Cloning,sequence analysis,and prokaryotic expression of cDNA encoding a putative non-specific lipid-transfer protein from the bracts of dovetree (<Emphasis Type="Italic">Davidia involucrata</Emphasis> Baill)

The 967-bp cloneP1A5 was isolated from a suppression subtractive hybridization cDNA library of dovetree bracts (Davidia involucrata Baill.). A complete cDNA of 1047 bp was obtained via 5-RACE (5′Rapid Amplification of cDNA End) techniques, using the gene-specific primer P1A5-1. Northern blot analyses showed that the gene was predominantly expressed in the bracts, while the blotting signal from the leaves was weak. Its deduced amino acid sequence was most highly homologous to the lipid-transfer protein 3 precursor isolated from upland cotton, the lipid-transfer protein SDi-9 from the common sunflower, and the non-specific lipid-transfer protein precursor allergen from sweet cherry. It also had features in common with plant nsLTPs (non-specific lipid-transfer proteins), including eight conserved cysteine residues, a high isoelectric point (8.9), and a lack of tryptophans. The deduced amino acid sequence had two transmembrane helices -- the first from Position 5 (Gly) to Position 35 (Val); the second, from Position 28 (Ala) to Position 46 (Leu). A cleavage site for the putative signal peptide was predicted to occur between Positions 28 (Ala) and 29 (Ala). Therefore, the putative mature form of the protein would comprise 92 amino acids, with a molecular weight of 9.2 kD. All these results provide compelling evidence that theP1A5 clone belongs to the nsLTP1 gene family, thus being named theP1A5 putative nsLTPI gene. This is the first nsLTP gene reported from Davidiaceae. The nucleotide sequence data reported here has been released in the GenBank, EMBL, and DDBJ Nucleotide Sequence Databases, under the accession number AY059472.     

Expression of gelatinases and their tissue inhibitors in rat corpus luteum during pregnancy and postpartum

Extensive tissue remodeling occurs in the corpus luteum (CL) during both formation and luteolysis. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play pivotal roles in these processes. In the present study, to evaluate the potential roles of matrix degrading proteases in luteal development and regression, we examined gelatinases and TIMP-1, -2, -3 mRNA expressions, as well as gelatinase activity in rat CL during pregnancy and postpartum using Northern blot, in situ hybridization, and gelatin zymography, respectively. The results showed that MMP-2 mRNA was only expressed at the early stages of pregnancy; TIMP-2 mRNA was highly expressed at the early and late pregnancy and day 1 postpartum, but could not be detected during the mid-phase of pregnancy; TIMP-3 mRNA expression was abundant during early pregnancy and peaked at day 7, but was absent from other time points examined. MMP-9 and TIMP-1 mRNAs in rat CL were below detectable level in the current study. Furthermore, the active MMP-2 was only present during the early stages of pregnancy, and no MMP-9 activity was observed in the zymogram. Taken together, our results suggest that MMP-2 and TIMP-3 may have functional roles in rat luteal formation, while TIMP-2 may be implicated in both formation and regression of the pregnant CL.     

Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat.

Pseudomonas sp. strain IST 103 (PCP103) capable of utilizing pentachlorophenol (PCP) was determined by utilization of a carbon source and release of the hydroxylating enzyme PCP-4 monooxygenase. The metabolites were extracted from the culture medium and analyzed by high-performance liquid chromatography. The enzyme purified to apparent homogeneity from an extract of PCP-grown cells indicated that a fraction of DEAE-cellulose ion exchange chromatography of molecular size of 30,000 kDa determined by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis was responsible for dechlorination of PCP. The plasmid isolated from the bacterium was subjected to Shotgun cloning by restriction digestion by BamHI, HindIII, and SalI, ligated to pUC19 vector, and transformed into Escherichia coli XLBlue1alpha. The recombinant clones having higher potentiality to degrade PCP were selected by utilization of a carbon source and release of intermediary metabolites during degradation of PCP as the sole source of carbon and energy. The recombinant clones, which contained an insert of 3.0 kb of SalI and HindIII sites, were sequenced and compared with gene sequences deposited in GenBank by BLAST search; this indicated homology with the thdf gene of monooxygenase of thiophene and furan. Southern blot analysis performed by developing gene probes indicated the presence of the PCP monooxygenase gene in plasmids of the bacterium.     

Overexpression of protein-tyrosine phosphatase PTP sigma is linked to impaired glucose-induced insulin secretion in hereditary diabetic Goto-Kakizaki rats

The impaired glucose-induced insulin release in type 2 diabetes mellitus may be accounted for by reduced B-cell ATP/ADP ratio or decreased phosphorylation of proteins regulating exocytosis of insulin. This, in turn, could be due to enhanced phosphatase activity. Using in situ hybridization techniques to assess the expression of 11 different phosphotyrosine phosphatases (PTPases), known to be present in the B-cells, overexpression by approximately 60% of PTP sigma (also known as LAR-PTP2 or PTP NE-3) was demonstrated in pancreatic islets and liver of spontaneously type 2 diabetic Goto-Kakizaki (GK) rats. In agreement with these findings Western blot of islet lysates, using a polyclonal PTP sigma antiserum, showed increased amounts of the protein in GK relative to control rat islets. Exposure of isolated islets for 20 h to 5 muM antisense to PTP sigma, composed of an antisense PNA sequence of 15 bases linked to the cell penetrating peptide transportan, increased glucose-induced insulin secretion from GK rat islets, but not from control islets. In parallel, the amounts of the phosphatase decreased. In conclusion, increased expression of PTP sigma may be of pathogenetic significance for the defective insulin secretion in GK rat islets.     

PCR和Southern Blot检测土拉弗氏菌气溶胶

为提高检测土拉弗氏菌的特异性和敏感性,建立了土拉菌ＰＣＲ及核酸杂交检测方法。运用平板计数、多聚酶链反应对土拉菌气溶胶稳定性进行了比较,结果表明ＰＣＲ具有较高灵敏度,并且在采样后３小时ＰＣＲ就可以得出定性结果,而平板计数则需要３～７天。采用ＰＣＲ法合成了土拉菌３７６－ｂｐ探针,分别对细菌菌液、５６８－ｂｐＰＣＲ产物和气溶胶样品进行杂交,结果表明菌悬液直接杂交可检出１０５ＣＦＵ左右的细菌,检测ＰＣＲ产物可达４０ｐｇ。ＰＣＲ和Ｓｏｕｔｈｅｒｎ印迹相结合有利于细菌的分离鉴定     

Cytochrome P450s of the 4A Subfamily in the Brain

Abstract: Members of the P450 4A subfamily are key enzymes in the synthesis and degradation of metabolites of arachidonic acid, which are of physiological importance in the brain. In the rat, four members of this subfamily, 4A1, 4A2, 4A3, and 4A8, have been described. In this study, the expression of members of the 4A subfamily in the rat brain has been examined by PCR amplification, by western and northern blotting, and by protein N-terminal sequencing. With PCR all four members of the subfamily were detectable in the liver and kidney. P450 4A1 was found exclusively in the liver and kidney, whereas P450 4A2 was detectable in all the tissues tested, including the lung, seminal vesicles, prostate, cerebral cortex, hypothalamic preoptic area, cerebellum, and brainstem. The tissue distribution of P450 4A3 was similar to that of 4A2 except that it was not detectable in seminal vesicles. A P450 4A8-specific fragment was amplified from the kidney, liver, and prostate and weakly from the cerebral cortex but not from other brain regions. Despite the evidence of their presence by PCR, no members of the 4A family were detectable on northern blots with mRNA from the brain. On western blots a P450 4A-specific antiserum recognized a band in P450 fractions prepared from the brain. The intensity of the signal with 30 pmol of P450 from the brain was similar to that with 10 pmol of liver microsomal P450. The brain P450 was extracted from 1 g of brain, whereas the 10 pmol of liver P450 is the equivalent of 1 mg of liver. This suggests a brain content of 4A P450 that is 0.1% of that in the liver. N-terminal sequencing of the protein bands in the brain P450 fraction revealed the presence of both P450 4A8 and 4A3. These data show the presence in the brain of forms of P450 whose level of mRNA is too low to be detected on northern blots. The specificity of tissue distribution shows that this is not just a nonspecific background level of expression and suggests a role of brain P450 in the synthesis and degradation of arachidonic acid metabolites.