Molecular genetic diversity and population structure in Lycium accessions using SSR markers

This study was conducted to assess the genetic diversity and population structure of 139 Lycium chinense accessions using 18 simple sequence repeat (SSR) markers. In total, 108 alleles were detected. The number of alleles per marker locus ranged from two to 17, with an average of six. The gene diversity and polymorphism information content value averaged 0.3792 and 0.3296, with ranges of 0.0793 to 0.8023 and 0.0775 to 0.7734, respectively. The average heterozygosity was 0.4394. The model-based structure analysis revealed the presence of three subpopulations, which was consistent with clustering based on genetic distance. An AMOVA analysis showed that the between-population component of genetic variance was less than 15.3%, in contrast to 84.7% for the within-population component. The overall F_ST value was 0.1178, indicating a moderate differentiation among groups. The results could be used for future L. chinense allele mining, association mapping, gene cloning, germplasm conservation, and designing effective breeding programs.     

Industrial scale of optimization for the production of carboxymethylcellulase from rice bran by a marine bacterium, Bacillus subtilis subsp. subtilis A-53

Rice bran and yeast extract were found to be the best combination of carbon and nitrogen sources for the production of carboxymethycellulase (CMCase) by Bacillus subtilis subsp. subtlis A-53. Optimal concentrations of rice bran and yeast extract for the production of CMCase were 5.0% (w/v) and 0.10% (w/v), respectively. Optimal temperature and initial pH of medium for cell growth of B. subtilus subsp. subtilis A-53 were 35 °C and 7.3, whereas those for the production of CMCase by B. subtilus subsp. subtilis A-53 were 30 °C and 6.8. Optimal agitation speed and aeration rate in a 7 L bioreactor were 300 rpm and 1.0 vvm, respectively. The optimal agitation speed and aeration rate for the production of CMCase by B. subtilus subsp. subtilis A-53 were lower than those for cell growth. The highest productions of CMCase by B. subtilus subsp. subtilis A-53 in 7 and 100 L bioreactors were 150.3 and 196.8 U mL⁻¹, respectively.     

Development of AFLP and STS markers linked to a waterlogging tolerance in Korean soybean landraces

Among the 400 soybean (Glycine max) landraces, we selected 3 tolerant (KAS150-9, KAS160-15, and KAS170-9) and 3 susceptible lines (KAS160-14, KAS160-20, and KAS201-6-1) by the survival percentage and injury scores. Susceptible lines showed decrease in chlorophyll content and increase in glucose and malondialdehyde (MDA) contents under waterlogging stress, while tolerant lines did not change significantly. For AFLP analysis, 8 EcoRI (+3) and 8 MseI (+3) primers used in 32 primer combinations generated a total of 2 566 bands with a mean of 80 bands per primer combination, of which 1 117 (43.5 %) were clearly polymorphic between the tolerant and susceptible lines. A genetic similarity coefficient, based on cluster analysis using an unweighted pair grouping method of average (UPGMA), was 0.79 for the tolerant group, while the susceptible landraces were genetically less related, with a genetic similarity coefficient of 0.17. The 10 reproducible polymorphic PCR products present in the 3 tolerant or susceptible lines were sequenced and converted into sequence tagged site (STS) markers. These STS primer sets were designated GmWT01-GmWT06 and GmWS01-GmWS04. Two STS primer sets, GmWT06 and GmWS02, generated a single monomorphic PCR product identical in size to the original AFLP fragments. For the broad application of these STS markers in marker-assisted selection (MAS) for soybean genotypes tolerant to waterlogging stress, two developed STS markers are being evaluated with putative waterlogging tolerant mutant lines induced by γ-radiation in soybean mutation breeding programs.     

Diversifying selection and functional analysis of interleukin-4 suggests antagonism-driven evolution at receptor-binding interfaces

Background  

Interleukin-4 (IL4) is a secreted immunoregulatory cytokine critically involved in host protection from parasitic helminths [1]. Reasoning that helminths may have evolved mechanisms to antagonize IL4 to maximize their dispersal, we explored mammalian IL4 evolution.     

Entactin, a novel basal lamina-associated sulfated glycoprotein

A sulfated glycoprotein, entactin, of apparent molecular weight 158,000 has been isolated from an extracellular basement membrane-like matrix. This matrix is elaborated in cell culture by a mouse endodermal cell line. Antibodies prepared in rabbits against this sulfated glycoprotein react with mouse and rat basement membranes from a variety of tissues. These antibodies also react in a specific manner with a discrete component of mouse and rat kidney glomeruli. The electrophoretic mobility of this component is identical to that of entactin. The mouse kidney antigen, as shown by immunoelectron microscopic studies, is predominantly localized at the surface of epithelial cells of tubules and glomeruli adjacent to the basement membrane. Some antigen is also present in the basal lamina adjacent to the epithelial cells. Entactin is distinct from the basement membrane-associated protein GP-2, a protein similar to laminin. Entactin differs from GP-2 in electrophoretic mobility, cyanogen bromide peptide fragmentation pattern, immunological cross-reactivity, and incorporation of H235SO4. Entactin is insensitive to treatment with chrondroitinase ABC. It is suggested that this molecule plays a role in the interaction of the extracellular matrix and the cell surface.     

Oxidative stress-induced apoptosis is mediated by ERK1/2 phosphorylation

Oxidative stress is known to induce apoptosis in a wide variety of cell types, apparently by modulating intracellular signaling pathways. High concentrations of H2O2 have been found to induce apoptosis in L929 mouse fibroblast cells. To elucidate the mechanisms of H2O2-mediated apoptosis, ERK1/2, p38-MAPK, and JNK1/2 phosphorylation was examined, and ERK1/2 and JNK1/2 were found to be activated by H2O2. Inhibition of ERK1/2 activation by treatment of L929 cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced apoptosis, while inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 or MKK4 or MKK7 transfection did not affect H2O2-mediated apoptosis. H2O2-mediated ERK1/2 activation was not only Ras-Raf dependent, but also both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta dependent. H2O2-mediated PKCdelta-dependent and tyrosine kinase-dependent ERK1/2 activations were independent from each other. Based on the above results, we suggest for the first time that oxidative damage-induced apoptosis is mediated by ERK1/2 phosphorylation which is not only Ras-Raf dependent, but also both tyrosine kinase and PKCdelta dependent.     

TNF-alpha activates death pathway in human aorta smooth muscle cell in the presence of 7-ketocholesterol

This study was undertaken to investigate whether a physiologically compatible concentration of 7-ketocholesterol had any effect on human vascular smooth muscle cells (HVSMCs). We found that 7-ketocholesterol changed the viability of human aorta smooth muscle cells (HAoSMC) not by cytotoxicity but by activation of tumor necrosis factor-alpha receptor (TNFR)-mediated death. Whereas TNF-alpha did not affect the viability in the presence of 7alpha-hydroxycholesterol or cholesterol, the cytokine induced HAoSMC death in the presence of 7-ketocholesterol as detected by morphology, viability, and fragmentation of chromosomal DNA. The HAoSMC death was inhibited by a neutralizing anti-TNF receptor 1 (TNFR1) antibody and by the caspase inhibitors of z-VAD and z-DEVD. Activations of caspase-8 and -3 were detected from dying HAoSMCs. 7-Ketocholesterol inhibited translocation of the nuclear factor kappaB (NF-kappaB) subunits of p65 and p50 from the cytosol into the nucleus, increase of NF-kappaB activity, and expression of caspase-8 homolog Fas ligand interleukin-1-converting enzyme inhibitory protein by TNF-alpha. We also found that X-chromosome-linked inhibitor of apoptosis protein was degraded in dying HAoSMC. The present study proposes that 7-ketocholesterol would contribute to the disappearance of HVSMC in the atherosclerotic lesions by enhancing receptor-mediated death. This is the first report demonstrating induction of TNF-alpha-mediated death by oxysterol in cells.     

Separation and purification of useful proteins using hydrogel ultrafiltration

The hydrogel process is a different form of ultrafiltration and has been used to separate biological molecules. In this study, the gel pore size was predicted by pulse NMR technique and neural network using a data base obtained from gel filtration chromatography and diffusion experiment. Recombinant alkaline phosphatase expressed in insect cells was concentrated 1.5 times by hydrogel ultrafiltration by swelling at 20°C and collapsing at 35°C at 53–65% separation efficiency and 78–83% enzyme recovery. Wild and recombinantAutographa californica nuclear polyhedrosis viruses (AcNPV) were also concentrated 1.4 and 1.6 times of the feed solution at 48.5 and 60.0% separation efficiency, respectively. Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of AcNPV and recombinant proteins from insect cells.