Validated application of a new high-performance liquid chromatographic method for the determination of selected flavonoids and phenolic acids in human plasma using electrochemical detection

We developed a sensitive method for determination of 23 flavonoids and phenolic acids, which represent phenolic acids and five subclasses of flavonoids. Plasma samples were extracted with selective solid-phase-extraction columns and separated by RP-high-performance liquid chromatography (HPLC). For detection an electrochemical detector was used. Identification and test of purity were carried out via retention times and spectra analyses. Limits of detection varied from 1.45 to 22.27 nmol/l. Recovery varied from 81% to 106%. Reproducibility for all analytes was below 10% (coefficient of variation, CV (%)) and ranged between 3.1% and 9.8%. This method can be applied to samples from interventional studies as well as observational studies.     

New developments in developmental biology

A report on the 15th International Society of Developmental Biologists Congress, Sydney, Australia, 3-7 September 2005.     

Complete nucleotide sequences of bovine alpha S2- and beta-casein cDNAs: comparisons with related sequences in other species

The nucleotide sequences corresponding to bovine alpha S2- and beta- casein mRNAs have been determined by cDNA analysis. Both sequences appear to be complete at their 5' ends. The nucleotide sequence of alpha S2-casein, when compared with the corresponding cavine A sequence, helps to define the boundaries of a large amino acid repeat (approximately 80 residues) whereas comparisons with the nucleotide sequences of rat gamma- and mouse epsilon-casein mRNAs also reveal extensive sequence similarities. An alignment of these four sequences shows that the divergence of their translated regions has been characterized by the duplication and deletion of discrete segments of sequence that probably correspond to exons. A high degree of nucleotide substitution is also found when the four sequences are compared, except for well-conserved leader-peptide and phosphorylation-site sequences and, to a lesser extent, the 5'-untranslated regions. Similar comparison of the bovine and rat beta-caseins shows that their divergence has involved a high rate of nucleotide substitution but that no major insertions or deletions of sequence have occurred. The several splice sites that have veen defined in the rat beta-casein gene are likely to have been conserved in the bovine. The contrasting evolutionary histories of the alpha- and beta-casein coding sequences correlate with the distinctive functions of these proteins in the casein micelle system in milk.      

Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background  

Paradoxical sleep deprivation (PSD) associated with cocaine has been shown to enhance genital reflexes (penile erection-PE and ejaculation-EJ) in Wistar rats. Since hypertension predisposes males to erectile dysfunction, the aim of the present study was to investigate the effects of PSD on genital reflexes in the spontaneously hypertensive rat (SHR) compared to the Wistar strain. We also extended our study to examine how PSD affect steroid hormone concentrations involved in genital events in both experimental models.     

Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background

The rate-limiting step in prostaglandin (PG) biosynthesis is catalyzed by phospholipase A2 (PLA2) enzymes which hydrolyze arachidonic acid from membrane phospholipids. Despite their importance in uterine PG production, little is known concerning the specific PLA2 enzymes that regulate arachidonic acid liberation in the uterine endometrium. The objectives of this study were to evaluate the expression and activities of calcium-independent Group VI and Group IVC PLA2 (PLA2G6 and PLA2G4C) and calcium-dependent Group IVA PLA2 (PLA2G4A) enzymes in the regulation of bovine uterine endometrial epithelial cell PG production.

Methods

Bovine endometrial epithelial cells in culture were treated with oxytocin, interferon-tau and the PLA2G6 inhibitor bromoenol lactone, alone and in combination. Concentrations of PGF2alpha and PGE2 released into the medium were analyzed. Western blot analysis was performed on cellular protein to determine the effects of treatments on expression of PLA2G4A, PLA2G6 and PLA2G4C. Group-specific PLA2 activity assays were performed on cell lysates following treatment with oxytocin, interferon-tau or vehicle (control), alone and in combination. To further evaluate the role of specific PLA2 enzymes in uterine cell PG biosynthesis, cells were transfected with cDNAs encoding human PLA2G6 and PLA24C, treated as described above and PG assays performed.

Results

Constitutive cell production of PGF2alpha was about two-fold higher than PGE2. Oxytocin stimulated production of both PGs but the increase of PGF2alpha was significantly greater. Interferon-tau diminished oxytocin stimulation of both PGs. The PLA2G6 inhibitor, bromoenol lactone, abolished oxytocin-stimulated production of PGF2alpha. Treatments had little effect on PLA2G4A protein expression. In contrast, oxytocin enhanced expression of PLA2G6 and this effect was diminished in the presence of interferon-tau. Expression of PLA2G4C was barely detectable in control and oxytocin treated cells but it was enhanced in cells treated with interferon-tau. Oxytocin stimulated PLA2 activity in assays designed to evaluate PLA2G6 activity and interferon-tau inhibited this response. In assays designed to measure PLA2G4C activity, only interferon-tau was stimulatory. Cells overexpressing PLA2G6 produced similar quantities of the two PGs and these values were significantly higher than PG production by non-transfected cells. Oxytocin stimulated production of both PGs and this response was inhibited by interferon-tau. Bromoenol lactone inhibited oxtocin stimulation of PGF2alpha production but stimulated PGE2 production, both in the absence and presence of oxytocin. Cells over-expressing PLA2G4C produced more PGE2 than PGF2alpha and interferon-tau stimulated PGE2 production.

Conclusion

Results from these studies indicate that oxytocin stimulation of uterine PGF2alpha production is mediated, at least in part, by up-regulation of PLA2G6 expression and activity. In addition to its known inhibitory effect on oxytocin receptor expression, interferon-tau represses oxytocin-stimulated PLA2G6 expression and activity and this contributes to diminished PGF2alpha production. Furthermore, endometrial cell PGE2 biosynthesis was associated with PLA2G4C expression and activity and interferon-tau was stimulatory to this process.     

Studies of genotoxicity and mutagenicity of nitroimidazoles: demystifying this critical relationship with the nitro group

Nitroimidazoles exhibit high microbicidal activity, but mutagenic, genotoxic and cytotoxic properties have been attributed to the presence of the nitro group. However, we synthesised nitroimidazoles with activity against the trypomastigotes of Trypanosoma cruzi, but that were not genotoxic. Herein, nitroimidazoles (11-19) bearing different substituent groups were investigated for their potential induction of genotoxicity (comet assay) and mutagenicity (Salmonella/Microsome assay) and the correlations of these effects with their trypanocidal effect and with megazol were investigated. The compounds were designed to analyse the role played by the position of the nitro group in the imidazole nucleus (C-4 or C-5) and the presence of oxidisable groups at N-1 as an anion receptor group and the role of a methyl group at C-2. Nitroimidazoles bearing NO2 at C-4 and CH3 at C-2 were not genotoxic compared to those bearing NO₂ at C-5. However, when there was a CH3 at C-2, the position of the NO2 group had no influence on the genotoxic activity. Fluorinated compounds exhibited higher genotoxicity regardless of the presence of CH3 at C-2 or NO2 at C-4 or C-5. However, in compounds 11 (2-CH3; 4-NO2; N-CH2OHCH2Cl) and 12 (2-CH3; 4-NO2; N-CH2OHCH2F), the fluorine atom had no influence on genotoxicity. This study contributes to the future search for new and safer prototypes and provide.     

Announcement

Abstract

The Gastrin content has been estimated in the antral mucosae of male and female rats in different phases of the estrous cycle. Gastrin content appears to be highest in the rats at di‐estrous cycle end lowest in the rats at pro‐estrous. There is not much difference between the gastrin content at estrous and pro‐estrous. The gastrin content in the antral mucosa of male rats is higher than in rats a both estrous and pro‐estrous but it is about the same amount in rats at di‐estrous. The possible role of the circulating sex hormones on the storage of gastrin in the antral mucosa of rats has been discussed.     

Costs and Cost-Effectiveness of Hypertension Screening and Treatment in Adults with Hypertension in Rural Nigeria in the Context of a Health Insurance Program

BackgroundHigh blood pressure is a leading risk factor for death and disability in sub-Saharan Africa (SSA). We evaluated the costs and cost-effectiveness of hypertension care provided within the Kwara State Health Insurance (KSHI) program in rural Nigeria.MethodsA Markov model was developed to assess the costs and cost-effectiveness of population-level hypertension screening and subsequent antihypertensive treatment for the population at-risk of cardiovascular disease (CVD) within the KSHI program. The primary outcome was the incremental cost per disability-adjusted life year (DALY) averted in the KSHI scenario compared to no access to hypertension care. We used setting-specific and empirically-collected data to inform the model. We defined two strategies to assess eligibility for antihypertensive treatment based on 1) presence of hypertension grade 1 and 10-year CVD risk of >20%, or grade 2 hypertension irrespective of 10-year CVD risk (hypertension and risk based strategy) and 2) presence of hypertension in combination with a CVD risk of >20% (risk based strategy). We generated 95% confidence intervals around the primary outcome through probabilistic sensitivity analysis. We conducted one-way sensitivity analyses across key model parameters and assessed the sensitivity of our results to the performance of the reference scenario.ResultsScreening and treatment for hypertension was potentially cost-effective but the results were sensitive to changes in underlying assumptions with a wide range of uncertainty. The incremental cost-effectiveness ratio for the first and second strategy respectively ranged from US$ 1,406 to US$ 7,815 and US$ 732 to US$ 2,959 per DALY averted, depending on the assumptions on risk reduction after treatment and compared to no access to antihypertensive treatment.ConclusionsHypertension care within a subsidized private health insurance program may be cost-effective in rural Nigeria and public-private partnerships such as the KSHI program may provide opportunities to finance CVD prevention care in SSA.