Biomedical subjects

S Zhang

Publications and source records attributed to S Zhang.

At least 343 records · Page 19Linked to original sources

PubMed2000

Selectively 2H-labeled Glu/Asp: application to pKa measurements in Abeta amyloid peptides.

Human Abeta peptides have been linked to Alzheimer's disease, and it is hypothesized that formation of amyloid as well as neurotoxicity are important events in the etiology of the disease. Previous studies have shown that the soluble precursor to Alzheimer's amyloid undergoes a pH-dependent folding transition as the self-assembly activity appears, and based upon inter-residue proximities, it was suspected that stabilization of the soluble form might rely upon formation of an intramolecular salt-bridge. However, pKa studies on a model 17-residue Abeta fragment supported an electrostatic model where a solvation imperative for charged side-chain atoms drives the folding process. To explore this model in an active 26-residue fragment as well as the full-length 40-residue Abeta peptide, pKa measurements were performed via 1H and 2H NMR. To overcome issues related to sensitivity and spin system degeneracy, specifically deuterated allyl protected-Fmoc amino acids were synthesized for incorporation into a series of peptides, and a high sensitivity 2H observe NMR probe was constructed.

Amino Acid Sequence↗

PubMed2000

Interaction of isoflurane and cromakalim, a KATP channel opener, on coronary and systemic haemodynamics in chronically instrumented dogs.

BACKGROUND: Although isoflurane has been shown to cause coronary and systemic vasodilation through KATP channel activation, the interaction of KATP channel openers and isoflurane has not been fully investigated. The present study was carried out to determine the haemodynamic actions of cromakalim, a KATP channel opener, under the conscious state and during isoflurane anaesthesia in chronically instrumented dogs. METHODS: Fourteen dogs were chronically instrumented to measure systemic and coronary haemodynamics. Each dog was randomly assigned to receive doses of either cromakalim, 4 and 10 microg x kg(-1) i.v., or isoflurane, 2.1% end-tidal (1.5 MAC), plus cromakalim, 4 and 10 microg x kg(-1) i.v. RESULTS: Cromakalim dose-relatedly decreased mean arterial pressure and systemic vascular resistance and increased coronary blood flow in both conscious and anaesthetized states. With isoflurane, the duration of effects of cromakalim were prolonged. Isoflurane exerted an additive effect on the increase in coronary blood flow induced by a low-dose cromakalim, whereas it did not influence the effect of a high-dose cromakalim. The maximum rate of increase in left ventricular pressure and segment shortening were increased by cromakalim in the conscious state but unchanged during isoflurane anaesthesia. CONCLUSION: The results suggest that the coronary vasodilating effects of isoflurane and cromakalim are basically additive until cromakalim exerts the maximal effect, and that the action of cromakalim on the coronary vasculature is prolonged by isoflurane.

ATP-Binding Cassette Transporters↗

PubMed2000

The ets protein PEA3 suppresses HER-2/neu overexpression and inhibits tumorigenesis.

Because HER-2/neu overexpression is important in cancer development, we looked for a method of suppressing the cell transformation mediated by HER-2/neu overexpression. We have identified that the DNA-binding protein PEA3, which is encoded by a previously isolated gene of the ets family, specifically targeted a DNA sequence on the HER-2/neu promoter and downregulated the promoter activity. Expression of PEA3 resulted in preferential inhibition of cell growth and tumor development of HER-2/neu-overexpressing cancer cells. This is a new approach to targeting HER-2/neu overexpression and also provides a rationale to the design for repressors of diseases caused by overexpression of pathogenic genes.

3T3 Cells↗

PubMed2000

Multiple levels of tobacco WIPK activation during the induction of cell death by fungal elicitins.

Three protein kinases of 48, 44 and 40 kDa are activated at different stages in tobacco cells treated with fungal elicitins. Previously we demonstrated that the rapidly activated 48 kDa protein kinase is encoded by SIPK. Here we report that the elicitin-activated 44 kDa kinase is encoded by WIPK. Activation of this kinase occurred 2-4 h after elicitin treatment and was preceded by dramatic increases in WIPK mRNA and protein levels. Studies using actinomycin D and cycloheximide demonstrated that de novo transcription and translation were required for this activation of the kinase activity. Strikingly, the kinetics of WIPK activation following elicitin treatment correlated with the onset of hypersensitive response (HR)-like cell death. Moreover, staurosporine and K-252a, two Ser/Thr protein kinase inhibitors that blocked WIPK activation, suppressed cell death. The timing for elicitin-treated cells to commit to a death program correlated with the appearance of high levels of WIPK activity. These correlative data suggest that WIPK may play a role during HR development in tobacco. Interestingly, a fungal cell-wall elicitor that does not cause cell death induced WIPK mRNA and protein to similar levels as those observed with the elicitins. However, no corresponding increase in WIPK activity was detected. Thus WIPK appears to be controlled at multiple levels.

Cell Death↗

PubMed2000

Effects of noncatalytic residue mutations on substrate specificity and ligand binding of Thermobifida fusca endocellulase cel6A.

The availability of a high-resolution structure of the Thermobifida fusca endocellulase Cel6A catalytic domain makes this enzyme ideal for structure-based efforts to engineer cellulases with high activity on native cellulose. In order to determine the role of conserved, noncatalytic residues in cellulose hydrolysis, 14 mutations of six conserved residues in or near the Cel6A active-site cleft were studied for their effects on catalytic activity, substrate specificity, processivity and ligand-binding affinity. Eleven mutations were generated by site-directed mutagenesis using PCR, while three were from previous studies. All the CD spectra of the mutant enzymes were indistinguishable from that of Cel6A indicating that the mutations did not dramatically change protein conformation. Seven mutations in four residues (H159, R237, K259 and E263) increased activity on carboxymethyl cellulose (CM-cellulose), with K259H (in glucosyl subsite -2) creating the highest activity (370%). Interestingly, the other mutations in these residues reduced CM-cellulose activity. Only the K259H enzyme retained more activity on acid-swollen cellulose than on filter paper, suggesting that this mutation affected the rate-limiting step in crystalline cellulose hydrolysis. All the mutations lowered activity on cellotriose and cellotetraose, but two mutations, both in subsite +1 (H159S and N190A), had higher kcat/Km values (6.6-fold and 5.0-fold, respectively) than Cel6A on 2,4-dinitrophenyl-beta-D-cellobioside. Measurement of enzyme : ligand dissociation constants for three methylumbelliferyl oligosaccharides and cellotriose showed that all mutant enzymes bound these ligands either to the same extent as or more weakly than Cel6A. These results show that conserved noncatalytic residues can profoundly affect Cel6A activity and specificity.

Actinomycetales↗

PubMed2000

Site-directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B.

Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.

Actinomycetales↗

PubMed2000

Cloning, expression and characterization of a family 48 exocellulase, Cel48A, from Thermobifida fusca.

The gene for a 104-kDa exocellulase, Cel48A, formerly E6, was cloned from Thermobifida fusca into Escherichia coli and Streptomyces lividans. The DNA sequence revealed a type II cellulose-binding domain at the N-terminus, followed by a FNIII-like domain and ending with a glycosyl hydrolase Family 48 catalytic domain. The enzyme and catalytic domain alone were each expressed in and purified from S. lividans and had very low catalytic activity on swollen cellulose, carboxymethyl cellulose, bacterial microcrystalline cellulose and filter paper. However, in synergistic assays on filter paper, the addition of Cel48A to a balanced mixture of T. fusca endocellulase and exocellulase increased the specific activity from 7.9 to 11.7 micromol cellobiose.min-1.mL-1, more than 15-fold higher than any single enzyme alone. Cel48A retained > 50% of its maximum activity from pH 5 to 9 and from 40 to 60 degrees C. Using SWISSMODEL, the amino-acid sequence of the Cel48Acd was modeled to the known structure of Clostridium cellulolyticum CelF. Family 48 enzymes are remarkably homologous at 35% identity for all their catalytic domains and some of the properties of the 10 members are discussed.

Actinomycetales↗

PubMed2000

Thiopurine methyltransferase polymorphic tandem repeat: genotype-phenotype correlation analysis.

BACKGROUND: Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. Recently, a variable number tandem repeat (VNTR) within the TPMT promoter has been reported to "modulate" levels of this enzyme activity. METHODS: We set out to perform genotype-phenotype correlation analysis for the polymorphic TPMT tandem repeat in 1211 clinical laboratory samples in which red blood cell (RBC) TPMT activity had been measured and to compare those results with data for 279 control DNA samples. RESULTS: TPMT VNTR length varied from three to nine repeats ( *V3 to *V9), but the most common alleles were *V4 and *V5, with frequencies in the control samples of 0.54 and 0.36, respectively. The clinical laboratory samples were then stratified into those with "low," "intermediate," or "high" levels of RBC TPMT activity; that is, samples presumed to be homozygous for open reading frame (ORF)-based variant alleles, heterozygous for those alleles, or homozygous for the "wild-type" ORF sequence, respectively. TPMT VNTR genotype *V4/*V5 was associated with significantly higher RBC TPMT activity than were *V4/*V4 or *V5/*V5. Lowest activity levels were associated with genotypes that included an allele with more than 5 repeat elements. However, all of these effects were quantitatively small. Finally, there was linkage disequilibrium between VNTR allele *V5 and TPMT*3A, the most common ORF-based polymorphism associated with very low TPMT activity in white persons. CONCLUSIONS: These observations suggest that, in addition to the striking effects of ORF-based single nucleotide polymorphisms on TPMT activity, the VNTR within the 5'-flanking region of the TPMT gene also may modulate levels of RBC TPMT activity.

Adolescent↗

PubMed2000

A comparison of newer tests for the diagnosis of onchocerciasis.

Samples of human serum, skin and urine, collected in Cameroon, were used to assess the value of some newer methods for the diagnosis of onchocerciasis. Parasite DNA was detected in skin snips and urine by PCR, and parasite antigen was detected in serum and urine by immunoblotting. Serum concentrations of IgG4 antibodies reacting with recombinant Onchocerca volvulus antigens (OC3.6 and OC9.3) were also measured, using an ELISA. The PCR-based tests of skin snips and the serological tests for antigen and antibody tests showed higher sensitivities (90%-100%) than the urine PCR (14%) or the urine antigen test (68%). Although antibody detection is much easier to perform than tests based on PCR or antigen detection, the latter have an advantage in that they are only positive in people with current infections. Thus, antibody testing may be more useful for screening populations for infection or exposure to O. volvulus, whereas PCR and antigen testing are potentially more useful for diagnosis of infections in individuals and for monitoring the success of therapy.

Adolescent↗

PubMed2000

Effects of neonatal axoplasmic transport attenuation on the response properties of vibrissae-sensitive neurons in the trigeminal principal sensory nucleus of the rat.

We have previously shown that attenuation of axoplasmic transport by application of vinblastine to the developing infraorbital nerve (ION) results in a loss of central vibrissae-related patterns that is not accompanied by changes in the receptive field sizes for the V primary afferents innervating the whisker follicles. The present study examines the relationship between the loss of central vibrissae-related patterns and alterations in the response properties of neurons in the V principal sensory nucleus (PrV) of adult rats that sustained application of vinblastine to the ION at birth. Absence of histochemically demonstrable vibrissae-related patterns in PrV resulted in only modest changes in the receptive fields and response properties of vibrissae-sensitive neurons in this nucleus that projected to the contralateral thalamus. Response latencies to electrical activation of the V ganglion were similar in treated and untreated animals. The mean receptive field size was significantly increased from 1.3 +/- 0.7 vibrissae in controls to 1.7 +/- 0.9 vibrissae in vinblastine-treated animals, and the percentage of cells yielding a tonic response to vibrissae deflection was markedly reduced (p < 0.01 for both measures). Phasically responding cells recorded in vinblastine-treated animals showed a significant reduction in the mean number of spikes per stimulus following deflection of the vibrissae in either the preferred or non-preferred direction relative to cells recorded in normal animals (p < 0.05). The present results indicate that disruption of the normal vibrissae-related aggregates of neurons in PrV by application of vinblastine to the ION has limited effects on the functional representation of the vibrissae in this nucleus.

Action Potentials↗

PubMed2000

Study on the growth inhibition of human multiple myeloma cells by an IL-6Ralpha mutant.

DM650, a soluble human IL-6Ralpha mutant with mutation of C277D/H280I, was previously shown to exhibit an antagonism to IL-6, which resulted in growth-inhibition of human multiple myeloma cell line AF-10 autocrining as the growth-stimulating factor. We investigated here the nature of the growth inhibition by examining cell apoptosis. Flow-cytometric analysis of the DNA fragmentation demonstrated that 7.2% of the AF-10 cells were apoptotic after 24 h of treatment with DM650. The constitutive gene expression of bcl-2 in AF-10 cells indicated that apoptosis suppressed by IL-6 was independent of bcl-2 regulation. The altered gene expression of c-myc and p53 suggested that a novel apoptosis pathway, other than that suppressed by IL-6, might be triggered by a complex of DM650 and IL-6.

Apoptosis↗

PubMed2000

Molecular cloning and characterization of a tobacco MAP kinase kinase that interacts with SIPK.

A tobacco MAP kinase termed SIPK (Salicylic acid-Induced Protein Kinase) is activated in response to a variety of stress signals, including pathogen attack and wounding (S. Zhang and D.F. Klessig, Proc. Natl. Acad. Sci. USA 95:7225-7230, 1998; S. Zhang and D.F. Klessig, Proc. Natl. Acad. Sci. USA 95:7433-7438, 1998). Using the yeast two-hybrid system, we have identified a gene encoding a protein that interacts with SIPK but not the wounding induced protein kinase (WIPK), which is another tobacco MAP kinase. Sequence analysis indicated that this SIPK-interacting protein is a member of the MAP kinase kinase family; thus, it was named SIPK kinase (SIPKK). Co-immunoprecipitation experiments demonstrated that SIPKK and SIPK interact in vitro. Consistent with its putative function as a kinase, SIPKK phosphorylated myelin basic protein in vitro. Interestingly, SIPKK was induced at the mRNA level after Tobacco mosaic virus (TMV) infection or wounding, albeit with kinetics that are too slow to account for the activation of SIPK following these stimuli.

Amino Acid Sequence↗

PubMed2000

A comparison of 99Tcm-MIBI myocardial SPET and electron beam computed tomography in the assessment of coronary artery disease in two different age groups.

The aim of this study was to compare the clinical value of 99Tcm-MIBI single photon emission tomography (SPET) and electron beam computed tomography (EBCT) in the assessment of coronary artery disease (CAD) in different age groups. 99Tcm-MIBI SPET (stress-rest), EBCT and coronary angiography studies were performed in 64 consecutive patients with suspected CAD. The patients were classified into two groups: Group A = 40 patients > 45 years of age and Group B = 24 patients < or = 45 years of age. There were 31 and 14 patients with coronary stenosis > or = 50% as determined by coronary angiography in Groups A and B, respectively. All patients (30 cases) with abnormal 99Tcm-MIBI myocardial SPET and coronary calcification detected by EBCT had significant coronary artery disease, and 93.3% of the patients with normal 99Tcm-MIBI SPET and normal EBCT had normal coronary angiography or < 50% lumen narrowing of the coronary arteries. In Group B, the sensitivity of SPET for detecting CAD was significantly higher than that of EBCT (92.9 vs 42.9%, P < 0.01); the specificity of SPET was comparable to that of EBCT. In Group A, there was no significant difference between SPET and EBCT in terms of sensitivity (93.6 vs 90.3%) or specificity (88.9 vs 55.6%). However, in the detection of individual coronary artery disease, the specificity of SPET was significantly higher than that of EBCT in Group A (94.1 vs 66.7%, P < 0.001). The sensitivity of SPET was again significantly higher than that of EBCT (85.7 vs 38.1%, P < 0.005) in Group B. The accuracy of SPET was higher than that of EBCT in both groups (82.5 vs 67.5%, P < 0.01 in Group A; 93.1 vs 76.4%, P < 0.01 in Group B, respectively). We conclude that 99Tcm-MIBI myocardial perfusion SPET has a higher sensitivity than EBCT in the detection of CAD in patients < or = 45 years old and a higher specificity in patients > 45 years of age. A combination of SPET and EBCT may assess CAD more accurately.

Adult↗

PubMed2000

Freedericksz transition in an anticlinic liquid crystal

The Freedericksz geometry is used to show experimentally that a very-long-pitch, surface stabilized, anticlinic liquid crystal undergoes a two-step electric-field-induced transition to the synclinic phase. The liquid crystal remains undistorted below the threshold field E(th). For E>E(th), a Freedericksz transition occurs, wherein molecules in adjacent smectic layers undergo unequal azimuthal rotations about the layer normal, resulting in a nonzero polarization that couples to the applied field. Measurements of E(th) as a function of temperature are reported. Related quasielastic light scattering measurements demonstrate that acoustic Goldstone mode fluctuations are quenched by a dc electric field E>E(th). At high fields a transition to the synclinic phase occurs via solitary waves.

Journal Article↗

PubMed2000

Electric field-induced acoustic-optic mode coupling in an anticlinic liquid crystal

A dc electric field was applied perpendicular to the tilt plane of a pitch-compensated (unwound helix) anticlinic liquid crystal. By means of quasielastic light scattering, the field was found to couple the acoustic and optic Goldstone modes, resulting in an increase of the relaxation time tau(beta) of the acousticlike eigenmode. Elastic constants were estimated from the relaxation time data.

Journal Article↗

PubMed2000

Calcium-independent activation of salicylic acid-induced protein kinase and a 40-kilodalton protein kinase by hyperosmotic stress.

Reversible protein phosphorylation/dephosphorylation plays important roles in signaling the plant adaptive responses to salinity/drought stresses. Two protein kinases with molecular masses of 48 and 40 kD are activated in tobacco cells exposed to NaCl. The 48-kD protein kinase was identified as SIPK (salicylic acid-induced protein kinase), a member of the tobacco MAPK (mitogen-activated protein kinase) family that is activated by various other stress stimuli. The activation of the 40-kD protein kinase is rapid and dose-dependent. Other osmolytes such as Pro and sorbitol activate these two kinases with similar kinetics. The activation of 40-kD protein kinase is specific for hyperosmotic stress, as hypotonic stress does not activate it. Therefore, this 40-kD kinase was named HOSAK (high osmotic stress-activated kinase). HOSAK is a Ca(2+)-independent kinase and uses myelin basic protein (MBP) and histone equally well as substrates. The kinase inhibitor K252a rapidly activates HOSAK in tobacco cells, implicating a dephosphorylation mechanism for HOSAK activation. Activation of both SIPK and HOSAK by high osmotic stress is Ca(2+) and abscisic acid (ABA) independent. Furthermore, mutation in SOS3 locus does not affect the activation of either kinase in Arabidopsis seedlings. These results suggest that SIPK and 40-kD HOSAK are two new components in a Ca(2+)- and ABA-independent pathway that may lead to plant adaptation to hyperosmotic stress.

Calcium↗

PubMed2000

Estimation of 3-D conduction velocity vector fields from cardiac mapping data.

A method to estimate three-dimensional (3-D) conduction velocity vector fields in cardiac tissue is presented. The speed and direction of propagation are found from polynomial "surfaces" fitted to space-time (x, y, z, t) coordinates of cardiac activity. The technique is applied to sinus rhythm and paced rhythm mapped with plunge needles at 396-466 sites in the canine myocardium. The method was validated on simulated 3-D plane and spherical waves. For simulated data, conduction velocities were estimated with an accuracy of 1%-2%. In experimental data, estimates of conduction speeds during paced rhythm were slower than those found during normal sinus rhythm. Vector directions were also found to differ between different types of beats. The technique was able to distinguish between premature ventricular contractions and sinus beats and between sinus and paced beats. The proposed approach to computing velocity vector fields provides an automated, physiological, and quantitative description of local electrical activity in 3-D tissue. This method may provide insight into abnormal conduction associated with fatal ventricular arrhythmias.

Animals↗

PubMed2000

Conformational behavior of ionic self-complementary peptides.

Several de novo designed ionic peptides that are able to undergo conformational change under the influence of temperature and pH were studied. These peptides have two distinct surfaces with regular repeats of alternating hydrophilic and hydrophobic side chains. This permits extensive ionic and hydrophobic interactions resulting in the formation of stable beta-sheet assemblies. The other defining characteristic of this type of peptide is a cluster of negatively charged aspartic or glutamic acid residues located toward the N-terminus and positively charged arginine or lysine residues located toward the C-terminus. This arrangement of charge balances the alpha-helical dipole moment (C --> N), resulting in a strong tendency to form stable alpha-helices as well. Therefore, these peptides can form both stable alpha-helices and beta-sheets. They are also able to undergo abrupt structural transformations between these structures induced by temperature and pH changes. The amino acid sequence of these peptides permits both stable beta-sheet and alpha-helix formation, resulting in a balance between these two forms as governed by the environment. Some segments in proteins may also undergo conformational changes in response to environmental changes. Analyzing the plasticity and dynamics of this type of peptide may provide insight into amyloid formation. Since these peptides have dynamic secondary structure, they will serve to refine our general understanding of protein structure.

Amino Acid Sequence↗