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.     

Quaternary structures of intermediately ligated human hemoglobin a and influences from strong allosteric effectors: resonance Raman investigation

The Fe-histidine stretching (nu(Fe-His)) frequency was determined for deoxy subunits of intermediately ligated human hemoglobin A in equilibrium and CO-photodissociated picosecond transient species in the presence and absence of strong allosteric effectors like inositol(hexakis)phosphate, bezafibrate, and 2,3-bisphosphoglycerate. The nu(Fe-His) frequency of deoxyHb A was unaltered by the effectors. The T-to-R transition occurred around m = 2-3 in the absence of effectors but m > 3.5 in their presence, where m is the average number of ligands bound to Hb and was determined from the intensity of the nu(4) band measured in the same experiment. The alpha1-beta2 subunit contacts revealed by ultraviolet resonance Raman spectra, which were distinctly different between the T and R states, remained unchanged by the effectors. This observation would solve the recent discrepancy that the strong effectors remove the cooperativity of oxygen binding in the low-affinity limit, whereas the (1)H NMR spectrum of fully ligated form exhibits the pattern of the R state.     

Structural characterization of the proximal and distal histidine environment of cytoglobin and neuroglobin

Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (nu(Fe-His)) bands at 229 and 221 cm(-1), respectively. No time-dependent shift in the nu(Fe-His) band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (nu(Fe-CO), nu(C-O), delta(Fe-C-O)) and (nu(Fe-NO), nu(N-O), delta(Fe-N-O)) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.     

Novel contribution of cell surface and intracellular M1‐muscarinic acetylcholine receptors to synaptic plasticity in hippocampus

Muscarinic acetylcholine receptors (mAChRs) are well known to transmit extracellular cholinergic signals into the cytoplasm from their position on the cell surface. However, we show here that M1‐mAChRs are also highly expressed on intracellular membranes in neurons of the telencephalon and activate signaling cascades distinct from those of cell surface receptors, contributing uniquely to synaptic plasticity. Radioligand‐binding experiments with cell‐permeable and ‐impermeable ligands and immunohistochemical observations revealed intracellular and surface distributions of M1‐mAChRs in the hippocampus and cortex of rats, mice, and humans, in contrast to the selective occurrence on the cell surface in other tissues. All intracellular muscarinic‐binding sites were abolished in M1‐mAChR‐gene‐knockout mice. Activation of cell surface M1‐mAChRs in rat hippocampal neurons evoked phosphatidylinositol hydrolysis and network oscillations at theta rhythm, and transiently enhanced long‐term potentiation. On the other hand, activation of intracellular M1‐mAChRs phosphorylated extracellular‐regulated kinase 1/2 and gradually enhanced long‐term potentiation. Our data thus demonstrate that M1‐mAChRs function at both surface and intracellular sites in telencephalon neurons including the hippocampus, suggesting a new mode of cholinergic transmission in the central nervous system.     

Spreading disease with transport-related infection

Transportation among regions is found as one of the main factors which affect the outbreak of diseases. It will change the disease dynamics and break infection out even if infectious diseases will go to extinction in each city without transport-related infection. In this paper, a mathematical model is proposed to demonstrate the dynamics of such disease propagation between two regions (or cities) due to the population dispersal and infection on transports. Further, our analysis shows that transport-related infection intensifies the disease spread if infectious diseases break out to cause an endemic situation in each region, in the sense that both the absolute and relative size of patients increase. This suggests that it is very essential to strengthen restrictions of passengers once we know infectious diseases appeared.     

Synthesis of D-Alanine Oligopeptides Catalyzed by D-Aminopeptidase in Non-Aqueous Media

Synthesis of D-alanine oligopeptides from D-alanine methylester hydrochloride has been demonstrated by use of immobilized D-aminopeptidase from Ochrobactrum anthropi (Achromobacter sp.) in non-aqueous media. D-Alanine dimer and trimer were obtained in 56% and 6% yield, respectively, when 250 mM of the substrate was incubated for 3 hours with urethane-prepolymer immobilized D-aminopeptidase (1.5 U/ml) and 3 equivalents of triethylamine in water-saturated toluene. The k_cat of this reaction was calculated to be 19,500 (min^-1), which is several ten thousand times greater than that of the known enzymatic syntheses of amino acid oligomers.     

Cryosurgical hemorrhoidectomy: How to prevent the postoperative swelling and prolapse

Cryosurgical hemorrhoidectomy was performed on 171 patients with excellent results achieved in the great majority. The key to successful results is based on the prevention of prolapse of the edematous tissue after freezing. To prevent the edema, it is important to pull out the hemorrhoid as much as possible during the freezing, to freeze only on the rectal side of the anal verge, to carefully avoid freezing the anal verge, and to freeze the hemorrhoids in two or three stages when they are large. Less than satisfactory results were achieved in 11.7% of the patients. The anal discomfort and discharge in these patients were related to prolapse of the frozen tissue. Cryosurgery is an effective method of treatment for hemorrhoids if care is taken to use the proper technique.     

Serum boron concentration from inhabitants of an urban area in Japan

Boron (B) levels were determined in the serum of 980 healthy inhabitants living in an urban area of Japan by means of inductively coupled plasma emission spectrometry (ICPES). The results showed a log-normal distribution of serum B for both sexes, although there are age-related differences. In male subjects, serum B increases rapidly up to 49 yr of age, reaching a plateau between ages 50 and 69 yr old, followed by a gradual increase up to 70 yr or older. Female subjects exhibit a gradual increase up to the age of 70 yr old. The reference value for male and female subjects was 79.8 μg/L and 67.9 μg/L, and the reference interval was 33.3–191.2 μg/L and 29.5–154.9 μg/L, respectively. The obtained reference value and interval of the nonexposed group may be useful for health screening for B exposure, either for people living in regions with high levels of B in the environment, or for workers who are exposed to this element.