Human FLT3/FLK2 gene: cDNA cloning and expression in hematopoietic cells

The human FLT3 cDNA was cloned from a pre-B cell line and characterized. The deduced amino acid sequence shows that FLT3 codes for a receptor-type tyrosine kinase of 993 residues, presenting a strong similarity with the corresponding mouse FLT3/FLK2 protein as well as with the receptors for colony-stimulating factor 1 (CSF1R/FMS) and steel locus factor (SLFR/KIT). An analysis of the expression of the gene using amplification of reverse transcribed FLT3 mRNA by polymerase chain reaction shows that FLT3 is expressed in various lymphohematopoietic cells and tissues, including a series of immature cell lines and leukemias of lymphocytic origin.     

特异性下调GRP78基因表达对卵巢癌细胞耐药性的影响

目的:探讨RNA干扰技术沉默葡萄糖调节蛋白78(GRP78)基因对人卵巢癌细胞耐药性的影响,阐明GRP78基因沉默逆转肿瘤细胞耐药性的生物学机制.方法:构建pSilencerTM3.0-H1-GRP78 siRNA重组质粒,脂质体介导转染至SKOV3/DDP细胞;RT-PCR和Western blotting法检测GRP78基因的蛋白的表达;Western blotting法检测caspase-4和caspase-3蛋白表达;流式细胞术检测细胞凋亡率.结果:转染GRP78 siRNA重组质粒的SKOV3/DDP细胞GRP78基因和蛋白表达较转染空质粒组明显降低(P<0.05);加用顺铂后,转染GRP78 siRNA重组质粒细胞组较未转染GRP78 siRNA重组质粒细胞组caspase-4和caspase-3表达明显增加(P<0.05),细胞凋亡率也明显增加(P<0.05).结论:抑制GRP78基因表达能通过上调caspase-4和caspase-3表达及增加顺铂诱导的SKOV3/DDP细胞凋亡率,降低SKOV3/DDP细胞对顺铂的耐药性.     

Developmental regulation of Na+ / myo-inositol cotransporter gene expression

myo-Inositol plays a role in many important aspects of cellular regulation including membrane structure, signal transduction and osmoregulation. It is taken up into the cells by the Na+ / myo-inositol cotransporter (SMIT). We investigated developmental changes in the expression of SMIT mRNA and protein in the rat. In the fetal rat brain, SMIT mRNA was abundantly and diffusely expressed throughout the whole brain and the spinal cord. Positive signals were expressed in neuronal and non-neuronal cells in these regions. SMIT is gradually down-regulated nearer birth, but intense signals were still detected in the brain at postnatal day one. In the adult rat brain, very weak hybridization signals were detected throughout whole brain except for the choroid plexus where SMIT mRNA expression remained high. In contrast, the pattern of developmental regulation of SMIT gene expression in the kidney was opposite to that seen in the brain. Signals in the kidney were very weak during embryonic stages, whereas SMIT expression increased significantly after birth. These results suggest that myo-inositol and its transporter play an important role in the CNS developmental stage.     

Botulinum ADP-ribosyltransferase C3 induces elevation of the vitelline coat of ascidian eggs

Botulinum exoenzyme C3 ADP-ribosylates a 23 kDa protein of unfertilized eggs of the ascidian, Halocynthia roretzi. Microinjection of C3 into the eggs induced elevation of the egg vitelline coat. Co-injection of heparin or EGTA with C3 inhibited the inducing effect of C3. The vitelline coat of eggs which had been previously co-injected with heparin and C3 was elevated by addition of calcium ionophore, but not by insemination. C3 also induced an increased formation of inositol 1,4,5-triphosphate (IP3) in ascidian egg membranes. Thus the ADP-ribosylation of small GTP-binding protein by C3 seems to be responsible for elevation of the vitelline coat of ascidian eggs through IP3 formation and intracellular calcium mobilization.     

Thiazolidinediones downregulate stearoyl-CoA desaturase 1 gene expression in 3T3-L1 adipocytes

Thiazolidinediones (TZDs) are known to have potent increases of insulin sensitivity. Because peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a receptor for TZDs, is mainly expressed in adipocytes, we tried to search the TZD-targeted genes in mouse 3T3-L1 adipocytes. By the mRNA differential display method, one band repressed by troglitazone was obtained, which corresponded to the partial sequences of the stearoyl-CoA desaturase 1 (SCD1) gene. Troglitazone dramatically decreased SCD1 mRNA levels in 3T3-L1 adipocytes in a dose-dependent manner. Pioglitazone also repressed the SCD1 mRNA expression, whereas WY-14,643 had no apparent effect. Both troglitazone and pioglitazone raised the composition (weight percentage) of myristic acid (C14:0), palmitic acid (C16:0), and stearic acid (C18:0), but lowered the composition of the delta9-cis desaturated fatty acids such as myristoleic acid (C14:1, delta9), palmitoleic acid (C16:1, delta9), oleic acid (C18:1, delta9), and linoleic acid (C18:2, delta9,12). These results indicate that TZDs repress SCD1 activity in 3T3-L1 adipocytes via downregulating SCD1 enzyme gene expression.     

Ice/ced-3 family gene and apoptosis

Apoptosis is a process by which cells carry out their own execution by activating an orderly set of genetic and biochemical program. A genetic pathway of apoptosis has been identified in the nematode Caenorhabditis elegans. The ced-3 gene is required for all programmed cell death in C. elegans. Mammalian homolog of ced-3 has been identified as Ice family which is newly identified cysteine protease. Overexpression of Ice/ced-3 family gene can induce apoptosis in a variety of mammalian cells, and inhibitors of Ice/ced-3 family effectively prevent apoptosis induced by a variety of stimulus. Several housekeeping genes have been shown to be targets of Ice/ced-3 family gene, indicating that activation of Ice/ced-3 can induce irreversible fatal changes of cells.     

The experimental study on expression of BMP3 gene during fracture healing

The role of BMP3 in fracture repair and its basic mechanisms at the molecular aspect has been studied. Fourty-eight New Zealand white rabbits with the fractures in the middle of bilateral radial shafts were used as animal models, and divided randomly into six groups for calluses at the 1st, 2nd, 3rd, 4th, 6th and 8th week after the onset of fracture. The levels and the cellular localizations of expression of BMP3 mRNA were investigated with the nucleotide hybridization techniques. The results revealed that BMP3 gene expression was highly increased in the early phase of fracture repair, and reached its peak at the second week (about 3.4-fold of that of the normal control). The strong expression of BMP3 gene was localized in mesenchymal cells, chondroblasts and osteoblasts. The results suggest that BMP3 plays an important role of bone-induction in the early stage of fracture repair and it works by the way of autocrine or/and paracrine pathway.     

Temporal and spatial expression of distinct troponin T genes in embryonic/larval tail striated muscle and adult body wall smooth muscle of ascidian

During development of the ascidian Halocynthia roretzi, the tadpole larva hatched from the tailbud embryo metamorphoses to the adult with a body wall muscle. Although the adult body wall muscle is morphologically nonsarcomeric smooth muscle, it contains a troponin complex consisting of three subunits (T, I, and C) as do vertebrate striated muscles. Different from vertebrate troponins, however, the smooth muscle troponin promotes actin-myosin interaction in the presence of high concentration of Ca2+, and this promoting property is attributable to troponin T. To address whether the embryonic/larval tail striated muscle and the adult smooth muscle utilize identical or different regulatory machinery, we cloned troponin T cDNAs from each cDNA library. The embryonic and the adult troponin Ts were encoded by distinct genes and shared only < 60% identity with each other. These isoforms were specifically expressed in the embryonic/larval tail striated muscle and the adult smooth muscle, respectively. These results may imply that these isoforms regulate actin-myosin interaction in different manners. The adult troponin T under forced expression in mouse fibroblasts was unexpectedly located in the nuclei. However, a truncated protein with a deletion including a cluster of basic amino acids colocalized with tropomyosin on actin filaments. Thus, complex formation with troponin I and C immediately after the synthesis is likely to be essential for the protein to properly localize on the thin filaments.