Cloning,sequencing and overexpression in Escherichia coli of a xylanase gene,xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor

 A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in λZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selective polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70^°C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp. Received: 18 April 1995/Received revision: 4 August 1995/Accepted: 22 August 1995     

Glucocorticoids Stimulate Inflammatory 5-Lipoxygenase Gene Expression and Protein Translocation in the Brain

In the brain, the expression of 5-lipoxygenase (5-LO), the enzyme responsible for the synthesis of inflammatory leukotrienes, increases during aging. Antiinflammatory drugs are currently being evaluated for the treatment of aging-associated neurodegenerative diseases such as Alzheimer's disease. Although generally considered antiinflammatory, glucocorticoids, whose production also increases during aging, are not particularly effective in this disease. In human monocytes, 5-LO mRNA content increases on exposure to the synthetic glucocorticoid dexamethasone, which prompted us to hypothesize that glucocorticoids might increase 5-LO expression in the brain as well. We treated rats for 10 days either with corticosterone (implanted subcutaneously) or with dexamethasone (injected daily); they were killed on day 10 after pellet implantation or 24 h after the 10th dexamethasone injection. We found increased levels of 5-LO mRNA and protein in hippocampus and cerebellum of glucocorticoid-treated rats; 5-LO-activating protein (FLAP) mRNA content was not affected. Using western immunobloting, we also observed the concurrent translocation of 5-LO protein from cytosol to membrane, an indication of its activation. Thus, glucocorticoid-mediated up-regulation of the neuronal 5-LO pathway may contribute to rendering an aging brain vulnerable to degeneration.     

Genetic structure,diversity, and allelic richness in composite collection and reference set in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis>L.)

Background  

Plant genetic resources (PGR) are the basic raw materials for future genetic progress and an insurance against unforeseen threats to agricultural production. An extensive characterization of PGR provides an opportunity to dissect structure, mine allelic variations, and identify diverse accessions for crop improvement. The Generation Challenge Program conceptualized the development of "composite collections" and extraction of "reference sets" from these for more efficient tapping of global crop-related genetic resources. In this study, we report the genetic structure, diversity and allelic richness in a composite collection of chickpea using SSR markers, and formation of a reference set of 300 accessions.     

Attenuated neuroprotective effect of riboflavin under UV-B irradiation via miR-203/c-Jun signaling pathway in vivo and in vitro

Background

Riboflavin (RF) or vitamin B2 is known to have neuroprotective effects. In the present study, we report the attenuation of the neuroprotective effects of RF under UV-B irradiation. Preconditioning of UV-B irradiated riboflavin (UV-B-RF) showed attenuated neuroprotective effects compared to that of RF in SH-SY5Y neuroblostoma cell line and primary cortical neurons in vitro and a rat model of cerebral ischemia in vivo.

Results

Results indicated that RF pretreatment significantly inhibited cell death and reduced LDH secretion compared to that of the UV-B-RF pretreatment in primary cortical neuron cultures subjected to oxygen glucose deprivation in vitro and cortical brain tissue subjected to ischemic injury in vivo. Further mechanistic studies using cortical neuron cultures revealed that RF treatment induced increased miR-203 expression which in turn inhibited c-Jun expression and increased neuronal cell survival. Functional assays clearly demonstrated that the UV-B-RF preconditioning failed to sustain the increased expression of miR-203 and the decreased levels of c-Jun, mediating the neuroprotective effects of RF. UV-B irradiation attenuated the neuroprotective effects of RF through modulation of the miR-203/c-Jun signaling pathway.

Conclusion

Thus, the ability of UV-B to serve as a modulator of this neuroprotective signaling pathway warrants further studies into its role as a regulator of other cytoprotective/neuroprotective signaling pathways.     

A spectroscopic study of the conformations of active and latent forms of recombinant plasminogen activator inhibitor-1

Recombinant plasminogen activator inhibitor-1 (rPAI-1) purified from Escherichia coli, like its natural counterpart, can exist in either active or latent form. To elucidate the structural basis for these two forms, both active and latent rPAI-1 have been studied using ultra-violet (UV), circular dichroism (CD), and fluorescence spectroscopy. The secondary structures determined by CD show no significant differences and indicate that both the forms are predominantly alpha helical and random. The UV spectra are also very similar with absorption maxima around 278 nm. The structures of the two forms were further characterized by measuring tryptophan fluorescence emissions and their quenching with ionic (iodide) and neutral (acrylamide) quenchers. These data indicate clear differences in the tertiary structures of the two forms with the latent form being more compact and folded in comparison with the active form.     

Toad radiation reveals into-India dispersal as a source of endemism in the Western Ghats-Sri Lanka biodiversity hotspot

Background  

High taxonomic level endemism in the Western Ghats-Sri Lanka biodiversity hotspot has been typically attributed to the subcontinent's geological history of long-term isolation. Subsequent out of – and into India dispersal of species after accretion to the Eurasian mainland is therefore often seen as a biogeographic factor that 'diluted' the composition of previously isolated Indian biota. However, few molecular studies have focussed on into-India dispersal as a possible source of endemism on the subcontinent. Using c. 6000 base pairs of mitochondrial and nuclear DNA, we investigated the evolutionary history and biogeography of true toads (Bufonidae), a group that colonized the Indian Subcontinent after the Indo-Asia collision.     

A probe for detection of G-rich target strands through fluorescence quenching

A modified fluorescent probe U^FAA AAT CTC CGC CGC was synthesized using the nucleoside analogue 3′-O-(N,N′-diisopropylamino-2-cyanoethoxyphosphinyl)-5′-O-(4,4′-dimethoxytrityl)-2′-O-(dansyl-1-sulfonamidohexylaminocarbonyl)uridine for hybridization studies with perfectly matched (U/A) complementary DNA and with a DNA strand having similar G-rich telomeric units at their 3′-ends. Data on the thermal stability and decrease in fluorescence intensity due to the presence of dG units clearly demonstrated the potential application of this approach in DNA diagnostics in homogeneous hybridization assays. The text was submitted by the authors in English.     

The keratocyte: corneal stromal cell with variable repair phenotypes

Keratocytes, also known as fibroblasts, are mesencyhmal-derived cells of the corneal stroma. These cells are normally quiescent, but they can readily respond and transition into repair phenotypes following injury. Cytokines and other growth factors that provide autocrine signals for stimulating wound responses in resident cells are typically presented by platelets at the site of an injury. However, due to the avascular nature of the cornea many of the environmental cues are derived from the overlying epithelium. Corneal epithelial-keratocyte cell interactions have thus been extensively studied in numerous in vivo corneal wound healing settings, as well as in in vitro culture models. Exposure to the different epithelial-derived factors, as well as the integrity of the epithelial substratum, are factors known to impact the keratocyte response and determine whether corneal repair will be regenerative or fibrotic in nature. Finally, the recent identification of bone-marrow derived stem cells in the corneal stroma suggests a further complexity in the regulation of the keratocyte phenotype following injury.