Biomedical subjects
Publications and source records attributed to J Kudoh.
Using a panel of somatic cell hybrids, we have mapped the locus for the AHNAK/desmoyokin gene (AHNAK) to human chromosome 11. Fluorescence in situ hybridization experiments independently confirmed the chromosomal localization and refined it to band 11q12.
PURPOSE: The maternal inheritance of Leber's hereditary optic neuropathy (LHON) is caused by defects in the genes of mitochondrial DNA (mtDNA). The most prevalent mtDNA mutation, present in 40% to 90% of families with this disease, is a G to A substitution at nucleotide position 11778. The rapid and accurate quantification of heteroplasmy of this mutation will help determine the relative risk for disease expression. METHODS: The authors conducted screening tests for heteroplasmy in 44 visually affected patients with the 11778 mutation and 34 unaffected members of 36 Japanese families with LHON using the single-strand conformation polymorphism analysis. This method can detect even a single base difference between the sequences of wild type and mutant DNA strands. The percentage of mutant mtDNA was calculated using an image analyzer. RESULTS: Single-strand conformation polymorphism analysis allowed the detection of heteroplasmy ranging from 5% to 95%. Five (14%) of the 36 families showed heteroplasmy, and 14 (18%) of the 78 persons tested had heteroplasmy ranging from 10% to 94%. Seven patients with heteroplasmy with visual loss had mutant mtDNA ranging from 62% to 94%. CONCLUSIONS: Single-strand conformation polymorphism analysis is rapid, efficient, and accurate for detecting point mutations and quantifying heteroplasmy in mtDNA. Individuals with heteroplasmy with less than 60% of mutant mtDNA in circulating leukocytes are probably at lesser risk for developing optic atrophy.
We have identified a point mutation at nucleotide position 3460 in the ND1 gene of complex I in a Japanese pedigree with Leber's hereditary optic neuropathy by sequencing the ND genes in mitochondrial DNA. None of the 60 healthy Japanese had the 3460 mutation. The proband and his mother also had the 7444 mutation in the COI gene of complex IV and became nearly blind at age 19 with visual acuities of 0.02 OD and 0.04 OS We screened 30 patients with bilateral optic atrophy for the 3460 mutation, and identified one male patient who had the 3460 mutation in heteroplasmic fashion without carrying the 7444 mutation. He lost his sight at age 14 but it recovered to 1.2 OD and 0.7 OS about two years and half after the onset. The difference in final visual acuity between these two patients may reflect the degree of reduction in mitochondrial energy production.
Surgical specimens from 30 patients with transitional cell carcinoma of the bladder were examined for the presence of mutation of the p53 tumor suppressor gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. All of the specimens were superficial, low grade cancer, and 10 patients had recurrences during an observation period of 24 months following the initial surgical treatment. Among the 30 patients with superficial bladder cancer, 4 were found to have the mutated p53 gene and 3 of them had recurrences, which involved either invasive or high grade tumors. Two of these recurrent tumors with the p53 gene mutation showed identical mutations with those primary tumors, whereas the other one did not have the same mutation. The data suggest that bladder cancers with a mutation of the p53 gene have a greater probability of poor prognosis than those which do not, even if the primary lesion was a superficial, low grade tumor. Therefore, the presence of the p53 gene mutation may provide important clues to the factors involved in bladder cancer.
The major antibody binding regions of desmoglein 1 (Dsg1) in pemphigus foliaceus and pemphigus vulgaris were examined using cDNA-encoded fusion proteins combining glutathione S-transferase with various domains of bovine Dsg1, namely, the extracellular regions EC1-2, EC3-5, EC1-5, and the entire intracellular region IC. In immunoblot analyses using these fusion proteins, EC1-2, as well as EC1-5, which comprises EC1-2, were recognized by 50% of the sporadic pemphigus foliaceus sera and 45% of Brazilian pemphigus foliaceus sera that reacted with Dsg1 in immunoblotting of bovine desmosome preparations. None of these fusion proteins reacted with any sera of pemphigus vulgaris. None of these sera showed reactivity with EC3-5. In contrast, the IC domain was recognized by 91% of pemphigus vulgaris sera reactive with Dsg1 in bovine desmosome preparations, and by certain pemphigus foliaceus and Brazilian pemphigus foliaceus sera. These results indicate that major epitopes of Dsg1 recognized by pemphigus foliaceus and Brazilian pemphigus foliaceus sera are located in the extracellular amino-terminal domain EC1-2, and that sera of the Dsg1-positive pemphigus vulgaris contain antibodies against the intracellular domain, which may not play a pathogenic role. Possible reasons for this selectivity of antigen binding site are discussed.
The PCR/restriction endonuclease digestion (RE) assay and PCR/SSCP analysis of the rhodopsin gene in 13 Japanese families with autosomal dominant retinitis pigmentosa (ad RP) revealed a G-A substitution of the first nucleotide of codon 181, replacing Glu (GAG) with Lys (AAG), in one family. The proband showed an early onset of symptoms in childhood with a diffuse loss of rod and cone function and a relatively good preservation of cone function, corresponding to the type with relatively rapid progression to blindness (type I category of ad RP).
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We report the case of a 64-year-old woman with plasma cytoid lymphoma of the ileum and gastric wall. Serum protein electrophoresis and immunoelectrophoresis showed a triclonal IgG kappa, IgA kappa, and IgM kappa gammopathy. Biopsy of the tumor revealed three different cell types, which were stained with antibodies specific for IgG, IgA, and IgM. However, DNA analysis of immunoglobulin gene rearrangements using JH, C mu, and C kappa gene probes showed a monoclonal pattern. These results suggest that the three types of malignant cells producing different classes of immunoglobulins were derived from a single clone of B-cells that had undergone a "class switch".
We have investigated the presence of a point mutation at position 11778 in the ND4 gene of mitochondrial DNA in 17 Japanese families with Leber's hereditary optic neuropathy (LHON), and have identified the mutation in 14 (82.4%) of the 17 families. The prevalence of this mutation appears to be much higher in Japanese patients with LHON than in patients of other ethnic origins, such as Finnish, Dutch, German, and English families.
We have screened for possible disease-causing mutations in the peripherin/retinal degeneration slow (RDS) gene in 13 Japanese families with autosomal dominant retinitis pigmentosa (ADRP). Using polymerase chain reaction-single strand conformation polymorphism analysis, a novel mutation at codon 214 was found in which the highly conserved cysteine was replaced with a serine in one family. The mutation at codon 214 was found in all three affected siblings of this family, but none of the 40 normal control individuals had this mutation. These results strongly suggest that the mutation is pathogenic for RP in this family. The clinical phenotype for this family is a late-onset form of ADRP.
There has been controversy concerning the nature of the bullous pemphigoid (BP) antigen: immunoprecipitation identified BP antigen as a single, unique 230-kDa protein, whereas immunoblot analysis showed multiple antigen molecules, mainly 230- and 170-kDa proteins. In this study, to further characterize the 170-kDa protein, we have examined whether the 170-kDa protein is detected by immunoprecipitation. Extracts of human squamous cell carcinoma cells revealed the 170-kDa protein with immunoblot analysis. Although the conventional immunoprecipitation detected only the 230-kDa protein, some BP sera that detected the 170-kDa protein with immunoblotting also precipitated the 170-kDa protein with our modified immunoprecipitation, in which the cells were extracted with 1% sodium dodecylsulfate (SDS) buffer and reacted with the sera under reduced SDS concentration. The 170-kDa protein-specific BP sera clearly showed hemidesmosomal plaque staining with immunofluorescence of cultured cells. These results indicate that the 170-kDa protein is indeed one of the BP antigens and that the 230- and 170-kDa BP antigens are integrated in different ways in hemidesmosomes.
We have obtained a monoclonal antibody (33A-3D) that specifically recognize desmoyokin, a 680 kDa desmosomal plaque protein that is well characterized in bovine muzzle epidermis. A cDNA clone (DY6, 3693 bp) was isolated by immunoscreening a mouse keratinocyte expression library with 33A-3D, and it was confirmed that DY6 has a partial coding sequence for desmoyokin. DY6 consists of highly homologous repeats about 128 residues long. Furthermore, the 128-residue repeats exhibit a quasi seven-residue substructure, which we believe will adopt an antiparallel beta-sheet structure. Surprisingly, the amino acid sequence showed a significant homology with AHNAK, a newly identified human gene encoding a 700 kDa protein, which was suggested to be down-regulated in neuroblastoma. From its extensive homology, the similarity in both size and structure, and the identical patterns on Southern blot analysis of genomic DNAs, desmoyokin and AHNAK protein are thought to be identical. Although the desmoyokin/AHNAK protein is detected in a variety of cell types at both protein and mRNA levels, its distribution in keratinocytes (associated closely with cell membrane) is quite different from that in cells other than keratinocytes (distributed diffusely in the cytoplasm). These findings suggest that the desmoyokin/AHNAK protein is a ubiquitous molecule with a unique structure and appears to have different distributions (and probably different functions) among different cells.
More than 30 human genes have been mapped by a combination of the fluorescent in situ hybridization (FISH) and spot blot hybridization in this laboratory for the past five years. Furthermore, the Keio strategy has been established for the molecular analysis of the human genome using flow-sorted human chromosomes.
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Southern blot analysis of the human acrocentric chromosomes that were flow-sorted from B-lymphoblastoid cell line GM130B revealed that the sensitivity of the ribosomal RNA (rDNA) gene clusters to the restriction enzyme NotI differs among these rDNA-containing chromosomes: the rDNA clusters of Chromosomes (Chr) 13, 14, and 15 are much more sensitive to NotI digestion than those of Chrs 21 and 22 in this particular cell line. Detailed analysis by use of methylation-sensitive enzymes HpaII and HhaI and methylation-insensitive enzyme MspI confirmed the significant variation in the methylation status of rDNA clusters among these chromosomes. Quantitative analysis by fluorescent in situ hybridization (FISH) indicated that copy number of rDNA varies among individual chromosomes, but the average copy number in the acrocentric Chrs 21 and 22 is significantly greater than that of the Chrs 13, 14, and 15 in GM130B cells. Similar analysis reveals that the methylation status of rDNA clusters in another B-lymphoblastoid cell line GM131 was different from that of GM130B. These data together indicate that the copy number and methylation patterns of rDNA clusters differ among individual acrocentric chromosomes in a given cell line, and they are different among cell lines.
We have developed the "shotgun polymerase chain reaction," a method for obtaining a large number of DNA markers specific to a giant DNA fragment, which facilitates analysis of a particular chromosomal region. We applied this method to a giant NotI fragment which carries the immunoglobulin lambda constant region on chromosome 22. NotI digests of chromosome 22 flow-sorted from human B-lymphoblastoid cell line GM130B were size fractionated by pulsed-field gel electrophoresis. Preliminary Southern hybridization analysis revealed that the immunoglobulin lambda constant region was conveyed on 1.4- and 1.3-Mb NotI fragments in this cell line. The agarose gel corresponding to 1.2 to 1.5 Mb in size was excised into slices and subjected to polymerase chain reaction to identify gel slices containing NotI fragments carrying Ke-Oz+, a subtype of the immunoglobulin lambda constant region. From the NotI fragment thus identified, a large number of small DNA segments were amplified through the ligation-mediated random polymerase chain reaction method. The amplified products were cloned and analyzed for chromosomal origin and localization to particular NotI fragments. Seven of eighteen clones originated from the 1.4-Mb NotI fragment of chromosome 22 in GM130B cells, which appears to be exactly the same as detected by a probe for the immunoglobulin lambda constant region.
We have isolated and sequenced complementary DNA (cDNA) for the human 80K-L protein, a major substrate for protein kinase C and the human homologue of an 80- to 87-kDa bovine protein named MARCKS (myristoylated alanine-rich C kinase substrate). The human 80K-L cDNA encodes a protein of 332 amino acids with a calculated molecular weight of 31,534. Homology comparisons of the nucleotide sequences of the cDNAs indicated that their 3'-untranslated regions are more homologous than the coding regions. Spot blot hybridization using flow-sorted human chromosomes indicated that the gene encoding the 80K-L protein, designated MACS, maps to the q15----qter region of human chromosome 6, and it also suggested that a genomic region with a sequence homologous to the 3'-untranslated region of the 80K-L mRNA exists on chromosome 21.
A 73-year-old man was admitted with severe abdominal fullness. Physical examination suggested much ascites. Laboratory data revealed that BUN was 39 mg/dl, Cr was 2.2 mg/dl, UA was 19.7 mg/dl, and LDH was 1,569 U/l. In ascites, there were many cells with large nuclei and many vacuoles. Immunocytological characterization indicated that most of these cells were B lineage. And diagnosis of malignant lymphoma, the patient was treated with VEPA therapy. However, he soon died with acute renal failure. We examined the possibility of gene rearrangements between the c-myc and immunoglobulin genes and found that c-myc gene was rearranged at the first intron, and joined with immunoglobulin JH locus as head to head. These results suggested that the patient carried Burkitt's lymphoma of the American type.