Insulin and insulin-like growth factor I in follicular fluid after induction of ovulation in women undergoing in vitro fertilization.

This study was undertaken to evaluate the relationship between concentrations of insulin and insulin-like growth factor I (IGF-I) in follicular fluid and fertilization and cleavage of human oocytes fertilized in vitro. The concentration of oestradiol, progesterone, luteinizing hormone, follicle-stimulating hormone, testosterone, insulin and IGF-I was determined in 36 follicular fluids, free of visible blood contamination and containing mature oocyte-corona-cumulus complexes, obtained from 12 women undergoing in vitro fertilization. Follicular development was induced by clomiphene citrate and human menopausal gonadotrophin, and follicular aspiration was performed 35 h after an ovulatory dose of human chorionic gonadotrophin. Concentrations of IGF-I were significantly higher in follicular fluids associated with mature oocytes that fertilized and cleaved, than in follicular fluid associated with mature oocytes that did not fertilize (P < 0.001). There was no difference in the concentration of insulin between follicular fluids from which fertilized oocytes were obtained and those with oocytes that remained unfertilized. No significant correlations were found between rates of embryo cleavage, concentrations of insulin and IGF-I. Multiple linear regression analysis demonstrated that the concentrations of IGF-I in follicular fluid were predicted statistically by a negative regression coefficient for the concentration of testosterone, and by a positive regression coefficient for the concentration of progesterone in follicular fluid. No candidate variable was included in the model to predict concentrations of insulin. These data suggest an important role for IGF-I in the mature follicle.     

Humanin inhibits apoptosis in pituitary tumor cells through several signaling pathways including NF-κB activation

Humanin (HN) and Rattin (HNr), its homologous in the rat, are peptides with cytoprotective action in several cell types such as neurons, lymphocytes and testicular germ cells. Previously, we have shown that HNr is expressed in pituitary cells and that HN inhibited the apoptotic effect of TNF-α in both normal and tumor pituitary cells. The aim of the present study was to identify signaling pathways that mediate the antiapoptotic effect of HN in anterior pituitary cells from ovariectomized rats and in GH3 cells, a somatolactotrope cell line. We assessed the role of STAT3, JNK, Akt and MAPKs as well as proteins of the Bcl-2 family, previously implicated in the antiapoptotic effect of HN. We also evaluated the participation of NF-κB in the antiapoptotic action of HN. STAT3 inhibition reversed the inhibitory effect of HN on TNF-α-induced apoptosis in normal and pituitary tumor cells, indicating that STAT3 signaling pathway mediates the antiapoptotic effect of HN on pituitary cells. Inhibition of NF-κB pathway did not affect action of HN on normal anterior pituitary cells but blocked the cytoprotective effect of HN on TNF-α-induced apoptosis of GH3 cells, suggesting that the NF-κB pathway is involved in HN action in tumor pituitary cells. HN also induced NF-κB-p65 nuclear translocation in these cells. In pituitary tumor cells, JNK and MEK inhibitors also impaired HN cytoprotective action. In addition, HN increased Bcl-2 expression and decreased Bax mitochondrial translocation. Since HN expression in GH3 cells is higher than in normal pituitary cells, we may suggest that through multiple pathways HN could be involved in pituitary tumorigenesis.     

Phenotypic plasticity in breeding plumage signals in both sexes of a migratory bird: responses to breeding conditions

Adaptive phenotypic plasticity may respond to present ambient conditions. Sexual and social signals in both sexes may express phenotype performance. Plumage signals that change discontinuously allow relating discrete variation to previous performance. Both sexes of the pied flycatcher Ficedula hypoleuca present white patches on the wings and on the forehead, which constitute sexual and social signals. Forehead patches are moulted together with body plumage in Africa, while wing patches are partly moulted in Africa and partly in the breeding area soon after breeding. We studied individual inter‐year changes (corrected for regression to the mean) in the size of forehead and wing patches of both sexes in seven years for females or six years for males in two nearby study areas in central Spain. We found that initial signal extent strongly delimits the possible subsequent changes negatively. There is a negative association of male age with forehead patch changes. Cold and rainy springs are associated in females with decreases in both patch areas and vice versa, while no association with climate is observed in male wing patch changes. Cold pre‐breeding conditions predict positive changes in female wing and male forehead patches. Breeding success is positively associated with forehead patch changes in females. Late‐breeding males experience more positive changes in forehead patch size than early‐breeding males. Some of these trends can be explained by variable costs of breeding in certain conditions for subsequent signal production and/or maintenance, while absence of trends in some cases may be explained by sex differences in costs of breeding and interactions with phenotypic quality of breeders.     

The -308 polymorphism in the promoter region of the tumor necrosis factor-alpha (TNF-alpha) gene and ex vivo lipopolysaccharide-induced TNF-alpha expression in patients with aggressive periodontitis and/or type 1 diabetes mellitus

Several single-nucleotide polymorphisms (SNPs) have been identified in the TNF-alpha gene promoter. The transition G-->A at position -308 generates the TNF-alpha1 (G/G) and TNF-alpha2 (G/A or A/A) alleles, where the polymorphic TNF-alpha2 allele is associated with a high, in vitro TNF-alpha expression and an increased susceptibility to diverse illnesses. Here we study the association of the -308 TNF-alpha SNP with the susceptibility for developing aggressive periodontitis (AP), AP combined with type 1 diabetes mellitus (DM) and DM. We also explore the TNF-alpha capability expression and the presence of the -308 polymorphism. For this purpose we recruited 27 individuals with AP (AP+ group), 27 individuals with AP combined with DM (AP+/DM+ group), and 27 individuals with DM without signs of periodontitis upon clinical examination (DM+ group). The control group was comprised of 30 subjects. Genotyping for TNF-alpha promoter was performed by PCR-RFLP analysis. For TNF-alpha expression we used a blood culture system.     

Characterization of a trap mutant of the AAA+ chaperone ClpB

The AAA+ protein ClpB mediates the solubilization of protein aggregates in cooperation with the DnaK chaperone system (KJE). The order of action of ClpB and KJE on aggregated proteins is unknown. We describe a ClpB variant with mutational alterations in the Walker B motif of both AAA domains (E279A/E678A), which binds but does not hydrolyze ATP. This variant associates in vitro and in vivo in a stable manner with protein substrates, demonstrating direct interaction of ClpB with protein aggregates for the first time. Substrate interaction is strictly dependent on ATP binding to both AAA domains of ClpB. The unique substrate binding properties of the double Walker B variant allowed to dissect the order of ClpB and DnaK action during disaggregation reactions. ClpB-E279A/E678A outcompetes the DnaK system for binding to the model substrate TrfA and inhibits the dissociation of small protein aggregates by DnaK only, indicating that ClpB acts prior to DnaK on protein substrates.     

Roles of individual domains and conserved motifs of the AAA+ chaperone ClpB in oligomerization,ATP hydrolysis,and chaperone activity

ClpB of Escherichia coli is an ATP-dependent ring-forming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the Hsp100/Clp subfamily of AAA+ proteins and is composed of an N-terminal domain and two AAA-domains that are separated by a "linker" region. Here we present a detailed structure-function analysis of ClpB, dissecting the individual roles of ClpB domains and conserved motifs in oligomerization, ATP hydrolysis, and chaperone activity. Our results show that ClpB oligomerization is strictly dependent on the presence of the C-terminal domain of the second AAA-domain, while ATP binding to the first AAA-domains stabilized the ClpB oligomer. Analysis of mutants of conserved residues in Walker A and B and sensor 2 motifs revealed that both AAA-domains contribute to the basal ATPase activity of ClpB and communicate in a complex manner. Chaperone activity strictly depends on ClpB oligomerization and the presence of a residual ATPase activity. The N-domain is dispensable for oligomerization and for the disaggregating activity in vitro and in vivo. In contrast the presence of the linker region, although not involved in oligomerization, is essential for ClpB chaperone activity.