Dynamics of Tandem Repetitive Afa-Family Sequences in Triticeae, Wheat-Related Species

The Afa-family sequences in wheat-related species, Triticeae, are tandem repetitive sequences of 340 bp. All the analyzed Triticeae species carried the sequences in their genomes, though the copy numbers varied about 100-fold among the species. The nucleotide fragments amplified by PCR were cloned and sequenced, and their behavior in the evolution of Triticeae was analyzed by the neighbor-joining (NJ) method. The sequences in genomes with many copies of this family clustered at independent branches of the phylogenic tree, whereas the sequences in genomes with a few copies did not. This may suggest that Afa-family sequences had amplified several times in the evolution of Triticeae, each using a limited number of different master copies. In addition, the sequences of the A and B genomes of hexaploid common wheat indicated that the Afa-family sequences had not evolved in a concerted manner between the genomes. Furthermore, the sequences of each chromosome of the D genome of this species indicated that the sequences had amplified on all over the D-genome chromosomes in a short period. Received: 1 September 1997 / Accepted: 19 January 1998     

Two sesquiterpene lactones from artemisia species

Two new sesquiterpene lactones, himeyoshin and montanone, were isolated from Artemisia feddei and A. montana, respectively and identified by chemical and spectral data as1α,2α-epoxy-3-oxo-5,6-dihydroalantolactone and 1,10-dihydro-11,13-dehydromatricarin.     

Interaction of S-sulfonated human IgG with human complement and its components.

S-sulfonated human IgG (S-sIgG) was prepared by treating IgG with sodium sulfite and sodium tetrathionate. The treatment resulted in the selective cleavage of interchain disulfide bonds of the IgG to give S-sulfonate groups. Complement fixing activities of aggregated S-sIgG and the immune complex formed with the S-sIgG antibody were very weak. S-sIgG at a high dose reduced the activity of the first complement component (C1) in normal human serum without any reduction of other complement components activites, but S-alkylated IgG at the same dose did not. Loss of C1 activity was not caused by either S-sulfonated myeloma proteins (IgA and IgE) or urea-treated S-sIgG, in which both inter- and intra-chain disulfide bonds were cleaved. These results suggest that the selective reduction of C1 by S-sIgG is due to a conformational change of the immunoglobulin.     

Immunoelectrophoretic Analysis of Wheat Flour Albumins

Albumin preparations from four kinds of wheat flour (Durum, Manitoba No. 2, Western White and Norin No. 26) were analyzed by Immunoelectrophoresis. Seven to eleven components were detected for each preparation. They were classified and designated on the basis of the electrophoretic mobility (R) and the character of precipitin lines formed by antigen-antibody reactions.     

Direct observation of Abeta amyloid fibril growth and inhibition

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.     

Blockade of neutrophil elastase attenuates severe liver injury in hepatitis B transgenic mice

Serine proteinases produced by polymorphonuclear neutrophils play important roles in neutrophil-mediated tissue injury at inflammatory sites. Although neutrophil recruitment to the liver has been shown to be involved in the exacerbation of liver inflammation, the function of neutrophil elastase (NE) in liver injury remains unclear. Here, we found that administration of an NE inhibitor (NEI) reduced serum alanine aminotransferase (sALT) activity and inflammatory cell infiltration into the liver from 8 to 24 h after injection of antigen-specific cytotoxic T lymphocytes (CTLs) into hepatitis B virus transgenic mice. Furthermore, the NEI treatment reduced the expressions of inflammatory cytokines and chemokines in the liver and tumor necrosis factor alpha production by macrophages. In addition, the NEI treatment suppressed the mRNA expressions of CC chemokine ligand 3 (CCL-3), CCL-4, and macrophage inflammatory protein 2 (MIP-2) in neutrophils in the liver at 8 h after the CTL injection. In support of these results, we confirmed that administration of anti-CCL-3, anti-CCL-4, and anti-MIP-2 monoclonal antibodies suppressed sALT activity and leukocyte migration into the liver. In conclusion, the present results suggest that NE contributes to the early step of the inflammatory cascade in acute viral hepatitis and that NEIs may have potential as therapeutic drugs against acute severe viral hepatitis.     

Kinetic analysis of a chitinase from red sea bream, Pagrus major

Kinetic analysis was done on the 46-kDa chitinase (EC 3.2.1.14) purified from the stomach of red sea bream, Pagrus major, using glycolchitin and N-acetylchitooligosaccharides (GlcNAc(n), n=2-6) as substrates. High activity was observed at two pHs, such as 2.5 and 9.0, toward glycolchitin as seen in other insect chitinases, and also at both pH 2.5 and 5.0 even toward a short substrate, N-acetylchitopentasaccharide. Allosamidin competitively inhibited chitinase with Ki value of 0.0214 microM at pH 2.5 and 0.0024 microM at pH 9.0 in the reaction of glycolchitin. Substrate inhibition was observed in the reaction of N-acetylchitopentasaccharide. The anomeric forms of the products from N-acetylchitooligosaccharides were analyzed to be beta anomer by the high pressure liquid chromatography (HPLC) method. The data for both beta-anomer formation and allosamidin inhibition suggest that red sea bream chitinase belongs to family 18 of glycosyl hydrolases. This suggestion is also supported by the results for the N-terminal amino acid sequence.     

EspB from enterohaemorrhagic Escherichia coli is a natively partially folded protein

The structural properties of EspB, a virulence factor of the Escherichia coli O157 type III secretion system, were characterized. Far-UV and near-UV CD spectra, recorded between pH 1.0 and pH 7.0, show that the protein assumes alpha-helical structures and that some tyrosine tertiary contacts may exist. All tyrosine side-chains are exposed to water, as determined by acrylamide fluorescence quenching spectroscopy. An increase in the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate was observed at pH 2.0 in the presence of EspB, whereas no such increase in fluorescence was observed at pH 7.0. These data suggest the formation of a molten globule state at pH 2.0. Destabilization of EspB at low pH was shown by urea-unfolding transitions, monitored by far-UV CD spectroscopy. The result from a sedimentation equilibrium study indicated that EspB assumes a monomeric form at pH 7.0, although its Stokes radius (estimated by multiangle laser light scattering) was twice as large as expected for a monomeric globular structure of EspB. These data suggest that EspB, at pH 7.0, assumes a relatively expanded conformation. The chemical shift patterns of EspB 15N-1H heteronuclear single quantum correlation spectra at pH 2.0 and 7.0 are qualitatively similar to that of urea-unfolded EspB. Taken together, the properties of EspB reported here provide evidence that EspB is a natively partially folded protein, but with less exposed hydrophobic surface than traditional molten globules. This structural feature of EspB may be advantageous when EspB interacts with various biomolecules during the bacterial infection of host cells.