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Biomedical subjects

K Ng

Publications and source records attributed to K Ng.

At least 73 records · Page 4Linked to original sources
PubMed1994

Biosynthesis of lipophosphoglycan from Leishmania major: characterization of (beta 1-3)-galactosyltransferase(s).

Lipophosphoglycan (LPG) is the major cell surface molecule of promastigotes of all Leishmania species. It is comprised of three domains: a conserved GPI anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6-Gal(beta 1-4)Man(alpha 1-) backbone variously substituted with galactose, glucose and arabinose residues in L.major and capped with a neutral oligosaccharide. Using a microsomal membrane preparation from L.major, we have been able to demonstrate that galactose from UDP-[14C]galactose can be transferred to an endogenous acceptor, characterized as LPG. An in vitro assay was established, based on anion-exchange HPLC, that concurrently identifies and quantitates the products of the galactosyltransferases. We show that the products formed are [14C]galactose-labelled P3 (PO4-6-[Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-), P4b (PO4-6-[Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1-4)Man(alpha 1-) and P5b(PO4-6-[Gal(beta 1-3)Gal(beta 1-3)Gal(beta 1-3)]Gal(beta 1- 4)Man(alpha 1-). These are major galactosylated repeating units of the backbone of L.major LPG. The same products are also formed when LPG from L.donovani, which contains an unbranched backbone of P2 repeats, is used as an exogenous acceptor with L.major microsomal membranes and UDP-[14C]galactose. In addition, no formation of radioactive backbone repeats (P2) was detected in membrane incubations containing UDP-[14C]galactose with or without added unlabelled GDP-mannose, indicating that the addition of the (beta 1-3)-linked galactose branches is independent of the synthesis of the repeating disaccharide (P2) backbone. Preliminary kinetic analyses suggest that the addition of multiple (beta 1-3)-linked galactose residues may be catalysed by more than one (beta 1-3) galactosyltransferase. The (beta 1-3)galactosyltransferase(s) activity was not detected in microsomal membrane preparations from promastigotes of L.donovani.

Animals↗
PubMed1994

Immunoscintigraphy of human colon cancer xenografts in nude mice using a second-generation TAG-72 monoclonal antibody labelled with 99Tcm.

Monoclonal antibody CC83 is a second-generation high-affinity antibody directed against the TAG-72 antigen in colorectal cancer. Our objectives were to evaluate the biodistribution, pharmacokinetics and imaging properties of CC83 labelled with 99Tcm via a modified Schwartz technique. The immunological integrity of 99Tcm-CC83 was evaluated by size-exclusion FPLC and by determining the immunoreactive fraction in vitro against bovine submaxillary mucin. The biodistribution of 99Tcm-CC83 up to 24 h postinjection was evaluated in nude mice bearing subcutaneous LS174T human colon cancer xenografts. Blood radioactivity data was fitted to a one-compartment pharmacokinetic model. Images of tumour-bearing mice were obtained at 17-24 h postinjection with 99Tcm-CC83. 99Tcm-CC83 was eluted as intact immunoglobulin by FPLC analysis and the mean immunoreactive fraction was 0.49 +/- 0.15. Tumour uptake at 24 h postinjection was 11.2 +/- 4.1% i.d.g-1. Radioactivity in the blood was eliminated rapidly with a half-life of 8 h and tumour:blood ratios were > 2:1 at 24 h postinjection. LS174T tumours were successfully imaged in 3/3 mice. In vitro studies showed instability of 99Tcm-CC83 when challenged with cysteine and glutathione but not metallothionein, suggesting a metabolic route for the 99Tcm antibody in vivo. We conclude that CC83 labelled directly with 99Tcm retains its immunological integrity and capability specifically to target subcutaneous LS174T human colon cancer tumours hosted in nude mice. These results further suggest that 99Tcm-CC83 may have potential for imaging colorectal cancer in humans.

Animals↗
PubMed1993

Analysis of the syndrome of unilateral neglect.

Using a variety of clinical measures of unilateral neglect 40 right hemisphere lesioned stroke subjects were compared in terms of neuropsychological deficit. The results of the study encourage the view that unilateral neglect is a complex syndrome and subjects who indicate the disorder may vary in systematic dimensions. There are at least two factors contributing to the neglect syndrome--a scanning factor of external stimuli and a disrupted internal representation of space factor. Both the obtained factors were correlated with functional independence at 6 months post-stroke in a further group of 27 right hemisphere lesioned stroke patients. It is probable that most commonly these factors co-exist and form the classical neglect syndrome but theoretically they may occur separately and this leads to implications for management.

Attention↗
PubMed1993

Evaluation of the polymerase chain reaction method for identifying members of the Anopheles gambiae (Diptera: Culicidae) complex in southern Africa.

Chromosomally and electrophoretically identified field samples of members of the Anopheles gambiae Giles complex from southern Africa and colony material were used to test the polymerase chain reaction (PCR) method of identification. The samples included An. arabiensis Patton, An. quadriannulatus Theobald, and An. merus Donitz. Only one of the 217 specimens examined was disputed. Twenty specimens (9%) were reamplified because of the presence of multiple bands or lack of amplification after the first assay. Specimens of An. merus from this region displayed both the diagnostic An. merus PCR band as well as the An. quadriannulatus band, possibly because of interspecific hybridization or random variation within the population. This work extends the geographical range over which the PCR technique has been verified to southern Africa.

Africa, Southern↗
PubMed1993

Characterization of the 1,3-beta-glucan synthase of Aspergillus fumigatus.

1,3-beta-Glucan synthase activity has been detected in a membrane fraction extracted from the mycelium of the filamentous fungus Aspergillus fumigatus. The enzyme was solubilized by CHAPS and stabilized by filtration on a Bio-gel P30 column. Highest activity was obtained in the early exponential phase of growth. Four factors--GTP, NaF, sucrose and EDTA--added during the extraction procedure, were essential for optimal 1,3-beta-glucan synthase activity. The soluble enzyme preparation was photolabelled with 5-azido-[32P]UDP-glucose and 5-125IASA-UDP-glucose which bind covalently to the enzyme after UV irradiation. These UDP-glucose substrate analogues were competitive inhibitors of the enzyme with a Ki of 1.42 mM and 0.3 mM for 5-azido-UDP-glucose and 5-ASA-UDP-glucose, respectively (Km for UDP-glucose = 1.9 mM). Potential UDP-glucose-binding polypeptides were identified with molecular masses of 31, 50 and 115 kDa.

Affinity Labels↗
PubMed1993

Immune reconstitution following peripheral blood stem cell transplantation, autologous bone marrow transplantation and allogeneic bone marrow transplantation.

The rate and pattern of recovery of total lymphocytes, T cell subsets, B cells and NK cells were compared for 12 months following recovery phase peripheral blood stem cell (PBSC) autotransplantation (n = 49), autologous (n = 7) and allogeneic BMT (n = 11). The PBSC group had a significantly faster recovery of total lymphocyte count, total T cells (CD3+ cells), CD8 cells and CD4 cells than the allogeneic BMT group. The pattern of earlier recovery of CD8 cells than CD4 cells was the same for each type of transplant. Reconstitution following autologous BMT was intermediate between PBSC and allogeneic BMT. Multivariate analysis identified type of transplant, number of mononuclear cells transplanted and conditioning regimen as significantly influencing immune recovery.

B-Lymphocyte Subsets↗
PubMed1993

Detection of MYCN gene amplification and deletions of chromosome 1p in neuroblastoma by in situ hybridization using routine histologic sections.

BACKGROUND: Amplification of the MYCN oncogene and partial deletion of chromosome 1p are genetic changes frequently seen in neuroblastoma that are indicators of poor prognosis. The identification techniques usually used--Southern blotting for MYCN gene amplification, and karyotypic analysis of viable tumor cells for large 1p deletions--are time-consuming and suited for specialized laboratories. EXPERIMENTAL DESIGN: We have developed an immunofluorescence in situ hybridization assay suitable for detection of MYCN amplification and 1p deletion in formalin-fixed, paraffin-embedded tissue sections. The technique is rapid, does not involve the use of radioactivity, and can be carried out in laboratories already familiar with conventional in situ hybridization and immunohistochemical detection methods. RESULTS: MYCN gene amplification appeared as multiple signals per nucleus, corresponding to double minute chromosomes. Deletion of 1p was detected by loss of one of the normal signals present within the nucleus of a neuroblastoma cell line. Use of different fluorophores enabled the simultaneous detection of MYCN gene amplification and 1p deletion at the individual cell level. Detection was found to be enhanced by RNase and pepsin treatment of tissue sections before hybridization and by a novel microwave denaturation method. CONCLUSIONS: Application of this methodology to formalin-fixed samples of neuroblastoma will permit comprehensive retrospective studies of these two genetic markers using archived tumors. In situ hybridization surveys will have widespread applications for studying known genetic aberrations in individual cells of a variety of solid tumors and for determining interrelationships and clinical significance in relation to tumor progression and patient outcome.

Chromosomes, Human, Pair 1↗
PubMed1992

Sorbitol dehydrogenase from Bacillus subtilis. Purification, characterization, and gene cloning.

Cloning of the sorbitol dehydrogenase gene (gutB) from Bacillus subtilis offers an excellent system for studying zinc binding, substrate specificity, and catalytic mechanism of this enzyme through protein engineering. As a first step to clone gutB, B. subtilis sorbitol dehydrogenase has been purified to homogeneity and characterized. It is a tetrameric enzyme with a molecular mass of 38 kDa for each subunit. Atomic absorption analysis shows the presence of 1 mol of zinc atom/subunit. Substrate specificity and stereospecificity of the enzyme toward C-2 and C-4 of hexitols were established. Sequence of the first 31 amino acids was determined, and a set of oligonucleotide probes was designed for gene cloning. A positive clone carrying a 5-kilobase pair HindIII insert was isolated and sequenced. Sequence alignment indicated that the deduced amino acid sequence of B. subtilis sorbitol dehydrogenase shows 36% identity in sequence with the liver sorbitol dehydrogenase from sheep, rat, and human. In reference to the sequence of alcohol dehydrogenase, two potential zinc binding sites were identified. Sequence information related to the structure-function relationships of the enzyme is discussed.

Amino Acid Sequence↗
PubMed1992

Sumatriptan and cerebral perfusion in healthy volunteers.

1. The effect of sumatriptan on regional cerebral perfusion was studied in healthy volunteers. 2. Intravenous sumatriptan (2 mg) had no detectable effect on regional cerebral perfusion as measured using a SPECT system with 99technetiumm labelled hexemethylpropyleneamineoxime. 3. Sumatriptan had no effect on pulse, blood pressure or ECG indices. 4. All six volunteers experienced minor adverse effects during the intravenous infusion.

Adult↗
PubMed1992

Extranodal Ki-1 lymphoma in a renal transplant patient.

THe Ki-1 (CD 30) antigen has been identified as a marker of a subset of large cell anaplastic non-Hodgkin's lymphoma that may have a more favourable prognosis. We describe an immunosuppressed renal transplant patient who developed primary extranodal large cell anaplastic Ki-1 lymphoma of T-cell phenotype confined to the sigmoid colon. The patient was treated with local resection of the sigmoid colon and reduction of immunosuppression and at two year follow-up remains free of recurrence. It is recognised that long term immunosuppression in post transplant patients increases the risk of certain malignancies. Localised gut lymphoma may be amenable to local resection as a curative procedure.

Colonic Neoplasms↗
PubMed1991

Dibasic amines as competitive ions improve the resolution between polyanionic nucleotides.

Aliphatic diamines when used as single ion pairing reagents were capable of resolving 3'-,5'- and 2'-,5'- nucleotidyl diphosphates from one another while conventional ion pairing reagents did not separate these positional isomers. The use of 1,2-diamines resulted in the greatest resolution while increasing spacing between the amino groups progressively reduced the resolution while increasing the retention volume. A competitive ion pairing system was also developed using triethylamine as an additional ion pairing reagent. Using this system ethylenediamine, 1,2- and 1,3-diaminopropane were nearly equivalent in their ability to resolve adenosine 3'-phosphate 5'-phosphate, from adenosine 2'-phosphate 5'-phosphate, and adenosine 3'-phosphate 5'-beta-methylenephosphosulfate (3'-mePAPS) from adenosine 2'-phosphate 5'-beta-methylenephosphosulfate (2'-mePAPS), respectively. The ability to easily resolve these positional isomers allows the use of a more simplified synthetic procedure that does not involve the use selective protecting groups to specifically phosphorylate the 2' or 3' hydroxyl group. We have used this procedure on a semipreparative scale to obtain small quantities of both mePAPS and 2'-mePAPS for use in enzymatic studies.

Adenine Nucleotides↗
PubMed1991

Synthesis and utilization of a nonhydrolyzable phosphoadenosine phosphosulfate analog.

3'-Phosphoadenosine 5'-phosphosulfate (PAPS) functions as the high-energy sulfate donor for sulfate ester synthesis in all higher organisms. This activated sulfate, like its adenosine 5'-phosphosulfate precursor, is both chemically labile and vulnerable to sulfohydrolase degradation. These obstacles have limited the utility of the native PAPS in the purification and mechanistic description of the numerous PAPS-utilizing enzymes. This paper describes the synthesis of the 2'- and 3'-isomers of a nonhydrolysable, and thus stable, PAPS analog, beta-methylene-PAPS, from the previously described beta-methylene-APS (L. Callahan et al., Anal. Biochem. 177, 67-71, 1989). The method involves phosphorylation of beta-methylene-APS with trimetaphosphate and separation of the resulting mixed 2'(3')-isomers by ion-pair reverse-phase HPLC. The utilization of this analog as an inhibitor of APS kinase and PAPS translocase, two of the numerous PAPS-utilizing activities, as well as an affinity ligand for purification of APS kinase, is described.

Animals↗
PubMed1991

Possible coupling of chemical to structural dynamics in subtilisin BPN' catalyzed hydrolysis.

The viscosity dependence of enzymatic catalysis was examined in subtilisin BPN' catalyzed hydrolysis of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-thiobenzyl ester. The viscosity of the reaction medium was varied by added glycerol, ethylene glycol, sucrose, glucose, fructose, poly(ethylene glycol) and Ficoll-400. Responses of the Michaelis-Menten parameters associated with hydrolysis were calculated from data obtained by spectrophotometric techniques. The reactions with these two substrates have catalytic rates well below the diffusion-controlled limit and thus enable us to study the viscosity effects on catalytic steps of non-transport nature. It was found that the Km values for both amide and ester reactions remained relatively independent of cosolvents. On the other hand, while the kcat values for amide were insensitive to cosolvents, those for ester were substantially attenuated except in the case of poly(ethylene glycol). The observed rate attenuations cannot be explained by changes in proton activity, water activity, dielectric constant of the reaction medium or shifts of any kinetically important pKa. Instead, the results can be adequately described by microviscosity effects on the unimolecular deacylation step with a coupling constant of 0.65 +/- 0.11. In addition, the different viscosity dependence in the acylation vs deacylation step can be rationalized in terms of fluctuation-dependent chemical dynamics of proton transfers in the context of the Bogris-Hynes model.

Amino Acid Sequence↗