Biomedical subjects

H A Liang

Publications and source records attributed to H A Liang.

5 recordsLinked to original sources

PubMed2004

Functional expression of L- and T-type Ca2+ channels in murine HL-1 cells.

In the search for a readily available source of native cardiac cells, we investigated the molecular and pharmacological properties of the immortalized cardiac atrial myocyte cell line, HL-1 cells. This work focused on the expression pattern of voltage-gated Ca2+ channels (VGCC). Reverse transcription-polymerase chain reaction analysis revealed that HL-1 cells have mRNA for several types of Ca2+ channels including the L-types, alpha1C and alpha1D, as well as T-types, alpha1H and alpha1G, but are lacking N-type, alpha1B and the T-type, alpha1I. Western blot analysis demonstrated significant alpha1C protein subunit expression, with less alpha1D subunit apparent, while alpha1A, alpha1B and alpha1E subunit expression was undetectable. Immunocytochemical staining showed that the alpha1C protein subunit is expressed predominantly on the cell surface, whereas the alpha1D protein is expressed mostly intracellularly. Whole-cell patch-clamp measurements demonstrated the presence of low (ICa,T) and high (ICa,L) voltage-activated Ca2+ currents, with preferential sensitivity to mibefradil and nimodipine, respectively. Addition of increasing external Ca2+ concentrations, [Ca2+]o, resulted in Ca2+ influx measured by fluorometric imaging with an EC50 of 0.8 mM [Ca2+]o. At a fixed [Ca2+]o of 0.125 mM, Ca2+ influx was also triggered by increasing the extracellular K+ concentration, [K+]o, with an EC50 of 3.7 mM [K+]o. As increasing [K+]o depolarizes the cell, this latter result is consistent with Ca2+ influx through a voltage-dependent mechanism. L-type (nimodipine and verapamil) and T-type (mibefradil and pimozide) Ca2+ channel blockers inhibited Ca2+ influx with IC50s of 1, 2, 0.4 and 0.2 microM, respectively. Antagonists of N-type (omega-conotoxins GVIA) and P/Q-type (MVIIC or omega-agatoxin IVA) did not inhibit Ca2+ influx, consistent with the lack of expression of N-, P-, or Q-type channels observed in the molecular studies. Taken together, these findings indicate that HL-1 cells express L- and T-subtypes of VGCC and are a unique in vitro model system for the study of native, mammalian cardiac Ca2+ channels.

Animals↗

PubMed2001

Vascular endothelial growth factor in porcine-derived extracellular matrix.

An extracellular matrix (ECM) derived from the submucosa of the porcine small intestine (SIS) has been shown to induce angiogenesis and host tissue remodeling when used as a xenogeneic bioscaffold in animal models of wound repair. In the present study, we compared the in vitro effects of SIS ECM extracts to several purified angiogenic growth factors on human dermal microvascular endothelial cell (HMEC) growth patterns. The SIS ECM was shown to induce tube formation from HMEC in a three-dimensional fibrin-based angiogenesis assay in a manner similar to that caused by the addition of vascular endothelial growth factor (VEGF). This tube formation was blocked in the presence of anti-VEGF neutralizing antibody. Western blots and ELISA procedures showed that the SIS ECM contains as much as 0.77 ng VEGF/g SIS. The closely related endothelial cell mitogen, platelet-derived growth factor (PDGF), was not detectable in the SIS extracts. We conclude that VEGF is present in the SIS extracellular matrix. The role of VEGF in SIS-induced wound repair remains unknown, but its presence in the ECM makes it a possible contributor to the angiogenic effect of SIS when this ECM is used as a tissue repair scaffold in animal models of wound repair.

Animals↗

PubMed1999

Prostate cancer expression profiling by cDNA sequencing analysis.

Prostate cancer is a frequently diagnosed solid tumor that is originated mostly from prostate epithelium. One of the key issues in prostate cancer research is to develop molecular markers that can effectively detect and distinguish the progression and malignancy of prostate tumors. Automated, single-pass cDNA sequencing was utilized to rapidly identify expressed genes in a number of cDNA libraries constructed from various normal and tumor prostatic tissues. These included cell lines as well as short-term epithelial culture. A total of 6604 expressed sequence tags (ESTs) were generated and searched against on-line nucleotide and protein databases. A relational database centric software system was constructed to process, store, and analyze EST data rapidly. cDNA contigs were also obtained by assembly of multiple EST sequences. Protein structural signatures were annotated using motif analysis tools including BLOCKS and an in-house-designed neural network. Cross-library comparisons revealed their unique gene expression profiles. Several differentially expressed cDNA clones were identified, and their expression patterns were confirmed by RNA dot blot and RT-PCR analyses.

Cell Line↗

PubMed1995

Formation of simian immunodeficiency virus long terminal repeat circles in resting T cells requires both T cell receptor- and IL-2-dependent activation.

Although immunodeficiency viruses can enter resting CD4+ T lymphocytes, activation of T cells is required for complete viral cDNA synthesis and transport of double-stranded viral DNA to the nucleus. Cross-linking T cell receptors (TCRs) on resting CD4+ T cells induces reverse transcription of full-length simian immunodeficiency virus (SIV) genomes, but TCR engagement alone is insufficient to stimulate SIV DNA to move to the nucleus and form long terminal repeat (LTR) circles. Neither ligation of TCR or CD28 receptors nor interleukin 2 (IL-2) alone induces formation of LTR circles; however, the combination of TCR ligation with either CD28 ligation or IL-2 doses. Anti-IL-2 serum inhibits the formation of LTR circles induced by cross-linking CD3 and CD28, but has no effect on the induction of increased viral reverse transcription. Thus, two signals appear to be required for immunodeficiency viruses to move to the T cell nucleus, one from the TCR to promote reverse transcription of the viral genome, the other through an IL-2-dependent process leading to formation of LTR circles.

Animals↗

PubMed1993

T-cell activation influences initial DNA synthesis of simian immunodeficiency virus in resting T lymphocytes from macaques.

The relationship between T-cell activation and early events in the replication cycle of simian immunodeficiency virus (SIV) was analyzed in resting T lymphocytes from macaques. We used the polymerase chain reaction to detect an early product of reverse transcription (R/U5) and almost complete viral DNA (long terminal repeat/gag). We found that SIV can enter resting T lymphocytes and initiate replication but that the reverse transcription process is not efficient and proceeds slowly in resting cells. Cross-linking the CD3/T-cell receptor complex with monoclonal antibodies, unlike cross-linking either the CD28 or CD2 accessory receptor and like phorbol myristate acetate, induced a rapid increase in viral R/U5 DNA detected within 3 to 6 h postinfection. Anti-CD3 or phorbol myristate acetate induced replication of full-length viral DNA within 6 to 9 h postinfection, but full-length SIV DNA was not detectable at earlier time points. We then compared various inhibitors of T-cell activation for their effects on viral initiation and complete replication. Cyclosporin A, an inhibitor of a distal step in T-cell activation, blocked anti-CD3-induced T-cell proliferation and completion of SIV DNA replication but had no effect on induced increases in SIV R/U5 DNA. By contrast, initial SIV DNA synthesis was partially blocked by inhibitors of very early steps in T-cell activation, including herbimycin A, an inhibitor of protein tyrosine kinases, and by two different inhibitors of protein kinase C, H7 and staurosporine. Since resting T cells do not efficiently complete SIV DNA synthesis and cyclosporin A can block the formation of complete viral DNA induced in activated T cells, a cellular factor(s) present in activated T cells appears to be required for the formation of full-length SIV DNA.

Animals↗