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Detection of CTP gene expression in the asexual erythrocytic stages of Plasmodium falciparum (FCC1 /HN) by reverse transcriptase- polymerase chain reaction and construction of eukaryotic expression vector of CTP gene

Huihong CHEN ; Xinbing YU ; Zhongdao WU ; Jin XU ; Jiahai LU

Journal of Tropical Medicine.2001;1(1):10-12.

Objectve To detect whether the CTP(phosphocholine cytidylyltransferase) gene was expressed in the asexual erythrocytic stages of Plasmodium falciparum (FCC 1/HN )by using the RT - PCR and to construct eukaryotic expression vector of CTP. Method The erythrocytic stage parasites of Plasmodium falciparum were cultured as described by Trager and Jensen. RNA from erythrocytic stage parasite was extracted by using Trizol reagent. The complete genes coding for CTP gene isolates FCCI/HN were amplified by reverse transcriptase -polymerase chain reaction(RT- PCR). CTP gene was cloned into eukaryotic expression vector pcDNA3. Results CTP encoding gene was amplified from the erythrocytic stages of Plasmodiumfalciparum (FCC 1/HN) and eukaryotic expression vector of CTP was constructed. Conclusion CTP gene was expressed in the erythrocytic stages of Plasmodium falciparum (FCC 1/HN) and eukaryotic expression vector of CTP was successfully constructed.

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The Importance of Helper T-cell Epitope in Vaccine Development

Waiyan Candy NG ; Mingzhao FUNG

Journal of Tropical Medicine.2002;2(3):207-214.

The major challenge in vaccine development a gainst various disease-causing organisms is to use defined antigen to stimulate appropriate immune responses that lead to resistance. The use of peptide-based vaccine is gaining greater attention asits flexibility in the incorporation of multiple defined and different epitopes into a single construct for eliciting d esirable arms of the immune system. It is generally safer than the use of live a ttenuated vaccine while it is relative ease of manufacture than subunit vaccine. However, development of peptide-based vaccine faces significant challenges. This approach has limited ability to elicit immune responses in a genetically dive rse outbred population due to the Major Histocompatibility Complexes (MHC) polym orphism. For the same reason, peptide immunizations often elicit inadequate cyto toxic T lymphocyte (CTL) and antibody (Ab) responses due to the lack of appropri ate helper Tlymphocyte (HTL) activity. Another possible disadvantage of using linear peptide construct is that, for eliciting appropriate antibody responses, s urface immunoglobulin (Ig) receptor clustering is needed in order to activate th e resting B cells. Problems caused by MHC polymorphism may be circumvented by the use of promiscuous T-cell epitopes. Promiscuous T-cell epitopes from the mea sles virus F protein (amino acid [aa] 288 to 302) and a murine defined T-helper cell epitope (V1E8, aa 191-209) that bind to multiple MHC molecules have bee n identified and have been used in highly immunogenic constructs to overcome hap lotype-restricted immune responses. Synthetic non-natural Pan DR Epitope (PADR E) which have degenerate binding capacity to several common HLA-DR can enhance immunogenicity of short-peptide immunogen, both in terms of absolute titers and quality of antibody responses. Besides, a number of so called "promiscuous" T -cell epitopes from Influenza virus hemagglutinin (HA), Plasmodium falciparum pre-erythrocytic stage antigens and mycobacterial proteins have been reporte d to be universally immunogenic. For promiscuous binding to several isotypic and allotypic forms of MHC class Ⅱ molecules, these peptides should display overla pping MHC-binding motifs or should use anchors that are conserved among ligands and should lack allele-specific anchor residues that would prevent binding wit h the other class Ⅱ molecules. Understanding the biophysical basis for both the promiscuity and the specificity of peptide recognition by MHC Ⅱ molecules will provide a molecular rationale for strategies to overcome genetic restriction in the context of vaccine design.

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Expression of Human Connexin26 in COS-7 Cells

Cong XU ; Guowei LI ; Zeyu LIU ; Jiawei LIAO ; Panpan XUE ; Binyan TAN ; Jiahai LU

Journal of Tropical Medicine.2007;7(5):401-407.

Objective To construct the eukaryotic expression plasmid for the expression of human Connexin26 in COS-7 cells.Methods Total RNA was isolated from human peripheral blood lymphocytes and used as template for the PCR cloning of the human Connexin26 gene.The human Cx26 cDNA containing the 678 bp whole coding region of the human Connexin26 gene was amplified by PCR using specific primers and cloned into the pCI-neo vector to construct the recombinant eukaryotic expression plasmid,pCI-Cx26.The recombinant plasmid was identified by restriction endonuclease digestion,and transfected into COS-7 cells by liposome.The expression of Cx26 mRNA and the protein were analyzed by RT-PCR and SDS-PAGE,respectively.Results Restriction endonuclease digestion analysis verified successful construction of the recombinant plasmid,pCI-Cx26.The expression of Cx26 mRNA and protein in the transfected COS-7 cells were detected by RT-PCR and SDS-PAGE,respectively.Conclusion The eukaryotic expression plasmid for human Cx26 has been constructed successfully with the capability of expression in COS-7 cells.

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Classification of A Medically Important Group of Gamasid Mites by Numerical Taxonomy in Yunnan, China

Lipu LUO ; Xianguo GUO

Journal of Tropical Medicine.2007;7(1):7-10.

Objective To determine the taxonomic position of medically important gamasid mites and to compare numerical taxonomy with the traditional systematics in the classification of gamasid mites. Methods Based on hierarchical cluster analysis, numerical taxonomy was applied to study fifty-seven species of medically important gamasid mites. Results The results of cluster analysis based on squared euclidean distance showed that Hirstionyssus Fonseca and Echinonyssus Hirst should be separated from Laelapidae Berlese and formed an independent family. The taxonomic position of the remaining species remains the same as those in the traditional systematics. Conclusion Numerical taxonomy can objectively reflect the taxonomic position of the medically important gamasid mites. The result of classification by numerical taxonomy is consistent with traditional systematics for gamasid mites.

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A New (2Fe-2S) Ferredoxin Gene from Trichomonas Vaginalis

Yucai FU ; Hong XU ; Jiaxin ZHANG ; Kehao ZHNAG ; Hong LIU ; Huirong LIN ; Xiaohong ZHENG

Journal of Tropical Medicine.2004;4(4):352-356,381.

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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Regulation of Th1 and Th2 Type Cytokines in the NCC Patients Treated with Praziquantel

Yajie LI ; Weizhe ZHANG ; Xiaoli ZHANG ; Suqin MA

Journal of Tropical Medicine.2004;4(3):227-229,233.

Objective To explore immunological mark and the outcome of disease after pharmacological treatment in human NCC with praziquantel. Methods 35 patients were treated with praziquantel for 6 months. Levels of serum IL-2,IFN-γ, IL-12, IL-5, IL-10 and TNF-α were measured before and after treatment. Results Th1 type cytokines IL-2, IFN-γand IL-12 were up regulated after treatment( P ＜ 0. 01 ) . Levels of TNF-α and Th2 type cytokines IL-5 and IL-10 were remarkable decreased after treatment( P ＜ 0. 01 ). The levels of IL-2, IFN-γ and IL-12 from the patients with good response to the treatment is higher than those with no response to the treatment. Conclusion Th1 type cytokines were up regulated while Th2 type cytokines were down regulated in the NCC patients treated with praziquantel. The protective immunity may be related to the Thl cell activation.

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Expression of Hox Genes in Murine Brain by Human Cytomegalovirus Infection

Ni XIE ; Liyu CHEN ; Minhua LUO

Journal of Tropical Medicine.2007;7(5):408-411,417.

Objective To study the effect of of human cytomegalovirus (HCMV) infection on the expressions of Hox genes.Methods Forty eight kunming mice were randomly divided into infection group (n=32) injected with HCMVAD169 and control group (n=16) injected with saline into their brain.After 7,15,30,and 60 days,the cerebral lesions were observed by pathological method.HCMV antigen was detected by immunohistochemical method and HCMV DNA was detected by polymerase chain reaction (PCR). On the basis of developing HCMV mouse model. reverse trancriptase-polymerase chain reaction (RT-PCR) was applied to determine the expressions of Hox gene in the brains of infected mice.The expression of Hox genes were also analysed with Northern-blot by isotope labelled Hox genes oligonucleotide probes. Results A HCMV infection model was developed and extensive pathological damages in brain tissue of infected mice were observed.Meanwhile.the HCMV-LA and HCMV-DNA were also found in brain tissues of HCMV infected mice.The expression level of Hox genes in control and infected mouse brain were determined by RT-PCR and Northern-blot.RT-PCR and Northern-blot showed that mouse brain expressed Hox-A9,Hox-A10,Hox-A11,Hox-A12,and Hox-A13,but they did not express Hox-B13.After HCMV infection,murine brain was induced to express Hox-B13 gene(P＜0.01),and reached the peak at 30 d after infection.Comparing with the control group,the expression of Hox-A9 and Hox-A11 were down-regulated in infected group (P＜0.05);the expression of Hox-A10 and Hox-A13 were significantly higher in infected groups (P＜0.05).Conclusion The results suggest that HCMVAD169 is able to cause mouse CNS infection and induced the abnormal expressions of Hox genes. which provides more information for understanding the mechanism of congenital abnormal due to HCMV infection and a valuable method of clinical prevention and treatment of HCMV infection.

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A Novel Rabl-like Gene with A 25 bp Intron in Trichomonas vaginalis

Lili LUO ; Xiaoyuan XU ; Mingyan XU ; Yucai FU

Journal of Tropical Medicine.2008;8(7):633-638,648.

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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Induction of Apoptotic-like Cell Death in Trichomonas vaginalis by Metronidazole

Petrus TANG ; Chingcheng HUANG ; Jyhwei SHIN ; Renli ZHANG ; Juli LIU ; Yucai FU

Journal of Tropical Medicine.2007;7(9):837-841,849.

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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Molecular Cloning and Sequence Analysis of Rab11 GTPase in Trichomonas vaginalis

Renli ZHANG ; Mingyan XU ; Jingjie XU ; Shitong GAO ; Dana HUANG ; Yijie GEN ; Yucai FU

Journal of Tropical Medicine.2006;6(3):267-270,283.

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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