[Molecular cloning and localization of Leishmania donovani expression site associated genes-like protein].

OBJECTIVE: To clone the novel gene that specifically expressed in the amastigotes of Leishmania donovani, and observe subcellular localization of the gene encoding protein. METHODS: mRNA from promastigotes and amastigotes of L. donovani were prepared. The novel expressed sequence tag of amastigotes was selected by suppression subtractive hybridization. The expression of the novel gene in different stages of L. donovani was detected by Northern hybridization and semi-quantitative RT-PCR. The subcellular localization of the novel gene encoding protein was observed. RESULTS: The subtractive library of the specifically expressed sequence tag of amastigotes was constructed, and a novel gene designated as expression site associated genes-like protein (ESAGLP) gene was cloned. The full length of ESAGLP cDNA was 2,258 bp. The open-reading frame encoded a polypeptide of 620 amino acid residues. ESAGLP gene expressed only in amastigotes, the encoding protein was localized in the mitochondria. CONCLUSION: The ESAGLP gene is identified as a novel gene which specifically expressed in Leishmania donovani amastigotes, and its encoding protein is localized in the mitochondria.

[Virulence-associated gene profiling of different Leishmania spp].

OBJECTIVE: To investigate the expression level of virulence-associated genes in promastigotes and amastigotes of different Leishmania spp. METHODS: Total RNA was extracted from the promastigotes and amastigotes of Leishmania donovani, L. infantum, L. tropica, L. major and L. mexicana, and relevant strains. According to the reported gene sequences in GenBank, primers were designed in relation to the virulence-associated genes [GDP-mannose pyrophosphorylase (GDPMP), 3'a2rel-related protein (A2rel), beta-galactofuranosyl transferase (LPG1), lipophosphoglycan biosynthetic protein (LPG2), kinetoplast membrane protein 11 (KMP-11), cpc gene for cysteine proteinase (CPC), hydrophilic acylated surface protein (HASPB1), cathepsin L-like cysteine protease (CPB2), cathepsin L-like cysteine proteinase lmcpb2.8 (CPB2.8), Mr 100 000 heat shock protein (CLP b)], and control genes (alpha tubulin gene and GAPDH). Semi-quantitative RT-PCR was performed to detect expression level of these genes in promastigotes and amastigotes of different Leishmania spp. RESULTS: There was a significant difference in the expression profiles of the genes among the promastigotes and amastigotes of different Leishmania spp. The HASPB1 was detected in the amastigotes of all strains and promastigotes of L. donovani, the GDPMP, LPG1, LPG2, CPB2.8, CPB2, CPC, A2rel and CLP b were expressed in the promastigotes and/or amastigotes of the specific Leishmania spp, respectively. None of the stains carried the KMP-11 gene, whereas the amastigotes of L. donovani SC10 strain and L. major 5ASKH strain possessed CPC. CONCLUSION: The expression profile of the virulence-associated genes shows species-specific and stage-specific differences.

[Comparative proteomic analysis of the promastigotes and amastigotes of Leishmania donovani].

OBJECTIVE: To explore the protein profile and identify developmentally regulated proteins of the promastigotes and axenic amastigotes with comparative proteomics technique. METHODS: The total proteins of promastigotes and axenic amastigotes of Leishmania donovani SC6 strain were separated by two-dimensional electrophoresis (2-DE) in a broad pH range (3-10), and the gel was stained with Coomassie blue. The images were analyzed by PDQuest 1.0 software, and the major developmentally regulated proteins were identified by electrospray mass spectrometry. RESULTS: Approximately 700 protein spots were revealed in equivalent proteins of the promastigotes and axenic amastigotes separated by 2-DE, among which more than 90% protein spots showed equivalent quantity and distribution, with 6 proteins up-regulated and 3 proteins down-regulated in axenic amastigotes compared with promastigotes. Five of the 6 up-regulated proteins were with known function, respectively ascribed as Rieske iron-sulfur protein precursor, alpha-tubulin, peroxidoxin 1, dihydrolipoamide acetyltransferase precursor, and mannose-1-phosphate guanylyltransferase. Two of the 3 down-regulated proteins were identified as heat shock protein 70 and beta-tubulin. The functions of the developmentally regulated proteins were related to the carbohydrate/energy metabolism, stress response, or formation of cell membrane/cytoskeleton. CONCLUSION: The findings demonstrate the differences in protein expression profiles between promastigotes and amastigotes.

[RAPD analysis and construction of specific DNA probes between Pinellia ternata and Typhonium flagelliforme].

OBJECTIVE: To establish a molecular marking method to identify Pinellia ternata and Typhonium flagelliforme. METHODS: Twenty-two random oligonucleotide primers were used in RAPD analysis on the genomic DNA of two types of Pinellia ternata in Sichuan and two types of Typhonium flagelliforme in Guangxi. The special fragments were sequenced, marked as probes and then conducted Southern blot. RESULTS: A great deal of special bands was found between Pinellia ternata and Typhonium flagelliforme. A Pinellia ternata specific molecule was screened. CONCLUSION: RAPD analysis and specific DNA probes show potential value in the identification of Pinellia ternata and Typhonium flagelliforme.

Effect of S1P5 on proliferation and migration of human esophageal cancer cells.

AIM: To investigate the sphingosine 1-phosphate (S1P) receptor expression profile in human esophageal cancer cells and the effects of S1P5 on proliferation and migration of human esophageal cancer cells. METHODS: S1P receptor expression profile in human esophageal squamous cell carcinoma cell line Eca109 was detected by semi-quantitative reverse transcription polymerase chain reaction. Eca109 cells were stably transfected with S1P5-EGFP or control-EGFP constructs. The relation between the responses of cell proliferation and migration to S1P and S1P5 expression was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and migration assay, respectively. RESULTS: Both normal human esophageal mucosal epithelium and Eca109 cells expressed S1P1, S1P2, S1P3 and S1P5, respectively. Esophageal mucosal epithelium expressed S1P5 at a higher level than Eca109 cell line. S1P5 over-expressing Eca109 cells displayed spindle cell morphology with elongated and extended filopodia-like projections. The proliferation response of S1P5-transfected Eca109 cells was lower than that of control vector-transfected cells with or without S1P stimulation (P < 0.05 or 0.01). S1P significantly inhibited the migration of S1P5-transfected Eca109 cells (P < 0.001). However, without S1P in transwell lower chamber, the number of migrated S1P5-transfected Eca109 cells was greater than that of control vector-transfected Eca109 cells (P < 0.001). CONCLUSION: S1P binding to S1P5 inhibits the proliferation and migration of S1P5-transfected Eca109 cells. Esophageal cancer cells may down-regulate the expression of S1P5 to escape the inhibitory effect.