A subunit of mammalian signal peptidase is homologous to yeast SEC11 protein

Canine signal peptidase consists of a complex of five proteins (Evans, A. E., Gilmore, R., and Blobel, G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 581-585). A cDNA encoding the 21-kDa subunit of the signal peptidase complex was isolated from a liver cDNA library using an 88-base pair probe, generated by the polymerase chain reaction. The 820-base pair cDNA was sequenced and found to encode a protein of 21,585 daltons. The deduced amino acid sequence from the canine cDNA was found to be 47% identical to the yeast SEC11 protein. SEC11 has been shown to be required for signal peptide cleavage, normal rate of secretion, and cell survival in Saccharomyces cerevisiae (B?hni, P. C., Deshaies, R. J., and Schekman, R. W. (1988) J. Cell Biol. 106, 1035-1042). It is, therefore, likely that the 21-kDa subunit of signal peptidase complex is the structural and functional homologue of the yeast SEC11 gene product.     

Lack of correlation between in vitro susceptibility to Benznidazole and phylogenetic diversity of Trypanosoma cruzi, the agent of Chagas disease

Chagas disease remains an important health problem in Central and South America. Nitroimidazole derivative drugs like Benznidazole are commonly used to treat Trypanosoma cruzi infection. Natural variation of drug susceptibility between various T. cruzi stocks has been proposed as a possible explanation of treatment failure. Thus, the aim of this work was to determine potential correlations between in vitro Benznidazole susceptibility of different T. cruzi stocks and their genetic diversity. For this purpose, 16 natural stocks representing the overall genetic diversity of the parasite were analysed. Genetic characterisation was assessed by both random amplified polymorphic DNA (RAPD) and multilocus enzyme electrophoresis (MLEE) analyses. Drug activity was determined by two complementary methods, the MTT-PMS micro-method and FACs analysis. The 50% inhibitory concentrations (IC(50)s) were determined. Important variation of IC(50) values (7.3-16.9 microM) among stocks belonging to different discrete typing units (DTUs) was recorded. Further, correlation analysis showed that natural susceptibility to Benznidazole in T. cruzi expressed as IC(50) level was not related with its genetic structure represented by the different DTUs. These results are discussed in relation with the proposed hypothesis establishing a link between genetic diversity and biological behaviour in T. cruzi.     

Identification and solubilization of a signal peptidase from the phototrophic bacterium Rhodobacter capsulatus.

In Gram-negative bacteria, exported proteins are synthesized with an amino-terminal signal sequence which is cleaved off by the signal peptidase during, or shortly after the translocation process. Here, we report the identification and solubilization of a signal peptidase from the phototrophic bacterium Rhodobacter capsulatus which cleaves homologous and heterologous precursor proteins at the authentic cleavage site. This signal peptidase is the first identified component of the R. capsulatus protein export machinery.     

Identification of the main ubiquitination site in human erythroid alpha-spectrin

Erythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of alpha-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in alpha-spectrin and may be present in several other proteins.     

Aldehyde dehydrogenase 1A1 and gelsolin identified as novel invasion-modulating factors in conditioned medium of pancreatic cancer cells

Conditioned medium (CM) from clonal sub-populations of the pancreatic cancer cell line, MiaPaCa-2 with differing invasive abilities, were examined for their effect on in vitro invasion. Conditioned medium from Clone #3 (CM#3) strongly promoted invasion, while CM from Clone #8 (CM#8) inhibited invasion in vitro. 2D DIGE followed by MALDI-TOF MS analysis of CM#3 and CM#8 identified 41 proteins which were differentially regulated; 27 proteins were down-regulated and 14 proteins up-regulated in the invasion-promoting CM#3 when compared to CM#8. Western blotting analysis confirmed the down-regulated expression of gelsolin and the up-regulation of aldehyde dehydrogenase 1A1 in CM#3. Down-regulation of aldehyde dehydrogenase 1A1 in Clone #3 CM and gelsolin levels in Clone #8 CM by siRNA transfection revealed an important involvement of these proteins in promoting and inhibiting invasion in these pancreatic cancer cell lines.     

Two putative BIN2 substrates are nuclear components of brassinosteroid signaling

GSK3 is a highly conserved kinase that negatively regulates many cellular processes by phosphorylating a variety of protein substrates. BIN2 is a GSK3-like kinase in Arabidopsis that functions as a negative regulator of brassinosteroid (BR) signaling. It was proposed that BR signals, perceived by a membrane BR receptor complex that contains the leucine (Leu)-rich repeat receptor-like kinase BRI1, inactivate BIN2 to relieve its inhibitory effect on unknown downstream BR-signaling components. Using a yeast (Saccharomyces cerevisiae) two-hybrid approach, we discovered a potential BIN2 substrate that is identical to a recently identified BR-signaling protein, BES1. BES1 and its closest homolog, BZR1, which was also uncovered as a potential BR-signaling protein, display specific interactions with BIN2 in yeast. Both BES1 and BZR1 contain many copies of a conserved GSK3 phosphorylation site and can be phosphorylated by BIN2 in vitro via a novel GSK3 phosphorylation mechanism that is independent of a priming phosphorylation or a scaffold protein. Five independent bes1 alleles containing the same proline-233-Leu mutation were identified as semidominant suppressors of two different bri1 mutations. Over-expression of the wild-type BZR1 gene partially complemented bin2/+ mutants and resulted in a BRI1 overexpression phenotype in a BIN2(+) background, whereas overexpression of a mutated BZR1 gene containing the corresponding proline-234-Leu mutation rescued a weak bri1 mutation and led to a bes1-like phenotype. Confocal microscopic analysis indicated that both BES1 and BZR1 proteins were mainly localized in the nucleus. We propose that BES1/BZR1 are two nuclear components of BR signaling that are negatively regulated by BIN2 through a phosphorylation-initiated process.     

Components of Resistance to Early Blight in Four Potato Cultivars: Effect of Leaf Position

Components of early blight resistance were quantified in leaves of different ages in four potato cultivars. The components of resistance: incubation period (IP), lesion number (LN), early blight severity, lesion expansion rate (LER), latent period (LP) and spore production by lesion area (SPLA), were evaluated separately in the lower, middle and upper leaves of four potato cultivars. Plants of cultivar Aracy (resistant), Delta (moderately resistant), Desirée (susceptible) and Bintje (susceptible) were inoculated with an Alternaria solani isolate at the beginning of the flowering stage. Disease severity varied in different plant parts. In all cultivars, regardless of resistance, the smallest values of LN, and severity were recorded on the upper leaves, suggesting that young tissues are less susceptible. In cultivar Aracy, the IP was long, with small values of LN and LER and consequently, low values of early blight severity in all leaf positions were recorded. Although IP was long in cultivar Aracy, no differences between the moderately resistant cultivar Delta and the susceptible cultivars Bintje and Desirée could be detected for this component. The IP was only influenced by leaf position in cultivar Aracy. Clear differences in resistance levels among cultivars could be detected regarding LN, severity and LER. However, neither LP nor SPLA were associated with resistance level of cultivars or with leaf position. Analyses according to plant part suggest that evaluations on leaves of the middle third part are most suitable for screening for early blight resistance in potato.     

神经递质3H-去甲肾上腺素释放与家蝇对拟除虫菊酯抗性的关系

在用K⁺去极化条件下,研究了溴氰菊酯和氯菊酯分别对敏感、抗溴氰菊酯和抗氯菊酯家蝇Musca domestica 品系脑突触体释放神经递质去甲肾上腺素的影响。结果表明：在用K⁺去极化后,神经递质去甲肾上腺素的释放在抗溴氰菊酯和抗氯菊酯家蝇品系中比敏感品系分别下降47.0%和51.0%;当用10^-5 mol/L溴氰菊酯预处理家蝇脑突触体,用K+去极化后对敏感、抗溴氰菊酯和抗氯菊酯家蝇品系释放去甲肾上腺素的加强作用分别提高80.3%、26.5%和70.5%;用10^-5 mol/L氯菊酯预处理3个家蝇品系的突触体对去甲肾上腺素释放均无加强作用。由此表明,家蝇对溴氰菊酯的抗性是与Na+通道的亲和性降低有关,而氯菊酯的抗性与Na+通道的亲和性关系不大。     

Thirty-three amino acids of the mature moiety of an unprocessed maltose-binding protein are sufficient for export in Escherichia coli.

Maltose-binding protein (MBP) is translocated across the cytoplasmic membrane of Escherichia coli; successful export depends on information in both the signal peptide and the mature moiety of the protein. To determine the shortest portion of the mature region that would maintain detectable entry of MBP into the export pathway, we took advantage of the properties of an MBP species with proline substituted in the +1 position relative to the cleavage site (MBP27-P). This protein efficiently crosses the cytoplasmic membrane but is not processed and acts as a competitive inhibitor of signal peptidase I (leader peptidase). Export of MBP27-P is measured by the inhibition of processing of other proteins, such as ribose-binding protein (RBP). A series of truncated derivatives of MBP27-P were tested for the ability to inhibit processing of RBP. An MBP27-P species with only 33 amino acids of the mature moiety inhibited processing of RBP, indicating that this truncated polypeptide was probably exported and interacted with signal peptidase I.