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Biomedical subjects

K Ng

Publications and source records attributed to K Ng.

At least 55 records · Page 3Linked to original sources
PubMed1996

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

Lipophosphoglycan (LPG), is the major cell surface molecule of promastigotes of all Leishmania species. It is comprised of three domains: a conserved glycosylphosphatidylinositol anchor linked to a repeating phosphorylated disaccharide (P2; PO4-6Gal beta 1-4Man alpha 1-) backbone and capped with a neutral oligosaccharide. In Leishmania major the backbone is substituted at the C(O)3 of the Galp residue with side chains containing Galp, Glcp and Arap residues whereas in Leishmania donovani the backbone is unsubstituted. We report the solubilization of a (beta 1-3) galactosyltransferase [(beta 1-3)GalT] from a L. major microsomal preparation using Triton X-100. Solubilization occurs with a 10-fold stimulation of enzyme activity. This (beta 1-3)GalT specifically transfers Gal residues from UDP-Gal to exogenously added L. donovani LPG acceptor. Depolymerization of the [14C]Gal-labelled LPG product with mild acid and analysis by high-performance anion-exchange chromatography detected only the phosphotrisaccharide. (P3; PO4-6([14C]Gal beta 1-3-4Man alpha 1-) found in L. major LPG. This contrasts with the activity of the membrane-bound enzyme which also synthesizes the larger phosphosaccharide units[Ng, Handman and Bacic (1994) Glycobiology 4, 845-853]. This suggests that more than one (beta 1-3)GalT is involved in the addition of these Gal units and that the solubilized activity is the (beta 1-3)GalT that adds the first beta Gal residue to the acceptor. The (beta 1-3)GalT was partially purified by lectin-affinity chromatography and used to establish the K(m) values for UDP-Gal (445 microM) and L. donovani acceptor (280 microM as P2 molar equivalent) in kinetic assays. Inhibition studies with various glycosides and mono- and di-saccharides established the P2 repeating unit as the minimum acceptor structure recognized by (beta 1-3)GalT. The detergent-solubilized (beta 1-3)GalT was reversibly inactivated by millimolar concentrations of univalent anionic salts. The (beta 1-3)GalT had an absolute requirement for Mn2+ and also required Mg2+ for optimum activity; Mg2+ cannot substitute for Mn2+, which is loosely bound to beta (1-3)GalT and is probably involved in the correct folding of the enzyme. The (beta 1-3)GalT was unaffected by Ca2+ ions, but were irreversibly inactivated by micromolar levels of transition metal ions (Cu2+ > Zn2+ > Ni2 > Co2+). The (beta 1-3)GalT activity was also inhibited by diethyl pyrocarbonate, but not by N-ethylmaleimide or iodoacetamide, suggesting that active-site histidine residues, rather than cysteine residue(s), are important for enzyme activity.

Animals↗
PubMed1996

Enhanced manipulation of directional coronary atherectomy and coronary angioplasty guide catheters.

Manipulation of thin-walled coronary guiding catheters, especially directional coronary atherectomy guides, may sometimes be difficult. This is particularly so in patients with tortuous or ectatic aorto-iliac arterial systems. To improve torque transmission and eliminate kinking, the introducer catheter or a smaller caliber diagnostic catheter is advanced to within 1.5 cm of the distal guide tip. Connecting the rotating hemostatic valve to the introducer hub or diagnostic catheter proximally allows distal contrast injections and pressure monitoring via this inner catheter. Other methods of enhancing guide catheter manipulation are also discussed.

Atherectomy, Coronary↗
PubMed1996

Characterization of lipophosphoglycan from a ricin-resistant mutant of Leishmania major.

One of the virulence factors of the protozoan parasite Leishmania major is the surface glycoconjugate, lipophosphoglycan (LPG). A Ricin-resistant mutant of L.major was generated and characterised with respect to its virulence in mice and the structure and expression of LPG. The LPG from this mutant (1F6-B5) retained the tripartite structure of wild-type LPG, comprising a glycosylphosphatidylinositol (GPI) anchor linked to a phosphorylated disaccharide backbone terminating in a nonreducing neutral oligosaccharide cap. The structure of the GPI anchor and the major capping oligosaccharide were identical to wild-type LPG. However, there were variations in the number of phosphorylated repeats (PO4-6Gal(beta 1-4)Man(alpha 1-) comprising the backbone of LPG, although the degree of substitution with side branches (approx. 95%) was similar to that of wild-type LPG. Thus, the mutant LPG was shorter in length having, on average, 15 repeat units per molecule compared with 30 in the wild-type LPG. The mutant LPG contained both arabinose (Ara(beta 1-2)[Gal(beta 1-3)-]1,2) and galactose ([Gal(beta 1-3)-]1-8) capped side branches linked to the backbone. In contrast to wild-type LPG, the number of arabinose-capped side chains was significantly reduced, and a new population of galactose-capped (Gal(beta 1-3)]5-8) side branches was present. The level of LPG expression in mutant parasites was approximately one-tenth of the wild-type parasite. The mutant parasites were avirulent in mice. Over a period of 18 months, they did not cause lesions and organisms could not be isolated from the draining lymph nodes.

Animals↗
PubMed1996

Specificity of binding of beta-glucoside activators of ryegrass (1-->3)-beta-glucan synthase and the synthesis of some potential photoaffinity activators.

Structure-activity relationships among glycoside activators of ryegrass (Lolium multiflorum) (1-->3)-beta-glucan synthase were investigated using a number of natural and synthetic glycosides, including some carrying photoaffinity functions. There is an absolute requirement for a beta-D-glycosyl moiety in the activator, both S- and N-glucosides are active, and the position of the glucosidic linkage in beta-glucose disaccharides has a significant effect on the affinity of binding. However, the binding requirement does not extend beyond a single beta-D-glucosyl residue, and beta-D-oligoglucosides are less effective than disaccharides. The nature of the aglycon has a major influence on the binding affinity. Hydrophobic aglycons lower the concentration required for half-maximal stimulation of the enzyme obtained from an Eadie-Hofstee plot of kinetic data (Ka) for activation, but charge aglycons increase Ka. Relative to methyl-beta-D-glucoside and cellobiose (Ka 1.1 mM), the most potent compounds tested were N-[4-(benzoyl)benzoyl]-beta-D-glucosylamine and 2'-[4-azidosalicylamino]ethyl-1-thio-beta-D-glucoside with K(a)s of approximately 30 microM. The latter also was tested for its potential to specifically label the beta-glucoside-binding site on the synthase, but under the conditions used the binding was found to be nonspecific.

Enzyme Activation↗
PubMed1995

Chondroitin sulfate A is a cell surface receptor for Plasmodium falciparum-infected erythrocytes.

Adherence of Plasmodium falciparum-infected erythrocytes to cerebral postcapillary venular endothelium is believed to be a critical step in the development of cerebral malaria. Some of the possible receptors mediating adherence have been identified, but the process of adherence in vivo is poorly understood. We investigated the role of carbohydrate ligands in adherence, and we identified chondroitin sulfate (CS) as a specific receptor for P. falciparum-infected erythrocytes. Parasitized cells bound to Chinese hamster ovary (CHO) cells and C32 melanoma cells in a chondroitin sulfate-dependent manner, whereas glycosylation mutants lacking chondroitin sulfate A (CSA) supported little or no binding. Chondroitinase treatment of wild-type CHO cells reduced binding by up to 90%. Soluble CSA inhibited binding to CHO cells by 99.2 +/- 0.2% at 10 mg/ml and by 72.5 +/- 3.8% at 1 mg/ml, whereas a range of other glycosaminoglycans such as heparan sulfate had no effect. Parasite lines selected for increased binding to CHO cells and most patient isolates bound specifically to immobilized CSA. We conclude that P. falciparum can express or expose proteins at the surface of the infected erythrocyte that mediate specific binding to CSA. This mechanism of adherence may contribute to the pathogenesis of P. falciparum malaria, but has wider implications as an example of an infectious agent with the capacity to bind specifically to cell-associated or immobilized CS.

Animals↗
PubMed1995

Optical studies of a bacterial photoreceptor protein, photoactive yellow protein, in single crystals.

Photoactive yellow protein (PYP), isolated from Ectothiorhodospira halophila, is a water soluble, 14 kDa photoreceptor protein with a fully reversible photocycle resembling that of sensory rhodopsin II. We have established the presence of photoactivity in PYP crystals and defined the relaxation kinetics of spectroscopically distinguishable species in quantitative terms. The PYP crystal has a bright yellow color and displays pronounced anisotropic absorption properties. Linear dichroism measurements show that the transition moment of the PYP chromophore makes an angle of 73 degrees (or 107 degrees) with respect to the six-fold crystallographic symmetry axis. The crystal absorbance can be bleached reversibly as indicated by absorption changes. A bleached photostationary state in the crystal can be established via CW laser illumination, and the extent of crystal bleaching is found to be clearly dependent on excitation laser wavelength, intensity and illumination time. These results provide the information for designing time-resolved crystallography experiments in which a minimum perturbation is applied to the PYP crystals. Global exponential fitting shows that the relaxation from the photostationary state in the crystal is biphasic at -4 degrees C; a slower component of 1.4 +/- 0.2 s-1 accounts for 60% of the absorbance change and a faster component of 5.2 +/- 0.9 s-1 for the other 40%. As a control, we found that the kinetics for the same relaxation in solution are well described by one exponential and agree quantitatively with previous studies. The two rate constants observed in the crystal show similar temperature dependences, with activation energies for the slow and fast components of 11.7 +/- 1.2 and 5.5 +/- 2.3 kcal/mol, respectively. However, the amplitudes associated with the two exponents show different and opposite temperature dependence. Our results show that the solution kinetic model is not directly applicable to crystals. A kinetic model consistent with the optical data is important to extract the underlying structural intermediates from the time-resolved X-ray diffraction data obtained in parallel with the optical data described here. We propose an alternative model for the photocycle in the crystal which contains an additional bleached intermediate in parallel with the last long-lived intermediate in the solution model.

Bacterial Proteins↗
PubMed1995

Modulation of cell phenotype in human osteoblast-like cells by the simian virus 40.

At present, the majority of in vitro research into bone metabolism is performed on either primary cultures of bone or osteosarcoma lines. A better model of the behaviour of normal bone cells would be a cell line derived from normal, adult bone that retained osteoblast-like characteristics. We infected a culture of bone cells from adult humans with simian virus 1613, a variant of the simian virus 40, and obtained 12 clones of variable morphology. The clones were maintained in culture for as long as 6 months. Population doubling times, synthesis of alkaline phosphatase and osteocalcin, secretion of mineral, morphology, and ability to withstand freezing were examined. SV/EC cell morphology varied from the polygonal, osteoblast-like to the bipolar, fibroblast-like. Population doubling times ranged from 0.55 to 2.8 days (compared with 3.9 days for the nontransformed human osteoblast-like cells). Synthesis of alkaline phosphatase varied but was less than that by the human osteoblast-like cells. With the exception of clone 11, all of the transformed clones synthesised mineral in vitro under mineralising conditions. Four clones showed increased synthesis of alkaline phosphatase and increased population doubling times after passaging. All of the clones were successfully frozen and thawed, but, unlike normal human osteoblast-like cells, none responded to stimulation with parathyroid hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed1995

Prediction of employment status 2 years after traumatic brain injury.

The present study used a multivariate approach to investigate which of a range of variables relating to demographic factors, injury severity and degree of disability on admission to rehabilitation were the best predictors of employment status 2 years after traumatic brain injury (TBI). Subjects were 74 TBI patients who had been working prior to injury, had undergone rehabilitation at Bethesda Hospital and attended a review clinic 2 years after injury. A cross-validation sample consisted of a further 50 such subjects. Following preliminary analysis four input variables were selected: age under or over 40 at time of injury, Glasgow Coma Scale score on acute hospital admission, duration of post-traumatic amnesia and total score on the Disability Rating Scale (DRS) on admission to rehabilitation. Stepwise discriminant function analysis resulted in a discriminant function consisting of three variables--total score on the Disability Rating Scale, Glasgow Coma Scale Score and age--which correctly classified 74% of grouped cases. A second analysis using the original discriminant function correctly classified 68% of the cross-validation sample. Chi-square analysis showed no significant difference between these results, thus confirming these variables, in combination, as predictors of employment status 2 years after TBI.

Activities of Daily Living↗
PubMed1994

Kinetic mechanism of ATP-sulphurylase from rat chondrosarcoma.

ATP-sulphurylase catalyses the production of adenosine 5'-phosphosulphate (APS) from ATP and free sulphate with the release of PPi. APS kinase phosphorylates the APS intermediate to produce adenosine 3'-phosphate 5'-phosphosulphate (PAPS). The kinetic mechanism of rat chondrosarcoma ATP-sulphurylase was investigated by steady-state methods in the physiologically forward direction as well as the reverse direction. The sulphurylase activity was coupled to APS kinase activity in order to overcome the thermodynamic constraints of the sulphurylase reaction in the forward direction. Double-reciprocal initial-velocity plots for the forward sulphurylase intersect to the left of the ordinate for this reaction. KmATP and Kmsulphate were found to be 200 and 97 microM respectively. Chlorate, a competitive inhibitor with respect to sulphate, showed uncompetitive inhibition with respect to ATP with an apparent Ki of 1.97 mM. Steady-state data from experiments in the physiologically reverse direction also yielded double-reciprocal initial-velocity patterns that intersect to the left of the ordinate axis, with a KmAPS of 39 microM and a Kmpyrophosphate of 18 microM. The results of steady-state experiments in which Mg2+ was varied indicated that the true substrate is the MgPPi complex. An analogue of APS, adenosine 5'-[beta-methylene]phosphosulphate, was a linear inhibitor competitive with APS and non-competitive with respect to MgPPi. The simplest formal mechanism that agrees with all the data is an ordered steady-state single displacement with MgATP as the leading substrate in the forward direction and APS as the leading substrate in the reverse direction.

Adenosine Phosphosulfate↗
PubMed1994

Kinetic mechanism of adenosine 5'-phosphosulphate kinase from rat chondrosarcoma.

Biosynthesis of the activated sulphate donor adenosine 3'-phosphate 5'-phosphosulphate (PAPS) involves the sequential action of two enzyme activities. ATP-sulphurylase catalyses the formation of APS (adenosine 5'-phosphosulphate) from ATP and free sulphate, and APS is then phosphorylated by APS kinase to produce PAPS. Initial-velocity patterns for rat chondrosarcoma APS kinase indicate a single-displacement formal mechanism with KmAPS 76 nM and KmATP = 24 microM. Inhibition studies using analogues of substrates and products were carried out to determine the reaction mechanism. An analogue of PAPS, adenosine 3'-phosphate 5'-[beta-methylene]phosphosulphate, exhibited competitive inhibition with APS and non-competitive inhibition with ATP. An analogue of APS, adenosine 5'-[beta-methylene]phosphosulphate was also competitive with APS and non-competitive with ATP. Adenosine 5'-[beta gamma-imido]triphosphate showed competitive inhibition with respect to ATP and produced mixed-type inhibition, with a pronounced intercept effect and a small slope effect, with respect to APS. These results are in accord with the formulation of the predominant pathway as a steady-state ordered mechanism with APS as the leading substrate and PAPS as the final product released.

Adenosine Phosphosulfate↗
PubMed1994

Rat chondrosarcoma ATP sulfurylase and adenosine 5'-phosphosulfate kinase reside on a single bifunctional protein.

The sulfate-activation pathway consists of the sequential action of ATP sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) and adenosine 5'-phosphosulfate kinase ATP:adenylylsulfate 3'-phosphotransferase, EC 2.7.1.25). Both sulfurylase and kinase from rat chondrosarcoma were copurified through substrate affinity chromatography using stable analogs of APS (adenosine 5'-phosphosulfate) and PAPS (3'-phosphoadenosine 5'-phosphosulfate). A 56-kDa protein, containing both activities, was then purified to apparent homogeneity through reversed-phase chromatography and observed on denaturing polyacrylamide gels. The molecular mass of the native active unit containing both activities in the purified preparation corresponded to approximately 60 kDa by analytical gel filtration. Coincident binding and elution of ATP sulfurylase and APS kinase by immunoaffinity chromatography, using polyclonal serum generated against the 56-kDa protein, also demonstrated that the enzyme contains both activities. Lastly, a single N-terminal amino acid sequence was obtained from the 56-kDa band isolated by gel electrophoresis. These results all suggest that ATP sulfurylase and APS kinase from rat chondrosarcoma residue on a single bifunctional protein.

Amino Acid Sequence↗
PubMed1994

Photoaffinity labeling of chloroquine-binding proteins in Plasmodium falciparum.

A photoreactive analog of chloroquine, N-(4-(4-diethylamino-1-methylbutylamino)quinolin-6-yl)-4- azi do-2- hydroxybenzamide (referred to as ASA-Q), has been synthesized and shown to mimic the action of chloroquine in possessing substantial antimalarial activity against a chloroquine-sensitive strain of Plasmodium falciparum. As for chloroquine, ASA-Q is less effective at killing drug-resistant strains of malaria, and the resistance can be modulated using the reagent verapamil. ASA-Q has been radiolabeled with Na125I and used as a photoaffinity probe for labeling chloroquine-binding proteins in malaria-infected erythrocytes. Two proteins have been identified with apparent molecular masses of 42 and 33 kDa in both chloroquine-sensitive and chloroquine-resistant strains of malaria. Photoaffinity labeling of the two proteins by iodo-ASA-Q was competitively inhibited by an excess of unlabeled chloroquine. The structurally related antimalarials amodiaquine and quinine also inhibited labeling of the two proteins, while verapamil and doxycycline had no effect. We suggest that the two labeled proteins are the macromolecular targets of chloroquine action in malaria parasites.

Affinity Labels↗
PubMed1994

Magnetic resonance imaging of urinary calculi.

Accurate prediction of the response of an individual patient to lithotripsy remains impossible. Certain factors such as the chemical composition, size, and position of the calculus are known to be important in determining the success rate. This paper reports the use of magnetic resonance imaging (MRI) to evaluate 141 urinary calculi in vitro. A wide range of signals for each chemical type of calculus was found on each of the three imaging sequences used (T1-weighted, T2-weighted, and proton density). None of the chemical groups examined showed a typical MRI profile allowing it to be distinguished from the other groups. Analysis of variance showed a statistical difference between signals for apatite and struvite on the T1-weighted sequence, and between struvite and uric acid on the proton density sequence (both, P < 0.05). These results show for the first time that MRI is capable of distinguishing between different chemical types of stones. This is particularly important for the comparison of struvite and apatite which appear to be similar in conventional investigations but have quite different hardness values. Further work is in progress correlating the results of this study with stone microhardness and extracorporeal shockwave lithotripsy fragility tests to determine whether MRI accurately predicts the success of lithotripsy.

Humans↗
PubMed1994

Polymerase chain reaction amplification of wildebeest-associated and cervine-derived malignant catarrhal fever virus DNA.

A polymerase chain reaction (PCR) assay was developed for the detection of alcelaphine herpesvirus 1 (AHV1), a causative agent of malignant catarrhal fever (MCF) of ruminants. A pair of 20-base primers was constructed based on the published nucleotide sequence of gene A of the WC11 isolate of AHV1 and was used to amplify a DNA fragment of 413 base pairs. The optimised PCR assay was highly sensitive, i.e. it detected 10 fg of genomic DNA of AHV1 (WC11 isolate). The amplified fragment was shown to be specific for AHV1 DNA by (i) cleavage with XbaI which yielded 2 subfragments of approximately 140 and 280 base pairs and (ii) chemiluminescence Southern blot hybridisation with a digoxigenin-labelled 25-base internal probe. The PCR assay also amplified AHV1 gene sequences in tissue samples from deer and rabbits experimentally infected with materials derived from deer with clinical sheep-associated MCF.

Animals↗
PubMed1994

Secondary hyperparathyroidism, vitamin D deficiency and hip fracture: importance of sampling times after fracture.

There is controversy about how often elevated parathyroid hormone (PTH) levels are found in hip fracture patients. The aim of this study was to determine whether changes in PTH levels after fracture and surgery could explain some of the variation in published data. Blood samples were obtained from 24 elderly patients with hip fracture before surgery, immediately after surgery and at 2 weeks and 3 months after fracture. PTH levels were elevated (> 5.5 pmol) in 33% initially and then fell significantly at 2 weeks in virtually all subjects (P < 0.001) and remained significantly lower after 3 months (n = 17). Although 25-hydroxyvitamin D levels were low (< 30 nmol) in 44% of the patients, the fall in PTH was not explained by alterations in vitamin D metabolites or other measured parameters. The cause of the variation in PTH levels is unknown but measurements immediately after fracture could overestimate the incidence of secondary hyperparathyroidism. Vitamin D deficiency is common in our hip fracture population and is not influenced by hospitalisation.

Aged↗