Effects of synthetic human gastrin-releasing peptide on pancreatic exocrine secretion and release of pancreatic polypeptide in conscious rats

The effects of a newly synthesized peptide, human gastrin-releasing peptide (hGRP), on the pancreatic exocrine secretion and the release of pancreatic polypeptide (PP) were examined in the conscious rat. Plasma PP concentrations were determined by a recently established specific radioimmunoassay for rat PP. Amounts of 0.18, 0.35, and 3.5 nmol/kg/h hGRP significantly stimulated both pancreatic exocrine secretion and 0.35 nmol/ kg/h of hGRP increased PP release. Simultaneously infused proglumide (300 mg/kg/h) did not affect either pancreatic exocrine secretion or PP release. However, simultaneous infusion of atropine (100 μg/kg/h) slightly inhibited PP release, but did not restrict the incremental response of pancreatic protein secretion to hGRP. These results suggest that hGRP directly stimulates pancreatic exocrine secretion and PP release.     

Inhibition of Histone Deacetylation and DNA Methylation Improves Gene Expression Mediated by the Adeno-Associated Virus/Phage in Cancer Cells

Bacteriophage (phage), viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.     

Molecular cloning and characterization of receptors for the mammalian bombesin-like peptides

The bombesin-like peptides comprise a large family of peptides common to both amphibians and mammals that function as growth factors, neurotransmitters, and paracrine hormones. GRP, the mammalian homolog of bombesin and its receptor, as well as NMB, the mammalian homolog of ranatensin, are expressed in human neoplasms and, in particular, in small cell lung carcinomas (SCLC). To better characterize the physiological roles of bombesin-like peptides, our laboratory has cloned the receptors for GRP in murines, rats, and humans. The 3T3 GRP receptor was isolated and characterized using the two-electrode-voltage-clamp analysis and acquorinemission methods in xenopus oocytes expression system. The rat and human GRP and NMB receptors were cloned by hybridization at low stringency, using the mouse cDNA receptor probe. Sequence analysis of the receptors showed 384 and 390 amino acids for GRP and NMB receptors, respectively. The homology between the two receptors is 60% and between species in the same receptor, 90%. The receptors belong to the 7-membrane spanning domains superfamily. The specific GRP-R antagonist blocked the response to bombesin in oocytes injected with GRP-R, but failed to do so in oocytes injected with NMB-R. The two receptors differ in their distribution of tissue expression. RNA blot and RNase protection analysis showed the same size of mRNA without alteration in the receptors. RT + PCR analysis performed on genomic DNA revealed similarity between normal and cell DNAs, suggesting no major gene deletion or rearrangement. Southern blot analysis indicated the absence of gene amplification. Sequence analysis of the exonic segments of the receptor genes displayed identical amino acids to the respective cDNAs. None of the genes had classic TATAA box. Somatic cell hybrids localized the GRP-R on the X-chromosome and the NMB-R on chromosome 6. The same sequence of normal genes and cDNAs of GRP and NMB receptors, together with the gene characterization, demonstrated that SCLC cell lines do not require a structural change in receptor protein or genomic rearrangement.     

烫伤大鼠结肠动力基本功能单位细胞损害和内质网应激研究

Objective To observe expressions of glucose-regulated protein (GRF78) and caspase-12 in nervous system-interstitial cells of Cajal (ICC) in smooth muscle in colonic wall in rats with scald inju-ry, as well as their relevant ultrastructural changes, so as to probe the possible mechanisms of dynamic dam-age in murine colon after a scald injury. Methods Fifty healthy Sprague-Dawley rats were randomly di-vided into scald (n =40) and control (n = 10) groups. Rats in scald group were inflicted with 30% TBSA full-thickness scald, and received an intraperitoneally injection of Ringer lactate solution (50 mg/kg) for re-suscitation, while those in control group had similar treatment with the exception of scald. Rats in control group and scald group were sacrificed at 3, 6, 12, 24 post scald hour ( PSH, 10 rats at each time point) for collection of 4 cm of colonic tissue, 5 cm proximal to the cecum. A segment of colonic wall, 1 cm in length, was obtained from the middle of the harvested segment of colon, and it was fixed with 3% glutaraldebyde or 10% formaldehyde. The samples fixed with glutaraldehyde were used to observe uhrastructural alterations under transmission electron microscope, while that with formaldehyde were used to observe expressions of GRP78 and caspase-12 in colonic wall by immunohistochemical assay. Results The colonic smooth mus-cle cells of rats in control group showed regular arrangment, their organelles were abundant, nucleus central-ly located, euchromatin distributed evenly with more abundant mitochondrial cristae and less smooth endo-plasmic reticulum, neuronal organelles were abundant in intermuscular plexus, and ICC could be seen in the neighborhood of neurons. The colonic smooth muscle cells appeared in irregular and disordered manner in scald group, perinuclear space was widened, intercellular vacuoles were observed, mitochondria showed vac-uolation degeneration with dissolved and condensed cristae, rough endoplasmic reticula were dilated with par-tial dissolution, and perinuclear cytoplasm of ICC was obviously decreased. The expression of GRP78 was increased in scald group at 3, 6, 12 PSH (4.3±0.9, 6.0±0.7,4.8±1.1 score) as compared with that in control group ( 2.4±0.7 score, P<0.05 ). The expression of caspase-12 in scald group at 6, 12, 24 PSH was higher than that in control group (P<0.05). GRP78 was consistantly expressed in cytoplasm in con-trol group, while in scald group, it mainly appeared in mucosa, myenteric plexus, and stromal cells, but on-ly moderately or lightly expressed in smooth muscle cells. The expression of GRP78 was positive in scald group at 3, 6, 12 PSH, strongly positive at 6 PSH, and it was also expressed in cytoplasm in control group. The expression of caspase-12 in scald group was not obviously positive at 3 PSH, and weakly positive at 6, 24 PSH, but strongly positive at 12 PSH, while no expression was shown in control group. Conclusions Marked pathological changes are observed in enteric nervous system-interstitial cells of Cajal-smooth muscle in rats with severe scald injury. It may be related with cellular injuries induced by caspase-12 apoptotic path-ways through activated endoplasmic reticulum stress.     

儿童空腹血浆胃泌素和胃泌素释放肽的水平

应用放射免疫法测定了125例儿童(年龄自2月至14岁)空腹血浆胃泌素(gastrin)和胃泌素释放肽(gastrin-releasing peptide,GRP)。胃泌素和GRP的均值和实测范围分别为14.0±14.9,0～98.0 pmol/L和8.8士8.2,0～48.0 pmol/L。两种激素的浓度无显著的性别差异。胃泌素以2月至3岁以下组的含量为最高,其均值为12～14岁组的4倍以上。结果表明,胃泌素的浓度与年龄呈负相关(r=-0.50,P<0.001),而GRP的浓度则与年龄无相关性(P>0.05)。本文讨论了在不同年龄组的儿童的空腹血浆浓度的变化。     

我国伤寒沙门氏菌的分子流行病学特征Ⅱ．部分伤寒沙门氏菌的16SrRNA基因多态性

利用ＰＣＲ扩增标记的Ｄｉｇ－ｄＵＴＰ－１６ＳｒＲＮＡ基因为探针，分析我国不同时间和地区分离的１１９株伤寒沙门氏菌和１株鼠伤寒沙门氏菌染色体经ＰｓｔⅠ消化后的１６ＳｒＲＮＡ基因限制性图谱。结果发现，各菌株的杂交片段范围为７．０～２６．５ｋｂ，每个菌株有５～１０条杂交带不等。通过对每个菌株的杂交结果进行数值分类，１１９株伤寒沙门氏菌可分为３８个ＲＴｓ，其中新疆伊犁１９９１年流行菌株和大连１９９０年爆发菌株大部分（１３／２０）为同一ＲＴ；从国内各高发省份分离的一些流行株也有相同的ＲＴ；而一些地区的散发菌株具有独特的ＲＴ；鼠伤寒沙门氏菌的ＲＴ则更为特别。对３９个ＲＴｓ，进行聚类分析发现：国内的一些流行菌株，爆发菌株在遗传距离０．５５处聚成一大类；而散发菌株，非流行菌株则在０．７０处聚成另一类。此外，从健康带菌者分离的菌株２５１所具有的ＲＴ单成一类。鼠伤寒沙门氏菌的距离更远。     

Push-pull perfusion reveals meal-dependent changes in the release of bombesin-like peptides in the rat paraventricular nucleus

Bombesin (BN)-like peptides have been implicated in the regulation of ingestive behavior. The main objective of the present study was to monitor the dynamics of central BN-like peptide release in relationship to spontaneous meal ingestion and termination. Peptide level fluctuations were determined using in vivo push-pull perfusion of the hypothalamic paraventricular nucleus (PVN) and off PVN sites, combined with ex vivo radioimmunoassay. Analysis across all meals revealed significant differences between preprandial, prandial and postprandial extracellular BN-like immunoreactivity (BLI) at the anterior aspect of the PVN, with about a 3-fold diminution during a meal as compared to before or after a meal. Meal-related fluctuations were not detected at more distal hypothalamic sites or at sites within the caudate nucleus. When the analysis was restricted exclusively to the first meal after dark onset, a similar pattern of change in the interstitial levels of PVN BLI was generally observed; levels being higher preprandially as compared to the prandially (albeit by a smaller magnitude), and the termination of the first meal being accompanied by a robust (about 3-fold) increase in BLI. This is the first demonstration of site specific in vivo release of BN-like peptides in relation to feeding status and it further supports the physiological role of this family of peptides in the regulation of food intake.     

The significance of cortisol for osmoregulation in carp (Cyprinus carpio) and tilapia (Sarotherodon mossambicus)

Changes in plasma cortisol and glucose concentration were studied in carp during acclimation from fresh water (FW) to 1.5% salt water and vice versa. There was an increase in cortisol and glucose concentration during acclimation from FW to salt water which lasted for several days. Reacclimation to FW did not cause clear changes in cortisol and glucose levels. One single injection of cortisol (0.2 mg/100 g or 1 mg/100 g) and additional transfer to salt water (1.5% for carp and 2.7% for tilapia) altered the changes caused by acclimation alone of cortisol, glucose, Na+ concentration, and the osmolality in plasma. Gill Na-K-ATPase activity was also influenced. The effects of cortisol on electrolyte concentrations during acclimation and on Na+-K+-ATPase activity differed in both types of fish. Cortisol clearly lowered the increase in plasma Na+ concentration of the stenohaline carp and increased the ATPase activity. The changes in plasma Na+ concentration of the euryhaline tilapia was not clearly altered and the enzyme activity was inhibited. The significance of these cortisol effects is discussed.     

Morphine induced saccharin aversion in α-methyltyrosine pretreated rats

Rats pretreated with l--methyltyrosine (AMT) and then given pairings of saccharin drinking and morphine infusion subsequently showed a reduced preference for saccharin over water, when compared with control rats. Control conditions were AMT pretreatment with saccharin-saline pairings, saline pretreatment with saccharin-morphine pairings, and saline pretreatment with saccharin-saline pairings. A second experiment also showed significantly greater saccharin aversion with morphine-infused, AMT-pretreated rats than with saline-infused, AMT-pretreated rats although the morphine dosage was lower than that used in Experiment 1. These results suggest that morphine is aversive to rats that have been treated with AMT.This research was supported in part by U.S. Public Health Service Grant MH 13570-03.