Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

结直肠癌P53蛋白定量分析及其与肿瘤生物学行为的关系

应用MD-20显微图象分析仪对30例结直肠癌的P53蛋白免疫组织化学反应产物进行定量测定,以观察P53蛋白相对含量与肿瘤生物学行为的关系.结果显示:结直肠癌P53蛋白MOD值与PCNA增殖指数呈正相关(P<0.05).P53蛋白MOD值大的肿瘤多里浸润性生长方式,且浸润至浆膜外者多(PM<0.01,P<0.05).淋巴结转移与MOD值无明显关系(P>0.05).结果提示:结直肠癌P53蛋白相对含量对肿瘤的生物学行为有重要影响.     

FATP1 silence inhibits the differentiation and induces the apoptosis in chicken preadipocytes

FATP1 plays an important role in the trafficking of free fatty acids in adipocytes, however, its precise function and relationship with other fatty acid transporters all remain poorly understood. In this study, FATP1 gene silencing was induced by transfecting siRNA of target sequence into chicken preadipocytes, then the expression of FABP was found down-regulated while the expression of FAT was raised. In addition, differential inhibition of the cells was observed and the expressions of PPARγ and C/EBPα were found down-regulated. Moreover, the silencing also induced the down-regulation of FAS and inhibited the adipogenesis in adipocytes. Of specific interest here was that FATP1 silencing significantly improved the expressions and activities of cell apoptotic factors Caspases 3 and BCL2 associated X protein (Bax). Consequently, FATP1 deficiency prevented the differentiation while induced apoptosis in chicken preadipocytes.     

Mechanisms of liquid flux across pulmonary alveolar epithelial cell monolayers

Summary Active transport of sodium by pulmonary alveolar epithelial cells (AEC) is believed to be an important component of edema clearance in the normal and injured lung. Data supporting this premise have come from measurements of sodium movement across AEC monolayers or from perfused lung model systems. However, direct measurement of fluid flux across AEC monolayers has not been reported. In the present work, AEC were studied with an experimental system for the measurement of fluid flux (Jv) across functionally intact cell monolayers. Primary adult rat type II alveolar epithelial cells were cultured on 0.8 μm nuleopore filters previously coated with gelatin and fibronectin. Intact monolayers were verified by high electrical resistance (> 1000 Θ) at 4–5 d of primary culture. At the same time interval, transmission electron microscopy revealed cells with type I cell-like morphology throughout the monolayer. These were characterized by both adherens and tight junctional attachments. Fluid flux across the monolayers was measured volumetrically over a period of 2 h in the presence of HEPES-buffered DMEM containing 3% fatty acid-free bovine serum albumin. Flux (Jv) was inhibited 39% by 1 × 10⁻⁴ M ouabain (P < 0.01) and 27% by 5 × 10⁻⁴ M amiloride (P < 0.05). These data support the concept that AEC Na⁺/K⁺-ATPase and Na⁺ transport systems are important determinants of AEC transepithelial fluid movement in vitro.     

Time-Series Clustering of lncRNA-mRNA Expression during the Adipogenic Transdifferentiation of Porcine Skeletal Muscle Satellite Cells

Skeletal muscle satellite cells (SMSCs), which are multifunctional muscle-derived stem cells, can differentiate into adipocytes. Long-chain non-coding RNA (lncRNA) has diverse biological functions, including the regulation of gene expression, chromosome silencing, and nuclear transport. However, the regulatory roles and mechanism of lncRNA during adipogenic transdifferentiation in muscle cells have not been thoroughly investigated. Here, porcine SMSCs were isolated, cultured, and induced for adipogenic differentiation. The expressions of lncRNA and mRNA at different time points during transdifferentiation were analysed using RNA-seq analysis. In total, 1005 lncRNAs and 7671 mRNAs showed significant changes in expression at differential differentiation stages. Time-series expression analysis showed that the differentially expressed (DE) lncRNAs and mRNAs were clustered into 5 and 11 different profiles with different changes, respectively. GO, KEGG, and REACTOME enrichment analyses revealed that DE mRNAs with increased expressions during the trans-differentiation were mainly enriched in the pathways for lipid metabolism and fat cell differentiation. The genes with decreased expressions were mainly enriched in the regulation of cell cycle and genetic information processing. In addition, 1883 DE mRNAs were regulated by 193 DE lncRNAs, and these genes were related to the controlling in cell cycle mainly. Notably, three genes in the fatty acid binding protein (FABP) family significantly and continuously increased during trans-differentiation, and 15, 13, and 11 lncRNAs may target FABP3, FABP4, and FABP5 genes by cis- or trans-regulation, respectively. In conclusion, these studies identify a set of new potential regulator for adipogenesis and cell fate and help us in better understanding the molecular mechanisms of trans-differentiation.     

Protein kinase Cbeta regulates heterologous desensitization of thrombin receptor (PAR-1) in endothelial cells

We studied the effects of protein kinase C (PKC) activation onendothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca²⁺ concentration inhuman dermal microvascular endothelial cells (HMEC) exposed to-thrombin or phorbol ester,12-O-tetradecanoylphorbol-13-acetate (TPA).Immunofluorescence showed that thrombin and TPA reduced the cellsurface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1desensitization. In contrast, prior activation of PKC with TPA produceddesensitization to thrombin and histamine, indicatingheterologous PAR-1 desensitization. Treatment of cells withstaurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC isozymes(PKC_I andPKC_II) by transducing cellswith antisense cDNA of PKC_Iprevented the TPA-induced decrease in cell surface PAR-1 expression andrestored ~60% of the cytosolic Ca²⁺ signal in response tothrombin. In contrast, depletion of PKC isozymes did not affect theloss of cell surface PAR-1 and induction of homologous PAR-1desensitization by thrombin. Therefore, homologous PAR-1desensitization by thrombin occurs independently of PKC isozymes,whereas the PKC-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.