Search PubMed⌕ Search

Biomedical subjects

S A Lewis

Publications and source records attributed to S A Lewis.

At least 91 records · Page 5Linked to original sources
PubMed1994

A novel cochaperonin that modulates the ATPase activity of cytoplasmic chaperonin.

The folding of alpha- and beta-tubulin requires three proteins: the heteromeric TCP-1-containing cytoplasmic chaperonin and two additional protein cofactors (A and B). We show that these cofactors participate in the folding process and do not merely trigger release, since in the presence of Mg-ATP alone, alpha- and beta-tubulin target proteins are discharged from cytoplasmic chaperonin in a nonnative form. Like the prokaryotic cochaperonin GroES, which interacts with the prototypical Escherichia coli chaperonin GroEL and regulates its ATPase activity, cofactor A modulates the ATPase activity of its cognate chaperonin. However, the sequence of cofactor A derived from a cloned cDNA defines a 13-kD polypeptide with no significant homology to other known proteins. Moreover, while GroES functions as a heptameric ring, cofactor A behaves as a dimer. Thus, cofactor A is a novel cochaperonin that is structurally unrelated to GroES.

Adenosine Triphosphatases↗
PubMed1994

Anti-inflammatory and bronchodilator properties of RP 73401, a novel and selective phosphodiesterase type IV inhibitor.

1. We have investigated the effects of RP 73401, a novel, potent and highly selective cyclic nucleotide phosphodiesterase (PDE) type IV inhibitor, in guinea-pig and rat models of bronchoconstriction and allergic inflammation. In some models, the effects of RP 73401 have been compared with those of the standard PDE type IV inhibitor, rolipram. 2. RP 73401 (0.4-400 micrograms kg-1, intratracheally (i.t.) on lactose) inhibited antigen-induced bronchospasm in previously sensitized conscious guinea-pigs (ID50: 7 +/- 1 micrograms kg-1) and in anaesthetized rats (ID50: 100 +/- 25 micrograms kg-1). Rolipram inhibited the antigen-induced bronchospasm in guinea-pigs with an ID50 of 5 +/- 1 micrograms kg-1. In guinea-pig bronchoalveolar lavage (BAL) fluid, total inflammatory cell and eosinophil numbers were reduced by RP 73401 (ID50s: 3.9 +/- 0.8 micrograms kg-1 and 3.2 +/- 0.7 micrograms kg-1, respectively). In the rat, inflammatory cell numbers are less affected. Only the highest dose of RP 73401 (400 micrograms kg-1) significantly inhibited eosinophil influx (41 +/- 16% inhibition). 3. RP 73401 (0.02-100 micrograms kg-1, i.v.) inhibited PAF-induced bronchial hyperreactivity to bombesin in the anaesthetized guinea-pig (ID50: 0.09 +/- 0.03 micrograms kg-1) and inhibited (0.4-40 micrograms kg-1, i.t.) histamine-induced airway microvascular leakage in the anaesthetized guinea-pig by approximately 60% at all doses. 4. RP 73401 relaxed guinea-pig isolated trachea under basal tone (EC50: 9 nM) and when precontracted with histamine (IC50: 2 nM), methacholine (IC50: 29 nM) or leukotriene D4 (LTD4, IC50: 4 nM). 5. RP 73401 (0.4-100 microg kg-1, i.t.) inhibited bronchospasm induced by histamine (ID.%: 34 +/- 6 microg kg-1), methacholine (ID50: 66 +/- 12 pg kg-1) and LTD4 (ID50: <4 microg kg-1) in the anaesthetized guinea pig.Against these same bronchoconstrictors, rolipram (i.t.) had ID5o values of 44 +/- 4, 72 +/- 18 and<4 pg kg- respectively. RP 73401 (4 and 40 pg kg-, i.t.) increased the magnitude and duration of bronchodilatation produced by salbutamol in the anaesthetized guinea-pig. At doses producing significant bronchodilatation, RP 73401 was without effect on heart rate or blood pressure in the anaesthetized guinea-pig. RP 73401 (0.01 -0.25 mg kg-1, i.v.) did not affect heart rate and produced only a small fall in blood pressure in the anaesthetized rat.6. These data demonstrate that RP 73401 and rolipram inhibit antigen- and mediator-induced bronchospasmin guinea-pigs with the same potency. Furthermore, RP 73401 administered directly into the airways, protects against allergic airway inflammation. These results indicate the importance of PDE IV in regulating smooth muscle and inflammatory cell activity. At doses suppressing the inflammatory response in the lung, RP 73401 had little effect in the cardiovascular system. RP 73401 may have a role as a bronchodilator and, more importantly, as a prophylactic anti-inflammatory agent in the treatment of asthma.

Albuterol↗
PubMed1994

Mammalian urinary bladder permeability is altered by cationic proteins: modulation by divalent cations.

It was previously demonstrated that protamine sulfate (PS, a cationic polypeptide) as well as synthetic cationic polypeptides (CpP, e.g., polylysine and polyarginine) caused an increase in the apical membrane conductance of the mammalian urinary bladder epithelium that was voltage dependent. The membrane conductance induced by these CpP was mediated by a saturable binding site and was partially blocked by CpP (self-inhibition). The PS-induced membrane conductance can be modified by polyvalent cations at three sites. The first site was to competitively inhibit the interaction of PS with an apical membrane binding site. The second site was to reversibly block the conductance induced by PS. The relative binding affinity (block of PS-induced conductance) sequence was as follows: UO2(2+) > La3+ > Mn2+ > Ba2+ > or = Ca2+ > Sr2+. Although La3+, Mn2+, Ba2+, Ca2+, and Sr2+ inhibited > or = 81% of the PS-induced conductance, UO2(2+) inhibited only 51% and Mg2+ was without effect. The third site was to increase the rate of loss of the PS-induced conductance from the apical membrane. Although neither carbodiimides (carboxyl group reactive reagents) nor neuraminidase (cleaves sialic acid residues) altered the effect of PS on the urinary bladder conductance, PS increased the conductance of lipid bilayers composed of negatively charged phospholipids. A candidate for the binding site might be the negatively charged phosphate groups of membrane lipids.

Animals↗
PubMed1994

Role of intracellular Ca2+ in modulation of tight junction resistance in A6 cells.

The role of intracellular Ca2+ in the development and maintenance of epithelial tight junctional integrity is poorly understood. We assessed tight junctional resistance (Rj) in confluent monolayers of A6 cells that were treated with mucosal amiloride such that the transepithelial resistance (Rt) reflects Rj. Solution Ca2+ concentration [Ca2+] was reduced by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) addition to the bathing solutions. Reduction of mucosal [Ca2+] to 1 microM or reduction of serosal Ca2+ to 100 microM did not significantly alter Rt. However, a further decrease of serosal Ca2+ to 40 microM caused the resistance to fall to < 12% of the control value. Following restoration of serosal [Ca2+], Rt increased to a new steady-state value within approximately 15 min. The magnitude of recovery of Rt was inversely correlated with the length of time the epithelium was exposed to low serosal [Ca2+]. To further test the effects of asymmetric Ca2+ removal, the serosal [Ca2+] was chelated using EGTA to reduce Rt. When the Ca2+ ionophore A-23187 was subsequently added to the mucosal solution, Rt increased from 20% to 60% of the control level. In addition, cells were loaded with the fluorescent Ca2+ indicator, Calcium Green, and the temporal relationship between changes in Rt and intracellular Ca2+ was determined. Following removal of serosal Ca2+, cell Ca2+ decreased, followed by a decrease in Rt. In contrast, returning Ca2+ to the serosal bathing solution resulted in a parallel increase of both Rt and cell [Ca2+]. These data strongly suggest that changes in intracellular [Ca2+] play an important role in the regulation of Rj.

Amiloride↗
PubMed1993

The mechanism of equilibrium binding of microtubule-associated protein 2 to microtubules. Binding is a multi-phasic process and exhibits positive cooperativity.

The mechanism of binding of microtubule-associated protein 2 (MAP2) to taxol-stabilized microtubules (MTs) was examined through Scatchard analysis of equilibrium binding and by immunoelectron microscopy. We demonstrate the following. 1) Binding is a cooperative process as indicated by sigmoidal binding curves, prominent humps in Scatchard plots, and an all-or-none response in binding during ligand titrations. At high tubulin/MAP2 ratios, the Kd for noncontiguous binding (5-25 microM) is estimated to be 100-1500 times greater than that predicted for contiguous binding, suggesting a high degree of cooperativity. 2) Cooperativity is indicated independently by a highly clustered or patchy distribution of MAP2 on MTs as revealed by immunoelectron microscopy. 3) The binding of truncated constructs of mouse MAP2 protein suggests that a domain of MAP2 conferring cooperativity is located in or near the MT binding site near the carboxyl terminus. We speculate that in the cell, cooperativity may generate MTs with uniform biochemical properties and contribute to the segregation of MAPs in neuronal cell processes.

Animals↗
PubMed1993

Effect of protamine sulfate on the permeability properties of the mammalian urinary bladder.

Protamine sulfate (PS, an arginine-rich protein of molecular weight 5,000) has been reported to affect the ionic permeability of gallbladder epithelium, the permeability of cultured epithelial cells to mannitol, and the permeability of endothelial cell layers to albumin. Although the effect of PS has been widely investigated, the mechanism of its action on membrane permeability is presently unknown. The effect of PS on the rabbit urinary bladder epithelium was studied using both transepithelial and intracellular microelectrode techniques in conjunction with equivalent circuit analysis. The addition of 100 micrograms/ml of PS to a NaCl-containing mucosal solution caused (over a 40-min period) a large increase in the transepithelial conductance (Gt) and a transient hyperpolarization of the transepithelial voltage (Vt) followed by a depolarization of Vt. This secondary depolarization of Vt was not present if the mucosal solution was a KCl or a K-gluconate Ringer. The PS-induced increase in Gt was due to an increase in the apical membrane permeability to both cations (Na+ or K+) and anions (Cl- or gluconate). Further studies revealed the following features of the PS-induced conductance. (i) Trypsin inhibits the PS effect; however, this was due to PS hydrolysis by trypsin and not a membrane effect. (ii) Mucosal PS partially inhibited the PS-induced apical membrane conductance. (iii) The ability of PS to increase the membrane conductance was enhanced when the apical membrane potential was cell interior negative. (iv) The rate of conductance change (at any given membrane potential) was a saturating function of the PS concentration. This finding suggests that PS must interact with a membrane binding site before it can induce a change in the membrane conductance. (v) Lanthanum inhibited the PS-dependent conductance by two different mechanisms. One was as a reversible blocker of the PS-induced conductance. The other was by inhibiting the interaction between PS and a membrane binding site. A kinetic model is developed to describe the steps involved in the increase in membrane conductance.

Animals↗
PubMed1993

Protection of swine against foot-and-mouth disease with viral capsid proteins expressed in heterologous systems.

Three groups of swine were each inoculated with a different antigen preparation of foot-and-mouth disease virus (FMDV) capsid proteins and challenged by contact exposure to animals infected with FMDV. One group of four animals was inoculated with an extract from cells infected with a recombinant baculovirus containing the FMDV P1-2A structural protein precursor gene and a portion of the P2 gene. Two out of four animals were protected from clinical disease, but not from virus replication. A second group of animals was inoculated with an extract from Escherichia coli that expressed FMDV proteins from a construct containing the P1-2A gene, a portion of the P2 gene and the 3C protease gene. Three out of four animals in this group did not develop clinical signs of FMD upon challenge and two of four were protected against virus replication. In contrast, inoculation of a third group of swine with an extract from E. coli expressing the same FMDV construct as present in the recombinant baculovirus failed to protect any of the four animals from generalized FMD.

Animals↗
PubMed1993

Mercury levels in eggs, tissues, and feathers of herring gulls Larus argentatus from the German Wadden Sea Coast.

The relationships between mercury levels in eggs, tissues, and feathers of male and female herring gulls caught at their nests at a colony on the German Wadden Sea Coast were investigated, and an assessment of mercury intake and excretion of these birds was made. Samples of the liver, ovary, pectoral muscle, and body feathers, as well as the primary feather (in some cases), and eggs were taken from 37 adult herring gulls. Analysis of total mercury in all samples showed that body-feather and tissue levels were independent of sex. There was, however, a significant difference between levels of mercury in the primary feathers of male and female birds. Egg levels were not correlated to pectoral muscle, ovary, or feather levels although they were positively correlated with mercury levels in the liver. Liver levels were in turn correlated with mercury levels in the ovary. The ratio of mean feather to mean egg concentration ranged from 3.7 to 5.5 according to which feather was used. It was estimated that herring gulls from the Wadden coast ingested between 825 and 1337 microg of mercury in the year prior to analysis. It was also estimated that female birds may excrete over 20% more mercury via their eggs than could be excreted by male birds.

Journal Article↗
PubMed1993

Modification of epithelial permeability by cationic polypeptides.

It has been demonstrated that protamine sulfate (PS; a cationic polypeptide composed of 70% arginine) increases the apical membrane conductance of the mammalian urinary bladder. In this report, synthetic cationic polypeptides (CpP; e.g., polyarginine) were used to determine whether the response of the bladder to PS was due to its cationic nature (i.e., its arginine content). We demonstrate that CpP induce a large increase in the cation and anion conductance of the apical membrane of the rabbit urinary bladder epithelium. The modulation of the membrane conductance by CpP is dependent upon a number of parameters. 1) The magnitude of the conductance change was voltage dependent. 2) An increase in the total charge per molecule increased the rate of conductance change. 3) An increase in the charge density (ratio of charged amino acids to total amino acids) increased the rate of change of conductance. 4) La3+ inhibited the ability of CpP to alter the membrane conductance. 5) The rate of reversal of the CpP-induced conductance was dependent upon the total charge per molecule as well as the charge density. 6) The level of self-inhibition (ability of solution CpP to inhibit the CpP-induced membrane conductance) was inversely correlated with the charge density and was also concentration dependent, with less inhibition occurring at low mucosal CpP concentrations. These data are consistent with a model developed to describe the effect of PS on the conductive properties of the urinary bladder epithelium.

Animals↗
PubMed1992

RP 59227, a novel PAF receptor antagonist: effects in guinea pig models of airway hyperreactivity.

The PAF antagonists RP 59227 and WEB 2086 (100 micrograms.kg-1 i.v., 10 min prior to platelet-activating factor (PAF) infusion) abolished or reduced (P less than 0.05) hyperreactivity to bombesin measured at 1 h. Similarly, RP 59227 and WEB 2086 (10 mg.kg-1 p.o., 1 h prior to PAF aerosol) abolished or reduced (P less than 0.01) hyperreactivity to bombesin measured at 24 h. Lower concentrations of RP 59227 and WEB 2086 (3 mg.kg-1 p.o.) were without effect. RP 59227 or WEB 2086 (3 or 10 mg.kg-1 p.o., 1 h prior to antigen aerosol) did not protect against antigen-induced hyperreactivity to histamine measured at 24 h. Antigen-(but not PAF)-induced hyperreactivity was accompanied by an increase in total cell number and, specifically, eosinophil number in bronchoalveolar lavage fluid. The PAF antagonists did not affect BALF cell populations. It is concluded that RP 59227 and WEB 2086 are potent PAF antagonists which inhibit PAF-but not antigen-induced airway hyperreactivity. These data suggest that endogenous PAF may not be involved in antigen-induced hyperreactivity in the guinea pig.

Animals↗
PubMed1992

Identification and characterization of the structural and nonstructural proteins of African horsesickness virus and determination of the genome coding assignments.

Proteins present in purified African horsesickness virus (AHSV) and in infected cells were analyzed by SDS-polyacrylamide gel electrophoresis. Twelve viral proteins were identified, one minor and four major structural proteins, three major and two minor nonstructural proteins, as well as variable amounts of two additional structural proteins. Cell-free translation of total AHS virion RNA in a rabbit reticulocyte system resulted in the synthesis of proteins which were qualitatively and quantitatively similar to those found in infected cells. The in vivo and in vitro synthesized proteins were viral specific as demonstrated by immunoprecipitation. The coding assignments of all the purified genome segments were determined by in vitro translation and confirmed by immunoprecipitation.

African Horse Sickness Virus↗
PubMed1992

RP 58802B, a long-acting beta 2-adrenoceptor agonist: assessment of antiasthma activity in the guinea-pig in vivo.

We have examined the protective actions of RP 58802B, a novel beta 2-adrenoceptor agonist, administered by the inhaled and oral routes in the anaesthetized and conscious guinea-pig against bronchospasm induced by histamine or antigen (ovalbumin). We have also examined the effects of RP 58802B on airway reactivity and inflammatory cell infiltration in platelet-activating factor (PAF) (aerosol)-induced bronchial hyperreactivity and on PAF (tracheal instillation)-induced microvascular leakage in the guinea-pig. Nebulized RP 58802B produced a rapid onset and long lasting inhibition of histamine-induced bronchospasm in the anaesthetized guinea-pig (EC50 = 3.2 +/- 0.9 micrograms/ml; duration greater than 90 min). Given orally, RP 58802B (5 mg/kg, 60 min before challenge) produced a greater than three-fold shift to the right of the dose-response curve and depressed the maximum response to histamine by 39 +/- 11%. Increasing the concentration to 25 mg/kg had no futher effect. Similar protection was still seen 4 h after oral dosing. In conscious guinea-pigs, RP 58802B (5 or 25 mg/kg, p.o. 60 min before challenge) significantly attenuated antigen-induced dyspnoea with the time to severe dyspnoea increasing from 170 +/- 32 to 325 +/- 32 s at the higher dose of drug. RP 58802B (10 or 25 mg/kg, p.o. 60 min before exposure to PAF) prevented the development of bronchial hyperreactivity. Although PAF-induced bronchial hyperreactivity was not accompanied by an increase in the number of pulmonary eosinophils, RP 58802B (25 mg/kg p.o.) reduced the numbers of eosinophils recovered by lavage. RP 58802B (10 mg/kg p.o.) significantly inhibited PAF-induced microvascular leakage into guinea-pig lung. These data suggest that RP 58802B, in addition to being a potent and long acting bronchodilator, may have a prophylactic role in preventing bronchial hyperreactivity and in reducing plasma exudation into the lungs.

Administration, Inhalation↗
PubMed1991

Transport-related modulation of the membrane properties of toad urinary bladder epithelium.

Impedance analysis and transepithelial electrical measurements were used to assess the effects of the apical membrane Na+ channel blocker amiloride and anion replacement on the apical and basolateral membrane conductances and areas of the toad urinary bladder (Bufo marinus). Mucosal amiloride addition decreased both apical and basolateral membrane conductances (Ga and Gbl, respectively) with no change in membrane capacitances (Ca and Cbl). Consequently, the specific conductances of these membranes decreased without significant changes in membrane area. Following amiloride removal, an increase was obtained in the steady-state rate of sodium transport compared to values before amiloride addition. This increase was independent of the initial transport rate, suggesting activation of a quiescent pool of apical sodium channels. Chloride replacement by acetate or gluconate had no significant effects on apical or basolateral membrane capacitances. The effects of these replacements on membrane conductances depended on the anion species. Gluconate (which induces cell shrinkage) decreased both membrane conductances. In contrast, acetate (which induces cell swelling) increased Ga and had no effect on Gbl. The increase in the apical membrane conductance was due to an increase in the amiloride-sensitive Na+ conductance of this membrane. In summary, mucosal amiloride addition or chloride replacements led to changes in membrane conductances without significant effects on net membrane areas.

Amiloride↗
PubMed1991

Urinary proteases degrade epithelial sodium channels.

The mammalian urinary bladder epithelium accommodates volume changes by the insertion and withdrawal of cytoplasmic vesicles. Both apical membrane (which is entirely composed of fused vesicles) and the cytoplasmic vesicles contain three types of ionic conductances, one amiloride sensitive, another a cation-selective conductance and the third a cation conductance which seems to partition between the apical membrane and the mucosal solution. The transport properties of the apical membrane (which has been exposed to urine in vivo) differ from the cytoplasmic vesicles by possessing a lower density of amiloride-sensitive channels and a variable level of leak conductance. It was previously shown that glandular kallikrein was able to hydrolyze epithelial sodium channels into the leak conductance and that this leak conductance was further degraded into a channel which partitioned between the apical membrane and the mucosal solution. This report investigates whether kallikrein is the only urinary constituent capable of altering the apical membrane ionic permeability or whether other proteases or ionic conditions also irreversible modify apical membrane permeability. Alterations of mucosal pH, urea concentrations, calcium concentrations or osmolarity did not irreversible affect the apical membrane ionic conductances. However, urokinase and plasmin (both serine proteases found in mammalian urine) were found to cause an irreversible loss of amiloride-sensitive current, a variable change in the leak current as well as the appearance of a third conductance which was unstable in the apical membrane and appears to partition between the apical membrane and the mucosal solution. Amiloride protects the amiloride-sensitive conductance from hydrolysis but does not protect the leak pathway. Neither channel is protected by sodium. Fluctuation analysis demonstrated that the loss of amiloride-sensitive current was due to a decrease in the sodium-channel density and not a change in the single-channel current. Assuming a simple model of sequential degradation, estimates of single-channel currents and conductances for both the leak channel and unstable leak channel are determined.

Amiloride↗