Fate of herpes simplex virus in lymphocytes from inflammatory joint effusions. ii. Mechanisms of non-permissiveness. Mononuclear cells which were isolated from rheumatoid synovial effusions and cultured without prior separation failed to support the growth of herpes simplex virus (HSV). However, subpopulations of T lymphocytes separated on the basis of cell size or receptors for sheep red cells were, in isolation, permissive for this virus. Virus growth in these permissive cells was blocked by cocultivation with the remaining mononuclear cell populations.


Lead-cadmium interaction: effects on viral-induced mortality and tissue residues in mice. Simultaneous exposure to lead and cadmium is highly probable due to common environmental sources of exposure. Mice were exposed simultaneously for 10 wek to lead and cadmium and effects were observed on accumulation of the metals in tissues and susceptibility to viral challenge. The incidence of viral-induced mortality was enhanced in all groups of mice exposed to lead and suppressed in groups of mice treated with cadmium. Mice exposed to both lead and cadmium had mortality rates intermediate to those observed after exposure to the individual metals. Lead and cadmium residues in tissues of mice exposed to both metals, as compared to residues in tissues of mice which received lead or cadmium only, indciated that interaction occurs in regard to metabolism, storage, and excretion of these elements. Chronic coexposure to lead and cadmium appears to alter certain effects produced by exposure to each metal alone.


Search for viral nucleic sequences in rheumatoid cells. DNA and RNA were extracted from synovial membranes, synovial fibroblast cells, peripheral blood lymphocytes, and synovial fibroblast cells strains derived from patients with rheumatoid arthritis and other joint conditions. They were hybridised after immobilisation on nitrocellulose filters with iodinated viral nucleic acids extracted from measles, rubella virus, SV--40, and a retrovirus, RD--114. In addition, in situ-hybridisation was carried out on sections of synovial membranes by means of iodinated measles and rubella virus RNA. In no case did any hybridisation occur. Positive control systems included synovial fibroblast strains transformed with SV--40, LLC--MK2 cells chronically infected with rubella virus and RD cells infected with RD--114. It was concluded tht the synovial cells did not contain viral genomes of measles, rubella virus, SV--40, or RD--114, or at least at a level equivalent to the positive control cells.


Reversal of protein-energy malnutrition in children during treatment of advanced neoplastic disease. The effectiveness of enteral and parenteral feeding in supporting a satisfactory nutritional status and/or reversing protein-energy malnutrition was evaluated in 28 children, ages 1-19 (14 female) with advanced malignant disease (21 solid tumors, 7 leukemia-lymphoma). At the onset of treatment, 21 patients received intensive nutritional counseling (INC) and oral supplementation while seven received total parenteral nutrition (TPN). Sixteen of 21 patients who received INC had a decreased intake (x 48 +/- 24%) Recommended Dietary Allowances (RDA) for kilocalories and dramatic weight loss (x 16.4 +/- 12.4%). A total of 18 patients received TPN for a mean of 24 days (7-60); kcal averaged 90 +/- 26% RDA during weight gain. At onset of TPN, the mean serum albumin, transferrin and total lymphocyte counts were 3.06 +/- 0.38 g/dl, 175 +/- 62 mg/dl, and 1102 +/- 966/mm3 respectively, 15/18 children had subnormal anthropometric measurements and 17/18 patients were anergic to recall skin test antigens. TPN for less than 9-14 days neither repleted weight, skinfold reserves, nor serum albumin concentrations (greater than 3.2 g/dl) although an early increase (p less than .02) in transferrin concentration was observed. However, TPN for 28 days supported weight gain (3.27 kg, 16 +/- 6%), increased serum albumin (0.62 +/- 0.43 g/dl, p less than .001) and transferrin (62 +/- 42, p less than .002) to normal concentrations and reversed anergy in 7/11 patients retested. This study documents the severity of protein energy malnutrition which accompanies intense treatment of children with cancer and the nutritional and immunological benefits of a 28 day course of TPN.


Prolactin and Leydig cell responsiveness to LH/hCG in the rat. The effects of prolactin and 2-bromo-alpha-ergocryptine (CB-154) on Leydig cell function in intact and hypophysectomized male rats were studied. The conclusions can be summarized as follows: prolactin (1) has a direct stimulatory effect on the number of LH receptors on rat Leydig cells, (2) has no effect on the characteristics of the dose-response curve of isolated Leydig cells (hCG stimulated androgen production) in vitro even after treatment with pharmacological doses in vivo, and (3) acts synergistically with LH to stimulate the quantity of androgen produced by the Leydig cells in response to hCG in vitro and to increase the sensitivity of the hCG-dose-response curve. Treatment of intact rats with CB-154 reduced the quantity of androgen produced by the Leydig cells in vitro after exposure to hCG and decreased LH binding to the same cells by 50%. These results suggest that under normal conditions, endogenous prolactin plays a key role in maintaining the functional integrity of rat Leydig cells.


Lack of adrenal androgen stimulation by ACTH in extreme hyperprolactinemia. Acute and prolonged alpha 1-24 corticotropin stimulation was performed on a treated chromophobe adenoma patient with partial ACTH deficiency and extreme hyperprolactinemia. Cortisol and aldosterone stimulated normally. However, the basal concentrations of androstenedione (A) and dehydroepiandrosterone (DHA) were low, and that of DHA-sulfate (DHAS) was undetectable. Furthermore, A and DHA did not stimulate normally, and DHAS did not stimulate at all. It has been claimed that adrenal androgen production is increased in hyperprolactinemia. However, the inability of prolactin (Prl) to maintain adrenal androgen (AA) secretion, with and without added ACTH, is demonstrated in this patient.


Serum antibody levels as risk factors in the dissemination of herpes zoster. Serum antibody levels against varicella-zoster virus (VZV) were examined by immune adherence hemagglutination assay (IAHA), indirect fluorescent antibody (IFA) assay, and complement fixation techniques in 67 immunocompromised patients with localized and disseminated herpes zoster. In the serum obtained initially, undetectable IAHA titers were found in 56.5% of the patients with disseminated zoster compared with 18.2% of those with localized zoster. When serum obtained within the first seven days of illness was analyzed, undetectable IAHA titers and IFA titers of less than 32 were noted in 77.8% of those with disseminated zoster but in only 18.5% of those with localized disease. Peak serum antibody titers in patients with disseminated zoster were eventually equal to or greater than those in localized zoster. The patient groups were comparable in age, underlying disease, and therapy, although Hodgkin's disease was more frequent in patients with disseminated zoster. Thus, the absent IAHA or low IFA levels of circulating antibody early in illness were highly significant risk factors in dissemination of virus in herpes zoster.


Herpes simplex virus and recurrent laryngeal nerve paralysis. Report of a case and review of the literature. A 61-year-old man experienced the abrupt onset of a nonspecific febrile illness followed by the acute development of bilateral vocal cord paralysis. There was no evidence for Guillain-Barré syndrome, multiple sclerosis, brainstem encephalitis, myasthenia gravis, metabolic encephalopathy, poliomyelitis, diphtheria, botulism, tumor, vasculitis, or extrinsic nerve compression. No cause for the fever was ascertained, and the vocal cord paralysis improved sponaneously. Acute and convalescent viral serological studies demonstrated a diagnostic complement-fixation antibody titer rise to herpes simplex virus (HSV) and no rise in titer to influenza A and B, cytomegalovirus, poliomyelitis, or Mycoplasma. This case is similar to several others reported in the literature that suggest a viral neuritis in tenth nerve paralyses in children. The difficulties involved in diagnosing HSV CNS disease before death are discussed, and the medical literature is reviewed for evidence that HSV is the etiological agent in selected cranial neuropathies.


Structure-function relationship in hemoproteins: the role of cytochrome c3 in the reduction of colloidal sulfur by sulfate-reducing bacteria. Cytochromes c3 of different strains of sulfate-reducing bacteria have been purified and tested for their capacity to reduce colloidal sulfur to hydrogen sulfide. The results are in good agreement with the activities reported for the whole cells. Cytochrome c3 is the sulfur reductase of some strains of sulfate-reducing bacteria such as Desulfovibrio desulfuricans Norway 4 and sulfate-reducing bacterium strain 9974 from which the sulfur reductase activity can be purified with the cytochrome c3. In contrast, Desulfovibrio vulgaris Hildenborough cytochrome c3 is inhibited by the product of the reaction namely hydrogen sulfide. Chloramphenicol has no effect on the sulfur reductase activity of D. desulfuricans Norway 4 when resting cells grown on lactate-sulfate medium are put in the presence of colloidal sulfur. This shows that the sulfur reductase activity is constitutive and corresponds to the fact that colloidal sulfur grown cells do not contain more cytochrome c3 (or another sulfur reductase) than lactate-sulfate-grown cells.


Effect of dithiocarb and dimethyl sulfoxide on irreversible binding of 14C-bromobenzene to rat liver microsomal protein. The irreversible binding of [14C]-bromobenzene to rat liver microsomal protein in vitro was inhibited by dithiocarb and DMSO. Dithiocarb suppressed this binding in a time- and concentration-dependent manner (I50 = 4.5 10(-5) M). DMSO reduced the degree of covalent binding by 61% from 5 x 10(-5) M to 8 x 10(-4) M. Dithiocarb was also effective in inhibiting irreversible binding of bromobenzene to liver protein in vivo. Our results are consistent with the hypothesis that dithiocarb exerts its antihepatotoxic efficacy by depressing microsomal mixed-function oxidase activity.


Viral interference phenomena induced by foot-and-mouth disease temperature-sensitive mutants in bovine kidney cells. Cultures of bovine kidney (BK) cells infected with temperature-sensitive (ts) mutants of foot-and-mouth disease virus (FMDV) were incubated at 38.5 degrees C, a temperature nonpermissive for mutant virus growth and RNA synthesis. The cells were subsequently resistant to viral growth and RNA synthesis when superinfected with wild-type FMDV and with heterologous fowl plague virus. The extent of interference was proportional to the multiplicity of infection of the ts mutant. It increased with time elapsed between infection with mutant and challenge infection, becoming greater than 99 percent after 24 hours. Interference was not proportional to decreased levels of cellular protein synthesis. The interference could be produced in the presence of actinomycin D, and thus was apparently mostly caused by the ts mutant itself rather than by interferon. The interference could not be produced in other less susceptible cell lines. Supernatant fluids from the BK cells infected with ts mutant virus interfered with wild-type FMD viral growth and RNA synthesis in fresh BK cells, and also showed low levels of activity in a vesicular stomatitis virus-plaque reduction assay. The properties of the supernatant fluid-interfering agent resembled to some extent those of an interferon. The ts mutant-mediated interference factor was apparently not able to diffuse into the supernatant fluid.


Influence of genetic and physiological properties of the host cell on the cytopathic expression of herpes simplex virus. Two plaque morphology variants, a cell aggregating variant and a syncytial variant, were isolated from MDBK cells infected with the MP mutant of herpes simplex virus, type 1. The variants differed in the polypeptides produced in infected MDBK cells. The properties of the variants were stable on passage in cells and both variants produced only syncytia in KB and Hep-2 cells. The physiological state of MDBK cells influenced the cytopathological expression of the infecting virus, so that, under certain conditions, each variant could shift from one type of plaque morphology to the other. However, attempts to correlate this plaque morphology shift with a difference in the glycopolypeptides synthesized in the infected cells were unsuccessful.


Immunogenicity of subviral herpes simplex virus preparations. I. Formation of neutralizing antibodies in different animal species after administration of herpes simplex virus solubilized antigens. Production of neutralizing antibodies was followed in guinea pigs, rabbits, hamsters and mice immunized with crude antigen extracts (AM) from human diploid cells infected with herpes simplex virus type 1. The AM induced relatively high levels of neutralizing antibodies in all four species. The antibodies were predominantly complement-requiring and remained so even after administration of repeated AM doses. With the strains used, the antibody response was predominantly type specific and, surprisingly, the type specificity of sera usually increased after administration of repeated doses of AM. Guinea pigs seemed to be the best responsive animal species. They developed the highest levels of antibodies and complement-nonrequiring antibodies were seen in them earlier than in the other animal species. The dose-response experiments carried out in guinea pigs indicated that after a single dose administration the ratio between complement-requiring and complement-nonrequiring antibodies was dependent on the amount of antigen administered. When AM was given without adjuvant less efficient antibody production wws observed than after the administration of the same amount of antigen with adjuvant.


Tryptic peptide analysis of the structural proteins of a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture. The individual structural polypeptides of HVJ (haemagglutinating virus of Japan--the Sendai strain of parainfluenza 1 virus) were examined by tryptic peptide analysis. [3H]-methionine-labelled structural proteins of the wild-type virus of HVJ (HVJ-W) and [35S]-methionine-labelled corresponding constituent proteins of a temperature-sensitive (ts) mutant (HVJ-pB) derived from an HVJ carrier culture were compared by ion-exchange chromatography on columns of P-type chromobeads. The tryptic peptides of the individual structural proteins showed characteristic elution profiles. In all the structural proteins tested, the chromatographic elution profiles of both strains generally showed a close resemblance. However, certain minor peaks which were present in one strain but absent in the other strain were detected in the preparations of the P, HN, and F polypeptides. Further, analysis of the NP polypeptide showed that a major peak of one strain appeared at a position in the pH gradient different from a seemingly corresponding major peak of the other strain. In the M protein some possibly homologous minor peaks were found to differ between the two strains.


Assembly of viral structural proteins in cells infected with a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture. Brief report. The processing of virus polypeptides synthesized in cells infected with HVJ (haemagglutinating virus of Japan--the Sendai strain of parainfluenza 1 virus) was studied. Maturation of a temperature-sensitive (ts) mutant (HVJ-pB) derived from an HVJ carrier culture was inhibited at 38 degrees C incubation. A considerable amount of viral components were made at the restrictive temperature. They were, with the exception of the polypeptide HN, well preserved without a great loss of their function and successfully incorporated into virus particles released after lowering the incubation temperature. The membrane (M) protein seems to be essential for virus morphogenesis.


Inhibition of foot and mouth disease virus and procapsid synthesis by zinc ions. Brief report. Zinc ions inhibit virus production and viral RNA synthesis in FMDV infected-BHK 21 cells. The degree of inhibition depends upon the zinc concentration and the time of addition of the drug. A differential inhibition on virus and procapsids synthesis was observed.


Characterization of the polypeptides synthesized in cells infected with a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture. The intracellular synthesis of virus-specific polypeptides in cells infected with the wild-type virus of HVJ (HVJ-W) (haemagglutinating virus of Japan--the Sendai strain of parainfluenza 1 virus) and with a temperature-sensitive (ts) mutant (HVJ-pB) derived from an HVJ carrier culture has been analysed by polyacrylamide gel electrophoresis. At the permissive temperature (32 degrees C), all of the known virus structural polypeptides were identified in cells infected with each strain of virus and in addition to the non-structural polypeptides B and C, another polypeptide at the region with a molecular weight of 26,000 to 27,000 (26 to 27K) could be detected in infected cells. At the non-permissive temperature (38 degrees C), the synthesis of the polypeptide M was markedly restrained in cells infected with HVJ-pB, while other major virus polypeptides were present in approximately comparable amounts to cells infected with the wild-type virus. A non-structural polypeptide with a molecular weight of 105K was dominant in ts mutant infected cells at higher temperatures and disappeared after temperature-shift from 38 degrees to 32 degrees C. The production of the non-structural polypeptides B and 27K was also temperature-sensitive. The molecular weights of the polypeptides B, M and 27K in HVJ-pB infected cells were larger than those of the corresponding polypeptides in HVJ-W infected cells. The synthesis of the M protein in HVJ-prinfected cells started just after lowering the incubation temperature and the newly made M protein was successfully incorporated into virus particles.


A morphological study of human rotavirus. Human rotavirus has a characteristic icosahedral structure which has a honeycomb-like appearance on the surface of the smooth particles and 42 polygonal capsomeres in the rough particles.


Clones of cells from a human embryo lung: their growth and susceptibility to respiratory viruses. Colonies of cells were obtained from human fetal lung tissue and exposed to recently isolated respiratory viruses. There was a considerable variation in the number of rounded cells found in different colonies exposed to rhinovirus types 2 and 9 (RV2 and 9), human coronavirus 229E (HCV), adenovirus type 3 (Ad3) and respiratory syncytial virus (RSV). Smaller colonies had more rounded cells than larger colonies. Clones were established from 9 out of 11 colonies. They varied in their rate of growth and the pattern formed on a plastic surface. They varied also in their virus susceptibility particularly to "difficult" rhinoviruses such as RV9 and SF1340. One cell clone (HL1/77 Clone 8), was highly susceptible to all these viruses. All cultures were more sensitive to RSV when maintained in F12K medium than in MEM, whereas there was no difference for rhinoviruses.  Influenza A and B and parainfluenza 3 viruses sometimes produced cytopathic effect, and always produced haemadsorption, but unlike the previous strains could not be passed serially and presumably produced little infectious virus. All clones were rather insusceptible to Ad3; but the virus could be passed, whereas coxsackie virus B3 produced no CPE. Substantial yields of coronavirus and rhinovirus were obtained in gelatin sponge cultures. Two "very difficult" respiratory viruses which had just been adapted to tissue culture; namely, HS rhinovirus and JK coronavirus grew in 7 of 9 and in 6 of 9 clones respectively.


Protection of mice against virulent virus infection by a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture. Experimental infection with HVJ (haemagglutinating virus of Japan-the Sendai strain of parainfluenza 1 virus) in mice was studied. Aerosol infection of newborn mice with the wild-type virus (HVJ-W) retarded the development of body weight and killed the animals within a few weeks. Large amounts of virus were isolated from both the lungs and the nasal turbinates of infected mice. In contrast, newborn mice exposed by inhalation to a temperature-sensitive (ts) mutant (HVJ-pB) derived from an HVJ carrier culture showed no clinical signs and grew equally well as mock-infected animals. No infectious virus could be recovered from the lungs although the ts mutant grew to moderate titre in the nasal turbinates. The prior inoculation of newborn mice with the ts mutant virus induced a state of significant resistance to subsequent challenge with the virulent wild-type virus. No replication of challenge virus in both lungs and nasal turbinates could be detected and the animals were protected a lethal infection. It is suggested that an avirulent temperature-sensitive mutant which has lost the capacity to replicate in the lower respiratory tract but is still capable of multiplying in the nasal turbinates may be a promising candidate for use in live vaccines especially against the infectious disease of the lower respiratory tract.


Rhinoviruses: kinetics of ultraviolet inactivation and effects of UV and heat on immunogenicity. The kinetics of ultraviolet inactivation for two human rhinoviruses and poliovirus were compared. No major differences in the rates of ultraviolet inactivation were detectable. All viral preparations inactivated by ultraviolet irradiation induced neutralizing antibody in guinea pigs. In contrast, when guinea pigs were immunized with a heat inactivated rhinovirus preparation, little or no neutralizing antibody was elicited. Immune electron microscopy of the heated rhinovirus preparations revealed the presence of particles resembling empty capsids. These results suggest that rhinoviruses and enteroviruses are affected in a similar manner when subjected to ultraviolet or heat inactivation.


Role of envelope proteins of paramyxoviruses in the modification of cell membrane antigens. Adsorption of paramyxoviruses to separated membranes of tumor cells produces neoantigens that are immunogenic in syngeneic mice (xenogenization). Thus it became possible to study this process by using modified virus or virus fractions. Membranes with adsorbed viral derivatives produced an immune response which was measured by cytotoxic and complement fixing antibodies and the appearance of autoimmune disease. The effect of viral preparations with reduced F (fusion) protein activity was compared to treatment of membranes with equivalent amounts of active or inactive virus as measured by hemagglutination. Viral preparations without hemolytic activity showed diminished adsorption to membranes and the immune response was reduced. Triton X 100 and desoxycholate extracted from membranes immunogenic material with associated paramyxovirus antigens.


Comparison of cowpox-like viruses isolated from European zoos. Brief report. Poxviruses isolated from captive carnivores in Russia (Moscow virus) and elephants in Germany (elephant virus) were very closely-related to cowpox virus. Immunological analysis with absorbed sera separated elephant virus but not cowpox and Moscow virus, whereas polypeptide analysis separated cowpox but not elephant and Moscow virus. A combination of biological tests separated all three. The epidemiological implications are briefly reviewed.


Polypeptide patterns of infectious bronchitis virus serotypes fall into two categories. Brief report. Molecular weights of six major polypeptides of infectious bronchitis virus (IBV) are: 1. 75,000; 2. 50,000; 3. 45,000; 4. 35,000; 5. 28,000 or 24,000, and 6. 22,000 dalton. According to the mobility of protein 5 the polypeptide patterns of IBV serotypes fall into two main categories.


Herpes simplex virus DNA polymerase, thymidine kinase and deoxyribonuclease activities in cells infected with wild type, ultraviolet-irradiated and defective virus. The DNA polymerase, thymidine kinase and deoxyribonuclease activities were studied in cells infected with wild type (wt), ultraviolet (UV)-irradiated and defective herpes simplex virus type 1. All three enzymatic activities were expressed in cells infected with wt virus. In cells infected with UV-irradiated virus, the thymidine kinase and deoxyribonuclease activities were inhibited and the DNA polymerase activity was markedly suppressed. In cells producing defective virus, there was thymidine kinase activity, but the viral deoxyribonuclease activity was considerably reduced. The DNA polymerase activity was fully expressed in cells producing defective virus at passage level 5, but at passage level 6, the activity of the viral DNA polymerase declined.


Effects of antimetabolites on the production of tubular structures in Vero cells infected with herpes simplex virus type 2. Treatment of Vero cells infected with herpes simplex virus type 2 with the proper concentrations of hydroxyurea (HU) reduced the production of infectious virus and markedly increased the accumulation of tubular structures in the nuclei when the drug was added within 6 hours after infection. Similar accumulation of tubular structures in the infected nuclei was also observed when infected cells were treated with phosphonoacetic acid at proper concentrations. The release of HU-treated, herpes simplex virus type 2-infected cells from the drug-induced blocking of synthesis of infectious virus resulted in the marked decrease of tubular structures coincidentally with the beginning of production of infectious virus. The data suggest the possibility that the disappearance of tubular structures may be related to the active production of infectious virus.


Comparisons of some bluetongue virus isolates by plaque neutralization and relatedness tests. Seven North American bluetongue virus isolates, cloned by plaquing, and sera to five of them were reacted in a plaque neutralization test. Using a paired controls system, each virus-serum reaction was studied in terms of the regression of percent neutralization on log serum dilution. Antigen-antibody interaction terms in the analysis of effective dose estimates were used to assess the relatedness of the virus isolates. The degree of cross reactivity formed a spectrum from virtually no evidence of unrelatedness to clear antigenic differences.


Classification of porcine enteroviruses by antigenic analysis and cytopathic effects in tissue culture: description of 3 new serotypes. Porcine enteroviruses isolated in the United Kingdom between 1972 and 1976 were compared with the 8 serotypes previously described and with human coxsackie B virus types 1 to 6 for ability to grow in different cell lines. This allowed the classification of all strains into 3 broad groups according to type of cytopathic effect in IBRS-2 cells and further subdivision on the basis of production of cytopathic effect in BHK 21, HeLa and VERO cells. None of the porcine enterovirus strains was neutralized by antisera to human enteroviruses (Lim Benyesh-Melnick Pools) with the exception of swine vesicular disease virus, which was neutralised by coxsackie B5 antiserum. Antisera prepared either in gnotobiotic pigs or in guinea pigs against the 8 porcine enterovirus serotypes failed to neutralize 9 isolates which could be classified into 3 new serotypes, for which Nos. 9, 10 and 11 are proposed. Guinea pig sera could be used as an alternative to gnotobiotic pig sera for type differentiation.


Production of interferon by human tumor cell lines. Fourteen continuous human cell lines, including nine derived from tumors and five from non-neoplastic tissues, produced interferon in response to induction with bluetongue virus (BTV), Newcastle disease virus (NDV), and poly(I) . poly(C) complexed with DEAE-dextran. The seven best interferon-producing cell lines (one from a melanoma, five derived from carcinomas, and one SV40-virus-transformed kidney cell line) responded to at least one of the viral inducers with yields of interferon over 1000 units/ml. Because the HT-1376 bladder carcinoma cell line produced high yields of interferon in this survey, and is easily propagated, the optimal conditions for interferon production were investigated, using BTV as the inducer. Interferon yields in 59 inductions over a period of about two years consistently fell within a 6-fold range, and had a geometric mean titer of about 2700 reference units (RU)/ml, representing the production of about 3 RU/10(3) cells. This yield is comparable to mean titers of 1 to 10 RU/10(3) cells obtained by others with human leukocytes, foreskin cell strains, or the Namalva lymphoblastoid cell line. UV-inactivated BTV at a multiplicity corresponding to 10 PFU/cell was as effective an inducer in the HT-1376 cell line as the fully infectious virus at a multiplicity of 1 PFU/cell. The interferon produced by the HT-1376 epithelial cell line has characteristics similar to the interferon induced by poly(I) . poly(C) in human diploid fibroblasts. These studies clearly demonstrate that many different types of tumor-derived cells have the capacity to produce interferon, and that some equal or surpass the efficiency of diploid cells.


A new method for the isolation of undegraded FMDV-specific RNA from infected BHK cells. Fractionation of Foot-and-Mouth disease virus infected cells by currently described procedures, leads to the appearance of variable amounts of heterogeneous single-stranded RNA fragments. A new method based upon the fractionation of cultured cells at extremely low temperatures has been developed to minimize the degradation of the viral RNAs by cellular nucleases. It was shown that the viral RNAs obtained by this procedure were almost non-degraded, and similar to those found in other picornavirus infected cells. More than 90 per cent of the polysomal RNAs were found as genome-size molecules, presumably being messenger RNA (mRNA). It was also found that the mRNA analyzed on sucrose gradients sedimented slightly ahead of the 35S genomic RNA. However, no differences were found when the analysis was performed on polyacrylamide gels.


Determination of antibodies against cytomegalovirus-induced early antigens by using rabbit lung fibroblasts. Brief report. The determination of antibodies against cytomegalovirus-induced antigens in 30 patients with acute cytomegalovirus infection and in 100 healthy control persons, using early antigen preparations of human lung fibroblasts and of rabbit lung fibroblasts, yielded results showing a high degree of agreement. For such investigations, however, the use of rabbit cells proved to be of advantage because the preparation of the early antigens can be carried out without inhibition of the DNA synthesis of the host cells.


[Ultrastructure of the human submandibular salivary glands]. By means of electron microscopy cells in the human submandibular glands were studied. It was demonstrated that in acini two types of glandular cells were present: mucosal and seromucosal. In the latter, secretory granules are descrete with electron opaque cores in most of them. Mucocytes are filled with an electron transparent secrete; secretory granules often confluent and their membranes rupture. The acini are surrounded with myoepithelial cells. Intercalated ducts consist of cells with moderately electron opaque granules. In some granules there are dense bodies excentrically situated. In these cells there occur lipid inclusions. Striated ducts are composed of basal (electron transparent) and high cylindric (light and dark) cells. The cylindrical cells have a large amount of mitochondria, deep folds in their basal plasmolemma protruding into cytoplasma. Most of the cells in these parts contain small apically accumulated secretory granules with a dense matrix and separate larger ones scattered in the cell. It is possible to suggest that some secretory granules of ductal or, perhaps, acinar origin contain hormonal products.


[Ultrastructural characteristics of soft tissue rhabdomyosarcomas]. Seven rhabdomyosarcomas of human soft tissues were studied electron microscopically. Certain ultrastructural features characteristic of tumour rhabdomyoblasts were revealed: atypical fibrillar apparatus, spatially uneven process of myofibrillogenesis, disturbed cyto- and histotypical differentiation ratio and others. These features may be used for electron microscopic diagnosis of rhabdomyosarcomas among soft tissue tumours of low differentiation.


Cavernous hemangioma of the retina. A four-generation pedigree with neurocutaneous manifestations and an example of bilateral retinal involvement. Cavernous hemangioma of the retina is an unusual vascular hamartoma whose coexistence with vascular anomalies of the skin and central nervous system has been recognized recently. A 39-year-old woman, who had an acute palsy of the right third cranial nerve, had a history of seizures, cutaneous vascular anomalies, and a cavernous hemangioma of the retina of the right eye. One of her daughters demonstrated bilateral retinal cavernous hemangiomas, and another daughter, who developed seizures when febrile, displayed cutaneous vascular anomalies. A four-generation pedigree showed a number of cutaneous vascular anomalies, seizures, and stroke-related deaths. The pedigree suggests further support for considering this disorder an authentic oculoneurocutaneous triad.


A new intermediate adenovirus type causing conjunctivitis. A new adenovirus of intermediate type involved in conjunctivitis has been isolated. The virus is related to adenovirus types 13 and 30 by neutralization and to types 10 and 19 by hemagglutination inhibition. Since 1976 the agent has been causing sporadic cases of conjunctivitis in different parts of The Netherlands. Twenty-three cases have been established by virus isolation from the affected eyes. The symptoms of the disease range from epidemic keratoconjunctivitis to pharyngoconjunctival fever. Also, adenovirus type 13-30/10-19 has been isolated from the cervix uteri of four women in a series of 1,477 women with cervicitis.


An outbreak of disease in pigeons associated with a herpesvirus. An outbreak of disease in young pigeons associated with a herpesvirus is reported. The clinical history, macroscopic and microscopic appearance of lesions and virus isolation are described. Most affected birds showed lesions in the upper alimentary tract epithelium as well as in skin, nasal mucosa and salivary glands. Lesions in liver, spleen and pancreas were uncommon. A herpesvirus capable of producing CPE on tissue culture and lesions on chorioallantoic membrane of developing chicken embryos was isolated and described. Inoculation of experimental pigeons with the virus failed to reproduce the disease.


Somatomedin-like activity, prolactin, growth hormone and insulin in human cord blood. In order to assess the possible influence of fetal polypeptide hormones on birth size, somatomedin-like receptor activity (SmLRA) (n = 281), prolactin (n = 158), growth hormone (n = 133) and insulin (n = 108) concentrations were measured in the cord blood of 281 singleton infants born after different complications of pregnancy. Infant sex did not significantly affect the concentration of any hormone. SmLRA concentrations appeared to rise from 25 to 38 weeks of gestation, but thereafter fell. Concentrations of prolactin, growth hormone and insulin correlated negatively with gestational age. Insulin emerged as the hormone most clearly related to fetal growth, since concentrations were high (mean +/- SD = 20.6 +/- 11.5 uU/ml) in serum from infants of diabetic mothers (IDM) and other large-for-dates infants (10.1 +/- 4.8 uU/ml), but low (5.3 +/- 0.5 uU/ml) in infants who were small-for-dates (SFD). In contrast, SmLRA concentrations were high in serum from SFD infants (0.63 +/- 0.29 U/ml) and low in IDM (0.43 +/- 0.16 U/ml). Prolactin concentrations were higher in serum from SFD infants (212 +/- 101 ng/ml) and from IDM (237 +/- 182 ng/ml) than from normal infants born at term (139 +/- 68 ng/ml). Administration of intramuscular betamethasone to pregnant women in premature labour resulted in significant elevations in the concentrations of prolactin and insulin in cord blood.


Structural aspects of the dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila. 1. A dye-linked alcohol dehydrogenase was purified 60-fold from extracts of Rhodopseudomonas acidophila 10050 grown aerobically on ethanol. 2. The properties of this enzyme were identical with those of the alcohol dehydrogenase synthesized by this organism during growth on methanol anaerobically in the light, and they are judged to be the same enzyme. 3. The enzyme gave a single protein band, coincident with alcohol dehydrogenase activity, during electrophoresis on polyacrylamide gel. 4. The amino acid composition, ioselectric point, u.v. and visible absorption spectra of the enzyme were determined and compared with those of other similar enzymes. 5. The presence of 0.7--1.0 g-atom of non-haem, acidlabile iron/mol of enzyme was shown by atomic absorption spectrophotometry and colorimetric assay. The iron could not be dissociated from the enzyme by dialysis against chelating agents. 6. E.p.r. spectroscopy of the enzyme did not indicate any redox function for the iron during alcohol dehydrogenation, but showed a signal at g = 2.00 consistent with the presence of a protein-bound organic free radical. 8. Antisera were raised against alcohol (methanol) dehydrogenases purified from Rhodopseudomonas acidophila, Paracoccus denitrificans and Methylophilus methylotrophus. 9. The antiserum to the Rhodopseudomonas acidophila enzyme cross-reacted with neither of the two other antisera, nor with crude extracts of methanol-grown Hyphomicrobium X and Pseudomonas AM1, thus emphasizing its singular biochemical properties.


Properties of mammalian nuclear-envelope nucleoside triphosphatase. The nucleoside triphosphatase activities of the nuclear envelopes from rat liver, pig liver and simian-virus-40-transformed mouse-embryo 3T3 cells were shown to exhibit similar parperties. All three preparations hydrolyse ATP, 2'-dATP, 3'-dATP, GTP, CTP and UTP in the presence of Mg2+, Ca2+, Mn2+ and Co2+ with a pH optimum of 8.0, are sensitive to inhibition by mercurials, arsenicals, quercetin, proflavin and adenosine 5'-[gamma-thio]triphosphate and are partially inactivated by exposure to high ionic strength. The kinetic behaviour is similar for all substrates irrespective of the source of material. The typical Eadie-Hofstee plot, which is concave upwards at pH 8.0 when the ionic strength is 20mM, becomes linear when the pH is increased to 8.5 or the ionic strength to 160mM. The overall evidence, particularly the labelling of only one polypeptide by [gamma-32P]ATP, suggests that under the conditions of preparation and assay used only one class of nucleoside triphosphatase active sites is detectable in nuclear envelopes. The importance of these results for an understanding of the role of the enzyme in vivo is discussed.


Affinity chromatography of human plasma low- and high-density lipoproteins. Elution by selective cleavage of a bond in the spacer. Human plasma low- and high-density lipoproteins were found to bind to Sepharose gels containing coupled cholesterol or cholic acid. The lipoproteins were bound very strongly, and it was not possible to elute them under non-denaturing conditions. The detergents Triton X-100 and sodium dodecyl sulphate eluted the lipoproteins in partly denatured form. Adsorbents were used where the steroid was coupled through a spacer containing a thiol ester bond. It was thus possible to elute bound lipoproteins by selective cleavage of the bond with hydroxylamine. A small proportion of albumin was the only contaminant detected, the amounts depending on which ligand was used. Low- and high-density lipoproteins were separated by gel filtration. They behaved as did the native molecules when analysed by gel filtration, immunodiffusion, immunoelectrophoresis and electrophoresis in polyacrylamide gradient gels. The high capacity and the selectivity of the adsorbents make them suitable for the removal of lipoproteins from protein solutions.


Polyamines and cellular adenosine 3' :5'-cyclic monophosphate. The effect of polyamines on the cellular concentrations of cyclic AMP was studied. It was shown that 1 microM-spermine caused a decrease in cyclic AMP in chick-embryo heart cells, chick-embryo fibroblasts, neuroblastoma, glioma and neuroblastoma-glioma hybrid cells, grown in culture. A similar decrease was observed when polyamines were added to cells in the presence of a phosphodiesterase inhibitor or after stimulating the cells with various hormones. Noradrenaline was used in cultures of heart cells, prostaglandin E1 and adenosine for neuroblastoma and neuroblastoma-glioma hybrids, whereas isoproterenol was used for the stimulation of glioma cells. Polyamines at higher concentrations were either without effect or caused a slight increase in cyclic AMP. Spermidine (10 microM) also caused a decrease in cellular cyclic AMP, as did 0.1 microM-putrescine. It is suggested that the effect of polyamines on cellular cyclic AMP may be explained by the effect of these polycations on the activity of cellular phosphodiesterase.


Phosphatidylinositol metabolism in rat hepatocytes stimulated by glycogenolytic hormones. Effects of angiotensin, vasopressin, adrenaline, ionophore A23187 and calcium-ion deprivation. 1. The effects on phosphatidylinositol metabolism of three Ca(2+)-mobilizing glycogenolytic hormones, namely angiotensin, vasopressin and adrenaline, have been investigated by using rat hepatocytes. 2. All three hormones stimulate both phosphatidylinositol breakdown and the labelling of this lipid with (32)P. 3. The response to angiotensin occurs quickly, requires a high concentration of the hormone and is prevented by [1-sarcosine, 8-isoleucine]angiotensin, a specific angiotensin antagonist that does not prevent the responses to vasopressin and to adrenaline. This response therefore seems to be mediated by angiotensin-specific receptors. 4. [1-Deaminocysteine,2-phenylalanine,7-(3,4-didehydroproline),8-arginine] vasopressin, a vasopressin analogue with enhanced antidiuretic potency, is relatively ineffective at stimulating phosphatidylinositol metabolism. This suggests that the hepatic vasopressin receptors that stimulate phosphatidylinositol breakdown are different in their ligand selectivity from the antidiuretic vasopressin receptors that activate renal adenylate cyclase. 5. Incubation of hepatocytes with ionophore A23187, a bivalent-cation ionophore, neither mimicked nor appreciably changed the effects of vasopressin on phosphatidylinositol metabolism, suggesting that phosphatidylinositol breakdown is not controlled by changes in the cytosol Ca(2+) concentration. This conclusion was supported by the observation that hormonal stimulation of phosphatidylinositol breakdown and resynthesis persists in cells incubated for a substantial period in EGTA, although this treatment somewhat decreased the phosphatidylinositol response of the hepatocyte. The phosphatidylinositol response of the hepatocyte therefore appears not to be controlled by changes in cytosol [Ca(2+)], despite the fact that this ion is thought to be the second messenger by which the same hormones control glycogenolysis. 6. These results may be an indication that phosphatidylinositol breakdown is an integral reaction in the stimulus-response coupling sequence(s) that link(s) activation of alpha-adrenergic, vasopressin and angiotensin receptors to mobilization of Ca(2+) in the rat hepatocyte.


Corticotropin regulation of the synthesis of a specific rat adrenal cytosolic protein. Effects of hypophysectomy and actinomycin D. The mechanism of corticotropin stimulation of the synthesis of a specific rat adrenal cytosolic protein was investigated. This protein (protein E) has a mol.wt. of approx. 30000. It is detected by polyacrylamide-gel electrophoresis of cytosol prepared from adrenal slices from rats treated with corticotropin in vivo and control rats, the slices being incubated with [(3)H]- and [(14)C]-leucine respectively. In rats 1-15 days after hypophysectomy, corticotropin, like dibutyryl cyclic AMP, induces an increase in protein E similar to that induced in control rats, even though both compounds no longer stimulate total protein synthesis. Corticotropin stimulation of protein E synthesis is mediated by cyclic AMP but not by corticosterone, since aminoglutethimide, a steroidogenic inhibitor, does not affect corticotropin stimulation, and dexamethasone alone has no effect. Actinomycin D, when injected in vivo 1h before or after corticotropin injection, prevents the effect of corticotropin on protein E synthesis, which is interpreted as evidence that mRNA synthesis is necessary for the stimulation of protein E synthesis. When injected more than 2h after corticotropin, actinomycin D does not prevent corticotropin stimulation of protein E synthesis, but completely blocks corticotropin stimulation of total protein synthesis. This is interpreted as meaning that, after stimulation of mRNA coding for protein E, corticotropin has no effect on the synthesis of protein E. On the other hand, corticotropin stimulation of protein E synthesis persists after hypophysectomy even though it no longer stimulates total protein synthesis. These data suggest that the factor(s) involved in the synthesis of protein E are more stable than those involved in total protein synthesis.


The metabolism of cholecalciferol in the liver of Japanese quail (Coturnix coturnix japonica) with particular reference to the effects of oestrogen. 1. Studies were carried out in vitro with the livers of Japanese quail that had been fed from hatching on diets supplying their full requirements for vitamin D. 2. 25-Hydroxycholecalciferol was the major metabolite when liver homogenates of egg-laying female and oestrogen-treated quail of both sexes were incubated with [3H]cholecalciferol. 3. Very little 25-hydroxycholecalciferol was generated from liver homogenates of adult male and immature quail. Instead the cholecalciferol was converted into one or more compounds less polar than 25-hydroxycholecalciferol and into a number of highly polar metabolites, some of which were water-soluble. 4. Oestrogen not only stimulated the 25-hydroxylation of cholecalciferol but also protected both cholecalciferol and 25-hydroxycholecalciferol from degradation by the enzymic pathways active in immature and male birds. 5. These actions of oestrogen may be of physiological significance in relation to the high requirements of laying birds for 1,25-dihydroxycholecalciferol to support the intense metabolism of calcium associated with egg-shell calcification.


Uridine kinase activities in developing, adult and neoplastic rat tissues. Uridine kinase activities were found chiefly in the soluble fractions of rat tissues. In normal adults the activities ranged from 13 munits/g in skeletal muscle to 178 munits/g in colon. Enzyme activities in several rat neoplasms were significantly higher (e.g. in a fibrosarcoma, mammary carcinoma, renal carcinoma, pancreatic carcinoma and lymphocytic lymphoma, but not in a fast-growing Morris hepatoma). The activities were not related to tumour growth rates or sizes. In normal foetal liver, lung, brain, heart and kidney, uridine kinase concentrations equalled or exceeded those in the adult homologous tissue, but maximal activities in liver were reached 3--5 days post partum. In suckling rats the intestinal activity decreased substantially immediately after birth and normally did not rise again until late in the third postnatal week. Premature upsurges could be evoked by an injection of cortisol or by starvation of the pups overnight. Pancreatic activity was absent from 1-day-old rats, and only about 5% of the adult activity was reached by day 20; adult activities were attained rapidly after weaning. In pancreas, precocious formation or uridine kinase was elicited by overnight starvation of 2-week-old rats.


Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins. The nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process.


Mössbauer spectra of photosystem-I-reaction centres from the blue-green alga Chlorogloea fritschii. Substantial amounts of iron have been shown by Mössbauer spectroscopy to be present in Photosystem-I preparations from the blue-green alga Chlorogloeta fritschii. Changes in the spectra on chemical reduction provide evidence that some of this iron is very similar to that found in the 4Fe-4S centres of ferredoxins. Such reduced samples also show e.p.r. signals consistent with maximum reduction of iron-sulphur centres A and B of Photosystem I. An unchanged component in the spectra indicates, assuming all centres A and B are reduced, the presence of another iron-containing species.


Chemotherapeutically active anthraquinones. I. Aminoanthraquinones. Basically substituted 2,6-diacetaminoanthraquinones proved to have an interesting in vivo effect against Entameba histolytica, but this effect was markedly exceeded by that of the series of the 2,6-diamidinoanthraquinones. A number of compounds of this series revealed a significantly stronger extraintestinal effect in the golden hamster than the standard amebicides metronidazole and tinidazole, but they were not superior to these compounds in their trichomonacidal effect, which was in general only a moderate one. In both substance classes, the amebicidal effect was confined to 2,6-disubstitution. Variations of the anthraquinone system resulted in a loss of activity. The structure-activity relationships are discussed.


[The influence of ether anesthesia in white mice on the contents of cyclic nucleotides in their brain and of cyclic nucleotides and glycogen in their liver (author's transl)]. During ether narcosis (10% diethyl ether v/v) of 30 min in white mice, the brain content of cAMP and cGMP is significantly diminished. By contrast, the liver cAMP and cGMP concentrations are increased 5 and 30 min after beginning narcosis but there is no or little difference, respectively, as compared to controls after 60 min. Liver glycogen content is elevated after 5 min but is diminished after 30 and 60 min as compared to controls. Pretreatment of the animals with the alpha-blocking agent phentolamine and the beta-blocking agent propranolol, respectively, prevents neither the increase in liver cAMP nor the decrease in liver glycogen during ether narcosis. It may be assumed that the changes in the liver and brain contents of cAMP and cGMP during ether application are due to physico-chemical alterations at the cell membrane which result in changes of the activity of the enzymes adenylate cyclase and guanylate cyclase in brain and liver tissue.


Collagenase-solubilized lipoprotein--glycosaminoglycan complexes of human aortic fibrous plaque lesions. The interaction of lipoproteins and arterial connective tissue macromolecules was studied using human atherosclerotic plaque tissues. After extraction with 0.15 M NaCl, the tissues were repeatedly digested with collagenase followed by elastase. The collagenase-solubilized lipoprotein--GAG complexes were isolated by gel-filtration and ultracentrifugation and analyzed for lipids, GAG and protein. While extraction by 0.15 M NaCl released only about 13% of the total cholesterol from the tissues, subsequent digestions by collagenase and elastase yielded 60% and 17% cholesterol, respectively. Both 0.15 M NaCl and collagenase treatment released equal amounts of GAG and accounted for 84% of the total GAG. Immunologically, lipoproteins resembled serum apoB-containing lipoproteins. Bio-Gel A-50m column chromatography of collagenase-extracted materials gave a single peak which contained lipoproteins of 1.006 and 1.063 floating densities, GAG and hydroxyproline. Hyaluronic acid (HA) and chondroitin 6-sulfate were identified; HA was the major GAG. Although the precise nature of the interaction of arterial connective tissue components with lipoproteins is not completely understood, isolation of such complexes indicates the importance of these macromolecules in sequestration of lipoproteins.


Effect of risk factors and antirheumatic drugs on the proliferation of aortic wall cells. The proliferation of aortic smooth muscle cells (ASMC) of Wistar rats, impaired by risk factors such as arterial hypertension, diabetes mellitus, atherogenic diet and staphylolysin injections and of normal Wistar rats treated with antirheumatic drugs such as prednisolone and acetylsalicylic acid was investigated. The cells of these animals were cultivated, subcultivated, and in the 2nd subcultures the cell numbers/5 ml medium were counted by means of Coulter Counter, and the cells were incubated with [3H]thymidine and the percentage of labelling in 100 or 1000 counted cells was stated. The effect of risk factors such as LDL and staphylolysin and of antirheumatic drugs such as prednisolone, acetylsalicylic acid, D-penicillamine and chloroquine added to the 2nd subcultures of cultivated ASMC of normal minipigs was investigated by the same method. The proliferation of cultivated ASMC of rats impaired by risk factors was accelerated. The proliferation of cultivated ASMC of rats treated with antirheumatic drugs was inhibited. The proliferation of ASMC of minipigs in the 2nd subcultures was activated by addition of risk factors and inhibited by addition of antirheumatic drugs. Antirheumatic drugs given to the rats and added to the medium of the 2nd subcultures of ASMC of normal minipigs inhibit the acceleration of ASMC proliferation induced by simultaneously given risk factors. The proposal to augment up our arsenal of the hitherto existing preventive and therapeutical measures by the application of antirheumatic drugs based on the experimental models referred to is supported by the result of a limited prospective double-blind-study of a sample of 133 male patients after myocardial infarction. The most remarkable result that the acceleration of the ASMC proliferation, the real pathologic process of arteriosclerosis, is inhibited by the application of antirheumatic drugs, at exactly the same time as the acceleration of the fibroblast proliferation, the real pathologic process in rheumatic diseases--ASMC and fibroblast, both being mesenchymal cells--recommends the use of these drugs in the prevention and therapy of human arteriosclerosis. The surprising result of our in-vivo experiments, that the acceleration of the growth of the ASMC induced by risk factors and the inhibition of the growth induced by antirheumatic drugs persist in the subcultures, is explained by the "selection theory" that there are dissimilar kinds of ASMC in normal arteries and that they react differently.


Cholesterol metabolism in non-obese women--Failure of physical conditioning to alter levels of high density lipoprotein cholesterol. The effects of a 6-week program of vigorous exercise were studied in 14 non-obese females aged 22--26. Preceding and following a regimen consisting of 30--45 min of jogging 5 days per week, treadmill performance, body weight, total plasma cholesterol, and plasma high density lipoprotein (HDL) cholesterol were assessed. Aerobic performance improved markedly after training as demonstrated by a reduced heart rate at each submaximal treadmill workload, and by an increase in maximal attainable workload. In the absence of a significant change in body weight, total cholesterol fell significantly after training (171 +/- 6 vs 161 +/- 5 mg/dl, P less than 0.05) whereas HDL cholesterol was not significantly altered (63 +/- 5 vs 58 +/- 3 mg/dl). The ratio of total cholesterol to HDL cholesterol fell insignificantly (2.92 +/- 0.19 vs 2.86 +/- 0.14). Our findings differ from prior reports of elevations of high density lipoprotein levels following physical conditioning in men. We suggest that hormonal or other factors leading to higher baseline levels of HDL in women counteract the expected alterations in lipoprotein metabolism induced by physical training.


Marked increase in high density lipoproteins in mountaineers. Apoprotein-AI concentrations were measured in whole blood in 13 male mountaineers before, during and after a strenuous climb of 8 weeks' duration. The AI concentration almost doubled in 3 weeks and remained elevated at that level for the full period of the climb. This study demonstrates the rapid rate at which high density lipoproteins can rise and the very high levels (148 +/- 29 mg/dl in whole blood) that can be reached. The rise can be attributed at least in part to exercise though other factors such as exposure to cold, hypoxia and mental stress might also have contributed.


Fibrinogen and low density lipoprotein in the development of cerebral atherosclerosis. Cerebral arteries from 65 human subjects were examined by immunofluorescence, using antisera against human fibrinogen and low density lipoprotein (LDL). Deposition of fibrinogen and LDL was most frequent at the bifurcation of the middle cerebral arteries and least in the basilar arteries in all age groups. In general, deposition of LDL was associated with deposition of fibrinogen, and lone deposition of LDL in the absence of fibrinogen was only rarely seen. Fibrinogen was scattered in the intercellular spaces, and located in the inner layer or edges of the thickened intima of the bifurcation with increasing plaque formation. Fibrinogen was observed even in the subendothelial region of the uninvolved intima at the bifurcations. LDL was present in the cytoplasm of the endothelial cells in the earliest stage, and it increased in the extracellular stroma with increase in intimal thickening, corresponding closely to the distribution of perifibous oil-red-O-stained lipids. No LDL was detected in the uninvolved intima. The observations suggest that deposition of fibrinogen in the intima might precede LDL deposition and possibly play a more important role than LDL in the development of atherosclerotic lesions in the cerebral arteries, especially in their early stage. Severe atherosclerosis at the bifurcations may be in part due to increased permeation of these plasma proteins, possibly as a result of hemodynamic stress.


Incorporation of polyenephosphatidylcholine into serum lipoproteins after oral or intravenous administration. Radioactive polyenephosphatidycholine (PPC) was injected i.v. in dogs, rats and rabbits. In addition, PPC was administered orally to dogs and rats. The incorporation of the PPC applied into serum lipoproteins was determined. After i.v. injection in dogs approximately 80% of the radioactive dose was transported by HDL. Only around 50% of the dose was found, however, in the HDL fraction of rats and rabbits. Radiochemical analyses provided a quantitative determination of the time course of PPC incorporation into HDL as well as of the metabolites of PPC. After in vitro incubation with rat and human serum PPC was incorporated into HDL by approximately 50% of the applied dose and by 5--14% into LDL, by approximately 15% into VLDL and by 20--30% into other serum constituents. In vitro incubation of PPC with human HDL yielded the complete incorporation into this lipoprotein fraction.


Increase of serum high density lipoprotein with progression and regression of aortic lipid deposition in rats. The progression and regression of aortic lipid deposition was studied in male ExHC rats. Rats in the progression group were fed an atherogenic diet containing 2% cholesterol, 0.4% sodium cholate and 10% olive oil for periods up to 32 weeks. Rats in the regression group were first fed the atherogenic diet for 16 weeks, and then maintained on a basal low cholesterol diet. Half the rats were killed at 4 weeks and the other half at 16 weeks after cessation of the atherogenic diet. Rat aortas of the progression group contained progressively more lipid pari passu with the duration of cholesterol feeding, but connective-tissue proliferation was absent. Deposited lipid in the aorta of cholesterol-fed rats disappeared very slowly after the rats were returned to the basal diet. serum HDL decreased in the hypercholesterolemic ExHC rats fed the atherogenic diet for 4 weeks. By contrast, serum HDL and apo A-I increased in both hypercholesterolemic ExHC rats fed the atherogenic diet for 32 weeks and slightly hypercholesterolemic ExHC rats fed the basal diet for 16 weeks in the regression period.


Effect of smoking on serum levels of HDL apoproteins. Quantitative determination of HDL apoproteins in serum from 97 healthy men showed a significantly lower apoA-I and apoA-II level in smokers than in non-smokers. Also a significant, negative correlation was found between the apoA-I or apoA-II levels, and the number of cigarettes smoked per day.


Lipoprotein lipids in chronic renal failure and haemodialysis. The influence of etiology and implications for atherogenesis. Lipoprotein lipid analysis has been carried out in 39 women and 28 men with chronic renal failure on haemodialysis. The results have been analysed in relation to the etiology of the renal disease and compared with those obtained in age- and sex-matched controls and in triglyceride-matched controls. Serum cholesterol was normal or low in glomerulonephritis but was normal in analgesic nephropathy. Serum triglycerides and VLDL lipids were raised uniformly regardless of the etiology of the renal disease. LDL triglyceride and HDL triglyceride were also raised. LDL cholesterol and phospholipid were low in glomerulonephritis but were normal in analgesic nephropathy. HDL cholesterol was reduced in both male and female patients regardless of etiology, statistical significance was not reached for the women. The ratio of esterified to free cholesterol tended to be reduced in all the lipoproteins regardless of sex or etiology but the changes were not significant in all groups. Comparison of the lipid abnormalities with those found in other hyperlipidaemic states suggests that the lipid disorders found in chronic renal failure are probably insufficient to explain the rapid development of vascular disease which has been reported.


HDL lipids in close relatives of coronary heart disease patients. Environmental and genetic influences. Female first-degree relatives of CHD patients differed, after the age of forty, from the normal control population by their low HDL cholesterol. Between the ages of 20 and 40 years a slight but significant increase in HDL triglycerides was observed. Except for hormonal contraception which induces significant lowering of HDL cholesterol in the first degree relatives, the observed differences in HDL lipids did not seem related to environmental factors but rather to be genetically determined. In contrast, the low HDL cholesterol observed in the wives of the CHD patients appeared to be related to differences in alcohol intake.


Sex hormones and high density lipoproteins in healthy males. The serum concentrations of testosterone, androstenedione, oestradiol and total low polar oestrogens, mainly oestradiol and oestrone, were measured in 26 healthy male subjects. The subjects were divided into two groups each of them with low (less than 1.04 mmol/l) and high (greater than 1.56 mmol/l) levels of serum HDL cholesterol. The group with high HDL cholesterol had significantly higher testosterone than the other group. Positive correlations were established between HDL cholesterol and testosterone and total cholesterol and testosterone concentration in serum.


In vivo metabolism of esterified cholesterol and apoproteins in rabbit plasma low density lipoproteins. Preparations of rabbit low density lipoproteins (LDL) labelled with 3H in the esterified and free cholesterol moieties and with 125I in the apoprotein moiety were injected intravenously into other rabbits. A substantial proportion of the esterified [3H]cholesterol removed from LDL during the first 30 min was recovered in the high density lipoprotein (HDL) fraction, while no such transfer of labelled apoprotein was observed. There was a clear cut differential in the decay of the different LDL components, slowest for the apoprotein, intermediate for esterified cholesterol and fastest for free cholesterol.


[Levels of specific antibodies against poliomyelitis virus and degree of immune protection in Umbria population]. The Authors studied neutralizing antibodies against poliomyelitis wild viruses in a sample of Umbria population. The number of seronegative results for each virus strain was very low (less than 1%). The geometric mean titres were 21.07, 26.72, 19.13 for poliovirus types 1, 2, 3, respectively. The results of this seroepidemiological study shows that the Umbria population is satisfactory protected against the poliomyelitis.


[Method for isolation of enteroviruses from edible mollusca]. A method for recovery of Poliovirus from experimentally inoculated mussels has been evaluated. The method, based on adsorption-elution-acid precipitation of virus particles, allows a recovery of more than 93% of the added virus.


Na+/K+-dependent adenosinetriphosphatase activity in mammalian kidney. Microsomal preparations with Na+/K+-dependent ATPase activity from the outer medulla of rabbit and pig kidney were obtained. Purifications were assaied by centrifugation on sucrose discontinuous gradients and gel-filtration on Sepharose 6B, after detergent incubation. Sodium dodecylsulfate, with ATP as protecting agent, can remove a maximal amount of non specific proteins from the membranes and allows the recovery of a fraction with very high specific activity. The approach to purification by affinity chromatography techniques leads to interesting results, which induce us to pursue the present researchs to applicate an affinity method to Na+/K+-ATPase.


[Modifications of liver content of cyclic nucleotides in the rat poisoned with white phosphorus]. In rat liver following white phorphorus poisoning a biphasic increase in cyclic AMP concentration was observed. After a lag period of 1 hour the cyclic AMP content rose to a first peak at 4 hours and to a second peak at 12 hours of intoxication. The cyclic AMP level fell to normal after 24 hours, by which time the cyclic nucleotide concentration was approaching control values. On the contrary, cyclic GMP content was found to the normal level during the different stages of intoxication. Only at 36 hours the cyclic GMP amount appeared significantly increased above the control values. Serum activity of alanine- and aspartate-amino transferases was found changed from 8 hours to 24 hours after poisoning. The serum level of the two enzymes was overlapping the control values after 36 hours. These results are discussed in relation to hepatocyte necrosis following white phosphorus intoxication.


Polymyxin B and heart muscle.

Polymyxin B and heart muscle. The effects of the polypeptide antibiotic, polymyxin B, on myocardial contractility were studied in t;e isolated rat heart muscle. Five different doses of polymyxin B were tested. There were no changes in contractility with does ranging from the clinical therapeutic value to three times greater. There was an initial increase and then depression with a dose six times greater than the therapeutic dose. There was no direct competitive interaction between polymyxin B and halothane or Ca2+. This suggests that polymyxin B does not depress the myocardium in clinical doses and does not interfere with Ca2+ influx at the myocardial cell membrane.


Uridine diphosphate galactose 4-epimerase. Alkylation of enzyme-bound diphosphopyridine nucleotide by p-(bromoacetamido)phenyl uridyl pyrophosphate, an active-site-directed irreversible inhibitor. When UDP-galactose 4-epimerase is inactivated by p-(bromoacetamido)phenyl uridyl pyrophosphate (BUP), the diphosphopyridine nucleotide (DPN) associated with this enzyme as a tightly bound coenzyme cannot be reduced by substrates or by UMP-activated reduction by glucose. Upon acid denaturation of the inactivated enzyme, the DPN released corresponded to 15-30% of that released from the native enzyme. When the enzyme is inactivated by [14C]BUP, about 80% of the radioactivity bound at the active site is released from the protein upon acid denaturation. When epimerase-[3H]DPN is inactivated with [14C]BUP, the 3H and 14C released from the protein upon denaturation of the complex cochromatograph on DEAE-Sephadex. Experiments with [nicotinamide-4-3H]DPN and [adenine-2,8-3H]DPN show that it is the adenine ring that is alkylated. The data suggest that the adenine ring of DPN in epimerase-DPN may be oriented near the glycosyl-binding subsite of this enzyme. Since the nicotinamide ring must also be near this site, it appears that the DPN may not be in an extended conformation when it is bound at the active site of UDP-galactose 4-epimerase from Escherichia coli.


Covalent structure of collagen: amino acid sequence of alpha 2-CB5 of chick skin collagen containing the animal collagenase cleavage site. The amino acid sequence of the 112 residues from the amino terminus of alpha 2-CB5 from chick skin collagen was determined by automated sequential degradation of intact alpha 2-CB5 and several chymotryptic and tryptic peptides. This segment of the peptide includes the site of the action of animal collagenases. As compared to the sequence around the alpha 1 cleavage site, the alpha 2 sequence is notable for the remarkable constancy of the residues to the amino side and the relative abundance of hydrophobic residues to the carboxyl side of the cleavage site, suggesting that these features are important in the recognition by the enzyme. The sequence of this region of the alpha 2 chain is consistent with the Gly-X-Y triplet structure and the preference of certain residues for either the X or Y position in distribution. However, three of the six residues of leucine were found in the Y position rather than the X position. Leucine residues were found only once in the Y position in the alpha 1 (I) chain. This preference does not appear to hold in the alpha 2 chain.


Interaction between cytochrome c and cytochrome b5. The reduction of cytochrome c by cytochrome b5 was studied over a wide range of ionic strengths in four different buffer systems. The reaction rate decreased linearly as the I1/2 was increased, suggesting that electrostatic interactions are important in the interaction. The ionic strength dependence of the reaction rate was in quantitative agreement with the theory of Wherland & Gray [Wherland, S., & Gray, H.B. (1976) Proc. Natl. Acad. Sci U.S.A. 73, 2950] only if the effective radius of the interaction was 2 A. This indicates that the interaction between the two proteins is best described as the sum of n complementary charge interactions, each involving a specific lysine on cytochrome c and a specific carboxyl group on cytochrome b5. The number of complementary charge interactions, n, was calculated to be five to seven, in agreement with the results of our specific modification studies. Ultracentrifugation and gel permeation techniques were used to demonstrate that cytochrome b5 and cytochrome c formed a stable complex at low ionic strength.


Inhibition of adenylate kinase by P1-(lin-benzo-5'-adenosyl)-P4-(5'-adenosyl) tetraphosphate and P1-(lin-benzo-5'-adenosyl)-P5-(5'-adenosyl pentaphosphate. P1-(lin-Benzo-5'-adenosyl)-P5-(5'-adenosyl) penraphosphate and P1-(lin-benzo-5'-adenosyl)-P4-(5'-adenosyl) tetraphosphate have been synthesized from lin-benzoadenosine 5'-monophosphoromorpholidate and adenosine 5'-tetraphosphate and adenosine 5'-triphosphate. These mixed dinucleoside polyphosphates are potent inhibitors of porcine muscle adenylate kinase, with association constants of 2 x 10(5) M-1 for the pentaphosphate and 2 x 10(6) M-1 for the tetraphosphate, respectively, as determined by kinetics and fluorescence experiments. The increase in fluorescence intensities and fluorescence lifetimes of both inhibitors upon binding to adenylate kinase results from a breaking of the intramolecular stacking interaction observed when these ligands are free in solution and implicates their binding to the enzyme in an "open" or "extended" form. These results and the dimensional requirements of these inhibitors are discussed in relation to our current knowledge of the active site of adenylate kinase and to the known inhibitors of adenylate kinase, P1,P5-bis(5'-adenosyl) pentaphosphate and P1,P4-bis-(5'-adenosyl) tetraphosphate.


Denaturation of thermophilic ferricytochrome c-552 by acid, guanidine hydrochloride, and heat. The denaturation of Thermus thermophilus cytochrome c-552 by acid, guanidine hydrochloride, and heat was studied by measuring the changes in absorption and circular dichroism. Cytochrome c-552 was remarkably resistant to acid; the pK of the transition from the low- to the high-spin form was roughly 0.3. The effect of guanidine hydrochloride on the heme iron-methionine bond of Thermus and horse cytochromes c was also investigated; a comparison of the free-energy changes for the displacement of the bond indicated that the coordination in cytochrome c-552 is highly stable. The spectra of guanidine hydrochloride unfolded cytochrome c-552 were dependent on the pH; the titration curve showed the presence of a cooperative single transition of pK = 4.7, with a one-proton dissociation, suggesting the ionization of a histidine residue. In the presence of guanidine hydrochloride, the influence of the heat on the ligand bond in cytochrome c-552 was studied. The van't Hoff plots of the reaction were biphasic. The enthalpy changes in the higher temperature range were independent on the guanidine hydrochloride concentration, while those in the lower range were not.


Binding of platinum(II) intercalation reagents to deoxyribnonucleic acid. Dependence on base-pair composition, nature of the intercalator, and ionic strength. The DNA binding of three platinum(II) intercalation reagents has been studied and found to depend upon base composition, the nature of the intercalator, and the ionic strength of the solvent medium. In 0.2 M NaCl, binding data for calf thymus DNA show the association constants to be approximately 10(4) M-1. The binding constants decrease in the order [(o-phen)Pt(en)]2+ greater than or equal to [(terpy)Pt(HET)]+ greater than [(bipy)Pt(en)]2+. The number of available intercalation sites for the doubly charged intercalators is only 70% of the number expected from the nearest-neighbor exclusion model. Binding of [(o-phen)Pt(en)]2+ and [(terpy)Pt(HET)]+ to various DNAs depends linearly on G.C content. Both reagents exhibit essentially the same degree of G.C specificity. Intercalative binding is a function of ionic strength. Increasing the salt concentration minimizes the importance of metallointercalator charge, and extrapolation to 1 M salt reveals the intercalative abilities, as reflected in binding constants, to be equivalent for [(terpy)Pt(HET)]+ and [o-phen)Pt(en)]2+ and about 1 order of magnitude less than that of ethidium.


Glutamyl transfer ribonucleic acid synthetase of Escherichia coli. Study of the interactions with its substrates. The binding of the various substrates to Escherichia coli glutamyl-tRNA synthetase has been investigated by using as experimental approaches the binding study under equilibrium conditions and the substrate-induced protection of the enzyme against its thermal inactivation. The results show that ATP and tRNAGlu bind to the free enzyme, whereas glutamate binds only to an enzyme form to which glutamate-accepting tRNAGlu is associated. By use of modified E. coli tRNAsGlu and heterologous tRNAsGlu, a correlation could be established between the ability of tRNAGlu to be aminoacylated by glutamyl-tRNA synthetase and its abilities to promote the [32P]PPi-ATP isotope exchange and the binding of glutamate to the synthetase. These results give a possible explanation for the inability of blutamyl-tRNA synthetase to catalyze the isotope exchange in the absence of amino acid accepting tRNAGlu and for the failure to detect an enzyme-adenylate complex for this synthetase by using the usual approaches. One binding site was detected for each substrate. The specificity of the interaction of the various substrates has been further investigated. Concerning ATP, inhibition studies of the aminoacylation reaction by various analogues showed the existence of a synergistic effect between the adenine and the ribose residues for the interaction of adenosine. The primary recognition of ATP involves the N-1 and the 6-amino group of adenine as well as the 2'-OH group of ribose. This first interaction is then strengthened by the phosphate groups- Inhibition studies by various analogues of glutamate showed a strong decrease in the affinity of this substrate for the synthetase after substitution of the alpha- or gamma-carboxyl groups. The enzyme exhibits a marked tendency to complex tRNAs of other specificities even in the presence of tRNAGlu. MgCl2 and spermidine favor the specific interactions. The influence of monovalent ions and of pH on the interaction between glutamyl-tRNA synthetase and tRNAGlu is similar to those reported for other synthetases not requiring their cognate tRNA to bind the amino acid. Finally, contrary to that reported for other monomeric synthetases, no dimerization of glutamyl-tRNA synthetase occurs during the catalytic process.


Determination of individual side-chain conformations, tertiary conformations, and molecular topography of tyrocidine A from scalar coupling constants and chemical shifts. We report for the decapeptide tyrocidine A: (a) H alpha and H beta chemical shifts and scalar coupling constants for most residues of tyrocidine A in methanol-d4 and dimethyl-d6 sulfoxide (Me2so-d6) and the H alpha and H beta chemical shifts for other residues; (b) scalar coupling constants 3J alpha beta for nine side chains in methanol-d4 but only seven side chains in Me2SO-d6, due to chemical shift degeneracy; the Gln9 and Tyr10 side chains in methanol-d4 were only approximately analyzed; (c) a total spin-spin analysis of Pro5 in Me2SO-d6 and, partly by comparison, also in methanol-d4; (d) conversion of 3J alpha beta values to side-chain conformations for all residues in methanol-d4; comparisons, where possible, led to the conclusion that side-chain conformations are similar in methanol-d4 and Me2SO-d6; (e) an absolute conformational analysis of Pro5 from 3J values and a method of assigning all pro-R,S protons; Pro5 has a Ramachandran B, C2-Cexo-Cendo conformation; (f) chi 1, chi 2 conformations of several aromatic residues based upon proton-chromophore distance measurement from anomalous chemical shifts and Johnson-Bovey diagrams; (g) pro-R and pro-S assignments of H beta's from anomalous chemical shifts, high-temperature dependence of anomalous chemical shifts, and backbone side-chain nuclear Overhauser effects; (h) most tertiary conformations of the whole tyrocidine A molecule possessing residues 4--8 and 10 in highly preferred (ca. 90%) chi 1 conformations, but residues 1--3 and 9 having at least two chi 1 rotamers; (2) description of three topographical regions of the molecule--a hydrophobic region, a flat hydrophilic surface on the other side of the molecule, and a hydrophilic region consisting of two peptide backbone units and the side chains of Asn8, Gln9, and Tyr10; (j) proposed side chain, beta-turn, and beta-pleated sheet conformations that readily account for all "normal" and anomalous chemical shifts.


Glucocorticoid regulation of mouse mammary tumor virus gene expression. Glucocorticoid hormones act rapidly and specifically to stimulate the synthesis of mouse mammary tumor virus RNA in a variety of mouse mammary tumor cells and infected heterologous cells. The increase in viral RNA production appears to be mediated by receptor proteins and requires the presence of basal levels of viral RNA. Infection of heterologous cells with MMTV may alter host cell responses to glucocorticoids; in addition, production of unintegrated viral DNA in these cells has provided reagents required for studying the structure and function of the viral DNA itself. The advent of new techniques for genetic manipulation of eukaryotic cells and for isolation of large amounts of specific DNA sequences should now permit detailed analyses of steroid hormone action in this system.


Oxidation of fatty alcohols to acids in the caecum of a gourami (Trichogaster cosby). Oxidation of fatty alcohols to acids in gourami caeca was investigated by measuring the reduction of NAD+ and the formation of labeled hexadecanoic acid from [1(-14)C]hexadecanol. Virtually all dehydrogenase activity is in the microsomal fraction. Maximal activity is obtained with NAD+ as cofactor whereas with NADP+ 60% of that activity is obtained. The enzyme is rather specific for long chain alcohols and 2 NADH are formed for each molecule of hexadecanol oxidized to acid. It is stabilized by mercaptoethanol, and completely inhibited by p-chloromercuribenzoate. The activity is optimal at pH 9.5. At higher pH, small amounts of aldehyde are found. The first reaction in the sequence, fatty alcohol leads to aldehyde leads to acid seems to occur under the more physiological condition at a much slower rate than the second reaction so that free aldehyde is not detected. Addition of palmitic acid indicated an uncompetitive product inhibition. The oxidation of alcohol to acid is reversible only to a very minor extent even in the presence of NADPH, CoA, ATP and Mg2+. Location, activity and properties of the enzyme are in agreement with the earlier observation from dietary experiments that in the gourami fatty alcohols of wax esters are oxidized to acids in the course of absorption.


A dolichyl phosphate-cleaving acid phosphatase from Tetrahymena pyriformis. Tetrahymena pyriformis contains an enzyme which hydrolyzed dolichyl phosphate. This activity was solubilized from lyophilized samples of this organism and was relatively stable when stored frozen. The soluble enzyme preparation had an acid pH optimum and hydrolyzed both dolichyl and phytanyl phosphates at equivalent rates. The polyprenylphosphate phosphatase activity was compared with the acid phosphatases which hydrolyzed p-nitrophenyl phosphate and marked differences were found. Dolichyl phosphate hydrolysis required Mg2+ for maximum activity while the bulk of the phosphatase activity was not effected by the absence of this ion. Other differences were that the polyprenylphosphate phosphatase was relatively insensitive to inhibitors such as tartrate and vanadium oxide sulfate which had a pronounced effect on the rate of p-nitrophenyl phosphate hydrolysis. The two activities also appeared to have different subcellular distributions. The polyprenylphosphate phosphatase was markedly inhibited by ethoxy formic anhydride, a reagent which is active against enzymes containing a histidine residue at their active site, while p-nitrophenyl phosphate hydrolysis was unaffected. The polyprenylphosphate phosphatase may be important in regulating the level of dolichyl phosphate in T. pyriformis and thus the rate of glycoprotein synthesis. It is also a useful tool which is capable of liberating dolichol from dolichyl phosphate under mild conditions which will permit the further characterization of the polyprenols.


Guinea pig very low density lipoproteins are a good substrate for lipoprotein lipase. In contrast to plasma from most other animals, guinea pig plasma causes little or no stimulation of lipoprotein lipase activity. Very low density lipoproteins (VLDL) isolated by ultracentrifugation of guinea pig serum caused a definite stimulation of lipase activity, whereas the infranatant inhibited the activity. Gel filtration in 5 M guanidinium hydrochloride of delipidated VLDL demonstrated that the activation was caused by a low molecular weight protein. The VLDL themselves were hydrolized at similar rates as human VLDL both by guinea pig and by bovine lipoprotein lipases. Thus, guinea pig VLDL contain an activator for lipoprotein lipase analogous to that in other animals and there is enough of the activator to support rapid hydrolysis of the VLDL lipids by the lipase.


Individual assignments of the heme resonances in the 360 MHz 1H NMR spectra of cytochrome c-557 from Crithidia oncopelti. With the use of nuclear Overhauser effects, spin decoupling and saturation transfer experiments individual assignments for numerous resonances of the heme group in the 360 MHz 1H NMR spectra of reduced and oxidized cytochrome c-557 from Crithidia oncopelti were obtained. These data provide direct evidence that the heme substituent in position 2 is a vinyl group. They further show that in spite of the different covalent structures of the heme groups the heme crevice and the electronic heme structure in the oxidized state are nearly identical in cytochrome c-557 and in mammalian cytochromes c.


Preparation and partial characterization of iron-sulfur, iron-selenium, and iron-tellurium complexes of bovine serum albumin. An artificial Fe-S* protein was prepared by the reaction of bovine serum albumin with FeSO4 and Na2S or with a synthetic Fe-S*-1,4-butanenedithiol complex. These improved methods enabled us to characterize the derivatives from serum albumin. The Fe-S* albumin complex has about 20 iron ions  and 14 labile sulfur atoms per molecule of the protein, whose absorption spectrum closely resembled that of 2Fe-2S* proteins. Its electron paramagnetic resonance spectrum exhibited signals different from those of ferredoxins. The addition of p-chloromercuriphenylsulfonate quenched the optical absorption in the visible region as well as the electron paramagnetic resonance signals. These properties of the albumin-iron complex are similar to those of iron-sulfur dithiothreitol and mercaptoethanol complexes, suggesting that the albumin-iron complex has one or more protein ligands besides sulfur lignads. Presumably, the oxygen atom of the tyrosine residue, or other hydroxyamino acids participates in the complex formation. In this context, the albumin polypeptide appears to be incapable of forming an iron-sulfur cluster identical to those of ferredoxins. Yet, from the albumin-iron derivative, the extrusion of the iron-sulfur core with benzenethiol provided products similar to those from ferredoxins. The iron-selenium and iron-tellurium derivatives of the bovine serum albumin were prepared and partially characterized by optical absorption and electron paramagnetic resonsnace spectroscopies. These results imply that both selenium and tellurium can be incorporated into the protein molecule as the respective labile components.


Contractile proteins in epidermis. Isolation and properties of guinea-pig epidermal myosin. Proteins of apparent molecular weights between 10 000 and 250 000 could be solubilized from guinea pig epidermis using a Tris/sucrose/ATP buffer. When the ionic concentration of the solubilized extract was made 75 mM with respect to KCl and 2 mM with respect to MgCl2, a protein complex precipitated which on SDS-polyacrylamide gel electrophoresis resolved into bands corresponding in migration to myosin, actin and a number of low molecular weight proteins. Myosin was dissociated from the complex with 0.6 M KI and purified by gel filtration chromatography on an agarose column. The purified epidermal myosin fraction contained a polypeptide of 200 000 molecular weight andtwo low molecular weight polypeptides of 16 500 and 13 000. The amino acid composition of the epidermal myosin heavy chain was similar to that of muscle myosin. At high ionic strength epidermal myosin had high specific (K+ + Ca2+)- and (K+ + EDTA)-ATPase activities and low specific (K+ + Mg2+)-ATPase activity. The pH activity curves of the (K+ + Ca2+)- and (K+ + EDTA)-ATPase were different. ATP was hydrolyzed faster than other nucleoside triphosphates. At low ionic strength, the (K+ + Mg2+)-ATPase activity of epidermal myosin was stimulated two fold by skeletal muscle actin. The myosin formed bipolar filaments in 50 mM KCl in the presence of 5 mM Mg2+.


Interaction of cytochrome c with the phosphorprotein phosvitin. Candida krusei cytochrome c forms a molecular complex with phosphorprotein phosvitin in weakly alkaline solution of low ionic strength. At most, about 22 molecules of cytochrome c bind to a phosvitin molecule. The complex at the binding ratio below about 11 (half of the maximum ratio) as a much higher binding strength. Several lines of evidence indicate that the marked difference in the binding strength is due to the difference in negative charges on phosvitin molecule concerned in the binding of a cytochrome c molecule. The phosvitin-bound cytochrome c seems to have a preferred orientation with the front surface of the molecule containing the exposed heme edge in contact with the phosvitin molecule.


Heme-linked properties of Pseudomonas cytochrome c peroxidase. Evidence for non-equivalence of the hemes. Pseudomonas cytochrome c peroxidase contains two hemes, one of which is shown to be in low-spin and one in high-spin state. The ferric enzyme reveals absorption maxima at 640 and 705 nm. The alkaline transition of these bands indicates the sixth iron-binding ligand of the low-spin and high-spin heme to be, respectively, a methionyl residue and a water molecule. The high-spin heme reacts with hydrogen peroxide to form a ferryl structure, which is the reactive intermediate in the peroxidatic reaction. The ferrous enzyme binds carbon monoxide in a 1:1 molar ratio, whereas the ferric form is unreactive towards small anionic ligands like F- and CN-. On this basis the peroxidase may also be classified as a cytochrome cc'.


On the evolution of blue proteins.

On the evolution of blue proteins. Amino acid sequences of 8 plastocyanins, 8 azurins, stellacyanin, two regions in human ceruloplasmin (ferroxidase)--all of which proteins are known to bind a blue (type 1) copper--and subunit II of bovine mitochondrial cytochrome c oxidase were compared by statistical methods to assess similarities and derive possible evolutionary relationships. It is suggested that all of the examined proteins are monophyletic. The two ceruloplasmin partial sequences clearly demonstrate that this protein has undergone a duplication. A calculated most parcimonious phylogenetic tree shows the divergence of the azurin and plastocyanin ancestor to be the earliest event. Stellacyanin and later the blue oxidase (ceruloplasmin) evolved from the plastocyanin branch, which the cytochrome c oxidase subunit evolved from the azurin ancestor.


[Reinnervation mechanism of skeletal muscles damaged by botulism toxin]. Reinnervation by botulinus toxin impaired motor nerve implantation was investigated in rat experiments with a purpose of elucidating conditions for regeneration of nerve fibers in the area of botulinic paralysis. Administration of the sublethal dose of toxin into the limb accelerated synaptogenesis of the nerve implanted. The latter exerts a normalizing effect on the muscular tissue and polarization level of its fibers.


[Effect of diazepam on the Na, K-ATPase state in a penicillin--induced hyperactivity focus in the cerebral cortex]. Intramuscular injection of diazepam to rats at doses of 0.01 and 2 mg/kg 25-30 min after penicillin application to the rat brain cortex leads to alteration of periodic appearance of epileptic seizures (ES), to changes in the seizure pattern, and to emergence of periodic acceleration of epileptiform discharges (ED). Injection of diazepam at a dose of 2 mg/kg 20 min before penicillin application results in the reduction of ED latency in the epileptogenic focus and in a decrease in their frequency before seizures as compared to the control animals without diazepam injection. ES appear irregularly, their quantity is markedly reduced while duration is increased. Diazepam injection leads to disappearance of the rat moving reaction during ER and ES. In vivo experiments diazepam (2 mg/kg) does not influence brain cortex Na, K-ATPase of crude synaptosomes. However, diazepam leads to an increase in Na, K-ATPase activity both in the primary and dependent secondary epileptogenic foci. It is suggested that the anticonvulsant action of diazepam may be underlain by its activating effect on Na, K-ATPase of neuronal membranes in the epileptogenic focus.


[Significance of beta-adrenoreception in realizing the glycogenolytic and lipolytic effects of excess thyroid hormones]. In experiments on rats the chronic administration of beta-adrenoblocker obsidan (propranolol) in doses blocking the glycogenolytic and lipolytic effects of exogenous epinephrine does not prevent a decrease in the liver and heart glycogen level and an increase of free fatty acids (FFA) in the serum as well as of lipolytic activity of adipose tissue in vitro, which are produced by high doses of thyroxine.


[Approximately hourly rhythm of 3',5'-cAMP in rat parotid gland slices]. 3',5'-CAMP concentration in rat parotid gland slices indubated in vitro for 12-14 hours was measured by radioimmunoassay. Clices for determination of cyclic nucleotide concentrations were taken at 10-minute intervals over a period of 2 hours. All slices used in a specific experiment originated from a single gland. Rhythmic changes in 3',5'-cAMP concentration in the rat parotid gland were found. The period of these changes (20-50 min) was similar to that of fluctuations in other parameters, such as dry weight, the rate of protein synthesis and ornithine decarboxylase activity, described for the same system elsewhere.


[Microionophoretic study of interaction of cyclic purine nucleotides and mediator substances on the cortical neurons]. Statistically significant data indicating selective interaction of noradenaline and cAMP as well as of acetylcholine and cGMP were obtained in experiments on the microionophoretic bringing of cyclic purine nucleotides and mediator substances acetylcholine and noradrenaline to the rabbit cortical neurons. Studies on the relationships between cAMP and cGMP at the single unit level suggest their multilevel functional interaction with other systems of intracellular regulators.


[Antistress effects of diazepam and activation of the positive reinforcing system upon changes on corticosteroid excretion and lipid level after peripheral electric pain stimulation]. It was shown in experiments on rats that the reaction of self-stimulation (SS) decreased the enhanced level of 11-hydrohycorticosteroids (11-HCS) in the blood after electrical pain stimulation(EPS) of the feet. Diazepam (1 mg/kg) decreased the levels of 11-HCS and beta-lipoproteids and increased the level of phospholipids after self-stimulation and pain stimulation. Diazepam and preliminary electrical pain stimulation induced activation of self-stimulation. It is concluded that diazepam and the activation of the positive reinforcing system exert similar effects on 11-HCS excretion and lipid levels after pain stimulation.


[Effect of obsidan on some mechanisms of the heart productivity regulation]. In acute experiments on anesthetized cats obsidan caused a decrease in the venous blood inflow to the heart and increased the volume of the vascular bed. It is suggested that the blockade of the myocardial beta-adrenoreceptors is not the only cause of such an effect.


[Multiphasic regulatory system mediating the effect of estradiol in rat uterus]. Estradiol is shown to induce histidine decarboxylase and histamine to activate adenylate cyclase in the rat uterus. Cyclic AMP like histamine simulates the effect of estradiol, intensifying RNA synthesis and inducing glycolytic enzymes and uterus inhibition. It was found by autoradiography that 3H-estradiol is accepted by the nuclei of some myometrium cells, 3H-histamine by their cytoplasm and 3H-cAMP is selectively bound by endothelium cells of the uterus capillaries. The estradiol messengers (histamine and cAMP) seem to mediate hormonal effect of some uterus heterofunctional cells forming a kind of multicellular functional system.


Effects of the histamine H2-receptor blocking drugs burimamide and cimetidine on noradrenergic transmission in the isolated aorta of the rabbit and atria of the guinea-pig. 1 In rabbit aortic strips, concentration-response curves to noradrenaline (NA) were shifted to the right in a parallel and concentration-dependent manner by the alpha-adrenoceptor blocking drug, phentolamine and also by the histamine H(2)-receptor blocking drugs, burimamide and cimetidine. Responses to 5-hydroxytryptamine were not affected by these drugs.2 Burimamide had the properties of a competitive antagonist of noradrenaline, possessing about one-hundredth the potency of phentolamine. Cimetidine was weaker than burimamide and did not fulfil the requirements for competitive antagonism of noradrenaline.3 In guinea-pig isolated atria, in which noradrenergic transmitter stores were labelled with [(3)H]-noradrenaline, phentolamine (3 muM), burimamide (30 muM) and cimetidine (30 muM), in decreasing order of effectiveness, each enhanced stimulation-induced efflux of [(3)H]-noradrenaline, indicating that their blocking effects on prejunctional alpha-adrenoceptors in this tissue are in the same order of relative potency as on postjunctional alpha-adrenoceptors in rabbit aortic strips.4 In the concentrations used (30 muM), neither burimamide nor cimetidine interfered with the neuronal uptake of noradrenaline. Burimamide, and to a much lesser extent, cimetidine, increased the resting efflux of [(3)H]-noradrenaline from guinea-pig atria.5 The effect of clonidine, a partial agonist on prejunctional alpha-adrenoceptors in guinea-pig atria, in increasing stimulation-induced efflux of [(3)H]-noradrenaline when stimulated with 150 pulses at 5 Hz was blocked by cimetidine (30 muM) and reversed by phentolamine (3 muM) and burimamide (30 muM).