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Biomedical subjects

J Peng

Publications and source records attributed to J Peng.

At least 55 records · Page 3Linked to original sources

bcl 10 gene mutation in hepatocellular carcinoma.

OBJECTIVE: To detect the mutation frequency of the bcl 10 gene in the early and advanced stages of hepatocellular carcinoma (HCC). METHODS: Genome DNA samples were extracted from 46 cases of fresh HCC tumor tissues and their non-tumor adjacent tissues. Polymerase chain reaction-single strand conformation polymorphism method was used to detect point mutations of the three exons of the bcl 10 gene. For each individual exon, six random samples from those showing abnormal DNA bands were sequenced to verify those mutations. The relationship between serum alpha-fetoprotein (AFP) level and bcl 10 mutation, between the tumor size and bcl 10 mutation was also analyzed. RESULTS: Among the 46 samples, 26 cases (56.5%) were found to have mutations in exon 1, 5 out of the 6 cases were shown to have 5744 C-->G mutation by sequencing; 25 cases (54.3%) were found to have mutations in exon 2, 4 out of the 6 cases were shown to have 11,311 T deletion mutation by sequencing. Twenty-one cases (45.7%) were found to have mutations in exon 3, all of the 6 cases selected for sequencing were shown to have 14,116 C-->T mutation. Statistical analysis showed that neither serum alpha-fetoprotein level nor the size of hepatocellular carcinoma has a significant relationship with bcl 10 mutation. CONCLUSION: The bcl 10 gene has a high mutation frequency in liver cancer.

Adaptor Proteins, Signal Transducing↗

[Apoptosis of bone marrow cells aplastic anemia patients treated with cyclosporin A].

OBJECTIVE: To evaluate the effects of cyclosporin A (CsA) on the apoptosis of bone marrow cells of aplastic anemia (AA) patients. METHODS: The apoptosis of and Fas antigen expression on the bone marrow mononuclear cells (MNC) in 25 AA patients and 10 normal controls were assayed by TUNEL and FACS, respectively. RESULT: The percentage of CD(34)(+) cells was decreased significantly in AA patients than in normal controls (P < 0.05), and so did the percentage of CD(34)(+)Fas(+) cells (P < 0.05). The percentage of apoptotic cells in AA patients was higher than that of controls (P < 0.01). After the patients were treated with CsA for 2 months, the percentage of apoptotic cells was decreased (P < 0.01). The percentage of CD(34)(+)Fas(+) cells was positively correlated with that of apoptotic cells in AA patients. CONCLUSION: Fas is involved in the apoptosis of CD(34)(+) cells of AA patients. CsA could reduce the percentage of apoptosis of bone marrow cells in AA patients.

Adolescent↗

[Vacuolation effect of Helicobacter pylori on gastric epithelial cells].

This study was designed to observe vacuolation effect of Helicobacter pylori on gastric epithelial cells. The H. pylori isolates derived from patients with peptic ulcer, chronic gastritis and gastric cancer were plated on common selective medium and their vacuolation effects on SGC-7901 cells were compared. The percentage of H. pylori(Tox+) strain in clinical isolates from different gastroduodenal diseases had no significant difference. The toxicity of the H. pylori (Tox+) strain from peptic ulcer was significantly higher than that from chronic gasritis.

Adolescent↗

Inhibition of cardiac tumor necrosis factor-alpha production by calcitonin gene-related peptide-mediated ischemic preconditioning in isolated rat hearts.

Previous investigations have demonstrated that calcitonin gene-related peptide (CGRP) plays an important role in the mediation of ischemic preconditioning in rats. In the present study, we examined signal transduction pathways of CGRP-mediated ischemic preconditioning. Thirty minutes of global ischemia and 40 min of reperfusion caused a dramatic decrease in myocardial function, and a significant increase in the release of cardiac creatine kinase in the coronary effluent and in the content of tumor necrosis factor-alpha (TNF-alpha) in myocardial tissues. However, ischemic preconditioning (three cycles of 5-min ischemia and 5-min reperfusion) or pretreatment with CGRP for 5 min dramatically improved the recovery of cardiac function, and reduced the release of cardiac creatine kinase and the TNF-alpha content. The effect of ischemic preconditioning was abolished by CGRP-(8-37), the selective CGRP receptor antagonist, and by capsaicin, which depletes sensory nerve neurotransmitter content, but was unaltered by treatment with glibenclamide, a blocker of the ATP-sensitive potassium (K(ATP)) channel. The protective effects of exogenous CGRP-induced preconditioning were also not blocked by glibenclamide. These results suggest that the cardioprotective effects afforded by CGRP-mediated ischemic preconditioning are related to inhibition of cardiac TNF-alpha production, but not to activation of the K(ATP) channel.

Animals↗

HCaRG, a novel calcium-regulated gene coding for a nuclear protein, is potentially involved in the regulation of cell proliferation.

Since a negative calcium balance is present in spontaneously hypertensive rats, we searched for the gene(s) involved in this dysregulation. A cDNA library was constructed from the spontaneously hypertensive rat parathyroid gland, which is a key regulator of serum-ionized calcium. From seven overlapping DNA fragments, a 1100-base pair novel cDNA containing an open reading frame of 224 codons was reconstituted. This novel gene, named HCaRG (hypertension-related, calcium-regulated gene), was negatively regulated by extracellular calcium concentration, and its basal mRNA levels were higher in hypertensive animals. The deduced protein showed no transmembrane domain, 67% alpha-helix content, a mutated calcium-binding site (EF-hand motif), four putative "leucine zipper" motifs, and a nuclear receptor-binding domain. At the subcellular level, HCaRG had a nuclear localization. We cloned the human homolog of this gene. Sequence comparison revealed 80% homology between rats and humans at the nucleotide and amino acid sequences. Tissue distribution showed a preponderance in the heart, stomach, jejunum, kidney (tubular fraction), liver, and adrenal gland (mainly in the medulla). HCaRG mRNA was significantly more expressed in adult than in fetal organs, and its levels were decreased in tumors and cancerous cell lines. We observed that after 60-min ischemia followed by reperfusion, HCaRG mRNA declined rapidly in contrast with an increase in c-myc mRNA. Its levels then rose steadily to exceed base line at 48 h of reperfusion. HEK293 cells stably transfected with HCaRG exhibited much lower proliferation, as shown by cell count and [(3)H]thymidine incorporation. Taken together, our results suggest that HCaRG is a nuclear protein potentially involved in the control of cell proliferation.

Adaptor Proteins, Signal Transducing↗

An in vitro transcription system that recapitulates equine infectious anemia virus tat-mediated inhibition of human immunodeficiency virus type 1 Tat activity demonstrates a role for positive transcription elongation factor b and associated proteins in the mechanism of Tat activation.

Equine infectious anemia virus (EIAV) activates transcription via a Tat protein, a TAR element, and the equine elongation factor positive transcription elongation factor b (P-TEFb). In human cells, EIAV Tat (eTat) can inhibit the ability of human immunodeficiency virus type 1 (HIV-1) Tat (hTat) to activate transcription from the HIV-1 long terminal repeat, demonstrating that EIAV Tat can interact nonproductively with human P-TEFb. To study the mechanism of EIAV Tat and HIV-1 Tat activation, we developed an in vitro elongation assay that recapitulates EIAV Tat-mediated inhibition of HIV-1 Tat trans-activation. We found that eTat specifically inhibits activation of elongation by HIV-1 Tat while having no effect on basal transcription elongation. The competitive inhibition of hTat activation was reversed by an activity present in HeLa cell nuclear extracts, most likely a form of P-TEFb. Recombinant P-TEFb (cyclin T1 and CDK9) overcame the inhibition of transcription by eTat but in a nonspecific manner. EIAV Tat affinity chromatography was used to purify the activity present in nuclear extract that was capable of reversing eTat inhibition. We characterized the protein components of this activity, which include cyclin T1, CDK9, Tat-SF1, and at least three unidentified proteins. These data suggest that additional factors are involved in the mechanism of Tat activation.

Binding, Competitive↗

The transcription factor EPAS-1/hypoxia-inducible factor 2alpha plays an important role in vascular remodeling.

We have studied the role of the basic helix-loop-helix-PAS transcription factor EPAS-1/hypoxia-inducible factor 2alpha in vascular development by gene targeting. In ICR/129 Sv outbred background, more than half of the mutants displayed varying degrees of vascular disorganization, typically in the yolk sac, and died in utero between embryonic day (E)9.5 and E13.5. In mutant embryos directly derived from EPAS-1(-/-) embryonic stem cells (hence in 129 Sv background), all embryos developed severe vascular defects both in the yolk sac and embryo proper and died between E9.5 and E12.5. Normal blood vessels were formed by vasculogenesis but they either fused improperly or failed to assemble into larger vessels later during development. Our results suggest that EPAS-1 plays an important role at postvasculogenesis stages and is required for the remodeling of the primary vascular network into a mature hierarchy pattern.

Animals↗

Stress proteins as biomarkers of oxidative stress: effects of antioxidant supplements.

The potential benefits to health of the supply of antioxidants, either through dietary intake or as supplements, is equivocal. There is a need to develop biomarkers that may act as monitors of cellular defense as influenced by antioxidant status. Thirty-two individuals participated in the project and 19 received supplements for 5 weeks in the form of a capsule containing a defined mixture of antioxidants. No change was noted in levels of superoxide dismutase and glutathione peroxidase following antioxidant supplementation. On the other hand, increase in total antioxidant status and decrease in malondialdehyde, protein carbonyl formation, and erythrocyte hemolysis were noted. In lymphocytes isolated from individuals receiving antioxidant supplements and subjected to a heat shock in the presence of the free radical generator 2, 2'-azobis-(2-amidinopropane)-dihydrochloride, enhanced synthesis of heat shock proteins hsp 105, hsp 90, hsp 70, and hsp 40 by contrast with decreased synthesis of heme oxygenase HO-1 (hsp 32) were noted. We conclude that antioxidant status modulates the synthesis of stress proteins.

Adult↗

Neurotoxicity induces cleavage of p35 to p25 by calpain.

Cyclin-dependent kinase 5 (cdk5) and its neuron-specific activator p35 are required for neurite outgrowth and cortical lamination. Proteolytic cleavage of p35 produces p25, which accumulates in the brains of patients with Alzheimer's disease. Conversion of p35 to p25 causes prolonged activation and mislocalization of cdk5. Consequently, the p25/cdk5 kinase hyperphosphorylates tau, disrupts the cytoskeleton and promotes the death (apoptosis) of primary neurons. Here we describe the mechanism of conversion of p35 to p25. In cultured primary cortical neurons, excitotoxins, hypoxic stress and calcium influx induce the production of p25. In fresh brain lysates, addition of calcium can stimulate cleavage of p35 to p25. Specific inhibitors of calpain, a calcium-dependent cysteine protease, effectively inhibit the calcium-induced cleavage of p35. In vitro, calpain directly cleaves p35 to release a fragment with relative molecular mass 25,000. The sequence of the calpain cleavage product corresponds precisely to that of p25. Application of the amyloid beta-peptide A beta(1-42) induces the conversion of p35 to p25 in primary cortical neurons. Furthermore, inhibition of cdk5 or calpain activity reduces cell death in A beta-treated cortical neurons. These observations indicate that cleavage of p35 to p25 by calpain may be involved in the pathogenesis of Alzheimer's disease.

Alzheimer Disease↗

Compromised kidney graft rejection response in Vervet monkeys after withdrawal of immunosuppressants tacrolimus and sirolimus.

BACKGROUND: In nonprimates, organ allografts are often not rejected after withdrawal of immunosuppression. In this study, we examined whether such a phenomenon also occurs in primates. METHODS: Vervet monkeys were transplanted with renal allografts and treated for 60 days with tacrolimus, or tacrolimus plus sirolimus. The drugs were totally withdrawn on day 61. The survival of the monkeys was monitored, and their response to donor- or third party-derived alloantigens was examined in vivo and in vitro. RESULTS: The majority (80-100%) of the grafts survived for at least additional 30 days with no signs of acute rejection. The compromised rejection is donor-specific, because recipient monkeys failed to reject a donor-derived skin graft, but a third-party skin graft was rejected. In vitro mixed lymphocyte reaction and interleukin-2 production in the mixed lymphocyte reaction between the recipients and their donors or between the recipients and a third party had no discernable patterns, and thus did not reflect the in vivo status of the immune system. Although the recipients could not reject the graft acutely after drug withdrawal, the kidney grafts and the donor-derived skin grafts had pathological findings of chronic rejection. CONCLUSIONS: The rejection response of the monkeys to an established graft after withdrawal of immunosuppression is compromised. The compromised rejection is specific and is not due to a permanent alteration of the immune system by the initial drug treatment. The allografts are not inert but have low levels of interaction with the recipient immune system.

Animals↗

Effect of tacrolimus (FK506) and sirolimus (rapamycin) mono- and combination therapy in prolongation of renal allograft survival in the monkey.

BACKGROUND: Our previous studies confirmed that tacrolimus (FK506) and sirolimus [rapamycin (RAPA)], in combination, are not antagonistic but are synergistic in the prolongation of heart and small bowel grafts in the rodent. The aim of this study was to confirm further the synergistic effect of combined FK506 and RAPA in the more clinically relevant model, kidney transplantation in monkeys. METHODS: A total of 60 male Vervet monkeys were randomly assigned to 10 groups (n> or =5). Monkeys with renal allografts were treated with different doses of FK506 and/or RAPA orally for 60 days. Graft survival, body weight, clinical biochemistry determinations, oral glucose tolerance test, trough levels of the two drugs, and histopathology were investigated. RESULTS: Low doses of FK506 (1 or 4 mg/kg) combined with RAPA (0.5 mg/kg) produced synergistic effect in the prolongation of renal graft survival [combination index (CI) = 0.292, 0.565]. There were no additive or synergistic drug-associated toxicities such as hyperglycemia, nephrotoxicity, and hyperlipidemia. There also was no pharmacological antagonism. CONCLUSION: Concomitant therapy of low-dose (drug-optimal) FK506 and RAPA produced a synergistic effect in the prolongation of kidney allograft survival in Vervet monkeys without additive drug-associated toxicities.

Animals↗

An NMR and molecular modeling study of the site-specific binding of histamine by heparin, chemically modified heparin, and heparin-derived oligosaccharides.

The diprotonated form of histamine binds site-specifically to heparin, a highly sulfated 1-->4 linked repeating copolymer comprised predominantly of 2-O-sulfo-alpha-L-iduronic acid (the I ring) and 2-deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl (the A ring). The binding is mediated by electrostatic interactions. The structural features of histamine and heparin, which are required for the site-specific binding, have been identified from the results of (1)H NMR studies of the binding of histamine by six heparin-derived oligosaccharides and four chemically modified heparins and molecular modeling studies. The results indicate that the imidazolium ring of diprotonated histamine is critical for directing site-specific binding, while the ammonium group increases the binding affinity. The imidazolium ring binds within a cleft, with the A ring of an IAI triad at the top of the cleft, and the I rings forming the two sides. The H3 proton of the A ring is in the shielding cone of the imidazolium ring. The carboxylate group of the I-ring at the reducing end of the IAI triad and possibly the sulfamido group of the A-ring are essential for site-specific binding, whereas the 2-O-sulfate group of the I ring and the 6-O-sulfate group of the A ring are not. The results indicate that histamine binds to the IAI triad with the I rings in the (1)C(4) conformation. Also, the configuration of the carboxylate group is critical, as indicated by the absence of site-specific binding of histamine by the related IAG sequence, where G is alpha-D-glucuronic acid. The molecular modeling results indicate that the N1H and N3H protons of the imidazolium ring of site-specifically bound histamine are hydrogen bonded to the carboxylates of the I rings at the nonreducing and reducing ends of the IAI trisaccharide sequence.

Animals↗

P-TEFb kinase recruitment and function at heat shock loci.

P-TEFb, a heterodimer of the kinase Cdk9 and cyclin T, was isolated as a factor that stimulates formation of productive transcription elongation complexes in vitro. Here, we show that P-TEFb is located at >200 distinct sites on Drosophila polytene chromosomes. Upon heat shock, P-TEFb, like the regulatory factor HSF, is rapidly recruited to heat shock loci, and this recruitment is blocked in an HSF mutant. Yet, HSF binding to DNA is not sufficient to recruit P-TEFb in vivo, and HSF and P-TEFb immunostainings within a heat shock locus are not coincident. Insight to the function of P-TEFb is offered by experiments showing that the direct recruitment of a Gal4-binding domain P-TEFb hybrid to an hsp70 promoter in Drosophila cells is sufficient to activate transcription in the absence of heat shock. Analyses of point mutants show this P-TEFb stimulation is dependent on Cdk9 kinase activity and on Cdk9's interaction with cyclin T. These results, coupled with the frequent colocalization of P-TEFb and the hypophosphorylated form of RNA polymerase II (Pol II) found at promoter-pause sites, support a model in which P-TEFb acts to stimulate promoter-paused Pol II to enter into productive elongation.

Animals↗

Substrate recognition through a PDZ domain in tail-specific protease.

Tail-specific protease (Tsp) is a periplasmic enzyme that selectively degrades proteins bearing a nonpolar C-terminus. Its substrate specificity suggests that Tsp may contain a substrate recognition domain, which selectively binds to the nonpolar C-termini of substrate proteins, separate from its catalytic site. In this work, we show that substrate recognition of Tsp is mediated by a PDZ domain, a small protein module that promotes protein-protein interactions by binding to internal or C-terminal sequences of their partner proteins. Partial proteolysis by V8 protease at a single peptide bond immediately N-terminal to the PDZ domain resulted in two distinct and relatively stable fragments and complete loss of catalytic activity. Photoaffinity labeling with a fluorescent nonpolar peptide caused the covalent attachment of the peptide to a single site on the Tsp protein. Systematic deletion mutagenesis of Tsp localized the binding site to amino acids 206-307, a region that completely encompasses the putative PDZ domain (217-301). The isolated PDZ domain (amino acids 206-334) is capable of folding into a well-behaved structure and binds to a nonpolar peptide with a dissociation constant (K(D)) of 1.9 microM, similar to that of the intact Tsp protein. Site-directed mutagenesis of a surface residue at the peptide binding site of the PDZ domain, valine 229, to Glu or Gln resulted in an increase in the K(M) value but had no effect on the k(cat) value. The use of a separate substrate recognition domain such as a PDZ domain may be a general mechanism for achieving selective protein degradation.

Binding Sites↗

Preparation and use of a photoactivatable glucose-6-phosphate analogue.

A benzophenone-containing derivative of glucose-6-phosphate, 6-[(3-([2,3-3H2]-p-benzoyl dihydrocinnamidoylpropyl-1-oxy)phosphoryl]-D-glucopyranose ([3H]BZDC-Glc-6-P) was synthesized and employed to photoaffinity label proteins on intact rat liver microsomes. The use of a non-photoactivatable, UV-transparent desoxy analogue of BZDC, named p-benzyldihydrocinnamoyl (BnDC), is introduced as a general method to achieve competition when hydrophilic ligands are modified with hydrophobic photophores.

Animals↗

Blocking of CD44-hyaluronic acid interaction prolongs rat allograft survival.

BACKGROUND: Lymphocyte activation and infiltration into a transplanted organ is an integral component of the rejection process. Graft infiltration of lymphocytes requires adhesion of leukocytes to the endothelium, diapedesis, and transmigration. One of several proteins involved in this process is CD44, which is known to interact with endothelial hyaluronan (HA). Blockade of cell-matrix and cell-cell interactions have been used extensively for modulation of immune responses and graft rejection. Based on these observations, we evaluated the effects of blocking CD44-HA interactions in a transplantation model. METHODS: We used a low molecular weight hyaluronic acid formulation (LMWHA) for the treatment of rat renal and cardiac allograft recipients. LMWHA was administered intraperitoneally at 0.5-5 mg/kg for 5-10 days after transplantation with or without a subtherapeutic dose of cyclosporine. RESULTS: LMWHA monotherapy prolonged allograft survival significantly, but only for a few days. In combination with low-dose cyclosporine, long-term survival of allografts was observed in some of recipients. CONCLUSION: Further definition of the underlying mechanism of LMWHA therapy may provide a rationale for the development of novel, nontoxic, nonimmunogenic immunotherapies.

Animals↗

Crystal structure and replacement reaction of coordinated water molecules of the heteropoly compounds of sandwich-type tungstoarsenates.

Six new heteropoly compounds in the [M4(H2O)2(As2W15O56)2]16- series (M = CuII, MnII, CoII, NiII, ZnII, CdII), previously unknown, were synthesized and characterized by means of IR, UV-vis, CV, 183W NMR, TG-DSC, and elemental analyses. The synthetic method used in preparing this type of heteropoly compounds was different from that in preparing the corresponding tungstophosphates in that the starting materials were transition metal chlorides in 1.5 times the stoichiometric amount and the required pH value is lower than 2. The crystal structure of Na16[Cu4(H2O)2(As2W15O56)2].47H2O was solved in triclinic, P1 symmetry, with a = 12.721(3) A, b = 24.516(5) A, c = 26.450(5) A, alpha = 89.90(3) degrees, beta = 77.32(3) degrees, gamma = 89.96(3)degrees, V = 8048(3) A3, Z = 2, and R = 0.0966. This anion is isostructural with the previously reported [Cu4(H2O)2(P2W15O56)2]16-, having a rhombic tetrameric cluster Cu4O16 sandwiched by two trivacant Dawson-Wells anions [As2W15O56]12-. The range of the bond lengths of the equatorial Cu-O bonds is 1.83-2.05 A, while that of the axial Cu-O bonds is 2.30-2.39 A. The distortion of the Cu4O16 cluster is smaller in the As species than in the P species. Two copper atoms in the Cu4O16 cluster are coordinated by water molecules. The replacement reactions of the coordinated water molecules of this series of heteropoly compounds in aqueous solutions and in selected organic solvents are also reported here for the first time. The results show that [Fe(CN)6]4-, [Fe(CN)6]3-, H2NCH2CH2NH2, etc., can replace the coordinated water to form its characteristic color in aqueous solutions, while in organic solvents the coordinated water molecules are lost, leaving unshared coordination positions that can be occupied by some organic ligands such as pyridine, lactic acid, and acetone to restore the octahedral coordination of M2+. The crystallographic morphologies of this series of heteropolyanions after phase transfer are dependent on different transition metal ions present in the central M4O16 clusters although the anions are isostructural with each other.

Journal Article↗