Mutual recognition between polymerized liposomes. IV. Polysaccharide-lectin system.

As a model of cell-cell recognition processes, the association processes of a polysaccharide (mannan)-carrying liposome with a lectin (Concanavalin A, Con A)-carrying polymerized liposome were followed by turbidimetry. The association process was strongly inhibited by the addition of a low molecular weight sugar, methyl-alpha-D-mannopyranoside, which shows that the association between the liposomes is due to the specific interaction between Con A and mannan. The association rate constant obtained was much smaller than the theoretical value for a diffusion-controlled binary association process. This implies that the association rate of liposomes is limited by the recognition between complementary ligands bound on the liposome surfaces. Another reason for the smaller association rate constant in the liposome-liposome system is the repulsive hydration effect. The effect of the surface density of the lectin immobilized on the liposome on the recognition was also examined.     

Mutagenicity of N4-aminocytidine and its derivatives in Chinese hamster lung V79 cells. Incorporation of N4-aminocytosine into cellular DNA

N4-Aminocytidine induced mutation to 6-thioguanine resistance in Chinese hamster lung V79 cells in culture. Previous studies with experimental systems of in vitro DNA synthesis and of phage and bacterial mutagenesis have shown that this nucleoside analog induces base-pair transitions through its incorporation into DNA, with its erroneous base-pairing property. Incorporation of exogenously added [5-3H]N4-aminocytidine into the DNA of V79 cells was in fact observed in the present study. N4-Aminodeoxycytidine was not mutagenic for the V79 cells. Several alkylated N4-aminocytidine derivatives were tested for their mutagenicity in this system. Those with an alkyl group on the N'-nitrogen of the hydrazino group at position 4 of N4-aminocytidine were mutagenic, but those having an alkyl on the N4-nitrogen were not. These results are consistent with those previously observed in the bacterial mutagenesis systems, and agree with a mechanism of mutation in which a tautomerization of N4-aminocytosine is the necessary step for causing the erroneous base pairing.     

Characterization of tumor-associated fucogangliosides from PC 12 pheochromocytoma cells

PC 12h pheochromocytoma cells were subcutaneously transplanted into rat. We found the transplanted tumors accumulated some fucogangliosides associated with PC 12 cells. These gangliosides were isolated and purified by DEAE-Sephadex A-25 and Iatrobeads column chromatographies. Their structures were determined by fast atom bombardment mass spectrometry, proton nuclear magnetic resonance spectrometry, permethylation study, and sequential degradation using various exoglycosidases and mild acid hydrolysis. Two tumor-associated fucogangliosides were found to possess the blood group B determinant as follows: G6: IV2Fuc alpha, IV3Gal alpha, II3NeuAc, GgOse4Cer; G11: IV2Fuc alpha, IV3Gal alpha, II3 (NeuAc)2, GgOse4Cer. A ganglioside with the similar structure as ganglioside G6 was isolated from rat hepatoma cells (Holmes, E.H., and Hakomori, S-I. (1982) J. Biol. Chem. 257, 7698-7703). However, ganglioside G11 has not previously been reported in the literature. These fucogangliosides reacted with the monoclonal antibody prepared by immunizing mice with PC 12h cells. Other fucogangliosides were also found to accumulate in the transplanted tumor tissues. They were identified as fucosyl-GM1 and fucosyl-GDlb. These fucogangliosides did not react with the monoclonal antibody against PC 12h cells.     

Kinetic analysis of the binding of guanine nucleotide to bovine brain smg p25A

Bovine brain smg p25A, a guanine nucleotide-binding protein with a Mr of about 25,000, bound specifically GTP, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and GDP. The initial velocities of the binding of GTP gamma S to GDP-bound smg p25A and the dissociation of GDP from this protein increased by decreasing Mg2+ concentrations or increasing NaCl concentrations. The initial velocity of the binding of GTP gamma S to GDP-free smg p25A was not affected by changing Mg2+ concentrations. These results indicate that the dissociation of GDP from smg p25A limits the binding of GTP to this protein, and suggest that there is a protein stimulating the dissociation of GDP from smg p25A and thereby stimulating the binding of GTP to this protein in mammalian tissues. In fact, the protein stimulating the dissociation of GDP, but not of GTP gamma S, from smg p25A was detected in bovine brain cytosol.     

Radioimmunoassay for non-enzymatically glycated serum proteins

We have developed a radioimmunoassay (RIA) for nonenzymatically glycated serum proteins. The polyclonal antibodies prepared against reduced glycated human albumin were specific for the glucitollysine residues of serum proteins. Serum proteins from diabetic patients (n = 25) contained 5.3 +/- 2.8 nmoles of glucitollysine/mg protein, compared to 2.0 +/- 0.2 in controls (n = 20). The intra- and inter-assay variables were 3.2-6.2% and 4.4-8.6%, respectively. Results from this assay procedure correlated well with those from the boronate affinity chromatography procedure (r = 0.94; P less than 0.001). The data suggested that diabetic serum proteins contained at least 2.5 times as much immunochemically detectable glucitollysine residures as normal serum proteins after reduction of the proteins with sodium borohydride.     

Studies on DNA markers (D4S10 and D4S43/S127) genetically linked to Huntington's disease in Japanese families

Summary This is the first full report on the genetic linkage between Japanese Huntington's disease and the DNA markers D4S10 and D4S43/S127. With use of the HindIII, BglI, and EcoRI polymorphisms detected at D4S10, and the combination of all these polymorphisms to give composite haplotypes, nine Japanese Huntington's disease families were found to be informative. Three recombinants for D4S10 were detected in these families, giving a maximum lod score of 1.662 at a of 0.10. Similarly, when we used the MspI and PvuII polymorphisms detected by D4S43/S127, five families gave informative results. No recombinant was detected in these families, giving a maximum lod score of 3.348 at a of 0.00. These results clearly support the view that the Japanese Huntington's disease gene may be identical with the Western gene, in spite of the lower prevalence rate in Japan.     

Antimutagenesis by factors affecting DNA repair in bacteria

The term 'antimutagen' was originally used to describe an agent that reduces the apparent yield of spontaneous and/or induced mutations, regardless of the mechanisms involved. The 'antimutagens' include 'desmutagens' and 'bio-antimutagens'. In this article, our attention was focused on the bio-antimutagens affecting DNA repair in bacteria. Cobaltous chloride reduced the frequency of mutations in Escherichia coli induced by MNNG. The possibility that metal compound inhibits the growth of mutagen-treated cells was examined. The results clearly showed that the antimutagen surely reduces the mutation rate. The target of cobaltous chloride was found to be cellular factors including Rec A. Vanillin and cinnamaldehyde had strong antimutagenic activities against UV, 4NQO and AF-2. They stimulated Rec A-dependent recombination repair functions in the mutagen-treated cells. Among plant materials, tannins possess antimutagenic activity against UV-induced mutations in E. coli. It has been found that tannic acid stimulates the excision repair encoded by the uvrA gene thereby reducing the yield of mutants. Substances which are antimutagenic in bacterial systems also had antimutagenic activity in cultured mammalian cell systems. Vanillin reduced the frequency of mutagen-induced chromosomal aberrations.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. II. Effect of the steel locus on spermatogenesis

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermatogonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/+ genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. I. Different distribution patterns between melanocytes and mast cells in the skin

In spite of their different origin, both melanocytes and mast cells are deficient in the skin of mutant mice of the Sl/Sld genotype. Since the neural crest and the liver of Sl/Sld embryos contain normal precursors of melanocytes and mast cells, respectively, the deficiency is attributed to a defect in tissue environment necessary for migration and/or differentiation of precursor cells. We investigated whether the tissue environment used for differentiation of melanocytes and mast cells was identical by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent pigmented and nonpigmented stripes were obtained. In the nonpigmented stripes of these Sl/Sld in equilibrium with +/+ chimaeras, melanocytes were not detectable in hair follicles but were detectable in the dermis. In contrast, melanocytes were detectable neither in hair follicles nor in the dermis of nonchimaeric Sl/Sld mice. Concentrations of mast cells were comparable in the pigmented and nonpigmented stripes of Sl/Sld in equilibrium with +/+ chimaeras, but the average concentration of mast cells significantly varied in the chimaeras (from 8% to 74% of the value observed in control +/+ mice). The present result suggests that mesodermal cells that support the migration and differentiation of both melanocyte precursors and mast-cell precursors mix homogeneously in the dermis and that ectodermal cells that influence the invasion of differentiating melanocytes into hair follicles make discrete patches.     

Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvovirus B19.

In this study, we identified a region in the human parvovirus structural protein which involves the neutralization of the virus by a monoclonal antibody and site-specific synthetic peptides. A newly established monoclonal antibody reacted with both viral capsid proteins VP1 and VP2. The epitope was found in six strains of independently isolated human parvovirus B19. The monoclonal antibody could protect colony-forming unit erythroid in human bone marrow cell culture from injury by the virus. The monoclonal antibody reacted with only 1 of 12 peptides that were synthesized according to a predicted amino acid sequence based on nucleotide sequences of the coding region for the structural protein of B19 virus. The sequence recognized by the antibody was a site corresponding to amino acids 328 to 344 from the amino-terminal portion of VP2. This evidence suggests that the epitope of the viral capsid protein is located on the surface of the virus and may be recognized by virus-neutralizing antibodies.