Improvement in fermentation characteristics of degermed ground corn by lipid supplementation

With rapid growth of fuel ethanol industry, and concomitant increase in distillers dried grains with solubles (DDGS), new corn fractionation technologies that reduce DDGS volume and produce higher value coproducts in dry grind ethanol process have been developed. One of the technologies, a dry degerm, defiber (3D) process (similar to conventional corn dry milling) was used to separate germ and pericarp fiber prior to the endosperm fraction fermentation. Recovery of germ and pericarp fiber in the 3D process results in removal of lipids from the fermentation medium. Biosynthesis of lipids, which is important for cell growth and viability, cannot proceed in strictly anaerobic fermentations. The effects of ten different lipid supplements on improving fermentation rates and ethanol yields were studied and compared to the conventional dry grind process. Endosperm fraction (from the 3D process) was mixed with water and liquefied by enzymatic hydrolysis and was fermented using simultaneous saccharification and fermentation. The highest ethanol concentration (13.7% v/v) was achieved with conventional dry grind process. Control treatment (endosperm fraction from 3D process without lipid supplementation) produced the lowest ethanol concentration (11.2% v/v). Three lipid treatments (fatty acid ester, alkylphenol, and ethoxylated sorbitan ester 1836) were most effective in improving final ethanol concentrations. Fatty acid ester treatment produced the highest final ethanol concentration (12.3% v/v) among all lipid supplementation treatments. Mean final ethanol concentrations of alkylphenol and ethoxylated sorbitan ester 1836 supplemented samples were 12.3 and 12.0% v/v, respectively.Mention of brand or firm names does not constitute an endorsement by University of Illinois or USDA above others of similar nature not mentioned     

The masking of peroxidase-catalysed oxidation of IAA in Vigna

Partially purified enzyme preparations of extracts of Vigna seedlings exhibited guaiacol-oxidase activity but not IAA-oxidase activity. However, by ageing the enzyme preparations, or by treating them with H₂O₂, it was possible to unmask IAA-oxidase activity. Gel filtration of Vigna extracts on Sepharose yielded separate peaks for IAA-oxidase, guaiacol-oxidase and auxin protectors. The appearance of a separate IAA-oxidase peak reflected the overlap of peroxidase and protector; the apparent difference in the migration rate of IAA-oxidase and guaiacol-oxidase activity proved to be an artifact. The data imply that previous reports of differences between peroxidase and IAA oxidase need to be reinvestigated to rule out the possible effect of contamination by endogenous, high MW auxin protectors. A rapid method for removing most of the auxin protectors and thereby unmasking IAA-oxidase activity is described.     

Chlamydia trachomatis pulmonary infection induces greater inflammatory pathology in immunoglobulin A deficient mice

Chlamydia trachomatis is an intracellular bacterial pathogen that primarily infects via mucosal surfaces. Using mice with a targeted disruption in IgA gene expression (IgA(-/-) mice), we have studied the contribution of IgA, the principal mucosal antibody isotype, in primary immune defenses against pulmonary C. trachomatis infection. Bacterial burden was comparable between IgA(-/-) and IgA(+/+) animals following C. trachomatis challenge. Serum and pulmonary anti-Chlamydia antibody levels were higher in IgA(-/-) animals, with the exception of IgA. Lung sections of challenged IgA(-/-) mice showed more extensive immunopathology than corresponding IgA(+/+) animals. Real-time PCR analysis demonstrated significantly greater IFN-gamma and TGF-beta mRNA expression in IgA(-/-) as compared to IgA(+/+) animals. Together, these results suggest that IgA may not be necessary for clearance of primary C. trachomatis infection. However, IgA(-/-) mice displayed exaggerated lung histopathology and altered cytokine production, indicating an important role for IgA in regulating C. trachomatis induced pulmonary inflammation and maintenance of mucosal homeostasis.     

The heterosis estimates for growth and survival traits in sterlet and Siberian sturgeon purebreds and hybrids

The aim of the present study was to estimate and compare the growth and survival traits of the hybrids and purebreds produced by crossing the Siberian sturgeon (Acipenser baerii) (S) and sterlet (Acipenser ruthenus) (St) in order to determine the heterosis effect in the F1 generation. We compared the breeding conditions, mean body weight (BW) and cumulative survival in the artificially produced hybrid crosses of sterlet and Siberian sturgeon with respect to their pure parental species in indoor and outdoor aquaculture systems at different developmental stages. Fertilization and hatching rates were found to be significantly higher in S × S purebred compared to St × S hybrid. The highest values of BW were recorded in St × S hybrid (557.54 ± 179.7 g) on 862 days post-hatch (dph) while the highest cumulative survival was recorded in S × S purebred (14.3%). The recorded cumulative survival and mean BW was significantly lower in St × St purebred. The highest positive heterosis was recorded for mean BW of St × S hybrid (51.3% on 862 dph) throughout the sampling points. The studied sturgeon hybrids had higher mean BW compared to St × St purebred under suboptimal rearing conditions. Although there was no clear demonstration of the superiority in performance of reciprocal hybrids over purebreds, the St × S hybrid can be used for achieving better productivity in aquaculture systems.     

Micropropagation of Salix pseudolasiogyne from nodal explants

The effect of phytohormones on the breaking of dormancy of axillary buds in Salix pseudolasiogyne and their subsequent proliferation from nodal explants were examined. Nodal explants obtained from a 20–year-old S. pseudolasiogyne tree were cultured either on woody plant basal medium (WPM) or WPM supplemented with benzyladenine (BA, 2.2/4.4 μM), zeatin (1.1/2.2 μM), gibberillic acid (GA₃, 2.9 and 14.5 μM), and GA₃ + BA (2.9 + 4.4 μM). Although axillary shoots developed in all the media, a higher percentage bud break occurred on BA supplemented media. To corroborate the results, endogenous levels of cytokinins [Cks, N ⁶-isopentenyladenine (iP), zeatin riboside (t-ZR), dihydrozeatinriboside (DHZR)] and abscisic acid (ABA) were determined. On BA supplemented media, the levels of zeatin type (Z-type) of Cks were higher than those of isopentenyladenine type of Ck in the explants, while the ABA level was low. Axillary shoots did not grow well and became necrotic upon subculture to fresh basal WPM. In order to improve shoot growth, they were subcultured twice at a 4-week interval on to WPM supplemented with BA (2.2/4.4 μM), GA₃ (1.4 μM), or GA₃ + BA (1.4 + 4.4/2.9 + 4.4 μM). Maximal shoot growth (93%) was achieved on WPM supplemented with 2.2 μM BA. Comparative analyses of endogenous Cks revealed that higher Cks (Z-type Cks) were present in actively growing shoots. Rooting was readily achieved when the shoots were subcultured to WPM without phytohormones. The rooted plants were acclimatized well upon transplantation.     

Identification of conserved domains in Salmonella muenchen flagellin that are essential for its ability to activate TLR5 and to induce an inflammatory response in vitro

The bacterial surface protein flagellin is widely distributed and well conserved among distant bacterial species. We and other investigators have reported recently that purified flagellin from Salmonella dublin or recombinant flagellin of Salmonella muenchen origin binds to the eukaryotic toll receptor TLR5 and activates the nuclear translocation of NF-kappaB and mitogen-activated protein kinase, resulting in the release of a host of pro-inflammatory mediators in vitro and in vivo. The amino acid sequence alignment of flagellins from various Gram-negative bacteria shows that the C and N termini are well conserved. It is possible that sequences within the N and C termini or both may regulate the pro-inflammatory activity of flagellin. Here we set out to map more precisely the regions in both termini that are required for TLR5 activation and pro-inflammatory signaling. Systematic deletion of amino acids from either terminus progressively reduced eukaryotic pro-inflammatory activation. However, deletion of amino acids 95-108 (motif N) in the N terminus and 441-449 (motif C) in the C terminus abolished pro-inflammatory activity completely. Site-directed mutagenesis analysis provided further evidence for the importance of motifs N and C. We also present evidence for the functional role of motifs N and C with the TLR5 receptor using a reporter assay system. Taken together, our results demonstrate that the pro-inflammatory activity of flagellin results from the interaction of motif N with the TLR5 receptor on the cell surface.     

Identification of an extracellular domain within the human PiT2 receptor that is required for amphotropic murine leukemia virus binding

Human PiT2 (PiT2) is a multiple-membrane-spanning protein that functions as a type III sodium phosphate cotransporter and as the receptor for amphotropic murine leukemia virus (A-MuLV). Human PiT1 (PiT1), another type III sodium phosphate cotransporter, is a highly related protein that functions as a receptor for gibbon ape leukemia virus but not for A-MuLV. The ability of PiT1 and PiT2 to function as discrete viral receptors with unique properties presumably is reflected in critical residue differences between these two proteins. Early efforts to map the region(s) within PiT2 that is important for virus binding and/or entry relied on infection results obtained with PiT1-PiT2 chimeric cDNAs expressed in Chinese hamster ovary (CHOK1) cells. These attempts to localize the PiT2 virus-binding site were hampered because they were based on infectivity, not binding, assays, and therefore, receptors that bound but failed to facilitate virus entry could not be distinguished from receptors that did not bind virus. Using a more accurate topological model for PiT2 as well as an A-MuLV receptor-binding assay, we have identified extracellular domain one (ECD1) of the human PiT2 receptor as being important for A-MuLV binding and infection.     

Gi-coupled receptors mediate phosphorylation of CPI-17 and MLC20 via preferential activation of the PI3K/ILK pathway

Sustained smooth-muscle contraction or its experimental counterpart, Ca2+ sensitization, by G(q/13)-coupled receptor agonists is mediated via RhoA-dependent inhibition of MLC (myosin light chain) phosphatase and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation by a Ca2+-independent MLCK (MLC kinase). The present study identified the corresponding pathways initiated by G(i)-coupled receptors. Somatostatin acting via G(i)1-coupled sstr3 receptor, DPDPE ([D-Pen2,D-Pen5]enkephalin; where Pen is penicillamine) acting via G(i)2-coupled delta-opioid receptors, and cyclopentyl adenosine acting via G(i)3-coupled adenosine A1 receptors preferentially activated PI3K (phosphoinositide 3-kinase) and ILK (integrin-linked kinase), whereas ACh (acetylcholine) acting via G(i)3-coupled M2 receptors preferentially activated PI3K, Cdc42 (cell division cycle 42)/Rac1, PAK1 (p21-activated kinase 1) and p38 MAPK (mitogen-activated protein kinase). Only agonists that activated ILK induced sustained CPI-17 (protein kinase C potentiated inhibitor 17 kDa protein) phosphorylation at Thr38, MLC20 phosphorylation at Ser19, and contraction, consistent with recent evidence that ILK can act as a Ca2+-independent MLCK capable of phosphorylating the MLC phosphatase inhibitor, CPI-17, at Thr38. ILK activity, and CPI-17 and MLC20 phosphorylation were inhibited by LY294002 and in muscle cells expressing ILK(R211A) or treated with siRNA (small interfering RNA) for ILK. ACh acting via M2 receptors activated ILK, and induced CPI-17 and MLC20 phosphorylation and muscle contraction, but only after inhibition of p38 MAPK; all these responses were inhibited in cells expressing ILK(R211A). Conversely, ACh activated PAK1, a step upstream of p38 MAPK, whereas the three other agonists did so only in cells transfected with ILK(R211A) or siRNA for ILK. The results demonstrate reciprocal inhibition between two pathways downstream of PI3K, with ILK inhibiting PAK1, and p38 MAPK inhibiting ILK. Sustained contraction via G(i)-coupled receptors is dependent on CPI-17 and MLC20 phosphorylation by ILK.