Nitro- and Amino-polysaccharide Formation from Dextran

Dextran was subjected to oxidative scission by periodate, followed by ring closure with nitromethane to form nitrodextran. The nitro group attached to the ring was reduced by LiAlH₄ to yield amino-polysaccharide of which the molecular weight was about 10,000. It became clear that nitrodextran consisted of 3-deoxy-3-nitro-mannopyranoside, -glucopyranoside, -galactopyranoside and -talopyranoside and their molar ratio was 6: 5: 1: 2 as determined by column Chromatographic separation and gas Chromatographic analysis of the methanolyzate of nitro-dextran.     

Physicochemical Studies on Sulfated Polysaccharides Extracted from Seaweeds at Various Temperatures

Sulfated polysaccharides (SPS) were extracted from three species of seaweeds of Ulvacea (Ulva pertusa, Ulva conglobata and Entromorpha prolifera) for 4 hr at various temperatures and their physicochemical properties were studied using viscometric and equilibrium sedimentation measurements in order to determine the optimum extracting condition.

Sulfated polysaccharides extracted at various temperatures from the seaweed of U. pertusa had the same physicochemical properties, while the larger molecular components of SPS was not extracted from U. conglobata and E. prolifera, at the low temperature of 30~40°C. This was confirmed by analyses of their viscosity and molecular weight and by gel filtration chromatography, in which each SPS showed two or three peaks.

The larger molecular component of SPS could be extracted at the high temperature of 80~90°C in the thermostable form.     

The Chlorinolysis of Methionine Derivatives

The chlorinolysis of l-methionine methyl ester hydrochloride with molecular chlorine was carried out under various conditions, resulting in methyl l-2-amino-4,4,4-trichlorobutanoate and methyl l-2-amino-3,4,4,4-tetrachlorobutanoate which were isolated as N-benzoyl and N-carbobenzoxy derivatives. The chlorinolysis of N-acylmethionine ester and methionine sulfoxide ester proceeded also without cleavage of the N-protecting group to give the same products as above. However, the reaction of methionine sulfone derivative with chlorine did not proceed in the same conditions.

It was proved that the resulting polychloroamino acid derivatives are optically pure. The possible chlorinolysis mechanism was also proposed.     

Breakage of Chromosomal DNA with Aromatic Reductones

Strand breakages of mammalian cellular chromosomal DNA with aromatic reductones were ascertained by use of a cultured cell strain of the rat fetal lung (RFL). The mode of the breakages was investigated by ultracentrifugal analyses. The reductones induced the breakages of the cellular DNA in two different fashions; one is single strand breaks and another double strand breaks. Although the single strand breaks were rapidly repaired, double strand breaks were only partially repaired. Both breaks were not cytocidal. Some physiological alterations were observed to follow the strand breaks.     

Method for Determination and Productivity of Sclerothionine in Sclerotinia Fungi

For determining sclerothionine (STH, S-2-hydroxyethylergothioneine), the method of P. C. Jocelyn which determined ergothioneine in blood was applied, and it was shown that STH was necessary to be degraded with 70% KOH for 1 hr at 100°C, in order to get a theoretical amount of trimethylamine. Trimethylamine produced was trapped by picric acid and spectrophotometrically measured as picrate at 410 mμ. By this method and using paper chromatography, STH in Sclerotinia culture could be determined successfully, and it was found that, among Sclerotinia fungi, a strain of Sclerotinia libertiana which can form sclerotium normally only produced STH, and other various strains of the same genus produced ergothioneine. The cultural condition for production of STH by the Sclerotinia libertiana strain was investigated. As a result, in the shaking liquid culture containing wheat bran, 1.0%; glucose, 1.0%; Polypepton, 0.6%; KH₂PO₄, 0.05%; MgCl₂, 0.05% and cystine-HCl, 0.003% as nutrient, the addition of methionine at a later period in about 0.01% concentration was found to stimulate the accumulation of STH in mycelium.     

Induction of Catalase Activity by Hydrocarbons in Candida tropicalis рK 233

1. The catalase activity of Candida tropicalis pK 233 was induced by hydrocarbons but not by glucose, galactose, ethanol, acetate or lauryl alcohol.

2. The induction of the catalase activity depending upon hydrocarbons was sensitive to cycloheximide but not to chloramphenicol.

3. Glucose repressed strongly the induction of the catalase activity by hydrocarbons but galactose did not affect seriously.

4. When C. tropicalis was incubated with hydrocarbons, the appearance of microbodies was observed electronmicroscopicaliy.

     

Structural Study of Cell Attachment Peptide Derived from Laminin by Molecular Dynamics Simulation

Peptides with cell attachment activity are beneficial component of biomaterials for tissue engineering. Conformational structure is one of the important factors for the biological activities. The EF1 peptide (DYATLQLQEGRLHFMFDLG) derived from laminin promotes cell spreading and cell attachment activity mediated by α2β1 integrin. Although the sequence of the EF2 peptide (DFATVQLRNGFPYFSYDLG) is homologous sequence to that of EF1, EF2 does not promote cell attachment activity. To determine whether there are structural differences between EF1 and EF2, we performed replica exchange molecular dynamics (REMD) simulations and conventional molecular dynamics (MD) simulations. We found that EF1 and EF2 had β-sheet structure as a secondary structure around the global minimum. However, EF2 had variety of structures around the global minimum compared with EF1 and has easily escaped from the bottom of free energy. The structural fluctuation of the EF1 is smaller than that of the EF2. The structural variation of EF2 is related to these differences in the structural fluctuation and the number of the hydrogen bonds (H-bonds). From the analysis of H-bonds in the β-sheet, the number of H-bonds in EF1 is larger than that in EF2 in the time scale of the conventional MD simulation, suggesting that the formation of H-bonds is related to the differences in the structural fluctuation between EF1 and EF2. From the analysis of other non-covalent interactions in the amino acid sequences of EF1 and EF2, EF1 has three pairs of residues with hydrophobic interaction, and EF2 has two pairs. These results indicate that several non-covalent interactions are important for structural stabilization. Consequently, the structure of EF1 is stabilized by H-bonds and pairs of hydrophobic amino acids in the terminals. Hence, we propose that non-covalent interactions around N-terminal and C-terminal of the peptides are crucial for maintaining the β-sheet structure of the peptides.     

Glycosylation Status of CD43 Protein Is Associated with Resistance of Leukemia Cells to CTL-Mediated Cytolysis

To improve cancer immunotherapy, it is important to understand how tumor cells counteract immune-surveillance. In this study, we sought to identify cell-surface molecules associated with resistance of leukemia cells to cytotoxic T cell (CTL)-mediated cytolysis. To this end, we first established thousands of monoclonal antibodies (mAbs) that react with MLL/AF9 mouse leukemia cells. Only two of these mAbs, designated R54 and B2, bound preferentially to leukemia cells resistant to cytolysis by a tumor cell antigen–specific CTLs. The antigens recognized by these mAbs were identified by expression cloning as the same protein, CD43, although their binding patterns to subsets of hematopoietic cells differed significantly from each other and from a pre-existing pan-CD43 mAb, S11. The epitopes of R54 and B2, but not S11, were sialidase-sensitive and expressed at various levels on leukemia cells, suggesting that binding of R54 or B2 is associated with the glycosylation status of CD43. R54^high leukemia cells, which are likely to express sialic acid-rich CD43, were highly resistant to CTL-mediated cytolysis. In addition, loss of CD43 in leukemia cells or neuraminidase treatment of leukemia cells sensitized leukemia cells to CTL-mediated cell lysis. These results suggest that sialic acid-rich CD43, which harbors multiple sialic acid residues that impart a net negative surface charge, protects leukemia cells from CTL-mediated cell lysis. Furthermore, R54^high or B2^high leukemia cells preferentially survived in vivo in the presence of adaptive immunity. Taken together, these results suggest that the glycosylation status of CD43 on leukemia is associated with sensitivity to CTL-mediated cytolysis in vitro and in vivo. Thus, regulation of CD43 glycosylation is a potential strategy for enhancing CTL-mediated immunotherapy.