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Biomedical subjects

S Barnes

Publications and source records attributed to S Barnes.

At least 127 records · Page 7Linked to original sources
PubMed1994

Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives.

Superoxide (O2-.), nitric oxide (.NO), and their reaction product peroxynitrite (ONOO-) have all been shown to independently exert toxic target molecule reactions. Because these reactive species are often generated in excess during diverse inflammatory and other pathologic circumstances, we assessed the influence of .NO on membrane lipid peroxidation induced by O2-., H2O2, and .OH derived from xanthine oxidase (XO) and by ONOO-. Experimental conditions in lipid oxidation systems were adjusted to yield different rates of delivery of .NO, relative to rates of O2-. and H2O2 generation, by infusion of either .NO or via .NO released from S-nitroso-N-acetylpenicillamine or S-nitrosoglutathione. Peroxidation of phosphatidylcholine liposomes was assessed by formation of thiobarbituric acid-reactive products and by liquid chromatography-mass spectrometry. Liposomes exposed to XO-derived reactive species in the presence of .NO exhibited both stimulation and inhibition of lipid peroxidation, depending on the ratio of the rates of reactive oxygen species production and .NO introduction into reaction systems. Nitric oxide alone did not induce lipid peroxidation. Linolenic acid emulsions peroxidized by XO-derived reactive species showed similar dose-dependent regulation of lipid peroxidation by .NO. Mass spectral analysis of oxidation products showed formation of nitrito-, nitro-, nitrosoperoxo-, and/or nitrated lipid oxidation adducts, demonstrating that .NO serves as a potent terminator of radical chain propagation reactions. Electron spin resonance (ESR) analysis of incubation mixtures provided no evidence for formation of paramagnetic iron-lipid-nitric oxide complexes in reaction systems. Peroxynitrite-dependent lipid peroxidation, which predominantly occurs by metal-independent mechanisms, was also inhibited by .NO. Peroxynitrite-mediated benzoate hydroxylation was partially inhibited by .NO, inferring reaction between .NO and ONOOH. It is concluded that .NO can both stimulate O2-./H2O2/.OH-induced lipid oxidation and mediate oxidant-protective reactions in membranes at higher rates of .NO production, with the prooxidant versus antioxidant outcome critically dependent on relative concentrations of individual reactive species. Prooxidant reactions of .NO will occur after O2-. reaction with .NO to yield potent secondary oxidants such as ONOO- and the antioxidant effects of .NO a consequence of direct reaction with alkoxyl and peroxyl radical intermediates during lipid peroxidation, thus terminating lipid radical chain propagation reactions.

Hydroxyl Radical↗
PubMed1994

Glycine and taurine conjugation of bile acids by a single enzyme. Molecular cloning and expression of human liver bile acid CoA:amino acid N-acyltransferase.

In order to establish whether a single enzyme in human liver was capable of conjugating bile acids with both glycine and taurine, a cDNA encoding human liver bile acid-CoA:amino acid N-acyltransferase (hBAT) has been isolated and characterized. A specific immunoaffinity-purified rabbit anti-hBAT polyclonal antibody was used to screen a lambda Zap XR human liver cDNA library resulting in the isolation of two unique clones. hBAT8 and hBAT9 (1669 and 1491 base pairs in length, respectively) were isolated following screening of 4 x 10(5) clones of the cDNA library. Restriction mapping and sequence analysis demonstrated that the cDNAs were identical except hBAT8 contained an additional 178 bases of 5' sequence; hBAT8 was completely sequenced, characterized, and used for all subsequent studies. hBAT8 consisted of a 184-nucleotide 5'-nontranslated region, an open reading frame of 1,254 bases predicting a protein of 418 amino acids with a molecular mass of 46,296 Da, and a 3'-nontranslated region of 209 nucleotides followed by a poly(A)+ tail. The identity of the cDNA was confirmed by the following findings: 1) the open reading frame began with an ATG codon and was followed by a nucleotide sequence which, when translated, corresponded exactly to the first 17 NH2-terminal amino acids of purified human liver BAT; 2) cytosol of Escherichia coli XL1-Blue cells transfected with hBAT8 subcloned into an expression vector, pKK233-2, demonstrated significant enzymatic activity for the conjugation of both taurine and glycine with cholic acid; 3) bacterial expression of hBAT8 generated a protein that comigrated with hBAT from human liver during SDS-polyacrylamide gel electrophoresis and cross-reacted with a specific polyclonal rabbit anti-hBAT antibody during immunoblot analysis; 4) kinetic characteristics of the expressed enzyme were very similar to those reported for purified liver BAT. These data demonstrate that a single cDNA is present in human liver which codes for a protein capable of catalyzing the conjugation of cholic acid with both glycine and taurine.

Acyltransferases↗
PubMed1994

Proton modulation of M-like potassium current (IKx) in rod photoreceptors.

The effect of extracellular pH (pHe 6.9-8.1) and intracellular pH (pHi 6.4-8.1) on the non-inactivating voltage-sensitive M-like potassium current (IKx) was studied in patch-clamped salamander rod photoreceptors. The midpoint of the IKx activation curve shifted by 6.6 mV per pHe unit, with acidification producing positive shifts and alkalinization producing negative shifts. The time constant of IKx activation shifted with pHe in a manner consistent with the shifts in the activation curve. Maximum conductance and gating charge were unaffected by changes in pHe. IKx did not depend on pHi. Given the importance of IKx in rod function, these results suggest that pHe could affect the signal transmitted from rods by changing IKx activation parameters.

Ambystoma↗
PubMed1994

After transduction: response shaping and control of transmission by ion channels of the photoreceptor inner segments.

Photoreceptors convert the elements of the visual image into the elements of a neural image. This process involves well-studied molecular events occurring at the outer segment, but also employs important molecular events in the proximal regions of the photoreceptor, including the synaptic terminal, encompassed here as the inner segment. Integral to neural processing at this level in the visual system, the inner segment mechanisms modify the visual signal before transmission to second order cells at the photoreceptor output synapse. This commentary, emphasizing the author's own work, discusses biophysical properties of the ensemble of ion channels in the photoreceptor inner segment that shape the light response and enable its transmission. Examples that illustrate ion channels whose biophysical properties seem well suited for their roles in photoreceptor function include: h channels, cation-selective channels activated by hyperpolarization, which carry current that counteracts the strong hyperpolarizing influence of cGMP-gated channel closure accompanying bright light; Kx channels, carrying potassium current which shares the kinetic properties of the M-current found in many other cell types, which shape responses to dim light and set the dark resting potential; and Ca channels that regulate calcium influx to control Ca-gated channel activity and synaptic output, "re-transducing" the neural signal now into a chemical one. The role of chloride current, carried in Ca-activated Cl channels dependent on the unknown chloride equilibrium potential in photoreceptors, is also discussed.

Animals↗
PubMed1994

Modulation of ion channels in rod photoreceptors by nitric oxide.

Subcellular compartments in the outer retina of the larval tiger salamander were identified as likely sites of production of nitric oxide (NO), a recently recognized intercellular messenger. NADPH diaphorase histochemistry and NO synthase immunocytochemistry labeled photoreceptor ellipsoids and the distal regions of bipolar and glial cells apposing photoreceptor inner segments, suggesting a role for NO in visual processing in the outer retina. We investigated the actions of NO on several rod photoreceptor ion channels. Application of the NO-generating compound S-nitrosocysteine increased Ca2+ channel current and a voltage-independent conductance, but had no affect on voltage-gated K+ or nonspecific cation currents. Given the steep relation between voltage-dependent Ca2+ influx and photoreceptor synaptic output, these results indicate that NO could modulate transmission of the photoresponse to second order cells.

Amino Acid Oxidoreductases↗
PubMed1994

Soy intake and cancer risk: a review of the in vitro and in vivo data.

International variations in cancer rates have been attributed, at least in part, to differences in dietary intake. Recently, it has been suggested that consumption of soyfoods may contribute to the relatively low rates of breast, colon, and prostate cancers in countries such as China and Japan. Soybeans contain a number of anticarcinogens, and a recent National Cancer Institute workshop recommended that the role of soyfoods in cancer prevention be investigated. In this review, the hypothesis that soy intake reduces cancer risk is considered by examining relevant in vitro, animal, and epidemiological data. Soybeans are a unique dietary source of the isoflavone genistein, which possesses weak estrogenic activity and has been shown to act in animal models as an antiestrogen. Genistein is also a specific inhibitor of protein tyrosine kinases; it also inhibits DNA topoisomerases and other critical enzymes involved in signal transduction. In vitro, genistein suppresses the growth of a wide range of cancer cells, with IC50 values ranging from 5 to 40 microM (1-10 micrograms/ml). Of the 26 animal studies of experimental carcinogenesis in which diets containing soy or soybean isoflavones were employed, 17 (65%) reported protective effects. No studies reported soy intake increased tumor development. The epidemiological data are also inconsistent, although consumption of nonfermented soy products, such as soymilk and tofu, tended to be either protective or not associated with cancer risk; however, no consistent pattern was evident with the fermented soy products, such as miso. Protective effects were observed for both hormone- and nonhormone-related cancers. While a definitive statement that soy reduces cancer risk cannot be made at this time, there is sufficient evidence of a protective effect to warrant continued investigation.

Animals↗
PubMed1994

Modulation of ionic currents by dopamine in an interneurone of the respiratory central pattern generator of Lymnaea stagnalis.

Dopamine elicits alternating bursts of activity in the respiratory interneurones of the snail Lymnaea stagnalis. One of the neurones (VD4) was isolated in culture, and the effects of dopamine on both membrane voltage and current were studied utilising the whole-cell tight-seal recording technique. Dopamine had little effect on resting potentials near -60 mV, nor did it affect spike threshold or input resistance measured near -60 mV. However, it did alter the excitability of the cell, changing the response to current injection from one of repetitive spiking to one of rapid accommodation. Under voltage-clamp, VD4 responded to dopamine (EC50 = 92 nmol l-1) with increased net outward current at all potentials more positive than -60 mV. This was due primarily to an increase in voltage-gated potassium current and a decrease in calcium current. A reduction of Cd(2+)-sensitive outward current, possibly calcium-gated potassium current, was also evident at potentials more positive than +60 mV. The physiological actions of dopamine on these cells in vivo are consistent with the inhibitory mechanisms presented in this study.

Action Potentials↗
PubMed1994

Role of bile salt hydrophobicity in hepatic microtubule-dependent bile salt secretion.

Under basal conditions, bile salt secretion by the liver is not affected by microtubule disruption. However, when a bile salt load is imposed on the liver, a microtubule-dependent secretion mechanism is recruited (J. Lipid Res. 1988. 29: 144-156). We tested the hypothesis that recruitment of this microtubule-dependent mechanism is influenced by the relative hydrophobicity of the bile salts being secreted. Intact male rats were depleted of bile salts by overnight biliary diversion, pretreated with colchicine (a microtubule inhibitor) or its inactive isomer, lumicolchicine (control), and reinfused intravenously with bile salts of increasing hydrophobicity (taurodehydrocholate < tauroursodeoxycholate < taurocholate) at 200 nmol/min.100 g. After 45 min, when steady-state bile salt secretion was achieved, tracer [3H]taurocholate was administered intravenously. The colchicine-insensitive component of bulk bile salt secretion was constant at approximately 130 nmol/min.100 g, and the colchicine-sensitive component increased from approximately 0 to 35 and 60 nmol/min.100 g, respectively, with reinfusion of the more hydrophobic bile salts. Retained bile salts accumulated in the liver and serum and were detectable in urine. Peak biliary secretion of [3H]taurocholate in control animals increased linearly from 15.3 to 18.0% administered dose/min with increasing hydrophobicity of the secreted bile salts (P < 0.002). In colchicine-pretreated animals, peak secretion rates decreased linearly from 13.8 to 9.2%/min (P < 0.001), with maximal inhibition in taurocholate-reinfused animals (P < 0.01). Utilization of a microtubule-dependent secretion mechanism increases with increasing bile salt hydrophobicity. This mechanism permits more efficient hepatic secretion of bile salts, but increases the susceptibility of bile salt secretion to microtubule disruption. We postulate that microtubule-dependent insertion of bile salt transporters into the canalicular membrane underlies the enhanced bile salt secretion observed when a bile salt load is imposed upon the liver.

Animals↗
PubMed1994

Ultrastructural localization of a fluorinated bile salt in hepatocytes.

BACKGROUND: Interactions of bile salts with hepatocellular organelles are critical for the formation of bile, yet these interactions remain poorly characterized. We present a novel approach for visualizing bile salts at the ultrastructural level within hepatocytes, using a unique fluorinated bile salt conjugate and electron energy loss spectroscopy. EXPERIMENTAL DESIGN: Isolated rat hepatocytes were incubated for 5 and 20 minutes with the 2-fluoro-beta-alanine (FBAL) N-acyl amidate conjugate of cholic acid (C-FBAL, 50 microM). FBAL is a byproduct of hepatic 5-fluorouracil catabolism, and when conjugated to cholic acid is excreted into bile in a manner similar to the naturally occurring N-acyl amidates of bile salts. Cells were subjected to rapid cryofixation and automated freeze-drying followed by vapor-phase fixation using the LifeCell system, thus avoiding exposure to the leaching action of liquid fixatives. After resin infiltration, the cellular distribution of fluorine was determined in ultrathin sections with a Zeiss CEM902 electron microscope equipped for electron energy loss spectroscopy. RESULTS: Fluorine was detected primarily in association with intracellular membranes, particularly membranes of the endoplasmic reticulum (p < 0.05 at 20 minutes by morphometric analysis). Fluorine also was detected in association with membranes of the Golgi apparatus. The fluorine signal was confirmed by serial spectra of cell regions containing these organelles (p < 0.01), but was not detectable in the free cytosol, mitochondria or extracellular medium, nor in hepatocytes not exposed to C-FBAL. CONCLUSIONS: We conclude that cryofixation and freeze-dry processing followed by electron microscopy with electron energy loss spectroscopy is a valuable technique for examining intracellular processing of bile salts. Our results suggest that bile salts localize to the membranes, but not lumena, of organelles during hepatocyte exposure to bile salts, calling into question the proposed role for vesicular transport of bile salts within hepatocytes.

Animals↗
PubMed1993

Photoresponsive pacemaker neurons from the dissociated retina of Aplysia.

Isolated retinal pacemaker neurons of Aplysia devoid of any morphologically specialized photoreceptive processes responded to illumination. Neurons were dissociated from other retinal cells and maintained in primary culture for whole cell recording. The membrane potential was depolarized and action potentials were evoked by illumination. Light adaptation diminished the response and prolonged the latency. Under voltage clamp neurons responded with increased inward current. Responses were slower and smaller than specialized photoreceptor responses. Pairs and small clusters of monopolar neurons also responded to light. These photoresponsive monopolar neurons are likely to be the output neurons of the ocular circadian clock. Although photoresponsive central neurons have been described in Aplysia, this is the first report of photoresponses from isolated retinal neurons.

Action Potentials↗
PubMed1993

Modulation of transmission gain by protons at the photoreceptor output synapse.

Synaptic transmission of the light response from photoreceptors to second-order cells of the retina was studied with the whole-cell patch-clamp technique in tiger salamander (Ambystoma tigrinum) retinal slices. Synaptic strength is modulated by extracellular pH in a striking manner: Light-sensitive postsynaptic currents in horizontal and bipolar cells were found to be exponential functions of pH, exhibiting an e-fold increase per 0.23 pH unit over the pH range from 7 to 8. Calcium channel currents in isolated photoreceptors were measured and also exhibited proton sensitivity. External alkalinization from pH 7 to 8 shifted the voltage dependence of channel activation negative by 12 mV. A model of the synaptic transfer function suggested that presynaptic Ca channels could be the primary sites of proton action. Increased Ca influx and transmitter release brought about by alkalinization give rise to larger postsynaptic currents. These results suggest that activity-dependent interstitial pH changes known to occur in the retina, while not alleviating signal clipping at this synapse, may provide an adaptative mechanism controlling gain at the photoreceptor output synapse.

Ambystoma↗
PubMed1993

Genistein and biochanin A inhibit the growth of human prostate cancer cells but not epidermal growth factor receptor tyrosine autophosphorylation.

The effect of the isoflavones, genistein, daidzein, and biochanin A on the growth of the LNCaP and DU-145 human prostate cancer cell lines has been examined. Genistein and biochanin A, but not daidzein, inhibit both serum and EGF-stimulated growth of LNCaP and DU-145 cells (IC50 values from 8.0 to 27 micrograms/ml for serum and 4.3 to 15 micrograms/ml for EGF), but have no significant effect of the EGF receptor tyrosine autophosphorylation. In contrast, tyrphostin 25, a specific EGF receptor tyrosine kinase inhibitor, inhibits EGF-stimulated growth and EGF receptor tyrosine autophosphorylation in these whole cells, but does not inhibit serum-stimulated growth. These data suggest that the mechanism of action of genistein and biochanin A does not depend on inhibition of EGF receptor tyrosine autophosphorylation, but on a more distal event in the EGF receptor-mediated signal transduction cascade.

Anticarcinogenic Agents↗
PubMed1993

Purification and properties of a human platelet inositol 1,4,5-trisphosphate 3-kinase.

An inositol 1,4,5-trisphosphate 3-kinase (Ins(1,4,5)P3 3-kinase) has been purified 943-fold from a 30,000g human platelet extract and has a specific activity of 283 nmol/min/mg protein and an apparent Km for inositol 1,4,5-trisphosphate of 0.76 microM; the optimal pH for the enzymatic activity was 7.2. Under both denaturing and nondenaturing conditions, the kinase preparation contained two polypeptides, both of which exhibited Ca2+/calmodulin-dependent Ins(1,4,5)P3 3-kinase activity. In the presence and absence of calmodulin, Ins(1,4,5)P3 3-kinase exhibited a biphasic response to Ca2+, being stimulated between 10(-7) and 10(-6) M Ca2+ and inhibited when the Ca2+ level was further increased. Ins(1,4,5)P3 3-kinase was stimulated by calmodulin approximately 10-fold, requiring 55 nM calmodulin for a half-maximal effect. Calmodulin stimulation was immediately reversed upon chelation of Ca2+ by ethylene glycol bis (beta-amino-ethyl ether) N,N'-tetraacetic acid consistent with a mechanism of activation involving a direct interaction of calmodulin with Ins(1,4,5)P3 3-kinase. Since we have previously shown that Ins(1,4,5)P3 3-kinase can also be phosphorylated and consequently inactivated by protein kinase C in vitro (Lin, A. N., Barnes, S., and Wallace, R. W., 1990, Biochem. Biophys. Res. Commun. 170, 1369-1376), Ins(1,4,5)P3 3-kinase appears to be a key enzyme in the inositol phosphate signaling pathway and as such may play an important role in human platelet function.

Animals↗
PubMed1993

Inhibition of tumor promoter-induced hydrogen peroxide formation in vitro and in vivo by genistein.

Here we report that genistein, a soybean isoflavone, strongly inhibits tumor promoter-induced H2O2 formation both in vivo and in vitro. Genistein suppressed H2O2 production by 12-O-tetradecanoylphorbol-13-acetate- (TPA) stimulated human polymorphonuclear leukocytes (PMNs) and HL-60 cells in a dose-dependent manner over the concentration range 1-150 microM. Human PMNs were more sensitive to the inhibitory effect of genistein than HL-60 cells (50% inhibitory concentration 14.8 and 30.2 microM, respectively). In addition, genistein moderately inhibited superoxide anion formation by HL-60 cells and scavenged exogenously added H2O2 under the same conditions as in cell culture. However, the H2O2-scavenging effect of genistein was about 50% lower than its inhibition of cell-derived H2O2 formation at all concentrations. In the CD-1 mouse skin model, genistein strongly inhibited TPA-induced oxidant formation, edema, and PMN infiltration in mouse skin. Inhibition of TPA-mediated H2O2 in vivo may result from decreased cell-derived H2O2 formation, scavenging of H2O2 produced, and/or suppression of PMN infiltration into the dermis. The antioxidant properties of genistein may be responsible for its anticarcinogenic effects, and the dietary availability of genistein makes it a promising candidate for the prevention of human cancers.

Animals↗
PubMed1993

Cholera toxin and pertussis toxin provoke differential effects on luteinizing hormone release, inositol phosphate production, and gonadotropin-releasing hormone (GnRH) receptor binding in the gonadotrope: evidence for multiple guanyl nucleotide binding proteins in GnRH action.

A growing body of evidence suggests a role for guanyl nucleotide binding proteins (G proteins) in GnRH action. G protein activation provokes LH release, inositol phosphate (IP) production, decreased gonadotrope responsiveness to GnRH, increased gonadotrope responsiveness to the calcium ionophore A23187, and decreased GnRH receptor binding. The specific G proteins involved in these actions, however, are not known. This study uses pertussis toxin (PTX) and cholera toxin (CTX), which affect the activity of a number of G proteins by ADP ribosylation of a Cys or an Arg residue, respectively, of the alpha-subunit. Although not an effective LH secretagogue in itself, CTX enhanced GnRH-, NaF-, and A23187-stimulated LH release after an 18-h pretreatment period. CTX pretreatment did not affect GnRH- or NaF-stimulated IP production. Conversely, 18 h pretreatment with PTX reduced GnRH- and NaF-provoked IP production compared to control values, but did not affect LH release. In addition, pretreatment with either CTX, PTX, or Bt2cAMP provoked a decrease in GnRH receptor binding compared to control. The results of this study suggest that: 1) GnRH stimulates IP production, but not LH release, through a PTX-sensitive G protein; 2) A distinct CTX-sensitive G protein appears to provoke gonadotrope sensitization by stimulating an increase in intracellular cAMP levels; and 3) there appears to be a distinct G protein, insensitive to PTX and CTX, capable of mediating LH release independent of IP production and cAMP.

Animals↗