Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells

Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo-pituitary-adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF(1) receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF(2) receptor) with little or no binding affinity for the CRF(1) receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF(1) receptor and CRF(2) receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF(1) receptor, while Ucn2 increased the levels via the CRF(2) receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.     

Structural and thermodynamic consequences of removal of a conserved disulfide bond from equine beta-lactoglobulin

A disulfide bond between cysteine 66 and cysteine 160 of equine beta-lactoglobulin was removed by substituting cysteine residues with alanine. This disulfide bond is conserved across the lipocalin family. The conformation and stability of the disulfide-deleted mutant protein was investigated by circular dichroism. The mutant protein assumes a native-like structure under physiological conditions and assumes a helix-rich molten globule structure at acid pH or at moderate concentrations of urea as the wild-type protein does. The urea-induced unfolding experiment shows that the stability of the native conformation was reduced but that of the molten globule intermediate is not significantly changed at pH 4 by removal of the disulfide bond. On the other hand, the molten globule at acid pH was destabilized by removal of the disulfide bond. This difference in the stabilizing effect of the disulfide bond was interpreted by the effect of the disulfide in keeping the molecule compact against the electrostatic repulsion at acid pH. In contrast to the wild-type protein, the circular dichroism spectrum in the molten globule state at acid pH depends on anion concentration, suggesting that the expansion of the molecule through electrostatic repulsion induces alpha-helices as observed in the cold denatured state of the wild-type protein.     

WNT pathways in the neonatal ovine uterus: potential specification of endometrial gland morphogenesis by SFRP2

Endometrial glands are critical for uterine function and develop between birth (Postnatal Day [P] 0) and P56 in the neonatal ewe. Endometrial gland morphogenesis or adenogenesis involves the site-specific budding differentiation of the glandular epithelium from the luminal epithelium followed by their coiling/branching development within the stroma of the intercaruncular areas of the endometrium. To determine whether WNT signaling regulates endometrial adenogenesis, the WNT signaling system was studied in the neonatal ovine uterus. WNT5A, WNT7A, and WNT11 were expressed in the uterine epithelia, whereas WNT2B was in the stroma. The WNT receptors FZD2 and FZD6 and coreceptor LRP6 were detected in all uterine cells, and FZD6 was particularly abundant in the endometrial epithelia. Secreted FZD-related protein-2 (SFRP2), a WNT antagonist, was not detected in the P0 uterus, but was abundant in the aglandular caruncular areas of the endometrium between P7 and P56. Exposure of ewes to estrogens during critical developmental periods inhibits or retards endometrial adenogenesis. Estrogen-induced disruption of endometrial adenogenesis was associated with reduction or ablation of WNT2B, WNT7A, and WNT11, and with an increase in WNT2 and SFRP2 mRNA, depending on exposure period. Collectively, results implicate the canonical and noncanonical WNT pathways in regulation of postnatal ovine uterine development and endometrial adenogenesis. Expression of SFRP2 in aglandular caruncular areas may inhibit the WNT signaling pathway, thereby concentrating WNT signaling and restricting endometrial adenogenesis in the intercaruncular areas of the uterus. Further, estrogen-induced inhibition of adenogenesis may be mediated by a reduction in WNT signaling caused by aberrant induction of SFRP2 and loss of several critical WNTs.     

Complete genome sequence of the denitrifying and N2O-reducing bacterium Azoarcus sp. strain KH32C

We report the finished and annotated genome sequence of a denitrifying and N(2)O-reducing betaproteobacterium, Azoarcus sp. strain KH32C. The genome is composed of one chromosome and one megaplasmid and contains genes for plant-microbe interactions and the gene clusters for aromatic-compound degradations.     

Protein phosphatase 2A regulatory subunit Bbeta promotes MAP kinase-mediated migration of A431 cells.

BACKGROUND/AIMS: Phosphatases are involved in regulation of MAP kinase (MAPK). A431 cells migrate on collagen after EGF stimulation using MAPK. To clarify the involvement of PP2A in this MAPK-dependent migration, the expression of an isoform of the B regulatory subunit was inhibited. METHODS: An antisense sequence corresponding to Bbeta cDNA was transfected into A431 cells. Their migratory activity on collagen was examined using Transwell, and MAPK phosphorylation and phosphatase activity were measured, and the results were compared with those obtained with mock-transfected cells. RESULTS: Antisense-transfected cells showed less Bbeta protein and phosphatase activity than mock-transfected controls. Migration of antisense-transfected cells showed a low response to EGF. The response of MAPK phosphorylation of antisense-transfected cells to EGF stimulation and adhesion to collagen in the presence or absence of EGF were markedly decreased. Phosphatase activity of PP2A-Bbeta also did not respond to EGF, collagen or EGF plus collagen, and remained at low levels. CONCLUSION: These results suggested that PP2A-Bbeta promotes cell migration through the MAPK cascade.     

Tea catechins reduce inflammatory reactions via mitogen-activated protein kinase pathways in toll-like receptor 2 ligand-stimulated dental pulp cells

AimsIn this study, we evaluated whether catechins could inhibit the expression of pro-inflammatory mediators induced by dental caries-related bacteria, Streptococci, or pathogen-associated molecular patterns (PAMPs) stimulation in human dental pulp fibroblasts (HDPF). We further determined the mechanisms of the anti-inflammatory activity of catechins.Main methodsStreptococci or PAMP-stimulated HDPF were treated with catechin, and then the expression and production of pro-inflammatory mediators were determined by RT-PCR and ELISA. Furthermore, the signal transduction pathways activated with toll-like receptor (TLR)2 ligand were assessed by Immunoblot and ELISA using blocking assay with specific inhibitors.Key findingsIncreased expressions of pro-inflammatory mediators are found in inflamed dental pulp, especially in HDPF. We recently reported that dental pulpal innate immune responses may mainly result from the predominantly-expressed TLR2 signaling. Catechins, polyphenolic compounds in green tea, exert protective and healing effects through multiple mechanisms, including antioxidative and anti-inflammatory effects. However, there are no reports concerning the effects of catechins on dental pulp. In this study, we demonstrated that the up-regulated expressions of IL-8 or PGE₂ in Streptococci or PAMP-stimulated HDPF were inhibited by catechins, (?)-epicatechin gallate (ECG) and (?)-epigallocatechin gallate (EGCG). In TLR2 ligand-stimulated HDPF, specific inhibitors of extracellular signal regulated kinase (ERK)1/2, p38, c-jun NH₂-terminal kinase (SAP/JNK), NF-κB or catechins markedly reduced the level of pro-inflammatory mediators and the phosphorylation of these signal transduction molecules was suppressed by catechins.SignificanceThese findings suggest that catechins might be useful therapeutically as an anti-inflammatory modulator of dental pulpal inflammation.     

Target proteins of the cytosolic thioredoxin in Plasmodium falciparum

The target proteins of a cytosolic Trx (PfTrx-1) in Plasmodium falciparum with Trx-affinity chromatography were examined. Based on the Trx protein reduction pathway, we generated a cysteine mutant of PfTrx-1, which captures the target protein as a mixed disulfide intermediate. A number of proteins were captured with PfTrx-1(C33S) immobilized on resin and were eluted by DTT treatment. The PfTrx-1(C33S) immobilized resin-captured proteins were trypsin-digested and analyzed on a liquid chromatography-mass spectrometry system. Analysis of the sequence data against databases assigned 20 proteins, four of which had been found previously in P. falciparum, with the remaining 16 being new targets. The potential Trx-target proteins included those in pathways such as the redox cycle, protein biosynthesis, energy metabolism and signal transduction. We captured 4 enzymes in the glycolysis pathway (hexokinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate mutase and l-lactate dehydrogenase (LDH)) as Trx-targets, and we found that PfTrx-1 enhanced the activity of PfGAPDH and PfLDH.     

A wheat homolog of MOTHER OF FT AND TFL1 acts in the regulation of germination

Seed dormancy is an adaptive mechanism and an important agronomic trait. Temperature during seed development strongly affects seed dormancy in wheat (Triticum aestivum) with lower temperatures producing higher levels of seed dormancy. To identify genes important for seed dormancy, we used a wheat microarray to analyze gene expression in embryos from mature seeds grown at lower and higher temperatures. We found that a wheat homolog of MOTHER OF FT AND TFL1 (MFT) was upregulated after physiological maturity in dormant seeds grown at the lower temperature. In situ hybridization analysis indicated that MFT was exclusively expressed in the scutellum and coleorhiza. Mapping analysis showed that MFT on chromosome 3A (MFT-3A) colocalized with the seed dormancy quantitative trait locus (QTL) QPhs.ocs-3A.1. MFT-3A expression levels in a dormant cultivar used for the detection of the QTL were higher after physiological maturity; this increased expression correlated with a single nucleotide polymorphism in the promoter region. In a complementation analysis, high levels of MFT expression were correlated with a low germination index in T1 seeds. Furthermore, precocious germination of isolated immature embryos was suppressed by transient introduction of MFT driven by the maize (Zea mays) ubiquitin promoter. Taken together, these results suggest that MFT plays an important role in the regulation of germination in wheat.     

Expression profile of two storage-protein gene families in hexaploid wheat revealed by large-scale analysis of expressed sequence tags

To discern expression patterns of individual storage-protein genes in hexaploid wheat (Triticum aestivum cv Chinese Spring), we analyzed comprehensive expressed sequence tags (ESTs) of common wheat using a bioinformatics technique. The gene families for alpha/beta-gliadins and low molecular-weight glutenin subunit were selected from the EST database. The alignment of these genes enabled us to trace the single nucleotide polymorphism sites among both genes. The combinations of single nucleotide polymorphisms allowed us to assign haplotypes into their homoeologous chromosomes by allele-specific PCR. Phylogenetic analysis of these genes showed that both storage-protein gene families rapidly diverged after differentiation of the three genomes (A, B, and D). Expression patterns of these genes were estimated based on the frequencies of ESTs. The storage-protein genes were expressed only during seed development stages. The alpha/beta-gliadin genes exhibited two distinct expression patterns during the course of seed maturation: early expression and late expression. Although the early expression genes among the alpha/beta-gliadin and low molecular-weight glutenin subunit genes showed similar expression patterns, and both genes from the D genome were preferentially expressed rather than those from the A or B genome, substantial expression of two early expression genes from the A genome was observed. The phylogenetic relationships of the genes and their expression patterns were not correlated. These lines of evidence suggest that expression of the two storage-protein genes is independently regulated, and that the alpha/beta-gliadin genes possess novel regulation systems in addition to the prolamin box.