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Biomedical subjects

Z Lu

Publications and source records attributed to Z Lu.

At least 235 records · Page 13Linked to original sources
PubMed1998

A novel high-affinity inhibitor for inward-rectifier K+ channels.

Inward-rectifier K+ channels are a group of highly specialized K+ channels that accomplish a variety of important biological tasks. Inward-rectifier K+ channels differ from voltage-activated K+ channels not only functionally but also structurally. Each of the four subunits of the inward-rectifier K+ channels has only two instead of six transmembrane segments compared to the voltage-activated K+ channels. Thus far, there are no high-affinity ligands that directly target any inward-rectifier K+ channel. In the present study, we identified, purified, and synthesized a protein inhibitor of the inward-rectifier K+ channels. The inhibitor, called tertiapin, blocks a G-protein-gated channel (GIRK1/4) and the ROMK1 channel with nanomolar affinities, but a closely related channel, IRK1, is insensitive to tertiapin. Mutagenesis studies show that teritapin inhibits the channel by binding to the external end of the ion conduction pore.

Amino Acid Sequence↗
PubMed1998

Evolution of an Escherichia coli protein with increased resistance to oxidative stress.

L-1,2-Propanediol:NAD+ 1-oxidoreductase of Escherichia coli is encoded by the fucO gene, a member of the regulon specifying dissimilation of L-fucose. The enzyme normally functions during fermentative growth to regenerate NAD from NADH by reducing the metabolic intermediate L-lactaldehyde to propanediol which is excreted. During aerobic growth L-lactaldehyde is converted to L-lactate and thence to the central metabolite pyruvate. The wasteful excretion of propanediol is minimized by oxidative inactivation of the oxidoreductase, an Fe2+-dependent enzyme which is subject to metal-catalyzed oxidation (MCO). Mutants acquiring the ability to grow aerobically on propanediol as sole carbon and energy source can be readily selected. These mutants express the fucO gene constitutively, as a result of an IS5 insertion in the promoter region. In this study we show that continued selection for aerobic growth on propanediol resulted in mutations in the oxidoreductase conferring increased resistance to MCO. In two independent mutants, the resistance of the protein was respectively conferred by an Ile7 --> Leu and a Leu8 --> Val substitution near the NAD-binding consensus amino acid sequence. A site-directed mutant protein with both substitutions showed an MCO resistance greater than either mutant protein with a single amino acid change.

Alcohol Oxidoreductases↗
PubMed1998

A plasmid expression system for quantitative in vivo biotinylation of thioredoxin fusion proteins in Escherichia coli.

The high affinity binding interaction of biotin to avidin or streptavidin has been used widely in biochemistry and molecular biology, often in sensitive protein detection or protein capture applications. However, in vitro chemical techniques for protein biotinylation are not always successful, with some common problems being a lack of reaction specificity, inactivation of amino acid residues critical for protein function and low levels of biotin incorporation. This report describes an improved expression system for the highly specific and quantitative in vivo biotinylation of fusion proteins. A short 'biotinylation peptide', described previously by Schatz, is linked to the N-terminus of Escherichia coli thioredoxin (TrxA) to form a new protein, called BIOTRX. The 'biotinylation peptide' serves as an in vivo substrate mimic for E. coli biotin holoenzyme synthetase (BirA), an enzyme which usually performs highly selective biotinylation of E.coli biotin carboxyl carrier protein (BCCP). A plasmid expression vector carrying the BIOTRX and birA genes arranged as a bacterial operon can be used to obtain high level production of soluble BIOTRX and BirA proteins and, under appropriate culture conditions, BIOTRX protein produced by this system is completely biotinylated. Fusions of BIOTRX to other proteins or peptides, whether these polypeptides are linked to the C-terminus or inserted into the BIOTRX active site loop, are also quantitatively biotinylated. Both types of BIOTRX fusion can be captured efficiently on avidin/streptavidin media for purification purposes or to facilitate interaction assays. We illustrate the utility of the system by measurements of antibody and soluble receptor protein binding to BIOTRX fusions immobilized on streptavidin-conjugated BIAcore chips.

Acetyl-CoA Carboxylase↗
PubMed1998

The conserved carboxyl terminus and zinc finger-like domain of the co-chaperone Ydj1 assist Hsp70 in protein folding.

Ydj1 is a member of the Hsp40 (DnaJ-related) chaperone family that facilitates cellular protein folding by regulating Hsp70 ATPase activity and binding unfolded polypeptides. Ydj1 contains four conserved subdomains that appear to represent functional units. To define the action of these regions, protease-resistant Ydj1 fragments and Ydj1 mutants were analyzed for activities exhibited by the unmodified protein. The Ydj1 mutant proteins analyzed were unable to support growth of yeast at elevated temperatures and were found to have alterations in the J-domain (Ydj1 H34Q), zinc finger-like region (Ydj1 C159T), and conserved carboxyl terminus (Ydj1 G315D). Fragment Ydj1 (1-90) contains the J-domain and a small portion of the G/F-rich region and could regulate Hsp70 ATPase activity but could not suppress the aggregation of the model protein rhodanese. Ydj1 H34Q could not regulate the ATPase activity of Hsp70 but could bind unfolded polypeptides. The J-domain functions independently and was sufficient to regulate Hsp70 ATPase activity. Fragment Ydj1 (179-384) could suppress rhodanese aggregation but was unable to regulate Hsp70. Ydj1 (179-384) contains the conserved carboxyl terminus of DnaJ but is missing the J-domain, G/F-rich region, and a major portion of the zinc finger-like region. Ydj1 G315D exhibited severe defects in its ability to suppress rhodanese aggregation and form complexes with unfolded luciferase. The conserved carboxyl terminus of Ydj1 appeared to participate in the binding of unfolded polypeptides. Ydj1 C159T could form stable complexes with unfolded proteins and suppress protein aggregation but was inefficient at refolding denatured luciferase. The zinc finger-like region of Ydj1 appeared to function in conjunction with the conserved carboxyl terminus to fold proteins. However, Ydj1 does not require an intact zinc finger-like region to bind unfolded polypeptides. These data suggest that the combined functions of the J-domain, zinc finger-like region, and the conserved carboxyl terminus are required for Ydj1 to cooperate with Hsp70 and facilitate protein folding in the cell.

Adenosine Triphosphatases↗
PubMed1998

Alteration of alpha1-adrenoceptor subtypes in aortas of 12-month-old spontaneously hypertensive rats.

Alterations in alpha1-adrenoceptor subtypes in aortas from 12-month-old spontaneously hypertensive rats (SHR) were studied in functional studies and RNase protection assays. The norepinephrine-induced contraction, including maximum response and pD2 values, was not significantly different between the SHR and age-matched Kyoto Wistar (WKY) rats. The pA2 values of the alpha1D-adrenoceptor subtype-selective antagonist BMY7378 (8-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-8-azaspiro(4.5)dec ane-7,9-dionedihydrochloride) were increased from 8.10 +/- 0.12 in WKY rats to 8.45 +/- 0.13 in SHR (P < 0.05). The pA2 values of the alpha1A-adrenoceptor subtype-selective antagonist RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dim ethyl-1H-indole-3-ethanamine hydrochloride) were reduced from 8.52 +/- 0.20 in WKY rats to 7.82 +/- 0.18 in SHR (P < 0.05), whereas the pA2 values of the alpha1A/alpha1D-adrenoceptor subtype-selective antagonist WB4101 (2-(2,6-dimethoxphenoxyethyl)-aminomethyl-1,4 benzodioxane) were not significantly different between WKY rats and SHR (9.05 +/- 0.22 versus 9.27 +/- 0.15, P > 0.05). Preincubation of preparations in 50 microM chloroethylclonidine for 30 min irreversibly inhibited the norepinephrine-induced response more profoundly in aortas from SHR than in aortas from WKY rats. The results of RNase protection assays showed that mRNAs for alpha1A- and alpha1B-adrenoceptor subtypes were decreased and that mRNA for the alpha1D-adrenoceptor subtype was increased in aortas from SHR compared with WKY rats. The results suggested that the alpha1A-adrenoceptor subtype was decreased and the alpha1D-adrenoceptor subtype was increased in aortas of 12-month-old SHR.

Adrenergic alpha-Antagonists↗
PubMed1998

The bovine herpesvirus type 1 UL3.5 open reading frame encodes a virion structural protein.

The bovine herpesvirus type 1 (BHV-1) open reading frame (ORF) UL3.5 is similar to ORFs found in pseudorabies virus, infectious laryngotracheitis virus, equine herpesvirus type 1, and varicella zoster virus, but clearly absent from herpes simplex virus. The published sequence for this ORF predicts a 126-amino-acid (13.2 kDa) protein product with an isoelectric point of 12.3. We confirmed the UL3.5 sequence, expressed the ORF as a glutathione-S-transferase fusion protein, and made rabbit antibodies against the purified fusion protein. The antiserum detected a 13-kDa protein in Western blots of MDBK cells infected with BHV-1, but not with other herpesviruses or uninfected cells. The BHV-1 UL3.5 protein was characterized as a component of the virion envelope or tegument because it was expressed as a late protein, it was present in the cytoplasm but not the nucleus of infected cells, and it was removed from purified virions by detergent extraction.

Animals↗
PubMed1998

High-Resolution Analysis of the nu6, nu7, nu8, and nu9 Bands of H15N16O3 Measured by Fourier Transform Spectroscopy.

The analysis of the nu6, nu7, nu8, and nu9 bands of H15N16O3 located at 646.9641, 578.4719, 743.6166, and 458.2917 cm-1, respectively, has been carried out in the 400-800 cm-1 region using high-resolution Fourier transform spectra recorded at Ottawa. Using the ground state energy levels calculated from the v = 0 rotational constants of H15N16O3 [A. P. Cox, M. C. Ellis, C. J. Attfield, and A. C. Ferris, J. Mol. Struct. 320, 91-106 (1994)], it was possible to assign the A-type nu6 and nu7 bands and the C-type nu8 and nu9 bands of H15N16O3 up to high J and Ka rotational quantum numbers. The v6 = 1, v7 = 1, v8 = 1, and v9 = 1 experimental energy levels were then introduced in a least-squares fit calculation and precise upper state Hamiltonian constants (band centers and rotational constants) were determined allowing one to reproduce the infrared data to within the experimental uncertainty. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

MAP-kinase dependent induction of monocytic procoagulant activity by beta2-integrins.

beta2-Integrin adhesion molecules play crucial roles in monocyte transmigration and adherence to the inflamed extracellular matrix. While integrin engagement contributes to inflammatory cell activation, little is known about the precise signaling pathways that are important to integrin-dependent monocyte activation. We examined the role of tyrosine phosphorylation and extracellular-signal regulated kinase (ERK) activity in beta2-integrin signaling in monocytes. Cross-linking of the LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) integrins on the surface of THP-1 monocytic cells induced the accumulation of tyrosine phosphoproteins. As part of this signal both ERK-1 and ERK-2 are tyrosine phosphorylated. In vitro kinase assays documented an increase in ERK-2 activity following both LFA-1 and MAC-1 cross-linking. beta2-Integrin cross-linking also led to a marked increase in 4-h procoagulant activity (PCA) in THP-1 cells and purified human monocytes. Inhibition of tyrosine phosphorylation by genistein (10 microg/ml), or selective ERK inhibition with PD98059 (10 microM), was able to block the integrin-dependent induction of PCA in both THP-1 cells and human monocytes. Thus, beta2 integrin signaling in monocytic cells can flow through the tyrosine phosphorylation and activation of the ERK mitogen activated protein kinases, which is essential for the subsequent expression of tissue factor. These results suggest that the ERK proteins likely function to integrate various adhesion-dependent signals during the process of monocyte transmigration.

Blood Coagulation Factors↗
PubMed1998

Ribosome association contributes to restricting mRNAs to the cell body of hippocampal neurons.

In neurons, mRNAs are differentially sorted to axons, dendrites, and the cell body. Recently, regions of certain mRNAs have been identified that target those mRNAs for translocation to the processes. However, the mechanism by which many, if not most mRNAs are retained in the cell body is not understood. Total inhibition of translation, by puromycin or cycloheximide, results in the mislocalization of cell body mRNAs to dendrites. We have examined the effect of translational inhibitors on the localization of ferritin mRNA, the translation of which can also be inhibited specifically by reducing iron levels. Using nonisotopic in situ hybridization, ferritin mRNA is found restricted to the cell body of cultured rat hippocampal neurons. Following treatment with either puromycin or cycloheximide, it migrates into dendrites. Control experiments reveal that the drugs affect neither the viability of the neuronal cultures, nor the steady-state level of ferritin mRNA. When transcription and protein synthesis are inhibited simultaneously, ferritin mRNA is found in the dendrites of puromycin, but not of cycloheximide-treated neurons. However, the localization of ferritin mRNA is unaffected by changes in iron concentration that regulate its translation rate specifically. We propose a model whereby cell body-restricted mRNAs are maintained in that location by association with ribosomes and with another cell component, which traps mRNAs when they are freed of ribosome association. The release of all mRNA species, as happens after total protein synthesis inhibition, floods the system and allows cell body mRNAs to diffuse into dendrites. In contrast, the partial release of the single ferritin mRNA species does not saturate the trapping system and the mRNA is retained in the cell body.

Actins↗
PubMed1998

Effects of micromolar concentrations of Mn, Mo, and Si on alpha1-adrenoceptor-mediated contraction in porcine coronary artery.

We studied the effects of trace elements, Mn, Mo, and Si, on vasoconstriction induced by norepinephrine (NE) or electrical field stimulation in isolated porcine right coronary arteries. Alpha1-adrenoceptor (AR) antagonist prazosin dose-despondently suppressed vasoconstriction in response to NE or field stimulation indicating an alpha1-AR mediated response. Mn, Mo, and Si at 0.3-3 micromol/L dose-despondently inhibited NE mediated contraction (all p < 0.05). In contrast, Mn, Mo, and Si at the same concentrations (0.3-3 micromol/L) enhanced the maximal contractile response to field stimulation in a dose-dependent manner (all p < 0.05), but these elements at 10 micromol/L suppressed the vasoconstrictive response. The results indicate that in porcine right coronary arteries, the alpha1-AR-mediated vasoconstriction by NE or electrical field stimulation was affected differently by micromolar concentrations of Mn, Mo, and Si and that the elements might facilitate NE release presynaptically but inhibit the contractile response postsynaptically.

Adrenergic alpha-Antagonists↗
PubMed1998

Differential effects of d, l-sotalol and d-sotalol on isoproterenol-increased delayed rectifier outward potassium current in guinea pig single ventricular myocytes.

The aim of this study was to compare the effects of d, l-sotalol and d-Sotalol on the delayed rectifier K+ outward current in the presence of isoproterenol at different concentrations. Time-dependent delayed rectifier K+ outward currents were measured in isolated guinea pig single myocytes using the whole-cell configuration of the patch-clamp technique. Currents were measured in response to 300 ms depolarizing pulses from a holding potential of -40 mV in three experimental protocols [control, isoproterenol (10(-9) mol/L-10(-6) mol/L), and isoproterenol (10(-9) mol/L-10(-6) mol/L) plus either d, l-Sotalol (10(-4) mol/L) or d-Sotalol (10(-4) mol/L)]. IK tail currents were measured upon repolarization to -40 mV. It was found that IK was significantly amplified in the presence of isoproterenol (10(-9) mol/L-10(-6) mol/L) plus d-Sotalol. At 10(-8) mol/L isoproterenol, IK was increased by 92.7% +/- 17.1% (P < 0.05) and 54.3% +/- 13.4% after d-Sotalol addition (P < 0.05). In contrast, d, l-Sotalol completely conteracted the increase of Ik by isoproterenol (< 10(-8) mol/L), and compared to control, IK was decreased by 35.6% +/- 8.1% at 10(-8) mol/L isoproterenol plus d, l-Sotalol (P < 0.05). It is concluded that the beta-adrenergic blocking property of d, l-Sotalol but not that of d-Sotalol maintains the delayed rectifier K+ outward current blockade in the presence of isoproterenol in guinea pig myocytes. This might contribute to a superior antiarrhythmic efficacy as compared to d-Sotalol.

Adrenergic beta-Agonists↗
PubMed1998

Expression of functional domain of chicken gizzard calponin.

A full-length cDNA of the function domain of wild-type chicken gizzard calponin was cloned into expression vector pAED4 and the recombinant function domain of wild-type calponin was expressed in an Escherichia coli expression system. The actin domain of calponin (CaP-B) can bind with actin and it is a requisite for its inhibition of ATPase and vasoconstriction of smooth muscle. In this study, the cDNA of CaP-B was inserted into vector pAED4 by direction-cloning method. The cDNA of CaP-B was obtained with PCR cloning technique. The recombinant DNA pAED4-Cap-B was transformed into E. coli BL21 (DE3) and identified with the restriction analysis. The bacterial clones containing transformants were induced to be highly expressed in E. coli BL21 (DE3). The target protein was detected and identified by Western Blot analysis. The content of target protein was as high as 10% of the whole protein after overnight (16 h) culture. The results confirmed that Cap-B was relatively highly expressed in E. coli.

Animals↗
PubMed1998

Encoding of acoustic directional information by saccular afferents of the sleeper goby, Dormitator latifrons.

This paper reports on directional response properties of saccular afferents of the sleeper goby, Dormitator latifrons, to 100-Hz acoustic particle motions with a focus on testing the hypothesis that the response directionality of a fish's auditory afferents derives from the morphological polarity of sensory hair cells in the otolithic organs. Spontaneous rates (SR) and best sensitivities (BS) of saccular afferents ranged from 0 to 162 spikes/sec and from 0.2-to 100-nm RMS displacement. SR did not vary with BS. Most saccular afferents were phase-locked to sinusoidal stimulation and had sustained temporal response patterns with some adaptation. All saccular afferents were directionally sensitive to the stimulus, and the sharpness of directional response curves was determined by a directionality index (DI). The DI ranged from 0.64 to 1.50 (mean = 1.02, SE = 0.02, n = 100) and gradually decreased with stimulus level throughout afferents' response dynamic range. Many afferents had approximately symmetric directional response curves relative to their best response axes (BRA). BRA of most afferents remained constant with stimulus level. The BRA distribution had a peak along an axis that correlates closely with the morphological polarity of saccular hair cells. Therefore, our results strongly support the hypothesis.

Acoustic Stimulation↗
PubMed1998

Mechanism of inhibition of tannic acid and related compounds on the growth of intestinal bacteria.

Tannic acid, propyl gallate and methyl gallate, but not gallic acid, were found to be inhibitory to the growth of intestinal bacteria Bacteroides fragilis ATCC 25285, Clostridium clostridiiforme ATCC 25537, C. perfringens ATCC 13124, C. paraputrificum ATCC 25780, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 13047, Salmonella typhimurium TA98 and S. typhimurium YG1041 at 100-1000 microg/ml in culture broth. Neither Bifidobacterium infantis ATCC 15697 nor Lactobacillus acidophilus ATCC 4356 was inhibited by any of the above compounds up to 500 microg/ml. Tannic acid has a much greater relative binding efficiency to iron than propyl gallate, methyl gallate or gallic acid. The inhibitory effect of tannic acid to the growth of intestinal bacteria may be due to the strong iron binding capacity of tannic acid; whereas the effect of propyl gallate and methyl gallate probably occurs by a different mechanism. The growth of E. coli was restored by the addition of iron to the medium after the precipitate caused by tannic acid was removed. Neither B. infantis nor L. acidophilus require iron for growth. This probably contributes to their resistance to tannic acid. Because tannins are abundant in the human diet, tannins may affect the growth of some intestinal bacteria and thus may have an impact on human health.

Bacteria↗
PubMed1998

Morphological polarizations of sensory hair cells in the three otolithic organs of a teleost fish: fluorescent imaging of ciliary bundles.

It has been suggested that the morphological polarity of sensory hair cells in the otolithic organs plays important roles in directional hearing in fish. In this study, we examined the hair cell polarization patterns in the saccule, utricle, and lagena of a teleost fish, the sleeper goby (Dormitator latifrons). In contrast to using traditional scanning electron microscopy, we employed a simple and rapid method that enabled us to map the hair cell polarization patterns using immunocytochemical and confocal imaging techniques. The hair cells in the caudal part of the saccular epithelium are oriented dorsally and ventrally, with some variations in the caudal end. The hair cells in the rostral part have diverse morphological polarizations. The utricular hair cells fall into internal and external groups which have opposing polarizations. The lagenar hair cells are found in anterior and posterior groups with approximately opposite polarizations. The saccular and lagenar epithelia are oriented perpendicular to the horizontal plane of the fish, while the utricular epithelium lies on the horizontal plane. Thus, the sleeper goby's ear is morphologically capable of being a three-dimensional sound detector.

Animals↗
PubMed1998

Altered subcellular localization patterns of ferritin and beta-actin mRNAs in muscle cultures, resulting from incomplete penetration of digoxigenin-labelled riboprobes.

Protocols for in situ hybridization (ISH) of cultured cells often include storage in alcohol at -20 degrees C between fixation of the cultures and the ISH procedure. In experiments aimed at localizing ferritin mRNA in C2 muscle cultures by ISH with digoxigenin-labelled riboprobes, we have noticed that omission of the ethanol storage dramatically changed the pattern of mRNA localization. In cultures stored in 50%, 70%, or 90% ethanol for at least 15 min, ferritin signal was stronger on myotubes than myoblasts but was uniformly distributed over both. In untreated cultures, the signal was patchy, concentrated on the extremities of the elongated myoblasts and very sparse in myotubes. Similar results were obtained with a probe to beta-actin used as a control, except that signal was higher in myoblasts in all conditions. When the probes were reduced in size to approximately 100 bases from 561 for ferritin and 1150 for actin, the pattern became uniform, regardless of prehybridization treatment. The patchy pattern disappeared when cells were treated with RNase A following hybridization, suggesting that it is non-specific, despite its absence in cultures hybridized with a sense probe. We conclude that incomplete access of RNA probes can result not only in a reduced ISH signal but also in artefactual patterns of mRNA localization.

Actins↗
PubMed1998

Coupled ion movement underlies rectification in an inward-rectifier K+ channel.

We studied block of the internal pore of the ROMK1 inward-rectifier K+ channel by Mg2+ and five quaternary ammoniums (tetramethylammonium, tetraethylammonium, tetrapropylammonium, tetrabutylammonium, and tetrapentylammonium). The apparent affinity of these blockers varied as a function of membrane voltage. As a consequence, the channel conducted K+ current more efficiently in the inward than the outward direction; i.e., inward rectification. Although the size of some monovalent quaternary ammoniums is rather large, the zdelta values (which measure voltage dependence of their binding to the pore) were near unity in symmetric 100 mM K+. Furthermore, we observed that not only the apparent affinities of the blockers themselves, but also their dependence on membrane voltage (or zdelta), varied as a function of the concentration of extracellular K+. These results suggest that there is energetic coupling between the binding of blocking and permeating (K+) ions, and that the voltage dependence of channel blockade results, at least in part, from the movement of K+ ions in the electrical field. A further quantitative analysis of the results explains why the complex phenomenon of inward rectification depends on both membrane voltage and the equilibrium potential for K+.

Animals↗