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Biomedical subjects

R Yagi

Publications and source records attributed to R Yagi.

At least 37 records · Page 2Linked to original sources
PubMed1997

Development of HIV-1 protease expression methods using the T7 phage promoter system.

New and simple human immunodeficiency virus type 1 (HIV-1) protease expression methods in Escherichia coli were developed using the T7 phage promoter system. In order to suppress leaky HIV-1 protease expression under the control of the T7 polymerase, two new methods were tested. One involved the introduction of supplementary T7 promoter regions into host cells [E. coli BL-21 (DE3)] containing the HIV-1 protease gene under the control of the T7 promoter. It was expected that the supplementary T7 promoter regions would compete with the HIV-1 protease expression vector for the T7 polymerase binding. The other involved the infection of late-log-phase cultures of E. coli JM109 harboring the same HIV-1 protease expression vector with the M13 phage expressing T7 polymerase. Both methods were effective, and transformants with the mature HIV-1 protease expression vector showed ten times higher HIV-1 protease activity than activities obtained with the autoprocessing vector. The expression systems described here are convenient and are also easily applicable for the expression of other proteins toxic for E. coli.

Bacteriophage T7↗
PubMed1997

In vitro and ex vivo anti-human immunodeficiency virus (HIV) activities of a new water-soluble HIV protease inhibitor, R-87366, containing (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid.

In a series of compounds containing (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid (AHPBA), a transitionstate mimetic, R-87366:(2S,3S)-3-[N-(quinoxaline-2-carbonyl)-L-asparaginyl]amino- 2-hydroxy-4-phenylbutanoyl-L-proline tert-butylamide, was found to be a potent human immunodeficiency virus protease inhibitor (Ki value was 11 nM) and anti-HIV agent (IC90 value was 0.5 microM for HIV-1IIIB acutely infected cells) with moderate water-solubility (4.2 mg/ml at 25 degrees C). The compound was also active in chronically infected Molt-4/HIV-1IIIB cells, and inhibited the proteolytic processing of p55 into p17, suggesting that its anti-HIV activity was derived from HIV protease inhibition. The compound showed more potent activity (IC90 value was 0.03-0.25 microM) against clinical isolates of HIV in 5 out of 6 patients examined with varying clinical status in an ex vivo assay. One isolate, however, from the sixth patient, was less sensitive to R-87366 (IC90 value was 0.5 microM). In experiments with this strain, R-87366 showed comparatively low efficacy in acutely infected peripheral blood mononuclear cell (PBMC). This result suggests that the diversity of sensitivity shown in the ex vivo assay could be caused by the viral property itself. As a result of the determination of nucleic acid sequences in the clinical isolates, some amino acids were found to be substituted in the protease region, in contrast to the HIV-1 clade B consensus sequence, and some of them have been reported to contribute to the susceptibility of HIV protease inhibitors.

Amino Acid Sequence↗
PubMed1997

EMG power spectrum and integrated EMG of ankle plantarflexors during stepwise and ramp contractions.

The aim of this study was to investigate whether the median frequencies (MF) of the electromyogram (EMG) and the integrated EMG (IEMG) of histochemically differentiated ankle plantarflexors, the gastrocnemius and soleus, were force dependent. Bipolar intramuscular wire electrodes were used to measure EMG of the soleus (SO), medial head of gastrocnemius (GM), and lateral head of gastrocnemius (GL) during ramp (single ongoing contractions) with the force increasing linearly from 0 to 100% of maximum voluntary contraction (MVC) and stepwise (steady force levels) ankle plantarflexion at 10, 20, 30, 40, 60, and 80% MVC. EMG and force were measured simultaneously. Power spectral analysis of these signals was performed to calculate MF on 1024-point by fast Fourier transform (FFT) technique. IEMG value of each muscle was also obtained at the same levels of force. While IEMG of three heads of triceps surae in both stepwise and ramp contractions increased significantly with increasing force, MF values of GL during stepwise contraction increased significantly (20, 40, 60, 80% MVC). These results suggest that the sensitivity of EMG power spectrum might be influenced by the proportion of fast twitch muscle fibers, which histochemically corresponds to type II fibers.

Adult↗
PubMed1996

Structure-activity relationships of HIV-1 PR inhibitors containing AHPBA--II. Modification of pyrrolidine ring at P1' proline.

Systematic replacement in the 3- or 4-position of the pyrrolidine ring at P1' proline was carried out. Compound 26, which has a Cl atom in the 4(S)-position was the most active among inhibitors substituted with other halogen atoms or other substituents. Furthermore, the replacement of the Z group in compound 26 with five- or six-membered fused aromatic heterocycle carbonyl groups produced more potent inhibitors. 7-Methoxybenzofuran-2-carbonyl derivative (44) was the best of these and showed Ki = 4.5 nM against HIV PR and IC90S 0.58 microM and 0.06 microM in chronic and acute infections, respectively. These results suggest that the combination of the 4(S)-CI atom and fused bicyclic heterocycles may be effective in improving their cellular penetration.

Cells, Cultured↗
PubMed1995

A clinical trial of therapeutic electrical stimulation for amyotrophic lateral sclerosis.

This paper describes the effects of therapeutic electrical stimulation (TES) on the wasting muscles in a patient with amyotrophic lateral sclerosis. The patient is a 47-year-old male, and he has a history of progressive muscle weakness and atrophy, affected more in the right side. Percutaneously indwelling intramuscular electrodes were implanted to the affected muscles in the right upper and lower extremities but no electrode in the corresponding left region. Within a month of TES therapy, a rapid improvement of extremity motion appeared in the TES treated side. Long-term application of TES more than 3 months increased the strength of the muscle which had been evidently weaker than the non-treated side. CT findings of both the upper and lower extremities with TES therapy showed an increase in the density and a reduction in the moth-eaten image. An increase in the thickness of the muscles was also observed in the TES treated side while deterioration was observed in the muscles on the non-treated side.

Amyotrophic Lateral Sclerosis↗
PubMed1995

Design and anti-HIV-1 activity of ribozymes that cleave HIV-1 LTR.

A hairpin ribozyme (HR112) and two hammerhead ribozymes (RZ115 and RZ119) containing a 5'C(UUCG)G3' loop were designed to cleave the long terminal repeat (LTR) of HIV-1. When the ribozyme catalyzed RNA cleavage reaction for a chemically synthesized 19 mer (LTR 19) was measured, the t 1/2 value of LTR 19 mediated by RZ115 was smaller than that of the RZ119 case. Moreover, the transformed CEM cells harboring the gene encoding these ribozymes were challenged with a HIV-1IIIB strain, two ribozymes, HR112 and RZ119 possessed strong anti-HIV-1 activity. However, the anti-HIV-1 activity displayed by RZ115 was weak. On the basis of secondary structure predictions of the RNA transcribed with the gene encoding ribozymes, the secondary structure of the transcribed RNA with RZ115 sequences was observed to be different from those with the other ribozymes. It has been demonstrated that the secondary structures of transcribed RNAs can possibly influence the anti-HIV-1 activity.

Antiviral Agents↗
PubMed1994

Structure-activity relationships of HIV-1 PR inhibitors containing AHPBA.

A series of Human Immunodeficiency Virus type-1 protease (HIV-1 PR) inhibitors that contain 3-amino-2-hydroxy-4-phenylbutanoic acid (AHPBA) at the scission site of the substrate were prepared and evaluated for their inhibitory activity. Preliminary studies on the chain length of inhibitors and the hydroxyl configuration of AHPBA indicated that small (2S,3S)-derivatives, composed of the regions between the P3 and P2' sites, showed enough inhibitory activity toward HIV-1 PR to become prototypes for further structural modification. Systematic replacement at the sites from P3 to P2' revealed that some bicyclic heteroarylcarbonyl derivatives possessed strong potency and good enzyme selectivity.

Amino Acid Sequence↗
PubMed1994

Studies of human immunodeficiency virus type 1 (HIV-1) protease inhibitors. III. Structure-activity relationship of HIV-1 protease inhibitors containing cyclohexylalanylalanine hydroxyethylene dipeptide isostere.

Systematic replacement of the P4-P2 subsites of substrate-based human immunodeficiency virus type 1 protease (HIV-1 PR) inhibitors containing cyclohexylalanylalanine hydroxyethylene dipeptide isostere (Cha-psi [H.E.]-Ala) at positions corresponding to the scissile sites of substrates was carried out. The structure-activity relationship revealed that compounds with the combination of hydrophilic P3 and beta-branched hydrophobic P2 amino acids generally showed strong inhibitory activity against HIV-1 PR. In particular, compounds 4 (Boc-Orn-Val-Cha-psi [H.E.]-Ala-NHBun; Bu(n) = n-butyl, Ki = 11 nM) and 6 (Z-Orn-Val-Cha-psi [H.E.]-Ala-NHBun, Ki = 8 nM) exhibited good enzyme selectivity, possessing no significant inhibitory activities toward closely related aspartic proteases, pepsin, cathepsin D, and renin. As a possible model system for (anti-Mo-MSV/MLV complex (Mo-MSV = Moloney murine sarcoma virus; MLV = murine leukemia virus)) activity was investigated. Both compounds were found to inhibit moderately the focus formation of Mo-MSV/MLV complex in NIH3T3 cells (compound 4, IC50 = 1.8 microM; compound 6, IC50 = 1.0 microM).

Amino Acid Sequence↗
PubMed1993

Studies of HIV-1 protease inhibitors. I. Incorporation of a reduced peptide, simple aminoalcohol, and statine analog at the scissile site of substrate sequences.

Inhibitors of the protease of human immunodeficiency virus type-1 (HIV-1) were designed and synthesized. A reduced peptide, simple aminoalcohol, and statine analog, 4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA), were inserted at the scissile site of substrate sequences of HIV-1 protease. While both reduced peptides and simple aminoalcohol derivatives were weak inhibitors, the peptides containing AHPPA demonstrated moderate inhibitory activity. The more potent alcohol configuration of AHPPA is (R), which is opposite to the configuration in potent inhibitors of other aspartic proteases. In particular, compound 28 ((3R,4S)-4-(N-tert-butoxycarbonyl- L-glutaminyl-L-asparaginyl)amino-3-hydroxy-5-phenylpentanoic acid 2'-methylbutylamide) had a Ki of 0.36 microM and exhibited excellent enzyme specificity.

Amino Acid Sequence↗
PubMed1993

Studies of HIV-1 protease inhibitors. II. Incorporation of four types of hydroxyethylene dipeptide isosteres at the scissile site of substrate sequences.

Human immunodeficiency virus type 1 (HIV-1) protease inhibitors containing four types of hydroxyethylene dipeptide isosteres were designed and synthesized. These inhibitors consist of eight stereoisomers of phenylalanylproline (Phe-psi[H.E.]-Pro), four stereoisomers of phenylalanylalanine [Phe-psi[H.E.]-Ala), and one stereoisomer each of phenylalanylglycine (Phe-psi[H.E.]-Gly) and cyclohexylalanylalanine (Cha-psi[H.E.]-Ala) hydroxyethylene dipeptide isosteres. For the synthesis of the latter two isosteres, a newly developed synthetic method for gamma-lactone was applied. The inhibitory activities of these peptides were evaluated by cleavage assay of partially purified gag proteins or purified synthetic peptide. Of the inhibitors examined, compounds 2c (Z-Asn-(2S,3R,4S,5S)-Phe-psi[H.E.]-Pro-NHB(un); Bu(n) = n-butyl, Ki = 0.50 microM), 21a (Z-Asn-(2R,4S,5S)-Phe-psi[H.E.]-Ala- NHBu(n), Ki = 0.34 microM) and 23 (Z-Asn-(2R,4S,5S)-Cha-psi[H.E.]-Ala- NHBu(n), Ki = 0.46 microM) were moderately potent inhibitors. The results revealed that the alkyl substituent at C2 is essential, and the stereochemistry of the hydroxyethylene dipeptide isosteres greatly affected their inhibitory activities.

Amino Acid Sequence↗
PubMed1993

Misincorporation of ribonucleotides by DNA polymerase during in vitro DNA replication.

The fidelity of DNA replication with respect to misincorporation of ribonucleotides into DNA has been studied. Primed M13 DNA was replicated by E. coli DNA polymerase I in the presence of four dNTPs, a single rNTP and Mg2+ as a cofactor. All four rNTPs were incorporated into DNA in place of the corresponding dNTPs when rNTP/dNTP ratio was 1000:1. The frequency of rNTP misincorporation was C > G > A > U.

Bacteriophage M13↗
PubMed1989

Reconstruction of the four major ligaments in an unstable knee joint after dislocation by solvent-preserved human fascia lata transplantation. A case report.

Once the opportunity for primary repair of injured knee ligaments after traumatic dislocation has been lost, ligamentous reconstruction is difficult using only autogenic tissues because of the risk of loss of function at the donor site, so other substitutes are needed. The four major ligaments in the unstable knee of a 35-year-old man were reconstructed by solvent-preserved human fascia lata three months after traumatic open dislocation. The clinical results were satisfactory. Arthroscopic examination one year later showed that the reconstructed ligaments had good thickness and tension and were composed of autologous connective tissue without evidence of rejection. The literature on dislocation of the knee and on cruciate ligament reconstruction by allograft was reviewed, and a brief introduction to solvent-preserved human fascia lata was presented. The commercialization of this material has solved some common problems concerned with using allogenic tissues.

Adult↗
PubMed1985

A new method for venous interposition grafts using fibrin glue.

We developed a new technique of venous interposition graft where the principle of sleeve anastomosis was applied at the proximal suture site and fibrin glue was used at both suture sites to prevent leakage. An advantage of this procedure was a reduction in the number of stitches, which reduced operative time and obtained good vascular healing. Since in an animal experiment a high patency rate of 97% was obtained, we applied the procedure to a clinical case with complete amputation at the PIP joint level of a long finger. Good recirculation was seen on angiography 6 weeks postoperatively.

Adhesives↗
PubMed1985

Primary hypomagnesemia with secondary hypocalcemia. Report of a case and review of the world literature.

Primary hypomagnesemia with secondary hypocalcemia (PHSH) is a rare type of hypocalcemic disorder which occurs in early infancy and is clinically characterized by recurrent tetany and/or convulsion. In this paper, a male infant with PHSH who had frequent seizures at the age of 9 days is described. Besides PHSH, several illnesses in infancy are manifested by hypomagnesemia and hypocalcemia, i.e. transient neonatal hypomagnesemic hypocalcemia, congenital renal or hepatic insufficiencies, magnesium-losing nephropathy, combined impairments of intestinal absorption and renal reabsorption of magnesium. PHSH is to be differentiated from these illnesses by the demonstration of a combination of the following findings; hypocalcemia refractory to calcium but responsive to magnesium, continuous requirement for magnesium supplementation to maintain normocalcemia, lack of hypermagnesiuria and/or impaired intestinal absorption of magnesium. Twenty cases from the literature were found to exhibit these characteristics. The clinical, biochemical, and endocrine features of PHSH are summarized on the basis of a review of the data of these and the present case. No associated illness was known in the afflicted infants or mothers. Both male and female infants were afflicted at a male to female ratio of 15:6. Some siblings were afflicted but none of the parents or relatives. The onset of tetany and/or convulsion was between the 9th day and 4th month, which is later than that of other neonatal hypocalcemic illnesses. Hypocalcemia was more pronounced than other infantile hypocalcemic illnesses. The role of the parathyroid hormone in the pathogenesis of hypocalcemia has been studied in several studies but no unifying concepts have yet been established.

Calcium↗
PubMed1985

[Basic and clinical study on functional electrical stimulation (FES) for the paralyzed upper extremity].

Functional electrical stimulation (FES) will become one of the useful reconstructive methods for the paralyzed upper extremity due to upper motor neuron lesion. As a basic study, the author analyzed the electromyogram of the grasp and the lateral pinch movements in three normal hands. On the basis of this electromyographic analysis, FES was applied for the hands of two tetraplegics and three hemiplegics using percutaneous wire electrodes. As a result, three types of holding patterns were obtained in the paralyzed hands by FES, namely, parallel extension grip, grasp and lateral pinch. Clinically, the author made a FES system for the tetraplegic patient, and utilized it for the activities of daily living. The patient could get hand opening, parallel extension grip, grasp and lateral pinch in his paralyzed hand by using this FES system, and he obtained a certain measure of independence of the activities of daily living such as eating, writing and so on.

Adult↗