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Biomedical subjects

T Kitagawa

Publications and source records attributed to T Kitagawa.

At least 55 records · Page 3Linked to original sources
PubMed2001

Cellular biology of cryopreserved allograft valves.

Although analyzing the precise mechanisms of cryopreserved allograft valve failure may be difficult due to a number of crucial reasons and the interrelationships between the overlapping mechanisms, there is some evidence that cryopreservation is currently the best method of storing allograft valves. The present review shows the basic cellular biology of cryopreserved allograft valves for long-term durability, particularly relevant to allograft valve cellular viability, the immune response mainly caused by viable donor cells, and the preservation and regeneration of the intrinsic extracellular matrix. The present findings are as follows. First, cryopreservation produces serious damage to cytosolic and mitochondrial functions of both endothelial cells and fibroblasts, which may cause valve failure after implantation. Second, although the collagen synthesis of cryopreserved valves was relatively maintained, total protein synthesis was highly diminished and the collagenolytic ability was activated immediately after the thawing process. These findings imply that the cryopreservation itself may cause the collagen metabolism to become degradable, which will lead to valve failure. Further examination of collagen metabolism and modulation of the collagenolytic activity will be necessary to improve the tissue preservation for improved clinical use.

Apoptosis↗
PubMed2001

Characteristics of Pseudomonas aeruginosa isolated from skin infections.

From January 1995 to June 2000, 19 Pseudomonas aeruginosa were detected in 19 cases of infectious skin diseases. Coagulase-negative staphylococci coexisted with P. aeruginosa in 10 cases and P. aeruginosa was also often associated with Enterococcus sp., Staphylococcus aureus, Streptococcus sp. or other species. Twelve cases carried P. aeruginosa more predominantly than the other species. Some coagulase-negative staphylococci, Enterococcus sp., S. aureus and Streptococcus sp. were also predominant. The frequency of predominant P. aeruginosa was significantly higher in secondary infections due to ulcers and decubitus than in secondary infections due to other skin diseases. P. aeruginosa was also often detected in the legs and feet, but not in the trunk. This organism showed high sensitivities to antibiotics, except minocycline. No significant differences in sensitivity or resistance to antibiotics were found between P. aeruginosa when detected as the predominant or single strain and this microorganism when detected as the non-predominant strain. Thus, the detection of P. aeruginosa important in choosing the appropriate antibiotics to be used and this is sometimes influenced by the patient's condition.

Adult↗
PubMed2001

Effect of lipase activities of Propionibacterium granulosum and Propionibacterium acnes.

We studied the lipase activities of Propionibacterium granulosum, P. acnes and the suppression of these activities by Jumi-haidoku-to (JHT). Lipase activity of P. acnes biotype III (BIII) was strongest, while that of P. granulosum was faintly expressed. Compared with the control medium, the production of propionic and butyric acids was suppressed by all the tested mediums combined with JHT. The decrease in these acids produced by JHT was significantly higher in P. granulosum than in P. acnes. Although P. acnes BIII may produce a strong effect on acne, the presence of P. granulosum should not be ignored. Further research is required on the correlation between P. acnes and P. granulosum.

Culture Media↗
PubMed2000

Heme structure of hemoglobin M Iwate [alpha 87(F8)His-->Tyr]: a UV and visible resonance Raman study.

Heme structures of a natural mutant hemoglobin (Hb), Hb M Iwate [alpha87(F8)His-->Tyr], and protonation of its F8-Tyr were examined with the 244-nm excited UV resonance Raman (UVRR) and the 406.7- and 441.6-nm excited visible resonance Raman (RR) spectroscopy. It was clarified from the UVRR bands at 1605 and 1166 cm(-)(1) characteristic of tyrosinate that the tyrosine (F8) of the abnormal subunit in Hb M Iwate adopts a deprotonated form. UV Raman bands of other Tyr residues indicated that the protein takes the T-quaternary structure even in the met form. Although both hemes of alpha and beta subunits in metHb A take a six-coordinate (6c) high-spin structure, the 406.7-nm excited RR spectrum of metHb M Iwate indicated that the abnormal alpha subunit adopts a 5c high-spin structure. The present results and our previous observation of the nu(Fe)(-)(O(tyrosine)) Raman band [Nagai et al. (1989) Biochemistry 28, 2418-2422] have proved that F8-tyrosinate is covalently bound to Fe(III) heme in the alpha subunit of Hb M Iwate. As a result, peripheral groups of porphyrin ring, especially the vinyl and the propionate side chains, were so strongly influenced that the RR spectrum in the low-frequency region excited at 406.7 nm is distinctly changed from the normal pattern. When Hb M Iwate was fully reduced, the characteristic UVRR bands of tyrosinate disappeared and the Raman bands of tyrosine at 1620 (Y8a), 1207 (Y7a), and 1177 cm(-)(1) (Y9a) increased in intensity. Coordination of distal His(E7) to the Fe(II) heme in the reduced alpha subunit of Hb M Iwate was proved by the observation of the nu(Fe)(-)(His) RR band in the 441.6-nm excited RR spectrum at the same frequency as that of its isolated alpha chain. The effects of the distal-His coordination on the heme appeared as a distortion of the peripheral groups of heme. A possible mechanism for the formation of a Fe(III)-tyrosinate bond in Hb M Iwate is discussed.

Amino Acid Substitution↗
PubMed2000

Identification of histidine 77 as the axial heme ligand of carbonmonoxy CooA by picosecond time-resolved resonance Raman spectroscopy.

The heme proximal ligand of carbonmonoxy CooA, a CO-sensing transcriptional activator, in the CO-bound form was identified to be His77 by using picosecond time-resolved resonance Raman spectroscopy. On the basis of the inverse correlation between Fe-CO and C-O stretching frequencies, we proposed previously that His77 is the axial ligand trans to CO [Uchida et al. (1998) J. Biol. Chem. 273, 19988-19992], whereas later a possibility of displacement of His77 by CO with retention of another unidentified axial ligand was reported [Vogel et al. (1999) Biochemistry 38, 2679-2687]. Although our previous resonance Raman study failed to detect the Fe-His stretching [nu(Fe-His)] mode of CO-photodissociated CooA of the carbonmonoxy adduct due to the rapid recombination, application of the picosecond time-resolved resonance Raman technique enabled us to observe a new intense line assignable to nu(Fe-His) at 211 cm(-)(1) immediately after photolysis, while it became nondiscernible after 100-ps delay. The low nu(Fe-His) frequency of photodissociated CooA indicates the presence of some strain in the Fe-His bond in CO-bound CooA. This and the rapid recombination of CO characterize the heme pocket of CooA. The 211 cm(-)(1) band was completely absent in the spectrum of the CO-photodissociated form of the His77-substituted mutant but the Fe-Im stretching band was observed in the presence of exogenous imidazole (Im). Thus, we conclude that His77 is the axial ligand of CO-bound CooA and CO displaces the axial ligand trans to His77 with retention of ligated His77 to activate CooA as the transcriptional activator.

Bacterial Proteins↗
PubMed2000

Characteristics and biodistribution of cationic liposomes and their DNA complexes.

We have developed some novel liposome formulations for gene transfection. The formulations consisting of O,O'-ditetradecanoyl-N-(alpha-trimethyl ammonio acetyl) diethanolamine chloride (DC-6-14) as a cationic lipid, phospholipid and cholesterol showed effective gene transfection activity in cultured cells with serum and in vivo, i.e., intraperitoneal injection in mice. In this report, the physicochemical characteristics and biodistribution of the liposomes containing DC-6-14 (DC-6-14 liposomes) as a drug (gene) carrier for gene therapy were investigated in vitro and in vivo. DC-6-14 liposome-DNA complexes were usually thought to have positive surface charge. However, depending on the ratio of DNA to liposomes, zeta-potential of the complexes became negative. The diameter of the complexes also depended on the DNA-liposome ratio, and showed a maximum when their surface potential was neutral. When biodistribution of the complexes was determined after intravenous injection, positively charged complexes showed an immediate lung accumulation. On the other hand, negatively charged complexes did not show lung accumulation. These results have suggested that biodistribution of the DNA-liposome complexes, prepared with DC-6-14 liposomes, depends on their surface charge. Therefore, some surface modification of DC-6-14 liposomes may improve the biodistribution and hence the targetability of their DNA complexes.

Animals↗
PubMed2000

Acid-catalyzed rearrangement of ethynylcycloheptatriene to phenylallene.

7-Ethynylcycloheptatriene (1) cleanly isomerizes to phenylallene in the presence of acid. A mechanism involving the protonation of ethynylnorcaradiene, which is in equilibrium with 1, followed by the cleavage of a three-membered ring to give an arenium ion, is proposed. The rearrangement is accelerated by a factor of 370 by introducing tert-butyl groups on C-2 and C-5, indicating the importance of the equilibrium concentration of the norcaradiene form as a rate-controlling factor.

Journal Article↗
PubMed2000

Synthesis, structure, and H2O2-dependent catalytic functions of disulfide-bridged dicopper(I) and related thioether-copper(I) and thioether-copper(II) complexes.

A disulfide-bridged dicopper(I) complex, [Cu2(Py2SSPy2)](ClO4)2 (1) (Py2SSPy2 = bis(2-[N,N-bis(2-pyridylethyl)-amino]-1,1- dimethylethyl)disulfide), a thioether-copper(I) complex, [Cu(iPrSPy2)](ClO4) (2) (iPrSPy2 = N-(2-isopropylthio-2-methyl)propyl-N,N-bis-2-(2-pyridyl)ethylamine, and a thioether-copper(II) complex, [Cu-(PheSPy2)(H2O)](ClO4)2 (3) (PheSPy2 = N-(2-methyl-2-phenethylthio)propyl-N,N-bis-2-(2- pyridyl)ethylamine), were newly synthesized by the reactions of Cu(ClO4)2.6H2O with a thiol ligand of Py2SH (N,N-bis[2-(2-pyridyl)-ethyl]-1,1-dimethyl-2- mercaptoethylamine) and thioether ligands of iPrSPy2 and PheSPy2, respectively. For complexes 1 and 2, X-ray analyses were performed. Complex 1 crystallizes in the triclinic space group P1, and complex 2 crystallizes in the orthorhombic space group Pbca with the following unit cell parameters: for 1, a = 15.165 (3) A, b = 22.185 (4) A, c = 14.989 (3) A, alpha = 105.76 (1) degrees, beta = 90.82 (2) degrees, gamma = 75.23 (1) degrees, and Z = 2; for 2, a = 17.78 (2) A, b = 17.70 (1) A, c = 15.75 (1) A, and Z = 8. Complex 1 is the first structurally characterized example obtained by the redox reaction Cu(II) + RSH-->Cu(I) + RSSR and has two independent structures (1a, 1b) which mainly differ in S-S bond distances, Cu(I)...Cu(I) separations, and C-S-S-C dihedral angles of the disulfide units. The S-S bond distances of 2.088(7) A in 1a and 2.070(7) A in 1b are indicative of significant activation of the S-S bonds by the dicopper centers. Fragment molecular orbital (FMO) analyses and molecular orbital overlap population (MOOP) analyses based on the extended Hückel method clarify the preferable formation of the disulfide S-S bond in 1 rather than the formation of a thiolate-copper(II) complex within the Py2S- ligand framework. Catalytic functions of complexes 1-3 were investigated with peroxides (H2O2 and tBuOOH) as oxidants. Complex 1 catalyzed the selective oxidation of cyclohexane to cyclohexanol and mediated the cyclohexene epoxidation in the presence of H2O2. A transient dark green intermediate observed in the reaction of 1 with H2O2 is characterized by UV-vis, EPR, and resonance Raman spectroscopies, identifying it as a Cu(II)-OOH species, 1(OOH). The resonance Raman features of the nu(O-O) bands at 822 and 836 cm-1, which are red-shifted to 781 and 791 cm-1, respectively, upon introduction of H2(18)O2, are indicative of formation of two kinds of Cu-OOH species rather than the Fermi doublet and the significant weakening of the O-O bonds. These mechanistic studies demonstrate that by virtue of the electron-donating ability of the disulfide unit the Cu-OOH species can be actually activated for one-electron oxidation, which has been reported so far unfavorable for other vibrationally characterized Cu-OOH species.

Journal Article↗
PubMed2000

A new cationic liposome for efficient gene delivery with serum into cultured human cells: a quantitative analysis using two independent fluorescent probes.

Cationic liposomes are useful to transfer genes into eukaryotic cells in vitro and in vivo. However, liposomes with good transfection efficiency are often cytotoxic, and also require serum-free conditions for optimal activity. In this report, we describe a new formulation of cationic liposome containing DC-6-14, O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl)diethan olamine chloride, dioleoylphosphatidylethanolamine and cholesterol for gene delivery into cultured human cells. This liposome, dispersed in 5% serum-containing growth medium, efficiently delivered a plasmid DNA for GFP (green fluorescent protein) into more than 80% of the cultured human cell hybrids derived from HeLa cells and normal fibroblasts. Flow cytometric analysis revealed that the efficiency of the GFP gene expression was 40-50% in a tumor-suppressed cell hybrid, while it was greatly reduced in the tumorigenic counterpart. The enhanced GFP expression in tumor-suppressed cell hybrids was quantitatively well correlated with a prolonged presence of the plasmid DNA, which had been labeled with another fluorescent probe, ethidium monoazide, within the cells. These results suggest that a newly developed cationic liposome is useful for gene delivery in serum-containing medium into human cells and the stability of the plasmid DNA inside the cell is a crucial step in this liposome-mediated gene expression. The mechanisms by which cationic liposome mediates gene transfer into eukaryotic cells are also discussed.

Cations↗
PubMed2000

Structural factors for the formation of propellane-type products in the solvolysis of bicyclic bridgehead compounds

Methanolyses of 2-oxobicyclo[3.3.1]non-1-yl triflate, 3, 3-dimethyl-2-oxobicyclo[3.3.1]non-1-yl triflate, and 2-oxobicyclo[4. 3.1]dec-1-yl mesylate gave the corresponding propellanone in 12%, 20%, or 3.2% yield, respectively, beside substitution or rearranged products under typical conditions. No propellane-type product was obtained in the solvolyses of 1-bromobicyclo[3.3.1]nonane, 2-methylidenebicyclo[3.3.1]non-1-yl heptafluorobutyrate, and 3, 3-dimethyl-2-thioxobicyclo[3.3.1]non-1-yl tosylate. The factors that permit the formation of the propellane-type product from the intermediate bridgehead cations are examined with the aid of theoretical calculations at PM3 and B3LYP/6-31G.

Journal Article↗
PubMed2000

Resonance raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide.

Cytochrome bo from Escherichia coli, a member of the heme-copper terminal oxidase superfamily, physiologically catalyzes reduction of O(2) by quinols and simultaneously translocates protons across the cytoplasmic membrane. The reaction of its ferric pulsed form with hydrogen peroxide was investigated with steady-state resonance Raman spectroscopy using a homemade microcirculating system. Three oxygen-isotope-sensitive Raman bands were observed at 805/X, 783/753, and (767)/730 cm(-)(1) for intermediates derived from H(2)(16)O(2)/H(2)(18)O(2). The experiments using H(2)(16)O(18)O yielded no new bands, indicating that all the bands arose from the Fe=O stretching (nu(Fe)(=)(O)) mode. Among them, the intensity of the 805/X cm(-)(1) pair increased at higher pH, and the species giving rise to this band seemed to correspond to the P intermediate of bovine cytochrome c oxidase (CcO) on the basis of the reported fact that the P intermediate of cytochrome bo appeared prior to the formation of the F species at higher pH. For this intermediate, a Raman band assignable to the C-O stretching mode of a tyrosyl radical was deduced at 1489 cm(-)(1) from difference spectra. This suggests that the P intermediate of cytochrome bo contains an Fe(IV)=O heme and a tyrosyl radical like compound I of prostaglandin H synthase. The 783/753 cm(-)(1) pair, which was dominant at neutral pH and close to the nu(Fe)(=)(O) frequency of the oxoferryl intermediate of CcO, presumably arises from the F intermediate. On the contrary, the (767)/730 cm(-)(1) species has no counterpart in CcO. Its presence may support the branched reaction scheme proposed previously for O(2) reduction by cytochrome bo.

Bacterial Proteins↗
PubMed2000

Solvolysis of 2-Bicyclo

The solvolysis rate of 2-bicyclo[3.2.2]nonyl p-toluenesulfonate (6-OTs) was nearly equal to that of cycloheptyl p-toluenesulfonate in 2,2,2-trifluoroethanol (TFE). This indicates that the ethylene bridge in 6-OTs does not significantly enhance the rate and that 6-OTs ionizes without anchimeric assistance. The solvolysis of [1-(13)C]-2-bicyclo[3.2.2]nonyl p-toluenesulfonate in methanol or TFE gave 2-substituted bicyclo[3.2.2]nonane, exo-2-substituted bicyclo[3.3.1]nonane, 2-bicyclo[3.2.2]nonene (10), and 2-bicyclo[3.3.1]nonene (11), whose distributions of (13)C labels were determined by quantitative (13)C NMR analysis using a relaxation reagent. The (13)C labels were exclusively placed at only two positions, the ratios of them were not unity, and the labels in 10 were less extensively scrambled than those in other products. These results indicate that the 2-bicyclo[3.2.2]nonyl cation is classical and that 10 is formed at a former ionization stage than 2-substituted bicyclo[3.2.2]nonane. The (13)C redistributions for both exo-2-substituted bicyclo[3.3.1]nonane and 11, which are yielded via 1,3-hydride shift, were similar to that of 2-substituted bicyclo[3.2.2]nonane, suggesting that 1,3-hydride shift occurs mainly at the solvent-separated ion pair.

Journal Article↗
PubMed2000

Malignant transformation and EGFR activation of immortalized mouse liver epithelial cells caused by HBV enhancer-X from a human hepatocellular carcinoma.

We have previously observed that all human hepatocellular carcinomas (HCCs) from HBV carriers examined had the integrated X region. In this study, HBV DNA was isolated from an integration site in one HCC that had a single, very small integrated viral DNA including the X region, but it had no expression of X gene as poly(A)RNA. It was found that HBV DNA was present between alphoid repetitive sequences, and it included Enhancer and X regions, encompassing the adr sequence from 910 to 1811. Nucleotides for 8 amino acids at the 3' end, a stop codon of X gene and a poly(A) signal downstream of X gene were lost by integration, and nucleotides for 7 amino acids and a stop codon were substituted by a connected alphoid sequence. When this cloned HBV DNA was transfected with an expression vector to an immortalized mouse liver epithelial cell line, MLE-10, malignant transformation occurred. Transformants having expressed poly(A)RNA of the X gene showed anchorage-independent growth in soft agar and tumor formation in the subcutis of nude mice. The mRNA level of EGFR was found to be remarkably enhanced in X-transformed cells, in contrast with the absence of this mRNA in parental and ras-transformed MLE-10. Our data provide evidence that the Enhancer-X region alone is the key contributor to the malignant change of pre-malignant liver cells in HBV carriers through activation of some specific genes, such as EGFR.

Amino Acid Sequence↗
PubMed2000

Determination of a sulfur-containing drug in human plasma by an improved method for sulfur chemiluminescence detection in combination with capillary gas chromatography.

An improved method for sulfur chemiluminescence detection in combination with capillary gas chromatography was established. The method was applied to the analysis of a sulfur-containing antiasthma drug, S-1452, and its nine metabolities in human plasma. The high selectivity and sensitivity of the method allowed accurate quantitation of trace levels of these compounds in human plasma with no interferences from the biological components present. To enable stable measurements and maintain reproducibility over a long period, the performance characteristics of a commercially available instrument were investigated. The gas seal in the injection port was found to easily corrode through interaction of the sulfur analyte with the metal gas seal. To prevent this, a disk made from an alloy of platinum and gold (60:40) was mounted on the gas seal. The reproducibility of the measurement was improved remarkably by using the disk. The use of compressed air of high purity significantly lowered the signal-to-noise ratio. The optical filter was kept clean by using a nickel catalyst to trap ozone in place of copper manganese oxide (CuMn2O4). These improvements raised the sensitivity and selectivity with the lower quantitation limits of 0.5-1.0 ng/mL in human plasma.

Air↗
PubMed2000

Age-dependent changes in lipid peroxide levels in peripheral organs, but not in brain, in senescence-accelerated mice.

The tissue concentration of lipid peroxides was determined in the brain, heart, liver, lung and kidney of accelerated senescence-prone (SAMP-8) and -resistant (SAMR-1) mice at 3, 6 and 9 months of age by a method involving chemical derivatization and high performance liquid chromatography. The level of lipid peroxides in the brain did not show an age-dependent change, but at each age the brain level of lipid peroxides was significantly higher in SAMP-8 than in SAMR-1. In contrast, the lipid peroxide levels in the peripheral organs showed increases with aging in both strains, and they were significantly higher in SAMP-8 than in SAMR-1 at both 3 and 6 months of age (except at 3 months of age in the kidney). These results suggest that increased oxidative stress in the brain and peripheral organs is a cause of the senescence-related degeneration and impairments seen in SAMP-8.

Age Factors↗
PubMed2000

Interactions of phosphatidylinositol 3-kinase Src homology 3 domain with its ligand peptide studied by absorption, circular dichroism, and UV resonance raman spectroscopies.

Absorption, circular dichroism (CD), and UV resonance Raman (UVRR) spectroscopies were applied to selectively examine the environmental and structural changes of Trp and Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain induced by ligand association. Comparison of the spectra of PI3K SH3 in the presence or absence of its ligand peptide RLP1 (RKLPPRPSK) indicated that RLP1 binding changed the environment of Trp55 of the SH3 to be more hydrophilic and its H bonding weaker and that of Tyr residues to be more hydrophobic. The D21N mutant (Asp21 --> Asn) of the SH3 yielded a UV CD distinct from that of the wild type, and its spectral changes induced by RLP1 binding were smaller and different from those of the wild type in absorption, CD, and UVRR spectra, suggesting that the mutation of conserved Asp21 affected the conformation of the ligand binding cleft and thus might lead to the decrease in the ligand affinity. These data provide direct evidence for the occurrence of environmental and structural changes of PI3K SH3 by the association of a ligand and the D21N mutation.

Absorption↗