A combinatorial library of beta-chlorovinyl chalcones (4) were synthesized by Claisen-Schmidt condensation reaction. Catalytic reaction of substituted 3-chloro-3-phenyl-propenal (2) and 1-(2,4-dimethoxy-phenyl)-ethanone or 1-(4-methoxy-phenyl)-ethanone (3) in alkaline conditions furnished the target compound 5-chloro-1-(2,4-dimethoxy-phenyl)-5-phenyl-penta-2,4-dien-1-one (4). The synthesized compounds were screened for their biological activity viz. anticancer, anti-inflammatory and antimicrobial activities. Synthesized compounds 4g and 4h revealed promising anti-inflammatory activity (66-67% TNF-alpha and 95-97% IL-6 inhibitory activity at 10 microM). Cytotoxicity of the compounds checked using CCK-8 cell lines and found to be nontoxic to slightly toxic. Furthermore, the anticancer activity (30-40%) was shown by compounds 4d, 4e, 4h and 4b at 10 microM concentrations against ACHN followed by Calu 1, Panc1, HCT116 and H460 cell lines. Some of the compounds 4d, 4e, 4a, 4i and 4b revealed promising antimicrobial activity at MIC 50-100 microg/mL against selected pathogenic bacteria and fungi.

The role of different cytokines in the pathogenesis of L-arginine (Arg)-induced acute pancreatitis in rat, and the ability of KSG-504, a novel cholecystokinin receptor antagonist, to exert protection in this type of acute pancreatitis was evaluated. Male Wistar rats received 250 mg/100 g body weight of Arg intraperitoneally twice, at an interval of 1 h. Control rats received instead the same amount of glycine at the same times. Fifty mg/kg KSG-504 was injected subcutaneously 0.5 h before and 6, 18 and 36 h after the first Arg administration. Rats were examined 12, 24 and 48 h after pancreatitis induction. To assess the severity of inflammation, the edema was quantified, the serum amylase level was measured, and histologic examinations were performed. Serum tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) levels were determined by bioassay, using the TNF-sensitive WEHI 164 and the IL-6-dependent B9 cell lines, respectively. In Arg-induced acute pancreatitis, the amylase level was increased significantly at 12 h (48.600 +/- 3.980 U/l) and 24 h (30.800 +/- 3.813 U/l) vs. the control group (6.382 +/- 184 U/l). No significant alteration in the ratio pancreatic weight/body weight was found in the different groups. However, in Arg-induced acute pancreatitis, both the TNF-alpha (15.1 +/- 6.9 U/ml) and the IL-6 (39.6 +/- 19.2 pg/ml) levels were already elevated significantly at 12 h vs. the controls (3.1 +/- 0.8 U/ml and 15.2 +/- 3.1 pg/ml, respectively) and remained elevated at 24 and 48 h. Simultaneous KSG-504 administration did not modify the measured cytokine levels. No significant changes in plasma CCK levels were observed. In Arg-induced acute pancreatitis, histological evaluation revealed diffuse but microfocal necrobiotic alterations. No marked protective effects of KSG-504 were observed on histological sections. These results suggest that excessive doses of Arg induce severe acute pancreatitis in rat, with a simultaneous cytokine level elevation. Endogenous CCK does not seem to play an essential role in the pathogenesis of Arg-induced acute pancreatitis.

BACKGROUND

Malnutrition is frequent in peritoneal dialysis (PD) patients, but the contribution of gastrointestinal (GI) dysfunction has not been well established.

METHODS

We studied GI function in 49 stable PD patients to ascertain its relationship with malnutrition. After an overload fat diet, fecal fat, sugar, starch and nitrogen, intestinal protein permeability (alpha(1)-antitrypsin fecal clearance [C-alpha(1)-AT]), fecal chymotrypsin (CT), GI hormones and gastrin, pepsinogen I and II, cholecystokinin (CCK), gastrin releasing peptide (GRP), and neuropeptide Y (NPY) were measured. Vasoactive intestinal polypeptide (VIP), substance P (SP), and tumor necrosis factor (TNF-alpha) and biochemical nutritional markers were evaluated.

RESULTS

All patients showed high fecal sugar. Elevated fecal nitrogen was found in 21 patients, 6 with high C-alpha(1)-AT. High fecal starch levels appeared in 21, fat in 20, and low fecal CT in 39 patients. These determinations showed inverse relation with nutritional markers. Increased fecal C-alpha(1)-AT values were associated with lower serum albumin. Fecal CT values showed a negative linear correlation with serum albumin and were inversely associated with retinol-binding protein, normalized protein nitrogen appearance, and serum iron. High plasma levels of pancreatic stimulating hormones were found: gastrin, CCK, and VIP. These levels were higher in patients with a worse pancreatic exocrine function. Higher values of other GI hormones, gastrin, pepsinogen I and II, CCK, GRP, and TNF-alpha. Normal concentrations of NPY, VIP, and PS were observed.

CONCLUSIONS

GI abnormalities (malabsorption, maldigestion, pancreatic dysfunction, and protein losing enteropathy) are present in an important number of PD patients. These features are negatively associated to nutrition.

The aim of the present study was to investigate the effects of cholecystokinin (CCK) CCK(A) and CCKB receptor antagonists SR 27897 B, devazepide, L 365260 and PD 135158 (CAM 1028) on exploratory behaviour in the elevated zero-maze in the rat. For further validation of the elevated zero-maze, the effects of a reference anxiolytic diazepam (0.25, 0.5, 1.0, 2.0 mg/kg), a non-benzodiazepine (BDZ) anxiolytic buspirone (0.04, 0.2, 1.0, 5.0 mg/kg), BDZ receptor inverse agonists FG 7142 (5, 10, 15, 20 mg/kg) and DMCM (0.1, 0.5, 1.0, 1.5 mg/kg), and a BDZ receptor antagonist flumazenil (10 mg/kg) were studied. Diazepam decreased the number of stretched-attend postures in all doses used and increased the percentage of time spent exploring in open parts at doses of 0.5 and 1.0 mg/kg. The effects of diazepam were blocked by flumazenil. FG 7142 and DMCM had effects only in subconvulsive doses (20 mg/kg and 1.5 mg/kg). Flumazenil and buspirone failed to show any effect. The CCK(A) receptor antagonists were also without any effect. The CCK(B) receptor antagonists L 365260 (1.0 and 5.0 mg/kg) and PD 135158 (100 microg/kg) had a significant anxiolytic-like effect. The CCK(B) receptor antagonists increased the number of open part entries, the number of head dips, the percentage of time spent exploring in the open part and decreased the number of stretched-attend postures. These data support the hypothesis of the involvement of the CCK(B) receptor subtype in the neurobiological mechanisms of anxiety.

OBJECTIVE

To identify the cholecystokinin (CCK)-A receptors (CCK-AR) on the guniea pig gallbladder interstitial cells of cajal (ICC) and to study CCK-8 induced gallbladder muscle strip contractions through the CCK-AR.

METHODS

The existence of CCK-AR was examined by immunohistofluorescence on sectioned tissue and cultured cells. In vitro contractile response of guinea pig gallbladder muscle strips and the strips with ICC removed were also studied with CCK-8 receptors added.

RESULTS

In tissue sections, intensely CCKAR-immunoreactive interstitial cells were found mainly in the muscular layers. In cultured cell sections, distinctive double staining of C-kit and CCK-AR ICCs were found. When we removed the ICC of the gallbladder, CCK-8 induced muscle strip contraction dose response curve significantly shifted to the right.

CONCLUSIONS

We proved that both the existence of CCK-AR on the guinea pig gallbladder ICC and CCK evoked contraction are mediated through direct action on CCK-AR on the gallbladder ICC.

Recent studies have suggested the critical roles of miRNAs for disease progression. miRNA-483-5p (miR-483-5p) was previously found to have a relationship with tumor cell behavior, but its biological function in Hirschsprung's disease (HSCR) remains undefined. Thus, we explored the role of miR-483-5p in the pathogenesis of HSCR. Histological changes of colonic tissues were evaluated by hematoxylin and eosin (HE) staining. Quantitative real-time PCR and western blotting were used to determine relative expression levels of miRNA, mRNA, and proteins in 20 HSCR patients and 20 normal colon tissues. In this study, we found that miR-483-5p expression in HSCR tissues was significantly increased and their downregulation promoted cell proliferation, cell cycle progression and invasion and inhibited cell apoptosis in human 293T and SH-SY5Y cell lines by the CCK-8, flow cytometry, and Transwell assay. GNDF family receptor alpha 4 (GFRA4) was confirmed as a downstream target of miR-483-5p by dual-luciferase reporter gene assay and inversely correlated with miR-483-5p expression in cell lines. Taken together, miR-483-5p may play a crucial role in the pathogenesis of HSCR by targeting GFRA4.

Calcitonin gene-related peptide (CGRP) is a potent inhibitor of pancreatic enzyme secretion in vivo. Recent studies have shown that CGRP exerts its inhibitory action at a central vagal site. The present study investigates the mechanism responsible for the central action of CGRP. Rats were fitted with lateral cerebroventricular cannulas, using stereotaxic instruments, 4 days before pancreatic secretion studies. In anesthetized rats, administration of 2-deoxy-D-glucose (2-DG) (75 mg/kg iv) or CCK-8 (40 pmol. kg-1. h-1) produced a 100 and 75% increase in protein secretion, respectively, which was completely blocked by atropine. Intracerebroventricular (ICV) administration of CGRP (0.03-0.6 nmol/h) resulted in a dose-related inhibition of pancreatic protein secretion evoked by 2-DG or CCK-8. CGRP administered by the ICV route was 10-40 times more potent than CGRP given by the intravenous route. In contrast, ICV administration of CGRP had no significant effect on pancreatic protein secretion evoked by electrical vagal stimulation or bethanechol, which directly activates the pancreatic muscarinic receptor. Chemical sympathectomy induced by pretreatment with guanethedine (20 mg/kg ip, 2 days) or alpha-adrenergic receptor blockade with phentolamine did not alter the inhibitory effects of CGRP. We recently demonstrated that CCK stimulated the enteropancreatic neural pathways to mediate pancreatic secretion in rats with a chronic vagotomy. ICV-administered CGRP did not affect CCK-stimulated pancreatic secretion in rats with a chronic vagotomy. In conclusion, CGRP in the central nervous system inhibits pancreatic enzyme secretion stimulated by 2-DG and CCK-8, which act through vagal pathways. The inhibitory action of CGRP is not mediated by the sympathetic nervous system but appears to depend on intact vagus nerves.

This study aimed to investigate the effect of non-small cell lung cancer (NSCLC) cell-derived exosome on cell proliferation and apoptosis in normal lung fibroblast cells and NSCLC cells, and whether it regulates cell functions through delivering alpha-smooth muscle actin (ASMA). NSCLC exosomes were extracted from A549 cells, then cocultured with normal lung fibroblasts (HLF1 cells) and NSCLC cells (A549 cells). Blank ShRNA and ASMA ShRNA plasmids were transferred into HLF1 cells/A549 cells with or without NSCLC exosomes, which were divided into 4 groups accordingly: Negative control (NC) group, SH group, Exosome group and Exosome+SH group. Western blot, immunofluorescence, qPCR, CCK-8 and AV/PI were used to detect protein level, gene expression, cell proliferation and cell apoptosis, respectively. In HLF1 cells, cell proliferation was enhanced while cell apoptosis rate was inhibited in Exosome group compared with NC group; and cell proliferation was attenuated while cell apoptosis rate was raised in Exosome+SH group than Exosome group in rescue experiment; the expressions of apoptotic markers C-caspase3 and Bcl-2 also revealed the same trends. Additionally, in A549 cells, cell proliferation was also increased while cell apoptosis was inhibited in Exosome group compared with NC group; and cell proliferation was reduced while cell apoptosis rate was elevated in Exosome+SH group than Exosome group in rescue experiment. In conclusion, NSCLC derived exosomes promote cell proliferation and inhibit cell apoptosis in both normal lung fibroblasts and NSCLC cells by delivering ASMA.

Gastrin, which induces gastric acid secretion, and a structurally similar hormone, cholecystokinin (CCK)-a potent acid inhibitor, may each play a role in gastric cancer. However, few studies have investigated this hypothesis in humans. We therefore investigated whether serum gastrin or CCK concentrations at baseline were associated with the incidence of gastric non-cardia adenocarcinomas (GNCA), oesophagogastric junctional adenocarcinomas (EGJA) or gastric carcinoid tumours over 24 years of follow-up in a study nested within the all-male Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study of Finnish smokers.

Totals of 283 incident GNCA, 96 EGJA and 10 gastric carcinoid cases, and 778 matched controls, were included in our analysis. Gastrin and CCK were measured using specific radioimmunoassays. Odds ratios (OR) and 95% confidence intervals (95% CI) were estimated by multivariable logistic regression with adjustment for all known or suspected confounding factors, including Helicobacter pylori seropositivity.

Those with high gastrin (Q4 vs Q1), had an increased risk of GNCA (fully adjusted OR: 1.92; 95% CI: 1.21, 3.05) and gastric carcinoids, though the small number of carcinoid cases meant the fully adjusted model was unstable (age-adjusted continuous model OR: 4.67; 95% CI: 2.67, 8.15). CCK was associated with risk of GNCA only for those in Q3 relative to Q1 (OR: 0.56; 95% CI: 0.33, 0.96), and no significant trend was observed.

Our data suggest that high serum concentrations of gastrin may be associated independently with an increased risk of gastric cancer; the role of CCK in cancer risk is less clear.

Thylakoids are chlorophyll-containing membranes in chloroplasts that have been isolated from green leaves. It has been previously shown that thylakoids supplemented with a high-fat meal can affect cholecystokinin (CCK), ghrelin, insulin and blood lipids in humans, and can act to suppress food intake and prevent body weight gain in rodents. This study investigates the addition of thylakoids to a high carbohydrate meal and its effects upon hunger motivation and fullness, and the levels of glucose, insulin, CCK, ghrelin and tumour necrosis factor (TNF)-alpha in overweight women. Twenty moderately overweight female subjects received test meals on three different occasions; two thylakoid enriched and one control, separated by 1 week. The test meals consisted of a high carbohydrate Swedish breakfast, with or without addition of thylakoids. Blood samples and VAS-questionnaires were evaluated over a 4-h period. Addition of thylakoids suppressed hunger motivation and increased secretion of CCK from 180 min, and prevented postprandial hypoglycaemia from 90 min following food intake. These effects indicate that thylakoids may intensify signals of satiety. This study therefore suggests that the dietary addition of thylakoids could aid efforts to reduce food intake and prevent compensational eating later in the day, which may help to reduce body weight over time.

Patients with obstructive jaundice (OJ) that requires surgery often have malnutrition associated with increased perioperative morbidity. This study investigated the factors influencing nutritional derangements in these patients. A series of 46 OJ patients were investigated prospectively (28 malignant tumors, 18 benign obstructions). A nutritional risk index of < 83.5 was used to define protein-calorie malnutrition. Liver function, cholecystokinin (CCK), tumor necrosis factor-alpha (TNFalpha), and endotoxin levels were determined. A multivariate analysis was performed, and an obstructive jaundice malnutrition index (OJMI) was obtained. Altogether, 22 (48%) OJ patients had malnutrition (33% with benign obstructions, 57% with malignant disease). Malnourished patients had higher serum bilirubin levels (258 +/- 120 vs. 154 +/- 62 mmol/L; p = 0.005), longer duration of jaundice (16 +/- 9 vs. 9 +/- 5 days; p = 0.03), and higher plasma levels of CCK (4.0 +/- 1.3 vs. 1.7 +/- 1.0 pmol/L; p = 0.005), alanine aminotransferase (ALT) (226 +/- 209 vs. 187 +/- 161 UI/L; p = 0.01), endotoxin (15 +/- 10 vs. 6.5 +/- 7.0 EU/L; p = 0.007), and TNFalpha (69 +/- 82 vs. 23 +/- 15 pg/ml; p = 0.008) than those without malnutrition. However, only serum bilirubin, CCK, ALT, and patient age were predictors for malnutrition by multivariate analysis. Malnutrition might be expected (95% confidence interval) in patients older than 68 years with increased bilirubin >> 290 mmol/L) and ALT >> 210 UI/L) levels that corresponded with an OJMI>> 55. It was concluded that nutritional alterations in patients with obstructive jaundice were determined by the intensity of the biliary obstruction correlated with increased plasma CCK levels as well as with liver dysfunction and patient age.

We have analyzed the effect of palmitoleic acid on short-term food intake in male rats. Administration of omega-7 palmitoleic acid by oral gavage significantly decreased food intake compared to palmitic acid, omega-9 oleic acid, or a vehicle control. Palmitoleic acid exhibited a dose-dependent effect in this context and did not cause general malaise. A triglyceride form of palmitoleate also decreased food intake, whereas olive oil, which is rich in oleic acid, did not. Palmitoleic acid accumulated within the small intestine in a dose-dependent fashion and elevated levels of the satiety hormone cholecystokinin (CCK). Both protein and mRNA levels of CCK were affected in this context. The suppression of food intake by palmitoleic acid was attenuated by intravenous injection of devazepide, a selective peripheral CCK receptor antagonist. Palmitoleic acid did not alter the expression of peroxisome proliferator-activated receptor alpha (PPARα) target genes, and a PPARα antagonist did not affect palmitoleic acid-induced satiety. This suggests that the PPARα pathway might not be involved in suppressing food intake in response to palmitoleic acid. We have shown that orally administered palmitoleic acid induced satiety, enhanced the release of satiety hormones in rats.

Necroptosis, oxidative stress, and inflammation are major contributors to the pathogenesis of ischemic acute kidney injury. Necrostatin-1 (Nec-1), an inhibitor of the kinase domain of receptor-interacting protein kinase-1 (RIP1), has been reported to regulate renal ischemia and reperfusion (I/R) injury; however, its underlying mechanism of action remains unclear. HK-2 cells were used to create an in vitro I/R model, in which the cells were subjected to hypoxia, followed by 2, 6, and 12 h of reoxygenation. For the in vivo study, a rat model of renal I/R was established in which samples of rat blood serum and kidney tissue were harvested after reperfusion to assess renal function and detect histological changes. Cell viability and necroptosis were analyzed using the Cell Counting Kit (CCK)-8 assay and flow cytometry, respectively. The expression levels of molecules associated with necroptosis, oxidative stress, and inflammation were determined by real-time PCR, western blotting, immunofluorescence staining, and ELISA. Luciferase and chromatin immunoprecipitation (ChIP) assays were performed to confirm the relevant downstream signaling pathway. We found that pretreatment with Nec-1 significantly decreased hypoxia-inducible factor-1α (HIF-1α) and miR-26a expression, as well as the levels of factors associated with necroptosis (RIP1, RIP3, and Sirtuin-2), oxidative stress (malondialdehyde [MDA], NADP⁺/NADPH ratio), and inflammation (interleukin [IL]-1β, IL-10, and tumor necrosis factor alpha [TNF-α]) in I/R injury cells and the rat model. However, these effects could be reversed by miR-26a overexpression or TRPC6 knockdown. Mechanistic studies demonstrated that HIF-1α directly binds to the promoter region of miR-26a, and that TRPC6 is a potential target gene for miR-26a. Our findings indicate that Nec-1 can effectively protect against renal I/R injury by inhibiting necroptosis, oxidative stress, and inflammation, and may exert its effects through mediation of the HIF-1α/miR-26a/TRPC6/PARP1 signaling pathway.

CCK exhibits a potent cytoprotective activity against acute gastric lesions, but its role in ulcer healing has been little examined. In this study we determined whether exogenous CCK or endogenously released CCK by camostate, an inhibitor of luminal proteases, or by the diversion of pancreatico-biliary secretion from the duodenum, could affect ulcer healing. In addition, the effects of antagonism of CCK-A receptors (by loxiglumide, LOX) or CCK-B receptors (by L-365,260), an inhibition of NO-synthase by N(G)-nitro-L-arginine (L-NNA), or sensory denervation by large neurotoxic dose of capsaicin on CCK-induced ulcer healing were examined. Gastric ulcers were produced by serosal application of acetic acid and animals were sacrificed 9 days after ulcer induction. The area of ulcers and blood flow at the ulcer area were determined. Plasma levels of gastrin and CCK and luminal somatostatin were measured by RIA and mucosal biopsy samples were taken for histological evaluation and measurement of DNA synthesis. CCK given s.c. reduced dose dependently the ulcer area; the threshold dose of CCK being 1 nmol/kg and the dose inhibiting this area by 50% being 5 nmol/kg. This healing effect of CCK was accompanied by a significant increase in the GBF at ulcer margin and the rise in luminal NO production, plasma gastrin level and DNA synthesis. Concurrent treatment with LOX, completely abolished the CCK-8-induced acceleration of the ulcer healing and the rise in the GBF at the ulcer margin, whereas L-365,260 remained without any influence. Treatment with camostate or diversion of pancreatic juice that raised plasma CCK level to that observed with administration of CCK-8, also accelerated ulcer healing and this effect was also attenuated by LOX but not by L-365,260. Inhibition of NO-synthase by L-NNA significantly delayed ulcer healing and reversed the CCK-8 induced acceleration of ulcer healing, hyperemia at the ulcer margin and luminal NO release, and these effects were restored by the addition to L-NNA of L-arginine but not D-arginine. Capsaicin denervation attenuated CCK-induced ulcer healing, and the accompanying rise in the GBF at the ulcer margin and decreased plasma gastrin and luminal release of somatostatin when compared to those in rats with intact sensory nerves. Detectable signals for CCK-A and B receptor mRNAs as well as for cNOS mRNA expression were recorded by RT-PCR in the vehicle control gastric mucosa. The expression of CCK-A receptor mRNA and cNOS mRNA was significantly increased in rats treated with CCK-8 and camostate, whereas CCK-B receptor mRNA remained unaffected. We conclude that CCK accelerates ulcer healing by the mechanism involving upregulation of specific CCK-A receptors, enhancement of somatostatin release, stimulation of sensory nerves and hyperemia in the ulcer area, possibly mediated by NO.

Colorectal cancer (CRC) is the most common type of behavioral cancers, miRNAs play a critical role in cancer development and progression. In the present study, we downloaded the original data from Gene Expression Omnibus (GEO) and conduct data analysis. has-mir-29c-3p mimic, inhibitor, negative control or si-SPARC (secreted protein acidic, rich in cysteine) were transfected into HCT116 cells, respectively. Quantitative real time PCR (qRT-PCR) was used to measure has-mir-29c-3p and SPARC mRNA expressions, western blot was used to detect ACAA1 (acetyl-CoA acyltransferase 1), ACOX1 (acyl-CoA oxidase 1), COL1A1(collagen, type I, alpha-1), COL1A2 (collagen, type I, alpha-2), COL4A1 (collagen, type IV, alpha-1), COL5A2 (collagen, type V, alpha-2), COL12A1 (collagen, type XII, alpha-1), CPT2 (carnitine palmitoyltransferase 2), ETHE1 (persulfide dioxygenase), HMGCS2 (3-hydroxy-3-methylglutaryl-CoA synthase 2), SPARC, SQRDL (sulfide quinone oxidoreductase), and TST (thiosulfate sulfurtransferase) protein expression. CCK-8 and wound healing assay were employed to verify cell proliferation and migration. The luciferase reporter assay data made sure the target correlation of has-mir-29c-3p and SPARC. Firstly, we found that the expression of has-mir-29c-3p was lower in CRC tissues than in their paired corresponding non-cancerous tissues and there was significant inversed correlation between has-mir-29c-3p and SPARC. Overexpression of has-mir-29c-3p reduced cell proliferation and migration. SPARC was identified as a direct target of has-mir-29c-3p, whose silencing reduced cell proliferation and migration. These data showed that has-mir-29c-3p regulates CRC cell functions through regulating SPARC expression. Taken together, has-mir-29c-3p may function as an oncogenic miRNA targeting SPARC, targeted modulation of has-mir-29c-3p expression may became a potential strategy for the treatment.

An immunocytochemical investigation was carried out on round and spreading hemocytes of Planorbarius corneus by using 20 antisera to vertebrate bioactive peptides. The immunotests showed the presence of alpha 1-antichymotrypsin-bombesin-, calcitonin-, CCK-8 (INC)-, CCK-39-, gastrin-, glucagon-, Met-enkephalin-, neurotensin-, oxytocin-, somatostatin-, substance P-, VIP-, and vasopressin-immunoreactive molecules in the spreading hemocytes. The round hemocytes were only positive to anti-bombesin, anticalcitonin, anti-CCK-8 (INC), anti-CCK-39, anti-neurotensin, anti-oxytocin, anti-substance P and anti-vasopressin antibodies. No immunostaining was observed with anti-CCK-8 (Peninsula), anti-insulin, anti-prolactin, anti-thyroglobulin and anti-thyroxin (T4) antibodies. As probably in vertebrates, these bioactive peptides may modulate immuno cell function.

The current study tested the hypothesis that cholecystokinin (CCK) A receptor (CCKAR) in areas supplied by the celiac artery (CA), stomach and upper duodenum, and the cranial mesenteric artery (CMA), small and parts of the large intestine, is necessary for reduction of meal size, prolongation of the intermeal interval (time between first and second meal) and increased satiety ratio (intermeal interval/meal size or amount of food consumed during any given unit of time) by the non-nutrient stimulator of endogenous CCK release camostat. Consistent with our previous findings camostat reduced meal size, prolonged the intermeal interval and increased the satiety ratio. Here, we report that blocking CCKAR in the area supplied by the celiac artery attenuated reduction of meal size by camostat more so than the cranial mesenteric artery route. Blocking CCKAR in the area supplied by the cranial mesenteric artery attenuated prolongation of the intermeal interval length and increased satiety ratio by camostat more so than the celiac artery route. Blocking CCKAR in the areas supplied by the femoral artery (control) failed to alter the feeding responses evoked by camostat. These results support the hypothesis that CCKAR in the area supplied by the CA is necessary for reduction of meal size by camostat whereas CCKAR in the area supplied by the CMA is necessary for prolongation of the intermeal interval and increased satiety ratio by this substance. Our results demonstrate that meal size and intermeal interval length by camostat are regulated through different gastrointestinal sites.

Cholecystokinin octapeptide (CCK-8) and ceruletide (1 microgram/kg) produced a pronounced increment of plasma corticosterone levels at 30 min after intraperitoneal administration. The response to these peptides was suppressed by pretreatment with a selective antagonist for CCK-A receptors, (-)L-364,718, in a dose-related manner, but not with an antagonist for CCK-B receptors, (+)L-365,260. However, (-)L-364,718 itself had no effect on basal levels of plasma corticosterone. These results indicate that peripheral administration of CCK-8 and ceruletide stimulates the hypothalamo-pituitary adrenal axis through the activation of CCK-A receptors, but not CCK-B receptors.

Cholecystokinin (CCK) plays a major role in the regulation of pancreatic enzyme secretion based on its binding to the CCK-A receptor (CCK-AR). While CCK-AR is known to be expressed in rat islet B cells, the localization of CCK-AR in rat pancreatic A and D cells remains poorly understood. The aim of this study was to identify the localization of CCK-AR in rat pancreatic islets by means of double immunofluorescence straining with antibodies against CCK-AR, glucagon, insulin and somatostatin and with in situ hybridization to detect its transcript. CCK-AR-like immunoreactive cells were found to overlap both with glucagon-like immunoreactive cells and insulin-like immunoreactive cells but not with somatostatin-like immunoreactive cells. An in situ hybridization study using a cRNA probe for CCK-AR revealed that CCK-AR mRNA was expressed in the center and periphery of the pancreatic islets. Further to this, immunofluorecsence staining using anti-glucagon antibody was carried out after in situ hybridization using the CCK-AR cRNA probe in order to identify CCK-AR mRNA expressing cells. CCK-AR mRNA exhibited a distribution pattern almost identical to that of glucagon-like immunoreactive cells. These results show clearly that CCK-AR exists not only in B but also in A cells of the rat pancreas, suggesting that CCK regulates the secretion of insulin and glucagon at least partly via CCK-AR.

A method of in vitro translation scanning was applied to a variety of polytopic integral membrane proteins, a transition metal P type ATPase from Helicobacter pylori, the SERCA 2 ATPase, the gastric H+,K+ ATPase, the CCK-A receptor and the human ileal bile acid transporter. This method used vectors containing the N terminal region of the gastric H+,K+ ATPase or the N terminal region of the CCK-A receptor, coupled via a linker region to the last 177 amino acids of the beta-subunit of the gastric H+,K+ ATPase. The latter contains 5 potential N-linked glycosylation sites. Translation of vectors containing the cDNA encoding one, two or more putative transmembrane domains in the absence or presence of microsomes allowed determination of signal anchor or stop transfer properties of the putative transmembrane domains by the molecular weight shift on SDS PAGE. The P type ATPase from Helicobacter pylori showed the presence of 8 transmembrane segments with this method. The SERCA 2 Ca2+ ATPase with this method had 9 transmembrane co-translational insertion domains and coupled with other evidence these data resulted in a 11 transmembrane segment model. Translation of segments of the gastric H+,K+ ATPase provided evidence for only 7 transmembrane segments but coupled with other data established a 10 membrane segment model. The G7 protein, the CCK-A receptor showed the presence of 6 of the 7 transmembrane segments postulated for this protein. Translation of segments of the human ileal bile acid transporter showed the presence of 8 membrane insertion domains. However, translation of the intact protein provided evidence for an odd number of transmembrane segments, resulting in a tentative model containing 7 or 9 transmembrane segments. Neither G7 type protein appeared to have an arrangement of sequential topogenic signals consistent with the final assembled protein. This method provides a useful addition to methods of determining membrane domains of integral membrane proteins but must in general be utilized with other methods to establish the number of transmembrane alpha-helices.

The purpose of this study was to develop poly(lactide-co-glycolide) (PLGA) based in situ forming implants (ISFI) for controlled release of thymosin <em>alpha</em> 1 (T<em>alpha</em>1). The ISFI was prepared by dissolving PLGA in N-methyl-2-pyrrolidone (NMP) or mixtures of NMP and triacetin. T<em>alpha</em>1 microparticles, prepared by spray-freeze drying method with chitosan or bovine serum albumin as a protectant, were suspended in PLGA solutions. The effects of T<em>alpha</em>1 pre-encapsulation, PLGA molecular weight, PLGA concentration and organic solvents composition on the in vivo T<em>alpha</em>1 release were evaluated by subcutaneously injecting T<em>alpha</em>1-loaded ISFI into Sprague-Dawley Rats. The pharmacological efficacy of T<em>alpha</em>1-loaded ISFI was examined using immunosuppressive BALB/c mice induced by cyclophosphamide. The ISFI composed of T<em>alpha</em>1 pre-encapsulated with chitosan, higher molecule-weight PLGA at higher concentration and more triacetin showed a lower initial release and a longer sustained release period. The optimal prescription of our study showed a low initial release of 29.3% (24 h), followed by a slow and continuous drug release up to 28 d in vivo. An in vitro release device was designed to mimic the in vivo release of T<em>alpha</em>1, and good correlation was observed between the in vitro and in vivo releases, with the linear correlation coefficient of 0.9899. T<em>alpha</em>1-loaded ISFI showed low cytotoxicity as tested by <em>CCK</em>-8 assay. T<em>alpha</em>1-loaded ISFI significantly increased the thymic index and spleen index of immunosuppressive mice. These results suggest that the ISFI is a suitable system for controlled release of T<em>alpha</em>1.