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<p><b>OBJECTIVE</b>To investigate the role of protein kinase C (PKC) in thrombin-induced monocyte chemoattractant protein-1(MCP-1) release by human lung fibroblasts (HLF-1).</p><p><b>METHODS</b>Cultured human lung fibroblasts HLF-1 were incubated with different concentrations of PKC inhibitors before by thrombin stimulation. MCP-l protein levels in the supernatants were assessed using ELISA, and MCP-1 mRNA levels in the cell lysate were measured by quantitative real-time PCR.</p><p><b>RESULTS</b>The broad spectrum PKC inhibitors bisindolylmaleimide I and RO-31-8220 obviously inhibited thrombin-induced MCP-l mRNA and protein expressions in HLF-1 cells, whereas Ca(2+)-dependent PKC inhibitor Go 6976 had no such effects.</p><p><b>CONCLUSION</b>Ca(2+)-independent PKC mediates thrombin-induced MCP-1 release in cultured HLF-1 cells.</p>
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Humans , Cell Line , Cells, Cultured , Chemokine CCL2 , Metabolism , Fibroblasts , Metabolism , Indoles , Pharmacology , Lung , Cell Biology , Metabolism , Protein Kinase C , Thrombin , PharmacologyABSTRACT
Objective The aim of this study is to clone and characterize a novel Trichomonas vaginalis Rabl-like geue (TvRabl-like) with a small intron. Methods The eDNA clone of TvRabl-like gene was isolated from a cDNA expression library and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST ClustalW programs.Phylogenetic analysis was carried out by MEGA3 program. The genomic DNA and mRNA of TvRab1-like gene were amplified using PCR and RT-PCR techniques respectively and also sequenced. Results The eDNA sequence of TvRab1-like gene had a length of 705 base pairs with an open reading frame of 603 bp. The deduced amino acid sequence from the open reading frame possessed 200 residuals corresponding to a putative M.W. 22532.2 and an estimated pl of 7.4. Sequence analysis demonstrated that TvRab1-like gene showed the highest homology to T. vaginalis Rabla (63% identity and 79% similarity) and the Rabl subfamily of other species, suggesting that the deduced amino acid sequence from this cDNA clone was a Rabl isoform. Phylogenetic analysis showed that TvRab1-like gene was clustered with T.vaginalis Rab1 subfamily in the phylogenetic tree. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA possessed a 25 bp intron which contained canonical 5' GT-AG-3' and branch site motifs as those larger introns found in T.vaginalis and other eukaryotes. The analysis of RT-PCR products demonstrated the presence of the unspliced mRNA and spliced mRNA, indicating that there was a intron. Conclusion These data suggest that TvRabl-like gene belongs to T.vaginalis Rabl subfamily. TvRabl-like gene possesses a 25 bp splieeosomal intron which is the smallest one of the introns identified in this deepest-branching protist and might be the shortest intron of eukaryotes. Study of those introns might provide more insights into the intron evolution of eukaryotes.
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Objective To screen cell growth and senescence-related genes of the parasitic pmtist Trichomonas vaginalis,we launched an EST program and isolated two cDNA clones from a T.vaginalis cDNA library,which showed high homology in deduced amino acid sequences to yeast Sir2 and designated as TvSir2 and TvSir2-like.Method The cDNA sequence of TvSIR2 had a length of 1034 base pairs (bp) with an open reading frame of 915 bp,and TvSIR2-like,1214 bp with an open reading frame of 1116 bp.Result The two deduced amino acid sequences shared all the three conserved cole domains with yeast Sir2 and its homologues,suggesting that the two clones were Sir2 homologues. A cDNA fragment from each cDNA clone was subvloned into the expression vector pET-41a.The expression of the fusion proteins in E.coli BL21 stains was induced by isopropylthio-β-D-galactoside (IPTG).Two anti-sera were prepared by immunizing two guinea pigs with the purified fusion proteins, Western-blot analysis demonstrated that each anti-serum reacted with the corresponding recombinant protein and detected a clear band (TvSir2,34 000 Mr;TvSir2-like,42 000 Mr)in protein extracts of the protist.Immunofluolescence techniques showed that TvSir2 and TvSir2-like proteins were both localized in the legions of perinuelear (ER) and Golgi complex.Conclusion Our data suggest that TvSir2 and TvSir2-like were two members of Sir2 family.Their biological functions in the protist would be further studied.
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Objective Apoptosis or programmed cell death(PCD) has been studied extensively in multicellular organisms,however,very little is known about the molecular mechanisms by which apoptosis occurs in unicellular protozoan parasites.The aim of this study is to characterize the apoptosis or PCD of Trichomonas vaginalis induced by metronidazole (MTZ).Methods T. Vaginalis strain cultures were treated with various concentrations of MTZ and the number of viable cells were determined at different time intervals.The genomic DNA of MTZ treated T. Vaginalis was extracted and DNA fragmentation was analyzed.TUNEL assay was carried out to detect the endonuclease activity in T. Vaginalis after MTZ treatment.Flow cytometric analysis was used to analyse the phosphatidylserine (PS) exposure of T. Vaginalis.Results Metronidazole (MTZ) induced an apoptotic-like cell death in T. Vaginalis.This apoptotic-like cell death was demonstrated by cell shrinkage,phosphatidylserine exposure,and nuclear chromatin condensation.However, no oligonucleosmal DNA laddering was detected.Conclusion The regulatory pathway of apoptotic cell death in T. Vaginalis may be different from multicellular organisms.The determination of protozoan apoptotic pathways leading to cell death might ultimately allow the identification of new therapeutic targets.
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Objective To evalute Qiliqiangxin capsule for improving cardiac function and identifiy Qiliqiangxin capsule for directing cytoprotective effects against apoptosis of cardiomyocytes. Methods Sprague Dawley (SD) rats were tested as subjects and the model rats were built by anterior descending coronary deligation, then the survival rats were devided into 6 groups randomly:sham-operated group(sham,n=8),fed with normal saline of same volume instead;control group(control,n=7),fed with normal saline of same volume instead;Qiliqiangxin great dosage group(great,n=9),Qiliqiangxin 1.2g/(kg?d) treatment;Qiliqiangxin medium dosage group(medium,n=9),Qiliqiangxin 0.6g/(kg?d) treatment;Qiliqiangxin small dosage group(small,n=8 rats),Qiliqiangxin 0.3g/(kg?d) treatment;the captopril group(captopril,n=9),captopril 25mg/(kg?d) treatment.24 hours later,rats in sham operated group and model group were given normal saline,rats in treating group were given Qiliqiangxin,rats in the contrasting group were given captopril.After being treated for 4 weeks, all rats′ heart function was measruated by right flank arteria carotis communis arterial cannula.The left ventricle mass index was calculated and the expression of protein caspase-3 was tested by immunohistochemical method and Westernblot. Results Compared with control group, Qiliqiangxin capsule could reduce effectively BW,HMI,LVMI,increase LVEDP and LVSP,+dp/dtmax and -dp/dtmax.Immunohistochemical analysis and Westernblot analysis showed that adminstration of Qiliqiangxin significantly inhibited caspase-3 expressions in myocardial cells in a dose-dependent manner.Conclusion Qiliqiangxin significantly improved the CHF rat′s cardiac function and reduced the left ventricle mass index and heart mass index 4 weeks after being treated with relative high dose Qiliqiangxin. It might be partially attributed to the suppression of caspase-3 expressions.
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Objective To explore the association of expression between the longevity gene SIRT1 and the forkhead transcription factor FOXO1 in the beta cell of islet in rat,and provide the basic prevention and cure rationale for the type 2 diabetes mellitus.Methods Eleven male SD rats of 18 months old were randomized into two groups:6 in calorie restriction(CR)group and 5 in the control group.After 6 months' breeding,the caudal end of islet was collected.The expression and location of SIRT1,FOXO1 and insulin were detected by immunohistochemistry;sections of pancreas were stained for senescence-associated ?-galactosidase(SA-?-Gal)to identify cell senescence of pancreatic islets.Results SIRT1 and FOXO1 were expressed in both the kytoplasm and nucleus,while insulin and ?-Gal were located in the kytoplasm.Compared with the control group rats,there was higher SIRT1 expression(P0.05)expression was detected in the beta cells of CR group rats.The lower positive rate of FOXO1 located in the nucleus the higher expression of SIRT1 in the rat beta cell of islet.Conclusion Calorie restriction could induce the high expression of SIRT1 in the rat beta cell of islet,and we think SIRT1 may delay the senescence process of rat beta cell through the repression of transcription factor FOXO1;it's beneficial to the prevention and cure for type 2 diabetes mellitus.
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Objective Rab11 GTPases play an essential role in regulating membrane trafficking pathways in eukaryotic cells. Nonetheless, there has been little work done on characterizing the transport machinery of Trichomonas. The aim of this study is to clone and characterize a Rab11 gene of Trichomonas vaginalis.Methods A cDNA expression library was constructed with T. vaginalis total RNA. A cDNA clone, which showed a high degree of homology with Rab proteins of different species, was isolated and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST and ClustalW programs. The genomic DNA corresponding to the cDNA sequence was amplified using PCR techniques and following by sequencing. Results cDNA with a length of 710 base pairs and an open reading frame of 636 bp was obtained. The deduced amino acid sequence from the open reading frame was found to possess 211 residuals. Sequence analysis demonstrated that this cDNA clone was homologous to the Rab11 subfamily of different species (60% identity and 79% similarity with Arabidopsis thaliana Rab11c, 58% identity and 78% similarity with human Rab11b), and that the amino acid sequence contains all the well known conserved sequence elements of Rab family. Specific Rab motifs were also detected in the deduced amino acid sequence. Phylogenetic analysis showed that its closest homologues are Rab11 proteins from other species. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA sequence encompassing the putative 5'-ATG and 3'-stop codon is identical to the cDNA sequence.Conclusion A cDNA clone corresponding to the T. vaginalis Rab11 gene was obtained.The function of this gene in regulating membrane trafficking pathways of the parasitic protist is still under investigation.
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LA G1 was identified as a gene that is differentially expressed during the yeast replicative life span and was shown to play a role in determining yeast longevity. The cDNA of rat LASS1, the mammalian homolog of yeast LA G1, was cloned from rat cerebral cortex and sequenced, which is different to the predicted sequence in the GenBank. Sequence analysis revealed that this cDNA clone contains an open reading frame of 1 053 bp. The deduced amino acid sequence has 350 residues and shares a predicted Laglp motif and a TLC domain conserved in Lag1 proteins. Total RNAs were isolated from rat cerebral cortices at varying ages: newborn, one month, six months, twelve months, and twenty-four months. Semi-quantitative RT-PCR and Northern blot analysis were performed to analyze the LASS1 expression level in rat cerebral cortex tissues at varying ages. Senescence-associated β-galactosidase (SA-β-gal) activity was firstly used as a biomarker for assessing senescence in rat neurons. The results showed that LASS1 expression was upregulated from newborn to adult rats (1～6 month) and declined in aged cortex. SA-β-gal staining positive neurons significantly increased in the aged cerebral cortex. The age-related expression alternation of LASS1 in rat cerebral cortex provides an important clue in exploring the role of LASS1 in mammalian neuron aging.
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Objectives The ferredoxins are iron-sulfur proteins, which function in electron transfer reactions in a variety of systems and participate in the activation of the antimicrobial agent metronidazole. The aim of this study is to clone and characterize ferredoxin genes of Trichomonas vaginalis. Methods A cDNA expression library was constructed with T. Vaginalis total RNA. Hundreds of cDNA clones were isolated and sequenced. Sequence analysis was performed using BLAST programs, ClustalW program, etc. Results One of the cDNA clones, which has homology with T.vaginalis ferredoxin, was further analyzed. This cDNA clone has an open reading frame of 312 base pairs. The deduced precursor protein contains 103 amino acid residues with a hydrogenosome targeting sequence (MLSQCSPLRF) at the N-terminal end. The primary sequence analysis revealed that this new ferredoxin (TvFd2) has a high homology (69% identity) to the previous reported T.vaginalis ferredoxin(TvFd). Interestingly, TvFd2 is homologous to both the two subclasses of (2Fe-2S) ferredoxins, the oxidase ferredoxins and the photosynthetic ferredoxins,but with low similarity. The conserved four-cysteine residues, which are predicted to form the iron-sulfur cluster,are arranged in a typical pattern of (2Fe-2S)ferredoxins(-C-X5-C-X2-C-Xn-C-). Conclusion These data show that TvFd2 is a putative new (2Fe-2S) ferredoxin of T.vaginalis. Its biological function remains to be studied.
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Objective To clone and characterize a RRas-like gene from Trichomonas vaginalis for studying cellular signal transduction pathways in the organism. Methods A cDNA clone, which showed homology with RRas proteins of human being, was isolated and sequenced from a cDNA expression library of T. vaginalis. The genomic DNA corresponding to the cDNA sequence was amplified using PCR technique and sequenced. Sequence analysis was per-formed using BLASTP, RPS-BLAST and ClustalW programs. Phylogenetic tree was constructed and bootstrapped with 1 050 replicates using the software MEGA3. Results The cDNA sequence showed a length of 705 bp with an open reading frame of 615 bp. The deduced amino acid sequence from the open reading frame possesses 205 residuals. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA sequence encompassing the putative 5′-ATG and 3′-stop codons was identical to the cDNA sequence. Sequence analysis demonstrated that this gene was most homologous to the RRas members of Homo sapiens and Mus musculus (both having 51% identity and 70% similarity), and the amino acid sequence contains highly conserved GTP-binding domains and a fully conserved effector domain of human RRas member. Phylogenetic analysis showed that TvRRas clustered with RAS oncoprotein branch and RRAS branch of human. Conclusion The encoding protein probably belongs to a RRas family of T. vaginalis.
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Total RNA was isolated from Trichomonas vaginalis and Tv-Sir2-like cDNA was amplified by RT-PCR and cloned into pGEM-T Easy plasmid. A fragment of Tv-Sir2-like cDNA was subcloned into the expression vector pET-41b and expressed in E.coli BL21 with induction of IPTG. The full-length of Tv-Sir2-like cDNA was cloned and sequenced. The prokaryotic expression system of pET-41b/Tv-Sir2-like was constructed. The fusion protein of Tv-Sir2-like was expressed in E.coli BL21, occupying 30% of the total bacterial protein after being induced by IPTG for 5 h. SDS-PAGE analysis showed that the fusion protein was about Mr 59 000. The recombinant protein of Tv-Sir2-like is efficiently expressed in E.coli BL21.