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Biomedical subjects

Y Ike

Publications and source records attributed to Y Ike.

At least 37 records · Page 2Linked to original sources
PubMed1996

Enhancement of catalytic activities of serine proteases by tripeptides compounds.

The tripeptide compounds, Glu-Arg-Pro-amide (ERPm), D-Pro-Thr-Trp-amide (dPTWm) and thioproline-Thr-Trp (tPTW), were obtained by screening of synthetic peptides for growth-inhibitory activity toward cultured transformed cells. The effects of these peptide compounds on proteases were investigated and the results showed that these compounds enhanced the amidolytic activity of serine proteases despite the fact that each reaction was carried out under optimal conditions. ERPm stimulated the activities of trypsin, chymotrypsin, thrombin, plasmin urokinase and elastase. dPTWm also showed similar effects except that toward chymotrypsin. tPTW elevated the activity only of trypsin, chymotrypsin and thrombin. Stimulation of trypsin activity by these compounds was also confirmed by using casein as a substrate. None of these compounds affected the amidolytic activities of metalloproteinases (MMP-1 and MMP-9), cysteine proteinases (m- and mu-calpains, cathepsin B and papain) or an exopeptidase (leucine aminopeptidase). The activation was at least partly due to the stabilization of the catalytic activity of proteases as well as prevention of autolysis.

Amino Acid Sequence↗
PubMed1996

The traA gene of the Enterococcus faecalis conjugative plasmid pPD1 encodes a negative regulator for the pheromone response.

The Enterococcus faecalis conjugative plasmid pPD1 (59 kb) produces an aggregation substance in response to pheromone. The traA gene (962 bp) is a member of a gene cluster involved in the regulation of the pPD1 pheromone response. A chimeric E. faecalis suicide plasmid containing a 762-bp DNA fragment from the middle portion of the traA gene was constructed. The pPD1 traA was disrupted by integration of this chimeric plasmid via homologous recombination. The E. faecalis strain containing pPD1 with the disrupted traA exhibited dry colony morphology, constitutive clumping, and an ability to transfer at high frequencies in a short (10-min) mating period, indicating that the traA product of pPD1 is a negative regulator for the pheromone response.

Bacterial Proteins↗
PubMed1996

Cloning and genetic organization of the bacteriocin 31 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pYI17.

The conjugative plasmid pYI17 (57.5 kb) isolated from Enterococcus faecalis YI717 confers a pheromone response on the host and encodes the bacteriocin 31 gene. Bacteriocin 31 is active against E. hirae 9790, E. faecium, and Listeria monocytogenes. pYI17 was mapped physically by restriction enzyme analysis and the relational clone method. Deletion mutant and sequence analyses of the EcoRI fragment B cloned from pYl17 revealed that a 1.0-kb fragment contained the bacteriocin gene (bacA) and an immunity gene (bacB). This fragment induced bacteriocin activity in E. faecalis OG1X and E. hirae 9790. The bacA gene is located on the pYI17 physical map between 3.37 and 3.57 kb, and bacB is located between 3.59 kb and 3.87 kb, bacA encodes 67 amino acids, and bacB encodes 94 amino acids. The deduced amino acid sequence of the bacA protein contained a series of hydrophobic residues typical of a signal sequence at its amino terminus. The predicted mature bacA protein (43 amino acids) showed sequence homology with the membrane-active class II bacteriocins of lactic acid bacteria. Analysis of Tn5 insertion mutants and the resulting transcripts indicated that these genes are transcribed as an operon composed of bacA, bacB, and an open reading frame located downstream of bacB designated ORF3.

Amino Acid Sequence↗
PubMed1996

Differential chemosensitivity in oncogene-transformed cells.

The effects of anticancer drugs on cell growth have been investigated by MTT colorimetric assay using mouse and rat cultured cells transformed by various oncogenes and tumor viruses. Aclarubicin, mitomycin C and 1-hexylcarbamoyl-5-fluorouracil showed higher growth-inhibitory activities toward transformed cells than those toward the normal counterparts. Nimustine, bleomycin and 5-fluorouracil also showed selective growth-suppressive activities toward transformed cells except for a few cell lines. ras-oncogene-transformed cells were more sensitive toward 5-fluorouracil and 1-hexylcarbamoyl-5-fluorouracil than the normal parent cells and other transformed cells. These drugs would thus be effective in the chemotherapy of ras-induced cancer.

Animals↗
PubMed1995

Increase of cyclin B by overexpression of cystatin alpha.

Degradation of cyclin B was effectively suppressed when cells were treated with ALLN (N-acetylleucylleucylnorleucinal) which inhibits proteasome, calpain and cysteine proteinase cathepsins. In order to examine which protease degrades cyclin B, the effect of a cathepsin inhibitor, cystatin alpha, was investigated. The cystatin alpha gene was inserted into an inducible expression vector, pMSG, and transfected into NIH3T3 mouse fibroblasts. The expression of cystatin alpha was induced effectively in the transfected cells after treatment with dexamethasone. Overexpression of cystatin alpha resulted in an increase of the amount of cyclin B, suggesting that cysteine proteinase cathepsins might be involved in the degradation of cyclin B.

3T3 Cells↗
PubMed1995

Physical mapping of the conjugative bacteriocin plasmid pPD1 of Enterococcus faecalis and identification of the determinant related to the pheromone response.

The pheromone-responding conjugative bacteriocin plasmid pPD1 (59 kb) of Enterococcus faecalis was mapped physically by using a relational clone approach, and transposon analysis with Tn917 (Emr) or Tn916 (Tcr) facilitated the location of the bacteriocin-related genes in a segment of about 6.7 kb. Tn917 insertions within a 3-kb region resulted in constitutive clumping. The nucleotide sequence of the region that included the insertions giving rise to constitutive clumping was determined. The region of pPD1 spanned about 8 kb and was found to contain a number of open reading frames, some of which were named on the basis of homologies with two other pheromone-responding plasmids, pAD1 and pCF10. The genes were arranged in the sequence repB-repA-traC-traB-traA-ipd-traE-traF- orfY-sea-1 with all but repB and traA oriented in the same (left-to-right) direction. traC and traB corresponded, respectively, to traC and traB of pAD1 and to prgY and prgZ of pCF10.

Amino Acid Sequence↗
PubMed1992

Incorporation and degradation by kidney lysosomes of cystatin alpha injected intravenously.

Cystatin alpha was purified from Escherichia coli transfected with a recombinant cystatin alpha gene and injected into the tail vein of rats. Its fate was then followed using a sensitive enzyme immunoassay for cystatin alpha. Results showed that it was rapidly removed from the blood and taken up by the kidney. Percoll density-gradient analysis showed that it was rapidly incorporated into lysosomes in the kidney. Its level in the kidney was maximal 30 min after its injection then rapidly decreased. The activity of cathepsin H in the kidney lysosomal fraction was markedly decreased 30 min after injection of cystatin alpha but recovered rapidly. Anti-(cystatin alpha) antibody precipitated cathepsin H and anti-(cathepsin H) antibody precipitated cystatin alpha in an extract of the lysosome-rich fraction of the kidney 30 min after injection of cystatin alpha. These results indicate that cystatin alpha was taken up by kidney lysosomes, formed a complex with cathepsin H and initially decreased the activity of cathepsin H, but that later the level of cystatin alpha in the kidney rapidly decreased.

Animals↗
PubMed1992

Evidence that the hemolysin/bacteriocin phenotype of Enterococcus faecalis subsp. zymogenes can be determined by plasmids in different incompatibility groups as well as by the chromosome.

The hemolysin (Hly/Bac) determinant in strains of Enterococcus faecalis was found to be present on plasmids in different incompatibility groups (conferring different sex pheromone responses) as well as on the chromosome. Of 33 Hly/Bac plasmids identified in clinical isolates, the related pheromone for 30 was cAD1; the related pheromone for another two (pYI1 and pYI3) or one (pYI2) was cOB1 or cY12, respectively. The representative Hly/Bac plasmids pAD1, pYI1, pOB1, and pYI2, which responded to pheromones cAD1, cOB1, cOB1, and cYI2, respectively, were compatible with one another. As additions to the incompatibility group IncHly of pAD1, groups for pOB1, pYI1, and pYI2 were designated IncHlyII, IncHlyIII, and IncHlyIV, respectively. Eleven of the 30 plasmids conferring a response to cAD1 were very similar to pAD1 on the basis of their restriction endonuclease profiles. EcoRI fragment D, F, or H containing parts of the Hly/Bac gene(s) of pAD1 hybridized to similar EcoRI fragments from each of the other three representatives of incompatibility groups (i.e., pOB1, pYI1, and pYI2) and to homologous DNA representing the chromosome of the plasmid-free Hly/Bac strain YI6-1.

Bacteriocins↗
PubMed1992

Hyperhemolytic phenomena associated with insertions of Tn916 into the hemolysin determinant of Enterococcus faecalis plasmid pAD1.

Members of the Tn916 family of conjugative transposons are able to insert themselves into Enterococcus faecalis hemolysin/bacteriocin plasmid pAD1 (and related elements) in such a way as to generate hyperexpression of the hemolysin/bacteriocin. To examine this phenomenon in more detail, E. faecalis (pAD1::Tn916) derivatives defective or altered in hemolysin expression were isolated and characterized with respect to production of the L (lytic) or A (activator) component (also known as CylA) and the specific location of the transposon. The mutants fell into five classes. Class 1 strains were nonhemolytic, and the related insertions mapped in a location known to affect expression of the L component. The other four classes varied from an inability to express hemolysin (class 2) to different degrees of hyperhemolytic expression (classes 3 to 5); the insertions in these classes mapped in a similar place within cylA, near the 3' end of the determinant. A previous study provided evidence that CylA is also necessary for bacteriocin immunity; however, these insertions did not destroy this function. (A Tn917 insertion in the 5' half of the determinant eliminates immunity.) In mutant classes 3 to 5, the presence of tetracycline enhanced hemolysin expression. In late-exponential-phase broth cultures, hemolysin could not be detected in supernatants of classes 2 to 5, in contrast to a wild-type control strain; however, different amounts of the L component could be detected, with the lowest in class 2 and greater-than-normal amounts in classes 3 to 5. Although nucleotide sequencing showed that the Tn916 insertions in classes 2 to 5 were at identical sites, the transposon junction sequences differed in some cases. The data indicated that cylA translation into the transposon would result in different truncation sites, and these differences were probably related to phenotype differences.

Amino Acid Sequence↗
PubMed1991

Low cost device for electrotransformation and its application to the highly efficient transformation of Escherichia coli and Enterococcus faecalis.

A simple, low cost device for electrotransformation has been designed and constructed. The cost of the circuit was only $150. Maximum field strength of 12,000 V/cm with an actual time constant up to 11 msec was obtained with a newly designed circuitry and a 0.1-cm electrode gap cuvette. Eschericia coli strains DH1, DH5 alpha, and LE392 were transformed at an efficiency of 10(9)/micrograms DNA with plasmids pUC18 and pBR322. E. faecalis strain OG1X was transformed at an efficiency of 0.9 x 10(5)/micrograms DNA without treatment with glycine.

DNA, Bacterial↗
PubMed1990

Genetic analysis of the pAD1 hemolysin/bacteriocin determinant in Enterococcus faecalis: Tn917 insertional mutagenesis and cloning.

Thirty-seven nonhemolytic/nonbacteriocinogenic mutations in Enterococcus (Streptococcus) faecalis plasmid pAD1 were generated by Tn917 insertion. All were found to belong to one of two complementation classes. Each class of mutants secreted either hemolysin/bacteriocin (Hly/Bac) component A or L into the culture medium. DNA encoding Hly/Bac was cloned in Escherichia coli in which both components of the hemolysin were expressed individually and collectively. The region encoding components A and L was further defined by deletion analysis and physically mapped. A total of approximately 8.4 kilobases of pAD1 DNA were observed to be required for hemolysin expression. Hly/Bac activity of the wild-type and the inactive L substance was observed to be heat stable. Active Hly/Bac resulting from incubating separately secreted components A and L was also found to be heat stable. The results indicate that component A activates component L and that activated component L possesses the Hly/Bac activity. Component A was also observed to be associated with host immunity to the Hly/Bac.

Bacteriocins↗
PubMed1989

Studies on the reactive site of the cystatin superfamily using recombinant cystatin A mutants. Evidence that the QVVAG region is not essential for cysteine proteinase inhibitory activities.

For study of the inhibition mechanism of the cystatin superfamily, cystatin A artificial mutants were obtained in which a well-conserved QVVAG region in the cystatin superfamily was changed to KVVAG or QVTAG and these mutants were then expressed in E. coli. For this, genes with these sequences were synthesized enzymatically from 11 oligodeoxynucleotides and expressed under the tac promoter gene of the E. coli plasmids. The products expressed were then purified on Sephadex G-50 and HPLC DEAE-5PW columns. The substitutions in cystatin A were confirmed by the amino acid compositions, N-terminal amino acid sequences and elution positions on ion-exchange chromatography of the products. The Ki values of these products for the cysteine proteinases, papain and cathepsins B, H and L, were determined in comparison with those of wild type recombinant cystatin A. Results showed that the cystatin A mutants had similar inhibitory activities to those of wild type recombinant cystatin A. Namely replacement of amino acids in the QVVAG sequence of cystatin A did not significantly affect the inhibitory activities on these proteinases. The results suggest that the QVVAG region is less important than the N-terminal region of cystatin for inhibitory activities on cysteine proteinases.

Amino Acid Sequence↗
PubMed1988

Total synthesis of the cystatin alpha gene and its expression in E. coli.

A gene encoding cystatin alpha has been chemically synthesized, cloned and expressed in E. coli. The gene of 318 base pairs was assembled by enzymatic ligation of 19 oligonucleotides and cloned into a pBR322-derived expression plasmid down stream of the tac promoter. The expression product of the synthetic gene has been purified by Sephadex G-50 column chromatography and shown to have the same properties as those of the authentic protein isolated from rat epidermis.

Amino Acid Sequence↗
PubMed1988

Sequence analysis of termini of conjugative transposon Tn916.

Transposon Tn916 is a 16.4-kilobase, broad-host-range, conjugative transposon originally identified on the chromosome of Enterococcus (Streptococcus) faecalis DS16. Its termini have been sequenced along with the junction regions for two different insertions. The ends were found to contain imperfect inverted repeat sequences with identity at 20 of 26 nucleotides. Further in from the ends, imperfect directly repeated sequences were present, with 24 of 27 nucleotides matching. The transposon junction regions contained homologous segments but of a nature not consistent with a direct duplication of the target sequence. Within the right terminus was a potential outwardly reading promoter. Tn916 is believed to transpose via an excision-insertion mechanism; based on the analyses of the termini, as well as two target sequences (before insertion and after excision), a possible model is suggested.

Base Sequence↗