Synaptosomal-associated protein 25 gene expression is hormonally regulated during ovulation and is involved in cytokine/chemokine exocytosis from granulosa cells

During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors including members of the IL cytokine family via the process of exocytosis. Exocytosis is controlled by the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex consisting of proteins residing in the vesicle membrane and the plasma membrane. One of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins, synaptosomal-associated protein (SNAP)25, is expressed abundantly in neuronal cells and is also induced transiently in the rat ovary in response to LH. Therefore, we sought to determine the molecular mechanisms controlling ovarian expression of the Snap25 gene, and the role of SNAP25 in exocytosis of secreted factors, such as ILs from cumulus cells and granulosa cells. In preovulatory follicles of equine (e) chorionic gonadotropin (CG)-primed mice, expression of Snap25 mRNA was negligible but was induced markedly 8 h after human (h) CG stimulation. In Pgr null mice Snap25 mRNA and protein levels were significantly lower at 8 h after hCG compared with wild-type mice. To analyze the molecular mechanisms by which progesterone receptor regulates this gene, a 1517-bp murine Snap25 promoter-luciferase reporter construct was generated and transfected into granulosa cell cultures. Three specificity protein (SP)-1/SP-3 sites, but not consensus activator protein 1 or cAMP response element sites, were essential for basal and forskolin/phorbol 12-myristate 13-acetate-induced promoter activity in granulosa cells. The induction was significantly suppressed by PGR antagonist, RU486. Treatment of cumulus oocyte complexes or granulosa cells with FSH/amphiregulin, LH, or forskolin/phorbol 12-myristate 13-acetate-induced elevated expression of Snap25 mRNA and increased the secretion of eight cytokine and chemokine factors. Transfection of granulosa cells with Snap25 small interfering RNA significantly reduced the levels of both SNAP25 protein and the secretion of cytokines. From these results, we conclude that progesterone-progesterone receptor-mediated SNAP25 expression in cumulus oocyte complexes and granulosa cells regulates cytokine and chemokine secretion via an exocytosis system.     

Distinct mechanisms underlie distinct polyphenol-induced neuroprotection

Glutamate excitotoxicity is mediated by intracellular Ca(2+) overload, caspase-3 activation, and ROS generation. Here, we show that curcumin, tannic acid (TA) and (+)-catechin hydrate (CA) all inhibited glutamate-induced excitotoxicity. Curcumin inhibited PKC activity, and subsequent phosphorylation of NR1 of the NMDA receptor. As a result, glutamate-mediated Ca(2+) influx was reduced. TA attenuated glutamate-mediated Ca(2+) influx only when simultaneously administered, directly interfering with Ca(2+). Both curcumin and TA inhibited glutamate-induced caspase-3 activation. Although Ca(2+) influx was not attenuated by CA, caspase-3 was reduced by direct inhibition of the enzyme. All polyphenols reduced glutamate-induced generation of ROS.     

Growth Promoting Activities of Sclerotinin A and Its Analogs

(±)-Sclerotinin A (Scl. A) showed the same promoting activity as natural (+)-Scl. A, which was isolated as a growth promoting substance of rice seedlings from Sclerotinia sclerotiorum along with sclerin. By the combined use of Scl. A and gibberellin, the synergistic effect on the growth of rice seedlings was noticed as in the case of sclerin. Correlation between the chemical structure and biological activity of Scl. A derivatives and analogs was discussed.     

Synthesis and evaluation of N-(4-benzylphenyl)piperazines as VGF inducers

A series of compounds was discovered that induce the production of VGF mRNA in SH-SY5Y cells and exhibit cytoprotection under tunicamycin induced endoplasmic reticulum (ER) stress. The aminophenol ring and linker chain of the template SUN N8075 (1) was modified to yield compounds with higher efficacy and lower propensity for adverse effects.     

Abeta-induced BACE-1 cleaves N-terminal sequence of mPGES-2

We previously indicated that amyloid beta (Abeta) augments protein levels of beta-site amyloid precursor protein cleaving enzyme-1 (BACE-1) through oxidative stress. In this study, we revealed that BACE-1 is involved in the cleavage of membrane-bound prostaglandin E2 synthase-2 (mPGES-2) in its N-terminal portion, which, in turn, enhanced the generation of prostaglandin E2 (PGE2). PGE2 results in increased Abeta production, initiating a cell-injuring cycle. Using rat primary cortical neurons, a 48 h treatment with Abeta 1-42 (5 μM) resulted in the enhanced extracellular PGE2 levels up to about 1 ng/mL, which was attenuated by treatment with a BACE-1 inhibitor (200 nM). A synthetic peptide sequence of 20-amino acids that included the cleavage site of mPGES-2 (HTARWHL RAQDLHERS AAQLSLSS) was cleaved by recombinant BACE-1, confirmed using reverse-phase high-performance liquid chromatography. Cleaved or activated mPGES-2 augments the generation of PGE2. In addition, mPGES-2 was determined to be colocalized with BACE-1 and cyclooxygenase-2 in the perinuclear region in cells after exposure to Abeta. Exposure of neurons to PGE2 led to cell death, and Abeta production was enhanced by PGE2 (1 ng/mL, 48 h). Collectively, these results suggest that Abeta might cause neuroinflammation that aggravates Alzheimer’s disease pathogenesis.     

Flavonols and flavones as BACE-1 inhibitors: structure-activity relationship in cell-free, cell-based and in silico studies reveal novel pharmacophore features

Generation and accumulation of the amyloid beta peptide (Abeta) following proteolytic processing of the amyloid precursor protein (APP) by BACE-1 (Beta-site APP Cleaving Enzyme-1, beta-secretase) and gamma-secretase is a main causal factor of Alzheimer's disease (AD). Consequently, inhibition of BACE-1, a rate-limiting enzyme in the production of Abeta, is an attractive therapeutic approach for the treatment of AD. In this study, we discovered that natural flavonoids act as non-peptidic BACE-1 inhibitors and potently inhibit BACE-1 activity and reduce the level of secreted Abeta in primary cortical neurons. In addition, we demonstrated the calculated docking poses of flavonoids to BACE-1 and revealed the interactions of flavonoids with the BACE-1 catalytic center. We firstly revealed novel pharmacophore features of flavonoids by using cell-free, cell-based and in silico docking studies. These results contribute to the development of new BACE-1 inhibitors for the treatment of AD.     

Age-related changes of alpha-crystallin aggregate in human lens

Summary. Lens alpha-crystallin, composed of two subunits alpha A- and alpha B-crystallin, forms large aggregates in the lens of the eye. The present study investigated the aggregate of human lens alpha-crystallin from elderly and young donors. Recombinant alpha A- and alpha B-crystallins in molar ratios of alpha A to alpha B at 1:1, corresponding to the aged sample, were also studied in detail. We found by ultra-centrifugation analysis that the alpha-crystallin aggregate from elderly donors was large and heterogeneous with an average sedimentation coefficient of 30 S and a range of 20–60 S at 37 °C. This was higher compared to the young samples that had an average sedimentation coefficient of 17 S. The sedimentation coefficients of recombinant alpha A- and alpha B-crystallins were approximately 12 S and 15 S, respectively. Even when recombinant alpha-crystallins were mixed in molar ratios equivalent to those found in vivo, similar S values as the native aged alpha-crystallin aggregates were not obtained. Changes in the self-association of alpha-crystallin aggregate were correlated to changes in chaperone activity. Alpha-crystallin from young donors, and recombinant alpha A- and alpha B-crystallin and their mixtures showed chaperone activity, which was markedly lost in samples from the aged alpha-crystallin aggregates.     

DIP (mDia interacting protein) is a key molecule regulating Rho and Rac in a Src-dependent manner

Cell movement is driven by the coordinated regulation of cytoskeletal reorganization through Rho GTPases downstream of integrin and growth-factor receptor signaling. We have reported that mDia, a target protein of Rho, interacts with Src and DIP. Here we show that DIP binds to p190RhoGAP and Vav2, and that DIP is phosphorylated by Src and mediates the phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation. When endogenous DIP was inhibited by expressing dominant-negative mutants of DIP or siRNA, phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation was diminished, and EGF-induced actin organization, distribution of p190RhoGAP and Vav2, and cell movement were affected. Therefore, DIP seems to transfer the complex of the three proteins from cytosol to beneath the membrane, and the three proteins, in turn, can be phosphorylated by Src. DIP inactivated Rho and activated Rac following EGF stimulation in the membrane fraction. Thus, DIP acts as a regulatory molecule causing Src kinase-dependent feedback modulation of Rho GTPases downstream of Rho-mDia upon EGF stimulation, and plays an important role in cell motility.     

Discovery of 1,2,3-triazole-based fibroblast growth factor receptor modulators

To avoid production of a phospholipidosis-inducing metabolite, we replaced the amide structure of SUN13837 (1) with a 1,2,3-triazole. The resulting 1,2,3-triazole analog of 1 (compound 2) displayed greater neuroprotective activity than 1. Structural modification of 2 yielded compound 10, which showed improved neuroprotective activity and negligible mechanism-based inactivation against CYP3A4. In addition, installation of a methyl group at the 5-position of 1,2,3-triazole of 10 significantly boosted the neuroprotective activity. These 1,2,3-triazole derivatives displayed reduced phospholipidosis risk, sufficient systemic exposure, and high central nervous system penetration, and therefore may be potentially useful agents for the treatment of neurodegenerative diseases.     

Hyaluronan fragments generated by sperm-secreted hyaluronidase stimulate cytokine/chemokine production via the TLR2 and TLR4 pathway in cumulus cells of ovulated COCs, which may enhance fertilization

The toll-like receptor (TLR) system is expressed in cumulus cells of ovulated cumulus-oocyte complexes (COCs) and is activated by bacterial lipopolysaccharides (LPS). However, the endogenous ligand(s) for the TLRs and the physiological role(s) in ovulated COCs remain to be defined. Based on reports that hyaluronan fragments can activate TLR2 and TLR4 in macrophages, and that ovulated COCs are characterized by a hyaluronan-rich matrix, we cultured ovulated mouse COCs with purified hyaluronan fragments, treated them with purified hyaluronidase or exposed them to sperm as a physiologically relevant source of hyaluronidase. Hyaluronan fragments or hyaluronidase activated the NFkappaB pathway and induced Il6, Ccl4 and Ccl5 mRNA expression within 2 hours. Anti-TLR2 and anti-TLR4 neutralizing antibodies significantly suppressed hyaluronan fragment- and hyaluronidase-induced activation of the NFkappaB pathway and the expression of these genes. When ovulated COCs were cultured with sperm, the expression and secretion of cytokine/chemokine family members were induced in a time-dependent manner that could be blocked by TLR2/TLR4 antibodies or by a hyaluronan-blocking peptide (Pep-1). The chemokines secreted from TLR2/TLR4-stimulated COCs activated cognate chemokine receptors (CCRs) localized on sperm and induced sperm protein tyrosine phosphorylation, which was used as an index of capacitation. Significantly, in vitro fertilization of COC-enclosed oocytes was reduced by the TLR2/TLR4 neutralizing antibodies or by Pep-1. From these results, we propose that TLR2 and TLR4 present on cumulus cells were activated by the co-culture with sperm in a hyaluronan fragment-dependent manner, and that chemokines secreted from COCs induced sperm capacitation and enhanced fertilization, providing evidence for a regulatory loop between sperm and COCs during fertilization.