Effects of bienzyme complex formation of cysteine synthetase from escherichia coli on some properties and kinetics

Some properties and kinetics of the free and bound serine acetyltransferases (SATs) and O-acetylserine sulfhydrylase-As (OASS-As) from Escherichia coli were investigated. In some cases, SATdeltaC20, deleting 20 amino acid residues from the C-terminus of the wild-type SAT (Biosci. Biotechnol. Biochem., 63, 168-179 (1999)) was tested for comparison. The optimum pH and stability against some reagents for the free and bound wild-type SATs were similar except for the resistance to cold inactivation. The kinetics for the wild-type SAT and SATdeltaC20 followed a Ping-Pong Bi Bi mechanism with a mixed-type inhibition by L-cysteine. The kinetics and kinetic constants for the wild-type SAT were not changed by the complex formation with OASS-A. The optimum pH for OASS-A was shifted towards an alkaline pH by the complex formation. Thermal stability and stability against some reagents for the free and bound OASS-As were almost the same. On the other hand, the maximum velocity for OASS-A was lowered and dissociation constants for the substrates and products were increased by forming the complex with the wild-type SAT, although the kinetics for the free and bound enzymes followed the same Ping-Pong Bi Bi mechanism. From comparisons of computed courses of L-cysteine formation from L-serine using SAT (wild-type SAT and SATdeltaC20) and OASS-A with the experimental results and changes in the stability of the wild-type SAT by the complex formation, we discuss the role and significance of a complex formation for the cysteine synthetase.     

Tumour necrosis factor alpha (TNF-alpha) interferes with Fas-mediated apoptotic cell death on rheumatoid arthritis (RA) synovial cells: a possible mechanism of rheumatoid synovial hyperplasia and a clinical benefit of anti-TNF-alpha therapy for RA

To investigate the mechanism of rheumatoid synovial hyperplasia (RASH), the influence of tumour necrosis factor alpha (TNF-alpha) on Fas-mediated apoptotic cell death (Fas-ACD) was examined on cultured rheumatoid synovial cells (RASCs). RASCs were obtained from the synovial tissues of eight patients with rheumatoid arthritis (RA) and SCs from eight patients with osteoarthritis (OA) were used as a control. To examine the influence of TNF-alpha on Fas-ACD, SCs were cultured with anti-Fas antibody (CH11) for 16 h in the absence or presence of different doses of recombinant TNF-alpha. ACD was determined by electron microscopic analysis and the percentage of apoptotic cells was calculated by trypan blue staining. In addition, the expression of Fas and Bcl-2 on RASCs was examined by flow cytometry. As a result, RASCs were more susceptible to Fas-ACD in vitro than OASCs. TNF-alpha interfered with Fas-ACD on RASCs in a dose-dependent manner. Moreover, removal of TNF-alpha activity by a neutralizing anti-TNF-alpha antibody (cA2) restored Fas-ACD. Flow cytometric analysis showed no significant changes in either Fas or Bcl-2 expression on RASCs after the culture with TNFalpha.These results suggest the following: (1) Fas-ACD might be diminished in vivo by local excessive TNF-alpha and this might contribute in part to RASH. (2) The inhibition of Fas-ACD on RASCs by TNF-alpha might not be associated with changes in the expression of Fas or Bcl-2. (3) In addition, considering a magnetic resonance imaging (MRI) finding of marked reduction in the RASH after cA2 treatment, blockade of TNF-alpha activity could restore Fas-ACD in RA synovium, implicating a clinical benefit of anti-TNF-alpha therapy for RA.     

Purification and some characteristics of enterocin ON-157, a bacteriocin produced by Enterococcus faecium NIAI 157

Bacteriocin-like activity (BLA) was screened in 690 strains of lactic acid bacteria isolated from plant materials such as silages and fermented vegetables. Among them, a strain identified as Enterococcus faecium NIAI 157 showed a clear BLA against the indicator strain, Ent. faecium IFO 13712. The proteinaceous nature and antimicrobial activity against closely related species strongly indicated that this BLA was a bacteriocin and was designated enterocin ON-157. The bacteriocin activity of this strain was extracellularly produced in the logarithmic growth phase in MRS broth and purified by ultrafiltration, ammonium sulphate precipitation and cation-exchange chromatography. Purified enterocin ON-157 had a molecular weight of approximately 2500 Da in SDS-PAGE analysis and was easily inhibited by treatment with alpha-amylase and proteolytic enzymes. Enterocin ON-157 had a bactericidal mode of action and inhibited the growth of the enterococci, Lactobacillus sake and Listeria monocytogenes. Enterococcus faecium NIAI 157 harboured two plasmids, 49.0 kb and 43.7 kb, and a variant missing a larger plasmid by curing with novobiocin lost the bactriocin activity.     

In vitro evolutionary thermostabilization of congerin II: a limited reproduction of natural protein evolution by artificial selection pressure

The thermostability of the conger eel galectin, congerin II, was improved by in vitro evolutionary protein engineering. Two rounds of random PCR mutagenesis and selection experiments increased the congerin II thermostability to a level comparative to its naturally thermostable isoform, congerin I. The crystal structures of the most thermostable double mutant, Y16S/T88I, and the related single mutants, Y16S and T88I, were determined at 2.0 angstroms, 1.8 angstroms, and 1.6 angstroms resolution, respectively. The exclusion of two interior water molecules by the Thr88Ile mutation, and the relief of adjacent conformational stress by the Tyr16Ser mutation were the major contributions to the thermostability. These features in the congerin II mutants are similar to those observed in congerin I. The natural evolution of congerin genes, with the K(A)/K(S) ratio of 2.6, was accelerated under natural selection pressures. The thermostabilizing selection pressure artificially applied to congerin II mimicked the implied natural pressure on congerin I. The results showed that the artificial pressure made congerin II partially reproduce the natural evolution of congerin I.     

Odor-active constituents in fresh pineapple (Ananas comosus [L.] Merr.) by quantitative and sensory evaluation

By application of aroma extract dilution analysis (AEDA) to an aroma distillate prepared from fresh pineapple using solvent-assisted flavor evaporation (SAFE), 29 odor-active compounds were detected in the flavor dilution (FD) factor range of 2 to 4,096. Quantitative measurements performed by stable isotope dilution assays (SIDA) and a calculation of odor activity values (OAVs) of 12 selected odorants revealed the following compounds as key odorants in fresh pineapple flavor: 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDF; sweet, pineapple-like, caramel-like), ethyl 2-methylpropanoate (fruity), ethyl 2-methylbutanoate (fruity) followed by methyl 2-methylbutanoate (fruity, apple-like) and 1-(E,Z)-3,5-undecatriene (fresh, pineapple-like). A mixture of these 12 odorants in concentrations equal to those in the fresh pineapple resulted in an odor profile similar to that of the fresh juice. Furthermore, the results of omission tests using the model mixture showed that HDF and ethyl 2-methylbutanoate are character impact odorants in fresh pineapple.     

Glycoprotein-specific ubiquitin ligases recognize N-glycans in unfolded substrates

Misfolded or unassembled polypeptides in the endoplasmic reticulum (ER) are retro-translocated into the cytosol and degraded by the ubiquitin-proteasome system. We reported previously that the SCF(Fbs1,2) ubiquitin-ligase complexes that contribute to ubiquitination of glycoproteins are involved in the ER-associated degradation pathway. Here we investigated how the SCF(Fbs1,2) complexes interact with unfolded glycoproteins. The SCF(Fbs1) complex was associated with p97/VCP AAA ATPase and bound to integrin-beta1, one of the SCF(Fbs1) substrates, in the cytosol in a manner dependent on p97 ATPase activity. Both Fbs1 and Fbs2 proteins interacted with denatured glycoproteins, which were modified with not only high-mannose but also complex-type oligosaccharides, more efficiently than native proteins. Given that Fbs proteins interact with innermost chitobiose in N-glycans, we propose that Fbs proteins distinguish native from unfolded glycoproteins by sensing the exposed chitobiose structure.     

Pilt, a novel peripheral membrane protein at tight junctions in epithelial cells

Tight junctions (TJs) serve as a barrier that prevents solutes and water from passing through the paracellular pathway, and as a fence between the apical and basolateral plasma membranes in epithelial cells. TJs consist of transmembrane proteins (claudin, occludin, and JAM) and many peripheral membrane proteins, including actin filament (F-actin)-binding scaffold proteins (ZO-1, -2, and -3), non-F-actin-binding scaffold proteins (MAGI-1), and cell polarity molecules (ASIP/PAR-3 and PAR-6). We identified here a novel peripheral membrane protein at TJs from a human cDNA library and named it Pilt (for protein incorporated later into TJs), because it was incorporated into TJs later after the claudin-based junctional strands were formed. Pilt consists of 547 amino acids with a calculated M(r) of 60,704. Pilt has a proline-rich domain. In cadherin-deficient L cells stably expressing claudin or JAM, Pilt was not recruited to claudin-based or JAM-based cell-cell contact sites, suggesting that Pilt does not directly interact with claudin or JAM. The present results indicate that Pilt is a novel component of TJs.     

Enhanced tolerance of rice to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes

One of the widest ranging abiotic stresses in world agriculture arises from low iron (Fe) availability due to high soil pH, with 30% of arable land too alkaline for optimal crop production. Rice is especially susceptible to low iron supply, whereas other graminaceous crops such as barley are not. A barley genomic DNA fragment containing two naat genes, which encode crucial enzymes involved in the biosynthesis of phytosiderophores, was introduced into rice using Agrobacterium-mediated transformation and pBIGRZ1. Phytosiderophores are natural iron chelators that graminaceous plants secrete from their roots to solubilize iron in the soil. The two transgenes were expressed in response to low iron nutritional status in both the shoots and roots of rice transformants. Transgenic rice expressing the two genes showed a higher nicotianamine aminotransferase activity and secreted larger amounts of phytosiderophores than nontransformants under iron-deficient conditions. Consequently, the transgenic rice showed an enhanced tolerance to low iron availability and had 4.1 times greater grain yields than that of the nontransformant rice in an alkaline soil.