Enhanced butanol production in Clostridium acetobutylicum ATCC 824 by double overexpression of 6-phosphofructokinase and pyruvate kinase genes

This study elucidated the importance of two critical enzymes in the regulation of butanol production in Clostridium acetobutylicum ATCC 824. Overexpression of both the 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA) genes increased intracellular concentrations of ATP and NADH and also resistance to butanol toxicity. Marked increases of butanol and ethanol production, but not acetone, were also observed in batch fermentation. The butanol and ethanol concentrations were 29.4 and 85.5 % higher, respectively, in the fermentation by double-overexpressed C. acetobutylicum ATCC 824/pfkA+pykA than the wild-type strain. Furthermore, when fed-batch fermentation using glucose was carried out, the butanol and total solvent (acetone, butanol, and ethanol) concentrations reached as high as 19.12 and 28.02 g/L, respectively. The reason for improved butanol formation was attributed to the enhanced NADH and ATP concentrations and increased tolerance to butanol in the double-overexpressed strain.     

Light-induced phosphorylation of crystallins in the retinal pigment epithelium

Protein phosphorylations have essential regulatory roles in visual signaling. Previously, we found that phosphorylation of several proteins in the retina and retinal pigment epithelium (RPE) is involved in anti-apoptotic signaling under oxidative stress conditions, including light exposure. In this study, we used a phosphoprotein enrichment strategy to evaluate the light-induced phosphoproteome of primary bovine RPE cells. Phosphoprotein-enriched extracts from bovine RPE cells exposed to light or dark conditions for 1h were separated by 2D SDS-PAGE. Serine and tyrosine phosphorylations were visualized by 2D phospho Western blotting and specific phosphorylation sites were analyzed by tandem mass spectrometry. Light induced a marked increase in tyrosine phosphorylation of beta crystallin A3 and A4. The most abundant light-induced up-regulated phosphoproteins were crystallins of 15-25 kDa, including beta crystallin S and zeta crystallin. Phosphorylation of beta crystallin suggests an anti-apoptotic chaperone function of crystallins in the RPE. Other chaperones, cytoskeletal proteins, and proteins involved in energy balance were expressed at higher levels in the dark. A detailed analysis of RPE phosphoproteins provides a molecular basis for understanding of light-induced signal transduction and anti-apoptosis mechanisms. Our data indicates that phosphorylation of crystallins likely represents an important mechanism for RPE shielding from physiological and pathophysiological light-induced oxidative injury.     

Neuroprotective effects of overexpressed cyclophilin B against Aβ-induced neurotoxicity in PC12 cells

Accumulated amyloid-β (Aβ) is a well-known cause of neuronal apoptosis in Alzheimer disease and functions in part by generating oxidative stress. Our previous work suggested that cyclophilin B (CypB) protects against endoplasmic reticulum (ER) stress. Therefore, in this study we examined the ability of CypB to protect against Aβ toxicity. CypB is present in the neurons of rat and mouse brains, and treating neural cells with Aβ_25-35 mediates apoptotic cell death. Aβ_25-35-induced neuronal toxicity was inhibited by the overexpression of CypB as measured by cell viability, apoptotic morphology, sub-G1 cell population, intracellular reactive oxygen species accumulation, activated caspase-3, PARP cleavage, Bcl-2 proteins, mitogen-activated protein kinase (MAPK) activation, and phosphoinositide 3-kinase (PI-3-K) activation. CypB/R95A PPIase mutants did not reduce Aβ_25-35 toxicity. We showed that Aβ_25-35-induced apoptosis is more severe in a CypB knockdown model, confirming that CypB protects against Aβ_25-35-induced toxicity. Consequently, these findings suggest that CypB may protect against Aβ toxicity by its antioxidant properties, by regulating MAPK and PI-3-K signaling, and through the ER stress pathway.     

Effect of 3,3',4',5-tetrachlorosalicylanilide on reduction of excess sludge and nitrogen removal in biological wastewater treatment process

A metabolic uncoupler, 3,3',4',5-tetrachlorosalicylanilide (TCS), was used to reduce excess sludge production in biological wastewater treatment processes. Batch experiments confirmed that 0.4 mg/l of TCS reduced the aerobic growth yield of activated sludge by over 60%. However, the growth yield remained virtually constant even at the increased concentrations of TCS when cultivations were carried out under the anoxic condition. Reduction of sludge production yield was confirmed in a laboratory-scale anoxic-oxic process operated for 6 months. However, it was found that ammonia oxidation efficiency was reduced by as much as 77% in the presence of 0.8 mg/l of TCS in the batch culture. Similar results were also obtained through batch inhibition tests with activated sludges and by bioluminescence assays using a recombinant Nitrosomonas europaea (pMJ217). Because of this inhibitory effect of TCS on nitrification, the TCS-fed continuous system failed to remove ammonia in the influent. When TCS feeding was stopped, the nitrification yield of the process was resumed. Therefore, it seems to be necessary to assess the nitrogen content of wastewater if TCS is used for reducing sludge generation.     

Hyperlipidemia affects multiscale structure and strength of murine femur

To improve bone strength prediction beyond limitations of assessment founded solely on the bone mineral component, we investigated the effect of hyperlipidemia, present in more than 40% of osteoporotic patients, on multiscale structure of murine bone. Our overarching purpose is to estimate bone strength accurately, to facilitate mitigating fracture morbidity and mortality in patients. Because (i) orientation of collagen type I affects, independently of degree of mineralization, cortical bone?s micro-structural strength; and, (ii) hyperlipidemia affects collagen orientation and μCT volumetric tissue mineral density (vTMD) in murine cortical bone, we have constructed the first multiscale finite element (mFE), mouse-specific femoral model to study the effect of collagen orientation and vTMD on strength in Ldlr^−/−, a mouse model of hyperlipidemia, and its control wild type, on either high fat diet or normal diet. Each µCT scan-based mFE model included either element-specific elastic orthotropic properties calculated from collagen orientation and vTMD (collagen-density model) by experimentally validated formulation, or usual element-specific elastic isotropic material properties dependent on vTMD-only (density-only model). We found that collagen orientation, assessed by circularly polarized light and confocal microscopies, and vTMD, differed among groups and that microindentation results strongly correlate with elastic modulus of collagen-density models (r²=0.85, p=10⁻⁵). Collagen-density models yielded (1) larger strains, and therefore lower strength, in simulations of 3-point bending and physiological loading; and (2) higher correlation between mFE-predicted strength and 3-point bending experimental strength, than density-only models. This novel method supports ongoing translational research to achieve the as yet elusive goal of accurate bone strength prediction.     

Enhanced Methane Production from Anaerobic Digestion of Disintegrated and Deproteinized Excess Sludge

To improve biogas yield and methane content in anaerobic digestion of excess sludge from the wastewater treatment plant, the sludge was disintegrated by using various methods (sonication, alkaline and thermal treatments). Since disintegrated sludge contains a high concentration of soluble proteins, the resulting metabolite, ammonia, may inhibit methane generation. Therefore, the effects of protein removal from disintegrated sludge on methane production were also studied. As a result, an obvious enhancement of biogas generation was observed by digesting disintegrated sludge (biogas yield increased from 15 to 36 ml/g COD_added·day for the raw excess sludge and the sonicated sludge, respectively). The quality of biogas was also improved by removing proteins from the disintegrated sludge. About 50% (w/w) of soluble proteins were removed from the suspension of disintegrated sludge by salting out using 35 g MgCl₂·6H₂O/l and also by isoelectric point precipitation at pH 3.3. For deproteinized sludge, methane production increased by 19%, and its yield increased from 145 ml/g COD_removed to 325 ml/g COD_removed. Therefore, the yield and quality of biogas produced from digestion of excess sludge can be enhanced by disintegrating the sludge and subsequent protein removal. Revisions requested 14 November 2005; Revisions received 13 January 2006     

Decreased hippocampal brain‐derived neurotrophic factor and impaired cognitive function by hypoglossal nerve transection in rats

The hypoglossal nerve controls tongue movements, and damages of it result in difficulty in mastication and food intake. Mastication has been reported to maintain hippocampus‐dependent cognitive function. This study was conducted to examine the effect of tongue motor loss on the hippocampus‐dependent cognitive function and its underlying mechanism. Male Sprague Dawley rats were subjected to the initial training of Morris water maze task before or after the bilateral transection of hypoglossal nerves (Hx). When the initial training was given before the surgery, the target quadrant dwelling time during the probe test performed at a week after the surgery was significantly reduced in Hx rats relative to sham‐operated controls. When the initial training was given after the surgery, Hx affected the initial and reversal trainings and probe tests. Brain‐derived neurotrophic factor (BDNF) expression, cell numbers and long‐term potentiation (LTP) were examined in the hippocampus on the 10^th day, and BrdU and doublecortin staining on the 14^th day, after the surgery. Hx decreased the hippocampal BDNF and cells in the CA1/CA3 regions and impaired LTP. BrdU and doublecortin staining was decreased in the dentate gyrus of Hx rats. Results suggest that tongue motor loss impairs hippocampus‐dependent cognitive function, and decreased BDNF expression in the hippocampus may be implicated in its underlying molecular mechanism in relation with decreased neurogenesis/proliferation and impaired LTP.     

Ice premelting during differential scanning calorimetry

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.     

Positions of multiple insertions in SSU rDNA of lichen-forming fungi

Lichen-forming fungi, in symbiotic associations with algae, frequently have nuclear small subunit ribosomal DNA (SSU rDNA) longer than the 1,800 nucleotides typical for eukaryotes. The lichen-forming ascomycetous fungus Lecanora dispersa contains insertions at eight distinct positions of its SSU rDNA; the lichen-forming fungi Calicium tricolor and Porpidia crustulata each contain one insertion. Insertions are not limited to fungi that form lichens; the lichen ally Mycocalicium albonigrum also contains two insertions. Of the 11 insertion positions now reported for lichen-forming fungi and this ally, 6 positions are known only from lichen-forming fungi. Including the 4 newly reported in this study, insertions are now known from at least 17 positions among all reported SSU rDNA sequences. Insertions, most of which are Group I introns, are reported in fungal and protistan lineages and occur at corresponding positions in genomes as phylogenetically distant as the nuclei of fungi, green algae, and red algae. Many of these positions are exposed in the mature rRNA tertiary structure and may be subject to independent insertion of introns. Insertion of introns, accompanied by their sporadic loss, accounts for the scattered distribution of insertions observed within the SSU rDNA of these diverse organisms.