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Biomedical subjects

G K Wong

Publications and source records attributed to G K Wong.

At least 19 recordsLinked to original sources
PubMed1999

Characterization of the human endothelial nitric-oxide synthase promoter.

Understanding transcription initiation of the endothelial nitric-oxide synthase (eNOS) gene appears pivotal to gaining a comprehensive view of NO biology in the blood vessel wall. The present study therefore focused upon a detailed dissection of the functionally important cis-DNA elements and the multiprotein complexes implicated in the cooperative control of constitutive expression of the human eNOS gene in vascular endothelium. Two tightly clustered cis-regulatory regions were identified in the proximal enhancer of the TATA-less eNOS promoter using deletion analysis and linker-scanning mutagenesis: positive regulatory domains I (-104/-95 relative to transcription initiation) and II (-144/-115). Analysis of trans-factor binding and functional expression studies revealed a surprising degree of cooperativity and complexity. The nucleoprotein complexes that form upon these regions in endothelial cells contained Ets family members, Sp1, variants of Sp3, MAZ, and YY1. Functional domain studies in Drosophila Schneider cells and endothelial cells revealed examples of positive and negative protein-protein cooperativity involving Sp1, variants of Sp3, Ets-1, Elf-1, and MAZ. Therefore, multiprotein complexes are formed on the activator recognition sites within this 50-base pair region of the human eNOS promoter in vascular endothelium.

Animals↗
PubMed1998

The complete genomic sequence of 424,015 bp at the centromeric end of the HLA class I region: gene content and polymorphism.

We report here the genomic sequence of the centromeric portion of HLA class I, extending 424,015 bp from tumor necrosis factor alpha to a newly identified gene approximately 20 kb telomeric of Otf-3. As a source of DNA, we used cosmids centromeric of HLA-B that had been mapped previously with conventional restriction digestion and fingerprinting and previously characterized yeast artificial chromosomes subcloned into cosmids and mapped with multiple complete digest methodologies. The data presented provide a description of the gene content of centromeric HLA class I including new data on intron, promoter and flanking sequences of previously described genes, and a description of putative new genes that remain to be characterized beyond the structural information uncovered. A complete accounting of the repeat structure including abundant di-, tri-, and tetranucleotide microsatellite loci yielded access to precisely localized mapping tools for the major histocompatibility complex. Comparative analysis of a highly polymorphic region between HLA-B and -C was carried out by sequencing over 40 kb of overlapping sequence from two haplotypes. The levels of variation observed were much higher than those seen in other regions of the genome and indeed were higher than those observed between allelic HLA class I loci.

Centromere↗
PubMed1998

Antifungal activities of rapamycin and its derivatives, prolylrapamycin, 32-desmethylrapamycin, and 32-desmethoxyrapamycin.

The antifungal agent rapamycin is highly effective in inhibiting growth of yeast and mold strains. This study demonstrates that in liquid medium, rapamycin is more active than its derivatives (prolylrapamycin, 32-desmethylrapamycin, 32-desmethoxyrapamycin) against Candida albicans, Saccharomyces cerevisiae, and Fusarium oxysporum. All the rapamycins were more active than amphotericin B. Although four other molds were not inhibited in liquid medium, they were very sensitive to rapamycin and its derivatives when tested on agar. The latter assay showed that rapamycin is the most active and 32-desmethylrapamycin is more active than prolylrapamycin and 32-desmethoxyrapamycin. The conclusion of this study is that rapamycin is the most active antifungal agent of the compounds examined. The unexpected finding of high activity of rapamycin and its derivatives against filamentous fungi when assayed by the agar diffusion assay suggests that rapamycin or a derivative may hold promise for chemotherapy against pathogenic molds as well as yeasts.

Amphotericin B↗
PubMed1997

Multiple-complete-digest restriction fragment mapping: generating sequence-ready maps for large-scale DNA sequencing.

Multiple-complete-digest mapping is a DNA mapping technique based on complete-restriction-digest fingerprints of a set of clones that provides highly redundant coverage of the mapping target. The maps assembled from these fingerprints order both the clones and the restriction fragments. Maps are coordinated across three enzymes in the examples presented. Starting with yeast artificial chromosome contigs from the 7q31.3 and 7p14 regions of the human genome, we have produced cosmid-based maps spanning more than one million base pairs. Each yeast artificial chromosome is first subcloned into cosmids at a redundancy of x15-30. Complete-digest fragments are electrophoresed on agarose gels, poststained, and imaged on a fluorescent scanner. Aberrant clones that are not representative of the underlying genome are rejected in the map construction process. Almost every restriction fragment is ordered, allowing selection of minimal tiling paths with clone-to-clone overlaps of only a few thousand base pairs. These maps demonstrate the practicality of applying the experimental and software-based steps in multiple-complete-digest mapping to a target of significant size and complexity. We present evidence that the maps are sufficiently accurate to validate both the clones selected for sequencing and the sequence assemblies obtained once these clones have been sequenced by a "shotgun" method.

Base Composition↗
PubMed1997

Dynamics of filamentous actin organization in the sea urchin egg cortex during early cleavage divisions: implications for the mechanism of cytokinesis.

We have used confocal laser scanning microscopy in conjunction with BODIPY-phallacidin staining of filamentous actin to investigate changes in the quantity and organization of cortical actin during the first two cell cycles following fertilization in eggs of the sea urchin Strongylocentrotus purpuratus. Quantification of fluorescent phallacidin staining reveals that the amount of filamentous actin (F-actin) in the cortex undergoes cyclical increases and decreases during early cleavage divisions, peaking near the beginning of the cell cycle and decreasing to a minimum at cytokinesis. Changes in the content of cortical F-actin are accompanied by the growth and disappearance of rootlet-like bundles of actin filaments which extend from the bases of microvilli that cover the surface of the egg. Actin rootlets reach their maximum degree of development by 20 min postfertilization, and then gradually decrease in number and length over the next 40 min. Small actin rootlets persist until cleavage, disappear during cytokinesis, and reform following division. The formation of actin rootlets requires cytoplasmic alkalization and is inhibited by cytochalasin D. Cytochalasin D washout experiments demonstrate that assembly of the cortical actin cytoskeleton can be blocked until 5 min before the onset of cleavage and still allow normal cytokinesis. These results illustrate the dynamic nature of cortical actin organization during early development and demonstrate that cytokinesis occurs at the point of minimum cortical F-actin content. They further demonstrate that cytokinesis can occur in embryos in which the normal developmental sequence of changes in cortical actin organization has been blocked by treatment with cytochalasin D, suggesting that these changes do not function in the establishment of the contractile apparatus for cytokinesis, but rather serve other developmental functions. Cell Motil. Cytoskeleton 36:30-42, 1997.

Actins↗
PubMed1996

Alteration of cell cycle timing and induction of surface instability in starfish blastomeres microinjected with antibodies to spectrin.

Spectrin has been implicated in a variety of different processes during late embryogenesis, after transcription of the zygotic genome has been activated. However, relatively little is known about the role of maternally derived spectrin during the early cleavage divisions that give rise to a multicellular embryo. To investigate the role of spectrin in early development, we have microinjected anti-spectrin antibodies into Patiria miniata starfish embryos to inhibit the activity of the maternal pool of spectrin. Microinjection of affinity-purified anti-spectrin antibody, or low to moderate doses of F(ab) fragments, into one blastomere of a two-cell-stage embryo caused a dose-dependent, progressive increase in the length of the cell cycle compared to the uninjected control blastomere. The progeny of injected blastomeres were unable to participate in the formation of a blastula epithelium, instead forming a loose aggregate of cells that eventually stopped dividing. When division stopped, the cells formed surface protrusions and became motile. At high doses of either whole antibody or F(ab) fragments, cells initiated, but failed to complete, cytokinesis. Blastomeres injected with high doses of F(ab) fragments also failed to reform nuclei and underwent variable periods of cell cycle arrest up to 12 hr. Injected embryos stained with BODIPY-phallacidin exhibited extensive disruption of the cortical actin cytoskeleton. These results support previous studies implicating spectrin in stabilizing the cell surface and maintaining the organization of the cortical cytoskeleton. They further suggest that spectrin is not required for the initiation or contraction of the cleavage furrow, but functions in the completion of cytokinesis. Most surprisingly, however, the results demonstrate that inhibition of spectrin function alters cell cycle timing, suggesting that disruption of the actin cytoskeleton inhibits progression through the cell cycle.

Animals↗
PubMed1996

Assembly of high-resolution restriction maps based on multiple complete digests of a redundant set of overlapping clones.

An approach to restriction-site mapping and contig building that uses fragment-size data from multiple complete digests of a set of clones that oversample a genomic region is presented. Maps containing both fragment-length data and clone-end data are maintained for each restriction enzyme. Synchronization between the maps for the different enzymes is achieved by requiring the clone-end maps for all enzymes to be compatible. Basic concepts that underlie multiple-complete-digest mapping--including the match/merge approach to map incorporation, extension vs assimilation, ambiguity, and clone-end compatibility--are presented. An initial application of multiple-complete-digest mapping to real data on a set of cosmid clones suggests that this mapping method has exceptional power to produce accurate maps that are well suited to the needs of large-scale DNA-sequencing projects.

Algorithms↗
PubMed1996

Stimulation of cortical actin polymerization in the sea urchin egg cortex by NH4Cl, procaine and urethane: elevation of cytoplasmic pH is not the common mechanism of action.

Previous studies have demonstrated that the penetrating weak base NH4Cl and the anesthetics procaine and urethane disrupt the normal attachment of cortical granules to the cortex of the sea urchin egg. Hylander and Summers (1981: Dev. Biol. 86:1-11) hypothesized that this effect may be caused by a pH-induced polymerization of cortical actin. We have tested this hypothesis by measuring the intracellular pH of eggs of the sea urchins S. purpuratus and A. punctulata treated with NH4Cl, procaine, or urethane, and determining the effects of these agents on the organization of cortical actin. Intracellular pH was determined by the ratiometric measurement of the fluorescent dye BCECF, and filamentous actin organization was examined by confocal laser scanning microscopy of BODIPY-phallocidin stained eggs. Treatment of eggs with either NH4Cl or procaine resulted in a rapid and reversible increase in cytoplasmic pH of up to 1 pH unit and a dose-dependent increase in the intensity of fluorescent staining of the cortex, indicating an increase in the content of filamentous actin. While urethane also induced a dramatic polymerization of cortical actin, no effect on cytoplasmic pH could be detected. These results demonstrate that NH4Cl, procaine and urethane all induce an increase in the amount of filamentous actin in the sea urchin egg cortex that may participate in the detachment of cortical granules. However, these compounds do not share a common mechanism of action based on the elevation of cytoplasmic pH.

Actins↗
PubMed1993

Development of the liver in the chicken embryo. II. Erythropoietic and granulopoietic cells.

Hepatic hemopoiesis is apparent in the chicken embryo on day 7 of incubation (Hamburger and Hamilton Stage 30), and a peak in hemopoietic activity occurs on day 14 (Stage 40). During this period, the differentiation of hemopoietic cells was examined by light microscopy and by transmission and scanning electron microscopy. Glycol methacrylate sections were used in lieu of smears to study hemopoietic cells, thus minimizing the problems of cell shrinkage and rupture. The sections were superior to smears for close examination of nuclear and cytoplasmic morphologies and for precise localization of hemopoietic cells to intravascular and extravascular sites. The avian liver is involved directly with erythropoiesis and granulopoiesis only. Erythropoietic cells, occurring in intravascular and extravascular locations, appear throughout the time frame examined. Blood islands with granulopoietic cells were not observed until days 8-9 (Stage 35). Granulopoiesis in the liver produces only eosinophilic leukocytes. Individual granulopoietic cells appear first in the connective tissue sheaths of hepatic vessels, and these cells subsequently congregate into blood islands. Endothelial cells of the sinusoidal linings, through asymmetric divisions, frequently release daughter cells into the circulation, and Kupffer cells are actively engaged in phagocytosis of erythrocytes. From a comparative standpoint, the elements deemed critical to hemopoiesis in the mammalian liver--prehepatocyte population, hepatic vasculature, and compartments for stem cell differentiation--may not hold the same importance in the bird, owing to an inordinate reliance on intravascular hemopoiesis in this vertebrate class.

Animals↗
PubMed1993

The immediate fate of cells in the epithelial somite of the chick embryo.

The precise origins and fates of cells forming the epithelial ball and those contained within the somitocoele at the centre of the somite in the chick embryo are unknown. In particular, it is not known whether the progeny of the proliferating cells in both the epithelial ball and the somitocoele simply mix with each other, or whether they represent two separate populations that remain isolated during proliferation. We tested whether cells found in both of these locations are the result of cell migration, cell division or both. To do this, we injected single cells in both the epithelial ball and the somitocoele with lysinated fluorescein dextran, a molecule that can only be transferred to other cells through cell division, and observed their development over the next 24 h. Control experiments were also performed to assess the incidence of error associated with the microinjection method and to provide reliable and valid comparative data. Our results showed that the progeny of the cells in the somitocoele, as well as any non-proliferating cells, appear to remain in the somitocoele. In contrast, the progeny of the cells in the epithelial ball either remain in the epithelial ball or migrate into the somitocoele to mix freely with the other cells present. These results suggest that the cells in the epithelial ball and the somitocoele develop as discrete groups, with the possibility of diverse fates accompanying further development.

Animals↗
PubMed1992

Development of the liver in the chicken embryo. I. Hepatic cords and sinusoids.

Hemopoiesis in the liver of the chicken embryo begins on day 7 of incubation (Hamburger and Hamilton Stage 30) and peaks on day 14 (Stage 40). During this time frame, the differentiation of hepatic cells was examined by light microscopy, transmission and scanning electron microscopy, and morphometry. The avian liver is a closely packed mass of dendriform cords and discontinuous sinusoids. Hepatocytes are pyramidal in shape, and they ring the bile canaliculi which run through the centers of the cords. Semithin sections, made possible by infiltration and embedding in glycol methacrylate, were stained with hematoxylin and eosin to assess the general architecture of the organ and the lipid content of the hepatocytes and by the periodic acid-Schiff reaction and hematoxylin to visualize the cytoplasmic stores of glycogen. The number of hepatocytes with demonstrable glycogen fluctuates erratically in early hemopoiesis, and the proportion of glycogen-containing cells progressively increases as hemopoiesis climbs to a peak. Most differentiating hepatocytes are devoid of lipid droplets until Stages 39 and 40. From Stage 30 to 35, hepatocyte volume falls to its lowest value. Subsequently (Stages 36 to 40), cell volume increases and hepatocytes achieve a relatively uniform size. Ultrastructural changes in the differentiating hepatocytes, including alterations to the mitochondria, endoplasmic reticulum, and Golgi apparatus, are documented. These morphological and morphometric findings on the prehepatocyte population and hepatic vasculature cover 2 of the 3 elements deemed critical to hepatic hemopoiesis in many vertebrates.

Animals↗
PubMed1992

The effect of a deprogrammer on the position of the terminal transverse horizontal axis of the mandible.

This study measured the amount of positional change in the terminal transverse horizontal axis of the mandible that results from altering undesirable neuromuscular influence. Centric relation records were made on 19 healthy subjects before and after deprogramming. The axis was located with a computerized axiograph, and the centric relation records were compared in vivo at the axis location. The positional change of the axis was quantified in three dimensions at an accuracy of +/- 0.05 mm. The net amount of positional change was compared to scores from the temporomandibular joint scale to test for correlation with the potential for developing a temporomandibular disorder. The findings showed that the amount of positional change in the terminal transverse horizontal axis in 18 of 19 healthy patients after 12 hours of deprogramming was less than 0.5 mm and did not correlate with scores from the TMJ Scale.

Adolescent↗
PubMed1986

Enumerating T-cell subsets on blood smears. An evaluation of an indirect immunoalkaline phosphatase method.

Peripheral blood T-lymphocyte subsets in 179 Chinese subjects were enumerated by an indirect immunoalkaline staining method done on ordinary blood smears. Absolute counts (X 10(9)/L) were 1.32 +/- 0.53 (T11), 0.73 +/- 0.31 (T4), 0.45 +/- 0.22 (T8) for males (n = 135) and 1.22 +/- 0.39 (T11), 0.69 +/- 0.20 (T4), 0.44 +/- 0.19 (T8) for females (n = 44). T4:T8 ratio for males and females were 1.79 and 1.58, respectively (0.05 less than P less than 0.10). Within-film area-to-area difference was 4.2 +/- 2.1% (n = 537 films). Interobserver discrepancy was 54 per 3,000 cells counted, or 1.8%. Film-to-film difference was 5.4 +/- 2.6% (30 pairs). T-lymphocyte subsets enumeration by immunostaining blood smears is a simple and reliable alternative to the immunofluorescence method. It avoids procedures that could artifactually affect lymphocyte subsets. The lower T4:T8 ratios revealed in this study could result because of technical reasons.

Adolescent↗
PubMed1983

Effects of dietary protein on the subacute toxicity of butylated hydroxytoluene (BHT) in rats.

Individual and combined effects of dietary protein and butylated hydroxytoluene (BHT) were investigated. Groups of young, female rats were fed diets containing 24, 8, or 4% protein for 5 weeks. During the fifth week, BHT was administered intragastrically to half the rats in each group at 250 mg/kg/day for 7 days. Reduction of protein intake, particularly to 4% dietary level, caused significant reduction in body weight gain, relative liver weight, hepatic microsomal cytochrome P-450, liver total protein, and serum albumin concentration as well as elevation of hepatic glucose-6-phosphatase activity, relative heart weight, and serum globulin concentration. BHT treatment, on the other hand, caused significant reduction in body weight gain and glucose-6-phosphatase activity as well as liver enlargement, induction of hepatic microsomal protein and cytochrome P-450, and elevation of serum total cholesterol level. Several effects of BHT intensified as protein intake was reduced. In addition, reduction of relative spleen weight and liver necrosis were observed only among the BHT-treated rats fed 4% dietary protein. It was concluded that combination of protein deprivation and BHT treatment produced responses that are significantly greater than the sum of the corresponding responses produced by the individual treatments.

Animals↗
PubMed1983

Effects of dietary protein on the pharmacokinetics of butylated hydroxytoluene (BHT) in rats.

The effects of dietary protein on the metabolism of butylated hydroxytoluene (BHT) were examined in the present study by comparison of the pharmacokinetics of BHT in rats fed different levels of dietary protein. Groups of young female rats were fed diets containing 24%, 8%, or 4% protein, as supplied from casein, for 4 weeks. At the end of the feeding period, a single oral dose of 500 mg BHT/kg was administered intragastrically to each rat. The serum BHT concentration was then measured at intervals during the first 12 hr following BHT administration. Several kinetic measurements were obtained from both the zero-order and first-order plots of serum BHT concentration versus time. Reduced BHT metabolism as indicated by increased concentration and delayed clearance of BHT in the circulation was found among rats fed 4% dietary protein compared to those fed 24% protein. On the other hand, no significant alteration was observed in any of the kinetic parameters obtained from rats fed 8% dietary protein. It was concluded that metabolism of BHT is reduced only when protein consumption is reduced to a 4% dietary level.

Animals↗