Expression of a thermostable xylanase gene from Bacillus coagulans ST-6 in Lactococcus lactis

AIMS: The aim of the study is to evaluate whether xylanase can be used as a potential reporter gene for cloning and expression studies in Lactococcus. METHODS AND RESULTS: The 750 bp xylanase gene was amplified and subcloned into the unique NheI restriction enzyme site of pMG36e and subsequently transformed into competent Escherichia coli XLI-blue MRF cells and Lactococcus lactis cells. Bacterial culture containing pMG36e-Xy has an enzyme activity of 390 microg xylose ml(-1) culture 30 min(-1), respectively, when compared with 40 microg xylose ml(-1) culture 30 min(-1) for the negative control (plasmidless strain). CONCLUSIONS: The thermostable xylanase gene was successfully expressed in both E. coli and L. lactis. The activity of xylanase can be easily detected by the formation of visible clearing zones around the transformed colonies on Remazol Brilliant Blue-Xylan (RBB-Xylan) agar media. However, there were some significant differences in the optimum growth temperature and plasmid stability in the new clones. SIGNIFICANCE AND IMPACT OF THE STUDY: The constructed reporter vector has the potential to be used as a reporter system for Lactococcus as well as E. coli, and it is an addition to the pool of lactococcal vector systems.     

Detection and phylogenetic profiling of nodavirus associated with white tail disease in Malaysian <Emphasis Type="Italic">Macrobrachium rosenbergii</Emphasis> de Man

White tail disease (WTD) is a serious viral disease in the hatcheries and nursery ponds of Macrobrachium rosenbergii in many parts of the world. A new disease similar to WTD was observed in larvae and post larvae of M. rosenbergii cultured in Malaysia. In the present study, RT-PCR assay was used to detect the causative agents of WTD, M. rosenbergii nodavirus (MrNV) and extra small virus (XSV) using specific primers for MrNV RNA2 and XSV. The results showed the presence of MrNV in the samples with or without signs of WTD. However, XSV was only detected in some of the MrNV-positive samples. Phylogenetic analysis showed that the RNA2 of our Malaysian isolates were significantly different from the other isolates. Histopathological studies revealed myofiber degeneration of the tail muscles and liquefactive myopathy in the infected prawns. This was the first report on the occurrence of MrNV in the Malaysian freshwater prawn.     

Structure of a novel phosphotyrosine-binding domain in Hakai that targets E-cadherin

Phosphotyrosine-binding domains, typified by the SH2 (Src homology 2) and PTB domains, are critical upstream components of signal transduction pathways. The E3 ubiquitin ligase Hakai targets tyrosine-phosphorylated E-cadherin via an uncharacterized domain. In this study, the crystal structure of Hakai (amino acids 106-206) revealed that it forms an atypical, zinc-coordinated homodimer by utilizing residues from the phosphotyrosine-binding domain of two Hakai monomers. Hakai dimerization allows the formation of a phosphotyrosine-binding pocket that recognizes specific phosphorylated tyrosines and flanking acidic amino acids of Src substrates, such as E-cadherin, cortactin and DOK1. NMR and mutational analysis identified the Hakai residues required for target binding within the binding pocket, now named the HYB domain. ZNF645 also possesses a HYB domain but demonstrates different target specificities. The HYB domain is structurally different from other phosphotyrosine-binding domains and is a potential drug target due to its novel structural features.     

Tesk1 interacts with Spry2 to abrogate its inhibition of ERK phosphorylation downstream of receptor tyrosine kinase signaling

The Sprouty (Spry) proteins function as inhibitors of the Ras-ERK pathway downstream of various receptor tyrosine kinases. In this study, we have identified Tesk1 (testicular protein kinase 1) as a novel regulator of Spry2 function. Endogenous Tesk1 and Spry2 exist in a complex in cell lines and mouse tissues. Tesk1 coexpression relocalizes Spry2 to vesicles including endosomes, inhibiting its translocation to membrane ruffles upon growth factor stimulation. Independent of its kinase activity, Tesk1 binding leads to a loss of Spry2 function as an inhibitor of ERK phosphorylation and reverses inhibition of basic fibroblast growth factor (bFGF)- and nerve growth factor-induced neurite outgrowth in PC12 cells by Spry2. Furthermore, depletion of endogenous Tesk1 in PC12 cells leads to a reduction in neurite outgrowth induced by bFGF. Tesk1 nullifies the inhibitory effect of Spry2 by abrogating its interaction with the adaptor protein Grb2 and interfering with its serine dephosphorylation upon bFGF and FGF receptor 1 stimulation by impeding its binding to the catalytic subunit of protein phosphatase 2A. A construct of Tesk1 that binds to Spry2 but does not localize to the vesicles does not interfere with its function, highlighting the importance of subcellular localization of Tesk1 in this context. Conversely, Tesk1 does not affect interaction of Spry2 with the E3 ubiquitin ligase, c-Cbl, and consequently, does not affect its inhibition of Cbl-mediated ubiquitination of the epidermal growth factor receptor. By selectively modulating the downstream effects of Spry2, Tesk1 may thus serve as a molecular determinant of the signaling outcome.     

Synergism of cellulase enzymes in mixed culture solid substrate fermentation

Sugar cane bagasse was subjected to a mixed culture, solid substrate fermentation with Trichoderma reesei QM9414 and Aspergillus terreus SUK-1 to produce cellulase and reducing sugars. The highest cellulase activity and reducing sugar amount were obtained in mixed culture. The percentage of substrate degradation achieved employing mixed culture was 26% compared to 50% using separate cultures of the two molds. This suggests that the synergism of enzymes in mixed culture solid substrate fermentation have lower synergism than in pure culture.     

Reduction in serum levels of adhesion molecules, interleukin-6 and C-reactive protein following short-term low-dose atorvastatin treatment in patients with non-familial hypercholesterolemia.

Hypercholesterolemia causes endothelial dysfunction, an early feature of atherosclerosis, leading to increased production of adhesion molecules and cytokines. The aim of this study was to investigate the effects of three months of treatment with low dose atorvastatin on serum levels of adhesion molecules, interleukin-6 (IL-6) and highly sensitive C-reactive protein (hs-CRP) in patients with non-familial hypercholesterolemia. Fifty-five patients with non-familial hypercholesterolemia were randomized to treatment with atorvastatin 10 mg/day or placebo for 3 months. Soluble intercellular adhesion molecules-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, IL-6 and hs-CRP levels were measured to assess the inflammatory activity of the endothelium. There was a significant reduction in ICAM-1 at 2 weeks (p<0.0001) with further reduction at 3 months (p<0.0001). At 3 months, there were significant reductions in VCAM-1 (p<0.02), IL-6 (p<0.0001) and hs-CRP (p<0.01), but an increase in E-selectin levels (p<0.002). Treatment with statin was an independent determinant of change in ICAM-1 (p<0.05) and IL-6 levels (p<0.05) after correcting for anthropometric indices, blood pressure and lipid profile. Low-dose atorvastatin treatment leads to reduction in proinflammatory markers of endothelial function, suggesting an attenuation of endothelial activation and improvement in endothelial function, independent of lipid lowering. This may lead to a reduction in the progression of atherosclerosis.     

Dockers at the crossroads.

The family of docker proteins containing phosphotyrosine-binding (PTB) domains appears to represent a family of critically positioned and exquisitely controlled signalling proteins that relay signals from the activated receptors to downstream pathways. These proteins all have a membrane attachment domain, a PTB domain that targets the protein to a subset of receptors and a number of phosphorylatable tyrosines that dock other signalling proteins. Evidence is accruing that suggests that the PTB domain has evolved from a pleckstrin homology (PH) domain to bind to a range of sequences that, while bestowing specificity, allows switching of the docker protein between receptors or signalling systems. The history of the PTB domain and how it influences the participation of docker protein in various signalling pathways are discussed.     

An efficient protocol for particle bombardment-mediated transformation of <Emphasis Type="Italic">Centella asiatica</Emphasis> callus

In this study, we report an optimization of particle bombardment transformation system for Centella asiatica callus. A total of eight parameters affecting the genetic transformation system were optimized using the synthetic green fluorescent protein (sGFP) as a reporter driven by the CaMV 35S promoter. The results indicated that DNA delivery conditions of 9-cm target distance, 1,100 psi helium pressure, 1.0 μm gold particles size, 27 mmHg chamber vacuum pressure, 2 times number of bombardment, spermidine as precipitation agent, 60 h post-bombardment incubation time and 2 μg plasmid DNA concentration were optimal for C. asiatica callus transformation. The expression of sGFP was monitored using fluorescence microscope and further confirmed using RT-PCR. This optimized genetic transformation system is applicable for rapid transient gene analysis and transgenic C. asiatica production.     

Cytolytic Effects and Apoptosis Induction of Newcastle Disease Virus Strain AF2240 on Anaplastic Astrocytoma Brain Tumor Cell Line

Newcastle disease virus (NDV) is a member of genus Avulavirus within the family Paramyxoviridae. Interest of using NDV as an anticancer agent has arisen from its ability to kill tumor cells with limited toxicity to normal cells. In this investigation, the cytotolytic properties of NDV strain AF2240 were evaluated on brain tumor cell line, anaplastic astrocytoma (U-87MG), by using MTT assay. Cytological observations were studied using fluorescence microscopy and transmission electron microscopy to show the apoptogenic features of NDV on U-87MG. DNA laddering in agarose gel electrophoresis and terminal deoxyribonucleotide transferase-mediated dUTP-X nick end-labeling staining assay confirmed that the mode of cell death was by apoptosis. However, analysis of the cellular DNA content by flowcytometery showed that there was a loss of treated U-87MG cells in all cell cycle phases (G1, S and G2/M) accompanied with increasing in sub-G1 region (apoptosis peak). Early apoptosis was observed 6 h post-inoculation by annexin-V flow-cytometry method. It could be concluded that NDV strain AF2240 is a potent antitumor agent that induce apoptosis and its cytotoxicity increasing while increasing of time and virus titer.