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M Zou

Publications and source records attributed to M Zou.

53 records · Page 3Linked to original sources
PubMed1995

Expression of human interleukin-11 cDNA in E. coli.

A 551-bp hIL-11 gene fragment that includes no nucleotide sequences encoding signal polypeptide and the initial 8 amino acids of the mature protein was cloned into a high-level expression vector pEx31B of E. coli. The authors identified the recombinant plasmid, designated pEx31-IL11, by restriction endonucleases digestion and DNA sequencing. The resulting recombinant plasmids were then used to transform E. coli strain HB101, and expression in the PL promoter system, which is temperature-regulated, was achieved. The expressed fusion protein amounts to 50% of total bacterial proteins. The hIL-11 protein expressed in E. coli was fused to the N-terminal 99 amino acids of the MS2 polymerase to form the inclusion body. These recombinant proteins can be purified to about 80% by extracting inclusion body with urea. One IL-6-dependent cell line 7 TD1 was used for bioassay. The recombinant hIL-11 protein was preliminarily purified and renatured to a specific activity of 10(5)U/mg, even in the presence of an excess of a neutralizing anti-IL-6 antibody.

DNA, Complementary↗
PubMed1995

Genetics of follicular thyroid cancer.

Cancer is a multistep phenomenon, and multiple cellular genetic lesions are involved in the emergence of the malignant neoplasm. Several early events, including ras mutations in follicular thyroid carcinoma and RET gene rearrangement in papillary tumors, have been implicated in the neoplastic transformation of thyrocytes. The stepwise array of mutations or deletions described to date in candidate oncogenes and tumor suppressor genes that are necessary for the advance to malignancy and for tumor progression await further investigation. The identification of additional, thyroid-cancer-specific lesions in these pathways is being further studied also.

Adenocarcinoma, Follicular↗
PubMed1995

Purification of recombinant human granulocyte-macrophage colony stimulating factor expressed in Escherichia coli.

Recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) was expressed as inclusion bodies (IB) in E. coli. A simple and effective protocol has been worked out for the purification. IB collected after the breakage of bacteria through sonication were subjected to repeated washing followed by solubilization in TE buffer (50 mmol/L of Tris.HCl, 1 mmol/L of EDTA, pH 8.3) containing 8 mol/L urea and 10 mmol/L DL-dithiothreitol. By means of Sephacryl-200 HR, refolding, and Q Sepharose Fast Flow, rhGM-CSF was obtained with a purity of 99%. The total protein recovery was 10% and specific activity of rhGM-CSF was 1 x 10(7) u/mg. The sequence of N-terminal 16 amino acid residues of purified rhGM-CSF was determined and found to be identical to the native protein. This study provided useful parameters for mass production of rhGM-CSF.

Amino Acid Sequence↗
PubMed1995

Cloning, expression and purification of the ligand-binding region of human IL-6R in E. coli and its preliminary functional identification.

The ligand-binding region of human IL-6R is taken as the target gene fragment to be cloned and expressed. With pET-3b as expressing vector, two recombinants pET-6R(B) and pET-6R(B)4 have been constructed encoding the ligand-binding region (28 kD) of hIL-6R and its dimmer (53 kD), respectively. After induction with IPTG, they produced two proteins rIL6R-28 of 28 kD and rIL6R-53 of 53 kD amounting to 50% and 30% of total bacteria proteins, respectively. The expressed products were mainly recovered as inclusion bodies. After purification and renaturation, both of them were capable of augmenting the growth-stimulating effect of IL-6 on 7TD1 cells, an IL-6 dependent cell line. The result of ELISA also revealed that both rIL6R-28 and rIL6R-53 had the obvious ligand-binding activity.

Antigens, CD↗
PubMed1994

Restoring allosterism with compensatory mutations in hemoglobin.

Abnormal human hemoglobins (HBs) with amino acid substitutions in the alpha 1 beta 2 interface have very high oxygen affinity and greatly reduced cooperativity in O2 binding compared to normal human Hb. In such abnormal Hbs with mutations at position beta 99, the intersubunit hydrogen bonds between Asp-beta 99 and Tyr-alpha 42 and between Asp-beta 99 and Asn-alpha 97 are broken, thus destabilizing the deoxyquaternary structure of these Hbs. A molecular dynamics method has been used to design compensatory amino acid substitutions in these Hbs that can restore their allosteric properties. We have designed a compensatory mutation in a naturally occurring mutant Hb, Hb Kempsey (Asp-beta 99-->Asn), and have produced it using our Escherichia coli expression plasmid pHE2. We have determined the O2 binding properties of this recombinant double mutant Hb, Hb(Asp-beta 99-->Asn and Tyr-alpha 42-->Asp) and have used 1H NMR spectroscopy to investigate the tertiary structures around the heme groups and the quaternary structure in the alpha 1 beta 2 subunit interface. Our results clearly show that the Tyr-alpha 42-->Asp replacement can substantially compensate for the functional defect of Hb Kempsey caused by the Asp-beta 99-->Asn substitution. The structural and functional information derived from this recombinant Hb provides insights into the structural basis of allosterism and the design of compensatory amino acid substitutions to restore the functional properties of other abnormal HBs associated with hemoglobinopathies.

Allosteric Regulation↗
PubMed1994

Low rate of ret proto-oncogene activation (PTC/retTPC) in papillary thyroid carcinomas from Saudi Arabia.

BACKGROUND: The ret proto-oncogene activation (PTC/retTPC oncogene) in thyroid papillary carcinoma has been reported in different populations with different frequencies. Thyroid papillary carcinoma appears to behave more aggressively in the Persian Gulf region than elsewhere. In the current study, the frequency of PTC/retTPC oncogene in thyroid tumors from Saudi Arabia was investigated. METHODS: PTC/retTPC oncogene transcripts were analyzed by the polymerase chain reaction amplification of cDNA synthesized by treatment of total RNA with reverse transcriptase. Seven multinodular goiters, 1 follicular adenoma, 4 follicular carcinomas, 40 papillary carcinomas, and 5 anaplastic carcinomas were studied. RESULTS: Only one papillary carcinoma specimen was found to have PTC/retTPC oncogene transcripts. The breakpoint of the rearranged PTC/retTPC oncogene is identical to that previously described. The PTC/retTPC-positive sample was also examined for p53 tumor suppressor gene mutations in exons 5-8. One transitional point mutation was detected at codon 161 (GCC to ACC), changing Ala to Thr. CONCLUSIONS: This study questions the relevance of PTC/retTPC oncogene in the carcinogenesis of thyroid papillary carcinomas in the Saudi population. Genetic background among races may contribute to the different frequencies of PTC/retTPC oncogene in thyroid papillary carcinoma.

Carcinoma, Papillary↗
PubMed1994

Differential nm23 gene expression at the fetal-maternal interface.

The product of the nm23 gene has been proposed as a candidate tumour metastasis suppressor protein. A strong association has been observed between reduced expression of the nm23 gene and acquisition of metastatic behaviour in some tumour cells, including breast cancer and melanoma, but not in others, such as neuroblastoma and colon, cervical and thyroid cancers. During the early gestation period both human and murine trophoblast cells exhibit in vitro invasive properties similar to those of neoplastic cells. Such invasive properties, however, disappear in the late stage of gestation. In the present study, we examined the abundance of nm23 mRNA from various fetal-maternal interface tissues (uterus, decidua, placenta and embryo) during early (day 8), mid (day 14) and late (day 18) stages of gestation in CD1 mice, in order to determine whether nm23 plays any anti-invasive and/or biological roles during gestation. nm23 was found to be expressed in all the tissues during the early and mid stages of gestation. The expression levels were, however, variable among different tissues and development stages. In the early stage, nm23 mRNA levels were the highest and similar among tissues from the uterus, decidua, placenta and embryo. In the mid stage, the mRNA levels were reduced significantly in the uterus, decidua and placenta, but not in the embryo. In the late stage, nm23 mRNA was further reduced to the extent that it could not be seen in the decidua, was barely seen in the uterus and was weakly present in the placenta. However, the mRNA level of the embryo in the late stage was still high and similar to the early stage. We also examined nm23 expression in trophoblast cells from normal human term placenta and a highly metastatic human choriocarcinoma cell line, JAR. nm23 expression was significantly higher in JAR than in normal placenta, indicating that nm23 does not appear to have an anti-metastatic function in this cell line. Several cytokines--interleukin 2 (IL-2), tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)--and prostaglandin E2 (PGE2) known to modulate tumour growth and metastasis were examined to determine whether they regulate nm23 expression in JAR in vitro. The B16F10 melanoma cell line was used as control. No effect was found in the JAR cell line, whereas TNF-alpha, IFN-gamma and PGE2 down-regulated nm23 expression in the B16F10 cell line.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Production of unmodified human adult hemoglobin in Escherichia coli.

We have constructed a plasmid (pHE2) in which the synthetic human alpha- and beta-globin genes and the methionine aminopeptidase (Met-AP) gene from Escherichia coli are coexpressed under the control of separate tac promoters. The Hbs were expressed in E. coli JM109 and purified by fast protein liquid chromatography, producing two major components, a and b. Electrospray mass spectrometry shows that at least 98% and about 90% of the expressed alpha and beta chains of component a, respectively, have the expected masses. The remaining 10% of the beta chain in component a corresponds in mass to the beta chain plus methionine. In component b, both alpha and beta chains have the correct masses without detectable N-terminal methionine (< 2%). These results have been confirmed by Edman degradation studies of the amino-terminal sequences of the alpha and beta chains of these two recombinant Hb (rHb) samples. rHbs from components a and b exhibit visible optical spectra identical to that of human normal adult Hb (Hb A). Component a and Hb A have very similar oxygen-binding properties, but component b shows somewhat altered oxygen binding, especially at low pH values. 1H-NMR spectra of component a and Hb A are essentially identical, whereas those of component b exhibit altered ring current-shifted and hyperfine-shifted proton resonances, indicating altered heme conformation in the beta chain. These altered resonance patterns can be changed to those of Hb A by converting component b to the ferric state and then to the deoxy state and finally back to either the carbonmonoxy or oxy form. Thus, our E. coli expression system produces native, unmodified Hb A in high yield and can be used to produce desired mutant Hbs.

Amino Acid Sequence↗
PubMed1993

High levels of Nm23 gene expression in advanced stage of thyroid carcinomas.

The product of Nm23 gene has been proposed as a candidate tumour metastasis suppressor protein. A strong association has been observed between reduced expression of Nm23 gene and acquisition of metastatic behaviour in some tumour cells including breast cancer and melanoma, but not in others such as colon cancer, neuroblastoma, and cervical cancer. In the present study, we examined the abundance of Nm23 mRNA in 39 thyroid tissue specimens including five multinodular goitres, one follicular adenoma, 26 papillary and three follicular carcinomas, and four anaplastic carcinomas. Nm23 was found to be expressed in all the tissue specimens. The expression was, however, variable in different stages of thyroid carcinoma. In stages I through III of differentiated thyroid carcinoma, the average level of Nm23 gene expression was comparable to that in multinodular goitres. In advanced stage of thyroid carcinoma (stage IV and anaplastic), 2-fold increase of Nm23 expression was noted. No mutations were found in the coding region of the gene. Nm23 mRNA level cannot, therefore, be used as a marker of low metastatic potential in thyroid carcinomas. The association of high level Nm23 expression with anaplastic thyroid carcinoma suggests its correlation with rapid cell proliferation.

Base Sequence↗
PubMed1993

High-affinity binding of thyrotropin to the extracellular domain of its receptor transfected in Chinese hamster ovary cells.

Thyrotropin (TSH) receptor is a cell surface receptor that shares a high degree of homology with other glycoprotein hormone receptors including lutropin-choriogonadotropin (LH/CG) and follicle-stimulating hormone (FSH) receptors. Although the extracellular domain of TSH receptor is important for ligand binding, no direct information is available on whether extracellular domain alone is sufficient for high-affinity binding. Moreover, mutations made in the second cytoplasmic loop or the cytoplasmic tail of TSH receptor were reported to reduce significantly the affinity of TSH binding. In an attempt to determine whether TSH receptor extracellular domain is sufficient for high-affinity TSH binding or whether it requires transmembrane regions, we made a construct (TSHR-EX/CMV) that encodes for only the extracellular domain plus a foreign hydrophobic tail. The TSHR-EX/CMV was transfected and stably expressed in Chinese hamster ovary (CHO) cells. The truncated receptor was anchored to the cell surface through the hydrophobic tail at the carboxyl terminus. High-affinity TSH binding was observed comparable to that of the cells transfected with full-length TSH receptor. The CHO cells transfected with TSHR-EX/CMV did not respond to TSH stimulation of adenylate cyclase, whereas the cells transfected with the full-length TSH receptor cDNA did. The data presented here show that the extracellular domain of TSH receptor is sufficient to confer high-affinity TSH binding.

Animals↗
PubMed1993

Expression of thyrotrophin receptor gene in thyroid carcinoma is associated with a good prognosis.

OBJECTIVE: The clinical course of thyroid carcinoma is very variable. It is well known that thyroid carcinomas of similar histology can behave differently in terms of local invasion and distant metastases: there is no reliable way to predict the disease course with confidence. In the present study we compared the TSH receptor and c-myc mRNA levels in different stages of thyroid carcinomas to identify whether they are useful markers for thyroid tumour biological behaviour and prognosis. DESIGN: Thyroid tumour specimens were used as the source of RNA. The TSH receptor and c-myc mRNA levels were detected by Northern blot analysis and quantitated by laser densitometry. PATIENTS: Thyroid tissues were obtained from five patients with multinodular goitres, 22 with differentiated and three with anaplastic carcinomas. MEASUREMENTS: Total cellular RNA was extracted from thyroid tissue specimens and blotted onto nylon membranes. Northern blot analysis was used to detect TSH receptor and c-myc mRNA. The mRNA levels were then quantitated by laser densitometry and compared with each disease stage. RESULTS: TSH receptor mRNA levels were significantly lower in carcinomas as compared to benign tumours. With advancing disease stage, the neoplastic tissues generally showed a progressive decline in TSH receptor mRNA levels. Interestingly, in two specimens from patients with distant metastases, TSH receptor mRNA levels were not significantly reduced and were comparable to those in benign tumours. Both patients are still alive, one of them 18 years after operation, indicating that tumour histology is dissociated from its biological behaviour. c-myc mRNA levels were increased but not significantly so in stage 1-3 carcinomas. However, in stage 4 carcinomas c-myc expression was significantly increased. Thus c-myc overexpression is associated with poor prognosis. Although there is a negative correlation between TSH receptor and c-myc mRNA levels, the correlation was not significant. CONCLUSIONS: These results indicate that decreased TSH receptor and increased c-myc gene expression levels are associated with thyroid cell de-differentiation. They are useful markers for thyroid tumour de-differentiation and disease prognosis.

Adult↗
PubMed1993

p53 mutations in all stages of thyroid carcinomas.

The p53 gene has been implicated as a tumor suppressor gene whose inactivation by mutations has been noted in a variety of human malignancies. Using single strand conformation polymorphism analysis of cDNA fragments amplified by reverse transcription-polymerase chain reaction, we analyzed 57 thyroid tumor specimens (8 follicular adenomas and 49 carcinomas) for the presence of mutations in exons 5, 6, 7, and 8 of p53 gene. Twelve of 49 (24.5%) of the thyroid carcinomas tested presented a mutated p53 allele, but none of the 8 benign thyroid tumors did. Mutations were found in 1 of 5 anaplastic carcinomas and 11 of 44 differentiated carcinomas. Three of these 11 differentiated tumor specimens showed foci of solid tissue with evidence of dedifferentiation. Two samples (1 with anaplastic carcinoma, the other with papillary carcinoma) had double mutations on the same allele resulting in a frameshift. Most mutations were point mutations, and 50% of those were G:C to A:T transitions. Seventy-five percent of the mutations were in exons 7 and 8. The presence of p53 mutations was not associated with tumor stage or histological type. Our data suggest that p53 mutations are involved in thyroid carcinogenesis and may play an important role in the malignant transformation of thyroid cells as well as thyroid tumor progression.

Adenocarcinoma, Follicular↗
PubMed1992

Typing for major histocompatibility complex class II antigens in thyroid tissue blocks: association of Hashimoto's thyroiditis with HLA-DQA0301 and DQB0201 alleles.

This study was undertaken 1) to find out whether we can type major histocompatibility class II antigens from the paraffin-embedded series of thyroid tissue, and 2) to investigate whether HLA-DQ genes are involved in conferring a risk of Hashimoto's thyroiditis. To this end we used the polymerase chain reaction to amplify DNA from paraffin-embedded thyroid tissue blocks of histologically proven Hashimoto's disease. We used 46 specimens for HLA-DQA and 32 for DQB typing. The alleles were identified by sequence-specific oligonucleotide hybridizations. Fifty controls from the same geographic region were also typed using peripheral leukocyte DNA. HLA-DQA0301 (in linkage disequilibrium with DR4) was significantly increased (58.7% vs. 32% in controls; chi 2 = 6.73; P less than 0.01) in patients compared to controls. DQB0201 (in linkage disequilibrium with DR3) was also increased in the patient group (66% vs. 36% in controls; chi 2 = 6.63; P less than 0.01). Although DQA0301/DQB0201 heterozygotes (18.8%) were increased in patients compared to controls (6%), the difference was not significant. However, 81% of the patients (26 of 32) were DQA0301 and/or DQB0201 positive compared to 48% of controls (chi 2 5.98; P less than 0.05). We conclude that it is feasible to type HLA antigens from tissue blocks and that susceptibility to Hashimoto's disease is probably mediated through two pathways: DQA0301/DR4 and DQB0201/DR3.

Alleles↗
PubMed1992

[The application of monoclonal antibodies (McAbs) against human serum IgA in single radial immunodiffusion (SRID)].

Among many McAbs to human IgA, two McAbs (A1A10 and A1C9) against two determinants were selected and mixed. They formed opaque precipitation rings with IgA in agarose containing 2% PEG 6000. SRID with our mixed McAbs or PcAbs was used to determine serum IgA concentration in 40 undiluted serum specimens: a comparison of the two showed similar results, with a correlation coefficient of 0.9493 (P < 0.001). We conclude that mixed McAbs A1A10 and A1C9 can replace PcAbs in SRID for the measurement of human total serum IgA.

Antibodies, Anti-Idiotypic↗