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Biomedical subjects

W Y Cheung

Publications and source records attributed to W Y Cheung.

At least 19 recordsLinked to original sources
PubMed1995

Brain L-glutamate decarboxylase. Inhibition by phosphorylation and activation by dephosphorylation.

Previously we showed that the activity of the gamma-aminobutyric acid-synthesizing enzyme L-glutamate decarboxylase (GAD) in crude brain extract is inhibited by ATP and protein phosphatase inhibitors. We suggested that GAD activity is regulated by protein phosphorylation. In this paper we further present evidence to support our hypothesis that protein kinase A and calcineurin may be involved in regulation of GAD activity through phosphorylation and dephosphorylation fo GAD, respectively. In addition, the effect of neuronal stimulation on GAD activity in cultured neurons is also included. A model to link neuronal excitation and activation of GAD by Ca(2+)-dependent phosphatase is proposed.

Animals↗
PubMed1995

Stimulation of myosin light-chain kinase by Cd2+ and Pb2+.

The effect of Cd2+ on myosin light chain kinase (MLCK) reported in the literature is controversial, apparently because the level of Ca2+ contaminating the reaction mixture could not be accurately controlled by the addition of a metal chelator when Cd2+ was also present. In the present study, we have reduced the contaminating Ca2+ to a trace level that did not interfere with the enzyme activity; thus the use of a metal chelator was not necessary. We showed that Cd2+, or Pb2+ had a biphasic effect on MLCK isolated from chicken gizzard: stimulation at low and inhibition at high concentrations. (The stimulatory effect of on the enzyme activity isolated from chicken gizzard: stimulation at low and inhibition at high concentrations). The stimulatory effect of Cd2+ or Pb2+ on MLCK activity was not seen in the absence of calmodulin, and was abolished by trifluoperazine, a calmodulin antagonist, indicating that the heavy metals exert their activation via calmodulin. The inhibition of the enzyme activity by Cd2+ or Pb2+ at higher concentrations was also seen with the calmodulin-independent catalytic fragment of MLCK, suggesting that the inhibition is probably through their binding to sulfhydryl groups that are essential for catalytic activity. Pb2+ was more effective than Cd2+ in stimulating the enzyme activity, but less potent in inhibition. The extent of stimulation by heavy metals most likely resulted from a combination of the biphasic effects. Dithiothreitol and N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine selectively chelated Cd2+ and Pb2+ over Ca2+, and reversed their stimulatory or inhibitory effect on MLCK.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A family of related sequences associated with (TTTAGGG)n repeats are located in the interstitial regions of wheat chromosomes.

A family of related sequences associated with (TTTAGGG)n repeats has been cloned from the wheat cultivar Chinese Spring. These sequences reveal a high level of polymorphism between wheat varieties when used as restriction fragment length polymorphism (RFLP) probes. Although this family of sequences contains motifs homologous to the repeats in the telomeres of wheat, they are located at interstitial sites on wheat chromosomes.

Blotting, Southern↗
PubMed1994

H+ is involved in the activation of calcineurin by calmodulin.

We have studied recently the surface hydrophobic properties of several calmodulin (CaM) target enzymes and found that a certain amphipathic domain(s) of the enzyme was exposed in response to a H+ increase within the neutral pH range. The exposed domain appeared to be related to their CaM-binding domains and associated with their activation by CaM (Huang, S., Carlson, G. M., and Cheung, W. Y. (1994) J. Biol. Chem. 269, 7631-7638). In this paper, the involvement of H+ in the activation of calcineurin (CaN), one of the CaM target enzymes, was further studied. Using dansylated CaM to monitor its conformational change, we found that the binding of Ca2+ to CaM occurred at a lower range of Ca2+ concentrations (pCa from 7 to 6) than the formation of CaM.CaN complex (pCa from 6 to 5). However, addition of H+ within the neutral pH range shifted the formation of CaM.CaN complex to the lower range of Ca2+ concentrations. Similarly, the addition of H+ shifted the Ca2+ requirement for enzyme activation to a lower Ca2+ range. These results show that the interaction of CaM with CaN could be initiated either by Ca2+ or by H+ in the presence of suboptimal Ca2+, suggesting a positive cooperative effect between Ca2+ and H+. The CaM-stimulated activity of CaN was accompanied by a time-dependent autoinactivation. Kinetic analysis showed that the autoinactivation initiated by Ca2+ or by H+ was biphasic and that the decay rate constants were comparable. In heavy water (D2O), the Ca(2+)-induced formation of CaM.CaN complex as well as the activity of CaN were decreased, indicating a solvent deuterium isotope effect. The same solvent isotope effect was observed in the H(+)-induced formation of CaM.CaN complex and in the H(+)-induced enzyme activity. When the enzyme was rendered CaM-independent by limited proteolysis, the isotope effect was abolished, suggesting that H+ was involved in the CaM-dependent and not the CaM-independent activity. Collectively, these data suggest that the interaction of CaM with CaN depends on the two cations: Ca2+ exposes an amphipathic domain on CaM, and H+ exposes the CaM-binding domain on the target enzyme.

Animals↗
PubMed1994

Calmodulin-dependent enzymes undergo a protein-induced conformational change that is associated with their interactions with calmodulin.

The anionic hydrophobic (amphipathic) fluorescent probe 2-(p-toluidinyl)-naphthalene-6-sulfonate was used to investigate the surface hydrophobic properties of calmodulin (CaM)-dependent enzymes as follows: calcineurin, myosin light chain kinase, cyclic nucleotide phosphodiesterase, CaM-dependent protein kinase II, and the gamma-subunit of phosphorylase kinase. We found that certain domains of these enzymes that interacted with 2-(p-toluidinyl)-naphthalene-6-sulfonate were exposed by a transient proton (H+) increase within the neutral pH range. This H(+)-induced exposure, which could be caused either by direct addition of H+ or by the release of H+ from metal chelators upon their binding of Ca2+, seemed to be more closely linked with a change in pH value (i.e. transient H+ increase) than with the actual equilibrium pH value of the system. Unlike the case with CaM-dependent enzymes, the H(+)-induced conformational change was uncommon in CaM-independent enzymes. When CaM-binding domains were removed from calcineurin and smooth muscle myosin light chain kinase, the resultant enzymes no longer exposed new domains in response to H+ increase. Using dansylated CaM to monitor the formation of CaM-enzyme complexes, we found that complex formation occurred with an uptake of H+ from solution. When CaM-dependent enzymes were evaluated at suboptimal concentrations of Ca2+, addition of H+ enhanced both the formation of CaM-enzyme complexes and the CaM-dependent catalytic activities, but this synergistic H+ effect occurred within only a narrow range of Ca2+ concentrations. These findings suggest that the H(+)-exposed domains in CaM-dependent enzymes are involved in the binding of CaM and that both conformational changes in CaM and its enzyme targets are necessary for complex formation. Further, the findings are consistent with the notion that CaM-binding domains are masked in the nonactivated (uncomplexed) conformations of CaM-dependent enzymes. The interplay between H+ and Ca2+ is discussed in relation to other systems that display interdependent effects of these two ions.

Animals↗
PubMed1994

Practice characteristics associated with audit activity: a medical audit advisory group survey.

AIM: A survey of general practices in West Glamorgan was carried out to assess the development of record systems and practice organization and to relate this to audit activity. METHOD: Following mutual agreement practices were visited by two assessors. Visits were conducted using an agreed protocol and practice data were recorded on a semi-structured questionnaire, which had been sent to the practices in advance of the visits. Practices varied from single handed practices to group practices with six partners. RESULTS: Fifty seven of the 63 practices in the county suitable for inclusion were visited (90%). Data were analysed from 54 practices (three visits were regarded as a pilot). The practices more likely to be involved in audit were those with three or more partners and which had modern medical record systems. Audit activity had occurred in 87% of practices who kept clinical summaries in the notes (compared with 38% who did not), in 87% of practices with long-term medication summaries in the notes (compared with 40% with no summaries) and in 85% of practices with a computerized age-sex register (compared with 50% with no register). All training practices had undertaken audit compared with 63% of non-training practices. CONCLUSION: The survey enabled the medical audit advisory group to identify the type and degree of audit undertaken locally and highlighted the characteristics which encourage this activity. As a consequence the group is able to target practices who have limited or no involvement in audit and to offer assistance and advice on record modification likely to enhance audit activity.

England↗
PubMed1993

Latent sites of Epstein-Barr virus infection.

Epstein-Barr virus (EBV) has been detected in a wide spectrum of tumors. This study investigates the detection rate of EBV-DNA by Southern blot hybridization analysis (SOBH) and polymerase chain reaction (PCR) in different tissues from persons without apparent EBV-related diseases. Of 20 tonsillectomy specimens studied, SOBH indicated positivity for EBV-DNA in 1 case, and PCR indicated positivity in 10. In autopsies performed on patients with no apparent evidence of EBV-related diseases, the viral DNA was only detected by PCR in the following: parotid gland (7/15), submandibular gland (8/20), nasopharynx (8/10), tonsil (8/10), larynx (5/6), lung (5/9), cervical lymph node (7/10), mediastinal lymph node (7/10), abdominal lymph node (4/10), spleen (6/10), thyroid (5/10), liver (1/10), pancreas (1/4), kidney (4/10), uterine cervix (1/4), ovary (1/5) and testis (1/3). These results provide a baseline for interpreting the role of EBV in carcinogenesis.

Adolescent↗
PubMed1991

Long-range physical mapping of the alpha-amylase-1 (alpha-Amy-1) loci on homoeologous group 6 chromosomes of wheat.

Long-range physical maps of the small multigene family of the malt alpha-amylase genes (alpha-Amy-1) located on the long arms of wheat chromosomes 6A (the alpha-Amy-A1 locus) and 6B (alpha-Amy-B1) were generated by pulsed-field gel electrophoresis analysis. By using three methylation-sensitive rare-cutter restriction endonucleases, NotI, NruI and MluI, and an alpha-Amy-1 cDNA probe and four gene-specific genomic probes from the alpha-Amy-B1 locus, the size of the alpha-Amy-A1 locus was estimated to be about 700 kb and of the alpha-Amy-B1 locus to be about approximately 4300 kb. These two maps indicate clustering of GC-rich and C-methylation-sensitive restriction enzyme recognition sites. At least five regions reminiscent of 'CpG islands' are apparent in alpha-Amy-B1, and three in alpha-Amy-A1. Correlation between recombination frequency and physical distance within the alpha-Amy-B1 locus suggests that 1 cM approximates to 1 Mb in physical distance.

Bacterial Proteins↗
PubMed1990

The isolation of high molecular weight DNA from wheat, barley and rye for analysis by pulse-field gel electrophoresis.

A method is presented for the preparation of large DNA molecules from protoplasts embedded in agarose blocks of three different cereals--hexaploid bread wheat (Triticum aestivum), barley (Hordeum vulgare) and rye (Secale cereale). Pulse-field gel electrophoresis (PFGE) analysis of these DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA molecules was greater than 6 Mb. DNA samples prepared by this method were shown to be useful for restriction analysis using both frequent and rare cutting enzymes.

DNA↗
PubMed1990

Activation of troponin C by Cd2+ and Pb2+.

Certain heavy metal actions such as Cd2+ and Pb2+ mimic Ca2+ effectively in stimulating calmodulin (CaM). We now show that these cations also activate skeletal muscle troponin C (TnC), a Ca2(+)-binding protein highly homologous to CaM. Like Ca2+, these cations allow TnC to alter its electrophoretic mobility on polyacrylamide gels, and to bind to phenyl-Sepharose. Moreover, they activate TnC to stimulate myofibrillar ATPase. When TnC was removed from the skeletal myofibrils by treatment with trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid (CDTA), the ATPase activity was no longer stimulated by the cations. However, after reconstitution of CDTA-treated skeletal myofibril with TnC, the response of ATPase to Ca2+, Cd2+ or Pb2+ was restored. These findings suggest that the activation of myofibrillar ATPase by Cd2+ and Pb2+ is mediated through TnC. The ability of the heavy metals to stimulate TnC-supported ATPase activity correlated quite well with the ability to increase the extent of the myofibrillar superprecipitation. The activation of TnC by Cd2+ or Pb2+ could constitute a possible molecular basis for their toxicity.

Adenosine Triphosphatases↗
PubMed1989

Phosphatidylinositol modulates the response of calmodulin-dependent phosphatase to calmodulin.

Phosphatidylinositol (PtdIns) and many other phospholipids activated calmodulin (CaM)-dependent phosphatase in the presence or absence of Ca2+, and the stimulation was more pronounced in the presence of Ca2+. In addition, PtdIns modulated the response of phosphatase to CaM: at low and nonstimulatory concentrations (less than 70 microM), PtdIns augmented the activity of phosphatase by a submaximum concentration of CaM, giving a synergistic effect; and at high concentrations (greater than 100 microM), PtdIns suppressed the synergistic effect. Kinetic experiments indicated that PtdIns (both nonstimulatory and stimulatory concentrations) increased the affinity of phosphatase for CaM. In addition to the CaM regulatory site, phosphatase appears to have two PtdIns regulatory sites: a high-affinity site the occupation of which does not stimulate enzyme activity, and a low-affinity site the occupation of which stimulates enzyme activity in the absence of CaM and inhibits it in the presence of CaM. Modulating the response of phosphatase to CaM is not unique to PtdIns, and was observed with other phospholipids, including some that did not stimulate the enzyme. This raises the possibility that certain phospholipids may regulate phosphatase in two ways: (i) direct activation of the enzyme and (ii) modulation of its response to CaM.

Animals↗
PubMed1989

Calmodulin-dependent phosphatase preferentially dephosphorylates a 28 kDa protein in human platelets.

1. Human platelets contain a calmodulin-dependent phosphatase (calcineurin) that has many properties similar to those of bovine brain calmodulin-dependent phosphatase. 2. The activity of calcineurin phosphatase accounts for a small fraction of the total phosphatase activity in human platelets. 3. Labeling of human platelets with 32P yielded many phosphoproteins. 4. Incubation of a lysate of the 32P-labeled platelets with bovine brain calmodulin-dependent phosphatase led to preferential dephosphorylation of a 28 kDa protein (P28), a minor component of platelet proteins. 5. P28 is one of several proteins that were rapidly labeled upon stimulation of platelets with thrombin. 6. Even though the enzyme is known to catalyze the dephosphorylation of many substrates in vitro, its apparent preference for P28 suggests that its activity is highly selective.

Animals↗
PubMed1988

Demonstration of endogenous inhibitors for the formation of 14,15-oxido-5,8,11-eicosatrienoic acid from phosphatidylinositol in bovine lung extract.

Bovine lung extract contained an enzyme or enzyme system that catalyzed the formation of 14,15-oxido-5,8,11-eicosatrienoic acid from phosphatidylinositol. The enzyme activity increased markedly during the course of purification, apparently due to the removal of two endogenous inhibitors from the enzyme. Using membranes with known molecular weight cut-offs, we estimated the Mr of Inhibitor 1 to be between 10,000 and 100,000 and Inhibitor 2 less than 1,000. Inhibitor 1 appeared to be partially inactivated by trypsin and was heat labile, whereas Inhibitor 2 was resistant to trypsin and was heat resistant. Both inhibitors were hydrophilic.

8,11,14-Eicosatrienoic Acid↗
PubMed1988

The structure and regulation of phosphoglucose isomerase in Saccharomyces cerevisiae.

We have cloned and sequenced the PGI1 gene, encoding phosphoglucose isomerase (E.C.5.3.1.9), from Saccharomyces cerevisiae. The nucleotide sequence predicts subunits of 554 amino acids with a molecular weight of 61,230. Both the size and amino acid composition correlate well with measurements from purified protein. We have compared the PGI1 protein with the predicted sequence for pig muscle PGI. In spite of some evolutionary divergence the proteins are very similar and there are some highly conserved regions, two of which have been implicated in the active site. It has been suggested that PGI exists in two or more isozyme forms in S. cerevisiae and analogy with ADR2/ADC1 suggests that such PGI isozymes might also be differentially regulated during glycolytic/gluconeogenic growth. We have used accurate quantitation of PGI1 mRNA and gene fusions of PGI1 to the lacZ gene of Escherichia coli to show that PGI1 transcription is regulated neither between glycolytic and gluconeogenic growth nor between exponential and stationary phase. The complete lack of PGI activity in PGI1 deletion mutants and of differential regulation suggests that the isozymes of PGI might result merely from processing of the PGI1 gene product.

Amino Acid Sequence↗