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R Othman

Publications and source records attributed to R Othman.

7 recordsLinked to original sources
PubMed1998

Tryptophan-containing mutant of human (group IIa) secreted phospholipase A2 has a dramatically increased ability to hydrolyze phosphatidylcholine vesicles and cell membranes.

Human nonpancreatic (group IIa) secreted phospholipase A2 (human sPLA2) is associated with a number of inflammatory disorders in which the extracellular concentrations of this enzyme can become highly elevated. It is probable that the enzyme normally acts as an acute-phase protein whose function is to facilitate the removal of infectious organisms or damaged host cells as part of the normal inflammatory response. The enzyme shows negligible activity with phosphatidylcholine (PC) vesicles and cell membranes, presumably reflecting the enzyme's lack of ability to bind productively to such condensed neutral interfaces. Mammalian pancreatic enzymes show modest activity with such interfaces and contain a unique tryptophan at position 3, which is part of the presumptive interfacial binding surface of these enzymes. Human sPLA2 does not contain tryptophan. The amphiphilic indole side chain of tryptophan is noted for its ability to penetrate the lipid interface of membranes, and tryptophan residues appear to be associated with the ability of lipases and phospholipases A2 to bind to and hydrolyze such interfaces. We have investigated in detail the properties of a V3W mutant of human sPLA2, which has a unique tryptophan on the interfacial binding surface of this enzyme. Although this enzyme shows a modest ( approximately 50%) reduction in activity when anionic substrates are used under standard assay conditions, the activity of the enzyme on phosphatidylcholine vesicles and cell membranes is dramatically increased compared with human sPLA2. This is particularly the case with small unilamellar vesicles of PC, where activity is enhanced over 250-fold compared to the almost zero activity expressed by human sPLA2. This enhanced activity is best explained by increased interfacial binding and activation of the V3W mutant and is not due to enhanced active-site binding and hydrolysis. The results highlight the important role that tryptophan residues can play in interfacial binding, particularly to condensed zwitterionic interfaces. The interfacial characteristics of the mutant human enzyme now resemble more closely the mammalian pancreatic enzymes that already have a tryptophan at position 3.

3T3 Cells↗
PubMed1998

Phospholipase D and phosphatidic acid enhance the hydrolysis of phospholipids in vesicles and in cell membranes by human secreted phospholipase A2.

Phosphatidyl-choline (PC) vesicles and normal cell membranes are resistant to hydrolysis by human group II secreted PLA2, an enzyme that can attain high concentrations in extracellular fluids during many inflammatory processes. This highly cationic enzyme (pI>10.5) has a marked preference for anionic phospholipid interfaces, normally present within the cell. Therefore, the ability of one such anionic phospholipid, phosphatidic acid (PA), to enhance the activity of this enzyme has been investigated in detail. Results using model membrane vesicles and a continuous fluorescence assay highlight the ability of low molar proportions of PA to stimulate vesicle hydrolysis and this stimulation with increasing PA was parallelled by enhanced interfacial binding. In contrast, no productive binding of this enzyme could be detected to the surface of pure PC vesicles. The enhancement of hydrolysis in the presence of PA could also be achieved by prior treatment of pure PC vesicles with PLD, an effect that was dependent on the concentration of PLD and the duration of exposure to this enzyme. The fluorescence assay also allowed cell membranes and whole cells to be used as substrates and whereas such membrane presentations were refractory to hydrolysis by the human enzyme, prior treatment with PLD allowed hydrolysis using concentrations of this PLA2 that would be found extracellularly under inflammatory conditions. These results highlight the potential for PA, generated at the surface of the cell membrane, to be hydrolysed by extracellular human sPLA2 with the generation of lysophosphatidic acid and other lipid mediators and provides one possible mechanism whereby this human sPLA2 could become pro-inflammatory.

Calcium↗
PubMed1997

Mapping the interfacial binding surface of human secretory group IIa phospholipase A2.

Human secretory group IIa phospholipase A2 (hIIa-PLA2) contains a large number of prominent cationic patches on its molecular surface and has exceptionally high affinity for anionic surfaces, including anionic membranes. To identify the cationic amino acid residues that support binding of hIIa-PLA2 to anionic membranes, we have performed extensive site-directed mutagenesis of this protein and measured vesicle binding and interfacial kinetic properties of the mutants using polymerized liposomes and nonpolymerized anionic vesicles. Unlike other secretory PLA2s, which have a few cationic residues that support binding of enzyme to anionic membranes, interfacial binding of hIIa-PLA2 is driven in part by electrostatic interactions involving a number of cationic residues forming patches on the putative interfacial binding surface. Among these residues, the amino-terminal patch composed of Arg-7, Lys-10, and Lys-16 makes the most significant contribution to interfacial adsorption, and this is supplemented by contributions from other patches, most notably Lys-74/Lys-87/Arg-92 and Lys-124/Arg-127. For these mutants, complete vesicle binding occurs in the presence of high vesicle concentrations, and under these conditions the mutants display specific activities comparable to that of wild-type enzyme. These studies indicate that electrostatic interactions between surface lysine and arginine residues and the interface contribute to interfacial binding of hIIa-PLA2 to anionic vesicles and that cationic residues closest to the opening of the active-site slot make the most important interactions with the membrane. However, because the wild type binds extremely tightly to anionic vesicles, it was not possible to exactly determine what fraction of the total interfacial binding energy is due to electrostatics.

Arginine↗
PubMed1996

Human non-pancreatic (group II) secreted phospholipase A2 expressed from a synthetic gene in Escherichia coli: characterisation of N-terminal mutants.

A gene coding for human non-pancreatic (group II) secreted phospholipase A2 (hnpsPLA2) has been constructed by the single-step ligation of twelve synthetic oligonucleotides. The gene has been cloned into a modification of the bacterial expression vector pET 11 which allows protein over-expression as inclusion bodies and enables about 3 mg/litre of pure refolded fully active enzyme to be obtained. The protein was expressed as a 1-Ala mutant (N1A) to allow removal of the initiator methionine by the Escherichia coli amino-peptidase. This mutant had very similar properties to the wild-type enzyme. A double mutant, N1A, V3W was also constructed and expressed in high yield. This tryptophan-containing mutant showed similar properties to the wild-type and N1A mutant but had about 40% of the activity under the assay conditions used. This tryptophan was used as a reporter group for interfacial binding and its properties were compared to those of the corresponding tryptophan in PLA2 from procine pancreas. Expression of the wild-type gene sequence for hnpsPLA2 in E. coli gave the expected mutant protein still with the initiator methionine and with much reduced activity. Interfacial binding of all hnpsPLA2 mutants to anionic phospholipids was very similar when assessed by fluorescence methods. Comparisons of these mutants with the pancreatic enzyme revealed significant differences in terms of the effect of calcium on interfacial binding. The ability to express reasonably large amounts of the N1A mutant in E. coli will provide a basis for future site directed mutagenesis studies of this important human enzyme.

Amino Acid Sequence↗
PubMed1995

Interactive database management (IDM).

Interactive database management (IDM) is a data editing software that provides complete data editing at the time of initial data entry when information is 'fresh at hand'. Under the new interactive system, initial data recording is subjected to instant data editing by the interactive computer software logic. Data are immediately entered in final form to the database and are available for analysis. IDM continuously checks all variables for acceptability, completeness, and consistency. IDM does not allow form duplication. Many functions including backups have been automated. The interactive system can export the database to other systems. The software has been implemented for two Department of Veterans Affairs Cooperative Studies (CCSHS #5 and CSP #385) which collect data for 1400 and 1000 variables, respectively at 28 VA medical centers. IDM is extremely user friendly and simple to operate. Researchers with no computer background can be trained quickly and easily to use the system. IDM is deployed on notebook microcomputers making it portable for use anywhere in the hospital setting.

Clinical Trials as Topic↗
PubMed1994

Traditional psychotherapeutic healing and healers in the Palestinian community.

The aim of this paper is to offer some general observations about traditional psychotherapeutic healing and healers in the Palestinian community. Ten healers, known as sheikhs or sheikhas, were interviewed, and their statements comprise the principal data on which this study is based. Observations are offered regarding (1) the background, training and some characteristics of the healers; (2) characteristics of the patients and illnesses treated; (3) therapeutic techniques and treatment rationale.

Adult↗