Characterization of xylanase from Lentinus edodes M290 cultured on waste mushroom logs

Extracellular enzymes from Lentinus edodes M290 on normal woods (Quercus mongolica) and waste logs from oak mushroom production were comparatively investigated. Endoglucanase, cellobiohydrolase, beta-glucosidase, and xylanase activities were higher on waste mushroom logs than on normal woods after L. edodes M290 inoculation. Xylanase activity was especially different, with a three times higher activity on waste mushroom logs. When the waste mushroom logs were used as a carbon source, a new 35 kDa protein appeared. After the purification, the optimal pH and temperature for xylanase activity were determined to be 4.0 and 50 degrees C, respectively. More than 50% of the optimal xylanase activity was retained when the temperature was increased from 20 to 60 degrees C, after a 240 min reaction. At 40 degrees C, the xylanase maintained 93% of the optimal activity, after a 240 min reaction. The purified xylanase showed a very high homology to the xylanase family 10 from Aspergillus terreus by LC/MS-MS analysis. The highest Xcorr (1.737) was obtained from the peptide KWI SQGIPIDGIG SQTHLGSGGS WTVK originated from Aspergillus terreus, indicating that the 35 kDa protein was xylanase. This protein showed low homology to a previously reported L. edodes xylanase sequence.     

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.     

Topological constraints in nucleic acid hybridization kinetics

A theoretical examination of kinetic mechanisms for forming knots and links in nucleic acid structures suggests that molecules involving base pairs between loops are likely to become topologically trapped in persistent frustrated states through the mechanism of ‘helix-driven wrapping’. Augmentation of the state space to include both secondary structure and topology in describing the free energy landscape illustrates the potential for topological effects to influence the kinetics and function of nucleic acid strands. An experimental study of metastable complementary ‘kissing hairpins’ demonstrates that the topological constraint of zero linking number between the loops effectively prevents conversion to the minimum free energy helical state. Introduction of short catalyst strands that break the topological constraint causes rapid conversion to full duplex.     

MCPIP1 ribonuclease antagonizes dicer and terminates microRNA biogenesis through precursor microRNA degradation

MicroRNAs (miRNAs) are versatile regulators of gene expression and undergo complex maturation processes. However, the mechanism(s) stabilizing or reducing these small RNAs remains poorly understood. Here we identify mammalian immune regulator MCPIP1 (Zc3h12a) ribonuclease as a broad suppressor of miRNA activity and biogenesis, which counteracts Dicer, a central ribonuclease in miRNA processing. MCPIP1 suppresses miRNA biosynthesis via cleavage of the terminal loops of precursor miRNAs (pre-miRNAs). MCPIP1 also carries a vertebrate-specific oligomerization domain important for pre-miRNA recognition, indicating its recent evolution. Furthermore, we observed potential antagonism between MCPIP1 and Dicer function in human cancer and found a regulatory role of MCPIP1 in the signaling axis comprising miR-155 and its target c-Maf. These results collectively suggest that the balance between processing and destroying ribonucleases modulates miRNA biogenesis and potentially affects pathological miRNA dysregulation. The presence of this abortive processing machinery and diversity of MCPIP1-related genes may imply a dynamic evolutional transition of the RNA silencing system.     

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.     

Characterizing the complexity of enzymes on the basis of their mechanisms and structures with a bio-computational analysis

Enzymes are basically composed of 20 naturally occurring amino acids, yet they catalyse a dizzying array of chemical reactions, with regiospecificity and stereospecificity and under physiological conditions. In this review, we attempt to gain some understanding of these complex proteins, from the chemical versatility of the catalytic toolkit, including the use of cofactors (both metal ions and organic molecules), to the complex mapping of reactions to proteins (which is rarely one-to-one), and finally the structural complexity of enzymes and their active sites, often involving multidomain or multisubunit assemblies. This work highlights how the enzymes that we see today reflect millions of years of evolution, involving de novo design followed by exquisite regulation and modulation to create optimal fitness for life.     

Draft Genome Sequence of Fusobacterium nucleatum subsp. fusiforme ATCC 51190T

Fusobacterium nucleatum, one of the major causative bacteria of periodontitis, is classified into five subspecies (nucleatum, polymorphum, vincentii, animalis, and fusiforme) on the basis of the several phenotypic characteristics and DNA homology. This is the first report of the draft genome sequence of F. nucleatum subsp. fusiforme ATCC 51190(T).     

Role of angiotensin II and oxidative stress on renal aquaporins expression in hypernatremic rats

The aim of this study was to assess whether endogenous Ang II and oxidative stress produced by acute hypertonic sodium overload may regulate the expression of aquaporin-1 (AQP-1) and aquaporin-2 (AQP-2) in the kidney. Groups of anesthetized male Sprague–Dawley rats were infused with isotonic saline solution (control) or with hypertonic saline solution (Na group, 1 M NaCl), either alone or with losartan (10 mg kg^?1) or tempol (0.5 mg min^?1 kg^?1) during 2 h. Renal function parameters were measured. Groups of unanesthetized animals were injected intraperitoneally with hypertonic saline solution, with or without free access to water intake, Na+W, and Na?W, respectively. The expression of AQP-1, AQP-2, Ang II, eNOS, and NF-kB were evaluated in the kidney by Western blot and immunohistochemistry. AQP-2 distribution was assessed by immunofluorescence. Na group showed increased natriuresis and diuresis, and Ang II and NF-kB expression, but decreased eNOS expression. Losartan or tempol enhanced further the diuresis, and AQP-2 and eNOS expression, as well as decreased Ang II and NF-kB expression. Confocal immunofluorescence imaging revealed labeling of AQP-2 in the apical plasma membrane with less labeling in the intracellular vesicles than the apical membrane in kidney medullary collecting duct principal cells both in C and Na groups. Importantly, our data also show that losartan and tempol induces a predominantly accumulation of AQP-2 in intracellular vesicles. In unanesthetized rats, Na+W group presented increased diuresis, natriuresis, and AQP-2 expression (112?±?25 vs 64?±?16; *p?<?0.05). Water deprivation increased plasma sodium and diuresis but decreased AQP-2 (46?±?22 vs 112?±?25; §p?<?0.05) and eNOS expression in the kidney. This study is a novel demonstration that renal endogenous Ang II–oxidative stress, induced in vivo in hypernatremic rats by an acute sodium overload, regulates AQP-2 expression.     

The Activity of Neutral α-Glucosidase and Selected Biochemical Parameters in the Annual Cycle of Breeding Carp (Cyprinus carpio L.)

The aim of the study was to demonstrate seasonal changes in the hydrolytic and transferase activity of neutral α-glucosidase, the level of glucose, cholesterol, triglycerides and total protein in the annual breeding cycle of the carp. The study was conducted on fish from a fish farm in Lower Silesia (Poland). Blood serum was collected from the heart in: June, September and December of two consecutive years. The results of the study show that the hydrolytic and transferase activity of neutral α-glucosidase, as well as the results of basic biochemical parameters are highest in summer, when the fish seek and intake food intensively. The lowest values were observed in spring, when carp have the lowest metabolism after the wintering period.