Molecular cloning of ATR5(Emoy2) from Hyaloperonospora arabidopsidis, an avirulence determinant that triggers RPP5-mediated defense in Arabidopsis

RPP5 is the seminal example of a cytoplasmic NB-LRR receptor-like protein that confers downy mildew resistance in Arabidopsis thaliana. In this study, we describe the cloning and molecular characterization of the gene encoding ATR5(Emoy2), an avirulence protein from the downy mildew pathogen Hyaloperonospora arabidopsidis isolate Emoy2. ATR5(Emoy2) triggers defense response in host lines expressing the functional RPP5 allele from Landsberg erecta (Ler-0). ATR5(Emoy2) is embedded in a cluster with two additional ATR5-like (ATR5L) genes, most likely resulting from gene duplications. ATR5L proteins do not trigger RPP5-mediated resistance and the copy number of ATR5L genes varies among H. arabidopsidis isolates. ATR5(Emoy2) and ATR5L proteins contain a signal peptide, canonical EER motif, and an RGD motif. However, they lack the canonical translocation motif RXLR, which characterizes most oomycete effectors identified so far. The signal peptide and the N-terminal regions including the EER motif of ATR5(Emoy2) are not required to trigger an RPP5-dependent immune response. Bioinformatics screen of H. arabidopsidis Emoy2 genome revealed the presence of 173 open reading frames that potentially encode for secreted proteins similar to ATR5(Emoy2), in which they share some motifs such as EER but there is no canonical RXLR motif.     

Inhibition of human breast cancer Matrigel invasion by Streptolysin O activation of the EGF receptor ErbB1

Streptolysin O (SLO) is a protein cytotoxin derived from Group A beta-hemolytic streptococci that associates with membranes and permeabilizes cells. Oxidation inactivates SLO, eliminating the characteristic hemolytic and cytotoxic activities. However, oxidized SLO produces beneficial therapeutic effects in vivo on scleroderma, scar formation and wound healing. Here we report that oxidized SLO also significantly inhibited invasion by human metastatic breast cancer MDA-MB-231 cells through Matrigel in an in vitro model of metastatic disease. This dose-dependent response corresponded to selective SLO activation of epidermal growth factor receptor (EGFR) ErbB1. SLO and EGF were equally selective in activation of EGFR, but EGF elicited larger relative increases in phosphorylation at various sites, especially pronounced for Tyr845. Addition of SLO did not affect either ERK1/2 or Akt kinases and altered the expression of only 10 of 84 metastasis-related genes in MDA-MB-231 cells. Neither SLO nor EGF promoted growth of several human breast cancer cell lines. Knockdown of EGFR by siRNA ablated the inhibitory effect of SLO on cancer cell invasion, showing SLO selectively activated ErbB1 kinase to reduce invasion without increasing cell growth. The results suggest SLO might have promise as a new therapy to inhibit metastasis.     

Steady-state kinetic properties of sorbitol dehydrogenase from chicken liver

The steady-state kinetic properties of partially purified chicken liver sorbitol dehydrogenase (SDH) were determined spectrophotometrically at 25 degrees C, in 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, pH 8.0. In the sorbitol-to-fructose direction, analysis was based on initial rate data obtained at [NAD(+)](o)=0.1-0.4 mM and [sorbitol](o)=1.25-10 mM. The reverse process was analyzed by recording progress curves for NADH consumption, starting with [NADH](o)=0.2 mM and [fructose](o)=66.7-267 mM. The kinetics conformed to an ordered sequential model, with the cofactors adding first. The steady-state parameters in the forward direction, K(NAD(+)), K(iNAD(+)) and K(sorbitol), were found to be 210+/-62 muM, 220+/-69 microM and 3.2+/-0.54 mM, respectively. The corresponding parameters in the reverse direction were K(NADH)=240+/-58 microM, K(iNADH)=10+/-2.8 microM and K(fructose)=1000+/-140 mM. The results indicated a close parallelism with human SDH, yet up to 40-fold differences were observed when compared to related reports on other mammalian species. The structural and adaptive bases of the variation in substrate and cofactor affinities need to be accounted for.     

Synthesis and characterization of polymeric soybean oil-g-methyl methacrylate (and n-butyl methacrylate) graft copolymers: biocompatibility and bacterial adhesion

Peroxidation, epoxidation, and/or perepoxidation reactions of soybean oil under air at room temperature resulted in cross-linked polymeric soybean oil peroxides on the surface along with the waxy soluble part, sPSB, with a molecular weight of 4690, containing up to 2.3 wt % peroxide. This soluble polymeric oil peroxide, sPSB, initiated the free radical polymerization of either methyl methacrylate (MMA) or n-butyl methacrylate (nBMA) to give PSB-g-PMMA and PSB-g-PnBMA graft copolymers. The polymers obtained were characterized by (1)H NMR, thermogravimetric analysis, differential scanning calorimetry, and gel permeation chromatography techniques. Polymeric oil as a plasticizer lowered the glass transition of the PSB-g-PMMA graft copolymers. PSB-g-PMMA and PSB-g-PnBMA graft copolymer film samples were also used in cell culture studies. Fibroblast and macrophage cells were strongly adhered and spread on the copolymer film surfaces, which is important in tissue engineering. Bacterial adhesion on PSB-g-PMMA graft copolymer was also studied. Both Staphylococcus epidermidis and Escherichia coli adhered on the graft copolymer better than on homo-PMMA. Furthermore, the latter adhered much better than the former.     

Neu4, a novel human lysosomal lumen sialidase, confers normal phenotype to sialidosis and galactosialidosis cells

Three different mammalian sialidases have been described as follows: lysosomal (Neu1, gene NEU1), cytoplasmic (Neu2, gene NEU2), and plasma membrane (Neu3, gene NEU3). Because of mutations in the NEU1 gene, the inherited deficiency of Neu1 in humans causes the severe multisystemic neurodegenerative disorder sialidosis. Galactosialidosis, a clinically similar disorder, is caused by the secondary Neu1 deficiency because of genetic defects in cathepsin A that form a complex with Neu1 and activate it. In this study we describe a novel lysosomal lumen sialidase encoded by the NEU4 gene on human chromosome 2. We demonstrate that Neu4 is ubiquitously expressed in human tissues and has broad substrate specificity by being active against sialylated oligosaccharides, glycoproteins, and gangliosides. In contrast to Neu1, Neu4 is targeted to lysosomes by the mannose 6-phosphate receptor and does not require association with other proteins for enzymatic activity. Expression of Neu4 in the cells of sialidosis and galactosialidosis patients results in clearance of storage materials from lysosomes suggesting that Neu4 may be useful for developing new therapies for these conditions.     

Mutations in TMEM76* cause mucopolysaccharidosis IIIC (Sanfilippo C syndrome)

Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.     

The effects of short-term training on platelet functions and total antioxidant capacity in rats

The purpose of the present study was to investigate the effect of short-term endurance training on plasma total antioxidant status (TAS) and on in vitro platelet aggregation and ATP release. Blood samples were collected from the abdominal aorta of rats following short-term treadmill exercise (25 m/min, 0 % grade, 30 min) for three consecutive days, as well as in non-exercised control group. Platelet aggregation and platelet ATP release were evaluated by impedance and bioluminescence techniques, respectively. Plasma TAS was measured spectrophotometrically. Plasma TAS was higher and ADP-induced platelet ATP release was lower in the short-term training group with respect to the control group (p<0.001). Significant negative correlation (r = -0.56, p<0.05) was found between plasma TAS and ADP-induced platelet ATP release. Neither ADP- and collagen-induced maximum aggregation rate nor collagen-induced platelet ATP release were significantly different between the groups. According to these results, short-term training caused an alteration in platelet functions limited to the secretion response, which may be related to the oxidant/antioxidant balance changes favoring the antioxidants. The improved plasma total antioxidant capacity was possibly sufficient to prevent exercise-induced oxidative damage, and the adaptive response of platelets might be associated with enhanced antioxidant status.     

Bioconversion of methanol to formaldehyde. II. By purified methanol oxidase from modified yeast, Hansenula polymorpha

Modified methylotrophic yeast Hansenula polymorpha (HP A16) that was obtained by repressing leucine oxotrophic yeast; a wild type of Hansenula polymorpha CB4732 was used in this study. The yeast is grown with methanol, which is used as a sole carbon source, using various methanol concentrations and temperatures, and methanol oxidase (MOX) which is a key enzyme of methanol metabolism; production is maximized. Whole yeast cells were cultivated under optimized inoculation conditions; they were separated into two portions. One portion of these cells was directly used in bioconversion of methanol to formaldehyde. The second portion of the free cells was broken into pieces and a crude enzyme extract was obtained. The MOX enzyme in this extract was purified via salt precipitation, dialysis, and chromatographic methods. The purified MOX enzyme of yeast (HP A16) oxidized the methanol to formaldehyde. Optimization of bioconversion conditions was studied to reach maximum activity of enzyme. The optimum temperature and pH were found to be 35 degrees C and pH 8.0 in boric acid/NaOH buffer, and it was stable over the pH range of 6-9, at the 20 degrees C 15 min. A suitable reaction period was found as 50 min. The enzyme indicated low carbon primary alcohols (C2 to C4), as well as methanol. Initially, MOX activity increased with the increase of methanol concentration, but enzyme activity decreased. The apparent Km and Vmax values for methanol substrate of HP A16 MOX were 0.25 mM and 30 U/mg, respectively. The purified MOX enzyme was applied onto sodium dodecyl sulphate-polyacrylamide gel electrophoresis; molecular weight of the enzyme was calculated to be about 670 kDa. Each MOX enzyme is composed of eight identical subunits, each of whose molecular weight is around 82 kDa and which contain eight moles of FAD as the prosthetic group, and the pI of the natural enzyme is found to be 6.4. The purified MOX enzyme was used in the bioconversion of methanol to formaldehyde as a catalyst; this conversion was compared to the conversion percentages of whole cells in our previous article in terms of catalytic performances.