Simulation of intrafollicular conditions prevents GVBD in bovine oocytes: a better alternative to affect their developmental capacity after two-step culture

To increase the developmental competence of bovine oocytes isolated from small, medium, and large follicles (2-3, 3-4, and 4-6 mm in diameter, respectively), we tried to modify the conditions for their in vitro culture. The first step involved conditions maintaining at least for 48 hr a reversible inhibition of the germinal vesicle breakdown (GVBD) and the second step stimulated the resumption of meiosis and completion of nuclear and cytoplasmic maturation during the subsequent 20-22 hr of culture. The effectiveness of this model depended mainly on the medium composition (reduced NaHCO3, substitution of serum with serum albumin, addition of antioxidants (curcumin), increased viscosity by agar, the reduction of oxygen concentration (within 6%-8%), the reduction of the proportion between the number of cumulus-oocyte complexes (COCs), and the reduction of the amount of a medium (within 6-7 mul per COC) to amplify the GVBD-inhibitory effect of oocyte surrounding granulosa cells. The COCs were incubated in clumps of 6-7 COCs. The effectiveness and reversibility of GVBD inhibition depended also on the duration of COCs isolation. The full reversibility of the GV block was controlled morphologically and also by measuring histone H1 and MAP kinase activities. The two-step versus one-step (24 hr) maturation technique was evaluated by the percentage of total and hatched blastocysts at day 9. When compared with one-step maturation, the two-step culture showed a slightly increased proportion of total and hatched blastocysts developed from growing follicles, mainly from the smallest category (13.9% vs. 7.1% and 9.2% vs. 3.3% for total blastocysts and hatched, respectively). However, the two-step culture of oocytes from large regressing follicles substantially reduced the blastocyst yield (9.7% vs. 39.1% and 4.9% vs. 26.7% for total blastocysts and hatched, respectively). The transfer of ten blastocysts (developed after two-step culture) to ten recipients resulted in seven pregnancies.     

Circulating beta-chemokines and matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 system in hemodialyzed patients--role of oxidative stress

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), beta-chemokines, increased oxidative stress (SOX) and inflammation have been implicated as important factors in atherosclerosis and vascular remodeling. We hypothesized the possible roles of beta-chemokines [monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory proteins (MIP-1alpha, MIP-1beta) and regulated upon activation, normal T-cell expressed and secreted (RANTES)] as regulators of the metabolism of the vascular extracellular matrix in conditions of increased SOX in hemodialysis (HD) patients. We compared pre-dialysis levels of MMP-9/TIMP-1 system, beta-chemokines, Cu/Zn superoxide dismutase (Cu/Zn SOD) as a marker of SOX and C-reactive protein (CRP) as a marker of inflammation in HD patients with and without cardiovascular disease (CVD) to those of controls. HD patients, particularly those with CVD, showed a significant increase in values of Cu/Zn SOD, CRP, TIMP-1, TIMP-1/MMP-9 ratio, MCP-1 and MIP-1beta, whereas RANTES levels were lower than in the controls. The levels of MIP-1alpha as well as MMP-9 in all HD groups were similar to the controls. The positive correlations were observed between the MMP-9/TIMP-1 system and beta-chemokines, SOX and inflammation in whole HD group and in the subgroup with CVD. Multivariate analysis showed that the duration of dialysis followed by Cu/Zn SOD, MIP-1alpha and beta levels were the significant positive predictors of TIMP-1. In conclusion, our data show that MMP-9/TIMP-1 system and beta-chemokines could cooperate in conditions of elevated SOX, which ultimately predisposes hemodialysis patients to accelerated atherosclerosis.     

Effects of triple antioxidant combination (vitamin E, vitamin C and alpha-lipoic acid) with insulin on lipid and cholesterol levels and fatty acid composition of brain tissue in experimental diabetic and non-diabetic rats

The aim of this research was to examine the effects of a triple antioxidant combination (vitamins E (VE) and C (VC) plus alpha-lipoic acid (LA)) on the total lipid and cholesterol levels and the fatty acid composition of brain tissues in experimental diabetic and non-diabetic rats. VE and LA were injected intraperitoneally (50 mg/kg) four times per week and VC was provided as a supplement dissolved in the drinking water (50 mg/kg). In addition, rats in the diabetes 1 and D+VELAVC groups were given daily by subcutaneous insulin injections (8 IU/kg), but no insulin was given to rats in the diabetes 2 group. The results indicate that the brain lipid levels in the D+VELAVC, diabetes 1 and diabetes 2 groups were higher than in the control group (P<0.01). Total lipid was also higher in the non-diabetic rats treated with LA and VC. Total cholesterol was higher in the diabetes 1 and diabetes 2 groups (P<0.05) than in controls. Cholesterol levels were similar in the D+VELAVC and LA groups but lower in the VC, VE and VELAVC groups of non-diabetic rats (P<0.05 and P<0.01). In respect of fatty acid composition, palmitic acid levels were lower in the diabetes 2 and non-diabetic VE groups than the control group (P<0.05), but higher in the non-diabetic LA group (P<0.05). Oleic acid (18:1 n-9) levels were lower in the diabetic and non-diabetic groups than the control group (P<0.01), but higher in the non-diabetic LA group. Arachidonic acid (20:4 n-6) levels were similar in the diabetes 1, D+VELAVC and control groups (P>0.05) but higher in the non-diabetic VE, VC, LA and VEVCLA groups (P<0.05) and lower in the diabetes 2 group (P<0.05). Docosahexaenoic acid (22:6 n-3) was elevated in the diabetes 2 and VEVCLA groups (P<0.01, P<0.05). In conclusion, the current study confirmed that treatment with a triple combination of VE, VC and LA protects the arachidonic acid level in the brains of diabetic and non-diabetic rats.     

Calreticulin, a Ca2+-binding chaperone of the endoplasmic reticulum

Calreticulin is a 46-kDa Ca2+-binding chaperone found across a diverse range of species. The protein is involved in the regulation of intracellular Ca2+ homeostasis and endoplasmic reticulum (ER) Ca2+ storage capacity. Calreticulin is also an important molecular chaperone involved in "quality control" within secretory pathways. The protein contains structurally and functionally unique domains with specialized functions. Studies on calreticulin knockout mice indicate that the protein is essential in early cardiac development. The protein also plays an important role in autoimmunity and cancer.     

Predicting fold novelty based on ProtoNet hierarchical classification

MOTIVATION: Structural genomics projects aim to solve a large number of protein structures with the ultimate objective of representing the entire protein space. The computational challenge is to identify and prioritize a small set of proteins with new, currently unknown, superfamilies or folds. RESULTS: We develop a method that assigns each protein a likelihood of it belonging to a new, yet undetermined, structural superfamily. The method relies on a variant of ProtoNet, an automatic hierarchical classification scheme of all protein sequences from SwissProt. Our results show that proteins that are remote from solved structures in the ProtoNet hierarchy are more likely to belong to new superfamilies. The results are validated against SCOP releases from recent years that account for about half of the solved structures known to date. We show that our new method and the representation of ProtoNet are superior in detecting new targets, compared to our previous method using ProtoMap classification. Furthermore, our method outperforms PSI-BLAST search in detecting potential new superfamilies.     

Effectiveness of statins for secondary prevention in elderly patients after acute myocardial infarction: an evaluation of class effect

Background

Clinical trials have shown the benefits of statins after acute myocardial infarction (AMI). However, it is unclear whether different statins exert a similar effect in reducing the incidence of recurrent AMI and death when used in clinical practice.

Methods

We conducted a retrospective cohort study (1997–2002) to compare 5 statins using data from medical administrative databases in 3 provinces (Quebec, Ontario and British Columbia). We included patients aged 65 years and over who were discharged alive after their first AMI-related hospital stay and who began statin treatment within 90 days after discharge. The primary end point was the combined outcome of recurrent AMI or death from any cause. The secondary end point was death from any cause. Adjusted hazard ratios (HRs) for each statin compared with atorvastatin as the reference drug were estimated using Cox proportional hazards regression analysis.

Results

A total of 18 637 patients were prescribed atorvastatin (n = 6420), pravastatin (n = 4480), simvastatin (n = 5518), lovastatin (n = 1736) or fluvastatin (n = 483). Users of different statins showed similar baseline characteristics and patterns of statin use. The adjusted HRs (and 95% confidence intervals) for the combined outcome of AMI or death showed that each statin had similar effects when compared with atorvastatin: pravastatin 1.00 (0.90–1.11), simvastatin 1.01 (0.91– 1.12), lovastatin 1.09 (0.95–1.24) and fluvastatin 1.01 (0.80– 1.27). The results did not change when death alone was the end point, nor did they change after adjustment for initial daily dose or after censoring of patients who switched or stopped the initial statin treatment.

Interpretation

Our results suggest that, under current usage, statins are equally effective for secocondary prevention in elderly patients after AMI.Randomized controlled trials (RCTs) have shown that the use of statins after acute myocardial infarction (AMI) are effective in reducing the incidence of both fatal and nonfatal cardiovascular events.^{1,2,3,4,5,6,7,8} Although these trials have significantly influenced post-AMI treatment,^9,10,11,12 it remains unclear whether all statins are equally effective in preventing recurrent AMI and death. Drugs in the same class are generally thought to be therapeutically equivalent because of similar mechanisms of action (class effect).^13,14,15 However, in the absence of comparative data, this assumption requires evaluation. Statins differ in multiple characteristics, including liver and renal metabolism, half-life, effect on other serum lipid components, bioavailability and potency.^16,17,18,19 These differences could potentially influence the extent to which the drugs are beneficial. Despite limited evidence in support of a differential benefit of statins for secondary prevention, preferential prescribing already occurs in practice and cannot be fully explained by the existing evidence or guidelines.²⁰ Comparative data of statins are thus required to inform health care decision-making.A number of RCTs have directly compared statins using surrogate end points, such as lipid reduction,^21,22,23 markers of hemostasis and inflammation^24,25,26 or reduction in number of atherotic plaques.²⁷ However, the extent to which these results can be extrapolated to clinically relevant outcomes remains to be established. The newly released PROVE IT– TIMI 22 trial²⁸ was the first trial to compare 2 statins for cardiovascular prevention. The study showed that atorvastatin used at a maximal dose of 80 mg (intensive therapy) was better than pravastatin at a dose of 40 mg (standard therapy) in decreasing the incidence of cardiovascular events and procedures. The study was, however, conducted to show the benefit associated with increased treatment intensity. It did not compare the drugs by milligram-equivalent doses or by cholesterol-lowering equivalent doses. Moreover, no difference was detected when death alone or the combined outcome of death or AMI was evaluated. Other than the PROVE IT–TIMI 22 trial, few data are currently available from RCTs that compare statins for cardiovascular prevention.²⁹We conducted a population-based study to examine the relative effectiveness of different statins for long-term secondary prevention after AMI. We used retrospective cohorts of elderly patients prescribed statins after AMI in 3 provinces. Five statins were studied: atorvastatin, pravastatin, simvastatin, lovastatin and fluvastatin. The newest statin, rosuvastatin, was not available during the study period and was not considered in this study.     

Hyphal growth and mycorrhiza formation by the arbuscular mycorrhizal fungus Glomus claroideum BEG 23 is stimulated by humic substances

Effects of humic substances (humic acid or fulvic soil extract) or saprophytic microorganisms (Paecilomyces lilacinus and an unidentified actinomycete) on growth of mycelium and mycorrhiza formation by Glomus claroideum BEG23 were studied in a hydroponic system. Humic substances stimulated root colonization and production of extraradical mycelium by the mycorrhizal fungus. Both humic and fulvic acids tended to decrease populations of culturable bacteria and fungi in the cultivation system, indicating a moderately antibiotic activity. The addition of saprophytic microorganisms able to use humic substances to the cultivation system further stimulated the development of the mycorrhizal fungus. However, stimulation of G. claroideum was also observed when the saprophytic microorganisms were heat-killed, suggesting that their effect was not linked to a specific action on humic substances. The results indicate that humic substances may represent a stimulatory component of the soil environment with respect to arbuscular mycorrhizal fungi.     

Environmental origins of sexually selected variation and a critique of the fluctuating asymmetry-sexual selection hypothesis

Identifying sources of phenotypic variability in secondary sexual traits is critical for understanding their signaling properties, role in sexual selection, and for predicting their evolutionary dynamics. The present study tests for the effects of genotype, developmental temperature, and their interaction, on size and fluctuating asymmetry of the male sex comb, a secondary sexual character, in Drosophila bipectinata Duda. Both the size and symmetry of elements of the sex comb have been shown previously to be under sexual selection in a natural population in northeastern Australia. Two independent reciprocal crosses were conducted at 25 degrees and 29 degrees C between genetic lines extracted from this population that differed in the size of the first (TC1) and third (TC3) comb segments. These temperatures are within the documented range experienced by the species in nature. Additive and dominance genetic effects were detected for TC1, whereas additive genetic, and Y-chromosomal effects were detected for TC3. TC2 and TC3 decreased sharply with increasing temperature, by 10% and 22%, respectively. In contrast, positional fluctuating asymmetry (PFA) significantly increased with temperature, by up to 38%. The results (1) document an important source of environmental variance in a sexual ornament expected to reduce trait heritability in field populations, and thus act to attenuate response to sexual selection, (2) suggest that variation in ornament size reflects differences in male condition; and (3) support the general hypothesis that asymmetry in a sexual ornament is indicative of developmental instability arising from environmental stress. The "environmental heterogeneity" (EH) hypothesis is proposed, and supportive evidence for it presented, to explain negative size-FA correlations in natural populations. Data and theory challenge the use of negative size-FA correlations observed in nature to support the FA-sexual selection hypothesis, which posits that such correlations are driven by differences in genetic quality among individuals.