Blochkirby4639

Of the novel sub-chemotypes, α- and β-naphthyl analogs (33 and 27) exhibited sub micromolar antiviral potencies comparable to that of PF74. Interestingly, although only moderately inhibiting HIV-1 (single-digit micromolar EC50s), analogs of the 2-indolone sub-chemotype consistently lowered the melting point (Tm) of CA hexamers, some with improved metabolic stability over PF74.Advanced maternal age is an emerging health problem which involves many functional and structural alterations in oocytes, and its study is relevant to design better approaches to improve the reproductive function in women of advanced age. A constraint to this type of studies is the limited amount of samples and the ethical problems of working with human gametes. This study aims to characterize the in vitro-induced age-related modifications in a bovine model, as well as to determine if this model is a reliable approach to study human aging. For this purpose, we have focused on aging-related alterations related to oocyte mitochondrial dysfunction, a key hallmark in aging. Morphological and bioenergetic in vitro-induced alterations in bovine oocytes were compared to an in vivo aged group and to the already reported information regarding humans and other animal models. Parameters monitored included ooplasmic volume; mitochondrial mass, distribution and aggregation, assessed by MitoTracker Green; mitochondrial activity, monitored by JC-1; and the mitochondrial levels of hydrogen peroxide (H2O2), quantified using MitoPY. Results show a significant decrease in oocyte cytoplasmic volume after both in vitro and in vivo aging (p less then 0.001). Additionally, the levels of H2O2 increased significantly after in vitro and in vivo aging (p less then 0.001) and mitochondrial aggregation patterns were significantly different after 30 h of in vitro maturation, with MII oocytes presenting small aggregates inside the cytoplasm, whereas aged oocytes had a lack of granularity (p less then 0.001). In contrast, there were no differences between the different aging groups in terms of mitochondrial mass, distribution and activity. In conclusion, this in vitro approach of inducing aging-related alterations may be considered as a reliable approach to study the aging process in human female gametes, since it causes the same types of alterations in both species.C-type natriuretic peptide (CNP) and its natriuretic peptide receptors subtype 2 (NPR2) are essential for the maintenance of oocyte meiotic arrest in different species. Extracellular vesicles (EVs) in bovine follicular fluid (FF) are important for cell communication within the ovarian follicle. This study investigated the involvement of EVs from FF of bovine ovarian follicles in the CNP-NPR2 system, first by analyzing the presence of CNP in the EV contents, followed by addition of EVs to in-vitro maturation (IVM) medium, to evaluate the effect on maintenance of oocyte meiosis arrest and improvements in in-vitro embryo production. As expected, CNP was observed in FF and granulosa cells from the ovarian follicles. To the best of our knowledge, this is the first time that CNP has been found in the EV contents. To evaluate the possible effect of EVs on the progression of oocyte meiosis, the IVM was performed under three conditions CNP and EV supplementation and control condition. Both the CNP and EV treatments instem seems to be involved in modulating the cGMP levels, while the contents of EVs might be involved in modulating the cAMP levels.To more clearly understand the equine gonadotrope response to kisspeptin and gonadotropin releasing hormone (GnRH), peripheral LH and FSH were quantified in diestrous mares after treatment with either equine kisspeptide (eKp-10, 0.5 mg iv), GnRH (25 μg iv), or a combination thereof every 4 h for 3 days. The following observations were made 1) a diminished LH and FSH response to eKp-10 and GnRH was observed by Day 3, but was not different by treatment, 2) a decrease in basal LH concentration was observed from Day 1 to Day 3 for the eKp-10, but not the GnRH treated mares, 3) there was no change in basal FSH with either treatment. Additionally, pre-treatment with GnRH antagonist (antide 1.0 mg iv) eliminated any measurable change in LH after eKp-10 (1.0 mg iv) treatment. Both GnRH and kisspeptin are Gαq/11 coupled receptors, therefore quantifying the rise in intracellular calcium following treatment with cognate ligand allows simultaneous assessment of receptor activation. Direct stimulation of equine primary pituitary cells with GnRH and/or eKp-10 demonstrates three distinct populations of pituitary cells one population responded to both eKp-10 and GnRH, a second, independent population, responded to only eKp-10, and a third population responded only to GnRH. These populations were confirmed using co-immunofluorescence of hemipituitaries from mares in diestrus. Although the rise in peripheral LH concentration elicited by eKp-10 is dependent on GnRH, this work suggests that kisspeptin also has a specific and direct effect on the equine gonadotrope, independent of GnRH.Studies on adipokines, substances that are produced in adipose tissue, indicate that they influence both metabolism and reproduction. GLPG0634 molecular weight Chemerin is a novel addition to the adipokine family. It is believed that chemerin receptors are expressed in different structures of the hypothalamic-pituitary-gonadal (HPG) axis, which are crucial for endocrine control of reproductive functions, including the pituitary. The aim of this study was to investigate the expression of chemerin receptors (CMKLR1, GPR1, CCRL2) genes and proteins in the porcine pituitary. The effect of chemerin on MAPK/Erk1/2, Akt and AMPK signalling pathways was also investigated. The anterior (AP) and posterior (PP) lobes of the pituitary were examined on days 2 to 3, 10 to 12, 14 to 16, and 17 to 19 of the oestrous cycle and on days 10 to 11, 12 to 13, 15 to 16, and 27 to 28 of pregnancy. This is the first study to demonstrate that CMKLR1, GPR1 and CCRL2 are expressed in the porcine AP and PP, which implies that this gland is sensitive to chemerin action. The expression of the studied chemerin receptors fluctuated during different phases of the cycle and early gestation, which could be related to changes in the endocrine status of female pigs. The study also revealed that CMKLR1 and CCRL2 proteins were present in gonadotrophs and thyrotrophs, whereas CCRL2 was also present in somatotrophs, during the cycle and early pregnancy. We observed that chemerin affected MAPK/Erk1/2, Akt and AMPK signalling pathways in the porcine AP. These results suggest that chemerin may participate in the regulation of reproductive functions at the level of the pituitary.Studies were conducted to evaluate an optimal concentration of roscovitine needed to maintain abattoir origin oocytes at germinal vesicle stage in experiment 1 and their subsequent maturation and developmental competence after chemical activation in experiments 2 and 3, respectively. The cumulus-oocyte complexes (COCs) aspirated from ovaries collected from a local slaughterhouse were cultured in TCM-199 based pre-maturation medium supplemented with 25, 50 or 75 μM roscovitine, depending on the experimental group. After 24 h, the COCs were denuded of cumulus, fixed and stained with aceto-orcein and examined for their nuclear status. They were classified as germinal vesicle, diakinesis, metaphase-I, metaphase-II and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. In experiment 2, the COCs pre-matured in media supplemented with 50 μM roscovitine for 24 h were washed and kept for in vitro maturation along with another group of freshly collected COCs for 30 h. All trs in the pre-maturation group when compared with the group having freshly collected oocytes. In experiment 3, no difference was observed in the proportion of oocytes cleaving and those developing to the blastocyst stage between the pre-matured and freshly matured groups. In conclusion, the present study, for the first time, demonstrates the possible use of roscovitine as a meiotic inhibitor for camel oocytes. Keeping in view the ability of these oocytes to mature and develop to the blastocyst stage at par with the fresh oocytes, more flexible schedules for maturation and manipulation of such oocytes could be developed.Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide and targeted therapeutics exhibit limited success. Polo-like kinase 1 (PLK1), a Ser/Thr kinase, plays a pivotal role in cell-cycle regulation and is considered a promising target in HCC. Here, via structural optimization using both biochemical kinase assays and cellular antiproliferation assays, we discovered a potent and selective PLK1 kinase inhibitor, compound 31. Compound 31 exhibited biochemical activity with IC50 of less then 0.508 nM against PLK1 and a KINOMEscan selectivity score (S(1)) of 0.02 at a concentration of 1 μM. Furthermore, 31 showed broad antiproliferative activity against a variety of cancer cell lines, with the lowest antiproliferative IC50 (11.1 nM) in the HCC cell line HepG2. A detailed mechanistic study of 31 revealed that inhibition of PLK1 by 31 induces mitotic arrest at the G2/M phase checkpoint, thus leading to cancer cell apoptosis. Moreover, 31 exhibited profound antitumor efficacy in a xenograft mouse model. Collectively, these results establish compound 31 as a good starting point for the development of PLK1 targeted therapeutics for HCC.Fractalkine (FKN) and its specific receptor CX3CR1 play a critical role in the pathogenesis of atherosclerosis including recruitment of vascular cells and the development of inflammation. However, its contribution to regulating the development of atherosclerotic calcification has not been well documented. Osteogenic transformation of vascular smooth muscle cells (VSMCs) is critical in the development of calcification in atherosclerotic lesions. In this study, for the first time, we evaluated the effect of FKN/CX3CR1 on the progression of VSMCs calcification and defined molecular signaling that is operative in the FKN/CX3CR1-induced osteogenic transformation of VSMCs. We found that high-fat diet induced atherosclerotic calcification in vivo was markedly inhibited in the Apolipoprotein E (ApoE) and CX3CR1 deficient (ApoE-/-/CX3CR1-/-) mice compared with their control littermates. FKN and CX3CR1 were both expressed in VSMCs and up-regulated by oxidized low-density lipoprotein (ox-LDL). FKN/CX3CR1 promoted the exication by activating RUNX2 through Jak2/Stat3 signaling pathway and suppressing OPG. Our findings suggest that targeting FKN/CX3CR1 may provide new strategies for the prevention and treatment of atherosclerotic calcification.Red mud (RM) contains large quantities of microscale particles less then 1 μm and high concentrations of potentially toxic elements. In this research, we have used two types of RM of similar chemical properties but containing different quantities of micro-particles, to test whether their size plays a role in the uptake of chemical elements by earthworm Eisenia fetida. Earthworms were exposed for seven days to artificial soils (prepared in the laboratory following a protocol) amended with increasing quantities of RM. Mortality of 86 % occurred when earthworms were exposed to amended soil containing 46 % of particles below 1 μm. Surprisingly, tissue analyses have shown decreased concentrations of metals instead of the expected toxic effect. SEM analysis revealed that micro-particles strongly adhere to the earthworm epidermis putting them under the large stress. Micro-particles in RM clog their minute dermal pores of 90 nm-735 nm in diameter, which size depends on whether the earthworm's body is contracted or stretched.