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Biomedical subjects

A Lim

Publications and source records attributed to A Lim.

At least 37 records · Page 2Linked to original sources
PubMed2000

Drug target validation: lethal infection blocked by inducible peptide.

Genome projects are generating large numbers of potential new targets for drug discovery. One challenge is target validation, proving the usefulness of a specific target in an animal model. In this paper, we demonstrate a new approach to validation and assay development. We selected in vitro specific peptide binders to a potential pathogen target. By inducing the expression of a selected peptide in pathogen cells causing a lethal infection in mice, the animals were rescued. Thus, by combining in vitro selection methods for peptide binders with inducible expression in animals, the target's validity was rigorously tested and demonstrated. This approach to validation can be generalized and has the potential to become a valuable tool in the drug discovery process.

Amino Acid Sequence↗
PubMed2000

Betabellins 15D and 16D, de Novo designed beta-sandwich proteins that have amyloidogenic properties.

The betabellin structure is a de novo designed beta-sandwich protein consisting of two 32-residue beta-sheets packed against one another by hydrophobic interactions. d-Amino acid residues are used to energetically favor formation of type-I' beta turns. Air oxidation of betabellin 15S (B15S) (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p denotes d-Pro, h denotes d-His, and k denotes d-Lys) yields betabellin 15D (B15D), a 64-residue disulfide-bridged protein. The amino acid sequence of B15D contains a conformationally constrained d-Pro residue at the i + 1 position of each type-I' beta turn. To test whether d-Pro residues are necessary for folding at these positions, the six d-Pro residues of B15D are replaced by d-Ala residues in betabellin 16D (B16D). Previously, transmission electron microscopy showed that B15D forms unbranched, 35-A wide fibrils that associate into bundles in 5.0 mM 3-(N-morpholino)propanesulfonate and 250 mM NaCl at pH 7; under these conditions, B16D forms ribbon-like assemblies. The B15D fibrils resemble the protofilaments that constitute amyloid fibrils. The present studies show that both B15D and B16D have characteristics of amyloidogenic proteins: the unbranched fibrils and ribbons stained with Congo red and displayed a green birefringence, exhibited a cross-beta structure, and bound 1-anilino-8-naphthalenesulfonate. Thus, these de novo designed beta-sandwich proteins should provide useful models for studying the mechanism of amyloid protofilament formation and assembly into amyloid fibrils and for designing potential inhibitors of amyloidogenesis.

Amino Acid Sequence↗
PubMed2000

Detection of small-molecule enzyme inhibitors with peptides isolated from phage-displayed combinatorial peptide libraries.

BACKGROUND: The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. RESULTS: We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range. CONCLUSIONS: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.

Alcohol Dehydrogenase↗
PubMed2000

High frequency of circulating gamma delta T cells with dominance of the v(delta)1 subset in a healthy population.

TCR gamma delta(+) cells constitute <5% of all circulating T cells in healthy, adult Caucasians, and V(delta)1(+) cells constitute a minority of these cells. In contrast to TCR alpha beta(+) cells, their repertoire is selected extrathymically by environmental antigens. Although increased frequencies of V(delta)1(+) cells are found in several diseases, their function remains obscure. Here we show that the frequency of peripheral blood gamma delta T cells in healthy West Africans is about twice that of Caucasians, mainly due to a 5-fold increase in V(delta)1(+) cells, which is consequently the dominant subset. No age dependency of V(delta)1 frequencies was identified and the V(delta)1(+) cells in the African donors did not show preferential V(gamma) chain usage. Analysis of the CDR3 region size did not reveal any particular skewing of the V(delta)1 repertoire, although oligoclonality was more pronounced in adults compared to children. The proportions of CD8(+), CD38(+) and CD45RA(hi)CD45RO(-) cells within the V(delta)1(+) subset were higher in the African than in the European donors, without obvious differences in expression of activation markers. No significant correlations between levels of V(delta)1(+) cells and environmental antigens or immunological parameters were identified. Taken together, the evidence argues against a CDR3-restricted, antigen-driven expansion of V(delta)1(+) cells in the African study population. Our study shows that high frequencies of TCR gamma delta(+) cells with dominance of the V(delta)1(+) subset can occur at the population level in healthy people, raising questions about the physiological role of V(delta)1(+) T cells in the function and regulation of the immune system.

Adult↗
PubMed2000

Proximal third femoral shaft fractures in children: prevention of angular deformities using bilateral Thomas splints.

This is a prospective study to look at the outcome of unilateral proximal third femoral shaft fractures in children treated with a bilateral Thomas splint in the Department of Orthopaedic Surgery Universiti Kebangsaan Malaysia between the period of January 1996 and June 1998. Eighteen children aged between 2 years and 12 years old with unilateral proximal third fractures of the femoral shaft were treated using a bilateral Thomas splint. Angular deformities were measured using a goniometer metric scale before and after Thomas splints. The percentage of varus tilt corrected ranged from 17% to 72% with an average correction of 29% from the initial deformity and the difference was statistically significant (p < 0.05). The percentage of posterior tilt corrected ranged from 19% to 60% with the average correction of 20% from the initial deformity. The difference was statistically significant (p < 0.05). From this study, we conclude that bilateral Thomas splints can give a better correction of angular deformity for proximal third femoral shaft fractures in children below twelve years of age.

Child↗
PubMed2000

Follow up of water use in a tin mining area affected with arsenic poisoning.

Ron Phibun district in southern Thailand has been known as an endemic area for arsenic contamination. The government has been trying to improve the situation by encouraging the use of rainwater and piped water. This study aimed to document the change of water use and to identify factors associated with safe water use in 1997 compared to that in 1994. Home visits and face-to-face questionnaire interviews were undertaken. Information on water use for drinking, cooking, washing food and washing utensils in 1994 and 1997 was obtained. Among 3,849 households from which data could be obtained (estimated 79% of total households), the percentages of using safe water (including water from bottled rain water, piped and artesian well water) for drinking and cooking rose from 72.5 and 57.9 in 1994 to 93.6 and 80.9 in 1997, respectively. The percentages for washing foods and for washing utensils rose from 28.6 and 20.5 to 59.1 and 53.8, respectively. In 1997, percentage of households using piped water for drinking and cooking was still low (3.6 and 12.3) compared to those using piped water for washing food and utensils (39.1 and 43.6). Multivariate analysis shows that independent factors of the household predicting safe water use are: high arsenic area, near main road and having piped water installed. The influence of these factors (as judged by the level of odds ratio) operates more or less equally on water use for all purposes, except that installation of piped water has more influence on washing water than drinking and cooking water. We conclude that safe water supply in the area is still inadequate. Even if piped water is installed, it is often not used for drinking and cooking. The reasons for not using piped water for drinking and cooking need to be identified.

Arsenic Poisoning↗
PubMed1999

Biophysical characterization of betabellin 16D: a beta-sandwich protein that forms narrow fibrils which associate into broad ribbons.

The betabellin structure is a de novo designed beta-sandwich protein consisting of two 32-residue beta sheets packed against one another by hydrophobic interactions. Betabellin 16S (B16S), a 32-residue peptide chain (HSLTAKIakLTFSIAahTYTCAVakYTAKVSH, where a is DAla, h is DHis, and k is DLys), did not have beta structure in water at pH 6.5. Air oxidation of B16S furnished betabellin 16D (B16D), a 64-residue disulfide-bridged two-chain protein, which also did not fold in water at pH 6.5. However, the extent of beta structure observed for B16D increased with pH and ionic strength of the solution and the B16D concentration as observed by circular dichroism spectropolarimetry. Transmission electron microscopy showed that B16D formed narrow fibrils that associated into broad ribbons in 5.0 mM Mops and 0.25 M NaCl at pH 6.9.

Circular Dichroism↗
PubMed1999

Implication of gammadelta T cells in the human immune response to cytomegalovirus.

In normal individuals, gammadelta T cells account for less than 6% of total peripheral T lymphocytes and mainly express T-cell receptor (TCR) Vdelta2-Vgamma9 chains. We have previously observed a dramatic expansion of gammadelta T cells in the peripheral blood of renal allograft recipients only when they developed cytomegalovirus (CMV) infection. This increase was long lasting (more than 1 year), was associated with an activation of gammadelta T cells, and concerned only Vdelta1 or Vdelta3 T-cell subpopulations. Analysis of gammadelta TCR junctional diversity revealed that CMV infection in these patients was accompanied by (a) a marked restriction of CDR3 size distribution in Vdelta3 and, to a lesser extent, in Vdelta1 chains; and (b) a selective expansion of Vdelta1 cells bearing recurrent junctional amino acid motifs. These features are highly suggestive of an in vivo antigen-driven selection of gammadelta T-cell subsets during the course of CMV infection. Furthermore, Vdelta1 and Vdelta3 T cells from CMV-infected kidney recipients were able to proliferate in vitro in the presence of free CMV or CMV-infected fibroblast lysates but not uninfected or other herpes virus-infected fibroblast lysates. This in vitro expansion was inhibited by anti-gammadelta TCR mAb's. These findings suggest that a population of gammadelta T cells might play an important role in the immune response of immunosuppressed patients to CMV infection.

Adolescent↗
PubMed1999

The utility of apolipoprotein E genotyping in the diagnosis of Alzheimer disease in a community-based case series.

CONTEXT: A recent collaborative study found that apolipoprotein E (APOE) genotype, in conjunction with the clinical diagnosis of Alzheimer disease (AD), was useful in improving diagnostic specificity (correctly not diagnosing AD) relative to the clinical diagnosis alone. Since these samples are particularly enriched with patients with AD and the APOE epsilon4 allele, results may not be generalizable to patients seen in the general medical community. OBJECTIVE: To evaluate the diagnostic utility of the APOE genotype in diagnosing AD in a community-based case series from the largest health maintenance organization in an urban area. DESIGN: We examined the effect of including APOE genotype on the diagnosis of AD in a community-based case series of patients presenting with memory complaints. PATIENTS: Clinical and neuropathologic diagnoses and APOE genotype were obtained from 132 patients who underwent evaluation for dementia and subsequent autopsy. MAIN OUTCOME MEASURES: Sensitivity, specificity, and positive and negative predictive values given various combinations of clinical diagnoses and the presence of an APOE epsilon4 allele. RESULTS: Of the 132 patients, 94 had neuropathologically confirmed AD, yielding a prevalence of 71%. The clinical diagnosis alone yielded a sensitivity of 84%, an estimated specificity of 50%, and positive and negative predictive values of 81% and 56%, respectively. The presence of an epsilon4 allele alone was associated with an estimated sensitivity of 59%, specificity of 71%, and positive and negative predictive values of 83% and 41%, respectively. Using the presence of clinical AD and an epsilon4 allele decreased the sensitivity to 49% and increased the specificity to 84%. The positive and negative predictive values were 88% and 40%, respectively. Alternatively, the clinical diagnosis of AD or the presence of an epsilon4 allele in individuals not meeting clinical criteria for AD increases the estimated sensitivity to 94% but decreases the specificity to 37%. The positive and negative predictive values were 79% and 70%, respectively. The changes in the sensitivity and specificity for the combined tests relative to clinical diagnosis alone offset each other. For lower prevalence communities, the positive predictive value will be much lower than those observed herein. CONCLUSIONS: Our findings do not support the use of APOE genotyping alone in the diagnosis of AD in the general medical community. Although the presence of an epsilon4 allele in older persons with clinical AD increased the probability of having AD and the absence of an epsilon4 allele in this group decreased the probability of having AD, the association is not strong enough in the differential diagnosis of non-Alzheimer dementia and AD.

Aged↗
PubMed1999

CD95 ligand expression in dedifferentiated breast cancer.

CD95 ligand expression has been observed in various malignancies. Studying the CD95 ligand (CD95L) and receptor (CD95) system in eight non-malignant mammary tissues and 40 breast cancer tissues, mRNA and protein expression was determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunofluorescence. mRNA levels of CD95L correlated positively (r=0.90; p< 0.01) and transmembrane CD95 inversely (r=-0.88; p< 0.01) with histopathological grading of the breast tumours: CD95L mRNA levels were low in adenomas, but increased by 20-fold in grade I, 120-fold in grade II, and 310-fold in grade III breast cancer. In contrast, CD95 mRNA levels were low in high-grade carcinomas, but high in benign mammary tissues. Since CD95L acts as an efficient inducer of apoptosis in CD95(+) cells, apoptotic cells were identified on the tissue sections. Tumour-infiltrating lymphocytes and stromal cells in close proximity to CD95L-expressing breast cancer underwent apoptosis. As a functional test, CD95(+) target cells were cultured on breast cancer tissue sections. The target cells underwent apoptosis when cultured on breast cancer sections, but could be rescued when CD95L was specifically blocked by a CD95-Fc fusion molecule. The data suggest an inverse regulation of CD95 ligand and receptor expression during dedifferentiation of breast cancer. Killing of bystander cells by the CD95L-expressing breast tumour could be involved in tissue invasion.

Adult↗
PubMed1999

Frequent enrichment for CD8 T cells reactive against common herpes viruses in chronic inflammatory lesions: towards a reassessment of the physiopathological significance of T cell clonal expansions found in autoimmune inflammatory processes.

We recently evidenced a dramatic enrichment for T cells reactive against Epstein-Barr virus (EBV) within inflamed joints of two rheumatoid arthritis patients. To assess the generality of this phenomenon and its relevance to autoimmunity, we studied the responses of CD8 T cells from patients with either acute or chronic inflammatory diseases (rheumatoid arthritis: n = 18, ankylosing spondylitis: n = 5, psoriatic arthritis: n = 4, Reiter's syndrome: n = 3, arthrosis: n = 2, uveitis: n = 2, multiple sclerosis: n = 2, encephalitis: n = 1) against viral proteins derived from EBV and another common herpes virus, human cytomegalovirus (CMV). T cell responses against EBV and/or CMV epitopes were frequently observed within CD8 T cells derived from chronic inflammatory lesions, irrespective of their location (knee, eye, brain) and autoimmune features. In most cases, CD8 T cells derived from affected organs yielded stronger anti-viral T cell responses than CD8 T cells derived from patients' PBL, even in chronic inflammatory diseases devoid of autoimmune features or induced by defined bacterial agents. Taken together, these results suggest that the presence of virus-specific T cells within inflamed lesions of patients suffering from autoimmune diseases is a general phenomenon associated with chronic inflammation rather than the initiating cause of the autoimmune process. Since this phenomenon was sometimes associated with long-term T repertoire biases within inflamed lesions, the physiopathological significance of T cell clonal expansions found in a recurrent fashion within chronically inflamed autoimmune lesions should be interpreted with caution.

Adult↗
PubMed1999

Renal, central nervous system and pancreatic overexpression of recombinant soluble Crry in transgenic mice. A novel means of protection from complement-mediated injury.

Crry is a potent complement regulator that inhibits classical and alternative pathway C3 convertases in rodents. We have produced transgenic animals expressing Crry as a recombinant soluble protein driven by the broadly active metallothionein-I promoter. These animals have high serum and urinary levels of rsCrry leading to inhibition of complement activity. In nephrotoxic serum nephritis (NSN), injected antibodies bind to glomeruli, leading to complement activation and subsequent glomerular injury and albuminuria. We have shown that rsCrry can block such injury and reduce albuminuria by as much as 75%. Corresponding to the reduction in albuminuria was the complete absence of C3 staining in glomeruli by immunofluorescence microscopy in 17/20 transgene positive animals. Support for a local source of protective rsCrry in this model is provided by the demonstration of Crry transgene mRNA in the glomerulus and a very high fractional excretion of rsCrry in the urine. Therefore, rsCrry expression markedly ameliorates an antibody-induced disease model in vivo. In addition, local synthesis of Crry in other organs that are targets of immune injury has been found. For example, Crry transgene mRNA is present throughout the central nervous system and in pancreatic islets. Thus, continuous complement inhibition at the C3 convertase step appears to be feasible and is effective in complement-mediated injury states. A number of disease models affecting these target organs can be tested using these mice.

Animals↗
PubMed1999

Metal-ion binding and limited proteolysis of betabellin 15D, a designed beta-sandwich protein.

Betabellin 15D is a 64-residue, disulfide-bridged homodimer. When folded into a beta structure, the protein is predicted to have two clusters of three histidine residues, each cluster able to bind a divalent metal ion. When the protein was incubated with Cu2+, Zn2+, Co2+, or Mn2+, metal complexes of betabellin 15D were observed by electrospray-ionization mass spectrometry. The relative abundances of the ionic complexes suggested an order of affinities of Cu2+ > Zn2+ > Co2+ > Mn2+, consistent with solution-phase affinities for nitrogen- or sulfur-containing ligands. Limited proteolysis of betabellin 15D by immobilized pepsin, as measured by nanoelectrospray-ionization mass spectrometry, showed that the Phe12-Ser13 peptide bond of betabellin 15D was cleaved more slowly in the presence of Cu2+ than in its absence. Because Cu2+ has little or no effect on the catalytic rate of pepsin, the slower cleavage of the Phe12-Ser13 peptide bond may be due to its decreased accessibility caused by Cu(2+)-induced folding of betabellin 15D.

Amino Acid Sequence↗
PubMed1999

WebPHYLIP: a web interface to PHYLIP.

A web interface to PHYLIP (version 3.57 C) is implemented using CGI/Perl programming. It enables users to do phylogenetic analysis through the Internet.

Data Display↗
PubMed1999

Clinico-neuropathological correlation of Alzheimer's disease in a community-based case series.

OBJECTIVES: Most clinico-neuropathological correlative studies of Alzheimer's Disease (AD) are based on research cohorts that are not necessarily generalizable to patients seen in the general medical community. In this study, we examine the accuracy of the criteria used in diagnosing AD in a community-based case series of patients with memory complaints. DESIGN AND PARTICIPANTS: Clinical and neuropathological diagnoses were obtained from 134 patients evaluated for dementia who subsequently underwent autopsy. SETTING: Subjects who exhibited new symptoms of dementia and were enrolled in the University of Washington/Group Health Cooperative Alzheimer's Disease Patient Registry were eligible for this study. MEASUREMENTS: Clinico-pathological correlation was performed using NINCDS-ADRDA (National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association) and CERAD (Consortium to Establish a Registry for Alzheimer's Disease) criteria. RESULTS: Ninety-five of the 134 cases studied met CERAD neuropathological criteria for AD. The sensitivity of NINCDS-ADRDA "probable AD" was 83% (diagnosing AD correctly) and overall clinical diagnostic accuracy was 75%. However, there was a high rate of additional neuropathological findings. Only 34 of the 94 cases had pure AD on neuropathology, whereas the remainder frequently had coexisting vascular or Parkinson's disease lesions. CONCLUSIONS: This study of a large series of community-based incident dementia cases provides a way of judging the adequacy of currently available clinical diagnostic criteria. It also shows that co-existing neuropathological findings are common in community-based AD.

Aged↗
PubMed1999

Oxygen depletion-induced dormancy in Mycobacterium bovis BCG.

Gradual depletion of oxygen causes the shift-down of aerobic growing Mycobacterium bovis BCG to an anaerobic synchronized state of nonreplicating persistence. The persistent culture shows induction of glycine dehydrogenase and alpha-crystallin-like protein and is sensitive to metronidazole.

Amino Acid Oxidoreductases↗