Biomedical subjects

J P Henry

Publications and source records attributed to J P Henry.

At least 19 recordsLinked to original sources

PubMed2001

Cholesterol regulates membrane binding and aggregation by annexin 2 at submicromolar Ca(2+) concentration.

Annexin 2 is a member of the annexin family which has been implicated in calcium-regulated exocytosis. This contention is largely based on Ca(2+)-dependent binding of the protein to anionic phospholipids. However, annexin 2 was shown to be associated with chromaffin granules in the presence of EGTA. A fraction of this bound annexin 2 was released by methyl-beta-cyclodextrin, a reagent which depletes cholesterol from membranes. Restoration of the cholesterol content of chromaffin granule membranes with cholesterol/methyl-beta-cyclodextrin complexes restored the Ca(2+)-independent binding of annexin 2. The binding of both, monomeric and tetrameric forms of annexin 2 was also tested on liposomes of different composition. In the absence of Ca(2+), annexin 2, especially in its tetrameric form, bound to liposomes containing phosphatidylserine, and the addition of cholesterol to these liposomes increased the binding. Consistent with this observation, liposomes containing phosphatidylserine and cholesterol were aggregated by the tetrameric form of annexin 2 at submicromolar Ca(2+) concentrations. These results indicate that the lipid composition of membranes, and especially their cholesterol content, is important in the control of the subcellular localization of annexin 2 in resting cells, at low Ca(2+) concentration. Annexin 2 might be associated with membrane domains enriched in phosphatidylserine and cholesterol.

Annexin A2↗

PubMed2001

Effects of combination of low doses of angiotensin-converting enzyme inhibitor and diuretics on renal function in spontaneously hypertensive rats: comparison between acute and chronic treatment.

The goal of this study was to assess the effect of acute or chronic treatment with S5590, a combination of the angiotensin-converting enzyme inhibitor perindopril (0.76 mg/kg/day) and the diuretic indapamide (0.24 mg/kg/day) on renal function in spontaneously hypertensive rats with moderate renal injury. Renal function was evaluated in conscious rats by clearance methods using labelled inulin and PAH, after catheterisation of the carotid artery, jugular vein and bladder. Both acute and chronic treatment normalised renal vascular resistance, although the effect on blood pressure was more marked after chronic than after acute treatment. Although acute treatment with S5590 increased glomerular filtration rate and renal blood flow, chronic treatment did not affect these parameters. Diuresis and natriuresis were only slightly modified and the results suggest a marked renal vasodilatation. In conclusion, the maintenance of renal function after chronic treatment, in a setting of normalisation of arterial pressure, suggest that such a combined treatment may exert marked renal functional protective effects in hypertension.

Animals↗

PubMed2001

[Diagnosis and treatment of gastroesophageal reflux in the adult: guidelines recommended by French and Belgian consensus].

Usual gastroesophageal reflux (GER) presentations are heartburn and acid regurgitation. The prevalence in occidental population ranges from 5 to 45% according to symptoms frequency. Oesophagitis is observed in 30 to 50% of examined patients and only erosive and ulcerative lesions must be considered. Distinction is made between non-severe oesophagitis (isolated loss of substance), severe oesophagitis (circonferential loss of substance) and complicated oesophagitis (stenosis, ulcerations, brachyoesophagus). 24-hour pH-monitoring analyses reflux duration and relations between symptoms and reflux specially in unusual extraoesophageal presentations. Symptoms and quality of life are the main criteria for staging. In few patients, oesophagitis is severe. Complications (stenosis, ulcerations, bleeding, endobrachyoesophagus) are observed in 10 to 15% of cases. Endobrachyoesophagus with intestinal metaplasia is a risk for neoplasia. The consensus conference proposes this initial therapeutic strategy. In cases of time-spaced symptoms: antiacids, alginic acid or low doses of anti-H2 with life style changes. In cases of typical frequent symptoms, in patients younger than 50 years: 4-weeks treatment with half dosed proton pump inhibitors (PPI) or standard doses of anti-H2 or prokinetics. Nowadays, the majority of the experts propose empiric full-dose treatment. This attitude is more logical as total symptoms suppression with full dose PPI brings positive clues for exact GOR diagnostic without endoscopy. In patients older than 50 years or with alarming symptoms (weight loss, dysplagia, bleeding, anemia): endoscopy must be performed. Patients with non severe oesophagitis: PPI without checking endoscopy. In patients with severe or complicated oesophagitis: 8-weeks treatment following by endoscopy; in non relieved patients: doses are increased. In cases of extraoesophageal presentations: standard PPI treatment during 4 to 8 weeks if GER is well established. In long term strategy, if recidives are rare: intermittent treatment. In early and frequent recidives: long term adapted PPI or surgery. Stenosis are treated by PPI, pneumatic dilatation or surgery if unsuccessful. Brachyoesophagus must be checked by endoscopy every 2 years (malignancy risk).

Adult↗

PubMed2001

[Biological factors influencing response to diuretics in patients with cirrhosis and ascites].

PURPOSES: To examine the biological factors influencing response to diuretics in patients with cirrhosis and ascites. METHODS: Sixty-nine patients were evaluated. Patients were classified into 3 groups: group 1: "good responders" (responding to spironolactone 200 mg/day), group 2: "bad responders" (responding to spironolactone doses above 200 mg/day or requiring addition of furosemide), and group 3: "non-responders" (not responding to spironolactone 400 mg/day and furosemide 160 mg/day). RESULTS: There were 30 patients in group 1, 24 in group 2 and 15 in group 3. The degree of activation of the renin-aldosterone and sympathetic system in group 2 was significantly higher than in group 1 and lower than in group 3. Natriuresis in group 2 (11 +/- 0.7 mEq/24h) was significantly below group 1 (20 +/- 2 mEq/24h) and above group 3 (5 +/- 0.6 mEq/24h). CONCLUSIONS: In patients with cirrhosis and ascites, the degree of activation of the renin-aldosterone and sympathetic nervous system influences diuretic response of ascites and is estimated by measured baseline natriuresis.

Aldosterone↗

PubMed2001

[Long term surveillance of polyps and colorectal cancers].

It is necessary to assume the colorectal cancer follow-up after curative operation to detect cancer recurrence and new polyps or cancers. A good follow-up by endoscopy is also necessary for patients with colorectal polyps and for patients with familial adenomatous polyposis.

Adenomatous Polyposis Coli↗

PubMed2000

N-Terminal domain of annexin 2 regulates Ca(2+)-dependent membrane aggregation by the core domain: a site directed mutagenesis study.

Annexin 2 binds and aggregates biological membranes in a Ca(2+)-dependent manner. This protein exists as a monomer (p36) or as a heterotetramer (p90) in which two p36 chains are associated with a dimer of p11, a member of the S100 protein family. Protein kinase C phosphorylates the protein at the level of the N-terminal tail on serines 11 and 25, thereby modifying its oligomeric structure and its properties of membrane aggregation. To analyze these effects, the properties of a series of mutants in which serines 11 and 25 were replaced by alanine and/or glutamic acid were investigated. The affinity for p11 light chain was decreased in the S11E mutants. Glutamic acid residues in positions 11 or 25 did not change membrane binding, either in the tetrameric or in the monomeric form. On the other hand, these mutations affected the aggregation properties of the two forms. For the tetramer, the aggregation efficiency was decreased but not the Ca(2+) sensitivity, whereas the latter was affected in the case of the monomer. The effects were stronger in the S11E mutants, and they were cumulative in the double mutant. They suggest a different conformation of the N-terminal domain in the mutants (and in the phosphorylated protein), a hypothesis which is supported by proteolysis experiments. This conformational change would affect aggregation by the monomer through a dimerization step.

Allosteric Regulation↗

PubMed2000

Biochemical characterization of Rab3-GTPase-activating protein reveals a mechanism similar to that of Ras-GAP.

Small G proteins of the Rab family are regulators of intracellular vesicle traffic. Their intrinsic rate of GTP hydrolysis is very low but is enhanced by specific GTPase-activating proteins (GAPs) that switch G proteins to their inactive form. We have characterized the activity of recombinant Rab3-GAP on Rab3A in solution. The K(m) and K(d) values (75 microm) indicate a low affinity of Rab3-GAP for its substrate. The affinity is higher for the transition state analog Rab3A:GDP:AlF(x) (15 microm). The k(cat) (1 s(-)(1)) is within the range of values reported for other GAPs. A mutation in the switch I region of Rab3A disrupted the interaction with Rab3-GAP. Furthermore, Rabphilin, a putative target of Rab3, inhibited the activity of Rab3-GAP on Rab3. Therefore, the Rab3-GAP-binding site involves the switch I region of Rab3 and overlaps with the Rabphilin-binding domain. Substitution of a single arginine residue (Arg-728) of Rab3-GAP disrupted its catalytic activity but not its interaction with Rab3A. We propose that Rab3-GAP, like Ras- and Rho-GAPs, stabilizes the transition state of Rab3 and provides a critical arginine residue to accelerate the GTPase reaction.

Aluminum Compounds↗

PubMed2000

Selective endothelin-A versus combined endothelin-A/endothelin-B receptor blockade in rat chronic heart failure.

BACKGROUND: The relative efficacy of endothelin-A (ET(A)) receptor blockade versus combined ET(A)-ET(B) receptor blockade in chronic heart failure (CHF) is still largely unknown. METHODS AND RESULTS: We compared, in a rat model of CHF (coronary ligation), the hemodynamic and structural effects of 1 month of treatment with the ET(A) antagonist ABT-627 (5 mg x kg(-1) x d(-1)), the ET(B) antagonist A-192621 (30 mg x kg(-1) x d(-1)) or a combination of the 2 drugs. Doses were chosen for their capacity to block the pressor response to ET-1 (for ET(A) blockade) or the depressor responses to sarafotoxin S6c or ET-1 (for ET(B) blockade). ET(A) and combined ET(A)-ET(B) blockade reduced systolic blood pressure to the same extent, whereas ET(B) blockade had no effect. In contrast, only combined ET(A)-ET(B) blockade significantly reduced heart rate. Both ET(A) and combined ET(A)-ET(B) blockade, but not ET(B) blockade alone, increased left ventricular (LV) fractional shortening and wall thickening and reduced LV end-diastolic pressure, as well as LV end-diastolic and end-systolic volumes. However, all treatments (including ET(B) blockade) decreased LV collagen accumulation. CONCLUSIONS: The chronic blockade of both ET(A) and ET(B) receptors improved systemic hemodynamics, as well as LV function and remodeling, to the same extent as ET(A) receptor blockade alone. However, only combined ET(A)-ET(B) receptor blockade decreased heart rate. Whether this differential effect on heart rate affects the long-term outcome after treatment with ET(A) or mixed ET(A)-ET(B) antagonists in CHF remains to be determined.

Animals↗

PubMed2000

Improvement of endothelial function by chronic angiotensin-converting enzyme inhibition in heart failure : role of nitric oxide, prostanoids, oxidant stress, and bradykinin.

BACKGROUND-Chronic heart failure (CHF) impairs the endothelium-dependent, flow-mediated dilation (FMD) of small arteries. However, whether chronic angiotensin-converting enzyme (ACE) inhibition affects the impairment of FMD in CHF is unknown. We investigated the effects of long-term ACE inhibition on the FMD of peripheral arteries in rats with CHF and the mechanism(s) involved. METHODS AND RESULTS-FMD was assessed in isolated, perfused gracilis muscle arteries from sham-operated, and untreated or ACE inhibitor-treated (perindopril 2 mg. kg(-1). day(-1) for 10 weeks) rats with CHF (coronary artery ligation). The role of nitric oxide (NO), prostaglandins, and free radicals was assessed by pretreating the vessels with the NO synthase inhibitor N(W)-nitro-L-arginine, the cyclooxygenase inhibitor diclofenac, or the free radical scavenger N-2-mercaptopropionyl-glycine (MPG). Endothelial NO synthase mRNA expression was determined by reverse transcriptase polymerase chain reaction. In animals with hemodynamic and echographic signs of CHF, FMD was converted into vasoconstriction, and this was prevented by ACE inhibition. FMD of arteries from sham-operated or ACE inhibitor-treated CHF rats was abolished by N(W)-nitro-L-arginine. In untreated CHF rats, FMD was increased by diclofenac and MPG. In contrast, in arteries from ACE inhibitor-treated rats, neither diclofenac nor MPG affected FMD. In parallel, ACE inhibition prevented the reduction of endothelial NO synthase mRNA by CHF. CONCLUSIONS-In CHF, ACE inhibition normalized NO-dependent dilatation and suppressed the production of vasoconstrictor prostanoid(s), resulting in improved FMD. The improvement of FMD might contribute to the beneficial effects of ACE inhibition during CHF.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed2000

Vesicular monoamine transporter 2 as a marker of gastric enterochromaffin-like cell tumors.

The vesicular monoamine transporter 2 (VMAT2) facilitates the ATP-dependent accumulation of biogenic amine inside the secretory granules of endocrine cells and neurons and was demonstrated in the histamine-producing enterochromaffin-like (ECL) cells of the stomach. In the present investigation, VMAT2 immunohistochemistry was tested in 85 endocrine tumors, of which 60 were well differentiated gastrointestinal and pancreatic growths, 5 poorly differentiated (neuro)endocrine carcinomas (PDEC) and 1 mixed PDEC/ECL cell carcinoma of the stomach, 12 pheochromocytomas/paragangliomas, 3 adrenocortical lesions, 2 parathyroid and 2 lung neuroendocrine tumors. Extensive and intense VMAT2 immunoreactivity was observed in 16 of 16 gastric ECL cell tumors, 6 of 6 adrenal pheochromocytomas, 2 of 2 chromaffin paragangliomas and in 3 of the 4 carotid body paragangliomas investigated. Rare VMAT2-positive cells were observed in 12 of 21 intestinal enterochromaffin (EC) cell tumors, in 9 of 11 pancreatic neuroendocrine tumors, and in the mixed PDEC/ ECL cell carcinoma of the stomach (differentiated cells only). No VMAT2 immunoreactivity was observed in five gastrin, four somatostatin and three enteroglucagon/peptideYY tumors of the gastrointestinal tract, in six gastric PDECs, in three adrenocortical growths, and two parathyroid and two lung neuroendocrine tumors. These data support VMAT2 immunohistochemistry as being a useful tool for the diagnosis of gastric ECL cell tumors, separating them from all other endocrine tumors arising in the gastroduodenal area i.e., gastrin, somatostatin, EC cell and PDEC tumors, all of which proved essentially negative.

Biomarkers, Tumor↗

PubMed2000

Evidence against a role of inducible nitric oxide synthase in the endothelial protective effects of delayed preconditioning.

Preconditioning the heart with brief periods of ischaemia induces delayed endothelial protection against reperfusion injury, but the precise mechanisms involved in this endogenous protein are still unclear. Induction of the type II-nitric oxide synthase (iNOS) acts as a mediator of the preconditioning against myocardial infarction and stunning. The present study was designed to assess whether iNOS also contributes to the delayed endothelial protective effects of preconditioning. Rats were subjected to 20 min ischaemia followed by 60 min reperfusion 24 h after sham surgery or preconditioning (one cycle or 2 min ischaemia/5 min reperfusion and two cycles of 5 min ischaemia/5 min reperfusion). At the end of the reperfusion, coronary segments were removed distal to the site of occlusion and mounted in wire myographs. Ischaemia-reperfusion (I/R) decreased the endothelium-dependent relaxations to acetylcholine (maximal relaxations: sham, 66+/-5%; I/R, 40+/-1%; P<0.05) and this impairment was prevented by preconditioning (maximal relaxation: 61+/-6%). Administration of N-(3-aminomethyl)benzyl)acetaminide (1400W), a highly selective inhibitor for iNOS, 10 min before prolonged ischaemia did not modify the beneficial effect of preconditioning (maximal relaxation: 66+/-5%). These data show that preconditioning induces delayed protection against reperfusion-injury. However, in contrast to the myocytes, these endothelial protective effects of delayed preconditioning do not involve iNOS.

Acetylcholine↗

PubMed2000

Constitutive phosphorylation of the vesicular inhibitory amino acid transporter in rat central nervous system.

gamma-Aminobutyric acid (GABA) and glycine are stored into synaptic vesicles by a recently identified vesicular inhibitory amino acid transporter [VIAAT, also called vesicular GABA transporter (VGAT)]. Immunoblotting analysis revealed that rat brain VIAAT migrated as a doublet during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a predominant slower band in all areas examined except olfactory bulb and retina. The slower band corresponded to a phosphorylated form of VIAAT as it was converted to the faster one by treating brain homogenates with alkaline phosphatase or with an endogenous phosphatase identified as type 2A protein-serine/threonine phosphatase using okadaic acid. In contrast, the recombinant protein expressed in COS-7 or PC12 cells co-migrated with the faster band of the brain doublet and was insensitive to alkaline phosphatase. To investigate the influence of VIAAT phosphorylation on vesicular neurotransmitter loading, purified synaptic vesicles were treated with alkaline phosphatase and assayed for amino acid uptake. However, neither GABA nor glycine uptake was affected by VIAAT phosphorylation. These results indicate that VIAAT is constitutively phosphorylated on cytosolic serine or threonine residues in most, but not all, regions of the rat brain. This phosphorylation does not regulate the vesicular loading of GABA or glycine, suggesting that it is involved at other stages of the synaptic vesicle life cycle.

Alkaline Phosphatase↗

PubMed2000

Increased endothelium--monocyte interactions in salt-sensitive hypertension: effect of L-arginine.

Although adhesion of monocytes to endothelial cells is considered as one of the initial factors leading in the long term to the development of atherosclerosis, the effects of hypertension on monocyte-endothelial cell interactions are still largely unknown. Thus we evaluated whether hypertension affects adhesion of monocytes on rat carotid endothelium, and whether this adhesion may be modified by long-term treatment with L-arginine, the physiologic precursor of nitric oxide (NO). Hypertension was induced in Dahl rats using a sodium-rich diet (8%), in the absence or the presence of L-arginine (1.25 g/L in drinking water). After 1 month, the carotid arteries were isolated, opened longitudinally, and incubated in the presence of 2 x 10(6) monocytes previously rendered fluorescent by incubation with tetramethyl rhodamine isothiocyanate (TRITC), and adherent cells were counted under fluorescence microscopy. In parallel, the production of NO was evaluated in vitro in isolated aorta and isolated hearts. Hypertension markedly increased adhesion of monocytes on carotid endothelium, and this was reduced by L-arginine. Hypertension also reduced an index of NO release at the level of the aorta and the coronary circulation. This impaired release of NO was partially prevented by L-arginine. Thus hypertension was associated with an increased adhesion of monocytes, which is probably due at least in part to a decreased production of NO. The increased adhesion was partly reduced by L-arginine, possibly secondary to an increased production of NO. Such an increased adhesion of monocytes may contribute the increased cardiovascular risk in hypertension.

Analysis of Variance↗

PubMed1999

Exercise improves flow-mediated vasodilatation of skeletal muscle arteries in rats with chronic heart failure. Role of nitric oxide, prostanoids, and oxidant stress.

BACKGROUND: Flow-mediated dilatation (FMD) of the peripheral arteries may be impaired in chronic heart failure (CHF), and this could contribute to the increased peripheral resistance and exercise intolerance that occur with this disease. Physical exercise improves the FMD of large conduit arteries in CHF, but whether a similar impairment also occurs in smaller arteries is unknown. The mechanisms of the changes in FMD after CHF or exercise are also unknown. METHODS AND RESULTS: FMD was assessed in isolated, perfused, and preconstricted gracilis muscle arteries from sham-operated rats or CHF rats (coronary artery ligation) who were either sedentary or exercised (30-minute swimming period twice a day for 10 weeks, starting 7 days after ligation). In animals with hemodynamic and echographic signs of CHF, FMD was abolished and converted into vasoconstriction (percent change in diameter after 370 microL/min flow: sham, 42+/-5%; CHF, -4+/-3%; P<0.05). Exercise partially restored FMD (18+/-3%; P<0.05 versus CHF). In sham rats, FMD was abolished by the nitric oxide-synthase inhibitor Nomega-nitro-L-arginine (L-NA) but unaffected by the cyclooxygenase inhibitor diclofenac or the free radical scavenger N-(2-mercaptopropionyl)-glycine (MPG). In arteries from sedentary CHF rats, FMD was not modified by L-NA, but it was partially restored by diclofenac or MPG. In exercised CHF rats, FMD was abolished by L-NA and only moderately improved by diclofenac or MPG. Likewise, endothelial nitric oxide synthase mRNA expression (determined by reverse transcription polymerase chain reaction at the level of the gracilis muscle) was reduced by CHF, and this was prevented by exercise. CONCLUSIONS: CHF abolishes the FMD of small arteries by impairing the nitric oxide pathway, increasing oxidant stress, and releasing a prostanoid-contracting factor. Exercise partially restores FMD by increasing expression of endothelial nitric oxide synthase and preventing the production of vasoconstrictor prostanoids and free radicals. Such restoration of FMD might contribute to the increase in exercise capacity after physical exercise in CHF.

Animals↗

PubMed1999

Molecular dissection of regulated secretory pathways in human gastric enterochromaffin-like cells: an immunohistochemical analysis.

Enterochromaffin-like (ECL) cells regulate gastric acid secretion through vesicular release of histamine. Until now, the molecular machinery of human ECL cells involved in the formation and release of vesicles is largely unknown. We analyzed tissue samples obtained from normal human gastric mucosa (n=4) and ECLomas (n=5) immunohistochemically using the APAAP method or double immunofluorescence confocal laser microscopy. Human pheochromocytomas (n=5) were investigated in parallel and compared to ECL cells. Secretory pathways were characterized using antibodies specific for marker proteins of large dense-core vesicles (LDCVs; islet cell antigen 512, chromogranin A, pancreastatin, and vesicular monoamine transporter 2) and small synaptic vesicle (SSV) analogues (synaptophysin). Tissues were also analyzed for expression of the peptide hormone processing enzymes, carboxypeptidase E and prohormone convertase 1, as well as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, 25-kDa synaptosome-associated protein (SNAP25), syntaxin, and synaptobrevin. Immunoreactivity for markers of LDCVs and SSV analogues were detected in normal ECL cells and ECLomas. Both tissues also showed expression of carboxypeptidase E and prohormone convertase 1. Analysis of vesicular SNARE (v-SNARE) and target membrane SNARE (t-SNARE) proteins revealed the presence of SNAP25, syntaxin, and synaptobrevin in normal and neoplastic ECL cells. Our data suggest that ECL cells possess the two vesicle types of regulated neuroendocrine secretory pathways, LDCVs and SSV analogues. Since ECL cells also contain typical SNARE proteins, the molecular machinery underlying secretory processes in this cell type appears to be identical to the secretory apparatus of neuroendocrine cells and neurons. In addition, our findings suggest that the secretory apparatus of ECL cells is maintained during neoplastic transformation.

Aspartic Acid Endopeptidases↗

PubMed1999

Presence of the vesicular inhibitory amino acid transporter in GABAergic and glycinergic synaptic terminal boutons.

The characterization of the Caenorhabditis elegans unc-47 gene recently allowed the identification of a mammalian (gamma)-amino butyric acid (GABA) transporter, presumed to be located in the synaptic vesicle membrane. In situ hybridization data in rat brain suggested that it might also take up glycine and thus represent a general Vesicular Inhibitory Amino Acid Transporter (VIAAT). In the present study, we have investigated the localization of VIAAT in neurons by using a polyclonal antibody raised against the hydrophilic N-terminal domain of the protein. Light microscopy and immunocytochemistry in primary cultures or tissue sections of the rat spinal cord revealed that VIAAT was localized in a subset (63-65%) of synaptophysin-immunoreactive terminal boutons; among the VIAAT-positive terminals around motoneuronal somata, 32.9% of them were also immunoreactive for GAD65, a marker of GABAergic presynaptic endings. Labelling was also found apposed to clusters positive for the glycine receptor or for its associated protein gephyrin. At the ultrastructural level, VIAAT immunoreactivity was restricted to presynaptic boutons exhibiting classical inhibitory features and, within the boutons, concentrated over synaptic vesicle clusters. Pre-embedding detection of VIAAT followed by post-embedding detection of GABA or glycine on serial sections of the spinal cord or cerebellar cortex indicated that VIAAT was present in glycine-, GABA- or GABA- and glycine-containing boutons. Taken together, these data further support the view of a common vesicular transporter for these two inhibitory transmitters, which would be responsible for their costorage in the same synaptic vesicle and subsequent corelease at mixed GABA-and-glycine synapses.

Amino Acid Transport Systems↗

PubMed1998

The isolated complex of the translocase of the outer membrane of mitochondria. Characterization of the cation-selective and voltage-gated preprotein-conducting pore.

The complex of the translocase mitochondrial outer membrane (TOM), mediates recognition, unfolding, and translocation of preproteins. We have used a combination of biochemical and electrophysiological methods to study the properties of the preprotein-conducting pore of the purified TOM complex. The pore is cation-selective and voltage-gated. It shows three main conductance levels with characteristic slow and fast kinetics transitions to states of lower conductance following application of transmembrane voltages. These electrical properties distinguish it from the mitochondrial voltage-dependent anion channel (porin) and are identical to those of the previously described peptide-sensitive channel. Binding of antibodies to the C terminus of Tom40 on the intermembrane space side of the outer membrane modifies the channel properties and allows determination of the orientation of the channel within the lipid bilayer. Mitochondrial presequence peptides specifically interact with the pore and decrease the ion flow through the channel in a voltage-dependent manner. We propose that the presequence-induced closures of the pore are related to structural alterations of the TOM complex observed during the various stages of preprotein movement across the mitochondrial outer membrane.

Biological Transport↗

PubMed1998

Increased UDP-glucuronosyltransferase activity and decreased prostate specific antigen production by biochanin A in prostate cancer cells.

Our laboratory has characterized androgen metabolism in an androgen-responsive prostate cancer cell line (LNCaP) and showed that these cells accumulated intracellular testosterone primarily as glucuronidated metabolites. Using a cell-free assay with testosterone as substrate, we showed that LNCaP had UDP-glucuronosyltransferase (UDPGT) activity. Because dietary factors, such as flavonoids in soy products, may reduce the risk for hormone-dependent cancers, we studied the effects of flavonoids on testosterone-UDPGT activity. LNCaP cells were exposed to selected flavonoids for up to 6 days. The increase in UDPGT-specific activity was linear over this period. Of the compounds tested, biochanin A was the most potent, with increased activity at concentration range 0.5-50 microM. Activities were linear for time and protein and were unaffected by flavonoids added directly to the assay. Kinetics studies showed no change in Km for testosterone in the face of these large increases in specific activity. Cellular metabolism of testosterone reflected the increase in enzyme activity. Intact cells treated with biochanin A produced testosterone-glucuronide from testosterone at twice the rate of controls. The steroid form of the UDPGT transcript was expressed in LNCaP cells and was enhanced in biochanin A-treated LNCaP cells. Additionally, biochanin A markedly decreased prostate specific antigen (PSA) level against the effect of testosterone on PSA production. Biochanin A significantly decreased the testosterone-stimulated release of PSA, presumably because biochanin A increased UDPGT and increased the intracellular glucuronidation of testosterone. These studies suggest that the modulation of hormone metabolism by dietary factors may be important in the prevention and treatment of prostate cancer.

Anticarcinogenic Agents↗