Development of mutants of <Emphasis Type="Italic">Melanocarpus albomyces</Emphasis> for hyperproduction of xylanase

The wild type filamentous fungus, Melanocarpus albomyces, produces many commercially valuable enzymes, including Xylanases and Xylan-debranching enzymes with low activity. In this paper, we report for the first time the development of M. albomyces mutants from vegetative spores. Profuse sporulation of M. albomyces was induced on Potato Carrot Agar medium. These spores, when subjected to chemical mutation, led to the isolation of the hyper-xylanase producing mutant, viz, M. albomyces IITD3A. Various parameters including number of spores, nitrogen source and C/N ratio of the medium were optimized for production of xylanase by the mutant in a shake flask culture. Under controlled pH at 7.8, the mutant produced highly active xylanase with 415 IU/mL after 36 h of growth on soluble alkaline lignocellulosic extract in a 14-L fermentor. The overall productivity of xylanase was 8-fold higher than the wild type culture with11, 530 IU/L/h. The enzyme can be easily stored at 37°C for 50 days by addition of a small amount of the preservative — thiomersal. Also, for long term storage, a lyophilized powder form of the enzyme can be used which retained 100% of its activity for > 50 days. When assayed at pH 7.5 and temperature 55°C, the xylanase retained 100% of its original activity, and also at pH 9.0, it retained > 50% of its activity for 2 h, which is promising for its application in the pulp and paper industry.     

ROCK- and myosin-dependent matrix deformation enables protease-independent tumor-cell invasion in vivo

Tumor cells invading three-dimensional matrices need to remodel the extracellular matrix (ECM) in their path. Many studies have focused on the role of extracellular proteases; however, cells with amoeboid or rounded morphologies are able to invade even when these enzymes are inhibited. Here, we describe the mechanism by which cells move through a dense ECM without proteolysis. Amoeboid tumor cells generate sufficient actomyosin force to deform collagen fibers and are able to push through the ECM. Force generation is elevated in metastatic MTLn3E cells, and this correlates with increased invasion and altered myosin light chain (MLC) organization. In metastatic cells, MLC is organized perpendicularly to the direction of movement behind the invading edge. Both the organization of MLC and force generation are dependent upon ROCK function. We demonstrate that ROCK regulates the phosphorylation of MLC just behind the invading margin of the cell. Imaging of live tumors shows that MLC is organized in a similar ROCK-dependent fashion in vivo and that inhibition of ROCK but not matrix-metalloproteases reduces cancer cell motility in vivo.     

Dose-finding study with nicotine as a proconvulsant agent in PTZ-induced seizure model in mice

The present study was conducted to investigate the possible interaction between low doses of nicotine and pentylenetetrazole (PTZ) in vivo and also to evaluate the influence of nicotine on the antiseizure efficacy of topiramate and sodium valproate in the PTZ-induced seizure model in mice. Graded dose–response study with nicotine showed the CD50 value for nicotine at 6.76 mg/kg. i.p. Subtheshold dose of nicotine (4 mg/kg, i.p.) pretreatment significantly decreased the CD50 value for PTZ from 47.86 mg/kg, i.p. (of PTZ per se) to 31.62 mg/kg, i.p. Sodium valproate but not topiramate, significantly inhibited PTZ-induced seizures in mice with an ED50 value of 177.83 mg/kg, i.p. Nonconvulsive dose of nicotine (1 mg/kg, i.p.) significantly antagonized the protective efficacy of sodium valproate against PTZ-induced seizures and increased the ED50 value to 338.84 mg/kg, i.p. PTZ-induced seizures significantly increased the mouse brain levels of MDA and reduced the level of GSH while sodium valproate reversed such changes. Nicotine pretreatment reversed the anti-lipid peroxidative action of sodium valproate in the PTZ-induced seizure model in mice. The study highlighted the convulsant as well as proconvulsant role of nicotine and established dose discrimination for nicotine as a proconvulsant agent and an anti-antiseizure agent. The study bears significant clinical relevance particularly amongst epileptic smokers who may show failure of efficacy of antiepileptic agents and present with breakthrough seizure attacks on exposure to nicotine.     

The yolk nucleus in the freshwater teleost Garra gotyla gotyla (Gray)

The structure and functional significance of the yolk nucleus in Garra are described. The study shows that the yolk nucleus appears in the cytoplasm of the early perinucleolus stage of the oocyte. It is present as a deeply stained structure enclosed in a vesicle. It disappears in the yolk vesicle stage and vitellogenesis begins soon afterwards. This probably indicates that it plays a role in the initiation of vitellogenesis. When yolk formation starts, the yolk nucleus disappears; this also suggests that it is of functional significance in yolk synthesis.     

Effect of second-leaf removal or kinetin treatment on the nucleic acid metabolism of senescing first seedling leaf of barley

1. Changes in nucleic acid metabolism in first seedling leaves of barley plants during aging (from 7 to 27 days) were followed, and the effect of continual removal of the second leaf and basal meristem or of treating the first leaf with 20p.p.m. kinetin on these changes was examined. During aging of the first seedling leaves the ribosomal RNA, DNA and soluble RNA declined, with ribosomal RNA showing the most rapid fall. This was, however, accompanied by increased incorporation of ³²P into RNA, which reached its peak on the fifteenth day. 2. Second-leaf removal partially suppressed first-leaf senescence as judged by retarded chlorophyll and nucleic acid decline and by a decreased extent of RNA labelling. Treatment with kinetin, however, did not prove effective. 3. No significant differences in the sucrose-gradient pattern of ³²P-labelled nucleic acids or in the ³²P-labelled nucleotide composition of RNA fractions during aging or during the two treatments were noted, except for a decrease in CMP content of soluble RNA during aging. 4. The results demonstrate that important changes in RNA metabolism are associated with leaf senescence.     

Terminal deoxynucleotidyl transferase activity in a cell line (molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia

A terminal deoxynucleotidyl transferase having a sedimentation coefficient of 3–4S has been found associated with the chromatin from a cell line (Molt-4) derived from the peripheral blood of a patient with acute lymphoblastic leukemia.     

Fate of pathological prion (PrPsc92–138) in soil and water: prion-clay nanoparticle molecular dynamics

Pathogenic prion protein scrapie (PrP^sc) may contaminate soils for decades and remain in water in colloidal suspension, providing infection pathways for animals through the inhalation of ingested dust and soil particles, and drinking water. We used molecular dynamics simulations to understand the strong binding mechanism of this pathogenic peptide with clay mineral surfaces and compared our results to experimental works. We restricted our model to the moiety PrP(92–138), which is a portion of the whole PrP^sc molecule responsible for infectivity and modeled it using explicit solvating water molecules in contact with a pyrophyllite cleavage plane. Pyrophyllite is taken as a model for common soil clay, but it has no permanent structural charge. However, partial residual negative charges occur on the cleavage plane slab surface due to a slab charge unbalance. The charge is isotropic in 2D and it was balanced with K⁺ ions. After partially removing potassium ions, the peptide anchors to the clay surface via up to 10 hydrogen bonds, between protonated lysine or histidine residues and the oxygen atoms of the siloxane cavities. Our results provide insight to the mechanism responsible for the strong association between the PrP^sc peptide and clay nanoparticles and the associations present in contaminated soil and water which may lead to the infection of animals.     

Shelf-life enhancement of bio-inoculant formulation by optimizing the trace metals ions in the culture medium for production of DAPG using fluorescent pseudomonad R62

Statistical experimental design was used to optimize the concentration of trace elements for production of antifungal compound, 2,4-diacetylphloroglucinol (DAPG), from fluorescent pseudomonad R62 in shake-flask cultivation. The selection of the trace metal ions, influencing DAPG production, was done using Plackett-Burman design (PBD). Only Zn(2+), Mn(2+) and MoO(4)(2-) were the most significant components (p<0.05). A quadratic model was used to fit the response. Application of response surface methodology (RSM) revealed that the optimum values of the salts of the trace elements Zn(2+) (ZnSO(4)·7H(2)O), Mn(2+) (MnCl(2)·4H(2)O), and MoO(4)(2-) (Na(2)MoO(4)·2H(2)O) were 83, 42 and 135μM, respectively, to achieve 125 mg/L of DAPG, which was nearly 13-fold more compared to its production in basal synthetic medium in shake flask. The studies in 14L bioreactor resulted in 135 mg/L of DAPG at the end of 36 h of cultivation. The culture broth containing 125 mg/L of DAPG was found to be sufficient for keeping the bio-inoculant viable in non-sterile talcum powder-based formulations (which contained 25μg DAPG/g carrier) when stored at 28°C for 6 months. The structure of the purified DAPG was confirmed using (1)H NMR and mass spectrometry.