New preparation of PM2 phage DNA and an endonuclease assay for a single-strand break

PM2 is a bacteriophage which has closed circular double-stranded DNA as a genome, which is the sole source for endonuclease assay for a single strand break in the fmol range. Therefore, it is important to isolate PM2 DNA with low control nicks for the endonuclease assay. Usually, the isolation method of phage DNA is to use ultracentrifugation which takes at least 4 days. In this report, a fast and effective method which takes only 2 days was developed to purify DNA using polyethylene glycol (PEG) 8000 and the yields of phage DNA isolated by these two methods were compared. The method using PEG 8000 increased the yield of PM2 DNA from 31.2% to 45.2%, and decreased the nick from 17.1% to 13.1%. Recently, the complete PM2 DNA genome sequence of 10,079 bp was published. The exact number of nucleotides of PM2 DNA is important for the correct enzyme assay which measures nicks generated by an endonuclease. The correct calculation of endonuclease activity of rpS3 for nick-circle assay was performed to measure single-strand breaks in this report. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative Biochemistry of the Oxidative Burst Produced by Rose and French Bean Cells Reveals Two Distinct Mechanisms

Cultured cells of rose (Rosa damascena) treated with an elicitor derived from Phytophthora spp. and suspension-cultured cells of French bean (Phaseolus vulgaris) treated with an elicitor derived from the cell walls of Colletotrichum lindemuthianum both produced H₂O₂. It has been hypothesized that in rose cells H₂O₂ is produced by a plasma membrane NAD(P)H oxidase (superoxide synthase), whereas in bean cells H₂O₂ is derived directly from cell wall peroxidases following extracellular alkalinization and the appearance of a reductant. In the rose/Phytophthora spp. system treated with N,N-diethyldithiocarbamate, superoxide was detected by a N,N′-dimethyl-9,9′-biacridium dinitrate-dependent chemiluminescence; in contrast, in the bean/C. lindemuthianum system, no superoxide was detected, with or without N,N-diethyldithiocarbamate. When rose cells were washed free of medium (containing cell wall peroxidase) and then treated with Phytophthora spp. elicitor, they accumulated a higher maximum concentration of H₂O₂ than when treated without the washing procedure. In contrast, a washing treatment reduced the H₂O₂ accumulated by French bean cells treated with C. lindemuthianum elicitor. Rose cells produced reductant capable of stimulating horseradish (Armoracia lapathifolia) peroxidase to form H₂O₂ but did not have a peroxidase capable of forming H₂O₂ in the presence of reductant. Rose and French bean cells thus appear to be responding by different mechanisms to generate the oxidative burst.     

Expression analysis of proline metabolism-related genes from halophyte Arabis stelleri under osmotic stress conditions

Arabis stelleri var.japonica evidenced stronger osmotic stress tolerance than Arabidopsis thaliana.Using an A.thaliana microarray chip,we determined changes in the expression of approximately 2 800genes between A.stelleri plants treated with 0.2 M mannitol versus mock-treated plants.The most significant changes in the gene expression patterns were in genes defining cellular components or in genes associated with the endomembrane system,stimulus response,stress response,chemical stimulus response,and defense response.The expression patterns of three de novo proline biosynthesis enzymes were evaluated in A.stelleri var.japonica seedlings treated with 0.2 M mannitol,0.2 M sorbitol,and 0.2 M NaCl.The expression of Δ1-pyrroline-5-carboxylate synthetase was not affected by NaCl stress but was similarly induced by mannitol and sorbitol.The proline dehydrogenase gene,which is known to be repressed by dehydration stress and induced by free L-proline,was induced at an early stage by mannitol treatment,but the level of proline dehydrogenase was increased later by treatment with both mannitol and NaCl.The level of free L-proline accumulation increased progressively in response to treatments with mannitol,sorbitol,and NaCl.Mannitol induced L-proline accumulation more rapidly than NaCl or sorbitol.These findings demonstrate that the osmotic tolerance of the novel halophyte,Arabis stelleri,is associated with the accumulation of L-proline.     

Antibodies against non-immunizing antigens derived from a large immune scFv library

Target-specific antibodies can be rapidly enriched and identified from an antibody library using phage display. Large, naïve antibody libraries derived from synthetic or unimmunized sources can yield antibodies against virtually any antigen, whereas libraries from immunized sources tend to be smaller and are used exclusively against the antigen of immunization. In this study, 25 scFv libraries made from the spleens of immunized rabbits, each with a size ranging from 10⁸ to higher than 10⁹, were combined into a single large library with > 10¹⁰ individual clones. Panning of this combined library yielded target-specific rabbit scFv clones against many non-immunizing antigens, including proteins, peptides, and a small molecule. Notably, specific scFv clones against a rabbit self-antigen (rabbit serum albumin) and a phosphorylated protein (epidermal growth factor receptor pTyr1173) could be isolated from the library. These results suggest that the immune library contained a significant number of unimmunized clones and that a sufficiently large immune library can be utilized similarly to a naïe library, i.e., against various non-immunizing antigens to yield specific antibodies.     

Altered cell shapes, hyperplasia, and secondary growth in Arabidopsis caused by beet curly top geminivirus infection

Arabidopsis Sei-O ecotype was found to be hypersusceptible to the BCTV-Logan strain in that it developed very severe symptoms, including severely deformed inflorescences with the callus-like structure, and accumulated high level of viral DNA. Microscopic studies of the BCTV-induced cell divisions demonstrated that the activation of cell divisions was preceded by the phloem disruption and the callus-like structure seemed to be originated from the cortex nearby disrupted phloem. We have further defined the callus-like structure formed by BCTV infection using molecular and histochemical analyses. Results indicate that BCTV infection causes the phloem disruption, following by cell enlargement and elongation in cortex and even epidermis. Finally, BCTV induced symptomatic secondary growth in cortex by de novo anticlinal and periclinal cell divisions. Expression of cdc2 and saur from BCTV-infected Arabidopsis correlates with symptom development. These results suggest a critical role of auxin in symptom development in the interactions between Arabidopsis and BCTV.     

SUMOylation participates in induction of ischemic tolerance

Ground squirrels in hibernation torpor have been shown to have striking increases in global SUMOylation on tissue immunoblots. Here, we find evidence that global SUMOylation is also involved in ischemic tolerance in primary cortical neuronal cultures (from rats and mice) and SHSY5Y human neuroblastoma cells. Cultured cortical neurons preconditioned by sublethal oxygen/glucose deprivation (OGD) were less vulnerable to severe OGD than non-preconditioned neurons. Preconditioned neurons maintained elevated SUMO-1 conjugation levels (and, to a lesser extent those of SUMO-2/3) on western blots in contrast to non-preconditioned cells. Further, cortical neurons and SHSY5Y cells in which transfected SUMO-1 or SUMO-2 were over-expressed showed increased survival after severe OGD. In contrast, cell cultures subjected to depletion of endogenous SUMO-1 protein by RNAi had reduced survival after exposure to this form of in vitro ischemia and an attenuated protective response to preconditioning. These findings suggest that maintenance of a globally elevated SUMO-1 (and maybe SUMO-2/3) conjugation level as revealed by immunoblot assays is a component of ischemic tolerance.     

Sodium hydrogen sulfide inhibits nicotine and lipopolysaccharide‐induced osteoclastic differentiation and reversed osteoblastic differentiation in human periodontal ligament cells

Although previous studies have demonstrated that hydrogen sulfide (H₂S) stimulated or inhibited osteoclastic differentiation, little is known about the effects of H₂S on the differentiation of osteoblasts and osteoclasts. To determine the possible bioactivities of H₂S on bone metabolism, we investigated the in vitro effects of H₂S on cytotoxicity, osteoblastic, and osteoclastic differentiation as well as the underlying mechanism in lipopolysaccharide (LPS) and nicotine‐stimulated human periodontal ligament cells (hPDLCs). The H₂S donor, NaHS, protected hPDLCs from nicotine and LPS‐induced cytotoxicity and recovered nicotine‐ and LPS‐downregulated osteoblastic differentiation, such as alkaline phosphatase (ALP) activity, mRNA expression of osteoblasts, including ALP, osteopontin (OPN), and osteocalcin (OCN), and mineralized nodule formation. Concomitantly, NaHS inhibited the differentiation of tartrate‐resistant acid phosphatase (TRAP)‐positive osteoclasts in mouse bone marrow cells and blocked nicotine‐ and LPS‐induced osteoclastogenesis regulatory molecules, such as RANKL, OPG, M‐CSF, MMP‐9, TRAP, and cathepsin K mRNA. NaHS blocked nicotine and LPS‐induced activation of p38, ERK, MKP‐1, PI3K, PKC, and PKC isoenzymes, and NF‐κB. The effects of H₂S on nicotine‐ and LPS‐induced osteoblastic and osteoclastic differentiation were remarkably reversed by MKP‐1 enzyme inhibitor (vanadate) and expression inhibitor (triptolide). Taken together, we report for the first time that H₂S inhibited cytotoxicity and osteoclastic differentiation and recovered osteoblastic differentiation in a nicotine‐ and periodontopathogen‐stimulated hPDLCs model, which has potential therapeutic value for treatment of periodontal and inflammatory bone diseases. J. Cell. Biochem. 114: 1183–1193, 2013. © 2012 Wiley Periodicals, Inc.     

Production of a new sucrose derivative by transglycosylation of recombinant Sulfolobus shibatae beta-glycosidase

The gene encoding beta-glycosidase of the hyperthermophilic archaea Sulfolobus shibatae (SSG) was expressed in Escherichia coli. Recombinant SSG (referred to as rSSG hereafter) was efficiently purified, and its transglycosylation activity was tested with lactose as a donor and various sugars as acceptors. When sucrose was used as an acceptor, we found a distinct intermolecular transglycosylation product and confirmed its presence by TLC and high performance anion exchange chromatography (HPAEC). The sucrose transglycosylation product was isolated by paper chromatography, and its chemical structure was determined by 1H and 13C NMR. The sucrose transfer product was determined to be beta-D-galactopyranosyl-(1-->6)-alpha-D-glucopyranosyl-beta-d-fructofuranoside with a galactose molecule linked to sucrose via a beta-(1-->6)-glycosidic bond.