Identification, characterization, and effects of Xenopus laevis PNAS-4 gene on embryonic development.

Apoptosis plays an important role in embryonic development. PNAS-4 has been demonstrated to induce apoptosis in several cancer cells. In this study, we cloned Xenopus laevis PNAS-4 (xPNAS-4), which is homologous to the human PNAS-4 gene. Bioinformatics analysis for PNAS-4 indicated that xPNAS-4 shared 87.6% identity with human PNAS-4 and 85.5% with mouse PNAS-4. The phylogenetic tree of PNAS-4 protein was also summarized. An analysis of cellular localization using an EGFP-fused protein demonstrated that xPNAS-4 was localized in the perinuclear region of the cytoplasm. RT-PCR analysis revealed that xPNAS-4, as a maternally expressed gene, was present in all stages of early embryo development. Whole-mount in situ hybridization showed that xPNAS-4 was mainly expressed in ectoderm and mesoderm. Furthermore, microinjection of xPNAS-4 mRNA in vivo caused developmental defects manifesting as a small eye phenotype in the Xenopous embryos, and as a small eye or one-eye phenotype in developing zebrafish embryos. In addition, embryos microinjected with xPNAS-4 antisense morpholino oligonucleotides (MOs) exhibited a failure of head development and shortened axis.

RNA interference against Biot2, a novel mouse testis-specific gene, inhibits the growth of tumor cells.

Biot2 is a novel murine testis-specific gene that was first identified using the SEREX technique, and named by our laboratory. Using conventional RT-PCR and real time RT-PCR, we tested the expression profile of Biot2 in normal tissues and various murine tumor cell lines. Using RNA interference, we studied the biological function of Biot2 in tumorigenesis. We applied various types of growth assay, such as the in vitro MTT, colony-forming and BrdU incorporation assays, along with in vivo tumorigenicity assays, to reveal its inhibition of tumor cell proliferation. The results revealed that the Biot2 transcript was detected only and strongly in the testis tissues and abundantly in five types of murine cancer cell line. Treating B16 murine melanoma, LL/2 murine Lewis lung carcinoma and CT26 murine colorectal adenocarcinoma with special shRNA targeting Biot2 can significantly reduce the proliferation rate of these three tumor cell lines in vitro, as measured by the MTT, colony-forming and BrdU incorporation assays. The tumorigenicity of the CT26 cells transfected with special shRNA targeting Biot2 was also decreased distinctly in vivo compared with the control. It was therefore concluded that Biot2 plays a key role in tumorigenesis and could be a potential target for biotherapy.

Nuclear FAM289-Galectin-1 interaction controls FAM289-mediated tumor promotion in malignant glioma.

BACKGROUND: FAM92A1-289(abbreviated FAM289) is recognized as one of the newly-discovered putative oncogenes. However, its role and molecular mechanisms in promoting cancer progression has not yet been elucidated. This study was performed to reveal its oncogenic functions and molecular mechanisms in human glioblastoma multiforme (GBM) cell models with knockdown or overexpression of FAM289 in vitro and in vivo. METHODS: To elucidate the molecular mechanisms underlying FAM289-mediated tumor progression, the protein-protein interaction between FAM289 and Galectin-1 was verified by co-immunoprecipitation, followed by an analysis of the expression and activity of Galectin-1-associated signaling molecules. Knockdown and overexpression of FAM289 in glioma cells were applied for investigating the effects of FAM289 on cell growth, migration and invasion. The determination of FAM289 expression was performed in specimens from various stages of human gliomas. RESULTS: FAM289-galectin-1 interaction and concomitant activation of the extracellular signal-regulated kinase (ERK) pathway participated in FAM289-mediated tumor-promoting function. Since the expression of DNA methyl transferase 1 (DNMT1) and DNA methyl transferase 3B (DNMT3B) was regulated by FAM289 in U251 and U87-MG glioma cells, Galectin-1 interaction with FAM289 may promote FAM289 protein into the cell nucleus and activate the ERK pathway, thereby upregulating DNMTs expression. Drug resistance tests indicated that FAM289-mediated TMZ resistance was through stem-like property acquisition by activating the ERK pathway. The correlation between FAM289, Galectin-1 expression and the clinical stage of gliomas was also verified in tissue samples from glioblastoma patients. CONCLUSIONS: Our results suggest that high expression of FAM289 in GBM tissues correlated with poor prognosis. FAM289 contributes to tumor progression in malignant glioma by interacting with Galectin-1 thereby promoting FAM289 protein translocation into the cell nucleus. FAM289 in the nucleus activated the ERK pathway, up regulated DNMTs expression and induced stem-like property gene expression which affects drug resistance of glioma cells to TMZ. This study provided functional evidence for FAM289 to be developed as a therapeutic target for cancer treatment.

Soluble expression of human DRR1 (down-regulated in renal cell carcinoma 1) in Escherichia coli and preparation of its polyclonal antibodies.

Human DRR1 (down-regulated in renal cell carcinoma 1) is widely expressed in normal tissues but dramatically reduced or even undetectable in a number of different cancer cell lines and primary tumour types. DRR1 from Homo sapiens was cloned into the pQE30 vector for fusion-protein expression with six histidine residues in Escherichia coli BL21(DE3). A soluble protein with a molecular mass of approx. 19 kDa on SDS/PAGE that matches the expected rDRR1 (recombinant DRR1) molecular mass (18.7 kDa) was obtained. The soluble and insoluble expression of recombinant protein DRR1 (rDRR1) was temperature-dependent. The expression rDRR1 was in soluble and insoluble forms at 37 degrees C, and approx. 80% of total rDRR1 was soluble at 37 degrees C, while rDRR1 was almost exclusively expressing in soluble form at 20 degrees C. The expressed rDRR1 at 20 degrees C was affinity-purified on Ni(2+)-charged resin under native conditions. The purified protein was further identified by ESI-MS (electrospray ionization MS). The purified recombinant protein rDRR1 was further used to raise anti-(human DRR1) polyclonal antibodies, which were suitable for detecting both the recombinant exogenous DRR1 and the endogenous DRR1 from tissues and cells by immunoblotting and immunohistochemistry. The purified rDRR1 and our prepared anti-(human DRR1) polyclonal antibodies may provide useful tools for future biological function studies on DRR1.

A facile synthesis and fungicidal activities of 2-(alkylamino)-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-ones.

The aza-Wittig reactions of iminophosphorane 3 with aromatic isocyanates generated carbodiimides 4, which were reacted with alkylamines under mild conditions to give a series of 2-(alkylamino)-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-ones 6 and 8 in satisfactory yield. Their structures were confirmed by (1)H NMR, EI-MS, IR and elementary analysis, and compound 8c was further analyzed by single-crystal X-ray diffraction. The preliminary bioassays indicated that these compounds showed excellent fungicidal activities against six kinds of fungi.

Identification and characterization of two novel variants of the DUF1208 protein FAM92A1.

FAM92A1 (named FAM92A1-271) belongs to the family of proteins with conserved DUF1208 domains. Its function remains elusive. We identified two novel transcript variants (FAM92A1-251, FAM92A1-289) of FAM92A1. The presence of these transcripts in cancerous and normal cells, as well as their influence on cell proliferation and apoptosis, were investigated. The subcellular location of FAM92A1 was determined by fluorescence microscopy. We found that FAM92A1-271 and FAM92A1- 289 were highly expressed in both normal and cancerous cells, but FAM92A1-251 was only expressed at a moderate level in both types of cell. Overexpression of FAM92A1-271, FAM92A1-251 and FAM92A1-289 inhibited cell proliferation, caused S-phase arrest and induced apoptosis. Subcellular localization showed that FAM92A1 localizes to the nucleus. Our results show that FAM92A1 has different splicing variants, and that it may take part in regulating cell proliferation and apoptosis.

Biomechanical properties of different segments of human umbilical cord vein and its value for clinical application.

No satisfactory effects have been obtained with the use of synthetic blood vessels (diameter <6 mm) as substitutes for human small arteries or veins for the purpose of clinical vascular reconstruction. Therefore, blood vessels of human origin, for example, umbilical cord blood vessels, with their wide availability, still should be considered. However, little information on biomechanical properties of human umbilical cord blood vessels is available. The objective was to provide a theoretical basis for the clinical application of umbilical cord veins as optional material for small-caliber grafts. This was a nonrandomized, noncontrolled in vitro study. The experiment was conducted in the Laboratory of Medical Biomechanics, Yunyang Medical College. Umbilical cord veins of 20 normal fetuses of spontaneous labor were collected by the Department of Obstetrics and Gynecology, Taihe Hospital in Shiyan City, Hubei Province. The fetuses aged 37-40 weeks, and the parturients were 20-30 years old. Umbilical cord veins of the 20 fetuses were used and the placental ends were treated as proximal ends while the fetal ends as distal ends. The fetal ends were divided into three segments: proximal, middle, and distal segments. The relationship between pressure of umbilical cord veins segments and the diameters was measured on the biomechanical experiment stand for soft tissues, and then the elastic modulus was calculated. The materials were transversely extracted, refrigerated, and sliced up before HE staining. The geometrical morphology indexes were measured by a computer image analysis system (Leica-Q500IW). The main outcome measures were: incremental elastic modulus (E(inc)), pressure-strain elastic modulus (E(p)), volume elastic modulus (E(v)), diameter, and wall thickness of the veins. E(inc), E(p), and E(v) of umbilical cord veins of proximal, middle, and distal segments increased with the pressure elevated. The three kinds of elastic modulus of proximal segments (E(inc): 26.98 +/- 3.21, E(p): 16.58 +/- 2.12, E(v): 8.31 +/- 2.35) were all lower than those of distal segments (E(inc): 33.20 +/- 4.21, E(p): 119.45 +/- 2.87, E(v): 9.71 +/- 1.32) (F = 95.74-126.52, p < 0.05), and a tendency to increase was shown from proximal segments to distal segments. Media thickness [(0.30 +/- 0.05)] mm, (0.24 +/- 0.03) mm] and the diameters [(3.07 +/- 0.12) mm, (2.30 +/- 0.13) mm] decreased gradually from proximal to distal segments (F = 12.76, p < 0.01). It is feasible to use umbilical cord veins as substitutes for the transplantation of small-caliber arteries in terms of basic biomechanical properties. On vascular grafting, different segments of umbilical cord veins should be chosen cautiously so that the biomechanical characteristics of umbilical cord vein grafts could be in accordance with those of host to increase the long-term patency rate of transplanted blood vessels.

The Tumor-promoting Effects of FAM92A1-289 in Cervical Carcinoma Cells.

BACKGROUND/AIM: FAM92A1-289 is recognized as one of the newly-discovered putative oncogenes. This study was performed to reveal its oncogenic functions in human cervical carcinoma cells. MATERIALS AND METHODS: The FAM92A1-289+ cell line was established with knock-in technique and selected by puromycin-resistance screening. Scratch assay, methylthiazol tetrazolium assay, colony forming assay and xenograft test were used to examine cell migration, cell proliferation, cell viability and tumor formation, respectively. RESULTS: FAM92A1-289+ cells showed higher migration rate (p<0.05), higher cell viability (p<0.01), higher colony formation and tumor growth. The FAM92A1-289 protein was pulled-down by antibodies against proliferating cell nuclear antigen (PCNA) in the co-immunoprecipitation assay. CONCLUSION: The up-regulated expression of FAM92A1-289 could facilitate cell migration, boost cell proliferation and promote colony formation in vitro and tumor growth in vivo. The interaction between FAM92A1-289 and PCNA was verified by co-immunoprecipitation. This study provided functional evidence for FAM92A1-289 to be developed as a therapeutic target for cancer treatment.

p120-catenin participates in the progress of gastric cancer through regulating the Rac1 and Pak1 signaling pathway.

p120-catenin (p120), an E-cadherin regulator, has been implicated as central to a series of genetic and epigenetic changes that ultimately lead to tumor progression and metastasis. Ras-related C3 botulinum toxin substrate 1 (Rac1)and p21-activated kinases (PAKs) are effectors of p120. In the present study, we examined the expression of p120, Rac1 and Pak1 using immunohistochemistry in human gastric cancer tissues. Then, we used the gastric cancer SGC7901 and AGS cell lines to explore the possible mechanism of p120, Rac1 and Pak1 in the progress of gastric cancer. Western blotting was used to detect the expression of p120, Rac1 and Pak1 in the two cell lines. Next, p120 was silenced using p120 siRNA or overexpression of p120 by transfection of the plasmid p120 1A into the two cell types, western blotting was used to investigate the expression changes of Rac1 and Pak1. Furthermore, the effects of p120 siRNA-mediated knockdown or overexpression on the proliferation and invasive ability of gastric cancer cells were investigated using wound healing test and Matrigel invasion assays. The results showed that p120 was downregulated in both poorly differentiated group and well differentiated human gastric cancer. However, Rac1 and Pak1 were upregulated in poorly differentiated tissues and remain low in well differentiated gastric cancer tissues. In the two gastric cancer cell lines, although the expression of Rac1 and Pak1 remained unchanged after the p120 knockdown, the expressions of Rac1 and Pak1 protein were decreased after p120 overexpression in both SGC7901 and AGS cells. Furthermore, knockdown of p120 promoted gastric cancer cell proliferation and invasion; overexpression of p120 reduced the proliferation and invasion of gastric cancer cells. In conclusion, based on our results, we speculate that p120 participates in the progress of gastric cancer through regulating Rac1 and Pak1, which provides a potential prevention and a promising therapeutical approach for the patients with gastric cancer.

Pim-1 is up-regulated by shear stress and is involved in shear stress-induced proliferation of rat mesenchymal stem cells.

AIMS: Investigation of the response of mesenchymal stem cells (MSCs) to vascular mechanical forces is very important in the field of cardiovascular intervention. Ser/Thr-protein kinase Pim-1 is a novel transducer of cell survival and the cell cycle that promotes signals in the hematopoietic cell system. Current studies aim to foster an understanding of Pim-1 expression and regulation in MSCs in response to different durations and strengths of laminar shear stress (SS) and to investigate the role of Pim-1 in SS-induced cell proliferation. MAIN METHODS: A parallel-plate flow chamber was used to control the strength and duration of SS. Proliferation was measured with the BrdU cell proliferation assay. The expressions of Pim-1 mRNA and protein were evaluated by reverse transcription-polymerase chain reaction and western blotting, respectively. RNA interference was used to knock down the Pim-1 gene. KEY FINDINGS: The results showed that SS up-regulation of Pim-1 mRNA and protein was time-dependent. Pim-1 induction was SS strength-dependent, and the expression level reached a maximum at 30 dynes/cm(2). Inhibitors of p38MAPK and ERK attenuated the SS-induced expression of Pim-1. In addition, SS significantly increased BrdU-uptake, which was effectively blocked by the silencing of Pim-1. SIGNIFICANCE: These results demonstrated that Pim-1 is expressed in MSCs and plays an important role in the SS-induced proliferation of MSCs.

[Effects of the novel tumor/testis antigen, Biot2, on proliferation of NIH3T3 cell].

AIM: To study the biological effects of the novel tumor/testis antigen Biot2 on proliferation of NIH3T3 cell and to identify the preliminary biological functions of it. METHODS: The Biot2 gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1(+) to construct the pcDNA3.1-Biot2 plasmid. The recombinant plasmid pcDNA3.1-Biot2 was transfected into NIH3T3 cell by lipofectamine 2000, and then the positive clones were screened by G418. The expression of pcDNA3.1(+) and Biot2 mRNA in positive clones were detected by RT-PCR, Realtime RT-PCR and Western blot, respectively. The cell growth curves were measured by MTT assay to study the changes of proliferation of the NIH3T3 cells stably transfected by pcDNA3.1-Biot2 plasmid and the controls. RESULTS: The eukaryotic expression vecter pcDNA3.1-Biot2 was successfully constructed. The expression of pcDNA3.1(+) vecter was found by RT-PCR. Biot2 mRNA transcription and protein were detected by Realtime RT-PCR and Western blot respectively. The growth rate of NIH3T3 cells stably transfected by the recombinant plasmid was faster than that of the controls. CONCLUSION: The stable pcDNA3.1-Biot2 transfected NIH3T3 cell line is successfully established, and the proliferation of the cells stadly transfected is promoted, which will provide experimental basis for further study on biotherapy using Biot2 target antigen.

Isolation and characterization of a novel Xenopus gene (xVAP019) encoding a DUF1208 domain containing protein.

We have identified a novel Xenopus gene (xVAP019) encoding a DUF1208 domain containing protein. Using whole-mount in situ hybridization and RT-PCR, we found abundant xVAP019 maternal transcripts in the animal hemisphere during the cleavage stages and blastula stages. During gastrulation xVAP019 is differentially expressed with higher levels in the animal helf and the highest in marginal zone, then further expressed widely at neuronal stages with strongest signals in the prospective CNS regions and the epidermal ectoderm. Subsequently xVAP019 was expressed predominantly in the head, the eyes, the otic vesicle, branchial arches, spinal cord, notochord, somites, and tailbud. It is absent or very weak in the endoderm. Injecting a morpholino oligo complementary to xVAP019 mRNA or injecting a caped xVAP019 mRNA caused most of embryos to die during gastrulation and neurulation. Overexpression of xVAP019 mRNA also led to eye defect, shorten interocular distance, small body size and abnormal pigment formation in parts of the survival embryos. Similar effects were induced by injecting the xVAP019 human homologous gene FAM92A1. Our results suggest that xVAP019 is essential for the normal ectoderm and axis mesoderm differentiation and embryos survival. This investigation is for the first time in vivo study examining the role of this novel gene and reveals an important role of xVAP019 in embryonic development.

Identification and preliminary function study of Xenopus laevis DRR1 gene.

Xenopus laevis has recently been determined as a novel study platform of gene function. In this study, we cloned Xenopus DRR1 (xDRR1), which is homologous to human down-regulated in renal carcinoma (DRR1) gene. Bioinformatics analysis for DRR1 indicated that xDRR1 shared 74% identity with human DRR1 and 66% with mouse DRR1, and the phlogenetic tree of DRR1 protein was summarized. The xDRR1 gene locates in nuclei determined by transfecting A549 cells with the recombinant plasmid pEGFP-N1/xDRR1. RT-PCR analysis revealed that xDRR1 gene was expressed in all stages of early embryo development and all kinds of detected tissues, and whole-mount in situ hybridization showed xDRR1 was mainly present along ectoderm and mesoderm. Furthermore, xDRR1 expression could suppress A549 cell growth by transfecting with plasmid pcDNA3.1(+)/xDRR1. xDRR1 probably plays important roles involving in cell growth regulation and Xenopus embryo development.