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Biomedical subjects

A Lim

Publications and source records attributed to A Lim.

At least 19 recordsLinked to original sources
PubMed2001

Identification and location of a cysteinyl posttranslational modification in an amyloidogenic kappa1 light chain protein by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry.

Amyloid-deposited light chain (AL) amyloidosis is correlated with the overproduction of a monoclonal immunoglobulin light chain protein by a B-lymphocyte clone. Since the amyloid fibril deposits in AL amyloidosis most often consist of the N-terminal fragments of the light chain, the majority of studies have focused on the determination of the primary structure of the protein, and reducing agents have been used routinely in the initial purification process. In this study, two light chain proteins were isolated and purified, without reduction, from the urine of a patient diagnosed with kappa 1 (kappa1) AL amyloidosis. One protein had a relative molecular mass of 12,000 and the other 24,000. Electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry, in combination with enzymatic digestions, were used to verify the amino acid sequences and identify and locate posttranslational modifications in these proteins. The 12-kDa protein was confirmed to be the N-terminal kappa1 light chain fragment (variable region) consisting of residues 1-108 or 1-109 and having one disulfide bond. The 24-kDa protein was determined to be the intact kappa1 light chain containing a cysteinyl posttranslational modification at Cys214 and disulfide bonds located at Cys23-Cys88, Cys134-Cys194, and Cys214-Cys. The methods used in this report enable high-sensitivity determination of amino acid sequence and variation in intact and truncated light chains as well as posttranslational modifications. This approach facilitates consideration of the effect of cysteinylation on the native protein structure and the potential involvement of this modification in AL amyloidosis.

Amino Acid Sequence↗
PubMed2001

Cross-presentation by dendritic cells of tumor antigen expressed in apoptotic recombinant canarypox virus-infected dendritic cells.

We have investigated the possible usefulness of recombinant canarypox virus (ALVAC) encoding the melanoma-associated Ag, Melan-A/MART-1 (MART-1), in cancer immunotherapy, using a dendritic cell (DC)-based approach. ALVAC MART-1-infected DC express, and are able to process and present, the Ag coded by the viral vector. One consistent feature of infection by ALVAC is that these viruses induce apoptosis, and we show cross-presentation of Ag when uninfected DC are cocultured with ALVAC MART-1-infected DC. Uptake of apoptotic virally infected DC by uninfected DC and subsequent expression of tumor Ag in the latter were verified by flow cytometry analysis, image cytometry, and confocal microscopy. Functional activity was monitored in vitro by the stimulation of a MART-1-specific cytotoxic T cell clone. Heightened efficiency in Ag presentation is evidenced in the 2- to 3-fold increase in IFN-gamma production by the T cell clone, as compared with the ALVAC-infected DC alone. Cocultures of ALVAC MART-1-infected and uninfected DC are able to induce MART-1-specific T cell immune responses, as assessed by HLA class I/peptide tetramer binding, IFN-gamma ELISPOT assays, and cytotoxicity tests. Overall, our data indicate that DC infected with recombinant canarypox viruses may represent an efficient presentation platform for tumor Ags, which can be exploited in clinical studies.

Antigen Presentation↗
PubMed2001

Plate-based dormancy culture system for Mycobacterium smegmatis and isolation of metronidazole-resistant mutants.

Mycobacterium smegmatis is an obligate aerobe. However, growth analyses in oxygen-limited liquid cultures have shown that the bacillus is able to survive anoxia with a half-life of 4 days by shifting down to a drug-resistant, dormant state. Metronidazole is the first lead against dormant bacilli and shows selective toxicity for this physiological state. Here, we report a plate-based dormancy culture system employing anoxic jars for M. smegmatis. Its usefulness for the genetic analysis of dormancy was demonstrated by isolating the first metronidazole-resistant mutants. Highly resistant mutants formed slightly yellow (as opposed to creamy) colonies. Furthermore, high-level metronidazole resistance correlated with an increased half-life of 12 days under anoxic conditions. This suggests a link between metronidazole susceptibility and anaerobic survival.

Anti-Bacterial Agents↗
PubMed2001

Genome sequence of enterohaemorrhagic Escherichia coli O157:H7.

The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance.

Base Sequence↗
PubMed2001

egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus.

The recently described staphylococcal enterotoxins (SE) G and I were originally identified in two separate strains of Staphylococcus aureus. We have previously shown that the corresponding genes seg and sei are present in S. aureus in tandem orientation, on a 3.2-kb DNA fragment (Jarraud, J. et al. 1999. J. Clin. Microbiol. 37:2446-2449). Sequence analysis of seg-sei intergenic DNA and flanking regions revealed three enterotoxin-like open reading frames related to seg and sei, designated sek, sel, and sem, and two pseudogenes, psi ent1 and psi ent2. RT-PCR analysis showed that all these genes, including seg and sei, belong to an operon, designated the enterotoxin gene cluster (egc). Recombinant SEG, SEI, SEK, SEL, and SEM showed superantigen activity, each with a specific V beta pattern. Distribution studies of genes encoding superantigens in clinical S. aureus isolates showed that most strains harbored such genes and in particular the enterotoxin gene cluster, whatever the disease they caused. Phylogenetic analysis of enterotoxin genes indicated that they all potentially derived from this cluster, identifying egc as a putative nursery of enterotoxin genes.

3' Untranslated Regions↗
PubMed2001

Post-traumatic arachnoiditis: an unusual cause of Brown-Sequard syndrome.

Brown-Sequard syndrome (BSS) is a unilateral cord injury characterised by an ipsilateral motor deficit with contralateral pain and temperature hypoaesthesia. Although there are a variety of causes, the majority of cases are generally of neoplastic origin or are traumatic in origin. We describe a rare cause of Brown-Sequard syndrome as a result of post-traumatic arachnoiditis. Magnetic resonance imaging with the use of thin-slice high-resolution constructive interference in steady state (CISS) and T2-weighted spin-echo sequence were used to demonstrate the cause and appearance of the lesion in the spinal canal and was useful in the assessment and management of the patient. This case illustrates the usefulness of the CISS sequence in MRI for elucidating arachnoiditis.

Adult↗
PubMed2001

Dorso-ulnar osteocutaneous reverse flow flap of the thumb.

Three cases of distal thumb reconstruction with a reverse pedicled osteocutaneous flap taken from the dorso-ulnar aspect of the first metacarpal are presented. Even though the indications are rare, this flap is useful for the reconstruction of distal osteocutaneous defects of the thumb where more complex procedures are not feasible or considered as excessive.

Adult↗
PubMed2001

Ten years of experience with the submental flap.

This article describes the authors' experience with the submental flap over the past 10 years. A brief review of the key points and some refinements in the operative technique are discussed. The results concern 31 patients with a mean age of 57 years. All flaps were pedicled except two. One case of composite flap with bone was used. The mean size of the flap was 11.8 x 5.5 cm, and the mean postoperative stay was 11.1 days. Complications encountered were one case of temporary palsy of the marginal mandibular branch of the facial nerve, one hematoma at the recipient site, and two cases of partial flap loss. Color and texture match were good. The authors believe this flap to have great clinical potential and to be a worthwhile addition to the existing surgical armamentarium.

Adult↗
PubMed2001

Shotgun optical maps of the whole Escherichia coli O157:H7 genome.

We have constructed NheI and XhoI optical maps of Escherichia coli O157:H7 solely from genomic DNA molecules to provide a uniquely valuable scaffold for contig closure and sequence validation. E. coli O157:H7 is a common pathogen found in contaminated food and water. Our approach obviated the need for the analysis of clones, PCR products, and hybridizations, because maps were constructed from ensembles of single DNA molecules. Shotgun sequencing of bacterial genomes remains labor-intensive, despite advances in sequencing technology. This is partly due to manual intervention required during the last stages of finishing. The applicability of optical mapping to this problem was enhanced by advances in machine vision techniques that improved mapping throughput and created a path to full automation of mapping. Comparisons were made between maps and sequence data that characterized sequence gaps and guided nascent assemblies.

Contig Mapping↗
PubMed2001

Perturbation and proinflammatory type activation of V delta 1(+) gamma delta T cells in African children with Plasmodium falciparum malaria.

gamma delta T cells have variously been implicated in the protection against, and the pathogenesis of, malaria, but few studies have examined the gamma delta T-cell response to malaria in African children, who suffer the large majority of malaria-associated morbidity and mortality. This is unfortunate, since available data suggest that simple extrapolation of conclusions drawn from studies of nonimmune adults ex vivo and in vitro is not always possible. Here we show that both the frequencies and the absolute numbers of gamma delta T cells are transiently increased following treatment of Plasmodium falciparum malaria in Ghanaian children and they can constitute 30 to 50% of all T cells shortly after initiation of antimalarial chemotherapy. The bulk of the gamma delta T cells involved in this perturbation expressed V delta 1 and had a highly activated phenotype. Analysis of the T-cell receptors (TCR) of the V delta 1(+) cell population at the peak of their increase showed that all expressed V gamma chains were used, and CDR3 length polymorphism indicated that the expanded V delta 1 population was highly polyclonal. A very high proportion of the V delta 1(+) T cells produced gamma interferon, while fewer V delta 1(+) cells than the average proportion of all CD3(+) cells produced tumor necrosis factor alpha. No interleukin 10 production was detected among TCR-gamma delta(+) cells in general or V delta 1(+) cells in particular. Taken together, our data point to an immunoregulatory role of the expanded V delta 1(+) T-cell population in this group of semi-immune P. falciparum malaria patients.

CD3 Complex↗
PubMed2001

Generation of a humanized, high affinity anti-tissue factor antibody for use as a novel antithrombotic therapeutic.

Blocking the cofactor function of human tissue factor may be beneficial in various coagulation-mediated diseases. The murine antibody D3 binds to the membrane proximal substrate interaction region of human tissue factor and blocks tissue factor function even in the presence of bound factor VIIa. The cloned murine D3 antibody was humanized and affinity matured by exchanging amino acids in the complementarity determining regions as well as in the antibody framework. The humanized antibody, D3H44, bound to tissue factor with a 100-fold increased affinity (KD 0.1 nM) as compared to the original murine and chimeric versions. Depending on the particular disease, different pharmacokinetic properties of the antibody may be required and, therefore, several antibody variants-- F(ab), F(ab')2, IgG2, IgG4 and IgG4b-were generated. In vitro, the humanized D3 antibodies displayed potent inhibition of plasma clotting and tissue factor: factor VIIa-mediated activation of factors IX and X (e.g. D3H44-F(ab')2, IC50(F.X) 47 pM). In addition, D3H44-F(ab')2 completely prevented fibrin deposition in a human ex vivo thrombosis model under venous blood flow conditions (IC50 37 nM). The humanized D3 antibodies may be utilized for treatment of cardiovascular diseases which involve tissue factor activity, e.g. acute coronary syndrome and venous thrombosis.

Amino Acid Sequence↗
PubMed2001

[Technological advances in immuno-oncology: from fundamental concepts to patient immunological monitoring].

Over the past decade, cancer immunology has known several advances due to both basic research and new technologies recently developed in this field. This review will illustrate the impact of some new immunological technologies and how the latter resulted in the exploration of new territories in cancer immunology and the emergence of new concepts that allowed to revisit the immunosurveillance concept and permitted to improve the patient monitoring.

Antigens, Neoplasm↗
PubMed2000

Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire.

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR beta-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vbeta subsets (Vbeta2, Vbeta4, and Vbeta16 positive) with highly conserved TCRbeta complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR beta-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR alpha-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR alpha-chain vs TCR beta-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.

Amino Acid Motifs↗
PubMed2000

Retrovirus-mediated gene transfer in primary T lymphocytes: influence of the transduction/selection process and of ex vivo expansion on the T cell receptor beta chain hypervariable region repertoire.

We have initiated a phase I/II clinical trial, involving the use of herpes simplex thymidine kinase gene (HS-tk)-expressing donor primary T cells, in order to modulate the graft-versus-host disease (GvHD) occurring after allogeneic hematopoietic stem cell transplantation. The preparation of gene-modified T cells (TkTCs) required a 12-day ex vivo culture comprising an initial OKT3 and IL-2 stimulation, a retrovirus-mediated transduction, and a 7-day selection step in the presence of G418 and IL-2. The low transduction efficiency as well as the culture conditions may significantly alter the diversity of the T cell repertoire. We therefore examined the T cell repertoire of HS-tk-expressing T cell samples from 11 different donors by the Immunoscope method. This method analyzes the hypervariable region of the T cell receptor beta chain (TCRBV) by amplifying the complementarity-determining region 3 (CDR3) and determining size diversity. In all examined samples (four of which were infused into patients), all TCRBV subfamilies were represented with, however, a significant skewing within a minority of subfamilies. Kinetic studies demonstrated that this skewing appeared between day 7 and day 12, with dates of appearance variable from one subfamily to another. In addition, the repertoire analysis of two different culture products, harvested and produced at different times from the same donors, suggested that some repertoire abnormalities could be donor specific. Quantitative analysis revealed no major modifications in gene usage, even in skewed TCRBV subfamilies, with a few clonal expansions concerning a limited number of TCRBV subfamilies. Importantly, identical abnormalities were found in control cells grown in parallel under similar conditions but not transduced or selected, thus demonstrating that these abnormalities were not related to the transduction or the selection process, but rather to the ex vivo culture. The initial stimulus used for T cell activation is a major source of TCRBV perturbation, since replacing the OKT3 + IL-2 stimulus by CD3 + CD28 monoclonal antibody-coated beads prevented the occurrence of alterations. Overall, the HS-tk-expressing T cells used in our clinical trial exhibit limited TCR repertoire skewing that is not due to the transduction/selection procedure. However, future T cell gene transfer protocols for clinical trials should be designed to take into account or possibly prevent such T cell repertoire alterations.

CD28 Antigens↗
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↗