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K Lai

Publications and source records attributed to K Lai.

At least 37 records · Page 2Linked to original sources
PubMed1999

Wedge factor dependence with depth, field size, and nominal distance--a general computational rule.

We have investigated the dependence of the wedge factors with field size, depth, nominal, and extended distances for 4, 6, 18, and 24 MV photon beams. Analysis of the experimental data suggests a general linear dependence of the wedge factors with field size and depth. The study shows that changes in wedge factors are insignificant (< or = +/-1.0%) with respect to measurements at nominal SSD, SAD, or extended SSD. This independence of the wedge factors on source-to-surface distance was studied for different photon energies (4-24 MV) and for different attenuating wedges (external and internal wedges). For clinical applications, an algorithm is presented to calculate the wedge factor dependence with field size and depth. The new algorithm has been successfully implemented to replace wedge look-up tables for dose and MU calculations in PRISM 1.2 treatment planning system used in our department.

Algorithms↗
PubMed1998

Probing the inhibitor-binding site of aldose reductase with site-directed mutagenesis.

Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes, and enzyme inhibitors have been proposed as therapeutic agents. While effectively preventing the development of diabetic complications in animals, results from clinical studies of AR inhibitors have been disappointing, possibly due to poor potency in man. To assist in the design of more potent and specific inhibitors, crystallographic studies have attempted to identify enzyme-inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. To confirm and extend these findings we quantified inhibitor activity with single, site-directed, mutant, human AR enzymes in which the apolar active-site residues tryptophan 20, -79, -111 and phenylalanine 115 were replaced with alanine or tyrosine, decreasing the potential for van der Waals interactions. Consistent with molecular models, the inhibitory activity of Tolrestat, Sorbinil and Zopolrestat decreased 800-2000-fold when tested with the mutant enzyme in which Trp20 was replaced with alanine. Further, alanine substitution for Trp111 decreased Zopolrestat's activity 400-fold, while mutations to Trp79 and Phe115 had little effect on the activity of any of the inhibitors. The alanine mutation at Trp111 had no effect on Tolrestat's activity but decreased the activity of Sorbinil by about 1000-fold. These latter effects were unanticipated based on the number of non-bonded interactions between the inhibitors, Tolrestat and Sorbinil, and Trp20 and Trp111 that have been identified in the crystal structures. In spite of these unexpected findings, our results are consistent with the hypothesis that AR inhibitors occupy the enzyme active site and that hydrophobic interactions between the enzyme and inhibitor contribute to inhibitor binding stability.

Aldehyde Reductase↗
PubMed1998

Vascular endothelial growth factor induces heparin-binding epidermal growth factor-like growth factor in vascular endothelial cells.

Although several cytokines and growth factors have been shown to regulate vascular endothelial growth factor (VEGF) production, little is known about how VEGF may regulate growth factors that have known mitogenic and chemotactic actions on mesenchymal cells (which are involved in the maturation of the angiogenic process). We investigated the effect of VEGF on heparin-binding epidermal growth factor-like growth factor (HB-EGF) expression in human umbilical vein endothelial cells. HB-EGF mRNA was induced by 8-fold after 2 h of VEGF stimulation, and it returned to base line within 6 h. VEGF did not alter the half-life of HB-EGF mRNA (55 min). Nuclear run-on experiments showed a 4.9-fold increase in HB-EGF gene transcription within 2 h of VEGF stimulation, and Western analysis demonstrated an associated increase in cellular HB-EGF protein. We found that platelet-derived growth factor-BB (PDGF-BB) mRNA was also induced 3-fold after 5 h of VEGF stimulation, whereas neither endothelin 1 nor transforming growth factor-beta1 was regulated by VEGF. Finally, conditioned medium from VEGF-stimulated endothelial cells produced an increase in DNA synthesis in vascular smooth muscle cells, and this effect was blocked by a neutralizing antibody to PDGF. The induction of HB-EGF and PDGF-BB expression in endothelial cells may represent the mechanism by which VEGF recruits mesenchymal cells to form the medial and adventitial layers of arterioles and venules during the course of angiogenesis.

Cell Movement↗
PubMed1998

Duarte allele impairs biostability of galactose-1-phosphate uridyltransferase in human lymphoblasts.

The Duarte allele (D) is a missense mutation (N314D) that produces a characteristic isoform and partial impairment of galactose-1-phosphate uridyltransferase (GALT) in human erythrocytes, fibroblasts, and transformed lymphoblasts. The position of this amino acid is distant, however, from presumptive catalytic site(s) as deduced from a three-dimensional model of crystallized Escherichia coli galT protein. To evaluate the mechanism(s) involved in the partial impairment of enzymatic activity, we compared the activity, abundance, biological stability, and mRNA of GALT in human lymphoblastoid cell lines cultured from individuals homozygous for wild-type (WT/WT) and Duarte alleles (N314D/N314D). No other nucleotide differences were present in their GALT genes. The apparent Vmax was reduced in N314D/N314D cells to 31 +/- 3.6 compared to WT/WT of 54 +/- 6.5 nmole UDP-galactose formed/g cell protein/hour. Both genotypes had similar apparent KMs for UDP-glucose of 0.142 +/- 0.057 mM and 0.133 +/- 0.056 mM. This reduced Vmax was associated with a reduced abundance of the 86kD GALT dimer as determined by Western blots and densitometry. Using RNase protection assays, this reduced GALT protein in the N314D/N314D cell lines was not associated with reduced abundance of GALT mRNA. Using cycloheximide (3-[2-(3,5-Dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide) inhibition of de novo protein synthesis, GALT enzyme activity, and its dimeric protein had a biological T1/2 of approximately 24 hours in N314D/N314D cell lines as compared to 50 hours for WT/WT lymphoblasts. Upon exposure to 50 degrees C for 15 minutes, N314D/ N314D lymphoblasts retained 45% of GALT activity, whereas controls retained 77% activity. Reduced activity and thermal sensitivity caused by the N314D mutation reverted to control values when a lysine was substituted for a glutamic acid at amino acid 203 in cis (E203K). In summary, N314D/N314D lymphoblasts have reduced GALT enzyme capacity, dimeric protein abundance, biological, and thermal stability. We conclude that the substitution of aspartate for asparagine at amino acid 314 in the human GALT protein reduces the biostability of the active enzyme in human lymphoblasts.

Alleles↗
PubMed1997

Inhibition of growth and p21ras methylation in vascular endothelial cells by homocysteine but not cysteine.

Although hyperhomocysteinemia has been recognized recently as a prevalent risk factor for myocardial infarction and stroke, the mechanisms by which it accelerates arteriosclerosis have not been elucidated, mostly because the biological effects of homocysteine can only be demonstrated at very high concentrations and can be mimicked by cysteine, which indicates a lack of specificity. We found that 10-50 microM of homocysteine (a range that overlaps levels observed clinically) but not cysteine inhibited DNA synthesis in vascular endothelial cells (VEC) and arrested their growth at the G1 phase of the cell cycle. Homocysteine in this same range had no effect on the growth of vascular smooth muscle cells (VSMC) or fibroblasts. Homocysteine decreased carboxyl methylation of p21(ras) (a G1 regulator whose activity is regulated by prenylation and methylation in addition to GTP-GDP exchange) by 50% in VEC but not VSMC, a difference that may be explained by the ability of homocysteine to dramatically increase levels of S-adenosylhomocysteine, a potent inhibitor of methyltransferase, in VEC but not VSMC. Moreover, homocysteine-induced hypomethylation in VEC was associated with a 66% reduction in membrane-associated p21(ras) and a 67% reduction in extracellular signal-regulated kinase 1/2, which is a member of the mitogen-activated protein (MAP) kinase family. Because the MAP kinases have been implicated in cell growth, the p21(ras)-MAP kinase pathway may represent one of the mechanisms that mediates homocysteine's effect on VEC growth. VEC damage is a hallmark of arteriosclerosis. Homocysteine-induced inhibition of VEC growth may play an important role in this disease process.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1997

Retinyl ester hydrolysis and retinol efflux from BFC-1beta adipocytes.

Adipose tissue is an important storage depot for retinol, but there are no data regarding retinol mobilization from adipose stores. To address this, dibutyryl cAMP was provided to murine BFC-1beta adipocytes and its effects on retinol efflux assessed. High performance liquid chromatography analysis of retinol and retinyl esters in adipocytes and media indicated that cAMP stimulated, in a time- and dose-dependent manner, retinol accumulation in the culture media and decreased cellular retinyl ester concentrations. Study of adipocyte retinol-binding protein synthesis and secretion indicated that cAMP-stimulated retinol efflux into the media did not result from increased retinol-retinol-binding protein secretion but was dependent on the presence of fetal bovine serum in the culture media. Since our data suggested that retinyl esters can be hydrolyzed by a cAMP-dependent enzyme like hormone-sensitive lipase (HSL), in separate studies, we purified a HSL-containing fraction from BFC-1beta adipocytes and demonstrated that it catalyzed retinyl palmitate hydrolysis. Homogenates of Chinese hamster ovary cells overexpressing HSL catalyzed retinyl palmitate hydrolysis in a time-, protein-, and substrate-dependent manner, with an apparent Km for retinyl palmitate of 161 microM, whereas homogenates from control Chinese hamster ovary cells did not.

Adipocytes↗
PubMed1997

Black children deficient in galactose 1-phosphate uridyltransferase: correlation of activity and immunoreactive protein in erythrocytes and leukocytes.

A recent study found a high prevalence of a missense mutation (S135L) in the gene for galactose 1-phosphate uridyltransferase (GALT) in black children with galactosemia (J Pediatr 1996; 128:89-95). In the present study, GALT activity and GALT protein content were measured in erythrocytes and leukocytes of eight black and seven white galactosemic (GALT-deficient) children, for correlation with the presence of the S135L and Q188R (highly prevalent in white galactosemic children) missense mutations. The S135L mutation was found in 9 of 16 alleles of black children but not in white children; the Q188R mutation was found in 10 of 14 alleles examined in white galactosemic children and in 4 of 16 alleles in black galactosemic children. The GALT activity was near zero in the erythrocytes of white and black galactosemic children (0.26 +/- 0.28 vs 0.33 +/- 0.25 mumol/hr per gram of hemoglobin, respectively; p = 0.61) (normal 17 to 26 mumol/hr per gram), and no correlation of erythrocyte activity with genotype was observed. The GALT activity was higher in the leukocytes of black galactosemic children compared with white children (5 +/- 6 vs 1 +/- 2 mumol/hr per gram, respectively) (normal 172 to 374 mumol/hr per gram), but the difference was not statistically significant (p = 0.11). Analysis by genotype revealed that the two S135L homozygotes had much more leukocyte activity (9 and 17 mumol/hr per gram) than Q188R homozygotes or than all non-S135L allelic genotypes. Compound heterozygotes (S135L/G) had intermediate activity. The GALT protein was not detectable by Western blot in the erythrocytes of either white or black galactosemic children, as determined by antibodies specific for both C- and N-terminal sequences. The GALT protein was undetectable in the leukocytes of white galactosemic children, but leukocytes from black galactosemic children with the S135L mutation contained reduced but readily detectable GALT protein. Erythrocyte galactose 1-phosphate levels were significantly lower in galactosemic children with an S135L mutant allele (1.1 +/- 0.2 gm/dl) compared with children who had other mutations (3.1 +/- 0.9 mg/dl; p = 0.0001). The correlation of protein content data with activity levels in the blood cells suggests that the S135L missense mutation affects the stability of GALT protein to produce a deficiency state.

Adolescent↗
PubMed1997

Molecular basis for Duarte and Los Angeles variant galactosemia.

Human orythrocytes that are homozygous for the Duarte enzyme variant of galactosemia (D/D) have a characteristic isoform on isoelectric focusing and 50% reduction in galactose-1-phosphate uridyltransferase (GALT) enzyme activity. The Duarte biochemical phenotype has a molecular genotype of N314D/N314D. The characteristic Duarte isoform is also associated with a variant called the "Los Angeles (LA) phenotype," which has increased GALT enzyme activity. We evaluated GALT enzyme activity and screened the GALT genes of 145 patients with one or more N314D-containing alleles. We found seven with the LA biochemical phenotype, and all had a 1721C-->T transition in exon 7 in cis with the N314D missense mutation. The 1721C-->T transition is a neutral polymorphism for leucine at amino acid 218 (L218L). In pedigree analyses, this 1721C-->T transition segregated with the LA phenotype of increased GALT activity in three different biochemical phenotypes (LA/N, LA/G, and LA/D). To determine the mechanism for increased activity of the LA variant, we compared GALT mRNA, protein abundance, and enzyme thermal stability in lymphoblast cell lines of D and LA phenotypes with comparable genotypes. GALT protein abundance was increased in LA compared to D alleles, but mRNA was similar among all genotypes. When LA/D and D/D GALT biochemical phenotypes were compared to N/N GALT phenotypes, both had 50%, as compared to 21%, reduction in GALT activity in the wild type (N/N) after exposure at identical initial enzyme activity to 50 degrees C for 15 min. We conclude that the codon change N314D in cis with the base-pair transition 1721C-->T produces the LA variant of galactosemia and that this nucleotide change increases GALT activity by increasing GALT protein abundance without increasing transcription or decreasing thermal lability. A favorable codon bias for the mutated codon with consequently increased translation rates is postulated as the mechanism.

Alleles↗
PubMed1996

Mitogen-activated protein kinase phosphatase-1 in rat arterial smooth muscle cell proliferation.

Smooth muscle cell proliferation and migration is important in arteriosclerosis. In this process, cytokines and growth factors are upregulated and bind to their respective receptors, which in turn stimulate mitogen-activated protein (MAP) kinases. MAP kinases then relay signals to the nucleus that activate quiescent smooth muscle cells. Phosphatases downregulate MAP kinases. We investigated the role of a dual-specificity tyrosine phosphatase, MAP kinase phosphatase-1 (MKP-1), in smooth muscle cell proliferation. MKP-1 expression was high in arterial tissue by Northern analysis, and MKP-1 message was detected mainly in the arterial smooth muscle layer by in situ hybridization. After balloon injury of the rat carotid artery, expression of MKP-1 decreased greatly, whereas that of MAP kinases, especially p44 MAP kinase, increased. The time course of the reduction in MKP-1 message correlated with increased tyrosine phosphorylation and elevated p44 MAP kinase enzymatic activity. In rat arterial smooth muscle cells overexpressing MKP-1, growth was arrested in the G1 phase and entry into the S phase was blocked. A reduction in MKP-1 expression may contribute in part to proliferation of smooth muscle cells after vascular injury, possibly through a decrease in dephosphorylation of p44 MAP kinase.

Angioplasty, Balloon↗
PubMed1996

A prevalent mutation for galactosemia among black Americans.

OBJECTIVE: To define the mutation causing galactosemia in patients of black American origin who have no galactose-1-phosphate uridyltransferase (GALT) activity in erythrocytes but good clinical outcome. METHODS: We discovered a mutation caused by a C-->T transition at base-pair 1158 of the GALT gene that results in a serine-to-leucine substitution at codon 135 (S135L). We developed a method with which to screen populations for its prevalence. We compared galactose-1-phosphate uridyltransferase among erythrocytes, leukocytes, and transformed lymphoblasts, as well as total body oxidation of D-(13C)-galactose to 13CO2 among three genotypes for GALT (S135L/S135L, Q188R/Q188R, and Normal/Normal). RESULTS: We found a 48% prevalence of the S135L mutation among 17 black American patients with classic galactosemia and a 1% prevalence in a population of 50 black Americans without galactosemia. The S135L mutation was not found in 84 white patients with G/G galactosemia nor in 87 white control subjects without galactosemia. We found normal whole body oxidation of D-(13C)-galactose by the patient homozygous for S135L and various degrees of enzyme impairment among different tissues. CONCLUSIONS: The S135L mutation in the GALT gene is a prevalent cause of galactosemia among black patients. Because GALT activity varies in different tissues of patients homozygous for S135L, they may have a better clinical outcome than patients who are homozygous for Q188R when both are treated from infancy.

Base Sequence↗
PubMed1996

Inositol transport in Saccharomyces cerevisiae is regulated by transcriptional and degradative endocytic mechanisms during the growth cycle that are distinct from inositol-induced regulation.

Regulation of inositol uptake activity in Saccharomyces cerevisiae during the growth cycle was examined. Activity increased as the cell population transited from lag phase to exponential growth, and continued to increase until late exponential phase. The increase in activity was due to increased transcription of the ITR1 gene and synthesis of the Itr1 permease. When the culture reached stationary phase, uptake activity decreased and dropped to a minimum within 4 h. The decrease was due to repression of ITR1 transcription, independent of the negative regulator Opi1p, and degradation of the existing permease. Degradation depended on delivery of the permease to the vacuole through the END3/END4 endocytic pathway. During exponential growth in inositol-containing medium the permease is also rapidly degraded, whereas in inositol-free medium the permease is highly stable. Rapid degradation of the permease at stationary phase occurred in inositol-free medium, indicating that there are two distinct mechanisms that trigger endocytosis and degradation in response to different physiological stimuli. In addition, the level of the enzyme required for inositol biosynthesis, inositol-1-phosphate synthase, encoded by INO1, is not reduced in stationary-phase cells, and this contrast in the regulation of inositol supply is discussed.

Biological Transport↗
PubMed1996

Querulous paranoia in Chinese patients: a cultural paradox.

OBJECTIVE: There has been no reported case of querulous paranoia from the Asian population. A prospective study was undertaken to identify patients with querulous paranoia in an outpatient clinic. METHOD: One thousand, five hundred and fifty-one new referrals to a university-affiliated psychiatric outpatient clinic in Hong Kong were screened for querulous paranoia during routine clinical work. RESULTS: Three patients with querulous paranoia (0.19%) were identified during 1 year. The case histories of these three patients are reported. CONCLUSION: Possible reasons for the low reporting rate are discussed and the importance of sociocultural traditions in the development and recognition of querulous paranoia is emphasised.

Adult↗
PubMed1995

Characterization of P-S bond hydrolysis in organophosphorothioate pesticides by organophosphorus hydrolase.

The extensive use of organophosphorothioate insecticides in agriculture has resulted in the risk of environmental contamination with a variety of broadly based neurotoxins that inhibit the acetylcholinesterases of many different animal species. Organophosphorus hydrolase (OPH, EC 3.1.8.1) is a broad-spectrum phosphotriesterase that is capable of detoxifying a variety of organophosphorus neurotoxins by hydrolyzing various phosphorus-ester bonds (P-O, P-F, P-CN, and P-S) between the phosphorus center and an electrophilic leaving group. OPH is capable of hydrolyzing the P-X bond of various organophosphorus compounds at quite different catalytic rates: P-O bonds (kcat = 67-5000 s-1), P-F bonds (kcat = 0.01-500 s-1), and P-S bonds (kcat = 0.0067 to 167 s-1). P-S bond cleavage was readily demonstrated and characterized in these studies by quantifying the released free thiol groups using 5,5'-dithio-bis-2-nitrobenzoic acid or by monitoring an upfield shift of approximately 31 ppm by 31P NMR. A decrease in the toxicity of hydrolyzed products was demonstrated by directly quantifying the loss of inhibition of acetylcholinesterase activity. Phosphorothiolate esters, such as demeton-S, provided noncompetitive inhibition for paraoxon (a P-O triester) hydrolysis, suggesting that the binding of these two different classes of substrates was not identical.

Aryldialkylphosphatase↗
PubMed1995

Regulation of inositol transport in Saccharomyces cerevisiae involves inositol-induced changes in permease stability and endocytic degradation in the vacuole.

Uptake of inositol by Saccharomyces cerevisiae is mediated by a specific inositol permease encoded by the ITR1 gene. Removal of inositol from the growth medium results in an increase in ITR1 mRNA abundance. The increase in ITR1 mRNA is accompanied by an increase in de novo synthesis of the Itr1 permease leading to an increased capacity for uptake. When inositol is added to the growth medium inactivation of uptake activity occurs, and both transcription of ITR1 and uptake activity are repressed to a basal level of function. The transcriptional regulation of ITR1 depends on the INO2, INO4, and OPI1 genes. In addition, repression is also achieved by regulation of ITR1 expression at the post-translational level. In this study, we show that there is a change in the stability of the Itr1 permease after the addition of inositol to the growth medium. Immunoblot analysis using a monoclonal antibody against an epitope attached to the Itr1 permease showed that the addition of inositol causes a dramatic increase in the rate of degradation of the permease. After the repressed (basal) level is achieved, turnover continues to be rapid. The increased rate of degradation was also observed in strains with mutations that block conjugation to ubiquitin. Degradation was not observed in strains defective in the END3/END4 endocytic pathway or in the production of vacuolar proteases (PEP4). Thus, inactivation of the Itr1 permease is accompanied by endocytic internalization followed by degradation in the vacuole. Inactivation may be a separate process that precedes and signals endocytic degradation. Since the end3/end4 mutations did not affect uptake activity under derepressed conditions, endocytosis is not required for normal inositol uptake.

Base Sequence↗
PubMed1995

Pseudologia fantastica and gender identity disturbance in a Chinese male.

A Chinese patient with pathological lying (pseudologia fantastica) and gender identity disturbance is described. The stories "save face" and lack a truly self-aggrandising quality. The importance of keeping "face" and maintaining modest behaviour in the Chinese culture is central to the psychopathology.

Adult↗
PubMed1994

Bimetallic binding motifs in organophosphorus hydrolase are important for catalysis and structural organization.

Organophosphorus hydrolase is a broad spectrum phosphoric acid hydrolase (EC 3.1.8.1) which appears to contain a binuclear metal center with two metals interactively involved in catalysis and/or structural functions. Site-directed mutagenesis has been employed to evaluate the participation of the various histidine and cysteine residues in metal coordination. The kinetic characteristics and metal binding stoichiometries of the purified site-directed substitutions of each of the histidine and cysteine residues in the catalytic domain of the protein to asparagine and serine residues, respectively, were determined. These data support the hypothesis that the histidines at positions 55, 57, and 201 are coordinated to a metal ion (M1) at the active center of the enzyme and that His254 and His257 are involved in the formation of a second structural metal center (M2). These and other unidentified amino acids may participate in a co-catalytic center. Although previous solution chemical studies concluded that cysteines are not involved in metal coordination, serine substitutions for Cys59 and Cys227 do affect metal content and catalytic activity. In contrast, substitution of asparagine for His230 does not affect the metal stoichiometry, but does reduce the kcat by 10(-4), indicating that it may be directly involved in the reaction chemistry. The H201N substitution eliminates activity but maintains one molar equivalent of metal and may function as a bridging ligand.

Amino Acid Sequence↗
PubMed1994

Dual control of inositol transport in Saccharomyces cerevisiae by irreversible inactivation of permease and regulation of permease synthesis by INO2, INO4, and OPI1.

Uptake of inositol by Saccharomyces cerevisiae is regulated through transcriptional control of the gene that encodes the major inositol permease, ITR1 (Nikawa, J., Tsukagoshi, Y., and Yamashita, S. (1991) J. Biol. Chem. 266, 11184-11191). ITR1 mRNA abundance decreases when cells are transferred from medium without inositol to medium with inositol. Here we demonstrate that the mechanism of transcriptional regulation of ITR1 is through the action of the INO2, INO4 and OPI1 genes. INO2 and INO4 are required for derepressed levels of ITR1 mRNA, and OPI1 is necessary for repression of transcript levels in response to inositol. The INO2, INO4, and OPI1 genes thus coordinate uptake of inositol to endogenous inositol biosynthesis and to phospholipid biosynthesis. Repression of transcription of ITR1 also requires ongoing synthesis of phosphatidylcholine, defining an additional link between synthesis of phospholipids and regulation of inositol uptake. Analysis showed that the INO1 gene, encoding a key enzyme in the inositol biosynthetic pathway, responded to decreases in permease activity with a graduated increase in the level of INO1 mRNA. We also found that, in addition to the transcriptional regulation, inositol permease activity is regulated by irreversible inactivation. Inactivation of the ITR1 permease occurs in response to the presence of inositol and involves a change in the functional half-life of the protein.

Biological Transport↗