hypersensitivity. The beta-SYM like features of THY were measured in 7 patients before and 7 days after beta-SYM blockade with propranolol (mean dose 411 +/- 32 mg/day [SEM]) which was shown to block the heart rate response to isoproterenol. These results were compared with those in a similar group of thyrotoxic patients rendered euthyroid with 131I. During beta-SYM blockade, heart rate decreased from 101 +/- 6.3 to 78 +/- 4.6 (P less than 0.01), but the elevated metabolic rate (V02), resting ventilation (VE), and increased hypoxic and hypercapnic ventilatory responses were not significantly affected. In the group rendered euthyroid with 131I, heart rate decreased from 110 +/- 3.5 to 76 +/- 7.8 (P less than 0.02), but in contrast to the result of beta-SYM blockade, a 28% decrease in VO2 (P less than 0.01), 41% decrease in VE (P less than 0.05), a 38% decrease in hypercapnic ventilatory response (P less than 0.05), and a 66% decrease in hypoxic response (P less than 0.03) occurred. During THY, beta-SYM mechanisms are responsible for part of the tachycardia, but the metabolic and ventilatory abnormalities are not beta-SYM mediated.


An infant with Cushing's disease due to an adrenocorticotropin-producing pituitary adenoma. An 8-month old male with Cushing's disease is presented; his clinical presentation and appearance were typical of infants with glucocorticoid excess. Concentrations of cortisol, 17-hydroxyprogesterone, and adrenal androgens were strikingly elevated. High doses of dexamethasone did not suppress the excretion of urinary free cortisol or 17-hydroxycorticoids, and administration of ACTH elicited no further rise in plasma cortisol. Responses of LH, FSH, and PRL to iv LRF and TRF were appropriate for age, but neither TSH nor ACTH rose significantly. Plasma ACTH values were elevated to 700 pg/ml. An intracranial mass lesion superior and anterior to the sella turcica was demonstrated by computerized axial tomography and angiography. An inoperable pituitary adenoma was a massive surrounding fibroblastic reaction was found at craniotomy. The pathological diagnosis of an ACTH-producing pituitary adenoma was confirmed by immunohistochemistry and by the in vitro secretion of ACTH by cells cultured from the tumor.


Localizaiton of insulinomas by radioimmunoassay of blood obtained by the transport route. Transhepatic catheterization of the portal system was performed to obtain blood for RIA of insulin. This technique localized insulinomas in two patients after negative celiac, superior mesenteric, and subselective gastroduodenal arteriograms. The tumors were found in the predicted locations at surgery.