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.     

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.     

New diterpene esters from Aleurites fordii fruits

A toxic diterpenoid diester and a monoester were isolated from the fruits of Aleurites fordii. The structure of the monoester was found to be 13-O-acetyl-16-hydroxyphorbol by transforming it into bisdehydrophorbol-(12,20)-diacetate. The structure of the toxic constituent was established as 12-O-palmityl-13-O-acetyl-16-hydroxyphorbol by partial synthesis from the monoester.     

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%).     

Halo-shape technique for leadless pacemaker implantations: A case report

A 92-year-old woman underwent an implantation of a leadless pacemaker (Micra; Medtronic, Inc, Minneapolis, MN) for complete atrioventricular block after a transvenous lead extraction due to a pocket infection of a dual chamber pacemaker. Marked scoliosis and a humpback due to an advanced age made it impossible to direct the tip of the pacemaker delivery catheter towards the right ventricular septum or apex and shape the catheter into a gooseneck-shape. Thus, by attaining a halo-catheter shape of the delivery catheter, the catheter tip could be directed toward the infero-basal portion of the right ventricular septum. The pacemaker was successfully deployed at that site without any complications, and good device parameters were achieved. The halo-shape technique may be also an alternative method for delivering a leadless pacemaker in patients with an unsuccessful delivery of a leadless pacemaker to the right ventricular septum using the conventional gooseneck-shape technique.     

Generation of cardiomyocytes by atrioventricular node cells in long-term cultures

Turnover of cardiac pacemaker cells may occur during the lifetime of the body, and we recently raised the hypothesis that specialized cardiac cells have in common the potential to generate cardiomyocytes from fibroblasts. To examine this hypothesis, we analyzed the ability of atrioventricular node cells (AVNCs) to generate functional cardiomyocytes in long-term culture. AVNCs were isolated from adult guinea pig hearts and cultured for up to three weeks. Under phase-contrast microscopic observation over time, it was found that within a week, a number of fibroblasts gathered around the AVNCs and formed cell clusters, and thereafter the cell clusters started to beat spontaneously. The nascent cell clusters expanded their area gradually by three weeks in culture and expressed specific cardiac genes and proteins. Maturation of newly formed cardiomyocytes seems to be slow in cultures of AVNCs compared with those of sinoatrial node cells. Stimulation of muscarinic receptors with acetylcholine induced a beating rate decrease which was blocked by atropine, and activation of adenylate cyclase activity with forskolin increased the beat rate, while stimulation of beta adrenoceptors by isoproterenol had no effect. These results indicate that AVNCs form a cluster of cells with properties of functional cardiomyocytes and provide evidence to support the hypothesis.