Biomedical subjects

Q Qian

Publications and source records attributed to Q Qian.

At least 55 records · Page 3Linked to original sources

PubMed1998

Derived protein and cDNA sequences of hamster amelogenin.

Hamster enamel protein extracts were analyzed by RP-HPLC and the isolated fractions by SDS-and Western blotting using polyclonal antibodies against recombinant mouse amelogenin and anti-peptide antibodies against the mouse exon 4-encoded sequence. Total RNA was extracted from enamel organ epithelia and, using a 3' rapid amplification of cDNA ends (3' RACE) technique, the coding regions for three different amelogenin isoforms were cloned along with the 3' non-coding region. DNA sequencing revealed that the hamster amelogenin isoforms are 180, 73 and 59 amino acids in length, respectively. The 59-residue amelogenin corresponds to the leucine-rich amelogenin protein (LRAP), the 73-residue amelogenin corresponds to LRAP with the inclusion of the exon 4-encoded sequence, while the 180-residue amelogenin is the most abundant amelogenin isoform. Edman degradation was performed on purified hamster amelogenin, which provided the amino acid sequence in the region encoded by the 5' PCR amplification primer used in cloning. Therefore, the entire derived amino acid sequence of hamster amelogenin was revealed. The hamster amelogenin amino acid sequence was aligned with all its known homologues. Hamster differs from rat and mouse amelogenin at only three amino acid positions. Southern blot analysis using a panel of restriction enzymes gave the same pattern for hamster DNA obtained from males and females, suggesting that in hamster, as in mouse, amelogenin is expressed from a single gene located on the X chromosome.

Amelogenin↗

PubMed1998

Proteolytic activity of opossum tooth extracts.

Amelogenins are the main component of the developing enamel matrix. In placental mammals, amelogenins are rapidly cleaved following their secretion. HPLC fractionation of tooth extracts produces a complex chromatographic profile. The fractions are rich in amelogenin cleavage products that generally retain the amino-terminus of the parent protein but have varying lengths of peptide removed from the original carboxyl-terminus. In contrast, HPLC fractionation of opossum tooth extracts produces a simple profile with a single major chromatographic peak. SDS-and Western blot analyses demonstrated that most of the amelogenin consisted of a prominent protein band that migrated at 28 kDa. Mass spectroscopy confirmed the presence of two uncleaved, alternatively spliced forms of opossum amelogenin, Op202 and Op57, but did not detect major amelogenin cleavage products evident in tooth extracts from placental mammals. Amino acid composition analysis supported the conclusion that uncleaved amelogenin is the major component in the developing enamel matrix. Enzymogram analyses using gelatin, casein and recombinant amelogenin as substrates, comparing porcine, rat and opossum tooth extracts, suggested that fewer proteinases are present in opossum. These results identify potentially significant differences in the proteolytic processing of amelogenins between metatherian and eutherian mammals.

Amelogenin↗

PubMed1998

The methyl group of the N6-methyl-N6-threonylcarbamoyladenosine in tRNA of Escherichia coli modestly improves the efficiency of the tRNA.

tRNA species that read codons starting with adenosine (A) contain N6-threonylcarbamoyladenosine (t6A) derivatives adjacent to and 3' of the anticodons from all organisms. In Escherichia coli there are 12 such tRNA species of which two (tRNA(Thr1)GGU and tRNA(Thr3)GGU) have the t6A derivative N6-methyl-N6-threonylcarbamoyladenosine (m6t6A37). We have isolated a mutant of E. coli that lacks the m6t6A37 in these two tRNA(Thr)GGU species. These tRNA species in the mutant are likely to have t6A37 instead of m6t6A37. We show that the methyl group of m6t6A37 originates from S-adenosyl-L-methionine and that the gene (tsaA) which most likely encodes tRNA(m6t6A37)methyltransferase is located at min 4.6 on the E. coli chromosomal map. The growth rate of the cell, the polypeptide chain elongation rate, and the selection of Thr-tRNA(Thr)GGU to the ribosomal A site programmed with either of the cognate codons ACC and ACU were the same for the tsaA1 mutant as for the congenic wild-type strain. The expression of the threonine operon is regulated by an attenuator which contains in its leader mRNA seven ACC codons that are read by these two m6t6A37-containing tRNA(Thr)GGU species. We show that the tsaA1 mutation resulted in a twofold derepression of this operon, suggesting that the lack of the methyl group of m6t6A37 in tRNA(Thr)GGU slightly reduces the efficiency of this tRNA to read cognate codon ACC.

Adenosine↗

PubMed1998

Cloning, characterization, and tissue expression pattern of mouse tuftelin cDNA.

Tuftelin is a protein that has been suggested to function during enamel crystal nucleation. Published sequences for bovine tuftelin cDNA and genomic clones proposed different reading frames that radically affected the derived amino acid sequence of the tuftelin carboxyl-terminus. We have isolated and characterized a full-length mouse cDNA clone and a partial porcine cDNA clone that include the region of the proposed frame-shift. The mouse tuftelin clone is 2572 nucleotides in length, exclusive of the poly(A+) tail. Translation from the 5'-most ATG yields a protein of 390 amino acids with an isotope-averaged molecular mass of 44.6 kDa and an isoelectric point of 5.9. Comparison of the bovine, mouse, and porcine cDNAs supports the revised bovine tuftelin amino acid sequence and suggests that the bovine tuftelin translation initiation codon be re-assigned to a more 5' ATG. Re-assigning the translation initiation codon lengthens the tuftelin protein by 52 amino acids, 51 of which are identical between bovine and mouse. At the carboxyl-terminus, the revised bovine and the mouse sequences match at 39 of the final 42 amino acid positions, compared with 2 identities with the originally published bovine reading frame. Northern blot analysis reveals that tuftelin is not ameloblast-specific but is expressed in multiple tissues, including kidney, lung, liver, and testis. Two tuftelin RNA messages, of 2.6 and 3.2 kb, were detected. DNA sequence characterization of an RT-PCR amplification product confirmed expression of tuftelin in kidney, and identified an alternatively spliced mouse tuftelin mRNA lacking exon 2.

Amino Acid Sequence↗

PubMed1998

Murine enamelin: cDNA and derived protein sequences.

Enamelin is the largest enamel protein. Recently we reported the characterization of a cDNA clone encoding porcine enamelin. The secreted protein has 1104 amino acids--over 6 times the length of amelogenin (173 amino acids) and almost 3 times the lengths of sheathlin (395 amino acids) and tuftelin (389 amino acids). Immunohistochemistry has shown that uncleaved porcine enamelin concentrates at the growing tips of the enamel crystallites while its cleavage products localize to rod and interrod enamel. Here we report the isolation and characterization of cDNA encoding murine amelogenin and demonstrate the tooth specificity of porcine enamelin. The murine clone is 4154 nucleotides in length and encodes a protein of 1274 amino acids. In the absence of post-translational modifications murine enamelin has an isotope averaged molecular mass of 137 kDa and an isoelectric point of 9.4. Multiple tissue Northern blot analyses detect porcine enamelin mRNA in developing teeth but not in liver, heart, brain, spleen, skeletal muscle and lung. Mouse and porcine enamelin share 61% amino acid identity and 75% DNA sequence identity. Mouse enamelin has 14 tandemly arranged copies of an 11 amino acid segment that is found only once in porcine enamelin.

Amino Acid Sequence↗

PubMed1998

[Fas mediated apoptosis inhibited by human bcl-2 gene in lymphoma cell line Jurkat].

OBJECTIVE: To understand the mechanism of bcl-2 gene in escaping the immune surveillance in the development of lymphoma. METHODS: A recombinant retroviral vector pLXSN-bcl-2 was constructed by cloning bcl-2 cDNA into the replication defective retroviral vector pLXSN, and transferred to packaging cell line PA317 by electroporation. The G418 resistant colonies were selected, and the supernatants of the colony cultures were used to infect the human lymphoma cell line Jurkat. Cells in G418 resistant Jurkat colonies were characterized by immunohistochemistry. Anti-Fas monoclonal antibody (McAb) was applied to Jurkat cells for inducing apoptosis which mimicked the cytotoxic activity of T lymphocyte. RESULTS: Expression of bcl-2 in pLXSN-bcl-2 transfected Jurkat cell (Jurkat-bcl-2) increased, while there was no change of Fas gene expression. Apoptosis was blocked in Jurkat-bcl-2 by anti-Fas McAb treatment. CONCLUSION: Overexpression of bcl-2 in lymphoma cell line could inhibit cell apoptosis induced by anti Fas McAb, suggesting that overexpression of bcl-2 is one of the mechanisms in escaping immune surveillance in the development of lymphoma.

Apoptosis↗

PubMed1998

[Perforin and Fas-ligand expression of tumor infiltrating lymphocytes in human hepatocellular carcinoma].

OBJECTIVE: To investigate perforin and Fas-ligand (Fas-L) expression of tumor infiltrating lymphocytes (TIL) in human hepatocellular carcinoma (HCC). METHODS: Expression of perforin and Fas-L of TIL was studied in 20 HCC cases using in situ hybridization and immunohistochemistry. RESULTS: Expression of perforin and Fas-L gene were detected in TIL in 80% of the cases studied. Among them, one patient (no. 14) in whom expression of perforin and Fas-L were noticed in the majority of the TIL, had no recurrence of HCC for one and one half (1.5) year after tumor resection. It indicates that presence of large number of activated T cells might be beneficial for the tumor therapy. In the remaining cases, only 10% of TIL were obtained able to express perforin and Fas-L indicating that only a few TIL were activated and cytotoxic to HCC. CONCLUSIONS: Even there were a multitude of T cells infiltrating in HCC, only few of them were immunoactived and to be cytotoxic to HCC. It seems important to adopt measures in order to promote further proliferation of these activated T cells either in vitro or in vivo.

Adult↗

PubMed1998

[The development of computer-assisted gastric function testing system].

This paper introduces a system consisting of the PC computer, sensing device, the card for electro-gastric signals amplifier and analog-digital converter. By using only one piece of 1.2 M 3" diskette, the system can realize the dialogue between human and the computer to sample, process, store and display the electro-gastric signals, and print the auxiliary analysis and diagnostic results for doctors to refer to. When the computer is more and more popular today, it is one of the convenient, painless and effective method to check up the gastric functional disease.

Adult↗

PubMed1998

The human cytomegalovirus IE2 86 kDa protein elevates p53 levels and transactivates the p53 promoter in human fibroblasts.

Human cytomegalovirus (HCMV), a herpesvirus, has previously been reported to elevate cellular p53 levels within infected human fibroblasts. Since this elevation was reported to be due to the expression of HCMV immediate early (IE) gene products within the fibroblasts, and the HCMV immediate early region encodes several predominant proteins, the identity of the protein responsible for this elevation was investigated. We report that the HCMV IE2 86 kDa protein was clearly found to be the major immediate early protein responsible for elevating p53 levels within the fibroblasts. We also report that the HCMV IE2 86 kDa protein was found to transactivate the p53 promoter 5-6 fold, thus explaining the 5-6 fold rate of increased p53 protein synthesis we find in the HCMV infected fibroblasts. Since transactivation of the p53 promoter was observed in IE2 86 kDa transfected as well as in HCMV infected fibroblasts, transactivation of the p53 gene, therefore, is part of the mechanism HCMV uses to elevate p53 levels in infected fibroblasts. This is the first report to implicate the HCMV IE2 86 kDa protein in the elevation of p53 levels in fibroblasts, as well as in the transactivation of the p53 promoter in these cells.

Cells, Cultured↗

PubMed1998

[Construction and application of retroviral vector carrying green fluorescent protein].

OBJECTIVE: To construct retroviral vector carrying rapidly selective marker. METHODS: The recombination retroviral vector GCGFPPXSN was constructed by cloning the green fluorescent protein (GFP) cDNA into the retroviral vector containing putative internal ribosome entry sites GCXPXSN and transferred in ecotropic packaging cell line PE501 by electroporation method. The supernatants of the PE501GCGFPPXSN were used to infect the amphotropic packaging cell line PA317. The G418 resistant clones were selected in 4 weeks and were detectable by fluorescence microscopy or by fluorescence-activated cell sorting(FACS). RESULTS: A recombination retroviral vector GCGFPPXSN carrying rapidly selective marker GFP was constructed. GFP expression in packaging cell line PA317-GCGFPPXSN transferred by GCGFPPXSN was detected by fluorescence microscopy of FACS. PA317-GCGFPPXSN grew to a titer of 1.2 x 10(5) cpu/ml. By cocultivating retroviral vector producing cells and T lymphocytes, expression of GFP was observed in T lymphocytes 2 days after the end of the cocultivation. T lymphocytes expressing GFP were separated. CONCLUSION: The mammalian cell can be efficient gene transfected by retroviral vector carrying GFP. The use of GFP for cell marking represents an important advantage over conventional strategies which typically involve the use of neomycin resistance. GFP, in fact, allows a rapid in vitro selection of transduced cell by FACS. The selection requires only two-day culture with this retroviral vector, compared with 10-14 day culture with a classical retroviral vector.

Cell Line↗

PubMed1998

[Genetic analysis on a new sterile locus discovered in hybrids between indica and japonica rice (Oryza sativa L.)].

Japonica line 02428, possessing wide compatibility gene S-5n, showed semi-sterility as crossed with indica variety IR24. Genetic analysis was conducted on the spikelet fertility in segregation generations from the crosses of 02428 with IR24, Pecos and Akihikara. The results indicated that the sterility in hybrids between 02428 and IR24 was controlled by a single non-allelic to S-5 gene and its inheritance fitted to one-locus sporo-gametophytic interaction model. Pecos, an American variety, has a neutral allele. The new sterility locus was preliminarily located near G24 marker on chromosome 11 detected by RFLP. The non-allelic S-locus in the study is named tentatively S-p(t). It was found that the sterility caused by the cross of Akihikara and IR24 was controlled by two S-loci, S-5 and S-p(t), and its genetical effect was additive.

Chromosome Mapping↗

PubMed1997

Structural alterations far from the anticodon of the tRNAProGGG of Salmonella typhimurium induce +1 frameshifting at the peptidyl-site.

A total of 12 Salmonella typhimurium mutants were selected with mutations in the minor tRNAProGGG which suppress a +1 frameshift mutation in the hisD gene. This tRNA normally has 1-methylguanosine (m1G37) next to and 3' of the anticodon (position 37). Since the presence of m1G37 prevents frameshifting, some of the +1 frameshift suppressor derivatives of tRNAProGGG had alterations in the primary sequence abolishing the formation of m1G37. However, several of the mutant tRNAProGGG species had a normal level of m1G37 and a normal-sized anticodon loop, showing that neither m1G37 deficiency, nor an oversized anticodon loop, is a prerequisite for +1 frameshifting. Moreover, base substitutions far from the anticodon, e.g. in the acceptor stem, DHU-loop and stem, and at the top of the anticodon stem, promoted +1 frameshifting. When the frameshifting site (CCC-Uaa; CCC is in the zero frame and a +1 frameshift moves the ribosome to the CC-U codon) is overlapped by a nonsense codon (UAA), the efficiency of frameshifting decreased when release factor 1 was over-expressed and increased at an elevated temperature in a mutant with a temperature-sensitive release factor 1. The frameshifting site (CCC-Uac) was also overlapped with the sense codon UAC, which is decoded by a tRNA species having a 2-methylthio-cis ribozeatin (ms2io6A) at position 37. Mutations in the miaA gene affect the formation of this modified nucleoside and result in an A instead of ms2io6A37 in the tRNA. Such an undermodified tRNA is very inefficient in translation and the efficiency of frameshifting increased in a miaA1 mutant. These results suggest that the frameshifting event occurs at the P-site, since the efficiency of frameshifting was sensitive to the decoding activity of the overlapping codon. We conclude that tRNA with mutations far from the anticodon, with a normal-sized anticodon loop and having m1G37 induce +1 frameshifting at the P-site.

Alcohol Oxidoreductases↗

PubMed1997

Structural requirements for the formation of 1-methylguanosine in vivo in tRNA(Pro)GGG of Salmonella typhimurium.

Maturation of tRNA and rRNA and the assembly of the ribosome in all organisms occurs in vivo in a complex pathway in which various proteins such as endo- and exonucleases, tRNA and rRNA modifying enzymes and ribosomal proteins, act concomitantly and temporarily during the maturation process. One class of RNA binding proteins are the tRNA modifying enzymes, which catalyse the formation of various modified nucleosides present in tRNA. Here we analyse the consequences of various alterations in a tRNA on the formation of modified nucleosides in the tRNA and the aminoacylation of it under true in vivo conditions, i.e. in a cell with normal amounts of the tRNA substrate and the tRNA binding protein. We have devised a selection method to obtain mutants of tRNA(Pro)GGG in Salmonella typhimurium that may no longer be a substrate inl vivo for the tRNA(m1G37)methyltransferase. These mutant tRNAs were purified from cells in balanced growth by a solid phase hybridisation technique and the presence of 1-methylguanosine (m1G) in position 37 next to the anticodon was monitored. Of 13 different mutant tRNA(Pro)GGG species analysed, eight of them had a drastically reduced level of m1G. Some of these mutant tRNA species had alterations far from the nucleotide G37 modified by the enzyme; e.g. base-pair disruptions in the first, fourth and eighth (last) base-pair of the acceptor stem, in the D-stem, and in the top of the anticodon stem. The structure of all the mutant tRNA(Pro)GGG species must deviate from the wild-type form, since they all induced +1 frameshifting. Still, tRNA(Pro)GGG from five of the mutants had normal levels of m1G. Thus, only a subset of mutations, all inducing an altered tRNA structure, resulted in m1G deficiency. However, those alterations in tRNA(Pro)GGG, which influenced the tRNA(m1G37)methyltransferase activity, did not affect in vivo the formation of four other modified nucleosides and the aminoacylation of tRNA(Pro)GGG, demonstrating the extreme dependence of the tRNA(m1G37)methyltransferase on an almost perfect three-dimensional structure of the tRNA. We discuss that the conformation of the anticodon loop may be a major determining element for the formation of m1G37 in vivo.

Base Sequence↗

PubMed1997

The study of iodine in Chinese total diets.

In this work, China was divided into four area groups according to their geographical positions and dietary habits. All foods were divided into 12 types and the iodine contents in various diets were determined using epithermal neutron activation analysis (NAA). The intakes for China were evaluated. The results indicate that the intakes of iodine in northern areas are slightly higher and in south areas lower than the lowest recommended intake, and the average intake in China is 166 micrograms/person per day, which is within the recommended range. In addition, one province were chosen from each area groups. The dietary intakes of iodine were investigated in different ages and sex using total mixed diet method. Our results indicate that the average iodine intake of four provinces was lower than the recommended value, which suggests that it is necessary to supplement iodine in foods in China.

Adult↗

PubMed1997

Determination of bromine and iodine in normal tissues from Beijing healthy adults.

The contents of bromine and iodine in samples of heart, liver, spleen, lung, muscle, and hair from healthy adults living in Beijing, China, were determined using epithermal neutron activation analysis. The results indicate that the contents of bromine in lung and iodine in liver are higher than those in other tissues, except human hair. The bromine contents in Beijing human tissues are significantly lower than those in other countries. The contents of iodine are slightly lower than those in other countries, but the difference is not significant. Three biological standard reference materials were simultaneously determined with the samples, and our results agree well with the certified values.

Adult↗

PubMed1997

Gamma interferon is not essential in host defense against disseminated candidiasis in mice.

In vitro studies have suggested a role for interferon gamma (IFN-gamma) in host defense against disseminated candidiasis, but in vivo studies are inconclusive. We utilized homozygous IFN-gamma knockout (GKO) mice to determine if the cytokine is essential in host defense against this disease. Genotypes of mice were determined by PCR with specific primers for the normal or disrupted IFN-gamma gene. The GKO status of the mice was confirmed by an enzyme-linked immunosorbent assay, which showed no detectable IFN-gamma produced by their splenocytes stimulated by concanavalin A. To test the susceptibility of GKO mice to candidiasis, the animals were infected either intravenously (i.v.) or intragastrically (i.g.) with Candida albicans. GKO mice infected i.v. survived as long as wild-type (WT) mice and showed no difference in Candida CFU counts in liver, spleen, or kidneys compared to those for WT mice. When animals were given Candida i.g., at 3 h or at 10 or 21 days after infection, there was no dissemination of Candida to the lung, liver, spleen, or kidneys for either GKO or WT mice. There was no difference in Candida CFU counts recovered from the stomach or intestines between GKO and WT mice. Histological examination of the stomach cardial-atrium fold, where the fungus was located, showed that GKO mice did not have evidence of more tissue damage or fungal invasion than WT mice. Finally, the jejunum for both types of mice showed no evidence of tissue damage or fungal invasion. These studies indicate that IFN-gamma is not essential in host defense against C. albicans that originates from a mucosal site or that is given directly into the bloodstream in a mouse model.

Animals↗

PubMed1997

Cloning, characterization, and heterologous expression of exon-4-containing amelogenin mRNAs.

The formation of dental enamel is dependent upon amelogenins, a family of proteins constituting most of the developing enamel matrix. Depending upon the species, these enamel proteins are expressed from either one or two copies of the amelogenin gene. Each gene directs the synthesis of a variety of amelogenin isoforms through alternative splicing of their pre-mRNA transcript(s). Before the role of amelogenins in dental enamel formation can be better understood, one must know the isoforms that are secreted and their biochemical properties. Previously, we cloned and characterized 7 mouse amelogenin RNA messages generated by alternative splicing. The largest amelogenin cDNA encoded a 194-residue amelogenin isoform which was the only clone to contain the 42-nucleotide exon 4 segment. Anti-peptide antibodies raised against the derived translation of this exon revealed an unexpectedly diverse assortment of murine amelogenins, suggesting that additional splicing variants could contain the exon 4 coding region. Using exon-4-specific oligonucleotide primers, we have amplified, cloned, and characterized three different amelogenin RNA messages. These messages encode amelogenin polypeptides (exclusive of signal peptides) 194, 170, and 73 amino acids in length. The isotope-averaged molecular weights for the deduced, single-phosphorylated, proteins are 21,897.1, 19,113.9, and 8176.5 Daltons, respectively. Splice-site selection for the generation of these mRNAs was identical to that of the previously characterized messages for the M180, M156, and M59 except for the inclusion of exon 4. The exon-4-containing amelogenin isoforms were heterologously expressed in E. coli by means of the pET11 expression system (Novagen, Madison, WI).

Amelogenin↗

PubMed1997

Sheathlin: cloning, cDNA/polypeptide sequences, and immunolocalization of porcine enamel sheath proteins.

Sheath proteins designate low-molecular-weight non-amelogenin enamel polypeptides and their parent protein, which concentrate in the sheath space separating rod and inter-rod enamel (Uchida et al., 1995). Two porcine sheath proteins, with apparent molecular weights of 13 and 15 kDa, are characterized by protein sequencing. The primary structures of these polypeptides match a portion of the derived amino acid sequences of clones isolated from a porcine enamel organ epithelia-specific cDNA library. Sheath protein RNA messages differ by the inclusion or deletion of a 45-nucleotide segment and by the use of three alternative polyadenylation/cleavage sites. The secreted proteins are 395 and 380 residues in length, with molecular masses of 42,358 and 40,279 Daltons and calculated isoelectric points of 6.3 and 6.7, respectively. Polyclonal antibodies were raised against a synthetic peptide having the sheathlin-specific sequence EHETQQYEYSGGC. Immunohistochemistry with this antibody demonstrates that the protein encoded by the sheathlin cDNA is preferentially localized in the sheath space. We propose that the porcine sheath proteins and their proteolytic cleavage products be designated "sheathlin".

Amino Acid Sequence↗