Polymerase C1 levels and poly(R-3-hydroxyalkanoate) synthesis in wild-type and recombinant Pseudomonas strains.

A functional antibody highly specific for polymerase C1 of Pseudomonas oleovorans GPo1 was raised and used to determine polymerase C1 levels in in vivo experiments. The polymerase C1 antibodies did not show a cross-reaction with polymerase C2 of P. oleovorans. In wild-type P. oleovorans GPo1 and Pseudomonas putida KT2442, amounts of 0.075 and 0.06% polymerase relative to total protein, respectively, were found. P. oleovorans GPo1(pGEc405), which contained additional copies of the polymerase C1-encoding gene under the control of its native promoter, contained 0.5% polymerase C1 relative to total protein. Polymerase C1 reached 10% of total cell protein when the polymerase C1-encoding gene was overexpressed through the P(alk) promoter in P. oleovorans GPo1(pET702, pGEc74). Amounts of poly(R-3-hydroxyalkanoate) (PHA) increased significantly under non-nitrogen-limiting conditions when additional polymerase C1 was expressed in P. oleovorans. Whereas P. oleovorans produced 34% (wt/wt) PHA under these conditions, a PHA level of 64% (wt/wt) could be reached for P. oleovorans GPo1(pGEc405) and a PHA level of 52% (wt/wt) could be reached for P. oleovorans GPo1(pET702, pGEc74) after induction, compared to a PHA level of 13% for the uninduced control. All recombinant Pseudomonas strains containing additional polymerase C1 showed small changes in their PHA composition. Larger amounts of 3-hydroxyhexanoate monomer and smaller amounts of 3-hydroxyoctanoate and -decanoate were found compared to those of the wild type. Two different methods were developed to quantify rates of incorporation of new monomers into preexisting PHA granules. P. oleovorans GPo1 cells grown under nitrogen-limiting conditions showed growth stage-dependent incorporation rates. The highest PHA synthesis rates of 9.5 nmol of C8/C6 monomers/mg of cell dry weight (CDW)/min were found during the mid-stationary phase, which equals a rate of production of 80 g of PHA/kg of CDW/h.     

Mutational analysis of human immunodeficiency virus type 1 (HIV-1) accessory genes: requirement of a site in the nef gene for HIV-1 replication in activated CD4+ T cells in vitro and in vivo.

Human immunodeficiency virus type 1 (HIV-1) accessory genes including nef, vif, and vpr are important factors that determine the replication and pathogenesis of HIV-1. The state of activation is also important for the replication of HIV-1. We evaluated the properties of nef-, vif-, and vpr-minus macrophage-tropic HIV-1(JR) CSF in primary CD4+ Th1- or Th2-like cell cultures which had been activated through CD3 molecules in the presence of interleukin-2 (IL-2) and IL-12 (Th1-like culture) or IL-4 (Th2-like culture), respectively. In activated Th1- or Th2-like cultures, replication of nef-minus HIV-1(JR-CSF) was markedly lower than that of wild-type HIV-1. Subsequent analysis by site-directed mutagenesis showed that (i) the presence of an acidic amino acid-rich domain (amino acid residues 72 to 75) in the Nef protein was critical for the enhancement of viral DNA synthesis, resulting in increased virus growth rate, and (ii) prolines that form part of Src homology 3 binding domain were not essential for viral replication. We also confirmed the importance of sites by using an HIV-1-infected animal model, the hu-PBL-SCID mouse system, representing HIV-1 replication and pathogenesis in activated CD4+ T cells in vivo. These results indicate that Nef accelerates viral replication in activated CD4+ T cells.     

Reproductive performance, population dynamics and anthropometrics of the free-ranging Cayo Santiago rhesus macaques

This report summarizes demographic data collected on the Cayo Santiago colony of rhesus monkeys from 1976-1983 and compares the results with those from 1959-1964 [8,9]. For males and nonpregnant/nonlactating, pregnant, and lactating females mean (+/- 1 SD), body weights, crown-rump lengths, and ponderal indices are tabulated for each age on a large (n = 586) single sampling of this free-ranging population of macaques.     

Accidental methyl methacrylate inhalation toxicity in a rhesus monkey (Macaca mulatta).

A rhesus monkey (Macaca mulatta), accidentally exposed to vapors of methyl methacrylate for 22 hours was found in a comatose condition. Attempts to revive the animal were unsuccessful. Necropsy revealed a diffusely mottled liver, pulmonary edema, and atelectasis. The thoracic cavities each contained 30 ml of clear yellow fluid. Histopathologic review of the tissues showed central lobular liver necrosis, pulmonary edema, pulmonary emphysema, and atelectasis. Analysis of a blood sample obtained from the monkey 1.5 hours prior to death showed a normal hemogram, but elevated values for serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, lactate dehydrogenase, phosphohexose isomerase, blood urea nitrogen, and serum sodium. The pathologic findings, laboratory results, and clinical history suggested a diagnosis of methyl methacrylate poisoning.     

Physiological and biochemical contributions to the taxonomy of the genus prototheca

Five physiological and biochemical characters, which had proved to be valuable for the taxonomy of the genus Chlorella, were studied in the genus Prototheca. There is no hydrogenase activity and no liquefaction of gelatin. Most strains are very acidtolerant (limit of growth at pH 2.0 or 2.5) and very salt-tolerant (limit of growth at 4 or 5% NaCl). Two strains grow well at 38°C. The 16 strains, which were previously assigned to seven taxa, fall into four different groups. Our results tend to support the assumption that Prototheca might be related to Chlorella protothecoides.     

Nucleotide Sequence Relationship Between Intracisternal Type A Particles of Mus musculus and an Endogenous Retrovirus (M432) of Mus cervicolor

Intracisternal type A particles are retrovirus-like structures found in embryonic cells and many tumors of Mus musculus but having no clear relationship with other retroviruses of this mouse species. We have observed a partial nucleotide sequence homology between the high-molecular-weight (32S and 35S) RNA components of intracisternal A-particles from a neuroblastoma cell line and the 70S RNA fraction from M432, a type of retrovirus endogenous to the Asian mouse Mus cervicolor. M432 complementary DNA (cDNA) was hybridized to the extent of 30% by the A-particle RNAs. The hybrids showed a lower thermal stability (DeltaT(m), 7 degrees C) than those formed with homologous RNA. The reaction was commensurate with that found between M432 cDNA and divergent sequences in the M. musculus genome. The capacity to hybridize M432 cDNA was closely correlated with the concentration of A-particle sequences in the cytoplasmic RNA of several M. musculus cell types. The major RNA fraction of M432 virus showed a reciprocal partial reaction with the A-particle cDNA's; the virus, which was grown in NIH/3T3 (M. musculus) cells, also contained a small proportion of apparently authentic A-particle nucleotide sequences. A subset of A-particle sequences seemed to be almost totally lacking in the main M432 RNA. The A-particle cDNA's hybridized extensively with divergent sequences in M. cervicolor cellular DNA, indicating that this mouse species may contain not only the partially homologous M432 virogene, but also a more complete genetic equivalent of the intracisternal A-particle.     

Fructose-2,6-bisphosphate increases the binding affinity of yeast phosphofructokinase to AMP

At saturating concentrations of AMP four molecules of this ligand are bound per octamer of yeast phosphofructokinase. Fructose-2,6-bisphosphate increases the binding affinity of the enzyme to AMP. This indicates synergistic cooperation of the two allosteric activators in the binding process. The stoichiometry of binding is not altered by fructose-2,6-bisphosphate.     

Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of the mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase.

The neutral metalloprotease elastase is one of the major proteins secreted into the culture medium by many Pseudomonas aeruginosa strains. Encoded by the lasB gene, the 33-kDa elastase is initially synthesized as a 53-kDa preproenzyme which is processed to the mature form via a 51-kDa proelastase intermediate. To facilitate studies on proteolytic processing of elastase precursors and on secretion, we developed systems for overexpression of lasB in Escherichia coli under the control of the inducible T7 and tac promoters. Although the 51-kDa proelastase form was detectable in E. coli under inducible conditions, most of the elastase produced under these conditions was found in an enzymatically active 33-kDa form. The amino-terminal sequence of the first 15 amino acid residues of this 33-kDa elastase species was identical to that of the mature P. aeruginosa enzyme, suggesting that processing was autocatalytic. To test this possibility, the codon in lasB encoding His-223, a presumed active-site residue, was changed to encode Asp-223 (lasB1) and Tyr-223 (lasB2). The effects of these mutations on enzyme activity and processing were examined. No proteolytic or elastolytic activities were detected in extracts of E. coli cells containing the lasB mutant alleles. Overexpression of the mutated lasB genes in E. coli resulted in the accumulation of the corresponding 51-kDa proelastase species. These were processed in vitro to the respective 33-kDa forms by incubation with exogenous purified elastase, without an increase in proteolytic activity. Molecular modeling studies suggest that the mutations have little or no effect on the conformation of the mutant elastases. In addition, wild-type elastase and the mutant proelastases were localized to the periplasm of E. coli. The present results confirm that His-223 is essential for elastase activity and provide evidence for autoproteolytic processing of proelastase.