Obtaining vacuolar protein sorting 34 gene in Sporothrix globosa by RACE method and analysis of its expression during the mycelium-to-yeast transition / 中华微生物学和免疫学杂志

Objective To obtain the full-length sequence of the vacuolar protein sorting 34 coding gene (vps34) of Sporothrix globosa (S. globosa) and to investigate the role of vps34 gene during the phase transition from mycelium to yeast in S. globosa. -ethods The 3′ end and 5′ end of the vps34 gene of S. globosa were amplified by rapid amplification of cDNA ends ( RACE ) . The obtained sequences were spliced and analyzed by bioinformatics software. Quantitative reverse transcription PCR ( qRT-PCR ) was used to analyze the expression of vps34 gene in mycelial and yeast phases. Results The vps34 gene of S. globosa was 3228 bp in length. The coding sequence was 3000 bp and encoded 999 amino acids with a mo-lecular mass of 111. 49×103 and an isoelectric point of 6. 38. It contained three domains including C2 PI3K class Ⅲ, PI3Ka Ⅲ and PI3Kc Ⅲ. The results of qRT-PCR showed that the expression of vps34 gene in yeast-phase S. globosa was higher than that in mycelial phase at 24 h (P<0. 05), and the greatest difference between them was observed at 48 h (P<0. 01). Conclusions Vps34p participates in the process of dimor-phic transformation of S. globosa. The obtainment of the full-length vps34 gene of S. globosa lays the founda-tions for further study on the function of Vps34p.

Effects of SPARC on expression of TGF-βl and collagen type Ⅰ in keloid fibroblasts / 中华医学美学美容杂志

Objective To observe the effects of secreted protein,acidic and rich in cysteine (SPARC) on the expression of TGF-β1 and collagen type Ⅰ in cultured human keloid fibroblasts by real-time fluorescence quantitative RT-PCR.Methods In vitro keloid fibroblasts were stimulated by different concentrations of recombinant human SPARC,and with the control group for comparison,real-time fluorescence quantitative RT-PCR to detect expression of TGFβ1 and collagen type Ⅰ.Results Compared with the control group,the expression of TGF-β1 and collagen type Ⅰ was significantly increased in the experimental group.Conclusions SPARC could enhance the expression of TGF-β1 and collagen type Ⅰ in keloid fibroblasts significantly.

Development of Quantitative Reverse Transcription-Polymerase Chain Reaction for the Mesurement of Angiotensin Converting Enzyme mRNA

BACKGROUND: The analysis of ACE gene expression in vital to study the role of angiotensin conveting enzyme(ACE) in the pathogenesis of cardiovascular disease. Traditionally, levels of individual mRNA expression have been analyzed by semiquantitative Northern blotting, which requires a large quantity of tissue. Therefore, gene expression of a little biopsy specimen from the human heart or atherectomy specimen from the blood vessel cannot be measured easily. Reverse transcription-polymerase chain reaction(RT-PCR) is very effective, sensitive and rapid method of detecting the method of quantitative RT-PCR(QRT-PCR) using recombinant RNA template as internal standard to measure the expression of ACE. METHOD: Recombinant RNA(rcRNA) was designed to yield PCR product which differs in size by about 200bp from that of the target RNA. Initially, spacer gene, which was composed of ACE sense primer, antisense primer, T7 promotor and poly(dT) tail with glutathione transferase(GSTM) gene of 180bp in the middle, was constructed. Then, standard rcRNA was obtained by in vitro transcription. Target RNA was mixed with rcRNA and amplified by PCR, togather with P-dCTP. PCR products were analyzed by gel electrophoresis. For quantitation, either gel was cut and radioactivity was counted or gel was dried and exposed to X-ray film and density was measured using image densitometer. We carried out semiquantitative RT-PCR to study the modulation of ACE expression in vascular smooth muscle cell(VSMC) by dexamethasone and basis FGF(bFGF). RESULT: The size difference of PCR products from the standard RNA and the extracted target RNA was matched as designed. By using QRT-PCR, there was 1.7*10(8) ACE mRNA molecules in 1 ng of rat lung total RNA. bFGF and dexamethasone upregulated ACE mRNA expression in cultured VSMC. CONCLUSION: These results suggest that RT-PCR using rcRNA as internal standard is a very useful method for quantitation or semiquantitation of ACE mRNA from a small amount of tissue or cultured cells. Expression of ACE in VSMC can be modulated by various stimuli such as basic FGF and dexamethasone. QRT-PCR could be widely used in the studies of expression of specific human genes.