Accelerated intestinal epithelial cell turnover after bowel resection in a rat is correlated with inhibited hedgehog signaling cascade.

PURPOSE: The hedgehog (Hh) signaling pathway is one of the key regulators of gastrointestinal tract development. Recent studies point to the role of hedgehog signaling in regulating adult stem cells involved in maintenance and regeneration of intestinal stem cells. The purpose of this study was to evaluate the role of Hh signaling during intestinal adaptation in a rat model of short bowel syndrome (SBS). METHODS: Male rats were divided into two groups: Sham rats underwent bowel transection and SBS rats underwent a 75 % bowel resection. Parameters of intestinal adaptation, enterocyte proliferation, and apoptosis were determined 2 weeks after operation. Illumina's Digital Gene Expression analysis was used to determine the Hh signaling gene expression profiling. Hh-related genes and protein expression were determined using Real-Time PCR, Western blotting, and immunohistochemistry. RESULTS: Massive small bowel resection resulted in a significant increase in enterocyte proliferation and concomitant increase in cell apoptosis. From the total number of 20,000 probes, 13 genes related to Hh signaling were investigated. In jejunum, eight genes were down-regulated, three genes up-regulated, and two genes remained unchanged. In ileum, five genes were down-regulated and six genes were unchanged in SBS vs sham animals. SBS rats also demonstrated a significant three- to fourfold decrease in SMO, GIL, and PTCH mRNA, and protein levels (determined by Real-Time PCR and Western blot) compared to control animals. CONCLUSION: Two weeks following massive bowel resection in rats, the accelerated cell turnover was accompanied by an inhibited Hh signaling pathway. Hh signaling may serve as an important mediator of reciprocal interactions between the epithelium and the underlying mesenchymal stroma during intestinal adaptation following massive bowel resection in a rat.

The role of the BMP signaling cascade in regulation of stem cell activity following massive small bowel resection in a rat.

PURPOSE: Bone morphogenetic proteins (BMPs) are a group of growth factors that are implicated in intestinal growth, morphogenesis, differentiation, and homeostasis. The role of the BMP signaling cascade in stimulation of cell proliferation after massive small bowel resection is unknown. The purpose of this study was to evaluate the role of BMP signaling during intestinal adaptation in a rat model of short bowel syndrome (SBS). METHODS: Male rats were divided into two groups: Sham rats underwent bowel transection and SBS rats underwent a 75 % bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined 2 weeks after operation. Illumina's Digital Gene Expression analysis was used to determine the BMP signaling gene expression profiling. BMP-related genes and protein expression were determined using real-time PCR, Western blotting and immunohistochemistry. RESULTS: From the total number of 20,000 probes, 8 genes related to BMP signaling were investigated. From these genes, five genes were found to be up-regulated in jejunum (BMP1-10 %, BMP2-twofold increase, BMP3-10 %, BMP2R-12 % and STAT3-28 %) and four genes to be up-regulated in ileum (BMP1-16 %, BMP2-27 %, BMP3-10 %, and STAT3-20 %) in SBS vs sham animals with a relative change in gene expression level of 10 % or more. SBS rats also demonstrated a significant increase in BMP2 and STAT3 mRNA and protein levels (determined by real-time PCR and Western blot) compared to control animals. CONCLUSION: Two weeks following massive bowel resection in rats, the BMP signaling pathway is stimulated. BMP signaling may serve as an important mediator of reciprocal interactions between the epithelium and the underlying mesenchymal stroma during intestinal adaptation following massive bowel resection in a rat.

Fenofibrate reduces intestinal damage and improves intestinal recovery following intestinal ischemia-reperfusion injury in a rat.

PURPOSE: Fenofibrate (FEN) is known as a nuclear receptor activator which regulates many pathophysiological processes, such as oxidative stress, inflammation, and leukocyte endothelium interactions. Recent studies have demonstrated an anti-oxidant, anti-inflammatory, and anti-ischemic role of FEN in the attenuation of ischemia-reperfusion (IR) injury in the kidney, liver, brain, and heart. The purpose of the present study was to examine the effect of FEN on intestinal recovery and enterocyte turnover after intestinal IR injury in rats. METHODS: Male Sprague-Dawley rats were divided into four experimental groups: (1) sham rats underwent laparotomy, (2) sham-FEN rats underwent laparotomy and were treated with intraperitoneal (IP) FEN (20 mg/kg); (3) IR rats underwent occlusion of both the superior mesenteric artery and the portal vein for 30 min followed by 24 h of reperfusion, and (4) IR-FEN rats underwent IR and were treated with IP FEN immediately before abdominal closure. Intestinal structural changes, Park's injury score, enterocyte proliferation, and enterocyte apoptosis were determined 24 h following IR. The expression of Bax, Bcl-2, p-ERK, and caspase-3 in the intestinal mucosa was determined using real-time PCR, Western blot, and immunohistochemistry. RESULTS: Treatment with FEN resulted in a significant decrease in Park's injury score in jejunum (32 %) and ileum (33 %) compared to IR animals. IR-FEN rats also demonstrated a significant increase in mucosal weight in jejunum (23 %) and ileum (22 %), mucosal DNA (38 %) and protein (65 %) in jejunum, villus height in jejunum (17 %) and ileum (21 %), and crypt depth in ileum (14 %) compared to IR animals. IR-FEN rats also experienced significant proliferation rates as well as lower apoptotic indices in jejunum and ileum which was accompanied with higher Bcl-2 levels compared to IR animals. CONCLUSIONS: Treatment with fenofibrate prevents intestinal mucosal damage and stimulates intestinal epithelial cell turnover following intestinal IR in a rat model.

Effect of taurine on intestinal recovery following intestinal ischemia-reperfusion injury in a rat.

PURPOSE: Taurine (TAU) is a sulfur-containing amino acid that is involved in a diverse array of biological and physiological functions, including bile salt conjugation, osmoregulation, membrane stabilization, calcium modulation, anti-oxidation, and immunomodulation. Several studies have established that treatment with TAU significantly protects cerebral, cardiac and testicular injury from ischemia-reperfusion (IR). The purpose of the present study was to examine the effect of TAU on intestinal recovery and enterocyte turnover after intestinal IR injury in rats. METHODS: Male Sprague-Dawley rats were divided into four experimental groups: (1) Sham rats that underwent laparotomy, (2) Sham-TAU rats that underwent laparotomy and were treated with intraperitoneal (IP) TAU (250 mg/kg); (3) IR-rats that underwent occlusion of both superior mesenteric artery and portal vein for 30 min followed by 48 h of reperfusion, and (4) IR-TAU rats that underwent IR and were treated with IP TAU (250 mg/kg) immediately before abdominal closure. Intestinal structural changes, Park's injury score, enterocyte proliferation and enterocyte apoptosis were determined 24 h following IR. The expression of Bax, Bcl-2, p-ERK and caspase-3 in the intestinal mucosa was determined using Western blot and immunohistochemistry. RESULTS: Treatment with TAU resulted in a significant decrease in Park's injury score compared to IR animals. IR-TAU rats also demonstrated a significant increase in mucosal weight in jejunum and ileum, villus height in jejunum and ileum and crypt depth in ileum compared to IR animals. IR-TAU rats also experienced significantly lower apoptotic indices in jejunum and ileum which was accompanied by a higher Bcl-2/Bax ratio compared to IR animals. CONCLUSIONS: Treatment with taurine prevents gut mucosal damage and inhibits intestinal epithelial cell apoptosis following intestinal IR in a rat.

The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA.

Mitochondrial DNA (mtDNA)-depletion syndromes (MDS; OMIM 251880) are phenotypically heterogeneous, autosomal-recessive disorders characterized by tissue-specific reduction in mtDNA copy number. Affected individuals with the hepatocerebral form of MDS have early progressive liver failure and neurological abnormalities, hypoglycemia and increased lactate in body fluids. Affected tissues show both decreased activity of the mtDNA-encoded respiratory chain complexes (I, III, IV, V) and mtDNA depletion. We used homozygosity mapping in three kindreds of Druze origin to map the gene causing hepatocerebral MDS to a region of 6.1 cM on chromosome 2p13, between markers D2S291 and D2S2116. This interval encompasses the gene (DGUOK) encoding the mitochondrial deoxyguanosine kinase (dGK). We identified a single-nucleotide deletion (204delA) within the coding region of DGUOK that segregates with the disease in the three kindreds studied. Western-blot analysis did not detect dGK protein in the liver of affected individuals. The main supply of deoxyribonucleotides (dNTPs) for mtDNA synthesis comes from the salvage pathway initiated by dGK and thymidine kinase-2 (TK2). The association of mtDNA depletion with mutated DGUOK suggests that the salvage-pathway enzymes are involved in the maintenance of balanced mitochondrial dNTP pools.

Leptin is essential in maintaining normal vascular compliance independent of body weight.

The adipocytokine leptin centrally regulates body weight by enhancing metabolic rate and signaling satiety, but it also has wide-ranging peripheral effects. Leptin receptors are expressed on vascular smooth muscle cells and have a role in maintaining vascular tone. We investigated the vascular effects of leptin repletion or calorie restriction on leptin-deficient mice (ob/ob) and a leptin antagonist on wild-type (WT) mice. Aortic compliance was assessed by the measurement of pulse wave velocity by noninvasive Doppler; blood pressure was measured by left ventricular catheterization. We found that ob/ob mice have much stiffer aortas than WT mice and that reduction in aortic stiffness was greater in ob/ob mice treated with leptin vs calorie restriction, despite similar weight loss. Interestingly, treating WT mice with a leptin antagonist increases aortic stiffness with no change in weight. Thus, we conclude that leptin is essential for maintaining normal aortic compliance independent of body weight.

Evidence-based recommendations for bowel cleansing before colonoscopy in children: a report from a national working group.

BACKGROUND: There are no current recommendations for bowel cleansing before colonoscopy in children. The Israeli Society of Pediatric Gastroenterology and Nutrition (ISPGAN) established an iterative working group to formulate evidence-based guidelines for bowel cleansing in children prior to colonoscopy. METHOD: Data were collected by systematic review of the literature and via a national-based survey of all endoscopy units in Israel. Based on the strength of evidence, the Committee reached consensus on six recommended protocols in children. Guidelines were finalized after an open audit of ISPGAN members. RESULTS: Data on 900 colonoscopies per year were accrued, which represents all annual pediatric colonoscopies performed in Israel. Based on the literature review, the national survey, and the open audit, several age-stratified pediatric cleansing protocols were proposed: two PEG-ELS protocols (polyethylene-glycol with electrolyte solution); Picolax-based protocol (sodium picosulphate with magnesium citrate); sodium phosphate protocol (only in children over the age of 12 years who are at low risk for renal damage); stimulant laxative-based protocol (e. g. bisacodyl); and a PEG 3350-based protocol. A population-based analysis estimated that the acute toxicity rate of oral sodium phosphate is at most 3/7320 colonoscopies (0.041 %). Recommendations on diet and enema use are provided in relation to each proposed protocol. CONCLUSION: There is no ideal bowel cleansing regimen and, thus, various protocols are in use. We propose several evidence-based protocols to optimize bowel cleansing in children prior to colonoscopy and minimize adverse events.

Irreversible inhibition of nuclear exoribonuclease by thymidine-3'-fluorophosphate and p-haloacetamidophenyl nucleotides.

Exoribonuclease purified from Ehrlich ascites tumor cell nuclei and in intact HeLa cell nuclei is irreversibly inactivated by tow concentrations of p-bromo- and p-iodoacetamidophenyl nucleotides and by thymidine-3'-fluorophosphate. Iodoacetate, bromoacetate, and thymidine-5'-fluorophosphate do not affect the enzyme. Although p-haloacetamidophenyl nucleotides inactivate ribonucleic acid polymerase of isolated HeLa cell nuclei, thymidine-3'-fluorophosphate does not affect the activity of this enzyme in vitro.

beta(3)-adrenoceptor deficiency blocks nitric oxide-dependent inhibition of myocardial contractility.

The cardiac beta-adrenergic pathway potently stimulates myocardial performance, thereby providing a mechanism for myocardial contractile reserve. beta-Adrenergic activation also increases cardiac nitric oxide (NO) production, which attenuates positive inotropy, suggesting a possible negative feedback mechanism. Recently, in vitro studies suggest that stimulation of the beta(3)-adrenoceptor results in a negative inotropic effect through NO signaling. In this study, using mice with homozygous beta(3)-adrenoceptor deletion mutations, we tested the hypothesis that the beta(3)-adrenoceptor is responsible for beta-adrenergic activation of NO. Although resting indices of myocardial contraction were similar, beta-adrenergic-stimulated inotropy was increased in beta(3)(-/-) mice, and similar hyper-responsiveness was seen in mice lacking endothelial NO synthase (NOS3). NOS inhibition augmented isoproterenol-stimulated inotropy in wild-type (WT), but not in beta(3)(-/-) mice. Moreover, isoproterenol increased myocardial cGMP in WT, but not beta(3)(-/-), mice. NOS3 protein abundance was not changed in beta(3)(-/-) mice, and cardiac beta(3)-adrenoceptor mRNA was detected in both NOS3(-/-) and WT mice. These findings indicate that the beta(3)-adrenergic subtype participates in NO-mediated negative feedback over beta-adrenergic stimulation.

Wireless video capsule in pediatric patients with functional abdominal pain.

OBJECTIVES: Upper endoscopy (esophagogastroduodenoscopy [EGD]) has a limited role, if any, in the evaluation of functional abdominal pain (FAP). Nevertheless, children with intractable FAP are occasionally referred to EGD to rule out intestinal pathology. We evaluated the role of wireless video capsule endoscopy (VCE) in children referred for EGD with a diagnosis of FAP. PATIENTS AND METHODS: Ten children older than 10 years of age were prospectively enrolled. Children were first studied with the PillCam SB (VCE; Given Imaging, Yokneam, Israel) followed by standard EGD within 2 weeks. After the completion of the study, a questionnaire of tolerance and content regarding the 2 procedures was completed by the patients. RESULTS: Physical examinations and laboratory tests were within normal limits in all of the patients. Patients swallowed the endoscopic capsules without difficulty. There were no complications. VCE identified gastritis in 4 patients (confirmed by biopsies), whereas EGD detected erosive gastritis in only 1 of the 4 children. EGD detected no duodenal abnormalities. VCE detected Crohn disease in the small intestine and cecum in 1 patient. VCE was ranked by 8 patients as convenient and as a preferable procedure compared with EGD. CONCLUSION: The results of this small cohort suggest that in children with FAP, VCE is more sensitive than EGD for detection of macroscopic gastric and small bowel pathologies.

A thiocyanopalladation/carbocyclization transformation identified through enzymatic screening: stereocontrolled tandem C-SCN and C-C bond formation.

Herein we describe a formal thiocyanopalladation/carbocyclization transformation and its parametrization and optimization using a new elevated temperature plate-based version of our visual colorimetric enzymatic screening method for reaction discovery. The carbocyclization step leads to C-SCN bond formation in tandem with C-C bond construction and is highly stereoselective, showing nearly absolute 1,2-anti-stereoinduction (5 examples) for substrates bearing allylic substitution, and nearly absolute 1,3-syn-stereoinduction (16 examples) for substrates bearing propargylic substitution. Based upon these high levels of stereoinduction, the dependence of the 1,2-stereoinduction upon cyclization substrate geometry, and the generally high preference for the transoid vinyl thiocyanate alkene geometry, a mechanistic model is proposed, involving (i) Pd(ii)-enyne coordination, (ii) thiocyanopalladation, (iii) migratory insertion and (iv) ß-elimination. Examples of transition metal-mediated C-SCN bond formation that proceed smoothly on unactivated substrates and allow for preservation of the SCN moiety are lacking. Yet, the thiocyanate functionality is of great value for biophysical chemistry (vibrational Stark effect) and medicinal chemistry (S,N-heterocycle construction). The title transformation accommodates C-, O-, N- and S-bridged substrates (6 examples), thereby providing the corresponding carbocyclic or heterocyclic scaffolds. The reaction is also shown to be compatible with a significant range of substituents, varying in steric and electronic demand, including a wide range of substituted aromatics, fused bicyclic and heterocyclic systems, and even biaryl systems. Combination of this new transformation with asymmetric allylation and Grubbs ring-closing metathesis provides for a streamlined enantio- and diastereoselective entry into the oxabicyclo[3.2.1]octyl core of the natural products massarilactone and annuionone A, as also evidenced by low temperature X-ray crystal structure determination. Utilizing this bicyclic scaffold, we demonstrate the versatility of the thiocyanate moiety for structural diversification post-cyclization. Thus, the bridging vinyl thiocyanate moiety is smoothly elaborated into a range of derivative functionalities utilizing transformations that cleave the S-CN bond, add the elements of RS-CN across a π-system and exploit the SCN moiety as a cycloaddition partner (7 diverse examples). Among the new functionalities thereby generated are thiotetrazole and sulfonyl tetrazole heterocycles that serve as carboxylate and phosphate surrogates, respectively, highlighting the potential of this approach for future applications in medicinal chemistry or chemical biology.

A simple cell-suspension method for transplantation of V2 carcinoma.

A simple and rapid method for the preparation of an essentially monodispersed cell suspension from the rabbit V2 carcinoma, suitable for either large or small amounts of tumor tissue, did not require specialized equipment and yielded relatively large numbers of viable cells. Cell suspensions prepared by this method could be used to approximate a dose-response curve relating the percentage of tumor takes to the number of cells inoculated and could allow quantitative correlations not possible when macroscopic tumor fragments are used for implantation. The method, or slight modifications of it, should prove suitable for most tissues with a significant connective tissue component.

Resting host and tumor perfusion as determinants of tumor vascular responses to norepinephrine.

Blood flow determinations and arteriograms were obtained in rat (Walker carcinoma) and rabbit (V2 carcinoma) liver tumors at rest and after norepinephrine administration. Resting tumor blood flow exceeded resting hepatic flow in both models, and both tumors responded with vasoconstriction and reduced blood flow. In tumors and the surrounding normal host tissue, the greater the perfusion prior to drug administration, the greater is the response (decrease in perfusion) to the vasoconstrictor. Although tumor perfusion decreased after vasoconstrictor, post-norepinephrine angiograms revealed an improved diagnostic image because of the enlarged but unresponsive tumor feeder vessels, persistent tumor blush, and simultaneous vasoconstriction in the normal liver. In these models, improved tumor visualization resulted even though a decrease in tumor blood flow had occurred. The angiographic image is related therefore to the lack of vasoconstriction in the tumor feeder vessel, which has, however, a decreased blood flow and the correspondingly greater volume of normally constricting hepatic arteries which results in a marked decrease in the background of vessels upon which the tumor image is superimposed.

Autoantibodies against bactericidal/permeability-increasing protein (BPI-ANCA) in cystic fibrosis patients treated with azithromycin.

Autoantibodies against bactericidal/permeability-increasing protein (BPI-ANCA) were found in patients with cystic fibrosis (CF). It is speculated that they represent a marker of the chronic endobronchial infection and sustained inflammatory response in CF. Our aim was to evaluate whether azithromycin (AZM), through its antiinflammatory effect, could affect the level of BPI-ANCA in CF patients. Eighteen patients with CF aged 5.5-36.3 years (median 15.1) were enrolled in a randomised, double-blind, placebo-controlled trial of AZM (250 mg twice a week to 10 patients) or placebo (8 patients) for 12 weeks. BPI-ANCA levels were recorded pre- and post-treatment and compared to a group of 18 matched healthy controls. Chi-square analysis, Kruskal-Wallis and Mann-Whitney tests were used to compare between the groups. Pre- and post-treatment values were compared using the Wilcoxon Signed-Ranked test. BPI-ANCA was found in 12 CF patients (67%) and four (22%) healthy subjects (P<0.001). The mean BPI-ANCA level was 3.94+/-6.15 U/ml (mean+/-SD) in healthy subjects and 38.11+/-42.34 U/ml in CF patients (P=0.023). The mean BPI-ANCA level was higher in patients with Pseudomonas aeruginosa compared to those without (64+/-35 U/ml and 25+/-41 U/ml respectively, P=0.032). No change in BPI-ANCA levels occurred in the AZM-treated patients [35 (0-127) U/ml (median (range) and 30 (0-120) U/ml, respectively] or in the placebo group [10 (0-66) U/ml and 13 (0-83) U/ml, respectively]. BPI-ANCA levels are significantly higher in patients with CF compared to healthy controls. BPIANCA levels are higher among patients colonised with P. aeruginosa. Twelve weeks of AZM therapy did not lower the BPI-ANCA level in patients with CF.

The esophageal motility disorder of polymyositis. A prospective study.

A prospective study of 12 patients with the diagnosis of polymyositis or dermatomyosiois was initiated to determine the characteristics of the esophageal manometric patterns in this group. All of the patients were women between the ages of 30 to 80 years and met three of four of Bohan's diagnostic criteria for these diseases. The patients' conditions were classified according to Rose and Walton's muscle weakness index. Almost two thirds of our patients exhibited distal esophageal dysfunction by manometry. Based on our roentgenographic and manometric data, we conclude that in addition to the well-described abnormalities in the striated portion of the esophagus, smooth-muscle dysfunction occurs with a high frequency in this patient population.

Weight loss is not associated with hyperleptinemia in humans with pancreatic cancer.

Pathological weight loss is a feature of many diseases and contributes to mortality and morbidity. Although cytokines have been implicated in some models of pathological weight loss, little is known about cellular mechanisms responsible for cachexia in patients with cancer. Leptin is a fat cell product that acts centrally to reduce appetite and decrease metabolism. Leptin synthesis is stimulated by cytokines, and circulating levels of cytokines are elevated in some cancer patients. We hypothesized that cytokine-induced hyperleptinemia contributes to pathological weight loss in patients with pancreatic cancer. To evaluate this hypothesis, fasting serum leptin concentrations were measured in 64 patients undergoing surgery for pancreatic cancer. Preoperative interviews were used to assess body weight and appetite history. Thirty of 64 pancreatic cancer patients had cachexia (weight loss of >10% over the 6 months before surgery). Self-reported loss of appetite was associated with the presence of cachexia. Leptin concentrations, when corrected for body mass index, were lower than levels reported in healthy humans. Six patients had leptin levels more than 2 times those predicted by body mass index. There was no association between patients with increased leptin concentration and weight loss or anorexia. We conclude that a reduced appetite contributes to weight loss in patients with pancreatic cancer. High plasma leptin levels, however, do not appear to contribute to cachexia in these patients.

Role of the beta 3-adrenergic receptor locus in obesity and noninsulin-dependent diabetes among members of Caucasian families with a diabetic sibling pair.

Obesity and insulin resistance are important risk factors for the development of noninsulin-dependent diabetes (NIDDM) and are prevalent among predisposed first degree relatives of diabetic individuals. Recent molecular screening and analysis of a common missense mutation of the beta 3-adrenergic receptor gene suggested this locus as a strong candidate for increased obesity, earlier age of diabetes onset, and insulin resistance. To test the hypothesis that the beta 3-adrenergic receptor locus affects diabetes susceptibility, obesity as measured by body mass index, and components of the insulin resistance syndrome, we examined the role of this region in families ascertained for two or more NIDDM siblings. Linkage analysis was conducted using both parametric and nonparametric analyses, including multipoint sibling pair analysis. We found no evidence for linkage to NIDDM as a dichotomous trait and no evidence for linkage to body mass index, waist/hip ratio, insulin levels, or glucose levels as quantitative traits or to reported age of onset among NIDDM individuals. The Trp64 Arg missense mutation was present in 11% of the population. The mutation was not associated with NIDDM, and Arg64 carriers did not have earlier NIDDM onset, higher body mass index, or higher waist/hip ratio than Trp64 homozygotes. Among relatives, Arg64 carriers had significantly lower fasting glucose levels and lower waist/hip ratios than Trp64 homozygotes, but no characteristics of the insulin resistance syndrome. We conclude that the beta 3-adrenergic receptor locus does not play an important role in NIDDM susceptibility or in the insulin resistance syndrome among members of families with a strong predisposition to NIDDM.

Interaction of levodopa and metoclopramide on gastric emptying.

Dopamine is postulated as an inhibitory neurotransmitter in the alimentary tract. The purposes of this study were to determine the effect of levodopa on the rate of gastric emptying in man and to investigate the interaction of levodopa and metoclopramide (a putative dopamine antagonist) on gastric emptying. On separate days, 7 normal male subjects received oral levodopa, 1,000 mg, and placebo in a randomized double-blind fashion 20 min before taking a mixed solid-liquid test meal labeled with 99mtechnetium(Tc)-diethylenetriamine pentaacetic acid. Gastric emptying was measured over 90 min by a gamma camera technique. At 90 min, the mean percent isotope remaining in the stomach after placebo, 54.7 +/- 5.0%, was less than after levodopa, 85.1 +/- 4.9% (p less than 0.01). Four of these normal subjects were studied further, and received levodopa, 1,000 mg, and metoclopramide, 10 mg, by intramuscular injection, 20 min before the same labeled test meal. In these 4 subjects, at 90 min, mean percent isotope remaining in the stomach after the combination of levodopa and metoclopramide, 48.5 +/- 2.2%, was less than after levodopa alone, 83.3 +/- 7.0% (p less than 0.05), and in the same range as mean percent after placebo, 47.3 +/- 3.6%. It is concluded that (1) levodopa inhibited gastric emptying of isotope in a mixed solid-liquid meal in normal subjects; (2) metoclopramide antagonized this effect returning gastric emptying to normal; and (3) the data suggest the possibility that dopaminergic receptors have an inhibitory effect on gastric emptying of the human stomach.

The inactivation of horseradish peroxidase by a polystyrene surface.

Horseradish peroxidase conjugates are inactivated by reaction with plastic enzyme immunoassay reaction vessels (Gilford Cuvette Paks). The loss of enzyme activity is prevented by Tween.

Intramolecular hydrogen bonding and conformational studies of bridged thebaine and oripavine opiate narcotic agonists and antagonists.

A conformational study of a series of oripavine derivatives is reported using the PCILO semiempirical quantum mechanical method. Low-energy conformers of carbinol substituents on C7-C19-R1R2OH are found with and without intramolecular hydrogen bonding to the C6-OCH3 group. The relative energies of these conformers depend on the R1 and R2 groups and the diastereoisomerism of the alcohol. The results are consistent with available NMR and IR studies of intramolecular hydrogen bonding and with crystallographic data. The importance of interaction between specific conformations of C19 carbinols and a lipophilic receptor site is suggested. A hypothesis is formulated to explain observed differences in pharmacological activity between diastereoisomers at C19 in the oripavine series and also to explain how these diastereoisomers alter the established pattern of N-substituent effects on relative agonist/antagonist potency found in other rigid opiates. By contrast, conformational studies of the C19 optical isomers of the C7-C8 etheno form of buprenorphine lead to the prediction of greatly reduced intrinsic potency differences between C19 diastereoisomers for this compound and for buprenorphine itself.