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Biomedical subjects

A Lau

Publications and source records attributed to A Lau.

At least 37 records · Page 2Linked to original sources
PubMed2000

Inhibition of neuronal apoptosis by docosahexaenoic acid (22:6n-3). Role of phosphatidylserine in antiapoptotic effect.

Enrichment of Neuro 2A cells with docosahexaenoic acid (22:6n-3) decreased apoptotic cell death induced by serum starvation as evidenced by the reduced DNA fragmentation and caspase-3 activity. The protective effect of 22:6n-3 became evident only after at least 24 h of enrichment before serum starvation and was potentiated as a function of the enrichment period. During enrichment 22:6n-3 incorporated into phosphatidylserine (PS) steadily, resulting in a significant increase in the total PS content. Similar treatment with oleic acid (18:1n-9) neither altered PS content nor resulted in protective effect. Hindering PS accumulation by enriching cells in a serine-free medium diminished the protective effect of 22:6n-3. Membrane translocation of Raf-1 was significantly enhanced by 22:6n-3 enrichment in Neuro 2A cells. Consistently, in vitro biomolecular interaction between PS/phosphatidylethanolamine /phosphatidylcholine liposomes, and Raf-1 increased in a PS concentration-dependent manner. Collectively, enrichment of neuronal cells with 22:6n-3 increases the PS content and Raf-1 translocation, down-regulates caspase-3 activity, and prevents apoptotic cell death. Both the antiapoptotic effect of 22:6n-3 and Raf-1 translocation are sensitive to 22:6n-3 enrichment-induced PS accumulation, strongly suggesting that the protective effect of 22:6n-3 may be mediated at least in part through the promoted accumulation of PS in neuronal membranes.

Animals↗
PubMed2000

Creatine kinase, an ATP-generating enzyme, is required for thrombin receptor signaling to the cytoskeleton.

Thrombin orchestrates cellular events after injury to the vascular system and extravasation of blood into surrounding tissues. The pathophysiological response to thrombin is mediated by protease-activated receptor-1 (PAR-1), a seven-transmembrane G protein-coupled receptor expressed in the nervous system that is identical to the thrombin receptor in platelets, fibroblasts, and endothelial cells. Once activated by thrombin, PAR-1 induces rapid and dramatic changes in cell morphology, notably the retraction of growth cones, axons, and dendrites in neurons and processes in astrocytes. The signal is conveyed by a series of localized ATP-dependent reactions directed to the actin cytoskeleton. How cells meet the dynamic and localized energy demands during signal transmission is unknown. Using the yeast two-hybrid system, we identified an interaction between PAR-1 cytoplasmic tail and the brain isoform of creatine kinase, a key ATP-generating enzyme that regulates ATP within subcellular compartments. The interaction was confirmed in vitro and in vivo. Reducing creatine kinase levels or its ATP-generating potential inhibited PAR-1-mediated cellular shape changes as well as a PAR-1 signaling pathway involving the activation of RhoA, a small G protein that relays signals to the cytoskeleton. Thrombin-stimulated intracellular calcium release was not affected. Our results suggest that creatine kinase is bound to PAR-1 where it may be poised to provide bursts of site-specific high-energy phosphate necessary for efficient receptor signal transduction during cytoskeletal reorganization.

Adenosine Triphosphate↗
PubMed2000

Structural studies of human alkyladenine glycosylase and E. coli 3-methyladenine glycosylase.

Human alkyladenine glycosylase (AAG) and Escherichia coli 3-methyladenine glycosylase (AlkA) are base excision repair glycosylases that recognize and excise a variety of alkylated bases from DNA. The crystal structures of these enzymes have provided insight into their substrate specificity and mechanisms of catalysis. Both enzymes utilize DNA bending and base-flipping mechanisms to expose and bind substrate bases. Crystal structures of AAG complexed to DNA suggest that the enzyme selects substrate bases through a combination of hydrogen bonding and the steric constraints of the active site, and that the enzyme activates a water molecule for an in-line backside attack of the N-glycosylic bond. In contrast to AAG, the structure of the AlkA-DNA complex suggests that AlkA substrate recognition and catalytic specificity are intimately integrated in a S(N)1 type mechanism in which the catalytic Asp238 directly promotes the release of modified bases.

Alkylation↗
PubMed2000

On the concentrations of cyclins and cyclin-dependent kinases in extracts of cultured human cells.

Cyclins and cyclin-dependent kinases (CDKs) are key regulators of the human cell cycle. Here we have directly measured the concentrations of the G(1) and G(2) cyclins and their CDK partners in highly synchronized human cervical carcinoma cells (HeLa). To determine the exact concentrations of cyclins and CDKs in the cell extracts, we developed a relatively simple method that combined the use of (35)S-labeled standards produced in rabbit reticulocyte lysates and immunoblotting with specific antibodies. Using this approach, we formally demonstrated that CDC2 and CDK2 are in excess of their cyclin partners. We found that the concentrations of cyclin A2 and cyclin B1 (at their peak levels in the G(2) phase) were about 30-fold less than that of their partner CDC2. The peak levels of cyclin A2 and cyclin E1, at the G(2) phase and G(1) phase, respectively, were only about 8-fold less than that of their partner CDK2. These ratios are in good agreement with size fractionation analysis of the relative amount of monomeric and complexed forms of CDC2 and CDK2 in the cell. All the cyclin A2 and cyclin E1 are in complexes with CDC2 and CDK2, but there are some indications that a significant portion of cyclin B1 may not be in complex with CDC2. Furthermore, we also demonstrated that the concentration of the CDK inhibitor p21(CIP1/WAF1) induced after DNA damage is sufficient to overcome the cyclin-CDK2 complexes in MCF-7 cells. These direct quantitations formally confirmed the long-held presumption that CDKs are in excess of the cyclins in the cell. Moreover, similar approaches can be used to measure the concentration of any protein in cell-free extracts.

Adenocarcinoma↗
PubMed2000

Identification of three alternative first exons and an intronic promoter of human PDE5A gene.

In the accompanying paper we present evidence for the existence of three PDE5A isoforms that differed only in the 5' end of the mRNAs. In this paper we present evidence that the three isoform-specific 5' ends were encoded by three alternative first exons that were arranged in the order of A1-A3-A2. Because the isoform-specific mRNAs could be transcribed from individual promoters, DNA fragments of the two intronic regions (A1-A3 and A3-A2) were tested for possible promoter activities. The intron between A1- and A3-specific exons did not exhibit any promoter activities even in smooth muscle cells that expressed the A3 isoform (see accompanying paper). In contrast, the intron between A3- and A2-specific exons had promoter activities in PDE5A2-expressing COS-7 and smooth muscle cells. This intronic promoter was bound by transcription factors AP-2 and Sp1, but not by AP-1, as shown by DNase I footprint analysis. However, the sequence bound by AP-2 (5'-GGGAAACGCTCGCGGGAGAGTTGG) is unusual in that it bears little resemblance to the consensus AP-2-binding sequence.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

Expression of three isoforms of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in human penile cavernosum.

Inhibition of cGMP-specific phosphodiesterase type V (PDE5) has been shown to improve penile erection in patients with erectile dysfunction. We report here the cloning of three PDE5 isoforms from human penile tissues. Two of the isoforms were identical to PDE5A1 and PDE5A2, respectively, which had been isolated from nonpenile tissues. The third isoform was novel and hence called PDE5A3. The deduced amino acid sequence of PDE5A3 was the same as the C-terminal 823-residue sequence of PDE5A1 and PDE5A2. While PDE5A1 and A2 isoforms were expressed in all tissues examined, the A3 isoform was confined to tissues with a smooth muscle or cardiac muscle component. When expressed in COS-7 cells, PDE5A1, A2, and A3 isoforms had similar cGMP-catalytic activities with K(m) of 6.2, 5.75, and 6.06 microM, respectively. Their cGMP-catalytic activities were inhibited by zaprinast with IC(50) values of 3.2 microM, 1.3 microM, and 1.6 microM, respectively, and by sildenafil with IC(50) of 28, 14, and 13 nM, respectively.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

Degradation of cyclin A does not require its phosphorylation by CDC2 and cyclin-dependent kinase 2.

Many cyclins are degraded by the ubiquitination/proteasome pathways involving the anaphase-promoting complex and SCF complexes. These degradations are frequently dependent on phosphorylation by cyclin-dependent kinases (CDKs), providing a self-limiting mechanism for CDK activity. Here we present evidence from in vitro and in vivo assay systems that the degradation of human cyclin A can be inhibited by kinase-inactive mutants of CDK2 and CDC2. One obvious interpretation of these results is that like other cyclins, CDK-dependent phosphorylation of the cyclin A may be involved in cyclin A degradation. Our data indicated that CDK2 can phosphorylate cyclin A on Ser-154. Site-directed mutagenesis of Ser-154 abolished the phosphorylation by recombinant CDK2 in vitro and the majority of cyclin A phosphorylation in the cell. Activation of CDK2 and binding to SKP2 or p27(KIP1) were not affected by the phosphorylation of Ser-154. Surprising, in marked contrast to cyclin E, where phosphorylation of Thr-380 by CDK2 is required for proteolysis, degradation of cyclin A was not affected by Ser-154 phosphorylation. It is likely that the stabilization of cyclin A by the kinase-inactive CDKs was mainly due to a cell cycle effect. These data suggest an important difference between the regulation of cyclin A and cyclin E.

CDC2 Protein Kinase↗
PubMed2000

The effect of pregnancy and delivery on the function and ultrastructure of the rat bladder and urethra.

OBJECTIVE: To examine the effect of pregnancy and delivery on the function and ultrastructure of the bladder and urethra in rats. Material and methods The study comprised six virgin and 18 pregnant rats; both groups underwent cystometry (at the 19th day of gestation, and 2 days and 6 weeks after parturition). Tissues from the bladder and urethra were collected for electron microscopy, western blotting and immunostaining for caveolin-1 and caveolin-3. RESULTS: The bladder capacity was greater and the modified leak-point pressures lower in pregnant and 2-day postpartum rats than in virgin and 6-week postpartum rats. The residual volume was significantly higher in the pregnant group. Electron microscopy showed more sarcolemmal caveolae in the smooth muscle cells of both the bladder and urethra of virgin rats than in the other groups. Lipid droplets and subsarcolemmal mitochondria accumulated in pregnant and 2-day postpartum rats. Caveolin-1 protein was detected in the cytoplasmic membrane of urethra and bladder smooth muscle cells. Caveolin-3 was detected in the membrane of striated muscle in the intrinsic sphincter. Western blotting showed increased caveolin-1 protein expression in the bladder and urethra of 2-day postpartum rats; in contrast, levels of caveolin-1 were lower in pregnant rats than in virgin and 6-week postpartum rats. CONCLUSION: s During pregnancy there was a significant decrease in sarcolemmal caveolae and caveolin-1 in the smooth muscle cells of the rat bladder and urethra. The changes in caveolae and the membrane protein caveolin may play a role in the functional changes associated with pregnancy and after delivery.

Animals↗
PubMed2000

Placental haemozoin and malaria in pregnancy.

Malaria infections in pregnant women cause poor birth outcomes. Malaria pigment (haemozoin) accumulates in the placenta within macrophages and extracellularly, but its pathological significance is not understood. In order to study the potential role of haemozoin in malaria pathogenesis, we enrolled primigravid women at a Malawian government antenatal clinic and followed them through delivery. One hundred and thirteen women (71 per cent) out of 159 women followed through delivery were parasitaemic at least once. Mean placental haemozoin concentrations were significantly higher in women with delivery parasitaemias (223 ng/mg protein) than in women who never had a detectable parasitaemia (43 ng/mg protein; P<0.05), but were not significantly higher in women who were parasitaemic only during the antenatal period (67 ng/mg protein). Haemozoin was not associated with preterm delivery (PTD) or intrauterine growth retardation (IUGR) (P -values, 0.307-0.787). Thus, placental haemozoin is associated with malaria infection at the time of delivery and does not seem to be associated with poor birth outcome.

Adult↗
PubMed2000

Upregulation of L-plastin gene by testosterone in breast and prostate cancer cells: identification of three cooperative androgen receptor-binding sequences.

L-Plastin is normally a leukocyte-specific actin-binding protein; it is also expressed in the majority of human cancer cell lines that are derived from many types of solid tumors. We have previously reported the isolation of the L-plastin gene promoter, in which we identified several potential steroid receptor-binding sequences. We now obtained evidence that L-plastin gene expression was positively regulated by testosterone in androgen receptor (AR)-positive prostate and breast cancer cells. DNase I footprint analysis identified three AR-binding elements (ARE) located in a 545-bp region approximately 1.1 kb upstream from the transcription initiation site. However, each of these three AREs exhibited very little testosterone/AR-responsive enhancer activities toward a test promoter (of the thymidine kinase gene) when tested in MCF-7 breast cancer cells. Their testosterone/AR responsiveness became evident only when two or three of them were combined. In PC-3 prostate cancer cells, cooperation among L-plastin AREs was still evident although individually they had moderate levels of testosterone/AR responsiveness. Thus, the three L-plastin AREs, despite their imperfect sequences compared with the consensus ARE, could cooperate with each other to become a potent testosterone/AR-responsive unit, which was likely responsible for the inducibility of the L-plastin gene by testosterone.

Base Sequence↗
PubMed2000

The environmental effects of dental amalgam.

Dental amalgam is one of the most commonly used materials in restorative dentistry. However, one of its major components, mercury, is of particular concern due to its potential adverse effects on humans and the environment. In this review, the environmental impact of dental amalgam will be discussed, with particular reference to the effects attributed to its mercury component. Mercury commonly occurs in nature as sulfides and in a number of minerals. Globally, between 20,000-30,000 tons of mercury are discharged into the environment each year as a result of human activities. According to a recent German report, approximately 46 per cent of the freshly triturated amalgam is inserted as new amalgam restorations and the rest is waste. Depending on the presence of an amalgam separating unit, some of the generated amalgam-contaminated sludge is discharged into the sewage system. Lost or extracted teeth with amalgam fillings and amalgam-contaminated waste, such as trituration capsules and cotton rolls are discharged with the solid waste and, in most instances, are incinerated. Use of disinfectants containing oxidizing substances in dental aspirator kits may contribute to remobilization of mercury and its subsequent release into the environment. Nevertheless, dental mercury contamination is only a small proportion of terrestrial mercury (3-4 per cent), which is quite insignificant compared with industrial pollution and combustion of fossil fuels by vehicles. The environmental impact of dental mercury is mainly due to the poor management of dental amalgam waste. Proper collection of mercury-contaminated solid waste prevents the release of mercury vapour during combustion. In addition, the use of amalgam separating devices reduces the amount of amalgam-contaminated water released from dental clinics.

Dental Amalgam↗
PubMed2000

The role of working memory in analogical mapping.

The impact of a working-memory load on analogical mapping was examined in two experiments, using a dual-task paradigm. In Experiment 1, we used a phonological working-memory load; in Experiment 2, we used a phonological working-memory load and an executive working-memory load. The subjects were required to identify correspondences between visual scenes, either for single objects or for three objects simultaneously. The results indicated that the imposition of either a phonological or an executive working-memory load decreased the frequency with which the subjects identified correspondences between scenes based on relations and increased the frequency with which they identified correspondences based on object attributes. The frequency with which subjects made relational mappings was increased by the instruction to find correspondences for multiple objects in a scene simultaneously, rather than for just one. These results indicate that mapping on the basis of relations places greater demands on both modality-specific buffers and executive components of working memory than does mapping on the basis of object attributes.

Association Learning↗
PubMed1999

MDM2 and MDMX inhibit the transcriptional activity of ectopically expressed SMAD proteins.

Transforming growth factor-beta (TGF-beta) inhibits cell proliferation in many cell types, and acquisition of TGF-beta resistance has been linked to tumorigenesis. One class of proteins that plays a key role in the TGF-beta signal transduction pathway is the SMAD protein family. MDM2, a key negative regulator of p53, has recently been shown to suppress TGF-beta-induced growth arrest in a p53-independent manner. Here we show that MDM2 and the structurally related protein MDMX can inhibit the transcriptional activity of ectopically expressed SMAD1, SMAD2, SMAD3, and SMAD4. Immunofluorescence staining indicated that ectopically expressed SMAD4 was present in both the cytoplasm and nucleus, and MDM2 and NIDMX were localized mainly to the nucleus and cytoplasm, respectively. When SMAD4 was coexpressed with either MDM2 or MDMX, nuclear accumulation of SMAD4 was strikingly inhibited. We have no evidence that SMAD4 binds directly to MDM2 or MDMX; hence, the inactivation and nuclear exclusion of SMAD4 by MDM2/MDMX may involve other indirect mechanisms.

Animals↗
PubMed1999

Frequency of meiotic trisomy depends on involved chromosome and mode of ascertainment.

Although maternal meiotic errors predominate in most studies of nonmosaic trisomy, studies of trisomy ascertained through confined placental mosaicism (CPM) have shown a high rate of somatic errors. However, origin of trisomy of many of the chromosomes involved in CPM has not been evaluated previously in cases ascertained through spontaneous abortions (SAs). Therefore, it was impossible to determine if the relative lack of meiotic errors in trisomy-CPM cases was a characteristic of the specific chromosome involved or due simply to ascertainment through a mosaic state. In the present study, parental and meiotic/somatic stages of origin of trisomy were determined in 89 SAs involving trisomy of chromosomes 2, 4 to 10, 12, 15, 17, and 20. Comparisons were then made to origin of trisomy in cases of confined and generalized trisomy mosaicism. Although somatic errors are generally more common in mosaic cases, this depends on the specific chromosome involved. The results suggest that there are chromosome-specific differences in the relative frequency of somatic chromosome gain or loss and/or the ability of an early somatic loss of one chromosome from a trisomic conceptus to "rescue" the pregnancy. As mean maternal age was less in the somatic than meiotic origin cases (P < 0.01), the age distribution of the study population should also influence the probability of detecting a somatic error. No phenotypic differences were apparent when cases were subdivided based on either parent or stage of origin of the trisomy.

Abortion, Spontaneous↗
PubMed1999

Choosing antithrombotic therapy for elderly patients with atrial fibrillation who are at risk for falls.

OBJECTIVE: To determine whether the risk of falling (with a possible increased chance of subdural hematoma) should influence the choice of antithrombotic therapy in elderly patients with atrial fibrillation. DESIGN: A Markov decision analytic model was used to determine the preferred treatment strategy (no antithrombotic therapy, long-term aspirin use, or long-term warfarin use) for patients with atrial fibrillation who are 65 years of age and older, are at risk for falling, and have no other contraindications to antithrombotic therapy. Input data were obtained by systematic review of MEDLINE. Outcomes were expressed as quality-adjusted life-years. RESULTS: For patients with average risks of stroke and falling, warfarin therapy was associated with 12.90 quality-adjusted life-years per patient; aspirin therapy, 11.17 quality-adjusted life-years; and no antithrombotic therapy, 10.15 quality-adjusted life-years. Sensitivity analysis demonstrated that, regardless of the patients' age or baseline risk of stroke, the risk of falling was not an important factor in determining their optimal antithrombotic therapy. CONCLUSIONS: For elderly patients with atrial fibrillation, the choice of optimal therapy to prevent stroke depends on many clinical factors, especially their baseline risk of stroke. However, patients' propensity to fall is not an important factor in this decision.

Accidental Falls↗
PubMed1999

Differential regulation of human T-plastin gene in leukocytes and non-leukocytes: identification of the promoter, enhancer, and CpG island.

Plastins (fimbrins) are a family of actin-bundling proteins conserved from yeast to humans. In humans, three tissue-specific plastin isoforms have been identified. The T isoform (T-plastin) is unique in that it is expressed in all tissues except leukocytes. To investigate how the T-plastin gene is differentially regulated in leukocytes and non-leukocytes, we isolated a genomic clone that included 9 kb of the upstream flanking region, 0.1 kb of the first exon, and 5.9 kb of the first intron. From this clone, we obtained a continuous sequence of 5535 bp, including 3138 bp of the upstream flanking region, the first exon, and 2286 bp of the first intron. A cluster of four transcription initiation sites was located by S1 mapping. A region spanning these sites and extending 1.4 kb into the first intron had the characteristics of a CpG island. Three CG-containing restriction sites within this island were analyzed and found all or variably methylated in four T-plastin-negative leukemia cell lines. In contrast, the same sites were not methylated in three T-plastin-expressing cell lines or in a sample of normal blood lymphocytes. A basal promoter was located 250 bp upstream from the transciption initiation sites. It comprised a CCAAT box, an Sp1 motif, and four AP2 motifs. No TATA or Inr sequence was found. The basal promoter exhibited weak activity when assayed in fibrosarcoma cells. Stronger promoter activities were found in the presence of the SV40 enhancer or a T-plastin enhancer located some 500 bp from the basal promoter. In T-plastin-negative leukemia cells, the T-plastin basal promoter could be activated by the SV40 enhancer but not by the T-plastin enhancer. DNA footprinting identified the T-plastin enhancer as two inverted symmetric octamers (AGATAACCTC and GAGGTCAGCT) separated by 17 nucleotides.

Base Sequence↗
PubMed1999

Transgenic models in the study of reproduction.

The development of techniques to manipulate genes within mouse embryonic stem (ES) cells has allowed investigators to study the functions of many genes in vivo. We have used these techniques to functionally mutate genes to study how the loss of the gene affects development, oncogenesis, and reproduction. Genes affecting development include members of the transforming growth factor-beta (TGF-beta) superfamily and their signaling pathway. We have shown that mutations in this complicated signaling network affect a wide range of embryonic developmental processes including craniofacial morphogenesis, dentition and muscle development. One specific member of TGF-beta family, inhibin alpha, has been identified as a novel tumor suppressor in the testes, ovaries, and adrenal glands. Another focus of research in the laboratory is the analysis of roles of proteins in the hypothalamic-pituitary-gonadal axis and the affect of disrupting this pathway on reproductive function. We have demonstrated that several genes expressed in the pituitary and gonads are required for folliculogenesis leading to female infertility and in two cases are important for Sertoli cell proliferation in males. The studies using ES cell technology has enabled us to dissect two complex networks in animal models.

Animals↗