Synthesis of bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis> by the purple nonsulfur bacterium <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis>

The ability of purple nonsulfur bacteria Rhodobacter capsulatus B10 to synthesize bacteriochlorophyll under phototrophic and dark conditions was studied. The modes for cultivation in the dark with oxygen limitation in a continuous culture at D = 0.1 h^?1 were selected. The yield of biomass reached 20 g/l; the bacteriochlorophyll a output of the process amounted to 16.6 mg/l h^?1.     

An Automated ELISA Using Recombinant Antigens for Serologic Diagnosis of B Virus Infections in Macaques

B virus (Macacine herpesvirus 1) occurs naturally in macaques and can cause lethal zoonotic infections in humans. Detection of B virus (BV) antibodies in macaques is essential for the development of SPF breeding colonies and for diagnosing infection in macaques that are involved in human exposures. Traditionally, BV infections are monitored for presence of antibodies by ELISA (a screening assay) and western blot analysis (WBA; a confirmatory test). Both tests use lysates of infected cells as antigens. Because WBA often fails to confirm the presence of low-titer serum antibodies detected by ELISA, we examined a recombinant-based ELISA as a potential alternative confirmatory test. We compared a high-throughput ELISA using 384-well plates for simultaneous antibody screening against 4 BV-related, recombinant proteins with the standard ELISA and WBA. The recombinant ELISA results confirmed more ELISA-positive sera than did WBA. The superiority of the recombinant ELISA over WBA was particularly prominent for sera with low (<500 ELISA units) antibody titers. Among low-titer sera, the relative sensitivity of the recombinant ELISA ranged from 36.7% to 45.0% as compared with 3.3% to 10.0% for WBA. In addition, the screening and confirmatory assays can be run simultaneously, providing results more rapidly. We conclude that the recombinant ELISA is an effective replacement for WBA as a confirmatory assay for the evaluation of macaque serum antibodies to BV.Abbreviations: BV, B virus (Macacine herpesvirus 1); EU, ELISA units; g, glycoprotein; HSV, herpes simplex virus; tELISA, titration ELISA; UN, uninfected; WBA, western blot analysisB virus (BV; Macacine herpesvirus 1) is a member of the genus Simplexvirus, subfamily Alphaherpesvirinae and family Herpesviridae. The virus occurs naturally in macaques (Macaca spp.) and causes a lethal zoonotic infection in 80% of untreated humans. Because biomedical professionals working with macaques, their cells, or tissues are at risk for becoming infected with BV, it is important to know the status of macaques involved in potential BV exposures. Although cases of BV infection after encounters between tourists and macaques have not been reported, any event that involves direct or fomite-associated contact with macaques has inherent risks. Identification of zoonotic BV infection through the detection of antibodies enables timely antiviral intervention, which is critical to reduce or prevent morbidity and mortality. Similarly rapid detection is important to maintain the biointegrity of SPF captive macaque colonies. The identification of BV in clinical specimens is achieved by using cell culture, PCR, or antibody detection methods. Because BV is shed only rarely from peripheral sites, the identification of BV infection in monkeys and humans currently is based on antibody detection (serology).^14,23,28In our laboratory, current serological diagnosis for B virus infections has been based on 2 principal tests: a titration-based (that is, traditional) ELISA (tELISA) as a screening test and western blot analysis (WBA) as a confirmatory test. Each test uses quality-controlled BV antigens that are prepared from lysates of infected cells.^20,22,23 Because BV is the only simplex virus in the Alphaherpesvirinae subfamily that is known to infect macaques,^14,28 antibodies interacting with BV antigens are used to indicate BV infection and not an infection due to a crossreacting virus. In practice, tELISA has identified numerous BV antibody-positive sera, the majority of which are low-titer sera from SPF colonies, which fail to be confirmed by WBA, and therefore, are classified as false positives.²³ We, therefore, searched for other approaches that could be used for confirmation of tELISA results. One reasonable option was the use of BV recombinant proteins as antigens. Numerous investigators have used recombinant-based assays for routine diagnosis of infections with viruses, including cytomegalovirus,³⁶ Epstein–Barr,⁶ herpes simplex (HSV1 and HSV2)^{2,3,17,31,32,34} Crimean–Congo hemorrhagic fever,¹⁰ HIV,³⁶ dengue,^5,11,27 hepatitis C,²⁴ hepatitis B,⁸ West Nile,²⁶ influenza,¹⁶ Ebola, and Marburg³³ viruses.Screening for the presence of serum IgG molecules against an array of defined and purified recombinant antigens has distinct advantages over assays that use the entire complement of viral antigens that are present in virus-infected cells. This is particularly true for pathogens that require BSL4 laboratories.^28,33 The pattern of reactivity obtained against each individual recombinant protein may have diagnostic value, by enabling identification of the stage of infection and the prediction of the prognosis of the disease.^3,4,18 However, using a single or only a few recombinant proteins as ELISA antigens can lead to a false-negative result if the antibody repertoire produced after BV infection reacts with other antigenic determinants that are not represented by the particular recombinant antigens used in the test.^{3,18,28,31,34}Several laboratories have examined the efficacy of using a single BV recombinant antigen (that is, glycoprotein D [gD]) for diagnosing BV infections in macaques^25,37 and humans,¹⁵ and we previously reported the diagnostic potential of an ELISA that incorporated several recombinant BV antigens.²⁸ We chose 4 recombinant BV glycoproteins as candidate antigens: peptides corresponding to the full-length extracellular domain of gB, gC, and gD and the membrane-associated segment of gG (gGm). Among these antigens, gGm was the most BV-specific, because it failed to crossreact with antibodies induced by HSV1 and HSV2. To validate the use of the recombinant BV antigens for the purpose of BV antibody detection, a panel of antibody-negative (n = 40) and antibody-positive (n = 75) macaque sera that were confirmed to be positive by tELISA and WBA were tested against the panel of the 4 B virus recombinant antigens, all of which showed fairly high sensitivity for detecting antibodies to BV.²⁸Here, we examine the performance of the recombinant-based ELISA (rELISA) for BV detection by using numerous (>1000) macaque sera, which have a broad range of antibody titers as determined by tELISA. Because manual ELISA to identify antibodies against an array of antigens are too laborious to be cost-effective, we adapted a previously described high-throughput automated single-antigen ELISA performed in 384-well plates to detect antibodies in macaque sera to multiple BV antigens.²³ This assay format has been adapted to include antigens from other alphaherpesviruses²³ and can be easily modified further for other viruses. We then compared the performance of the rELISA with that of whole-virus tELISA and WBA. The main goal of this study was to determine whether the 384-well rELISA is an effective alternative to WBA as a confirmatory assay for tELISA.     

Release of mtDNA from mitochondria and activation of its replication in tissues of irradiated mice

A nessessary condition for normal functioning of mitochondria is the maintenance of certain numbers of intact mtDNA molecules. In the present study, we investigasted changes in the number of mtDNA copies in brain and spleen cells of mice subjected to irradiation. For the first time, we observed the irradiation-induced output of mtDNA fragments into brain and spleen cell cytosol. In the cytosol of these cells, examined in mice 5 h after 5 Gy irradiation, 1841 h.p. mtDNA fragments were detected able to persist for at 3 weeks. In addition, larger fragments of mtDNA (10,090 b.p.) were detected in the cytosol of brain cells of irradiated mice. The occurrence of mtDNA fragments in the cytosol of brain cells is accompanied with an increase in the number of mtDNA copies in the mitochondrial matrix. The induction of mtDNA replication in brain cells of irradiated animals may be considered as a compensatory reaction in response to mtDNA damage. A sharp decrease in the amount of mtDNA copies in the mitochondrial matrix of spleen cells on the first day after irradiation may be considered as apoptosis development. However, the compensatory reaction in brain cells was also noticed but in later terms.     

Importance of signaling via the IFN-α/β receptor on host cells for the realization of the therapeutic benefits of cyclophosphamide for mice bearing a large MOPC-315 tumor

Here we show that low-dose cyclophosphamide (CY), that depends for its therapeutic effectiveness on the immunopotentiating activity of the drug for T cell-mediated tumor-eradicating immunity, is curative for ~80% of wild-type (WT) mice bearing a large s.c. MOPC-315 tumor, but only for ~10% of IFN-α/βR^−/− mice bearing a large s.c. MOPC-315 tumor. Histopathological examination of the s.c. tumors of such mice on day 4 after the chemotherapy revealed that the low dose of CY led to accumulation of T lymphocytes in both the WT and the IFN-α/βR^−/− mice. However, in the CY treated tumor bearing WT mice the T lymphocytes were present throughout the tumor mass and in direct contact with tumor cells, but in the CY treated tumor bearing IFN-α/βR^−/− mice most of the T lymphocytes remained in blood vessels. In addition to being important for CY-induced transendothelial migration of T lymphocytes into the tumor mass, we show here that signaling via the IFN-α/βR is also important for CY-induced control of metastatic tumor progression in the spleen and liver of the tumor bearing mice. Finally, CY cured tumor bearing WT mice were resistant to a subsequent challenge with MOPC-315 tumor cells, but the few CY cured tumor bearing IFN-α/βR^−/− mice were not. Thus, signaling via the IFN-α/βR on host cells in MOPC-315 tumor bearers is important for CY-induced: (a) transendothelial migration of T lymphocytes into the tumor mass and the eradication of the primary tumor, (b) control of metastatic tumor progression, and (c) resistance to a subsequent tumor challenge. This work was supported by Research Grant 03-19 from the American Cancer Society-Illinois Division.