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Biomedical subjects

L Chiang

Publications and source records attributed to L Chiang.

At least 19 recordsLinked to original sources
PubMed2000

Dually incontinent nursing home residents: clinical characteristics and treatment differences.

OBJECTIVE: Previous studies have described urinary and fecal incontinence in nursing homes and their separate effects on healthcare utilization. However, little is known about those who are incontinent of both. DESIGN: Retrospective chart review. SETTINGS: Twenty sites in three states PARTICIPANTS: A total of 413 nursing home residents were categorized as having neither fecal nor urinary incontinence (C, n = 114), urinary incontinence only (UI, n = 53), fecal incontinence only (FI, n = 9), or were dually incontinent (DI, n = 237). MEASUREMENTS: Charts were abstracted for sociodemographic information and health status information as well as utilization for the year before the date of abstraction. We then compared these characteristics across groups using ANOVA with pairwise comparisons and multiply adjusted regression. RESULTS: Almost all patients with DI were cognitively and mobility impaired. However, there were no significant differences between the groups with respect to age and number of diagnoses. A diagnosis of stroke was also more common among those with DI compared with C. When examining healthcare utilization in multiply adjusted regression, dually incontinent residents received significantly fewer days of hospital care than those with UI. CONCLUSIONS: Dual incontinence in NH residents is likely to have an important functional component. These residents seem to be treated less aggressively with respect to hospitalization compared with those with UI alone. The reasons for these differences need to be explored further.

Aged↗
PubMed1997

Targeting of U2AF65 to sites of active splicing in the nucleus.

U2AF65 is an essential splicing factor that promotes binding of U2 small nuclear (sn)RNP at the pre-mRNA branchpoint. Here we describe a novel monoclonal antibody that reacts specifically with U2AF65. Using this antibody, we show that U2AF65 is diffusely distributed in the nucleoplasm with additional concentration in nuclear speckles, which represent subnuclear compartments enriched in splicing snRNPs and other splicing factors. Furthermore, transient expression assays using epitope-tagged deletion mutants of U2AF65 indicate that targeting of the protein to nuclear speckles is not affected by removing either the RNA binding domain, the RS domain, or the region required for interaction with U2AF35. The association of U2AF65 with speckles persists during mitosis, when transcription and splicing are downregulated. Moreover, U2AF65 is localized to nuclear speckles in early G1 cells that were treated with transcription inhibitors during mitosis, suggesting that the localization of U2AF65 in speckles is independent of the presence of pre-mRNA in the nucleus, which is consistent with the idea that speckles represent storage sites for inactive splicing factors. After adenovirus infection, U2AF65 redistributes from the speckles and is prefferentially detected at sites of viral transcription. By combining adenoviral infection with transient expression of deletion mutants, we show a specific requirement of the RS domain for recruitment of U2AF65 to sites of active splicing in the nucleus. This suggests that interactions involving the RS region of U2AF65 may play an important role in targeting this protein to spliceosomes in vivo.

Amino Acid Sequence↗
PubMed1996

Efficient random subcloning of DNA sheared in a recirculating point-sink flow system.

Based on a high-performance liquid chromatographic pump, we have built a device that allows recirculation of DNA through a 63-microm orifice with ensuing fractionation to a minimum fragment size of approximately 300 base pairs. Residence time of the DNA fragments in the converging flow created by a sudden contraction was found to be sufficiently long to allow extension of the DNA molecules into a highly extended conformation and, hence, breakage to occur at midpoint. In most instances, 30 passages sufficed to obtain a narrow size distribution, with >90% of the fragments lying within a 2-fold size distribution. The shear rate required to achieve breakage was found to be inversely proportional to the 1.0 power of the molecular weight. Compared with a restriction digest, up to 40% of all fragments could be cloned directly, with only marginal improvements in cloning efficiency having been observed upon prior end repair with Klenow, T4 polymerase or T4 polynucleotide kinase. Sequencing revealed a fairly random distribution of the fragments.

Bacteriophage M13↗
PubMed1996

Translational control of programmed cell death: eukaryotic translation initiation factor 4E blocks apoptosis in growth-factor-restricted fibroblasts with physiologically expressed or deregulated Myc.

There is increasing evidence that cell cycle transit is potentially lethal, with survival depending on the activation of metabolic pathways which block apoptosis. However, the identities of those pathways coupling cell cycle transit to survival remain undefined. Here we show that the eukaryotic translation initiation factor 4E (eIF4E) can mediate both proliferative and survival signaling. Overexpression of eIF4E completely substituted for serum or individual growth factors in preserving the viability of established NIH 3T3 fibroblasts. An eIF4E mutant (Ser-53 changed to Ala) defective in mediating its growth-factor-regulated functions was also defective in its survival signaling. Survival signaling by enforced expression of eIF4E did not result from autocrine release of survival factors, nor did it lead to increased expression of the apoptosis antagonists Bcl-2 and Bcl-XL. In addition, the execution apparatus of the apoptotic response in eIF4E-overexpressing cells was found to be intact. Increased expression of eIF4E was sufficient to inhibit apoptosis in serum-restricted primary fibroblasts with enforced expression of Myc. In contrast, activation of Ha-Ras, which is required for eIF4E proliferative signaling, did not suppress Myc-induced apoptosis. These data suggest that the eIF4E-activated pathways leading to survival and cell cycle progression are distinct. This dual signaling of proliferation and survival might be the basis for the potency of eIF4E as an inducer of neoplastic transformation.

3T3 Cells↗
PubMed1995

Analysis of trans-dominant mutants of the HIV type 1 Rev protein for their ability to inhibit Rev function, HIV type 1 replication, and their use as anti-HIV gene therapeutics.

The HIV-1 rev gene product facilitates the transport of singly spliced and unspliced HIV-1 transcripts and is necessary for productive HIV-1 infection. On the basis of the previously described trans-dominant Rev mutant M10, four point mutants and one frameshift mutant of the Rev protein were constructed. The mutants were inserted into retroviral expression vectors and analyzed for their ability to inhibit Rev-mediated gene expression. Transient transfection systems were used to screen these new mutants, and each was shown to inhibit expression of a Rev-dependent CAT reporter plasmid. Inhibition of HIV-1 envelope gene expression was tested in the HeLa-T4 cell line and was also shown to be inhibited by the trans-dominant Rev mutants. Retroviral vector producer cell lines were constructed and used to transduce Rev trans-dominant genes into the human T-cell line SupT1. The engineered SupT1 cell lines were then challenged with HIV-1 IIIB and HIV-1 expression was monitored by Northern blot analysis and in situ hybridization. SupT1 cells expressing either a Rev point mutant or the frameshift mutant showed greatly reduced HIV-1 mRNA accumulation and the Rev-dependent singly spliced and unspliced HIV-1 mRNAs were reduced. The kinetics of viral replication following challenge of Rev trans-dominant-engineered SupT1 cells with both HIV-1 IIIB and MN strains was significantly reduced and cells were protected from viral lysis. Viruses that emerge late in infection from Rev trans-dominant-engineered cultures are not resistant to Rev-mediated inhibition. Last, trans-dominant Rev-mediated protection of human CD4+ lymphocytes from challenge with primary HIV-1 patient isolates confirms the potential utility of this system as an anti-HIV-1 gene therapy approach.

Amino Acid Sequence↗
PubMed1995

Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes.

Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vectors expressing either HIV-1-specific antisense-TAR or antisense-Tat/Rev RNA, transdominant HIV-1 Rev protein, and a combination of antisense-TAR and transdominant Rev. The engineered CD4+ T lymphocytes were then infected with four different clinical HIV-1 isolates. We found that replication of all HIV-1 isolates was inhibited by all the anti-HIV vectors tested. Greater inhibition of HIV-1 was observed with transdominant Rev than with antisense RNA. We hereby demonstrated effective protection by antisense RNA or transdominant mutant proteins against HIV-1 infection in primary CD4+ T lymphocytes using clinical HIV-1 isolates, and this represents an essential step toward clinical anti-HIV-1 gene therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Macrophage production of basic fibroblast growth factor in the fibroproliferative disorder of alveolar fibrosis after lung injury.

In organ repair following injury, macrophages accumulate and granulation tissue, comprised of fibroblasts and endothelial cells, develops in the injured area. Basic fibroblast growth factor (bFGF), a potent stimulator of fibroblast and endothelial cell growth, has been linked to the fibroproliferative process. Macrophages are thought to play a central role in the fibroproliferative response, and prior studies indicate that they produce bFGF. Whereas it is plausible that macrophages produce bFGF in a fibroproliferative process, currently no data exists that directly identifies the macrophage as a source of bFGF in a fibroproliferative disorder. We used the model of acute intraalveolar granulation tissue formation following lung injury to determine if the macrophage was a cellular source of bFGF in a naturally occurring fibroproliferative process. To examine this hypothesis, patients with severe acute lung injury underwent bronchoalveolar lavage during the phase of lung repair. Polymerase chain reaction and Northern analysis of macrophage RNA revealed the presence of two species of bFGF messenger RNA (4.4 kb and 1.9 kb). Metabolic labeling studies of recovered macrophages revealed a newly synthesized 18-kd protein with antigenic similarity to bFGF. Immunohistochemical evaluation of lung tissue from patients who died following acute lung injury, showed numerous bFGF immunoreactive macrophages present within airspaces containing fibroblastic and vascular tissue proliferation. This investigation has identified the alveolar macrophage as a cellular source of bFGF in the fibroproliferative disorder of intraalveolar fibrosis following acute lung injury.

Autoradiography↗
PubMed1992

DNA sequence characterization of the G gene region of bacteriophage Mu.

The nucleotide sequence of a 1.2 kb region of bacteriophage Mu DNA was determined. This region contains the 3' end of the F gene, the complete G gene, and the 5' end of the I gene, all late genes involved in Mu virion morphogenesis. Identity of the G gene open reading frame was confirmed by sequencing four Gam mutations. The G open reading frame is predicted to encode proteins of 16.7 or 17.2 kDa, depending on which of two possible start codons are used to initiate translation. Four new nuB mutations in the DNA gyrase-binding site between the G and I genes were also sequenced and found to be identical to the nuB103 mutation sequenced previously.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of a dideoxyguanosine triphosphate-resistant mutant of human immunodeficiency virus reverse transcriptase.

The appearance of drug-resistant strains of viral pathogens is a major difficulty confounding current efforts to block viral infections. The identification and analysis of mutations responsible for drug resistance can provide important clues helpful in understanding the mechanisms of resistance and in the eventual development of better therapies. We have used a direct screening method to scan libraries of mutagenized genes encoding the reverse transcriptase of human immunodeficiency virus type 1, and have recovered a variant enzyme that is resistant to the chain-terminator inhibitor 2',3'-dideoxyguanosine triphosphate. The single substitution mutation in this variant conferred broad crossresistance to a variety of other antiviral compounds currently in clinical trials. Virus carrying the mutation was fully infectious in cultured human lymphocytes. The replication of the mutant virus was highly resistant to phosphonoformic acid but did not show increased resistance to the prodrug dideoxyguanosine.

Antiviral Agents↗
PubMed1990

Characterization of cellular dermal infiltrates in human cutaneous mastocytosis.

Biopsies of lesional and nonlesional skin from 14 patients with localized cutaneous or associated systemic mastocytosis were examined by ultrastructural and immunohistochemical techniques. Mast cells within lesions of the dermis were highly variable between patients with regard to cell number and extent of degranulation, although lesional sites consistently contained more mast cells than did nonlesional sites. Two mast cell patterns were identified based upon granule morphology. In biopsies from 8 patients, the majority of granules contained electron-dense amorphous zones; crystalline lattices; and indistinct, incomplete solid scrolls forming parallel lamellae. In biopsies from 6 patients, in addition to these granules, there were also granules composed of electron-dense amorphous zones, reticulated matrices, and/or distinct scrolls with lucent cores interrupted by dense spheres. The granule morphology for the first group (N = 8) was identical with that seen in the preponderant type of skin mast cell of 6 normal control subjects, whereas the granule morphology of the second group (N = 6) displayed an abnormal ultrastructural phenotype for skin that included granule types normally found not only in skin but also in intestinal lamina propria and lung. For individual patients, the patterns of granule ultrastructure were consistent between clinically nonlesional and lesional skin. A minority of cells in both patient groups appeared primitive ultrastructurally, exhibiting rudimentary, Golgi-associated progranules; monocyte-like morphologic characteristics; and mitotic activity. Moreover, when mast cells in lesional skin were screened for a limited panel of surface antigens, they displayed common patterns of reactivity (M718+, HLA-DR/DQ+, CD4+), and in a selected case, immunoelectron microscopy confirmed the presence of these antigens on mast cell plasma membranes. Dermal mast cells from normal donors (N = 6) lack these epitopes. These observations suggest that infiltrates in cutaneous mastocytosis may exhibit phenotypic characteristics not only of cutaneous mast cells, but in some patients also of mucosal mast cells. In either circumstance, the mast cells may display antigenic determinants common to monocyte/macrophages. Concordance of granule phenotype between lesional and clinically uninvolved skin of individual patients furthers the notion that even localized mastocytosis reflects covertly defective systemic mast cell homeostasis.

Adult↗