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J Zhu

Publications and source records attributed to J Zhu.

At least 649 records · Page 36Linked to original sources
PubMed1995

Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action.

Tubulin polymerization into microtubules is a dynamic process, with the equilibrium between growth and shrinkage being essential for many cellular processes. The antineoplastic agent taxol hyperstabilizes polymerized microtubules, leading to mitotic arrest and cytotoxicity in proliferating cells. Using a sensitive filtration-calorimetric assay to detect microtubule nucleating activity, we have identified epothilones A and B as compounds that possess all the biological effects of taxol both in vitro and in cultured cells. The epothilones are equipotent and exhibit kinetics similar to taxol in inducing tubulin polymerization into microtubules in vitro (filtration, light scattering, sedimentation, and electron microscopy) and in producing enhanced microtubule stability and bundling in cultured cells. Furthermore, these 16-membered macrolides are competitive inhibitors of [3H]taxol binding, exhibiting a 50% inhibitory concentration almost identical to that of taxol in displacement competition assays. Epothilones also cause cell cycle arrest at the G2-M transition leading to cytotoxicity, similar to taxol. In contrast to taxol, epothilones retain a much greater toxicity against P-glycoprotein-expressing multiple drug resistant cells. Epothilones, therefore, represent a novel structural class of compounds, the first to be described since the original discovery of taxol, which not only mimic the biological effects of taxol but also appear to bind to the same microtubule-binding site as taxol.

Animals↗
PubMed1995

Characterization of Rad, a new member of Ras/GTPase superfamily, and its regulation by a unique GTPase-activating protein (GAP)-like activity.

We have recently identified a new member of the Ras/GTPase superfamily termed Rad which has unique sequence features and is overexpressed in the skeletal muscle of humans with type II diabetes (Reynet, C., and Kahn, C. R. (1993) Science, 262, 1441-1444). When expressed in bacteria as a glutathione S-transferase fusion protein, Rad bound [alpha-32P]GTP quickly and saturably. Binding was specific for guanine nucleotides and displayed unique magnesium dependence such that both GTP and GDP binding were optimal at relatively high Mg2+ concentrations (1-10 mM). Rad had low intrinsic GTPase activity which was greatly enhanced by a GTPase-activating protein (GAP) activity present in various tissues and cell lines. Several known GAPs had no stimulatory effect toward Rad. Conversion of Ser to Asn at position 66 in Rad (equivalent to position 12 in Ras) resulted in a total loss of GTP binding. Mutation of Pro61 (equivalent to Gly12 in Ras) or Gln109 (equivalent to Gln61 in Ras) had no effect on Rad GTPase activity, whereas creation of a double mutation at these positions resulted in exceptionally high intrinsic GTPase activity. In vitro, Rad was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (PK). Phosphopeptide mapping indicated two PKA phosphorylation sites near the COOH terminus. Rad also co-precipitated a serine/threonine kinase activity from extracts of various tissues and cell lines which catalyzed phosphorylation on Rad but was not inhibited by PKA inhibitor. Thus, Rad is a GTP-binding protein and a GTPase which has some structure/function similarities to Ras, but displays unique features. Rad may also be phosphorylated on serine/threonine residues by PKA and other kinases, as well as regulated by its own GAP which is present in many tissues and cell types.

Amino Acid Sequence↗
PubMed1995

Deletion of lysine 121 creates a temperature-sensitive alteration in insulin binding by the insulin receptor.

Recently we reported the deletion of Lys-121 in one allele of the insulin receptor gene from a child with severe insulin resistance. In the present work, this mutant receptor (M121) was shown to have an abnormal sensitivity to temperature and an alteration in "negative cooperativity." In contrast to the wild-type receptor (HIRC), insulin binding by the M121 receptor was rapidly and irreversibly lost at temperatures above 30 degrees C with the phosphorylated form of the receptor being more temperature-sensitive than the nonphosphorylated form. Although insulin binding activity was lost, Western analysis and other studies showed that the mutant receptor remained intact. Measurements of 125I-insulin dissociation at 21 degrees C in the presence of native insulin (an estimate of negative cooperativity) demonstrated a difference between the mutant and wild-type receptor. Insulin dissociation from the mutant receptor was not as pronounced as that found with the wild-type receptor. Thus, an abnormality in insulin binding by the mutation was evident at lower "permissive" temperatures. The results of these and other studies argue that Lys-121 occupies an important position for the regulation of insulin receptor conformation. This regulation apparently influences negative cooperative interactions with insulin and modulates signal transduction.

Alkylating Agents↗
PubMed1995

Deletion analysis of protein kinase C inactivation by calphostin C.

Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C. Both PKC-alpha (a Ca(2+)-dependent conventional isoform) and PKC-epsilon (a Ca(2+)-independent novel isoform) are similarly inactivated by calphostin C (75-100 nM produced 50% inhibition), suggesting that inactivation requires a site common to both classes of PKC. We therefore performed studies to identify a critical region in the regulatory domain of PKC-alpha required for inactivation by calphostin C. A series of N-terminal-truncation mutants of bovine PKC-alpha expressed in Saccharomyces cerevisiae was tested with 500 nM calphostin C, a concentration sufficient to inactivate wild-type PKC-alpha by 80-90%. This concentration was as effective with mutant proteins containing deletions of up to 91 amino acid (aa) residues from the amino terminus (ND91), whereas a mutant protein truncated by 140 aa (ND140) was inactivated by only 20%. These findings imply that the aa sequence 92-140 is a structural determinant of PKC-alpha inactivation by calphostin C. This sequence contains one of the phorbol ester-binding sites (aa 102-144), which is highly conserved among most PKC isoforms including PKC-epsilon. In addition to aa 92-140, PKC-stimulating cofactors (phosphatidylserine, phorbol ester, and Ca2+) are required for inactivation by calphostin C even in the case of PKC mutants that do not require these cofactors for enzymatic activity. These results suggest that cofactors provide a template that is required for productive interaction of PKC and the inhibitor. The significance of the proposed proximity effect to calphostin C action is discussed.

Animals↗
PubMed1995

Differential protein kinase C ligand regulation detected in vivo by a phenotypic yeast assay.

The molecular dissection of protein kinase C (PKC) action has been based in part on time-consuming functional assays such as the mouse skin model for testing the tumor promoter activity of phorbol esters and related PKC activators. To help overcome the limitations imposed by the complexity of such assays, we developed the yeast Saccharomyces cerevisiae as an alternative, rapid, and simple experimental system. This model has a specific phenotype, an increase in the cell doubling time, that is proportional to the level of enzymatic activity of expressed mammalian PKC isoforms. We used this phenotype to assay and compare the regulation of native bovine PKC alpha and mutants in the conserved regulatory region C1 in vivo by various activators: two diterpenes, the phorbol ester phorbol-12-myristate-13-acetate (PMA) and mezerein, and the indole alkaloid indolactam V. We found that PMA activated PKC mutants lacking either Cys-rich, zinc finger-like repeat of the conserved region C1 to comparably reduced levels, whereas indolactam V activated native PKC alpha but none of the mutants at normal doses. In contrast, mezerein activated native PKC alpha and a mutant lacking the second Cys repeat equally well but mutants lacking the first Cys repeat of C1 at a greatly reduced level. These differential responses were supported by the observed in vitro PKC catalytic activities. Therefore, PMA regulates PKC alpha activity comparably well via either Cys repeat, whereas mezerein regulation predominantly occurs via the first Cys repeat of C1. Indolactam V activation was less potent, it was greatly reduced in the absence of either Cys repeat, and displayed no preference. We introduce this phenotypic assay as a rapid and general screen for the PKC-activating or possibly inhibitory potential of drug candidates and to identify the PKC regulatory sites involved in these interactions.

Animals↗
PubMed1995

Application of sciatic functional index in nerve functional assessment.

In order to confirm the reliability of the sciatic functional index (SFI) in the rat, SFI, muscle strength, electrophysiological, and morphometric assessments were carried out from the 10th day to the sixth month after nerve injury or repair. The results showed that the SFI has a positive correlation with all tested indices of muscle strength, electrophysiology, and morphology (r = 0.925-0.996, P < 0.01 or P < 0.001). These results indicate that the SFI is a reliable index for evaluating rat sciatic nerve regeneration and can be widely used.

Animals↗
PubMed1995

Detection of kappa-opioid receptor mRNA in immature T cells.

The R1.1 cell line has been shown to express kappa-opioid receptors on the cell surface. Our analysis shows that the R1.1 cell line exhibits a CD4NEG CD8NEG CD3LOW CD25LOW cell surface phenotype, characteristic of thymocytes in one of the early stages of differentiation. We have developed reverse transcriptase polymerase chain reaction (RT-PCR) conditions that permit the detection of mRNA coding for the kappa-receptor. Using cell fractionation techniques we have isolated CD4NEG CD8NEG thymocytes, and analysis by RT-PCR shows that these primary immature thymocytes also express the kappa-opioid receptor. We hypothesize that the expression of kappa-opioid receptor may be a marker which is characteristic of immature T development.

Animals↗
PubMed1995

Cloning of a human kappa opioid receptor from the brain.

By using a rat kappa opioid receptor cDNA as a probe to screen a human brain cDNA library, we isolated a 4.0-kb clone (z115) which encompasses a major portion of a human kappa opioid receptor (hkor), extending from the amino acid residue #6 to the 3'-untranslated region. The extreme 5'-region 232-bp fragment of z115 was used as a probe to screen a human genomic DNA library. A 1.6-kb fragment (d2) of one positive clone was found to extend from 5'-untranslated region to beyond the exon/intron junction at residue Arg86. The genomic DNA fragment d2 and the cDNA clone z115 were assembled to generate a clone (d2-z115) containing the entire coding sequence of hkor. Clone d2-z115 has an open reading frame of 1140 bp, which encodes for a 380-amino acid protein. The deduced amino acid sequence has 93.9% and 93.2% identity to rat and mouse kappa receptors, respectively. It also displays approximately 60% identity to both human mu and delta receptors. Northern blot analysis showed that in the human brain there was a single hkor mRNA transcript of 6.0 kb. Among brain regions examined, the amygdala, caudate nucleus, hypothalamus and subthalamic nucleus contained high levels of hkor mRNA. Hkor was cloned into the expression vector pBK-CMV and transiently expressed in COS-1 cells. Hkor had high affinity for [3H] diprenorphine, a nonselective opioid antagonist, and displayed stereospecific binding to naloxone. kappa selective ligands (U50,488H and nor-BNI) had high affinities, whereas mu and delta selective ligands bound with much lower affinities. Dynorphin A (1-17) and alpha-neoendorphin, both endogenous kappa peptides, bound with high affinities. These binding characteristics confirmed that hkor is a kappa receptor, most likely kappa 1 type. Cloning of the human kappa receptor allows investigation of interactions of compounds with the human receptor, instead of rodent receptors, for development of better therapeutic agents.

Amino Acid Sequence↗
PubMed1995

Sequence of kappa-opioid receptor cDNA in the R1.1 thymoma cell line.

To our knowledge, this is the first demonstration of kappa-opioid receptor mRNA in cells of the immune system. While the presence of opioid receptors on cells of the immune system has been controversial, cell-binding analysis has indicated that the kappa-opioid receptor is expressed by the immature T cell line R1.1. We have developed a reverse transcriptase-polymerase chain reaction protocol to amplify the mRNA extracted from R1.1 cells with primers derived from the cDNA sequence of the mouse kappa-opioid receptor. Nucleotide sequences of the amplified products were examined and two populations of cDNA were detected which differ in the 5' region upstream of the ATG start codon. Comparison of these sequences to the previously published kappa-opioid receptor cDNA sequence suggests the presence of an intron-exon junction in the 5' non-coding region.

Animals↗
PubMed1995

Experimental study using a direct current electrical field to promote peripheral nerve regeneration.

A direct current (DC) electrical field was used to stimulate transected and sutured rat sciatic nerve. Electrophysiologic, morphometric, and functional assessments were carried out on days 10, 20, 30, and 60 postoperatively. The results showed that the electrophysiologic index, the diameter and myelin-sheath thickness of regenerating nerve fibers, the sciatic functional index (SFI), and the strength of the triceps surae muscle in the experimental group were much better than those in the control group. This indicated that DC electrical stimulation could effectively promote peripheral nerve regeneration and produce positive effects after 3 weeks, and that the stimulator was not rejected by the host for a relatively long period of time.

Action Potentials↗
PubMed1995

Analysis of conditional genetic effects and variance components in developmental genetics.

A genetic model with additive-dominance effects and genotype x environment interactions is presented for quantitative traits with time-dependent measures. The genetic model for phenotypic means at time t conditional on phenotypic means measured at previous time (t-1) is defined. Statistical methods are proposed for analyzing conditional genetic effects and conditional genetic variance components. Conditional variances can be estimated by minimum norm quadratic unbiased estimation (MINQUE) method. An adjusted unbiased prediction (AUP) procedure is suggested for predicting conditional genetic effects. A worked example from cotton fruiting data is given for comparison of unconditional and conditional genetic variances and additive effects.

Gossypium↗
PubMed1995

Intravenous propofol vs thiamylal-isoflurane for caesarean section, comparative maternal and neonatal effects.

Several studies on propofol (Diprivan) for induction of anaesthesia during caesarean section have demonstrated its safety, however, it safety during maintenance of anaesthesia is not yet fully evaluated. The present study was undertaken to compare the maternal and neonatal effects of propofol or isoflurane in 74 term parturients undergoing primary or repeat caesarean section. Patients were randomly assigned to two groups, propofol group (n = 37) received propofol 1.5-2.5 mg.kg-1 for induction followed by a continuous infusion of propofol of 0.05-0.2 mg.kg-1.min-1. The isoflurane group (n = 37) received thiamylal 3-4mg.kg-1 for induction followed by isoflurane 0.25-0.75% for maintenance. All patients had rapid sequence induction using succinylcholine and endotracheal intubation, 50% N2O and O2 were used in all patients until delivery. After delivery N2O concentration was increased to 67% and intravenous butorphanol (Stadol) was given as needed. Patients in the propofol group had less hypertension after intubation (P < 0.05) and this was also of shorter duration compared to patients in the isoflurane group (5 min vs 10 min respectively). Maternal blood loss as well as intraoperative awareness and recovery time did not differ significantly between the two groups. Neonatal status as ascertained by Apgar scores, cord acid base status and the neurological and adaptive capacity scores (NACS) was equally good in both groups. It is concluded that propofol used for induction and maintenance of anaesthesia is a safe alternative to thiamylal/isoflurane for patients undergoing caesarean section and is associated with less hypertensive response during laryngoscopy and intubation.

Acid-Base Equilibrium↗
PubMed1995

Desflurane analgesia for vaginal delivery.

The use of subanaesthetic concentration of inhalational anaesthetic for vaginal delivery offers many advantages to the mother and newborn. Desflurane, with the characteristics of rapid onset and minimal metabolism, may provide better analgesia and safety for labour pain control. Eighty healthy parturients were randomly assigned to receive either desflurane 1.0-4.5% and oxygen (n = 40) or nitrous oxide 30-60% in oxygen (n = 40). Analgesia was assessed using a score from 0 (no relief) to 4+ (excellent analgesia), amnesia for the delivery, blood loss were recorded. Neonates were evaluated by Apgar scores and neurologic and adaptive capacity scores (NACS). Data were analyzed for statistical significance using Student's t-test or Chi-square when appropriate. Analgesia scores were similar for both groups with more amnesia in desflurane group (23% vs 0% P < 0.05). Blood loss did not differ significantly, 364 ml for the desflurane group and 335 ml for the nitrous oxide group. There were no significant differences for neonatal Apgar score at 1 min or at 5 min or the NACS at 2 hr or 24 hr between the two groups. We conclude that desflurane in subanaesthetic doses is safe and effective inhalation agent for normal delivery but might be associated with amnesia.

Acid-Base Equilibrium↗
PubMed1995

Desflurane: a new volatile anesthetic for cesarean section. Maternal and neonatal effects.

Desflurane, a new volatile anesthetic agent with low blood/gas solubility, has recently been studied in clinical and animal trials but its use in obstetrics has not been adequately evaluated. This prospective study was undertaken to evaluate the maternal and neonatal effects of desflurane in obstetrical patients. Seventy-five healthy parturients undergoing primary or repeat cesarean section were randomly assigned to one of three groups of 25 each, end-tidal 3% desflurane, 6% desflurane or 0.6% enflurane, combined with 50% N2O and O2. All patients had rapid sequence induction of anesthesia with thiopentone sodium followed by succinylcholine for tracheal intubation. After delivery, anesthesia was maintained with reduced concentration of desflurane or enflurane with 67% N2O in O2, supplemented by butorphanol tartrate. Maternal hemodynamic parameters, blood loss and maternal awareness during surgery were monitored. Neonatal outcome was evaluated by Apgar scores, neurological and adaptive capacity scores (NACS), cord blood gas and acid-base status, and time to sustained respiration (TSR). Maternal blood loss did not differ significantly between the three groups and none of the patients developed intraoperative awareness. All three groups responded to psychomotor performance equally fast. Patients in all three groups developed transient hypertension and tachycardia during induction of anesthesia which returned to baseline values in approximately 5 min. Neonatal outcome was equally good in the three groups. More neonates in the 6% desflurane group had TSR > 90 s compared to the 3% desflurane group (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Anesthesia, Obstetrical↗
PubMed1995

Startle reflex modulation during exposure to pleasant and unpleasant odors.

The eyeblink component of the startle response to acoustic probes was measured while subjects smelled pleasant odors, unpleasant odors, or no odor. Peak electromyogram (EMG) 20-90 ms after probe onset was greater during unpleasant than during no odor conditions; EMGs for pleasant odors did not differ from those for no odor. Base tension in orbicularis oculi muscles was also higher during unpleasant odors. The results for unpleasant odors parallel those found in previous studies that have used a variety of methods for producing negatively valenced experiences. The results for pleasant odors diverge from those of most previous studies, which have found startle attenuation during positively valenced experiences. However, the results may be compatible with the suggestion (Lang, Bradley, & Cuthbert, 1992) that the motivational state that modulates startle involves both valence and arousal. Pleasant odors may require appropriate contexts to produce the positive motivational state required for startle attenuation.

Acoustic Stimulation↗
PubMed1995

A novel translational regulation function for the simian virus 40 large-T antigen gene.

Cells use the interferon-induced, double-stranded-RNA-dependent protein kinase PKR as a defense against virus infections. Upon activation, PKR phosphorylates and thereby inactivates the protein synthesis initiation factor eIF-2, resulting in the cessation of protein synthesis. Viruses have evolved various strategies to counteract this cellular defense. In this paper, we show that simian virus 40 (SV40) large-T antigen can antagonize the translational inhibitory effect resulting from the activation of PKR in virus-infected cells. Unlike the situation with other virus-host cell interactions, SV40 large-T antigen does not block the activation of PKR, suggesting that SV40 counteracts the cellular antiviral response mediated by PKR at a step downstream of PKR activation. Mutational analysis of large-T antigen indicates that a domain located between amino acids 400 and 600 of large-T antigen is responsible for this function. These results define a novel translational regulatory function for the SV40 large-T antigen.

Adenosine Triphosphatases↗
PubMed1995

Myocardial ischemia and reperfusion: a murine model.

Myocardial ischemia followed by reperfusion promotes a complex series of inflammatory reactions as noted in a variety of large animal studies. With development of genetically altered mice, there is intense interest in developing murine models to study mechanisms operative in cardiovascular disease. We developed a mouse model to study coronary artery occlusion and reperfusion effects and the method required to perform these studies both acutely and chronically. In mice, we applied a left anterior descending coronary artery occlusion either permanently or for 30 or 60 min followed by reperfusion allowing flow through the previously occluded coronary artery bed. Reperfusion was documented visually as well as by using Doppler ultrasound and histopathological techniques. The area at risk (AAR) and infarct size (IS) were assessed by EVans blue dye and triphenyltetrazolium chloride staining with computerized planimetry using an image analysis software program. The infarct as percentage of AAR and IS as percentage of the left ventricle in 13 mice with permanent occlusion was 68.6 +/- 4.4 and 28.0 +/- 2.8%, respectively. Reperfusion after occlusions of 60 and 30 min resulted in a significant decrease in IS as a percentage of the AAR compared with permanent occlusion. Histological examination of the ischemic and reperfused myocardium shows infiltration of leukocytes into the ischemic region as well as contraction bands classically associated with reperfusion. This new model allows assessment of AAR, IS, cardiac function, and pathophysiology in the mouse. With the current technology to develop genetically altered mice for overexpression or targeted mutations of various genes, this model is used to understand the complex pathophysiology of ischemia and reperfusion injury.

Animals↗