Successful infection of the common marmoset (Callithrix jacchus) with human varicella-zoster virus.

The common marmoset, Callithrix jacchus, can be infected with human varicella-zoster virus (VZV), both wild-type strain KMcC and attenuated vaccine strain Oka/Merck. Infection was accomplished with either whole-cell-associated or cell extract VZV by combined oral-nasal-conjunctival application and was characterized by substantial and persistent anti-VZV antibody responses. The infectivity of VZV for marmosets was destroyed by treatment of inocula with heat or UV light. Diluted inocula with as few as 40 PFU/ml were infectious for marmosets. The lungs were demonstrated to be a major site of viral replication; both the presence of viral antigens and signs of pneumonia were demonstrated in lung tissues. Four serial passages of VZV KMcC were carried out in C. jacchus by a process of in vitro isolation and culturing of VZV from infected lung tissue and reapplication of the cultured isolates to fresh animals. The isolated viruses were identified as VZV both serologically and by restriction endonuclease analyses. The C. jacchus infectivity model should prove useful for determining the efficacy of subunit and live recombinant VZV vaccines as well as for the study of zoster.     

Activities of isolated mitochondria and mitochondrial enzymes from aerobically and anaerobically germinated barnyard grass (echinochloa) seedlings

Activity of mitochondria isolated from whole seedlings of Echinochloa crus-galli (L.) Beauv. var oryzicola germinated under aerobic and anaerobic conditions for 5 to 7 days was investigated. Mitochondria from both treatments exhibited good respiratory control and ADP/O ratios. Although O₂ uptake was low in anaerobic mitochondria, activity rapidly increased when the seedlings were transferred to air. Mitochondria from both aerobically and anaerobically grown seedlings of E. crus-galli var oryzicola maintained up to 66% of their initial respiration rate in the presence of both cyanide and salicylhydroxamic acid, and the inhibitory effects of cyanide and azide were additive. In addition, antimycin A was not an effective inhibitor of respiration. Reduced-minus-oxidized absorption spectra revealed that cytochromes a, a₃, and b were reduced to a greater extent and cytochrome c was reduced to a lesser extent in anaerobically germinated seedlings relative to that in aerobically germinated seedlings. An absorption maximum in the cytochrome d region of the spectrum was reduced to the same extent under both germination conditions and an absorption maximum at 577 nm was present only in anaerobically germinated seedlings. Anaerobically germinated seedlings contained 70% of the cytochrome c oxidase activity found in air grown seedlings. Upon exposure to air, the developmental pattern of this enzyme in anaerobically germinated seedlings was similar to air controls. Succinate dehydrogenase activity in anaerobic seedlings was only 45% of the activity found in aerobically germinated seeds, but within 1 hour of exposure to air, the activity had increased to control levels. The results suggest that mitochondria isolated from E. crus-galli var oryzicola differ from other plants studied and that the potential for mitochondrial function during anaerobiosis exists.     

The in vitro lifespan of MRC-5 cells is shortened by 5-azacytidine-induced demethylation

The minor base 5-methylcytosine (5mC) in DNA may be important for the regulation of gene expression. Random loss of 5mC may occur during pre-replicative DNA synthesis in mortal cell strains, and thus give rise to biochemical aberrations in aging cells. 5-Azacytidine (5azaC) was used to induce loss of 5mC in DNA of human diploid fibroblasts (MRC-5) in an attempt to accelerate in vitro senescence. The 5mC content of DNA was measured by incorporation of [3H]uridine into dividing cells, hydrolysis of DNA and separation of bases by HPLC. In untreated MRC-5 cells, 5mC was 3.6% of the total cytosine (C+5mC) at population doubling (PD) 20 (28% of lifespan) and fell to 1.6% at PD 67 (97% of lifespan). A single pulse treatment with 5azaC (1 microgram/ml) induced demethylation and shortened the lifespan by 10% (6.8 PDs loss). Pulse-treated cells showed temporary growth inhibition, though they subsequently regained normal growth rate and morphology. However, uniform treatment with 0.1 microgram/ml 5azaC between PD 20 and 23 produced no immediate growth inhibition, but a 22% loss of 5mC and 25% decrement in lifespan (16.6 PDs loss). The present results indicate that 5mC levels fall during normal aging of MRC-5 cells and accelerated 5mC loss shortens the in vitro lifespan of these cells. Hypomethylation may thus be responsible for some aspects of in vitro aging.     

Separation of multiple components of HeLa cell nuclear extracts required for pre-messenger RNA splicing

Components essential for nuclear pre-messenger RNA splicing have been partially purified from HeLa cell nuclear extracts by chromatography on DEAE-Sepharose, heparin-Sepharose, Mono Q, and Mono S. We have obtained six fractions which, when combined, efficiently splice a synthetic adenovirus 2 major late RNA substrate in vitro. All fractions contain components that support the formation of splicing intermediates (the cleaved 5' exon and the intron-exon 2 lariat). At least one of the fractions also contains an activity that is essential for the second step in the splicing reaction, namely cleavage at the 3' splice site and exon ligation. Two of the fractions are enriched in the major small nuclear ribonucleoprotein particles U1, U2, U4/U6, and U5. They participate in the formation of the splicing complexes which precedes the cleavage and ligation reactions. The remaining four fractions appear to contain protein factors, as suggested by their resistance to micrococcal nuclease.     

In vivo regulation of hepatic LDL receptor mRNA in the baboon. Differential effects of saturated and unsaturated fat

The effects of diets enriched with cholesterol and different fats upon plasma lipoproteins and hepatic low density lipoprotein (LDL) receptor mRNA levels were studied in a group of 18 normal baboons. Animals were fed diets containing 1% cholesterol and 25% fat as either coconut oil, peanut oil, or olive oil for a period of 20 weeks. Plasma total cholesterol, high density lipoprotein (HDL) cholesterol, beta-lipoprotein (LDL + very low density lipoprotein) cholesterol, apolipoprotein B and apolipoprotein A-I were measured in samples obtained at 4-week intervals. All three diet groups demonstrated a statistically significant increase in plasma cholesterol as compared to base line throughout the experiment. Hepatic LDL receptor (LDL-R) mRNA levels were quantified by dot blot hybridization in serial liver biopsies. Animals fed saturated fat sustained a significant reduction in hepatic LDL-R mRNA as compared to those fed either monounsaturated or polyunsaturated fat. A strong negative correlation between LDL-R mRNA and plasma total cholesterol (r = -0.71), HDL cholesterol (r = -0.76), and plasma apo A-I (r = -0.77) was observed only in those animals fed coconut oil. Weak negative correlations between LDL-R mRNA and other plasma parameters did not achieve statistical significance. We conclude that saturated and unsaturated oils may influence plasma cholesterol levels in part through differential effects on LDL receptor biosynthesis in baboons.     

Actinomycin synthetases. Multifunctional enzymes responsible for the synthesis of the peptide chains of actinomycin

Two enzymes were purified from actinomycin-synthesizing Streptomyces chrysomallus which could be identified as peptide synthetases involved in the biosynthesis of actinomycin. Actinomycin synthetase II activates the first two amino acids of the peptide chains of the peptide lactone antibiotic, threonine and valine (or isoleucine), as thioesters via their corresponding adenylates. It is a single polypeptide chain of Mr 225,000. Similarly, actinomycin synthetase III activates proline, glycine, and valine (the remaining three amino acids in the antibiotic) as thioesters and is a single polypeptide chain of about Mr 280,000. It also carries the methyltransferase function(s) for N-methylation of thioesterified glycine and valine. In addition, it catalyzes the formation of cyclo(sarcosyl-N-methyl-L-valine) from glycine, L-valine, and S-adenosyl-L-methionine at the expense of ATP. Although the cell-free synthesis of the peptide lactone was not as yet accomplished, the data provide evidence that together with the 4-methyl-3-hydroxyanthranilic acid-activating enzyme (now designated as actinomycin synthetase I) all amino acid-activating protein components of the actinomycin-synthesizing enzyme complex are identified.     

Comparative 16S rRNA oligonucleotide analyses and murein types of round-spore-forming bacilli and non-spore-forming relatives

The phylogenetic incoherency of the genus Bacillus as presently described is demonstrated by analysis of both published and new data from comparative 16S rRNA cataloguing of nine Bacillus species and a number of related non-Bacillus taxa, i.e. Caryophanon latum, Filibacter limicola and Planococcus citreus. While the ellipsoidal-spore-forming bacilli, e.g. B. subtilis and allied species, formed a coherent cluster, the round-spore-forming bacilli showed a higher degree of relationship to the non-spore-forming organisms than these bacilli show among each other. Thus B. sphaericus clustered with C. latum, B. globisporus grouped with F. limicola, B. pasteurii with Sporosarcina ureae, and 'B. aminovorans' with P. citreus, respectively. These organisms formed two related subclusters which, in their phylogenetic depth, are comparable to that of the B. subtilis subline. With the exception of 'B. aminovorans', the 16S rRNA phylogeny was entirely consistent with the distribution of murein types. Even more distantly related to and grouping outside the main Bacillus cluster was B. stearothermophilus, which displayed a moderate relationship to Thermoactinomyces vulgaris. Taxonomic problems arising from the new insights into the intrageneric relationships of Bacillus are discussed.     

Dissociation between lymphocyte activation for proliferation and for the capacity to adoptively transfer uveoretinitis

We have shown previously that immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) induces in rats severe eye disease, experimental autoimmune uveoretinitis (EAU). This study examined the uveitogenic capacity of IRBP of another species, the monkey, and tested the cross-antigenicity between these two proteins by a battery of immunological assays. Monkey IRBP was found to be approximately 20 times less uveitogenic in Lewis rats than bovine IRBP. High levels of cross-reactivity between bovine and monkey IRBP were demonstrated by antibodies as measured by the enzyme-linked immunosorbent assay, and by the radiometric ear test of delayed-type hypersensitivity, by using rats immunized with either one of the IRBP. On the other hand, lymphocytes from these rats failed to detect the cross-reactivity between the two IRBP by the proliferation response in culture. Yet, such lymphocytes did recognize the nonimmunizing IRBP when activated in culture for acquiring the capacity to adoptively transfer EAU into naive recipients. The data are discussed with regard to the limited usefulness of the lymphocyte proliferation assay for detection of immunopathogenic processes and the role of cross-reacting antigens in initiation of autoimmune responses.     

Luteal morphology, atresia, and plasma progesterone concentrations during the reproductive cycle of two oviparous lizards, Crotaphytus collaris and Eumeces obsoletus

From ovulation to oviposition, the corpora lutea of the oviparous lizards Crotaphytus collaris and Eumeces obsoletus exhibit three stages of luteal development: 1) luteogenesis, 2) luteal maturity, and 3) luteal regression. Each stage exhibits distinct characteristics, involving changes in: 1) luteal volume, 2) nuclear diameter of cells within the luteal cell mass, and 3) thecal development. Plasma progesterone concentration is greatest during luteogenesis and is positively correlated with ovarian atresia, although atresia occurred throughout the period of gravidity. These data suggest that in these two species, the corpora lutea secrete high amounts of progesterone immediately following ovulation and exhibit morphologically distinct stages of growth and regression.