PubMed Health⌕ Search

Biomedical subjects

E Nakajima

Publications and source records attributed to E Nakajima.

At least 19 recordsLinked to original sources
PubMed2006

Amelioration of retinal degeneration and proteolysis in acute ocular hypertensive rats by calpain inhibitor ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester.

BACKGROUND: Our recent study suggested involvement of calpain-induced proteolysis in retinal degeneration and dysfunction in acute ocular hypertensive rats. The purpose of the present study was to determine if an orally available form of calpain inhibitor, ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), ameliorated retinal degeneration induced by acute hypertension in rats. To help extrapolate the effect of SNJ-1945 from the rat model to the human glaucomatous patient, in vitro inhibition of calpain-induced proteolysis by SNJ-1945 in monkey and human retinal proteins was compared with proteolysis in rat proteins. METHODS: Intraocular pressure (IOP) in rats was elevated to 110 mm Hg for 50 min. SNJ-1945 was administrated i.p. or orally before ocular hypertension. Retinal degeneration was evaluated by hematoxylin and eosin (H&E) staining and cell counting. Transcripts for calpains and calpastatin in rat, monkey, and human retinas were measured by quantitative RT-PCR. Calpain activities were determined by casein zymography. Soluble retinal proteins from rat, monkey, and humans were incubated with calcium to activate calpains, with or without SNJ-1945. Proteolysis of calpain substrate alpha-spectrin was analyzed by immunoblotting. RESULTS: Elevated IOP caused retinal degeneration and proteolysis of alpha-spectrin. Both i.p. and oral administration of SNJ-1945 inhibited proteolysis of alpha-spectrin and ameliorated retinal degeneration. Transcript levels for calpain 1 and calpastatin were similar in rat, monkey, and human retinas. Calpain 2 transcript levels were higher in rats compared with monkey and human. Appreciable caseinolytic activities due to calpains were observed in monkey and human retinas. Incubation of retinal soluble proteins with calcium led to proteolysis of alpha-spectrin due to calpains in rat, monkey, and human samples. SNJ-1945 similarly inhibited proteolysis in all species. CONCLUSION: Our results suggested that orally available calpain inhibitor SNJ-1945 might be a possible candidate drug for testing in preventing progression of glaucomatous retinal degeneration.

Animals↗
PubMed2006

Low activity by the calpain system in primate lenses causes resistance to calcium-induced proteolysis.

The human genome contains 14 genes for 80 kDa catalytic subunit of the calcium-activated protease calpain (EC 34.22.17), yet no calpain-like cleavage sites have been detected on human lens crystallins in vivo. The purpose of the present study was to provide a comprehensive study of calpain activation in human and macaque lenses developing experimental cataract due to lens culture in ionophore A23187. Zymography was used to measure calpain activity; SDS-PAGE and immunoblotting were used to detect hydrolysis of potential lens protein substrates. Quantitative PCR was used to measure transcripts for calpains and the endogenous inhibitor calpastatin. We found that the lack of appreciable calpain-induced proteolysis in primate lenses is most likely due to relatively low levels of endogenous calpain activity compared to the high levels of endogenous calpain inhibitor, calpastatin.

Aged↗
PubMed2005

Biochemical properties of lens-specific calpain Lp85.

Lens-specific Lp82 and ubiquitous m-calpain are neutral, calcium-activated, cysteine proteases. Both calpains are activated during rodent lens maturation and cataract formation. Lp85 calpain (Lens protein with MW=85 kDa) is a slightly larger splice variant of Lp82. Lp85 contains a 28 amino acid insert peptide (IS3) in calcium binding domain IV. Theoretically, the insert could alter the properties of Lp85 and influence proteolytic activity. The purpose of the present experiment was to compare the biochemical properties of Lp85 to Lp82 and m-calpain. Recombinant Lp85 and Lp82 were separately expressed using the baculovirus system and partially purified using Co2+ affinity and DEAE chromatographies. Calcium activation, pH dependency, and susceptibility to calpain inhibitors were assessed in a protease assay using BODIPY fluorescence-labeled casein substrate. Hydrolysis of lens proteins was assessed by SDS-PAGE and immunoblotting. Cleavage site analysis was performed by mass spectroscopy and Edman sequencing. Computer-based homology modeling was used to predict the influence of the IS3 region on the 3-dimensional structure of Lp85. Compared to m-calpain, Lp85 showed a lower calcium-activation requirement (K(50%act)=20 microM), marked insensitivity to, and cleavage of, the endogenous tissue inhibitor of calpains-calpastatin, and different preferred cleavage sites on alphaA-crystallin (five amino acid C-terminal truncation) and on aquaporin 0 (G239 and N246). Although the IS3 insert was predicted to form a loop protruding from the calcium binding region of Lp85, the biochemical properties of Lp85 studied were nearly identical to those of Lp82. Lp85 and Lp82 did not catalyze hydrolysis of each other, but both hydrolyzed m-calpain. Lp85 seems to be the enzymatic equivalent of Lp82. Both calpains could become active at lower cellular calcium levels than m-calpain. Lp85/Lp82 may have different functions than m-calpain since they cleave substrates at different sites. Lp85/Lp82 may regulate m-calpain activity by catalyzing the hydrolysis of calpastatin. The function of the IS3 insert on Lp85 remains unknown but is speculated to control subcellular distribution.

Animals↗
PubMed2001

Three types of polymorphisms in exon 14 in porcine Mx1 gene.

Much is known about the antiviral activity of Mx proteins in species such as mouse and human. In the mouse, loss of resistability to influenza virus has been shown to be due to specific polymorphisms in the Mx gene. This gene is therefore an interesting candidate gene for disease resistance in farm animals. The porcine Mx1 gene has already been identified and characterized based on its homology with mouse Mx1; however, until now no evidence of polymorphisms in the porcine gene has been reported. In this study, we have found two new polymorphisms in exon 14 of porcine Mx1 by DNA sequencing and confirmed their presence in different breeds, using polymerase chain reaction (PCR)-restriction fragment length polymorphisms (RFLP) with NarI and NaeI restriction enzymes. On the basis of the deduced amino acid sequence, one allele contains a deletion that may result in a frameshift to yield several amino acid substitutions and extension of the carboxyl terminal region of Mx1 protein. The deletion allele, Mx1c, was found to be segregating in Landrace, Berkshire, Duroc, Hampshire, and Yucatan miniature pig. A second point mutation, Mx1b, was detected in Meishan and two Vietnamese native pig breeds. All other breeds tested were fixed for the Mx1a allele that is identical to the sequence reported previously. It will be interesting to determine if the Mx1c deletion is associated with variation in resistance to the myxovirus family in the pig.

Amino Acid Sequence↗
PubMed2001

Specific inhibition of HIV-1 gene expression by double-stranded RNA.

RNA interference (RNAi) is phenomenon in which introduced double-stranded RNAs (ds-RNAs) silence gene expression through specific degradation of their cognate mRNAs. RNAi has been observed in a wide variety of organisms and is considered to be a feature of nearly all eukaryotes. The mediators of sequence specific messenger RNA degradation are 21-23 nucleotide small interfering RNAs generated by ribonucleaseIII cleavage from longer dsRNAs. To investigate the potential of dsRNA to interfere with the function of HIV-1 genes, we have designed six longer dsRNAs containing the HIV-1 gag and env genes. Double-stranded RNAs were tested for inhibitory effects using monkey COS cells. In these anti-HIV-1 activity tests, the dsRNAs targeted to the HIV-1 env sequence showed more than 90% anti-HIV-1 efficiency. Our results show that dsRNA interference can be used as a powerful method for the inhibition of HIV-1 gene expression.

Animals↗
PubMed2001

Establishment of enzyme immunoassay for measuring serum sultopride levels.

Measurement of serum sultopride levels was performed using an enzyme immunoassay. Little or no cross-reactivity with metabolites of sultopride and other drugs was found. The results of reproducibility, recovery, and dilution testing were all good enough for clinical use. A comparison between the measurement values of this method (y) with that of high-performance liquid chromatography (x) showed high correlation (n = 211, r = 0.991, p < 0.0001, y = 0.99x + 107.5). In a comparison between the sultopride dose and serum levels in 161 patients, interindividual differences were large (19 times for same doses), implying that the serum level cannot be predicted from the dosage. The method was found to be reliable for serum level measurements of sultopride and useful for monitoring compliance and assessing the optimal dose.

Adult↗
PubMed2001

Radical cyclization in heterocycle synthesis. 12. Sulfanyl radical addition-addition-cyclization (SRAAC) of unbranched diynes and its application to the synthesis of A-ring fragment of 1alpha,25-dihydroxyvitamin D3.

Sulfanyl radical addition-addition-cyclization (SRAAC) of unbranched diynes proceeded smoothly to give cyclized exo-olefins, while the sulfanyl radical addition-cyclization-addition (SRACA) of diynes having a quaternary carbon gave cyclized endo-olefins. This method was successfully applied to the synthesis of A-ring fragment of 1alpha,25-dihydroxyvitamin D3.

Calcitriol↗
PubMed2001

K-ras mutation in sputum of primary lung cancer patients does not always reflect that of cancerous cells.

K-ras mutation in sputum was examined using mutant-allele-specific amplification method among 100 primary lung cancer and 15 non-oncological patients. K-ras mutation was detected in 11 out of 59 adenocarcinoma cases (18.6%), 5 out of 32 squamous cell carcinoma cases (15.6%), 2 out of 4 large cell carcinoma cases (50.0%) and 3 out of 15 non-oncological disease cases (20.0%). In the 18 cases of primary lung cancer K-ras mutation was examined in both sputum and the resected specimen of the primary lesion. In 5 cases K-ras mutation in sputum was detected without K-ras mutation in primary lesion. Therefore, these findings suggested that K-ras mutation in sputum may not be directly related to that of the primary lesion.

Adult↗
PubMed2000

Association between single nucleotide polymorphisms in the hMSH3 gene and sporadic colon cancer with microsatellite instability.

The association between three single nucleotide polymorphisms (SNPs) in the hMSH3 gene and sporadic colon cancer with microsatellite instability (MSI) was analyzed. Of the three SNPs observed in this population, SNPs at residues 235 and 693 were novel, while that at residue 3133 was previously described. The SNPs at residues 235 and 3133 caused amino acid substitutions, V79I and T1045A, respectively. We analyzed the allele frequencies of the three SNPs in samples from 19 patients with sporadic colon cancer with MSI and 90 healthy controls. We found that the V79 allele frequency was significantly higher in the tumor samples than in controls. In addition, the frequency of the G693 allele showed a higher trend in the tumor samples than in controls. These results indicated that some SNPs in the hMSH3 gene were associated with colon cancer with MSI.

Alleles↗
PubMed2000

Involvement of apoptosis in the endotoxemic lesions of the liver and kidneys of piglets.

The involvement of apoptosis was evaluated in lesions of endotoxemic piglets. A single injection with E. coli O111:B4 lipopolysaccharide (LPS) induced foci of coagulative necrosis in the liver and kidneys. No significant change was observed in these organs at 1.5 hr after LPS injection, but at 6 hr, epithelial cells with chromatin condensation or fragmentation and apoptotic bodies were visible. Foci of coagulative necrosis were formed within 24 hr after LPS inoculation. In and adjacent to the necrotic foci, dead hepatocytes with nuclear condensation or fragmentation were scattered. These dead cells were positively stained by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) methods. Electronmicroscopy revealed apoptotic cells with condensed or fragmented homogeneous nuclear chromatin, and necrotic cells with irregularly destroyed nuclei and cytoplasmic membranes. Apoptotic cell death were also observed in parietal cells of the stomach and lymphocytes in the lymphatic system. DNA ladders with approximately 200-bp multimers were observed in hepatic, renal and thymic samples prepared after 6 and 24 hr of LPS injection by agarose gel electrophoresis. These results suggest that apoptosis is involved in the pathology of swine endotoxemia.

Animals↗
PubMed1999

Identification of the protein components of mismatch binding complexes in human cells using a gel-shift assay.

In eukaryotes, mismatch recognition is thought to be mediated by two heterodimers, hMutSalpha (hMSH2+hMSH6), which preferentially binds to base-base mismatches and hMutSbeta (hMSH2+hMSH3), which binds to insertion/deletion loops. We studied these mismatch binding activities in several human cell lines with a gel-shift assay using various mismatch oligonucleotides as substrates. Both hMutSalpha and hMutSbeta activities could be detected in various human cell lines. In cells with amplified copies of the hMSH3 gene, a large increase in hMutSbeta and a reduction in hMutSalpha were observed. To identify the composition of each mismatch binding complex, the protein-DNA complexes were transferred from gel-shift polyacrylamide gel to a polyvinylidene difluoride membrane and were subjected to immunoblot analysis with an enhanced chemiluminescence protein detection system. The results clearly demonstrated that hMutSalpha detected by the gel-shift assay was composed of hMSH2 and hMSH6, while hMutSbeta was composed of hMSH2 and hMSH3. Our data, therefore, support a model whereby formation of hMutSalpha and hMutSbeta is mutually regulated. Combination of a gel-shift assay with immunoblotting (shift-Western assay) proved to be a highly sensitive technique and should be useful for studying the interactions between DNA and binding proteins, including DNA mismatch recognition.

Base Pair Mismatch↗
PubMed1999

Frameshift mutations and a length polymorphism in the hMSH3 gene and the spectrum of microsatellite instability in sporadic colon cancer.

Mutations in the hMVSH3 gene in sporadic colon cancer with microsatellite instability (MSI) were investigated, since several mismatch repair genes were known to be mutated in cancers with MSI, but only deletions in the (A)8 region in the hMSH3 gene have been reported. We also analyzed the relationships between hMSH3 mutations and the spectrum of MSI. We screened MSI in 79 sporadic colon cancer samples using mono- and dinucleotide repeat markers and the samples with MSI were further analyzed for tri- and tetranucleotide repeat instability and mutations in the hMSH3 gene by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Five (6%) out of 79 tumors were MSI-H and 15 (19%) were MSI-L. Two MSI-H tumors showed insertion in the (C)8 region in the hMSH6 gene and one tumor showed insertion and deletion in the (A)8 region in the hMSH3 gene, and two of the three above tumors showed MSI in tri-and tetranucleotide repeats. One MSI-L tumor showed somatic alteration in a 9-bp repeat sequence in hMSH3. No frameshift mutations were found in the (A)7 and (A)6 regions in hMSH3. Thus, we confirmed that the (A)8 region in hMSH3 is a hot spot and mutations in the (A)7 and (A)6 regions in hMSH3 are not common. The hMSH3 mutation may enhance genomic instability in some colorectal cancers.

Base Pair Mismatch↗
PubMed1999

[Effects of olprynone hydrochloride and milrinone on the Ca(2+)-related functions of skinned skeletal muscle fibers from the guinea pigs].

It was reported that phosphodiesterase (PDE) inhibitors such as caffeine, theophylline and amrinone accelerate Ca(2+)-induced Ca2+ release (CICR) rate from sarcoplasmic reticulum (SR) in skeletal muscle. In this study, the effects of PDE III inhibitors, olprynone hydrochloride and milrinone, on the Ca(2+)-related functions (CICR rate from SR, Ca2+ uptake into SR and Ca2+ sensitivity of the contractile system) of the skeletal muscle were examined using the chemically skinned fiber technique (Endo's method) in the extensor digitorum longus of gunia pigs. Olprynone hydrochloride and milrinone accelerated CICR rate in a dose-dependent manner (at higher concentrations than their clinical concentrations). Initial rate of Ca2+ uptake into SR and Ca2+ sensitivity of the contractile system were not affected at 1 mM of olprynone hydrochloride and 0.3 mM of milrinone. These results suggest that olprynone hydrochloride and milrinone may be safe to use within the limits of the clinically recommended concentrations in a MH susceptable patient, especially in a case in which CICR rate is accelerated compared with that in a normal patient using the skinned fiber test.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Close correlation between mutations of E2F4 and hMSH3 genes in colorectal cancers with microsatellite instability.

Defects in mismatch repair function can lead to the microsatellite instability (MI+; replication error) phenotype in certain human cancers. We previously reported that MI+ tumor-specific repeat number alteration at 13 consecutive trinucleotide (CAG) repeats within a coding exon of the E2F4 gene is a possible target of the defective repair pathway. Additional investigations revealed that E2F4 mutations are common (11 of 17 cases, 65%, mostly deletions) in a subset of human colorectal cancers with extensive MI+ phenotype, with respect to the proportion of loci affected and that most of these E2F4-mutated tumors (9 of 11, 82%) were accompanied by frameshift mutations in a polyadenine stretch within the seventh exon of the hMSH3 gene, a known mismatch repair gene that is responsible for repair of mismatch loops of two to four nucleotides. However, neither of these mutations was detected in 15 tumors with a lower incidence of MI+ loci. Similar repeat number alterations were less frequent in CAG repeats from other genes in all of the MI+ tumors we examined. These results indicate the presence of a novel cascade of mutational events that may be involved in acquisition of the malignant phenotype of human colorectal cancers with genetic instability.

Colorectal Neoplasms↗
PubMed1998

A novel missense mutation and frameshift mutations in the type II receptor of transforming growth factor-beta gene in sporadic colon cancer with microsatellite instability.

Microsatellite instability of DNA samples of 79 sporadic colon cancer patients were analyzed. These samples were also screened to search mutations in the repeat sequences in the gene for the type II receptor of transforming growth factor-beta (TGF-beta RII) using polymerase chain reaction (PCR), electrophoresis with urea gel, and PCR-single strand conformation polymorphism (PCR-SSCP) method. The incidence of microsatellite instability, defined as severe replication error phenotype (RER) with microsatellite alterations in more than three loci, was 6%. Deletion and insertion of an A residue in the (A)10 region, which cause frameshift mutation, were found in four samples and their incidence in the samples with microsatellite instability was 80%. A novel nucleotide substitution of T for G at 1918, which causes missense mutation of arginine to leucine at codon 528, was found in a sample with microsatellite instability. The mutation at 1918 was in highly conservative amino acid residue.

Colonic Neoplasms↗
PubMed1998

Characterization, chromosomal localization, and genetic variation of the alpha subunit of porcine eighth component of complement.

The complete amino acid sequence of the porcine alpha subunit of the eighth component of complement (C8A) was determined by characterizing the full length cDNA clone isolated from a porcine liver cDNA library. Porcine C8A was found to be similar to human and rabbit C8A in length, leader sequence, conserved cysteine residues, cysteine-rich modules, and overall sequence. Differences in the amino acid sequence among the three species were detected in the proposed candidate site for CD59 recognition (amino acids 352-389). The porcine C8A gene was physically mapped to chromosome 6q33-35 by in situ hybridization using the porcine bacterial artificial chromosome (BAC) clone as a hybridization probe. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of C8A was performed using the restriction enzyme Hha I. Distribution of the alleles was determined in pigs (n = 173) of several different breeds. Estimates of allele frequency of the 201 bp fragment were 0.22,.0.43,.0.04,.0.50,.0.58,.0.50,.0.98, and 0.91 in Landrace, Large White, Duroc, Berkshire, Jinhua, Crown Miniature Pig, wild boar, and Meishan, respectively.

Alleles↗
PubMed1996

hMutSbeta, a heterodimer of hMSH2 and hMSH3, binds to insertion/deletion loops in DNA.

In human cells, mismatch recognition is mediated by a heterodimeric complex, hMutSalpha, comprised of two members of the MutS homolog (MSH) family of proteins, hMSH2 and GTBP [1,2]. Correspondingly, tumour-derived cell lines defective in hMSH2 and GTBP have a mutator phenotype [3,4], and extracts prepared from these cells lack mismatch-binding activity [1]. However, although hMSH2 mutant cell lines showed considerable microsatellite instability in tracts of mononucleotide and dinucleotide repeats [4,5], only mononucleotide repeats were somewhat unstable in GTBP mutants [4,6]. These findings, together with data showing that extracts of cells lacking GTBP are partially proficient in the repair of two-nucleotide loops [2], suggested that loop repair can be GTBP-independent. We show here that hMSH2 can also heterodimerize with a third human MSH family member, hMSH3, and that this complex, hMutSbeta, binds loops of one to four extrahelical bases. Our data further suggest that hMSH3 and GTBP are redundant in loop repair, and help explain why only mutations in hMSH2, and not in GTBP or hMSH3, segregate with hereditary non-polyposis colorectal cancer (HNPCC) [7].

Base Sequence↗