Genetic regulation of amniotic fluid TNF-alpha and soluble TNF receptor concentrations affected by race and preterm birth

Racial disparity in spontaneous preterm birth (PTB) between African Americans and Caucasians in the US is unexplained, but is probably related to differences in amniotic fluid (AF) inflammatory cytokine profiles. Therefore, this study analyzed the association of 34 single nucleotide polymorphisms (SNPs) in TNF-α and its receptor genes (TNFR1 and TNFR2) with AF TNF-α and soluble TNF receptor (R1 and R2) concentrations in PTB. Samples consisted of African American and Caucasian cases (PTB), and controls (term birth) for which both cytokine, and maternal and fetal genotype data were available. Analyses were performed with genotype, case, and maker-status interaction in the model for log transformed cytokine concentrations. In Caucasians, two interactions between genotype and pregnancy outcome associated with cytokine concentrations, whereas 14 gene variants in African Americans showed interactions with pregnancy outcome, and 13 showed association with genetic markers. In conclusion, cytokine concentrations in African American preterm births can be partially explained by interactions between pregnancy outcome, SNPs and infection. This does not appear to be the case in Caucasians. These findings may be important in understanding disparity in rates of PTB between the two populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA recombination and RNA cleavage activities of the Flp protein: roles of two histidine residues in the orientation and activation of the nucleophile for strand cleavage.

Using a combination of DNA and hybrid DNA-RNA substrates, we have analyzed the mechanism of phosphoryl transfer by the Flp site-specific recombinase in three different reactions: DNA strand breakage and joining, and two types of RNA cleavage activities. These reactions were then used to characterize Flp variants altered at His309 and His345, amino acid residues that are in close proximity to two key catalytic residues (Arg308 and Tyr343). These histidine residues are important for strand cutting by Tyr343, the active-site nucleophile of Flp, but neither residue contributes to the type II RNA cleavage activity or to the strand-joining reaction in a pre-cleaved substrate. Strand cleavage reactions using small, diffusible nucleophiles indicate that this histidine pair contributes to the correct positioning and activation of Tyr343 within the shared active site of Flp. The implications of these results are evaluated against the recently solved crystal structure of Flp in association with a Holliday junction.     

Reactions of Cre with Methylphosphonate DNA: Similarities and Contrasts with Flp and Vaccinia Topoisomerase

Background

Reactions of vaccinia topoisomerase and the tyrosine site-specific recombinase Flp with methylphosphonate (MeP) substituted DNA substrates, have provided important insights into the electrostatic features of the strand cleavage and strand joining steps catalyzed by them. A conserved arginine residue in the catalytic pentad, Arg-223 in topoisomerase and Arg-308 in Flp, is not essential for stabilizing the MeP transition state. Topoisomerase or its R223A variant promotes cleavage of the MeP bond by the active site nucleophile Tyr-274, followed by the rapid hydrolysis of the MeP-tyrosyl intermediate. Flp(R308A), but not wild type Flp, mediates direct hydrolysis of the activated MeP bond. These findings are consistent with a potential role for phosphate electrostatics and active site electrostatics in protecting DNA relaxation and site-specific recombination, respectively, against abortive hydrolysis.

Methodology/Principal Findings

We have examined the effects of DNA containing MeP substitution in the Flp related Cre recombination system. Neutralizing the negative charge at the scissile position does not render the tyrosyl intermediate formed by Cre susceptible to rapid hydrolysis. Furthermore, combining the active site R292A mutation in Cre (equivalent to the R223A and R308A mutations in topoisomerase and Flp, respectively) with MeP substitution does not lead to direct hydrolysis of the scissile MeP bond in DNA. Whereas Cre follows the topoisomerase paradigm during the strand cleavage step, it follows the Flp paradigm during the strand joining step.

Conclusions/Significance

Collectively, the Cre, Flp and topoisomerase results highlight the contribution of conserved electrostatic complementarity between substrate and active site towards transition state stabilization during site-specific recombination and DNA relaxation. They have potential implications for how transesterification reactions in nucleic acids are protected against undesirable abortive side reactions. Such protective mechanisms are significant, given the very real threat of hydrolytic genome damage or disruption of RNA processing due to the cellular abundance and nucleophilicity of water.     

A rapid procedure for the isolation of phycobilisomes from cyanobacteria

This paper describes a method for the rapid isolation of phycobilisomes using a cationic detergent, CTAB (cetyltrimethylammonium bromide). The method has distinct advantages over those currently in use in that (i) release of intact phycobilisomes from cells in the presence of CTAB occurs in 40 s (as compared to 40-60 min of incubation required with Triton X-100), thereby reducing the chances of proteolysis of the component phycobiliproteins; and (ii) these phycobilisome preparations have reduced chlorophyll contamination in the initial stages. In addition this method also helps retain the structural and functional properties, as evidenced by spectroscopy and sodium dodecyl sulfate-polyacrylamide gel analysis.     

Adverse effects associated with selective serotonin reuptake inhibitors and tricyclic antidepressants: a meta-analysis

BACKGROUND: The use of antidepressant medications and the resulting costs have increased dramatically in recent years, partly because of the introduction of selective serotonin reuptake inhibitors (SSRIs). An assessment of the clinical and economic aspects of SSRIs compared with the older tricyclic antidepressants (TCAs) was initiated to generate information for purchasers of these drugs as well as clinicians. One component of this study was an examination of the adverse effects associated with the use of these drugs. METHODS: Searches of bibliographic databases (for January 1980 through May 1996) and manual scanning of both peer-reviewed publications and other documents were used to identify double-blind, randomized controlled trials involving at least one SSRI and one TCA. For the study of adverse effects, only trials that had at least 20 patients in each trial arm and that reported rates of adverse effects in both arms were retained. In total 84 trials reporting on 18 adverse effects were available. Meta-analyses were undertaken to calculate pooled differences in rates of adverse effects. The question of whether the method of eliciting information from patients about adverse effects made a difference in the findings was also examined. Finally, differences in drop-out rates due to adverse effects were calculated. RESULTS: The crude rates of occurrence of adverse effects ranged from 4% (palpitations) to 26% (nausea) for SSRIs and from 4% (diarrhea) to 27% (dry mouth) for TCAs. The differences in the rates of adverse effects between the 2 types of drugs ranged from 14% more with SSRIs (for nausea) to 11% more with TCAs (for constipation). The results did not depend on the method of eliciting information from patients. There were no statistically significant differences between drug classes in terms of drop-outs due to adverse effects. INTERPRETATION: SSRIs and TCAs are both associated with adverse effects, although the key effects differ between the drug classes. Further explanation of the adverse effects and their relation to discontinuation of medication will require better studies involving prospective collection of quality-of-life data.     

Microfilariae in fine needle aspirates from epididymal lesions

Microfilariae of Wuchereria bancrofti were observed in fine needle aspiration smears from three epididymal nodules, and degenerating microfilariae suggestive of Brugia malayi were found in the smears from a fourth case. The smears in all four cases showed a polymorphonuclear inflammatory cell component as well as epithelioid cell granulomata. While blood eosinophilia was present in all four cases, eosinophilia was present in the aspiration smears in only one case. Microfilariae could be demonstrated in the peripheral blood in only one case.