[von Willebrand disease type 1].
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Diagnosis, Differential↗
Biomedical subjects
M Sugimoto
Publications and source records attributed to M Sugimoto.
Abnormal telomere dynamics of B-lymphoblastoid cell strains from Werner's syndrome patients transformed by Epstein-Barr virus.
The characteristics of B-lymphoblastoid cell strains transformed by Epstein-Barr virus (EBV) from normal individuals and Werner's syndrome (WRN) patients were compared. We continuously passaged cell strains from 28 WRN patients and 20 normal individuals for about 2 years corresponding to over 160 population doubling levels (PDLs). First, the WRN mutation significantly suppressed the immortalization: all the 28 cell strains from WRN patients, as well as 15 out of 20 cell strains from normal individuals, died out before 160 PDLs mostly without developing a significant telomerase activity. The remaining five cell strains from normal individuals became moderately/strongly telomerase-positive and, three of them were apparently immortalized with an infinitively proliferating activity. Second, the monitoring of the telomere length of both normal and WRN cell strains during the culture period suggests that the WRN gene mutation causes abnormal dynamics of the telomere: (1) a significant proportion of WRN cell strains showed drastic shortening or lengthening of telomere lengths during cell passages compared with normal cell strains, and (2) WRN cell strains terminated their life-span at a wide range of telomere length (between 3.5 and 18.5 Kbp), whereas normal cell strains terminated within a narrow telomere length range (between 5.5 and 9 Kbp). The chromosomal aberration characteristic of WRN cells, including translocation was confirmed in our experiment. We discussed the correlation between the chromosomal instability, abnormal telomere dynamics and inability of immortalization of the WRN B-lymphobloastoid cell strains.
B-Lymphocytes↗
Down-regulation of the defective transcripts of the Werner's syndrome gene in the cells of patients.
Werner's syndrome (WS), an adult progeria, is a recessive genetic disorder caused by the mutations in the DNA helicase gene (WRN). In this study, a comparative northern blot analysis was made for poly(A)+ RNAs extracted from fibroblasts and B-lymphoblastoid cells of WS patients, relatives of patients, and normal individuals. The levels of mutant WRN mRNA from patient cells were significantly lower than those of intact mRNA from the cells of normal individuals by an average of 70%. Furthermore, an extremely low level of WRN mRNA(s), presumably a mixture of mutant and intact mRNAs, was observed for the patient's family members who carry one mutated allele. These results strongly suggest that a relatively low level of helicase mRNA is sufficient to prevent the onset of Werner's syndrome.
Adult↗
Cloning and characterization of a novel gene, WS-3, in human chromosome 8p11-p12.
A novel human gene referred to as the WS-3 gene, in the short arm of human chromosome 8, was cloned by a combination of exon trapping, thermal asymmetric interlaced-PCR (TAIL-PCR) and the Marathon-Ready cDNA amplification method. The gene consists of 7 exons separated by 6 introns, and is at the telomere side of the STS marker, D8S1055. The full-length WS-3 gene contains 1052 nucleotides and codes for a protein of 190 amino acids with a calculated mol. wt. of 20,747. Southern blot experiments showed that the WS-3 gene exists as a single copy in the human genome. A protein encoded by the WS-3 gene has an R-G-D (Arg-Gly-Asp) motif in the N-terminal region, which seems to confer adhesive properties to macromolecular proteins like fibronectin. Although WS-3 is a small gene with unknown biological function, its ubiquitous expression in various tissues and organs suggests that the encoded protein is one of the essential components of all organs and tissues.
Amino Acid Sequence↗
Polyamines prevent apoptotic cell death in cultured cerebellar granule neurons.
Polyamines play critical roles during the development of brain neurons. In the present study we examined the effects of polyamines on neuronal apoptotic death. Rat cerebellar granule neurons were cultured in the presence of a depolarizing concentration of KCl (25 mM) in the medium. Apoptotic neuronal death was induced by changing the medium to that containing 5.6 mM KCl without serum. Spermine as well as spermidine and putrescine prevented cell death in a concentration-dependent manner with the order of potency being spermine > spermidine > putrescine. The effect of spermine was partially blocked by several NMDA-type glutamate receptor antagonists including (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK-801). MK-801-sensitive neuroprotection by spermine depended on cell density. Activation of CPP32 (caspase-3/Yama/apopain)-like proteolytic activity, a key mediator of apoptosis, precedes neuronal death, and polyamines prevented an increase in this activity. These results demonstrate that polyamines protect neurons from apoptotic cell death through both NMDA receptor-dependent and -independent mechanisms, acting upstream from the activation of CPP32-like protease(s).
Animals↗
Gene expressions of transferred human chromosome 8 in mouse cell lines.
Gene expressions were studied for the human chromosome 8 which were introduced by microcell fusion to three mouse tumor cell lines: A9, mouse melanoma B16F10 and SCVA2 derived from SV40-transformed scid fibroblasts. Nineteen genes in the human chromosome 8 were chosen, and the presence of their transcripts in these cells was examined by RT-PCR. The data showed that most of the human genes were expressed, with a few exceptions: the indoleamine 2, 3-dioxygenase gene was expressed in none of these cell lines, while two other genes, calbindin and neuronal nicotinic acetylcholine receptor subunit (Ach Rbeta3) genes, were not expressed in the A9 and SCVA2 cell lines, respectively, suggesting some cell- or species-specific transcriptional regulations exist. These results show that the mouse cell lines carrying a human chromosome are powerful tools for chromosome-specific cDNA cloning.
Animals↗
Diagnostic and prognostic usefulness of N1,N8-diacetylspermidine and N1,N12-diacetylspermine in urine as novel markers of malignancy.
Recently, we found N1,N8-diacetylspermidine (Ac2Spd) and N1,N12-diacetylspermine (Ac2Spm) in human urine, and noted that their amount increased significantly in patients with urogenital malignancies. Previous findings that simultaneous reference to these diacetylpolyamines is useful in distinguishing cancer patients from healthy persons were confirmed by more recent analytical data on urine samples from several cancer patients. Further examination revealed that urinary Ac2Spm and Ac2Spd tended to decrease when cancer patients were treated and entered partial remission. In cases where the Ac2Spm and Ac2Spd levels were normal or near-normal after treatments, the prognosis of the patients was generally good. In contrast, when their level remained far above the normal limits after apparently effective treatment, the prognosis of the patients was poor. When a patient is in remission for more than 3 years, urinary levels of both Ac2Spm and Ac2Spd are stabilized and stay below the normal limits, with rare exceptions. The recurrence of a cancer as well as the complication of a second one during the period of follow-up examination was accompanied by elevation of urinary diacetylpolyamines. These observations indicate that urinary Ac2Spm and Ac2Spd are useful as prognostic indicators after treatment and during follow-up examination of cancer patients.
Adult↗
Analysis of helicase gene mutations in Japanese Werner's syndrome patients.
The profile of helicase gene mutations was studied in 89 Japanese Werner's syndrome (WRN) patients by examining the previously described mutations 1-4 as well as a new mutation found during this study, designated mutation 5. Of 178 chromosomes (89 patients), 89 chromosomes (50%) had mutation 4, 11 (6.2%) chromosomes had mutation 1, and two chromosomes (1.1%) contained mutation 5. Mutations 2 and 3 were not observed in this patient population. The remaining 76 (42.7%) chromosomes had none of these mutations. A significant fraction of all patients (22 total patients, 24.7%) appear to be compound heterozygotes, including those carrying mutations of both types 1 and 4. The genotypes analysis of the markers surrounding the. WRN helicase gene strongly suggests that most of the chromosomes carrying either mutation 1 or 4 were derived from two single founders.
DNA Helicases↗
Mutation and haplotype analyses of the Werner's syndrome gene based on its genomic structure: genetic epidemiology in the Japanese population.
The correlation between mutations in the Werner's syndrome (WRN) gene and the haplotypes of surrounding markers was studied in Japanese patients. We have elucidated the genomic structure of WRN helicase, and found five additional mutations, designated mutations 6-10. Mutations 4 and 6 were found to be the two major mutations in this population; these mutations comprised 50.8% and 17.5%, respectively, of the total in a sample of 126 apparently unrelated chromosomes. Almost all the patients homozygous for mutation 4 shared a haplotype around the WRN gene, consistent with the view that they are derived from a single ancestor. This important advantage demonstrated in the identification of the WRN gene suggests that the Japanese present a unique population for the cloning of other disease genes. The conserved haplotype was observed across 19 loci, extending a distance estimated to be more than 1.4 Mbp around the WRN gene. This haplotype is rare among random Japanese individuals. Unexpectedly, all the nine patients homozygous for mutation 6 shared a haplotype that was identical to this haplotype at 18 of these 19 markers. These results suggest that mutations 4 and 6 arose independently in almost identical rare haplotypes. The remaining mutations (1, 5, 7, 8, 9, and 10) occurred rarely, and were each associated with different haplotypes.
DNA Helicases↗
Selective allosteric enhancement of the binding and actions of acetylcholine at muscarinic receptor subtypes.
The ternary allosteric model predicts the possibility of discovering molecules with novel and highly subtype-selective modes of action. This approach has been applied to muscarinic receptors. The alkaloid brucine is capable of selectively enhancing by an allosteric mechanism the effects of low but not high concentrations of acetylcholine at only the m1 subtype of muscarinic receptors. A simple derivative of brucine, N-chloromethylbrucine, enhances acetylcholine actions selectively at only m3 receptors. In addition it binds to, but does not affect, the properties of m4 receptors, thereby demonstrating neutral cooperativity and an 'absolute' selectivity of action at m3 receptors over m4 receptors. Brucine N-oxide enhances acetylcholine binding at m3 and m4 receptors and is neutral at m1 and m5 receptors. These findings allow the possibility of developing muscarinic agents that have a novel and highly targeted mode of action; they may act only on a single muscarinic receptor subtype which is functioning sub-optimally and therefore be of use therapeutically in the early stages of Alzheimer's Disease.
Acetylcholine↗
Regulation of melanophore responsiveness in the background-adapted medaka, Oryzias latipes: change in the intracellular signaling system.
The responsiveness of melanophores of the medaka fish (wild type, Oryzias latipes) to a neurotransmitter and hormones is changed differentially after long-term adaptation to a black or white background. In the present study, we further examined whether this phenomenon involved some change in the intracellular signaling system. Using a permeabilized melanophore model, in which pigment granules could be dispersed by exogenously applied cAMP, the requirement of cAMP for pigment-dispersing reaction was revealed to be higher in melanophores of fish adapted to a black background (B cells) than in those of white background-adapted fish (W cells). Specific inhibitors of cAMP-dependent protein kinase (PKA) and cyclic nucleotide phosphodiesterase did not reduce the difference in the pigment dispersion level between B and W cells. A similar result was obtained with the free catalytic subunit of PKA. In contrast, the inhibition of protein phosphatase activity by okadaic acid diminished the difference in the responsiveness between B and W cells. These results suggest that the activity of protein phosphatase in B cell is higher than that in W cells, and that the change in the melanophore responsiveness by long-term chromatic adaptation to a background involves the change in the enzyme activity in the intracellular signaling system.
1-Methyl-3-isobutylxanthine↗
Molecular cloning and characterization of a cDNA encoding alpha-glucosidase from spinach.
A cDNA encoding spinach alpha-glucosidase was cloned and sequenced by the reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA comprised 2867 bp, and included an open reading frame which encodes a polypeptide of 903 amino acid residues. The calculated molecular mass of 101 kDa was larger than those of native alpha-glucosidases in spinach seeds, which are 78, 78, 82, and 82 kDa by SDS-PAGE for alpha-glucosidase I, II, III, and IV, respectively. The deduced amino acid sequence included those of tryptic peptides from native enzymes. Southern blot analysis suggested that the alpha-glucosidase gene was a single-copy gene. These results indicate the possibility that the multiplicity of alpha-glucosidase in spinach occurs via post-translational modification.
Amino Acid Sequence↗
Immortalization of immunologically committed Epstein-Barr virus-transformed human B-lymphoblastoid cell lines accompanied by a strong telomerase activity.
The immunological characteristics and immortalization processes of three EBV-transformed human B-lymphoblastoid cell lines, N0003, N0005 and N6803, with strong telomerase and infinitively proliferating activities are described. The three cell lines were apparently immortalized: they developed a strong telomerase activity at the population doubling levels (PDLs) between 11 and 135, and continued proliferation over 250 PDLs. All the cell lines expressed CD22, CD19 and CD20 antigens. They were uniformly stained with IgM (N0005), IgG (N6803) or IgA (N0003) at early PDLs between 17 and 20, and they secreted the corresponding class of Ig into the medium; the N6803 and N0003 cell lines continued to secrete each class of Ig at decreased levels while the N0005 cell line expressed or secreted virtually no Ig after immortalization. Karyotype analysis of the immortalized cell lines showed that they were derived from a single cell because they shared a set of abnormal chromosomes within each cell population, and two of the cell lines attained clonal characteristics before they developed a strong telomerase activity. These results indicate that the three immortalized cell lines with a strong telomerase activity correspond to the intermediate stages of B-cell differentiation naturally committed to a specific Ig class, and suggest that they were derived from a B-lymphoblastoid cell committed to a specific class of Ig with poor telomerase activity, rather than from a strongly telomerase-positive B-lymphoblastoid cell either committed or multipotential.
B-Lymphocytes↗
Tension pneumopericardium following tracheoplasty for congenital tracheal stenosis.
Tension pneumopericardium is a rare complication of mechanical ventilation following tracheoplasty for congenital tracheal stenosis. This case report describes fatal tension pneumopericardium in a three-month-old male infant some 24 hours following tracheoplasty for this condition. Because of persisting stenosis of the left main bronchus, ventilation pressures of PIP 23 cm H2O and PEEP 5 cm, progressing to PIP 28 cm and PEEP 7 cm were needed to maintain adequate respiratory exchange. Partial relief of the pneumopericardium was achieved by vigorous cardiac massage, but three days later the infant died of massive airway haemorrhage.
Acidosis, Respiratory↗
Putative phospholipid hydroperoxide glutathione peroxidase gene from Arabidopsis thaliana induced by oxidative stress.
An Arabidopsis cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase (PHGPX) was cloned and sequenced. The cDNA comprised 803 bp and included an open reading frame which encodes a polypeptide of 169 amino acid residues. The deduced amino acid sequence showed about 80 and 50% homology with plant putative PHGPXs and mammalian PHGPXs, respectively. Southern blot analysis suggested that putative PHGPX gene was a single-copy gene. The expression profile of the putative PHGPX in Arabidopsis under NaCl and Al/Fe treatments, which generate oxidative stress, was analyzed. Northern blot analysis revealed that the Arabidopsis putative PHGPX mRNA levels were increased about 3 and 4.5 times after exposure to NaCl and Al/Fe, respectively. These results suggest that the putative PHGPX gene is induced by oxidative stress in Arabidopsis.
Aluminum↗
Molecular cloning and characterization of a cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase from spinach.
A cDNA encoding spinach putative phospholipid hydroperoxide glutathione peroxidase (PHGPX) was cloned and sequenced. The cDNA included an open reading frame that encoded a polypeptide of 171 amino acid residues. The deduced amino acid sequence showed about 77 and 50% similarity to plant putative PHGPXs and mammalian PHGPXs, respectively. PCR product with the same size as that of the spinach putative PHGPX were obtained from maize, soybeans, and Arabidopsis, suggesting the expression of putative PHGPX genes in other plants.
Amino Acid Sequence↗
Sequence analysis of functional regions of homoserine dehydrogenase genes from L-lysine and L-threonine-producing mutants of Brevibacterium lactofermentum.
The homoserine dehydrogenase (HD) genes from Brevibacterium lactofermentum lysine- and threonine-producing mutants were cloned, using the polymerase chain reaction, and sequenced. We found the amino acid substitutions, Val104Ile in the lysine-producing mutants in which HD may cause leaky mutation and Ser393Phe in the threonine-producing mutant with feedback-insensitive HD.
Amino Acid Sequence↗
Monoclonal antibodies against pig ovarian follicular granulosa cells induce apoptotic cell death in cultured granulosa cells.
Two monoclonal antibodies capable of inducing granulosa cell apoptosis were produced against granulosa cells prepared from antral follicles of pig ovaries. The healthy follicles, 4-5 mm in diameter, were dissected from the ovaries of gilts, and then granulosa cells were isolated. BALB/c female mice were immunized with the isolated granulosa cells. Antibodies against the granulosa cells were detected by immunofluorescent staining using frozen ovarian sections. The isolated spleen cells prepared from immunized mice producing antibodies against the granulosa cells were fused with Sp2/O-Ag 14 mouse myeloma cells by standard hybridization techniques. Two hybridoma clones, PFG-1 and PFG-2, which produced specific IgM antibodies against granulosa cells were selected. Western blotting analysis revealed that PFG-1 and PFG-2 antibodies specifically recognized cell-membrane proteins with molecular weights of 55 and 70 kD and isoelectric points of 5.9 and 5.4, respectively. The monoclonal antibodies immunohistochemically reacted with granulosa cells of healthy follicles. When the isolated granulosa cells prepared from healthy follicles were cultured in medium containing 0.1 or 10 micrograms/m/PFG-1 or PFG-2 antibodies, respectively, the cells underwent apoptosis as determined by nuclear morphology, DNA electrophoresis and flow cytometric analysis. In conclusion, these two monoclonal antibodies against granulosa cells have cell-killing activity in cultured granulosa cells.
Animals↗