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Biomedical subjects

S Huang

Publications and source records attributed to S Huang.

At least 577 records · Page 32Linked to original sources
PubMed1992

Proliferation of human colon cancer cells: role of epidermal growth factor and transforming growth factor alpha.

Human colon cancer cells produce and secrete a variety of polypeptide growth factors. The functional role of these growth factors, however, is poorly understood. Though the secretion of epidermal growth factor (EGF)-like activity and EGF-related molecules by human colon cancer cells in culture has been reported, it is not known whether colon cancer cells produce and secrete EGF, and the functional role of EGF in the growth control of these cells is also unknown. We have shown that EGF acts as a potent growth stimulator on the moderately differentiated Moser colon cancer cell line and as an inhibitor on the highly metastatic KM12SM cell line. In the present study, we show that EGF is produced by human colon cancer cells and characterize the levels of EGF mRNA expression and EGF protein secretion from 8 human colon cancer cell lines. The cell-surface EGF receptors on these cell lines were also characterized by radiolabeled ligand binding and Scatchard analyses. All the cell lines expressed EGF mRNA and secreted EGF. Both high- and low-affinity subtypes of EGF receptor were detected on 7 of the cell lines. These lines also secreted transforming growth factor (TGF)alpha. Some cell lines exhibited a proliferative response to treatment with either exogenous EGF or TGF alpha, while others did not respond to treatment with these growth factors. Antibody-blocking experiments, using anti-EGF or anti-EGF receptor antibody, suggested that these cell lines could be broadly classified into 2 groups in terms of their autocrine or paracrine growth regulation via the cell-surface EGF receptor: (1) cells that utilized EGF and/or TGF alpha; and (2) cells that did not utilize EGF or TGF alpha (via the cell-surface receptor), even though they secreted abundant amounts of these growth factors.

Blotting, Northern↗
PubMed1992

Comparison of cobalamin-independent and cobalamin-dependent methionine synthases from Escherichia coli: two solutions to the same chemical problem.

In Escherichia coli, two enzymes catalyze the synthesis of methionine from homocysteine using methyltetrahydrofolate as the donor of the required methyl group: cobalamin-dependent and cobalamin-independent methionine synthases. Comparison of the mechanisms of these two enzymes offers the opportunity to examine two different solutions to the same chemical problem. We initiated the research described here to determine whether the two enzymes were evolutionarily related by comparing the deduced amino acid sequences of the two proteins. We have determined the nucleotide sequence for the metE gene, encoding the cobalamin-independent methionine synthase. Our results reveal an absence of similarity between the deduced amino acid sequences of the cobalamin-dependent and cobalamin-independent proteins and suggest that the two have arisen by convergent evolution. We have developed a rapid one-step purification of the recombinant cobalamin-independent methionine synthase (MetE) that yields homogeneous protein in high yield for mechanistic and structural studies. In the course of these studies, we identified a highly reactive thiol in MetE that is alkylated by chloromethyl ketones and by iodoacetamide. We demonstrated that alkylation of this residue, shown to be cysteine 726, results in complete loss of activity. While we are unable to deduce the role of cysteine 726 in catalysis at this time, the identification of this reactive residue suggests the possibility that this thiol functions as an intermediate methyl acceptor in catalysis, analogous to the role of cobalamin in the reaction catalyzed by the cobalamin-dependent enzyme.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1992

Procedure for development of an enzyme-linked immunosorbent assay. Development of an assay for human apolipoprotein A-I.

A set of criteria for selection of antibodies during the development of enzyme-linked immunosorbent assay (ELISA) is described. Using these criteria, a competitive ELISA for human apo A-I using a polyclonal goat antibody was developed. The assay recognizes apo A-I from plasma and high-density lipoprotein (HDL), as well as the pure delipidated apo A-I, equally. Intra- and inter-assay variations were 5.2% and 3.5%, respectively. Recovery rate, as determined by spiking a known quantity of pure delipidated apo A-I into a reference plasma, was determined to be 101.3%. The assay was validated by comparing the concentration of apo A-I in HDL with the dye elution method. The apo A-I ELISA to apo A-I dye elution ratio was 1.01. Apo A-I concentration in Centers for Disease Control reference material determined by this method was in agreement with the reported consensus value. Repeated freezing and thawing of the samples (three freeze-thaw cycles at -20 degrees C) as well as long-term freezing (up to 1 year at -70 degrees C) did not affect the concentration of apo A-I in the samples. The assay was applicable both to normolipidemic and dyslipidemic plasmas. No immunologic difference was noted when plasma from dyslipidemic subjects was assayed. A frequent problem of long-term storage is deamidation. The values found for apo A-I in a deamidated plasma were the same as those for the corresponding fresh plasma. Plasma apo A-I values were also positively correlated with that of HDL-cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies↗
PubMed1992

Rat KC cDNA cloning and mRNA expression in lung macrophages and fibroblasts.

We have isolated and sequenced overlapping cDNA clones for rat KC*. The 0.93 kb cDNA has a single open reading frame of 288 nucleotides, and substantial sequence identity with the platelet-factor 4 family members mouse KC, hamster gro, and human gro. Using cloned cDNA as a probe, expression of KC mRNA in lavaged rat alveolar macrophages (AMs) increased after lipopolysaccharide (LPS) treatment. We also studied expression in vitro by a rat fetal lung fibroblast cell line, RFL-6. Expression of KC mRNA in RFL-6 cells increased after treatment with interleukin 1 or with conditioned medium from rat AMs treated with LPS.

Amino Acid Sequence↗
PubMed1992

Crystal structure of the neutral form of fructose 1,6-bisphosphatase complexed with regulatory inhibitor fructose 2,6-bisphosphate at 2.6-A resolution.

The three-dimensional structure of the complex between fructose 1,6-bisphosphatase (EC 3.1.3.11) and the physiological inhibitor beta-D-fructose 2,6-bisphosphate (Fru-2,6-P2), an analogue of the substrate (fructose 1,6-bisphosphate), has been refined at 2.6-A resolution to a residual error (R) factor of 0.171. The rms deviations are 0.012 A and 2.88 degrees from ideal geometries of bond lengths and angles, respectively. The Fru-2,6-P2 occupies the active sites of both polypeptides C1 and C2 in the crystallographic asymmetric unit in the space group P3(2)21. The furanose and 6-phosphate of Fru-2,6-P2 are located at the fructose 6-phosphate site established earlier, and the 2-phosphate binds to the OH of Ser-124, the NH3+ of Lys-274, and the backbone NH of Gly-122 and Ser-123. Backbone displacements of 1 A occur for residues from Asp-121 to Asn-125. Model building of substrate alpha-D-Fru-1,6-P2 based on the binding structure of Fru-2,6-P2 in the active site shows interactions of the 1-phosphate with the backbone NH of Ser-123 and Ser-124. In the AMP sites, density peaks attributed to Fru-2,6-P2 are seen in C1 (and C4) but not in C2 (and C3). This minor binding of Fru-2,6-P2 to AMP sites partially explains the synergistic interaction between AMP and Fru-2,6-P2 and the protection of the AMP site from acetylation in the presence of Fru-2,6-P2. In the synergistic interaction between AMP and Fru-2,6-P2, inhibition of catalytic metal binding by the presence of Fru-2,6-P2 at the active site, and propagation of structural changes over some 28 A along beta-strand B3 from residues 121 to 125 in the active site to Lys-112 and Tyr-113 in the AMP site, as well as movement of helices across the interdimeric interfaces, may affect AMP binding and the subsequent R-to-T transition. In addition, occupancy of Fru-2,6-P2 at the AMP sites of C1 and C4 may favor binding of AMP to the remaining unoccupied AMP sites and thus promote the accompanying quaternary conformational changes.

Amino Acid Sequence↗
PubMed1992

Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow.

Using multidimensional flow cytometry we have defined and quantified the human T-cell differentiation pathway, focusing on those events occurring among the most immature thymocytes and putative bone marrow (BM) T-precursors. Early thymocytes were found to express the CD34 antigen and consisted of a mean 1.2% of cells within human pediatric (n = 9) and 2.0% in fetal thymi (n = 4). All CD34+ thymocytes were atypical blast by morphology, expressed intracytoplasmatic, but not cell surface, CD3, and were cell surface CD2+, CD5+, CD7+, CD38+, CD45+, CD45RA+, CD49d+, and LECAM-1(Leu8)high. CD34high thymocytes lacked surface expression of CD4 and CD8, but as CD34 expression diminished there was a coordinate increase in CD4 levels, followed by the appearance of CD8. The expression of CD1 and CD10 also increased concomitant with the loss of CD34, whereas expression of LECAM-1 diminished with CD34 downregulation. The differential expression of these antigens on early thymocytes (as well as the number of thymocytes displaying these patterns) was highly reproducible among the nine pediatric and four fetal specimens examined, suggesting a precise, stereotyped regulation of early differentiation events. Cell populations with antigen expression patterns suggestive of pluripotent stem cell (CD34high, CD38-), or non-T-lineage committed stem cells (CD34+, CD33+ or CD34+, CD19+) were not identified in either fetal or pediatric thymi (sensitivity = 1/10(4)). The presence of cells with the antigenic profile of the earliest CD34+ thymocytes was explored in human BM. Putative BM T-cell precursors with the appropriate phenotype (CD34+, CD7+, CD5+, CD2+, LECAM-1high) were readily identified in fetal specimens (constituting +/- 2% of the CD34+ population), but could not be reliably detected in adults. In contrast with thymi, only 13% of these cells expressed cytoplasmatic CD3, suggesting the presence of the immediate precursor of the putative prothymocyte population. This was further supported by the detection of CD34bright, CD7+, CD2-, CD5-, LECAM-1moderate cells in fetal specimens. Our results document the flow of cell surface differentiation during T-lymphopoiesis and suggest that T-lineage features are first acquired in the BM. The ability to reproducibly identify and isolate T-cell precursor populations of precisely defined maturational stage in marrow and thymus by multiparameter flow cytometry will facilitate characterization of the molecular events controlling T-lineage differentiation.

Antibodies, Monoclonal↗
PubMed1992

U1 and U2 small nuclear RNAs are present in nuclear speckles.

The localization of U1 and U2 small nuclear RNAs (snRNAs) has been examined by in situ hybridization using 2'-O-alkyl oligonucleotide probes. We have found that these snRNAs, which are essential for pre-mRNA splicing, localize in a speckled distribution, in addition to being present in three of four foci, in HeLa cell nuclei. However, in cells of defined passage, such as Detroit 551 and WI-38 fibroblasts, these snRNAs are concentrated in nuclear speckles, and foci are not observed. The speckled distribution of U1 and U2 snRNAs is coincident with the speckled regions enriched in small nuclear ribonucleoprotein particle (snRNP) proteins and the essential non-snRNP splicing factor SC-35. The localization of these key components of the pre-mRNA splicing machinery to speckled nuclear regions suggests that these regions may be involved in pre-mRNA splicing.

Cell Compartmentation↗
PubMed1992

A method to determine shear adhesive strength of fibrin sealants.

The adhesive strength of fibrin sealants has not been rigorously evaluated to date. The adhesive strength of six different concentrations of cryoprecipitated fibrinogen as well as the commercially available fibrin tissue adhesive Tissucol was tested under controlled conditions utilizing split-thickness skin grafts as the test adherand. This test configuration permitted the modeling of bonding strength for attachment of skin grafts as well as incorporate established engineering test standards for adhesives. An increase in fibrin concentration corresponded with an increase in shear adhesive strength. No significant increases in adhesive strength were attained after 5 min of bonding for all tested concentrations, except for the commercial adhesive, which attained the adhesive strength of an equivalent concentration of cryoprecipitated adhesive after 90 min. The adhesive strength, however, was an order of magnitude less than reported values of the tensile strength of fibrin material for similar concentrations. Therefore, it is important that the surgeon use a sufficiently high fibrinogen concentration for the specific clinical indication. The method of fibrin sealant preparation and/or the compounding adjuncts appear to have an effect on the development of adhesive strength.

Adhesiveness↗