Biomedical subjects

T Kohno

Publications and source records attributed to T Kohno.

At least 271 records · Page 15Linked to original sources

PubMed1994

Formation of sheared G:A base pairs in an RNA duplex modelled after ribozymes, as revealed by NMR.

The thermal stability and structure of an RNA duplex, r(GGACGAGUCC)2, the base sequence of which was modelled after both a hammerhead ribozyme and a lead ribozyme, were studied by CD and NMR. We previously demonstrated that the corresponding DNA duplex, d(GGACGAGTCC)2, formed unique 'sheared' G:A base pairs, where an amino proton, instead of an imino proton, of G is involved in the hydrogen bonding, and G and A bases are arranged 'side by side' instead of 'head to head' (Nucleic Acids Res. (1993) 21, 5418-5424). CD melting profiles showed that the RNA duplex is thermally more stable than the corresponding DNA duplex. NMR studies revealed that sheared G:A base pairs are formed in the RNA duplex, too, although the overall structure of the RNA is the A form, which differs from the B form taken on by the corresponding DNA. A model building study confirmed that sheared G:A base pairs can be accommodated in the double helical structure of the A form. A difference between the RNA and DNA duplexes in the stacking interaction involving G:A mismatch bases is also suggested. The demonstration that sheared G:A base pairs can be formed not only in DNA but also in RNA suggests that this base pairing plays an important role regarding the RNA structure.

Adenine↗

PubMed1994

Receptor-binding affinities of human epidermal growth factor variants having unnatural amino acid residues in position 23.

Ile-23 of human epidermal growth factor (hEGF) has been indicated, by mutagenesis and NMR studies, to be directly recognized by the receptor. In the present study, an unnatural phenylalanine analog, either 2-azaphenylalanine (2aF), 3-azaphenylalanine (3aF), 4-azaphenylalanine (4aF), or 4-fluorophenylalanine (4fF), was incorporated by an in vivo protein synthesis system into position 23 of [Phe23]hEGF, which retains appreciable receptor-binding affinity (about 20% of the wild type). We compared the receptor-binding affinities of the variants with that of [Phe23]hEGF and found that substitution of Phe-23 with 2aF or 3aF raised the affinity, while substitution with 4aF or 4fF remarkably reduced the affinity. The tertiary structure of [Phe23]hEGF was not significantly affected by the substitution of Phe-23 with 2aF, as shown by the two-dimensional nuclear magnetic resonance analysis. In addition, the substitution of residue 23 with His or Tyr produced an hEGF variant with a slightly higher receptor-binding affinity than that of [Phe23]hEGF. Our results suggest that the receptor has an asymmetric hydrophobic pocket for recognition of the side chain in position 23 of hEGF. Furthermore, on the receptor surface, this pocket seems to be adjacent to a less hydrophobic region with a hydrogen-bond acceptor and donor. Thus, the use of unnatural amino acids in addition to the 20 natural ones allows analyses of the structure-function relationship of a protein at a higher resolution than conventional site-directed substitution by only natural amino acid residues.

Amino Acids↗

PubMed1994

Long-term follow-up studies of open-door expansive laminoplasty for cervical stenotic myelopathy.

Follow-up at an average time of 7.8 years postoperatively on open-door expansive laminoplasty (EL) was carried out to determine the long-term results of surgery. Thirty-three patients had ossification of the posterior longitudinal ligament and 18 had cervical spondylotic myelopathy. The average age at operation was 54.7 years. Japanese Orthopaedic Association scores and recovery rates increased during the 3 years after surgery and then plateaued. Radiographically, average spinal canal diameter remained enlarged past 5 years' follow-up. Factors leading to worsening of clinical symptoms included age greater than 60 years (4 patients), loss of sagittal canal diameter (2 patients), progression of ossification (4 patients), and minor trauma (1 patient). Postoperative motor paresis due to C5 and C6 root damage recovered to 4 (manual muscle testing) in all patients within 6 years. The conclusion is that open-door EL is safe and leads to good results that are maintained for over 5 years.

Cervical Vertebrae↗

PubMed1994

Molecular recognition of the identity-determinant set of isoleucine transfer RNA from Escherichia coli.

Molecular recognition of Escherichia coli tRNA(Ile) by the cognate isoleucyl-tRNA synthetase (IleRS) was studied by analyses of chemical footprinting with N-nitroso-N-ethylurea and aminoacylation kinetics of variant tRNA(Ile) transcripts prepared with bacteriophage T7 RNA polymerase. IleRS binds to the acceptor, dihydrouridine (D), and anticodon stems as well as to the anticodon loop. The "complete set" of determinants for the tRNA(Ile) identity consists of most of the nucleotides in the anticodon loop (G34, A35, U36, t6A37 and A38), the discriminator nucleotide (A73), and the base-pairs in the middle of the anticodon, D and acceptor stems (C29.G41, U12.A23 and C4.G69, respectively). As for the tertiary base-pairs, two are indispensable for the isoleucylation activity, whereas the others are dispensable. Correspondingly, some of the phosphate groups of these dispensable tertiary base-pair residues were shown to be exposed to N-nitroso-N-ethylurea when tRNA(Ile) was bound with IleRS. Furthermore, deletion of the T psi C-arm only slightly impaired the tRNA(Ile) activity. Thus, it is proposed that the recognition by IleRS of all the widely distributed identity determinants is coupled with a global conformational change that involves the loosening of a particular set of tertiary base-pairs of tRNA(Ile).

Anticodon↗

PubMed1994

Anti-HTLV-I IgG in urine detected by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using a synthetic peptide, Cys-env gp46(188-224), as antigen.

Antibody IgG to human T-cell leukemia virus type I (HTLV-I) in urine was detected by a sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using a synthetic peptide, Cys-env gp46(188-224), as antigen, the sensitivity and specificity of which were 100 and 98.5%, respectively, using serum samples. Anti-HTLV-I IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-Cys-env gp46(188-224) conjugate and Cys-env gp46(188-224)-beta-D-galactosidase (Escherichia coli) conjugate. The complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Finally, bound beta-D-galactosidase activity was assayed by fluorometry. Thirty-one urine samples from seropositive subjects and 100 urine samples from seronegative subjects were tested. The sensitivity and specificity were 87 and 100%, respectively, with unconcentrated urine samples and 94 and 100%, respectively, with approximately 10-fold concentrated urine samples. These results were superior to those by the conventional ELISA and gelatin particle agglutination test.

Adult↗

PubMed1994

Diagnosis of HIV-1 infection by detection of antibody IgG to HIV-1 in urine with ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant proteins as antigens.

Anti-HIV-1 IgG in urine was detected by an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant reverse transcriptase (RT), p17 and p24 as antigens, and beta-D-galactosidase from Escherichia coli as label. Anti-HIV-1 IgG in urine was reacted simultaneously with 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate and recombinant protein-beta-D-galactosidase conjugate. The immune complex formed, consisting of the three components, was trapped onto polystyrene balls coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex was eluted from the polystyrene balls with excess of epsilon N-2,4-dinitrophenyl-L-lysine and transferred to clean polystyrene balls coated with affinity-purified (anti-human IgG gamma-chain) IgG. Finally, the enzyme activity bound to the last solid phase was assayed by fluorometry. Using recombinant RT as antigen, the sensitivity and specificity for 83 seropositives and 100 seronegatives were both 100%, and the lowest signal for 60 asymptomatic carriers was 8.2-fold higher than the highest signal for the seronegatives. The positivity with recombinant RT as antigen could be confirmed by using recombinant p17 and p24 as antigens. The sensitivity could be improved by a longer assay of bound beta-D-galactosidase activity by using concentrated urine samples and by the combined use of recombinant RT, p17, and p24. Thus, reliable diagnosis of HIV-1 infection was possible for asymptomatic carriers.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Use of microplates and fluororeader for ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of anti-HTLV-I IgG.

Previously, an ultrasensitive solid phase enzyme immunoassay (immune complex transfer enzyme immunoassay) was described to detect low levels of anti-HTLV-I IgG in serum below those detectable by conventional methods. In this method, polystyrene balls as solid phase were transferred from test tube to test tube with tweezers. This was not only tedious but also causative of false-positivity by carryover, unless tips of the tweezers were washed carefully after each transfer of polystyrene balls. Bound enzyme activities for many samples were measured one by one by fluorometry using a spectrofluorophotometer. As a result, the assay of many samples was difficult. In the present study, microplates and a fluororeader were used in place to test tubes and a spectrofluorophotometer. Polystyrene balls were transferred quickly and easily from well to well by placing a microplate upside down on that containing polystyrene balls, and turning the two plates together upside down. Tweezers were not used for transfer of polystyrene balls, minimizing the possibility of false-positivity. Fluorescence intensities of bound enzyme activities for 96 samples were measured within a minute by using a fluororeader. Thus, it became easy to test many samples, although the sensitivity was lowered to some extent.

Amino Acid Sequence↗

PubMed1994

Subretinal neovascularization in the rat induced by IRBP synthetic peptides.

The present study was undertaken to develop a new animal model of subretinal neovascularization that does not involve traumatic manipulation of the eye. Using this model, the mechanism of subretinal neovascularization and its penetration through Bruch's membrane, and the various factors that contribute to this process were then examined. Male Lewis rats were immunized with interphotoreceptor retinoid binding protein (IRBP) peptide R-4, and the eyes histologically examined at various times up to 45 days after immunization. On day 12 after immunization, inflammatory cells were identified primarily in the anterior segment of the eye, with scattered cells in the retina and choroid. The inflammation was most prominent on day 14, by which time many eyes showed serous retinal detachment. By day 18 the inflammation had declined in intensity, but branches of the retinal vessels were seen extending into the choroid. Examination on day 30 revealed even fewer inflammatory cells but an accumulation of retinal pigment epithelial cells and mononuclear cells was present in the subretinal space. Examination on day 45 revealed no appreciable inflammation, but typical new vessels were found in the eyes from five of the 13 rats (38%) examined at that point. Mild inflammation of the retinochoroidal tissue can induce subretinal new vessels in rats, and this model will be useful for further study of subretinal new vessel formation.

Amino Acid Sequence↗

PubMed1994

The hypocholesterolemic action of TA-7552 and its effects on cholesterol metabolism in the rat.

The hypocholesterolemic property of 1-(3,4-dimethoxyphenyl)-2,3- bis(methoxycarbonyl)-4-hydroxy-6,7,8-trimethoxynaphthalene (TA-7552) and its effects on cholesterol metabolism were investigated in the rat. TA-7552 incorporated into a hypercholesterolemic diet at a concentration of 0.2% and administered for 7 days reduced serum cholesterol by 72% and liver cholesterol by 90%, and its minimal effective dose was 0.01% in the diet. Its hypocholesterolemic effect was associated with an elevation of serum HDL-cholesterol. Inclusion of 0.1% TA-7552 in the normal laboratory chow accelerated fecal excretion of 14C derived from orally administered 4-[14C]cholesterol or carbonyl-[14C]taurocholate. The net amounts of fecal neutral sterols and bile acids were markedly increased by the same treatment. Hepatic bile acid production and hepatic and intestinal cholesterol biosynthesis as measured by cholesterol 7 alpha-hydroxylase activity and 1-[14C]acetate incorporation into tissue cholesterol, respectively, were both stimulated by the drug treatment. All these data indicate that this hypocholesterolemic agent inhibits intestinal absorption of both cholesterol and bile acids and compensatorily stimulates hepatic production of bile acids and cholesterol.

Animals↗

PubMed1994

Thyroglobulin antibodies in type 1 diabetic patients and their relatives--measurement with highly sensitive assay.

Autoimmunity in Type 1 (insulin-dependent) diabetes mellitus was assessed by measuring thyroglobulin antibodies (TGA) using a highly sensitive enzyme immunoassay in 65 young patients with Type 1 diabetes mellitus, 83 healthy first-degree relatives of the patients, 37 healthy control subjects and 67 healthy parents of the control subjects. TGA were found in 78.5% (51/65) of patients and were significantly more frequent in patients than in control subjects (40.5%, 15/37; P < 0.01). The prevalence of TGA in patients showed no correlation with age at onset, duration of diabetes or sex. Among the first-degree relatives the prevalence of TGA was significantly increased in mothers of patients than in mothers of the control subjects (80.0% vs. 54.3%, P < 0.05), while not significantly between fathers of patients and fathers of control subjects or between siblings of patients and control subjects. Comparing the TGA levels of TGA-positive subjects, the TGA levels in patients, their parents and their siblings were significantly higher than those in the corresponding control subjects (P < 0.05, P < 0.05 and P < 0.01, respectively). In the present study we thus more clearly demonstrated autoimmune diathesis in patients with Type 1 diabetes and in their first-degree relatives.

Adolescent↗

PubMed1994

Established IL-2-dependent double-negative (CD4- CD8-) TCR alpha beta/CD3+ ATL cells: induction of CD4 expression.

We established IL-2-dependent T cells from an adult T-cell leukaemia (ATL) patient whose leukaemic cells changed from CD4 single-positive in the initial phase to double-negative (CD4- CD8-) at the time of exacerbation. The cells termed SO-4 were of ATL cell origin and showed the double-negative TCR alpha beta/CD3+ T-cell phenotype. SO-4 cells acquired CD4 antigen expression following stimulation with concanavalin A (ConA) or immobilized anti-CD3 antibody. The induction was inhibited by herbimycin A, an inhibitor of protein tyrosine kinase (PTK) activity. No CD4 mRNA was detectable in unstimulated SO-4 cells but a 3.0 kb signal specific for CD4 mRNA was detected after stimulation. These findings indicate that SO-4 cells return to their original phenotype (CD4 single-positive) by stimulation involving PTK. The results indicate that there is a pathway of phenotypic cycling between CD4 single-positive and double-negative T cells.

Benzoquinones↗

PubMed1994

[Transurethral hyperthermic treatment in patients with benign prostatic hypertrophy].

Transurethral hyperthermic treatment was performed on 30 patients with benign prostatic hypertrophy (BPH). Two patients had a urethral catheter because of urinary retention. The prostate was heated transurethrally. The treatment consisted of 3-8 sessions of 60 min. each. To evaluate this treatment, the following parameters were determined before and 1-5 weeks after the last hyperthermia session; subjective symptoms score, and as objective data residual urine volume and uroflowmetry. The symptoms score improved in 25 (83%) patients. Of 2 patients with a catheter, the catheter could be removed from 1 patient. Although there was no change in prostatic volume, significant decreases in residual urine volume, and increases of maximum flow rate and mean flow rate were observed. No adverse reactions were seen. Judging from the above results, this treatment is considered to be useful for patients with BPH.

Aged↗

PubMed1994

[Thoracoscopic treatment of intrathoracic diseases].

Recently developed thoracoscopic surgical equipment and related instruments have enabled us to perform two groups of advanced thoracoscopic surgical procedures. Group 1: procedures formerly performed by open thoracotomy, and group 2: procedures performed by thoracoscopic surgery only. Group 1 includes bullectomy for spontaneous pneumothorax, wedge resection for lung nodules, resection of benign tumors of the mediastium or chest wall, lung lobectomy, and resection of giant bullae. Because thoracoscopic surgery is minimally invasive, intrathoracic surgical procedures should be performed by thoracoscopic surgery if the results are the same. Group 2 consists of surgery in patients with poor pulmonary reserve and laser ablation of the bullous lung in advanced emphysema. Until recently, surgical intervention was not an option in the patients in groups 2 because of the invasiveness of open thoracotomy. Thoracoscopic surgery allows resection of tumors in early stage lung cancer in patients with poor pulmonary reserve. Severely emphysematous lungs can be ablated with a laser and shrunk, to normalize pulmonary compliance, residual volume and total lung capacity, and thus relieve symptoms.

Humans↗

PubMed1994

Homozygous deletion at chromosome 2q33 in human small-cell lung carcinoma identified by arbitrarily primed PCR genomic fingerprinting.

We have searched for novel genetic alterations in human cancer cell lines by using the arbitrarily primed polymerase chain reaction (AP-PCR), which is a PCR-based genomic fingerprinting. A homozygous deletion was detected in a small-cell lung carcinoma (SCLC) cell line, NCI-H82. Since homozygous deletion is a critical genetic alteration for the inactivation of tumor suppressor gene, we defined the locus of homozygous deletion. Fluorescence in situ hybridization analysis revealed that the deletion was localized at chromosome 2q33. Allelic loss on chromosome 2q was detected in 29.4% (5/17) of SCLC and 37.5% (12/32) one non-SCLC by restriction fragment length polymorphism analysis. Considerably high incidence of allelic loss on chromosome 2q was also detected in colorectal carcinoma and neuroblastoma. These results suggest the presence of a novel tumor suppressor gene at 2q33, which is involved in the development of several human cancers. The size of the homozygous deletion in the NCI-H82 cell line was more than 20 kilobase pairs. Seven loci mapped to 2q32-qter were all retained in this cell line, suggesting the presence of submicroscopic interstitial deletion in this cell line. Homozygous deletion detected in this study should be an invaluable tool for positional cloning of the target tumor suppressor gene at 2q33.

Base Sequence↗

PubMed1994

[Mitral valve replacement through right thoracotomy in a patient with a functioning ITA graft].

A 51-year-old male was admitted to our Institution with a complaint of dyspnea on effort. He had undergone coronary artery bypass grafting and mitral valve annuloplasty three years previously. The left ventriculograms showed severe mitral regurgitation. The coronary angiograms revealed an ITA graft and two SVG grafts were fully functioning. Considering the risk of injury of ITA/SVG graft during redo median sternotomy, we approached the heart through right anterolateral thoracotomy. Mitral valve replacement was performed under profound hypothermia without aortic cross clamping. Temporary reduction of perfusion flow was useful in order to obtain a better visual field. His postoperative course was uneventful. Right thoracotomy has an advantage over median sternotomy when reoperation is to be done for patients with a functioning ITA graft.

Coronary Artery Bypass↗

PubMed1994

[Pulmonary root reconstruction with a cryopreserved pulmonary allograft for mechanical pulmonary valve thrombosis].

A 58-year-old woman suffering from congestive heart failure caused by a mechanical pulmonary valve thrombosis was operated on with a pulmonary allograft. She had experienced pulmonary valvotomy and resection of infundibular stenotic muscle for congenital pulmonary stenosis at the age of 23 years old. She got congestive heart failure caused by pulmonary regurgitation, and underwent pulmonary valve replacement with a St. Jude Medical (SJM) valve when she was 48 years old. She suffered from episodes of a thrombosed SJM valve in 1984 and 1993. Each time, thrombolytic treatment with urokinase or recombinant tissue-type plasminogen activator was effective. She suffered from the third episode of SJM valve thrombosis in January 1994. As thrombolytic treatment was not effective this time, the thrombosed SJM valve was resected and her pulmonary root was reconstructed with a cryopreserved pulmonary allograft. Postoperative course was uneventful, and she now enjoys her life without anticoagulant therapy.

Cryopreservation↗

PubMed1994

[Thoracoscopic resection of schwannoma: a report of two cases].

We have performed thoracoscopic resection of schwannoma without intraspinal extension in two asymptomatic cases confirmed by chest roentgenogram and computed tomography: the case 1 with the tumor as large as 4.0 x 4.0 x 3.5 cm found in the paravertebral fifth Intercostal space, the case 2 with the tumor as large as 3.2 x 2.5 x 1.8 cm found on the first rib in contact with the supreme intercostal vein. In the operation of case 1, the dilated fifth intercostal vein in contact with the tumor which could not be controlled by Endo-Clip was doubly ligated by the use of Knot-Pusher. The tumor was successfully dissected from the chest wall and proved to arise from the fifth intercostal nerve. The tumor was brought into Endo-Pouch and extracted through one of the skin incisions which was minimally enlarged to accommodate the pouched lesion. In the operation of case 2, curved forceps facilitated the procedure because the tumor located near the apex of thorax. The tumor was found to originate from sympathetic nerve and removed through the minimally enlarged incision as case 1. We conclude as the following. The benign tumor found in the mediastinum or the chest wall is amenable to thoracoscopic treatment. In thoracoscopic procedure, as the operation under thoracotomy, we must acquire skills of standard operative technique, i.e., suture or ligation, because those skills are necessary when Endo-Clip or Endo-GIA cannot be used. The skin incision should be minimally enlarged finally when the specimen is extracted to minimize operative intervention.

Adult↗

PubMed1993

Role of basic residues for the binding of omega-conotoxin GVIA to N-type calcium channels.

Each of four basic residues of omega-conotoxin GVIA was replaced with alanine to study the role of basic residues for the binding of this toxin to N-type calcium channels. The activities of these analogs were estimated from the inhibitory action on 125I-omega-conotoxin GVIA binding to chick brain synaptic plasma membranes. The replacement of Arg17, Lys24 and Arg25 resulted in no significant change in the activity and all of the analogs gave the same IC50 value (0.15 nM) as that of native omega-conotoxin GVIA. The inhibitory action of [Ala2]omega-conotoxin GVIA (K2A) was 40-times less potent (IC50 = 5.5 nM); however, full inhibition was achieved at a concentration above 0.1 microM. These results indicate that the Arg residue is not essential for the activity of omega-conotoxin GVIA. The nature of association to ion channels may be different between omega-conotoxin GVIA and mu-conotoxin GIIIA.

Amino Acid Sequence↗