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L Chen

Publications and source records attributed to L Chen.

At least 1,243 records · Page 69Linked to original sources
PubMed1993

Metastatic conversion of cells by expression of human papillomavirus type 16 E6 and E7 genes.

The human papillomavirus type 16 (HPV-16) is a DNA tumor virus highly associated with cervical carcinoma. Viral DNA from HPV-16 is found in primary tumors and their metastatic lesions. To investigate the role of HPV-16 oncoproteins in the development of cancer metastasis, the E6 and E7 genes from HPV-16 were inserted into retrovirus and introduced into nonmetastatic mouse cell lines. Expression of either of the viral genes from HPV-16 made the cells metastatic in nude mice. In contrast, expression of the E6 and E7 genes of HPV type 6 (HPV-6b), which is frequently found in nonmalignant HPV-associated diseases, did not. The metastatic ability of cells transduced with viral genes of HPV-16 did not correlate with their growth rate or sensitivity to destruction by natural killer cells. Our results demonstrate that expression of oncogenic proteins of HPV-16 can cause tumor metastasis and implicate HPV-16 in an important role regarding the progression of HPV-associated human cancers.

Animals↗
PubMed1993

Evolution of polysaccharide hydrolase substrate specificity. Catalytic amino acids are conserved in barley 1,3-1,4- and 1,3-beta-glucanases.

Catalytic amino acid residues in a 1,3-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) and a homologous 1,3-1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73) from barley have been investigated. To identify amino acids responsible for protonation of the glycosidic oxygen during hydrolysis, carbodiimide-mediated labeling of the enzymes with [14C]glycine ethyl ester was performed. This resulted in loss of activity and specific modification of the Glu288 residues in both enzymes. The stoichiometry of labeling was approximately 1:1, and modification was reduced in the presence of substrate analogues. Based on these data, the Glu288 residues are likely to be present at the active sites of the respective enzymes and may represent the catalytic acids in the hydrolytic reaction. The catalytic nucleophiles of the two enzymes were investigated by labeling with specific, mechanism-based epoxyalkyl-beta-oligoglucosides. Amino acid residues Glu232 and Glu231 were identified as the likely catalytic nucleophiles in the 1,3-1,4- and 1,3-beta-glucanases, respectively. Thus the position of the catalytic nucleophile and the putative proton donating amino acids in the two classes of beta-glucan endohydrolases are conserved. The acquisition of distinct substrate specificities in the evolution of these related enzymes may therefore not require the recruitment of novel catalytic amino acids but rather differences in their positioning at the active site and/or changes in substrate binding residues.

Amino Acid Sequence↗
PubMed1993

RNA recognition by an isolated alpha helix.

A 17 amino acid peptide containing the arginine-rich region of the HIV Rev protein binds specifically to Rev response element (RRE) RNA. Even though it is highly charged, the peptide forms an alpha helix in solution, but only when its N- and C-termini are modified to provide favorable electrostatic interactions with the helix macrodipole. Binding affinity for IIB RNA (the primary binding site within the RRE) increases with alpha helix content, whereas nonspecific binding affinity is independent of helix content. Binding of mutant peptides demonstrates that one threonine, one asparagine, and four arginine side chains are important for sequence-specific recognition. Transactivation of the HIV LTR using Tat-Rev peptide hybrids and the RRE IIB site indicates that the peptide adopts an alpha-helical conformation in vivo. The results suggest that interactions with the RNA backbone may help to orient the alpha helix in the major groove of RNA.

Amino Acid Sequence↗
PubMed1993

Rubredoxin oxidase, a new flavo-hemo-protein, is the site of oxygen reduction to water by the "strict anaerobe" Desulfovibrio gigas.

A rubredoxin-oxygen oxidoreductase, a homodimer with a molecular weight of 43 kDa per monomer, was found to be a component of an electron transfer chain that couples the reduction of oxygen to water with NADH oxidation. This FAD-containing protein appears to contain a new type of heme group. The electron transfer chain is not inhibited by cyanide and azide. In contrast, CO decreases NADH oxidation rate and also induces release of the prosthetic groups from the native terminal reductase.

Chromatography, Gel↗
PubMed1993

Isolation and characterization of flavoredoxin, a new flavoprotein that permits in vitro reconstitution of an electron transfer chain from molecular hydrogen to sulfite reduction in the bacterium Desulfovibrio gigas.

A new FMN-containing flavoprotein isolated from Desulfovibrio gigas provided maximum coupling efficiency for the reduction of bisulfite from molecular H2. This protein, which is distinct from flavodoxin and for which the name flavoredoxin is proposed, is required for reconstitution of an electron transfer chain between hydrogenase and bisulfite reductase. A Ca(2+)-binding protein functions as a modulator in the presence of Ca2+ in the process. The finding of a membrane-bound cytochrome c with a molecular weight of 104,000 Da that is also active in this electron transfer chain provides an explanation for the energetic linkage between periplasmic and cytoplasmic proteins in this sulfate-reducing bacterium.

Amino Acid Sequence↗
PubMed1993

Role of RNA structure in arginine recognition of TAR RNA.

The human immunodeficiency virus Tat protein binds specifically to an RNA stem-loop structure (TAR) that contains two helical stem regions separated by a three-nucleotide bulge. A single arginine within the basic region of Tat mediates specific binding to TAR, and arginine as the free amino acid also binds specifically to TAR. We have previously proposed a model in which interaction of the arginine guanidinium group with guanosine-26 (G26) and with a pair of phosphates is stabilized by formation of a base triple between U23 in the bulge and A27.U38 in the upper helix. Here we show by NMR spectroscopy that formation of the base triple is critical for arginine binding to TAR. Mutants of TAR that cannot form the base triple or that remove the guanine contact do not bind arginine specifically. These mutants also showed reduced transactivation by Tat. A triple mutant designed to form an isomorphous base triple between C23 and G27.C38 binds arginine and adopts the same conformation as wild-type TAR. These results demonstrate the importance of RNA structure for arginine binding and further demonstrate the direct correspondence between arginine and Tat binding.

Arginine↗
PubMed1993

Autoantibodies to the GLUT-2 glucose transporter of beta cells in insulin-dependent diabetes mellitus of recent onset.

Purified immunoglobulin G (IgG) from the serum of patients with insulin-dependent diabetes mellitus (IDDM) of recent onset inhibits high-Km uptake of 3-O-methyl-beta-D-glucose by rat pancreatic islets. To determine if the inhibition is the result of antibodies against GLUT-2, the high-Km glucose transporter of beta cells, we incubated IDDM sera with rat islet cells and with AtT-20ins cells transfected to express GLUT-2. IDDM sera inhibited glucose uptake in islet cells and in GLUT-2-expressing AtT-20ins cells but not in AtT-20ins cells transfected to express the low-Km isoform, GLUT-1. In 24 of 30 (77%) patients with newly diagnosed IDDM, IgG binding as measured by immunofluorescence and flow cytometry of the cells transfected to express GLUT-2 was > 2 standard deviations from the mean of the nondiabetic population; 29 of 31 (96%) of nondiabetic children were negative (P < 0.0001). Increased IgG binding could be removed by absorption with GLUT-2-expressing cells but not with GLUT-1-expressing cells. We conclude that most patients with IDDM of recent onset have autoantibodies to GLUT-2.

3-O-Methylglucose↗
PubMed1993

A murine CDC25/ras-GRF-related protein implicated in Ras regulation.

A partial cDNA encoding a novel putative p2, ras guanine nucleotide release-inducing factor (GRF), GRF2, was amplified from murine embryonic stem cells. The presumptive catalytic region of GRF2 is related to the yeast Ras GRF encoded by CDC25. GRF2 is 80% identical to murine CDC25Mm/ras-GRF, but is more similar to yeast CDC25 than to other ras GRFs related to the Drosophila son of sevenless gene product. A 9-kb GRF2 messenger RNA was highly expressed in brain, but GRF2-specific antibodies recognized apparent GRF2 proteins in various mouse tissues in addition to brain. Thus GRF2 represents a novel widely-expressed protein that is highly related to CDC25Mm/ras-GRF, at least in its catalytic domain. Both GRF2 and CDC25Mm/ras-GRF are expressed in murine embryonic stem cells, suggesting that different Ras activators may regulate ras-dependent proliferation and differentiation in early mouse development.

Amino Acid Sequence↗
PubMed1993

The lack of CD8 alpha cytoplasmic domain resulted in a dramatic decrease in efficiency in thymic maturation but only a moderate reduction in cytotoxic function of CD8+ T lymphocytes.

The glycoprotein CD8 is believed to play an important role in the maturation and function of MHC class I-restricted T lymphocytes. CD8 has been proposed to function as a co-receptor of the TcR to participate in signal transduction, possibly through its cytoplasmic domain that binds to protein tyrosine kinase p56lck. A T cell-specific transgene encoding CD8 alpha truncated at the cytoplasmic domain ("tailless CD8 alpha"), was introduced into CD8 alpha-deficient mice. This animal model was used to study the role of the CD8 cytoplasmic domain in T cell ontogeny and function. "Tailless CD8 alpha" was expressed on the cell surface of thymocytes and peripheral T cells. A small population of peripheral CD4- T cells (6% of T lymphocytes) was found to have cell surface expression of "tailless CD8 alpha" and endogenous CD8 beta, indicating that these cells may belong to the CD8+ T cell lineage. A consistent result was obtained from CD8 alpha-deficient mice bearing the "tailless CD8 alpha" and the MHC class I-restricted 2C TcR transgenes. A small population of CD4- T cells expressing CD8 beta, the "tailless CD8 alpha" and the 2C TcR transgenes was present in the periphery of these mice in a selecting background, but was absent in a deleting background. When "tailless CD8 alpha" mice were infected with lymphocytic choriomeningitis virus (LCMV), the peripheral CD8+ CD4- T cell subset expanded dramatically and a significant LCMV-specific cytolytic activity was detected. The results suggest that the cytoplasmic portion of CD8 alpha is not absolutely required but dramatically enhances the efficiency of thymic maturation of CD8+ T cells. The lack of CD8 alpha cytoplasmic domain in peripheral CD8+ T cells does not abolish the generation of cytotoxicity in response to an in vivo LCMV infection, although the cytolytic activity is slightly reduced compared to that in control mice.

Animals↗
PubMed1993

Effect of denervation on endothelium-derived relaxing factor-dependent relaxation in the rat cremaster muscle.

Endothelium-derived relaxation mediated primarily by endothelium-derived relaxing factor/nitric oxide (EDRF/NO) is essential in the maintenance of vascular tone. However, little is known about the effects of denervation on EDRF-mediated relaxation in the microcirculation after reimplantation. Using intravital videomicroscopy in the rat cremaster model, this study assessed the effect of acute and chronic denervation of the muscle, produced by severing the genitofemoral nerve, on EDRF-mediated vasorelaxation. The results demonstrated that acetylcholine-induced EDRF relaxation (10(-6)M) significantly relieved norepinephrine-induced vasoconstriction in small arteries (40-80 microns diameter) in both the acute and chronic denervated muscles. There was no significant difference in the relaxant response between the denervated or innervated controls in each group or between the acute or chronic groups. This relaxation was prevented by methylene blue (10(-4) M). These findings suggest that EDRF-mediated relaxation in the microcirculation is not dependent on tissue innervation. Thus the use of potent endothelial dependent agonists may be of use in cases of vasospasm postreimplantation.

Acetylcholine↗
PubMed1993

Functional significance of cumulus expansion in the mouse: roles for the preovulatory synthesis of hyaluronic acid within the cumulus mass.

Gonadotropin-stimulated expansion of the mouse cumulus oocyte complex (COC) in vitro, measured with a quantitative videographic method, is comparable to that observed to occur in vivo when medium is supplemented with porcine follicle stimulating hormone (pFSH), 10% fetal bovine serum (FBS), and 2.5 mM glucosamine or optimal concentrations of glutamine and glucose. In the absence of glucosamine, the volumetric expansion of COCs in vitro is never more than 25% of that occurring in its presence. The addition of 6-diazo-5-oxo-1-norleucine (DON), an inhibitor of glucosamine synthesis to medium supplemented with glutamine and glucose, completely inhibits cumulus expansion in vitro. This system was utilized to examine the relationship between cumulus expansion and fertilization rates, and the maintenance of fertilizability in culture. Successful fertilization (as determined by development to the 2-cell stage) was correlated with the quantity and quality of the expanded cumulus mass, and conversely, the spontaneous loss or mechanical removal of the cumulus was correlated with a loss of fertilizability following additional incubation in culture medium. In addition, the i.p. injection of DON inhibited cumulus expansion within the intact follicle and suppressed ovulation.

Animals↗
PubMed1993

Preliminary crystal structure studies of a ternary electron transfer complex between a quinoprotein, a blue copper protein, and a c-type cytochrome.

A ternary electron transfer protein complex has been crystallized and a preliminary structure investigation has been carried out. The complex is composed of a quinoprotein, methylamine dehydrogenase (MADH), a blue copper protein, amicyanin, and a c-type cytochrome (c551i). All three proteins were isolated from Paracoccus denitrificans. The crystals of the complex are orthorhombic, space group C222(1) with cell dimensions a = 148.81 A, b = 68.85 A, and c = 187.18 A. Two types of isomorphous crystals were prepared: one using native amicyanin and the other copper-free apo-amicyanin. The diffraction data were collected at 2.75 A resolution from the former and at 2.4 A resolution from the latter. The location of the MADH portion was determined by molecular replacement. The copper site of the amicyanin molecule was located in an isomorphous difference Fourier while the iron site of the cytochrome was found in an anomalous difference Fourier. The MADH from P. denitrificans (PD-MADH) is an H2L2 hetero-tetramer with the H subunit containing 373 residues and the L subunit 131 residues, the latter containing a novel redox cofactor, tryptophan tryptophylquinone (TTQ). The amicyanin of P. denitrificans contains 105 residues and the cytochrome c551i contains 155 residues. The ternary complex consists of one MADH tetramer with two molecules of amicyanin and two of c551i, forming a hetero-octamer; the octamer is located on a crystallographic diad. The relative positions of the three redox centers--i.e., the TTQ of MADH, the copper of amicyanin, and the heme group of c55li--are presented.

Bacterial Proteins↗
PubMed1993

Crystal structure analysis of amicyanin and apoamicyanin from Paracoccus denitrificans at 2.0 A and 1.8 A resolution.

The crystal structure of amicyanin, a cupredoxin isolated from Paracoccus denitrificans, has been determined by molecular replacement. The structure has been refined at 2.0 A resolution using energy-restrained least-squares procedures to a crystallographic residual of 15.7%. The copper-free protein, apoamicyanin, has also been refined to 1.8 A resolution with residual 15.5%. The protein is found to have a beta-sandwich topology with nine beta-strands forming two mixed beta-sheets. The secondary structure is very similar to that observed in the other classes of cupredoxins, such as plastocyanin and azurin. Amicyanin has approximately 20 residues at the N-terminus that have no equivalents in the other proteins; a portion of these residues forms the first beta-strand of the structure. The copper atom is located in a pocket between the beta-sheets and is found to have four coordinating ligands: two histidine nitrogens, one cysteine sulfur, and, at a longer distance, one methionine sulfur. The geometry of the copper coordination is very similar to that in the plant plastocyanins. Three of the four copper ligands are located in the loop between beta-strands eight and nine. This loop is shorter than that in the other cupredoxins, having only two residues each between the cysteine and histidine and the histidine and methionine ligands. The amicyanin and apoamicyanin structures are very similar; in particular, there is little difference in the positions of the coordinating ligands with or without copper. One of the copper ligands, a histidine, lies close to the protein surface and is surrounded on that surface by seven hydrophobic residues. This hydrophobic patch is thought to be important as an electron transfer site.

Amino Acid Sequence↗
PubMed1993

Identification of a new P450 subfamily, CYP4F1, expressed in rat hepatic tumors.

The expression of the rat cytochrome P450 CYP4 family was studied in hepatic tumors. In most of the primary and transplantable hepatic tumors studied, lauric acid omega-hydroxylase activity associated with the CYP4A subfamily enzymes decreased. The expression of CYP4A proteins and mRNAs in these tumors as assessed by Western and Northern blot was undetectable. However, while RNA analysis revealed the absence of 4A1, 4A2, and 4A3 mRNAs, the expression of CYP4 gene(s) was detected. A Uni-ZAP cDNA library was constructed from a 2-acetylaminofluorene-induced transplantable rat hepatic tumor and screened with a CYP4 family probe. A full-length sequenced cDNA of 1977 bp isolated contained a 23-bp 5' untranslated region, a 1572-bp open reading frame, and a 382-bp 3' untranslated region. This cDNA sequence deduced amino acid sequence encodes a P450 protein having a conserved region of the CYP4 family exhibiting 44-45% amino acid identity to rat CYP4A subfamily members, 43% to human CYP4B1, 35 and 32% to insect CYP4C1 and CYP4D1, respectively. This new P450 was thus named CYP4F1. RNA blot analysis with CYP4F1 cDNA and CYP4F1-specific oligonucleotide probes revealed the expression of CYP4F1 in all tumors. This is the first example of a P450 constitutively expressed in rat hepatomas at levels exceeding those in the parental liver tissue. These results suggest that there is differential regulation of CYP4 genes during hepatic carcinogenesis.

2-Acetylaminofluorene↗