Biomedical subjects

P Loke

Publications and source records attributed to P Loke.

26 records · Page 2Linked to original sources

PubMed1999

Suppressive antigen-presenting cells in Helminth infection.

Infection with filarial nematodes is commonly associated with a failure of T cells to proliferate in response to parasite antigen. We have investigated the possibility that antigen-presenting cells recruited during filarial infection are responsible for impairment of T cell function. We have found that the human filarial parasite Brugia malayi, when implanted into the peritoneal cavity of mice, recruits a population of adherent cells that actively block the proliferation of T cells. Phenotypic analysis of the recruited cells reveals large numbers of both macrophages and eosinophils and cell sorting experiments demonstrate that these antiproliferative cells are Mac-1-positive. Studies in gene-deficient mice have demonstrated that proliferative suppression is dependent on the in vivo production of IL-4 but not IL-5 or IL-10, while suppression in vitro is not mediated by any known antiproliferative factor. Our results suggest that helminth infection can lead to the development and/or recruitment of an IL-4-dependent macrophage population that mediates suppression via a novel mechanism.

Animals↗

PubMed1998

Analysis of glutamines in catalysis in Cephalosporium acremonium isopenicillin N synthase by site-directed mutagenesis.

Isopenicillin N synthase (IPNS), an important enzyme in the beta-lactam antibiotic biosynthetic pathway, is responsible for the catalytic conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. Three catalytic ligands essential for IPNS activity have already been determined. Based on an Aspergillus nidulans IPNS crystal structure, the probable involvement of a fourth amino acid as a catalytic ligand was previously revealed. To continue the search for the fourth catalytic ligand, we report investigations on whether or not glutamines play a role in the catalytic action of Cephalosporium acremonium IPNS (cIPNS). Three glutamine residues were targeted for modification based on the previous revelation of one (Q337) via crystal structure coordinates, the conservation of one (Q234) in isozyme alignment and the proximity of one (Q227) to the catalytic centre. Analysis of the biotransformed mutant enzymes showed retention of activity, thereby rejecting the involvement of a possible glutamine as a catalytic ligand in cIPNS catalysis.

Acremonium↗

PubMed1998

Mutational evidence for the role of serine-283 in Cephalosporium acremonium isopenicillin N synthase.

Creation of isopenicillin N from delta-(L-alpha-aminodipyl)-L-cysteinyl-D-valine (ACV) in the penicillin and cephalosporin biosynthetic pathway is catalysed by isopenicillin N synthase (IPNS), a non-heme iron-containing dioxygenase. A tripeptide R-X-S motif which consists of arginine-281 and serine-283 (Cephalosporium acremonium IPNS numbering) was found to be conserved in IPNS and other related proteins. These two amino acids mentioned were proposed to have a role in ACV substrate binding by the recent Aspergillus nidulans IPNS crystal structure. Using site-directed mutagenesis arginine-281 in C. acremonium IPNS (cIPNS) was earlier found to be essential for catalysis by our group. Similarly, serine-283 in cIPNS was also altered by site-directed mutagenesis to determine its role in cIPNS. No measurable activity was detected from the resultant mutant using enzyme bioassays. It is most likely that the eliminatin of the mutant's substrate-binding capability similar to that of arginine-281 lead to the abolishment of the catalytic reaction. This highlights the importance of the R-X-S motif in the functionality of cIPNS.

Acremonium↗

PubMed1998

Catalytic activity in Cephalosporium acremonium isopenicillin N synthase does not involve glutamine-234.

The catalytic activity of isopenicillin N synthase (IPNS), a crucial enzyme which converts delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N in the beta-lactam antibiotic biosynthetic pathway, is known to be dependent upon the ligation of two histidines and an aspartate to the iron active centre. Recent studies have ruled out the suggested requirement of the penultimate glutamine, Q330 and Q328 in Aspergillus nidulans and Streptomyces jumonjinensis IPNS respectively, for catalysis. As a counter proposal, glutamine-230 from S. jumonjinensis IPNS was presented to be crucial for activity. However, we report differing results from the site-directed mutagenesis of the corresponding glutamine-234 in Cephalosporium acremonium IPNS. Based on IPNS enzymatic assays, we conclude that glutamine-234 is not essential for catalysis in cIPNS. Furthermore, we advocate the use of soluble proteins over solubilized proteins especially for studies which involve enzymatic catalysis.

Acremonium↗

PubMed1997

Functional analysis of a conserved aspartate D218 in Cephalosporium acremonium isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is instrumental in the catalytic conversion of a tripeptide precursor delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to a bioactive intermediate isopenicillin N in the beta-lactam antibiotic biosynthetic pathway. It has recently been shown that this reaction is dependent on a conserved aspartate, D214, in a bacterial Streptomyces jumonjinensis IPNS. Thus, this study was carried out to provide the experimental evidence for the involvement of a similarly conserved aspartate residue, D218, in a fungal Cephalosporium acremonium IPNS (cIPNS). Initially, alteration of the aspartate residue to generate the mutant D218L cIPNS protein was achieved by site-directed mutagenesis. Subsequent enzyme assays indicated that the catalytic property of the mutant protein was lost, attesting to the need for the corresponding conserved aspartate to maintain IPNS functionality. It is also evident from the observed results that site-directed mutagenesis of this particular aspartate residue in cIPNS can affect its solubility. It is therefore important to take these potential changes into consideration when site-directed mutant proteins are analysed for catalytic function.

Acremonium↗

PubMedDate not supplied

Site-directed mutagenesis of proline-285 to leucine in Cephalosporium acremonium isopenicillin-N-synthase affects catalysis and increases soluble expression at higher temperatures.

The conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N is dependant on the catalytic action of isopenicillin N-synthase (IPNS), an important enzyme in the penicillin and cephalosporin biosynthetic pathway. One of the amino acid residues suggested by the Aspergillus nidulans IPNS crystal structure for interaction with the valine isopropyl group of ACV is proline-283. Site-directed mutagenesis of the corresponding proline-285 to leucine in Cephalosporium acremonium IPNS resulted in non-measurable activity but an increased soluble expression at higher temperatures in a heterologous E. coli host.

Acremonium↗

PubMedDate not supplied

Replacement of tyrosine-197 and the corresponding tyrosine-195 to isoleucine in Cephalosporium acremonium and Streptomyces clavuligerus isopenicillin N synthase.

Isopenicillin N synthase (IPNS) is one of the key enzymes in the penicillin and cephalosporin biosynthetic pathway which catalyses the conversion of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine to isopenicillin N. The IPNS from Penicillium chrysogenum 23X-80-269-37-2, a high penicillin V-producer, was found to possess an isoleucine residue instead of tyrosine at position 195. An attempt to increase the specific activity of IPNS from Cephalosporium acremonium and Streptomyces clavuligerus was undertaken by altering the corresponding tyrosine residue to an isoleucine at the corresponding location. Unfortunately, no apparent increase in specific activity was encountered when the purified mutant enzymes were analysed and thus, this amino acid difference is likely not responsible for high specific activity in IPNS.

Acremonium↗

PubMedDate not supplied

A comparison of three site-directed mutagenesis kits.

In this comparative study, three different mutagenesis kits, namely the MutaGene phagemid in vitro mutagenesis kit (Bio-Rad), the Transformerä Site-Directed mutagenesis kit (Clontech) and the Quik-change site-directed mutagenesis kit (Stratagene) were used for the mutagenesis of IPNS genes. However, a large difference in mutation efficiencies among these kits was encountered. Furthermore, these kits employ different strategies with its own individual strengths and weaknesses. Thus, a comparison among these three kits to evaluate their usefulness and improvements on the strategy adopted by the Quik-change site-directed mutagenesis kit, which was the kit of choice for our work, are presented for the benefit of research work.

Base Pair Mismatch↗