Fujio Takeuchi

Structural changes in the oligosaccharide moiety of human IgG with aging

In order to elucidate the relationship between glycosylation of IgG and aging, oligosaccharide structures of human IgG purified from sera of men and women aged 18 to 73 years were investigated. Oligosaccharides were liberated quantitatively from IgG by hydrazinolysis followed by N-acetylation and were tagged with p-aminobenzoic acid ethyl ester. The oligosaccharide structures were then analyzed by HPLC in conjunction with sequential exoglycosidase digestion. All IgG samples were shown to contain a series of biantennary complex type oligosaccharides which consisted of ±Galβ1-4GlcNAcβ1-2Manα1-6(±GlcNAcβ1-4)(±Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(±Fucα1-6)GlcNAc and their mono- and di-sialo glycoforms in different ratios. In female IgG samples only, the incidence of non-galactosylated oligosaccharides with non-reducing terminal GlcNAc residues increased with aging (r>0.8), whereas that of digalactosylated oligosaccharides decreased (r<−0.8). A weaker correlation was observed between aging and the incidence of neutral and monosialo oligosaccharides in female IgG (r:0.461 and r=−0.538, respectively) and between aging and the incidence of oligosaccharides with a bisecting GlcNAc in both male and female IgG samples (r=0.566 and r=0.440, respectively). In addition, a significant change with aging in the galactosylation of IgG oligosaccharides was observed in females in their thirties, fifties, and sixties (p<0.02, p<0.01, and p<0.04, respectively). These findings may contribute to our understanding of autoimmune diseases such as rheumatoid arthritis in which glycosylation is involved.

Altered frequency of initiation sites of DNA replication in Werner's syndrome cells

SummaryDNA replication of cultured fibroblasts of early passage derived from Werners syndrome (adult progeria) patients and from normal subjects were compared by DNA fiber autoradiography. The frequency of replication initiation was decreased in Werners syndrome cells derived from five patients compared with that in normal cells derived from three persons of different ages. The rate of DNA chain elongation did not differ between Werners syndrome cells and normal cells.

Prolongation of S phase and whole cell cycle in Werner's syndrome fibroblasts

Abstract The cell cycle was determined in early passage fibroblasts from Werners syndrome and normal subjects. The average cell cycle time was prolonged in Werners syndrome cells due to changes in the duration of S phase. Using alkaline sucrose gradient sedimentation, the rate of DNA elongation was examined, and no difference was observed between Werners syndrome cells and normal cells.

Localization of a gene for familial juvenile hyperuricemic nephropathy causing underexcretion-type gout to 16p12 by genome-wide linkage analysis of a large family.

OBJECTIVE Familial juvenile hyperuricemic nephropathy (FJHN, MIM 162000) is an autosomal-dominant disease characterized by underexcretion-type hyperuricemia, gout, and chronic renal failure. No loci responsible for this disease or any underexcretion-type hyperuricemia/gout have ever been identified. The aim of the study was to localize a gene responsible for FJHN by linkage analysis. METHODS A single large family with at least 20 affected members was analyzed. DNA was obtained from 13 affected and 18 non-affected members after lymphoblastoid cell lines were established. Initially, polymorphic data were obtained for 343 microsatellite loci covering all chromosomes except the X chromosome. Parametric linkage analysis was performed using the obtained data with LINKAGE package software. RESULTS Following a genome-wide search using a set of highly polymorphic microsatellite markers, initial evidence for linkage was obtained for a marker on chromosome 16p. We subsequently genotyped the same subjects for 12 additional markers spanning approximately 30 cM on the short arm of chromosome 16. We obtained a maximum 2-point logarithm of odds (LOD) score of 6.04 at theta = 0 with the marker D16S401; multipoint linkage analysis yielded a maximum LOD score of 6.14 with markers D16S401 and D16S3113, and established a minimum candidate interval of approximately 9 cM. CONCLUSION A gene for FJHN was localized to a candidate interval of approximately 9 cM at 16p12. These findings will be useful for the presymptomatic diagnosis of FJHN in some families and for testing genetic heterogeneity of FJHN in general.

Autoradiographic Studies of DNA Replication in Werner’s Syndrome Cells

We have compared cultured fibroblasts of early passage derived from patients with the Werner syndrome and from normal subjects in several aspects of cell cycle time and DNA replication. The average cycle time was prolonged in Werners syndrome cells compared with normal cells because of changes in the duration of S phase. The durations of G1 and G2 were unchanged. In addition, the labeling index was lower in Werners syndrome cells, suggesting that there are two cell-cycle abnormalities in Werners syndrome cells. The cause of the prolongation of S phase was investigated by DNA fiber autoradiography and alkaline sucrose density gradient sedimentation. The rate of DNA chain elongation was not different in Werners syndrome cells from that in normal cells, but the frequency of replication initiation was decreased in Werners syndrome cells.

TNF-α and TNF-β gene polymorphisms in systemic sclerosis

Tumor necrosis factor (TNF)-alpha and TNF-beta, mediators of inflammatory responses, have been implicated in the pathogenesis of autoimmune diseases. The aim of this investigation was to determine whether two promoter region polymorphisms of the TNF-alpha gene (TNF-alpha -308 and TNF-alpha -238) and a determinant in the first intron of the TNF-beta gene (TNF-beta +252) affect susceptibility to systemic sclerosis (scleroderma) (SSc). Fifty patients and 60 healthy blood donors from Japan were genotyped for these markers by polymerase chain reaction-based methods. Fishers exact test was used to test for significant associations. Because of very limited variation at the TNF-alpha -308 and TNF-alpha -238 loci in the Japanese people, statistical analyses with sufficient power could not be done for these genotypes. However, the two homozygous genotypes of the TNF-beta +252 locus were found to be significantly associated with SSc. Compared to controls, the frequency of the TNF-1 genotype was decreased, whereas that of TNF-2 was increased in SSc patients. The former implies an association with resistance, while the latter suggests an association with susceptibility to the disease. These results show that the TNF-beta +252 locus plays an important role in the etiopathogenesis of SSc.

Severe impairment in adenine metabolism with a partial deficiency of adenine phosphoribosyltransferase

Among three unrelated patients with recurrent 2,8-dihydroxyadenine urolithiasis, two completely lacked adenine phosphoribosyltransferase (APRT) in both erythrocytes and proliferative T cells. The third patient possessed significant enzyme activities in both hemolysates and T-cell extracts at levels comparable to heterozygotes for complete APRT deficiency. Despite significant APRT activities in cell extracts, cultured T cells from the third patient were at least 100-fold more resistant than normal T cells to an adenine analog, 6-methylpurine, whose cytotoxicity is dependent on APRT. These data indicate that APRT activity in T cells from the third patient is positive in cell extracts, but apparently not operating in viable cells. Although the cells from the patients with complete APRT deficiency were as resistant to 6-methylpurine as the cells from the third patient, the cells from the heterozygotes for complete APRT deficiency were almost as sensitive as normal T cells. Therefore, adenine metabolism in the third patient but not in the heterozygotes seems to be as severely impaired as in the patients with complete APRT deficiency, which is quite consistent with the clinical manifestations in these individuals.

Associations between the HLA-A polymorphism and the clinical manifestations of Behcet's disease.

IntroductionThe objective was to investigate associations between the HLA-A gene and Behcets disease (BD) and its clinical manifestations.MethodsGenotyping for the HLA-A locus was performed using the polymerase chain reaction-Luminex typing method in 223 BD patients and 1,398 healthy controls.ResultsThe phenotypic frequencies of HLA-A*02:07 (odds ratio (OR) = 2.03, P = 0.002), A*26:01 (OR = 1.85, P = 0.008), and A*30:04 (OR = 2.51, P = 0.006) tended to be higher in BD patients than in normal controls, but the frequency of A*33:03 (OR = 0.59, P = 0.003) tended to be lower in BD patients. A meta-analysis adopting our and the Japanese data confirmed the associations of HLA-A*02:07, A*26:01, and A*33:03 with BD. Furthermore, the frequencies of the HLA-A*02:07, A*26:01, and A*30:04 were significantly higher in patients with skin lesions (OR = 2.37, P < 0.0005, Pc < 0.012) and arthritis (OR = 2.32, P = 0.002, Pc = 0.048), with uveitis (OR = 3.01, P < 0.0005, Pc < 0.012), and with vascular lesions (OR = 9.80, P < 0.0005, Pc < 0.012) and a positive pathergy test (OR = 4.10, P = 0.002, Pc = 0.048), respectively, than in controls. In HLA-B*51 non-carriers, these associations were also significant, being much stronger between HLA-A*26:01 and uveitis (OR = 4.19, P < 0.0005, Pc < 0.012) and between HLA-A*30:04 and vascular lesions (OR = 13.97, P < 0.00005, Pc < 0.0012). In addition, HLA-A*30:04 was associated with genital ulcers in HLA-B*51 non-carriers (OR = 3.89, P = 0.002, Pc = 0.048).ConclusionsHLA-A*02:07, A*26:01, and A*30:04 were associated with increased risk for BD, while HLA-A*33:03 with decreased risk. HLA-A*02:07, A*26:01, and A*30:04 were associated with skin lesions and arthritis, with uveitis, and with vascular lesions, genital ulcers, and a positive pathergy test, respectively.

Changes in chromatin structure during aging of human skin fibroblasts

Human skin fibroblasts from embryo, 16-, 30- and 60-year-old adults were cultivated and passaged in vitro. Their chromatin structures were examined by the sensitivity to micrococcal nuclease and by electron microscopy. When the mode of DNA degradation by the nuclease was analysed during in vitro aging of the embryo skin fibroblasts, the discrete ladder of nucleosomal DNA became obscure in old cells. Analogous change of chromatin structure was also observed even in young cells as their donor ages increased. From the observation with electron microscopy, it became clear that chromatin of fibroblasts from 30-year-old adults does not have regularly spaced nucleosomes, compared with chromatin from embryo. These results suggest that the length of the linker DNA which connects core particles becomes to be heterogeneous by aging, both in vivo and in vitro in human skin fibroblasts.

Decrease in the average size of replicons in a Werner syndrome cell line by Simian Virus 40 infection

We have measured the distance between replicon initiation sites as well as the rate of DNA chain elongation in Simian Virus 40 (SV40)-infected and uninfected Werner syndrome (WS) and normal cell lines by DNA fiber-autoradiography. There was no difference in the rate of chain elongation among these cell lines. On the other hand, the replicon center-to-center distance was clearly longer in WS fibroblasts than that in normal fibroblasts. SV40 infection changed the center-to-center distance in WS cells toward that in normal cells.