The study was aimed to investigate the expression of Rac1 in human acute leukemic cell line HL-60 and effect of Rac1 on cell cycle progression and apoptosis. The mRNA expression of Rac1 in HL-60 cell line and normal human peripheral blood mononuclear cells (PBMNC) were examined by semi-quantitative RT-PCR. After transfection of HL-60 cells with different concentrations of Rac1 antisense oligodeoxynucleotide (ASODN) by means of FuGENE6, the survival, cell cycle, apoptosis of HL-60 cells were observed through MTT assay, FCM test, Wright-Giemsa, acridine orange/ethidium bromide (AO/EB) and Annexin V-FITC/PI staining test respectively. The results showed that Rac1 relative amount in HL-60 was 0.84 +/- 0.13, while it in the normal PBMNC was 0.26 +/- 0.1 (P < 0.01); the expression of Rac1 in HL-60 cells was significantly upregulated. Compared with sense oligodeoxynucleotide (SODN), HL-60 cell viability, after exposure to ASODN at a concentration of 2.0 g/L decreased, (73.7 +/- 5.0)% vs (93.2 +/- 3.0)% (P < 0.01), while the proportion of G(1) cells increased as (52.1 +/- 6.8)% vs (31.6 +/- 4.7)% (P < 0.05), the percentage of Annexin V positive cells increased, (19.2 +/- 2.1)% vs (4.1 +/- 1.7)% (P < 0.01), and HL-60 cells were observed to have formation of apoptotic bodies. The data presented indicate that Rac1 may be involved in regulation of HL-60 cell cycle and apoptosis, promote overproliferation of HL-60 cells and inhibit their apoptosis.
Apoptosis ; physiology ; Cell Cycle ; physiology ; HL-60 Cells ; Humans ; Oligonucleotides, Antisense ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; rac1 GTP-Binding Protein ; biosynthesis ; genetics ; physiology

OBJECTIVETo investigate the role of antiapoptotic Bcl-x(L) protein in megakaryocyte differentiation and maturation.

METHODSRNA interference was used to block the expression of Bcl-x(L) when K562 cells were induced to differentiate into megakaryocyte (CD61 + cells) by PDBu, and the expression of Bcl-x(L) was evaluated with flow cytometry and reverse transcription polymerase chain reaction (RT-PCR). The CD34 + cell fraction was positively isolated by using the MiniMACS system from normal bone marrow. Immunochemical staining and flow cytometry were used to detect the expression of Bcl-x(L) in the differentiation (CD41 + cells) of CD34 + cells induced by trombopoietin (TPO).

RESULTSAmong K562 cells induced by PDBu, the percentage of CD6L + cells rapidly increased in 24 hours and maintained at a high positive level in 72 hours. When exposured to si-Bcl-x(L), the percentage of CD6 1 + cells only slightly increased in 72 hours. The expression of Bcl-x(L) mRNA was significantly decreased after transfection compared with that of control group, and Bcl-x(L) protein expression decreased correspondingly. After the CD34 + bone marrow cells having been treated with TPO for 5 days to 20 days, the Bcl-x(L)-megakaryocytes increased as the culture time prolonged, and there was a strong expression of Bcl-x(L) in immature megakaryocyte and an obviously decreased expression in degenerating megakaryocytes maturation.

CONCLUSIONSIncreased expression of antiapoptotic Bcl-x(L) may be essential to mature megakaryocyte. The down-regulation of antiapoptotic Bcl-x(L) in mature megakaryocyte may be crucial to platelets formation.

Cell Differentiation ; Humans ; K562 Cells ; Megakaryocytes ; physiology ; RNA Interference ; bcl-X Protein ; biosynthesis ; genetics ; physiology

Spermiogenesis is a complex process leading to the formation of motile spermatozoa characterized by a highly stable chromatin compaction that transfers the paternal genome into the oocyte. It is commonly held that these haploid cells are devoid of transcriptional and translational activities and that the transcripts represent remnants of stored mRNAs. Recently, the chromatin organization of mature spermatozoa has been revisited as a double nucleoprotamine-nucleohistone structure possessing less-condensed regions sensitive to nuclease activity, which could be implicated in the expression of genes involved in the early embryo development. The existence of a complex population of mRNAs in human sperm is well-documented, but their role is not yet elucidated. Evidence for a latent transcriptional capacity and/or a potential de novo translation in mature spermatozoa from fertile men are essential for understanding the last steps of sperm maturation, such as capacitation and acrosome reaction. As such, we have documented the relationship between sperm quality and the distribution of sperm RNAs by showing divergent levels of transcripts encoding for proteins involved in either nuclear condensation (protamines 1 and 2) or in capacitation (eNOS and nNOS, c-myc) or in motility and sperm survival (aromatase) between low and high motile sperm issued from the same sample. Therefore, analyzing the profile of mRNAs could be helpful either as a diagnostic tool for evaluating male fertility after spermatogenesis or for prognosis use for fertilization.
Chromatin ; ultrastructure ; Humans ; Male ; Protein Biosynthesis ; RNA, Messenger ; genetics ; Spermatogenesis ; genetics ; physiology ; Spermatozoa ; physiology ; Transcription, Genetic

OBJECTIVETo investigate whether the sonic hedgehog signaling pathway is involved in the development of human fetal prostate, and to evaluate the changing staining patterns of its molecules, sonic hedgehog (SHH), patchedl (PTC1), smoothened (SMO), and GLI1, in the human fetal prostate at various gestation stages.

METHODSFifteen human fetal prostate specimens at various developmental stages (10 - 39 weeks) were included in this study. SHH, PTC1, SMO and GLI1 were detected in all the specimens by immunohistochemical technique. All the slides were observed and assessed under the light microscope.

RESULTSSHH, PTC1, SMO and GLI1 could be detected in human fetal prostate tissues, and their expression formed two surges, the former at week 16, and the latter at week 28. The staining of SHH and SMO was distributed only in the ductal epithelium but not in the stroma. The expression of PTC1 and GLI1 could be found mainly in the epithelium, with minimal staining in the stroma.

CONCLUSIONThe sonic hedgehog signaling pathway is involved in the development of the human fetal prostate. The high expression of its molecules at early gestation stages might be associated with the induction of prostatic buds, while their abundant expression at later gestation stages might be related to the prostate ductal branching, growth, differentiation and morphogenesis.

Gene Expression Regulation, Developmental ; physiology ; Hedgehog Proteins ; biosynthesis ; Humans ; Male ; Oncogene Proteins ; biosynthesis ; Patched Receptors ; Prostate ; embryology ; metabolism ; Receptors, Cell Surface ; biosynthesis ; Receptors, G-Protein-Coupled ; biosynthesis ; Signal Transduction ; physiology ; Smoothened Receptor ; Trans-Activators ; biosynthesis ; Zinc Finger Protein GLI1

Animals ; Fatty Liver ; metabolism ; Humans ; Ion Channels ; biosynthesis ; genetics ; physiology ; Mitochondrial Proteins ; biosynthesis ; genetics ; physiology ; RNA, Messenger ; biosynthesis ; genetics ; Uncoupling Protein 2

OBJECTIVETo study the significance of Fas and Fas-L expression in adenocarcinoma of uterine cervix.

METHODSBoth carcinoma tissue and their surrounding tissues from 36 patients with adenocarcinoma of uterine cervix, previously untreated either by radiation or chemotherapy, were studied for the expression of Fas and Fas-L by immunohistochemical stain with DNA apoptosis fragment detected by TUNEL.

RESULTSThe TUNEL labeling index was negatively correlated with differentiation of adenocarcinoma of cervix. Compared to highly differentiated and moderately differentiated tumor, the TUNEL labeling index was reduced obviously in poorly differentiated adenocarcinoma (P < 0.01). Fas expression was detected in 31 cases (86%) while there were only 3 weakly stained in the normal endocervical glands around the carcinoma. The 5 unstained carcinomas were 3 highly differentiated and 2 moderately differentiated. The positively stained Fas was associated with differentiation; the stronger the stain, the less differentiation there was. The Fas-L expression was detected in all adenocarcinomas while there was only 1 weakly stained in the normal ones. No significant difference was found in the expression of Fas-L in carcinomas with different degrees of differentiation. No correlation was observed between Fas and Fas-L expression.

CONCLUSIONSThe Fas expression is positively correlated with the different degrees of differentiation and Fas-L expression may be associated with the escape from of immunal surveillance.

Adenocarcinoma ; diagnosis ; metabolism ; Apoptosis ; physiology ; Biomarkers, Tumor ; biosynthesis ; Cell Differentiation ; physiology ; Fas Ligand Protein ; Female ; Humans ; Immunohistochemistry ; Membrane Glycoproteins ; biosynthesis ; Uterine Cervical Neoplasms ; diagnosis ; metabolism ; fas Receptor ; biosynthesis

Hepcidin is a small cystein-rich cationic peptide produced mainly by the liver. It was initially isolated from human plasma and exhibited antimicrobial activity. Recently, several lines of evidence have suggested that hepcidin is a key regulator of iron metabolism at the whole body level and is relative to inflammation, infection, hypoxia and anemia. Hepcidin, is implicated in duodenal iron absorption and iron mobilization from reticuloendothelial macrophages. The major mechanism of hepcidin function seems to be the regulation of transmembrane iron transport. As both iron deficiency and iron excess are associated with cellular dysfunction, so hepcidin or hepcidin-related therapeutics could find a place in the treatment of various diseases such as hemochromatosis and anemia of chronic disease. To elucidate biological function of hepcidin further and use it for other research, it is necessary to produce enough hepcidin through DNA recombinant technique. As a highly successful system for the production of a variety of heterologous proteins, the methylotrophic Pichia pastoris system has the probability for a high level production of hepcidin. The subject of this paper is to summarize the regulation of hepcidin gene expression and the understanding of functions of hepcidin. At last, giving a prospect of production hepcidin by gene engineer.
Antimicrobial Cationic Peptides ; biosynthesis ; genetics ; physiology ; Hepcidins ; Humans ; Iron ; metabolism ; Protein Engineering ; methods

Noncoding RNAs (ncRNAs) have played a critical role in cellular biological functions. Recently, some peptides or proteins originating from annotated ncRNAs were identified in organism development and various diseases. Here, we briefly review several novel peptides translated by annotated ncRNAs and related key functions. In addition, we summarize the potential mechanism of bifunctional ncRNAs and propose a specific "switch" triggering the transformation from the noncoding to the coding state under certain stimuli or cellular stress. The coding properties of ncRNAs and their peptide products may provide a novel horizon in proteomic research and can be regarded as a potential therapeutic target for the treatment of various diseases.
Animals ; Calcium/metabolism* ; Humans ; Open Reading Frames ; Protein Biosynthesis ; RNA, Messenger/genetics* ; RNA, Untranslated/physiology*

OBJECTIVETo observe the effect of small interfering RNA (siRNA)-induced MAPK p42 silencing on the survival of HeLa cells.

METHODSTwo siRNAs targeting at the MAPK p42 gene and one random siRNA were synthesized respectively by Silencer siRNA Construction Kit and transfected into HeLa cells by Lipofectamin 2000. The expression of p42(MAPK) in the transfected HeLa cells was analyzed by Western blotting and immunohistochemistry, and the morphology of cells were observed with electron microscope. TUNEL assay and Annexin V/PI staining were employed for detecting the cell apoptosis.

RESULTSThe expression of p42(MAPK) in the HeLa cells was remarkably suppressed after transfection with the two siRNAs, reduced by about 2.5 and 3.2 folds respectively in comparison with the negative control. Chromatin margination in the cell nuclei were observed in the transfected cells, and TUNEL assay and Annexin V/PI staining further confirmed the occurrence of cell apoptosis.

CONCLUSIONIn vitro MAPK p42 siRNA-1 and siRNA-2 transfection can specifically silence the gene expression and induce apoptosis of HeLa cells.

Apoptosis ; physiology ; Gene Silencing ; physiology ; HeLa Cells ; Humans ; Mitogen-Activated Protein Kinase 1 ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Transfection

OBJECTIVESTo identify whether Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) can induce tumor necrosis factor receptor-associated factor 1 (TRAF1) expression and promote its anti-apoptosis activity via the NF-kappaB signaling pathway, and assess that LMP1 suppresses apoptosis in nasopharyngeal carcinoma (NPC).

METHODSA stable transfected cell line HNE2-LMP1 was established by introducing LMP1 cDNA into HNE2 cells. Transactivation of TRAF1 was determined by luciferase reporter assay, while expression of TRAF1 mRNA was detected by RT-PCR and expression of TRAF1 protein and caspase 3 by Western blot analysis. Apoptosis activity was observed through fluorescence staining.

RESULTSLMP1 induced TRAF1 expression in NPC cells and caused a decrease in apoptosis. This induction could be blocked by antisense LMP1. Moreover, LMP1-mediated induction of a TRAF1 promoter-driven reporter gene was significantly impaired when the kappaB site kappaB1 or kappaB5 was disrupted, whereas mutation of kappaB3 had only a minor effect on LMP1 dependent up-regulation of the reporter gene.

CONCLUSIONLMP1 induces TRAF1 expression and promotes its anti-apoptosis activity via the NF-kappaB signaling pathway, which may be one of the mechanisms that LMP1 uses to suppress apoptosis in NPC cells.

Apoptosis ; physiology ; Humans ; NF-kappa B ; physiology ; Nasopharyngeal Neoplasms ; physiopathology ; Protein Biosynthesis ; Signal Transduction ; physiology ; TNF Receptor-Associated Factor 1 ; Tumor Cells, Cultured ; Viral Matrix Proteins ; physiology