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Biomedical subjects

E Lim

Publications and source records attributed to E Lim.

At least 55 records · Page 3Linked to original sources
PubMed1997

Calcineurin is required for virulence of Cryptococcus neoformans.

Cyclosporin A (CsA) and FK506 are antimicrobial, immunosuppressive natural products that inhibit signal transduction. In T cells and Saccharomyces cerevisiae, CsA and FK506 bind to the immunophilins cyclophilin A and FKBP12 and the resulting complexes inhibit the Ca2+-regulated protein phosphatase calcineurin. We find that growth of the opportunistic fungal pathogen Cryptococcus neoformans is sensitive to CsA and FK506 at 37 degrees C but not at 24 degrees C, suggesting that CsA and FK506 inhibit a protein required for C. neoformans growth at elevated temperature. Genetic evidence supports a model in which immunophilin-drug complexes inhibit calcineurin to prevent growth at 37 degrees C. The gene encoding the C. neoformans calcineurin A catalytic subunit was cloned and disrupted by homologous recombination. Calcineurin mutant strains are viable but do not survive in vitro conditions that mimic the host environment (elevated temperature, 5% CO2 or alkaline pH) and are no longer pathogenic in an animal model of cryptococcal meningitis. Introduction of the wild-type calcineurin A gene complemented these growth defects and restored virulence. Our findings demonstrate that calcineurin is required for C. neoformans virulence and may define signal transduction elements required for fungal pathogenesis that could be targets for therapeutic intervention.

Amino Acid Sequence↗
PubMed1997

The effect of early versus late fasciotomy in the management of extremity trauma.

BACKGROUND: Recent reports have demonstrated an increase in the number of complications associated with delayed timing of fasciotomy for trauma. This study examines the effectiveness of early (less than 12 hours) versus late (more than 12 hours) fasciotomy in the injured extremity. METHODS: This is a retrospective review of 88 patients undergoing fasciotomy for extremity trauma admitted to the University of Cincinnati from January 1990 through December 1995. Records were reviewed for demographics, compartment pressures, time and type of fasciotomy, complications, limb salvage, and mortality. Statistical analysis was determined with chi-squared, multivariant regression analysis, and Student's t test with significance at p less than 0.05. RESULTS: Sixty-one (69%) patients had fasciotomy performed before 12 hours and twenty-seven (31%) after 12 hours. Although the rates of infection differed significantly between the two groups (7.3% for early versus 28% for late), the rates of limb salvage and neurologic sequelae were similar. Age, mechanism, shock, associated injuries, and time to fasciotomy were not predictive of complications. CONCLUSIONS: Fasciotomy for trauma is most efficacious when performed early. However, when performed late, it results in similar rates of limb salvage as compared with early fasciotomy but at the increased risk of infection. These results support aggressive use of fasciotomy in extremity trauma regardless of time of diagnosis.

Adult↗
PubMed1995

Mutations that perturb cyclophilin A ligand binding pocket confer cyclosporin A resistance in Saccharomyces cerevisiae.

In complex with the peptidyl-prolyl isomerase cyclophilin A, the immunosuppressive antifungal drug cyclosporin A (CsA) inhibits a Ca2+/calmodulin-dependent protein phosphatase, calcineurin, which regulates signal transduction. We isolated and characterized cyclophilin A mutations that confer CsA resistance in a Saccharomyces cerevisiae strain whose growth is CsA-sensitive. Three mutations (G70S, H90Y, and G102A) alter single amino acids conserved between yeast and human cyclophilin A, which structural analyses implicate in CsA binding to human cyclophilin A. By Western analysis, all three mutant proteins are expressed in yeast. In vitro, two purified mutant cyclophilins (G70S, G102A) retain prolyl isomerase activity and have moderately reduced affinity for CsA and calcineurin but, when bound to CsA, do bind and inhibit calcineurin phosphatase activity. In contrast, the purified H90Y mutant cyclophilin is dramatically decreased in prolyl isomerase activity, CsA affinity, and calcineurin binding and inhibition. These studies identify conserved cyclophilin A residues that participate in CsA binding and catalysis.

Amino Acid Isomerases↗
PubMed1995

Gene disruption with PCR products in Saccharomyces cerevisiae.

We describe here the generation of gene disruption constructs using PCR amplification of selectable markers with primers that provide homology to the target gene of interest. We find that regions of homology as short as 38 to 50 bp suffice to mediate homologous recombination in yeast. We describe applications of this technology to three specific yeast genes that would have been difficult to disrupt with current methods. By dispensing with the need to either clone the gene of interest or engineer a standard disruption construct, this method should facilitate analysis of sequenced genes of unknown function, which will soon include the entire yeast genome.

Base Sequence↗
PubMed1995

Directed tagging of the Arabidopsis FATTY ACID ELONGATION1 (FAE1) gene with the maize transposon activator.

The FATTY ACID ELONGATION1 (FAE1) gene of Arabidopsis is required for the synthesis of very long chain fatty acids in the seed. The product of the FAE1 gene is presumed to be a condensing enzyme that extends the chain length of fatty acids from C18 to C20 and C22. We report here the cloning of FAE1 by directed transposon tagging with the maize element Activator (Ac). An unstable fae1 mutant was isolated in a line carrying Ac linked to the FAE1 locus on chromosome 4. Cosegregation and reversion analyses established that the new mutant was tagged by Ac. A DNA fragment flanking Ac was cloned by inverse polymerase chain reaction and used to isolate FAE1 genomic clones and a cDNA clone from a library made from immature siliques. The predicted amino acid sequence of the FAE1 protein shares homology with those of other condensing enzymes (chalcone synthase, stilbene synthases, and beta-ketoacyl-acyl carrier protein synthase III), supporting the notion that FAE1 is the structural gene for a synthase or condensing enzyme. FAE1 is expressed in developing seed, but not in leaves, as expected from the effect of the fae1 mutation on the fatty acid compositions of those tissues.

Acetyltransferases↗
PubMed1995

Perforation of the sigmoid colon following ingestion of a dental plate.

The authors report on the perforation of the sigmoid colon in a 48-year-old male following the accidental ingestion of a partial dental plate. Sharp, pointed objects may perforate the bowel and surgery should be considered. A review of the literature revealed no previously reported cases.

Colon, Sigmoid↗
PubMed1994

Functional domains of recombinant bactericidal/permeability increasing protein (rBPI23).

The 23-kDa recombinant amino-terminal bactericidal/permeability increasing protein fragment (rBPI23) has all of the antibacterial and antiendotoxin properties of the holoprotein. In the current studies, we have identified multiple active domains within rBPI23 with chemical and proteolytic cleavage fragments and with synthetic overlapping peptides. We also demonstrate a novel, high affinity heparin binding property for rBPI23, in addition to its established bactericidal and lipopolysaccharide binding properties. Cleavage fragments and synthetic, overlapping peptides of rBPI23 were analyzed for inhibition of the lipopolysaccharide-induced Limulus amebocyte lysate reaction, for bactericidal activity, and for heparin binding. Three separate, active domains were identified in amino acid regions 17-45, 65-99, and 142-169. A single synthetic peptide (85-99) was bactericidal. These results indicate that rBPI23 is comprised of three separate functional domains which contribute to the high affinity interaction of rBPI23 with Gram-negative bacteria. The individual activity of each domain and the cooperative interaction among domains provide the basis for developing rBPI23 analogues with increased biologic efficacy.

Amino Acid Sequence↗
PubMed1994

Competition between rBPI23, a recombinant fragment of bactericidal/permeability-increasing protein, and lipopolysaccharide (LPS)-binding protein for binding to LPS and gram-negative bacteria.

Lipopolysaccharide (LPS)-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are two structurally related lipid A-binding proteins with divergent functional activities. LBP mediates activation of macrophage and other proinflammatory cells. In contrast, BPI has potent bactericidal and LPS-neutralizing activities. A recombinant fragment of BPI (rBPI23) retains the potent biological activities of the holo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis, and endotoxemia. For therapeutic effectiveness in many clinical situations, rBPI23 will have to successfully compete with high serum levels of LBP for binding to endotoxin and gram-negative bacteria. The relative binding affinities of rBPI23 and human recombinant LBP (rLBP) for lipid A and gram-negative bacteria were evaluated. The binding of both proteins to lipid A was specific and saturable with apparent Kds of 2.6 nM for rBPI23 and 58 nM for rLBP. rBPI23 was approximately 75-fold more potent than rLBP in inhibiting the binding of 125I-rLBP to lipid A. The binding affinity of rBPI23 (Kd = 70 nM) for Escherichia coli J5 bacteria was also significantly higher than that of rLBP (Kd = 1,050 nM). In addition, rBPI23 at 0.2 micrograms/ml was able to inhibit LPS-induced tumor necrosis factor release from monocytes in the presence of 20 micrograms of rLBP per ml. These results demonstrate that rBPI23 binds more avidly to endotoxin than does rLBP and that, even in the presence of a 100-fold weight excess of rLBP, rBPI23 effectively blocks the proinflammatory response of peripheral blood mononuclear cells to endotoxin.

Acute-Phase Proteins↗
PubMed1993

Effects of gene dosage and sequence modification on the frequency and timing of transposition of the maize element Activator (Ac) in tobacco.

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3' end of the element on germinal excision frequencies. Alteration of three bases to create a Bgl II site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.

Agrobacterium tumefaciens↗
PubMed1993

The role of IgA anti-HLA class I antibodies in kidney transplant survival.

The unusually high 88% one-year cadaver kidney graft survival rate in patients with IgA nephropathy (IgAN) prompted us to investigate the influence of IgA anti-HLA class I antibodies on subsequent graft survival. We found that patients with various original diseases with IgA antibodies to the HLA molecule had high 91% one-year graft survival compared with 58% one-year survival for those who did not have preformed IgA antibodies against the HLA molecule prior to transplantation (P < 0.0005). The IgA antibodies were detected by reaction with class I HLA molecules isolated by capture with monoclonal antibody and detected with an enzyme-linked immunosorbent assay. In contrast, IgG antibodies to the HLA molecule resulted in a lower one-year graft survival rate (74%) than in those patients without IgG antibodies (87%) (p = 0.08). IgA antibodies to the HLA molecule, when present, tended to react at a high frequency on a random lymphocyte panel. These findings suggest that sensitization resulting in IgA anti-HLA antibodies may counteract the deleterious effect of an IgG antibody response in clinical kidney transplantation.

Cells, Cultured↗
PubMed1993

Tidal volume, lung hyperinflation and arterial oxygenation during general anaesthesia.

Impaired pulmonary oxygen (O2) exchange is common during general anaesthesia but there is no clinical unanimity as to methods of prevention or treatment. We studied 14 patients at risk for pulmonary dysfunction because of increased age, obesity, cigarette smoking, or chronic lung disease. Pulmonary O2 exchange was measured during four conditions of ventilation: awake spontaneous, conventional tidal volume (CVT, 7 ml.kg-1) or high tidal volume (HVT, 12 ml.kg-1) controlled ventilation, and five min after manual hyperinflation (HI) of the lungs. The FIO2 was controlled at 0.5, and FETCO2 was kept constant by adding dead space during HVT. Eight patients were ventilated with N2O/O2 and six with air/O2. Arterial blood gases were used to calculate the (A-a)DO2. In seven patients (A-a)DO2 worsened after induction of anaesthesia, while in seven there was no change or an improvement. Manual HI significantly reduced (A-a)DO2, but changing tidal volume (VT) had no effect. Using a multivariate model to predict O2 exchange, obesity and type of surgery were significantly associated with worsening, while level of VT and inspiratory gas (N2O or N2) were not significant predictors. Thus patient and surgical factors were more important determinants of pulmonary gas exchange during anaesthesia than were tidal volume or inspiratory gas. Manual HI is a simple and effective manoeuvre to improve gas exchange.

Adult↗
PubMed1993

Ambulance response time to emergency departments.

The study was conducted to ascertain the time it takes an ambulance team to reach a patient and transport the patient to an emergency department after a 995 call. One hundred and sixty-two cases brought to two emergency departments (Singapore General Hospital and Tan Tock Seng Hospital) between 11 March 1992 and 16 March 1992 were studied. The information was obtained from ambulance officers of the Singapore Civil Defence Force. It took an average of 11.40 +/- 4.88 minutes for an ambulance team to reach a patient and 30.50 +/- 10.62 minutes for the patient to reach an emergency department after a 995 call. With the present level of staff in Singapore, basic life support care starts 11.40 minutes and advanced life support care 30.50 minutes after a 995 call. These times are unacceptable if it involves a cardiac arrest or a trauma patient. Factors which cause these long time intervals include traffic congestion, inadequate public education, location of patient (whether on ground level or highrise) and distance from the emergency departments.

Ambulances↗
PubMed1992

Effects of home bleaching preparations on composite resin color.

The lightening of teeth is done professionally by exposure to warm hydrogen peroxide solutions or at home with an active bleaching gel of perhydrol urea or carbamide peroxide. This study compared the effects of two commercial bleaching gels on the color of composite resin with those in a previous study using the dental office technique. Commercial composite resins test specimens were exposed to the bleaching gels or water (control) for 312 hours at 37 degrees C at 100% relative humidity. Color change value was calculated with before-and-after solution exposure L*a*b* color values. ANOVA (alpha = 0.01) revealed no differences in the color of the specimens after use of the test solutions. All color change values were less than 2 or the normal limit of visual acuity. No visual color change was evident for these composite resins. The at-home method recorded no shade-altering effects compared with the professional technique, which created a noticeable color change.

Analysis of Variance↗
PubMed1992

Germinal and somatic activity of the maize element Activator (Ac) in Arabidopsis.

We have investigated the germinal and somatic activity of the maize Activator (Ac) element in Arabidopsis with the objective of developing an efficient transposon-based system for gene isolation in that plant. Transposition activity was assayed with a chimeric marker that consists of the cauliflower mosaic virus 35S promoter and a bacterial streptomycin phosphotransferase gene (SPT). Somatic activity was detected in seedlings germinated on plates containing streptomycin as green-resistant sectors against a background of white-sensitive cells. Germinal excisions resulted in fully green seedlings. The transposition frequency was extremely low when a single copy of the transposon was present, but appeared to increase with an increase in Ac copy number. Plants that were selected as variegated produced an increased number of green progeny. The methylation state of the Ac elements in lines with either low or high levels of excision was assessed by restriction analysis. No difference was found between these lines, indicating that the degree of methylation did not contribute to the level of Ac activity. Germinal excision events were analyzed molecularly and shown to carry reinserted transposons in about 50% of the cases. In several instances, streptomycin-resistant siblings carried the same transposed Ac element, indicating that excision had occurred prior to meiosis in the parent. We discuss parameters that need to be considered to optimize the use of Ac as a transposon tag in Arabidopsis.

DNA↗
PubMed1992

Obstetric admissions to the intensive care unit--a retrospective review.

A five-year retrospective study of obstetric admissions to the Surgical Intensive Care Unit (SICU) in the National University Hospital, Singapore was carried out with the aim of determining the incidence, causes and outcome of these admissions. Most of the patients were admitted following emergency caesarean sections. Obstetric complications was the reason for admission in 56.8% with hypertensive disease of pregnancy being the major cause and haemorrhage accounting for the rest. Anaesthetic complications accounted for 21.6% of admissions and these included difficult intubation, aspiration pneumonitis, cardiac arrhythmias and respiratory depression. Medical complications due to cardiovascular disease, autoimmune disease and malignancy also accounted for 21.6% of admissions. Only 37 out of 16264 deliveries (0.22%) required intensive care support. The median of duration of stay was one day.

Adult↗
PubMed1992

Reactivity of monoclonal antibody E5 with endotoxin. II. Binding to short- and long-chain smooth lipopolysaccharides.

The murine monoclonal IgM antibody E5 has been shown to significantly reduce the mortality and morbidity of patients with Gram-negative sepsis in a multicenter randomized placebo-controlled clinical trial. The in vitro binding characteristics of monoclonal antibody (mAb) E5 were studied using highly purified smooth lipopolysaccharide (LPS) isolated from a variety of clinically relevant, wild-type Gram-negative bacteria. Using a sensitive antibody-capture assay which involves immobilized mAb E5 and a chromogenic Limulus amebocyte lysate (LAL) LPS-detection system, mAb E5 was shown to bind to all 15 smooth LPS preparations tested, including LPS isolated from Escherichia, Klebsiella, Proteus, Pseudomonas, Salmonella, Serratia and Yersinia species. When LPS was fractionated according to size by size-exclusion chromatography, mAb E5 was shown to bind to smooth LPS molecules that have long as well as short O-polysaccharide chains. These results confirm and extend those reported previously and demonstrate that the anti-lipid A mAb E5 binds specifically to a diverse spectrum of smooth LPS isolated from wild-type Gram-negative bacteria.

Animals↗
PubMed1992

Reactivity of monoclonal antibody E5 with endotoxin. I. Binding to lipid A and rough lipopolysaccharides.

The murine IgM monoclonal antibody (mAb) E5 was produced by a hybridoma derived from spleen cells of a mouse immunized with the J5 rough mutant of Escherichia coli O111:B4. In a multicenter randomized placebo-controlled clinical trial, E5 has been shown to reduce significantly the mortality and morbidity of patients with Gram-negative sepsis. The characteristics of E5 binding to endotoxin were studied in vitro. We report here the results of binding to an extensive panel of rough lipopolysaccharide (LPS) and lipid A preparations. Using standard immunologic techniques, including enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), as well as an antibody capture assay using immobilized antibody and a chromogenic Limulus amebocyte lysate (LAL) detection system, E5 was shown to bind to all rough LPS (chemotypes Ra through Re from Salmonella minnesota and E. coli J5) and lipid A preparations tested. E5 displayed a Kd for Ra LPS of approximately 6.5 nM. These results confirm and extend those reported previously and provide evidence that E5 binds specifically to lipid A and to the lipid A moiety of rough LPS.

Animals↗