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Biomedical subjects

S Paul

Publications and source records attributed to S Paul.

At least 163 records · Page 9Linked to original sources
PubMed1995

Expression of multidrug resistance-associated protein in NIH/3T3 cells confers multidrug resistance associated with increased drug efflux and altered intracellular drug distribution.

Multidrug resistance is a major obstacle to cancer treatment. Using an expression cDNA library transfer approach to elucidating the molecular basis of non-P-glycoprotein-mediated multidrug resistance, we previously established that expression of multidrug resistance protein (MRP), an ATP-binding cassette superfamily transporter, confers multidrug resistance (G. D. Kruh et al., Cancer Res., 54: 1649-1652, 1994). In the present study, we generated NIH/3T3 MRP transfectants without using chemotherapeutic drugs to facilitate the pharmacological analysis of the MRP phenotype. MRP transfectants displayed increased resistance to several lipophilic drugs, including doxorubicin, daunorubicin, etoposide, actinomycin D, vincristine, and vinblastine. However, increased resistance was not observed for Taxol, a drug for which transfection of MDR1 confers high levels of resistance. Verapamil increased the sensitivity of MRP transfectants relative to control transfectants, but reversal was incomplete for doxorubicin and etoposide, the drugs for which MRP conferred the highest resistance levels. For the latter two drugs, MRP transfectants, which were approximately 8- and approximately 10-fold more sensitive than control cells in the absence of verapamil, exhibited 3.8- and 3.3-fold relative sensitization with 10 microM verapamil, respectively, but remained approximately 2 and approximately 3-fold more resistant than control cells. Analysis of drug kinetics using radiolabeled daunorubicin revealed decreased accumulation and increased efflux in MRP transfectants. Confocal microscopic analysis of intracellular daunorubicin in MRP transfectants was consistent with reduced intracellular drug concentrations, and also revealed an altered pattern of intracellular drug distribution characterized by the initial accumulation of drug in a perinuclear location, followed by the development of a punctate pattern of drug scattered throughout the cytoplasm. This pattern was suggestive of a process of drug sequestration, possibly followed by vesicle transport. Both increased drug efflux and perinuclear drug accumulation are consistent with the reported localization of MRP in plasma and cytosolic membranes (N. Krishnamachary and M. S. Center, Cancer Res., 53: 3658-3663, 1993; M. J. Flens et al., Cancer Res., 54: 4557-4563, 1994). These results thus indicate that the drug specificity of MRP is quite similar to that of MDR1, but also suggest potential differences in Taxol specificity and the level of verapamil sensitivity. In addition, these results indicate that MRP functions to extrude drug from the cell, but additionally suggest the intriguing possibility that drug sequestration contributes to drug resistance by protecting cellular targets and/or contributing to drug efflux.

3T3 Cells↗
PubMed1995

Site-directed mutagenesis of proteolytic antibody light chain.

Amino acid residues in a proteolytic antibody light chain selected by molecular modeling were substituted with Ala by site-directed mutagenesis. Hydrolysis of vasoactive intestinal polypeptide (VIP), the immunogen employed to elicit the antibody light chain, was reduced by > 95% by replacement of Ser27a or His93 by Ala residues. Similar reductions in the activity were observed using synthetic protease substrates containing Arg-methylcoumarinamide (MCA) and Lys-MCA bonds. Turnover of the Ser27a and His93 mutants was lower than that of wild-type protein by about two orders of magnitude. The activity of the wild-type protein was inhibited selectively by diisopropylfluorophosphate (DFP), a serine protease inhibitor, but the residual activity of the Ser26 mutant was refractory to DFP. The affinity of the wild-type light chain for the substrate ground state was nearly unaffected by mutations at Ser27a and His93. In contrast, a Ser26 single mutant and a His27d/Asp28 double mutant displayed increased Km (by about tenfold) and increased turnover (by about tenfold) using VIP as substrate. The kinetic constants for these mutants and the wild type protein were essentially identical with Boc-Glu-Ala-Arg as substrate. Thus, two types of residues participating in catalysis by the light chain have been identified. Ser27a and His93 are essential for catalysis but not for initial high affinity complexation and substrate. Ser26 and His27d or Asp28 participate in VIP binding and limit turnover indirectly.

Amino Acid Sequence↗
PubMed1995

Unexpected presence of polyreactive catalytic antibodies in IgG from unimmunized donors and decreased levels in rheumatoid arthritis.

Polyclonal IgG from healthy humans and unimmunized mice was screened for peptide-methylcoumarinamide (peptide-MCA) hydrolyzing activity. The activity was detected in every IgG sample examined. Contaminant enzymes were precluded as an explanation, as the activity tracked exactly with the 150-kDa IgG peak separated by gel filtration in denaturing solvent (6 M guanidine hydrochloride) and with the 50-kDa Fab fragment peak produced by papain-digestion of the IgG. Patients with rheumatoid arthritis displayed a 3.2-fold reduced peptide-MCA hydrolyzing activity (mean) compared to healthy subjects. Control osteoarthritis patients showed no diminution in activity. A progressive decrease in the activity by 7.4-fold of the pre-immune levels was observed in mice over the course of hyperimmunization with SRBC, indicating that exogenous Ag challenge, like rheumatoid arthritis, is associated with decreased catalytic activity. Apparent Km values of the IgG for Pro-Phe-Arg-MCA were 0.39 to 0.53 mM, values approximately 3-orders of magnitude greater than observed previously for Ag-specific catalysis by Abs. The only common structural feature in peptide-MCA conjugates utilized by the Abs as substrates was the presence of Arg-MCA and Lys-MCA bonds. The IgG hydrolyzed Pro-Phe-Arg-Phe at the Arg-Phe peptide bond, showing that the activity is relevant to cleavage of peptide bonds in natural Ags. The universal occurrence of this polyreactive catalytic activity in unimmunized donors and its diminution in an autoimmune disease and nonspecific Ag challenge suggest that it may possess an important, but as yet unidentified, biologic role.

Adult↗
PubMed1995

In vivo reconstitution of an active siderophore transport system by a binding protein derivative lacking a signal sequence.

Transport of iron (III) hydroxamates across the inner membrane of Escherichia coli depends on a binding protein-dependent transport system composed of the FhuB, C and D proteins. The FhuD protein, which is synthesized as a precursor and exported through the cytoplasmic membrane, represents the periplasmic binding protein of the system, accepting as substrates a number of hydroxamate siderophores and the antibiotic albomycin. A FhuD derivative, carrying an N-terminal His-tag sequence instead of its signal sequence and therefore not exported through the inner membrane, was purified from the cytoplasm. Functional activity, comparable to that of wild-type FhuD, was demonstrated for this His-tag-FhuD in vitro by protease protection experiments in the presence of different substrates, and in vivo by reconstitution of iron transport in a fhuD mutant strain. The experimental data demonstrate that the primary sequence of the portion corresponding to the mature FhuD contains all the information required for proper folding of the polypeptide chain into a functional solute-binding protein. Moreover, purification of modified periplasmic proteins from the cytosol may be a useful approach for recovery of many polypeptides which are normally exported across the inner membrane and can cause toxicity problems when overproduced.

Amino Acid Sequence↗
PubMed1995

Natural catalytic antibodies: peptide-hydrolyzing activities of Bence Jones proteins and VL fragment.

Monoclonal human light chains, i.e. Bence Jones proteins, and their recombinant variable fragments (VL) were screened for proteolytic activity using peptide-methylcoumarinamide (peptide-MCA) conjugates and vasoactive intestinal polypeptide (VIP) as substrates. Sixteen of 21 Bence Jones proteins and one of three VL fragments were capable of detectable cleavage of one or more substrates. The magnitude and kinetic characteristics of the activity varied with different substrates. Among the peptide-MCA substrates, the presence of tripeptide or tetrapeptide moieties with a basic residue at the scissile bond generally favored expression of the activity. The influence of N-terminal flanking residue recognition was evident from differing values of Km and kcat (turnover number) observed using different Arg-containing peptide-MCA substrates. Different light chains displayed different kinetic parameters for the same substrate, suggesting unique catalytic sites. Hydrolysis of VIP was characterized by nanomolar Michaelis-Menten constants (Km), suggesting comparatively high affinity recognition of this peptide. The 25-kDa monomer and the 50-kDa dimer forms of one light chain preparation were resolved by gel filtration in 6 M guanidine hydrochloride. Following renaturation, the monomer displayed 51-fold greater peptide-MCA-hydrolyzing activity than the dimer. A renatured VL domain prepared by gel filtration in 6 M guanidine hydrochloride displayed VIP-hydrolyzing activity in the 12.5-kDa peak fractions. These results provide evidence for the proteolytic activity of certain human light chains and imply that this phenomenon may have a pathophysiological significance.

Amino Acid Sequence↗
PubMed1995

Catalytic activity of anti-thyroglobulin antibodies.

Thyroglobulin (Tg)-specific autoantibodies from a patient with Hashimoto's thyroiditis hydrolyzed radiolabeled Tg, shown by production of several smaller sized products on SDS electrophoresis gels. The apparent Km value for Tg was in the nanomolar range, a property typical of an Ab combining site. The Tg antibodies also hydrolyzed tripeptide-methylcoumarinamide (MCA) substrates with lower affinity, displaying a preference for Arg-MCA and Lys-MCA containing conjugates. The hydrolysis of one of these conjugates, Pro-Phe-Arg-MCA, was inhibited competitively by Tg, suggesting a catalytic site located in the Ab combining site. In control experiments, 1) the hydrolytic activities were removed by immunoadsorption with immobilized anti-human IgG; 2) IgG depleted of the Tg-specific Abs by affinity chromatography did not display Tg and Pro-Phe-Arg-MCA hydrolyzing activities; and 3) the peptide-MCA hydrolyzing activity tracked exactly with the 150-kDa IgG peak on a gel filtration column run in denaturing solvent (6 M guanidine chloride).

Amino Acid Sequence↗
PubMed1995

Encapsulation of vasoactive intestinal peptide into liposomes: effects on vasodilation in vivo.

The purpose of this study was to determine whether encapsulation of vasoactive intestinal peptide (VIP) into liposomes potentiated its vasorelaxant effects in vivo. Using intravital microscopy, we measured the diameter of second-order arterioles (53 +/- 1 microns) in the hamster cheek pouch before, during and after suffusion of VIP, liposomes and VIP encapsulated into liposomes for 7 min. We found that VIP (0.05, 0.1 & 1.0 nmol) induced significant, time- and concentration-dependent vasodilation (9 +/- 1%, 13 +/- 3% and 14 +/- 1% increase from baseline values, respectively; mean +/- SEM; n = 12; p < 0.05). Arteriolar diameter returned to baseline values within 1-4 min after suffusion was stopped. These effects were significantly potentiated when VIP (0.05, 0.1 & 1.0 nmol) was encapsulated into liposomes (26 +/- 6%, 38 +/- 7% and 34 +/- 3% increase from baseline values, respectively; n = 12; p < 0.05). In addition, arteriolar diameter returned to baseline values 5-13 min after suffusion was stopped. Suffusion of liposomes alone had no significant effects on arteriolar diameter (n = 12; p > 0.5). We conclude that encapsulation of VIP into liposomes potentiates and prolongs of its vasorelaxant effects in the peripheral microcirculation in vivo.

Acetylcholine↗
PubMed1995

Enhancement of morphine analgesia by the GABAB agonist baclofen.

Opioid-GABAergic interactions for the treatment of post-operative pain were investigated in two double-blind, placebo-controlled experiments. We first studied the effect of pre-operatively administered baclofen, a GABAB receptor agonist, on the analgesia produced by intravenously administered morphine, a predominantly mu-opioid analgesic. In a separate trial, we studied the effect of baclofen on the analgesia produced by pentazocine, a predominantly kappa-opioid analgesic. While baclofen alone did not affect the level of post-operative pain, morphine analgesia was significantly enhanced by baclofen compared to placebo. In contrast, baclofen did not affect the level of pentazocine analgesia: however, females receiving pentazocine showed significantly greater analgesia than males.

Adult↗
PubMed1995

The pathophysiologic process of ventricular remodeling: from infarct to failure.

In the past, hypertensive heart disease was the principal cause of congestive heart failure, but currently ischemic heart disease is the major etiologic factor. In the last 20 years, the role of myocardial infarction (MI) and the subsequent alteration in ventricular architecture of the infarcted and noninfarcted myocardium have become increasingly associated with a phenomenon known as ventricular remodelling. This process consists of left ventricular wall thinning in the infarction area, ventricular chamber dilatation, and compensatory hypertrophy of the noninfarcted portion of the myocardium. This article describes the pathophysiologic transformation that begins with MI and ventricular remodeling and ends in congestive heart failure.

Heart Failure↗
PubMed1995

Culture and its influence on occupational therapy evaluation.

In the increasingly multicultural society of north America, occupational therapists have a responsibility to develop awareness and knowledge concerning different cultural groups. By accepting and understanding clients' customs, values and beliefs, clinicians have a better chance of assessing and producing more effective outcomes. Since occupational therapy has incorporated western middle-class values into its theory and practice, many evaluation tools used are based on norms developed for a white middle-class population. Using these evaluations with minority groups brings the danger of improper interpretation of test results. Consequently, increasing emphasis is being placed on the importance of culture fairness and the development of culture-fair evaluation tools for usage across different cultural groups. This paper will present a discussion concerning the importance, advantages and disadvantages of both culture-fair and culture-specific tests and evaluation tools.

Attitude to Health↗