Preparation and characterization of recombinant prolactin receptor extracellular domain from rat

Zinc is an essential trace element necessary to life. This metal may exert some of its physiological effects by acting directly on cellular membranes, either by altering permeability or by modulating the activity of membrane-bound enzymes. On the other hand, calcium is an essential element in a wide variety of cellular activities. The aim of the present work was to study a possible interaction between zinc and calcium on intestinal transport of D-galactose in jejunum of rabbit in vitro. In media with Ca2+, when ZnCl2 was present at 0.5 or 1 mM, zinc was found to reduce the D-galactose absorption significantly. In Ca(2+)-free media, where CaCl2 was omitted and replaced isotonically with choline chloride, the sugar transport was not modified by zinc. Verapamil at 10(-6) M (blocking mainly Ca2+ transport) did not modify the inhibitory effect of zinc on D-galactose transport. When 10(-6) M of A 23187 (Ca(2+)-specific ionophore) was added with/without Ca2+ to the media, ZnCl2 produced no change in sugar transport. These results could suggest a possible interaction of calcium and zinc for the same chemical groups of membrane, which could affect the intestinal absorption of sugars.     

Macrophage inflammatory protein-2: chromosomal regulation in rat small intestinal epithelial cells

Nonpathogenic, resident bacteria participate in the pathogenesis of inflammation in the small intestine, but the molecular messages produced by such bacteria are unknown. Inflammatory responses involve the recruitment of specific leukocyte subsets. We, therefore, hypothesized that butyrate, a normal bacterial metabolite, may modulate chemokine secretion by epithelial cells, by amplifying their response to proinflammatory signals. We studied the expression of the chemokine, macrophage inflammatory protein-2 (MIP-2) by the rat small intestinal epithelial cell line, IEC-6. Cells were stimulated with lipopolysaccharide or with interleukin 1beta (IL-1beta) and incubated with sodium butyrate. Acetylation of histones was examined in Triton X acetic acid-urea gels by PAGE. Unstimulated IEC-6 cells did not secrete MIP-2. However, lipopolysaccharide and IL-1beta induced MIP-2 expression. Butyrate enhanced MIP-2 secretion both in lipopolysaccharide-stimulated and IL-1beta-stimulated enterocytes; but butyrate alone did not induce MIP-2 expression. Butyrate increased the acetylation of histones extracted from the nuclei of IEC-6 cells. Furthermore, acetylation of histones (induced by trichostatin A, a specific inhibitor of histone deacetylase) enhanced MIP-2 expression by cells stimulated with IL-1beta. In conclusion, trichostatin A reproduced the effects of butyrate on MIP-2 secretion. Butyrate, therefore, increases MIP-2 secretion in stimulated cells by increasing histone acetylation. We speculate that butyrate carries information from bacteria to epithelial cells. Epithelial cells transduce this signal through histone deacetylase, modulating the secretion of chemokines.     

Molecular characterization of the GTPase-activating domain of ADP-ribosylation factor domain protein 1 (ARD1)

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins recognized as critical components in intracellular vesicular transport and phospholipase D activation. Both guanine nucleotide-exchange proteins and GTPase-activating proteins (GAPs) for ARFs have been cloned recently. A zinc finger motif near the amino terminus of the ARF1 GAP was required for stimulation of GTP hydrolysis. ARD1 is an ARF family member that differs from other ARFs by the presence of a 46-kDa amino-terminal extension. We had reported that the ARF domain of ARD1 binds specifically GDP and GTP and that the amino-terminal extension acts as a GAP for the ARF domain of ARD1 but not for ARF proteins. The GAP domain of ARD1, synthesized in Escherichia coli, stimulated hydrolysis of GTP bound to the ARF domain of ARD1. Using ARD1 truncations, it appears that amino acids 101-190 are critical for GAP activity, whereas residues 190-333 are involved in physical interaction between the two domains of ARD1 and are required for GTP hydrolysis. The GAP function of the amino-terminal extension of ARD1 required two arginines, an intact zinc finger motif, and a group of residues which resembles a sequence present in Rho/Rac GAPs. Interaction between the two domains of ARD1 required two negatively charged residues (Asp427 and Glu428) located in the effector region of the ARF domain and two basic amino acids (Arg249 and Lys250) found in the amino-terminal extension. The GAP domain of ARD1 thus is similar to ARF GAPs but differs from other GAPs in its covalent association with the GTP-binding domain.     

Absorption of methochlorpromazine in rat small intestinal everted sac

The absorption of methochlorpromazine in rat small intestinal everted sac was investigated. 0.22% of the drug added in mucosal fluid was transferred to serosal fluid for 30 min at 37 degrees C. The absorption of the drug was slightly inhibited by choline, and more markedly inhibited by more hydrophobic quaternary ammonium cations such as tetraethylammonium and cetyltrimethylammonium. Moderate inhibition was observed by a polyamine, spermine. The absorption rate of methochlorpromazine markedly depended on temperature. The Arrhenius plot of the apparent transfer rate constant revealed high activation energy (117 kJ/mol) for the transport. Uptake of methochlorpromazine to a small intestinal segment was also inhibited by other quaternary ammonium cations corresponding with their inhibitory effects on its transport. These results suggest that methochlorpromazine binds to the relatively hydrophobic region of small intestinal epithelial cells and transfers by passing through a high energy barrier.     

Small intestinal dysmotility following abdominal irradiation in the rat small intestine

Abdominal symptoms such as diarrhoea, abdominal cramps and vomiting are common during and after abdominal radiotherapy for gynaecological and pelvic malignancy. It has recently been recognized that small intestinal dysmotility may contribute to these symptoms but the underlying mechanisms are unclear in part because of the technical difficulties inherent in performing studies in irradiated small intestine. The aim of the current study was to evaluate small intestinal motor activity using perfused micromanometric techniques in 6-8-cm segments of ileum during arterial perfusion with isotonic oxygenated fluorocarbon solution. Intestinal segments from six rats were studied 4 days after treatment with 10 Gy abdominal irradiation. Ileal segments from nine nonirradiated animals acted as controls. For each experiment the total number of pressure waves, high-amplitude (> 20 mmHg, long-duration > 6 sec) pressure waves, and long (> 20 associated) bursts of pressure waves were determined. Irradiation had no effect on the overall number of pressure waves, but increased high-amplitude long-duration (HALD) pressure waves (248 vs 7, P < 0.01). In control animals HALD waves were localized to a single recording site but after radiotherapy 74% of HALD waves were temporally associated with similar pressure waves in other manometric channels. Forty-seven per cent of associated HALD waves migrated aborally. Retrograde migration of HALD waves was seen in five segments following irradiation. Irradiation abolished bursts of > 20 pressure waves.     

Studies on the composition of phospholipids in rat small intestinal smooth muscle

The phospholipid composition of rat small intestinal smooth muscle was investigated in comparison with those of the mucosa and liver. Phospholipid content per g of the wet smooth muscle was almost identical with that of the mucosa and was about 1/4 of that in the liver. The phospholipid/protein ratio of the smooth muscle was about 1/2 of the value in the liver. Sphingomyelin content was significantly high and amounted to 18% of total phospholipids. This value was about twice that in the mucosa and 4 times higher than that in the liver. On the other hand, the percent distribution of phosphatidylcholine was lowest in the smooth muscle. Distribution patterns of phosphatidylserine and phosphatidylinositol in the smooth muscle as well as in the mucosa were different from those in the liver. The occurrence of vinyl-ether and ether phospholipids was clearly demonstrated in the smooth muscle as well as in the mucosa. A major part of the ether lipids was detected in the phosphatidylethanolamine fraction, in which they amounted to about 50%; 40% as alkenyl-acyl type and 12% as alkyl-acyl type. A high content of ether lipids was also observed in the phosphatidylethanolamine fraction from mucosa, but the distribution was reversed, that is, 14% alkenyl-acyl type and 28% alkyl-acyl type. Fatty aldehydes, fatty alcohols, and fatty acids were also determined by gas-liquid chromatography. The compositions of fatty aldehydes in the phosphatidylethanolamine fraction from smooth muscle and from mucosa were similar, whereas the compositions of long chain fatty alcohol and fatty acids were clearly different. The compositions of fatty alcohols and fatty acids of the phosphatidylcholine fraction from smooth muscle showed significantly different patterns from those of the phosphatidylethanolamine fraction and from those of the same phospholipid fraction in the mucosa.     

Delta3,5-delta2,4-dienoyl-CoA isomerase from rat liver. Molecular characterization

rECH1, a recently identified rat cDNA (FitzPatrick, D. R., Germain-Lee, E., and Valle, D. (1995) Genomics 27, 457-466) encodes a polypeptide belonging to the hydratase/isomerase superfamily. We modeled the structure of rECH1 based on rat mitochondrial 2-enoyl-CoA hydratase 1. The model predicts that rECH1p has the hydratase fold in the core domain and two domains for interaction with other subunits. When we incubated 3,5,8,11, 14-eicosapentaenoyl-CoA with purified rECH1p, the spectral data suggested a switching of the double bonds from the Delta3-Delta5 to the Delta2-Delta4 positions. This was confirmed by demonstrating that the product was a valid substrate for 2,4-dienoyl-CoA reductase. These results indicate that rECH1p is Delta3,5-Delta2,4-dienoyl-CoA isomerase. Subcellular fractionation and immunoelectron microscopy using antibodies to a synthetic polypeptide derived from the C terminus of rECH1p showed that rECH1p is located in the matrix of both mitochondria and peroxisomes in rat liver. Consistent with these observations, the 36,000-Da rECH1p has a potential N-terminal mitochondrial targeting signal as well as a C-terminal peroxisomal targeting signal type 1. Transport of the protein into the mitochondria with cleavage of the targeting signal results in a mature mitochondrial form with a molecular mass of 32,000 Da; transport to peroxisomes yields a protein of 36,000 Da.     

Molecular cloning and characterization of a mitochondrial selenocysteine-containing thioredoxin reductase from rat liver

A thioredoxin reductase (TrxR), named here TrxR2, that did not react with antibodies to the previously identified TrxR (now named TrxR1) was purified from rat liver. Like TrxR1, TrxR2 was a dimeric enzyme containing selenocysteine (Secys) as the COOH-terminal penultimate residue. A cDNA encoding TrxR2 was cloned from rat liver; the open reading frame predicts a polypeptide of 526 amino acids with a COOH-terminal Gly-Cys-Secys-Gly motif provided that an in-frame TGA codon encodes Secys. The 3'-untranslated region of the cDNA contains a canonical Secys insertion sequence element. The deduced amino acid sequence of TrxR2 shows 54% identity to that of TrxR1 and contained 36 additional residues upstream of the experimentally determined NH2-terminal sequence. The sequence of this 36-residue region is typical of that of a mitochondrial leader peptide. Immunoblot analysis confirmed that TrxR2 is localized almost exclusively in mitochondria, whereas TrxR1 is a cytosolic protein. Unlike TrxR1, which was expressed at a level of 0.6 to 1.6 microgram/milligram of total soluble protein in all rat tissues examined, TrxR2 was relatively abundant (0.3 to 0.6 microgram/mg) only in liver, kidney, adrenal gland, and heart. The specific localization of TrxR2 in mitochondria, together with the previous identification of mitochondria-specific thioredoxin and thioredoxin-dependent peroxidase, suggest that these three proteins provide a primary line of defense against H2O2 produced by the mitochondrial respiratory chain.     

Molecular cloning and characterization of rat lin-10

In Caenorhabditis elegans, the vulval induction is mediated by tyrosine kinase receptor/Ras signal transduction pathway composed of the lin-3, let-23, and let-60 products. In addition to these gene products, the lin-2, lin-7, and lin-10 products are also implicated in this pathway. Lin-2 encodes a MAGUK and lin-7 encodes a small protein with one PDZ domain. The lin-10 product has no homology to known proteins. Here, we have cloned a rat homologue of the lin-10 product and characterized it. Rat lin-10 is ubiquitously expressed in various rat tissues and distributed in both the cytosol and membrane fractions. In brain, however, rat lin-10 is distributed only in the membrane fraction and enriched in the synaptic plasma membrane and postsynaptic density fractions. These results suggest that rat lin-10 is involved at least in synaptic functions in brain.     

Molecular and pharmacological characterization of GABAA receptors in the rat pituitary

Levels of mRNA for the major subunits of the GABAA receptor were assayed in the rat pituitary anterior and neurointermediate lobes by ribonuclease protection assay. alpha 1, beta 1, beta 2, beta 3, and gamma 2s were found to be the predominant subunits in the anterior lobe, whereas alpha 2, alpha 3, beta 1, beta 3, gamma 2s, and gamma 1 were the predominant subunits expressed in the neurointermediate lobe. alpha 5, alpha 6, and delta subunits were not detectable. Hill and Scatchard analysis of [3H] muscimol binding to anterior and neurointermediate lobe membranes showed high-affinity binding sites with dissociation constants of 5.6 and 4.5 nM, respectively, and Hill coefficients near 1. Muscimol sites were present at a maximum of 126 fmol/mg in the anterior lobe and 138 fmol/mg in the neurointermediate lobe. The central-type benzodiazepine antagonist [3H]Ro 15-1788 bound to a high-affinity site with a dissociation constant of 1.5 nM in both tissues, at a maximum of 60 fmol/mg in anterior pituitary and 72 fmol/mg in neurointermediate lobe. A Hill coefficient of 1 was measured for this site in both tissues. Assays of CL 218,872 displacement of Ro 15-1788 were consistent with a pure type I benzodiazepine site in the anterior lobe and a pure type II site in the intermediate lobe. These results are consistent with both tissue-specific expression of particular GABAA receptor subunits and receptor heterogeneity within individual cells in the pituitary.     

Effect of dietary corn starch intake on ruminal, small intestinal and large intestinal starch digestion in cattle

In Exp. 1, 24 yearling Holstein steers averaging 340 kg were fed either an alfalfa hay diet at a maintenance level of metabolizable energy (ME) intake or corn silage-corn diets at one, two or three times maintenance ME intake. After a 42-day adjustment period, steers were fed individually, and digestibilities of total alpha-glucosides, starch oligosaccharides and glucose were determined at 2-week intervals, with chromic oxide used as an indicator. Steers fed the alfalfa hay diet had higher (P less than .05) total tract digestibilities of total alpha-glucosides and starch than steers fed the corn diets. Fecal starch (percentage of dry matter) in steers fed the corn diets increased (P less than .05) from approximately 11 to 31% as level of ME intake increased from one to three times maintenance. Starch digestibilities for the corn diets fed at one, two and three times maintenance were 81.4, 76.4 and 76.0%, respectively. However, these trends toward reduced starch digestibilities were not significant. There appeared to be no apparent adaption of alpha-glucoside digestibility in the total digestive tract among steers fed different levels of corn over the intervals observed. In Exp. 2, four Holstein steers (350 KH) were each fitted with duodenal and ileal reentrant cannulas and fed either a low or a high level of corn. Alpha-glucoside intakes for animals given the low and high levels of cord averaged 1.7 and 3.2 kg, respectively. Steers fed the high level of corn digested more (p less than .05) alpha-glucoside in the total tract (2.9 vs 1.6 kg), reticulo-rumen (2.3 vs 1.2 kg) and large intestine level of corn. Steers fed the high level of corn also digested more corn in the small intestine (.415 vs .221 kg) than steers fed the lower level; however, differences were not significant. Although there were trends toward lower partial digestion coefficients (expressed as a percentage of alpha-glucoside presented to that segment) in the total tract, reticulo-rumen and small intestine for steers fed the high corn diet, the magnitude of the differences was not significant.     

UDP-glucuronosyltransferase UGT1A7 induced in rat small intestinal mucosa by oral administration of 2-naphthoflavone

Cerebellar basket cells form highly specialized inhibitory synaptic contacts with Purkinje cells, namely the pericellular basket and pinceau nerve terminal structures, wrapping around the Purkinje cell somatic and axon hillock regions. These inhibitory synaptic contacts are ideally located to control the ultimate output of the cerebellar cortex. Previous immunohistochemical studies have shown that these synaptic structures possess a very high density of the dendrotoxin (DTX)-sensitive potassium channel subunit, Kv1.2. We have taken advantage of this unique anatomical arrangement offering a high concentration of identified Kv channel subunits by combining whole-cell patch-clamp recording and fluorescence microscopy to establish a novel preparation and perform the first recordings from unambiguously identified mammalian CNS inhibitory presynaptic terminals. We report that DTX-sensitive potassium channels are present in basket cell terminals but not in the basket cell soma. This selective cellular distribution suggests that these channels play an important role in modulating cerebellar inhibitory synaptic transmission.     

Effect of aging on the intestinal transport of hydrophilic drugs in the rat small intestine

The effect of aging on the intestinal transport of hydrophilic drugs (and probe compounds) was investigated in the rat small intestine. Passive transport was suggested to be unchanged with aging from 8 (young) to 54 (old) and further to 101 (very old) weeks old, as shown for D-xylose and urea in single-pass intestinal perfusion (under urethane anesthesia), where steady-state transport across the intestinal membrane into the blood stream was evaluated. The passive transports of cephradine, 5-fluorouracil (5-FU) and L-glucose were also unchanged, though they were compared only between the young and the old. Consistently, the passive uptake in the intestinal everted sacs, where the entry process into the membrane was evaluated for 5-FU, D-xylose, urea and polyethylene glycol (PEG) 900, was unchanged with aging from the young to the very old. The carrier-mediated transport of cephradine was also unchanged with aging from the young to the old in perfusion under anesthesia, though that of D-glucose was declined by about 50% with aging from the young to the old and thereafter remained constant in the very old. In perfusion in unanesthetized rats, age independency in passive transport (examined for cephradine, L-glucose and D-xylose) and an age-dependent decline in D-glucose transport were also observed, suggesting that the findings under anesthesia are not qualitatively distorted. These results suggest that, although carrier-mediated transport may moderately decline with aging, the barrier function of the intestinal membrane to passive permeation of hydrophilic drugs (with molecular weight below 1000) may be unaffected by aging, supporting the suggestion from our previous in vivo studies that age-dependent increases in the orally absorbed fraction may be predicted for incompletely absorbed drugs because of delayed intestinal transit rather than increased intestinal transport (membrane permeability).     

Identification of extractable growth factors from small intestinal submucosa

When implanted as a biomaterial for tissue replacement, selected submucosal layers of porcine small intestine induce site-specific tissue remodeling. Small intestinal submucosa (SIS), as isolated, is primarily an acellular extracellular matrix material. In an attempt to discover the components of small intestinal submucosa which are able to induce this tissue remodeling, the material was extracted and extracts were tested for the ability to stimulate Swiss 3T3 fibroblasts to synthesize DNA and proliferate. Each of the four different extracts of small intestinal submucosa had measurable cell-stimulating activity when analyzed in both a whole cell proliferation assay (alamarBlue dye reduction) and a DNA synthesis assay ([3H]-thymidine incorporation). Proteins extracted from SIS with 2 M urea induced activity profiles in the two assays which were very similar to the activity profiles of basic fibroblast growth factor (FGF-2) in the assays. As well, the changes in cell morphology in response to the extracted proteins mimicked the changes induced by FGF-2. Neutralization experiments with specific antibodies to this growth factor confirmed the presence of FGF-2 and indicated that it was responsible for 60% of the fibroblast-stimulating activity of the urea extract of small intestinal submucosa. Western blot analysis with a monoclonal antibody specific for FGF-2 detected a reactive doublet at approximately 19 kDa and further confirmed the presence of FGF-2. Cell stimulating activity of proteins extracted from SIS with 4 M guanidine was neutralized by an antibody specific for transforming growth factor beta (TGF beta). Changes in the morphology of the fibroblasts exposed to this extract were nearly identical to changes induced by TGF beta. Although no reactive protein band was detected at 25 kDa in nonreduced western blot analysis, several bands were reactive at higher molecular weight. The identity of this TGF beta-related component of small intestinal submucosa is unknown. Identification of FGF-2 and TGF beta-related activities in SIS, two growth factors known to significantly affect critical processes of tissue development and differentiation, provides the opportunity to further elucidate the mechanisms by which this extracellular matrix biomaterial modulates wound healing and tissue remodeling.     

Molecular characterization of Cyclospora-like organisms from baboons

Cyclospora organisms are intestinal pathogens of humans that are increasingly recognized in many parts of the world; yet, the reservoirs and host range remain poorly defined. Analysis of 18S ribosomal DNA (rDNA) suggests that the human-associated Cyclospora species (Cyc-hu) is most closely related to the Eimeria species, which are host species-specific. Recently, oocysts identical to those of Cyc-hu were detected in baboon fecal specimens from Tanzania. The 18S rDNA from 3 of these baboon-associated oocyst specimens was amplified and sequenced. Phylogenetic analysis indicated that these baboon-associated Cyclospora-like organisms (Cyc-bab) are nearly identical to each other and are distinct from Cyc-hu (1.6%-1.7% dissimilar); however, these Cyc-bab organisms are the closest known relatives of Cyc-hu. Together, these primate-associated cyclosporans constitute a coherent clade within the diverse group of Eimeria species. These findings raise important questions about the evolutionary relationships of the eimeriids and Cyc-hu host range and should lead to improved polymerase chain reaction-based diagnostics.