Biomedical subjects

E Battaglia

Publications and source records attributed to E Battaglia.

At least 19 recordsLinked to original sources

PubMed2001

Regioselective and stereospecific glucuronidation of trans- and cis-resveratrol in human.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a polyphenol present in wine, which has been reported to have anti-inflammatory, anti-platelet, and anti-carcinogenic effects. The glucuronidation of this compound and that of the cis-isomer also naturally present, has been investigated in human liver microsomes. Both isomers were actively glucuronidated. The reaction led to the formation of two glucuronides (3-O- and 4'-O-glucuronides), whose structure was characterized by LC-MS and proton NMR. Glucuronidation was regio- and stereoselective. It occurred at a faster rate with the cis-isomer and preferred the 3-position on both isomers. In addition, the glucuronidation of resveratrol was tested using several recombinant UDP-glucuronosyltransferase (UGT) isoforms. The reaction was catalyzed by UGT of the family 1A (UGT1A1, 1A6, 1A7, 1A9, 1A10). The bilirubin conjugating UGT1A1 was mainly involved in the 3-O-glucuronidation of trans-resveratrol, whereas the phenol conjugating UGT1A6 activity was restricted to cis-resveratrol. The UGT1A9 and 1A10 were active toward both isomers. The activity supported by UGT2B7 and UGT2B15 was very low and restricted to cis-resveratrol. UGT1A3, 1A4, 2B4, and 2B11 were unable to form resveratrol glucuronides.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed2001

A unique multifunctional transporter translocates estradiol-17beta -glucuronide in rat liver microsomal vesicles.

A wide array of drugs, xenobiotics, and endogenous compounds undergo detoxification by conjugation with glucuronic acid in the liver via the action of UDP-glucuronosyltransferases. The mechanism whereby glucuronides, generated by this enzyme system in the lumen of the endoplasmic reticulum (ER), are exported to the cytosol prior to excretion is unknown. We examined this process in purified rat liver microsomes using a rapid filtration technique and [(3)H]estradiol-17beta-d-glucuronide ([(3)H]E(2)17betaG) as model substrate. Time-dependent uptake of intact [(3)H]E(2)17betaG was observed and shrinkage of ER vesicles by raffinose lowered the steady-state level of [(3)H]E(2)17betaG accumulation. In addition, rapid efflux of [(3)H]E(2)17betaG from rat liver microsomal vesicles suggested that the transport process is bidirectional. Microsomal uptake was saturable with an apparent K(m) and V(max) of 3.29 +/- 0.58 microm and 0.19 +/- 0.02 nmol.min(-1).mg protein(-1), respectively. Transport of [(3)H]E(2)17betaG was inhibited by the anion transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and probenecid. Specificity of the transport process was investigated by studying the cis-inhibitory effect of anionic metabolites, as well as substrates of the plasma membrane multidrug resistance-associated proteins on the uptake of [(3)H]E(2)17betaG. Collectively, these data are indicative of a novel multifunctional and bidirectional protein carrier for E(2)17betaG and other anionic compounds in the hepatic ER. This intracellular membrane transporter may contribute to the phenomenon of multidrug resistance.

Animals↗

PubMed2000

Manometric investigation of anorectal function in early and late stage Parkinson's disease.

Abnormal gastrointestinal function is relatively frequent in Parkinson's disease, and constipation is a disturbing symptom in many patients. However, it remains to be established whether anorectal abnormalities are characteristic of the late stages of the disease. Clinical and anorectal manometric function were investigated in groups of early and late stage parkinsonian patients. Thirty one patients (19 men, 12 women, age range 22 to 89 years) entered the study. The disease severity was assessed by Hoehn and Yahr staging: there were four (12.9%) stage I, seven (22.6%) stage II, 10 (32.2%) stage III, and 10 (32.2%) stage IV patients. Anorectal variables were measured by standard manometric equipment and techniques. Values obtained in early stage patients (Hoehn and Yahr stage I and II) were compared with those obtained in late stage patients (Hoehn and Yahr stage III and IV). Overall, more than 70% of patients complained of chronic constipation, with chronic laxative use reported in more than 30%. Late stage patients were slightly older than their early stage counterparts. Pelvic floor dyssynergia was documented in more than 60% of patients. Manometric variables were not different in the two groups. In conclusion, defecatory dysfunction is frequent in Parkinson's disease, it is not confined to late stage patients, and it is found early in the course of the disease. This has potential implications for a targeted therapeutic approach.

Adult↗

PubMed2000

Platelet-activating factor enhances vascular endothelial growth factor-induced endothelial cell motility and neoangiogenesis in a murine matrigel model.

We previously reported that platelet-activating factor (PAF) enhances the angiogenic activity of certain polypeptide mediators such as tumor necrosis factor and hepatocyte growth factor by promoting endothelial cell motility. The purpose of the present study was to evaluate whether the synthesis of PAF induced by vascular endothelial growth factor (VEGF) might affect endothelial cell motility, microvascular permeability, and angiogenesis. The neoangiogenesis and synthesis of PAF induced by VEGF were studied in vivo in a murine Matrigel model. Dermal permeability was studied in mice by injection of (125)I-albumin. The synthesis of PAF, cell motility, and the increased (125)I-albumin transfer across endothelial monolayers were studied in vitro by using cultures of human umbilical cord vein-derived endothelial cells (HUVECs). The results obtained demonstrate that the neoangiogenesis induced by VEGF in vivo was associated with a local synthesis of PAF and was inhibited by WEB2170 and CV3988, 2 chemically unrelated, specific PAF-receptor antagonists. In contrast, WEB2170 did not inhibit VEGF-enhanced dermal permeability, suggesting that the latter was independent of the synthesis of PAF. In vitro, it was found that VEGF induced the synthesis of PAF by HUVECs in a dose- and time-dependent manner. The cell motility induced by VEGF was inhibited by PAF-receptor antagonists. In contrast, VEGF-induced proliferation of HUVECs and albumin transfer through HUVEC monolayer were unaffected by PAF-receptor antagonists. These results suggest that the synthesis of PAF induced by VEGF enhances endothelial cell migration and contributes to the angiogenic effect of VEGF in the in vivo Matrigel model.

Animals↗

PubMed1999

Photoaffinity labeling of the aglycon binding site of the recombinant human liver UDP-glucuronosyltransferase UGT1A6 with 7-azido-4-methylcoumarin.

7-Azido-4-methylcoumarin (AzMC) is a fluorescent photoactive compound structurally related to 4-methylumbelliferone (4-MU), a marker substrate of the human liver recombinant UDP-glucuronosyltransferase (UGT) 1A6. AzMC was synthesized and utilized to label the substrate binding site of UGT1A6. AzMC exhibits a fluorescence spectrum with maximum excitation and emission wavelengths of 380 and 442 nm, respectively. Upon irradiation, the probe irreversibly inhibited glucuronidation activity measured with para-nitrophenol (pNP) as substrate and interacted with UGT1A6 according to a saturable process indicative of reversible binding before covalent incorporation of the photoaffinity label. This inhibition was both time and concentration dependent and led to the calculation of an inhibition constant, k(2) = 0.113 mM min(-1), and dissociation constant, K(d) = 2.89 mM, for the reaction. Partial photoinactivation of UGT1A6 with AzMC revealed that the probe decreased the apparent V(max) of the pNP glucuronidation reaction, but not the K(m). Moreover, inhibition was partially prevented by 1-naphthol, a surrogate substrate for the enzyme, or by preincubation with an active-site directed inhibitor, 5'-O-[[(2-decanoylamino-3-phenyl-propyloxycarbonyl)amino]-su lfonyl]-2 ',3'-O-isopropylideneuridine. In contrast, UDP-glucuronic acid (UDP-GlcUA) did not have any protective effect against photoinactivation and AzMC did not affect the photoaffinity labeling of UGT1A6 by 5-[beta-(32)P]N(3)UDP-GlcUA, a photoaffinity analog of UDP-GlcUA. Additionally, in the absence of irradiation, AzMC was found to be a competitive inhibitor of 4MU glucuronidation. Collectively, these results strongly indicate that AzMC specifically binds to the UGT1A6 aglycon binding site. Amino acid alignment of phenol-binding proteins revealed a conserved motif, YXXXKXXPXP. It is possible that this motif is involved in phenol binding to UGT1A6 and other phenol-accepting proteins.

Affinity Labels↗

PubMed1999

Thrombopoietin stimulates endothelial cell motility and neoangiogenesis by a platelet-activating factor-dependent mechanism.

In this study, we demonstrate that human umbilical cord vein-derived endothelial cells (HUVECs) expressed c-Mpl, the thrombopoietin (TPO) receptor, and that TPO activates HUVECs in vitro, as indicated by directional migration, synthesis of 1-alkyl-/1-acyl-platelet-activating factor (PAF) and interleukin-8 (IL-8), and phosphorylation of the signal transducers and activators of transcription (STAT) STAT1 and STAT5B. The observation that WEB 2170 and CV3988, 2 structurally unrelated PAF receptor antagonists, prevented the motility of HUVECs induced by TPO suggests a role of PAF as secondary mediator. Moreover, kinetic analysis of TPO-induced tyrosine phosphorylation of STAT demonstrated that STAT5B activation temporally correlated with the synthesis of PAF. PAF, in turn, induced a rapid tyrosine phosphorylation of STAT5B and PAF receptor blockade, by WEB 2170, preventing both TPO- and PAF-mediated STAT5B activation. The in vivo angiogenic effect of TPO, studied in a mouse model of Matrigel implantation, demonstrated that TPO induced a dose-dependent angiogenic response that required the presence of heparin. Moreover, the in vivo angiogenic effect of TPO was inhibited by the PAF receptor antagonist WEB 2170 but not by the anti-basic fibroblast growth factor neutralizing antibody. These results indicate that the effects of TPO are not restricted to cells of hematopoietic lineages, because TPO is able to activate endothelial cells and to induce an angiogenic response in which the recruitment of endothelial cells is mediated by the synthesis of PAF. Moreover, biochemical analysis supports the hypothesis that STAT5B may be involved in the signaling pathway leading to PAF-dependent angiogenesis.

Animals↗

PubMed1999

Structural and functional studies of UDP-glucuronosyltransferases.

UDP-Glucuronosyltransferases (UGTs) are glycoproteins localized in the endoplasmic reticulum (ER) which catalyze the conjugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GIcUA) as the sugar donor. Glucuronidation is a major factor in the elimination of lipophilic compounds from the body. In this review, current information on the substrate specificities of UGT1A and 2B family isoforms is discussed. Recent findings with regard to UGT structure and topology are presented, including a dynamic topological model of UGTs in the ER. Evidence from experiments on UGT interactions with inhibitors directed at specific amino acids, photoaffinity labeling, and analysis of amino acid alignments suggest that UDP-GIcUA interacts with residues in both the N- and C-terminal domains, whereas aglycon binding sites are localized in the N-terminal domain. The amino acids identified so far as crucial for substrate binding and catalysis are arginine, lysine, histidine, proline, and residues containing carboxylic acid. Site-directed mutagenesis experiments are critical for unambiguous identification of the active-site architecture.

Amino Acids↗

PubMed1999

Identification of thiopurine methyltransferase (TPMT) polymorphisms cannot predict myelosuppression in systemic lupus erythematosus patients taking azathioprine.

OBJECTIVE: To determine whether the presence of polymorphisms associated with reduced or absent activity of thiopurine methyltransferase (TPMT), an enzyme involved in azathioprine metabolism, can predict side-effects, particularly myelosuppression, in patients taking this drug. METHODS: The TPMT genotype was determined in 120 patients with systemic lupus erythematosus (SLE) together with 15 patients with inflammatory bowel disease (IBD) and correlated with the effects of clinical exposure to azathioprine. RESULTS: TPMT polymorphisms were detected in eight patients. Severe marrow toxicity occurred in the single homozygote identified. Azathioprine was generally well tolerated, but 11 drug-associated neutropenias were detected. In only one of the 11 cases was a TPMT polymorphism identified. CONCLUSION: Homozygous TPMT deficiency was associated with severe marrow suppression. In the majority of cases, however, TPMT genotyping prior to azathioprine therapy would not have predicted myelosuppressive events and may augment, but not replace, regular blood monitoring.

Azathioprine↗

PubMed1999

Photoaffinity labeling probe for the substrate binding site of human phenol sulfotransferase (SULT1A1): 7-azido-4-methylcoumarin.

A novel fluorescent photoactive probe 7-azido-4-methylcoumarin (AzMC) has been characterized for use in photoaffinity labeling of the substrate binding site of human phenol sulfotransferase (SULT1A1 or P-PST-1). For the photoaffinity labeling experiments, SULT1A1 cDNA was expressed in Escherichia coli as a fusion protein to maltose binding protein (MBP) and purified to apparent homogeneity over an amylose column. The maltose moiety was removed by Factor Xa cleavage. Both MBSULT1A1 and SULT1A1 were efficiently photolabeled with AzMC. This labeling was concentration dependent. In the absence of light, AzMC competitively inhibited the sulfation of 4MU catalyzed by SULT1A1 (Ki = 0.47 +/- 0.05 mM). Moreover, enzyme activity toward 2-naphthol was inactivated in a time- and concentration-dependent manner. SULT1A1 inactivation by AzMC was protected by substrate but was not protected by cosubstrate. These results indicate that photoaffinity labeling with AzMC is highly suitable for the identification of the substrate binding site of SULT1A1. Further studies are aimed at identifying which amino acids modified by AzMC are localized in the binding site.

Amino Acid Sequence↗

PubMed1999

Expression of CD40 and its ligand, CD40L, in intestinal lesions of Crohn's disease.

OBJECTIVE: Selected mechanisms of the immune system participate in the development of inflammatory bowel disease. Recently, overexpression of the ligand for CD40 (CD40L), a lymphocyte costimulatory molecule, was shown to induce severe inflammatory bowel disease in transgenic mice. In the present study, we examined the expression of CD40 and CD40L on surgical specimens of ileum from 12 patients with Crohn's disease and 10 patients with diverticulitis. METHODS: Several CD40L+ cells were present in the affected tissue of patients with Crohn's disease, whereas few scattered CD40L+ cells were detected in sections of histologically normal ileum, resected distantly from the affected tissue, in patients with diverticulitis and in normal ileum portions obtained from colorectal cancer undergoing extensive surgery. The phenotype of CD40L+ cells was mainly CD4+. RESULTS: In patients with Crohn's disease, several CD40+ cells were detectable in the same areas of lymphocytes expressing CD40L, whereas in patients with diverticulitis, the number of CD40+ cells was significantly lower. Most of the CD40+ cells costained with CD20, thus showing to be B-lymphocytes, and only a few were CD14+ macrophages. Several von Willebrand-positive vessels were also positive for CD40. In addition, several infiltrating macrophages were found to express B7-1 and B7-2 molecules, the ligands of CD28 and CTLA-4, which cooperate with the CD40-CD40L pathway in lymphocyte activation. Staining of ileal lesions with anti-CTLA-4 antibodies resulted in detection of none or very few positive cells. In contrast, in patients with diverticulitis, an enhanced number of B7-1 and B7-2 and CTLA-4 was observed. CONCLUSION: The local accumulation of CD40L+ together with CD40+ cells within intestinal lesions of Crohn's disease suggests the involvement of this co-stimulatory pathway.

Abatacept↗

PubMed1998

A functional role for histidyl residues of the UDP-glucuronic acid carrier in rat liver endoplasmic reticulum membranes.

Previous studies have documented the presence of protein-mediated transport of UDP-glucuronic acid (UDP-GlcUA) in rat liver endoplasmic reticulum (ER). To determine the crucial amino acids of the membrane transporter and evaluate their function in regulating the glucuronidation reaction, we examined the effect of histidyl-specific irreversible inhibitors on the uptake of radiolabeled UDP-GlcUA in rat liver ER. Inactivation of uptake (initial rate) was more pronounced with hydrophobic reagents [diethyl pyrocarbonate (DEPC), p-bromophenacyl bromide] as compared to the more hydrophilic reagent (p-nitrobenzenesulfonic acid methyl ester). DEPC was used to further characterize the inhibition because of its greater specificity for protein histidyl residues. While initial [14C]UDP-GlcUA uptake rates were diminished by DEPC treatment of intact microsomes, the accumulation of isotope at equilibrium was not significantly affected, indicating no loss of vesicle integrity. A pKa of approximately 7 for the modified residue(s) of the transporter supported the alkylation of imidazole moieties. Protection against inactivation was observed with UDP-GlcUA as well as other nucleotide-sugars known for their interaction with this transporter. Uptake activity of the transporter (Vmax) but not UDP-GlcUA binding (Km) was affected by a limited inactivation. Furthermore, a partial inactivation of the transporter impaired the binding of the photoaffinity label [beta-32P]5-azido-UDP-GlcUA to UDP-glucuronosyltransferases (UGTs) in intact, but not in detergent-disrupted, ER vesicles. These results demonstrate the involvement of histidyl residue(s) in the UDP-GlcUA uptake process in rat liver ER, provide additional evidence for the lumenal orientation of the UGT active site, and support the view that translocation of the UGT cosubstrate is a rate-limiting step of the glucuronidation reaction.

Acetophenones↗

PubMed1998

Mechanism of inhibition of rat liver bilirubin UDP-glucuronosyltransferase by triphenylalkyl derivatives.

A series of potent and competitive inhibitors of UDP-glucuronosyltransferase derived from 7,7,7-triphenylheptanoic acid has been synthesized in order to probe the active site of the isozyme involved in the glucuronidation of the endogenous toxic compound, bilirubin IX alpha. Like triphenylalkylcarboxylic acids, triphenyl alcohols were found to be very effective competitive inhibitors of the reaction (Ki 12 to 180 microM). Superimposition of the best inhibitors with bilirubin by computer modeling showed a marked spatial similarity, which accounts for the observed competitive-type inhibition. The bulky triphenylmethyl moiety of the inhibitor superimposed well on the part of the bilirubin molecule containing three of the four pyrrole rings. In agreement, substitution of the triphenylmethyl moiety by planar structures such as fluorenyl or indenyl rings completely suppressed the inhibition. In addition, the weak inhibition exerted by the shortest carboxylic acids could be related to the higher acidity of these molecules. The inhibition potency depended on the acidity of the molecules; the more acidic, the less inhibitory, suggesting that the presence of a negative charge on the inhibitor molecule prevents bilirubin glucuronidation. Based on these results, a reaction mechanism for bilirubin glucuronidation is postulated.

Animals↗

PubMed1998

The nutcracker esophagus: a late diagnostic yield notwithstanding chest pain and dysphagia.

The nutcracker esophagus, a primary motor disorder, is frequently associated with noncardiac chest pain. However, there are no data on whether its diagnosis, as in other esophageal motility disorders, is delayed. Since the disorder is frequently heralded by alarming symptoms such as chest pain and dysphagia, diagnosis should be made as soon as possible. In this study we assessed the diagnostic delay, if any, in patients with the nutcracker esophagus. Moreover, we were interested in whether the abnormalities described in the distal esophagus could also involve the entire viscus. Fifty-four subjects (age range 23-78 yr) with the nutcracker esophagus were assessed for clinical and manometric variables as an overall group and after dividing them into subgroups according to their symptoms. The manometric variables were compared with those obtained in 61 controls (age range 21-67 yr). Overall, a diagnosis of nutcracker esophagus was made after an average period of 36 +/- 6 months, and surprisingly, this was not different in the various subgroups complaining of either chest pain, dysphagia, or both. Analysis of manometric variables showed that the mean amplitude of contractions was significantly higher in the patients' group at all esophageal body levels, even in the proximal portions. Again, there were no significant differences among the subgroups of nutcracker esophagus with respect to the symptoms. Notwithstanding the presence of alarming symptoms, such as chest pain and dysphagia, the nutcracker esophagus is diagnosed on average after 3 years from the onset of symptoms. Manometric assessment seems to confirm that this entity may indeed represent a primary esophageal motor disorder. The major dysfunction is due to an abnormal increase of contraction amplitude of the entire esophageal body.

Adult↗

PubMed1998

Interaction of periodate-oxidized UDP-glucuronic acid with recombinant human liver UDP-glucuronosyltransferase 1A6.

Sodium periodate reacts with UDP-glucuronic acid (UDP-GlcUA) to generate a reactive derivative [periodate-oxidized UDP-GlcUA (o-UDP-GlcUA)]. The ability of this analog of UDP-GlcUA to inactivate and label the human recombinant UDP-glucuronosyltransferase (UGT) UGT1A6 via the UDP-GlcUA binding site was investigated. At an o-UDP-GlcUA concentration of 20 mM, the enzymatic activity of UGT1A6 was totally inactivated after 30 min of incubation at pH 7.4. Inhibition was irreversible, time-dependent, and concentration-dependent and exhibited pseudo-first order kinetics (kinact = 4.0 M-1.min-1). Cosubstrate protection with UDP-GlcUA was biphasic, with no protection in the first phase and almost total protection in the second phase, suggesting that at least 65% of the cross-linking occurs at the cosubstrate binding site. Partial inactivation by o-UDP-GlcUA led to a decrease in Vmax, suggesting that o-UDP-GlcUA can act as an active site-directed inhibitor. Furthermore, proteins, including the UGTs, from membrane fractions of a recombinant V79 cell line expressing the UGT1A6 enzyme and from rat liver microsomes were cross-linked by in situ periodate oxidation of [beta-32P]UDP-GlcUA. The present results suggest that periodate-oxidized UDP-GlcUA, which inactivates UGT1A6 by the possible formation of a Schiff base adduct with active site lysyl residues, can be used as a new affinity label for the UDP-GlcUA binding site.

Animals↗

PubMed1997

Regulation of polymorphonuclear cell activation by thrombopoietin.

Thrombopoietin (TPO) regulates early and late stages of platelet formation as well as platelet activation. TPO exerts its effects by binding to the receptor, encoded by the protooncogene c-mpl, that is expressed in a large number of cells of hematopoietic origin. In this study, we evaluated the expression of c-Mpl and the effects of TPO on human polymorphonuclear cells (PMN). We demonstrate that PMN express the TPO receptor c-Mpl and that TPO induces STAT1 tyrosine phosphorylation and the formation of a serum inducible element complex containing STAT1. The analysis of biological effects of TPO on PMN demonstrated that TPO, at concentrations of 1-10 ng/ml, primes the response of PMN to n-formyl-met-leu-phe (FMLP) by inducing an early oxidative burst. TPO-induced priming on FMLP-stimulated PMN was also detected on the tyrosine phosphorylation of a protein with a molecular mass of approximately 28 kD. Moreover, we demonstrated that TPO by itself was able to stimulate, at doses ranging from 0.05 to 10 ng/ml, early release and delayed synthesis of interleukin 8 (IL-8). Thus, our data indicate that, in addition to sustaining megakaryocytopoiesis, TPO may have an important role in regulating PMN activation.

Animals↗

PubMed1997

Esophageal dysfunction in scleroderma: relationship with disease subsets.

OBJECTIVE: To investigate the relationship between esophageal function and the extent of disease in a nonselected group of scleroderma patients, and to study gastric and small bowel motility in a group of scleroderma patients with more severe clinical manifestations. METHODS: Esophageal function in 125 scleroderma patients was investigated by radiologic, endoscopic, manometric, and pH-metric techniques. Ten patients also underwent gastrointestinal (GI) manometric recording, both during fasting and after a standard meal. RESULTS: Radiologic abnormalities of the esophagus were found in 55 of 81 patients (68%) and esophagitis in 45 of 125 (36%). No significant relationship was disclosed between GI symptoms, radiologic abnormalities, esophagitis grade, and the various disease subsets. However, the overall incidence of endoscopic esophagitis (irrespective of the degree) was significantly (P < 0.05) correlated with the patient subgroups, with 100% incidence of esophagitis in those having the more severe cutaneous involvement (type III). Manometric abnormalities were documented in 80% of patients, and pathologic reflux in 78%. The severity of esophageal abnormalities on manometry significantly correlated with the severity of the disease, whereas no correlations were found with pH-metric data. Ninety percent of the 10 female patients undergoing antroduodenal manometry displayed abnormal findings; of these, 60% showed neuropathic, and 30% myopathic, patterns. The latter were recorded in patients with a more severe stage of the disease (type III). CONCLUSION: A direct relationship was observed between scleroderma subsets and the severity of esophageal (and, probably, more distal gut) motor involvement. Since no correlation was found between esophageal symptoms and the severity of manometric abnormalities, manometry should be considered the single most important GI test to document the severity of the "esophageal" disease. Gastric and small bowel manometry may also offer evidence of widespread gut involvement, and provide a rationale for a more targeted therapeutic approach.

Adult↗

PubMed1997

Gluten-free diet normalizes mouth-to-cecum transit of a caloric meal in adult patients with celiac disease.

The mechanisms responsible for bowel disturbances in celiac disease are still relatively unknown. Recent reports suggested that small bowel motor abnormalities may be involved in this pathological condition; however, there are no studies addressing small bowel transit in celiac disease before and after a gluten-free diet. We studied the mouth-to-cecum transit time of a caloric liquid meal in a homogeneous group of celiac patients presenting with clinical and biochemical evidence of malabsorption and complaining of diarrhea. Sixteen patients were recruited and investigated by means of hydrogen breath test through ingestion of 20 g lactulose together with an enteral gluten-free diet formula. A urinary D-xylose test was also done in each patient. Both breath tests and D-xylose tests were carried out basally and after a period of gluten-free diet. Twenty healthy volunteers were recruited as a control group and underwent the same breath testing. At the time of the diagnosis, mouth-to-cecum transit time was significantly prolonged in celiacs with respect to controls (243 +/- 10 vs 117 +/- 6 min, P = 0.0001). The D-xylose test was also abnormal (average urinary concentration 2.8 +/- 0.25 g, normal values >4.5). No correlation was found in patients between mouth-to-cecum transit time and urinary D-xylose output (r = 0.22). After the gluten-free diet period, mouth-to-cecum transit time in celiacs was significantly reduced compared to prediet transit (134 +/- 8 vs 243 +/- 10 min, P = 0.0001) and did not show statistical difference when compared to that found in controls (P = 0.1). The D-xylose test reverted to normal in all but two subjects, who were found to be noncompliant with the diet. Mouth-to-cecum transit time is significantly prolonged in patients affected by untreated celiac disease when compared to healthy controls. This alteration might not be correlated to intestinal malabsorption, and the prolonged orocecal transit could be due to impaired small bowel function (deranged motility?). Since intestinal transit returned to normal values after an adequate gluten-free period, a link with severe active mucosal lesions is suggestive.

Adult↗

PubMed1997

Arginine 52 and histidine 54 located in a conserved amino-terminal hydrophobic region (LX2-R52-G-H54-X3-V-L) are important amino acids for the functional and structural integrity of the human liver UDP-glucuronosyltransferase UGT1*6.

The hepatic UDP-glucuronosyltransferase UGT1*6 is actively involved in the glucuronidation of short and planar phenols in humans. Based on the irreversible inhibition of the enzyme on chemical modification by 2,3-butanedione and diethyl pyrocarbonate, the roles of His54 and Arg52 were investigated by oligonucleotide site-directed mutagenesis. These amino acids belong to a consensus sequence LX2-R52-G-H54-X3-V-L located in a conserved hydrophobic region of the variable amino-terminal domain of UGT. Arg52 was replaced by alanine (mutant R52A), and His54 was replaced by alanine or glutamine (mutants H54A and H54Q). The immunological and catalytic properties of UGT1*6 and mutants were examined after stable expression in V79 cell lines. Immunoblots and immunoprecipitation studies revealed that the mutant and UGT1*6 proteins were expressed in the microsomal membranes in similar amounts. However, replacement of His54 by glutamine led to a complete loss of activity toward 4-methylumbelliferone, and the Vmax value was decreased 4-5-fold in the mutants R52A and H54A compared with the wild-type enzyme. The dissociation constants that characterize the binding of 4-methylumbelliferone and UDP-glucuronic acid to UGT1*6 were not greatly affected by the mutations. Interestingly, H54Q was not recognized by specific antibodies to the amino-terminal portion of UGT1*6, thereby indicating that this amino acid was critical to antibody recognition. In contrast, the mutants R52A and H54A could not be differentiated from the wild-type protein by pH optimum or thermal denaturation. Furthermore, these mutants were still sensitive to irreversible inhibition by diethyl pyrocarbonate and 2,3-butanedione, with second-order inactivation constant values similar to those obtained for UGT1*6. Altogether, the strict conservation of His54 and Arg52 and the mutational analysis of these residues suggest that these amino acids in the hydrophobic amino-terminal consensus sequence LX2-R52-G-H54-X3-V-L are important for the function and the structure required for optimal catalytic efficiency of UGT1*6.

Animals↗