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Biomedical subjects

R Koga

Publications and source records attributed to R Koga.

At least 37 records · Page 2Linked to original sources
PubMed1998

Biologically active oligodeoxyribonucleotides. 5. 5'-End-substituted d(TGGGAG) possesses anti-human immunodeficiency virus type 1 activity by forming a G-quadruplex structure.

A series of hexadeoxyribonucleotides (6-mers), d(TGGGAG), substituted with a variety of aromatic groups at the 5'-end were synthesized and tested for anti-human immunodeficiency virus type 1 (HIV-1) activity. While unmodified d(TGGGAG) (31) had no anti-HIV-1 activity, compound 23 with a 3,4-di(benzyloxy)benzyl (DBB) group at the 5'-end potently inhibited the HIV-1IIIB-induced cytopathicity of MT-4 cells in vitro (IC50 = 0.37 microM) without cytotoxicity up to 40 microM. A thermal denaturation study on the 5'-end-substituted 6-mers by means of the circular dichroism (CD) spectra demonstrated that the aromatic substituent attached at the 5'-end of the 6-mer strongly enhanced the formation of a parallel helical structure consisting of four strands (quadruplex). On the contrary, compound 36, in which one of the guanosines of 23 was replaced by a thymidine, did not form a quadruplex, thus exhibiting no anti-HIV-1 activity. Moreover, both compound 15, with a tert-butyldiphenylsilyl group solely at its 3'-end, and compound 21, with a relatively small substituent, a benzyl group, at the 5'-end, formed quadruplexes but had no anti-HIV-1 activity. These findings led us to the conclusion that both the quadruplex structure and the aromatic substituent with adequate size at the 5'-end are crucial for the interaction of the 5'-end-substituted 6-mers with the V3 loop as well as the CD4 binding site on viral gp120, resulting in anti-HIV-1 activity.

Anti-HIV Agents↗
PubMed1998

Biologically active oligodeoxyribonucleotides. Part 11: The least phosphate-modification of quadruplex-forming hexadeoxyribonucleotide TGGGAG, bearing 3-and 5-end-modification, with anti-HIV-1 activity.

We have found that a hexadeoxyribonucleotide (5'TGGGAG3', R-95288), Koizumi, M. et al. Bioorganic & Medicinal Chemistry, 1997, 5, 2235, bearing a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5'-end and a 2-hydroxyethylphosphate at the 3'-end, has high anti-HIV-1 activity and the least cytotoxicity in vitro and in vivo. In order to synthesize more potent hexadeoxyribonucleotides, we substituted phosphodiester (P-O) bonds in the 6-mer with the least phosphorothioate (P-S), phosphoramidate (P-N), or methylphosphonate (P-Me) bonds. When more than two P-N or P-Me bonds were introduced into a 6-mer, the phosphate-modified 6-mers had weak or no anti-HIV- activity, in spite of quadruplex structure formation. However, when P-S bonds were substituted for P-O bonds, anti-HIV-1 activity of their 6-mers did not dramatically decrease, compared with compounds substituted with P-N or P-Me bonds. The results suggest that the formation of a quadruplex structure is not always sufficient for anti-HIV-1 activity of the 6-mer, and that net negative charges derived from P-O or P-S bonds in the quadruplex are important for anti-HIV-1 activity. Moreover, among various phosphate-modified ODNs, we found that the anti-HIV-1 activity of ODN PS7 with only one P-S bond was the same as that of R-95288, both having a high stability in human plasma.

Anti-HIV Agents↗
PubMed1998

Molecular characterization of a single mitochondria-associated double-stranded RNA in the green alga Bryopsis.

Mitochondria from the green alga Bryopsis sp. very often contained a 4.5 kb double-stranded RNA (dsRNA) at a defined level. Complementary DNA probes derived from the mitochondrial dsRNA hybridized with none of the algal chloroplast dsRNAs of 1.7 to 2.2 kb, but did hybridize with a similar-sized dsRNA among several dsRNAs from the mitochondria of B. maxima. Sequence analysis of the mitochondrial dsRNA from Bryopsis sp. revealed only two large, overlapping, open reading frames (ORFs) on one strand if UGA was taken as a non-termination codon, suggesting the independent phylogenetic evolution of the mitochondrial dsRNA. Consensus sequence for RNA-dependent RNA polymerase was found within the longer ORF (2472 bp) of the dsRNA. The overlapping 52 bp of the ORFs in different reading frames is suggestive of the occurrence of a -1 ribosomal frameshift in the mitochondrial translation system. The observed simple genetic structures suggest that the algal mitochondrial dsRNA might be deficient in a gene for movement from cell to cell in host plants and, hence, has a plasmid-like nature that is distinct from that of infectious plant viruses. The nature and origin of the endogenous dsRNAs of various sizes and their relationships are discussed.

Amino Acid Sequence↗
PubMed1997

Protection of hu-PBL-SCID/beige mice from HIV-1 infection by a 6-mer modified oligonucleotide, R-95288.

We analyzed the anti-HIV-1 activity of an oligonucleotide derivative, R-95288, in severe combined immunodeficient (SCID/beige) mice transplanted with normal human peripheral blood leukocytes (PBLs), designated hu-PBL-SCID/beige mice. The human chimeric mice were inoculated with HIV-1(CC1) 3 weeks after the transplantation and sacrificed 2 weeks later. Virus infection was determined by coculture of splenocytes with fresh human PBLs and also by detection of HIV- specific DNA sequences using the polymerase chain reaction. No evidence of infection was observed in mice treated with R-95288 (100 mg/kg/day) using intraperitoneal delivery by osmotic minipumps starting 1 day before virus challenge. In contrast, virus infection was observed in over 80% of the saline-treated control mice. In addition, partial inhibition of HIV-1 infection was obtained in mice treated subcutaneously with R-95288 (100 mg/kg/day). Toxicity towards the engrafted human cells was not observed by flow cytometric analysis. Moreover, R-95288 failed to inhibit lymphocyte proliferation (CC50 > 400 microg/ml), while 90% inhibition of HIV-1 replication was achieved at 3.1 microg/ml in vitro. These results suggest the ability of R-95288 to protect the human chimeric mice against HIV-1 infection.

Animals↗
PubMed1997

Biologically active oligodeoxyribonucleotides--IX. Synthesis and anti-HIV-1 activity of hexadeoxyribonucleotides, TGGGAG, bearing 3'- and 5'-end-modification.

We have determined that hexadeoxyribonucleotides (5'TGGGAG3'), with modified aromatic groups such as a trityl group at the 5'-end, have anti-HIV-1 activity in vitro. The 6-mer bearing a 3,4-dibenzyloxybenzyl (3,4-DBB) group at the 5'-end had the most potent activity and the least cytotoxicity. When the 3'-end of the 5'-(3,4-DBB)-modified 6-mer was substituted with a 2-hydroxyethylphosphate, a 2-hydroxyethylthiophosphate, or a methylphosphate group at the 3'-end, anti-HIV-1 activity increased. Moreover, among various 3'- and 5'-end-modified 6-mers that were tested, the 6-mer (R-95288) bearing a 3,4-DBB group at the 5'-end and a 2-hydroxyethylphosphate group at the 3'-end was the most stable, when incubated with mouse, rat, or human plasma. Therefore, R-95288 was chosen as the best candidate for possible use in therapy on the basis of its anti-HIV-1 activity.

Animals↗
PubMed1996

Emerin deficiency at the nuclear membrane in patients with Emery-Dreifuss muscular dystrophy.

Mutations in the STA gene at the Xq28 locus have been found in patients with X-linked Emery-Dreifuss muscular dystrophy (EDMD). This gene encodes a hitherto unknown protein named 'emerin'. To elucidate the subcellular localization of emerin, we raised two antisera against synthetic peptide fragments predicted from emerin cDNA. Using both antisera, we found positive nuclear membrane staining in skeletal, cardiac and smooth muscles in the normal controls and in patients with neuromuscular diseases other than EDMD. In contrast, a deficiency in immunofluorescent staining of skeletal and cardiac muscle from EDMD patients was observed. A 34 kD protein is immunoreactive with the antisera--the protein is equivalent to that predicted for emerin. Together, our findings suggest the specific deficiency of emerin in the nuclear membrane of muscle cells in patients with EDMD.

Adolescent↗
PubMed1995

A severe muscular dystrophy patient with an internally deleted very short (110 kD) dystrophin: presence of the binding site for dystrophin-associated glycoprotein (DAG) may not be enough for physiological function of dystrophin.

We report a 4-yr and 5-month-old boy with severe clinical features of an early-onset Duchenne muscular dystrophy, who had a very short (110 kDa) dystrophin at the sarcolemma. The patient had a large deletion (exons 2-44) of the dystrophin gene which was predicted to cause a reading frame shift. Sequence analysis of dystrophin mRNA in muscle revealed an alternatively spliced gene product from exons 1 to 51 that caused restoration of the reading frame, in addition to an mRNA corresponding to the DNA deletion. A consistent result was obtained by immunocytochemical analysis of muscle; i.e. positive staining for dystrophin at the sarcolemma using antibodies against the C-terminus, cysteine-rich region and last three of 24 repeat units of the central rod-domain, but not for the remaining antibodies for dystrophin that recognize the N-terminal and proximal rod-domains. Immunostaining for dystrophin-associated glycoproteins (DAGs: 43 and 50 K) and merosin were preserved. Utrophin staining was positive but fainter than other DMD muscles. These results suggest that an extremely short dystrophin lacking the entire actin-binding site in the N-terminus cannot function properly even if the protein possesses the putative DAG-binding cysteine-rich and the C-terminal domains, and still has an ability to associate with sarcolemmal membrane.

Base Sequence↗
PubMed1994

Decreased myotonin-protein kinase in the skeletal and cardiac muscles in myotonic dystrophy.

To investigate the role of myotonin-protein kinase (MT-PK) in the pathophysiology of myotonic dystrophy (DM), we developed specific antibodies against synthetic MT-PK peptides. The antibody identified a 53kDa protein in skeletal muscle and recognized decreases in the amount of the protein in both adult and congenital DM patients, compared with amounts in controls and in patients with other muscle diseases. In cardiac muscle, this antibody identified a 62kDa protein, and in brain, both the 53 and 62kDa proteins were detected. These results suggest the presence of tissue-specific isoforms of MT-PK.

Adult↗
PubMed1994

Immunohistochemical analysis of perforin and granzyme A in inflammatory myopathies.

Perforin (PF) and granzyme A (GA) are candidates suspected of being cytolytic proteins of the granules of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. We analysed PF and GA in muscles from patients with inflammatory myopathies. Five cases of polymyositis (PM), two cases of inclusion body myositis (IBM), and five cases of dermatomyositis (DM) were studied immunohistochemically using anti-PF and GA antibodies raised against each synthetic peptide of human PF and mouse GA, together with a panel of monoclonal antibodies reactive for lymphocyte subsets. In PM and IBM, PF positive cells were colocalized with GA positive cells and occasionally invaded into the non-necrotic muscle fibres. The percentage of PF positive cells among the endomysial CD8 positive cell population was 9.9% (PM) and 12.5% (IBM), and the majority of the endomysial CD8 positive cells were alpha/beta T cells. In contrast, in DM, both PF and GA positive cells were very few in all cases. Only few inflammatory cells were CD16+ or CD57+ NK cells among these diseases. Our results suggest that PF and GA are secreted mainly from alpha/beta T cells, and may play a key role in muscle fibre damage in at least some PM and IBM, but not in DM.

Aged↗
PubMed1994

Monoallelic expression of normal mRNA in the PIT1 mutation heterozygotes with normal phenotype and biallelic expression in the abnormal phenotype.

The combined deficiency of thyrotropin, growth hormone and prolactin, caused by PIT1 abnormality manifests in the homozygous or heterozygous state. We studied a patient having an allele with Arg271Trp mutation, which produces clinical symptoms in heterozygotes by a dominant-negative effect. However, in the family, her father, grandmother and aunts had the same mutation without clinical symptoms, although the proband had typical phenotypic expression. We analyzed the PIT1 transcript in peripheral lymphocytes by reverse transcription-polymerase chain reaction and found monoallelic expression of normal allele in the father and grandmother and skewed pattern of biallelic expression in the proband. The phenotypic expression of PIT1 abnormality may depend on different transcription of the PIT1 gene.

Alleles↗
PubMed1994

[Dystrophin-related protein in diaphragm, limb and myoblast transferred muscles of mdx mouse].

Expression of the dystrophin-related protein (DRP or Utrophin) was examined with Western blot and immunohistochemical methods in diaphragm, limb and also in myoblast transferred muscles of the mdx mouse. Although we have hypothesized that progressive fibrosis in the diaphragm of the mdx mouse has been due to a smaller amount of DRP expression compared with limb muscles, we could not find any difference in the amount of DRP or in the DRP localization pattern between the two muscle sites. In limb muscles treated with myoblast transfer, dystrophin-positive muscle fibers had no DRP on their surface membrane, although dystrophin-negative muscle fibers were DRP-positive. These findings suggest that excessive expression of DRP is suppressed in the normalized muscle fiber with dystrophin. It also appears that the histological differences seen in the different muscles of the mdx mouse are not due to the amount of DRP present.

Animals↗
PubMed1993

Immunoblot analysis of dystrophin-related protein (DRP).

Polyclonal antibodies against the carboxy-terminal portion of dystrophin-related protein (DRP), the putative autosomal gene product which shares sequence homology with dystrophin, show the clear expression of DRP in mouse fetal muscle and in cultured human muscle cells, but not in mature mouse or human muscle. DRP has the same molecular mass as X-linked dystrophin and is recovered from the membrane fraction, but is associated with membranes more loosely than dystrophin.

Amino Acid Sequence↗
PubMed1993

Abnormal localization of laminin subunits in muscular dystrophies.

To address potential involvement of muscle basal lamina and membrane cytoskeleton proteins in the etiology of non-dystrophinopathy muscular dystrophies, we examined the immunostaining intensity and distribution of laminin subunits (A, B1, B2 and M), type IV collagen, dystrophin and spectrin in skeletal muscle biopsies from 64 myopathic patients (17 Fukuyama congenital muscular dystrophy: FCMD, 13 congenital muscular dystrophy unrelated to FCMD: other CMD, 16 Duchenne muscular dystrophy: DMD, and 18 other neuromuscular diseases. In FCMD muscle, we found a significant reduction of laminin M (merosin; a striated muscle specific basal lamina-associated protein) with approximately 26% of levels seen in controls by quantitative immunofluorescence. Other CMD and DMD muscles showed less dramatic reductions (78%, 80%, respectively). The localization of laminin M was also abnormal in FCMD muscle. Laminin B1 and B2 showed abnormalities similar to those observed with laminin M, but were less marked. Laminin A was only detected in rare regenerating fibers in control biopsies, whereas it was seen around most muscle fibers in FCMD patients, and in dystrophin deficient muscle fibers from DMD patients and its carrier. Staining intensity of type IV collagen in FCMD muscle was not significantly different from the other diseases. These findings may implicate a primary or central role for the basal lamina in FCMD muscle.

Adolescent↗
PubMed1991

Dystrophin-related protein in the fetal and denervated skeletal muscles of normal and mdx mice.

The amino acid sequence of the polyclonal antibodies we developed against the carboxyl terminus of the dystrophin-related protein, the putative gene product of B3 cDNA, had no homologous sequence to the dystrophin molecule except for two amino acids located at its ends for immunization. By immunohistochemical examination in C57B1/10ScSn and C57B1/10ScSn-mdx mice we found that the DRP was expressed on the surface membrane of fetal muscle fibers, was assembled at the neuromuscular junctions of the mature muscle fibers, and reappeared on the surface membrane of muscle fibers after denervation. Its localization was similar to that of the acetylcholine receptor, suggesting that DRP is one of the cytoskeletons which organize and stabilize the cytoplasmic domain of the acetylcholine receptor.

Aging↗
PubMed1990

Antibody against the C-terminal portion of dystrophin crossreacts with the 400 kDa protein in the pia mater of dystrophin-deficient mdx mouse brain.

The mdx mouse is an animal model for X-linked Duchenne muscular dystrophy. A polyclonal antibody against a synthetic peptide IV equivalent to the C-terminal portion (amino acids 3495-3544) of dystrophin crossreacted with a 400 kDa protein in the brain and the spinal cord of mdx mouse, as well as in the control B10 mouse. However, the protein did not crossreact with the polyclonal antibody raised against the N-terminal portion of dystrophin peptide I (amino acids 215-264). Immunofluorescent micrography revealed that the outside of the small arteries and the pia mater of the brain strongly reacted with the anti-peptide IV antibody. These results strongly suggest the presence of a crossreactive protein other than dystrophin, possibly a dystrophin-related autosomal gene product, in the pia mater.

Animals↗
PubMed1990

[Chronological change of EEG findings in a case of pyridoxine dependency seizures].

A patient of pyridoxine dependent seizures was reported. He was born at 34 weeks' gestation and weighted 2,760 g. Apgar scores were 6 and 9 at 1 and 5 minutes, respectively. He showed the first seizure 2 hours after his birth. Phenobarbital, phenytoin, sodium valproate, diazepam and clonazepam were not effective. Pyridoxal phosphate (50 mg) was given intravenously, resulting in suppression of convulsions. However, muscle tonus was severely depressed. In EEG, a discontinuous pattern was found in quiet and indeterminate sleep on the 2nd day of life. At 5th week multifocal spikes were found, and the discontinuous pattern persisted. Ictal discharges at 13th week showed generalized, continuous, irregular and high voltage slow waves with multifocal spikes. At 27th week of life, high voltage slow waves disappeared and multifocal spike discharges decreased. At 2 years and 10 months of age, the patient was suffering from athetotic cerebral palsy and severe mental retardation. Pyridoxal phosphate at the doses of 35-40 mg/kg/day had been administered. Irritability sometimes occurred and additional 50 mg of pyridoxal phosphate controlled this irritability effectively.

Administration, Oral↗
PubMed1988

Determination of deoxyspergualin by gas chromatography-selected-ion monitoring.

A gas chromatographic-selected-ion monitoring method was developed for the sensitive and specific determination of deoxyspergualin in dog plasma using a capillary column and a C8-amide homologue of deoxyspergualin as an internal standard. Extraction and purification of deoxyspergualin from its constituents, 7-guanidinoheptanamide and glyoxylylspermidine, and its possible metabolites was achieved by using CM-Sephadex C-25 column chromatography with stepwise elution with sodium chloride solution. Treatment of deoxyspergualin with acetylacetone resulted in the formation of the volatile pyrimidine derivative of 7-guanidinoheptanamide accompanying hydrolytic cleavage in the alpha-hydroxyglycine moiety of deoxyspergualin. Deoxyspergualin could be determined in a concentration of 10 ng/ml in plasma. The method was applied to the determination of deoxyspergualin in plasma during and after intravenous infusion into dogs.

Animals↗