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G Wang

Publications and source records attributed to G Wang.

At least 19 recordsLinked to original sources
PubMed2026

Development of RS1-specific ACMG/AMP variant classification criteria with pilot variant curation.

Gene-based therapies are being developed for retinal diseases, including RS1-related X-linked retinoschisis. Therefore it is essential to determine which variants are pathogenic and which are benign when enrolling patients. The Clinical Genome Resource (ClinGen) X-Linked Inherited Retinal Diseases (XLRD) Variant Curation Expert Panel (VCEP) brings together clinician scientists, molecular biologists, and geneticists to apply their expertise and review the clinical, genetic, population, and functional evidence for variants. American College of Medical Genetics (ACMG) guidelines have been modified for RS1 to develop a highly systematic and conservative framework for evaluating variants. The curation process involves applying 28 different codes, each with 4 strength levels (very strong, strong, moderate, supporting) across different domains of phenotype, population data, computational assessment, functional impact, and segregation. With RS1-specific rules, a total of 54 pilot variants were tested. These included 47 variants in ClinVar. Of these 21 variants were re-classified: 2 likely pathogenic variants and one likely benign were changed to variants of uncertain significance and 4 previously unclassified variants were changed to pathogenic, likely pathogenic and likely benign. Other changes resolved conflicts or multiple classifications.

Humans↗
PubMed1999

Role of DNAS1L3 in Ca2+- and Mg2+-dependent cleavage of DNA into oligonucleosomal and high molecular mass fragments.

Ca2+- and Mg2+-dependent endonucleases have been implicated in DNA fragmentation during apoptosis. We have demonstrated that particular nucleases of this type are inhibited by poly(ADP-ribosyl)ation and suggested that subsequent cleavage of PARP by caspase-3 might release these nucleases from poly(ADP-ribosyl)ation-induced inhibition. Hence, we purified and partially sequenced such a nuclease isolated from bovine seminal plasma and identified human, rat and mouse homologs of this enzyme. The extent of sequence homology among these nucleases indicates that these four proteins are orthologous members of the family of DNase I-related enzymes. We demonstrate that the activation of the human homolog previously specified as DNAS1L3 can induce Ca2+- and Mg2+-dependent DNA fragmentation in vitro and in vivo. RT-PCR analysis failed to detect DNAS1L3 mRNA in HeLa cells and nuclei isolated from these cells did not exhibit internucleosomal DNA fragmentation when incubated in the presence of Ca2+and Mg2+. However, nuclei isolated from HeLa cells that had been stably transfected with DNAS1L3 cDNA underwent such DNA fragmentation in the presence of both ions. The Ca2+ionophore ionomycin also induced internucleosomal DNA degradation in transfected but not in control HeLa cells. Transverse alternating field electrophoresis revealed that in nuclei from transfected HeLa cells, but not in those from control cells, DNA was cleaved into fragments of >1000 kb in the presence of Mg2+; addition of Ca2+in the presence of Mg2+resulted in processing of the >1000 kb fragments into 50 kb and oligonucleosomal fragments. These results demonstrate that DNAS1L3 is necessary for Ca2+- and Mg2+-dependent cleavage of DNA into both oligonucleosomal and high molecular mass fragments in specific cell types.

Amino Acid Sequence↗
PubMed1999

Effects of various numbers and positions of cis double bonds in the sn-2 acyl chain of phosphatidylethanolamine on the chain-melting temperature.

In an attempt to investigate systematically the effects of various single and multiple cis carbon-carbon double bonds in the sn-2 acyl chains of natural phospholipids on membrane properties, we have de novo synthesized unsaturated C20 fatty acids comprised of single or multiple methylene-interrupted cis double bonds. Subsequently, 15 molecular species of phosphatidylethanolamine (PE) with sn-1 C20-saturated and sn-2 C20-unsaturated acyl chains were semi-synthesized by acylation of C20-lysophosphatidylcholine with unsaturated C20 fatty acids followed by phospholipase D-catalyzed base-exchange reaction in the presence of excess ethanolamine. The gel-to-liquid crystalline phase transitions of these 15 mixed-chain PE, in excess H2O, were investigated by high resolution differential scanning calorimetry. In addition, the energy-minimized structures of these sn-1 C20-saturated/sn-2 C20-unsaturated PE were simulated by molecular mechanics calculations. It is shown that the successive introduction of cis double bonds into the sn-2 acyl chain of C(20):C(20)PE can affect the gel-to-liquid crystalline phase transition temperature, Tm, of the lipid bilayer in some characteristic ways; moreover, the effect depends critically on the position of cis double bonds in the sn-2 acyl chain. Specifically, we have constructed a novel Tm diagram for the 15 species of unsaturated PE, from which the effects of the number and the position of cis double bonds on Tm can be examined simultaneously in a simple, direct, and unifying manner. Interestingly, the characteristic Tm profiles exhibited by different series of mixed-chain PE with increasing degree of unsaturation can be interpreted in terms of structural changes associated with acyl chain unsaturation.

Calorimetry, Differential Scanning↗
PubMed1999

Identification and characterization of receptor for mammalian hepatopoietin that is homologous to yeast ERV1.

Hepatopoietin (HPO) is a novel polypeptide mitogen specific for hepatocytes and hepatoma cell lines, which is derived from liver and supports its regeneration. To determine whether HPO acts via a receptor-based signal transduction, recombinant human hepatopoietin was labeled by iodination and used to characterize its binding activity by specific displacement test and Scatchard analysis in primarily cultured rat hepatocytes and human hepatoma Hep-G2 cells. The binding was saturable and specific because it was replaceable by HPO but not by epidermal growth factor, transforming growth factor-alpha, or insulin. Scatchard analysis indicated the presence of a single class of high affinity receptor with dissociation constant (Kd) of 2 and 0.7 pM, and a receptor density of about 10, 000 sites/cell and 55,000 sites/cell in the rat hepatocytes and human hepatoma cells, respectively. The Kd values were consistent with the half-maximum dose of HPO activity. Affinity cross-linking of the receptor with 125I-HPO revealed a polypeptide of molecular mass approximately 90 kDa by SDS-polyacrylamide gel electrophoresis. Thus, the molecular mass of the HPO receptor was calculated to be about 75 kDa. These data demonstrated the existence of an HPO receptor in hepatocytes and hepatoma cells, which may account for biological effect.

Animals↗
PubMed1999

Independent development of sensory and motor innervation patterns in embryonic chick hindlimbs.

Previous studies suggest that sensory axon outgrowth is guided by motoneurons, which are specified to innervate particular target muscles. Here we present evidence that questions this conclusion. We have used a new approach to assess the pathfinding abilities of bona fide sensory neurons, first by eliminating motoneurons after neural crest cells have coalesced into dorsal root ganglia (DRG) and second by challenging sensory neurons to innervate muscles in a novel environment created by shifting a limb bud rostrally. The resulting sensory innervation patterns mapped with the lipophilic dyes DiI and DiA showed that sensory axons projected robustly to muscles in the absence of motoneurons, if motoneurons were eliminated after DRG formation. Moreover, sensory neurons projected appropriately to their usual target muscles under these conditions. In contrast, following limb shifts, muscle sensory innervation was often derived from inappropriate segments. In this novel environment, sensory neurons tended to make more "mistakes" than motoneurons. Whereas motoneurons tended to innervate their embryologically correct muscles, sensory innervation was more widespread and was generally from more rostral segments than normal. Similar results were obtained when motoneurons were eliminated in embryos with limb shifts. These findings show that sensory neurons are capable of navigating through their usual terrain without guidance from motor axons. However, unlike motor axons, sensory axons do not appear to actively seek out appropriate target muscles when confronted with a novel terrain. These findings suggest that sensory neuron identity with regard to pathway and target choice may be unspecified or quite plastic at the time of initial axon outgrowth.

Animals↗
PubMed1999

Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells.

Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control (psoSCR30) psoralen-conjugated TFOs in the presence and absence of UVA irradiation. The results demonstrated a 6- to 10-fold induction of supFG1 mutations in the psoAG30-treated cells as compared with psoSCR30-treated or untreated control cells. Interestingly, UVA irradiation had no effect onthe mutation frequencies induced by the psoralen-conjugated TFOs, suggesting a triplex-mediated but photoproduct-independent process of mutagenesis. Sequencing data were consistent with this finding since the expected T.A-->A.T transversions at the predicted psoralen crosslinking site were not detected. However, insertions and deletions were detected within the triplex binding site, indicating a TFO-specific induction of mutagenesis. This result demonstrates the ability of triplex-forming oligonucleotides to influence mutation frequencies at a specific site in a mammalian chromosome.

Animals↗
PubMed1999

A field test of the quotient method for predicting risk to Microtus canicaudus in grasslands.

We used gray-tailed voles, Microtus canicaudus, as an experimental model species to field test a prediction of the Quotient Method (QM) for risk to small mammals of an insecticide in grasslands. In May 1997, we placed voles into 12 0.2-ha enclosures planted with a mixture of pasture grasses. In late July, we applied 1.55 kg/ha of the insecticide Guthion(R) 2S (azinphos-methyl) in three treatments: a control (all habitat sprayed with water), full spray (all of the habitat sprayed with Guthion 2S), and half-spray (one-half of the habitat sprayed with Guthion 2S and one half with water). Five replicates were used for the half-spray and control, and two replicates for the full-spray. The Guthion 2S treatment did not depress population size, growth rate, or survival of voles in half-spray or full-spray enclosures. Our results were inconsistent with the QM prediction for use of Guthion 2S in grass habitats. These results also differed from our previous studies in alfalfa habitats in which measurable responses were detected at the same application rate. The differences probably are due to the different types of vegetation between current and previous studies. The grass habitat in our enclosures was an average of 60 cm high and was very thick from ground level to about 40 cm. Much of the residue likely accumulated in the upper strata of vegetation and did not reach ground level at this application rate. Under the conditions of this study, voles were less affected in grassland habitat than they were in alfalfa habitat.

Animals↗
PubMed1999

Does thin filament compliance diminish the cross-bridge kinetics? A study in rabbit psoas fibers.

The effect of thin filament compliance on our ability to detect the cross-bridge kinetics was examined. Our experiment is based on the facts that in rabbit psoas the thin filament (1.12 micrometer) is longer than half the thick filament length (0.82 micrometer) and that the thick filament has a central bare zone (0.16 micrometer). Consequently, when sarcomere length is increased from 2.1 to 2.4 micrometer, the same number of cross-bridges is involved in force generation but extra series compliance is introduced in the I-band. Three apparent rate constants (2pia, 2pib, and 2pic) were characterized by sinusoidal analysis at pCa 4.66. Our results demonstrate that 2pia and 2pib increased 13-16% when sarcomere length was increased from 2.0 to 2.5 micrometer, and 2pic decreased slightly (9%). This slight decrease can be explained by compression of the lattice spacing. These observations are at variance with the expectation based on increased series compliance, which predicts that the rate constants will decrease. We also determined compliance of the I-band during rigor. I-band compliance during rigor induction was 35% of sarcomere compliance at sarcomere length 2.4 micrometer, and 24% at sarcomere length 2.1 micrometer. We conclude that the presence of thin filament compliance does not seriously interfere with our ability to detect cross-bridge kinetics using sinusoidal analysis.

Animals↗
PubMed1999

Iterative X-ray Cone-Beam Tomography for Metal Artifact Reduction and Local Region Reconstruction.

: X-ray cone-beam reconstruction from incomplete projection data has important practical applications, especially in microtomography. We developed expectation maximization (EM)-type and algebraic reconstruction technique (ART)-type iterative cone-beam reconstruction algorithms for metal artifact reduction and local reconstruction from truncated data. These iterative algorithms are adapted from the emission computerized tomography (CT) EM formula and the ART. A key step in our iterative algorithms is introduction of a projection mask and computation of a 3-D spatially varying relaxation factor that allows compensation for beam divergence and data incompleteness. The algorithms are simulated with projection data synthesized from mathematical phantoms. In simulation, the EM-type and ART-type iterative algorithms are demonstrated to be effective for metal artifact reduction and local region reconstruction. They perform similarly in terms of visual quality, image noise, and discrepancy between measured and reprojected data. The EM-type and ART-type iterative cone-beam reconstruction algorithms have potential for metal artifact reduction and local region reconstruction in X-ray CT.

Journal Article↗
PubMed1999

Molecular genetic basis for the variable expression of Lewis Y antigen in Helicobacter pylori: analysis of the alpha (1,2) fucosyltransferase gene.

Helicobacter pylori lipopolysaccharides (LPS) express human oncofetal antigens Lewis X and Lewis Y. The synthesis of Lewis Y involves the actions of alpha (1,3) and alpha (1,2) fucosyltransferases (FucTs). Here, we report the molecular cloning and characterization of genes encoding H. pylori alpha (1,2) FucT (Hp fucT2) from various H. pylori strains. We constructed Hp fucT2 knock-out mutants and demonstrated the loss of Lewis Y production in these mutants by enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. The Hp fucT2 gene contains a hypermutable sequence [poly (C) and TAA repeats], which provides a possibility of frequent shifting into and out of coding frame by a polymerase slippage mechanism. Thus, the Hp fucT2 gene displays two major genotypes, consisting of either a single full-length open reading frame (ORF; as in the strain UA802) or truncated ORFs (as in the strain 26695). In vitro expression of Hp fucT2 genes demonstrated that both types of the gene have the potential to produce the full-length protein. The production of the full-length protein by the 26695 fucT2 gene could be attributed to translational-1 frameshifting, as a perfect translation frameshift cassette resembling that of the Escherichia coli dnaX gene is present. Examination of the strain UA1174 revealed that its fucT2 gene has a frameshifted ORF at the DNA level, which cannot be compensated by translation frameshifting, accounting for its Lewis Y off phenotype. In another strain, UA1218, the fucT2 gene is apparently turned off because of the loss of its promoter. Based on these data, we proposed a model for the variable expression of Lewis Y by H. pylori, in which regulation at the level of replication slippage (mutation), transcription and translation of the fucT2 gene may all be involved.

Amino Acid Sequence↗
PubMed1999

Comparative sequence analysis and predictions for the envelope glycoproteins of foamy viruses.

The foamy viruses (FVs) are a genus of complex retroviruses that has recently been found to possess several novel molecular features. There is increasing interest in the development of FVs as novel vectors for gene delivery. As there are remarkably few published studies of FV proteins, these recent findings prompted us to predict the structural features of FV glycoproteins with the aid of computer programs. We analysed all seven available FV Env sequences, a greater number of sequences than in previously published analyses. The relative rates of change for FV structural proteins were Pol < Env < Gag in increasing order, which differs from all other retroviruses. We determined that this difference is primarily caused by a higher relative rate of change for FV Gag proteins. We analysed the functional domains of FV glycoproteins and found that their structural organization was generally similar to other retroviruses. Putative structures were identified for the signal peptide, cleavage site, fusion peptide, membrane-spanning domain and the unique endoplasmic reticulum retrieval signal. Based on the predicted secondary structure of the transmembrane glycoprotein (TM) subunit, gp47, we also identified a unique prolonged central 'sheets and loops' region as the dominant feature of an unusually lengthy TM ectodomain. This lengthy central domain was flanked at each end by alpha-helices. The predictions reported here will stimulate and facilitate experimental approaches to better understand the structure and function of FV glycoproteins, and should assist in the planning and development of FV vectors.

Amino Acid Sequence↗
PubMed1999

Multiplex sequence analysis demonstrates the competitive growth advantage of the A-to-G mutants of clarithromycin-resistant Helicobacter pylori.

Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates.

Alanine↗
PubMed1999

Functional domains of the Rsp5 ubiquitin-protein ligase.

RSP5, an essential gene of Saccharomyces cerevisiae, encodes a hect domain E3 ubiquitin-protein ligase. Hect E3 proteins have been proposed to consist of two broad functional domains: a conserved catalytic carboxyl-terminal domain of approximately 350 amino acids (the hect domain) and a large, nonconserved amino-terminal domain containing determinants of substrate specificity. We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably interact with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II), and the finding that the hect domain, by itself, is sufficient for formation of the ubiquitin-thioester intermediate. Mutations within the hect domain that affect either the ability to form a ubiquitin-thioester or to catalyze substrate ubiquitination abrogate in vivo function, strongly suggesting that the ubiquitin-protein ligase activity of Rsp5 is intrinsically linked to its essential function. The amino-terminal region of Rsp5 contains three WW domains and a C2 calcium-binding domain. Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not, and requirements for Rpb1 binding and ubiquitination lie within the region required for in vivo function. Together, these results support the two-domain model for hect E3 function and indicate that the WW domains play a role in the recognition of at least some of the substrates of Rsp5, including those related to its essential function. In addition, we show that haploid yeast strains bearing complete disruptions of either of two other hect E3 genes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.

Alleles↗
PubMed1998

Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas.

We describe the first potent and selective blocker of the class E Ca2+channel. SNX-482, a novel 41 amino acid peptide present in the venom of the African tarantula, Hysterocrates gigas, was identified through its ability to inhibit human class E Ca2+ channels stably expressed in a mammalian cell line. An IC50 of 15-30 nM was obtained for block of the class E Ca2+ channel, using either patch clamp electrophysiology or K+-evoked Ca2+ flux. At low nanomolar concentrations, SNX-482 also blocked a native resistant or R-type Ca2+ current in rat neurohypophyseal nerve terminals, but concentrations of 200-500 nM had no effect on R-type Ca2+ currents in several types of rat central neurons. The peptide has the sequence GVDKAGCRYMFGGCSVNDDCCPRLGCHSLFSYCAWDLTFSD-OH and is homologous to the spider peptides grammatoxin S1A and hanatoxin, both peptides with very different ion channel blocking selectivities. No effect of SNX-482 was observed on the following ion channel activities: Na+ or K+ currents in several cultured cell types (up to 500 nM); K+ current through cloned potassium channels Kv1.1 and Kv1. 4 expressed in Xenopus oocytes (up to 140 nM); Ca2+ flux through L- and T-type Ca2+ channels in an anterior pituitary cell line (GH3, up to 500 nM); and Ba2+ current through class A Ca2+ channels expressed in Xenopus oocytes (up to 280 nM). A weak effect was noted on Ca2+ current through cloned and stably expressed class B Ca2+ channels (IC50 > 500 nM). The unique selectivity of SNX-482 suggests its usefulness in studying the diversity, function, and pharmacology of class E and/or R-type Ca2+ channels.

Amino Acid Sequence↗
PubMed1998

The fission yeast chromo domain encoding gene chp1(+) is required for chromosome segregation and shows a genetic interaction with alpha-tubulin.

In eukaryotes, the segregation of chromosomes is co-ordinated by the centromere and must proceed accurately if aneuploidy and cell death are to be avoided. The fission yeast centromere is complex, containing highly repetitive regions of DNA showing the characteristics of heterochromatin. Two proteins, Swi6p and Clr4p, that are associated with the fission yeast centromere also contain a chromo (chromatin organisation modifier) domain and are required for centromere function. We have analysed a novel fission yeast gene encoding a putative chromo domain called chp 1(+) (chromo domain protein in Schizosaccharomyces p ombe ). In the absence of Chp1p protein, cells are viable but show chromosome segregation defects such as lagging chromosomes on the spindle during anaphase and high rates of minichromosome loss, phenotypes which are also displayed by swi 6 and clr 4. A fusion protein between green fluorescent protein (GFP) and Chp1p, like Swi6p, is localized to discrete sites within the nucleus. In contrast to Swi6p and Clr4p, Chp1p is not required to repress silent mating-type genes. We demonstrate a genetic interaction between chp 1(+) and alpha-tubulin ( nda 2(+)) and between swi 6(+) and beta-tubulin ( nda 3(+)). Chp1p and Swi6p proteins may be components of the kinetochore which captures and stabilizes the microtubules of the spindle.

Amino Acid Sequence↗
PubMed1998

A unifying Tm diagram for phosphatidylethanolamines with sn-1 C20 saturated and sn-2 C18 unsaturated acyl chains.

We have determined calorimetrically the phase transition temperature (Tm) values of five sn-1 saturated/sn-2 unsaturated phosphatidylethanolamines (PE) in which the sn-1 acyl chain has 20 carbons and the sn-2 acyl chain has 18 carbons with different number and position of the cis double bond. When these Tm values are combined with the five published Tm values of related unsaturated PE, a unifying Tm diagram is generated for the first time. Moreover, as the molecular mechanics simulated structures of these lipids are taken into consideration, this unifying Tm diagram provides insight into how variations in the number and position of the cis double bond in the lipid's sn-2 acyl chain can influence the phase transition behavior of the lipid bilayer.

Calorimetry, Differential Scanning↗