Mutual dependence of VIP/PACAP and CCK receptor signaling for a physiological role in duck exocrine pancreatic secretion

Unlike in rodents, CCK has not been established as a physiological regulator in avian exocrine pancreatic secretion. In the isolated duck pancreatic acini, 1 nM CCK was required for stimulation of amylase secretion, maximal effect being achieved at 10 nM; picomolar CCK was without effect. Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 (10(-12)-10(-7) M) alone had no effect, but made picomolar CCK effective. VPAC agonist VIP 10(-10)-10(-7) M stimulated amylase secretion marginally, but made CCK 10(-12)-10(-10) M effective also. PACAP-27 and VIP both shifted the maximal CCK concentration from 10(-8) to 10(-9) M. This sensitizing effect was mimicked by forskolin. CCK dose dependently induced intracellular Ca2+ concentration ([Ca2+]i) oscillations. PACAP-38 (1 nM), PACAP-27 (1 nM), VIP (10 nM), or forskolin (10 microM) alone did not stimulate [Ca2+]i increase, neither did they modulate CCK (1 nM)-induced oscillations; but when they were added to cells simultaneously exposed to subthreshold CCK (10 pM), calcium spikes emerged. Amylase secretion induced by the simultaneous presence of 10 pM CCK and VPAC agonists was completely blocked by removing extracellular calcium, but the protein kinase C inhibitor staurosporine (1 microM) was without effect. CCK (10 nM)-induced secretion was inhibited by CCK1 receptor antagonist FK480 (1 microM). Gastrin from 10(-12) to 10(-6) M did not stimulate amylase secretion nor did it (100 nM) induce [Ca2+]i increase. The above data suggest that duck pancreatic acini possess both CCK1 and VPAC receptors; simultaneous activation of both is required for each to play a physiological role.     

Enzymatic enantioselective transcyanation of silicon-containing aliphatic ketone with (S)-hydroxynitrile lyase from Manihot esculenta

(S)-Hydroxynitrile lyase from Manihot esculenta (MeHNL) was shown for the first time to be able to catalyze the enantioselective transcyanation of acetyltrimethylsilane (ATMS) with acetone cyanohydrin to form (S)-2-trimethylsilyl-2-hydroxyl-propionitrile in an aqueous/organic biphasic system. To better understand the reaction, various influential variables were examined. The most suitable organic phase, optimal buffer pH, aqueous phase content, shaking rate, temperature, concentration of ATMS, acetone cyanohydrin and crude enzyme were diisopropyl ether (DIPE), 5.4, 13% (v/v), 190 rpm, 40°C, 10 mM, 20 mM, and 35 U/ml, respectively, under which the initial reaction rate, substrate conversion and product enantiomeric excess (e.e.) were 19.5 mM/h, 99.0% and 93.5%, respectively. A comparative study demonstrated that silicon atoms in the substrate had a great effect on the reaction, and that ATMS was a much better substrate for MeHNL than its carbon analogue 3,3-dimethyl-2-butanone (DMBO) with respect to the initial reaction rate, substrate conversion and product e.e. MeHNL has greater affinity towards ATMS than its carbon analogue as indicated by the much lower K_m. The activation energy of MeHNL-catalyzed transcyanation of ATMS was also markedly lower than that of DMBO. The silicon effect on the reaction was rationalized on the basis of the special characteristics of silicon atoms and the catalytic mechanism of MeHNL.     

Genetic variations at urotensin II and urotensin II receptor genes and risk of type 2 diabetes mellitus in Japanese

Urotensin II is among the most potent vasoactive hormones known and the urotensin II (UTS2) gene is localized to 1p36-p32, one of the regions reported to show possible linkage with type 2 diabetes in Japanese. When we surveyed genetic polymorphisms in the UTS2 and urotensin II receptor (GPR14) gene, we identified two SNPs with amino acid substitutions (designated T21M and S89N and an SNP in the promotor region (-605G>A) of the UTS2 gene, and two SNPs in the non-coding region of the GPR14 gene. We then studied these three SNPs in the UTS2 gene and two SNPs in the GPR14 gene in 152 Japanese subjects with type 2 diabetes mellitus and two control Japanese populations. The allele frequency of 89N was significantly higher in type 2 diabetic patients than in both elderly normal subjects (P = 0.0018) and subjects with normal glucose tolerance (P = 0.0011), whereas the allele frequency of T21M and -605G>A in the UTS2 gene and those of two SNPs in the GPR14 gene were essentially identical in these three groups. Furthermore, in the subjects with normal glucose tolerance, 89N was associated with significantly higher insulin levels on oral glucose tolerance test, suggesting reduced insulin sensitivity in subjects with 89N. These results strongly suggest that subjects with S89N in the UTS2 gene are more insulin-resistant and thus more susceptible to type 2 diabetes mellitus development.     

The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH(2)-terminal kinase

Targeted gene disruption studies have established that the c-Jun NH(2)-terminal kinase (JNK) signaling pathway is required for stress-induced release of mitochondrial cytochrome c and apoptosis. Here we demonstrate that activated JNK is sufficient to induce rapid cytochrome c release and apoptosis. However, activated JNK fails to cause death in cells deficient of members of the Bax subfamily of proapoptotic Bcl2-related proteins. Furthermore, exposure to stress fails to activate Bax, cause cytochrome c release, and induce death in JNK-deficient cells. These data demonstrate that proapoptotic members of the Bax protein subfamily are essential for JNK-dependent apoptosis.     

Lipid phosphate phosphatases regulate signal transduction through glycerolipids and sphingolipids

Lipid phosphate esters including lysophosphatidate (LPA), phosphatidate (PA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) are bioactive in mammalian cells and serve as mediators of signal transduction. LPA and S1P are present in biological fluids and activate cells through stimulation of their respective G-protein-coupled receptors, LPA(1-3) and S1P(1-5). LPA stimulates fibroblast division and is important in wound repair. It is also active in maintaining the growth of ovarian cancers. S1P stimulates chemotaxis, proliferation and differentiation of vascular endothelial and smooth muscle cells and is an important participant in the angiogenic response and neovessel maturation. PA and C1P are believed to act primarily inside the cell where they facilitate vesicle transport. The lipid phosphates are substrates for a family of lipid phosphate phosphatases (LPPs) that dramatically alter the signaling balance between the phosphate esters and their dephosphorylated products. In the case of PA, S1P and C1P, the products are diacylglycerol (DAG), sphingosine and ceramide, respectively. These latter lipids are also bioactive and, thus, the LPPs change signals that the cell receives. The LPPs are integral membrane proteins that act both inside and outside the cell. The "ecto-activity" of the LPPs regulates the circulating and locally effective concentrations of LPA and S1P. Conversely, the internal activity controls the relative accumulation of PA or C1P in response to stimulation by various agonists thereby affecting cell signaling downstream of EDG and other receptors. This article will review the various LPPs and discuss how these enzymes could regulate signal transduction by lipid mediators.     

Nitrate respiratory metabolism in an obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6

Hydrogenobacter thermophilus strain TK-6 was observed to grow anaerobically on nitrate as an electron acceptor when molecular hydrogen was used as an energy source. Nitrite was detected as the product of a respiratory reaction. 15NO, 15N2O, and 15N2 were detected with Na15NO3 as an electron acceptor. Western immunoblot analysis showed that cell-free extracts from cells grown on nitrate reacted with antibodies against heme cd1-type nitrite reductase from Pseudomonas aeruginosa. The positive bands, which had molecular masses similar to that of the heme cd1-type nitrite reductase, were also stained by heme staining. These results indicate that nitrite reductase of strain TK-6 is a heme cd1-type enzyme. Activity of ATP:citrate lyase, one of the key enzymes of the reductive TCA cycle, was detected in cell-free extract of cells cultivated on nitrate, which indicates that the cycle operates during anaerobic growth.     

Structure and chromosomal localization of the human glycogenin-2 gene GYG2

Glycogenin-2 is one of two self-glucosylating proteins involved in the initiation phase of the synthesis of the storage polysaccharide glycogen. Cloning of the human glycogenin-2 gene, GYG2, has revealed the presence of 11 exons and a gene of more than 46 kb in size. The structure of the gene explains much of the observed diversity in glycogenin-2 cDNA sequences as being due to alternate exon usage. In some cases, there is variation in the splice junctions used. Over regions of protein sequence similarity, the GYG2 gene structure is similar to that of the other glycogenin gene, GYG. A genomic GYG2 clone was used to localize the gene to Xp22.3 by fluorescence in-situ hybridization. Localization close to the telomere of the short arm of the X chromosome is consistent with mapping information obtained from glycogenin-2 STS sequences. Glycogenin-2 maps between the microsatellite anchor markers AFM319te9 (DXS7100) and AFM205tf2 (DXS1060), and its 3' end is 34.5 kb from the 3' end of the arylsulphatase gene ARSD. GYG2 is outside the pseudoautosomal region PAR1 but still in a region of X-Y shared genes. As is true for several other genes in this location, an inactive remnant of GYG2, consisting of exons 1-3, may be present on the Y chromosome.     

耐热性碱性磷酸酯酶作为非放射性标记物的研究

通过生物素与亲和素－酶复合物系统或地高辛与抗地高辛－酶复合物系统可把酶间接标记到探针上．Ｒｅｎｚ等通过不同的化学方法直接把酶标记到探针上［１～３］．耐热性碱性磷酸酯酶ＦＤ－ＴＡＰ（ｔｈｅｒｍｏｓｔａｂｌｅａｌｋａｌｉｎｅｐｈｏｓｐｈａｔａｓｅ）具有耐...     

Regulation of the G2/M phase of the cell cycle by sperm associated antigen 8 (SPAG8) protein

Sperm associated antigen 8 (SPAG8), a testis‐specific protein produced during male germ cell differentiation, was isolated from a human testis expression library using antibodies found in the serum obtained from an infertile woman. It was found to have a close functional relationship with microtubules. In this study, we generated a stably expressing SPAG8 CHO‐K1 cell line. Immunofluorescence confocal microscopy showed that SPAG8 was concentrated at the microtubule‐organizing center (MTOC) during prophase. As the cells progressed into metaphase, it co‐localized with α‐tubulin on the spindle. In anaphase, it was detected on both astral microtubules and mid‐zone. Following cytokinesis, SPAG8 resumed its localization on the MTOC. Meanwhile, flow cytometry analysis found that SPAG8 prolonged the G2/M phase of CHO‐K1 cells stably expressing SPAG8. Furthermore, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay showed that SPAG8 inhibited the proliferation of the stable cells. SPAG8 might be involved in the regulation of cell cycle by changing the phosphorylation level of Tyr15 on cdc2. These results suggest that SPAG8 might play a role in cell division during spermatogenesis. Copyright © 2009 John Wiley & Sons, Ltd.