Shaking of the intact rat and intestinal angulation diminish the jejunal unstirred layer. A sizeable pre-epithelial diffusion barrier (unstirred layer) is present during perfusion of the rat jejunum. In the present study, three rapidly transported compounds, CO, [14C]warfarin, and glucose (5.5 mmol/L), were used as probes to assess the ability of manipulations to reduce the unstirred layer. This layer was 700-800 microns thick in a 30-cm jejunal segment perfused in conventional fashion on the abdominal wall. Placement of four sharp angulations in the segment or replacement in the abdominal cavity reduced the maximal unstirred layer to 200-400 microns. Increasingly rapid shaking of the anesthetized, intact rat on a platform shaker produced progressively thinner unstirred layers. At 250 revolutions per minute, the maximal layer ranged from 32 to 68 microns for the three probes and may have been appreciably less if the epithelium offered appreciable resistance. Shaking yields a > 15-fold reduction in unstirred layer resistance and provides a means for measuring this resistance and for obtaining more accurate assessment of the true in vivo transport Michaelis constant (Km) of any compound.


Transforming growth factor alpha receptor binding sites in the canine gastrointestinal tract. Transforming growth factor alpha (TGF-alpha) interacts with the same plasma membrane receptor as epidermal growth factor and is likely to play a role in proliferative and trophic processes of gastrointestinal tissues. The distribution of receptor binding sites for TGF-alpha was examined in the canine gastrointestinal tract (distal esophagus, stomach fundus, descending duodenum, jejunum, ileum, transverse colon) by tissue section autoradiography. 125I-TGF-alpha yielded a labeling pattern comparable to that of 125I-epidermal growth factor. Specific binding sites were particularly abundant in the mucosa in each region, with the highest concentration in the esophagus, colon, and stomach, as assessed by computer assisted densitometry. The density of binding sites was moderate in the stomach muscularis mucosae, low in the external muscle layer, and very low to undetectable in the submucosa throughout the gastrointestinal tract. In most cases, the greatest density within the individual regions was detected in the area characterized by the highest proliferative rate. Lymphoid aggregates were not labeled. In conclusion, TGF-alpha receptor binding sites are present throughout the gastrointestinal tract with differential patterns in the various regions; they are principally distributed to the mucosa and predominantly located to proliferative cell areas. These results are consistent with a role of this factor in regional regulation of proliferation and differentiation in the gut.


Importance of gastric acid in gastric ulcer formation in rabbits with antibody-induced prostaglandin deficiency. The role of gastric acid in the development of gastroduodenal ulcers in prostaglandin-deficient conditions is unclear. In the current study, the effect of the proton pump inhibitor omeprazole on the formation of gastric ulcers was examined in a previously validated rabbit model of antibody-induced prostaglandin deficiency. Intragastric administration of 20 mg/kg omeprazole every 12 hours caused a profound suppression of gastric acidity (i.e., pH above 5 continuously). This same dose of omeprazole significantly reduced gastric ulcer formation induced by passive immunization with 6-keto-prostaglandin F1 alpha antibodies. It is concluded from these observations that gastric acid plays a critical role in the formation of gastric ulcers in rabbits with antibody-induced prostaglandin deficiency.


Surface hydrophobicity of gastric mucosa in Helicobacter pylori infection: effect of clearance and eradication. Surface hydrophobicity of the gastric mucosa is reduced in peptic ulcer disease and Helicobacter pylori infection. This abnormality may be caused by H. pylori or may be an inherent defect. The aim of the present study was to clarify the relationship between H. pylori infection and mucosal hydrophobicity by examining the effect of eradication of the organism. H. pylori-positive patients with (n = 42) or without (n = 42) duodenal ulcer were randomized to receive ranitidine, bismuth, or bismuth plus antibiotics. Surface hydrophobicity of gastric mucosa was assessed by measurement of plateau-advancing contact angle. Measurements were performed at presentation, end of treatment, and 1 month later. Contact angle was unchanged after ranitidine (55 degrees vs. 56 degrees) but increased with bismuth (57 degrees-62 degrees; P < 0.05) and bismuth plus antibiotics (56 degrees-67 degrees; P < 0.0001). One month after treatment ended, contact angles in patients in whom H. pylori was not eradicated were not different from those before treatment (56 degrees vs. 56 degrees) but increased to a value similar to H. pylori-negative controls in patients in whom H. pylori was eradicated (56 degrees-69 degrees; P < 0.0001). It is concluded that reduced mucosal hydrophobicity in peptic ulcer disease is secondary to H. pylori infection and that this impaired mucosal defense provides a possible mechanism whereby H. pylori infection predisposes to acid/peptic digestion.


Adrenergic blockage does not restore the canine gastric migrating motor complex during vagal blockade. Bilateral cervical cooling blockade of the vagi abolishes the gastric migrating motility complex (MMC), except for the pylorus. The present study investigated if this finding is caused by adrenergic inhibition of the stomach via sympathetic nerves entering the vagi below the level of the blockade. Stomach and upper small bowel motor activity was monitored in five chronic dogs whose vagosympathetic nerve trunks were previously isolated in bilateral, cervical skin loops to permit blockade by cooling. Adrenergic blockade was performed by an initial bolus injection of phentolamine (0.3 mg/kg) and propranolol (0.3 mg/kg) followed by a continuous intravenous infusion of the combined drugs at a rate of 1.5 mg.kg-1.h-1 for phentolamine and 0.3 mg.kg-1.h-1 for propranolol. Bilateral cervical vagal blockade abolished the gastric MMC, with persistence of phase III of the MMC in the upper small bowel. During combined vagal and adrenergic blockade, gastric contractions were absent in 4 of the 22 duodenal complexes observed. In the other duodenal complexes, intermittent gastric contractions were associated with all phases of the intestinal MMC, including phase I. At times, this contractile activity was accentuated during phase III but never reached the intensity of activity or showed the typical coordination features of the gastric MMC seen with the vagi intact. Therefore, unopposed adrenergic inhibition of the stomach does not explain the absence of the gastric MMC during vagal cooling blockade. Under normal circumstances, the vagus nerve is the most important pathway for central control of the appearance of the gastric MMC.


Currently used doses of omeprazole in Zollinger-Ellison syndrome are too high. The efficacy of omeprazole increases during the first few days of administration, suggesting that long-term maintenance dose requirements in patients with Zollinger-Ellison syndrome may be lower than those initially established by upward titration. Long-term maintenance doses of omeprazole were prospectively reduced in 37 patients who had been taking omeprazole for 22 +/- 4 months. Successful reduction was defined as reduction to 20 mg once or twice daily with an absence of symptoms, endoscopy without evidence of active acid-peptic disease, and a gastric acid output of < 10 mEq/h. Sixty-eight percent of patients (25/37) were successfully reduced to 20 mg of omeprazole once (18/24) or twice daily (7/13). Ninety-five percent of patients (20/21) without multiple endocrine neoplasia type I, severe gastroesophageal reflux disease, or previous partial gastrectomy had safe reductions of doses. It is concluded that the currently used omeprazole maintenance doses in patients with Zollinger-Ellison syndrome are too high and advocated that the initial dose still be established by acute daily upward titration followed by gradual reduction once control of acid output has been achieved.


Induction of cytochrome P450IA genes (CYP1A) by omeprazole in the human alimentary tract. Cytochrome P450 enzymes are capable of converting procarcinogens into either active mutagens or inactive metabolites. Because the distribution of these enzymes may be important for tissue susceptibility to procarcinogens, the expression and induction of CYP1A genes in the human alimentary tract were investigated. Endoscopic biopsy specimens were obtained from buccal mucosa, esophagus, gastric body, antrum, duodenum, and colon of 6 healthy volunteers before and 1 week after taking 20 mg of omeprazole daily. Tissue specimens were analyzed for the presence of CYP1A1 and 1A2 transcripts using hybridization methods and the polymerase chain reaction. P450-dependent enzymatic activity was assessed by deethylation of ethoxyresorufin. CYP1A1 messenger RNA (mRNA) and ethoxyresorufin activity were present constitutively in the duodenum of each volunteer. Omeprazole (20 mg/day for 1 week) induced CYP1A1 mRNA and enzymatic activity in 5 of 6 volunteers. The one individual who did not initially respond had a marked increase in both mRNA and enzymatic activity after receiving 60 mg of omeprazole daily for 1 week. After treatment with omeprazole, two individuals had low levels of CYP1A1 mRNA in several other alimentary tissues as well as low levels of CYP1A2 mRNA in the duodenum. The expression and induction by a pharmaceutical agent of CYP1A genes may have implications for intestinal metabolism of ingested xenobiotics including procarcinogens.


Gliadin immune reactivity is associated with overt and latent enteropathy in relatives of celiac patients. Antigliadin antibodies have been widely used in the screening of celiac disease. Using this test, candidates for jejunal biopsy were selected from 328 first-degree relatives of 128 adult celiac patients. All relatives geographically available were willing to participate in the study. Twenty-one turned out to be positive for antigliadin antibodies, and in 13 jejunal histology was consistent with celiac disease. In the remaining 8, the morphometric evaluation of jejunal biopsy specimens showed a mean surface to volume ratio that, although in the normal range, was significantly lower than that of other 10 relatives negative for antigliadin antibodies and 16 biopsied controls. It was concluded that antigliadin antibody testing is a valuable method for the screening of celiac disease among family members and that relatives with genetically predisposed gliadin sensitivity, without gross histological lesions but with minor morphometric abnormalities of the jejunal mucosa, may be regarded as subjects with latent celiac disease.


Manometry can predict feeding readiness in preterm infants. To assess the usefulness of duodenojejunal manometry in predicting when neonates will tolerate enteral feeding, small intestinal manometry was performed in 48 preterm infants. Characteristics of motor activity during fasting and feeding differed in 40 infants who were identified to be tolerant and 8 who were intolerant of feeding. Among infants who were feeding intolerant, motor quiescence was less pronounced and clustered motor activity more prominent than it was in infants who were able to tolerate feedings (P < 0.005). Those infants who tolerated feedings changed their pattern of motor activity in response to feeding (P < 0.01), and infants who did not tolerate feedings had no "fed response." Infants who were initially feeding intolerant became tolerant of feedings coincident with the appearance of motor activity that was similar to that of infants who were initially tolerant to food. The sensitivity of manometry to predict feeding intolerance was 1.0, and its specificity was 0.13.


Rabbit gut permeability in response to histamine chloramines and chemotactic peptide. Granulocyte-derived chlorinated amines and bacterial formyl peptides are thought to enhance epithelial permeability. In the current study, gut permeability to [51Cr]ethylenediaminetetraacetic acid (EDTA) was monitored in response to luminal formyl-methionyl-leucyl-phenylalanine (fMLP) and histamine monochloramine and dichloramine. Responses were determined in rabbits during states of basal and elevated permeability. Luminal fMLP had minimal effects of gut permeability in control and injured states. Histamine monochloramine or dichloramine enhanced epithelial permeability under basal conditions; this effect was exaggerated by a pre-existing injury. Both histamine monochloramine and dichloramine retained full histamine agonist properties, and a combination of antioxidant and antihistamine therapy was required to block this increase in gut permeability. Whereas histamine chloramines caused a dose-dependent cytotoxicity in rat-cultured enterocytes, marked histological changes to the mucosa were not evident, nor were mucosal glutathione levels depleted. As histamine chloramines retain the histaminergic and oxidizing potential of their precursors, they represent a unique form of inflammatory mediator, although their highly reactive nature precludes in vivo confirmation of their formation.


Biomechanical wall properties and collagen content in the partially obstructed opossum esophagus. A specially designed probe was used to investigate biomechanical wall properties of the esophagus in opossums with distal esophageal banding. A balloon was stepwise inflated and deflated 6 cm above the lower esophageal sphincter before the banding and at 2 weeks postoperatively. After animals were killed, collagen content of the esophageal wall was determined. Preoperatively, cross-sectional area and wall tension at maximal inflation of the balloon, compliance, and hysteresis were 108.04 +/- 7.23 mm2, 5.8 +/- 0.3 cm H2O/m, 0.664 +/- 0.081 mm2/cm H2O, and 101.14 +/- 18.89 mm2, respectively. Postoperatively, the parameters increased to 357.45 +/- 54.22 mm2 (P < 0.001), 10.5 +/- 1.4 cm H2O/m (P < 0.001), 2.402 +/- 0.420 mm2/cm H2O (P < 0.001), and 225.86 +/- 44.56 mm2 (P < 0.05), respectively. The collagen content was 0.065 +/- 0.004 and 0.104 +/- 0.009 mg/mg dry defatted weight in the mid and distal normal esophagus, respectively, compared with 0.110 +/- 0.012 and 0.121 +/- 0.012 mg/mg in the banded group (P < 0.05 in the middle part). A positive correlation was shown between the maximal cross-sectional area and the collagen content in the mid-esophagus (P < 0.05). In conclusion, biomechanical wall properties were altered significantly, indicating dilatation and decreased ability of the esophageal wall to resist intraluminal pressure.


Blood group-related antigen expression in normal and metaplastic human upper gastrointestinal mucosa. Lewis antigen expression in upper gastrointestinal epithelium was studied using four monoclonal antibodies to determine the relationship between aberrant differentiation and antigen expression. Specific patterns of type 1 and type 2 Lewis blood group antigen expression were found in the surface and glands of the esophagus, gastric fundus, and duodenum. In biopsy specimens of Barrett's esophagus, gastric fundic-type columnar metaplasia expressed Lewis antigens indistinguishable from those in the normal stomach. In Barrett's junctional and specialized columnar metaplasia, Lewis a antigen was aberrantly expressed on the surface in secretors and in the glands independent of secretor state. Lewis x reactivity was markedly diminished in the glands of Barrett's junctional and specialized columnar epithelium irrespective of secretor state. There was no significant aberrancy observed in the expression of Lewis b and y antigens. The observed aberrant expression of Lewis antigens may be caused by an altered differentiation program in Barrett's metaplastic epithelium and may define a role for these glycoconjugates in the process of metaplasia and carcinogenesis in Barrett's epithelium.


Failure of rectal ornithine decarboxylase to identify adenomatous polyp status. Rectal mucosal ornithine decarboxylase (ODC) activity has been reported to distinguish between patients with and without adenomatous polyps (AP). In the present investigation, ODC activity has been measured in 28 patients with AP and 34 patients without AP. To assess the intraindividual variation in ODC activity, repeat biopsies were performed on 11 patients. In addition, the effect of postbiopsy sample handling was investigated by storage of samples on either dry or wet ice during transport to the laboratory. The mean rectal mucosal ODC activity in patients with AP was 196.0 +/- 195.5, whereas that in AP negative patients was 182.2 +/- 320.5. The rectal mucosal ODC activity in patients with colorectal cancer was 388.2 +/- 581. Repeat samples in individuals were generally within the same range as the original samples. The method of sample transport did not significantly affect the level of ODC measured in a particular biopsy. Because of high variability in rectal mucosal ODC activity within the population, there was wide overlap in ODC values between those patients with and without AP in an unselected general population. Thus, the measurement of flat rectal mucosal ODC activity is not a good predictor of the presence or absence of AP. Additional studies of the factors affecting mucosal ODC activity are necessary before the potential clinical utility of the method can be realized in the general population.


Effect of sorbin on duodenal absorption of water and electrolytes in the rat. Sorbin is a newly isolated intestinal peptide that has been purified because of its ability to induce water absorption. The effects that sorbin and some synthetic peptides corresponding to its C-terminal sequence have on duodenal absorption of water, chloride, and sodium were studied in comparison with the effects of vasoactive intestinal peptide (VIP), [D-Ala,Met]-enkephalinamide (DAMA), and angiotensin II. The technique of an in situ ligated duodenal loop in the rat was used for all peptides. Under the experimental conditions used, a low basal secretion of water, chloride, and sodium was obtained; VIP induced an increase of the secretion, whereas DAMA induced an absorption, both in the nanomolar dose range. Angiotensin II and sorbin induced an absorption in the picomolar dose range. The most effective doses of sorbin peptides but not of angiotensin induced the lowest final concentrations of Na+ and Cl- obtainable without inducing secondary water secretion. All synthetic peptides containing the C-terminal heptapeptide of sorbin were active in the picomolar dose range. Contrary to angiotensin, they had no effect on blood pressure.


Alteration of the upper esophageal sphincter belch reflex in patients with achalasia. Some patients with achalasia have been reported to develop airway obstruction from a massively air-distended esophagus, which may represent an abnormality in the upper esophageal sphincter belch reflex. When questioned carefully, 95% of our achalasia patients reported difficulty with belching. The upper esophageal belch reflex in 23 consecutive achalasia patients and 12 healthy controls was studied using an upper esophageal sphincter sleeve manometry catheter and rapid injection of 20-50 mL of air into the midesophagus. Compared with normal subjects, achalasia patients were significantly less likely to have an esophageal belch for all volumes tested and were more likely to have an increase rather than a decrease in upper esophageal sphincter pressure in response to air injection. This study systematically documents that many achalasia patients have an alteration in the upper esophageal sphincter belch reflex that may be a contributory mechanism for some of the chest and upper airway symptoms reported by some patients during acute esophageal distension.


Chronic active gastritis due to Helicobacter pylori in immunized gnotobiotic piglets. In gnotobiotic piglets, parenteral vaccination with formalin-killed Helicobacter pylori and oral vaccination with live bacteria induced H. pylori-specific serum immunoglobulins G, M, and A. Vaccination reduced but did not prevent infection by subsequent challenge with viable H. pylori. Oral vaccination with killed bacteria was less effective in inducing serum antibody and had no effect on bacterial colonization. Immunization status influenced the histologic response of piglets to challenge by H. pylori. Lymphoplasmacytic gastritis was more severe in parenterally vaccinated piglets than in the other groups. In addition, neutrophilic infiltrates and neutrophilic gland abscesses in the gastric mucosa were present in 5 of 7 parenterally immunized piglets, none of the orally immunized piglets, and only 1 of 8 infected nonimmune control piglets. Furthermore, there was a positive correlation between immunoglobulin G, M, and A titer at challenge and severity of both neutrophilic and lymphocytic inflammation. These results suggest that although vaccination does not prevent infection by H. pylori, infection of an immune host leads to increased severity and activity of gastritis.


Cytokine messenger RNA profiles in inflammatory bowel disease mucosa detected by polymerase chain reaction amplification. Immunoregulatory properties of cytokines may mediate disordered inflammatory events in ulcerative colitis (UC) and Crohn's disease (CD). In the present study, profiles of cytokines produced by activated macrophages were studied in colonic tissue from 43 patients with and without inflammatory bowel disease (IBD). Cytokine messenger RNA (mRNA) extracted from mucosal biopsy specimens was studied using polymerase chain reaction assay techniques. A greater percentage of active UC samples had detectable levels of mRNA for interleukins (IL) 1, 6, and 8 and gro than samples in inactive UC and noninflammatory controls. These cytokines were comparable in active UC and inflammatory controls. Expression of gro mRNA in active UC tissue was significantly higher than in active CD. Tumor necrosis factor was detected in only 7 of 43 samples with no difference between groups. Active and inactive CD did not differ in percentage of cytokine mRNA expression. IL-1 receptor antagonist (IL-1ra) was detected in more inflammatory controls than in CD and was expressed in fewer IBD patients than IL-1. Expression of proinflammatory cytokines in grossly inactive CD and possible defective production of IL-1ra may explain disease reactivation and chronicity.


Effect of gastrin receptor blockade on endocrine cells in rats during achlorhydria. Hyperplasia of the oxyntic enterochromaffinlike cells in response to long-lasting blockade of acid secretion is closely related to hypergastrinemia. In the present study, the effect of a specific gastrin receptor antagonist on proton pump inhibitor-induced changes on serum gastrin levels, mucosal height, as well as gastrin- and enterochromaffin-like cells was investigated in rats. The proton pump inhibitor BY 308 or the vehicle methylcellulose [Methocel (controls)] was administered for 2 weeks in the presence and absence of the gastrin receptor antagonist PD 136450 (CAM 1189). BY 308 significantly increased serum gastrin levels, gastrin cell density, and antral gastrin concentration. Concomitant application of PD 136450 did not alter this response. In the oxyntic stomach, mucosal height, enterochromaffinlike cell density, labeling index of enterochromaffinlike cells, and histamine concentration were elevated after treatment with BY 308. These increases were almost completely abolished by PD 136450. Even in normogastrinemic control rats, PD 136450 significantly decreased mucosal height of the oxyntic part of the stomach and the labeling index of enterochromaffinlike cells. The results show that (a) trophic effects of drug-induced achlorhydria are mediated by gastrin; (b) even in control rats (normogastrinemic), gastrin is a trophic factor for the oxyntic mucosa; and (c) antral gastrin cell hyperplasia in states of chronic achlorhydria is not mediated by gastrin itself.


DNA aneuploidy in colonic biopsies predicts future development of dysplasia in ulcerative colitis. The objective of the present study was to determine whether abnormal epithelial DNA content (aneuploidy) in colonic biopsy specimens from ulcerative colitis (UC) patients correlated with and predicted histological progression to dysplasia. Aneuploidy was absent in 20 low-cancer risk patients. In 81 high-cancer risk patients aneuploidy correlated significantly with the severity of histological abnormality (negative, indefinite, dysplasia, or cancer). Statistically our data suggest that many more biopsy specimens than are usually taken are needed to detect focal dysplastic lesions. Prospective study of 25 high risk patients without dysplasia revealed 5 with aneuploidy, all of whom progressed to dysplasia in 1-2.5 years, whereas 19 patients without aneuploidy did not progress to either aneuploidy or dysplasia within 2-9 years. Our data indicate that aneuploidy in mucosal biopsy specimens correlates with histological grade and identifies a subset of patients without dysplasia who are more likely to develop it. It was concluded that more frequent and extensive colonoscopic surveillance of this minority subset of high risk patients and less frequent surveillance in the remaining majority may reduce cost and detect more curable lesions.


Serum hepatocyte growth factor levels in hepatectomized and nonhepatectomized surgical patients. Serum hepatocyte growth factor levels were measured in hepatectomized and nonhepatectomized surgical patients. The levels were significantly increased and reached a maximum within 7 days after surgery in both groups, returning to preoperative levels 28 days after partial hepatectomy and 7 days after other operations. Multiple regression analysis showed that such maximal hepatocyte growth factor levels were significantly related to having liver cirrhosis and postoperative maximal serum total bilirubin and alanine aminotransferase levels and peripheral white blood cell counts in the hepatectomized group and to postoperative maximal peripheral white blood cell counts and serum C-reactive protein levels in the nonhepatectomized group. However, the levels showed no relation to the resected liver volume and increment of the remaining liver volume 28 days after partial hepatectomy. It is concluded that serum hepatocyte growth factor levels were increased after partial hepatectomy in association with hepatocellular dysfunction and necrosis and systemic inflammation. It is unlikely that the increase was related to liver regeneration.


Antioxidant defenses in the bile duct-ligated rat. Changes in pro-oxidant and antioxidant balance in the serum and liver were studied in an experimental model of obstructive jaundice in the rat. The results showed a decrease in plasma vitamin E concentration (P < 0.01) and a threefold reduction in liver vitamin E concentration (P < 0.001). There was also a threefold reduction in levels of the liver enzymes glutathione peroxidase (P < 0.01) and glutathione transferase (P < 0.001), together with a six-fold reduction in catalase activity (P < 0.001). The serum selenium level decreased by 35% in the jaundiced rats (P < 0.05). The total liver glutathione level decreased to half the control value (P < 0.01). The malonyldialdehyde level, the measure of lipid peroxidation used in this study, doubled (P < 0.01). The results suggest a shift in the pro-oxidant/antioxidant balance in favor of lipid peroxidation. The possible etiology of this change and its relationship to human cholestasis are discussed.


Survival and prognostic indicators in hepatitis B surface antigen-positive cirrhosis of the liver. To evaluate indications for new therapies such as liver transplantation and antiviral therapy, survival of histologically proven hepatitis B surface antigen (HBsAg)-positive cirrhosis of the liver was assessed in a cohort of 98 patients followed up for a mean of 4.3 years. The overall survival probability was 92% at 1 year, 79% at 3 years, and 71% at 5 years. Variables significantly associated with the duration of survival were age, serum aspartate aminotransferase levels, presence of esophageal varices, and all five components of the Child-Pugh index (bilirubin, albumin, coagulation factors, ascites, encephalopathy). Multivariate analysis showed that only age, bilirubin, and ascites were independently related to survival. Survival of patients with decompensated cirrhosis (determined by the presence of ascites, jaundice, encephalopathy, and/or a history of variceal bleeding) and those with compensated cirrhosis at 5 years was 14% and 84%, respectively. For patients with compensated liver cirrhosis, hepatitis B e antigen (HBeAg) positivity was also a prognostic factor with a 5-year survival of 72% for HBeAg-positive cirrhosis and 97% for HBeAg-negative cirrhosis; the risk of death was decreased by a factor of 2.2 when HBeAg seroconversion occurred during follow-up. It is concluded that liver transplantation should be considered for patients with decompensated HBsAg-positive liver cirrhosis and antiviral therapy for patients with HBeAg-positive compensated cirrhosis.


Prognostic factors in sterile pancreatic necrosis. Although the overall mortality in sterile pancreatic necrosis is low, patients who experience systemic complications may have a higher mortality. To study the impact of systemic complications and other factors on survival, possible prognostic factors were evaluated among 26 patients who experienced at least one systemic complication. Mortality was 38%. Factors that correlated with a fatal outcome were high Ranson's scores during the first 48 hours (P = 0.01), high APACHE-II scores at admission (P = 0.04) and at 48 hours (P = 0.03), shock (P < 0.001), renal insufficiency (P < 0.05), multiple systemic complications (P < 0.001), and high body mass index (P = 0.01). Most systemic complications occurred during the first 2 weeks of illness. Logistic regression analysis showed that shock was the best predictor of a fatal outcome. Patients with favorable prognostic factors survived whether treated medically or surgically, whereas those with unfavorable factors had a fatal outcome whether treated medically or surgically. It is concluded that patients with severe sterile necrosis have a high mortality rate and that shock and other prognostic factors identify which patients are most likely to have a fatal outcome.


Hepatic cholesterol metabolism in estrogen-treated men. Operative liver biopsies were obtained from two male patients who developed gallstone disease during estrogen treatment of metastatic prostatic carcinoma. The heparin-sensitive binding of 125I-low-density lipoprotein (LDL) to liver homogenates (reflecting the expression of the LDL receptor) was determined, together with the activities of the rate-limiting enzymes in cholesterol synthesis [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase], bile acid production (cholesterol 7 alpha-hydroxylase), and cholesterol esterification (acyl CoA:cholesterol acyl transferase). The results were related to data available in 18 patients (5 male, 13 female) who underwent cholecystectomy because of gallstone disease. The hepatic 125I-LDL-binding activity was increased threefold compared with five controls, and the activity of HMG-CoA reductase was increased twofold. There was no major difference in the activities of cholesterol 7 alpha-hydroxylase or acyl CoA:cholesterol acyl transferase. The concentration of free and total cholesterol in liver microsomes was approximately 30% lower in the estrogen-treated men than in 11 controls. The results indicate that estrogen at pharmacological doses stimulates hepatic LDL-receptor expression and HMG-CoA reductase activity in men. The increased LDL-receptor expression could in part explain the enhanced plasma clearance of injected 125I-LDL and hence the reduction in plasma LDL cholesterol previously shown to occur in estrogen-treated men.


Menetrier's disease associated with formula protein allergy and small intestinal injury in an infant. Menetrier's disease in infancy is extremely rare, and its natural course has not been studied in detail. The present case report describes an infant whose initial diagnosis was formula protein allergy and who developed gastric outlet obstruction by 3 months of age. The diagnosis of Menetrier's disease was suggested by characteristic radiological, pathological, and functional abnormalities of the stomach. Small intestinal partial villous atrophy, malabsorption, and protein loss from both the stomach and the intestine were documented. Cytomegalovirus infection was excluded. There was no evidence for an immune deficiency. The described features and an unrelenting course suggest that infantile Menetrier's disease may be an entity distinct from the childhood and adult forms.


[Suppressive effect of circulating immune complexes on cytotoxicity of blood lymphocytes in patients with chronic myeloid leukemia]. Concentration of circulating immune complexes (CIC) and cytotoxicity (natural and antibody-dependent) of blood lymphocytes were studied in 52 patients at the developed stage of chronic myeloid leukemia (CML). Elevation of CIC level and reduction of cytotoxicity detectable in all CML patients were most manifest in those with high tumor mass and during exacerbation of the leukemia process. A conclusion has been made that CIC are suppressors of natural and antibody-dependent cytotoxicity of blood lymphocytes in CML patients.


[Blood protein-volume indicators in massive plasmapheresis]. Hyperproteinemia values were studied before and after plasmapheresis in 32 rheumatoid arthritis patients with no manifest circulatory disorders. Mathematical patterns enabling prediction of changes in protein concentration depending on the volume of removed plasma and the type of substitution have been proposed for 3 alternative regimens of the procedure (removal of 0.5 or 1.0 volume of the circulating plasma, and substitution for rheopolyglucin or albumin). The data of independent "examining" sampling (15 patients) have proved adequacy of the linear regression patterns.


[Preparation of universal anti-rhesus sera using soluble synthetic group A and B substances]. Isohemagglutinins alpha and beta were neutralized by soluble synthetic group A and B substances in group ++anti-rhesus sera. The investigations conducted have shown that water-soluble polymers A and B are efficient agents for the preparation of universal ++anti-rhesus sera. The neutralizing properties of synthetic and natural group A and B substances were comparatively studied.


[Refractory effect and alloimmunization after transfusion of thrombocytes]. The authors discuss possible ways for prevention of alloimmunization with HLA-antigen in acute leukemia patients after platelet transfusions. Prevention or delay of HLA-alloimmunization is possible when platelets of single random donor are used instead of pooled random donor platelets for transfusions. The effect of leucocyte removal from platelet concentrates and ultraviolet irradiation of platelet concentrates under conditions of blood bank on alloimmunization and refractoriness incidence has been considered.


[The role of plasma fibronectin in the pathogenesis of post- aggressive conditions]. The interrelations of changes in plasma fibronectin (FN) level, hemostatic parameters and RES function were studied in the time course of traumatic or burn disease. A combination of interdependent disorders in the systems studied reflecting features of varying periods of extreme states was established in rat experiments. The analysis of the data obtained has suggested that FN is involved in the pathologic process and participates in the pathogenesis of shock and its complications by interacting with hemostasis and RES as a mediator thus playing an important role in maintaining the level of nonspecific body resistance.


[Stomatologic aid to patients with hereditary coagulopathies under ambulatory conditions in the hemophilia center]. Simple and available for wide clinical practice methods of treatment and prevention of complicated forms of dental caries in patients with hemophilia and Willebrand's disease have been presented, as well as effective methods for prevention of hemorrhage from the small cavity after a tooth removal in these patients under conditions of hematological centres.


[Transferrin iron-binding capacity in hypersideremia]. Three methods for evaluation of serum iron-binding capacity have been described: biochemical, immunological based on transferrin assay, and biophysical based on electron-paramagnetic resonance (EPR) spectroscopy of transferrin. Interrelation has been shown between transferrin and general serum iron-binding capacity. Basing on the data presented it is suggested that in secondary hemochromatosis plasma contains an iron pool that is not specifically bound with transferrin, while in primary hemochromatosis such pool was not detected.


[Influence of mast cells on the regulatory mechanisms of hemopoietic bone marrow in inflammation]. Acute infectious peritonitis simulated in mice was used to show that previous osmotic deterioration of the abdominal mast cell population significantly influenced the function of hemopoiesis-inducing microenvironment (HIM). The earlier production of the colony-stimulating and erythropoietic activities by the non-adhesive myelokaryocytes and the decreased production of these activities by the adhesive cells have been established. The results obtained evidence that mast cells modulate HIM mediating their influence on hemopoiesis under natural conditions of inflammation.


[Erythrocytic ferritin in pregnant women and infants in the first year of life]. Erythrocytic ferritin levels were followed up in pregnant women with reference to its course, in newborns, and in infants during the first year of life. It is shown that the level of erythrocytic ferritin remains high enough in women with complicated pregnancy attended by a sharp reduction of iron reserves. No clear dependence of erythrocytic ferritin concentration on the character of the antenatal period course was observed in infants. The highest values of erythrocytic ferritin parameters were recorded by the first month and they decreased constantly by the first year of life.


[Combined evaluation of iron reserves in women]. Peripheral blood hemoglobin levels, serum iron, general serum iron-binding capacity, transferrin saturation with iron, transferrin, serum ferritin and erythrocytic ferritin were studied in 101 clinically normal women. Apparent and latent iron deficiency was rather frequently detected among them. Information on transferrin saturation with iron, erythrocytic ferritins and, particularly, serum ferritin, was most valuable in the estimation of iron deficiency. The combined evaluation of erythrocytic and serum ferritins has permitted the authors to distinguish 5 variants of iron reserves in the body and to avoid discrepancy arising in their individual estimations.


[Intravascular autorosette-formation in hemolytic anemia]. It has been established that monocyte and neutrophil autorosette-formation resultant in exocytic lysis of erythrocytes--constituents of autorosettes--takes a low-intensive course in the blood of children with chronic non-hematologic diseases. This process is significantly activated in hereditary microspherocytic and elliptocytic anemias. It has been suggested that autorosette-formation culminating in exocytic lysis of erythrocytes is a mechanism of eliminating pathological, damaged and old erythrocytes from the blood, both under physiological conditions and in varying diseases.


[Iron metabolism in anemia patients with no deficiency of iron reserves]. Iron metabolism was studied in anemia patients with no iron deficiency. The data obtained have suggested that iron transport to blood-synthesizing cells i disturbed in such patients with normal iron reserves. Basing on the study of iron metabolism and red blood parameters in the patients after the treatment by hypoxic hypoxia it is shown that this method combined with iron therapy can be successfully used for the treatment of this form of iron deficiency anemia.


[Diagnostic value of serum iron metabolism parameters in patients with malignant and reactive histiocytosis]. Difficulties in the morphological diagnosis of the diseases due to proliferation of mononuclear phagocyte system (MPS) cells have stimulated the search for additional differential-diagnostic criteria. The investigations were conducted in 10 malignant histiocytosis, 16 histiocytosis-X, and 34 reactive histiocytosis patients. The assay of serum ferritin was shown to have high informative value for clinical practice. The presence of extremely high hyperferritinemia in patients with morphologically verified histiocytic proliferations has evidenced a malignant character of the process.


[Clinical criteria for evaluation of hemophilia severity in expert estimation of patient's working capacity]. Data are presented on the evaluation of hemophilia severity during expert estimation of the patient's working capacity according to the information of the Republican Centre. The authors have developed the most optimal classification for the evaluation of the disease severity; the influence of hemorrhagic symptoms and concomitant diseases on hemophilia course and prognosis has been considered.


Actions of two novel prostaglandin analogs, SC-29169 and SC-31391, on guinea pig and human isolated urinary bladder. 1. We examined the effects of two novel PGE1 analogs, SC-29169 and SC-31391, on bladder muscle isolated preparations from guinea pig and man, in comparison with some naturally-occurring prostanoids and misoprostol. 2. In the guinea pig detrusor muscle, both prostaglandin analogs enhanced twitch responses elicited by field stimulation in the following order of potency: SC-31391 greater than SC-29169 greater than PGF2 alpha greater than or equal to PGE2, while a well-defined contractile effect was elicited only by SC-31391. 3. In the human detrusor muscle, nerve-mediated responses were not modified by prostaglandin analogs, as well as by PGF2 alpha or PGE2, while a contractile effect was observed with the same compounds: PGF2 alpha = SC-31391 greater than SC-29169 = PGE2 = PGE1. 4. The selective EP1-agonist, misoprostol did not induce any effect, in both guinea pig and human bladder. 5. These data suggest that the effect of prostaglandins in the bladder muscle differs according to the animal species and that, in the human detrusor muscle, SC-31391 and SC-29169 probably stimulate FP receptors.


Mast cells mediators evoke contractility and potentiate each other in mouse uterine horns. 1. The ability of prostaglandin F2 alpha (PGF2 alpha), histamine and serotonin to induce and/or potentiate contractions in mouse uterine horns was studied in an attempt to analyze whether uterine mast cells degranulation could favor contractions during labor. 2. PGF2 alpha was the most potent of the three compounds (EC50 = 0.7 microM), being followed by serotonin (EC50 = 1.2 microM) and histamine. Between 10 and 100 microM histamine only evoked weak contractions, not higher than 50% of maximal amplitude of contractions. 3. Serotonin (0.1 microM) potentiated the contractile effect of both histamine and PGF2 alpha when added simultaneously with the corresponding compound to the isolated organ bath. Also, histamine at threshold contractile concentration (3 microM) was able to potentiate the contractile effect of both serotonin and PGF2 alpha. 4. The potentiating effect of PGF2 alpha on both histamine and serotonin evoked contractions was recorded after treating the preparation with 10 microM of the compound for at least 10 min before the corresponding concentration-effect curves were performed. 5. It is postulated that the simultaneous presence of these mast cells mediators in myometrium could be an important stimulus for both to trigger and/or to maintain contractions during labor.


The responses to phorbol esters which stimulated protein kinase C in canine Purkinje fibers. 1. The effects of phorbol esters on canine Purkinje fibers were examined using conventional microelectrode techniques. 2. 12-O-Tetradecanoylphorbol-13-acetate (TPA) and 4-beta-phorbol-12,13-dibutyrate (PDB), which are specific activators of protein kinase C (PKC), decreased the action potential amplitude and the maximum rate of depolarization (Vmax) at 3 x 10(-7) M or higher. These phorbol esters had little effect on the resting potential. 3. PDB (1-3 x 10(-7) M) also reduced the contractile force, accompanied with initial increase (in 5 out of 8 experiments), whereas TPA did not decrease it to any significant extent. 4. An inactive analog of phorbol esters, 4-alpha-phorbol-12,13-didecanoate (PDD), decreased the action potential amplitude and Vmax, and slightly increased the action potential duration. However, PDD failed to produce any inotropic effect. 5. Post-rest potentiation of the contractile force after a rest from stimulation for 30 sec was inhibited in the presence of 3-10 x 10(-7) M TPA or 3 x 10(-7) M PDB. 6. Isoproterenol 10(-7) M augmented the action of PDB 3 x 10(-7) M. 7. These results suggest that activation of PKC may modulate myocardial Ca2+ homeostasis and influence the excitation-contraction process.


Repeated treatment with quinolones potentiates the seizures induced by aminophylline in genetically epilepsy-prone rats. 1. The effects of a chronic treatment with several quinolone derivatives on on the aminophylline-induced convulsions in the genetically epilepsy-prone rat have been investigated. 2. Two series of experiments have been performed: in the first one animals received the quinolone twice a day for 5 days, then were given aminophylline (80-140 mg.kg-1, i.p.); in the second series of experiments the rats were treated once a day with the quinolone plus 120 mg.kg-1 of aminophylline for 5 days. The changes induced by both treatment protocols on electrocortical activity and on the occurrance of seizures have been evaluated. 3. Enoxacin reduced the dose of aminophylline necessary for the induction of seizures in a higher degree with respect to the other quinolone derivatives. The derivatives which showed minor proconvulsant properties were ofloxacin, ciprofloxacin and cinoxacin. The potentiation of seizures induced by quinolones appeared a dose-dependent phenomenon which was more evident when high doses of quinolones were used. 4. The chronic treatment carried out daily with quinolones and aminophylline suggests that additive neurotoxic effects of both classes of drugs may contribute to the increase of severity of seizure scores. 5. The possible role of GABA-benzodiazepine, excitatory amino acid, purinergic mechanisms as well as the role of pharmacokynetic factors are discussed.


Pharmacological characterization of ATP receptors mediating vasodilation on isolated rabbit aorta. 1. The effects of adenosine 5'-triphosphate (ATP), 2-methyl-thio-ATP and adenosine on rabbit aorta were examined in isolated preparations precontracted by noradrenaline, both in vessels where the endothelium was present and mechanically removed. 2. In the presence of endothelium, ATP (30 microM-3 mM) induced a relaxation that was reduced by removal of the endothelium. 3. The maximum endothelium-dependent relaxation of ATP was twice the maximum endothelial activity of the potent agonist at P2y-purinoceptors, 2-methyl-thio-ATP. 4. Adenosine which acts on P1 purinoceptors, induced a relaxant effect at 1 mM concentration, both in the vessels with and without endothelium. 5. It is concluded that relaxation by ATP is induced both via the endothelial P2y-purinoceptor and via a "nucleotide" receptor that is located on endothelium and on smooth muscle of rabbit aorta.


Effects of S14001 on adrenergic neuroeffector interaction in isolated canine saphenous veins. 1. The effects of (S) fluoro-6 (morpholinyl-2 methoxy)-8-tetrahydro-1,2,3,4 quinoleine (S14001) on adrenergic neurotransmission in isolated canine saphenous veins were investigated in experiments which measured the accumulation, overflow and metabolism of 3H-norepinephrine. 2. S14001 inhibited the accumulation of total tritium (3H-norepinephrine and 3H-metabolites of norepinephrine) in a concentration-dependent manner. 3. Under basal conditions, S14001 increased tension and basal effiux of total tritium; the latter consisted predominantly of 3H-DOPEG. The increases in these parameters were not inhibited by desmethylimipramine (DMI). 4. During electrical stimulation, S14001 increased the contraction and overflow of total tritium; the latter consisted predominantly of 3H-DOPEG. The increases in these parameters were inhibited by DMI. 5. These experiments suggest that S14001 has dual effects on adrenergic neurotransmission in the canine saphenous vein: (a) an inhibitory action on the neuronal accumulation; and (b) a pharmacological displacement of the transmitter from adrenergic nerve terminals.


Comparison of acute effects of mitoxantrone and doxorubicin in guinea-pig atria. 1. The acute effects of doxorubicin (DOX) and mitoxantrone (MTX) on basal rate and on positive chronotropic activity induced by 1-noradrenaline (1-NA) were investigated in isolated guinea-pig atria. 2. DOX (10(-5)-10(-4)M) progressively depressed atrial rate after a short latency period. Only 10(-4) M MTX reduced the spontaneous frequency after 120 and 180 min incubation. This effect was significantly lower to that elicited by DOX (10(-4)M). 3. Atropine (1.5 x 10(-6) M) and reserpine pretreatment did not affect the negative chronotropic action induced by DOX or MTX. 4. DOX (10(-5)-10(-4) M) produced a significant reduction of the maximal chronotropic response (Emax) to 1-noradrenaline (1-NA) after 60, 120 and 180 min of exposure. 5. MTX (10(-5)-10(-4) M) after 60 and 120 min incubation induced a beta-adrenergic, concentration- and time-dependent, competitive blocking effect. After 180 min of exposure, MTX (10(-4) M) reduced the Emax to 1-NA which was of less magnitude to that produced by DOX (10(-4) M). 6. Although both DOX and MTX depressed spontaneous and 1-NA induced chronotropic activity, MTX effects were of a slower onset and development compared to those exerted by DOX.


Alterations of G protein function in cardiac tissues from streptozotocin-induced chronic diabetic rats. 1. This study was performed to investigate G protein function in cardiac tissues from chronic diabetic rats by using pertussis toxin (PTX) and cholera toxin (CTX) as probes for G(i) and Gs proteins, respectively. 2. In the 10-week control group, i.v. injection of PTX significantly elevated the basal heart rate without having any effect on the chronotropic response of right atria to increasing concentrations of isoproterenol (ISO). In the 10-week diabetic rats, PTX treatment had no effect on the basal heart rate or on the response of right atria to ISO. In the 6-month groups, PTX did not exert any effects on basal or ISO-stimulated heart rate in either control or diabetic rat. 3. The inhibitory effect of carbachol (CCH) on cardiac tension in ISO-stimulated left atria was completely abolished by i.v. injection of PTX in the 10-week groups (both control and diabetic rats). The same treatment, however, only slightly reduced the effect of CCH on left atria contraction in rats from 6-month groups. 4. In both control and diabetic rats in the 10-week groups, incubation with CTX caused a significant increase in heart rate in right atria, and in developed cardiac tension in left atria preparations. The magnitude of the increase was the same in both control and diabetic rats. 5. Studies carried out using ADP-ribosylation technique indicated that the amount of G(i) protein was not changed in the ventricular muscle of the 10-week diabetic rat. Labelling of Gs protein could not be detected in either control or diabetic rat heart.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of androgens and antiandrogens on the inotropism induced by ouabain and isoproterenol on the left atrium of the rat in vitro. 1. The effect of androgens 5 beta- and 5 alpha-dihydrotestosterone (DHT, 10(-9) M), and the antiandrogens cyproterone acetate (CPA, 10(-8)-10(-6) M), chlormadinone acetate (CMA, 10(-8)-10(-6) M), medroxyprogesterone acetate (MPA, 10(-8)-10(-6) M), spironolactone (SPI, 10(-5) M), flutamide (F, 10(-5) M) and cimetidine (C, 10(-5) M), on inotropic positive effect induced by ouabain (10(-8)-10(-5) M) and isoproterenol (10(-8)-10(-6) M), on electrically stimulated left atria of rat, has been assayed. 2. Ouabain (10(-6) M) did not modify the inotropic effect of isoproterenol (10(-8)-10(-6) M). 3. The androgens 5 beta- and 5 alpha-DHT (10(-9) M) and the antiandrogens SPI (10(-6) M), F (10(-5) M) and C (10(-5) M) inhibit the inotropic effect of ouabain and isoproterenol on electrically stimulated left atria of the rat. 4. The antiandrogens CPA, MPA and CMA to 10(-7) M, inhibit the inotropic effect of ouabain. The CPA (10(-8)-10(-6) M) inhibit, in a dose-dependent way the positive inotropic effect of isoproterenol. MPA and CMA (10(-8)-10(-6) M) also inhibit the inotropic effect isoproterenol but the inhibitory effect is greater with 10(-8) M than 10(-6) M of both drugs. 5. Taken together, our results suggest that steroidal hormones could modulate the cardiac contractility through interference with Na-pump in a non-digitalic site and/or with intracellular mediators in left atrium.


Effects of secretagogues on intracellular free calcium and magnesium concentrations in rat pancreatic acinar cells. 1. Rat pancreatic acinar cells were loaded with Fura 2 AM or Magfura AM and levels of cytosolic Ca2+ ([Ca2+]i) and Mg2+ ([Mg2+]i) were observed. 2. Addition of acetylcholine (ACh) evoked a transient rise in [Ca2+]i. The component of the rise dependent on extracellular Ca2+ sources, but not intracellular sources, was seen to be enhanced when both ACh and 5 mM Ca2+ were present in the medium. In the presence of elevated extracellular Mg2+ (10 mM) and ACh both components of the Ca2+ transient were inhibited. 3. Both GTP gamma S and fluoroaluminate, which can directly stimulate G-proteins, evoked a transient rise in [Ca2+]i in acinar cells. These responses were inhibited in the presence of elevated Mg2+. 4. Resting [Mg2+]i was seen to be 1.36 mM +/- 0.08 (n = 29) for cells in normal medium, 1.8 mM +/- 0.08 (n = 6) in elevated Mg2+ medium and 0.93 mM +/- 0.02 (n = 5) in cells bathed and Mg(2+)-free medium. Addition of ACh led to reductions in [Mg2+]i in cells bathed in normal medium and Mg(2+)-free medium but not elevated Mg2+ medium. 5. It is concluded that levels of extracellular Mg2+ strongly influence [Mg2+]i and [Ca2+]i mobilization during ACh-evoked responses. Mg2+ does not appear to be exerting its effects by influencing receptor-agonist interactions or by competing with Ca2+ at extracellular sites of Ca2+ uptake.


Effect of ATP and verapamil as cardioplegic additives in the isolated guinea pig heart. 1. A comparative study on isolated guinea pig hearts was carried out to determine the effects of ATP and verapamil as cardioplegic additives. 2. The hearts were arrested by one of the plegic solutions: I, potassium 20 mmol/l; II, potassium 20 mmol/l+verapamil 1.1 mumol/l; III, potassium 20 mmol/l+ATP 10 mmol/l. After 45 min of hypothermic ischemia, the hearts were reperfused by Krebs-Henseleit buffer. 3. Postischemic percentage change of myocardial functions (heart rate, contractility, heart work) and tissue enzymes (LDH, SGOT, SGPT) were compared between the groups. 4. Although a rapid cardiac arrest could be obtained by verapamil added cardioplegia. Postischemic myocardial recovery was much better with ATP added cardioplegic solutions.


Influence of ATP-sensitive potassium channel blocker on hypoxia-induced damage of isolated guinea pig heart. 1. Isolated guinea pig hearts were perfused under constant flow conditions with Krebs-Henseleit buffer. Hearts were subjected to 15 min of hypoxia followed by reoxygenation in the presence and in the absence of 100 microns glyburide, an ATP-sensitive potassium channel blocker. Heart rate, left ventricular pressure and lactate dehydrogenase (LDH) release were measured at 5 min intervals. 2. Small decreases in heart rate and left ventricular pressure were observed during glyburide infusion with hypoxia, however LDH release, which was used as an index of cellular damage, was dramatically elevated. 3. Neither glyburide, nor the vehicle in which it was dissolved, appeared to produce myocardial damage under normoxic conditions. 4. It is concluded that ATP-sensitive potassium channels are important in the protection of the myocardium during hypoxia in isolated guinea pig hearts perfused under constant flow conditions.


Maturational gonadotropin from the African catfish, Clarias gariepinus: purification, characterization, localization, and biological activity. A gonadotropic hormone of the African catfish, Clarias gariepinus, was was purified and chemically characterized. Its biological activity was tested and its localization in the gonadotropic cells of the pituitary demonstrated. An ethanolic extract of 500 pituitaries of adult male and female African catfish was subjected to ion-exchange chromatography on DE-52. The 31- to 38-kDa fraction was further purified on Sephadex G-75. On rpHPLC over an ODS 120T column two major components appeared as single bands after SDS-PAGE. From the amino acid composition and sequence analysis of these fractions, compared with those of salmon and carp GTH II-alpha and salmon GTH II-beta it was concluded that they represent catfish GTH alpha- and II-beta-subunits. The biological activity of the complete hormone (the 31- to 38-kDa fraction from the G-75 column) was tested on the production of 11 beta-hydroxyandrostenedione and 17 alpha-hydroxy-20 beta-dihydroprogesterone by catfish testis in vitro. Polyclonal antibodies were raised against the purified beta-subunit. Immunocytochemical study using these showed them to bind specifically to hypophysial gonadotropic cells. To date only one form of GTH has been demonstrated in the African catfish.


Light and electron microscopic immunocytochemical demonstration of synthesis, storage, and release sites of the neuropeptide calfluxin in Lymnaea stagnalis. The cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis control egg-laying and associated behaviors. They produce various peptides derived from two precursor molecules, proCDCH-I and II, one of which is calfuxin (CaFl). CaFL is involved in the control of the activity of a female accessory sex gland, the albumen gland. At the light microscope level, using an antibody raised against synthetic CaFl, immunoreactivity was demonstrated in all CDC somata as well as in the neurohemal CDC terminals in the periphery of the cerebral commissure and in the CDC axon collaterals in the inner region of the commissure. A group of small neurons in each cerebral ganglion was also immunopositive. At the ultrastructural level, secretory granules (SG) and large electron-dense granules (LG), formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were clearly immunolabeled. The density of immunolabeling of LG was 3.3 times greater than that of SG, indicating that CaFl is preferentially packed into LG. In the LG, the density of immunolabeling with anti-alpha CDCP (alpha CDCP is also a peptide derived from proCDCH-I and II) was 10 times greater than in SG, suggesting that CaFl and alpha CDCP are processed and sorted in (quantitatively) different ways. In the neurohemal terminals SG release their CaFl-immunopositive contents into the hemolymph by the process of exocytosis, whereas collaterals release such contents into the intracellular space of the intercerebral commissure. It is proposed that neurohemally released CaFl acts upon the albumen gland, whereas CaFl released from the collaterals may influence the activity of central neurons.(ABSTRACT TRUNCATED AT 250 WORDS)


Chronic fasting reduces the response of the thyroid to growth hormone and TSH, and alters the growth hormone-related changes in hepatic 5'-monodeiodinase activity in rainbow trout, Oncorhynchus mykiss. Chronically fasted rainbow trout (Oncorhynchus mykiss) had significantly lower plasma L-thyroxine (T4) and triiodo-L-thyronine (T3), and higher plasma growth hormone (GH) concentrations than fed animals. Fasted and fed trout were administered bovine thyrotropic hormone (bTSH), native ovine GH (oGH), or recombinant human GH (rhGH) alone, or GH in combination with bTSH to further study the effects of food deprivation on the activity of the pituitary-thyroid axis and on the control of hepatic T3 production. Although the fasted rainbow trout retained the ability to respond to bTSH challenge, the resultant elevation in plasma T4 concentration was significantly lower than that of fed animals; there was no plasma T3 response to bTSH challenge in either fed or fasted trout, except for a significant elevation in fed bTSH-injected fish and a significant depression in fed saline-injected fish sampled 2.5 hr after the injection. GH when administered alone had no significant effect on plasma T4 concentrations of either fed or fasted animals, and stimulated an increase in plasma T3 concentration and an increased hepatic T3 content only in the fed fish, despite a significant stimulation by both oGH and rhGH of in vitro hepatic 5'-monodeiodinase activity (MDA) in both fed and fasted groups. bTSH appeared to suppress rhGH- and oGH-stimulated MDA in fasted groups, and rhGH-stimulated MDA in fed trout. The data suggest that chronic fasting induced a down-regulation of the response of thyroid tissue to bTSH challenge, and of the GH-stimulation of T3 production, in vivo, although in vitro hepatic MDA was elevated following GH administration to both fed and fasted rainbow trout.


In vitro biosynthesis of steroids, including 11-deoxycortisol and 5 alpha-pregnane-3 beta,7 alpha,17,20 beta-tetrol, by ovaries of the goldfish Carassius auratus during the stage of oocyte final maturation. To examine the production of steroids with potential oocyte maturation-inducing or pheromonal activity in the goldfish (Carassius auratus) we have incubated mature ovaries of this species with 17-[3H]hydroxyprogesterone. The metabolites in the unconjugated, glucuronide, and sulfate fractions were identified by chromatography, microchemical reaction, and, in most cases, crystallization to constant specific activity. A major metabolite, present in all three fractions, was tentatively identified as 5 alpha-pregnane-3 beta,7 alpha,17,20 beta-tetrol. Although 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) was found in only low yield (as a sulfate), the presence of the tetrol indicates that it is synthesized in high yield but very rapidly metabolized. The relative proportions of 17,20 alpha-dihydroxy-4-pregnen-3-one (17,20 alpha-P), 11-deoxycortisol (17,21-dihydroxy-4-pregnen-3,20-dione) and 17,20 beta,21-trihydroxy-4-pregnen-3-one (17,20 beta,21-P) varied significantly between incubations and may be affected by the maturational state of the ovary or the method used to stimulate oocyte maturation. Testosterone was present predominantly as its glucuronide. Significant production of glucuronides and sulfates was observed in all incubations. Twenty-five to 30% of the radioactivity remained associated with the tissue, but the distribution of activity between the metabolites did not differ greatly from that found in the medium. These results indicate that 11-deoxycortisol and its 20 beta-reduced derivative (17,20 beta,21-P) may be significant in spawning female goldfish.


The primary structures of growth hormones of three cyprinid species: bighead carp, silver carp, and grass carp. The primary structures of growth hormone (GH) of three cyprinid species, bighead carp, silver carp, and grass carp, were determined by a chemical method and/or by molecular cloning. The bighead carp GH was extracted from pituitary tissue by use of an alkaline medium, then successively purified by gel filtration, hydrophobic interaction column chromatography, and reverse-phase high-pressure liquid chromatography. The primary structure of bighead carp GH determined chemically is identical to that deduced from the cDNA nucleotide sequence. By molecular cloning, the primary structures of silver carp and grass carp GHs were also determined. The GHs of these three cyprinid species all contain 188 amino acid residues and their sequences are identical. When four of the five cysteines of cyprinid GHs were arranged to match the same positions of cysteines of other vertebrate GHs, a maximally matched alignment was achieved. Among fishes, GHs are relatively conserved within the same order (82 to 100% identity) but they are more diversified between orders (49 to 68% identity). In further comparison, fish GHs are even more different from tetrapod GHs (37 to 58% identity). Although the primary structures of vertebrate GHs are relatively variable, four homologous sequences, notably one located at the C-terminal, are found.


Photorefractory Harris' sparrows (Zonotrichia querula) exposed to a winter-like daylength gradually regain photosensitivity after a lag. The time course of regaining photosensitivity was monitored in intact and in castrated Harris' sparrows (Zonotrichia querula) held on short days. Measured endpoints, assumed to be equivalent indicators of the photosensitive state, were photoinduced testicular growth in intact males and a photoinduced elevation in plasma LH (luteinizing hormone) concentration in castrated males. Intact males were initially photorefractory when they were moved from long days to short days, and they remained so for at least 3 weeks thereafter. Photosensitivity was partially restored between Weeks 3 and 5 on short days and then gradually increased to a maximum by Week 13. No further change was detected through Week 25 on short days, when the experiment ended. Castrated males, also photorefractory when moved from long days to short days, regained photosensitivity as early as Week 7 on short days, but not uniformly until Week 16. As reflected by linear regression analysis, recovery of photosensitivity was a gradual process through Week 22 on short days, when the experiment ended. Discrepancies between intact and castrated males raise the possibility that the measured endpoints are not equivalent indicators of the photosensitive state. In accordance with an earlier report (Wilson, 1990), recovery of photosensitivity in castrated Harris' sparrows held on short days was not signaled by a spontaneous increase in plasma LH concentration. Indeed, plasma LH concentrations of castrated males remained suppressed even after photosensitivity had been fully restored in intact males.


Testosterone-LH response and episodic secretion in the male marsupial, Dasyurus viverrinus. The pattern of testosterone-LH secretion, feedback mechanism, and periodicity of release were investigated in the male marsupial eastern quoll. Testosterone secretion is controlled directly by LH and in the wild, the secretion of both are synchronous and show a major peak at breeding time. An inverse relationship between the secretion of LH and testosterone occurs after castration which supports the hypothesis that a negative feedback mechanism exists between the gonads and the pituitary gland as in other mammals. These hormones fluctuate in a cyclic manner over 24 hr.


Plasma levels of sex steroids in the North Island brown kiwi (Apteryx australis mantelli) in relation to time of year and stages of breeding. North Island brown kiwi (Apteryx australis mantelli) have a seasonal pattern of egg laying with a peak in mid to late winter. This study is the first description of changes in plasma steroid concentrations during the annual reproductive cycle of this species. Blood samples were collected at intervals over a 2 1/2-year period from a population of kiwi at Tangiteroria, Northland, New Zealand. Male kiwi had annual cycles of plasma levels of testosterone and estradiol. Mean testosterone levels were low (less than 0.18 ng/ml) during February to April, rose in May to a broad peak lasting 4 months (maximum levels 1.90 +/- 0.76 ng/ml), declined in September, and reached low levels in November to January. With respect to the breeding cycle, testosterone levels were low in the nonbreeding period, rose to high levels over 2 to 4 months before egg laying, then declined steadily toward the start of incubation. Plasma testosterone levels were very low in brooding males. Mean estradiol levels in males showed a pattern similar to testosterone levels, except that the rise to peak levels (1750 +/- 680 pg/ml) started in April. In female kiwi mean testosterone levels were low (less than 0.10 ng/ml) throughout the year. There was an annual cycle of mean estradiol levels in females which in timing and amplitude was similar to the male cycle. Estradiol levels in relation to stages of the breeding cycle in females were basal in the nonbreeding period, increased over a 3-month period before egg laying, declined in the 2 weeks before laying, and were low thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)


Estradiol-17 beta secretion by the newly formed corpus luteum of a macropodid marsupial, the quokka (Setonix brachyurus). The production of significant amounts of estradiol-17 beta from the 2-day-old corpus luteum of the macropodid marsupial, Setonix brachyurus (quokka), was shown using an in vitro perifusion system. Sliced corpora lutea and finely chopped extra-luteal ovarian tissue (cortex plus interstitial tissue) were perifused separately for 2 hr, in tandem for the next hour (with the perifusate from the ovarian tissue directed into the chamber containing the corpora lutea), and separately again for a further 2 hr. Overall production rates of estradiol measured by radioimmunoassay were 1.0 pg (mg hr)-1 from luteal tissue and 0.64 pg (mg hr)-1 from the extra-luteal tissue. Androgen production rates were 0.18 ng (mg hr)-1 and 0.06 ng (mg hr)-1 from luteal and extra-luteal tissues, respectively, and progesterone was produced at a rate of 0.80 ng (mg hr)-1 from luteal tissue and 16.5 pg (mg hr)-1 from extra-luteal tissue. Production rates of all three steroids from the perifused tissue increased significantly when the ovary and corpus luteum were perfused in tandem. The secretion of C19 and C18 steroids from the young corpus luteum suggests that luteinization is not yet complete at this stage in the reproductive cycle of the quokka, and the tissue would be better described as a "postovulatory follicle." This is supported in the literature by the occurrence of mitoses in granulosa cells for 6-7 days after resumption of the delayed cycle. This delayed maturation of the macropodid corpus luteum, which is associated with the secretion of significant quantities of estradiol and only a transient spike of progesterone, is discussed in relation to the longer cycles and gestation periods characteristic of macropodid marsupials.


Evidence of a role for the turkey posterior pituitary in prolactin release. The aims of this study were: (1) to examine whether the posterior pituitary contains prolactin releasing factor (PRF) activity, (2) to determine to what extent known neurohypophyseal peptides contribute to this activity, and (3) to compare posterior pituitary PRF activities of hens in different reproductive stages. Anterior pituitary cells derived from juvenile female turkeys were incubated with posterior pituitary extracts or test substances for 3 hr. Posterior pituitary extracts (0.1-0.8 equivalent) contained a potent substance(s) which stimulated PRL release in a concentration-dependent manner (2.4 +/- 0.08 to 6.5 +/- 0.23 micrograms/500 k cells). Arginine vasotocin (AVT) and vasoactive intestinal peptide (VIP) antisera (1:500) completely abolished the PRL-releasing activities of their respective peptides but partially reduced (P less than 0.05) the PRF activity of the posterior pituitary (AVT, 19.9%; VIP, 55.1%). Mesotocin antiserum did not alter (P greater than 0.05) PRL release induced by posterior pituitary extract. Posterior pituitary extract (0.01-0.5 equivalent) from hens in each of the various stages of the reproductive cycle induced a concentration dependent PRL release. The 0.5 posterior pituitary equivalent dose from reproductively quiescent (nonphotostimulated), laying, photorefractory, and incubating hens increased PRL release 2.4-, 2.9-, 3.8-, and 11.1-fold, respectively. The turkey posterior pituitary contains a potent PRF activity, partially accounted for by VIP and AVT, at the assayed concentrations, which varies with the reproductive cycle.


Detection of the mRNA encoding vitellogenesis inhibiting hormone in neurosecretory cells of the X-organ in Homarus americanus by in situ hybridization. Vitellogenesis inhibiting hormone (VIH)-mRNA in secretory cells of the eyestalk of Homarus americanus was detected by nonradioactive in situ hybridization (ISH) using two digoxigenin-tailed oligonucleotide probes deduced from the peptide sequence. Two distinct clusters of positive cells were observed in the medulla terminalis ganglionic X-organ (MGTX). Only one of them gave a strong immunoreaction after incubation with a specific polyclonal anti-VIH serum and corresponded to the conventionally described VIH producing cells. The significance of the cells reacting positively in ISH but not in immunocytochemistry (ICC) is discussed. Northern blot analysis using 32P-labeling confirms the specificity of the probes and indicates an approximate size of 2.5 kb for VIH mRNA.


Sex steroid levels in the testes, ovaries, and pyloric caeca during gametogenesis in the sea star Asterias vulgaris. The concentrations of progesterone, testosterone, and estradiol were determined via radioimmunoassay in testes, ovaries, and pyloric caeca of the sea star Asterias vulgaris during one complete and two partial gametogenic cycles. These compounds were found in all tissues examined and were present in quantities similar to those reported previously in other echinoderms and in vertebrates. Testes and ovaries exhibited annual growth cycles during which testicular and ovarian mass increased up to 100-fold as gametes were produced and stored until spawning. Pyloric caecal mass varied during the annual reproductive season; however, no seasonal trends were apparent. In the testes, sex steroid levels were highest at the onset of spermatogenesis. Transient increases in the levels of estradiol coincided with spermatogonial mitotic proliferation. Transient increases in the levels of testosterone and progesterone in the testes coincided with spermatogenic column formation and with spermiogenesis, respectively. In the ovaries, estradiol and testosterone levels were highest at the onset of oogenesis while progesterone levels did not change significantly throughout the annual gametogenic cycle. Male and female pyloric caeca exhibited similar seasonal variations in levels of sex steroids as compared with the gonads. It is hypothesized that transient increases in the levels of sex steroids during gametogenesis may serve as endogenous modulators of reproduction.


Changes in brain gonadotropin-releasing hormone, pituitary and plasma gonadotropins, and plasma thyroxine during smoltification in chinook salmon (Oncorhynchus tschawytscha). Concentrations of brain salmon gonadotropin-releasing hormone (sGnRH), plasma gonadotropin I (GTH I), and pituitary GTH I and GTH II were determined in yearling chinook salmon (Oncorhynchus tschawytscha) during the parr-smolt transformation in two successive seasons. There were significant elevations in brain sGnRH content from February to March in 1988, and from February to April in 1989. Increases in brain sGnRH content coincided with elevations in plasma thyroxine levels that occurred from February to March, 1988 and 1989. Plasma GTH levels were relatively constant (1-2 ng/ml) throughout the period of sampling. However, during 1988, plasma concentrations of GTH I decreased significantly between late March and early April. During 1989, plasma GTH I levels appeared to reach a peak (2 ng/ml) in mid-February, but otherwise remained near 1 ng/ml. Previous studies have shown that GTH II was not detectable in plasma at this stage. During 1989, pituitary GTH I concentrations were 50- to 70-fold higher than that of GTH II, and increased, though not significantly, from February through April. Although GTH II was detected in the pituitary by RIA, it is likely that the measurable levels are due to GTH I cross-reaction in the GTH II RIA. Histological examination of the gonads indicated that throughout smoltification the oocytes remained in the perinucleolar stage of oogenesis and the testes were in the spermatogonial stage of spermatogenesis. Although no observable changes in gametogenesis occurred, the changes in brain sGnRH content, plasma GTH I levels, and pituitary GTH content suggest that some changes in the hypothalamic-pituitary axis may occur during smoltification.


Reproduction in worker honey bees is associated with low juvenile hormone titers and rates of biosynthesis. Three experiments were performed to determine the role of juvenile hormone (JH) in worker reproduction in queenless colonies of honey bees. In Experiment 1, egg-laying workers had low hemolymph titers of JH, as did bees engaged in brood care, while foragers had significantly higher titers. Experiment 2 confirmed these findings by demonstrating that laying workers have significantly lower rates of JH biosynthesis than foragers do. In Experiment 3, ovary development was inhibited slightly by application of the JH analog methoprene to 1-day-old bees, but was not affected by application to older bees, at least some already displaying egg-laying behavior. These results, which are consistent with earlier findings for queen honey bees, are contrary to a common model of insect reproduction, in which elevated JH titers trigger ovary development, which then leads to oviposition. Previous experiments have demonstrated that JH regulates nonreproductive behavior in workers that is associated with colony division of labor; perhaps this function is incompatible with a traditional role for JH in reproduction.


Effects of reproductive status, ovariectomy, and photoperiod on vasoactive intestinal peptide in the female turkey hypothalamus. Vasoactive intestinal peptide (VIP) appears to be a physiologically relevant prolactin (PRL)-releasing factor during the avian reproductive cycle, yet little is known of the factors involved in modulating the hypothalamic concentrations of this neuropeptide. A heterologous chicken VIP radioimmunoassay was developed to examine the effects of reproductive status, ovariectomy, and photoperiod on hypothalamic VIP immunoreactivity in the female turkey. VIP concentrations were highest in the median eminence/infundibular nuclear complex (ME/INF) relative to other subregions of the hypothalamus and changed only in this region during the reproductive cycle. Quiescent, nonphotostimulated hens subjected to stimulatory photoperiod exhibited a 1.6-fold increase in VIP in the ME/INF (quiescent 59.9 +/- 6.0 vs photostimulated 95.8 +/- 7.1 pg/microgram protein). An additional 2-fold increase in ME/INF VIP concentrations was observed in laying hens (183.0 +/- 28.5 pg/microgram protein). Coincident increases in plasma PRL were also observed. In contrast, during incubation and the photorefractory stage, a dissociation between hypothalamic VIP and plasma PRL occurred. No changes were observed in VIP in incubating hens, yet a 6-fold increase in PRL was noted, compared to layers. In addition, ME/INF VIP concentrations exhibited no change during the photorefractory stage, whereas a 28-fold decrease in plasma PRL occurred. VIP concentrations in the ME/INF of laying hens were unaffected by ovariectomy, whereas exposure to short photoperiod reduced VIP by 44%. The inhibitory effects of short photoperiod could not be reversed by administration of exogenous steroids, while steroid treatment reduced VIP concentrations by 45% in the ovariectomized hens. These results provide additional correlative evidence for a modulatory role of VIP in PRL secretion and suggest that the expression of this neuropeptide in the INF may serve as a neural link between photoperiodic mechanisms and PRL release during the avian reproductive cycle.


Purification, partial characterization, and heterologous radioimmunoassay of growth hormone (cGH) in red deer. Red deer growth hormone (cGH; 3.3 mg) was purified from an aqueous extract of seven pituitary glands (4.01 g wet weight) by preparative gel filtration on Sephadex G-100, gel filtration on Sephadex G-100 SF, and anion exchange chromatography on DEAE-Sepharose CL-6B. Purified cGH gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight under reducing conditions of 20,000 Da and gave a single peak on reverse-phase high-performance liquid chromatography. N-Terminal amino acid determination of 42 residues gave a sequence identical with those published for bovine and ovine GH. In a radioreceptor assay based on binding of iodinated recombinant bovine GH (rbGH) to liver microsomes prepared from a pregnant ewe, cGH was equipotent with an ovine GH (oGH) standard. In an oGH radioimmunoassay, cGH diluted in parallel with oGH and rbGH. Using this assay plasma GH concentrations were determined in adult nonpregnant red deer hinds over a 12-month period. There was a significant seasonality in plasma GH concentrations with concentrations consistently low between mid-May and mid-September. This is the period when voluntary food intake and liveweight gain are greatest. It is suggested that in the presence of low plasma GH concentrations nutrients may be diverted toward lipogenesis and hence promote fat deposition.


Adrenal-kidney and gonadal steroidogenesis during sexual differentiation of a reptile with temperature-dependent sex determination. Adrenal-kidney and gonadal steroidogenesis were studied during early development in the red-eared slider turtle, Trachemys scripta, which exhibits temperature-dependent sex determination. In vitro steroid secretion by adrenal-kidney-gonad complexes (AKGs) incubated for 6 hr was determined by radioimmunoassay (RIA). AKGs from presumptive males and females secreted progesterone at developmental stages before (stage 15), during (stages 17 and 19), and after (stage 21) the temperature-sensitive period for sex determination, and progesterone secretion increased significantly throughout the period from stage 15 to 21. Presumptive male AKGs secreted significantly more progesterone than female AKGs at stage 19. Corticosterone secretion by AKGs was observed at stage 17 in males only, but in both sexes at stages 19 and 21. Testosterone, estradiol, androstenedione, and dehydroepiandrosterone secretion by AKGs was detected only at stage 21. Of the steroids measured, progesterone and corticosterone were consistently secreted at the highest levels. Although some sex differences were observed, no obvious patterns of sexually dimorphic steroid secretion from AKGs were apparent. Gonads from stage 21, stage 24, and 10-day-old hatchlings from both presumptive sexes incubated with [7-3H]pregnenolone showed only weak precursor conversion, primarily to polar metabolites, in incubations as long as 24 hr. None of the steroids assessed by RIA of AKG incubates could be identified by TLC or HPLC analysis of the stage 21 and stage 24 gonadal incubates. However, progesterone was tentatively identified in incubates of 10-day post-hatch female gonads. For stage 21 females, AKGs were separated into gonadal and adrenal-kidney tissue (AK) components and incubated in vitro for 1, 3, and 18 hr.(ABSTRACT TRUNCATED AT 250 WORDS)


Isolation of maturational gonadotropin subunits from spotted seatrout (Cynoscion nebulosus) and development of a beta-subunit-directed radioimmunoassay for gonadotropin measurement in sciaenid fishes. Maturational gonadotropin (GTH) subunits were isolated from pituitaries of the spotted seatrout (Cynoscion nebulosus), a marine perciform teleost, by ethanolic extraction and ion-exchange, gel-filtration, and reverse-phase chromatography. Partial amino acid sequencing of the N-terminal regions of the alpha and beta subunits indicated 60-80% identities with various carp and salmon GTH subunits. The spotted seatrout GTH beta-subunit was used as radioligand in a radioimmunoassay (RIA) with Atlantic croaker (Micropogonias undulatus) GTH antiserum. Pituitary extracts and plasmas from a variety of sciaenid fishes diluted parallel to the croaker GTH standard in the RIA. These data suggest that there is a high degree of immunological similarity among the GTH beta subunits of sciaenid fishes. The RIA measured increased levels of GTH in the plasmas of three species of sciaenid fishes, spotted seatrout, orangemouth corvina (Cynoscion xanthulus), and red drum (Sciaenops ocellatus), following injections of a luteinizing hormone-releasing hormone analog. The beta-subunit-directed GTH RIA increases considerably the number of species in which studies of GTH physiology can now be conducted.


Temperature, but not photoperiod, influences gonadotropin-releasing hormone binding in the pituitary of the three-spined stickleback, Gasterosteus aculeatus. Gonadotropin-releasing hormone (GnRH) binding characteristics in pituitaries of stickle-backs under different physiological conditions were studied using D-Arg6-Pro9-salmonGnRH-NEt as labeled ligand. Both males and females displayed marked seasonal changes in the capacity of high-affinity GnRH binding sites; there was a high content in the breeding season (summer) (800-1500 pmol/pituitary) and no detectable high affinity (< 150 pmol) binding in late winter-early spring. The binding capacity was lower in postbreeding fish (ca. 400 pmol/pituitary in females, ca. 900 pmol in males) than in breeding fish (females: ca. 1850, males ca. 1400 pmol). GnRH binding sites were also studied in fish exposed to long and short photoperiod in combination with high and low temperature in winter. Only long photoperiod in combination with high temperature stimulated sexual maturation. The capacity of the GnRH binding sites was similar in fish exposed to long (females 1550 pmol, males 1000 pmol) and short (females 1800, males 900) photoperiod in combination with high temperature. In fish exposed to low temperature, binding was nondetectable irrespective of the photoperiod.


Ovarian development and correlated changes in hemolymphatic ecdysteroid levels in two spiders, Coelotes terrestris and Tegenaria domestica (Araneae, Agelenidae). Oocyte development during the first vitellogenic cycle of Coelotes terrestris and Tegenaria domestica is described. Under the present conditions, this development took about 40 days during which the oocytes went through six stages of maturation. For the first time presence of ecdysteroids is reported in adult females of spiders. Hemolymphatic ecdysteroid peaks were observed in both species at the transition between previtellogenesis and vitellogenesis. 20-Hydroxyecdysone and ecdysone were present in slightly different ratios in C. terrestris as well as T. domestica. These data largely agree with current views of ovarian development in Arthropods.


Immunoreactive Met-enkephalin-like material in the testis of Rana esculenta: identification and localization. Methionine-enkephalin (Met-Enk) has been detected in the testis of the frog, Rana esculenta, using a reverse-phase high-performance liquid chromatography system coupled with a specific radioimmunoassay. By means of immunocytochemical techniques Met-Enk positive cells have been localized in interstitial and germinal compartments. Particularly, spermatogonia, spermatocytes, and spermatozoa were stained in seminiferous tubules, and numerous interstitial cells showed strong cytoplasmic immunoreactivity in summer animals. Variations in the concentration of Met-Enk immunoreactive material occurred during the annual cycle. Our data show that Met-Enk is present in testes of nonmammalian vertebrate species. These results suggest that autocrine and/or paracrine mechanisms may regulate testicular activity in amphibians.


The complete amino acid sequence of the follitropin beta-subunit of the bullfrog, Rana catesbeiana. The amino acid sequence of the bullfrog, Rana catesbeiana, follitropin beta-subunit has been determined by sequencing the intact protein (residues 1-39) and peptides originated by lysyl endopeptidase and pepsin. Twelve cysteine residues and two sugar chain binding sites at Asn-5 and Asn-22 are positional identities with bullfrog and mammalian beta-subunits. The bullfrog FSH beta-subunit is composed of 107 amino acid residues with a molecular mass of 11,782 Da, including the six cystine bridges and excepting the sugar chain. The bullfrog FSH beta-subunit has approximately 60% sequence identity with that of mammals and 40% with the fish gonadotropin beta-subunit. Conserved sequences among mammals (residue numbers 33-55 and 66-71) extensively differed from those of the bullfrog.


In vitro steroid secretion by staged spermatocysts (Sertoli/germ cell units) of dogfish (Squalus acanthias) testis. Using the dogfish shark (Squalus acanthias) testis model in which different germ cell stages are topographically separated, we previously observed that steroidogenic enzyme activities vary qualitatively and quantitatively during spermatogenesis. To determine whether these data, obtained by radiolabeled tracer analysis of testicular microsomes, accurately predict steroid secretion by intact cultured spermatocysts (germ cell/Sertoli cell units), steroids were radioimmunoassayed directly in spent medium. Steroid output in basal medium was low, but after addition of 25-hydroxycholesterol (25-OH chol, 60 microM), large amounts of progestins (P) and testosterone (T) accumulated. Dehydroepiandrosterone (DHEA), androstenedione (A), and estradiol (E2) were low or nondetectable in the presence or absence of 25-OH chol; however, addition of T (1 microM) as substrate elevated E2 above assay limits. T and P contents of media increased progressively over a 24-hr culture period (Day 1). Replacement and analysis of spent media at 24-hr intervals indicated that secretion was continuous up to 6 days after seeding, although secretory rates (T plus P) and T/P ratios changed with time in culture. Spermatocysts in premeiotic (PrM), meiotic (M), and post-meiotic (PoM) stages all secreted T and P; however, absolute values, T/P ratios, and stage-dependent patterns varied from one experiment to another with no obvious seasonal pattern. Also steroid secretion by staged cysts did not consistently agree with predictions based on steroidogenic enzyme activities measured in earlier cell-free assays, whether or not 25-OH chol was present. This discrepancy was not explained by stage-related patterns of steroid secretion vs retention or by addition of putative in vivo regulators. Agents that elevated P and/or T were dibutyryl cyclic AMP, 1-isobutyl-3-methylxanthine (IBMX), and forskolin, whereas ventral lobe extract and Ca2+ ionophore (A23817) were inhibitory. An autoregulatory feedback loop was indicated by a negative relationship between cyst concentration per well and steroid secretion rate. In contrast to T and P, E2 secretion in the presence of substrate (T, 1 microM) was as predicted from previously determined patterns of aromatase activity (M > PrM > PoM) and increased in response to IBMX. Added [3H]P, [3H]T and [3H]E2 all were extensively metabolized to polar products and P metabolism was stage related (PoM > M > PrM). These data indicate that analysis of free P, T, and E2 in static cyst cultures may underestimate true steroidogenic potential. Assuming further characterization of the system, we conclude that cultured spermatocysts from dogfish testis have potential for studying the multifactorial regulation of steroid synthesis and secretion stage-by-stage during spermatogenesis.


Abnormal growth hormone receptor gene expression in the sex-linked dwarf chicken. Sex-linked dwarfism is a recessive mutation that causes a reduction in body weight gain and long bone growth of chickens. We examined the effect of the dwarfing gene on body weight, hepatic GH-binding activity, and the structure and expression of the growth hormone receptor (GHR) gene in two different lines of sex-linked dwarf (SLD) broiler chickens. Liver samples from one line of dwarf chicken were obtained from Arbor Acres Farm, Inc. (Glastonbury, CT) and fertile eggs from the second line of SLD were obtained from the University of Georgia. In the GA line, the average body weight of homozygous (dwdw) males at 11 weeks of age was 43% lower than that of normal (DwDw) males, while heterozygous (Dwdw) males were only 9% below normal. In the CT line, hepatic GH-binding activity of 35-week-old chickens was high (20% specific binding) in normal (DwDw) males and undetectable in liver membranes prepared from dwdw males. At 11 weeks of age, hepatic GH-binding activity of Dwdw males (3.9% specific binding) in the GA line was reduced by 44% and that of dwdw males was almost undetectable (0.34% specific binding) when compared to the average of normal GA males (7.1% specific binding). Southern and Northern blot analyses revealed different abnormalities in the GHR gene from the two separate lines of SLD. A restriction fragment length polymorphism in DNA and an aberrantly sized transcript (mRNA) were detected in the CT line of SLD chickens.(ABSTRACT TRUNCATED AT 250 WORDS)


Induction by beta-estradiol of vitellogenin in striped bass (Morone saxatilis): characterization and quantification in plasma and mucus. Striped bass (Morone saxatilis) were implanted with beta-estradiol to induce the production of vitellogenin, the egg yolk precursor produced by the liver. Electrophoretic analysis revealed that beta-estradiol caused marked production of a plasma protein of apparent molecular mass 170 kDa. Size exclusion chromatography suggested that the estradiol-induced protein circulated as a dimer. This protein was purified from the plasma of estradiol-treated fish by DEAE-agarose column chromatography and used to induce antibodies in rabbits and goats. Western blots revealed that the antiserum bound to the putative vitellogenin in plasma from estradiol-treated fish and adult females, but not with any proteins in male plasma. Western blot of ovarian extract revealed several smaller immunoreactive protein bands and supported the identity of the purified protein as vitellogenin. A competitive ELISA was developed with sensitivity in a range from 8 to 1000 ng/ml. Plasma concentrations of adult females during their spawning migration ranged from 100 to 600 micrograms/ml. Western blot of mucus extract revealed the presence of a 170-kDa protein in vitellogenic female fish along with several minor bands ranging from 50 to 110 kDa. Positive immunoreactivity was present in the surface mucus of all females and in none of the males collected during a spawning migration in the Hudson River.


Evidence for the colocalization of gastrin/CCK- and PYY/PP-immunoreactive substances in the small intestine of the lizard Podarcis hispanica: immunocytochemical and ultrastructural study. Peptide tyrosine tyrosine/pancreatic polypeptide (PYY/PP)- and C-terminal gastrin/cholecystokinin (G/CCK)-immunoreactive cells were investigated in the intestine of the lizard Podarcis hispanica, using immunocytochemistry with light and electron microscopy. Immunolabeling of consecutive semithin sections revealed coexistence of PYY/PP- and C-terminal G/CCK-like substances in some cells, while in others only PYY/PP or G/CCK immunoreactivity was found. Appropriate absorption controls excluded cross-reactivity between the antisera used. Ultrastructurally G/CCK+, PYY/PP+ cells were similar to G/CCK+, PYY/PP- cells but different from PYY/PP+, G/CCK- cells. Although virtually nothing is known concerning the physiological effects of these peptides in reptiles, their colocalization in the same cells in the intestine of Podarcis hispanica suggests a close relationship between them in the regulation of the digestive process.


Effect of breeding state, moulting, dehydration, exposure to saturated atmosphere, and arginine vasotocin on cutaneous water permeability in the toad Bufo bufo. Cutaneous water permeability was assessed in hydrated male toads under a variety of conditions, including dehydration and rehydration, and the effects of exogenous arginine vasotocin (AVT) were determined. Cutaneous water permeability (the rate of water uptake by toads in water) was high in toads collected in the breeding pond and declined steeply during the first week, coincident with reduced activity of the cutaneous mucus glands. The slopes of the dose-response curves relating AVT to cutaneous water influx were about the same at the transition from the breeding to the nonbreeding state, but the level of influx was higher in the breeding state. The dose-response relationship in long-term terrestrial-acclimated toads was similar to that in water-acclimated toads. The threshold dose for effect on the cutaneous water permeability was about 1 ng AVT. Dehydration had a substantially greater effect on the cutaneous water permeability than AVT. The ratio between dehydration and AVT responses tended to increase with increasing water transport capacity of the skin. Moulting and acclimation to a saturated atmosphere in fully hydrated toads more than doubled the water permeability and abolished the response to AVT. It is suggested that AVT and other factors increase the cutaneous water permeability by similar mechanisms, such as insertion of water channels in the apical membrane of the epidermal cells. The effect of AVT on the toad skin is interpreted as reflecting the general high potency of neurohypophysial hormones in stimulating the water permeability of membranes of variable permeability.