Improved ethanol tolerance and production in strains of Clostridium thermocellum

Two Clostridium thermocellum strains were improved for ethanol tolerance, to 5% (v/v), by gradual adaptation and mutation. The best mutant gave an ethanol yield of 0.37 g/g substrate, with a growth yield 1.5 times more than its parent. Accumulation of acids and reducing sugars by the mutant strain with 5% (v/v) ethanol was lower than that of the parent strain with 1.5% (v/v) ethanol.     

Efficacy of Indolicidin,Cecropin A (1-7)-Melittin (CAMA) and Their Combination Against Biofilm-Forming Multidrug-Resistant Enteroaggregative Escherichia coli

The present study examined the anti-biofilm efficacy of two short-chain antimicrobial peptides (AMPs), namely, indolicidin and cecropin A (1-7)-melittin (CAMA) against biofilm-forming multidrug-resistant enteroaggregative Escherichia coli (MDR-EAEC) isolates. The typical EAEC isolates re-validated by PCR and confirmed using HEp-2 cell adherence assay was subjected to antibiotic susceptibility testing to confirm its MDR status. The biofilm-forming ability of MDR-EAEC isolates was assessed by Congo red binding, microtitre plate assays and hydrophobicity index; broth microdilution technique was employed to determine minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs). The obtained MIC and MBEC values for both AMPs were evaluated alone and in combination against MDR-EAEC biofilms using crystal violet (CV) staining and confocal microscopy-based live/dead cell quantification methods. All the three MDR-EAEC strains revealed weak to strong biofilm-forming ability and were found to be electron-donating and weakly electron-accepting (hydrophobicity index). Also, highly significant (P < 0.001) time-dependent hydrodynamic growth of the three MDR-EAEC strains was observed at 48 h of incubation in Dulbecco’s modified Eagle’s medium (DMEM) containing 0.45% D-glucose. AMPs and their combination were able to inhibit the initial biofilm formation at 24 h and 48 h as evidenced by CV staining and confocal quantification. Further, the application of AMPs (individually and combination) against the preformed MDR-EAEC biofilms resulted in highly significant eradication (P < 0.001) at 24 h post treatment. However, significant differences were not observed between AMP treatments (individually or in combination). The AMPs seem to be an effective candidates for further investigations such as safety, stability and appropriate biofilm-forming MDR-EAEC animal models.

     

The contribution of trees to ecosystem methane emissions in a temperate forested wetland

Wetland‐adapted trees are known to transport soil‐produced methane (CH₄), an important greenhouse gas to the atmosphere, yet seasonal variations and controls on the magnitude of tree‐mediated CH₄ emissions remain unknown for mature forests. We examined the spatial and temporal variability in stem CH₄ emissions in situ and their controls in two wetland‐adapted tree species (Alnus glutinosa and Betula pubescens) located in a temperate forested wetland. Soil and herbaceous plant‐mediated CH₄ emissions from hollows and hummocks also were measured, thus enabling an estimate of contributions from each pathway to total ecosystem flux. Stem CH₄ emissions varied significantly between the two tree species, with Alnus glutinosa displaying minimal seasonal variations, while substantial seasonal variations were observed in Betula pubescens. Trees from each species emitted similar quantities of CH₄ from their stems regardless of whether they were situated in hollows or hummocks. Soil temperature and pore‐water CH₄ concentrations best explained annual variability in stem emissions, while wood‐specific density and pore‐water CH₄ concentrations best accounted for between‐species variations in stem CH₄ emission. Our study demonstrates that tree‐mediated CH₄ emissions contribute up to 27% of seasonal ecosystem CH₄ flux in temperate forested wetland, with the largest relative contributions occurring in spring and winter. Tree‐mediated CH₄ emissions currently are not included in trace gas budgets of forested wetland. Further work is required to quantify and integrate this transport pathway into CH₄ inventories and process‐based models.     

Identification of anaplastic lymphoma kinase as a receptor for the growth factor pleiotrophin

Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Here we show in cell-free assays as well as in radioligand receptor binding studies in intact cells that PTN binds to the ALK ECD with an apparent Kd of 32 +/- 9 pm. This receptor binding is inhibited by an excess of PTN, by the ALK ECD, and by anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells induces phosphorylation of both ALK and of the downstream effector molecules IRS-1, Shc, phospholipase C-gamma, and phosphatidylinositol 3-kinase. Furthermore, the growth stimulatory effect of PTN on different cell lines in culture coincides with the endogenous expression of ALK mRNA, and the effect of PTN is enhanced by ALK overexpression. From this we conclude that ALK is a receptor that transduces PTN-mediated signals and propose that the PTN-ALK axis can play a significant role during development and during disease processes.     

Biochemical association of poly(ADP-ribose) polymerase-1 and its apoptotic peptide fragments with DNA polymerase beta

We have characterized the biochemical association of two DNA damage-dependent enzymes, poly(ADP-ribose) polymerase-1 (PARP-1) [EC 2.4.2.30] and DNA polymerase beta (pol beta) [2.7.7.7]. We reproducibly observed that pol beta is an efficient covalent target for ADP-ribose polymers under standard conditions of enzymatically catalyzed ADP-ribosylation of betaNAD+ as a substrate. The efficiency of poly(ADP-ribosyl)ation increased as a function of the pol beta and betaNAD+ concentrations. To further characterize the molecular interactions between these two unique polymerases, we also subjected human recombinant PARP-1 to peptide-specific enzymatic degradation with either caspase-3 or caspase-7 in vitro. This proteolytic treatment, commonly referred to as 'a hallmark of apoptosis', generated the two physiologically relevant peptide fragments of PARP-1, e.g., a 24-kDa amino-terminus and an 89-kDa carboxy-terminal domain. Interestingly, co-incubation of the two peptide fragments of PARP-1 with full-length pol beta resulted in their domain-specific molecular association as determined by co-immunoprecipitation and reciprocal immunoblotting. Therefore, our data strongly suggest that, once PARP-1 is proteolyzed by either caspase-3 or caspase-7 during cell death, the specific association of its apoptotic fragments with DNA repair enzymes, such as pol beta, may serve a regulatory molecular role in the execution phase of apoptosis.     

Expression of thermostable alkaline protease gene from Thermoactinomyces sp. E79 in E. coli and heat activation of the gene product

The expression of Thermoactinomyces sp. E79 protease gene cloned into E. coli was highly host-dependent and the levels of protease expression was most stable in E. coli RR1 and E. coli HB101. Heating the intracellular extract at 70°C for 15 min converted the inactive recombinant E79 protease to its active mature form and also resulted in purification of the enzyme in a single step. Addition of 10 mM CaCl2 to the E79 protease decreased its autolysis and increased its thermal stability. © Rapid Science Ltd. 1998     

Mutations in exons 3 and 7 resulting in truncated expression of human ATP6V1B1 gene showing structural variations contributing to poor substrate binding-causative reason for distal renal tubular acidosis with sensorineural deafness

Distal renal tubular acidosis (dRTA) is an autosomal recessive syndrome results defect in either proximal tubule bicarbonate reabsorption or in distal tubule H⁺ secretion and is characterized by severe hyperchloraemic metabolic acidosis in childhood. dRTA is associated with functional variations in the ATP6V1B1 gene encoding β1 subunit of H⁺-ATPase, key membrane transporters for net acid excretion of α-intercalated cells of medullary collecting ducts. In the present study, a 13-year-old male patient suffering with nephropathy and sensorineural deafness was reported in the Department of Nephrology. We predicted improper functioning of ATP6V1B1 gene could be the reason for diseased condition. Therefore, exons 3, 4, and 7 contributing active site of ATP6V1B1 gene was amplified and sequenced (Accession numbers: KF571726, KM222653). The obtained sequences were BLAST searched against the wild type ATP6V1B1 gene which showed novel mutations c.307 A > G, c.308 C > A, c.310 C > G, c.704 T > C, c.705 G > T, c.709 A > G, c.710 A > G, c.714 G > A, c.716 C > A, c.717delC, c.722 C > G, c.728insG, c.741insT, c.753G > C. These mutations resulted in the expression of truncated protein terminating at Lys 209. The mutated ATP6V1B1structure superimposed with wild type showed extensive variations with RMSD 1.336 Å and could not bind to substrate ADP leading to non-functional ATPase. These results conclusively explain these mutations in ATP6V1B1 gene resulted in structural changes causing accumulation of H⁺ ions contributing to dRTA with sensorineural deafness.     

MicroRNA signatures differentiate melanoma subtypes

Melanoma is an aggressive cancer that is highly resistance to therapies once metastasized. We studied microRNA (miRNA) expression in clinical melanoma subtypes and evaluated different miRNA signatures in the background of gain of function somatic and inherited mutations associated with melanoma. Total RNA from 42 patient derived primary melanoma cell lines and three independent normal primary melanocyte cell cultures was evaluated by miRNA array. MiRNA expression was then analyzed comparing subtypes and additional clinicopathologic criteria including somatic mutations. The prevalence and association of an inherited variant in a miRNA binding site in the 3′UTR of the KRAS oncogene, referred to as the KRAS-variant, was also evaluated. We show that seven miRNAs, miR-142-3p, miR-486, miR-214, miR-218, miR-362, miR-650 and miR-31, were significantly correlated with acral as compared to non-acral melanomas (p < 0.04). In addition, we discovered that the KRAS-variant was enriched in non-acral melanoma (25%), and that miR-137 under expression was significantly associated with melanomas with the KRAS-variant. Our findings indicate that miRNAs are differentially expressed in melanoma subtypes and that their misregulation can be impacted by inherited gene variants, supporting the hypothesis that miRNA misregulation reflects biological differences in melanoma.Key words: melanoma, microRNA profiling, biomarker, acral, KRAS-variant, SNP     

An overview on genus garcinia: phytochemical and therapeutical aspects

The genus Garcinia belongs to the family Clusiaceae and has been involved in ayurvedic preparations to medicate various pathophysiological disorders. The bioactive molecules like hydroxycitric acid (HCA), flavonoids, terpenes, polysaccharides, procyanidines and polyisoprenylated benzophenone derivatives like garcinol, xanthochymol and guttiferone isoforms have been isolated from the genus Garcinia. The genus has received the attention of pharmaceutical industries due to their immense remedial qualities. The HCA has been known for its hypolipidemic property. The polyisoprenylated benzophenone and xanthone derivatives are known for their antioxidant, apoptotic, anti-cancer, anti-inflammatory, anti-bacterial, anti-viral, anti-fungal, anti-ulcer, anti-protozoal, and HAT inhibiting properties. Future studies on the synthesis of therapeutically important products and their analogs and evaluation of their safety and efficacy would be of great interest. Though the genus includes more than 300 species, we have made an effort to conceive the curative qualities of bioactive compounds of selected plants to the best of our knowledge.