Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.     

Decreased expression of high-molecular-weight calmodulin-binding protein and its correlation with apoptosis in ischemia-reperfused rat heart

A cardiac high-molecular-weight calmodulin-binding protein (HMWCaMBP) was previously identified as a homologue of the calpain inhibitor, calpastatin. In the present study, we investigated the expression of HMWCaMBP and calpains in rat heart after ischemia and reperfusion. Western blot analysis of normal rat heart extract with a polyclonal antibody raised against bovine HMWCaMBP indicated a prominent immunoreactive band of 140kDa. Both the expression and the activity of HMWCaMBP were decreased by ischemia reperfusion. Immunohistochemical studies showed strong-to-moderate HMWCaMBP immunoreactivity in normal heart and poor immunoreactivity in ischemia-reperfused heart muscle. However, the expression of micro-calpain and m-calpain in ischemia-reperfused heart was increased as compared to normal heart. The calpain inhibitory activity of ischemia-reperfused heart tissues was significantly lower as compared to normal heart tissues. The pre-ischemic and post-ischemic perfusion of hearts with a cell-permeable calpain inhibitor suppressed the increase in calpain expression but increased the HMWCaMBP expression. In-vitro HMWCaMBP was proteolyzed by micro-calpain and m-calpain. We also measured apoptosis in normal and ischemia-reperfused tissues. An increase in the number of apoptotic bodies was observed with increased duration of ischemia and reperfusion. Bcl-2 expression did not change in any of the groups, whereas Bax expression increased with ischemia-reperfusion and correlated well with the degree of apoptosis. Our findings suggest that HMWCaMBP may sequester calpains from its substrates in the normal myocardium, but it is susceptible to proteolysis by calpains during ischemia-reperfusion. Thus, decreased expression of HMWCaMBP may play an important role in myocardial injury.     

Synthesis, structural elucidation, DNA-PK inhibition, homology modelling and anti-platelet activity of morpholino-substituted-1,3-naphth-oxazines

A number of new angular 2-morpholino-(substituted)-naphth-1,3-oxazines (compound 10b), linear 2-morpholino-(substituted)-naphth-1,3-oxazines (compounds 13b-c), linear 6, 7 and 9-O-substituted-2-morpholino-(substituted)-naphth-1,3-oxazines (compounds 17-22, 24, and 25) and angular compounds 14-16 and 23 were synthesised. The O-substituent was pyridin-2yl-methyl (15, 18, and 21) pyridin-3yl-methyl (16, 19, and 22) and 4-methylpipreazin-1-yl-ethoxy (23-25). Twelve compounds were tested for their inhibitory effect on collagen induced platelet aggregation and it was found that the most active compounds were compounds 19 and 22 with IC(50)=55±4 and 85±4 μM, respectively. Furthermore, the compounds were also assayed for their ability to inhibit DNA-dependent protein kinase (DNA-PK) activity. The most active compounds were 18 IC(50)=0.091 μM, 24 IC(50)=0.191 μM, and 22 IC(50)=0.331 μM. Homology modelling was used to build a 3D model of DNA-PK based on the X-ray structure of phosphatidylinositol 3-kinases (PI3Ks). Docking of synthesised compounds within the binding pocket and structure-activity relationships (SAR) analyses of the poses were performed and results agreed well with observed activity.     

Alterations in sucrose metabolizing enzyme activities and total phenol content of Curcuma Ionga L. as affected by different triazole compounds

Changes in the sucrose metabolism of Cur-cuma longa L. plants were studied under treatment with different triazole compounds viz., triadimefon (TDM) and propiconazole (PCZ). Plants were treated with TDM at 15 mg/L and PCZ at 10 mg/L separately by soil drenching on 80, 110, and 140 days after planting (DAP). The plants were harvested randomly on 90, 120, and 150DAP to determine the effect of both the triazoles on sucrose metabolizing enzymes and phenol content. The sucrose metabolism was studied by analyzing sucrose metaboliz-ing enzymes like sucrose synthase and sucrose phosphate synthase. All the analyses were assayed in leaves and tubers of both control and treated plants. It was found that both of the triazole compounds had profound effects on these parameters.     

Some aspects of the biology of a cyprinid, Aspius vorax Heckel

Scales were used for the determination of age with back-calculation of length. The oldest fish was VII + years old. Back-calculation did not exhibit Lee's phenomenon. The most rapid growth occurred in summer at water temperatures over 25°C. The growth in weight was c . 331 g year^-1 after IV vears of life. Growth was well described by von Bertalanffy equation : l_l - 91·0 [ l= ^{0·122(l0·25)}] The length-weight relationship followed the cube law (b = 3·0601) K_n ranged from 0·74 to 1·18 with a mean value of 1·0. Spawning occurred in January, fecundity was 74 509 with a mean of 1157 eggs -¹ body weight. Mean diameter of eggs was 1071 (pm). A developed ovary had ova of two sizes, immature oocytes and mature ova. The fish is a carnivorous feeder.     

Renal function in the freshwater rainbow trout after dietary cadmium acclimation and waterborne cadmium challenge

Renal function was examined in adult rainbow trout (Oncorhynchus mykiss) after chronic exposure to a sublethal level of dietary Cd (500 mg/kg diet) for 52 d and during a subsequent challenge to waterborne Cd (10 microg/L) for 72 h. Dietary Cd had no major effects on UFR (urine flow rate) and GFR (glomerular filtration rate) but caused increased renal excretion of glucose, protein, and major ions (Mg(2+), Zn(2+), K(+), Na(+), Cl(-) but Ca(2+)). However, dietary Cd did not affect any plasma ions except Na(+) which was significantly elevated in the Cd-acclimated trout. Plasma glucose and ammonia levels fell by 25% and 36% respectively, but neither plasma nor urine urea was affected in Cd-acclimated fish. Dietary Cd exposure resulted in a remarkable increase of Cd load in the plasma (48-fold, approximately 22 ng/mL) and urine (60-fold, 8.9 ng/mL), but Cd excretion via the kidney was negligible on a mass-balance basis. Clearance ratio analysis indicates that all ions, Cd, and metabolites were reabsorbed strongly (58-100%) in both na?ve and dietary Cd exposed fish, except ammonia which was secreted in both groups. Mg(2+), Na(+), Cl(-) and K(+) reabsorption decreased significantly (3-15%) in the Cd-exposed fish relative to the control. Following waterborne Cd challenge, GFR and UFR were affected transiently, and only Mg(2+) and protein excretion remained elevated with no recovery with time in Cd-acclimated trout. Urinary Ca(2+) and Zn(2+) excretion rates dropped with an indication of renal compensation towards plasma declines of both ions. Cadmium challenge did not cause any notable effects on urinary excretion rates of metabolites. However, a significant decrease in Mg(2+) reabsorption but an increase in total ammonia secretion was observed in the Cd-acclimated fish. The study suggests that dietary Cd acclimation involves physiological costs in terms of renal dysfunction and elevated urinary losses.     

Visualization of Mycobacterium avium in Crohn's tissue by oil-immersion microscopy

Studies seeking Mycobacterium avium subsp. paratuberculosis in Crohn's disease by PCR have generated inconsistent findings. As an alternative, microscopy offers a number of advantages, including direct visualization of organisms in tissue. Experimental infections have demonstrated that M. avium organisms can be seen by both acid-fast staining and species-specific in situ hybridization, but because they are smaller than M. tuberculosis, oil-immersion microscopy (x 1,000 magnification) is needed. We performed a blinded search for M. avium in paraffin-embedded surgical resections from Crohn's and control subjects at two centres. Specimens were coded and subjected to acid-fast staining and ribosomal RNA in situ hybridization for M. avium rRNA. Agreement between these two methods was good (42/52 patients, kappa=0.60) and similar results were observed for patients from two centers. Together, both methods provided positive results in 10 of 17 Crohn's subjects (59%, 95% CI: 36-78), contrasting with only 5 of 35 control subjects (Odds ratio for Crohn's vs. controls=8.6, p=0.002). M. avium organisms had an intracellular localization within inflammatory lesions, but were often observed as lone organisms outside of granulomas. Using two assays in two settings, presence of M. avium organisms was strongly associated with Crohn's disease.     

Oral health status in elders from South Brazil: a population-based study

doi: 10.1111/j.1741‐2358.2011.00617.x Oral health status in elders from South Brazil: a population‐based study Objective: To assess the oral health status of community‐dwelling adults aged 60 years and older from southern Brazil and to determine demographics, socioeconomic, behavioural and dental risk indicators. Materials and methods: This cross‐sectional study used a multistage, probability sampling method to draw a representative sample of the metropolitan area of Porto Alegre, Brazil. A subsample of 217 subjects was included in this analysis. Oral mucosal lesions, dental caries, tooth loss and periodontal status (full‐mouth, six sites per tooth exam) were assessed by calibrated examiners. Results: Prevalence of edentulism was 39.5%, and mean tooth loss was 20.2 (SE = 0.6). Older individuals [Odds Ratio (OR) = 2.2], women (OR = 2.3), white people (OR = 5.9), individuals of lower socioeconomic status (OR = 5.6) and smokers (OR = 3.5) had higher likelihood of being edentulous. Approximately 36% of dentate individuals had caries and/or restoration affecting, in average, 5.0 teeth. Periodontitis affected 79% of subjects, and it was associated with older age (OR = 4.0), men (OR = 3.4) and large amounts of supragingival plaque (OR = 3.0). Conclusion: Poor oral health was observed in this elderly population from South Brazil. Sociodemographic disparities accounted for most of the burden of disease and treatment needs.     

Antioxidant defense responses: physiological plasticity in higher plants under abiotic constraints

Environmental stresses (salinity, drought, heat/cold, light and other hostile conditions) may trigger in plants oxidative stress, generating the formation of reactive oxygen species (ROS). These species are partially reduced or activated derivatives of oxygen, comprising both free radical and non-radical (H₂O₂) forms, leading to cellular damage, metabolic disorders and senescence processes. In order to overcome oxidative stress, plants have developed two main antioxidants defense mechanisms that can be classified as non-enzymatic and enzymatic systems. The first class (non-enzymatic) consists of small molecules such as vitamin (A, C and E), glutathione, carotenoids and phenolics that can react directly with the ROS by scavenging them. Second class is represented by enzymes among them superoxide dismutase, peroxidase and catalase which have the capacity to eliminate superoxide and hydrogen peroxide. In this review, we have tried to explore the related works, which have revealed the changes in the basic antioxidant metabolism of plants under various abiotic constraints.