Studies on the Cereal Starches

Significant correlations among amylograph characteristics or other relating properties of rice starch and flour were recognized by orderly rearrangement of previously obtained data. Linear regression of peak viscosity on breakdown was available for detecting a deteriorative damage of rice starch and grain. Alpha-amylase in white rice did not have close relation with peak viscosity of the flour. The ratio of breakdown to consistency and the corrected peak viscosity calculating the coincidence of the slurry concentration were significantly correlated with the starch iodine blue value and rigidity of the cold paste body respectively.     

Chitin Coated Cellulose as an Adsorbent of lysozyme like Enzymes Preparation and Properties

As a new adsorbent of lysozyme-like enzymes, chitin coated (CC-)cellulose was prepared. CC-cellulose was stable and had good flow properties for use in column chromatography. Affinity chromatography with CC-cellulose showed that 3~5 mg of lysozyme/ml resin was adsorbed specifically and desorbed quantitatively under mild conditions. The utilities of the method of affinity chromatography with CC-cellulose are discussed.     

Establishment of laurel forest vegetation in adjacent secondary forest in central Japan

Distributions of lucidophyllous species are limited due to the fragmentation of laurel forest. On Komayama Hill in central Japan, we evaluated the colonization of typical lucidophyllous vascular plants from a 350-year-old laurel forest into adjacent abandoned secondary forest for conservation and restoration purposes. A total of 14 consecutive subplots were established along the vegetation border between the two forests (length, 30 m; width, 5 m), extending 70 m into the secondary forest; 18 quadrats of old-growth forest were surveyed. Edge effects of old-growth forest were found to play an important role in re-establishing lucidophyllous saplings and seedlings in the secondary forest. In particular, the abundances of the four dominant canopy species of the old-growth forest significantly decreased with increasing distance. Hence, they are expected to colonize further into the secondary forest and, ultimately, to dominate the canopy. However, the number of lucidophyllous species did not change with distance. Species such as Ficus nipponica, Damnacanthus indicus, Ilex integra, and Lemmaphyllum microphyllum were near-completely or completely limited to the old-growth forest. They are known to be negatively affected by forest fragmentation and were observed to be struggling to colonize the exterior of the old-growth forest even after 60 years of abandonment. Their absence highlighted the limited colonization capacities of some old-growth forest species and underlined the time required for habitat restoration following human disturbance. We conclude that it is important to consider the population dynamics of dominant canopy species and the colonization of these interior species when assessing the habitat expansion of lucidophyllous species and hence the restoration of degraded lands.     

Effective protein folding in simple random search

Here I show the following facts using a simple random search model without including any sophisticated energy term. As the size of elements exponentially affects the efficiency of folding, it can be remarkably enhanced by dividing the elements into small blocks. As the folding of the blocks is completely independent, the total folding time can be reduced to the folding time of the single hardest block. This result gives the simplest and most straightforward answer to the Levinthal paradox.     

Resolution and characterization of tryptophyl fluorescence of hen egg-white lysozyme by quenching- and time-resolved spectroscopy

The fluorescence spectral distributions of four tryptophan residues of hen egg-white lysozyme were analyzed using time-resolved and quenching-resolved fluorescence spectroscopy. Trp62 and Trp108 gave the fluorescence maxima at 352 nm and 342 nm, respectively. The fluorescence of Trp28 and Trp111 occurred only at 300-360 nm and they were observed as an unresolved emission band with a maximum and shoulder at 320 nm and 330 nm. The fluorescence quenching and decay parameters of each tryptophan residue reconfirmed that Trp62 was fully exposed to the solvent but Trp108 was sealed in the cage of the peptide chains and furthermore showed that Trp28 and Trp111 are under the influence of the larger fluctuational motion at the hydrophobic matrix box. The fluorescence responses of each tryptophan residue to the lysozyme-ligand interaction suggested that the internal fluctuation was reduced by the binding of ligand to give a distorted conformation to the hydrophobic matrix box region.     

Stability of the dimerization domain effects the cooperative DNA binding of short peptides

The basic region peptide derived from the basic leucine zipper protein GCN4 bound specifically to the native GCN4 binding sequences in a dimeric form when the beta-cyclodextrin/adamantane dimerization domain was introduced at the C-terminus of the GCN4 basic region peptide. We describe here how the structure and stability of the dimerization domain affect the cooperative formation of the peptide dimer-DNA complex. The basic region peptides with five different guest molecules were synthesized, and their equilibrium dissociation constants with a peptide possessing beta-cyclodextrin were determined. These values, ranging from 1.3 to 15 microM, were used to estimate the stability of the complexes between the dimers with various guest/cyclodextrin dimerization domains and GCN4 target sequences. An efficient cooperative formation of the dimer complexes at the GCN4 binding sequence was observed when the adamantyl group was replaced with the norbornyl or noradamantyl group, but not with the cyclohexyl group that formed a beta-cyclodextrin complex with a stability that was 1 order of magnitude lower than that of the adamantyl group. Thus, cooperative formation of the stable dimer-DNA complex appeared to be effected by the stability of the dimerization domain. For the peptides that cooperatively formed dimer-DNA complexes, there was no linear correlation between the stability of the inclusion complex and that of the dimer-DNA complex. With the beta-cyclodextrin/adamantane dimerization domain, the basic region peptide dimer preferred to bind to a palindromic 5'-ATGACGTCAT-3' sequence over the sequence lacking the central G.C base pair and that with an additional G.C base pair in the middle. Changing the adamantyl group into a norbornyl group did not alter the preferential binding of the peptide dimers to the palindromic sequence, but slightly affected the selectivity of the dimer for other nonpalindromic sequences. The helical contents of the peptides in the DNA-bound dimer with the adamantyl group were decreased by reducing the stability of the dimer-DNA complex, which was possibly caused by deformation of the helical structure proximal to the dimerization domain.     

Vertical and temporal shifts in microbial communities in the water column and sediment of saline meromictic Lake Kaiike (Japan), as determined by a 16S rDNA-based analysis, and related to physicochemical gradients

The vertical and temporal changes in microbial communities were investigated throughout the water column and sediment of the saline meromictic Lake Kaiike by PCR-denaturing gradient gel electrophoresis (DGGE) of 16S rDNA. Marked depth-related changes in microbial communities were observed at the chemocline and the sediment-water interface. However, no major temporal changes in the microbial community below the chemocline were observed during the sampling period, suggesting that the ecosystem in the anoxic zone of Lake Kaiike was nearly stable. Although the sequence of the most conspicuous DGGE band throughout the anoxic water and in the top of the microbial mat was most similar to that of an anoxic, photosynthetic, green sulphur bacterium, Pelodyction luteolum DSM273 (97% similarity), it represented a new phylotype. A comparison of DGGE banding patterns of the water column and sediment samples demonstrated that specific bacteria accumulated on the bottom from the anoxic water layers, and that indigenous microbial populations were present in the sediment. The measurements of bicarbonate assimilation rates showed significant phototrophic assimilation in the chemocline and lithoautotrophic assimilation throughout the anoxic water, but were not clearly linked with net sulphide turnover rates, indicating that sulphur and carbon metabolisms were not directly correlated.     

The role of the CD134-CD134 ligand costimulatory pathway in alloimmune responses in vivo

The CD134-CD134 ligand (CD134L) costimulatory pathway has been shown to be critical for both T and B cell activation; however, its role in regulating the alloimmune response remains unexplored. Furthermore, its interactions with other costimulatory pathways and immunosuppressive agents are unclear. We investigated the effect of CD134-CD134L pathway blockade on allograft rejection in fully MHC-mismatched rat cardiac and skin transplantation models. CD134L blockade alone did not prolong graft survival compared with that of untreated recipients, and in combination with donor-specific transfusion, cyclosporine, or rapamycin, was less effective than B7 blockade in prolonging allograft survival. However, in combination with B7 blockade, long-term allograft survival was achieved in all recipients (>200 days). Moreover, this was synergistic in reducing the frequency of IFN-gamma-producing alloreactive lymphocytes and inhibiting the generation of activated/effector lymphocytes. Most impressively, this combination prevented rejection in a presensitized model using adoptive transfer of primed lymphocytes into athymic heart transplant recipients. In comparison to untreated recipients (mean survival time (MST): 5.3 +/- 0.5 days), anti-CD134L mAb alone modestly prolonged allograft survival (MST: 14 +/- 2.8 days) as did CTLA4Ig (MST: 21.5 +/- 1.7 days), but all grafts were rejected within 24 days. Importantly, combined blockade further and significantly prolonged allograft survival (MST: 75.3 +/- 12.7 days) and prevented the expansion and/or persistence of primed/effector alloreactive T cells. Our data suggest that CD134-CD134L is a critical pathway in alloimmune responses, especially recall/primed responses, and is synergistic with CD28-B7 in mediating T cell effector responses during allograft rejection. Understanding the mechanisms of collaboration between these different pathways is important for the development of novel strategies to promote long-term allograft survival.     

Cutting edge: a potent adjuvant effect of ligand to receptor activator of NF-kappa B gene for inducing antigen-specific CD8+ T cell response by DNA and viral vector vaccination

The ligand to receptor activator of NF-kappaB (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8(+) T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8(+) T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8(+) T cell-mediated immunity against infectious agents.     

A method for the detection of asparagine deamidation and aspartate isomerization of proteins by MALDI/TOF-mass spectrometry using endoproteinase Asp-N

A method was established for evaluating Asn deamidation and Asp isomerization/racemization. To detect the subtle changes in mass that accompany these chemical modifications, we used a combination of enzyme digestion by endoproteinase Asp-N, which selectively cleaves the N-terminus of L-alpha-Asp, and MALDI/TOF-mass spectrometry. To achieve better resolution, we employed digests of (15)N-labeled protein as an internal standard. To demonstrate the advantages of this method, we applied it to identify deamidated sites in mutant lysozymes in which the Asn residue is mutated to Asp. We also identified the deamidation or isomerization site of the lysozyme samples after incubating them under acidic or basic conditions.