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Biomedical subjects

H T Ho

Publications and source records attributed to H T Ho.

At least 19 recordsLinked to original sources
PubMed2006

Evaluation of a new transcutaneous bilirubinometer in Chinese newborns.

OBJECTIVE: To evaluate the use of a new transcutaneous bilirubinometer (JM-103 Minolta Airshields) for detection of hyperbilirubinaemia in term or near-term healthy Chinese newborns. METHODS: Transcutaneous bilirubin (TcB) was used to screen for severe hyperbilirubinaemia in newborn infants. Blood was taken for total serum bilirubin (TSB) measurement if the initial TcB level was higher than the 40th centile in Bhutani's nomogram. Paired TcB and TSB results were then reviewed over 6 months. The correlation as well as the mean difference between the two methods were calculated. The clinical application of TcB with Bhutani's nomogram in the prediction of severe hyperbilirubinaemia in low-risk, medium-risk and high-risk thresholds for phototherapy was also analysed. RESULTS: 997 paired TcB and TSB measurements were evaluated in term or near-term newborns. TcB was significantly correlated with TSB, with a correlation coefficient of 0.83 (p<0.001). Their mean difference was 21.7 micromol/l (SD 21.2, p<0.001), with the 95% limits of agreement between -19.9 and 63.3 micromol/l. In both low-risk and medium-risk thresholds for phototherapy, using the 75th centile of Bhutani's nomogram as threshold, TcB could identify all cases and had a sensitivity and negative predictive value of 100% each, a specificity of 56% and positive predictive value of 23%. For high-risk cases, using the 75th centile as cut-off, the sensitivity and negative predictive value were reduced to 86.7% and 97.0%, respectively. CONCLUSION: An accurate point-of-care bilirubin analyser facilitates bilirubin screening and avoids unnecessary blood tests. Although using the transcutaneous bilirubinometer JM-103 might result in a significant difference between TcB and TSB measured in Chinese newborns, combining the use of TcB and the 75th centile in Bhutani's nomogram as the cut-off level can identify all cases of significant hyperbilirubinaemia.

Bilirubin↗
PubMed2004

Evaluation of "point of care" devices in the measurement of low blood glucose in neonatal practice.

BACKGROUND: Low blood glucose in newborns is difficult to detect clinically. Hence a reliable "point of care" device (glucometer) for early detection and treatment of low glucose is needed. OBJECTIVE: To evaluate the performance of five readily available glucometers for the detection of low blood glucose in newborn infants. METHOD: Glucostix measurements were taken for newborns with risk factors using a Reflolux S (Boehringer) glucometer. If the initial reading was low (< 2.6 mmol/l), further measurements were taken with two other glucometers (phase I, Advantage and Glucotrend (Roche); phase II, Elite XL (Bayer) and Precision (Abbott)), and plasma glucose was measured in the laboratory (Aeroset; Abbott). RESULTS: Over 10 months, 101 specimens were collected from 71 newborns (57 in phase I; 44 in phase II). The Advantage glucometer usually overestimated blood glucose with a mean difference of 1.07 mmol/l (p < 0.01) at all low glucose ranges. The Glucotrend, Precision, and Elite XL glucometers performed better; the mean differences were not significantly different from the laboratory measured value (0.17 mmol/l (p = 0.37); -0.12 mmol/l (p = 0.13), and 0.24 mmol/l (p = 0.13) respectively). For detection of glucose concentrations < 2.6 mmol/l, the Precision glucometer had the highest sensitivity (96.4%) and negative predictive value (90%). For lower glucose concentrations (< 2.0 mmol/l), the Glucotrend glucometer performed even better (sensitivity 92.3%, negative predictive value 96.3%). CONCLUSION: Point of care devices should have good precision in the low glucose concentration range, sensitivity, and accuracy for early detection of neonatal hypoglycaemia. None of the five glucometers was satisfactory as the sole measuring device. The Glucotrend and Precision glucometers have the greatest sensitivity and negative predictive value. However, confirmation with laboratory measurements of plasma glucose and clinical assessment are still of the utmost importance.

Blood Glucose↗
PubMed2001

Progesterone receptor in patients with hepatolithiasis.

Altered gallbladder motility by progesterone has been recognized as an important factor in the development of gallstones. There are two types of hepatolithiasis, that occurs de novo in the intrahepatic ducts with an intact gallbladder (primary hepatolithiasis) and that which originates in the gallbladder and the stones migrate into the intrahepatic duct (secondary hepatolithiasis). This study was designed to evaluate the possible role of the progesterone receptor of gallbladder in the pathogenesis of hepatolithiasis. Eighty-four patients with hepatolithiasis (34 patients had primary hepatolithiasis and the other 50 patients secondary hepatolithiasis) were included. Paraffin-embedded specimens of gallbladder were processed to have an immunohistochemical staining for progesterone receptor. Positivity for progesterone receptor of gallbladder specimens was noted in eight patients (23.5%) with primary hepatolithiasis and in 23 patients (46%) with secondary hepatolithiasis. There is significant difference (P = 0.031) in positive rate for progesterone receptor between the primary hepatolithiasis and secondary hepatolithiasis groups. In conclusion, many more patients (46%) with secondary hepatolithiasis show positivity for progesterone receptor of gallbladder specimens than patients with primary hepatolithiasis (23.5%) (P = 0.031). This phenomenon is intriguing and should be of further evaluation and elucidated.

Bile Ducts, Intrahepatic↗
PubMed2000

On statistical analysis for placebo-challenging designs in clinical trials.

In clinical trials, appropriate designs are often chosen to address scientific/medical questions of particular interest to the investigator. For a chosen statistical design, however, standard statistical procedures may not be applicable owing to the nature of the design. In this paper we examine statistical methods for analysis of data collected from a placebo-challenging design which is often considered for assessment of the efficacy of drug products for indication of erectile dysfunction. An example concerning a clinical trial conducted with 120 male patients with erectile dysfunction is used to illustrate the derived statistical methods. Some recommendations to the randomization procedure for the study design of this kind are also made.

Cross-Over Studies↗
PubMed2000

4-Thiazolidinones: novel inhibitors of the bacterial enzyme MurB.

4-Thiazolidinones were synthesized and evaluated for their ability to inhibit the bacterial enzyme MurB. Selected 4-thiazolidinones displayed activity against the enzyme in vitro. This activity, coupled with the design principles of the thiazolidinones, supports the postulate that 4-thiazolidinones may be recognized as diphosphate mimics by a biological selector.

Bacteria↗
PubMed2000

Evaluation of autoSCAN-W/A and the Vitek GNI+ AutoMicrobic system for identification of non-glucose-fermenting gram-negative bacilli.

The autoSCAN-W/A (W/A; Dade Behring Microscan Inc., West Sacramento, Calif.) and Vitek AutoMicrobic System (Vitek AMS; bioMérieux Vitek Systems, Inc., Hazelwood, Mo.) are both fully automated microbiology systems. We evaluated the accuracy of these two systems in identifying nonglucose-fermenting gram-negative bacilli. We used the W/A with conventional-panel Neg Combo type 12 and Vitek GNI+ identification systems. A total of 301 isolates from 25 different species were tested. Of these, 299 isolates were identified in the databases of both systems. The conventional biochemical methods were used for reference. The W/A correctly identified 215 isolates (71. 4%) to the species level at initial testing with a high probability of >/=85%. The Vitek GNI+ correctly identified 216 isolates (71.8%) to the species level at initial testing with a high probability of >/=90%. After additional testing that was recommended by the manufacturer's protocol, the correct identifications of the W/A and Vitek GNI+ improved to 96.0 and 92.3%, respectively. The major misidentified species were Sphingomonas paucimobilis and Agrobacterium radiobacter in the W/A system and Acinetobacter lwoffii, Chryseobacterium indologenes, and Comamonas acidovorans in the Vitek GNI+ system. The error rates were 4.0 and 7.6%, respectively. The overall accuracy for both systems was above 90% if the supplemental tests were applied. There was no significant difference in accuracy (P > 0.05) between the two systems.

Automation↗
PubMed2000

Aldose reductase-deficient mice develop nephrogenic diabetes insipidus.

Aldose reductase (ALR2) is thought to be involved in the pathogenesis of various diseases associated with diabetes mellitus, such as cataract, retinopathy, neuropathy, and nephropathy. However, its physiological functions are not well understood. We developed mice deficient in this enzyme and found that they had no apparent developmental or reproductive abnormality except that they drank and urinated significantly more than their wild-type littermates. These ALR2-deficient mice exhibited a partially defective urine-concentrating ability, having a phenotype resembling that of nephrogenic diabetes insipidus.

Aldehyde Reductase↗
PubMed1999

Comparisons of genomic structures and chromosomal locations of the mouse aldose reductase and aldose reductase-like genes.

Aldose reductase (AR), best known as the first enzyme in the polyol pathway of sugar metabolism, has been implicated in a wide variety of physiological functions and in the etiology of diabetic complications. We have determined the structures and chromosomal locations of the mouse AR gene (Aldor1) and of two genes highly homologous to Aldor1: the fibroblast growth factor regulated protein gene (Fgfrp) and the androgen regulated vas deferens protein gene (Avdp). The number of introns and their locations in the mouse Aldor1 gene are identical to those of rat and human AR genes and also to those of Fgfrp and Avdp. Mouse Aldor1 gene was found to be located near the Cald1 (Caldesmon) and Ptn (Pleiotropin) loci at the proximal end of chromosome 6. The closely related genes Fgfrp and Avdp were also mapped in this region of the chromosome, suggesting that these three genes may have arisen by a gene duplication event.

Aldehyde Reductase↗
PubMed1999

Comparison of the D-glutamate-adding enzymes from selected gram-positive and gram-negative bacteria.

The biochemical properties of the D-glutamate-adding enzymes (MurD) from Escherichia coli, Haemophilus influenzae, Enterococcus faecalis, and Staphylococcus aureus were investigated to detect any differences in the activity of this enzyme between gram-positive and gram-negative bacteria. The genes (murD) that encode these enzymes were cloned into pMAL-c2 fusion vector and overexpressed as maltose-binding protein-MurD fusion proteins. Each fusion protein was purified to homogeneity by affinity to amylose resin. Proteolytic treatments of the fusion proteins with factor Xa regenerated the individual MurD proteins. It was found that these fusion proteins retain D-glutamate-adding activity and have Km and Vmax values similar to those of the regenerated MurDs, except for the H. influenzae enzyme. Substrate inhibition by UDP-N-acetylmuramyl-L-alanine, the acceptor substrate, was observed at concentrations greater than 15 and 30 microM for E. coli and H. influenzae MurD, respectively. Such substrate inhibition was not observed with the E. faecalis and S. aureus enzymes, up to a substrate concentration of 1 to 2 mM. In addition, the two MurDs of gram-negative origin were shown to require monocations such as NH4+ and/or K+, but not Na+, for optimal activity, while anions such as Cl- and SO4(2-) had no effect on the enzyme activities. The activities of the two MurDs of gram-positive origin, on the other hand, were not affected by any of the ions tested. All four enzymes required Mg2+ for the ligase activity and exhibited optimal activities around pH 8. These differences observed between the gram-positive and gram-negative MurDs indicated that the two gram-negative bacteria may apply a more stringent regulation of cell wall biosynthesis at the early stage of peptidoglycan biosynthesis pathway than do the two gram-positive bacteria. Therefore, the MurD-catalyzed reaction may constitute a fine-tuning step necessary for the gram-negative bacteria to optimally maintain its relatively thin yet essential cell wall structure during all stages of growth.

Cations, Divalent↗
PubMed1996

Biochemical evidence for the formation of a covalent acyl-phosphate linkage between UDP-N-acetylmuramate and ATP in the Escherichia coli UDP-N-acetylmuramate:L-alanine ligase-catalyzed reaction.

In the peptidoglycan biosynthesis pathway in Escherichia coli, UDP-N-acetylmuramate:L-alanine ligase (MurC) catalyzes the formation of UDP-N-acetylmuramyl-L-alanine. A peptide bond is formed in this reaction and an ATP molecule is hydrolyzed concomitantly to produce ADP and orthophosphate. A biochemical approach was devised to elucidate the role of ATP in this reaction. A fusion construct pMAL::murC was prepared and the maltose binding protein--UDP-N-acetylmuramyl:L-alanine ligase fusion protein was overproduced in E. coli/pMal::murC upon isopropyl beta-thiogalactoside induction. The fusion protein was purified to > or = 90% homogeneity by a single-step affinity chromatography. Subsequently, the ligase was released from the maltose binding protein by proteolytic cleavage and was purified to > or = 95% homogeneity by an ion-exchange chromatographic step. The kinetic parameters of the regenerated ligase are comparable to those of the purified native enzyme. This ligase was used to investigate the role that ATP plays in the formation of UDP-N-acetylmuramyl-L-alanine. UDP-N-acetyl[18O]muramate (with 18O located at the carboxylate function only) was prepared by a combination of chemical and enzymatic processes and was used as the substrate of the ligase to probe the reaction mechanism. All reaction products were purified and subjected to liquid chromatographic-mass spectrometric analysis. A single [18O]oxygen was transferred from UDP-N-acetyl[18O]muramate to the orthophosphate produced in the reaction. No [18O]oxygen was detected in the adenosine nucleotides recovered from the reaction. These results strongly suggest that this ligase-catalyzed peptide formation proceeds through an activated acyl-phosphate linkage during the reaction process. ATP therefore assists in the process of the peptide bond formation by donating its gamma-phosphoryl group to activate the carboxyl group of UDP-N-acetylmuramic acid.

Adenosine Triphosphate↗
PubMed1996

Structural studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.

Uridine diphosphate N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) adds the first amino acid to the sugar moiety of the peptidoglycan precursor, catalyzing one of the essential steps in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Here, we report our studies on the secondary and quaternary structures of UNAM:L-Ala ligase from Escherichia coli. The molecular weight of the purified recombinant enzyme determined by electrospray ionization mass spectrometry agreed well with the molecular weight deduced from the DNA sequence. Through sedimentation equilibrium analysis, we show that the enzyme exists in equilibrium between monomeric and dimeric forms and that the dissociation constant of the dimer, Kd, was determined to be 1.1 +/- 0.4 microM at 37 degrees C and 0.58 +/- 0.30 microM at 4 degrees C. A very similar Kd value was also obtained at 37 degrees C by gel filtration chromatography. The secondary structure of the enzyme was characterized by circular dichroism spectroscopy. No change in the secondary structure was observed between the monomeric and dimeric forms of the enzyme. The activity assays at enzyme concentrations both below and above the determined Kd value lead to the conclusion that the enzyme is active both as dimers and as monomers and that the specific activity is independent of the oligomerization state.

Centrifugation, Isopycnic↗
PubMed1995

Steady-state kinetic mechanism of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase.

The Escherichia coli MurB gene encoding UDP-N-acetylenolpyruvylglucosamine reductase was expressed to a level of approximately 100 mg/L as a fusion construct with maltose binding protein. Rapid affinity purification, proteolysis, and anion exchange chromatography yielded homogeneous enzyme containing 1 mol/mol bound FAD. Enzyme was maximally activated by K+, NH4+, and Rb+ at cation concentrations between 10 and 50 mM. Steady-state enzyme kinetics at pH 8.0 and 37 degrees C revealed weak and strong substrate inhibition by NADPH and UDP-N-acetylenolpyruvylglucosamine, respectively, where the KiS were 910 microM and 73 microM. Substrate inhibition was pH dependent for both substrates. Initial velocity measurements as a function of both substrates produced patterns consistent with a ping pong bi bi double competitive substrate inhibition mechanism. Data at pH 8.0 yielded kinetic constants corresponding to Km,UNAGEP = 24 +/- 3 microM, Ki,UNAGEP = 73 +/- 19 microM, Km,NADPH = 17 +/- 3 microM, Ki,NADPH = 910 +/- 670 microM, and kcat = 62 +/- 3 s-1. A slow anaerobic exchange reaction with thio-NADP+ provided evidence for release of NADP+ in the absence of UNAGEP. Alternate reduced nicotinamide dinucleotides, including NHXDPH, 3'-NADPH, and alpha-NADPH, were substrates, whereas NADH was not. Several nucleotides, including ADP and UDP, were weak inhibitors of the enzyme with inhibition constants between 5 and 97 mM. Various analogs of NADP+, including 3'-NADP+, thio-NADP+, APADP+, NEthDP+, and NHXDP+, were inhibitors of the enzyme with respect to NADPH and yielded inhibition constants in the range of 110-1100 microM. Analogs without the 2'- or 3'-phosphate of NADPH or NADP+ were not substrates or inhibitors. Double inhibition experiments with varied APADP+ and UNAG produced inhibition patterns consistent with mutually exclusive inhibitor binding. The data suggest that NADPH and UNAGEP share a subsite that prevents both molecules from binding at once.

Anaerobiosis↗
PubMed1995

UDP-N-acetylmuramyl-L-alanine functions as an activator in the regulation of the Escherichia coli glutamate racemase activity.

D-Glutamate is an essential component of the bacterial peptidoglycan. In Escherichia coli, the biosynthesis of D-glutamate is catalyzed by a glutamate racemase (encoded by the dga gene) and is regulated by UDP-N-acetylmuramyl-L-alanine [Doublet et al. (1994) Biochemistry 33, 5285], a bacterial peptidoglycan subunit precursor. Investigation was conducted to elucidate the interaction between the enzyme and its regulator. Whole and N-terminal truncated enzymes, encoded by individual constructs containing either a full-length or an N-terminal truncated dga gene, were evaluated. In the absence of the regulator, the purified whole enzyme showed a low-level basal racemase activity for which a Km value of 18.9 mM and a Vmax of 0.4 mumol/(min.mg) were determined, using D-glutamate as the substrate. Using the same substrate, in the presence of 6.5 microM UDP-N-acetylmuramyl-L-alanine, a Km value of 4.2 mM and a Vmax of 34 mumol/(min.mg) were measured. Similar kinetic parameters for the activated enzyme were obtained using L-glutamate as the substrate. The N-terminal truncated E. coli enzyme, with a 21 amino acid region removed, is similar in size to the Pediococcus pentosaceus glutamate racemase. Effects of the regulator on the full-length and the N-terminal truncated enzyme in the dialyzed cell lysate were compared. A host cell line, E. coli WM335 delta recA, containing a nonfunctional chromosomal dga gene was used to minimize the background interference. With 6.5 microM regulator added, the N-terminal truncated enzyme displayed a loss of more than 80% of the activity compared to the full-length enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Isomerases↗
PubMed1995

Staphylococcus haemolyticus contains two D-glutamic acid biosynthetic activities, a glutamate racemase and a D-amino acid transaminase.

Two D-glutamic acid biosynthetic activities, glutamate racemase and D-amino acid transaminase, have been described previously for bacteria. To date, no bacterial species has been reported to possess both activities. Genetic complementation studies using Escherichia coli WM335, a D-glutamic acid auxotroph, and cloned chromosomal DNA fragments from Staphylococcus haemolyticus revealed two distinct DNA fragments containing open reading frames which, when present, allowed growth on medium without exogenous D-glutamic acid. Amino acid sequences of the two open reading frames derived from the DNA nucleotide sequences indicated extensive identity with the amino acid sequence of Pediococcus pentosaceous glutamate racemase in one case and with that of the D-amino acid transaminase of Bacillus spp. in the second case. Enzymatic assays of lysates of E. coli WM335 strains containing either the cloned staphylococcal racemase or transminase verified the identities of these activities. Subsequent DNA hybridization experiments indicated that Staphylococcus aureus, in addition to S. haemolyticus, contained homologous chromosomal DNA for each of these genes. These data suggest that S. haemolyticus, and probably S. aureus, contains genes for two D-glutamic acid biosynthetic activities, a glutamate racemase (dga gene) and a D-amino acid transaminase (dat gene).

Amino Acid Isomerases↗