Biomedical subjects
Publications and source records attributed to M Zou.
Human interleukin-17 (hIL-17) gene without the signal sequence was isolated from activated peripheral blood lymphocytes by RT-PCR, then highly expressed in the yeast Pichia pastoris in the form of the glycosylated monomer. The monomer of rhIL-17 stimulated mouse fibroblast 3T3 cells to secrete IL-6 and was specifically bound to its receptors on 3T3 cells.
Cyclin D1 plays a key role in the regulation of the G1/S transition through the cell cycle. Deregulation of cyclin D1, most often leading to overexpression of the gene, has been reported in many tumor types. It has been suggested that cyclin D1 overexpression could be an alternative mechanism for pRb inactivation. We have previously found Rb gene mutations in 55% of malignant thyroid tumors. In the present study, we examined the cyclin D1 gene expression and amplification in 24 tumor samples (two of them are benign goiters) randomly selected from the same series of thyroid tumors, to see whether cyclin D1 overexpression is present in those specimens without Rb gene mutations. We found a four- to fivefold increase in cyclin D1 expression in 7 of 22 thyroid carcinomas as compared with that in benign nodular goiters. Six of them were found in carcinomas without Rb gene mutations. Among the remaining 15 thyroid carcinoma samples, 11 were found previously to have Rb gene mutations. The association between increased cyclin D1 expression and absence of Rb mutation is statistically significant (p < 0.05). We found no evidence of the cyclin D1 gene amplification or rearrangement to account for such an increase in cyclin D1 expression. We conclude that cyclin D1 overexpression may be relevant to thyroid carcinogenesis. Two mechanisms may be involved in the inactivation of pRb: one is through Rb gene mutations, and the other is by cyclin D1 overexpression.
To increase the sensitivity and specificity and decrease the cost on determining the isoenzymes of creatine kinase (CK), including CK-MM, CK-MB, and CK-BB, we used the electrophoresis bath with different buffers to isolate the isoenzymes and used the fluorescent scanner to determine the content of each isoenzyme. We found that the concordance appeared in sensitivity and specificity in comparison with the MOPS electrophoretic system. There was a positive correlation between our method and MOPS, and the cost was much less than that of the MOPS.
OBJECTIVE: Humulus scandens pollen (HSP) has been proved to be the one of the most important causative factors of autumnal hay fever in China. We established an enzyme immunoassay for specific IgE against HSP by using monoclonal anti-IgE antibody to detect sIgE in 143 patients with autumnal hay fever and 30 healthy volunteers. METHODS: An indirect method of ELISA was developed. The optimal conditions for the test were: coating the allergen at 4 degrees C overnight with a concentration of 12 micrograms/ml; the test serum was diluted 1:16 and incubated with allergen overnight at 37 degrees C; the most suitable dilution and incubative time of the peroxidase-conjugated mouse anti-human IgE antibody were 1:3,000 and 2 hours respectively. RESULTS: The patients' serum level of sIgE was significantly higher than controls (P < 0.001) and correlated significantly with the degree of skin test reaction (P < 0.01). The concordance between the results of skin test and ELISA was 74%-83%. The specificity of the assay was confirmed by heat inactivation and multiple absorption experiments. The coefficients of variation for the intraassay and interassay reproducibility ranged from 1.20%-8.30% and 8.63%-9.22% respectively. CONCLUSIONS: Determination of specific HSP IgE with ELISA assay shows good specificity, sensitivity and reproducibility. It favours clinical practice due to its lower cost.
In this paper, the determination of trace lead in foods by optical temperature control GFAAS was studied. The method was certified by the analysis of the national standard reference materials.
The determination of trace mercury using the self-developed circulating enrichment apparatus with cold vapour atomic absorption spectrometry was investigated. Various experimental conditions were discussed and optimized. The effect of interferences was examined. The developed method is sensitive, accurate, simple and available and has already been used in the analysis of various kinds of food samples and national standard reference materials, and the results obtained were satisfactory.
We have applied site-directed mutagenesis to our Escherichia coli hemoglobin expression plasmid and constructed five recombinant mutant hemoglobins (r Hbs): r Hb(alpha20His-->Gln or alpha:H20Q); r Hb(alpha:H50Q); r Hb(alpha:H72Q); r Hb(alpha:H89Q); and r Hb(alpha:H112Q). We have constructed these r Hbs to help us assess the contribution of the surface histidyl residues in the alpha-chain to the alkaline Bohr effect of human normal adult hemoglobin (Hb A). In our laboratory, we have monitored the variation of proton nuclear magnetic resonances arising from the C2 protons of the histidyl residues of Hb A as a function of pH and buffer conditions. Several of these resonances have been assigned to the individual histidyl residues on the surface of the hemoglobin molecule using naturally occurring mutant hemoglobins and chemically modified hemoglobins. In the present work, we have identified the C2 proton resonances of five surface histidyl residues of the alpha-chain, alpha20, alpha50, alpha72, alpha89, and alpha112, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance spectra of Hb A with those of the r Hbs. For the assignment of the C2 proton resonances of alpha20His and alpha112His, we have used combinations of mutations to compensate for the spectral perturbations resulting from the single mutations, which obscure the resonance assignment. On the basis of the new findings, in solvent containing 0.1 M chloride, the overall contributions from surface histidyl residues of both the alpha- and beta-chain and from other previously identified alkaline Bohr groups account for approximately 75% of the observed Bohr effect at pH 7.3 (the maximum Bohr effect under the prescribed solvent conditions). Our results show that some histidyl residues contribute to the Bohr effect and some oppose the net Bohr effect. In some cases, the addition of anions can diminish or reverse the contributions of specific histidyl residues to the overall Bohr effect. Thus, the Bohr effect, a heterotropic effect, depends on the intricate arrangement and interactions of all hydrogen and anion binding sites in the hemoglobin molecule. It is an excellent example of global electrostatic effects in proteins.
Transcellular biosynthesis of thromboxane (Tx) A2 between vascular smooth muscle cells (SMC) and platelets has been investigated by using 14C-arachidonic acid (AA) radiolabeled rat SMC (or platelets) and the fate of the label in phospholipids and eicosanoid fractions was studied using radioimmunoassay (RIA) and thin-layer chromatography (TLC). Stimulation of SMC with interleukin-1 beta (IL-1 beta) resulted in production of cyclooxygenase metabolites (e.g. 6-keto-PGF1 alpha, PGE2, PGF2 alpha, PGD2), 15-, 11-, 5-HETE, and free AA1 with a coincident decline of phosphatidylcholine (PC) in SMC. IL-1 beta did not induce TXB2 production, a stable metabolite of TXA2 measured by TLC and radioimmunoassay, either in human platelets from 0.01-100 U/ml for 1 h or in SMC for 24 h. However, human platelets converted exogenous PGH2 to TXA2 despite cyclooxygenase inhibition or PGH2 receptor blockade. Furthermore, TXB2 was produced in large quantities during co-incubation of IL-1 beta-stimulated SMC with human platelets for 30 min in concert with a significant decrease of 6-keto-PGF1 alpha and eicosanoids (PGE2, PGF2 alpha and PGD2) compared with control (P < 0.01). Pretreatment of SMC with cycloheximide and actinomycin not only inhibited IL-1 beta-induced eicosanoid synthesis and phospholipid breakdown but also diminished TXB2 production when co-incubated with platelets. These data suggest that a cell-cell interaction, i.e. platelet utilizing SMC-derived endoperoxides for its TXA2 production, might cause an excess thromboxane A2 synthesis.
The pleiotropic cytokine interleukin-6(IL-6) triggers the formation of a high affinity receptor complex constituted by the ligand-binding subunit IL-6 receptor alpha (IL-6R alpha) and the signal transducer gp130. To construct the antagonist of IL-6, a DNA segment encoding the soluble human IL-6R alpha (shIL-6R alpha) mutant with two substitutions C277D/H280I was generated by overlap extension polymerase chain reaction. The DNA segment was then subcloned into vector pET-3b and expressed in E.coli at a high level. The refolded and purified recombinant protein, which was designated as DM650, exhibited an increased IL-6-binding capability by 8-fold. DM650 antagonized IL-6 perfectly, resulting in the growth-inhibition of human myeloma AF-10 cells. DNA fragmentation assay proved that the growth of AF-10 cells was inhibited through the induction of apoptosis suppressed by IL-6.
Internal deletion K128-Q175 of the human interleukin-6 (hIL-6) has been generated at the cDNA level. With pBV220 as expressing vector, the recombinant pBV*-DIL-6 encoding the deletion mutant (12 kD) of hIL-6 has been constructed. The resulted recombinant plasmids were then used to transform E. coli strain HB101, and the expression in the PLPR promoter system, which is temperature-regulatable, was achieved. After purification and renaturation, the biological activity of the expressed product, designated as DM120, was measured by MTT method in an IL-6-dependent cell line 7TD1. The results show that the amino acid residues of IL-6 128 to 175 are crucial for IL-6 activity. Receptor binding assay in vitro indicates that the entire region is not involved in forming the receptor binding surface.
A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha-globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma-globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.
Vascular tone critically depends on the endothelial release of nitric oxide and prostacyclin. Superoxide anions counteract these relaxations by trapping nitric oxide under formation of peroxynitrite. As we have recently reported, peroxynitrite is able to inhibit prostacyclin formation in aortic microsomes (Zou et al., 1996). Here we show that peroxynitrite also blocks purified prostacyclin synthase with an IC50 value of about 50 nM and with a similar sensitivity also inhibits the enzyme activity in the EaHy 926 endothelial cell line. Thromboxane synthase, having the same heme-thiolate (P450) structure and a closely-related mechanism was unaffected by peroxynitrite. Anti-nitrotyrosine antibodies reacted positive by a Western blot after treatment of the purified enzyme with 1 microM peroxynitrite. Tetranitromethane also inhibited the enzyme activity which, like the inhibition by peroxynitrite, could be partially prevented in the presence of the substrate analog U46619. The simultaneous generation of superoxide and nitric oxide proved to be as efficient as a bolus of peroxynitrite which supports a possible inactivation of prostacyclin synthase under in vivo conditions. This substantiates an often suggested crucial role of superoxide in the pathophysiology of the cardiovascular system.
According to the X-ray crystallographic results from human deoxyhemoglobin, beta 99Asp at the alpha 1 Beta 2 interface forms hydrogen bonds with alpha 42Tyr and alpha 97Asn. To clarify the structural and functional roles of the hydrogen bond between alpha 97Asn and beta 99Asp, we have engineered a recombinant hemoglobin in which alpha 97Asn is replaced by Ala, and have investigated its oxygen-binding properties, and have used proton nuclear magnetic resonance spectroscopy to determine the structural consequences of the mutation. Recombinant Hb (alpha 97Asn-->Ala) shows a milder alteration of functional properties compared to the severely impaired beta 99 mutants of the human abnormal hemoglobins. The addition of inositol hexaphosphate, an allosteric effector, causes recovery of the functional properties of recombinant Hb (alpha 97 Asn-->Ala) almost to the level of human normal adult hemoglobin without this allosteric effector. r Hb (alpha 97 Asn-->Ala) shows very similar tertiary structure around the heme pockets and quaternary structure in the alpha 1 beta 2 interface compared to those of human normal adult hemoglobin. The proton nuclear magnetic resonance spectrum of the deoxy form of this recombinant hemoglobin shows the existence of an altered hydrogen bond which is believed to be between alpha 42Tyr and beta 99Asp at the alpha 1 beta 2 interface. Thus, the present results suggest that the intersubunit hydrogen bond between alpha 97 Asn and beta 99Asp at the alpha 1 beta 2 interface is not as crucial as the one between alpha 42Tyr and beta 99Asp in the deoxy quaternary structure. Preliminary molecular dynamics simulations have been used to calculate the contributions of specific interactions of several amino acid residues in r Hb (alpha 97Asn-->Ala) to the free energy of cooperativity of this recombinant hemoglobin. The results of these calculations are consistent with the experimental results.
Eukaryotic cell cycle progression is controlled by a host of cyclin/cyclin-dependent kinases (Cdks), that are themselves regulated by multiple factors, including a group of small cyclin-Cdk inhibitor proteins (p15, p16, p21 and p27). The involvement of Cdk inhibitors in carcinogenesis has been demonstrated by the studies of p16. p53 is frequently mutated in thyroid carcinomas and p21/Waf1 is a downstream effector of p53. It is conceivable that genetic defects of genes downstream in the p53 pathway could also be oncogenic. We, therefore, examined a series of 57 thyroid tumour specimens (eight follicular adenomas and 49 carcinomas) for deletion and point mutation of the p21/Waf1 gene. Three different kinds of deletions ranging from 349 to 450 bp were detected in five papillary carcinoma specimens by reverse transcription-polymerase chain reaction (RT-PCR). All the deletions were involved in the second exon of the p21/Waf1 gene. RT-PCR single strand conformational polymorphism (SSCP) analysis of remaining samples failed to reveal any point mutations in the coding region of the gene, except for a polymorphism at codon 31 (Ser to Arg). Genomic Southern blot analysis did not demonstrate any gene deletion or rearrangement in these samples, indicating abnormal RNA splicing may be involved. Analysis of intron-exon boundary and the coding region of the second exon did not reveal any mutation except for a point mutation (C to G) located 16 bp downstream from the splice donor site of the second intron in three out of five samples with p21/Waf1 deletions. Whether the mutation plays any role in aberrant RNA splicing remains to be determined. Among the five samples with p21/Waf1 gene deletions, none of them simultaneously carried a p53 or retinoblastoma (Rb) gene mutation. No p21/Waf1 abnormality was found in the benign adenomas. Thus, 12.5% (5/40) of thyroid papillary carcinoma specimens harboured p21/Waf1 gene deletions. Our data suggest that p21/Waf1 gene deletion is involved in thyroid carcinogenesis and may play an important role in thyroid cell transformation.
BACKGROUND: The authors previously found p53 mutations in 24% of malignant thyroid tumors, representing a wide stating spectrum. Overexpression of MDM2, most often due to gene amplification, has been suggested to be an additional mechanism for abrogation of the p53 function. In the current study, MDM2 gene expression and amplification were examined in a randomly selected subset of these tumors to explore the possibility that wild-type p53 may be inactivated by complexing with MDM2 in specimens without p53 mutations. METHODS: MDM2 gene expression and amplification were studied by Northern and Southern blot analysis, respectively. Twenty-two thyroid tumors were included: 16 papillary carcinomas, 1 follicular carcinoma, 3 anaplastic carcinomas, and 2 multinodular goiters (adenomatous goiters). RESULTS: A two- to threefold increase in MDM2 expression in 4 of 20 thyroid carcinomas was found. It was noteworthy that all of these four samples harbored p53 mutations. The association between increased MDM2 expression and p53 mutation was statistically significant (P < 0.005). No evidence of MDM2 gene amplification or rearrangement accounting for such an increase in MDM2 expression was found. CONCLUSIONS: Genetic and/or environmental factors contributing to random p53 mutations also may cause increased MDM2 expression. Given the moderate increase in MDM2 expression without associated genetic alterations such as gene amplification and rearrangement, MDM2 may not play any significant role in the development and progression of thyroid carcinoma.
The molecular mechanisms of tumor invasion and metastasis are yet to be fully elucidated. A potential tumor-metastasis-suppressor gene nm23 has been described in certain rodent and human tumors. In the present study, we examined the potential anti-invasive and anti-metastatic effect of nm23 gene in B16F10 cells, a malignant murine melanoma cell line. Transfection of nm23 gene into B16F10 melanoma cells resulted in significant suppression of the invasiveness and metastatic ability of melanoma cells and significantly enhanced the survival of tumor-bearing mice. B16F10 melanoma cells transfected with nm23 produced significantly less soluble ICAM-I and were more susceptible to LAK-cell-mediated cytotoxicity. Co-culture of B16F10 melanoma cells with IL-2 had no effect on nm23 expression, whereas treatment with PGE2, TNF-alpha and IFN-gamma resulted in down-regulation of nm23 expression. Concomitantly, in vivo treatment with TNF-alpha or IFN-gamma in experimental mice increased pulmonary metastases and lowered the overall survival period, as compared with IL-2 treatment alone. These results provide evidence that nm23, in addition to its anti-metastatic function, could also be involved in modulating tumor-target-structure expression, in down-regulating invasive potential and in production of soluble intracellular adhesion molecules. The down-regulation of nm23 by TNF-alpha, IFN-gamma and particularly by PGE2 warrants re-examination of current immunotherapeutic protocols and of the role played by PGE2 in tumor progression.
Human granulocyte-macrophage stimulating factor (hGM-CSF) is an important hematopoietic growth factor. hGM-CSF is able to stimulate hematopoietic progenitors to produce granulocytes, macrophages and eosinophils. hGM-CSF also shows the potential to treat myeloid leukemias. In order to prepare enough hGM-CSF, a genetically engineered strain E. coli HB101/pZW.GM47 was constructed by DNA recombination technologies in the present study, and characterized by restriction analysis, DNA sequencing, SDS-PAGE, Western blotting and bioassays. The strain has been shown to produce a 14kDa mature hGM-CSF protein with expression level higher than 40% and a specific activity of 5.0 x 10(7) u/mg which shows a good potential for clinical applications.
Since 1989 twenty-one thin flaps with preserved subdermal vascular network of the neck, shoulder or pectoral region have been used in 18 cases with severe scar contracture of the neck. The size of the flaps ranged from 10 cm x 2 cm to 20 cm x 8 cm and the pedicles were 3.5 to 4 cm in width. Our results proved the reliability of this method. The blood supply of the flap, the design and indications of the operation are discussed.