Rate of divergence of cellular sequences homologous to segments of Moloney sarcoma virus. The RNA genome of the Moloney isolate of murine sarcoma virus (M-MSV) consists of two parts--a sarcoma-specific region with no homology to known leukemia viral RNAs, and a shared region present also in Moloney murine leukemia virus RNA. Complementary DNA was isolated which was specific for each part of the M-MSV genome. The DNA of a number of mammalian species was examined for the presence of nucleotide sequences homologous with the two M-MSV regions. Both sets of viral sequences had homologous nucleotide sequences present in normal mouse cellular DNA. MSV-specific sequences found in mouse cellular DNA closely matched those nucleotide sequences found in M-MSV as seen by comparisons of thermal denaturation profiles. In all normal mouse cells tested, the cellular set of M-MSV-specific nucleotide sequences was present in DNA as one to a few copies per cell. The rate of base substitution of M-MSV nucleotide sequences was compared with the rate of evolution of both unique sequences and the hemoglobin gene of various species. Conservation of MSV-specific nucleotide sequences among species was similar to that of mouse globin gene(s) and greater than that of average unique cellular sequences. In contrast, cellular nucleotide sequences that are homologous to the M-MSV-murine leukemia virus "common" nucleotide region were present in multiple copies in mouse cells and were less well matched, as seen by reduced melting profiles of the hybrids. The cellular common nucleotide sequences diverged very rapidly during evolution, with a base substitution rate similar to that reported for some primate and avian endogenous virogenes. The observation that two sets of covalently linked viral sequences evolved at very different rates suggests that the origin of M-MSV may be different from endogenous helper viruses and that cellular sequences homologous to MSV-specific nucleotide sequences may be important to survival.


Photodynamic treatment of herpes simplex virus during its replicative cycle. Photodynamic treatment of herpes simplex virus type 1-infected hamster embryo fibroblasts (LSH strain) with a low concentration of proflavine (0.08 mug/10(5) cells per ml), a 3-9-diamine acridine dye, inhibited production not only of infectious progeny but also of virion particles. However, there was no appreciable inhibition of viral or cellular DNA synthesis, even when the infected cells were repeatedly exposed to this low concentration of dye and light during the replication cycle of the virus. It thus appears that photodynamic treatment of infected cells interferes with the processes involved in virus maturation.


Effects of temperature and antibody on the cyclid growth of vesicular stomatitis virus. To see the effects of temperature on the interrelated cyclic production of standard and defective interfering (DI) particles of vesicular stomatitis virus, a temperature-sensitive (ts) G114 mutant was passaged successively at different temperatures and the production of the two types of viral particles as well as the ability of Chinese hamster ovary cells to survive each passage was continuously monitored. When the temperature was nonpermissive for standard virus, the synthesis of both standard and defective interfering particles was inhibited. When revertants appeared in the population, their ability to take over the infection depended on the permissiveness of the temperature for the temperature-sensitive mutant. At permissive temperatures periodic inhibition of both types of standard viruses was maintained by the production of defective interfering particles. Reverents did not become a majority of the population due to this periodic inhibition. When the conditions were nonpermissive for the mutant, revertants became the major standard virus in the population within a few passages. These findings can be understood if conditions of high and low multiplicities are dissected out together with a thorough understanding of the individual properties of each of the viral particles and of the result of interactions between them. In the presence of antiserum which neutralized only 90% of the viral particles, cyclic production of standard virus occurred, with a decline in the total amount of virus produced after each cycle. Therefore, in the presence of limiting concentrations of antiserum, the virus appeared to be able to establish a persistent cyclic growth pattern.


Cell-free synthesis of polyoma virus capsid proteins VP1 and VP2. Polyadenylated RNA isolated from the cytoplasm of mouse 3T6 cells 28 h after infection with polyoma virus has been isolated and translated in vitro. Polyoma capsid proteins VP1 and VP2 have been identified in the cell-free product by polyacrylamide gel electrophoresis, specific immunoprecipitation, and tryptic peptide fingerprinting. Polyoma mRNA species have been isolated by preparative hybridization to purified viral DNA immobilized on cellulose nitrate filters and shown to code for both VP1 and VP2. These experiments establish conditions for the isolation of late polyoma mRNA and the cell-free synthesis of polyoma capsid proteins and indicate that the active mRNA species are at least partially virus coded.


Transmembrane phospholipid motions induced by F glycoprotein in hemagglutinating virus of Japan. Transfer of phospholipid from the envelope of hemagglutinating virus of Japan (HVJ) to erythrocyte (RBC) membrane and the virus-induced transfer of phospholipid between RBC membranes were studied using spin-labeled phosphatidylcholine (PC). The transfer of PC from membranes labeled densely with PC to unlabeled membranes was followed by the peak height increase in the electron spin resonance spectrum. The two kinds of transfer reactions took place very rapidly as reported previously. To obtain further details, the transfer reactions were studied with HVJ, HVJ inactivated by trypsin, HVJ harvested early, HVJ grown in fibroblast cells, the fibroblast HVJ activated by trypsin, influenza virus, and glutaraldehyde-treated RBCs. The results demonstrated that the viral F glycoprotein played a crucial role in the transmembrane phospholipid movements as well as in the fusion and hemolysis of RBCs. The transfer from HVJ to RBC's occurred partially through an exchange mechanism not accompanying the envelope fusion. This was shown by a decrease in the exchange broadening of the electron spin resonance spectrum of released spin-labeled HVJ (HVJ) and also by an increase in the ratio of PC to viral proteins incorporated into RBC membranes. HVJ modified RBC membrane so as to be able to exchange its phospholipids with those of inactive membranes such as fibroblast HVJ, influenza virus, glutaraldehyde-treated RBC'S, and phosphatidylcholine vesicles. HVJ affected the fluidity of RBC membranes markedly, the environments around PC being much fluidized. The virus-induced fusion was discussed based on close apposition of the membranes by HANA proteins and on the destabilization and fluidization of RBC membranes by F glycoproteins.


Inhibition of host cell protein synthesis by UV-inactivated poliovirus. The ability of poliovirus that was irradiated with UV light at energies up to 2,160 ergs/mm2 to subsequently inhibit host cell protein synthesis was measured. The inactivation of the host cell shutoff function followed one-hit kinetics. Increasing irradiation did not affect the rate of inhibition until the multiplicity of infection after irradiation was reduced to approximately 1 PFU/cell. At higher functional multiplicities, the rate was unchanged, but an increasing lag before the onset of inhibition was observed with increasing irradiation. The energy levels required to inactivate virus-induced inhibition of host cell protein synthesis suggest that damage to virus RNA rather than to virus capsid proteins is responsible for the loss of function. When the inactivation of host cell shutoff was compared with the inactivation of other viral functions by UV irradiation, it correlated exactly with the loss of infectivity but not with other viral functions measured. Guanidine treatment, which prevents detectable viral RNA and protein synthesis, completely inhibited host cell shutoff by low multiplicities of unirradiated virus infection but not higher multiplicities. When a high multiplicity of virus was first reduced to a low titer by irradiation, host cell shutoff was still evident in the presence of guanidine. The results demonstrate that the complete inhibition of host cell protein synthesis can be accomplished by one infectious viral genome per cell.


Anatomy of herpes simplex virus DNA VII. alpha-RNA is homologous to noncontiguous sites in both the L and S components of viral DNA. Previous reports from this laboratory (Honess and Roizman, 1974) have operationally defined alpha polypeptides as the viral proteins that are synthesized first in HEp-2 cells treated with cycloheximide from the time of infection with herpes simplex virus type 1 until the withdrawal of the drug 12 to 15 h after infection. It has also been shown that the viral RNA (designated alpha RNA) that accumulates in the cytoplasm during cycloheximide treatment and on polyribosomes immediately upon withdrawal of the drug is homologous to 10 to 12% of viral DNA, whereas the viral RNA accumulating in the cytoplasm of untreated cells at 8 to 14 h after infection is homologous to 43% of viral DNA (Kozak and Roizman, 1974). In the present study, alpha RNA and cytoplasmic RNA extracted from untreated cells 8 h after infection were each hybridized in liquid to in vitro labeled restriction endonuclease fragments generated by cleavage of herpes simplex virus type 1 DNA with Hsu I, with Bgl II, and with both enzymes simultaneously. The data show that only a subset of the fragments hybridized to alpha RNA, and these are scattered within both the L and S components of the DNA. There are at least five noncontiguous regions in the DNA homologous to alpha RNA; two of these are located partially within the reiterated sequences in the S component. All fragments tested hybridized more extensively with 8-h cytoplasmic RNA than with alpha RNA. Four adjacent fragments, corresponding to 30% of the DNA and mapping within the L component, hybridized exclusively with the cytoplasmic RNA extracted from cells 8 h after infection.


Six-NB-tropic murine leukemia viruses derived from a B-tropic virus of BALB/c have altered p30. We have examined the electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels of three virion proteins of B-tropic murine leukemia virus from BALB/c and six of its NB-tropic derivatives. The gp70 protein and a 13,000-molecular-weight virion protein tentatively identified as p15 of the NB-tropic viruses migrated with the corresponding B virus proteins. However, the major internal structural protein of type C virions, p30, of all the NB-tropic viruses migrated more rapidly than the p30 of their B virus progenitor. Although this change in p30 raises the possibility that p30 may be involved in determining the N-, B-, or NB-tropism of MuLV's, it is also possible that the change accompanies but does not directly determine the change in tropsim.


Application of freeze-drying intact cells to studies of murine oncornavirus morphogenesis. Using a method for freeze-drying intact cells, uninfected and murine leukemia virus (MuLV)-infected JLSV9 cell surfaces, as well as murine mammary tumor virus (MuMTV)-infected cell surfaces, were examined by electron microscopy. The 10-nm knobs of MuLV and the 5-nm spikes of MuMTV were clearly revealed on the surfaces of budding viruses and were also found dispersed over the cell surface. The MuLV knobs are randomly arranged on the virus surface, whereas the MuMTV spikes are much more ordered. Because freeze-fractured budding viral envelopes are devoid of intramembranous particles, the observed surface particles do not appear to be merely accentuated intramembranous particles. This technique should permit further analysis of the morphogenesis of viral envelopes without the need for externally applied labels.


Tunicamycin inhibits glycosylation and multiplication of Sindbis and vesicular stomatitis viruses. Tunicamycin (TM), an antibiotic that inhibits the formation of N-acetylglucosamine-lipid intermediates, thereby preventing the glycosylation of newly synthesized glycoproteins, inhibits the growth of Sindbis virus and vesicular stomatitis virus in BHK cells. At 0.5 mug of TM per ml, the replication of both viruses is inhibited 99.9%. Noninfectious particles were not detected. All the viral proteins were synthesized in the presence of TM, but the glycoproteins were selectively altered in that they migrated faster than normal viral glycoproteins when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting defective glycosylation. Within 1 h after TM addition, [14C]glucosamine incorporation into glycoproteins was inhibited 20%, whereas [35S]methionine incorporation was unaffected. By 2 to 3 h after TM addition, glucosamine incorporation had fallen to 15% of control value, with methionine incorporation being 60% of normal. TM did not affect the growth of the nomenveloped encephalomyocarditis virus in BHK cells, demonstrating that TM is not a general inhibitor of protein synthesis. These data demonstrate that TM specifically inhibits the glycosylation of viral glycoproteins and that glycosylation may be essential for the normal assembly of enveloped viral particles.


Complexity and polyadenylic acid content of visna virus 60-70S RNA. The genomic complexity of visna virus was measured by quantitative analysis of 18 RNase T1-resistant oligonucleotides from 60-70S RNA. T1-resistant oligonucleotides were separated by two-dimensional polyacrylamide gel electrophoresis. Visna virus had a genomic complexity of 3.6 X 10(6) daltons, very close to the size of a single 30-40S RNA subunit. It was therefore concluded that the visna virus genome is largely polyploid. Visna virus 60-70S RNA polyadenylic acid segment was purified by T1 RNase digestion followed by oligodeoxythymidylic acid-cellulose column chromatography. It contained over 99% AMP and had a size of about 200 nucleotides. The binding capacities on oligodeoxythymidylic acid-cellulose of native 60-70S RNA and purified 30-40S RNA subunits were examined. It was concluded that two out of three intact subunits contain a polyadenylic acid segment.


Rifampin-susceptible mutant of vesicular stomatitis virus: protein and RNA synthesis. A rifampin-susceptible strain (VSV Rif+) was selected from the wild vesicular stomatitis virus (VSV) population unsusceptible to rifampin. The VSV Rif+ was blocked in its intracellular replication in the presence of rifampin. In cells, rifampin affected primarily VSV Rif+ transcription, but to a different extent than in a cell-free system. In addition, a decrease in the amount of VSV Rif+ protein M was detected, linked to a stimulation of protein NS. In the absence of rifampin, protein M, although synthesized, was not immediately incorporated into the cell membrane. An interpretation of these observations is proposed.


Transcriptional mapping of vesicular stomatitis virus in vivo. Synthesis of the proteins of vesicular stomatitis virus (Indiana serotype) was studied in mouse L cells infected with virus that had been exposed to UV radiation. The UV target sizes measured during primary transcription indicated that the five genes occupy a single transcriptional unit. Thus, in infected cells, as a cell-free system, transcription of vesicular stomatitis virus RNA initiates at a single point and proceeds in the order N. NS, M, G, and L.


Presence of deletion molecules in human wart virus DNA. Deletion mutants of human wart virus were found to occur occasionally in the virus population transmitted in vivo, although the majority of virion DNA molecules were homogeneous in length and base sequence. Human wart virus DNA was 1.48 times as long as nondefective simian virus 40 DNA.


Influence of defective virion core proteins on RNA maturation with an avian sarcoma virus. The RNA of the avian sarcoma virus B77 temperature-sensitive mutant LA334 was investigated using electrophoretic analysis. The RNA from mutant virus grown at the nonpermissive temperature (42degrees C) showed a heterogeneous peak between 80 and 125S, and another at about 35S. The RNA of the mutant virus grown at the permissive temperature (35 degrees C) behaved like wild-type B77 virus RNA, exhibiting a major peak at 70S. The homology between the various RNA fractions and virus-specific DNA probe was determined, indicating that mutant virus grown at the nonpermissive temperature contains relatively large amounts of nonviral-specific RNA.


Loss of supercoiled viral DNA after infection of permissive cells by simian virus 40. During infection of BS-C-1 cells (a permissive line of monkey kidney cells) with simian virus 40, there was a loss of infecting viral supercoiled (component I) DNA molecules which was partially inhibited by cycloheximide. The kinetics of loss of component I in the presence of cycloheximide suggested that the loss was the result of at least two activities acting on the DNA, one cycloheximide sensitive and the second cycloheximide insensitive. Nucleotides from degraded infecting viral DNA were reincorporated into cell DNA that was synthesized during the infection.


Use of defective interfering particle RNA probes in the determination of the order of in vitro transcription of vesicular stomatitis virus genes. Annealing of various defective interfering particle RNAs of vesicular stomatitis virus to early in vitro viral transcription products has shown that transcription is sequential, with 13S to 18S mRNA's being produced first. Based on this finding, the 30S mRNA cistron is located at the 5' end of the viral genome.


Synthesis of Epstein-Barr virus DNA in vitro: effects of phosphonoacetic acid, N-ethylmaleimide, and ATP. Nuclei from superinfected Raji cells synthesized Epstein-Barr Virus (EBV) DNA in vitro in the absence of cell DNA synthesis. The synthesis of EBV DNA in vitro was inhibited by phosphonoacetic acid and N-ethylmaleimide, and maximum synthesis was achieved in the absence of an ATP-regenerating system. Nuclei from mock-infected cells required an ATP-regenerating system for maximum DNA synthesis.


Possible in vitro repair of viral RNA by ligase-like enzyme(s) in poliovirus-infected cells. A soluble polymerase-template complex prepared from poliovirus-infected cells was found to incorporate radioactive UTP into trichloroacetic acid-insoluble RNA linearly for 8 h in the presence of ATP and Mg2+. Radioactive CTP or GTP was not incorporated under identical conditions. Nearest-neighbor analysis of the in vitro product demonstrated that ATP was added to the viral RNA in the form of polyadenylic acid; UTP was added internally to the 3'-OH group of all four nucelotides. The data can best be explained by the addition of the UTP to the 3'-OH groups of single-stranded breaks in the double-stranded viral RNA and ligation to the adjacent 5'-phosphate groups. The enzymatic activity was also found in encephalomyocarditis virus- and rhinovirus type 1A-infected cells but not in uninfected cells.


Evidence for a diffusible T4 bacteriophage protein governing the initiation of delayed early RNA synthesis. Two forms of prereplicative phage RNA can be discerned in Escherichia coli early after infection with bacteriophage T4, immediate early and delayed early RNA. The transition from immediate early to delayed early RNA synthesis is inhibited by chloramphenicol. The present work presents evidence for the existence of a phage-specific protein, which effects this transition. Delayed early RNA formation was measured by a cistron-specific enzyme-forming-capacity method, in which RNA synthesized in the presence of chloramphenicol was allowed to express itself into enzyme activity after (i) the addition of rifampin to inhibit further transcription and (ii) subsequent removal of chloramphenicol. As representatives of delayed early transcription, the two phage-specific enzymes dCTPase and deoxynucleotide kinase were chosen. Primary infection with a phage mutant defective in one of these two enzymes was found to induce a diffusible factor, which in the presence of chloramphenicol could effect the formation of delayed early RNA corresponding to the missing enzyme, upon superinfection with wild-type phage. The activity of this factor, acting in trans, was abolished by the amino acid analogue ethionine. Mutation in the suA gene of the host did not relieve phage of the apparent need for protein synthesis in the transition from immediate early to delayed early phage RNA synthesis.


Assembly of viral membranes. I. Association of vesicular stomatitis virus membrane proteins and membranes in a cell-free system. We report here an in vitro system designed to study the interactions of vesicular stomatitis virus (VSV) proteins with cellular membranes. We have synthesized the VSV nucleocapsid (N) protein, nonstructural (NS) protein, glycoprotein (G protein), and membrane (M) protein in a wheat germ, cell-free, protein-synthesizing system directed by VSV 12 to 18S RNA. When incubated at low salt concentrations with purified cytoplasmic membranes derived from Chinese hamster ovary cells, the VSV M andG proteins bind to membranes, whereas the VSV N and NS proteins do not. The VSV M protein binds to membranes in low or high divalent cation concentrations, whereas binding of significant amounts of G protein requires at least 5 mM magnesium acetate concentrations.


Expression and thermal stability of simian virus 40 tumor-specific transplantation antigen and tumor antigen in wild type- and tsA mutant-transformed cells. We have explored aspects of a suggested relationship between the expression of simian virus 40 tumor-specific transplantation antigen (TSTA) and tumor antigen (TA). A unique rat embryo cell line transformed by a temperature-sensitive A mutant that loses TA during exposure to the nonpermissive temperature (A28-RE) was found to lose TSTA. On the other hand, a typical control tsA-transformed cell line (A239-MB) expressed both TA and TSTA at the non-permissive temperature. TA in lysates obtained from A239-MB cells was found to be three to four times more thermolabile by covwt-mb) when incubated at either 33 or 40 degrees C. These data complement previous reports using TA from lytic infection and are consistent with the suggestion that TA is virus encoded. In contrast to TA, which even in wild-type-transformed cells was completely destroyed in less than 10 min at 50 degrees C, TSTA, assayed in vivo by tumor rejection, and tumor-specific surface antigen(s) TSSA) defined by an in vitro cytolytic assay, were thermostabile. Even after 24 h of incubation of extracts of 50 degrees C, high levels of TSTA and TSSA activity were present. Since these surface antigens when obtained from cells transformed by tsA mutants were also thermostabile, they could not be distinguished from the wild-type antigens. These results (i) indicate a coordinate expression of TA and TSTA in transformed cells; (ii) confirm that TA is virus encoded; and (iii) confirm that tha antigenic and immunogenic determinants that characterize TA and TSTA activities are distinct. However, the possibility that TSTA, like TA, is of viral rather than cellular origin is not excluded.


Purification of DNA complementary to the env gene of avian sarcoma virus and analysis of relationships among the env genes of avian leukosis-sarcoma viruses. The env gene of avian leukosis-sarcoma viruses encodes a glycoprotein that determines the host range and surface antigenicitiy of virions. We have purified radioactive DNA (cDNAgp) complementary to at least a portion of the env gene for viral subgroups A and C; complementary DNA was synthesized with purified virions of wild-type avian sarcoma virus, and RNA from a mutant with a deletion in env was used to select DNA specific to env by molecular hybridization. The genetic complexity of cDNAgp for subgroup A (ca. 2,000 nucleotides) was sufficient to represent the entire deletion and most or all of the env cistron. The deletions in env in two independently isolated strains of virus (Bryan and rdNY8SR) overlap, and cDNAgp represents nucleotide sequences common to both deletions. By contrast, we could detect no overlap between deletions in env and deletions in the adjacent viral gene src. Laboratory stocks of viral subgroups A, B, C, D and E do not contain detectable amounts of env deletions when tested by molecular hybridization; hence, segregation of deletions in env is a less frequent event that the segregation of deletions in the viral transforming gene src (Vogt, 1971). We found extensive homology among the nucleotide sequences encoding the env genes of virus strains indigenous to chickens (subgroups A, B, C, D, and E) although subgorups B, D and E appear to differ slightly from subgroups A and C at the env locus. By contrast, viruses obtained from pheasant cells (subgroups F and G) have env genes with little or no relationship to env genes of chikcen viruses. According to available data, viruses of subgroup F arose by recombination between an avarian sarcoma virus and viral genes in the genome of ring-necked pheasants, whereas subgroup G viruses may be entirely endogenous to golden pheasants.


Variations in integration site of avian oncornaviruses in different hosts. We examined the integration site of avian oncornaviruses in the genome of different hosts with respect to the repetitive frequency of the cellular DNA sequences adjacent to the integrated proviral DNA. The following systems were studied: avian sarcoma virus (B-77) and avian leukosis virus (Rous-associated virus-61) in cultured duck embryonic cells and B-77 in cultured mouse 3T3 cells. These systems represent different host responses to viral infection, i.e., one in which both cellular transformation and viral replication occur (B-77-infected duck cells), one in which viral replication, but not transformation, occurs (Rous-associated virus-61-infected duck cells), and one in which transformation, but not viral replication, occurs (B-77-infected 3T3 cells). Two sequential hybridizations were used. First, large denatured DNA fragments (2.8 X 10(6) daltons) were reassociated to different C0t (mole-seconds per liter) values. Next, DNA remaining single stranded at different C0t values was isolated by hydroxylapatite column chromatography, immobilized on nitrocellulose filters, and hybridized with an excess of 3H-labeled 35S viral RNA to titrate the concentration of proviral DNA. Results show that B-77 sarcoma virus and Rous-associated virus-61 integrate in the unique region of duck DNA, whereas B-77 proviral DNA is associated with both repeated and unique host DNA sequences in transformed mouse 3T3 cells.


Picornaviral capsid assembly: similarity of rhinovirus and enterovirus precursor subunits. Cytoplasmic extracts of rhinovirus 1A-infected HeLa cells, pulsed 15 min with [3H]leucine, contained a 13S subunit which was rich in the capsid precursor, peptide 92. After a 30-min chase, most of the capsid-related protein sedimented in a 14S peak that contained equimolar amounts of the capsid peptides epsilon, alpha, and gamma, and some residual chain 92. The 14S subunit could be dissociated at pH 4.8 into 6S subunits containing only epsilon, alpha, and gamma chains in equal proportions, indicating that the 14S subunit is an oligomer of (epsilon gamma alpha) protomers. These subunits resemble subunits previously identified in the assembly of enteroviruses. These observations support the idea that rhinovirus assembly is basically similar to that of enteroviruses. Comparative studies on the peptide stoichiometry of the virion and the capsid precursor subunits indicate that rhinovirus 1A can contain as many as 11 immature protomers per virion.


Infectious linear DNA sequences replicating in simian virus 40-infected cells. A new class of linear duplex DNA structures that contain simian virus 40 (SV40) DNA sequences and that are replicated during productive infection of cells with SV40 is described. These structures comprise up to 35% of the radioactively labeled DNA molecules that can be isolated by selective extraction. These molecules represent a unique size class corresponding to the length of an open SV40 DNA molecule (FO III), and they contain a heterogeneous population of DNA sequences either of host or of viral origin, as shown by restriction endonuclease analysis and nucleic acid hybridization. Part of the FO III DNA molecules contain viral-host DNA sequences covalently linked with each other. They start to replicate with the onset of SV40 superhelix replication 1 day after infection. Their rate of synthesis is most pronounced 3 days after infection when superhelix replication is already declining. Furthermore, they cannot be chased into other structures. At least a fraction of these molecules is infectious when administered together with DEAE-dextran to permissive cells. After intracellular circularization, superhelical DNA FO I with an aberrant cleavage pattern accumulates. In addition, tumor and viral capsid antigen are induced, and infectious viral progeny is obtained. Infection of cells with purified SV40 FO I DNA does not result in FO III DNA molecules in the infected cells or in the viral progeny. It is suggested, therefore, that these FO III DNA molecules are perpetuated within SV40 virus pools by encapsidation into pseudovirions.


Junin virus structural proteins.

Junin virus structural proteins. Polyacrylamide gel electrophoresis of purified Junin virus revealed six distinct structural polypeptides, two major and four minor ones. Four of these polypeptides appeared to be covalently linked with carbohydrate. The molecular weights of the six proteins, estimated by coelectrophoresis with marker proteins, ranged from 25,000 to 91,000. One of the two major components (number 3) was identified as a nucleoprotein and had a molecular weight of 64,000. It was the most prominent protein and was nonglycosylated. The other major protein (number 5), with a molecular weight of 38,000, was a glucoprotein and a component of the viral envelope. The location on the virion of three additional glycopeptides with molecular weights of 91,000, 72,000, and 52,000, together with a protein with a molecular weight of 25,000, was not well defined.


Herpes simplex virus resistance and sensitivity to phosphonoacetic acid. Phosphonoacetic acid (PAA) inhibited the synthesis of herpes simplex virus DNA in infected cells and the activity of the virus-specific DNA polymerase in vitro. In the presence of concentrations of PAA sufficient to prevent virus growth and virus DNA synthesis, normal amounts of early virus proteins (alpha- and beta-groups) were made, but late virus proteins (gamma-group) were reduced to less than 15% of amounts made in untreated infected cells. This residual PAA-insensitive synthesis of gamma-polypeptides occurred early in the virus growth cycle when rates were identical in PAA-treated and untreated infected cells. Passage of virus in the presence of PAA resulted in selection of mutants resistant to the drug. Stable clones of mutant viruses with a range of drug sensitivities were isolated and the emergence of variants resistant to high concentrations of PAA involved the sequential selection of mutants progressively better adapted to growth in the presence of the drug. Increased drug resistance of virus yield or plaque formation was correlated with increased resistance of virus DNA synthesis, gamma-protein synthesis, and resistance of the virus DNA polymerase reaction in vitro to the inhibitory effects of the drug. PAA-resistant strains of herpes simplex virus type 1 (HSV-1) complemented the growth of sensitive strains of homologous and heterologous types in mixed infections in the presence of the drug. Complementation was markedly dependent upon the proportions of the resistant and sensitive partners participating in the mixed infection. Intratypic (HSV-1A X HSV-1B) recombination of the PAA resistance marker(s), Pr, occurred at high frequency relative to plaque morphology (syn) and bromodeoxyuridine resistance (Br, thymidine kinase-negative phenotype) markers, with the most likely order being syn-Br-Pr. Recombinant viruses were as resistant or sensitive to PAA as the parental viruses, and viruses recombinant for their PAA resistance phenotype were also recombinant for the PAA resistance character of the virus DNA polymerase. The results provide additional evidence that the herpesvirus DNA polymerase is the site of action of PAA and illustrate the potential usefulness of PAA-resistant mutants in genetic studies of herpesviruses.


Morphogenesis of vesicular stomatitis virus: electron microscope observations with freeze-fracture techniques. The morphogenesis of vesicular stomatitis virus was examined using freeze-fracture techniques, and the results obtained were compared with those from previously published experiments carried out with influenza viruses and togaviruses. The process of conversion of the host cell plasma membrane into the vesicular stomatitis virus envelope was accompanied by a loss of the intramembranal particles abundant in cell membranes. Frequently a dense accumulation of intramembranal particles could be seen at the base of the developing virion, suggesting that these structures might play some role in the generation of viral envelope. In addition to the viral structures that were seen to develop in the classical fashion, with their long axis perpendicular to the cell surface, structures were also found that suggested the initiation of a process similar to budding, with the long axis of the viral capsid parallel to the plasma membrane. In this situation, as in the "perpendicular" process, intramembranal particles were excluded from the viral structure, and an accumulation of these particles could be seen adjacent to the developing viral membrane.


Identification of simian virus 40 protein A. A large simian virus 40 (SV40)-specific protein can be efficiently immunoprecipitated from infected cell extracts with antisera obtained from hamsters bearing SV40-induced tumors. The protein has an apparent molecular weight of 88,000 to 100,000 with respect to markers with known molecular weights, but behaves anomalously on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Cell lines infected by two different strains of SV40 synthesize immunoreactive proteins that differ slightly in mobility during SDS-polyacrylamide gel electrophoresis, evidence that the protein is coded for by the virus. These differences in protein size correlate with differences in the electrophoretic mobility of viral DNA fragments obtained by digestion with HindII and III restriction enzymes. The size of the viral capsid proteins VP2 and VP3 also varies with the strain of virus. dl-1001, a constructed deletion mutant that lacks part of the SV40A gene, directs the synthesis of a 33,000-dalton polypeptide that is not detected in cells infected with wild-type virus. The deletion fragment, like the larger protein, is phosphorylated. Maps of tryptic peptides from the 88,000- to 100,000-dalton protein and the 33,000-dalton fragment show common peptides and provide strong direct evidence that the proteins are products of the SV40 A gene. The deletion fragment reacts with antitumor sera and binds to double-stranded DNA in the presence of the complete A protein.


Modification of simian virus 40 protein A. The A protein of simian virus 40 is phosphorylated in both productive and transforming infection. The phosphorylated amino acid has been identified as serine and has been localized in a single tryptic peptide of the protein. Because the A protein synthesized in infection by A mutants is phosphorylated to the same extent and in the same peptide as in infection by wild-type virus, the functional defect of the A mutants is apparently unrelated to phosphorylation. At least three distinct forms of the A protein with apparent molecular weights of 85,000, 88,000, and 100,000 can be identified in extracts of cells infected by wild-type virus. After exposure of cells to Nonidet P-40, the 85,000- and 88,000-dalton proteins were found in varying amounts in extracts of permissive cells but not in extracts of transformed cells. This finding raised the question of the possible functional importance of the smaller proteins in productive infection. However, the virtual absence of the 85,000- and 88,000-dalton proteins in some extracts of the fully permissive CV-1 cell line indicates that a conversion of the larger to the smaller forms of the A protein is not required in significant quantity for productive infection. Furthermore, a study of extraction conditions shows that the smaller proteins are easily generated during extraction and provides an explanation for the appearance of these proteins in some cells after extraction under unfavorable conditions.


Giant heterogeneous polyadenylic acid on vesicular stomatitis virus mRNA synthesized in vitro in the presence of S-adenosylhomocysteine. An in vitro transcription system in which vesicular stomatitis virus (VSV) mRNA species have been synthesized is described. In addition to purified VSV virions, which contain an RNA-dependent RNA polymerase, this system contained a cytoplasmic cell extract that enhanced correct transcription. Gel electrophoretic analysis of the methylated polyadenylic acid [poly(A)]-containing VSV mRNA produced in this system in the presenct of S-adenosylmethionine showed the discrete VSV mRNA species. However, when unmethylated mRNA was synthesized in the presence of S-adenosylhomocysteine, the poly(A)-containing transcripts were large and heterogeneous in molecular weight and did not contain discrete VSV mRNA species. Two-dimensional fingerprint analysis of the methylated and unmethylated products suggested that identical nucleotide sequences were present in the RNAs. Further analysis showed the presence of very large heterogeneous poly(A), 200 to 2,000 nucleotides in lenght, in the unmethylated transcript. Proof that this large poly(A) was covalently linked to the correct VSV mRNA transcripts was obtained by removal of the poly(A) by hybirdization with oligodeoxythymidylic acid and digestion with RNase H. This digestion produced unmethylated VSV mRNA transcripts with the same discrete sizes as the deadenylated RNAs produced from VSV mRNA initially isolated from VSV-infected cells. The results suggest that there is a relationship between methylation at the 5'-end and polyadenylation at the 3'-end of VSV mRNA's. Furthermore, addition of the very large poly(A) does not affect the normal process of sequential transcription of the VSV genome, suggesting that this poly(A) addition is occurring independently of further transcription.


Evidence that a precursor glycoprotein is cleaved to yield the major glycoprotein of avian tumor virus. A glycoprotein designated pr90, which is recognized by anti-gp85 serum, is present in lysates of pulse-labeled transformed cells. Under chase conditions, a reduction in the level of labeled pr90 is observed concomitant with the appearance of labeled, cell-associated viral glycoprotein.


Cyclic AMP regulation of mammary tumor virus production. Addition of dibutyryl cyclic AMP (cAMP) and agents (isoproterenol and epinephrine) that stimulate the activities of adenylate cyclase enhance the stimulation  of mammary tumor virus (MTV) production two- to threefold by glucocorticoid in short-term primary cultures of mammary tumors. This cAMP potentiation seems to depend on the stimulated level of MTV production by glucocorticoid alone, which increases MTV production 5- to 10-fold over basal level but varies greatly in absolute terms. When the stimulated level by glucocorticoid alone is suboptimal, cAMP seems to restore the sensitivity of the cells to the stimulatory effect of glucocorticoid to its maximum.


5' termini of poliovirus RNA: difference between virion and nonencapsidated 35S RNA. Poliovirus cytoplasmic, nonencapsidated 35S RNA yields approximately one pUp per molecule upon T2 RNase digestion, indicating that this RNA has the same 5' end as the polyribosome-associated viral RNA fraction. Double-stranded, replicative form RNA after the same treatment yielded approximately four pNp structures per molecule, 65% of which was pUp. In contrast, the 35S RNA from mature virions contained no detectable pNp, indicating that the 5' end of the virion RNA is different from that of the nonencapsidated RNA. None of the above molecules contained pppNp, ppNp, or GpppNp structures present in host mRNA. The virion RNA molecules, as we have shown previously for thenonencapsidated 35S viral RNA (Fernandez-Muñoz and Darnell, 1976), is not labeled with [methyl-3H]methionine.


Cyclic adenosine monophosphate: relationship to calcium metabolism and renal lithiasis. Recent concepts in calcium metabolism are being applied to the renal stone-forming patient. As our understanding of physiological mechanisms improves urinary cyclic nucleotide determinations are becoming useful in applied patient care. The changes in urinary cyclic adenosine monophosphate excretion as related to the different forms of hypercalciuria are reviewed.


Testicular lymphography: clinical study. Direct injection of lipiodol into the parenchyma of the human testis to study retroperitoneal lymphatics and lymph nodes is a potentially dangerous investigation. The information obtained with this study is incomplete and far inferior when compared to results obtained with standard procedures, such as pedal lymphography or cannulation of testicular lymphatics.


Infectious mononucleosis in children. Evaluation of Epstein-Barr virus-specific serological data. Epstein-Barr virus (EBV)-specific antibody responses were determined in 43 consecutive pediatric patients who had signs and symptoms of inectious mononucleosis (IM) and positive diagnostic tests for mononucleosis (Monspot). Thirty patients gave clear-cut serologic evidence of primary EBV infections; of the remaining 13 patients, seven had no antibodies to EBV in the acute- or convalescent-phase sera and six showed serologic patterns of past EBV infections. Further testing proved that the initial Monospot results were either false-positive or were incorrectly interpreted in all 13 patients with unidentifiable illnesses but in only two of the patients with current EBV infections. The data confirm the occurrence of classical IM in children and show that the disease and the EBV-specific antibody responses can be virtually indistinguishable from adult cases.


Nasopharyngeal carcinoma and Epstein-Barr virus. III. The detection of anti-nuclear antibodies. The presence of anti-nuclear antibodies (ANA) has been studied in 433 sera from 305 patients with anaplastic nasopharyngeal carcinoma (NPC) in Taiwan, by means of indirect fluorescent antibody method. Another 134 sera from normal adults or from patients with other diseases served as controls. Patients with NPC showed positive rate of 23.1%. Forty-two patients with other malignant and 25 patients with benign tumors of the head and neck showed positive rates of 9.5% and 8.0%, respectively. Forty-eight patients with inflammatory diseases and 19 normal adults were negative. The presence of ANA in 103 NPC patients before treatment was analyzed in detail. The positive rate was not significantly influenced by the age and sex of the patients, by the duration of the disease, by the extent of primary tumor and the presence of neck metastasis. But the positive rate rose as the anti-EB-VCA titer increased. This positive correlation of ANA and anti-EB-VCA antibodies was also significant in all the NPC sera. During and at the end of irradiation therapy, the ANA positive rates showed no remarkable variation. After treatment of the disease, ANA positive rate decreased gradually and this reduction became apparent after three years of remission of the disease. But the presence of ANA in NPC patients was not related to the prognosis of the patients. The possible mechanism in production of ANA in NPC patients was discussed with special reference to EB virus.


In vitro aggregation of platelets induced by alpha-toxin (phospholipase C) of Clostridium perfringens. Highly purified alpha-toxin (phospholipase C) of Clostridium perfringens prepared by affinity chromatography on agarose-linked egg-yolk lipoprotein induced the in vitro aggregation of platelets of an irreversible type. The aggregation started after a time lag, the length of which depended on the concentration of the toxin; the reciprocal of the time lag was found to be directly proportional to the toxin concentration. Using this assay method, we demonstrated that the platelet-aggregating activity of alpha-toxin reached minimum at around 70 C but heating at higher temperatures inactivated it to a lesser extent; the same anomaly in heat inactivation was observed with phospholipase C activity possessed by the toxin. By subjecting purified alpha-toxin to isoelectric focusing, four molecular forms were isolated, all of which were associated with both the platelet-aggregating and phospholipase C activities. From all these results we concluded that the entity responsible for the platelet-aggregating activity is identical with alpha-toxin (phospholipase C).


Comparative investigations of alpha- and beta-effects on the longitudinal and circular muscles of the pregnant rat myometrium. Comparative effects of noradrenaline (10(-8)-10(-6) g/ml) and isoprenaline (10(-10)-10(-6) g/ml) on electrical and contractile activity were investigated in longitudinal and circular muscle strips of rat myometrium (13-19 days pregnant). When recorded with an intracellular microelectrode, spike potentials of longitudinal muscle discharged spontaneously in bursts. Treatment with either noradrenaline or isoprenaline hyperpolarized the membrane and increased membrane conductance, causing the cessation of spontaneous activity. Slow potential was dominant in the circular muscle, and it became prolonged after treatment with noradrenaline, leading to an increase in tension. Isoprenaline reduced the duration of the slow potential and depressed the contraction. The minimal concentration of isoprenaline needed to suppress the spontaneous activity in circular muscle was much higher than in longitudinal muscle. The excitatory effect of noradrenaline on circular muscle was antagonized by treatment with phentolamine, and the inhibitory effect of catecholamines on both longitudinal and circular muscles by propranolol. alpha-Adrenoceptor appears predominant in circular muscle, while beta-adrenoceptor in longitudinal muscle in rat myometrium during late-pregnancy.


Effects of disulfide bond reduction on the excitatory and inhibitory postsynaptic responses of Aplysia ganglion cells. Three kinds of the cholinoceptive neurons, nicotinic depolarizing (D)-, nicotinic hyperpolarizing (H)-, and muscarinic H-tyes, as well as two other kinds of neurons, GABA H- and dopamine H-types, were identified in Aplysia abdominal ganglion, and the effects of disulfide bond reduction and reoxidation on their postsynape acetylcholine-induced responses of both nicotinic types (D- and H-) were depressed by reducing the disulfide bonds with dithiothreitol (DTT) and restored by reoxidizing with 5, 5' -dithiobis-(2-nitrobenzoic acid): (DTNB), whereas the responses of the muscarinic H-, GABA H-, and dopamine H-cells were not affected at all by either DTT or DTNB. In contrast to the results obtained from the electroplax, the cholinergic receptors in our preparation showed neither the activation by hexamethonium nor the augmentation of decamethonium-induced responses after reduction of disulfide bonds. In addition, our preparation did not demonstrate the long-lasting responses to bromoaTT-induced depression of the nicotinic responses was studied on the dose-response curves; the mode of receptor inhibition was rather complexed, being neither type of competitive nor non-competitive. We concluded that the disulfide bond is a crucial element in both types of nicotinic receptors (D and H), and that this bond is related to the activation process of the receptors regardless of their ionic specificities.


Experimental study on renal and hepatic glucose metabolism in total gastrectomized dogs with special reference to glycolysis and glyconeogenesis. In spite of the extensive studies on glucose metabolism in surgical field, the postoperative renal and hepatic glucose metabolism and their interrelation have not been reported. The present study was undertaken to interrelate the metabolic states in the kidney and the liver in total gastrectomized dogs. In the kidney, the glycolysis was inhibited in the early postoperative stage whereas the gluconeogenesis was activated. In the late postoperative stage the glycolysis remained slightly inhibited, while the gluconeogenesis remained activated. In the liver, in the early postoperative stage the glycolysis was inhibited, whereas the gluconeogenesis was activated. The glycolysis in the late postoperative stage returned to the preoperative stage, while the gluconeogenesis remained inhibited. The gluconeogenesis in both kidney and liver was disturbed in the early postoperative stage while they were restored, but were reversed, in the late postoperative stage.


Autonomous peripheral nerve activity causing generalized muscle stiffness and fasciculations: report of a case with physiological, pharmacological, and morphological observations. A 14-year-old boy with generalized muscle weakness, stiffness and fasciculations associated with profuse and continuous electromyographic (EMG) activity is described. The spontaneous mechanical and electrical muscle activity was unaffected by sleep, general anesthesia, or spinal anesthesia but was abolished by small doses of curare, succinyl-choline, and gallamine. Proximal and distal peripheral nerve block caused moderate and marked reduction of EMG activity, respectively, thus indicating that the disorder is due to autonomous peripheral nerve activity. The delayed motor nerve conduction velocities and the structural abnormalities seen in some of the myelin sheaths by light and electron microscopic studies on sural nerve biopsy preparations constitute further evidence that the peripheral nerve is the site of abnormality in this disorder. Diphenyl hydantoin and carbamazepine maintenance therapy produced adequate clinical relief.


Ambient temperature of hypoxia: a differential study of liver catalase and cytochrome oxidase in guinea pig. The activities of liver catalase and cytochrome oxidase have been determined in sea-level and high-altitude native guinea pigs exposed to different ambient temperatures. The activities of both of these enzymatic systems have been found to increase as ambient temperature is reduced, and this occurs in the sea-level and the high-altitude animals. At equal temperatures, cytochrome oxidase activity is identical in the liver of guinea pigs from sea-level and high-altitude. Catalase activity is approximately 50% lower in the high-altitude animals than in the sea-level ones maintained at the same ambient temperature. It is necessary to reassess current data on hypoxia-induced enzymatic and hormonal changes measured under conditions where the ambient temperature was not controlled, especially in those cases involving volunteer human subjects.


[Functional state of the cortical and medullary layers of the adrenals in primary arterial hypotension]. The functional state of the cortical and medullar layers of the adrenal glands was studied in 127 patients with primary arterial hypotension (109 females and 18 males, aged 17 to 48 years). As shown by daily urine excretion, hypotonic patients had a decreased basal activity of the glucocorticoid and androgenic function of the adrenal cortex in comparison with those in normal individuals. The potential of the adrenal cortex was preserved in these patients within sufficient limits. The adrenocorticotrophic function of the anterior lobe of the pituitary gland was found to decrease. Against the background of a reduction of the total excretion of all fractions a relative delay was noted in the process of glucocorticoids transformation into their precursors, as well as a relative predominance of glucocorticoids over mineralcorticoids, the content of desoxycorticosterone being reduced especially low. The content of adrenalin, noradrenaline and dophamine was below the normal level, the decrease being statistically significant. At the same time the excretion of DOPA was normal, on the average, the whole group of patients. The changes in the daily urine excretion of catecholamines and DOPA, as well as in their ratio indicate that the activity of both links of the sympathodrenal system is reduced in primary arterial hypotension.


[State of the neurohumoral regulatory system in circulatory insufficiency]. A moderate elevation of the daily excretion of free noradrenaline and adrenalin is observed in chronic circulatory insufficiency, beginning with Stage IIA. The catecholamines metabolism is elevated, as shown by the daily excretion of normethanpherine and methanpherine and of vanillyl-mandelic acid. The activity of renin and angiotensinases was growing along with the progressing cardiac insufficiency. The blood level of angiotensinogen was decreasing, especially in patients with Stage IIB and III of decompensation. The daily excretion of aldosterone was growing along with the development of cardiac insufficiency. The functional state of the glucocorticoid function of the adrenal cortex was of a phased nature in cases of circulatory insufficiency. The study of the functional state of the epiphysis was conducted by way of determining the blood level of melatonine and of its daily excretion. In Stages I and IIA the level of this hormone was clearly elevated, in Stages IIB and III -- decreased as compared with the initial and normal levels. The plasma level of the antidiuretic hormone was distinctly growing, beginning with Stage IIB, reaching its maximal values in Stage III.


[Effect of antikinin agents on the course of experimental myocarditis]. The therapeutic action of 5 preparations: specific antikinin substances -- anginin and contrical, and nonspecific -- epsilon-aminocapronic acid, aspirin and indometacine, was tested in 71 rabbits with allergic myocarditis. It has been concluded that in the allergic distruction of the myocardium plasma kinins, which are formed and accumulated in large quantities, take part in the pathogenesis of the disease serving as mediators of the tissue distruction, therefore specific antagonists of kinins and inhibitors of the kinin system should be included as pathogenetic agents into the complex of allergic myocarditis treatment.


Acceleration and weakness in parkinsonian dysarthria. This report presents some of the underlying neuromuscular mechanisms of dysarthric speech production as they are manifested in selected individuals with parkinsonism. Earlier explanations of parkinsonian dysarthria emphasized that the balanced hypertonus of rigidity formed the basis of reduced range of movements in speech. The present data reveal two problems in the neuromuscular control signals that can result in a reduced range of speech movements in the absence of rigidity. These neuromuscular problems are described as acceleration and weakness. The implication of these findings for the evaluation and treatment of parkinsonian dysarthria is discussed.


Effect of ethanol on the cyclic AMP system in rat brain. The effects of ethanol on adenylate cyclase and phosphodiesterase activity in vitro, and on cyclic AMP, ATP and adenosine levels in vivo were studied. Ethanol appeared to affect the cyclic AMP system indirectly, probably through its effect on neurotransmitter release or on adenosine formation.


Differentiation and aging of the intestinal mucosa of the rat. I. Morphological, enzyme-histochemical and disc electrophoretic aspects of the prenatal and postnatal differentiation of the intestinal mucosa. The large and small intestines of fetal, newborn, juvenile and adult rats were investigated at various points by means of enzyme histochemistry, disc electrophoretic separations of enzymes and electron microscopy. The results obtained permit division of the morphological and enzymatic differentiation of the rat intestinal epithelium into four periods of development. 1. The period of prenatal development from the 19th to the 21st fetal day with a continuous increase of the enzyme activities in the enterocytes of the entire intestinal mucosa. 2. The period of postnatal development from the first day of life until the end of the second week with enzyme activity remaining roughly the same compared with the 21st day of life. 3. The period of adaptation from the beginning of the third week of life up to the middle of the fourth week of life. During this phase the transition into the stage of adult maturity occurs both morphologically and enzymatically. 4. The stage of adult maturity, which is fully developed in the middle of the fourth week of life.


Effect of aging on cyclic AMP levels and adenylate cyclase and phosphodiesterase activities in the rat corpus striatum. Cyclic AMP levels and activities of adenylate cyclase and phosphodiesterase in the striatum were measured in 4, 12, 24 and 30 month-old rats. No age-related changes were observed either in cyclic AMP levels or basal adenylate cyclase activity. However, there was an age-related decline in dopamine sensitivity of adenylate cyclase without affecting NaF-induced stimulation. There was also an age-dependent decrease in phosphodiesterase activity at low substrate concentrations (3 X 10(-7) and 3 X 10(-6) M) but not at high substrate concentrations (3 X 10(-5) and 3 X 10(-4) M). These results suggest that the functions of dopaminergic neurons may have been altered by aging.


An effect of age on the phosphodiesterase activator protein of rat cerebral cortex. A brief note. Measurements were made of the effectiveness of phosphodiesterase activator protein isolated from the cerebral cortex of rats of various ages in stimulating the phosphodiesterase-catalyzed conversion of cyclic-AMP into 5'-AMP. An age dependent increase in the potency of preparations of activator protein was observed, with the activator isolated from animals six months of age and older being generally more efficacious than that from younger animals.


Comparative effect of glucagon, dibutyryl cyclic AMP, and epinephrine on the desaturation and elongation of linoleic acid by rat liver microsomes. The effect of glucagon, dibutyryl cyclic adenosine 3',5'-monophosphate, and epinephrine on the biosynthesis of polyunsaturated fatty acids of the linoleic acid family was studied. The incubations were performed with rat liver microsomes and labeled linoleic acid under desaturating and elongating conditions. Under desaturating conditions linoleic acid was converted to gamma-linolenic acid, whereas under elongating conditions it was converted to 20:2omega6. Glucagon, dibutyryl cyclic AMP, and epinephrine decreased the oxidative desaturation of linoleic acid to gamma-linolenic acid while the elongating reaction was not modified in the experimental conditions tested. Consequently, the results support the hypothesis that the oxidative desaturation of linoleic acid to gamma-linolenic acid is the main controllable step in the biosynthesis of polyunsaturated fatty acids of the linoleic acid family in the microsomes.


Response to the hypobetalipoproteinemic agent adamantyloxyphenyl piperidine in hyperlipemic rats. The hypobetalipoproteinemic activity of U-41,792 (1-[p-(1-adamantyloxy)-phenyl]-piperidine) is a marked and selective reduction of heparin precipitating lipoproteins (low density plus very low density lipoproteins) in cholesterol-cholic acid induced hypercholesterolemic rats. This activity consists of both a reduction in heparin precipitating lipoproteins (HPL) and an increase in high density lipoproteins that are not precipitated by heparin. The increase in high density lipoproteins is routinely noted by decreases in HPL/cholesterol ratios. The pattern of response following single 100 mg/kg doses of U-418792 was determined. After an I.V. dose was administered in a cottonseed oil emulsion, serum cholesterol levels were reduced, beginning at 8 hr after administration and persisting for 96 hr. Similar results, though delayed somewhat, were obtained after a single oral dose. Activity was accompanied by increases in weight and cholesterol content of livers. After multiple, daily, oral doses, liver weights, total lipids, and cholesterol contents were reduced. Hypobetalipopreteinmeic activity was enhanced by prolonged treatments as demonstrated by analyses of serum obtained weekly throughout 7 wk.


An overview of the biochemical pharmacology of probucol. Probucol was effective in lowering serum total cholesterol in mice at dietary livels as low as 0.0075%. It was also effective after a single 100 mg/kg I.V. dose in mice. The incorporation of acetate-(14)C into liver lipids of rats and mice was not significantly affected by probucol, although the results, especially in mice, make it impossible to rule out such an effect. Cholesterol absorption was estimated in rats using a dual isotope technique. The observed reductions were not statistically significant. Several liver enzyme activities were determined after probucol treatment in rats, and a significant elevation (32%) was observed in only one, glutamic dehydrogenase. Serum cholesterol was lowered markedly in cholesterol-fed cynomolgus monkeys by probucol. There was no effect on the excretion of neutral steroids and the observed increase in fecal bile acids after drug treatment could not be confirmed statistically.


Regulation of fatty acid biosynthesis in Ehrlich cells by ascites tumor plasma lipoproteins. Fatty acid biosynthesis in Ehrlich cells in vitro was reduced when very low density lipoproteins (VLDL) isolated from the ascites tumor plasma were added to the incubation medium. The degree of inhibition was dependent on the VLDL concentration. At the VLDL concentrations usually present in the ascites plasma, there was a 30% decrease in biosynthesis as measured by (3)H(2)O incorporation into fatty acids. Analysis of the labeled fatty acids by gas liquid chromatography indicated that this decrease was due to a reduction in fatty acid de novo biosynthesis and that chain elongation actually was increased when VLDL were present. Although ascites plasma low- and high density lipoproteins also produced a concentration-dependent inhibition of fatty acid biosynthesis, their effects were much smaller than those of the VLDL. Studies employing VLDL and radioactive free fatty acids indicated that the cells took up utlilzed fatty acids derived from these lipoproteins. When VLDL were present, labeled free fatty acid incorporation into cell phospholipids, cholesteryl esters, and CO(2) decreased, whereas its incorporation into the cell free fatty acid pool increased. By contrast, the cells incorporated only very small amounts of fatty acid from either low- or high density lipoproteins. This suggests that the VLDL exert their inhibitory effect on fatty acid synthesis by supplying exogenous fatty acids to the cells.


Serum lipoproteins as inhibitors of haemafflutination by rubella virus. The role of serum lipoproteins as non-antibody-like inhibitors of haemagglutination for rubella virus was investigated. Dissociation of lipoproteins into their respective lipid and protein components significantly reduced their inhibitory titre. This reduction was more prominent with the protein component. When mixtures of pure lipids were tested for their haemagglutination inhibitory activities, no inhibition was observed. Sonication of the three major lipoprotein fractions significantly increased the inhibitory activities of low density lipoproteins (LDL) and very low density lipoproteins (VLDL) but not high density lipoproteins (HDL). Succinylation, acetylation, and methylation of the lipoproteins did not appear to affect their inhibitory titre. On the other hand, phospholipase A treatment significantly increased the inhibitory properties of all lipoproteins fractions. These results are discussed in terms of the possible mechanisms by which the lipoproteins interact with the rubella haemagglutinins.


Turnover of apolipoprotein-B in two subjects with familial hypobetalipoproteinemia. The metabolism of the apoprotein of low-density apoliporprotein-B was studied in a father and son with familial hypobetalipoproteinemia. The synthetic rate of apolipoprotein-B was below the normal range in both subjects, while the fractional removal rate was normal. It is concluded that the subnormal concentration of low-density lipoprotein (LDL) inthese two subjects is due to a low synthetic rate. The synthetic rate of apolipoprotein-B of very low-density lipoprotein (BLDL-apoB) measured in the father was subnormal. The fraction of the VLDL-apoB pool which was oncverted into LDL-apoB was within the normal range. It is suggested that a low synthetic rate of VLDL may underlie the disorder.


Inheritance of type-III hyperlipoproteinemia. Lipoprotein patterns in first-degree relatives. The lipoprotein (LP) patterns were studied in the families of 19 index cases with type-III hyperlipoproteinemia (HLP). Seventy adult first-degree relatives (93% ascertainment) to the 19 probands were analyzed. The diagnosis of HLP type III among the first-degree relatives was based on three criteria, all of which had to be fulfilled to make the diagnosis: (1) presence of a slow-moving band in very-low-density LP (VLDL) on agarose gel electrophoresis migrating in beta or close to beta position; (2) A cholesterol/triglyceride ratio (mg/100 ml: mmoles/liter) in VLDL greater than 29.0; and (3) A "III-index" [cholesterol/triglycerides in VLDL x 10 divided by cholesterol/triglycerides in low-density LP (LDL)] greater than 1.30. When defined according to these criteria there was a marked over-representation of HLP type III among the relatives (27%). There was also an increased frequency of hypertriglyceridemia (28% against expected 15%), mainly because of a high prevalence of HLP type IV (24%). On agarose gel electrophoresis a "late pre-beta" band, probably indicative of an increased amount of intermediary LP particles, was frequently present (47%) among relatives not classified as HLP type III. Type-III patients with hypertriglyceridemia were characterized by a significantly higher body weight than those with normotriglyceridemic type III. However, there was no qualitative difference in the composition of the lipoproteins in normotriglyceridemic and hypertriglyceridemic type-III patients. A genetic analysis of the LP patterns within the families showed several examples of vertical transmission of HLP type III. There was no sex linkage. Six of thirteen analyzed parents showed LP patterns classified as HLP type III. Another two parents were most probably carriers of the gene. Of the siblings to the probands, 23% showed a type-III pattern and another four (7%) showed LP patterns very similar to type III, fulfilling two of three criteria for HLP type III. The data support the concept that HLP type III is inherited as an autosomal dominant gene. It was indicated that HLP type IV with a late pre-beta band in VLDL may represent another expression of the gene for HLP type III. It is suggested that HLP type III may be a pathogenetically heterogenous group of lipid disorders. A separation of type III into two subgroups with low or normal and high LDL cholesterol concentration, respectively, may facilitate the understanding of the inheritance of type III as well as of the pathogenesis behind this LP abnormality.


Effect of saline on the releasability of alkaline phosphatase from cells of Serratia marcescens. The effect of 0.9% sodium chloride solution on the release of alkaline phosphatases from cells of four strains of Serratia marcescens was studied. Saline had a greater action in the releasability of the enzyme on cells of the polymyxin B sensitive strains than those of the polymyxin B resistant strains. SDS-polyacrylamide gel electrophoresis of the released materials showed the presence of proteins and lipopolysaccharide components of the outer membrane as well as enzyme activity in all four strains. Cells from strains harvested under higher temperatures contained more releasable activity in the salin wash fraction than those harvested under refrigerated condition. Active components with molecular weights of 190,000 and 110,000 daltons were either absent or present to a lesser degree in the extracts released by the polymyxin B treatment of the washed cells. However, active components not released by saline were found in the polymyxin B extracts. Contrary to other reports, results of this study clearly showed the ubiquitous nature of alkaline phosphatase in S. marcescens. It appears that their releasability is related to the polymyxin B susceptibility as well as the instability of the outer membrane of the cell envelope.


Comparative release of alkaline phosphatases from Serratia marcescens by osmotic shock and polymyxin B treatment. The comparative release of periplasmic enzymes and proteins from two strains of Serratia marcescens by osmotic shock and polymyxin B treatment was studied. There were significant qualitative and quantitative differences in the materials released by these two techniques. The osmotic shock procedure released a higher level of alkaline phosphatase activity and a greater number of protein components than the polymyxin B treatment. The molecular weights of the active components released by the two techniques were shown to be 190,000 +/- 10,000 (A'), 140,000 +/- 10,000 (A) and 110,000 +/- 10,000 (B) daltons. Components released by polymyxin B were also released by osmotic shock. However, the reverse was not true. Component B in the osmotic shock fluids was by far the most active. The differences in the release mechanisms of the two techniques were discussed. It is suggested that polymyxin B treatment is the method of choice because of its selectiveness and mildness, despite the rather low level of activity of alkaline phosphatase released.


Radiolabelling of Bordetella pertussis envelope proteins by the 125 I-lactoperoxidase method. Bordetella pertussis strain number 18334 was grown in media which yielded cells with either a normal complement of surface antigens (X-model), or cells which were phenotypically altered (C-model). Neither X- nor C-mode bacteria incorporated more than traces of radioactivity when exposed to Na 125 I, lactoperoxidase and a source of H2O2 under conditions which gave substantial labelling of BSA and other soluble proteins. In contrast, envelope preparations were readily labelled. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that several polypeptides had incorporated 125 I but not in amounts proportional to their abundance in the envelopes. Envelopes from C-mode cells gave a labelling pattern similar to those of X-mode except in one region. Control experiments suggested that the failure of intact cells to become labelled may be due to bacterial inhibition of the reagents.


[Comparative studies of mitochondria of Torulopsis candida growth on glucose and hexadecane]. The oxidative activity of mitochondria of Torulopsis candida was compared during its growth on glucose and hexadecane. No principal differences were found in the tricarboxylic acid cycle and the electron transport chain in the mitochondria of "glucose" and "hexadecane" cells (and in the rate of functioning too). The activity of enzyme systems involved in oxidation of higher alcohols, aldehydes and higher fatty acids was higher in the mitochondria of the "hexadecane" cells as compared to the "glucose" cells; oxidation of aldehydes, contrary to oxidation of aldehydes, contrary to oxidation of alcohols, was found to be susceptible to inhibitors of the respiration chain and phosphorylation. The rate of oxidation of exogenous NADH is much lower and the oxidative activity towards NADPH is practically absent in the mitochondria of the "hexadecane" cells as compared to the mitochondria of the "glucose" cells.


[Chances in the serum lipid values after conversion to a "reasonable" diet (author's transl)]. Six to ten days after conversion from usual alimentary habits to a so-called "reasonable" regimen, the serum concentrations of different lipid fractions were examined under standardized conditions, in a total of 325 in-patients, who had not been pre-treated, and 268 of whom showed - after diverse examinations - a hyperlipoproteinaemia; the other 57 patients had normal lipid values. This diet food is vitamin-rich, poor in calories, protein-rich with a 20 per cent to 25 per cent part of calories, poor in carbohydrates (maximum 120 g/day), and extensively renouncing monomeric and dimeric carbohydrates; furthermore, it contains a percentage of 40 to 45 calories of lipids with a standardized part of multiple-unsaturated fatty acids. During the period of observation, there was in none of the patients an alteration of the body-weight exceeding 1 kg. During the period of the regimen, the following highly significant decreases - without any medicamentous measures - were stated with regard to the serum concentrations: Total cholesterol (average) 9.9 per cent, triglycerides (average) 23.1 per cent, beta-cholesterol (average) 17.6 per cent, and phosphatides (average) 14.2 per cent - all percentages being in relation to the reference values. A hyperproteinaemia (type IIa/Fredrickson) disappeared in 14 out of 18 cases, a hyperlipoproteinaemia (type IIb) in 14 out of 44 cases, and the same happened with this disease type IV in 114 out of 206 cases. A change of type was stated 16 times.


[Various physiological aspects of Serratia marcescens pigmented strains and their pigmentless variants with an elevated nuclease activity]. Some aspects of physiology of Serratia marcescens pigmentless variants with elevated nuclease activity were studied. The variants are characterized by a higher respiration rate when glucose, glycerol, inositol or maltose are used as an energy substrate, a higher respiration quotient, a lower growth rate, a lower economic coefficient, and a lower thermogenesis. The growth of Serratia marcescens pigmentless strains is presumed to be unbalanced.


[Errors in practising a so-called "reasonable" regimen (author's transl)]. The significant decrease of the serum concentrations of total cholesterol, triglycerides, phosphatides, beta-cholesterol, and beta-lipoproteins, which usually is observed after conversion from normal eating habits to a "reasonable" diet, is prevented by supplementary and unverifiable consumption of confectionery and/or by alcohol abuse in irregular intervals. The so-called "reasonable" regimen is a high-fat and protein-rich diet food containing more than 50 per cent of multiple-unsaturated fatty acid in the fat supply. The fact that the average serum concentrations of the divers lipid fractions within the entire group of the 30 patients examined (patients who - admittedly - were practising a luxury form of consumption) showed merely insignificant fluctuations, is in no way a justification for the conclusion that a "reasonable" diet will at any time ostensively represent a protection against the effect of luxury consumption on the serum lipids.


[Effect of growth conditions on substrate phosphorylation during sulfite oxidation in Thiocapsa roseopersicina]. Cell extracts of Thiocapsa roseopersicina, strains BBS and SL, grown in the light under anaerobic conditions and in the dark under aerobic conditions on a medium containing thiosulphate produce ATP during oxidation of sulphite in the presence of AMP cytochrome c and inorganic phosphate. The uncoupling agent (m-chlorocarbonylcyanidephenylhydrazone) has no effect on the rate of ATP production, this being an evidence of substrate phosphorylation. The rate of ATP synthesis is higher in extracts of the cells grown in the dark under autotrophic conditions than in extracts of the cells cultivated in the light under anaerobic conditions or in the dark in the presence of thiosulphate and glucose.


[Flavinogenesis in methylotrophic yeasts]. Methylotrophic yeasts Candida boidinii and Hansenula polymorpha requiring thiamine and biotin accumulate more flavins in the cells during growth on media containing methanol than during oxidation of ethanol and glucose, at the account of increased production of FAD whose percentage in the total flavin content of the cell rises sharply. The level of flavin production depends on growth conditions: the intracellular content of flavins and FAD during utilization of methanol by the yeast cells is higher under conditions of continuous cultivation than in periodic cultures. The content of NAD, another coenzyme of the respiration chain, in the cells during their growth on media containing methanol almost does not differ from its concentration in the cells cultivated on media containing glucose and ethanol. Elevated production of flavins and FAD by the yeast cells on media containing methanol may be caused by an increased requirement in FAD and its specific participation in the first stage of methanol oxidation.


[Differentiation of Blakeslea trispora mycelium in relation to carotene production]. Carotene synthesis by Blakeslea trispora is accompanied by a decrease in the rate of the main metabolic processes (protein synthesis, respiration) and changes in the bioenergetic pool of the mycelium. Considerable changes occur at the same time in the cytology of the microscopic fungus: the cell wall of the culture containing considerable amounts of carotene consists of many layers and peculiar thickened regions; partitions are formed; the nuclei are in the stage of interphase; lipid inclusions are larger and their number is higher.


Genes involved in the regulation of the neutral phosphatase in Chlamydomonas reinhardi. In Chlamydomonas reinhardi, mutations in either of two unlinked genes (PD2 and PD3) abolish the activity of the derepressible neutral phosphatase. The question arose whether these genes (or one of them) specify the structure of the enzyme or whether they have a regulatory function. Three mutants producing an active phosphatase at 25 degrees C but not at 35 degrees C were isolated and investigated. One of these mutants (PDts11) was allelic with PD2, another one (PDts12) was linked to PD3 and the third one (PDts13) was linked to PD2. PDts11 and PDts13 affected the formation of the neutral phosphatase only whereas PDts12 interfered with the formation of both neutral and alkaline phosphatases at 35 degrees C. The neutral phosphatase produced by the three mutants at low temperature was not more thermosensitive in vitro than the wild enzyme. Moreover, quite similar Km values were found in WT, PDts11 and PDts12 using naphthyl phosphate as a substrate. On the other hand, revertants of PD-2 and PD-3 were isolated: their neutral phosphatases could not be distinguished from the wild enzyme on the basis of their thermosensitivities and Km values for naphthyl phosphate. These results are consistent with the idea that PD2 and PD3 are regulatory genes. Other possible regulatory genes were revealed through PDts12 and PDts13 mutations.


Assembly of the mitochondrial membrane system. XIX. Genetic characterization of mit- mutants with deficiencies in cytochrome oxidase and coenzyme qh2-cytochrome c reductase. Nineteen mutants of S. cerevisiae exhibiting a double deficiency in cytochrome oxidase and coenzyme QH2-cytochrome c reductase (also cytochrome b deficient) have been studied. The mutants have been crossed to a set of rho- tester strains with different segments of mitochondrial DNA. The mutants have also been crossed to mit- testers with defined genetic lesions. In addition, crosses were performed with a respiratory competent strain to ascertain whether mitotic and meiotic segregants could be isolated with only one of the two enzymatic deficiencies. The rho- testers allowed the doubly deficient mutants to be separated into two classes. Mutants in class 1 were not restored by any of the rho- testers and appeared to have separate mutations, one in cytochrome oxidase and the other in cytochrome b. Mutants in class 2 were restored by a set of rho- clones whose retained segments of mitochondrial DNA contained the cytochrome b but not the cytochrome oxidase loci. These appeared to behave as single hit mutations. Further studies, however, indicated that both class 1 and class 2 mutants carried separate mutations in two different loci. Mitotic and meiotic segregants with a single enzymatic deficiency could be isolated. In a number of strains, the mutations were mapped in known cytochrome oxidase and cytochrome b loci. The apparent discrepancy of the rho- tests for the class 2 mutants was shown to be probably due to a high unstability in one of the mutations. It has been concluded that all the doubly deficient strains carry two mutations in previously described cytochrome oxidase and cytochrome b loci. This conclusion argues against the existence of a single gene on mitochondrial DNA that controls the biosynthesis of the two respiratory enzymes.


Mutagen sensitivity of Drosophila melanogaster. III. X-linked loci governing sensitivity to methyl methanesulfonate. Twenty-eight X-linked, recessive mutations of Drosophila melanogaster conferring enhanced sensitivity to the monofunctional alkylating agent, methyl methanesulfonate, have been recoered and assigned to five complementation groups. These groups can be distinguished on the basis of map location and variations in the pattern of mutagen sensitivity. Allelism of members of one complementation group with the previously described meiotic mutant, mei-41, (Baker and Carpenter, 1972) as well as the frequent appearance of female infertility with mutagen sensitivity suggests associated defects in meiotic chromosome behavior or early embryogenesis. Examination of the mutagen sensitivity of double mutants has led to the formulation of a working model of DNA repair for this organism. Studies  of a similar nature (Boyd et al., 1976) have identified five additional X chromosome complementation groups, suggesting that the genome of Drosophila melanogaster may contain many loci involved with mutagen sensitivity. The continued isolation and characterization of conditional mutants of this type promises future insights into the mechanisms of DNA replication, DNA repair and recombination in this complex higher eucaryote.


Glutamine utilization in both the arginine-specific and pyrimidine-specific carbamoyl phosphate synthase enzymes of eurospora crassa. As glutamine-dependent carbamoyl phasphate synthetase (CPS) activity in some organisms is composed of a glutaminase and an ammonium-dependent CPS, CPS- mutants in Neurospora crassa were examined for glutamine- and ammonium-dependent CPS activities. No evidence was found that the genetic location of these two functions were separable. This is discussed with reference to the close genetic proximity of the CPSpyr and aspartate carbamoyltransferase (ACT) structural gene (pyr-3) and the arg-2 gene which appears to specify a subunit responsible for glutamine utilisation in CPSarg.


Synthesis of cytochrome c oxidase polypeptides in an Escherichia coli cell-free system directed by Saccharomyces cerevisiae mitochondrial DNA. Using purified yeast mitochondrial DNA as a template for E. coli RNA polymerase (holoenzyme) complementary mitochondrial RNA has been synthesized in vitro. This RNA has been used to direct a low background E. coli S-30 protein-synthesizing system. The synthesis of mitochondrial polypeptides has been detected by using antiserum raised against purified cytochrome c oxidase holoenzyme and shown to be specific for this antigen. The antiserum-antigen complex was dissociated and subject to SDS-polyacrylamide gel electrophoresis and the presence of 3 polypeptides of 39, 31, and 26 X 10(3) daltons molecular weight demonstrated, which correspond to the subunits synthesized by mitochondria in whole cells which are inhibited with cycloheximide.


The existence of triphosphorylated 5'-ends in virus-specific RNA isolated from SV-40 transformed cells. The question about the nature of promoters in the transcriptional units containing SV-40 sequences in transformed cells was analyzed. It was found that the pulse-labeled RNA hybridizing to SV-40 DNA contains small but significant amounts of triphosphorylated 5'-ends detected as pppGp in alkaline hydrolyzates of this RNA. In another series of experiments the fragments of RNA containing triphosphorylated 5'-ends about 100 nucleotides in length have been isolated by hydroxyapatite chromatography. Some of them form hybrids with SV-40 DNA. The conclusion is drawn that at least some of SV-40 promoters are used for transcription initiation in SV-40 transformed cells.


[Anesthesia for short or outpatient operations (author's transl)]. It was intended to find out for which operations and operative investigations ambulant anesthesia offered advantages, what conditions must be fulfilled and which methods of anesthesia are suitable. The question arose not only because of increasing hospital costs, but also because of personal advantages of the out-patient operation for the patients and their families and for other reasons. The importance of consultation with the anesthetist in which the ambulant patient is prepared is emphasized and from there communications to the participants and questions of postoperative safety are examined.


Dose response at low doses of X-irradiation and MMS on the induction of micronuclei in mouse erythroblasts. The test of induced micronuclei in erythrocytes of mammalian bone marrow constitutes, because of its high experimental resolution power, a suitable method for the screening of induced chromosomal lesions at very low dosages of chemicals or irradiations. This test was used for a comparative investigation of the effect of low dose levels of X-irradiation and of the alkylating agent methyl methanesulphonate (MMS). The dose-effect curve of X-irradiation indicated a deviation from linearity at 10 rad, showing a significantly stronger effect than expected on extrapolation from the control to 100 rad. This deviation from linarity, however, only appeared at a low dose rate (18 R/min), whereas a linear dose-effect relation was indicated with a high dose rate (95 R/min). Experiments at 10 rad with different dose rates at two different current potentials suggested that this effect of the dose rate is more pronounced with soft than with hard X-irradiation. The induction of micronuclei with MMS follows a drastically different dose-effect curve as compared with X-irradiation. The relative efficiency of the treatment is lowest at low concentrations, presumably as a result of the efficient repair process at such dose levels. Simultaneous treatment with X-rays and MMS at low dose levels only resulted in an additive effect. This suggests that X-irradiation does not interfere with the repair process operating with MMS. The difference in the dose-effect relations of X-irradiation as compared with MMS may be brought back to the fact that X-rays, in contrast with MMS, produce double-strand breaks.


Heritable translocation test and dominant-lethal assay in mice with methyl methanesulfonate. A dominant-lethal test and a heritable translocation test were performed with methyl methanesulphonate (MMS) at 40 mg/kg by treating the sensitive periods of post-meiotic spermatogenesis i.e. spermatozoa and spermatids. In the dominant-lethal test 25 to 60% dominant-lethal mutations were obtained depending on the mating intervals. In the heritable translocation test 11% sterile and partially sterile F1 males were observed in 250 offspring of the MMS group. All of the 14 partially sterile and 6 of the 14 sterile F1 males were demonstrated to be translocation carriers. Fertility of the partial steriles was about 40% of normal fertility. The translocation frequencies in the primary spermatocytes of the partially sterile F1 males varied between 2 and 99%. Transmission of partial sterility and translocations was confirmed in the F2 generation. There were no partially sterile or sterile males among the 245 controls.


Familial hyperglucagonemia--an autosomal dominant disorder. Basal immunoreactive glucagon was elevated in four of nine asymptomatic relatives of a patient with glucagonoma. Immunoreactive glucagon remained elevated throughout 22 to 25 hours of continuous observation. Glucagon responses to intravenous glucose and arginine or mixed meals (or both) were abnormal, whereas glucose and insulin responses were normal. Gel filtration of plasma revealed that over 85 per cent of the four relatives' immunoreactive glucagon had a molecular weight of greater than 9000 daltons whereas that of 70 per cent of the patients with glucagonoma had a molecular weight of 3500 daltons, with the remainder eluting in the area of 9000 daltons. Pancreatic angiograms and hepatic scintiscans were normal in all four relatives. The data suggest an autosomal dominant transmission of hyperglucagonemia in this family. Immunoreactive glucagon with a molecular weight of 3500 or 9000 daltons appears to be required for the development of the clinical glucagonoma syndrome.


Effects of growth temperature and caffeine on genetic responses of Candida albicans to ethyl methanesulfonate, nitrous acid and ultraviolet radiation. Ultraviolet radiation is more effective than either ethyl methanesulfonate or nitrous acid in inducing reverse mutation from auxotrophy to prototrophy in C. albicans. The killing effect of each of the mutagens is greater for cells grown at 37 C than at 25 C after treatment; mutation frequencies are unaffected by post-treatment growth temperatures. Though caffeine depresses survival of mutagen treated cells at both 25 C or 37 C, its effect is more pronounced at 37 C. Caffeine has no effect on mutagenesis by nitrous acid or ethyl methanesulfonate; it depresses UV mutagenesis, but only at 37 C and at high UV dosages. These findings indicate that UV mutagenesis in C. albicans is mediated by a caffeine-sensitive, recombinational system for DNA repair analogous to those known to occur in other species of yeasts. The repair system of C. albicans is unique in being susceptible to caffeine only at high temperature and when the number of DNA lesions to be repaired is large. The caffeine-sensitive steps in repair critical to UV mutagenesis are not involved in fixing mutations induced by the chemical mutagens tested.


Inoculation experiment of Marek's disease vaccine contaminated with a reticuloendotheliosis virus. Two-day-old chicks were inoculated with one or ten doses of Marek's disease (MD) vaccine originated from the herpesvirus of turkeys (HVT) and contaminated with a reticuloendotheliosis virus (REV). As a result, they presented such symptoms as abnormality in the vane of remiges, undergrowth, anemia, and leg paralysis. These symptoms were the same as those induced by the same vaccine among chicks in the field. Control chicks which had been placed in the same house as those inoculated with the vaccine exhibited no abnormal signs. A persistent infection with REV was noticed in the vaccine-inoculated group. A horizontal infection with REV was the highest in the control group, which was followed by the group inoculated with one dose and that inoculated with ten doses in the order listed. The antibody response of chicks to HVT and MD virus was also inhibited by REV.


Isolation of a reticuloendotheliosis virus from chickens inoculated with Marek's disease vaccine. Over a period from spring to fall in 1974, a disease with delayed growth, anemia, abnormal feathers, and leg paralysis as main symptoms broke out in flocks of chickens inoculated with Marek's disease vaccine. A virus was isolated from affected birds in the field and the same lot of Marek's disease vaccine as inoculated into these birds. It had a common antigenicity to the T strain of reticuloendotheliosis virus (REV) and could not be discriminated from this strain on the basis of morphology or property. When chicks were inoculated with it, they presented essentially the same symptoms as the birds affected in the field. Since the disease was reproduced in this manner, it was presumed to have been caused by REV contained in the vaccine as contaminant. The virus persisted in the body for long time and also induced horizontal infection.


Typing foot-and-mouth disease virus by fluorescent antibody technique. Typing of foot-and-mouth disease (FMD) virus was performed by the direct fluorescent antibody (FA) technique. Type-specific FA was prepared from the following two sorts of procedures: (1) FA against live virus (FA-live) was prepared from hyperimmune serum taken from guinea pigs having received live FMD virus. Then it was adsorbed with concentrated heterotype antigen. (2) FA against inactivated virus (FA-Inact) was prepared from antiserum taken from guinea pigs immunized with purified FMD virus inactivated with acetylethyleneimine. Seventeen strains of FMD virus (seven strains of type A, seven strains of type O, and three strains of thpe C) were used. Type-specific FMD virus antigen was detected distinctly from the monolayer of BHK cells infected with each type of virus and fixed in acetone, in spite of negative results obtained from the cells fixed in methyl alcohol. All the 17 strains were typed successfully by the implementation of these two FA methods.


Fine structure of epithelial cells of Lieberkühn's crypts in feline panleukopenia. Electron microscopic observation was carried out on epithelial cells of Lieberkühn's crypts of cats naturally affected with feline panleukopenia. The most important change was the replication of feline panleukopenia. The most important change was the replication of feline panleukopenia virus in the nucleus with associated alterations in the lining epithelial cells of the crypts. In these cells in the early stage of infection, virus particles 20 nm in average diameter were found either singly or in small regularly arrayed clusters everywhere in the markedly swollen nucleus. In the course of infection, the nucleus of infected cells became rather atrophic with a marked margination of chromatin granules. Its major portion was occupied with masses of fine fibrillar substance. It was a "viral matrix area" in which appeared a large compact aggregate of virus particles showing a crystalline array. At the same time, the outer membrane of the nuclear envelope partially extended and disrupted. Membranous elements related to it in the cytoplasm were regularly distributed almost always with particles indistinguishable from the virus particles in the nucleus. From these results it was suggested that the major portion of the infected nucleus, or the site of viral replication, might correspond to the amphophilic intranuclear inclusion body revealed by light microscopy.


Adrenal cortex adenylate cyclase. In vitro acitivity of ACTH fragments and analogues. The ability of ACTH fragments and of an ACTH analogue [9-tryptophan(o-nitrophenylsulfenyl)] corticotropin-(1-24)-tetracosapeptide[Trp-(Nps)9 ACTH1-24] to stimulate adenylate cyclase in bovine adrenal cortex membranes and a crude membrane fraction from rat adrenals has been determined. Partial agonists like Trp (Nps)9 ACTH1-24 displayed intrinsic activity in the rat adrenal preparation only if tested in the presence of 5'-guanylylimidodiphosphate [Gpp(NH)p]. On the other hand, no addition of Gpp(NH)p was necessary to demonstrate intrinsic activity of Trp(Nps)9 ACTH1-24 for bovine adrenal cortex adenylate cyclase. A large decrease (15-fold) of the apparent Km values for ACTH1-24, ACTH1-23 and ACTH1-17 was observed with the rat adrenal preparation when Gpp(NH)p was added. The shift in apparent Km values for ACTH1-24 and ACTH1-23 for the bovine adrenal cortex adenylate cyclase system was small or insignificant when Gpp(NH)p was added. The observations suggest that the hormone receptor facilitates the action of guanylnucleotide sites in the membrane. When guanylnucleotide sites are occupied by Gpp(NH)p even weak interactions of the hormone receptor with e.g. partial agonists are propagated to the catalytic subunits of the adenylate cyclase complex resulting in enhanced activity. The differences in adenylate cyclase activation with hormone fragments or analogues and different target tissues may rather reflect the state of the coupling process involving guanylnucleotide binding sites of the isolated membrane fraction than differences in the receptor itself.