raf/myc-infected erythroid cells are restricted in their ability to terminally differentiate.

A comparison was made of the in vitro erythroid colony-forming abilities of v-raf-, v-myc-, and v-raf/v-myc-containing retroviruses. In methylcellulose, v-raf efficiently produced colonies of well-differentiated hemoglobin-synthesizing erythroid cells, whereas v-raf/v-myc-infected erythroid cells were inhibited from terminally differentiating but retained the ability to replicate extensively. In contrast, v-myc was unable to stimulate the formation of erythroid colonies.     

Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases.

Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites.     

Systematics of theLactobacillus population on rat intestinal mucosa with special reference toLactobacillus reuteri

The systematics of theLactobacillus population of the intestines of 88 different rats was studied; 80 rats had been fed on fermented oat-meal soup (Molin et al. 1992). One-hundred-twenty-twoLactobacillus strains from the intestinal mucosa were phenotypically classified together with twenty-eight reference strains ofLactobacillus andLeuconostoc, using 49 unit characters. Data were examined using Jaccard coefficient, and unweighted pair group algorithm with arithmetic averages. Two major and eleven minor clusters were defined at the 76% S_J-similarity level: Cluster 1 included thirty isolates which could not be identified further, but had resemblance to the type strains ofL. jensenii, L. gasseri, L. crispatus, and to some extent toL. acidophilus. Cluster 12 including fifty-four intestinal isolates was identified asL. reuteri; and so was cluster 13 (five isolates). Isolates of the major clusters were found in all parts of the intestines. The genomic homogeneity of theL. reuteri isolates was scrutinized by endonuclease restriction analysis of the chromosomal DNA, and the isolates could be divided into six genomic strains.     

Establishment of polychlorinated biphenyl-degrading enrichment culture with predominantly meta dechlorination.

Enrichment of polychlorinated biphenyl (PCB)-dechlorinating microorganisms from PCB-contaminated sediments from the Upper Hudson River, N.Y., was attempted. The enrichment strategy was to use pyruvate as the electron donor and dechlorination of Aroclor 1242 as the electron acceptor. The enrichment medium also contained non-PCB-contaminated Hudson River sediments, which were required for the PCB-dechlorinating activity. An enrichment culture (that had stable PCBT-dechlorinating activity over nine serial transfers during 1 year) was established under these conditions; however, the rate of dechlorination did not increase after the second serial transfer. Dechlorination occurred primarily from the meta positions of the biphenyl molecule. Hydrogen could be substituted for pyruvate as the electron donor with equal activity, but when acetate was used as the electron donor a delay in dechlorination was observed. Sulfate and bromethane sulfonate inhibited dechlorination activity. The pyruvate-Aroclor 1242 enrichment also dechlorinated Aroclors 1248, 1254, and 1260; the extent of chlorine removed was the greatest for Aroclor 1254. For comparison, nonautoclaved non-PCB-contaminated Hudson River sediments used in the assay also dechlorinated Aroclors, but only after 12 to 16 weeks of incubation. This suggests that PCB-dechlorinating organisms were also present in these sediments but in numbers lower than those in the enrichment culture.     

Production of an extracellular polyethylene-degrading enzyme(s) by Streptomyces species.

Extracellular culture concentrates were prepared from Streptomyces viridosporus T7A, Streptomyces badius 252, and Streptomyces setonii 75Vi2 shake flask cultures. Ten-day-heat-treated (70 degrees C) starch-polyethylene degradable plastic films were incubated with shaking with active or inactive enzyme for 3 weeks (37 degrees C). Active enzyme illustrated changes in the films' Fourier transform infrared spectra, mechanical properties, and polyethylene molecular weight distributions.     

Survival and replication of male-specific bacteriophages in molluscan shellfish.

The survival and replication of male-specific bacteriophages in hard-shelled clams (Mercenaria mercenaria) and their homogenates were examined to further assess their potential utility as indicator organisms. Trials were conducted in the presence and absence of a suitable bacterial host, Escherichia coli HS[pFamp]R. Results of this study demonstrated that male-specific bacteriophages were unable to replicate in hard-shelled clams, with or without added host cells. In addition, the densities of these bacteriophages were stable for up to 7 days in shellfish held at ambient seawater temperatures (less than 25 degrees C). Evidence of replication, although not observed in live shellfish, was found to occur in temperature-abused shellfish homogenates and supernatants, but only when a suitable bacterial host was present.     

Oxygen transport in the blood of arctic mammals: adaptation to local heterothermia

Summary The oxygen binding of whole blood from humans and two arctic mammals, reindeer and muskox, has been studied as a function of carbon dioxide and temperature. All bloods display a marked Bohr effect with Bohr coefficients in the range –0.44––0.73. The Bohr effect is more pronounced at 20°C. The temperature sensitivity of reindeer and muskox blood expressed by the apparent heat of oxygenation, H, is almost three times lower than that of human HbA under the same experimental conditions. This thermodynamic difference gives special benefits to arctic mammals with large heterothermy by safeguarding oxygen unloading at very low ambient temperatures.     

Separate functional domains of the herpes simplex virus type 1 protease: evidence for cleavage inside capsids.

The herpes simplex virus type 1 (HSV-1) protease (Pra) and related proteins are involved in the assembly of viral capsids and virion maturation. Pra is a serine protease, and the active-site residue has been mapped to amino acid (aa) 129 (Ser). This 635-aa protease, encoded by the UL26 gene, is autoproteolytically processed at two sites, the release (R) site between amino acid residues 247 and 248 and the maturation (M) site between residues 610 and 611. When the protease cleaves itself at both sites, it releases Nb, the catalytic domain (N0), and the C-terminal 25 aa. ICP35, a substrate of the HSV-1 protease, is the product of the UL26.5 gene. As it is translated from a Met codon within the UL26 gene, ICP35 cd are identical to the C-terminal 329-aa sequence of the protease and are trans cleaved at an identical C-terminal site to generate ICP35 e,f and a 25-aa peptide. Only fully processed Pra (N0 and Nb) and ICP35 (ICP35 e,f) are present in B capsids, which are believed to be precursors of mature virions. Using an R-site mutant A247S virus, we have recently shown that this mutant protease retains enzymatic activity but fails to support viral growth, suggesting that the release of N0 is required for viral replication. Here we report that another mutant protease, with an amino acid substitution (Ser to Cys) at the active site, can complement the A247S mutant but not a protease deletion mutant. Cell lines expressing the active-site mutant protease were isolated and shown to complement the A247S mutant at the levels of capsid assembly, DNA packaging, and viral growth. Therefore, the complementation between the R-site mutant and the active-site mutant reconstituted wild-type Pra function. One feature of this intragenic complementation is that following sedimentation of infected-cell lysates on sucrose gradients, both N-terminally unprocessed and processed proteases were isolated from the fractions where normal B capsids sediment, suggesting that proteolytic processing occurs inside capsids. Our results demonstrate that the HSV-1 protease has distinct functional domains and some of these functions can complement in trans.     

In vitro antigen challenge of human antibody libraries for vaccine evaluation: the human immunodeficiency virus type 1 envelope.

Human antibody responses, or versions thereof, can be cloned as phage display libraries. In vaccine evaluation, the possibility therefore exists of challenging the human response in vitro, rather than in vivo, in order to assist in establishing the most promising vaccine leads. The characteristics of the antibodies retrieved directly indicate the strengths and weaknesses of the vaccine at the molecular level. We applied this approach to compare recombinant and native human immunodeficiency virus type 1 envelope preparations. We conclude that recombinant gp160, gp140, and, to a lesser extent, gp120 present epitopes around the CD4 binding site in a conformation different from that of the native multimer and contrary to expected vaccine requirements. Antibodies to the potently neutralizing b12 epitope were selected preferentially from an immune library by purified human immunodeficiency virus type 1 virions. This suggests that b12 is a major epitope on the virions, in contrast to recombinant envelope preparations, in which related, weakly neutralizing epitopes predominate. Although the majority of virions in the preparation used are expected to be noninfective, it appears that they predominantly express a native envelope configuration and would be able to elicit potent neutralizing antibodies.