Biomedical subjects

M L Wu

Publications and source records attributed to M L Wu.

At least 73 records · Page 4Linked to original sources

PubMed1996

Kinetic resolution of amino acid esters catalyzed by lipases.

Racemic amino acids were resolved by lipase via hydrolysis of their esters. Lipases (Pseudomonas lipase from Amano PS, Rhizopus lipase from Serva, and porcine pancrease lipase from Sigma) could selectively hydrolyze the L-amino acid esters in aqueous solution with high reactivities and selectivities. The effect of the structural changes in the ester moiety on the stereoselectivity of the lipases was also investigated using D,L-homophenylalanine as a model. Procedures were developed for the resolution of natural and unnatural amino acids.

Amino Acids↗

PubMed1996

Mechanism of hydrogen peroxide and hydroxyl free radical-induced intracellular acidification in cultured rat cardiac myoblasts.

After a transient ischemic attack of the cardiac vascular system, reactive oxygen-derived free radicals, including the superoxide (O2-.) and hydroxyl (.OH) radicals can be easily produced during reperfusion. These free radicals have been suggested to be responsible for reperfusion-induced cardiac stunning and reperfusion-induced arrhythmia. Hydrogen peroxide (H2O2) is often used as an experimental source of oxygen-derived free radicals. Using freshly dissociated single rat cardiac myocytes and the rat cardiac myoblast cell line, H9c2, we have shown, for the first time, that an intriguing pHiota acidification (approximately 0.24 pH unit) is induced by the addition of 100 micromol/L H2O2 and that this dose is without effect on the intracellular free Ca2+ levels or viability of the cells. Using H9c2 as a model cardiac cell, we have shown that it is the intracellular production of .OH, and not O2-. or H2O2, that results in this acidification. We have excluded any involvement of (1) the three known cardiac pHi regulators (the Na+-H+ exchanger, the Cl--HCO3 exchanger, and the Na+-HCO3 co-transporter), (2) a rise in intracellular Ca2+ levels, and (3) inhibition of oxidative phosphorylation. However, we have found that H2O2-induced acidosis is due to inhibition of the glycolytic pathway, with hydrolysis of intracellular ATP and the resultant intracellular acidification. In cardiac muscle and in skinned cardiac muscle fiber, it has been shown that a small intracellular acidification may severely inhibit contractility. Therefore, the sustained pHi decrease caused by hydroxyl radicals may contribute, in some part, to the well-documented impairment of cardiac mechanical function (ie, reperfusion cardiac stunning) seen during reperfusion ischemia.

Acidosis↗

PubMed1995

Sexual isolation in Drosophila melanogaster: a possible case of incipient speciation.

It is generally believed that Drosophila melanogaster has no closely related species with which it can produce the viable and fertile hybrids that are essential for the genetic analysis of speciation. Following the recent report of molecular differentiation between a Zimbabwe, Africa, population and two United States populations, we provide evidence that strong sexual isolation exists between the D. melanogaster population in Zimbabwe and populations of other continents. In the presence of males of their own kind, females from most isofemale lines of Zimbabwe would not mate with males from elsewhere; the reciprocal mating is also significantly reduced, but to a lesser degree. The genes for sexual behaviors are apparently polymorphic in Zimbabwe and postmating reproductive isolation between this and other populations has not yet evolved. Whole chromosome substitutions indicate significant genetic contributions to male mating success by both major autosomes, whereas the X chromosome effect is too weak to measure. In addition, the relative mating success between hybrid and pure line males supports the interpretation of strong female choice. These observations suggest that we are seeing the early stages of speciation in this group and that it is driven by sexual selection. The genetic and molecular tractability of D. melanogaster offers great promise for the detailed analysis of this apparent case of incipient speciation.

Animals↗

PubMed1995

Thermodynamics of heme-induced conformational changes in hemopexin: role of domain-domain interactions.

Hemopexin is a serum glycoprotein that binds heme with high affinity and delivers heme to the liver cells via receptor-mediated endocytosis. A hinge region connects the two non-disulfide-linked domains of hemopexin, a 35-kDa N-terminal domain (domain I) that binds heme, and a 25-kDa C-terminal domain (domain II). Although domain II does not bind heme, it assumes one structural state in apo-hemopexin and another in heme-hemopexin, and this change is important in facilitating the association of heme-hemopexin with its receptor. In order to elucidate the structure and function of hemopexin, it is important to understand how structural information is transmitted to domain II when domain I binds heme. Here we report a study of the protein-protein interactions between domain I and domain II using analytical ultracentrifugation and isothermal titration calorimetry. Sedimentation equilibrium analysis showed that domain I associates with domain II both in the presence and absence of heme with Kd values of 0.8 microM and 55 microM, respectively. The interaction between heme-domain I and domain II has a calorimetric enthalpy of +11 kcal/mol, a heat capacity (delta Cp) of -720 cal/mol.K, and a calculated entropy of +65 cal/mol.K. By varying the temperature of the centrifugation equilibrium runs, a van't Hoff plot with an apparent change in enthalpy (delta H) of -3.6 kcal/mol and change in entropy (delta S) of +8.1 cal/mol.K for the association of apo-domain I with domain II was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Protein kinase C isoform delta is involved in the stimulation of the Na(+)-H+ exchanger in C6 glioma cells.

Protein kinase C (PKC), the major receptor for tumor-promoting phorbol esters, is a family consisting of at least 12 distinct isoforms. In C6 glioma cells, short term (10 min) treatment with 12-O-tetradecanoyl phorbol-13-acetate (TPA) results in a dose-dependent translocation of PKC alpha, PKC delta and PKC theta. Long term (24 hr) treatment with appropriate doses of TPA results in the complete down-regulation of PKC delta but not of PKC alpha PKC theta. This property was used to determine which isoform might be involved in the activation of the glial cell Na(+)-H+ exchanger. It was found that (a) TPA has dose-dependent effects on PKC alpha, PKC delta, and PKC theta translocation and on Na(+)-dependent, EIPA-sensitive Na(+)-H+ exchanger stimulation; (b) the antiporter is blocked both by staurosporine and in cells in which PKC alpha, PKC delta, and PKC theta have been completely down-regulated; (c) the inactive form (alpha-TPA) of TPA, which does not include translocation of the three isoforms (alpha, delta, and theta) has no effect on the antiporter; (d) Western blot analysis demonstrated dose-dependent TPA (10, 30, 100, and 1000 nM)-induced translocation of PKC alpha, PKC delta, and PKC theta. TPA (10, 30, 100, and 1000 nM)-induced Na(+)-H+ exchanger activation was also found to be dose dependent. There was no difference in TPA-induced Na(+)-H+ exchanger activation between 30 and 100 nM; it correlated with the extent of TPA-induced PKC delta translocation over the same concentration range, suggesting that isoform responsible for the exchanger activation is PKC delta; (e) When 1 microM TPA is added after prior treatment of the cells with 10 nM TPA, an additive effect on Na(+)-H+ exchanger is seen that is not observed when the initial stimulus in 30 nM TPA, as would be expected if the PKC delta isoform were responsible for exchanger activation; (f) Finally, when PKC delta, but not PKC alpha and PKC theta, is completely down-regulated by 24-hr pretreatment with 10, 30, or 100 nM TPA, the Na(+)-H+ exchanger can no longer be stimulated by 1 microM TPA; when 1 nM TPA pretreatment is used, no down-regulation occurs and the exchanger still responds to 1 microM TPA. We have shown that the Na(+)-H+ exchanger in C6 glioma cells can be stimulated by TPA-induced PKC activation and, for the first time, that PKC delta is involved in the activation of this antiporter. Our results also suggest that different members of the PKC family within a single cell elicit specific physiological responses.

Dose-Response Relationship, Drug↗

PubMed1994

Effect of metabolic inhibitors and second messengers upon Na(+)-H+ exchange in the sheep cardiac Purkinje fibre.

1. Acid extrusion through Na(+)-H+ exchange was studied in the sheep cardiac Purkinje fibre (bathed in Hepes-buffered solution, nominally free of CO2-HCO3-) by examining (i) intracellular pH (pHi) recovery from an intracellular acid load (induced by 20 mM NH4Cl prepulse) and (ii) the rate of rise of intracellular Na+ activity (aiNa) following the ammonium prepulse (used as an estimate of apparent Na+ influx on Na(+)-H+ exchange). The pHi and aiNa were recorded using ion-selective microelectrodes. 2. The pHi recovery and rise of aiNa were both greatly slowed in the presence of 2-deoxyglucose (DOG; glucose-free solution), an inhibitor of glycolysis, indicating inhibition of Na(+)-H+ exchange. 3. Cyanide moderately slowed pHi recovery rate but did not significantly affect the rise of aiNa. Estimates of beta 1 (intracellular buffering power) indicated an increase of approximately 50% in the presence of cyanide; such an increase accounts for most of the observed slowing of pHi recovery. It is concluded that oxidative inhibition with cyanide does not inhibit Na(+)-H+ exchange. 4. Intracellular ATP, measured from luciferin-luciferase luminescence, was reduced by a similar amount (approximately 70%) by either DOG or cyanide. This suggests that, if intracellular ATP (ATPi) reduction is the cause of exchanger inhibition by metabolic inhibitors, then ATPi generated glycolytically is more important for activation of the exchange. 5. 3-Isobutyl-1-methylxanthine (IBMX; a non-specific phosphodiesterase inhibitor which can elevate intracellular [cAMP]) slowed acid extrusion and reduced apparent Na+ influx by a similar amount, whereas addition of sodium nitroprusside (to elevate intracellular [cGMP]) had no effect, suggesting that raising intracellular [cAMP] downregulates Na(+)-H+ exchange, whereas raising intracellular [cGMP] does not. 6. Application of trifluorperazine (TFP; a non-specific calcium-calmodulin inhibitor) completely reversed the inhibitory effects of IBMX upon pHi recovery and aiNa. Under control conditions (no IBMX), TFP had no effect on pHi recovery or upon resting pHi. 7. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) had no significant effect on pHi recovery or apparent Na+ efflux. 8. We conclude that inhibition of glycolysis or elevation of cAMP produces downregulation of Na(+)-H+ exchange in the cardiac Purkinje fibre. Possible reasons for the lack of inhibitory effect of oxidative inhibitors are discussed.

1-Methyl-3-isobutylxanthine↗

PubMed1994

Conformational analysis of hemopexin by Fourier-transform infrared and circular dichroism spectroscopy.

Hemopexin is a serum glycoprotein that binds heme with the highest known affinity of any characterized heme-binding protein and plays an important role in receptor-mediated cellular heme uptake. Complete understanding of the function of hemopexin will require the elucidation of its molecular structure. Previous analysis of the secondary structure of hemopexin by far-UV circular dichroism (CD) failed due to the unusual positive ellipticity of this protein at 233 nm. In this paper, we present an examination of the structure of hemopexin by both Fourier-transform infrared (FTIR) and circular dichroism spectroscopy. Our studies show that hemopexin contains about 55% beta-structure, 15% alpha-helix, and 20% turns. The two isolated structural domains of hemopexin each have secondary structures similar to hemopexin. Although there are significant tertiary conformational changes indicated by the CD spectra, the overall secondary structure of hemopexin is not affected by binding heme. However, moderate changes in secondary structure do occur when the heme-binding domain of hemopexin associates with heme. In spite of the exceptionally tight binding at neutral pH, heme is released from the bis-histidyl heme-hemopexin complex at pH 5.0. Under this acidic condition, hemopexin maintains the same overall secondary structure as the native protein and is able to resume the heme-binding function and the native structure of the heme-protein (as indicated by the CD spectra) when returned to neutral pH. We propose that the state of hemopexin identified in vitro at pH 5.0 resembles that of this protein in the acidic environment of the endosomes in vivo when hemopexin releases heme during receptor-mediated endocytosis.

Circular Dichroism↗

PubMed1994

Extracellular ATP activates calcium entry and mobilization via P2U-purinoceptors in rat lactotrophs.

Extracellular ATP has been previously shown to activate calcium signalling in pituitary cell populations [1] but the particular cell types involved have not been identified. We used video imaging of Fura-2 loaded into single rat pituitary cells and identified as lactotrophs to study the effects of extracellular ATP on [Ca2+]i. ATP does not permeabilize the cells as shown by exclusion of propidium iodide. ATP causes two types of calcium transients in lactotrophs. The most common response is a rapid increase in [Ca2+]i that decays slowly and is terminated by washout of ATP. This type of response is also seen in calcium-free medium, demonstrating mobilization of calcium stores dependent upon the presence of the agonist. ATP also stimulates calcium entry as detected by Mn(2+)-quenching of Fura-2. ATP in Mg(2+)-free medium and ATP gamma S are effective agonists suggesting ATP4- is the active form. The presence of P2-purinoceptors is apparent because ATP, ADP and AMP increase [Ca2+]i in decreasing order of potency and adenosine has no effect. ATP-induced calcium transients are reduced by the P2-purinoceptor antagonists suramin and quinidine. UTP is equipotent with ATP and defines the receptor subtype as P2U. We conclude that ATP4- acts on rat lactotrophs via P2U-purinoceptors to elevate [Ca2+]i from intracellular and extracellular sources.

Adenosine Triphosphate↗

PubMed1994

DIDS-sensitive pHi regulation in single rat cardiac myocytes in nominally HCO3-free conditions.

The fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pHi in the spontaneously hypertensive rat (SHR) and in normal rat cardiac myocytes under nominally HCO3-free (20 mmol/L HEPES-buffered) conditions. When only the Na-H exchanger was blocked, the intrinsic buffering power (beta i) in SHR myocytes was significantly higher than when both the Na-H exchanger and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)-sensitive pHi regulators (the Na-HCO3 cotransporter and the Cl-HCO3 exchanger) were blocked. Similar low values for beta i were also found for normal rat myocytes in Na(+)-free conditions. In Cl(-)-free solution under nominally HCO3-free conditions, in both normal and SHR myocytes, the pHi slowly alkalinized (by 0.16 +/- 0.02 and 0.11 +/- 0.02 pH units, respectively); this alkalinization was also DIDS sensitive. The reacidification during NH4+ perfusion was inhibited 30.2 +/- 7.4% by DIDS. In addition, in the nominal absence of HCO3-, 100 mumol/L ATP acidified the pHi in both normal and SHR myocytes (by 0.21 +/- 0.03 and 0.33 +/- 0.03 pH units, respectively); this acidification was totally inhibited by 0.1 mmol/L DIDS. It has been shown, in rat cardiac myocytes, that ATP acidifies the pHi by 0.35 pH unit via stimulation of a DIDS-sensitive Cl-HCO3 exchanger in HCO3-containing solutions. Finally, we have shown, in normal cardiac myocytes, that two potent Na-H exchanger blockers, N-5-ethylisopropyl amiloride (EIPA) and N-5-methyl-N-isobutyl amiloride (MIA), only partially inhibited the pHi recovery from internal acidosis under nominally bicarbonate-free conditions. When DIDS was added at the same time as EIPA, pHi recovery from an internal acid loading was completely inhibited. Our results clearly demonstrate that in both normal and SHR cardiac myocytes, bicarbonate-dependent pHi regulators can be significantly activated under resting or acidified pHi in HEPES-buffered medium, probably because of the cellular production of CO2. The contribution of these bicarbonate-dependent pHi regulators, ie, the Na-HCO3 cotransporter and the Cl-HCO3 exchanger, cannot therefore be ignored even under nominally HCO3-free conditions.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1993

Characterization of hemopexin and its interaction with heme by differential scanning calorimetry and circular dichroism.

Hemopexin is a plasma glycoprotein that has two structural domains (I and II) and binds and transports heme particularly to liver cells. Differential scanning calorimetry (DSC) studies show that hemopexin is largely stabilized by heme, which binds exclusively to domain I. The melting temperature (Tm) of heme-hemopexin is 66.4 +/- 0.7 degrees C as compared with 53.9 +/- 0.3 degrees C for apohemopexin, and this Tm increase is accompanied by a 100 kcal increase in molar enthalpy. Heme stabilizes hemopexin by stabilizing domain I. This is demonstrated by the 26 degrees C increase in Tm from 51.9 +/- 0.3 to 77.6 +/- 0.6 degrees C and the over 3-fold increase in molar enthalpy when domain I associates with heme. A moderate change in domain I secondary structure is indicated by an increase in negative molar ellipticity at 206 nm. However, there is no net effect on the secondary structure of holo-hemopexin caused by heme binding as indicated by both far-UV circular dichroism (CD) and Fourier-transform infrared spectra. The characteristic positive ellipticity of hemopexin at 233 nm, ascribed to tryptophan residues in domain II, is dramatically increased, suggesting a change in teritary structure for domain II of hemopexin. DSC and CD results show that isolated domain I and domain II interact both in the presence and absence of heme. Moreover, domain II destabilizes heme-domain I, which may be an important factor in facilitating heme release to the hemopexin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Genetics of reproductive isolation in the Drosophila simulans clade: DNA marker-assisted mapping and characterization of a hybrid-male sterility gene, Odysseus (Ods).

In this study, we address the question of whether there exist major genes that cause complete male sterility in the interspecific hybrids of Drosophila and, if they do, how these genes may be characterized at the molecular level. Our approach is to introgress small segments of the X chromosome from Drosophila mauritiana (or Drosophila sechellia) into Drosophila simulans by repeated backcrosses for more than 20 generations. The introgressions are monitored by both visible mutations and a series of DNA markers. We compare the extent of introgressions that cause male sterility with those that do not. If a major sterility factor exists, there should be a sharp boundary between these two classes of introgressions and their breakpoints should demarcate such a gene. Furthermore, if male sterility is the only major fitness effect associated with the introgression, recombination analysis should yield a pattern predicted by the classical three-point cross. Both the genetic and molecular analyses suggest the presence of a major sterility factor from D. mauritiana, which we named Odysseus (Ods), in the cytological interval of 16D. We thus formalize three criteria for inferring the existence of a major gene within an introgression: (1) complete penetrance of sterility, (2) complementarity in recombination analysis, and (3) physical demarcation. Introgressions of Ods from D. sechellia do not cause sterility. Twenty-two introgressions in our collection have breakpoints in this interval of about 500 kb, making it possible to delineate Ods more precisely for molecular identification. The recombination analysis also reveals the complexity of the introgressed segments--even relatively short ones may contain a second male sterility factor and partial viability genes and may also interfere with crossovers. The spermatogenic defects associated with Ods and/or a second factor were characterized by phase-contrast microscopy.

Animals↗

PubMed1993

Population genetics of tandem repeats in centromeric heterochromatin: unequal crossing over and chromosomal divergence at the Responder locus of Drosophila melanogaster.

The Responder (Rsp) locus in Drosophila melanogaster is the target locus of segregation distortion and is known to be comprised of a tandem array of 120-bp repetitive sequences. In this study, we first determined the large scale molecular structure of the Rsp locus, which extends over a region of 600 kb on the standard sensitive (cn bw) chromosome. Within the region, small Rsp repeat arrays are interspersed with non-Rsp sequences and account for 10-20% of the total sequences. We isolated and sequenced 32 Rsp clones from three different chromosomes. The main results are: (1) Rsp repeats isolated from the same chromosome are not more similar than those from different chromosomes. This implies either that there are more homologous exchanges at the Rsp locus than expected or, alternatively, that the second chromosomes of D. melanogaster have diverged from one another more recently at the centromeric heterochromatin than at the nearby euchromatin. (2) The repeats usually have a dimeric structure with an average difference of 16% between the left and right halves. The differences allow us to easily identify the products of unequal exchanges. Despite the large differences between the two halves, exchanges have occurred frequently and the majority of them fall within a 29-bp interval of identity between the two halves. Our data thus support the suggestion that recombination depends on short stretches of complete identity rather than long stretches of general homology. (3) Frequent unequal crossover events obscure the phylogenetic relationships between repeats; therefore, different parts of any single repeat could often have different phylogenetic histories. The high rate of unequal crossing over may also help explain the evolutionary dynamics of the Rsp locus.

Animals↗

PubMed1993

The modulatory effects of endothelin-1, carbachol and isoprenaline upon Na(+)-H+ exchange in dog cardiac Purkinje fibres.

1. The modulatory effects of carbachol, endothelin-1 and isoprenaline upon Na(+)-H+ exchange were examined in dog cardiac Purkinje fibres. Intracellular pH (pHi) and intracellular sodium activity (aiNa) were recorded using pH and Na(+)-selective microelectrodes. Acid extrusion via Na(+)-H+ exchange was estimated from the pHi recovery rate (multiplied by intrinsic buffering power (beta i) and adding mean background acid load) in response to an internal acid load induced by the removal of 20 mM NH4Cl. All experiments in this work were performed in Hepes-buffered solutions at 37 degrees C. 2. beta i was estimated at various values of pHi in the range of 7.4-6.4 and was calculated from the fall of pHi induced by the addition and removal of NH4Cl. Experiments were performed when Na(+)-H+ exchange was blocked. The values of beta i in this tissue were only slightly dependent on pHi in the range of 7.4-6.4 with an empirical relationship: beta i = -4.69 pHi + 64.59. 3. Endothelin-1 (10(-8) M) alkalinized the resting pHi by approximately 0.1 pH unit and accelerated acid extrusion, by approximately 96%, at pHi approximately 6.9. A reduction of background acid loading within the cell cannot account for the augmentation of pHi recovery, since the rate of acid extrusion was not changed either at resting pHi or at internal acidification in Na(+)-free solution (a measure of background loading) by the addition of endothelin-1. The protein kinase C inhibitors staurosporin (10(-6) M) and 1-(5-isoquinolinylsulphonyl)-2-methyl-piperazine (H-7, 50 microM) could not block the effect of endothelin-1 on the antiporter. 4. At pHi approximately 6.8, carbachol (7.5 x 10(-4) M) accelerated pHi recovery by approximately 68% and alkalinized the resting pHi by approximately 0.1 pH unit. This stimulatory effect of carbachol was completely blocked by pretreatment with atropine (10(-4) M) and staurosporine 10(-6) M. The background acid load was not reduced by adding carbachol, since the acid extrusion during pHi recovery or at the resting state was not affected by the addition of carbachol to a sodium-free solution. 5. Isoprenaline (10(-6) M) slowed pHi recovery by approximately 45% measured at pHi 6.9 with no change in resting pHi. A rise in background acid loading could not account for the reduction of acid extrusion. Pretreated with atenolol (10(-6) M), a beta 1-selective antagonist, completely blocked the effect of isoprenaline.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

pH dependence of intrinsic H+ buffering power in the sheep cardiac Purkinje fibre.

1. Intrinsic, intracellular H+ buffering power (beta) was estimated in the isolated sheep cardiac Purkinje fibre at various values of intracellular pH (pHi) in the range 6.2-7.5 and for various values of extracellular pH (pHo) in the range 6.5-8.5. Buffering power was calculated from the fall of pHi (recorded with an intracellular pH-selective microelectrode) induced by addition and removal of extracellular, permeant weak acids and bases (NH4Cl, trimethylamine chloride, sodium propionate). Experiments were performed under conditions nominally free of CO2-HCO3. 2. beta was estimated firstly following acid loads induced by NH4Cl removal (10-20 mM) under conditions where Na(+)-H+ exchange was operational (i.e. in Na(+)-containing Tyrode solution). At constant pHi, the value of beta appeared to double (from a control level of 39.7 mM) as pHo was increased from 7.5 to 8.5. Notably, raising pHo in this range greatly accelerated pHi recovery from an intracellular acid load, indicating stimulation of acid extrusion. It is likely that this stimulation results in an overestimation of beta because it blunts the intracellular acid load. The apparent elevation of beta at high pHo may therefore be an artifact. 3. Estimates of beta were compared (NH4Cl removal) before and after inhibiting Na(+)-H+ exchange in Na(+)-free solution or with amiloride (1 mM). The acid load was larger and in many (but not all) cases the apparent value of beta decreased after inhibition of acid extrusion. This indicates that, if Na(+)-H+ exchange is operational, it can result in an overestimate of beta. In amiloride, beta was 26.6 +/- 1.4 mM (n = 8) at a mean pHi of 6.84 +/- 0.03. 4. Small stepwise reductions of external NH4Cl (from 40 to 0 mM), in the presence of Na(+)-free solution plus 5 mM-BaCl2 at constant pHo, resulted in small stepwise reductions of pHi (approximately 0.1 units). When these were used to calculate beta, we observed that beta increased roughly linearly as pHi became more acid. For a pHi of 7.2, beta approximately 20 mM. 5. An almost identical relationship between beta and pHi was found when using the method of sodium propionate addition (10-50 mM): amiloride (1 mM) was present and pHi was manipulated to various test levels by changing pHo. This confirms that beta varies inversely with pHi and also that it is independent of pHo. We conclude that the apparent variation of beta with pHo observed earlier was indeed an artifact.(ABSTRACT TRUNCATED AT 400 WORDS)

Amiloride↗

PubMed1990

Extracellular H+ inactivation of Na(+)-H+ exchange in the sheep cardiac Purkinje fibre.

1. The inhibition of acid extrusion via Na(+)-H+ exchange caused by reducing pHo (extracellular pH) was examined in the sheep cardiac Purkinje fibre. Intracellular pH (pHi) and intracellular Na+ activity (alpha 1 Na) were recorded using ion-selective microelectrodes. Acid extrusion via Na(+)-H+ exchange was estimated from the pHi recovery rate (multiplied by intracellular buffering power, beta) in response to an internal acid load induced by 20 mM-NH4Cl removal (nominally CO2-HCO3-free media). 2. At a given pHi, acid extrusion decreased sigmoidally with decreases of pHo in the range 8.5 to 6.5 (50% inhibition of efflux occurred at a pHo between 7.0 and 7.5). This inhibition was associated with a parallel decrease in Na+ influx as evidenced from a decrease in the rise of alpha i Na measured during acid extrusion, suggesting inhibition of Na(+)-H+ exchange. 3. The background acid-loading rate (estimated by adding 1 mM-amiloride to inhibit Na(+)-H+ exchange and recording the initial rate of fall of pHi) was found to be unaffected in the steady state by changes of pHo. We therefore conclude that the slowing of pHi recovery at low pHo is due to direct inhibition of Na(+)-H+ exchange rather than to an increase of background acid loading. 4. Reducing pHo (constant pHi) inhibited acid efflux by producing a parallel shift of the efflux versus pHi relationship to lower values of pHi, consistent with a decrease in the apparent internal H+ ion affinity (pKi) of the system. 5. Raising pHi (constant pHo) also inhibited acid efflux, but this was associated with a rise in the pHo required for 50% maximal inhibition of acid efflux (pKo), consistent with an increase in apparent affinity for external H ions. Thus reduction of pHo reduces pKi (point 4) while reduction of pHi reduces pKo (point 5). 6. Inhibition by elevated Ho+ was not linearly related to the decrease in chemical driving force for Na(+)-H+ exchange, nor was it related to a reversal of the transmembrane H+ gradient. We found that efflux still occurred when pHo less than pHi. 7. Efflux was not a unique function of the transmembrane H+ ratio (i.e. pHo-pHi). At appropriate values of pHi and pHo, acid efflux could be kept constant despite a four-fold change in the transmembrane H+ ratio. 8. Inhibition by low pHo was a saturating function of Ho+ ions with a Hill coefficient of 1.2.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Total synthesis of Trichosanthes trypsin inhibitor and its analogue.

Trichosanthes trypsin inhibitor (TTI) is a peptide consisting of 27 amino acid residues with three pairs of disulfide bonds. This paper reports the total synthesis and disulfide bond refolding of this inhibitor and its analogue. After purification, the amino acid sequence and stoichiometrical inhibitory activity against trypsin of the synthetic inhibitor were compatible with those of the natural inhibitor. The analogue of this inhibitor in which residue Met in position 6 was replaced by Ala was also synthesized. The antitrypsin activity of this synthetic analogue was also approximate to that of the natural inhibitor.

Amino Acid Sequence↗

PubMed1989

Sodium-hydrogen exchange and its role in controlling contractility during acidosis in cardiac muscle.

Intracellular pH (pHi) and Na (aina) were recorded in isolated sheep cardiac Purkinje fibres using ion-selective microelectrodes while simultaneously recording twitch tension. A fall of pHi stimulated acid-extrusion via sarcolemmal Na-H exchange but the extrusion was inhibited by reducing extracellular pH (pHo), indicating an inhibitory effect of external H ions upon the exchanger. Intracellular acidosis can reduce contraction by directly reducing myofibrillar Ca2+ sensitivity. The activation of Na-H exchange at low pHi can offset this direct inhibitory effect of H+ ions since exchange-activation elevates aina which then indirectly elevates Ca2+i (via Na-Ca exchange) thus tending to restore tension. This protection of contraction during intracellular acidosis can be removed if extracellular pH is also allowed to fall since, under these conditions, Na-H exchange is inhibited.

Acidosis↗

PubMed1989

Epithelial ovarian cancer and the ability to conceive.

Relationships between ovarian cancer and ability to conceive were explored in a case-control study of 188 women with histologically confirmed epithelial ovarian cancer and 539 control women in the San Francisco Bay Area. Control women consisted of two groups: those hospitalized without cancer, matched to cases by age, race, and hospital of diagnosis (n = 280); and those selected from the general population by random digital dialing, matched to cases by age, race, and telephone prefix (n = 259). Ovarian cancer risk among nulliparous (but not parous) women was positively associated with a history of unsuccessful attempts to conceive, of physician-diagnosed infertility, and of doubts about ability to conceive. Among all women, risk increased with increasing years of unprotected intercourse (P value for trend = 0.02). Risk among women having 10 or more yr of unprotected intercourse was 1.8 relative to that among women having less than 2 such yr (P = 0.01). This association was independent of parity, oral contraceptive use, and estimated years of ovulation, each associated with ovarian cancer. Further, duration of unprotected intercourse combined multiplicatively with each of these latter characteristics in increasing ovarian cancer risk. For example, while cancer risk exhibited a 2-fold range from lowest to highest years of unprotected intercourse and a 4-fold range from lowest to highest years of ovulation, risk among women in the highest joint category of these characteristics was 8 times that of women in the lowest category. We believe that some abnormality of ovulation that reduces the likelihood of conception plays a role in epithelial ovarian cancer.

Adult↗