Differential expression of InlB and ActA in Listeria monocytogenes in selective and nonselective enrichment broths.

AIM: To investigate the effect of selective and nonselective media on the expression of ActA and InlB proteins in Listeria monocytogenes. METHODS AND RESULTS: Polyclonal antibodies to InlB and ActA were used in western blotting to determine the effect of selective (BLEB, UVM, and FB) or nonselective (BHI and LB) enrichment broths or hotdog exudates. Of the 13 L. monocytogenes serotypes tested, 11 and 12 serotypes showed a strong InlB expression in brain heart infusion (BHI) and Luria-Bertani (LB), respectively, while only seven and one serotypes showed a strong ActA expression in these two respective broths, and others showed a weaker or no expression. On the contrary, in selective broths, expression of InlB was either very weak or undetectable. However, ActA expression was stronger in 12 serotypes when grown in buffered Listeria enrichment broth (BLEB), 11 in University of Vermont medium (UVM), and 10 in Fraser broth (FB). When tested in hotdog exudates, InlB and ActA were detected in serotypes grown at 37 degrees C but not at 4 degrees C. Transmission electron microscopy, enzyme-linked immunosorbent assay, and mRNA analysis further supported these observations. CONCLUSION: Overall, selective enrichment broths promote ActA while nonselective broths promote InlB expression. SIGNIFICANCE AND IMPACT OF THE STUDY: As commonly recommended enrichment broths show differential InlB and ActA expression, proper media must be selected to avoid false results during antibody-based detection of L. monocytogenes.

Phylogeny of east asian bufonids inferred from mitochondrial DNA sequences (Anura: amphibia).

We investigated the relationships of Asian bufonids using partial sequences of mitochondrial DNA genes. Twenty-six samples representing 14 species of Bufo from China and Vietnam and 2 species of Torrentophryne from China were examined. Three samples of Bufo viridis from Armenia and Georgia were also sequenced to make a comparison to its sibling tetraploid species B. danatensis. Bufo americanus, from Canada, was used as the outgroup. Sequences from the 12S ribosomal RNA, 16S ribosomal RNA, cytochrome b, and the control region were analyzed using parsimony. East Asian bufonids were grouped into two major clades. One clade included B. andrewsi, B. bankorensis, B. gargarizans, B. tibetanus, B. tuberculatus, its sister clade B. cryptotympanicus, and the 2 species of Torrentophryne. The second clade consisted of B. galeatus, B. himalayanus, B. melanostictus, and a new species from Vietnam. The placement of three taxa (B. raddei, B. viridis, and its sister species, B. danatensis) was problematic. The genus Torrentophryne should be synonymized with Bufo to remove paraphyly. Because B. raddei does not belong to the clade that includes B. viridis and B. danatensis, it was removed from the viridis species group. The species status of B. bankorensis from Taiwan is evaluated.

Pregnancy resulting from rape.

Pregnancy resulting from rape is more prevalent than generally recognized, and violations of women's sexual and reproductive self-determination take many forms. Four themes--relationship rape, power dynamics, maternal ambivalence, and social reactions and support--can be identified in one woman's experiences and the literature. Recommended interventions, based on a woman-centered empowerment framework, include safety assessment, formulating a safety plan, and facilitating social support. Emergency postcoital contraception is a preventive option.

Four new coccidia (Apicomplexia: Eimeriidae) from anoles (Lacertilia: Polychrotidae) in the Dominican Republic.

Fecal samples from 25 Anolis armouri, 2 Anolis bahorucoensis, 48 Anolis cybotes, and 21 Anolis olssoni (Lacertilia: Polychrotidae) from southern Hispaniola were examined for coccidian oocysts. Two eimerians and 2 isosporans are herein described as new species. Sporulated oocysts of Eimeria schwartzi n. sp. from A. armouri are ellipsoidal, 22.7 (20.8-25.0) x 15.7 (14.6-17.7) microns, with spherical to subspherical sporocysts, 7.9 (6.2-9.4) x 7.4 (6.2-8.3) microns. Sporulated oocysts of Isospora reui n. sp. from A. bahorucoensis are spherical to subspherical, 18.2 (15.6-20.0) x 17.8 (15.6-19.8) microns, with ovoid sporocysts, 11.9 (10.4-12.7) x 8.5 (7.5-9.4) microns. Sporulated oocysts of Isospora hendersoni n. sp. from A. armouri and A. cybotes are spherical to subspherical, 23.2 (20.8-26.0) x 21.1 (18.4-23.9) microns, with ellipsoidal sporocysts, 14.7 (12.5-15.6) x 10.0 (9.2-11.4) microns. Sporulated oocysts of Eimeria avilae n. sp. from A. olssoni are cylindrical, 29.3 (26.0-33.3) x 15.9 (13.5-18.9) microns, with ellipsoidal sporocysts 10.2 (9.4-11.4) x 6.8 (5.2-8.0) microns.

Expression of tandem glutathione S-transferase recombinant genes in COS cells for analysis of efficiency of protein expression and associated drug resistance.

Expression vectors were designed and constructed to achieve optimum production of two different isozymes of rat glutathione S-transferase (GST) (EC 2.5.1.18) in COS cells, for studies of drug resistance. Promoter-enhancer elements from the simian virus 40 (SV40) early-region or the mouse alpha 2(I)-collagen gene, GST cDNAs encoding the rat Ya or Yb1 isozymes, and an SV40 replicative origin (ori) were positioned in the vector to express two GSTs at high levels in the same cell. The optimized construct yielded levels of both GST proteins (1% of postmitochondrial protein fraction) that were up to 1.3-fold greater than the sum of those produced individually by two single-unit expression constructs. The best production of the tandem recombinant gene products was observed when the genes were placed in a head to head orientation in close proximity (1 kilobase). With the recombinant genes configured in this way, the plasmid DNA was also amplified in COS cells to higher levels (30% increase over single-unit expression constructs), as ori elements were placed on both DNA strands. Cells expressing the recombinant GSTs were viably sorted by flow cytometry on the basis of a GST-catalyzed conjugation of glutathione to monochlorobimane. Sorted COS cells that expressed both GST Ya and Yb1 from recombinant genes in a tandem, head to head configuration were 25 or 70% more resistant to the alkylating agent chlorambucil than cells that expressed GST Ya or Yb1 alone.

Recombinant glutathione S-transferase (GST) expressing cells purified by flow cytometry on the basis of a GST-catalyzed intracellular conjugation of glutathione to monochlorobimane.

COS cells transiently expressing glutathione S-transferase (GST) pi, Ya, or Yb1 (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony-forming assays to show that GST confers cellular resistance to the carcinogen benzo[a]pyrene (+/-)-anti-diol epoxide (anti-BPDE). We developed a sorting technique to viably separate recombinant GST+ cells (20%) from the nonexpressing electroporated population (80%) on the basis of a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane (mClB). The concentration of mClB, length of time cells are exposed to mClB, and activity of the expressed GST isozyme determined the degree to which recombinant GST+ cells fluoresced more intensely than controls. On-line reagent addition ensured that all cells were exposed to 25 microM mClB for 30-35 s during transit before being analyzed for fluorescence intensity and sorted. The apparent Km for mClB of the endogenous COS cell GST-catalyzed intracellular reaction was 88 microM. Stained GST Ya+ or Yb1+ cells catalyzed the conjugation 2 or 5 times more effectively than GST pi+ cells. Enzyme activity in cytosolic fractions prepared from sorted recombinant GST+ cells was 1.8 +/- 0.3-fold greater than that of the control (80 +/- 4 nmol/min/mg protein). Upon a 5-fold purification of GST pi+ cells in the electroporated population, resistance to anti-BPDE in colony-forming assays increased 5 times, from 1.1-fold (unsorted) to 1.5-fold (sorted) (P less than 0.001).