Genes encoding Pir51,Beclin 1,RbAp48 and aldolase b are up or down-regulated in human primary hepatocellular carcinoma

AIM:To reveal new tumor markers and target genes from differentially expressed genes of primary tumor samples using cDNA microarray.METHODS: The ^33p labeled cDNAs were synthesized by reverse transcription of message RNA from the liver cancerous tissue and adjacent non-cancerous liver tissue from the same patient and used to hybridize to LifeGrid 1.0 cDNA microarray blot containing 8400 known and unique human cDNA gene targets, and an expression profile of genes was produced in one paired human liver tumor tissue.After a global analysis of gene expression of 8400 genes,we selected some genes to confirm the differential expression using Northern blot and RT-PCR.RESULTS:Parallel analysis of the hybridized signals enabled us to get an expression profile of genes in which about 500genes were differentially expressed in the paired liver tumor tissues. We identified 4 genes, the expression of three-(Beclin 1, RbAp48 and Pir51) were increased and one (aldolase b) was decreased in liver tumor tissues. In addition,the expression of these genes in 6 hepatoma cell lines was also showed by RT-PCR analysis.CONCLUSION:cDNA microarray permits a high throughput identification of changes in gene expression. The genes encoding Beclin 1, RbAp48, Pir51 and aldolase b are first reported that may be related with hepatocarcinoma.

Expression of germ cell nuclear factor in mouse germ cells and sperm during postnatal period

Aim: To assess the spatial and temporal expression of germ cell nuclear factor (GCNF) in male mouse germ cells during postnatal development and in sperm before and after capacitation. Methods: The indirect immun-ofluorescence method with anti-GCNF antiserum was used to investigate the GCNF expression in mice at day 8, 10, 14, 17, 20, 28, 35, 70, and 420 after birth and in sperm before and after capacitation. Results: With the proceeding of spermatogenesis, GCNF was first detected in the nuclei of spermatogonia and a few early stage primary sperma-tocytes at day 8, which was increased gradually at day 10 to 14 inclusive. From day 17 to day 20, the GCNF was concentrated in round spermatids, while both spermatogonia and early stage primary spermatocytes became GCNF negative. From day 28 until day 420, strong GCNF expression was shown in round spermatids and pachytene spermatocytes, while spermatogonia, early primary spermatocytes and elongating spermatids were all GCNF negative. In addition, it was also found that GCNF was localized on the acrosomal cap region of spermatozoa and there was a big change in GCNF expression during capacitation, from 98 % GCNF positive before capacitation to about 20 % positive following capacitation. The localization of GCNF in caput and cauda spermatozoa was similar. Conclusion: GCNF may play important roles in spermatogenesis, capacitation and fertilization.

Spatial and temporal expression of germ cell nuclear factorin murine epididymis

Aim: To investigate the spatial and temporal expression of germ cell nuclear factor (GCNF) in mouse and rat epididymis during postnatal period. Methods: The epididymal sections from different postnatal days were stained for GCNF by the indirect immunofluorescence technique and digital photographs were taken by a Carl Zeiss confocal microscope. Results: GCNF was first detected on day 12 in mouse epididymis and day 14 in rat epididymis. The highest expression of GCNF was observed on day 35 in both mouse and rat epididymis. In adults, GCNF exhibited a region-specific expression pattern, i.e., it was expressed predominantly in the initial segment, caput and proximal corpus of rat epididymis and was abundant in the proximal corpus of mouse epididymis. GCNF could be found in the nuclei of the principal, apical, narrow, clear and halo cells. Conclusion: GCNF may play an important role in epididymal differentiation and development and in sperm maturation.

Solubility of disulfide-bonded proteins in the cytoplasm of Escherichia co/i and its “oxidizing” mutant

AIM: To study the influence of redox environment of Escherichia coli ( E. coli) cytoplasm on disulfide bond formation of recombinant proteins.METHODS: Bovine fibroblast growth factor (BbFGF) was selected as a model of simple proteins with a single disulfide bond and free cysteines. Anti-HBsAg single-chain Fv (HBscFv), an artificial multidomain protein, was selected as the model molecule of complex protein with 2 disulfide bonds. A BbFGF-producing plasmid, pJN-BbFGF,and a HBscFv producing-plasmid, pQE-HBscFv, were constructed and transformed into E. coli strains BL21(DE3)and M15[pREP4]respectively. At the same time, both plasmids were transformedinto a reductase-deficient host strain, E. coli Origami(DE3). The 4 recombinant E. coli strains were cultured and the target proteins were purified. Solubility and bioactivity of recombinant BbFGF and HBscFv produced in different host strains were analyzed and compared respectively.RESULTS: All recombinant E. colistrains could efficiently produce target proteins. The level of BbFGF in BL21(DE3)was 15-23% of the total protein, and was 5-10% in Origami (DE3). In addition, 65% of the BbFGF produced in BL21(DE3) formed into inclusion body in the cytoplasm,and all the target proteins became soluble in Origami (DE3). The bioactivity of BbFGF purified from Origami(DE3)was higher than its counterpart from BL21(DE3). The ED50of BbFGF from Origami(DE3) and BL21(DE3) was 1.6 μg/L and 2.2 μg/L, respectively. Both HBscFv formed into inclusion body in the cytoplasm of M15[pQE-HBscFv] or Origami[pQE-HBscFv]. But the supernatant of Origami[pQE-HBscFv] lysate displayed weak bioactivity and its counterpart from M15[pQE-HBscFv] did not display any bioactivity. The soluble HBscFv in Origami[pQE-HBscFv]was purified to be 1-2 mg/L and its affinity constant was determined to be 2.62×107 mol/L. The yield of native HBscFv refolded from indusion body in M15[pQE-H Fv] was30-35 mg/L and the affinity constant was 1.98×107 mol/L.There was no significant difference between the bioactivity of HBscFvs refolded from the inclusion bodies produced in different host strains.CONCLUSION: Modification of the redox environment of E. coli cytoplasm can significantly improve the folding of recombinant disulfide-bonded proteins produced in it.