Characterization of glucose oxidase-negative mutants of a lignin degrading basidiomycete Phanerochaete chrysosporium

The isolation and characterization of glucose oxidase-negative (gox ^-) mutants of Phanerochaete chrysosporium, is described. These mutants are deficient not only in their ability to produce hydrogen peroxide (H₂O₂) but also in lignin degradation (2-¹⁴C-synthetic lignin¹⁴CO₂), ligninase and peroxidase activities, decolorization of the dye poly-R 481, and production of ethylene from -oxo--methylthiobutyric acid (KTBA). The gox ^- mutants retained, albeit at a lower level, the capacity to produce veratryl alcohol, a typical secondary metabolite, and produced conidia at a level comparable to that of the wild type. The addition of ligninase and/or glucose oxidase to a gox ^- mutant (GOX-10) did not enhance its capacity to degrade lignin. The Gox⁺ revertant strains regained glucose oxidase activity, the ability to degrade lignin, as well as the other characteristics that were missing in the gox ^- mutants. The results suggest that the genetic lesion in these mutants affects the regulation of a set of secondary metabolic characteristics.Abbreviations Gox glucose oxidase - KTBA -oxo--methylthiobutyric acid Journal article no. 11740 from the Michigan Agricultural Experiment Station     

The surface topography of a monogenean Heterapta chorinemi from the gills of Scomberoides commersonianus

and 1986. The surface topography of a monogenean Heterapta chorinemi from the gills of Scomberoides commersonianus. International Journal for Parasitology 16: 595–600. The dorsal and ventral surfaces of H. chorinemi bear microvilli and boss-like structures. These may increase the surface area for respiratory gaseous exchange and absorption of nutrients from the surrounding medium. Uniciliated sensory endings, presumably rheoreceptors, are present over the entire surface whereas non-ciliated structures, possibly mechanoreceptors, are present only on the ventral surface of the haptor. The haptor possesses 6–10 pairs of pincer-type clamps and 30–40 pairs of open sucker-type clamps with scleritized jaws. The common genital atrium is situated on the ventral surface, the vaginal pore opens on the dorsal surface and the mouth is subterminal as described in previous light microscope studies.     

NOTPME: a 31P NMR probe for measurement of divalent cations in biological systems

1,4,7-Triazacyclononane-N,N',N'-tris(methylenephosphonate monoethylester) (NOTPME) has been synthesized, characterized and analyzed for use as a 31P NMR indicator of intracellular Mg2+ and Zn2+ ions. The 31P NMR spectrum of this chelate in the presence of metal ions shows characteristic resonances for the free chelate, Mg(NOTPME)-, Zn(NOTPME)-, and Ca(NOTPME)-. The Kd values indicate that this chelate has a 10-fold higher affinity for Mg2+ than for Ca2+ at physiological pH values. In the presence of Mg2+, NOTPME is readily loaded into red blood cells. A 31P NMR spectrum of red cells taken after several washings shows resonances characteristic of entrapped NOTPME and the Mg(NOTPME)- complex, the relative areas of which report an intracellular free Mg2+ concentration of 0.32 mM. The 31P chemical shifts of the free chelate and its metal complexes are far downfield from the typical phosphorus-containing metabolites observed in biological systems, thus making it possible to monitor intracellular cation concentrations and cell energetics simultaneously.     

Cytokine-induced production of IFN-beta 2/IL-6 by freshly explanted human endometrial stromal cells. Modulation by estradiol-17 beta

The cytokine IFN-beta 2/IL-6 has emerged as an important means of communication between cells--both within the immune system as well as outside it. In exploring the link between the endocrine and the immune systems, we have studied the secretion of IFN-beta 2/IL-6 by freshly explanted human endometrial stromal cells and its modulation by estrogens. Endometrial stromal cells produced IFN-beta 2/IL-6 in response to other inflammation-associated cytokines such as IL-1 alpha or beta, TNF, and IFN-gamma. This secretion was strongly inhibited by estradiol-17 beta at concentrations as low as 10(-9) M. Multiple species of stromal cell IFN-beta 2/IL-6 in the size range 23 to 30 kDa were detected using immunoprecipitation or immunoblotting procedures. The endometrial stromal cell IFN-beta 2/IL-6 species were phosphorylated and differentially glycosylated in a manner comparable to IFN-beta 2/IL-6 secreted by induced human peripheral blood monocytes or foreskin fibroblasts. However, in contrast to peripheral blood monocytes and fibroblasts, bacterial LPS did not induce IFN-beta 2/IL-6 production in endometrial stromal cells. Additionally, the IFN-beta 2/IL-6 identified in medium from IL-1 alpha-induced stromal cells is biologically active on hepatocytes. These observations, taken together with the observation that IFN-beta 2/IL-6 strongly inhibits the proliferation of human epithelial cells, suggest the possibility that stromal cell secreted IFN-beta 2/IL-6 may affect the physiology of the overlying epithelium in an hormonally modulated manner. Estrogen-regulated production of endometrial IFN-beta 2/IL-6 may participate in gender-specific systemic immunomodulation.     

Impedance spectroscopy as a tool for non‐intrusive detection of extracellular mediators in microbial fuel cells

Endogenously produced, diffusible redox mediators can act as electron shuttles for bacterial respiration. Accordingly, the mediators also serve a critical role in microbial fuel cells (MFCs), as they assist extracellular electron transfer from the bacteria to the anode serving as the intermediate electron sink. Electrochemical impedance spectroscopy (EIS) may be a valuable tool for evaluating the role of mediators in an operating MFC. EIS offers distinct advantages over some conventional analytical methods for the investigation of MFC systems because EIS can elucidate the electrochemical properties of various charge transfer processes in the bio‐energetic pathway. Preliminary investigations of Shewanella oneidensis DSP10‐based MFCs revealved that even low quantities of extracellular mediators significantly influence the impedance behavior of MFCs. EIS results also suggested that for the model MFC studied, electron transfer from the mediator to the anode may be up to 15 times faster than the electron transfer from bacteria to the mediator. When a simple carbonate membrane separated the anode and cathode chambers, the extracellular mediators were also detected at the cathode, indicating diffusion from the anode under open circuit conditions. The findings demonstrated that EIS can be used as a tool to indicate presence of extracellular redox mediators produced by microorganisms and their participation in extracellular electron shuttling. Biotechnol. Bioeng. 2009; 104: 882–891. © 2009 Wiley Periodicals, Inc.     

Identification of Potential Probiotics in the Midgut of Mulberry Silkworm,Bombyx mori Through Metagenomic Approach

Microorganisms play an important role in the growth and development of numerous insect species. The mulberry silkworm, Bombyx mori (Lepidoptera), harbors several bacteria in its midgut aiding the metabolic processes; however, the variability of bacterial spp. present in the midgut and their role(s) in the growth and development of the silkworm are poorly understood. The present work compares the diversity of midgut bacterial communities in silkworms of variable voltinism (Pure Mysore, PM: multivoltine; CSR2: bivoltine and PM × CSR2: crossbreed) through metagenomics. The predominance of Enterococcus (30.30%) followed by Bacillus (16.96%) was observed in PM, whereas Lactobacillus (56.56%) followed by Enterococcus (10.58%) was seen only in CSR2. Interestingly, crossbreed midgut harbored diverse bacterial communities (36.21% Lactobacillus, 25.94% Bacillus, 8.1% Enterococcus, and 18.37% uncultured bacteria). Metagenomic profiles indicate variability in the gut bacterial population in different kinds of silkworms influencing the physiological activities accordingly. The dominant bacteria, particularly lactobacilli, bacilli, and enterococci could be further explored for identifying the potential probiotic consortia based on a literature survey and potential involvement in nutrient absorption, disease/stress tolerance, and improved economic traits.

     

Double-beam flying spot scanner for two-dimensional polyacrylamide gel electrophoresis

The construction of a double-beam photometer in which the light source is a cathode ray oscilloscope is described. The light spot from the oscilloscope was focused and reduced in size at the gel plane to give a diameter of less than 0.15 mm and make it possible to scan over a 50 X 59-mm rectangle; using reduced spatial resolution (spot less than 0.2 mm) the area scanned becomes 70 X 90 mm. The light from the CRT was divided into two beams; one was directed through the transparent object to a photomultiplier and the other to a reference photomultiplier. The signals from these two detectors were converted to the logarithm of the ratio by a logging amplifier to give a direct measure of absorbance. Positioning of the spot, control of light intensity, and measurement of absorbance were carried out through an interface to a 16-bit computer. The relationship between measured and actual absorbance was linear over the range of absorbance 0 to 2, which could be raised to 1 to 3 by placing a neutral filter in the reference beam. The system generated an image containing 256 X 256 pixels in about 5 min, the scanning speed was determined by the persistence time of the P4 phosphor on the cathode ray tube, and faster scans can be made using A6 phosphor.     

Alteration of the protein kinase binding domain enhances function of the Saccharomyces cerevisiae molecular chaperone Cdc37

Cdc37 is a molecular chaperone that has a general function in the biogenesis of protein kinases. We identified mutations within the putative "protein kinase binding domain" of Cdc37 that alleviate the conditional growth defect of a strain containing a temperature-sensitive allele, tpk2(Ts), of the cyclic AMP-dependent protein kinase (PKA). These dominant mutations alleviate the temperature-sensitive growth defect by elevating PKA activity, as judged by their effects on PKA-regulated processes, localization and phosphorylation of the PKA effector Msn2, as well as in vitro PKA activity. Although the tpk2(Ts) growth defect is also alleviated by Cdc37 overproduction, the CDC37 dominant mutants contain wild-type Cdc37 protein levels. In addition, Saccharomyces cerevisiae Ste11 protein kinase has an elevated physical interaction with the altered Cdc37 protein. These results implicate specific amino-terminal residues in the interaction between Cdc37 and client protein kinases and provide further genetic and biochemical support for a model in which Cdc37 functions as a molecular chaperone for protein kinases.     

Receptor for AGE (RAGE): weaving tangled webs within the inflammatory response

The family of RAGE ligands, including Advanced Glycation Endproducts (AGEs), S100/calgranulins, High Mobility Group Box-1 (HMGB1) and amyloid beta peptide (Abeta) and beta-sheet fibrils are highly enriched in immune and inflammatory foci. In parallel, upregulation of Receptor for AGE (RAGE) is noted in diverse forms of inflammation and autoimmunity, based on experiments examining human tissues as well as animal models. Indeed, prior to the demonstration that S100/calgranulins were signal transduction ligands of RAGE, these molecules were considered "biomarkers" of disease and disease activity in disorders such as colitis and arthritis. Premiere roles for RAGE in advancing cellular migration implicate this receptor in targeting immune cells to vulnerable foci. Once engaged, ligand-RAGE interaction in inflammatory and vascular cells amplifies upregulation of inflammatory cytokines, adhesion molecules and matrix metalloproteinases (MMPs). Discerning the primal versus chronic injury-provoking roles for this ligand-receptor interaction is a challenge in delineating the functions of the ligand/RAGE axis. As RAGE is expressed by many of the key cell types linked integrally to the immune response, we propose that the sites and time course of ligand-RAGE stimulation determine the phenotype produced by this axis. Ultimately, drawing the fine line between antagonism versus stimulation of the receptor in health and disease will depend on the full characterization of RAGE in repair versus injury.