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Cloning, Molecular Analysis, and Expression of the Polyhydroxyalkanoic Acid Synthase (phaC) Gene from Chromobacterium violaceum

DANA KOLIBACHUK, ANDREA MILLER, AND DOUGLAS DENNIS

91KB

The polyhydroxyalkanoic acid synthase gene from Chromobacterium violaceum (phaCCv) was cloned and characterized. A 6.3-kb BamHI fragment was found to contain both phaCCv and the polyhydroxyalkanoic acid (PHA)-specific 3-ketothiolase (phaACv). Escherichia coli strains harboring this fragment produced significant levels of PHA synthase and 3-ketothiolase, as judged by their activities. While C. violaceum accumulated poly(3-hydroxybutyrate) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when grown on a fatty acid carbon source, Klebsiella aerogenes and Ralstonia eutropha (formerly Alcaligenes eutrophus), harboring phaCCv, accumulated the above-mentioned polymers and, additionally, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) when even-chain-length fatty acids were utilized as the carbon source. This finding suggests that the metabolic environments of these organisms are sufficiently different to alter the product range of the C. violaceum PHA synthase. Neither recombinant E. coli nor recombinant Pseudomonas putida harboring phaCCv accumulated significant levels of PHA.

ESR studies of order and dynamics in a nematic liquid crystal containing a dispersed hydrophobic aerosil

ALBERTO ARCIONIA, CORRADO BACCHIOCCHIA, LORIS GROSSIB, ALESSANDRO NICOLINIA AND CLAUDIO ZANNONIA

We have studied a system composed of an hydrophobic aerosil (R812) dispersed in the liquid crystal 4-n-octyl 4’-cyanobiphenyl (8CB) using the spin probe ESR technique and, in particular, we have determined, for dierent aerosil concentrations, the temperature dependence of the orientational order parameter hP2i and the rotational diusion coefcient of the probe 5-doxyl stearic acid in the ordered and isotropic phases of the system. We have found that increasing the silica concentration up to 10 wt % does not significantly change the transition temperatures of the system. The probe order parameter is instead depressed and we found that the exponent of an empirical Haller-type equation, used to fit its temperature dependence, changes roughly linearly with the aerosil concentration. The concentration eect on the probe dynamics is relatively small in the isotropic phase, where the D? temperature dependence is well fitted for all the systems with an Arrheniustype equation. In the nematic phase the dynamical behavior is more complex: we found that while local probe motion is still rather fast even when the macroscopic behavior is gel-like, the temperature dependence of D? is still of Arrhenius-type up to 3 wt % aerosil concentration, but it becomes of Vogel-Fulcher-Tammann type for the 10 wt % R812 system.

In situ atomic force microscopy study of hectorite and nontronite dissolution: Implications for phyllosilicate edge surface structures and dissolution mechanisms

BARRY R. BICKMORE, DIRK BOSBACH, MICHAEL F. HOCHELLA JR., LAURENT CHARLET, AND ERIC RUFE

194KB

The dissolution behavior of two smectite minerals, hectorite (trioctahedral) and nontronite (dioctahedral), was observed in situ, in acid solutions, using atomic force microscopy. As expected, the crystallites dissolved inward from the edges, and the basal surfaces appeared to be unreactive during the timescale of the experiments. The hectorite (010) faces appeared to dissolve about 6× more slowly than the lath ends, usually broken edges. The edges visibly dissolved on all sides, and appeared to roughen somewhat. On the other hand, the (010), (110), and (11–0) faces on nontronite crystals were exceptionally stable, so that any dissolution fronts originating at broken edges or defects would quickly become pinned along these faces, after which no more dissolution was observable. These observations can be explained by using periodic bond chain theory to predict the topology of the surface functional groups on the edge faces of these minerals. If a certain amount of predicted surface relaxation is allowed on the (110) and (11–0) faces of nontronite, an important difference between the exceptionally stable faces and the others becomes apparent. That is, the oxygen sites connecting the octahedral and tetrahedral sheets are all fully bonded on the nontronite (010), (110), and (11–0) edge faces, whereas all hectorite edge faces and nontronite broken edges would have coordinatively unsaturated connecting O atoms.

Variable retention of diatoms on screens during size separations

BRUCE E. LOGAN, UTA PASSOW, ALICE L. ALLDREDGE

Particles smaller than filter mesh pores can collide and stick to mesh fibers, biasing size separations based only on pore diameter. The removal of flocculating diatoms (Chaetoceros gracilis, Nitzschia angularis) was greater than removal of a nonfloc-forming species (Thalassiosira weissflogii) even after including the effects of cell size. Over a 5-d period an average of 1.4% of cells of N. angularis were removed by 230~pm pore-diameter screens; on day 6, 7.5% of cells were removed as cells began to flocculate. The percentage of C. gracilis removed continually increased from 0.4 to 28% during the same period, while the removal of T. weissfrogii was constant at 0.19% over a 10-d period. Prior to the onset of flocculation, sticking coefficients (rate of cell attachment to mesh fibers/rate striking fibers) were 0.05 for T. weissjlogii, 0.26 for N. angularis, and 0.73 for C. gracilis. Size separations will therefore tend to concentrate more flocculent species of phytoplankton into size fractions much larger than cell diameters.

Microbial utilization of the neurotoxin domoic acid: blue mussels (Mytilus edulis) and soft shell clams (Mya arenaria) as sources of the microorganisms

JAMES E. STEWART, L.J. MARKS, M.W. GILGAN, E. PFEIFFER, AND B.M. ZWICKER

The neurotoxin domoic acid is produced in quantity by the diatom Pseudo-nitzschia multiseries and is released to the environment directly and indirectly via food chains. Presumably there is a mechanism for the biodegradation and disposal of domoic acid and as bacteria are logical candidates for such an activity, a search for bacteria competent to carry out biodegradation of domoic acid was initiated. Extensive trials with a wide variety of bacteria isolated mainly from muds and waters taken from the marine environment showed that the ability to grow on or degrade domoic acid was rare; in fact, domoic acid was inhibitory to resting cells or growing cultures of most of these bacteria. In contrast, using enrichment techniques, it was possible to isolate from molluscan species that eliminate domoic acid readily, i.e., blue mussels (Mytilus edulis) and soft-shell clams (Mya arenaria), bacteria that exhibited growth with and biodegradation of domoic acid when supplemented with low concentrations of growth factors. The species that retain domoic acid for lengthy periods, such as sea scallops (Placopecten magellanicus) and red mussels (Modiolus modiolus), only occasionally yielded bacteria with this capability. The differences may be a result of the mechanisms used by the different shellfish in dealing with domoic acid, i.e., freely available in the blue mussels and soft shell clams but likely sequestered in the digestive glands of sea scallops and red mussels and thus, largely unavailable for bacterial utilization. The results show that Mytilus edulis and Mya arenaria, almost uniquely, are prime and reliable sources of domoic acid utilizing bacteria.

Uterine tubal cells remain uninfected after culture with in vitro-produced embryos exposed to bovine viral diarrhea virus

M.D. GIVENS, P.K. GALIK, K.P. RIDDELL, D.A. STRINGFELLOW

Bovine viral diarrhea virus (BVDV) has been isolated from washed and sonicated, in vitroproduced embryos, but the infectivity of BVDVassociated with intact, developing, embryos has not been demonstrated. The objective of this study was to determine if a dose of BVDV infective for coculture cells was associated with individual, developing embryos, following artificial exposure to the virus and washing. In 5 replicates, zona pellucida-intact, in vitro-produced embryos were assigned to a negative control embryo group, or were incubated in 105±106 cell culture infective doses (50%, CCID50) per milliliter of a type I, noncytopathic (strain SD-1) BVDV for 2 h. Unexposed negative control embryos and exposed positive control embryos were washed, sonicated and assayed for BVDVusing virus isolation with immunoperoxidase monolayer assay. Immediately or following cryopreservation, remaining virally-exposed, washed embryos were co-cultured individually with BVDV-negative cultures of bovine uterine tubal cells in a medium free of BVDVneutralizing activity. After two days in culture, uterine tubal cells and embryos (including the zona pellucida) were separated and washed. The culture medium, uterine tubal cells and embryos were then assayed for BVDV. Bovine viral diarrhea virus was not isolated from any negative control embryo group, but was isolated from all positive control embryo groups. Although all uterine tubal cell populations were confirmed to be susceptible to BVDV, virus was never isolated from uterine tubal cells or embryos from post-exposure culture.

The Chk1-Cdc25C regulation is involved in sensitizing A253 cells to a novel topoisomerase I inhibitor BNP1350 by bax gene transfer

MING-BIAO YIN, GUNNAR HAPKE, BIN GUO, RAMI G AZRAK, CHERYL FRANK AND YOUCEF M RUSTUM

Promotion of apoptosis may potentiate the sensitivity of tumor cells to chemotherapeutic agents, thus improving the efficacy of cancer treatment. The transfection of the proapoptotic bax gene, which results in the overexpression of bax protein, augments the growth inhibition of A253 cells by BNP1350. Increased drug response was associated with the induction of DNA fragmentation in the size of 30 ± 200 Kb, generating a cleaved fragment of 18 kDa from full-length 21 kDa bax and the cleavage of PARP. A253/vec cells treated with 0.07 mM(IC50) of BNP1350 accumulated in G2 phase at 24 h after drugremoval. In contrast, A253/Bax cells treated with an equimolar concentration of BNP1350 primarily displayed a G1 phase accumulation with a concurrent decrease in G2 phase. Certain cell cycle regulatory protein expression and activities were altered following drug exposure in both cell lines under similar conditions. Cdk2- and cdc2-associated H1 kinase activities were markedly increased in the A253/Bax cell line with marginal increased activity in the A253/vec cell line. A chk1 activity assay was performed with GST-cdc25C (200 ± 256) or GST-cdc25CS216A (200 ± 256) fusion proteins as the substrate. Increased chk1 activity was observed in the A253/vec cell line, with little change in the A253/Bax cell line, when exposed to equimolar concentrations of BNP1350 (0.07 mM). A Western blot of immunoprecipi- tated chk1 indicated that increased chk1 phosphorylation following DNA damage induced by BNP1350 was accompanied by the observed G2 accumulation in the A253/vec cell line, while only a slight increase in chk1 phosphorylation was seen in the A253/Bax cell line.

Toxicological Effects of Fine Particulate Matter Derived from the Destruction of the World Trade Center

STEPHEN H. GAVETT, NAJWA HAYKAL-COATES, JOHN K. MCGEE, JERRY W. HIGHFILL, ALLEN D. LEDBETTER, AND DANIEL L. COSTA

The goal of the experiments described in this report was to evaluate the toxicity of fine particulate matter (PM) derived from the destruction of the World Trade Center (WTC) on the respiratory tract of mice, and thereby contribute to the short-term health risk assessment of WTC PM being conducted by the Environmental Protection Agency. The adopted approach allowed a comparison of the intrinsic acute toxicity of fine WTC PM in the respiratory tract to well-studied PM reference samples that range in toxicity from essentially inert to quite toxic. The fundamental question was whether fine WTC PM was uniquely highly toxic. This toxicological research complements efforts by EPA and other organizations to assess the extent and level of worker and public exposures to PM derived from the WTC disaster and recovery efforts. This research is informative, but it is of limited scope, with a focus on the toxicological effects of the fine fraction of WTC dust from a single exposure. A more complete characterization of potential health effects would include consideration of other size fractions, repeated exposures, additional doses and endpoints, and responses in species or strains of differing sensitivity. It was not possible to assess these other considerations in the present study. Fallen dust samples were collected on September 12 and 13 from various sites around Ground Zero, and the fine PM fraction (< 2.5 microns in diameter; PM2.5) was isolated on filters. PM2.5 was extracted from the filters and extensively analyzed by several chemical and physical techniques.

Deletion of the Central Proline-Rich Repeat Domain Results in Altered Antigenicity and Lack of Surface Expression of the Streptococcus mutans P1 Adhesin Molecule

L. J. BRADY, D. G. CVITKOVITCH,C. M. GERIC, M. N. ADDISON, J. C. JOYCE, P. J. CROWLEY, AND A. S. BLEIWEIS

Abstract TextMembers of the family of surface adhesins of oral streptococci, including P1 of Streptococcus mutans, contain two highly conserved repeat domains, one rich in alanine (A region) and the other rich in proline (P region). To assess the contribution of the P region to the biological properties of P1, an internal deletion in spaP was engineered. In addition, the P region was subcloned and expressed as a fusion partner with the maltose binding protein of Escherichia coli and liberated by digestion with factor Xa. Results of Western blot experiments in which recombinant polypeptides were probed with a panel of 11 monoclonal antibodies indicated that the P region is a necessary component of conformational epitopes within the central portion of P1. Antibodies reactive with the P region were detected in a polyclonal rabbit antiserum generated against whole S. mutans cells but not in two rabbit antisera generated against purified P1 (Mr ; 185,000), suggesting that this domain is immunogenic on the surface of intact bacteria but not as part of a soluble full-length molecule. Finally, transformation of a spaP-negative mutant with a shuttle vector containing an internally deleted spaP lacking P-region DNA resulted in a complete absence of surface-localized P1 and substantially less P1 in sonicated cells compared to the case for the mutant complemented with the full-length gene. These results suggest that the P region is an integral component contributing to the conformation of the central region of P1 and indicate that its presence is necessary for surface expression of the molecule on S. mutans.

Absence of Macrophage Inflammatory Protein-1a Prevents the Development of Blinding Herpes Stromal Keratitis

TERRENCE M. TUMPEY, HAO CHENG, DONALD N. COOK, OLIVER SMITHIES, JOHN E. OAKES, AND ROBERT N. LAUSCH

Prior studies in our laboratory have suggested that the CC chemokine macrophage inflammatory protein-1a (MIP-1a) may be an important mediator in the blinding ocular inflammation which develops following herpes simplex virus type 1 (HSV-1) infection of the murine cornea. To directly test this hypothesis, MIP-1a-deficient (-/-) mice and their wild-type (+/+) counterparts were infected topically on the scarified cornea with 2.5 x 105 PFU of HSV-1 strain RE and subsequently graded for corneal opacity. Four weeks postinfection (p.i.), the
mean corneal opacity score of -/- mice was 1.1 + - 0.3 while that of the +/+ mice was 3.7 + - 0.5. No detectable infiltrating CD4+ T cells were seen histologically at 14 or 21 days p.i. in -/- animals, whereas the mean CD4+ T-cell count per field (36 fields counted) in +/+ hosts was 26 6 2 (P < 0.001). In addition, neutrophil counts in the -/- mouse corneas were reduced by >80% in comparison to the wild-type controls. At 2 weeks p.i., no interleukin-2 or gamma interferon could be detected in six of seven -/- mice, whereas both T-cell cytokines were readily demonstrable in +/+ mouse corneas. Also, MIP-2 and monocyte chemoattractant protein-1 protein levels were significantly lower in MIP-1a -/- mouse corneas than in +/+ host corneas, suggesting that MIP-1a directly, or more likely indirectly, influences the expression of other chemokines.