Search PubMed⌕ Search

Biomedical subjects

A Shirahata

Publications and source records attributed to A Shirahata.

At least 55 records · Page 3Linked to original sources
PubMed1999

Selectivity of spermine homologs on triplex DNA stabilization.

We synthesized seven homologs of spermine (H2N(CH2)3NH(CH2)nNH(CH2)3NH2, where n = 2-9; n = 4 for spermine) and studied their effects on melting temperature (Tm), conformation, and precipitation of poly(dA).2poly(dT). The triplex DNA melting temperature, Tm1 was 34.4 degrees C in the presence of 150 mM KCl. Addition of spermine homologs increased Tm1 in a concentration-dependent and structure-dependent manner, with 3-6-3 (n = 6) exerting optimal stabilization. The dTm1/dlog[polyamine] values were 9-24 for these compounds. The duplex melting temperature, Tm2 was insensitive to homolog concentration and structure, suggesting their ability to stabilize triplex DNA without altering the stability of the underlying duplex. Circular dichroism spectral studies revealed psi-DNA formation in a concentration-dependent and structure-dependent manner. Phase diagrams were constructed showing the critical ionic/polyamine concentrations stabilizing different structures. These compounds also exerted structural specificity effects on precipitating triplex DNA. These data provide new insights into the ionic/structural determinants affecting triplex DNA stability and indicate that 3-6-3 is an excellent ligand to stabilize poly(dA).2poly(dT) triplex DNA under physiologic ionic conditions for antigene therapeutics.

Circular Dichroism↗
PubMed1999

Primary structure of rat spermidine synthase: an example of refining the cDNA-derived amino acid sequence.

The primary structure of rat spermidine synthase having the N-terminal acetylated methionine and 98.7% homology with that of the mouse enzyme is presented using a limited amount of the homogeneous enzyme. The study strategy was principally to compare the molecular masses of liberated peptides determined by three specific cleavage methods with those expected from known cDNA-derived amino acid sequences of mouse and human enzymes using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS). The cleavage methods involved two enzymatic methods using lysylendopeptidase and arginylendopeptidase, and a chemical method for cleaving at the cysteine residue using 2-nitro-5-thiocyanobenzoic acid. Their usefulness was clearly demonstrated. Column-switching semimicro reversed-phase HPLC, which permits application of the entire reaction mixture, was useful for collecting a small amount of peptides containing the N-terminal amino acid, to confirm acetylation of the N-terminal methionine by MALDI TOF-MS. It was necessary in this approach to examine the amino acid sequence of certain peptides. The Edman method was used for the sequence analysis, and this will be replaced by an improved MALDI TOF-MS now available in a few laboratories.

Acetylation↗
PubMed1999

[Causes of death (1983-1997) in patients infected with human immunodeficiency virus by contaminated blood coagulation factor products: report of the clinical study group on prevention and treatments of HIV infection].

Our group conducted a national survey of patients who had died of acquired immunodeficiency syndrome (AIDS) after being infected with human immunodeficiency virus type 1 (HIV-1) by contaminated blood coagulation factor products. A total of 1446 hospitals, clinics, and other health-care institutions throughout Japan were enrolled in the study, which was supported by the Japanese government Ministry of Health and Welfare with a health sciences grant for research on HIV/AIDS. Of the 1434 registered patients who had been infected with HIV-1 by contaminated blood coagulation factor products, 493 (34%) had died as of Oct. 30, 1997. Most were hemophiliacs. The average CD4+ cell count was 25/microliter for the 398 who died of complications from AIDS, compared to an average of 158/microliter for those who died of other causes. Pneumocystis carinii pneumonia, cytomegalovirus infection, and atypical mycobacterial disease were the main causes of death in patients with AIDS. The annual death rate for AIDS patients in Japan dropped dramatically in 1997, 1 year later than a similar trend observed in the United States. The introduction and widespread adoption of new and effective drugs and treatment regimens for HIV-1 and opportunistic infections will be essential life-saving measures.

Acquired Immunodeficiency Syndrome↗
PubMed1999

[Hepatobiliary and pancreatic disorders as risk factors for fat-soluble vitamin deficiencies].

The aim of this article is to describe the fat-soluble vitamin status in patients with hepatobiliary and pancreatic diseases, and the contribution of these vitamin deficiency or excess to hepatic injury. A considerable number of patients with advanced liver disease and cholestasis might actually be fat-soluble vitamin deficient, although clinical signs of deficiency are uncommonly seen in patients with vitamin A and E deficiency. Increased bone resorption may be the predominant cause of hepatic osteodystrophy. On the other hand, the possible causes of vitamin K deficiency seen in patients with hepatobiliary disease are the decrease of vitamin K absorption from intestine, the disturbance of vitamin K cycle and the decrease of pool area for vitamin K storage. The intake of vitamin A may be associated with the risk of liver cirrhosis in lifetime teetotalers, although the retinyl palmitate reduces hepatic fibrosis in rats.

Avitaminosis↗
PubMed1999

Hemostatic alterations in beta-thalassemia/hemoglobin E patients.

To search for evidence of coagulation activation ex vivo, the levels of human prothrombin fragment 1+2 (F1+2) were examined in 69 beta-thalassemia/Hb E patients. Levels of protein C inhibitor (PCI) and activated protein C - PCI (APC:PCI) complex were also determined in 9 of the above patients in conjunction with protein C (PC) antigen and activity, in an attempt to detect increased consumption of PC. In mean level of F1+2, there was a statistically significant difference between normal control and post-splenectomized patients (p < 0.05) but not between normal control and non-splenectomized patients (p > 0.05). The mean levels of PC activity and PC antigen in the patients were much lower than in normal controls. However, the mean levels of PCI and the mean level of APC:PCI complex in the patients were not significantly different from those in normal controls (p > 0.05). The high level of F1+2 in post-splenectomized patients found in this study agreed well with clinical and other laboratory findings. The normal level of PC inhibitor and APC:PCI complex found in this study provided no evidence of increased consumption of protein C in thalassemia patients.

Adult↗
PubMed1998

Ionspray ionization-mass spectrometric separation and determination of heptafluorobutyryl derivatives of polyamines.

We describe here an application of ionspray ionization-mass spectrometry (IS-MS) for simultaneous determination of putrescine, spermidine, and spermine without the need for coupling with other separation techniques such as high-performance liquid chromatography or capillary electrophoresis. Because it was essential to increase the mass of these compounds to eliminate contaminated low-mass ions, heptafluorobutyryl (HFB) derivatives were chosen. It was found that each HFB-polyamine appeared as a single major cation carrying an ammonium ion in a solution of 50% acetonitrile-water containing 0.5% ammonium acetate at an orifice potential of 20 V. Under these conditions, the three polyamines after addition of 15N-labeled polyamines as internal standards, followed by heptafluorobutylation, were determined simultaneously. It was shown, using a sample injection bulb set between the syringe drive and ionspray probe, that the analysis time for data acquisition was about 3 min and the sensitivity was about 10 fmol. In addition, polyamines in rat liver were determined by this method after simple fractionation with a small column of CM-cellulose with reasonable accuracy and precision. Behaviors of acetylated polyamines were also described, and the results showed that the acetylated polyamines would become additional target compounds in the present method.

Animals↗
PubMed1998

Genetic analysis of protein C deficiency in nineteen Japanese families: five recurrent defects can explain half of the deficiencies.

We have been studying the molecular basis of protein C deficiency. In this study, we determined the molecular defects of protein C deficiency in 19 Japanese families by using a strategy combining polymerase chain reaction (PCR) and single-strand conformational polymorphism (SSCP) analysis. We identified 10 missense mutations, 1 in-frame deletion, 1 frameshift deletion, 1 frameshift addition, and 1 splice site mutation, 5 of which were novel. From the results of genetic analysis of 67 Japanese families with protein C deficiency reported in this and previous studies, the recurrent defects including Phe139Val and Met365Ile substitutions and a Lys150 d letion, a G8857 deletion, and a splice site mutation of G3079A were only found in Japanese subjects and seemed to be a founder effect. In contrast, Arg169Trp, Arg286His, Val297Met, and Asp359Asn substitutions, all occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These 9 recurrent molecular defects were found in 43 families in total, accounting 64% of Japanese families with protein C deficiency. In particular, the recurrent defects of Phe139Val, Arg169Trp, Va1297Met, and Met36-4Ile substitutions and a G8857 deletion were found in 33 families in total, accounting for 49% of Japanese families with protein C deficiency. For the identification of the genetic defect in Japanese patients with protein C deficiency, screening of these recurrent defects by using restriction enzyme cleavage is a rational method.

Exons↗
PubMed1998

Structure and activity of mouse S-adenosylmethionine decarboxylase gene promoters and properties of the encoded proteins.

The promoter regions of two S-adenosylmethionine decarboxylase genes (AMD genes) were isolated from a mouse genomic library. One promoter was that of the bona fide mouse AMD gene (AMD1) whereas the other was that of the intronless AMD gene (AMD2). There was no sequence identity between the two promoters. The sequence of the AMD1 promoter was highly homologous to the human AMD1 and rat Amd1B promoters. After transient transfection in various cell lines, the AMD1 promoter was one to two orders of magnitude stronger than the AMD2 promoter. Similar results were obtained by using stably transfected mouse FM3A cells. In S-adenosylmethionine decarboxylase (AdoMetDC)-overproducing SAM-1 cells, the AMD1 gene was amplified over 5-fold. AdoMetDC encoded by the intronless AMD2 gene had two amino acid replacements (Met to Ile at codon 70 and Ala to Val at codon 139), compared with the protein encoded by the AMD1 gene, and exhibited decreased catalytic activity (<50%) and decreased processing activity when expressed in AdoMetDC-deficient Escherichia coli cells. When Ile-70 of the protein encoded by AMD2 was converted into Met, both the catalytic and processing activities recovered markedly, indicating that Met-70 adjacent to the proenzyme-processing site is important for both activities. The third AMD locus (AMD3) in FM3A cells contains a pseudogene, in which deletion of two bases generates a premature termination codon at position 57. Since the AMD2 promoter had only 1-10% of the strength of the bona fide AMD1 gene and AMD2 protein possessed lower specific activity, the relative contribution of the AMD2-encoded enzyme to total AdoMetDC activity is small. Thus AdoMetDC activity in murine cells is thought to be due mainly to the product of the AMD1 gene.

Adenosylmethionine Decarboxylase↗
PubMed1998

Natural course of HGV infection in haemophiliacs.

The natural course of hepatitis G virus (HGV) infection was clarified in 70 haemophiliacs by testing for HGV RNA and antibodies against HGV envelope protein (anti-E2). None of 12 patients treated with only virus-inactivated coagulation factors were infected with HGV. Of 58 patients who received non-inactivated products, 28 (48%) were positive for HGV RNA and/or anti-E2. Of 16 patients with anti-E2, 14 were negative for the viral RNA, and had recovered from HGV infections. HCV antibodies were detected in 59 patients, and eight patients were successively negative for HCV RNA. Thus, the recovery rate of HGV infection (14/28, 50%) was higher than that of HCV (8/59, 14%) (P<0.001). Longitudinal study revealed that anti-E2 developed either during viraemia or some years after seronegativity for HGV RNA. Hence the antibody response itself seemed not to play a major role in the clearance of HGV though anti-E2 was associated with the clearance of HGV RNA. In conclusion, HGV and HCV are prevalent in patients treated with unsterilized coagulation factor concentrates. However, in contrast to HCV, spontaneous recovery is frequently observed in HGV infections.

Adolescent↗
PubMed1998

Diagnosis of DIC in very low birth weight infants.

Disseminated intravascular coagulation (DIC) occurs most frequently during the neonatal period. We have already established criteria for the early diagnosis of DIC in newborn infants, based on the analysis of clinical and laboratory findings in neonates with and without DIC. In that study, laboratory findings for subjects without DIC were evaluated based on the results obtained from neonates whose birth weights were more than 1500 g. Accordingly, these criteria are not necessarily applicable to the diagnosis of DIC in very low birth weight infants (VLBWI) since the physiological and pathological states of coagulation and fibrinolysis in VLBWI may differ from those of term newborn infants. Therefore, we measured platelet counts, plasma fibrinogen contents and the fibrin degradation product (D-dimer) concentrations in VLBWI with and without DIC. These findings indicate that the scoring system for platelet counts in our diagnostic criteria of DIC in newborn infants is applicable to the diagnosis of DIC in VLBWI. However, our scoring systems for fibrinogen and D-dimer were not usable for the diagnostic criteria of DIC in VLBWI since fibrinogen and D-dimer concentrations in VLBWI without DIC were lower than those in non-DIC neonates whose birth weights were above 1500 g. We devised criteria for the diagnosis of DIC in VLBWI based on those findings.

Biomarkers↗
PubMed1998

Effects of YM175, a new-generation bisphosphonate, on hypercalcemia induced by tumor-derived bone resorbing factors in rats.

YM175 (disodium cycloheptylaminomethylenediphosphonate monohydrate) is a new-generation bisphosphonate with stronger inhibitory activity on bone resorption than first-generation bisphosphonates. In the present study, the effect of YM175 on hypercalcemia induced in rats by single administration of either parathyroid hormone-related protein (PTHrP) or concomitant administration of PTHrP and interleukin 1beta (IL-1beta) was investigated. YM175 (0.01-1 mg/kg, i.v.) inhibited the increase in serum free calcium concentration induced by continuous administration of PTHrP alone (3 microg/rat/day, s.c., 7 days) dose-dependently. The inhibitory effect of YM175 appeared the day after administration and remained 3 days after administration. The effect of YM175 reached a maximum 2 days after administration, at which time the ED50 value of YM175 was calculated to be 0.041 mg/kg, i.v., revealing a potency approximately 50- and 10-fold stronger than those of either pamidronate or alendronate, respectively. In contrast, elcatonin (1-10 units/kg, s.c.) only transiently inhibited PTHrP-induced free calcium increase. YM175 (0.1-3 mg/kg, i.v.) also inhibited the increase in the serum free calcium concentration induced by continuous concomitant administration of both PTHrP and IL-1beta in a dose-dependent manner. These results indicated that YM175 is expected to be a useful drug for hypercalcemia associated with malignant tumors due to its efficacy and range of effect.

Animals↗
PubMed1998

[Prospective matched control study concerning the treatment and quality of life of hemophiliacs with inhibitors].

Factor VIII (IX) inhibitors represent one of the most serious problems for the treatment of patients with hemophilia. The Blood Products Research Organization (Japan) has supported a study group for treatment of hemophiliacs with inhibitors. In 1995 the study group started a prospective matched control study of hemophiliacs with and without inhibitors and compared such factors as quality of life and economic cost. Each inhibitor patient was matched with a control patient in terms of age, type of hemophilia, and severity of hemophilia. A total of 136 patient-pairs were enrolled. Bleeding episodes were more frequent in the control group than in the inhibitor group. Days of hospitalization, days in wheelchairs, and the number of impaired joints were significantly higher for the inhibitor group. Number of blood-product infusions, days of bed rest at home, and days of brace use were the same for both groups. Blood-product expenditures were significantly higher for the inhibitor group than for the control group (yen 10,872,283/patient/year vs. yen 4,327,542/patient/year). Our study highlighted the higher cost of treatment and lower quality of life for hemophiliaes with Factor VII inhibitors.

Adult↗