Repressor activity of CCAAT displacement protein in HL-60 myeloid leukemia cells.

CCAAT displacement protein (CDP)/cut is implicated in several systems as a transcriptional repressor of developmentally regulated genes. In myeloid leukemia cells, CDP/cut binding activity as assayed on the promoter of the phagocyte-specific cytochrome heavy chain gene gp91-phox varies inversely with expression of gp91-phox mRNA. We used two approaches to ascertain whether CDP/cut serves as a repressor of gp91-phox gene expression. First, we used transient transfection assays in 3T3 cells to demonstrate that the CDP/cut binding site from the gp91-phox promoter acts as a negative regulatory element in artificial promoter constructs. Second, we isolated a stable transformant of HL-60 myeloid cells constitutively expressing transfected CDP/cut cDNA. Stable transformants carrying expression vector alone or expressing CDP/cut mRNA were induced to differentiate along the macrophage lineage with phorbol ester or along the neutrophil lineage with dimethyl sulfoxide or retinoic acid/dimethylformamide. Northern blot analysis was used to assess induction of mRNAs encoding gp91-phox, and the myeloid oxidase cytosolic components, p47 and p67. In the stable transformant expressing transfected CDP/cut cDNA, gp91-phox induction was selectively reduced, whereas morphologic differentiation and induction of mRNA for myeloid oxidase components p47 and p67 were unaffected. These data provide persuasive evidence that CDP/cut acts to repress the gp91-phox gene

Method and parameters for genetic transformation of Streptococcus sanguis Challis.

A simple procedure for genetic transformation of Streptococcus sanguis Challis was developed and standardized. During the exponential phase of growth, cells became competent while growing as diplococci in broth con- taining 10 % foetal calf serum. High levels of competence were maintained by the cultures for 60 min. Competent cells could be stored frozen without loss of competence for at least three years. Using total chromosomal DNA as donor, the dose-response curve for transformation of a point mutation (streptomycin resistance) showed one-hit kinetics, as the DNA concentration varied from 0.000001 to 10 ~tg/ml. At 10 ~tg/ml, more than 2.2 % of the colony-forming units were transformed to streptomycin resistance, while trans- forming activity remained detectable with 1 pg of DNA/ml. Optimal time of exposure of competent cells to transforming DNA was 30 min. The trans- formation reaction was inhibited at 0 and 4\ub0C, whereas it occurred efficient- ly both at 25 and 37\ub0C

FGF receptor-mediated tyrosine phosphorylation of DHHC palmitoyltransferases: a mechanism to regulate the levels of NCAM palmitoylation?

Protein palmitoylation, the common posttranslational modification with the lipid palmitate, plays a pivotal role in protein trafficking and function. Our previous studies revealed that activation of fibroblast growth factor (FGF) receptor(s) by FGF2 leads to palmitoylation of the neural cell adhesion molecule (NCAM), its translocation to GM1 ganglioside-enriched lipid rafts, and stimulation of neurite outgrowth of cultured hippocampal neurons (1). Now, we used the Acyl-Biotin-Exchange (ABE) method and show a 1.3-fold increase in NCAM palmitoylation in mice injected with FGF2 in vivo. Furthermore, we analyzed the mechanisms of this regulation. Two members of the DHHC family, DHHC3 and DHHC7, were identified to mediate palmitoylation of NCAM in heterologous N2a cells (1). Because FGF:FGFR interaction leads to the activation of the canonical Ras/MAPK pathway and to the recruitment and activation of PLC\u3b3 and Src family tyrosine kinases (2), we investigated the tyrosine phosphorylation of DHHCs. Here, we present data showing that DHHC3 is highly tyrosine phosphorylated by activated FGFR1, suggesting a potential role of DHHC3 phosphorylation for regulation of its palmitoylating activity. We also report that treatment with the Src inhibitor PP2 significantly reduces DHHC3 tyrosine phosphorylation. Based on this evidence, we performed co-immunoprecipitation assays and found that Src but not FGFR1 is pulled-down by DHHC3, suggesting a possible direct interaction between the two molecules. Furthermore, DHHC3 isolated from brain homogenates appeared tyrosine phosphorylated, thus confirming the data obtained in the cell lines. Ongoing mutagenesis experiments aim to identify DHHC3 regulatory tyrosines. The generated DHHC3 mutants are assayed with the non-radioactive Click-IT palmitoylation assay that we specifically developed for NCAM. In summary, we demonstrated the FGFR-dependent phosphorylation of DHHC proteins, which might regulate palmitoylation activities of these enzymes towards to their substrates, including NCAM

Valorização da biomassa lenhocelulósica: estudo de sacarificação enzimática do cardo e da esteva

Dissertação de mestrado em Engenharia do ambiente. Instituto Politécnico de Beja, Escola Superior Agrária, 2013.A Cynara cardunculus e o Cistus ladanifer, são duas espécies bem adaptadas às condições edafoclimáticas do Alentejo, e uma alternativa aos materiais fósseis no contexto de biorrefinaria, visto serem biomassas lenhocelulósicas. No presente trabalho desenvolveu-se um estudo com a finalidade de utilizar as duas biomassas e perceber como se comportam na sacarificação enzimática da celulose, que efeitos têm os prétratamentos na eficiência da mesma e que efeito tem o recipiente onde se realiza a hidrólise. Foi ainda elaborado um desenho experimental com o cardo, com o objetivo de otimizar a sacarificação enzimática quanto à quantidade de sólidos, e enzimas a usar. Os resultados mostram que o cardo é mais sacarificável que a esteva e que os prétratamentos aumentaram a acessibilidade das enzimas, para a fração celulósica relativamente ao material não tratado. Para ambas as biomassas o pré-tratamento mais eficaz foi a explosão de vapor lavado com NaOH, tendo-se produzido 27,8 e 20,9 g/L de glucose para o cardo e esteva, respetivamente. A sacarificação da biomassa nos tubos “falcon”, foi menor do que nos tubos centrífuga e Erlenmeyer, que não apresentaram diferenças significativas. O valor máximo previsto para o CHA 130 pelo desenho experimental é de 31,7±1,0 g/L para CS=8, FPU=44,6, CBU=51,9

Method and parameters for genetic transformation of Streptococcus sanguis Challis.

A simple procedure for genetic transformation of Streptococcus sanguis Challis was developed and standardized. During the exponential phase of growth, cells became competent while growing as diplococci in broth containing 10% foetal calf serum. High levels of competence were maintained by the cultures for 60 min. Competent cells could be stored frozen without loss of competence for at least three years. Using total chromosomal DNA as donor, the dose-response curve for transformation of a point mutation (streptomycin resistance) showed one-hit kinetics, as the DNA concentration varied from 0.000001 to 10 micrograms/ml. At 10 micrograms/ml, more than 2.2% of the colony-forming units were transformed to streptomycin resistance, while transforming activity remained detectable with 1 pg of DNA/ml. Optimal time of exposure of competent cells to transforming DNA was 30 min. The transformation reaction was inhibited at 0 and 4 degrees C, whereas it occurred efficiently both at 25 and 37 degrees C

Protein-DNA interactions in the 5'-flanking region of the bovine pancreatic ribonuclease gene

In the 5'-flanking region of the bovine pancreatic ribonuclease gene a sequence has been identified which specifically binds one or more factors present in nuclear protein extracts prepared from bovine pancreas. The binding site, as delineated by footprinting analysis, is located in a region extending from positions -113 to -146 relative to the transcription initiation site of the ribonuclease gene. This region contains consensus sequences for known control transcriptional elements. The observed pattern of protein-DNA interactions is likely to be pancreas-specific as it could not be detected with nuclear extracts prepared from HeLa or bovine aorta endothelium cells

Naturally occurring C-terminally truncated STAT5 is a negative regulator of Human Immunodeficiency Virus-type1 expression.

CD4(+) cells of most individuals infected with HIV-1 harbor a C-terminally truncated and constitutively activated form of signal transducer and activator of transcription-5 (STAT5 Delta). We report that the chronically HIV-infected U1 cell line expresses STAT5 Delta but not full-length STAT5. Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of U1 cells promoted early activation of STAT5 Delta and of extracellular signal regulated kinases (ERKs), followed by later activation of activator protein 1 (AP-1) and HIV expression. Inhibition of ERK/AP-1 by PD98,059 abolished, whereas either tyrphostin AG490 or a STAT5 small interfering RNA (siRNA) enhanced, virion production in GM-CSF-stimulated U1 cells. Chromatin immunoprecipitation demonstrated the induction of STAT5 Delta binding to STAT consensus sequences in the HIV-1 promoter together with a decreased recruitment of RNA polymerase II after 1 hour of GM-CSF stimulation of U1 cells. Down-regulation of STAT5 Delta by siRNA resulted in the up-regulation of both HIV-1 gag-pol RNA and p24 Gag antigen expression in CD8-depleted leukocytes of several HIV-positive individuals cultivated ex vivo in the presence of interleukin-2 but not of interleukin-7. Thus, the constitutively activated STAT5 Delta present in the leukocytes of most HIV-positive individuals acts as a negative regulator of HIV expression