Low molecular weight Cooperia oncophora antigens. Potential to discriminate between susceptible and resistant calves after infection

The recognition of low molecular weight proteins by sera obtained during a single oral (primary) infection with 100,000 3rd-stage Cooperia oncophora larvae was studied in calves. Three groups of 6 or 7 calves were selected based on different egg excretion patterns. SDS-gel electrophoresis of adult Cooperia antigen under reducing conditions, followed by Western blotting, revealed that resistance of individual calves to C. oncophora might be related with antibody responses (42 days post infection) against at least 2 protein fragments (14-16 kDa and 27 kDa). The 14-16-kDa protein complex was bound, to some extent, by individual sera from all calves. The intensity of staining was negatively correlated with egg excretion on Day 42 p.i. Calves with high egg counts on Day 21 p.i. either did not or only weakly recognized the 27-kDa band. It has to be established whether the 14-16 kDa (or recombinant 14.2 kDa) provides a tool for immunodiagnostics and whether the 27-kDa fragment can help further unravel immune-mediated resistance to Cooperia.     

Retinoid-X receptors and the effects of 9-cis-retinoic acid on insulin secretion from RINm5F cells

OBJECTIVE: We evaluated the influence of chronic blockade of the renin-angiotensin system on hypertension induced by long-term thyroxin (T4) administration. To this end, we determined the effects of chronic treatment with captopril on blood pressure, cardiac hypertrophy and other renal and metabolic variables of hypertensive hyperthyroid rats. METHODS: T4 was administered s.c. at 0.38 mumol/kg per day and captopril was given in the drinking water (1.38 mmol/l). Both treatments were maintained for 6 weeks. Control rats received tap water. After the treatment period, the rats were placed in metabolic cages. Later, blood pressure was measured in conscious rats by intra-arterial determination. RESULTS: T4-treated rats showed an increased mean arterial pressure (MAP) whereas, in rats treated with T4 plus captopril, MAP was similar to that of the control group. Captopril did not affect the increased heart rate or ventricular weight/body weight ratio of hyperthyroid rats, but it improved the reduced creatinine clearance of these animals. CONCLUSIONS: The elevation in blood pressure produced by long-term T4 administration was prevented by chronic blockade of the renin-angiotensin system. Captopril improved the renal function of hyperthyroid rats, but did not affect the relative cardiac hypertrophy of these animals.     

Dose escalation of cyclophosphamide in patients with breast cancer: consequences for pharmacokinetics and metabolism

PURPOSE: The alkylating anticancer agent cyclophosphamide (CP) is a prodrug that undergoes a complex metabolism in humans producing both active and inactive metabolites. In parallel, unchanged CP is excreted via the kidneys. The aim of this study was to investigate the influence of dose escalation on CP pharmacokinetics and relative contribution of activating and inactivating elimination pathways. PATIENTS AND METHODS: Pharmacokinetics of CP were assessed in 12 patients with high-risk primary breast cancer who received an adjuvant chemotherapy regimen that included four courses of conventional-dose CP (500 mg/m2 over 1 hour every 3 weeks) followed by one final course of high-dose CP (100 mg/kg over 1 hour). Plasma concentrations of CP were analyzed by high-performance liquid chromatography (HPLC), 24-hour urinary concentrations of CP, and its inactive metabolites (carboxyphosphamide, dechloroethylcyclophosphamide [dechlorethylCP], ketocyclophosphamide [ketoCP]) were determined by 31-phosphorus-nuclear magnetic resonance (31P-NMR)-spectroscopy. RESULTS: There was no difference in dose-corrected area under the concentration-time curve (AUC) (216 v 223 [mumol.h/[mL.g]), elimination half-life (4.8 v 4.8 hours), systemic clearance (79 v 77 mL/min) and volume of distribution (0.49 v 0.45 L/kg) of CP between conventional- and high-dose therapy, respectively. However, during high-dose chemotherapy, we observed a significant increase in the renal clearance of CP (15 v 23 mL/min; P < .01) and in the formation clearance of carboxyphosphamide (7 v 12 mL/min; P < .05) and dechloroethylCP (3.2 v 4.2 mL/min; P < .05), whereas metabolic clearance to ketoCP remained unchanged (1.3 v 1.2 mL/min). Consequently, metabolic clearance to the remaining (reactive) metabolites decreased from 52 to 38 mL/min (P < .001). The relative contribution of the different elimination pathways to overall clearance of CP demonstrated wide interindividual variability. CONCLUSION: Overall pharmacokinetics of CP are apparently not affected during eightfold dose escalation. However, there is a shift in the relative contribution of different clearances to systemic CP clearance in favor of inactivating elimination pathways, thereby indicating saturation of bioactivating enzymes during dose escalation. Besides individual enzyme capacity, hydration and concomitant medication with dexamethasone modulated CP disposition.     

Molecular characterization of a novel short-chain dehydrogenase/reductase that reduces all-trans-retinal

The reduction of all-trans-retinal in photoreceptor outer segments is the first step in the regeneration of bleached visual pigments. We report here the cloning of a dehydrogenase, retSDR1, that belongs to the short-chain dehydrogenase/reductase superfamily and localizes predominantly in cone photoreceptors. retSDR1 expressed in insect cells displayed substrate specificities of the photoreceptor all-trans-retinol dehydrogenase. Homology modeling of retSDR1 using the carbonyl reductase structure as a scaffold predicted a classical Rossmann fold for the nucleotide binding, and an N-terminal extension that could facilitate binding of the enzyme to the cell membranes. The presence of retSDR1 in a subset of inner retinal neurons and in other tissues suggests that the enzyme may also be involved in retinol metabolism outside of photoreceptors.     

Detection and effects of helicobacters in healthy dogs and dogs with signs of gastritis

OBJECTIVES: To determine prevalence, colonization density, and distribution of helicobacters and gastric histologic findings in healthy dogs and dogs with signs of gastritis; to evaluate association of colonization density and gastric inflammation; and to compare the number of Helicobacter spp with degree of inflammation. DESIGN: Cross-sectional prevalence survey. ANIMALS: 25 healthy dogs and 21 dogs with signs of gastritis. PROCEDURE: During endoscopy, gastric mucosal biopsy specimens were obtained from healthy and affected client-owned dogs. Histologic and cytologic evaluation and results of a urease test were used for detecting helicobacters, which were identified definitively by use of transmission electron microscopy and bacterial culture. RESULTS: Helicobacters were detected in all 25 healthy and 20 of 21 affected dogs. Cytologic examination was a more sensitive method than histologic examination or the urease test. Helicobacters were found least frequently and in fewest number in the antrum in both groups of dogs. Gastric inflammation was evident in both groups of dogs and did not differ significantly between groups. A significant association was not detected between colonization density or the number of Helicobacter spp and degree of gastric inflammation. In both groups, H bizzozeronii, H felis, and H salomonis were cultured. CLINICAL IMPLICATIONS: Histologically verified chronic gastritis is common in dogs with signs of gastritis as well as in healthy dogs. Colonization density of helicobacters was not associated with degree of gastric inflammation in the dogs of our study. It remains to be determined whether certain strains of Helicobacter spp can induce gastritis in dogs.     

Cellular retinaldehyde-binding protein ligand interactions. Gln-210 and Lys-221 are in the retinoid binding pocket

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the retinal pigment epithelium (RPE) and Müller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa. Ligand interactions determine the physiological role of CRALBP in the RPE where the protein is thought to function as a substrate carrier for 11-cis-retinol dehydrogenase in the synthesis of 11-cis-retinal for visual pigment regeneration. However, CRALBP is also present in optic nerve and brain where its natural ligand and function are not yet known. We have characterized the interactions of retinoids with native bovine CRALBP, human recombinant CRALBP (rCRALBP) and five mutant rCRALBPs. Efforts to trap and/or identify a Schiff base in the dark, under a variety of reducing, denaturing, and pH conditions were unsuccessful, suggesting the lack of covalent interactions between CRALBP and retinoid. Buried and solvent-exposed lysine residues were identified in bovine CRALBP by reductive methylation of the holoprotein followed by denaturation and reaction with [3H]acetic anhydride. Radioactive lysine residues were identified by Edman degradation and electrospray mass spectrometry following proteolysis and purification of modified peptides. Human rCRALBP mutants K152A, K221A, and K294A were prepared to investigate possible retinoid interactions with buried or partially buried lysines. Two other rCRALBP mutants, I162V and Q210R, were also prepared to identify substitutions altering the retinoid binding properties of a random mutant. The structures of all the mutants were verified by amino acid and mass spectral analyses and retinoid binding properties evaluated by UV-visible and fluorescence spectroscopy. All of the mutants bound 11-cis-retinal essentially like the wild type protein, indicating that the proteins were not grossly misfolded. Three of the mutants bound 9-cis-retinal like the wild type protein; however, Q210R and K221A bound less than stoichiometric amounts of the 9-cis-isomer and exhibited lower affinity for this retinoid relative to wild type rCRALBP. Residues Gln-210 and Lys-221 are located within a region of CRALBP exhibiting sequence homology with the ligand binding cavity of yeast phosphatidylinositol-transfer protein. The data implicate Gln-210 and Lys-221 as components of the CRALBP retinoid binding cavity and are discussed in the context of ligand interactions in structurally or functionally related proteins with known crystallographic structures.     

The contribution of classical (beta1/2-) and atypical beta-adrenoceptors to the stimulation of human white adipocyte lipolysis and right atrial appendage contraction by novel beta3-adrenoceptor agonists of differing selectivities

The role of beta3- and other putative atypical beta-adrenoceptors in human white adipocytes and right atrial appendage has been investigated using CGP 12177 and novel phenylethanolamine and aryloxypropanolamine beta3-adrenoceptor (beta3AR) agonists with varying intrinsic activities and selectivities for human cloned betaAR subtypes. The ability to demonstrate beta1/2AR antagonist-insensitive (beta3 or other atypical betaAR-mediated) responses to CGP 12177 was critically dependent on the albumin batch used to prepare and incubate the adipocytes. Four aryloxypropanolamine selective beta3AR agonists (SB-226552, SB-229432, SB-236923, SB-246982) consistently elicited beta1/2AR antagonist-insensitive lipolysis. However, a phenylethanolamine (SB-220646) that was a selective full beta3AR agonist elicited full lipolytic and inotropic responses that were sensitive to beta1/2AR antagonism, despite it having very low efficacies at cloned beta1- and beta2ARs. A component of the response to another phenylethanolamine selective beta3AR agonist (SB-215691) was insensitive to beta1/2AR antagonism in some experiments. Because no [corrected] novel aryloxypropanolamine had a beta1/2AR antagonist-insensitive inotropic effect, these results establish more firmly that beta3ARs mediate lipolysis in human white adipocytes, and suggest that putative 'beta4ARs' mediate inotropic responses to CGP 12177. The results also illustrate the difficulty of predicting from studies on cloned betaARs which betaARs will mediate responses to agonists in tissues that have a high number of beta1- and beta2ARs or a low number of beta3ARs.