Biomedical subjects

T Ihara

Publications and source records attributed to T Ihara.

At least 73 records · Page 4Linked to original sources

PubMed1997

Affinity capillary electrophoresis using DNA conjugates.

An oligonucleotide ((dT)12)-immobilized capillary was prepared for the affinity capillary electrophoresis of DNA. Oligonucleotides ((dA)6 and (dT)6) which had the same chain length but had different base composition were separated in high resolution in the presence of Mg2+ ion. Whereas (dA)6 eluted slightly earlier than (dT)6 in a conventional polyacrylamide-coated capillary, the order of their mobilities was reversed in the (dT)12-modified system, suggesting the complementary hydrogen bondings between (dA)6 and the immobilized (dT)12.

Base Composition↗

PubMed1996

Ferrocene-oligonucleotide conjugates for electrochemical probing of DNA.

Toward the development of a universal, sensitive and convenient method of DNA (or RNA) detection, electrochemically active oligonucleotides were prepared by covalent linkage of a ferrocenyl group to the 5'-aminohexyl-terminated synthetic oligonucleotides. Using these electrochemically active probes, we have been able to demonstrate the detection of DNA and RNA at femtomole levels by HPLC equipped with an ordinary electrochemical detector (ECD) [Takenaka,S., Uto,Y., Kondo,H., Ihara,T. and Takagi,M. (1994) Anal. Biochem., 218, 436-443]. Thermodynamic and electrochemical studies of the interaction between the probes and the targets are presented here. The thermodynamics obtained revealed that the conjugation stabilizes the triple-helix complexes by 2-3 kcal mol-1 (1-2 orders increment in binding constant) at 298 K, which corresponds to the effect of elongation of additional several base triplets. The main cause of this thermodynamic stabilization by the conjugation is likely to be the overall conformational change of whole structure of the conjugate rather than the additional local interaction. The redox potential of the probe was independent of the target structure, which is either single- or double stranded. However, the potential is slightly dependent (with a 10-30 mV negative shift on complexation) on the extra sequence in the target, probably because the individual sequence is capable of contacting or interacting with the ferrocenyl group in a slightly different way from each other. This small potential shift itself, however, does not cause any inconvenience on practical applications in detecting the probes by using ECD. These results lead to the conclusion that the redox-active probes are very useful for the microanalysis of nucleic acids due to the stability of the complexes, high detection sensitivity and wide applicability to the target structures (DNA and RNA; single- and double strands) and the sequences.

DNA↗

PubMed1996

Effectiveness of combined anticoagulant therapy for extending portal vein thrombosis in Crohn's disease. Report of a case.

PURPOSE: Portal vein thrombosis is a rare complication of Crohn's disease, and its precise cause and appropriate treatment are not known. We describe a patient with extending portal vein thrombosis in Crohn's disease who was successfully treated with combined anticoagulant therapy. METHOD: Urokinase and tissue plasminogen activator were administered from a catheter inserted into the superior mesenteric artery, and heparin and a serine protease inhibitor also were given intravenously. RESULTS: On admission, thromboembolic occlusion was observed throughout the entire portal venous system in association with massive ascites and remarkable intestinal edema. After administration of combined anticoagulant therapy, thrombus rapidly decreased in size, and color Doppler ultrasonography showed a gradual increase in portal venous flow. The patient had no recurrence of symptoms while receiving warfarin after resolution of thrombus. CONCLUSION: This case report suggests that combined anticoagulant therapy is effective for patients with severe portal vein thrombosis in Crohn's disease and that color Doppler ultrasonography is useful for evaluation of portal venous flow.

Adult↗

PubMed1996

Detection by polymerase chain reaction of wild-type measles virus genome in the cerebrospinal fluid of a patient with SSPE who had received measles vaccine.

BACKGROUND: Previous studies have reported that approximately 4-5% of patients with subacute sclerosing panencephalitis (SSPE) were given measles vaccination but had no history of natural measles. However, in the case who received measles vaccine, it has been extremely difficult to determine whether the actual cause of SSPE is the inoculated vaccine virus or not. OBJECTIVES: To detect the measles virus genome in a patient with SSPE and to analyze its nucleotide and deduced amino acid sequence. STUDY DESIGN: We applied the polymerase chain reaction (PCR) to detect the measles virus genome in specimens from a 12-year-old boy with SSPE who had received measles vaccine 10 years before and had no history of apparent natural measles. The oligonucleotide primers for PCR were prepared based on the nucleotide sequence of the F and NP genes of the measles virus Edmonston strain. RESULTS: F and NP genes were detected in both the cerebrospinal fluid and peripheral blood lymphocytes. Nucleotide and deduced amino acid sequence analysis of the F gene showed that the patient's virus was different from that of the vaccine strain. Judging from these results, it was likely that the SSPE-associated strain in this case was derived from the wild-type rather than the vaccine strain. CONCLUSIONS: PCR is a useful method to establish a definitive diagnosis of SSPE and to study the nature of the SSPE-associated virus.

Journal Article↗

PubMed1996

Feedback signal from medial temporal lobe mediates visual associative mnemonic codes of inferotemporal neurons.

Functional roles of the cortical backward signal in long-term memory formation were studied in monkeys performing a visual pair-association task. Before learning of the task, the anterior commissure of the monkeys was transected, disconnecting the anterior temporal cortex of each hemisphere. After training with 12 pairs of pictures, we injected a grid of ibotenic acid unilaterally into the entorhinal and perirhinal cortex that provide massive backward projections ipsilaterally to the inferotemporal cortex. According to the histological examination, the lesions covered medial and lateral banks of the rhinal sulcus completely and most the entorhinal and perirhinal cortex. After the injections, the monkeys fixated the cue stimulus normally, relearned the preoperatively learned set (set-A) and learned a new set (set-B) of paired associates. Then single units were recorded from the same area as that for the prelesion control. We found that (i) in spite of the lesion, the sampled neurons responded strongly and selectively to both the set-A and set-B patterns, and that (ii) the paired associates elicited significantly correlated responses in the control neurons but not in the cells tested after the lesion either for set-A or set-B stimuli. We conclude that the ability of inferotemporal neurons to represent association between picture pairs was lost after disruption of backward neural signals from the limbic cortex to the inferotemporal neurons, while the ability of the neurons to respond to a particular visual stimulus was left intact.

Analysis of Variance↗

PubMed1996

Clinical features of measles in immunocompromised children.

Measles is often fatal for immunocompromised hosts. Protective immunity against measles has been studied but is still not completely understood. Recently, five cases of measles were encountered in immunocompromised children. Two of these were allogeneic bone marrow transplanted cases (one common variable immunodeficiency and one severe aplastic anemia) in remission, one Wilms' tumor case in remission, one hepatoblastoma case after cytotoxic therapy at disease onset and one exaggerating hemophagocytic syndrome case with suppressed natural killer cell activity. Clinical symptoms, laboratory findings and the immunologic backgrounds of these five patients were investigated. One of the patients, an 8 year old boy with hemophagocytic syndrome, died of giant cell pneumonia which was confirmed in the section of necropsy lung specimen. Two other patients who received allogeneic bone marrow transplants were not immune to measles, despite their own and their donors' immunizations. Their clinical symptoms were rather severe but both patients recovered and have remained seropositive for as long as 13 months. This fatality from measles is the first reported in a patient with hemophagocytic syndrome. Suppressed natural killer cell activity may be a poor prognostic factor. Also, secondary immunization failure for measles can occur in bone marrow transplanted patients with rather severe clinical symptoms.

Adult↗

PubMed1996

Detection of measles virus-induced apoptosis of human monocytic cell line (THP-1) by DNA fragmentation ELISA.

Measles virus (wild strain, Toyoshima strain)-induced cell death is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation in a human monocytic cell line (THP-1). DNA fragmentation of measles virus-infected THP-1 cells was demonstrated by DNA agarose gel electrophoresis as well as by DNA fragmentation ELISA. When measles virus-infected THP-1 cells were cultured on monolayers of fibroblasts or human umbilical vein endothelial cells (HUVEC), the percentage of measles virus antigen-positive THP-1 cells and DNA fragmentation were significantly decreased. Addition of anti-intercellular adhesion molecule (ICAM)-1 (CD54) monoclonal antibody to culture of measles virus-infected THP-1 cells reduced significantly DNA fragmentation induced by measles virus. These findings suggest that inhibition of virus spread by fibroblasts and HUVEC reduces apoptosis, and ICAM-1 (CD54) may participate in the DNA fragmentation pathway.

Antibodies, Monoclonal↗

PubMed1996

[Single and 4-week oral toxicity studies of prulifloxacin (NM441) in aged dogs].

Single-dose and repeated dose toxicity studies of prulifloxacin, a new antibacterial agent, were conducted in aged beagle dogs. I. A single-dose toxicity study Prulifloxacin was administered orally to aged female dogs at a single dose of 2500 and 5000 mg/kg. No death occurred in any group. Vomiting was observed in one of two animals at 2500 mg/kg and in both animals at 5000 mg/kg 3-4 hr after dosing. At 5000 mg/kg, vomiting was observed in both animals after feeding on the day after dosing. One animal also showed soft stool. Thereafter, no abnormalities were observed in any animal. No test article related changes were noted in food consumption, water consumption, body weight or pathological examination in any group. The results show that the lethal dose of prulifloxacin is judged to be greater than 5000 mg/kg in aged female dogs. II. A repeated dose toxicity study Aged male and female dogs were given the test article orally for 4 weeks at doses of 0 (control), 20, 100 and 500 mg/kg. No death occurred in any group. At 500 mg/kg, vomiting was observed every day or intermittently throughout the dosing period and salivation was observed almost every day from day 6 to the end of the dosing onward. Decreases or lack of food and water consumption, and decrease of body weight were noted at 500 mg/kg. At 100 mg/kg, slight decreases in food consumption and body weight were noted in the females. No abnormalities were noted in ophthalmoscopic or electrocardio-graphic examination. In urinalysis, decreases in Na+, K+ and Cl- concentrations and the total excretion amount were noted mostly at 500 mg/kg. A low specific gravity was noted in males at 500 mg/kg. In hematology and serum biochemistry, high GPT, BUN and creatinine, and decreases in WBC were noted in both sexes at 500 mg/kg. A high GOT was noted in males, and low Cl- in females at 500 mg/kg. At 100 mg/kg, a high GPT was noted. Rough surface in the kidney and chronic interstitial nephritis (basophilic change of tubule, atrophy of tubule, thickening of tubular basement membrane, interstitial mononuclear cell infiltration, interstitial focal fibrosis) were increased at 500 mg/kg. No toxicological findings were seen in the 20 mg/kg group. The results show that the NOAEL of prulifloxacin is 20 mg/kg for 4-week repeated dose toxicity in aged dogs.

Administration, Oral↗

PubMed1996

Severe anemia in a patient with isolated adrenocorticotropin deficiency.

A 64-year-old man was referred to our hospital for evaluation of progressive anemia. On admission, he had a severe normocytic normochromic anemia [hemoglobin 7.5 g/dl] requiring a blood transfusion. Endocrinological studies demonstrated an isolated ACTH deficiency. After receiving glucocorticoid replacement therapy, his anemia was rapidly corrected, his hematocrit and hemoglobin remained elevated for approximately 4 months. We present evidence that glucocorticoid plays an important role in the physiological regulation of human erythropoiesis.

Adrenocorticotropic Hormone↗

PubMed1995

Nitroglycerin inhibits the phosphorylation of intermediate filament proteins rather than myosin light chain on porcine coronary artery sustained contraction.

The smooth muscle relaxation induced by nitroglycerin is hypothesized to be mediated by an increase in the cytoplasmic concentration of guanosine 3',5'-monophosphate (cGMP) and subsequent dephosphorylation of the 20-kilodalton myosin light chain (MLC). We investigated this hypothesis in procine coronary arterial smooth muscle stimulated with histamine (3 microM) or K+ (30 mM). Stimulation of [32P]Pi-labeled muscle with histamine or K+ for 2 min resulted in a four- or 6.2-fold increase, respectively, in the incorporation of 32P into MLC. After 48 min of exposure to histamine, MLC phosphorylation decreased to the basal level and the phosphorylation of desmin, synemin, and of three unidentified cytosolic proteins was increased. K+ stimulation resulted in a sustained increase of MLC phosphorylation but had no effect on the phosphorylation of desmin, synemin, or the three unidentified cytosolic proteins. Application of nitroglycerin (1 microM) 48 min after histamine stimulation inhibited the phosphorylation of desmin, synemin, and the three cytosolic proteins. The sustained phase of histamine-induced contraction was also inhibited to a greater extent then the acute phase of histamine-induced contraction and both the acute and sustained phases of K(+)-induced contraction. These results suggest that MLC phosphorylation is required for both phases of K(+)-induced contraction, whereas phosphorylation of intermediate filament proteins is required for the sustained phase of histamine-induced contraction. Intermediate filament proteins, rather than MLC, may also be the target for the relaxant action of nitroglycerin during histamine-induced sustained contraction.

Animals↗

PubMed1995

Induction of apoptosis by T-2 toxin and other natural toxins in HL-60 human promyelotic leukemia cells.

Based on the DNA fragmentation profile in gel electrophoresis and the morphological changes in electron microscopy, the induction of apoptotic nuclear changes by mycotoxins and other microbial products, in total 31 chemicals, was investigated in HL-60 human promyelotic leukemia cells, along with the cytotoxicity tests with 3-[4,5-dimethylthiazol-zyl]-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion. Among the chemicals tested, trichothecenes (T-2 toxin, roridin A, nivalenol, deoxynivalenol), certain anthraquinones (luteoskyrin, skyrin, 2-hydroxyemodin), diketopiperazines (emethallicin A, emestrin), isocoumarins (ochratoxin A, citrinin), lactone (penicillic acid), dihydrobisfuran (aflatoxin B1), potassium ionophore (valinomycin), and an inhibitor of interleukin-2 synthesis (cyclosporin A) were positive for the induction of DNA fragmentation. No DNA fragmentation was observed under the present conditions with fumonisin B1, cyclic peptides (cyclochlorotine, phalloidin, microcystin-LR), certain anthraquinones (emodin, chrysophanol, rugulosin), and others (sterigmatocystin, cytochalasin A, griseofulvin, fusaric acid, kojic acid, rubratoxin B, butenolide, wortmannin, FK506, and sphingosine). The apoptotic changes in the cells exposed to T-2 toxin and luteoskyrin were confirmed by electron microscopic observation. Detailed experiments on dose and time dependencies revealed that T-2 toxin induced the apoptosis at 10 ng/ml (= 4 x 10(-8) M) levels within 2-6 hr without significant cytotoxicity evaluated by the dye exclusion and MTT.

Anthraquinones↗

PubMed1995

Fas antigen and bcl-2 expression on lymphocytes cultured with cytomegalovirus and varicella-zoster virus antigen.

Fas antigen and bcl-2 expression on peripheral blood mononuclear cells (PBMC) cultured with cytomegalovirus (CMV) and varicella-zoster virus (VZV) antigen was analyzed by three-color flow cytometry. Expression of Fas antigen increased significantly in CD45RO+ populations of both CD4+ and CD8+ cells obtained from CMV and VZV seropositive donors after culture with CMV and VZV antigen for 6 days. Fas antigen expression did not increase in PBMC cultured with control antigen. In contrast, bcl-2 expression decreased both in CD4+CD45RO+ and CD8+CD45RO+ cells from the same donors. Fas antigen and bcl-2 expression in CD45RA+ populations did not change. Cell viability of cultured cells with CMV and VZV antigen decreased after treatment with anti-Fas antibody and DNA fragments indicating apoptosis were detected in the cell lysate of these cultured cells after treatment with anti-Fas antibody. These data suggest that Fas antigen and bcl-2 protein may interact in regulating the cell death process of activated memory lymphocytes and eliminating lymphocytes activated by viral infection.

Adult↗

PubMed1995

Characterization of pseudorabies virus glycoprotein gII expressed by recombinant baculovirus.

The gene encoding the complete glycoprotein gII (homologue of gB of herpes simplex virus) of pseudorabies virus (PrV) was inserted into a baculovirus transfer vector, and a recombinant virus expressing gII was isolated. Three gII-related recombinant baculovirus-expressed peptides of 100, 60, and 45 to 50 kDa were detected with a polyclonal antibody against gII; these correspond to the authentic subunits gIIa and its cleavage products gIIb and gIIc, respectively. These proteins were subjected to N-terminal sequencing, and the results showed that the protease cleavage sites were identical to those of authentic gII. The expressed gII was shown to be transported to the surface of infected cells as judged by an indirect immunofluorescence test. Antibodies raised in mice immunized with the recombinant gII neutralized the infection of PrV in vitro. Mice inoculated with the recombinant gII were completely protected from lethal challenge with PrV.

Amino Acid Sequence↗

PubMed1995

Detection of human cytomegalovirus DNA in immunocompromised children by polymerase chain reaction.

BACKGROUND: Cytomegalovirus (CMV) infection is a major complication in immunocompromised patients, such as those with leukemia, acquired immunodeficiency syndrome (AIDS) and allograft recipients. A sensitive and specific diagnostic procedure of CMV infection is required. The polymerase chain reaction (PCR) procedure has been demonstrated to be more sensitive than conventional virus isolation. However, CMV DNA can be detected in patients with latent or asymptomatic infections by PCR because of its extreme sensitivity. OBJECTIVES: In this study, we used PCR and reverse transcription PCR (RT-PCR) to detect CMV DNA and mRNA in immunocompromised patients and evaluated the value of PCR and RT-PCR for diagnosis of active CMV infection. STUDY DESIGN: We examined thirty immunocompromised children for CMV DNA using peripheral blood mononuclear cells (PBMC), polymorphonuclear leukocytes (PMNL). urine and throat swabs from October 1991 to March 1992. Eighteen of the patients had acute lymphoblastic leukemia, four had non-Hodgkin's lymphoma, two had Wilms' tumor and six were recipients of an allogeneic bone marrow transplantation. Four patients developed CMV-related clinical symptoms during the observation period: two patients with pneumonia and two patients with hepatitis. RESULTS: CMV DNA was detected in four patients with clinical symptoms and in five patients without evidence of CMV infection. The incidence of CMV DNA was significantly higher in symptomatic patients than asymptomatic patients. CMV mRNA encoding the late antigen was detected by RT-PCR only in patients with clinical symptoms of CMV infection. CONCLUSIONS: We concluded that detection of CMV DNA by PCR is of little clinical significance for distinguishing latent form active CMV infection. It is suggested that detection of CMV mRNA by RT-PCR is useful for diagnosis of active CMV infection.

Journal Article↗

PubMed1995

Detection of varicella-zoster virus (VZV) DNA in throat swabs and peripheral blood mononuclear cells of immunocompromised patients with herpes zoster by polymerase chain reaction.

BACKGROUND: Varicella-zoster virus (VZV) is rarely isolated from throat swabs and peripheral blood leukocytes from patients with herpes zoster by conventional virus isolation methods. The polymerase chain reaction (PCR) is a highly sensitive method to detect VZV genomes. It has been reported that VZV DNA was detected in the cerebrospinal fluid (Puchhammerstockl et al., 1991) and peripheral blood mononuclear cells (PBMC) of patients with VZV-associated neurological symptoms (Gilden et al., 1992) by PCR. OBJECTIVES: We used the nested double PCR to detect VZV DNA in patients with herpes zoster. STUDY DESIGN: Sixteen patients with herpes zoster, ten immunocompromised and six immunocompetent patients, were studied. Throat swabs and PBMC were collected weekly and examined for VZV DNA by the nested double PCR. RESULTS: VZV DNA was detected in 60% (6/10) of throat swabs and in 60% (6/10) of PBMC of immunocompromised patients, and in 16.7% (1/6) of throat swabs and in 33% (2/6) of PBMC of immunocompetent patients within two weeks after the onset of skin rash. VZV DNA was detected in throat swabs or PBMC of two patients 5 and 7 days after cessation of acyclovir. CONCLUSION: VZV DNA was detected in throat swabs and PBMC-associated viremia exist in patients with herpes zoster. It is suggested that VZV spread from sensory ganglia to the skin or pharyngeal area along the nerve fiber or hematogenously and local cutaneous replication of VZV can lead to viremia with subsequent hematogenous dissemination in patients with herpes zoster.

Journal Article↗

PubMed1995

Markedly elevated levels of beta2-microglobulin in urine with measles viruria in patients with measles.

BACKGROUND: Measles is a highly contagious infectious disease in children. However, the pathogenesis has not yet been fully defined since susceptible cells to measles virus (MV) had not been developed. Recently, B95a cells, which are very susceptible to MV, have been reported. OBJECTIVES: To evaluate measles viruria in patients with measles, isolation of MV from urine was performed using B95a cells. STUDY DESIGN: Isolation of MV from supernates or sediments of urine in patients with measles was performed using B95a cells. The levels of beta2-microglobulin (MG) in urine and serum were also measured. RESULTS: MV was isolated from either supernates or sediments of urine. MV was isolated from 10 of 11 samples (91%) within 2 days of the appearance of a rash. MV was isolated from supernates of urine for up to 4 days after the appearance of a rash, and from sediments for up to 5 days. The levels of urinary beta2-MG were elevated within 2 days of the appearance of a rash. The levels of urinary beta2-MG with measles viruria were significantly higher compared to those without measles viruria. CONCLUSIONS: Measles viruria may occur early in all patients with measles and elevated levels of urinary beta2-MG in patients with measles may be the consequence of tubular injury.

Journal Article↗

PubMed1995

Contractile state is the major determinant of functional outcome in patients with left ventricular dysfunction treated with enalapril.

Large-scale drug trials have focused primarily on mortality and morbidity and less on the functional state of the myocardium. A model was developed to assess myocardial contractile state in patients with left ventricular (LV) dysfunction and to address the questions of differences in function between patients with and without overt heart failure, effects of enalapril, and best predictors of functional outcome. Pressure-angiographic data were obtained from 16 patients with overt heart failure and 47 without heart failure. Repeat studies were conducted in 41 patients following 1 year's treatment with enalapril or placebo. Left ventricular silhouettes were divided into 18 segments to estimate regional ejection fraction, wall stress and myocardial damage (% myocardial damage). Contractile state was assessed and ranked by ejection rate-preload-afterload relationships and by a score method based on 10 myocardial and ventricular function parameters. End-diastolic and end-systolic volumes (EDV, ESV) were significantly greater (P < 0.001), ejection fraction (EF) lower (P < 0.009), % myocardial damage greater (P < 0.008) and contractile state more depressed in patients with overt heart failure. Changes in EDV and ESV (delta placebo vs delta enalapril) were significant (delta EDV, P < 0.003; delta ESV, P < 0.014). Directional shifts in the diastolic pressure-volume relationships with enalapril or placebo depended primarily on the basal contractile state and diastolic volume range. The best single predictors of post-treatment EF were the score index (a surrogate parameter for the contractile state) and ESV. Added benefits of enalapril include the prevention of further dilatation in patients with less depressed contractile state and delay in the onset of heart failure. Asymptomatic patients with LV dysfunction should also be considered for angiotensin converting enzyme (ACE) inhibitor therapy.

Blood Pressure↗

PubMed1995

Increased expression of adhesion molecules (CD54, CD29 and CD44) on fibroblasts infected with cytomegalovirus.

The expression of ICAM-1 (CD54), beta 1 integrin (CD29), and CD44 on cytomegalovirus (CMV)-infected human embryonic fibroblasts (HEF) was analyzed by flow cytometry. The expression of these adhesion molecules increased significantly on CMV-infected HEF, on days 2 and 5 after inoculation, compared to uninfected HEF. However, the expression of these adhesion molecules decreased on herpes simplex virus (HSV)-1 and varicella-zoster virus (VZV)-infected HEF. Increased expression was not observed on HEF treated either with inactivated CMV or with supernatant fluid of CMV-infected cells. The addition of anti-cytokine (TNF-alpha, IL-1 beta, or IFN-gamma) antibodies had no effect on the increase of these adhesion molecules. This suggests that the increase in CD54, CD29, and CD44 on CMV-infected cells requires active virus replication and was not mediated by a soluble factor released from CMV-infected cells. Changes in adhesion molecules on CMV-infected fibroblasts may contribute to inflammation induced by CMV infection.

Animals↗