Biomedical subjects
Z Chen
Publications and source records attributed to Z Chen.
PCR-based cloning, isolation, and IgE-binding properties of recombinant latex profilin (rHev b 8).
BACKGROUND: Profilin (Hev b 8) in natural rubber latex (NRL) has been assumed to be an important allergen. Since latex profilin has a molecular mass similar to two other latex allergens (Hev b 1 and Hev b 6.03) in the 14-kDa range, it is difficult to obtain sufficient amounts of purified native profilin for investigations and diagnostics. The present study aimed to produce recombinant latex profilin (rHev b 8) and study its IgE-binding reactivity. METHODS: A profilin-specific cDNA encoding the latex profilin from Hevea brasiliensis leaves was synthesized and subcloned, and the rHev b 8 was overexpressed in fusion with the maltose-binding protein (MBP) in E. coli. The IgE-binding reactivity of rHev b 8 was studied by immunoblotting, immunoblot inhibition experiments, and the Pharmacia CAP method, with 25 sera from health-care workers with latex allergy and 17 sera from latex-sensitive spina bifida patients. RESULTS: rHev b 8 was found to have 131 amino acids and a sequence identity of 75% with birch profilin (Bet v 2). Analysis by the CAP system revealed the presence of rHev b 8-specific IgE antibodies in two out of 17 sera from spina bifida patients and in five out of 25 sera (20%) from health-care workers. Two subjects of the latter group with rHev b 8-specific IgE showed negative results in the skin prick tests with tree-pollen extracts and had no IgE to rBet v 2, indicating the presence of IgE-binding epitopes on the Hev b 8-molecule which do not cross-react with birch profilin. Immunoblot inhibition assays using MBP-rHev b 8 as inhibitor confirmed the presence of latex profilin in the NRL extract. IgE binding to the native latex profilin could be completely inhibited by the MBP-rHev b 8. CONCLUSIONS: Latex profilin represents a minor allergen in NRL and may have IgE-binding epitopes different from Bet v 2.
Association of the R485K polymorphism of the factor V gene with poor response to activated protein C and increased risk of coronary artery disease in the Chinese population.
Inherited predisposition to thrombosis contributes to the initiation and progression of coronary artery disease (CAD). The present study was designed to explore the relationship between genetic variation of coagulation factor V and occurrence of CAD. A total of 141 unrelated patients with CAD and 175 healthy controls were analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) for variation detection in all 25 exons of the factor V gene. Among the study subjects, 55 CAD patients and 73 controls were evaluated at random for response to activated protein C (APC) by Coatest APC resistance test. Polymorphisms in exon 4, 10, 13 and 16 of factor V gene were documented [642G-->T(S156), 1628--> A(R485K), 4070A-->G(H1299R) and 5380G A(V1736M), respectively]. The study also identified a novel polymorphism 327A G in exon 2 which did not alter the amino acid residue. Leiden mutation (R506Q) was not detected in any of our 316 subjects. Among the five polymorphisms, the allele frequency of 1628G--> A was significantly different between the CAD patients and the controls (0.36 vs. 0.21, p < 0.05). Subjects homozygous or heterozygous for the A allele of 1628G-->A polymorphism had lower normalized APC ratios than those with the GG genotype in the CAD group (1.16+/-0.13 and 1.18+/-0.23 vs. 1.36+/-0.33, p <0.05) and in the controls, indicating that A(1628) allele was associated with a poor response to APC. We conclude that the 1628G-->A (R485K) polymorphism of factor V is associated with a poor response to APC and increased risk for CAD.
Regulation of humoral immune responses by CD21/CD35.
Before antigen-specific immunity arises, the complement system responds by activation through the classical and/or alternative pathways leading to the covalent deposition of complement fragments. Three models, not mutually exclusive, have been proposed to explain how these complement fragments interact with their receptors, CD21/CD35, to enhance humoral immune responses: i) CD21/CD35 retain and focus antigens for optimal presentation; ii) CD21/CD35 on B cells serve as enhancing co-receptors for B-cell antigen receptor (BCR) signaling; iii) CD21/CD35 regulate B-cell responses, by CD19 aggregation. The coreceptor model led us to predict that CD21/CD3 5 may lower the threshold of BCR affinity to diversify the repertoire of humoral immune responses, but surprisingly, CD21/CD3 5-deficient mice expressing a transgenic BCR with very low affinity (Kalpha approximately =1.2 x 10(5) M(-1)) for the (4-hydroxy-3-nitrophenyl)acetyl hapten generated significant antibody and germinal center responses to even low doses of antigens in alum. The magnitudes of these responses were much below those of normal controls but higher doses of antigens substantially rectified these deficits. Thus, while CD21/CD35 play important roles in humoral immunity, our observations provide little support to the hypothesis that CD21/CD35 promote clonal diversity in immune responses by helping recruit low-affinity B cells.
Arsenic trioxide-induced apoptosis and differentiation are associated respectively with mitochondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia.
Recent studies showed that arsenic trioxide (As2O3) could induce apoptosis and partial differentiation of leukemic promyelocytes. Here, we addressed the possible mechanisms underlying these two different effects. 1.0 microM As2O3-induced apoptosis was associated with condensation of the mitochondrial matrix, disruption of mitochondrial transmembrane potentials (DeltaPsim) and activation of caspase-3 in acute promyelocytic leukemia (APL) cells regardless of their sensitivity to all-trans retinoic acid (ATRA). All these effects were inhibited by dithiothreitol (DTT) and enhanced by buthionine sulfoximine (BSO). Furthermore, BSO could also render HL60 and U937 cells, which had the higher cellular catalase activity, sensitive to As2O3-induced apoptosis. Surprisingly, 1.0 microM As2O3 did not induce the DeltaPsim collapse and apoptosis, while 0.1 microM As2O3 induced partial differentiation of fresh BM cells from a de novo APL patient. In this study, we also showed that 0.2 mM DTT did not block low-dose As2O3-induced NB4 cell differentiation, and 0. 10.5 microM As2O3 did not induce differentiation of ATRA-resistant NB4-derived sublines, which were confirmed by cytomorphology, expression of CD11b, CD33 and CD14 as well as NBT reduction. Another interesting finding was that 0.10.5 microM As2O3 could also induce differentiation-related changes in ATRA-sensitive HL60 cells. However, the differentiation-inducing effect could not be seen in ATRA-resistant HL60 sublines with RARalpha mutation. Moreover, low-dose As2O3 and ATRA yielded similar gene expression profiles in APL cells. These results encouraged us to hypothesize that As2O3 induces APL cell differentiation through direct or indirect activation of retinoic acid receptor-related signaling pathway(s), while DeltaPsim collapse is the common mechanism of As2O3-induced apoptosis.
Gene expression in CD34(+) cells from normal bone marrow and leukemic origins.
INTRODUCTION: To address the molecular regulation of hematopoiesis and the complex mechanism in leukemogenesis, we established the first catalogs of genes expressed in normal bone marrow and leukemia CD34(+) cells. MATERIALS AND METHODS: CD34(+) cell cDNA libraries were constructed using mRNA from adult bone marrow and from a case of acute myeloid leukemia-M5 transformed from myelodysplastic syndrome (MDS-AML). Expressed sequence tags (ESTs) and full-length cDNAs were generated by sequencing and were annotated using bioinformatic tools. RESULTS: From a total of 4142 ESTs obtained from normal bone marrow, 3424 meaningful tags were integrated into 1630 clusters, representing 622 known genes, 522 dbEST entries and 486 novel sequences. Out of 5382 ESTs from MDS-AML, 1985 clusters were produced based on the analysis of 4321 useful ESTs, including 711 known genes, 657 known ESTs and 617 novel sequences. Among 251 transcripts found in both bone marrow and MDS-AML EST datasets and those present in only one dataset, 58 showed statistically significant differences in EST copy numbers between the two tissues (P<0.05). Twenty putative full-length cDNAs for novel genes were also cloned from the MDS-AML library. CONCLUSION: The distinct gene expression patterns in MDS-AML-CD34(+) cells as compared to normal control cells may contribute to the development and/or maintenance of the malignant phenotypes of leukemia cells.
Enantioselective uptake of amino acid with overoxidized polypyrrole colloid templated with L-lactate.
An overoxidized polypyrrole colloid, which can recognise enantiomers of amino acids has been prepared by a newly developed molecular imprinting technique. A polypyrrole colloid, which had been polymerised from a mixture of pyrrole (monomer), polyvinylpyrrolidone (steric stabiliser), peroxodisulfate (oxidant) and L-lactate (dopant), was overoxidized to create a dopant-complementary cavity. The enantioselectivity of the overoxidized colloid was evaluated by comparing the uptake of L-alanine and L-cysteine with that of the respective D-enantiomers. The L/D uptake ratios for these amino acids were about 2, while phenylalanine showed suppressed uptake for both the enantiomers. The absence of phenylalanine uptake can be explained in terms of the molecular size, which is too large to be accommodated by the cavity created by L-lactate. In contrast, a colloid templated with L-phenyllactate took up L-phenylalanine with a higher enantioselectivity of about 7. A colloid templated with L-lactate was applied to surface chirality analysis through enantioselective adsorption on cysteine-modified gold surfaces. Quartz microbalance experiments and scanning electron microscope observation of the gold surface revealed that the colloidal particle has higher affinity to a surface modified with L-cysteine than to one modified with D-cysteine.
Recombinant Hev b 1: large-scale production and immunological characterization.
BACKGROUND: Hev b 1 represents one of the most important allergens in Hevea brasiliensis latex. It is difficult to get an appropriate amount of native Hev b 1 (nHev b 1) for research purposes. OBJECTIVE: The aim of this study was to produce sufficient amounts of Hev b 1 by recombinant methods to prove its suitability for latex allergy diagnostics. METHODS: We isolated total RNA of Hevea brasiliensis leaves and synthesized cDNA by RT PCR. Recombinant Hev b 1 (rHev b 1) as well as three fragments (amino acid residues 29-137, 48-137, 78-137) were subcloned and expressed as fusion proteins with Maltose-binding protein (MBP) in Escherichia coli. The MBP-rHev b 1 fusion protein was examined by RAST with the CAP method, histamine release test and immunoblots with human sera from spina bifida patients as well as from health care workers with latex allergy and monoclonal antibodies. RESULTS: Histamine release test and immunoblots revealed the high allergenicity of the MBP-rHev b 1 construct. By the CAP method, 54 out of 58 serum samples (93%) from latex-sensitized spina bifida patients previously showing immunoglobulin (Ig) E to nHev b 1 exhibited IgE-binding to rHev b 1. Among 71 latex-allergic health care workers tested, 16 (22.5%) had IgE antibodies to rHev b 1. The analysis of the fusion proteins carrying rHev b 1 fragments revealed that the loss of the N-terminal 28 amino acid residues did not affect IgE-binding. In contrast, the lack of the first 47 amino acid residues led to decreased IgE-binding reactivity in two out of four sera tested, whereas the absence of the N-terminal 77 residues abolished IgE-binding in these two sera. CONCLUSION: The MBP-rHev b 1 fusion protein exhibits a corresponding IgE-binding reactivity to nHev b 1 and may therefore substitute natural Hev b 1 for both in vitro diagnostics and research purposes.
Identification of genes encoding receptor-like protein kinases as possible targets of pathogen- and salicylic acid-induced WRKY DNA-binding proteins in Arabidopsis.
To understand how plant host genes are regulated during the activation of plant defence responses, we are studying a group of pathogen- and salicylic acid (SA)-induced DNA-binding proteins containing the novel WRKY domain. To identify downstream target genes of these WRKY proteins, we have searched the Arabidopsis genome and identified four closely linked genes on chromosome IV that contain an unusually large number of the W-box sequences [(T)TGAC(C/T)] recognized by WRKY proteins within a few hundred base pairs upstream of their coding regions. All four genes encode proteins characteristic of receptor-like protein kinases (RLK), each consisting of an N-terminal signal sequence, an extracellular receptor domain, a single transmembrane domain and a C-terminal cytoplasmic serine/threonine protein kinase domain. All four RLK genes were induced by treatment with SA or infection by a bacterial pathogen. Studies with one of the RLK genes (RLK4) indicated that a cluster of W-box elements in its promoter region were recognized by both purified WRKY proteins and SA-induced W-box binding activities from SA-treated Arabidopsis plants. Further analysis using the RLK4 gene promoter fused to a reporter gene in transgenic Arabidopsis indicated that the consensus WRKY protein-binding sites in the RLK4 gene promoter were important for the inducible expression of the reporter gene. These results indicate that pathogen- and SA-induced W-box binding proteins regulate not only genes encoding defence proteins with direct or indirect anti-microbial activities, but also genes encoding proteins with regulatory functions.
Effects of androgen and ageing on gene expression of vasoactive intestinal polypeptide in rat corpus cavernosum.
OBJECTIVE: To clarify the effects of androgens (testosterone and dihydrotestosterone, DHT) and ageing on gene expression of vasoactive intestinal polypeptide (VIP), assessed as the expression of VIP mRNA, in rat corpus cavernosum. Materials and methods The study comprised 160 male Sprague Dawley rats divided into group A (56 rats, 5 weeks old), group B (50 rats, 10 weeks old) and group C (54 rats, 58 weeks old). Groups A-C were subdivided, respectively, into subgroups 1 (intact controls), 2 (castrated), 3 (castrated but given testosterone undecanoate 25 mg/kg per month by intramuscular injection), 4 (castrated with testosterone undecanoate 50 mg/kg per month) and 5 (treated with finasteride 4.5 mg/kg per day, orally). At 4 and 10 weeks after these treatments half the rats were killed. Serum samples were taken for the measurement of total and free testosterone and DHT, using a radioimmunoassay. Penile samples (corpus cavernosum) were frozen in liquid nitrogen and stored at -80 degrees C. VIP mRNA was estimated using a semi-quantitative reverse-transcription polymerase chain reaction. RESULTS: There was no significant difference in VIP mRNA in rat corpus cavernosum between intact control and castrated subgroups, or subgroups treated with finasteride, in groups A-C, including both the 4- and 10-week old animals (P > 0.05). Penile VIP mRNA was unchanged at any dose of testosterone in the castrated subgroups in all groups (P > 0.05). There was no significant relationship between penile VIP mRNA and ageing (P > 0.05). CONCLUSIONS: The gene expression of VIP in rat corpus cavernosum is independent of androgens (testosterone and DHT) and ageing. Androgens probably induce penile erection by pathways other than VIP; ageing may have little effect on penile VIP mRNA.
Inhibition of transcription factor nuclear factor-kappaB by a mutant inhibitor-kappaBalpha attenuates resistance of human head and neck squamous cell carcinoma to TNF-alpha caspase-mediated cell death.
Tumour necrosis factor-alpha (TNF-alpha) is a cytokine that can induce cell death of different cancers via a cellular cascade of proteases, the caspases. However, TNF-alpha has been detected in tumour and serum of patients with head and neck squamous cell carcinoma (HNSCC), and tumour cell lines derived from this environment often exhibit resistance to TNF-alpha-induced cell death. Cell death mediated by TNF-alpha and caspases may be inhibited by cytoprotective genes regulated by transcription factor nuclear factor-kappaB (NF-kappaB). We recently showed that NF-kappaB is constitutively activated in HNSCC, and that inhibition of NF-kappaB by expression of a nondegradable mutant inhibitor of NF-kappaB, IkappaBalphaM, markedly decreased survival and growth of HNSCC cells in vivo. In the present study, we examined the TNF-alpha sensitivity and response of HNSCC with constitutively active NF-kappaB, and of HNSCC cells in which NF-kappaB is inhibited by stable expression of a dominant negative mutant inhibitor, IkappaBalphaM. Human lines UM-SCC-9, 11B and 38, previously shown to exhibit constitutive activation of NF-kappaB, were found to be highly resistant to growth inhibition by up to 10(4)U/ml of TNF-alpha in 5 day MTT assay. These TNF-alpha resistant HNSCC lines expressed TNF receptor I, and exhibited constitutive and TNF-alpha-inducible activation of NF-kappaB as demonstrated by nuclear localization of NF-kappaB p65 by immunohistochemistry. UM-SCC-9 I11 cells which stably expressed an inhibitor of NF-kappaB, IkappaBalpham, were susceptible to TNF-alpha-induced growth inhibition. DNA cell cycle analysis revealed that TNF-alpha induced growth inhibition was associated with accumulation of cells with sub-G0/G1 DNA content. Cell death was demonstrated by trypan blue staining, and was blocked by caspase inhibitor. We conclude that HNSCC that exhibit constitutive and TNF-alpha-inducible activation of transcription factor NF-kappaB are resistant to TNF-alpha, and that inhibition of NF-kappaB sensitizes HNSCC to TNF-alpha caspase-mediated cytotoxicity. The demonstration of the role of activation of NF-kappaB in resistance of HNSCC to TNF-alpha may be helpful in the identification of potential targets for pharmacological, molecular and immune therapy of HNSCC.
DNA of nonhuman primates harbors hepatitis C-virus-specific sequences of its 5'-non-coding region (5'-NCR).
The DNA from PBMCs of both hepatitis C virus (HCV)-positive patients and healthy HCV-negative human individuals tested thus far contains essential parts--up to 272/341 nucleotides--of the HCV 5'-non-coding region (5'-NCR). These findings bring up the question of the possible evolutionary background of these sequences. Therefore, using the same methodology, we looked for the same sequences in animals closely related to man, i.e., in nonhuman primates (two chimpanzees, one orang-utan, one Debrazza monkey, two New World monkey species and a prosimian). The DNA from PBMCs of the studied animals belonging to nonhuman primates contains essential parts--up to 272/341 nucleotides--of the HCV 5'-non-coding region (5'-NCR). A common sequence of 82 nucleotides is contained in the DNA of all the tested animals but only the chimpanzee's DNA harbors the same, longer sequence region of 272/341 nucleotides of the 5'-NCR found in human DNA. The results may provide a clue as to the possible origin of parts of the IRES containing sequence area of the HCV.
Antiplatelet and antithrombotic effects of the diterpene spiramine Q from Spiraea japonica var. incisa.
Spiramine Q, a diterpene, was isolated from a Chinese herbal plant Spiraea japonica var. incisa Yu. Born's and Wan HY's methods were used to investigate effects of spiramine Q on rabbit platelet aggregation and serotonin release, respectively. Its antithrombotic effect in mice was also evaluated by Myers' method. Spiramine Q selectively inhibited arachidonic acid-induced platelet aggregation in vitro or ex vivo, and decreased serotonin secretion from rabbit platelets. Spiramine Q (5 mg/kg) decreased the mouse mortality caused by injection of 80 mg/kg arachidonic acid in the tail vein. The results suggested that spiramine Q showed potent antiplatelet and antithrombotic activites.
Extrinsic allergic alveolitis caused by cabreuva wood dust.
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The Pseudomonas syringae avrRpt2 gene product promotes pathogen virulence from inside plant cells.
Several bacterial avr genes have been shown to contribute to virulence on susceptible plants lacking the corresponding resistance (R) gene. The mechanisms by which avr genes promote parasitism and disease, however, are not well understood. We investigated the role of the Pseudomonas syringae pv. tomato avrRpt2 gene in pathogenesis by studying the interaction of P. syringae pv. tomato strain PstDC3000 expressing avrRpt2 with several Arabidopsis thaliana lines lacking the corresponding R gene, RPS2. We found that PstDC3000 expressing avrRpt2 grew to significantly higher levels and often resulted in the formation of more severe disease symptoms in ecotype No-0 plants carrying a mutant RPS2 allele, as well as in two Col-0 mutant lines, cpr5 rps2 and coil rps2, that exhibit enhanced resistance. We also generated transgenic A. thaliana lines expressing avrRpt2 and demonstrated, by using several different assays, that expression of avrRpt2 within the plant also promotes virulence of PstDC3000. Thus, AvrRpt2 appears to promote pathogen virulence from within the plant cell.
Harpin-induced hypersensitive cell death is associated with altered mitochondrial functions in tobacco cells.
Mitochondria play important roles in animal apoptosis and are implicated in salicylic acid (SA)-induced plant resistance to viral pathogens. In a previous study, we demonstrated that SA induces rapid inhibition of mitochondrial electron transport and oxidative phosphorylation in tobacco cells. In the present study, we report that plant programmed cell death induced during pathogen elicitor-induced hypersensitive response (HR) is also associated with altered mitochondrial functions. Harpin, an HR elicitor produced by Erwinia amylovora, induced inhibition of ATP synthesis in tobacco cell cultures. Inhibition of ATP synthesis occurred almost immediately after incubation with harpin and preceded hypersensitive cell death induced by the elicitor. Diphenylene iodonium, an inhibitor of the oxidative burst, did not block harpin-induced inhibition of ATP synthesis or cell death, suggesting that oxidative burst was not the direct cause for these two harpin-induced processes. Unlike SA, harpin had no significant effect on total respiratory O2 uptake of treated cells. However, respiration of harpin-treated tobacco cells became very sensitive to the alternative oxidase inhibitors salicyl-hydroxamic acid and n-propyl gallate. Thus, harpin treatment resulted in reduced capacity of mitochondrial cytochrome pathway electron transport, which could lead to the observed inhibition of ATP synthesis. Given the recently demonstrated roles of mitochondria in apoptosis, this rapid inhibition of mitochondrial functions may play a role in harpin-induced hypersensitive cell death.
Cyclooxygenase regulates human oropharyngeal carcinomas via the proinflammatory cytokine IL-6: a general role for inflammation?
High levels of prostaglandins are produced in human oropharyngeal carcinoma (OPC). Five human OPC cell lines tested expressed both isoforms of cyclooxygenases (COX). The pan-COX inhibitor ketorolac continuously and significantly decreased PGE(2) production and IL-6 and IL-8 levels in all OPC cell lines tested, but did not affect IL-1alpha, GM-CSF levels, or in vitro tumor cell growth. In contrast, ketorolac reduced OPC growth in vivo. The OPC cell lines used express the IL-6 receptor, and IL-6 stimulation of these cells causes transduction to occur via STAT3 pathway activation. Coincubation with OPC cell lines with conditioned medium from a TPA-exposed HL-60 cells stimulated growth proportional to the IL-6 levels measured in the conditioned medium. This growth effect was specifically inhibited by anti-IL-6 antibody. These results are consistent with cytokine products of inflammatory cells having paracrine growth effects on OPC. If chronic inflammation plays a role in promoting the development of OPC, this mechanism may also apply to other epithelial tumor systems modulated by COX activity.
A radiation hybrid map of the cat genome: implications for comparative mapping.
Ordered gene maps of mammalian species are becoming increasingly valued in assigning gene variants to function in human and animal models, as well as recapitulating the natural history of genome organization. To extend this power to the domestic cat, a radiation hybrid (RH) map of the cat was constructed integrating 424 Type I-coding genes with 176 microsatellite markers, providing coverage over all 20 feline chromosomes. Alignment of parallel RH maps of human and cat reveal 100 conserved segments ordered (CSOs) between the species, nearly three times the number observed with reciprocal chromosome painting analyses. The observed number is equivalent to theoretical predictions of the number of conserved segments to be found between cat and human, implying that 300-400 Type I gene markers is sufficient to reveal nearly all conserved segments for species that exhibit the most frequently observed "slow" rate of genome reorganization. The cat-human RH map comparisons provide a new genomic tool for comparative gene mapping in the cat and related Felidae, and provide confirmation that the cat genome organization is remarkably conserved compared with human. These data demonstrate that ordered RH-based gene maps provide the most precise assessment of comparing genomes, short of contig construction or full-sequence determination.