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D Chen

Publications and source records attributed to D Chen.

At least 685 records · Page 38Linked to original sources
PubMed1994

Cloning, sequence analysis, and expression of the genes encoding the two subunits of the methylotrophic bacterium W3A1 electron transfer flavoprotein.

The genes encoding the two different subunits of the electron transfer flavoprotein (ETF) from the methylotrophic bacterium W3A1 have been identified, cloned, and sequenced. A 0.8-kilobase pair DNA fragment was generated for use as a molecular probe by the amplification of genomic sequences using the polymerase chain reaction and a primer pair with degenerate sequences derived from the NH2-terminal amino acid sequences determined for the ETF subunits purified from W3A1. The screening of a partial genomic minilibrary containing size-selected BamHI-SalI fragments using this probe identified a 2.2-kilobase pair insert containing the complete coding sequences for both W3A1 ETF subunits. The genes are arranged in tandem in the genomic DNA with only 2 bases between the TAG translation termination codon of the small subunit and the ATG translation initiation codon of the large subunit. The deduced amino acid sequences of each of the W3A1 ETF subunits exhibit only approximately 30% identity with the corresponding subunits of the ETF from human, rat, and Paracoccus denitrificans, which as a group are greater than 50% identical. Thus, the ETF from W3A1 may exhibit some unique structural features that, like other differences in some of its physical and functional properties, may distinguish this ETF from others in this family. A highly homologous region near the COOH terminus of the large subunit in all the ETF proteins was found to contain a sequence that matches in several ways the ADP-binding motif of flavoproteins and other dinucleotide-binding proteins, suggesting that the large subunit forms a portion of the FAD (or AMP) binding site in these proteins. Under control of the tac promoter, the cloned ETF subunit genes were co-expressed in Escherichia coli producing the heterodimeric holoprotein with physical, spectral, and electron-accepting properties essentially identical to the ETF isolated from W3A1. The recombinant ETF serves as the electron acceptor for W3A1 trimethylamine dehydrogenase in vitro, accumulating as the air-stable anionic semiquinone in the presence of excess trimethylamine. Fully reduced ETF could not be obtained even after prolonged enzymatic reduction.

Amino Acid Sequence↗
PubMed1994

Resetting the biological clock: mediation of nocturnal circadian shifts by glutamate and NO.

Circadian rhythms of mammals are timed by an endogenous clock with a period of about 24 hours located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Light synchronizes this clock to the external environment by daily adjustments in the phase of the circadian oscillation. The mechanism has been thought to involve the release of excitatory amino acids from retinal afferents to the SCN. Brief treatment of rat SCN in vitro with glutamate (Glu), N-methyl-D-aspartate (NMDA), or nitric oxide (NO) generators produced lightlike phase shifts of circadian rhythms. The SCN exhibited calcium-dependent nitric oxide synthase (NOS) activity. Antagonists of NMDA or NOS pathways blocked Glu effects in vitro, and intracerebroventricular injection of a NOS inhibitor in vivo blocked the light-induced resetting of behavioral rhythms. Together, these data indicate that Glu release, NMDA receptor activation, NOS stimulation, and NO production link light activation of the retina to cellular changes within the SCN mediating the phase resetting of the biological clock.

Amino Acid Oxidoreductases↗
PubMed1994

Modulation of tyrT promoter activity by template supercoiling in vivo.

We have found that initiation of RNA synthesis at the tyrT promoter of Escherichia coli can be stimulated on a plasmid by a factor of 4-6 by elevation of DNA supercoiling in vivo. Increased unconstrained plasmid supercoiling was achieved by inserting the tyrT promoter upstream of the tetracycline resistance gene tetA and transformation into a topA host. Under these conditions there is marked oversupercoiling of the plasmid DNA and we have shown previously that this can lead to increased promoter activity in the topological domain created. A critical element in the formation of this domain is the coupled transcription, translation and membrane insertion of tetA and we show that all of these events are important in the stimulation of tyrT promoter activity. The magnitude of the stimulation is in reasonable agreement with that measured in vitro as a function of plasmid supercoiling, if the unconstrained level of negative supercoiling in vivo is increased from a basal level of -sigma approximately 0.022 to -sigma approximately -0.052 by transcription-induced supercoiling. The induced supercoiling is very efficient, indicating that the tyrT promoter is itself contributing to the steady-state level despite a total lack of membrane anchorage for the tyrT transcription unit. This study provides a new example of the topological coupling of promoters.

Antiporters↗
PubMed1994

Elevated unconstrained supercoiling of plasmid DNA generated by transcription and translation of the tetracycline resistance gene in eubacteria.

Our previous studies have indicated that the leu-500 promoter of Salmonella typhimurium is activated by local supercoiling arising from the transcription of a divergent promoter (Chen et al., 1992). For this to occur on a plasmid, we have shown that the transcribing RNA polymerase must be anchored to the cell membrane by transcription, translation, and export of the tetA gene and that the cell background must be topA. In this study we have used (AT)n reporter sequences to analyze changes in unconstrained supercoiling of plasmid DNA under the circumstances in which the leu-500 promoter becomes activated. (AT)n sequences undergo a structural transition to a cruciform at a threshold level of negative supercoiling that is determined by the length of the tract, and this can be detected in the cellular DNA by in situ chemical probing. These studies have shown that there is elevated unconstrained supercoiling in tetA-carrying plasmids in either Escherichia coli or S. typhimurium cells in exponential growth. This oversupercoiling depends on the function of the tetA gene in cis and the delta topA cell background. These are exactly the conditions that lead to the activation of the leu-500 promoter, supporting the proposed mechanism for the suppression of the leu-500 mutation by topA. Use of (AT)n sequences of different lengths has permitted us to estimate the extent of oversupercoiling. When the tetA gene was initiated using the strong tac promoter, we were able to detect increased unconstrained DNA supercoiling even in topA+ E. coli cells.

Base Sequence↗
PubMed1994

Characterization of rotavirus VP2 particles.

Rotavirus particles consist of three concentric proteinaceous capsid layers. The innermost capsid (core) is made of VP2. The genomic RNA and the two minor proteins VP1 and VP3 are encapsidated within this layer. Empty rVP2 particles are produced when insect cells are infected with a recombinant baculovirus which contains the bovine Rf rotavirus gene 2 (Labbé et al., 1991, J. Virol. 65, 2946-2952). Analysis of expressed rVP2 particles by SDS-PAGE showed these particles were composed of three major VP2-related proteins, called bands A, B, and C, with apparent molecular weights of 94K, 85K, and 77K, respectively. N-Terminal amino acid sequence analysis of each band showed that band A and band B were blocked, and band C lacked 92 amino acids from the N terminus. Bands B and C were predicted to also lack an approximately 10K peptide fragment from the C terminus. Electron microscopy (EM) showed negatively stained rVP2 particles to be spherical with icosahedral symmetry, 520 +/- 20 A in diameter. Highly concentrated rVP2 particles were converted to unusual forms, including elongated bristly structures, helix-like structures, and sheet-like helix structures. These unusual forms apparently resulted from a structural conversion of individual rVP2 particles. This conversion was reversible both in solution or on a collodion-carbon-coated grid support. The reconstituted rVP2 particles possessed normal morphology and reacted with purified VP6 to form rVP2/6 empty double-layered (previously called single-shelled) virus-like particles with an association constant Ka approximately 10(11) M-1. Native viral core particles lacking RNA were obtained by dialysis of full cores prepared from purified SA11-4F rotavirus double-layered particles against a hypotonic buffer in the presence of EDTA. EM showed both the full and empty native viral cores to be spherical with icosahedral symmetry. Highly concentrated SA11-4F full and empty cores also were converted into elongated and bead-like structures. However, in contrast to rVP2 particles, the conversion of SA11-4F cores was not reversible. These results provide some helpful clues to understanding VP2 functions, the assembly of VP2 particles, the assembly of VP2/6 double-layered particles, and the transport of metabolites inside and outside of the core particle.

Amino Acid Sequence↗
PubMed1994

Adhesion of sickle cells to vascular endothelium is critically dependent on changes in density and shape of the cells.

Experiments were performed to explore whether adhesion differences among different density sickle (SS) cells are mainly caused by deformability characteristics attendant to density variations, which could affect their surface contacts with the endothelium, or caused by any changes in their membrane surface (eg, receptors). Also, what is the contribution of morphologic attributes of SS cells in adhesive and obstructive events? To resolve these issues, controlled modifications of cell density were performed using Nystatin and sucrose. This allowed elevation of the mean corpuscular hemoglobin concentration (MCHC) of light-density SS2 discocytes to that of SS4 native dense cells, as well as its normalization in both native and artificially dehydrated cells. Hemodynamic and adhesive characteristics of these individual SS cell populations and their defined mixtures were investigated in the ex vivo mesocecum vasculature. Dehydrated (high MCHC) SS2 discocytes, alone or with SS2 control cells, caused a higher peripheral resistance but no persistent microvascular blockage. Nevertheless, dehydrated SS2 discocytes resulted in a significantly decreased adhesion in venules (adhesion sites). Rehydration of these high MCHC discocytes completely restored their adhesivity, similar to that of SS2 control cells. In contrast, irreversibly sickled cell (ISC)-rich SS4 dense cell class (dense discocytes and 66% to 72% ISCs) was less adherent than dehydrated SS2 discocytes, but caused a persistent blockage of small diameter postcapillary venules when infused with SS2 control or dehydrated discocytes. In the areas of adhesion, SS4 dense discocytes outnumbered ISCs by 4 to 1, demonstrating that ISCs, because of their shape characteristics, are minimally adherent but play a distinct role in postcapillary obstruction. When MCHC (density) of SS4 cells was decreased by rehydration, it resulted in an almost complete reversal of their hemodynamic and adhesive behavior, confirming a profound influence of cell density. These findings demonstrate for the first time that, under shear flow, adhesion of different density SS cells to the endothelium is dependent on the inverse of cell density rather than any changes in their adhesion potential. Finally, the trapping of ISC-rich native dense cells, but not of dehydrated discocytes, in the areas of adhesion shows a distinct contribution of ISC in adhesion-initiated vasoocclusion.

Adult↗
PubMed1994

The hormone-binding role of 2 cysteines near the C terminus of the mouse glucocorticoid receptor.

Previous biochemical analyses with covalent affinity labels and thiol-blocking reagents suggested possible roles for one methionine residue and multiple cysteine residues in binding of steroid to the 250-amino acid hormone-binding domain at the C-terminal end of mammalian glucocorticoid receptors. To test the functional roles of these residues in the receptor's ability to bind hormone and active transcription of target genes, the mouse glucocorticoid receptor cDNA was specifically mutated to cause single amino acid substitutions for methionine 610 and for each of the 5 cysteines (at positions 628, 644, 649, 671, and 742) in the hormone-binding domain. Among these 6 residues, only mutations in cysteine 671 and cysteine 742 caused substantial reductions in function. In transient transfection assays, the concentration of dexamethasone required for half-maximal activation of a glucocorticoid-responsive reporter gene was increased by 10-40-fold by changing cysteine 742 to serine or cysteine 671 to serine or alanine. At saturating concentrations of dexamethasone, the mutant receptors activated the reporter gene to the same extent as the wild type receptor, indicating that the mutations affected only the hormone-binding function of the receptor and not its ability to bind DNA or activate transcription once the hormone was bound.

Amino Acid Sequence↗
PubMed1994

Interleukin 2 transcription factors as molecular targets of cAMP inhibition: delayed inhibition kinetics and combinatorial transcription roles.

Elevation of cAMP can cause gene-specific inhibition of interleukin 2 (IL-2) expression. To investigate the mechanism of this effect, we have combined electrophoretic mobility shift assays and in vivo genomic footprinting to assess both the availability of putative IL-2 transcription factors in forskolin-treated cells and the functional capacity of these factors to engage their sites in vivo. All observed effects of forskolin depended upon protein kinase A, for they were blocked by introduction of a dominant negative mutant subunit of protein kinase A. In the EL4.E1 cell line, we report specific inhibitory effects of cAMP elevation both on NF-kappa B/Rel family factors binding at -200 bp, and on a novel, biochemically distinct "TGGGC" factor binding at -225 bp with respect to the IL-2 transcriptional start site. Neither NF-AT nor AP-1 binding activities are detectably inhibited in gel mobility shift assays. Elevation of cAMP inhibits NF-kappa B activity with delayed kinetics in association with a delayed inhibition of IL-2 RNA accumulation. Activation of cells in the presence of forskolin prevents the maintenance of stable protein-DNA interactions in vivo, not only at the NF-kappa B and TGGGC sites of the IL-2 enhancer, but also at the NF-AT, AP-1, and other sites. This result, and similar results in cyclosporin A-treated cells, imply that individual IL-2 transcription factors cannot stably bind their target sequences in vivo without coengagement of all other distinct factors at neighboring sites. It is proposed that nonhierarchical, cooperative enhancement of binding is a structural basis of combinatorial transcription factor action at the IL-2 locus.

Base Sequence↗
PubMed1994

[Surgery for acute arterial emboli to the arm].

During a 5-year period (1987-1992) we removed under local anesthesia 20 emboli and 2 thrombi from the arms of 18 patients aged 60-90 using a Fogarty catheter. 18 of the emboli (90%) were of cardiac origin. 1 patient had thrombosis of the axillary artery (2 episodes). The occlusions were brachial in 14 (70%), axillary in 5 (25%), and radial in 1. From 1.5 hours to 1 week elapsed between onset of symptoms and operation. 9 patients underwent arteriography. Surgery was successful in 19 (95%). Limb necrosis developed in a patient operated on 1 week after onset of symptoms. Necrosis progressed despite a palpable radial pulse. 33 patients (15%) died of causes unrelated to the operation: 2 had terminal cardiac disease and the third concomitant occlusion of the superior mesenteric artery. Early surgical exploration is advocated for acute arterial embolism of the upper extremity. Ischemic changes may be reversible even later than 24 hours from onset of symptoms. Routine arteriography is not needed before surgical exploration of these cases.

Aged↗