Biomedical subjects
Publications and source records attributed to S Zhang.
A method to determine the concentration of atmospheric pollutant, sulfur dioxide, was studied by fluorophotometric method. The composition and concentration of reaction solution were optimized. This method can be developed to be a fibre optic fluorometric method for routine monitoring.
The glycine codons GGA or GGG, on the 5' side of stop codons UAG and UGA, are associated with a uniquely low termination efficiency in Escherichia coli, as compared to other codons, including the two glycine codons GGU and GGC. In contrast to the wild-type strain, all four glycine codons have a similar effect on termination at UAG in a strain with a mutant release factor 1 (RF1). Thus, these two glycine codon pairs, when present at the ribosomal P-site, affect termination efficiency of mutant or wild-type RF1 at UAG differently. If reading of GGA/G by tRNAGly2 is eliminated in the RF1 wild-type strain and replaced by a mutant form of tRNAGly3, termination efficiency is increased to the same level as for GGU/C, normally read by tRNAGly3. The results suggest an unusual interaction between the P-site tRNAGly2 and wild-type RF1 at the ribosomal A-site that is not present with mutant RF1.
The transcription factor NF-kappaB is a ubiquitously expressed inducible regulator of a broad range of genes. Recent studies have shown that activation of NF-kappaB predominantly is associated with protecting cells from apoptosis, but in some cell models, it is associated with promoting cell death. We used a rabbit spinal cord model of reversible ischemia to determine whether NF-kappaB was activated by ischemic and reperfusion injury. DNA binding activity of NF-kappaB was analyzed by an electrophoretic mobility shift assay in animals subjected to varying durations of ischemia and reperfusion. A low level of constitutive NF-kappaB DNA binding was detected in normal lumbar spinal cord extracts. Animals subjected to a short ischemic insult of 15 min, from which they usually recover neurologic function, had a significant increase in the amount of active NF-kappaB in nuclear extracts after 18 h reperfusion. There was no change in nuclear NF-kappaB DNA binding in animals occluded for 60 min that are permanently paraplegic and exhibit extensive neuropathological damage. The amount of deoxycholate-releasable NF-kappaB sequestered in the cytosol, however, decreased after 18 h reperfusion in rabbits occluded for 60 min. This correlated with a decrease in the amount of RelA(p65) NF-kappaB subunit. The results suggest that activation of NF-kappaB after a limited ischemic injury may participate in a neuroprotective response and not in cell death.
In previous studies, we have shown that electrically or chemically evoked activation of the ventrolateral orbital cortex (VLO) depresses the rat tail-flick (TF) reflex, and this antinociceptive effect is mediated by the periaqueductal gray (PAG). The aim of the present study was to examine whether electrical stimulation of the VLO could inhibit the rat jaw-opening reflex (JOR), and to determine whether electrolytic lesions of the PAG could attenuate this VLO-evoked inhibition. Unilateral electrical stimulation of the VLO significantly depressed the JOR elicited by tooth pulp or facial skin stimuli, with a mean threshold of 30.5+/-2.3 microA (n=22). Increasing stimulation intensities from 30 to 80 microA resulted in greater reduction of the dEMG amplitude from 22.9+/-5.0% to 69.7+/-3.7% of the baseline value (P<0.01, n=22). The inhibitory effect appeared 50 ms after the beginning of VLO stimulation and lasted about 150 ms, as determined by varying the conditioning-test (C-T) time interval. Unilateral lateral or ventrolateral lesions of the PAG produced only a small attenuation of the VLO-evoked inhibition of the JOR, but bilateral lesions eliminated this inhibition. These findings suggest that the VLO plays an important role in modulation of orofacial nociceptive inputs, and provide further support for the hypothesis that the antinociceptive effect of VLO is mediated by PAG leading to activation of a brainstem descending inhibitory system and depression of nociceptive inputs at the trigeminal level. The role played by VLO in pain modulation is discussed in association with the proposed endogenous analgesic system consisting of medullary cord-Sm-VLO-PAG-medullary cord.
OBJECTIVE: The purpose of this study was to evaluate acute angiographic success, in-hospital complications and long-term outcome after intracoronary stenting in patients with cardiac allograft vasculopathy. BACKGROUND: The application of conventional interventional modalities to treat discrete lesions in patients with cardiac allograft vasculopathy is associated with higher procedural morbidity, mortality and higher restenosis compared to atherosclerotic coronary artery disease. Elective coronary stenting has been shown to lower restenosis rates and improve long-term outcome in selected patients with native coronary artery disease; however, its safety and efficacy in reducing restenosis in patients with cardiac allograft vasculopathy is unknown. METHODS: Ten patients with 19 discrete lesions in a major coronary artery without diffuse distal disease underwent intracoronary stenting using Palmaz-Schatz stents. The average stent size was 3.4 mm, and the stent/artery ratio was 0.99+/-0.07. Eight of ten (80%) patients received antiplatelet therapy (aspirin plus ticlopidine) only. RESULTS: Procedural success was 100% with no in-hospital stent thrombosis, Q-wave myocardial infarction or death. Minimal luminal diameter increased from 0.83+/-0.38 mm to 3.23+/-0.49 mm after stenting. Diameter stenosis decreased from 74.91+/-11.52% to 5.90+/-4.09% after stenting. Follow-up angiography was performed in 8 of 10 (80%) patients and 16 of 19 (84%) lesions. Target lesion revascularization was required in 2 of 10 (20%) patients and 3 of 16 (19%) lesions. Allograft survival was 7 of 10 (70%) at the end of 22+/-11 months follow-up. CONCLUSIONS: Intracoronary stenting can be performed safely with excellent angiographic success in selected patients with cardiac allograft vasculopathy. The restenosis rate appears to be low despite the aggressive nature of the disease. A multicenter study with a larger number of patients is required to assess its efficacy in reducing restenosis and improving allograft survival.
Staphylococcus aureus enterotoxin D is one of the serotypes most commonly associated with food poisoning. Further characterization of the enterotoxin D-encoding plasmid revealed the presence of an open reading frame which encodes a previously unidentified enterotoxin, designated staphylococcal enterotoxin J (SEJ). SEJ is a protein of 269 amino acid residues which has substantial sequence similarity to the staphylococcal A, E, D family of enterotoxins. The enterotoxin D and J open reading frames are transcribed in opposite directions and are separated by an 895 nucleotide intergenic region which contains a perfect inverted repeat, with each arm of the repeat having a length of 21 nucleotides. Chloramphenicol acetyl transferase (cat) transcriptional fusions were used to quantify expression from the enterotoxin gene promoters. Both enterotoxin genes are expressed in S. aureus. However, only sed is regulated by the agr virulence gene signal transduction pathway. Western blot analyses utilizing anti-enterotoxin antisera have confirmed the results obtained with the cat reporter system. PCR amplification studies suggest that the sej determinant may be present on all sed-encoding plasmids.
Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin-1 and endomorphin-2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin-stimulated increase of cAMP in a dose-dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein-activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose-dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G-protein-activated inwardly rectifying potassium (GIRK) channels.
TFIIF (RAP30/74) is a general initiation factor that also increases the rate of elongation by RNA polymerase II. A two-hybrid screen for RAP74-interacting proteins produced cDNAs encoding FCP1a, a novel, ubiquitously expressed human protein that interacts with the carboxyl-terminal evolutionarily conserved domain of RAP74. Related cDNAs encoding FCP1b lack a carboxyl-terminal RAP74-binding domain of FCP1a. FCP1 is an essential subunit of a RAP74-stimulated phosphatase that processively dephosphorylates the carboxyl-terminal domain of the largest RNA polymerase II subunit. FCP1 is also a stoichiometric component of a human RNA polymerase II holoenzyme complex.
The human helicase gene SKI2W is located between RD and RP1 in the class III region of the major histocompatibility complex. Transcripts of SKI2W are detectable in RNA samples isolated from multiple tissues. The protein product Ski2w shares striking amino acid sequence similarities to the yeast antiviral protein Ski2p that controls the translation of mRNAs, probably based on the mRNA structural integrity. Whether this translational regulation mechanism for cellular and viral RNAs exists in mammals is under investigation. Antisera against human Ski2w were generated using fusion proteins produced in bacteria or insect cells. Western blot analysis showed that the endogenous Ski2w protein is approximately 140 kDa in size and is enriched in polysomal fractions of cytoplasmic extracts from HeLa cells. Ribosomal profile studies revealed that Ski2w distributed throughout the entire sucrose gradient in the presence of Mg2+, but co-sedimented with the 18S rRNA-containing 40S subunit and the small ribosomal subunit protein S27a in the presence of EDTA. The co-sedimentation of Ski2w with the 40S subunit is not affected by RNase A treatment of the cell extract, or the addition of KCl to 0.5 M, suggesting that Ski2w is associated with the 40S ribosomal subunit. Indirect immunofluorescence experiments showed that human Ski2w is localized in the nucleoli and in the cytoplasm. In essence, human Ski2w is present at the sites of ribosome biogenesis and protein synthesis.
Changes of nitric oxide (NO)-producing neurons in the brain following learning is not yet clear. In present study, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry and neuronal NO synthase (nNOS) immunohistochemistry were used to detect NOS neurons in rat brain. Results demonstrated that expression of NOS neurons in dentate gyrus and frontal cortex was significantly increased after a water-rewarded spatial alternation task when compared with that after sham training. The elevated expression of NOS neurons occurred not only in the earlier memory stage, but also in the later memory stage. In addition, the expression location and cell counts of NOS neurons in dentate gyrus and frontal cortex with NADPH-d staining or nNOS immunoreactivity resembled each other, but the cell counts of NADPH-d positive neurons were a little more than those of nNOS immunoreactive neurons. The involvement of NO in the processes of spatial learning and memory is further suggested.
Opioid drugs play important roles in the clinical management of pain, as well as in the development and treatment of drug abuse. The mu opioid receptor is the primary site of action for the most commonly used opioids, including morphine, heroin, fentanyl, and methadone. By sequencing DNA from 113 former heroin addicts in methadone maintenance and 39 individuals with no history of drug or alcohol abuse or dependence, we have identified five different single-nucleotide polymorphisms (SNPs) in the coding region of the mu opioid receptor gene. The most prevalent SNP is a nucleotide substitution at position 118 (A118G), predicting an amino acid change at a putative N-glycosylation site. This SNP displays an allelic frequency of approximately 10% in our study population. Significant differences in allele distribution were observed among ethnic groups studied. The variant receptor resulting from the A118G SNP did not show altered binding affinities for most opioid peptides and alkaloids tested. However, the A118G variant receptor binds beta-endorphin, an endogenous opioid that activates the mu opioid receptor, approximately three times more tightly than the most common allelic form of the receptor. Furthermore, beta-endorphin is approximately three times more potent at the A118G variant receptor than at the most common allelic form in agonist-induced activation of G protein-coupled potassium channels. These results show that SNPs in the mu opioid receptor gene can alter binding and signal transduction in the resulting receptor and may have implications for normal physiology, therapeutics, and vulnerability to develop or protection from diverse diseases including the addictive diseases.
Adhesion of hematopoietic cells to extracellular matrix components is important for blood cell development. However, little is known regarding the potential influence of IL-3 on this process for precursor B cells and Flt3-ligand has not yet been implicated in induction of adhesion of any blood cell types to extracellular matrix components. Therefore, we examined the characteristics of cytokine-induced cell adhesion to fibronectin (FN), using as a model the murine precursor B cell line, Baf3, a factor-dependent cell line requiring IL-3 for both growth and survival. Since factor-dependent hematopoietic cell lines expressing Flt3 receptor are extremely rare, we also studied Baf3/Flt3, a subline of Baf3 transduced with the Flt3 receptor gene. IL-3 induced adhesion of Baf3 and Baf3/Flt3 cells to FN, while Flt3-ligand induced adhesion of Baf3/Flt3 cells only. Whereas both Baf3 and Baf3/Flt3 cells expressed VLA-4 and -5 integrins as FN receptors, expression levels of VLA-4 and -5 were not affected by IL-3 or Flt3-ligand treatment. However, blocking experiments using anti-integrin antibodies showed that cytokine-induced adhesion of cells depended on both VLA-4 and -5 suggesting that IL-3 and Flt3-ligand activated these integrins. PI-3 kinase inhibitor wortmannin, PKC inhibitor H-7, or PKA inhibitor HA1004 did not suppress adhesion induced by IL-3 or Flt3-ligand; in contrast, PLC inhibitor U-73122 did suppress adhesion, suggesting the possibility that PLC, but not PI-3 kinase, PKC, or PKA, may be involved in this process. Since it is known that IL-3 and Flt3-ligand receptors are expressed on precursor B cells, and these receptors are downregulated during B cell maturation of primary cells, the induction of precursor B cell adhesion to FN by IL-3 and Flt3-ligand may contribute a mechanism by which precursor B cells adhere to bone marrow stroma, thereby influencing their development.
A novel technique has been used to determine the effective diffusion coefficients for 1,1,2-trichloroethane (TCE), a nonreacting tracer, in biofilms growing on the external surface of a silicone rubber membrane tube during degradation of 1,2-dichloroethane (DCE) by Xanthobacter autotrophicus GJ10 and monochlorobenzene (MCB) by Pseudomonas JS150. Experiments were carried out in a single tube extractive membrane bioreactor (STEMB), whose configuration makes it possible to measure the transmembrane flux of substrates. A video imaging technique (VIT) was employed for in situ biofilm thickness measurement and recording. Diffusion coefficients of TCE in the biofilms and TCE mass transfer coefficients in the liquid films adjacent to the biofilms were determined simultaneously using a resistances-in-series diffusion model. It was found that the flux and overall mass transfer coefficient of TCE decrease with increasing biofilm thickness, showing the importance of biofilm diffusion on the mass transfer process. Similar fluxes were observed for the nonreacting tracer (TCE) and the reactive substrates (MCB or DCE), suggesting that membrane-attached biofilm systems can be rate controlled primarily by substrate diffusion. The TCE diffusion coefficient in the JS150 biofilm appeared to be dependent on biofilm thickness, decreasing markedly for biofilm thicknesses of >1 mm. The values of the TCE diffusion coefficients in the JS150 biofilms <1-mm thick are approximately twice those in water and fall to around 30% of the water value for biofilms >1-mm thick. The TCE diffusion coefficients in the GJ10 biofilms were apparently constant at about the water value. The change in the diffusion coefficient for the JS150 biofilms is attributed to the influence of eddy diffusion and convective flow on transport in the thinner (<1-mm thick) biofilms.
After 10 years of clinical trials in patients with advanced cancer, monoclonal antibodies (mAbs) against cell surface antigens have not lived up to their initial promise. One such cell surface antigen is the ganglioside GD2. GD2 is richly expressed at the cell surfaces of human neuroblastomas, sarcomas, and melanomas. We have described a murine lymphoma (EL4) that is syngeneic in C57BL/6 mice and expresses GD2, a mAb against GD2 (mAb 3F8), and we have prepared a conjugate vaccine (GD2-keyhole limpet hemocyanin plus immunological adjuvant QS-21) that consistently induces antibodies against GD2. We demonstrate here, for the first time in a syngeneic murine model, that passively administered and vaccine-induced antiganglioside antibodies prevent outgrowth of micrometastases, and we use this model to establish some of the parameters of this protection. The level of protection was proportional to antibody titer. Treatment regimens resulting in the highest titer antibodies induced the most protection, and protection was demonstrated even when immunization was initiated after tumor challenge. Treatment with 3F8 1, 2, or 4 days after i.v. tumor challenge was highly protective, but waiting until 7 or 10 days after challenge resulted in minimal protection. The results were similar whether number of liver metastases or survival was used as the end point. These results suggest that unmodified mAbs or antibody-inducing vaccines against GD2 (and possibly other cancer cell surface antigens) should be used exclusively in the adjuvant setting, where circulating tumor cells and micrometastases are the primary targets.
Electrostatic interactions play important roles in the catalysis of chorismate to prephenate by chorismate mutase. Mutation of Gln88 to glutamate in the monofunctional chorismate mutase from Escherichia coli results in an enzyme with a pH profile of activity significantly different from that of the wild type protein. To investigate whether the mutation alters the substrate binding process or the catalysis, we have directly determined the thermodynamic parameters of a transition state analogue inhibitor binding to the wild-type chorismate mutase and its Q88E mutant using isothermal titration calorimetry. The results demonstrate that solvent reorganization and hydrophobic interactions contribute the predominant free energy to inhibitor binding. The charge state of Glu88 in the Q88E mutant was experimentally determined and was shown to be protonated at pH 4.5 and ionized at pH 7.8, consistent with earlier hypotheses. Most surprisingly, inhibitor binding energetics do not exhibit significant pH dependency for both enzymes. Our findings indicate that the charge state of Glu88 has a small impact on inhibitor binding but plays an important role in the catalytic process.
Salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two distinct members of the mitogen-activated protein (MAP) kinase family, are activated in tobacco resisting infection by tobacco mosaic virus (TMV). WIPK activation by TMV depends on the disease-resistance gene N because infection of susceptible tobacco not carrying the N gene failed to activate WIPK. Activation of WIPK required not only posttranslational phosphorylation but also a preceding rise in its mRNA and de novo synthesis of WIPK protein. The induction by TMV of WIPK mRNA and protein also occurred systemically. Its activation at the mRNA, protein, and enzyme levels was independent of salicylic acid. The regulation of WIPK at multiple levels by an N gene-mediated signal(s) suggests that this MAP kinase may be an important component upstream of salicylic acid in the signal-transduction pathway(s) leading to local and systemic resistance to TMV.
By searching the Expressed Sequence Tag (EST) data base, we identified a partial cDNA sequence encoding a novel human CC chemokine. The entire cDNA sequence was determined and revealed a CC chemokine whose mature protein consisted of 100 amino acids with predicted molecular weight of 11 kd. The chemokine preferantially chemoattracted lymphocytes and monocytes but not neutrophils. It was, therefore, named LMC (Lymphocyte and Monocyte Chemoattractant). LMC exhibited potent myelosuppressive activity, which was comparable to that of MIP-1alpha. We identified several bacterial artificial clones (BAC) containing the LMC gene along with two human CC chemokine subfamily members; leukotactin-1 (Lkn-1) and CKbeta8-1/CKbeta8. This data suggests that the LMC gene is located at human chromosome 17q which encompasses a human CC chemokine gene cluster.
It has been demonstrated that both salicylic acid and fungal elicitors activate a 48-kDa mitogen-activated protein kinase termed salicylic acid-induced protein kinase (SIPK) in tobacco suspension cells. Here, we show that infiltration of these agents into tobacco leaves also activates SIPK. Of particular interest, infiltration of water alone activated a kinase of the same size, possibly because of wounding and/or osmotic stresses. The kinetics of kinase activation, however, differ for these different treatments. Various mechanical stresses, including cutting and wounding by abrasion, also activated a 48-kDa kinase. By using an immune-complex kinase assay with antibodies specific for SIPK or wounding-induced protein kinase, we demonstrate that this wounding-activated 48-kDa kinase is SIPK, rather than wounding-induced protein kinase, as reported [Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H. & Ohashi, Y. (1995) Science 270, 1988-1992]. Activation of SIPK after wounding was associated with tyrosine phosphorylation but not with increases in SIPK mRNA or protein levels. Thus, the same mitogen-activated protein kinase, SIPK, appears to facilitate signaling for two distinct pathways that lead to disease resistance responses and wounding responses.