Biomedical subjects

Jinliang Li

Publications and source records attributed to Jinliang Li.

14 recordsLinked to original sources

PubMed2005

Requirement of an additional Sam68 domain for inhibition of human immunodeficiency virus type 1 replication by Sam68 dominant negative mutants lacking the nuclear localization signal.

Human immunodeficiency virus type 1 (HIV-1) replication requires active nuclear export of unspliced and incompletely spliced HIV-1 RNA transcripts. This process is evolutionally made possible by expression of HIV-1 Rev, one of the three HIV-1 proteins encoded by completely spliced HIV-1 RNAs. Evidence has accumulated to suggest that Sam68 plays an important role in HIV-1 replication through HIV-1 Rev protein. In the present study, we further examined the structure-function relationship of Sam68 protein in relation to HIV-1 replication. We identified a Sam68 domain located between aa269 and aa321 to be involved in the HIV-inhibitory effects of Sam68 dominant negative mutants lacking the nuclear localization signal (NLS). Deletion of this domain abrogated inhibition of HIV-1 replication by these mutants. HIV-1 Rev protein appeared to mediate the HIV-inhibitory effects of these mutants and by this domain, as assessed by Rev-dependent chloramphenicol acetyltransferase reporter gene assay, in trans rev-defective HIV-1 complementation assay, and RNase protection assay. The HIV-inhibitory mutants containing this domain were further found to have diminished binding affinity to the wild-type Sam68 and to be associated with cytoplasmic retention of exclusively nuclear localized wild type Sam68. Taken together, these results further ascertain the important role of Sam68 in HIV-1 Rev function and viral replication, and suggest that the HIV-inhibitory effects of Sam68 dominant negative mutants directly result from their binding to endogenous Sam68 and their interference with nuclear localization of endogenous Sam68.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Functional domains of APOBEC3G required for antiviral activity.

The viral protein, Vif, is essential for the production of infectious progeny virions in natural target cells of human immunodeficiency virus type 1 (HIV-1). Several recent reports indicate that Vif acts by antagonizing the activity of an endogenous human antiviral protein, APOBEC3G. To investigate this route to restrict HIV-1 infection, we employed mutagenesis to assess APOBEC3G function during HIV-1 infection including interaction with Vif, localization, and activity in virions. We found that APOBEC3G binds Vif in infected cells and the C'-terminal region is required for this interaction. APOBEC3G was only incorporated into virions in the absence of Vif and deletion of either the N'-terminal or C'-terminal regions of APOBEC3G abrogated virion localization. Assaying endogenous reverse transcription we found that APOBEC3G and its C'-terminal deletion mutant inhibited full-length cDNA synthesis, possibly through binding to viral RNA, a function revealed through gel-shift assays. Taken together, our studies suggest that APOBEC3G inhibits HIV-1 infection through interference with reverse transcription and that Vif counteracts APOBEC3G by impeding its entry into virions.

APOBEC-3G Deaminase↗

PubMed2004

Microarray analysis of changes in cellular gene expression induced by productive infection of primary human astrocytes: implications for HAD.

The role of astrocytes in HIV-1 associated dementia (HAD) is not well understood. HIV-1 binds efficiently to astrocytes but infects only a small fraction of the cells in vitro and in vivo. To gain insight into the biology of HIV-1-expressing astrocytes, we productively infected human fetal astrocytes with pseudotyped HIV-1 and employed Affymetrix oligonucleotide microarrays to determine global changes in cellular gene expression at the peak of virus production. With a twofold change as a cutoff, HIV-1 increased transcription of 266 genes in astrocytes and suppressed expression of 468. The functions of highly expressed genes included interferon-mediated antiviral responses (OAS1, IFIT1), intercellular contacts (SH3, glia-derived nexin), cell homing/adhesion (matrix metalloproteinases), and cell-cell signaling (neuropilin 1 and 2). Surprisingly, genes involved in innate immune responses of astrocytes were largely unaffected. The single most significant effect of HIV-1, however, was down-modulation of at least 55 genes involved in control of cell cycle, DNA replication, and cell proliferation, which were overrepresented in these categories with probability scores of 10(-10)-10(-26). Our data suggest that HIV-1 expression in astrocytes profoundly alters host cell biology, with potential consequences for the physiological function of astrocytes during HIV-1 infection in the brain.

Astrocytes↗

PubMed2004

Productive infection of primary murine astrocytes, lymphocytes, and macrophages by human immunodeficiency virus type 1 in culture.

A mouse model of human immunodeficiency virus type 1 (HIV-1) infection would be extremely valuable for evaluation of therapies and vaccines; however, multiple blocks to productive infection of NIH 3T3 and other mouse cell lines have been reported. The authors investigated the replication of HIV-1 in primary mouse astrocytes, lymphocytes, and macrophages in culture by infection with intact HIV-1 pseudotyped with the vesicular stomatitis virus G envelope glycoprotein (VSV-G) or with the envelope glycoprotein of amphotropic murine leukemia virus. Astrocytes, lymphocytes, and macrophages were susceptible to productive infection as variously assayed by detection of p24 and Tat proteins, viral protease-mediated processing of Gag, appropriately spliced viral RNA, and infectious progeny virus. As expected, NIH 3T3 cells were not susceptible to productive infection by VSV/NL4. Susceptibility mapped neither to the Fv locus nor to a possible polymorphism in cyclin T1. This study indicates that there are no intrinsic intracellular barriers to HIV-1 replication in primary mouse cells when virus entry is efficient.

Animals↗

PubMed2004

Gene expression profiles in response to the activation of adrenoceptors in A7r5 aortic smooth muscle cells.

1. Vascular adrenoceptors play an important role in vascular physiology and pathophysiology, such as hypertension, atherosclerosis and restenosis after angioplasty. To define the changes in the ene expression in vascular smooth muscle cells in response to the activation of alpha1- or beta-adrenoceptors, a DNA microarray was used. 2. First, the existence of alpha1- and beta-adrenoceptors in A7r5 aortic smooth muscle cells was confirmed by radioligand binding. Then, the inhibitory effects of phenylephrine (an alpha1-adrenoceptor agonist) and isoproterenol (a beta-adrenoceptor agonist) on the proliferation of A7r5 cells were determined by [3H]-thymidine incorporation. 3. The A7r5 cells were treated with 10 micromol/L phenylephrine or 1 micromol/L isoproterenol for 24 h and changes in gene expression were detected with the DNA microarray. Only 14 and 20 genes were identified after treatment of cells with phenylephrine and isoproterenol, respectively, and most genes displayed decreased expression. The changed genes could be grouped into five major functional categories: cell signalling/communication, cell structure/motility, cell/organism defence, gene/protein expression and metabolism. The gene expression profile in response to the activation of alpha1-adrenoceptors was very different from that following activation of beta-adrenoceptors. Interestingly, many phenylephrine-responsive genes were associated with metabolism, whereas many isoproterenol-responsive genes encoded cell signalling and structure proteins. This means that adrenoceptors may modulate multiple aspects of biological function in vascular smooth muscle cells. 4. Collectively, the activation of both alpha1-adrenoceptors (with phenylephrine) and beta-adrenoceptors (with isoproterenol) inhibited the proliferation of A7r5 cells, but microarray data revealed that the mechanisms may be different: the activation of alpha1-adrenoceptors could induce the expression of metabolic genes, resulting in the inhibition of proliferation, whereas activation of beta-adrenoceptors altered the expression of genes that encoded cell signalling and structure proteins to inhibit cell proliferation.

Adrenergic alpha-1 Receptor Agonists↗

PubMed2004

CD4-independent infection of astrocytes by human immunodeficiency virus type 1: requirement for the human mannose receptor.

Human immunodeficiency virus type 1 (HIV-1) infection occurs in the central nervous system and causes a variety of neurobehavioral and neuropathological disorders. Both microglia, the residential macrophages in the brain, and astrocytes are susceptible to HIV-1 infection. Unlike microglia that express and utilize CD4 and chemokine coreceptors CCR5 and CCR3 for HIV-1 infection, astrocytes fail to express CD4. Astrocytes express several chemokine coreceptors; however, the involvement of these receptors in astrocyte HIV-1 infection appears to be insignificant. In the present study using an expression cloning strategy, the cDNA for the human mannose receptor (hMR) was found to be essential for CD4-independent HIV-1 infectivity. Ectopic expression of functional hMR rendered U87.MG astrocytic cells susceptible to HIV-1 infection, whereas anti-hMR serum and hMR-specific siRNA blocked HIV-1 infection in human primary astrocytes. In agreement with these findings, hMR bound to HIV-1 virions via the abundant and highly mannosylated sugar moieties of HIV-1 envelope glycoprotein gp120 in a Ca(2+)-dependent fashion. Moreover, hMR-mediated HIV-1 infection was dependent upon endocytic trafficking as assessed by transmission electron microscopy, as well as inhibition of viral entry by endosomo- and lysosomotropic drugs. Taken together, these results demonstrate the direct involvement of hMR in HIV-1 infection of astrocytes and suggest that HIV-1 interaction with hMR plays an important role in HIV-1 neuropathogenesis.

Animals↗

PubMed2003

Complete sequence of the mitochondrial genome of Tetrahymena thermophila and comparative methods for identifying highly divergent genes.

The complete sequence of the mitochondrial genome of Tetrahymena thermophila has been determined and compared with the mitochondrial genome of Tetrahymena pyriformis. The sequence similarity clearly indicates homology of the entire T.thermophila and T.pyriformis mitochondrial genomes. The T.thermophila genome is very compact, most of the intergenic regions are short (only three are longer than 63 bp) and comprise only 3.8% of the genome. The nad9 gene is tandemly duplicated in T.thermophila. Long terminal inverted repeats and the nad9 genes are undergoing concerted evolution. There are 55 putative genes: three ribosomal RNA genes, eight transfer RNA genes, 22 proteins with putatively assigned functions and 22 additional open reading frames of unknown function. In order to extend indications of homology beyond amino acid sequence similarity we have examined a number of physico-chemical properties of the mitochondrial proteins, including theoretical pI, molecular weight and particularly the predicted transmembrane spanning regions. This approach has allowed us to identify homologs to ymf58 (nad4L), ymf62 (nad6) and ymf60 (rpl6).

Amino Acid Sequence↗

PubMed2003

Effects of losartan on pressure overload-induced cardiac gene expression profiling in rats.

1. In the present study, the effects of losartan on myocardial gene expression changes following cardiac hypertrophy were investigated. 2. Male Wistar rats were randomized to receive 5 or 30 mg/kg per day losartan (i.p.) 1 day after suprarenal abdominal aortic constriction. Two weeks later, cardiac morphology and function were recorded with echocardiography and mean arterial central pressure was measured using carotid catheters. Myocardial gene expression was assessed with cDNA microarrays. 3. The ratios of left ventricular weights to bodyweights, the posterior thickness of the left ventricle and mean arterial central pressure were significantly increased by aortic constriction and attenuated by losartan in a dose-related manner. Genes in different functional categories were regulated in pressure overload-induced cardiac hypertrophy and the majority of changes in gene expression were inhibited by losartan in a dose-dependent manner. 4. However, there were still some genes that were unaffected by losartan, even at a higher dose. In contrast, losartan, especially at a lower dose, was able to induce changes in the expression of several additional genes that were unregulated in simple aortic constriction. 5. In conclusion, losartan is able to inhibit pressure overload-induced cardiac hypertrophy, as well as the majority of pressure overload-related changes in gene expression. The genes that remained unaffected or those that were additionally induced by losartan are likely to be new targets for investigation or therapy.

Animals↗

PubMed2002

HIV-1 Tat protein-mediated transactivation of the HIV-1 long terminal repeat promoter is potentiated by a novel nuclear Tat-interacting protein of 110 kDa, Tip110.

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication is highly dependent on and modulated by interactions between viral and host cellular factors. Tat protein, encoded by one of the HIV-1 regulatory genes, tat, is essential for HIV-1 gene expression. A number of host cellular factors have been shown to interact with Tat in this process. During our attempts to determine the molecular mechanisms of Tat interaction with brain cells, we isolated a cDNA clone that encodes a novel Tat-interacting protein of 110 kDa or Tip110 from a human fetal brain cDNA library. GenBank BLAST search revealed that Tip110 was almost identical to a previously cloned KIAA0156 gene with unknown functions. In vivo binding of Tip110 with Tat was confirmed by immunoprecipitation and Western blotting, in combination with mutagenesis. The yeast three-hybrid RNA-protein interaction assay indicated no direct interaction of Tip110 with Tat transactivating response element RNA. Nevertheless, Tip110 strongly synergized with Tat on Tat-mediated chloramphenicol acetyltransferase reporter gene expression and HIV-1 virus production, whereas down-modulation of constitutive Tip110 expression inhibited HIV-1 virus production. Northern blot analysis showed that Tip110 mRNA was expressed in a variety of human tissues and cells. Moreover, digital fluorescence microscopic imaging revealed that Tip110 was expressed exclusively in the nucleus, and within a nuclear speckle structure that has recently been described for human cyclin T and CDK9, two critical components for Tat transactivation function on HIV-1 long terminal repeat promoter. Taken together, these data demonstrate that Tip110 regulates Tat transactivation activity through direct interaction, and suggest that Tip110 is an important cellular factor for HIV-1 gene expression and viral replication.

Amino Acid Sequence↗

PubMed2002

Direct participation of Sam68, the 68-kilodalton Src-associated protein in mitosis, in the CRM1-mediated Rev nuclear export pathway.

Human immunodeficiency virus type 1 (HIV-1) replication requires efficient nuclear export of incompletely spliced and unspliced HIV-1 mRNA transcripts, which is achieved by Rev expression at an early stage of the viral life cycle. We have recently shown that expression of Sam68, the 68-kDa Src-associated protein in mitosis, is able to alleviate Rev function block in astrocytes by promoting Rev nuclear export. In the present study, we utilized an antisense RNA expression strategy to down-modulate constitutive Sam68 expression and examined its effect on Rev function, HIV-1 gene expression, and viral replication. These results showed that down-modulation of constitutive Sam68 expression markedly inhibited HIV-1 production in 293T cells and viral replication in T lymphocytes such as Jurkat and CEM cells, as well as human peripheral blood mononuclear cells (PBMCs). Rev-dependent in trans complementation and reporter gene assays further demonstrated that inhibition of HIV-1 gene expression by Sam68 down-modulation was due to impeded Rev activity. Moreover, digital fluorescence microscopic imaging revealed that down-modulation of Sam68 expression caused exclusive nuclear retention and colocalization of both Rev and CRM1. Taken together, these data suggest that adequate Sam68 expression is required for Rev function and, thereby, for HIV-1 gene expression and viral replication, and they support the notion that Sam68 is directly involved in the CRM1-mediated Rev nuclear export pathway.

Active Transport, Cell Nucleus↗

PubMed2002

Expression of exogenous Sam68, the 68-kilodalton SRC-associated protein in mitosis, is able to alleviate impaired Rev function in astrocytes.

Human immunodeficiency virus type 1 (HIV-1) gene expression in astrocytes is restricted, resulting in a brief and limited synthesis of HIV-1 viral structural proteins. Impaired Rev function has been documented in these cells. However, the molecular mechanisms underlying the impaired Rev function are not fully understood. Using the astroglial cell line U87.MG as a model, we report here that HIV-1 gene expression down-regulated expression of Sam68, the 68-kDa Src-associated protein in mitosis, which was constitutively expressed at a lower level in astrocytes. Elevating the endogenous level of Sam68 expression considerably restored HIV-1 Rev function in astrocytes, as determined by a Rev-dependent reporter gene assay. However, elevation of Sam68 expression achieved only a modest increase in HIV-1 production, further supporting the notion that there are multiple cellular restrictions of HIV-1 gene expression in astrocytes. Mutagenesis analysis identified the region between amino acids 321 and 410 of Sam68 as being directly involved in the binding of Sam68 to Rev, while a double mutation in Rev, L78D and E79L, like those in the dominant-negative Rev mutant M10, eliminated Rev binding to Sam68. Moreover, subcellular fractionation and digital fluorescence microscopic imaging revealed that Sam68 expression promoted Rev nuclear export. Taken together, our studies demonstrate that a lower level of constitutive Sam68 expression, followed by further down-regulation by HIV-1 infection, contributes to impaired Rev function in astrocytes, and they suggest that Sam68 may play an important role in Rev nuclear export.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Analysis of specific bacteria from environmental samples using a quantitative polymerase chain reaction.

This article describes the use of quantitative PCR for measuring bacterial abundance in environmental samples. The two approaches discussed are: 1) The use of an internal PCR standard constructed to be the same size and have the same sequence as the primary amplification target, but differing from the primary target by 2-3 bases, corresponding to a unique restriction site. This allows the amount of target amplicon to be compared with the internal standard and circumvents the problem of differential amplification efficiencies when using dissimilar targets and standard amplicons. 2) The use of Taqman technology (Applied Biosystems, Foster City, California) with a dual labeled oligonucleotide probe which binds internal to the PCR primers. The detection of Bacteroides is used as an example for both approaches.

Bacteria↗

PubMed2002

[Recent outcome of bladder augmentation using deepithelialiezed segment of small intestine lined with urothelium].

OBJECTIVE: To evaluate urodynamically and clinically bladder augmentation using double-deepithelialized segment of the small intestine lined with urothelium to analyse the recent outcome to treatment of hyperreflexia neurogenical bladder. METHODS: 25 patients with neurogenical bladder underwent bladder augmentation using deepithelialized segment of the small intestine lined with urothelium (4 - 14 years old, 16 males and 9 females) from October 1998 to October 2000. 21 patients were followed up. Voiding cystoureterography and urodynamic and clinical evaluation were performed, and serum electrolyte, urea nitrogen and creatinine were tested before and 6 months to 2 years after operation. RESULTS: After operation, bladder volume, maximum urine flow rate and compliance were increased, but residual urine/bladder volume and detrusor pressure decreased. Uninhibitory contraction decreased in 15 patients. In 12 patients associated vesicoureteral reflux, clinical evaluation revealed poor outcomes before operation, but excellent (4 patients), good (9), effective (4), poor outcomes (4) after operation. Seven patients had urinary sensation recovered. Vesicoureteral reflux decreased in 9 patients. There were no electrolyte unbalance and abnormal renal function in all patients. CONCLUSIONS: Bladder augmentation using double-deepithelialized segment of the small intestine lined with urothelium shows a better result currently.

Adolescent↗

PubMedDate not supplied

Embryonic stem cells and cardiomyocyte differentiation: phenotypic and molecular analyses.

Embryonic stem (ES) cell lines, derived from the inner cell mass (ICM) of blastocyst-stage embryos, are pluripotent and have a virtually unlimited capacity for self-renewal and differentiation into all cell types of an embryoproper. Both human and mouse ES cell lines are the subject of intensive investigation for potential applications in developmental biology and medicine. ES cells from both sources differentiate in vitro into cells of ecto-, endoand meso-dermal lineages, and robust cardiomyogenic differentiation is readily observed in spontaneously differentiating ES cells when cultured under appropriate conditions. Molecular, cellular and physiologic analyses demonstrate that ES cell-derived cardiomyocytes are functionally viable and that these cell derivatives exhibit characteristics typical of heart cells in early stages of cardiac development. Because terminal heart failure is characterized by a significant loss of cardiomyocytes, the use of human ES cell-derived progeny represents one possible source for cell transplantation therapies. With these issues in mind, this review will focus on the differentiation of pluripotent embryonic stem cells into cardiomyocytes as a developmental model, and the possible use of ES cell-derived cardiomyocytes as source of donor cells.

Animals↗