NaCl differently interferes with Cd and Zn toxicities in the wetland halophyte species Kosteletzkya virginica (L.) Presl.

Hydroponic experiments were carried out using seedlings of the wetland halophyte species Kosteletzkya virginica (L.) Presl. exposed to 10???M Cd or 100???M Zn in the absence or presence of 50?mM NaCl. Interaction between salinity and heavy metals was analysed in relation to plant growth, water status and tissue ion contents (Na, K and Ca). Results showed a strong inhibition effect of Cd on leaf emergence, lateral branch development and leaf expansion. Heavy metals induced a significant decrease in plant dry weight, water content, osmotic potential (?? _S) and leaf water potential (?? _w). Cadmium and Zn accumulated to higher extent in the roots than in the shoots. Cadmium increased the leaf K concentration while Zn had an opposite effect. Salinity strongly reduced Cd uptake and translocation from roots to shoots: it mitigated the Cd impact on lateral branch emergence but had no effects on plant dry weight and water status. Cadmium drastically reduced Na translocation in salt-treated plants while Zn increased it. It is concluded that complex interactions exist between heavy metals and monovalent cations in salt conditions and that Cd and Zn display contrasting behaviour in this respect. Stress-induced modification of ion content did not fully explain growth inhibition in Kosteletzkya virginica.     

Microcalorimetric studies on the polyhydroxyalkanoates production of recombinant Escherichia Coli

The thermogenic curves of metabolism of two strains of Escherichia coli pUC19cab/XL-IBlue and XL-IBlue have been determined by using a LKB-2277 bioActivity Monitor and ampoule method at 37°C. pUC19cab/XL-IBlue was a recombinant E. coli strain bearing a foreign plasmid pUC19cab which brought the polyhydroxyalkanoates (PHAs) production. XL-IBlue was a host bacterium without any foreign DNA. Our studies reveal that the PHA production of recombinant E. coli has an apparent influence on their thermogenic curves of metabolism and therefore the initial time of PHAs production can be determined from these thermogenic curves. The text was submitted by the authors in English.     

Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog

Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs.     

Subtyping animal influenza virus with general multiplex RT-PCR and Liquichip high throughput (GMPLex)

This study developed a multiplex RT-PCR integrated with luminex technology to rapidly subtype simultaneously multiple influenza viruses. Primers and probes were designed to amplify NS and M genes of influenza A viruses HA gene of H1, H3, H5, H7, H9 subtypes, and NA gene of the N1 and N2 subtypes. Universal super primers were introduced to establish a multiplex RT-PCR (GM RT-PCR). It included three stages of RT-PCR amplification, and then the RT-PCR products were further tested by LiquiChip probe, combined to give an influenza virus (IV) rapid high throughput subtyping test, designated as GMPLex. The IV GMPLex rapid high throughput subtyping test presents the following features: high throughput, able to determine the subtypes of 9 target genes in H1, H3, H5, H7, H9, N1, and N2 subtypes of the influenza A virus at one time; rapid, completing the influenza subtyping within 6 hours; high specificity, ensured the specificity of the different subtypes by using two nested degenerate primers and one probe, no cross reaction occurring between the subtypes, no non-specific reactions with other pathogens and high sensitivity. When used separately to detect the product of single GM RT-PCR for single H5 or N1 gene, the GMPLex test showed a sensitivity of 10⁻⁵(= 280ELD₅₀) forboth tests and the Luminex qualitative ratio results were 3.08 and 3.12, respectively. When used to detect the product of GM RT-PCR for H5N1 strain at the same time, both showed a sensitivity of 10⁻⁴(=2800 ELD₅₀). The GMPLex rapid high throughput subtyping test can satisfy the needs of influenza rapid testing.Key words: Influenza Virus, General multiplex RT-PCR, Iuminex assay, Subtyping, HA and NA genes     

PAK1 is a novel MEK-independent raf target controlling expression of the IAP survivin in M-CSF-mediated osteoclast survival

As activation of the Ras/Raf/MEK/ERK pathway is a critical component of M-CSF-promoted osteoclast survival, determining specific mechanism by which M-CSF activates this signal transduction pathway is paramount towards advancing treatment of pathological conditions resulting in increased bone turnover. The p21 activated kinase PAK1 modulates activation of the Raf/MEK/ERK pathway by either directly activating Raf or priming MEK for activation by Raf. Therefore a role for PAK1 in M-CSF-mediated activation of the MEK/ERK pathway controlling osteoclast survival was assessed. Here we show that PAK1 is activated by M-CSF in a Ras-dependent mechanism that promotes osteoclast survival. Surprisingly, PAK1 did not modulate Raf activation or Raf-mediated MEK activation. M-CSF mediated activation of Raf was required for PAK1 activation and osteoclast survival promoted by PAK1. This survival response was MEK-independent as expression of constitutively active MEK did not rescue osteoclasts from apoptosis induced by blocking PAK1 function. Functionally, PAK1 promoted osteoclast survival by modulating expression of the IAP family member Survivin. M-CSF therefore functions to promote PAK1 activation as a novel MEK-independent Raf target to control Survivin-mediated osteoclast survival.     

Large-scale expression, purification, and characterization of an engineered prostacyclin-synthesizing enzyme with therapeutic potential

Recently, we reported that a novel hybrid enzyme (TriCat enzyme), engineered by linking human cyclooxygenase-2 (COX-2) with prostacyclin (PGI₂) synthase (PGIS) together through a transmembrane domain, was able to directly integrate the triple catalytic (TripCat) functions of COX-2 and PGIS and effectively convert arachidonic acid (AA) into the vascular protector, PGI₂ [K.H. Ruan, H. Deng, S.P. So, Biochemistry 45 (2006) 14003-14011]. In order to confirm the important biological activity and evaluate its therapeutic potential, it is critical to characterize the properties of the enzyme using the purified protein. The TriCat enzyme cDNA was subcloned into a baculovirus vector and its protein was expressed in Sf-9 cells in large-scale with a high-yield (∼4% of the total membrane protein), as confirmed by Western blot and protein staining. The Sf-9 cells’ membrane fraction, rich in TriCat enzyme, exhibited strong TriCat functions (K_m = 3 μM and K_cat = 100 molecules/min) for the TriCat enzyme and was 3-folds faster in converting AA to PGI₂ than the combination of the individual COX-2 and PGIS. Another superiority of the TriCat enzyme is its dual effect on platelet aggregation: it completely inhibited platelet aggregation at the low concentration of 2 μg/ml and then displayed the ability to reverse the initially aggregated platelets to their non-aggregated state. Furthermore, multiple substrate-binding sites were confirmed in the single protein by high-resolution NMR spectroscopy, using partially purified TriCat enzyme. These studies have clearly demonstrated that the isolated TriCat enzyme protein functions in the selective biosynthesis of the vascular protector, PGI₂, and revealed its potential for anti-thrombosis therapeutics.     

Molecular characterization of two novel isoforms and a soluble form of mouse CLEC-2

CLEC-2 was first identified by sequence similarity to C-type lectin-like molecules with immune functions. Recently, human CLEC-2 has been reported as a receptor for the platelet-aggregating snake venom toxin rhodocytin and the endogenous sialoglycoprotein podoplanin. It has also been reported to facilitate the capture of HIV-1. However, investigation of mouse CLEC-2 (mCLEC-2) has little progressed after its identification. In this study, we identified two novel splicing variants of mCLEC-2 derived from omission of exon 2 and 2/4, respectively. These two variants had different expression profiles and subcellular localization from full-length mCLEC-2. Moreover, we observed that full-length mCLEC-2 could be cleaved probably by proteases sensitive to aprotinin and PMSF into a soluble form that partially existed as a disulfide-linked homodimer. The results presented here represent a further advancement toward the understanding of mCLEC-2.