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.     

Two amino acid residues confer type specificity to a neutralizing, conformationally dependent epitope on human papillomavirus type 11.

Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism.     

Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial alpha-crystallin homolog.

The majority of active tuberculosis cases arise as a result of reactivation of latent organisms which are quiescent within the host. The ability of mycobacteria to survive extended periods without active replication is a complex process whose details await elucidation. We used two-dimensional gel electrophoresis to examine both steady-state protein composition and time-dependent protein synthetic profiles in aging cultures of virulent Mycobacterium tuberculosis. At least seven proteins were maximally synthesized 1 to 2 weeks following the end of log-phase growth. One of these proteins accumulated to become a predominant stationary-phase protein. N-terminal amino acid sequencing and immunoreactivity identified this protein as the 16-kDa alpha-crystallin-like small heat shock protein. The gene for this protein was shown to be limited to the slowly growing M. tuberculosis complex of organisms as assessed by Southern blotting. Overexpression of this protein in wild-type M. tuberculosis resulted in a slower decline in viability following the end of log-phase growth. Accumulation of this protein was observed in log-phase cultures following a shift to oxygen-limiting conditions but not by other external stimuli. The protein was purified to homogeneity from overexpressing M. smegmatis in two steps and shown to have a significant ability to suppress the thermal denaturation of alcohol dehydrogenase. Collectively, these results suggest that the mycobacterial alpha-crystallin protein may play a role in enhancing long-term protein stability and therefore long-term survival of M. tuberculosis.     

Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

 A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab₂). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab₃), were induced by the mAb17-1A therapy. Patients with detectable ab₃ after treatment had significantly higher ab₂ levels than those not developing ab₃. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab₂ as well as induction of anti-anti-idiotypic antibodies (ab₃), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab₃) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab₃ (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. Received: 20 October 1995 / Accepted: 18 December 1995