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Biomedical subjects

M M White

Publications and source records attributed to M M White.

At least 37 records · Page 2Linked to original sources
PubMed1998

Has the California tobacco control program reduced smoking?

CONTEXT: Comprehensive community-wide tobacco control programs are considered appropriate public health approaches to reduce population smoking prevalence. OBJECTIVE: To examine trends in smoking behavior before, during, and after the California Tobacco Control Program. DESIGN: Per capita cigarette consumption data (1983-1997) were derived from tobacco industry sales figures. Adult (> or =18 years) smoking prevalence data were obtained from the National Health Interview Surveys (1978-1994), the California Tobacco Surveys (1990-1996), the Current Population Surveys (1992-1996), and the California Behavioral Risk Factor Survey and its supplement (1991-1997). Trends were compared before and after introduction of the program, with the period after the program being divided into 2 parts (early, 1989-1993; late, 1994-1996). MAIN OUTCOME MEASURES: Change in cigarette consumption and smoking prevalence in California compared with the rest of the United States. RESULTS: Per capita cigarette consumption declined 52% faster in California in the early period than previously (from 9.7 packs per person per month at the beginning of the program to 6.5 packs per person per month in 1993), and the decline was significantly greater in California than in the rest of the United States (P<.001). In the late period, the decline in California slowed to 28% of the early program so that in 1996 an average of 6.0 packs per person per month were consumed. No decline occurred in the rest of the United States, and in 1996, 10.5 packs per person per month were consumed. Smoking prevalence showed a similar pattern, but in the late period, there was no significant decline in prevalence in either California or the rest of the United States. In 1996, smoking prevalence was 18.0% in California and 22.4% in the rest of the United States. CONCLUSIONS: The initial effect of the program to reduce smoking in California did not persist. Possible reasons include reduced program funding, increased tobacco industry expenditures for advertising and promotion, and industry pricing and political activities. The question remains how the public health community can modify the program to regain its original momentum.

California↗
PubMed1998

A single intravenous dose of murine megakaryocyte growth and development factor potently stimulates platelet production, challenging the necessity for daily administration.

The thrombopoietic efficacy of recombinant forms of c-mpl ligand is being actively investigated in preclinical studies using daily dosing schedules. However, a comprehensive kinetic study of the thrombopoietic response to a single injection of recombinant c-mpl ligand has not been performed. Here, we present the results of a detailed kinetic analysis of the platelet response to a single intravenous administration of pegylated recombinant murine megakaryocyte growth and development factor (PEG-rmMGDF) in mice. In addition, we compare the efficacy of single versus daily dosing in stimulating platelet production. A single intravenous injection of PEG-rmMGDF produced a marked and dose-dependent elevation in platelet number and a moderate increase in mean platelet volume (MPV). After administration of 25 or 250 micrograms/kg of PEG-rmMGDF, platelet number was first increased on day 3 and peaked at 2.7-fold (25 micrograms/kg) and 5.7-fold of normal (250 micrograms/kg) on day 5. Thereafter, platelet number declined and returned to baseline by days 9 and 14, with the 25 and 250 micrograms/kg doses, respectively. MPV began to increase on day 2 after PEG-rmMGDF, reaching maximum values of 1.2-fold (25 micrograms/kg) and 1.5-fold of normal (250 micrograms/kg) on day 4. Subsequently, MPV declined and was downregulated on days 6 to 7 (25 micrograms/kg) and day 8 (250 micrograms/kg). Based on these results, we evaluated the platelet response to PEG-rmMGDF administered intravenously as a single dose versus daily for 5 days. A single administration of 100 micrograms/kg produced a higher platelet number on day 5 than daily administration of 100 or 20 micrograms/kg for 5 days. However, the thrombocytosis was less sustained after single versus daily dosing. The smaller platelet number increase on day 5 after daily dosing reflected the production of larger platelets, rather than suppression of thrombopoiesis. Our results indicate that PEG-rmMGDF administered as a single intravenous dose potently stimulates platelet production in mice, challenging the need for its daily administration. Adoption of an intermittent administration schedule of this cytokine could be more efficacious and is merited in future clinical trials.

Animals↗
PubMed1998

Expression of ligand-induced binding sites on glycoprotein IIb/IIIa complexes and the effect of various inhibitors.

Glycoprotein (GP) IIb/IIIa is a therapeutic target for the blockade of platelet aggregation during acute coronary syndromes. Peptides, peptidomimetics, and antibodies are generated that competitively block the binding of fibrinogen or von Willebrand factor to the activated GPIIb/IIIa complex. Binding of these receptor blockades to GPIIb/IIIa effectively inhibits the formation of the platelet aggregate because ligand binding to the activated GPIIb/IIIa is the final common pathway to thrombus formation. In addition, bound antagonists induce a conformational change in the receptor. This conformational change, also called a ligand-induced binding site, can be used as a marker for receptor occupancy by GPIIb/IIIa receptor blockades. Using the binding properties of the D3 monoclonal antibody that binds with high affinity to the antagonist bound GPIIb/IIIa, we have developed a new method for monitoring the extent of receptor blockade by GPIIb/IIIa antagonists. This method has specific advantages over the interpatient variability of the aggregation assay and provides a method for the evaluation of appropriate target levels of GPIIb/IIIa blockade.

Antibodies, Monoclonal↗
PubMed1998

Interaction of D-tubocurarine analogs with the 5HT3 receptor.

D-Tubocurarine is a potent competitive antagonist of two members of the ligand-gated ion channel family, the muscle-type nicotinic acetylcholine receptor (AChR) and serotonin type-3 receptor (5HT3R). We have used a series of analogs of D-tubocurarine to determine the effects of methylation, stereoisomerization and halogenation on the interaction of D-tubocurarine with the 5HT3R. The affinities of the analogs for the 5HT3R span a 200-fold concentration range and fall into three broad groups. The first group, with affinity constants (Ki) < 150 nM, consists of D-tubocurarine and analogs modified at the nitrogens or 7' hydroxyl. The fact that these compounds all have high affinity for the 5HT3R suggests that these portions of the ligand do not make interactions with the receptor that are critical for high-affinity binding. The second group, with Ki's in the 1-5 microM range, consists of analogs modified at the 12'-hydroxyl or the adjacent 13'-carbon, which suggests that this portion of the ligand makes interactions that are important for high-affinity binding. The third, very low affinity, group is a compound with altered stereoconfiguration at the 1 carbon, demonstrating the importance of proper configuration of the antagonist in ligand-receptor interactions. For the most part, this pattern of selectivity is similar to that for the AChR, suggesting that the structures of the ligand-binding sites of these two receptors share common structural features.

Animals↗
PubMed1998

Effects of pregnancy and chronic hypoxia on contractile responsiveness to alpha1-adrenergic stimulation.

Decreased contractile response to vasoconstrictors in uterine and nonuterine vessels contributes to increased blood flow to the uterine circulation during normal pregnancy. Pregnancies complicated by preeclampsia and/or chronic hypoxia show a reversal or diminution of these pregnancy-associated changes. We sought to determine whether chronic hypoxia opposes the reduction in contractile response in uterine and nonuterine vessels during normal pregnancy and, if so, whether decreased basal nitric oxide (NO) activity was involved. We examined the contractile response to phenylephrine (PE) in guinea pig uterine artery (UA), mesenteric artery (MA), and thoracic aorta (TA) rings isolated from nonpregnant or pregnant guinea pigs that had been exposed throughout gestation to either low (1,600 m, n = 47) or high (3,962 m, n = 43) altitude. In the UA, pregnancy reduced contractile sensitivity to PE and did so similarly at low and high altitude (EC50: 4.0 x 10(-8) nonpregnant, 9.3 x 10(-8) pregnant at low altitude; 4.8 x 10(-8) nonpregnant, 1.0 x10(-8) pregnant at high altitude; both P < 0.05). Addition of the NO synthase inhibitor nitro-L-arginine (NLA; 200 mM) to the vessel bath increased contractile sensitivity in the pregnant UA (P < 0.05) and eliminated the effect of pregnancy at both altitutes. NLA also raised contractile sensitivity in the nonpregnant high-altitude UA, but contractile response without NLA did not differ in the high- and low-altitude animals. In the MA, pregnancy decreased contractile sensitivity to PE at high altitude only, and this shift was reversed by NO inhibition. In the TA, neither pregnancy nor altitude affected contractile response, but NO inhibition raised contractile response in nonpregnant and pregnant TA at both altitudes. We concluded that pregnancy diminished contractile response to PE in the UA, likely as a result of increased NO activity, and that these changes were similar at low and high altitude. Counter to our hypothesis, chronic hypoxia did not diminish the pregnancy-associated reduction in contractile sensitivity to PE or inhibit basal NO activity in the UA; rather it enhanced, not diminished, basal NO activity in the nonpregnant UA and the pregnant MA.

Adrenergic alpha-Agonists↗
PubMed1998

A Ser162-->Leu mutation within glycoprotein (GP) IIIa (integrin beta3) results in an unstable alphaIIbbeta3 complex that retains partial function in a novel form of type II Glanzmann thrombasthenia.

Platelets from Glanzmann thrombasthenia patient BL express approximately 30% of the normal alphaIIbbeta3 content and support fibrin-mediated clot retraction, but fail to bind fibrinogen or aggregate following cellular activation. BL platelets bind neither activation-dependent nor activation-independent ligands. DNA sequence analysis of BL platelet mRNA revealed a homozygous C583-->T point mutation in a conserved region of beta3, resulting in a Ser162Leu amino acid substitution. This mutation appears to produce destabilizing effects on the alphaIIbbeta3 complex, as evidenced by the fact that (1) the BL alphaIIbbeta3 complex exhibited altered sedimentation velocity through sucrose gradients, (2) alphaIIb and beta3 was not recognized by complex-dependent monoclonal antibodies or co-precipitated by integrin subunit-specific antibodies, and (3) biosynthesis and trafficking of the alphaIIbbeta3Leu162 complex was delayed relative to that of the wild-type control. Taken together, these data implicate the region encompassing Ser162 in the stabilization and ligand binding properties of the alphaIIbbeta3 complex.

Amino Acid Sequence↗
PubMed1997

Effect of Ca2+ on GP IIb-IIIa interactions with integrilin: enhanced GP IIb-IIIa binding and inhibition of platelet aggregation by reductions in the concentration of ionized calcium in plasma anticoagulated with citrate.

BACKGROUND: Integrilin (eptifibatide), a potent inhibitor of the fibrinogen binding function of GP IIb-llla, has been shown to reduce the thrombotic complications of angioplasty and of acute coronary syndromes. The present study was designed to determine whether the reduced Ca2+ concentrations in plasma anticoagulated with citrate affect Integrilin binding to GP IIb-IIIa and the ex vivo pharmacodynamic measurements for this drug. METHODS AND RESULTS: Lower concentrations of Integrilin were found to inhibit platelet aggregation in plasma anticoagulated with citrate (for ADP, mean+/-SD IC(50)=140+/-40 nmol/L, n=6; Ca2+ =40 to 50 micromol/L) than with PPACK (IC(50)=570+/-70 nmol/L, P<.0001, n=6; Ca2+ approximately 1 mmol/L). Chelation of Ca2+ with EDTA or citrate caused a similar degree of enhancement in the inhibitory activity of Integrilin. Measurements of D3 LIBS epitope expression showed that the enhanced inhibitory activity was caused by enhanced GP IIb-IIIa occupancy by Integrilin. Citrate anticoagulation decreased the amounts of Integrilin required to inhibit the binding of PAC1, a monoclonal antibody that mimics the GP IIb-IIIa binding activity of fibrinogen. Reduced Ca2+ also increased Integrilin inhibition of the binding of biotinylated fibrinogen to purified, immobilized GP IIb-IIIa. CONCLUSIONS: These data suggest that citrate anticoagulation removes Ca2+ from GP IIb-IIIa and enhances the apparent inhibitory activity of Integrilin. This finding indicates that the inhibitory activity of Integrilin is overestimated in blood samples collected with citrate, suggesting that it may be possible to achieve greater antithrombotic efficacy beyond that observed in clinical trials to date with Integrilin.

Antibodies, Monoclonal↗
PubMed1996

Selective induction of a glycoprotein IIIa ligand-induced binding site by fibrinogen and von Willebrand factor.

Ligand-induced binding sites (LIBS) are neoantigenic regions of glycoprotein (GP)IIb-IIIa that are exposed upon interaction of the receptor with the ligand fibrinogen or the ligand recognition sequence (RGDS). LIBS have been suggested to contribute to postreceptor occupancy events such as full-scale platelet aggregation, adhesion to collagen, and clot retraction. This study examined the induction requirements of a GPIIIa LIBS with regard to ligand specificity. Through the use of the anti-LIBS D3, we report that this complex-activating antibody induces fibrinogen- and von Willebrand factor-binding to GPIIb-IIIa on intact platelets. Bound ligand was detected by flow cytometric analysis and platelet aggregation assays. These bound ligands increased the number of D3-binding sites and altered the affinity of D3 for GPIIb-IIIa on platelets. In contrast, activation of platelet GPIIb-IIIa by D3 did not increase the binding of another RGD-containing ligand, vitronectin. Furthermore, bound vitronectin on thrombin-stimulated platelets did not cause the expression of the D3 LIBS epitope. We conclude direct activation of GPIIb-IIIa in the absence of platelet activation results in selective ligand interaction and that D3 LIBS induction requires the binding of the multivalent ligands, fibrinogen or von Willebrand factor. Thus, the region of GPIIIa recognized by D3 may be an important regulatory domain in ligand-receptor interactions that directly mediate platelet aggregation.

Animals↗
PubMed1996

cAMP promotes gap junctional coupling in T84 cells.

The effect of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on dye and electrical coupling was studied in T84 cells, a cell line often used as a model for epithelial cell fluid secretion. Injections of lucifer yellow (LY) into single cells within a cluster of control cells resulted in LY localization to 1.3 +/- 0.2 (mean +/- SE) cells within a cluster. Twenty-six percent of control T84 cell pairs were electrically coupled as assayed by the dual patch-clamp technique. Treatment of cells with agents that either increase intracellular cAMP and/or activate protein kinase A (PKA) increased dye localization to 3.8 +/- 0.6 cells and the proportion of electrically coupled cell pairs to 65%. No electrical coupling was observed in the presence of the Rp diastereomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), a PKA antagonist. Excess of Rp-cAMPS prevented cell coupling elicited by 20 microM of the Sp diastereomer of adenosine-3',5'-cyclic monophosphothioate. Expression of connexin 32 mRNA, but not of connexins 26, 43, or 45, was detected by reverse transcription-polymerase chain reaction. These results suggest that communication between T84 cells is modulated by PKA, providing a mechanism for regulating multicellular activity, such as fluid secretion.

Cell Line↗
PubMed1995

A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents.

Diagnosis of platelet dense granule storage pool disease and release defects at present requires a combination of studies including lumiaggregometry, conventional platelet aggregation, radioactive serotonin uptake and release, and electron microscopy. Flow cytometric methods have been developed to study platelet activation, aggregation, and alpha-granule protein release. Here, we have investigated the use of flow cytometry for analysis of platelet dense granule content uptake and release using mepacrine as a fluorescent marker. Mepacrine (quinacrine) is rapidly taken up and localized in dense granules of platelets. For the assay, as little as 20 microliters of blood from a fingerstick collected without anticoagulant or venous blood collected in 3.8% sodium citrate were diluted 1:40 with 2 ml Hanks balanced salt solution (BSS). 300 microliters of this cell suspension were incubated with mepacrine alone, or simultaneously with a mouse monoclonal antibody to human platelet glycoprotein IIb (Tab), used as a platelet-specific marker. The bound monoclonal antibody was then indirectly labelled with the fluorochrome, RED670. 100 microliters of the sample were further diluted with Hanks BSS for one- or two-colour flow cytometric analysis. To verify that mepacrine uptake was related to platelet dense granule content, platelets of beige mice, a strain with dense granule deficiency, were examined. Their mepacrine uptake was substantially decreased compared to that of normal mice. Decreased mepacrine uptake also was demonstrated in platelets of a patient with Hermansky-Pudlak syndrome in which a deficiency of platelet dense granules is characteristic. In both human and mouse platelets, mepacrine uptake was proportional to platelet size. Thrombin induced mepacrine release in a dose-dependent manner from 0.003 to 0.4 U/ml. Therefore both platelet uptake and release of mepacrine can be readily detected by flow cytometry. Flow cytometry provides an attractive alternative to aggregation and radioactive serotonin as methods to study defects in platelet dense granule function.

Animals↗
PubMed1995

The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating.

A highly conserved leucine is found in the middle of the porelining (M2) domain of the members of the ligand-gated ion channel family. Two very different roles have been proposed for this leucine. In one model, this residue swings into the lumen of the channel during desensitization to form the nonconducting desensitized state, whereas in the other model, the leucines from each subunit interact with each other to form a constriction in the channel that constitutes the actual gate of the channel. We examined the role of this leucine in the muscle-type acetylcholine receptor by replacing it with the polar amino acid threonine. Replacement of the leucine in any one subunit slows desensitization and shifts the dose-response relationship toward lower concentrations. Replacement of leucines in additional subunits leads to progressively larger shifts in the dose-response curves. The shift depends only on the number of leucines replaced, not on which particular subunits contain the mutation; in other words, the mutations act independently. At the single-channel level, the mutation greatly increases the channel mean open time. We conclude that the role of the conserved leucine is to set the mean open time of the channel through interactions with other regions of the receptor rather than to serve as the gate per se of the ion channel.

Animals↗
PubMed1995

Interspecies comparison of platelet aggregation, LIBS expression and clot retraction: observed differences in GPIIb-IIIa functional activity.

We examined interspecies differences in the function of the platelet fibrinogen receptor, GPIIb-IIIa, by comparing platelet aggregation responses to adenosine diphosphate (ADP) added alone or in combination with a GPIIIa specific monoclonal antibody (mAb), D3. D3 can activate the GPIIb-IIIa receptor in the absence of platelet activation, and it preferentially binds to a region on the GPIIIa subunit after the GPIIb-IIIa complex is occupied by ligand. Using human, monkey, dog, rabbit and pig platelets, we examined whether all species' platelets bound the D3 mAb similarly, and if the binding of Arg-Gly-Asp-Ser (RGDS) peptides induced the exposure of the anti-LIBS (D3) epitope as previously described for human platelets. We also evaluated how blocking of this neoantigenic region by the D3 mAb affected clot retraction, a process that requires linkage of GPIIb-IIIa with fibrin(ogen) and the platelet cytoskeleton. We found that all species tested bound the D3 mAb. Only in human and monkey platelets did D3 cause aggregation as well as inhibit clot retraction. However, in all species tested, except for pig, D3 prevented disaggregation of platelets typically observed when platelets are treated with low dose ADP. With the exception of pig platelets, there was increased D3 binding to platelets in the presence of RGDS peptides. We propose that this region of GPIIIa is important in the conformational changes that GPIIb-IIIa undergoes during the binding of ligand in most species tested. Our studies suggest 1) there are measurable inter-species differences in GPIIb-IIIa mediated platelet aggregation and clot retraction, 2) LIBS expression due to receptor occupancy is a common but not all-inclusive response and 3) interspecies comparisons may be useful in understanding structural and functional aspects of platelet GPIIb-IIIa.

Adenosine Diphosphate↗
PubMed1994

Ligand-receptor interactions in the nicotinic acetylcholine receptor probed using multiple substitutions at conserved tyrosines on the alpha subunit.

Affinity labeling studies have identified several conserved tyrosine residues in the alpha subunit of the nicotinic acetylcholine receptor (alpha Y93, alpha Y190, and alpha Y198) as being in or near the ligand binding site. Mutagenesis studies from several laboratories have shown that substitution of phenylalanine for tyrosine at these positions reduces the apparent affinity for ACh. We have examined this apparent reduction in affinity further through the use of multiple substitutions at each position. Substitution of either phenylalanine, tryptophan, or serine resulted in an apparent decrease in agonist affinity, but the degree of reduction depended on both the position and the nature of the substitution. Analysis of the effects of each substitution suggests that each residue interacts with the quaternary N of ACh, and that each residue may make a different type of interaction with the agonist.

Acetylcholine↗
PubMed1994

Characterization of d-tubocurarine binding site of Torpedo acetylcholine receptor.

d-Tubocurarine (curare) is a well-characterized competitive antagonist of nicotinic acetylcholine receptors (AChRs), and it is usually assumed that curare and agonists share a common binding site. We have examined the role of several highly conserved residues of the alpha-, gamma-, and delta-subunits in the interaction of curare with the Torpedo acetylcholine receptor (AChR). Curare inhibition of wild-type receptors is consistent with curare binding to a single high-affinity binding site [inhibitor constant (Ki) = 20 nM]. Phenylalanine substitutions for two tyrosine residues implicated as being in the ligand binding site (alpha Y93F, alpha Y190F) reduce curare affinity, indicating that these residues are also important for high-affinity curare binding. Phenylalanine substitution for alpha Y198 [alpha Y198F (notation used here: subunit/amino acid in wild-type/residue number/substitution)] causes a 10-fold increase in curare affinity (Ki = 3.1 nM), and measurement of the recovery from curare inhibition indicates that this increase in affinity is due to a reduction in the rate of curare dissociation from the receptor. In addition to the alpha-subunits, portions of the ligand binding sites also reside on the gamma- and delta-subunits, and photoaffinity studies have implicated two residues (gamma W55 and delta W57) as forming part of the curare sites. The gamma W55L mutation results in an eightfold decrease in curare affinity (Ki = 170 nM), whereas the delta W57L mutation has no effect. These data support the notion that the high-affinity curare binding site is formed by segments of the alpha- and gamma-subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Gating properties of mutant acetylcholine receptors.

A number of affinity labeling studies have identified several tyrosine residues in the alpha subunit of the nicotinic acetylcholine receptor as being in or near the ligand binding site. Studies employing site-directed mutagenesis of these residues (alpha Y93, alpha Y190, and alpha Y198; the notation used is subunit/amino acid/position in the Torpedo receptor/substitution) in mouse muscle, Torpedo electroplax, and alpha 7 neuronal acetylcholine receptors have demonstrated that substitution of phenylalanine for tyrosine results in a shift towards higher concentrations in the macroscopic dose-response curves for acetylcholine-elicited currents from voltage-clamped Xenopus oocytes that express the receptors. This decrease in apparent affinity has been ascribed to either a reduction in binding affinity or a reduction in the coupling of agonist binding to ion channel opening; both mechanisms would give rise to shifts in the dose-response curves. We have used kinetic analysis of ion channel gating at the single-channel level to obtain estimates for the rate constants associated with the ligand binding and channel opening steps for wild-type, alpha Y93F, and alpha Y198F receptors. The results suggest that the underlying cause of the shifts in the macroscopic dose-response curves is a reduction in acetylcholine affinity for the resting activatable state of the receptor. Furthermore, it is the association rate for agonist binding, rather than the dissociation rate, that is most affected by the mutations.

Animals↗