Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica最新文献

As the sole ubiquitous signal GTP-binding protein family in higher plants, Rac genes act as pivotal molecular switches and participate in many regulations of life activities. In order to study the biochemical characteristics of rice Rac protein osRACB, the complete coding sequence of osRACB was cloned into expression vector pET28a and expressed in E. coli BL21(DE3). After induced by 1 mmol/L IPTG at 37 degrees C for 4 h, the fusion protein His-osRACB was produced in a large amount. The fusion protein was purified by Ni(2+)-NTA column and digested by thrombin. After a series of processes including separation and recovery by electrophoresis, renaturation by glutathione and concentration by ultrafiltration, pure osRACB protein in an active form was obtained. The GTP-binding and hydrolyzing assay showed that osRACB has strong GTP special binding and hydrolysis activity regulated by Mg(2+). By comparing it with another rice Rac protein osRACD, it can be concluded that osRACB has stronger GTP-binding activity and weaker hydrolysis activity than osRACD.

Myocyte's contraction is regulated by signal transduction pathway composed with many protein factors, but the definite mechanism is still uncertain. A novel cardiac-specific kinase gene which participates in regulation of signal transduction, p93 named, was cloned from adult heart cDNA library. p93, coding a family member of MAPKKK, localized on 1p31.1 based on bioinformatics analyses. Northern blot and 76-tissue array analyses determined that p93 was merely expressed in heart, but was undetectable in other tissues. Immunohistochemical study showed that p93 predominantly localized in the nucleus of cardiac myocytes. In vitro kinase assay indicated that p93 was a functional kinase capable of autophosphorylation. p93 could directly interact with cardiac troponin I by yeast two-hybrid system assessed utilizing bait plasmid containing p93 C-terminus (733 835 aa) and results were further confirmed by co-immunoprecipitation in vivo. Our data suggest that p93 is a cardiac-specific kinase and may play important role in regulation of sarcomeric contraction protein with signal transduction pathway similar ILK.

Beginning from a mouse EST (GenBank Accession No: BE644537) which was significantly changed in cryptorchidism and represented a novel gene, GeneScan program was performed and a predicted mouse novel gene full-length cDNA sequence containing the BE644537 sequence was attained. Gene-specific primers were designed for PCR in mouse testis cDNA library. The sequencing result of the PCR product showed that we obtain a new gene mTSARG3 (GenBank Accession No: AF419292) whose full cDNA length is 1328 bp containing 8 exons and 7 introns. The predicted open reading frame encodes a protein of 316 amino acid residues. The protein encoded by the new gene is a new member of HSP40 protein family because the sequence contains the highly conserved J domain which is present in all DnaJ-like proteins and is considered to have a critical role in DnaJ-DnaK protein-protein interactions. mTSARG3 protein displays a 46% identity in a 336-amino-acid overlap with DJB4-MOUSE protein. The result of RT-PCR analysis and Northern blot showed that mTSARG3 is specifically expressed in mouse testis. Southern blot showed that there were no loss and rearrangement in cryptorchid. Based upon all these observations, it is considered that the function of the new gene is related to inhibit mouse testis spermatogenesis apoptosis.

To study the effect of oleate on ATP binding cassette transporter A1 (ABCA1) expression and cholesterol efflux in THP-1 macrophage-derived foam cells, after exposure of the cultured THP-1 macrophage-derived foam cells to oleate for different time, cholesterol efflux was determined by FJ-2107P type liquid scintillator. ABCA1 mRNA and its protein level were determined by RT-PCR and Western blot, respectively. The mean ABCA1 fluorescence intensity of THP-1 macrophage-derived foam cells was detected by flow cytometry. The results showed that oleate markedly inhibited ABCA1-mediated cholesterol efflux from THP-1 macrophage-derived foam cells. This was accompanied by a reduction in the membrane content of ABCA1. Oleate did not alter ABCA1 mRNA abundance, indicating that decreased ABCA1 transcription, enhanced mRNA decay, or impaired translation efficiency did not account for these inhibitory effects. Oleate, however, increased ABCA1 turnover when protein synthesis was blocked by cycloheximide. Oleate reduces cholesterol efflux and the level of ABCA1 protein in THP-1 macrophage-derived foam cells.

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFalpha transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

Leukemia inhibitory factor (LIF), a cytokine belonging to IL-6 family, which was discovered to inhibit the proliferation of murine myeloid leukemic cell line M1, has multiple functions in various biological processes. This factor is highly glycosylated when it binds to receptor to activate the signal transduction. Therefore, expression of LIF through eukaryotic system is the best way to obtain the correct glycosylation. In this study, human LIF cDNA with the sequence of signal peptide was cloned from adult blood cells by RT-PCR, and then subcloned into pcDNA3 for expression in HEK-293T cells. After transfection of the recombinant plasmid pcDNA3/LIF into HEK-293T cells, the conditioned medium containing the secreted LIF was obtained. The activity of the secreted LIF in the conditioned medium was detected through the phosphorylation of STAT3, EMSA and luciferase reporter (pGL2-APRE-luc) assays in Hep3B and 293T cells. Furthermore, to investigate the biological functions of the overexpressed recombinant LIF, a [(3)H]-thymedine incorporation assay was performed, and the results showed that the recombinant LIF inhibited the growth of M1 cells strongly. Taken together, all the function assays suggest that the recombinant LIF has the normal functions, suggesting that the recombinant LIF could be used for further studies.

A novel coronavirus (SARS-coronavirus, SARS-CoV) was discovered as the pathogen of the severe acute respiratory syndrome (SARS). According to studies with other coronaviruses, the membrane protein (M protein) is the main structural protein and the recombinant M protein may be useful as an antigen for detecting antibodies against coronavirus and for preparing vaccine. In this work, the M protein of SARS-CoV was expressed in E. coli as fusion protein with maltose binding protein at N-terminus and MxeGyrA intein CBD at C-terminus. The recombinant protein was identified by Western blot and mass spectrometry. The soluble parts of the cell crude extract were then partially purified by MBP affinity chromatography. The purified protein will be used for the studies on M protein's structure and the development of diagnostic method of SARS.

FtsZ protein plays a key role in the division of bacteria and chloroplast. To investigate the evolution of the chloroplast dividing apparatus, cloning and molecular characterization of a second chloroplast division gene, CrFtsZ3, from Chlamydomonas reinhardtii is performed. As there are two ftsZ genes in Chlamydomonas reinhardtii, duplication and divergence of the ftsZ genes might occur in an early stage before the emergence of green algae during the course of plant evolution. Sequence analysis showed that CrFtsZ3 gene had significant sequence homology with other ftsZs. It encoded a precursor of 479 amino acid residues with a putative transit peptide in its N-terminal. To study the function of CrFtsZ3, a recombinant plasmid expressing the full length CrFtsZ3/EGFP fusion protein was constructed. By using IPTG inducing, overexpression of CrFtsZ3/EGFP in E.coli was achieved, and this overexpression blocked cell division and resulted in filament formation. GFP-derived fluorescence showed regularly spaced dots along the bacterial filaments. This suggests that CrFtsZ3 could still recognize the signals of cell division site in E.coli and could take part in the process of bacterial division.

3T3-L1-adipocytes produce the adipocyte complement related protein of 30 kD (ACRP30), which is exclusively expressed in differentiated adipocytes. Decreased expression of ACRP30 correlates with insulin resistance in mouse models of altered insulin sensitivity. Adiponectin, the human homologue of ACRP30, circulates in human plasma at high levels. Plasma adiponectin levels have been reported to be decreased in some insulin-resistant states, such as obesity and type II diabetes mellitus. Here, full-length adiponectin and its C-terminal globular head domain (gAdiponectin) were expressed in Escherichia coli and gAdiponectin was used to immunize a rabbit to obtain polyclonal antiserum with titer of 10,000. Adiponectin was detected in human plasma with the use of gAdiponectin anti-serum by Western blot analysis, which was also detected by gACRP30 anti-serum. Injection in alloxan-treated rats with purified recombinant fusion adiponectin or gAdiponectin transiently abolished hyperglycemia. So adiponectin and gAdiponectin might have activity as a glucose lowering agent and potentially as a therapeutic for metabolic disease. All these results suggested that the recombinant protein had biological activity, and provided a useful tool in further studies.

A gene fragment encoding three copies of proinsulin C-peptide was synthesized and expressed in E. coli and the recombinant proinsulin C-peptide was produced through site-specific cleavage of the resulting gene products. The fusion protein was expressed at high level, about 80 mg/L, as a soluble product in the cytoplasm. Ni-NTA affinity chromatography efficiently separated the expressed fusion protein from the supernatant, to obtain about 37.5 mg/L of the fusion protein with 70% purity. Enzymatic digestion by trypsin and carboxypeptidase B of the fusion protein efficiently released native C-peptide, the overall yield of recombinant C-peptide at a purity over 95% was 1.5 mg/L. The good agreement of amino acids composition, together with shown similarities of the recombinant C-peptide to C-peptide standard in the comparative RP-HPLC analysis and IMMULITE C-Peptide quantitative assay, suggested that the recombinant C-peptide obtained in this report was the native human C-peptide. The investigation of the chemical stability of recombinant human C-peptide in aqueous solutions by RP-HPLC was also reported. The degradation of the recombinant C-peptide showed a marked dependence on pH and temperature. The degradation reaction of C-peptide occurred immediately in pH 3 or pH 9 buffered solution. The degradation reaction of C-peptide followed first-order kinetics in pH 3 buffered solution at 37 degrees C or 70 degrees C, only 40.3% of C-peptide was remained after 10 h at 70 degrees C. The maximum stability was achieved at pH 7.4, more than 90% of C-peptide were detected at pH 7.4 and 37 degrees C after 10 h and at pH 7.4 and 70 degrees C after 5 h. 99% and 96% of C-peptide was remained at pH 7.4 and 37 degrees C after 10 h with and without 10 g/L BSA respectively.