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E C Lin

Publications and source records attributed to E C Lin.

At least 55 records · Page 3Linked to original sources
PubMed1997

Identification of a region in the integrin beta3 subunit that confers ligand binding specificity.

Many integrin adhesion receptors bind ligands containing the Arg-Gly-Asp (RGD) peptide motif. Most integrins exhibit considerable specificity for particular ligands and can distinguish among the many conformations of RGD. In this study we identify the domain of the integrin beta subunit involved in determining ligand binding specificity. Chimeras of beta3 and beta5, the most homologous integrin beta subunits, were expressed with alphav on the surface of human 293 cells. The ligand binding phenotype of each chimera was assessed using the ligands Fab-9 and fibrinogen, both of which have a binding preference for alphavbeta3. The results of the study show that when exons C and D of the beta3 subunit (residues 95-233) are substituted into beta5, the chimera gained the ability to bind Fab-9 with an affinity close to that of wild-type alphavbeta3. This chimera was able to mediate cell adhesion to fibrinogen. Furthermore, the swap of only a 39-residue segment of this larger domain, beta3 residues 164-202, into the backbone of beta5 enabled the chimeric integrin to bind soluble Fab-9. This small domain is highly divergent among the integrin beta subunits, suggesting that it may play a role in determining ligand selection by all integrins.

Amino Acid Sequence↗
PubMed1997

Evidence that the integrin beta3 and beta5 subunits contain a metal ion-dependent adhesion site-like motif but lack an I domain.

The amino-terminal domain of each integrin beta subunit is hypothesized to contain an ion binding site that is key to cell adhesion. A new hypothesis regarding the structure of this site is suggested by the crystallization of the I domains of the integrin alphaL and alphaM subunits (Lee, J.-O., Rieu, P., Arnaout, M. A., and Liddington, R. (1995) Cell 80, 631-638; Qu, A., and Leahy, D. J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10277-10281). In those proteins, an essential metal ion is bound by a metal ion-dependent adhesion site (MIDAS). The MIDAS is presented at the apex of a larger protein module called an I domain. The metal ligands in the MIDAS can be separated into three distantly spaced clusters of oxygenated residues. These three coordination sites also appear to exist in the integrin beta3 and beta5 subunits. Here, we examined the putative metal binding site within beta3 and beta5 using site-directed mutagenesis and ligand binding studies. We also investigated the fold of the domain containing the putative metal binding site using the PHD structural algorithm. The results of the study point to the similarity between the integrin beta subunits and the MIDAS motif at two of three key coordination points. Importantly though, the study failed to identify a residue in either beta subunit that corresponds to the second metal coordination group in the MIDAS. Moreover, structural algorithms indicate that the fold of the beta subunits is considerably different than the I domains. Thus, the integrin beta subunits appear to present a MIDAS-like motif in the context of a protein module that is structurally distinct from known I domains.

Amino Acid Sequence↗
PubMed1997

Regulation of Escherichia coli cell envelope proteins involved in protein folding and degradation by the Cpx two-component system.

We show that the two-component signal transduction system of Escherichia coli, CpxA-CpxR, controls the expression of genes encoding cell envelope proteins involved in protein folding and degradation. These findings are based on three lines of evidence. First, activation of the Cpx pathway induces 5- to 10-fold the synthesis of DsbA, required for disulfide bond formation, and DegP, a major periplasmic protease. Second, using electrophoretic mobility shift and DNase I protection assays, we have shown that phosphorylated CpxR binds to elements upstream of the transcription start sites of dsbA, degP, and ppiA (rotA), the latter coding for a peptidyl-prolyl cis/trans isomerase. Third, we have demonstrated increased in vivo transcription of all three genes, dsbA, degP, and ppiA, when the Cpx pathway is activated. We have identified a putative CpxR consensus binding site that is found upstream of a number of other E. coli genes. These findings suggest a potentially extensive Cpx regulon including genes transcribed by sigma70 and sigma(E), which encode factors involved in protein folding as well as other cellular functions.

Amino Acid Isomerases↗
PubMed1997

In vitro phosphorylation study of the arc two-component signal transduction system of Escherichia coli.

The ArcB and ArcA proteins constitute a two-component signal transduction system that plays a broad role in transcriptional regulation. Under anoxic or environmentally reducing conditions, the sensor kinase (ArcB) is stimulated to autophosphorylate at the expense of ATP and subsequently transphosphorylates the response regulator (ArcA). ArcB is a complex, membrane-bound protein comprising at least three cytoplasmic domains, an N-terminal transmitter domain with a conserved His292 residue (H1), a central receiver domain with a conserved Asp576 residue (D1), and a C-terminal alternative transmitter domain with a conserved His717 residue (H2). To study the phosphoryl transfer pathways of the Arc system, we prepared the following His-tagged proteins: H1, D1, H2, H1-D1, D1-H2, H1-D1-H2, and ArcA. Incubations of various combinations of Arc proteins with [gamma-32P]ATP indicated that H1, but not D1 or H2, catalyzes autophosphorylation; that H1-P transfers the phosphoryl group to D1 much more rapidly than to ArcA; and that D1 accelerates the transphosphorylation of H2. Finally, ArcA is phosphorylated much more rapidly by H2-P than by H1-P. Available data are consistent with a signal transduction model in which (i) reception of a membrane signal(s) triggers autophosphorylation of H1 at His292, (ii) the phosphoryl group can migrate to D1 at Asp576 and subsequently to H2 at His717, and (iii) ArcA receives the phosphoryl group from either His292 or His717, the relative contribution of which is regulated by cytosolic effectors.

Bacterial Outer Membrane Proteins↗
PubMed1997

Regulation of expression of the ethanol dehydrogenase gene (adhE) in Escherichia coli by catabolite repressor activator protein Cra.

The adhE gene encodes ethanol dehydrogenase and is located at min 27.9 of the Escherichia coli chromosome. Expression of adhE is about 10-fold higher in cells grown anaerobically than in cells grown aerobically and is dependent on both transcriptional and posttranscriptional factors. In this study, a trans-regulatory element repressing adhE expression was characterized by genetic and biochemical approaches. A mutation downregulating adhE expression was mapped at min 2 of the chromosome. DNA sequence analysis revealed a missense mutation in the cra gene, formerly known as fruR. The cra gene encodes a catabolite repressor-activator protein (Cra) involved in the modulation of carbon flow in E. coli. The mutant protein (Cra*) sustained an Arg148-->His substitution causing 1.5- and 3-fold stronger repression of adhE transcription under anaerobic and aerobic conditions, respectively. By contrast, cra null mutants displayed 1.5- and 4-fold increased adhE transcription under those conditions. Disruption of the cra gene did not abolish the anaerobic activation of the adhE gene but diminished it twofold. Cra and Cra* were purified as fusion proteins tagged with an N-terminal 6xHis element. In vitro, both fusion proteins showed binding to the adhE promoter region and to the control fruB promoter region, which is a known Cra target. However, only 6xHis-tagged Cra, and not 6xHis-Cra*, was displaced from the DNA target by the effector, fructose-1-phosphate (F1P), suggesting that the mutant protein is locked in a promoter-binding conformation and is no longer responsive to F1P. We suggest that Cra helps to tighten the control of adhE transcription under aerobic conditions by its repression.

Aerobiosis↗
PubMed1997

Maternal performance differences between porcine stress syndrome-normal and -carrier Landrace females.

Differences between porcine stress syndrome (PSS) normal (NN) and carrier (Nn) Landrace dams were determined for adjusted number of pigs born alive, adjusted number of pigs at 21 d, adjusted 21-d litter weight, proportion of pigs surviving to 21 d, and farrowing interval. Data were analyzed from a total of 841 females, 623 normal (NN) and 218 carriers (Nn) having 2,231 and 869 records, respectively. Three susceptible (nn) females from two herds were dropped from the analysis because of their small contribution to the total number of records. Frequency of the recessive PSS allele ranged from .07 to .28 in the nine herds involved in this study. Data were adjusted using Landrace breed-specific adjustments and analyzed with mixed-model derivative-free REML procedures fitting the dams' PSS genotype as a fixed effect in the model. Only females having two or more successive parities were used in the analysis of farrowing interval, resulting in a reduction of total records analyzed to 2,201 (1,564 NN and 637 Nn records) from 632 females (445 NN and 187 Nn females). No differences between NN and Nn dams were observed for adjusted number of pigs born alive, adjusted number of pigs at 21 d, adjusted 21-d litter weight, proportion of pigs surviving to 21 d, and farrowing interval. The results of this investigation indicate no significant maternal performance differences between PSS NN or Nn Landrace dams.

Alleles↗
PubMed1996

Migration behavior and separation of sulfonamides in capillary zone electrophoresis. I. Influence of buffer pH and electrolyte modifier.

The influence of buffer pH and electrolyte modifier on the migration behavior and separation of thirteen sulfonamides was investigated by capillary zone electrophoresis. The results indicate that precise optimization of buffer pH is crucial in improving the separation of some closely migrating sulfonamides. On the addition of either an appropriate amount of an organic modifier (methanol or acetonitrile) or a low concentration of beta-cyclodextrin (beta-CD, 0.5 mM) to a phosphate-borate buffer at pH 6.85 and an applied voltage of 20 kV, the resolution of peaks between sulfathiazole and sulfamethoxypyridarine is markedly enhanced and effective separations of thirteen sulfonamides are achieved within a relatively short time. Methanol gives better resolution than acetonitrile as an organic modifier. Weak inclusion complexation occurs between beta-CD and sulfonamides, with the exception of sulfathiazole. The formation constants of thirteen sulfonamides with beta-CD are reported.

Animals↗
PubMed1996

Translation of the adhE transcript to produce ethanol dehydrogenase requires RNase III cleavage in Escherichia coli.

Previous studies have shown that the adhE gene, which encodes a multifunctional protein with ethanol dehydrogenase activity, is under transcriptional regulation. The level of dehydrogenase activity in cells grown fermentatively is about 10-fold higher than that in cells grown aerobically. In these studies, we mapped the promoter to a region well upstream of the protein-coding region of adhE. Unexpectedly, in mutants lacking the endoribonuclease RNase III, no significant ethanol dehydrogenase activity was detected in cells grown anaerobically on rich (Luria-Bertani) medium supplemented with glucose, even though adhE mRNA levels were high. Indeed, like Delta adhE mutants, strains lacking RNase III failed to grow fermentatively on glucose but grew on the more oxidized carbon source glucuronate. Computer-generated secondary structures of the putative 5' untranslated region of adhE mRNA suggest that the ribosome binding site is occluded by intramolecular base pairing. It seems likely that cleavage of this secondary structure by RNase III is necessary for efficient translation initiation.

Alcohol Dehydrogenase↗
PubMed1996

Transcriptional control mediated by the ArcA two-component response regulator protein of Escherichia coli: characterization of DNA binding at target promoters.

ArcA protein bearing an amino-terminal, oligohistidine extension has been purified, and its DNA binding activity has been characterized with or without prior incubation with carbamoyl phosphate. Electrophoretic mobility shift assays and DNase I protection assays indicate that where the phosphorylated form of the ArcA protein (ArcA-P) is expected to act as a transcriptional repressor (e.g., of lctPRD and gltA-sdhCDAB), the effect is likely to be mediated by sequestration of cis-controlling transcriptional regulatory elements. In contrast, in the case of cydAB, for which ArcA-P is expected to function as a transcriptional activator, two discrete binding sites have been identified upstream of a known promoter, and activation from these sites is likely to be mediated by a mechanism typical of the type I class of prokaryotic transcriptional activators. An additional ArcA-P binding site has also been located downstream of the known promoter, and a distinct role for this site in the regulation of the cydAB operon during anoxic growth transitions is suggested. These results are discussed within the framework of an overall model of signaling by the Arc two-component signal transduction system in response to changes in aerobiosis.

Bacterial Outer Membrane Proteins↗
PubMed1995

A biochemical characterization of the binding of osteopontin to integrins alpha v beta 1 and alpha v beta 5.

Osteopontin (OPN) is an extracellular matrix protein that binds to integrin alpha v beta 3. Here we demonstrate that two other integrins, alpha v beta 1 and alpha v beta 5, are also receptors for OPN. Human embryonic kidney 293 cells adhere to human recombinant osteopontin (glutathione S-transferase-osteopontin; GST-OPN) using integrin alpha v beta 1. When the 293 cells are transfected with the beta 5 subunit, they can also adhere to GST-OPN using integrin alpha v beta 5. Divalent cations regulate the binding of GST-OPN to both alpha v beta 1 and alpha v beta 5. Mg2+ and Mn2+ support the binding of GST-OPN to these integrins but Ca2+ does not. The highest affinity is observed in Mn2+. In the presence of this ion, the affinity of GST-OPN for alpha v beta 1 is 18 nM and the affinity for alpha v beta 5 is 48 nM. The antibody 8A2, which is an agonist for beta 1, promotes the adhesion of 293 cells to GST-OPN even when Ca2+ is present. This observation suggests that cellular events could modulate the affinity of alpha v beta 1 for OPN. Collectively, these findings prove that integrins alpha v beta 1, alpha v beta 3, and alpha v beta 5 have similar affinity for OPN. Therefore, all three integrins must be considered when evaluating the biological affects of OPN.

Amino Acid Sequence↗
PubMed1995

High density lipoprotein metabolism is altered by dietary cholesterol but not fat saturation in guinea pigs.

To study dietary fat and cholesterol effects on plasma high density lipoprotein (HDL) metabolism and rates of apolipoprotein (apo) A-I catabolism, guinea pigs were fed 15% (wt/wt) lard- or corn oil-based diets with 0.01% (basal), 0.08%, 0.17% or 0.33% cholesterol. Absorbed dietary cholesterol provided 6%, 50%, 100% and 200%, respectively, of the daily endogenous cholesterol synthetic mass. While total plasma cholesterol concentrations increased significantly above basal levels at the 0.17% and 0.33% cholesterol intakes, plasma apo E-free HDL (EoHDL) cholesterol concentrations did not increase significantly until the 0.33% cholesterol level (P < 0.001). Fractional catabolic rates (FCR) of injected [131I]-apo A-I were not altered by dietary treatment, either fat saturation or cholesterol, but were inversely correlated with plasma EoHDL cholesterol levels (r = -0.622), suggestive of a regulatory role of turnover rates on HDL cholesterol levels independent of dietary treatment. Analysis of the high affinity EoHDL binding to isolated hepatic membranes suggested that hepatic binding was not a determinant of HDL catabolism, as dietary cholesterol-induced decreases in Bmax (binding capacity) were not correlated with changes in apo A-I FCR. Even though dietary cholesterol was associated with increased plasma EoHDL cholesterol and with decreased HDL binding protein Bmax, these values did not correlate with each other nor with effects on apo A-I FCR.

Animals↗
PubMed1994

Prickly pear (Opuntia sp.) pectin alters hepatic cholesterol metabolism without affecting cholesterol absorption in guinea pigs fed a hypercholesterolemic diet.

Prickly pear pectin intake decreases plasma LDL concentrations by increasing hepatic apolipoprotein B/E receptor expression in guinea pigs fed a hypercholesterolemic diet. To investigate whether prickly pear pectin has an effect on cholesterol absorption and on enzymes responsible for hepatic cholesterol homeostasis, guinea pigs were fed one of three semipurified diets, each containing 15 g lard/100 g diet: 1) the lard-basal diet with no added cholesterol or prickly pear pectin (LB diet); 2) the LB diet with 0.25 g added cholesterol/100 g diet (LC diet); or 3) the LC diet containing 2.5 g prickly pear pectin/100 g diet, added at the expense of cellulose (LC-P diet). Animals fed the LB diet had the lowest plasma LDL and hepatic cholesterol concentrations, followed by animals fed the LC-P diet (P < 0.001). Hepatic 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase activity was highest in the group fed the LB diet, with similar values for animals in the other two groups. A positive correlation existed between plasma LDL cholesterol concentration and hepatic acyl CoA:cholesterol acyltransferase activity (r = 0.87, P < 0.001). Cholesterol absorption was not different among the three dietary groups. These results indicate that the decreased plasma and hepatic cholesterol concentrations of animals fed prickly pear pectin are not explained by differences in cholesterol absorption but rather are due to mechanisms that alter hepatic cholesterol homeostasis, resulting in lower plasma LDL concentrations.

Animals↗
PubMed1994

Effects of nitrate respiration on expression of the Arc-controlled operons encoding succinate dehydrogenase and flavin-linked L-lactate dehydrogenase.

Expression of sdhCDAB (encoding succinate dehydrogenase) and lctD (encoding the flavin-linked L-lactate dehydrogenase) is elevated aerobically and repressed anaerobically in Escherichia coli. The repression is initiated by autophosphorylation of the sensor protein ArcB, followed by phosphoryl group transfer to the regulator ArcA. ArcA-P, a global transcriptional regulator, then prevents sdh and lct expression. The stimulus for ArcB is not O2 deficiency per se. In vitro experiments showed that ArcB phosphorylation is enhanced by pyruvate, D-lactate, acetate, and NADH, the concentrations of which are likely to increase with the lack of an effective exogenous electron sink. In addition to their aerobic function, the two primary dehydrogenases also have roles in anaerobic nitrate respiration. Results presented here indicate that the increase of sdh and lct expression by nitrate depended on its chemical reduction, which in turn diminished the ArcA-P pool. Unexpectedly, a mutation in the fnr gene (encoding a global regulator involved in anaerobic metabolism) also alleviated the anaerobic repressions. Mutations in arcB or arcA were epistatic over that of fnr. Moreover, since this relief was counteracted by pyruvate in the growth medium, Fnr appears to affect formation of stimuli for ArcB. It is possible that Fnr also indirectly affects some of the other members of the arcA modulon, e.g., cyoABCDE (encoding the cytochrome o complex), cydAB (encoding the cytochrome d complex), and sodA (encoding the manganese-dependent superoxide dismutase).

Anaerobiosis↗
PubMed1994

Regulation of hepatic LDL metabolism in the guinea pig by dietary fat and cholesterol.

Studies were carried out to determine the independent and interactive effects of dietary fat and cholesterol on the regulation of hepatic apoB/E receptor expression and its relationship to hepatic cholesterol concentrations and low density lipoprotein (LDL) particle characteristics. Guinea pigs were fed 15% (w/w) fat diets (lard, olive oil, or corn oil) with cholesterol levels corresponding to absorbed intakes of 6 (basal), 50, 100, or 200% endogenous cholesterol synthesis. Guinea pigs maintained stable plasma cholesterol levels until cholesterol intake equaled or exceeded endogenous synthesis (P < 0.001). Fat type independently affected plasma total and LDL cholesterol levels such that lard > corn oil, with olive oil being intermediate (P < 0.05). Hepatic membrane apoB/E receptor number (Bmax) decreased as dietary cholesterol increased (P < 0.001) without an independent effect of dietary fat saturation. Bmax values were significantly correlated with plasma LDL cholesterol levels (r = -0.632), and with hepatic free (r = 0.527) and esterified cholesterol (r = -0.512) concentrations, which were both increased with dietary cholesterol (P < 0.001). Significant interactions between dietary fat type and cholesterol mediated the extent of hepatic free and esterified cholesterol accumulation. Dietary fat and cholesterol interactions also contributed to changes in LDL particle composition and peak density. The results of these studies do not support the thesis that dietary cholesterol-mediated suppression of apoB/E receptor expression is ameliorated by intake of polyunsaturated fatty acids. Dietary fat type and cholesterol amount interactively affect hepatic cholesterol concentrations and LDL composition and size, which in part determine plasma LDL cholesterol levels.

Animals↗
PubMed1993

The deduced amino-acid sequence of the cloned cpxR gene suggests the protein is the cognate regulator for the membrane sensor, CpxA, in a two-component signal transduction system of Escherichia coli.

The cpxA gene of Escherichia coli K-12 encodes a membrane-associated sensor element of a two-component signal transduction system in bacteria. The cognate regulator element, however, has not yet been definitively identified. A 2.1-kb segment upstream from cpxA was amplified by polymerase chain reaction, cloned and sequenced. An open reading frame encoding 232 amino acids was found. It showed high homology to the regulator elements of two-component transduction systems. The newly identified gene, designated as cpxR, may encode the cognate protein receiving signals from CpxA.

Amino Acid Sequence↗
PubMed1993

A novel method to study the electrodynamic behavior of actin filaments. Evidence for cable-like properties of actin.

Actin, one of the most abundant intracellular proteins, forms long linear polyelectrolytic polymers in solution. A novel technique to handle single actin filaments in solution was developed that allows the study of ionic currents elicited along the surface of electrically stimulated actin filaments. Electrical currents were observed about the polymer's surface under both high (100 mM KCl) and low (1 mM KCl) ionic strength conditions. The data are consistent with a dynamic behavior of the counterionic cloud surrounding the actin filaments that support ionic movements along their longitudinal axis upon electrical stimulation. Counterionic waves were highly nonlinear in nature and remained long after the electrical stimulation of the actin filaments ceased. In this report therefore, we demonstrate that actin filaments can function as biological "electrical wires" and can thus be conceptualized as nonlinear inhomogeneous transmission lines. This ability of actin filaments to conduct electrical signals may have important implications in the coupling of intracellular signals.

Actins↗
PubMed1993

Adaptation of Escherichia coli to redox environments by gene expression.

Escherichia coli is adroit in exploiting environmental energy sources to its greatest profit. A key strategy is to channel electron transport from donor to a terminal acceptor(s) so that the voltage drop is maximal. At the level of transcription, the goal is achieved by the interaction of three global regulatory systems, Fnr, NarL/NarX and ArcB/ArcA. In addition, the regulator FhlA is involved in a cascade-controlled pathway for the formate branch of the pyruvate fermentation pathway.

Adaptation, Physiological↗