Role of neurofibromin in modulation of expression of the tyrosinase-related protein 2 gene

Tyrosinase-related protein 2 (TRP-2)/DOPAchrome tautomerase is an enzyme involved in melanin biosynthesis and plays an important role in cytoprotection by preventing the production of a toxic melanin precursor, 5,6-dihydroxyindole. Neurofibromin is the protein product of a gene linked to neurofibromatosis type 1 (NF1), which is characterized by multiple neurofibromas and abnormalities in skin pigmentation. To explore the pathogenesis of NF1, we studied the role of neurofibromin in the regulation of TRP-2 gene expression. By means of transient cotransfection assays, we show that the expression of a reporter gene under the control of the TRP-2 gene promoter was increased by a neurofibromin-dependent signal through the 71-bp region (positions -415 to -345). A Lys-to-Glu substitution at position 1425 in neurofibromin abrogated this activating function. A dominant negative Ki-ras inhibitor mimics neurofibromin's function, and additively increases TRP-2 promoter activity when coexpressed with neurofibromin. Therefore, we suggest that neurofibromin is involved in the regulation of TRP-2 gene expression. Moreover, we found a single case of a glioblastoma multiforme that expresses TRP-2 mRNA but not tyrosinase mRNA, suggesting that TRP-2 may function in human neural tissues under certain conditions.     

人甘露糖结合凝集素相关丝氨酸蛋白激酶-2C端基因的克隆与鉴定

目的:获得人甘露糖结合凝集素相关丝氨酸蛋白激酶-2(MASP-2)C端编码基因,并表达人MASP-2 C端片段,为制备单克隆抗体及应用于临床相关疾病的检测奠定分子学基础.方法:采用RT-PCR技术从人胎肝组织总RNA中扩增人MASP-2 C端的cDNA,克隆入pGEX-6p-2表达载体,酶切图谱分析和序列分析鉴定.结果:获得编码人MASP-2 C端片段的cDNA,并且与pGEX-6p-2载体连接,转化大肠杆菌XL1-blue,成功构建人MASP-2 C端片段cDNA的重组克隆.重组质粒酶切图谱分析和DNA测序分析,人MASP-2 C端片段cDNA的重组克隆与GenBank中人MASP-2 C端的cDNA序列一致.结论:成功克隆了人MASP-2 C端片段cDNA,并在大肠杆菌中表达人MASP-2 C端多肽片段.     

Cloning and expression of human endothelial-monocyte-activating polypeptide 2 (EMAP-2) and identification of its putative precursor

Endothelial-monocyte-activating polypeptide 2 (EMAP-2) modulates a range of properties of endothelial cells, monocytes and neutrophils in vitro, and induces an acute inflammatory reaction and tumour regression in vivo. We generated the full-length human cDNA sequences of EMAP-2 and its putative precursor pro-EMAP-2 as PCR products. These were cloned into the pCR3 vector and subcloned into pGEX-2T for expression as fusion products with glutathione-S-transferase (GST). Recombinant EMAP-2 (rEMAP-2) was isolated by thrombin cleavage of the fusion protein, followed by affinity chromatography. rEMAP-2 retained biological activity, which was blocked by polyclonal antibodies raised against GST-EMAP-2. By Western blotting, a 34-kDa product corresponding to the predicted precursor proEMAP-2 was detected in lysates of the U937 monocytic cell line, while supernatants contained higher levels of the mature 22-kDa molecule.     

Adeno-associated virus 2-mediated transduction and erythroid cell-specific expression of a human beta-globin gene

Two strategies for crossbreeding of indigenous and exotic cattle for milk production in the tropics, viz. rotational crossing and formation of a composite breed, have been compared. Genetic considerations suggest that rotational crossing would lead to somewhat better dairy performance, mainly because of more heterozygosity. Predictions based on the performance of adjacent genetic groups as obtained from a comprehensive literature review point in the same direction. Rotational crossbreeding depends on a continuous introduction of bulls of both parental breeds. The herd will consist of 2 (or more) genetic groups, which might be inconvenient for breeding arrangements, but provides more flexibility. The system requires good organisation and is most suitable in large farms. In small scale dairying the composite breed strategy is the most practical approach to dairy cattle breeding in the tropics.     

Cloning and expression of the red visual pigment gene of goat (Capra hircus)

Cerebral cortical maps in adult primates reorganize within minutes-hours after peripheral injuries, but subcortical versus intracortical contributions to this rapid reorganization remain controversial. The present results show that injury of nerves to the hands of adult monkeys triggers rapid (minutes-hours) changes in maps of the hand in the brainstem main cuneate nucleus. These findings suggest that peripheral injury causes an initial concurrent reorganization of brainstem and cortical substrates and that early sensory changes emerge from reorganization involving multiple central levels.     

Cloning of chicken interferon regulatory factor-2 (IRF-2) cDNA: expression and mapping of the IRF-2 gene

The Visual Evoked Potential (VEP) is an electrophysiological commonly used test in investigating various neurophysiological disorders. Through the years many methods have been developed, but there are not many objective criteria in distinguishing a normal VEP waveform from an abnormal. In this communication we use the phase characteristics of the power spectrum as a criterion of distinguishing normals and abnormals. From our analysis, it was shown that the phase spectrum of a VEP has a certain periodicity in the 0- to 40-Hz region. By studying these periodicities we were able to determine the range of the period that characterizes normal and abnormal populations and to establish an experimental method for objectively examining any kind of VEP waveforms.     

Early myeloid cell-specific expression of the human cathepsin G gene in transgenic mice

The human cathepsin G (CG) gene is expressed only in promyelocytes and encodes a neutral serine protease that is packaged in the azurophil (primary) granules of myeloid cells. To define the cis-acting DNA elements that are responsible for promyelocyte-specific "targeting," we injected a 6-kb transgene containing the entire human CG gene, including coding sequences contained in a 2.7-kb region, approximately 2.5 kb of 5' flanking sequence, and approximately 0.8 kb of 3' flanking sequence. Seven of seven "transient transgenic" murine embryos revealed human CG expression in the fetal livers at embryonic day 15. Stable transgenic founder lines were created with the same 6-kb fragment; four of five founder lines expressed human CG in the bone marrow. The level of human CG expression was relatively low per gene copy when compared with the endogenous murine CG gene, and expression was integration-site dependent; however, the level of gene expression correlated roughly with gene copy number. The human CG transgene and the endogenous murine CG gene were coordinately expressed in the bone marrow and the spleen. Immunohistochemical analysis of transgenic bone marrow revealed that the human CG protein was expressed exclusively in myeloid cells. Expression of human CG protein was highest in myeloid precursors and declined in mature myeloid cells. These data suggest that the human CG gene was appropriately targeted and developmentally regulated, demonstrating that the cis-acting DNA sequences required for the early myeloid cell-specific expression of human CG are present in this small genomic fragment.     

Multiple control elements are required for expression of the human CD34 gene

Two cis regulatory elements of the human CD34 gene, the promoter and a 3' enhancer, have previously been described. In transient transfection assays, the promoter was not sufficient to direct cell type specific expression. In contrast, the 3' enhancer was active only in CD34+ cell lines, suggesting that this element might be responsible for stem cell-restricted expression of the CD34 gene. In the current work, through deletion and transient transfection experiments, we delineated the core enhancer sequence. We examined the role of this element upon stable integration. Our data suggested the presence of additional control elements. In order to identify them, using DNaseI hypersensitivity and methylation studies, we determined the chromatin structure of the entire CD34 locus. Amongst a number of DNaseI hypersensitive sites, we detected a strong CD34+ cell type-specific site in intron 4. This region, however, did not work as an enhancer by itself. By analyzing stable transfectants and transgenic animals, we demonstrated that the 3' enhancer and intron 4 hypersensitive regions, either alone or together, did not function as a locus control region upon chromosomal integration. In contrast, a 160kb genomic fragment encompassing the entire CD34 gene contained regulatory elements sufficient for high-level CD34 mRNA expression in murine stable lines. Our data indicate that combinatorial action of multiple, proximal and long-range, cis elements is necessary for proper regulation of CD34 expression.     

Cloning of the Escherichia coli radC gene: identification of the RadC protein

1. The DNA sequence of the radC gene suggests an open reading frame of 297-bp. 2. To identify the gene product, radC was subcloned in an expression vector, pKK223-3 and the RadC protein identified by the maxicell method as a polypeptide of approximately 11 kDa.     

Developmental and environmental regulation of tissue- and cell-specific expression for a pea protein farnesyltransferase gene in transgenic plants

Farnesylation mediates membrane targeting and in vivo activities of several key regulatory proteins such as Ras and Ras-related GTPases and protein kinases in yeast and mammals, and is implicated in cell cycle control and abscisic acid (ABA) signaling in plants. In this study, the developmental expression of a pea protein farnesyltransferase (FTase) gene was examined using transgenic expression of the beta-glucuronidase (GUS) gene fused to a 3.2 kb 5' upstream sequence of the gene encoding the pea FTase beta subunit. Coordinate expression of the GUS transgene and endogenous tobacco FTase beta subunit gene in tobacco cell lines suggests that the 3.2 kb region contains the key FTase promoter elements. In transgenic tobacco plants, GUS expression is most prominent in meristematic tissues such as root tips, lateral root primordia and the shoot apex, supporting a role for FTase in the control of the cell cycle in plants. GUS activity was also detected in mature embryos and imbibed embryos, in accordance with a role for FTase in ABA signaling that modulates seed dormancy and germination. In addition, GUS activity was detected in regions that border two organs, e.g. junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls, and primary and secondary roots. GUS is expressed in phloem complexes that are adjacent to actively growing tissues such as young leaves, roots of light-grown seedlings, and hypocotyls of dark-grown seedlings. Both light and sugar (e.g. sucrose) treatments repressed GUS expression in dark-grown seedlings. These expression patterns suggest a potential involvement of FTase in the regulation of nutrient allocation into actively growing tissues.     

Cloning and expression of the human galanin receptor GalR2

We have previously shown that the novel anticonvulsant levetiracetam exerts potent anticonvulsant activity against both focal and secondarily generalized seizures in fully amygdala-kindled rats, i.e. , a model of temporal lobe epilepsy. We examined whether levetiracetam also exhibits antiepileptogenic activity, i.e., prevents or retards acquisition or development of amygdala-kindling in rats. Before the experiments with chronic administration of levetiracetam at different doses, we determined the pharmacokinetics of the drug after i.p. injection. Levetiracetam had a relatively short half-life (about 2-3 hr) in rats, so that any lasting effects of the drug after chronic administration were certainly not due to drug accumulation. When rats were treated with levetiracetam during kindling acquisition at daily i.p. doses of 13, 27 or 54 mg/kg, the drug dose-dependently suppressed the increase in seizure severity and duration induced by repeated amygdala stimulation. After termination of daily treatment with 54 mg/kg, duration of behavioral seizures and of afterdischarges recorded from the amygdala remained to be significantly shorter compared to vehicle controls, although amygdala stimulations were continued in the absence of drug. These data thus indicate that levetiracetam not simply masked the expression of kindled seizures through an anticonvulsant action, but exerted a true antiepileptogenic effect. Adverse effects were not observed at any dose of levetiracetam tested in kindled rats. The powerful antiepileptogenic activity of levetiracetam in the kindling model indicates that levetiracetam is not only an interesting novel drug for symptomatic treatment of epilepsy but might be suited for pharmacological prevention of this disease in patients with a high prospective risk of the development of epilepsy.     

Cloning and expression of the chicken protein tyrosine phosphatase SH-PTP2

SH-PTP2 is a protein tyrosine phosphatase which contains two src homology 2 (SH2) domains. A partial cDNA clone encoding chicken SH-PTP2 was generated by RT-PCR and used as a probe to screen several chicken cDNA libraries. Two overlapping cDNA clones were identified and the nucleotide sequence of chicken SH-PTP2 containing the entire protein-coding region was determined. The deduced amino acid sequence shares 98% and 94% identity, respectively with the corresponding human and Xenopus proteins. Northern and Western blot analyses show that chicken SH-PTP2 is expressed ubiquitously like those of mammals and Xenopus. This suggests that chicken SH-PTP2 may have analogous biological roles to those of mammals.