Reconstitution of photosynthetic charge accumulation and oxygen evolution in CaCl2-treated PS II particles: II: EPR evidence for reactivation of the S1 → S2 transition in CaCl2-treated PS II particles with the 17-, 23-, and 34-kDa proteins

Extraction of PS II particles with 1 M CaCl₂ caused complete disappearance of the light-induced signal of the possible Kok S₂ state of the water-splitting complex and total loss of the O₂, evolving activity, concomitant with perfect removal of the 17-, 23- and 34-kDa proteins from the particles. The recovery of the multiline signal in the CaCl₂-treated PS II was performed by reinserting the 34-kDa protein, when CI^? was present in the solution for the EPR measurement. However, in the absence of Cl^?, besides the 34-kDa protein, the 17- and 23-kDa proteins were required for the recovery of the signal. These results are compared with the results on the recovery of the O₂, evolution in the reconstituted PS II to examine the role of these three proteins on the water splitting.     

A morphological study of ferritin synthesis in macrophages with ingested ferric hydroxide-potassium polyvinyl sulfate complexes

A ferric hydroxide-polyvinyl sulfate colloidal solution (Fe-PVS), prepared by mixing potassium polyvinyl sulfate (PVSK) and ferric hydroxide colloidal solution was used to study ferritin synthesis in rat peritoneal macrophages. The colloidal particles had spherical electron opaque ferric hydroxide cores with diameters of about 250 nm surrounded by radially arranged fibrous PVS molecules. They also had strong negative electric charges. Fe-PVS particles injected into the peritoneal cavity were taken up by the macrophages then disintegrated rapidly. In the phagolysosomes the electron opaque ferric hydroxide cores of Fe-PVS were denuded of their PVS frames then decomposed into small 5-6 nm granules 24 to 48 h after injection. These small granules were released from the lysosomes into the hyaloplasm and the myelin figures were found in the lysosomal vacuoles. No reaccumulation of granules in lysosomes was found even 3 months later. The intracellular distribution of ferritin in macrophages demonstrated by the immunocytochemical method showed a pattern similar to that of the small granules formed by the disintegration of Fe-PVS. This means that in rat peritoneal macrophages that contain ingested Fe-PVS particles ferritin first is synthesized in phagolysosomes by the ferric hydroxide cores that conjugate with apoferritin or protein subunits then they are dispersed into the cytoplasm. Two possible pathways for the biosynthesis of ferritin are discussed.     

Quantitative β-elimination-reduction of O-glycosyl linkages in chondroitin sulfates

The chondroitin 4-sulfate-peptide from whale cartilage contains serine, xylose, and galactose in ～1:1:2 molar ratio. Deamination with nitrous acid showed that about 50% of the serine is at the amino terminus. Various conditions of β-elimination-reduction were employed with the preparation to provide quantitative data on the linkage region between protein and carbohydrate. The optimal conditions used, 0.4m sodium hydroxide in the presence of 0.3m sodium borohydride and 0.01m PdCl₂·2H₂O for 24 h at 25°, resulted in an increase of alanine content and concomitant decrease of serine and conversion of xylose into xylitol, all in equimolar amounts. Furthermore, substitution of both the terminal amino and carboxyl groups, and elimination-reduction, brought about cleavage of most of the linkages; over 90% of the amino acids originally present were lost after re-isolation of the polymer fraction. These results indicate that β-elimination-reduction alone, under the optimal conditions, allows the mode of linkage to be quantitatively determined as an O-xylosylserine linkage. Under these optimal conditions, the linkage region between protein and a chondroitin 4- and 6-sulfate hybrid (1:1) from bovine tracheal cartilage was determined to be Gal-Gal-Xyl-O-Ser, thus being similar to that found in chondroitin 4-sulfate-peptide.     

Mass spectrometric and kinetic studies on slow progression of papain-catalyzed polymerization of l-glutamic acid diethyl ester

Papain polymerizes l-glutamic acid diethyl ester (Glu-di-OEt) regioselectively, resulting in the formation of poly (γ-ethyl α-l-glutamic acid) with various degrees of polymerization of less than 13. Reaction temperatures below 20 °C were appropriate for the reaction in terms of suppression of non-enzymatic degradation of Glu-di-OEt and an increase in the peptide yield, while the reaction was preceded by a pronounced induction period. Mass spectrometric analyses of the reaction conducted at 0 °C revealed that the accumulation of the initial dimerization product, l-glutamyl-l-glutamic acid triethyl ester (Glu-Glu-tri-OEt), was limited during the induction period, and that a sequential polymer derived from a further elongation of the dimer was the tetramer, but not the trimer. Kinetic analyses of acyl transfer reactions with Glu-di-OEt and Glu-Glu-tri-OEt as acyl acceptors and Nα-benzoyl-l-arginine ethyl ester as an acyl donor affirmed that Glu-Glu-tri-OEt bound more strongly than Glu-di-OEt both to the S- and S′-subsites of papain. Therefore, what occurred during the initial stage of the polymerization was interpreted as follows: the rate of the papain-catalyzed dimerization of Glu-di-OEt was extremely slow, once Glu-Glu-tri-OEt was initially synthesized it exclusively bound to the active site of papain, and then papain utilized the dimer in polymerization effectively rather than the monomer.     

Lectin binding sites related with rat ascites hepatoma cell adhesion

In order to elucidate the correlation between cell surface lectin binding sites and the degree of cell adhesiveness, quantitative lectin binding assays were performed using three types of rat ascites hepatoma cell lines (free cell, mixed cell, and island-forming cell types). The lectin binding site patterns showed no remarkable differences among the intact tumor cell lines, but treatment of the cells with L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-trypsin or neuraminidase induced remarkable differences in the modulation of the number of lectin binding sites. TPCK-trypsin treatment caused a marked decrease in the number of peanut agglutinin binding sites on the island-forming and mixed cell types, concomitant with disaggregation of the cells, showing that trypsin sensitive binding sites are involved in the cell-cell adhesion. Neuraminidase treatment caused a decrease in wheat germ agglutinin binding sites and an increase in castor bean agglutinin binding sites, and these effects were greater for the free cell type. These results indicated that alpha-sialyl-beta-D-galactosyl residues are more abundant on the cell surface of the free cell type than the other cell types. Therefore, it was suggested that electrostatic repulsion due to negative charges of the cell surface sialic acid contributes to the low cell adhesiveness of the free cell type.     

Isolation and characterization of a novel Enterococcus faecalis bacteriophage φEF24C as a therapeutic candidate

Vancomycin-resistant Enterococcus faecalis (VRE) has become a significant threat in nosocomial settings. Bacteriophage (phage) therapy is frequently proposed as a potential alternative therapy for infections caused by this bacterium. To search for candidate therapeutic phages against Enterococcus faecalis infections, 30 Enterococcus faecalis phages were isolated from the environment. One of these, virulent phage φEF24C, which has a broad host range, was selected for analysis. The plaque-forming ability of φEF24C was virtually unaffected by differences in the clinical host strains. Furthermore, the phage had a shorter latent period and a larger burst size than ordinary tailed phages, indicating that φEF24C has effective lytic activity against many Enterococcus faecalis strains, including VRE. Morphological and genomic analyses revealed that φEF24C is a large myovirus (classified as family Myoviridae morphotype A1) with a linear double-stranded DNA genome of c . 143 kbp. Analyses of the N-terminal amino acid sequences of the virion proteins, together with the morphology and the genome size, speculated that φEF24C is closely related to other myoviruses of Gram-positive bacteria that have been used experimentally or practically for therapy or prophylaxis. Considering these results, φEF24C may be a potential candidate therapeutic phage against Enterococcus faecalis infections.     

Mechanistic investigation of capability of enzymatically synthesized polycysteine to cross-link proteins

BackgroundPreviously, we had reported that α-chymotrypsin–catalyzed polymerization of l-cysteine ethyl ester in a frozen buffer provided poly-l-cysteine (PLCys) in good yield, of which degree of polymerization had been determined to be 6–11. Almost all of SH groups in PLCys were in free forms. Such a multi-thiol peptide may cross-link proteins through thiol/disulfide (SH/SS) exchange reactions, considering the knowledge that other synthetic multi-thiol additives changes properties of protein materials.MethodsThis study explored the capability of PLCys to cross-link proteins using lysozyme as a model protein which has four disulfide bonds but no free SH group. The protein was incubated with PLCys at neutral pH and at below 70 °C to avoid PLCys-independent, β-elimination-mediated cross-linkings. Protein polymerization was analyzed by SDS-PAGE and SEC. PLCys peptides involved in the protein polymer, which were released by reduction with dithiothreitol, were analyzed by RP-HPLC.ConclusionsAddition of urea and thermal treatment at 60 °C caused PLCys-induced lysozyme polymerization. Compared with free cysteine, a higher level of PLCys was required for the polymerization probably due to its low water solubility. RP-HPLC analyses suggested that PLCys played a role in the protein polymerization as a cross-linker.General significanceEnzymatically synthesized PLCys shows promise as a peptidic cross-linker for the production of protein polymers with novel physiochemical properties and functionalities.     

Defective DNA replication and repair associated with decreases in deoxyribonucleotide pools in a mouse cell mutant with thermolabile ubiquitin-activating enzyme E1.

Upon shift-up in temperature, mouse tsFS20 mutant cells with thermolabile ubiquitin-activating enzyme E1 immediately stopped DNA replication and showed cell cycle arrest in S-phase. In contrast, when the cells were permeabilized with lysolecithin after culture at the nonpermissive temperature, they exhibited a normal level of replicative DNA synthesis in vitro. In agreement with this, intracellular pools of deoxyribonucleoside triphosphates were significantly reduced in the cells cultured at the nonpermissive temperature. Even under the permissive conditions, tsFS20 cells were more sensitive to hydroxyurea and alkylating agents, and induced less mutation than the wild-type cells. These results suggest that the ubiquitin system affects DNA replication and repair.     

The NFLD antigen in Japan

The low-frequency red cell antigen NFLD was identified in 2 Japanese donors. A family study showed that the antigen is not part of the P1 blood group system. Anti-NFLD was found in serum of several donors (frequency of 0.044%).