Biomedical subjects

K Kita

Publications and source records attributed to K Kita.

At least 91 records · Page 5Linked to original sources

PubMed1999

Stimulation of ultraviolet-induced apoptosis of human fibroblast UVr-1 cells by tyrosine kinase inhibitors.

Damnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray-induced apoptosis in ultraviolet-resistant human UVr-1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet-induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet-induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal-regulated kinases but stimulated phosphorylation of stress-activated protein kinases. Consequently, the ultraviolet-induced concurrent increase in both phosphorylated extracellular signal-regulated kinases and stress-activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet-induced apoptosis.

Anthraquinones↗

PubMed1999

Growth inhibition of human pancreatic cancer cell lines by anti-sense oligonucleotides specific to mutated K-ras genes.

About 90% of human pancreatic cancers carry K-ras point mutation, which may play an important role in tumorigenesis. We investigated the inhibitory effects of anti-sense oligonucleotides targeting K-ras point mutation on the growth of cultured human pancreatic cancer cells. Eight human pancreatic cancer cell lines were screened for K-ras codon 12 point mutations by PCR-RFLP analysis and direct sequencing. Then, 3 cell lines with the major types of K-ras point mutation, i.e.,HuP-T1, HuP-T3 and PANC-1, and 1 without mutation, BxPC-3, were used for the experiments. Seventeen mer anti-sense oligonucleotides were designed, targeting the point mutation of K-ras codon 12, and transfected into the cells by the liposome-mediated method. Cell-growth activities were estimated by MTT assay. Levels of K-ras mRNA expression were determined using quantitative RT-PCR, and K-ras p21 protein synthesis was evaluated with Western blotting. Mutation-matched anti-sense oligonucleotides effectively inhibited the growth of these pancreatic cancer cell lines, except for BxPC-3, by suppressing K-ras mRNA expression and K-ras p21 protein synthesis. Moreover, mutation-matched anti-sense oligonucleotides showed stronger anti-proliferative effects than did mutation-mismatched ones. Our results suggest that anti-sense therapy specific to point mutations of K-ras mRNA is a practical approach to selective suppression of tumor growth, with little effect on normal cells.

Cell Division↗

PubMed1999

Rapid recovery from anorexia nervosa after a life-threatening episode with severe thrombocytopenia: report of three cases.

We report the cases of three patients with anorexia nervosa (AN) who each recovered rapidly after experiencing a life-threatening episode with severe thrombocytopenia. All three cases were the typical restricting-type of AN, occurring in adolescence. They refused to be admitted to a hospital until their general condition had been severely deteriorated. Their lowest platelet counts were 2.9, 4.6, and 2.3 x 10(4)/mm3, respectively. Apparent hemorrhagic tendencies, such as purpura, gingival and nasal bleeding, and gastrointestinal bleeding were observed. The bone marrow examination showed apparent hypoplasia in two patients. No evidence of disseminated intravascular coagulation or autoantibody to platelets was detected. The platelet counts recovered rapidly by water and nutritional supplementation. The recovery from the AN itself was excellent in all three patients without specific psychotherapy.

Adolescent↗

PubMed1999

Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells coexpressing the aldehyde reductase and glucose dehydrogenase genes.

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate [(R)-CHBE] using Escherichia coli cells, which coexpress both the aldehyde reductase gene from Sporobolomyces salmonicolor and the glucose dehydrogenase (GDH) gene from Bacillus megaterium as a catalyst was investigated. In an organic solvent-water two-phase system, (R)-CHBE formed in the organic phase amounted to 1610 mM (268 mg/ml), with a molar yield of 94.1% and an optical purity of 91.7% enantiomeric excess. The calculated turnover number of NADP+ to CHBE formed was 13,500 mol/mol. Since the use of E. coli JM109 cells harboring pKAR and pACGD as a catalyst is simple, and does not require the addition of GDH or the isolation of the enzymes, it is highly advantageous for the practical synthesis of (R)-CHBE.

Acetoacetates↗

PubMed1999

Increase in nucleoli after x-radiation of fibroblasts of patients with Gorlin syndrome.

Gorlin syndrome (GS) is an autosomal dominant disorder in which patients are abnormally susceptible to ionizing radiation with radiotherapeutic doses. Radiogenic basal cell carcinomas may develop with a short latent period in patients. The mechanisms underlying the abnormal radiosusceptibility of cells in patients with GS has not been well characterized. In this study we report an increase in the number of nucleoli in fibroblast cells from 3 patients with GS after x-radiation. In GS fibroblasts, the increase in nucleolus number concomitant with the increase of ribonucleoprotein immunoreactive aggregates within the nucleus was observed after x-radiation, whereas significant change was not found in normal fibroblasts derived from healthy donors. This increase disappeared when cells were cultured with the RNA synthesis inhibitor actinomycin D after x-radiation but not when they were cultured with cycloheximide or aphydicolin, which are protein and DNA synthesis inhibitors, respectively. Ultraviolet exposure did not induce remarkable changes in the GS nucleoli. Thus the increase in nucleoli was induced after x-radiation of GS fibroblasts, and this increase seemed to be related to RNA synthesis metabolism.

Basal Cell Nevus Syndrome↗

PubMed1999

Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli.

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms (LS forms) of African trypanosoma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase and these properties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little information concerning the molecular structure and enzymatic features of TAO have been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was cloned into the expression vector pET15b (pTAO) and recombinant TAO was expressed in Escherichia coli. The growth of the transformant carrying pTAO was cyanide-resistant. A peptide with a molecular mass of 37 kDa was found in the cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauromatum guttatum and Hansenula anomala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained vector alone, were inhibited. This cyanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transformant was sensitive to ascofuranone. These results clearly show the functional expression of TAO in E. coli and indicate that ubiquinol-8 in the E. coli membrane is able to serve as an electron donor to the recombinant enzyme and confer cyanide-resistant and ascofuranone-sensitive growth to E. coli. This system will facilitate the biochemical characterization of the novel terminal oxidase, TAO, and the understanding on the mechanism of the trypanocidal effect of ascofuranone.

Animals↗

PubMed1999

Effects of D1 and D2 dopamine receptor antagonists on cocaine-induced self-stimulation and locomotor activity in rats.

To clarify the involvement of D1 and D2 dopamine systems in intracranial self-stimulation (ICSS) and locomotor activity in rats, we studied the acute effects of cocaine and the interaction between cocaine and dopamine antagonists with respect to these behaviors. Although cocaine (5.0, 10.0, or 20.0 mg/kg) dose-dependently increased locomotor activity, it augmented the rate of ICSS only at 5.0 mg/kg. The failure of high doses of cocaine to augment purpose-oriented behavior such as ICSS may result from its induction of a manic-like state. The D1 dopamine receptor antagonist SCH23390 (0.02, 0.1, or 0.5 mg/kg) or the D2 antagonist nemonapride (0.04, 0.2, or 1.0 mg/kg) significantly decreased cocaine augmentation of ICSS. The higher two doses of either antagonist also produced a significant decrease in cocaine-induced locomotor activity. We therefore suspect that cocaine's augmentative effect on those behaviors, especially ICSS, requires activation of both D1 and D2 dopamine receptors.

Animals↗

PubMed1999

Diversity of 4-chloroacetoacetate ethyl ester-reducing enzymes in yeasts and their application to chiral alcohol synthesis.

Enzymes which reduce 4-chloroacetoacetate ethyl ester (CAAE) to (R)- or (S)-4-chloro-3-hydroxybutanoate ethyl ester (CHBE) were investigated. Several microorganisms which can reduce CAAE with high yields were discovered. An NADPH-dependent aldehyde reductase, ARI, and an NADPH-dependent carbonyl reductase, S1, were isolated from Sporobolomyces salmonicolor and Candida magnoliae, respectively, and enzymatic synthesis of chiral CHBE was performed through the reduction of CAAE. When ARI-overproducing Escherichia coli transformant cells or C. magnoliae cells were incubated in an organic solvent-water diphasic system. CAAE was stoichiometrically converted to (R)- or (S)-CHBE (> 92% enantiomeric excess), respectively. Multiple CAAE-reducing enzymes were present in S. salmonicolor, C. magnoliae and bakers' yeast. Comparison of the primary structures of these CAAE-reducing enzymes with other protein sequences showed that CAAE-reducing enzymes are widely distributed in various protein families, and various physiological roles of these enzymes in the cell were speculated.

Journal Article↗

PubMed1999

Low BCL-2 expression in acute leukemia with t(8;21) chromosomal abnormality.

In de novo t(8;21) AML which shows terminal neutrophilic differentiation, the BCL-2 expression was found to be significantly lower than that in types of other AML regardless of the phenotypic differentiation status. An inverse correlation between BCL-2 expression and the S/G2/M population cells was observed in AML. The S/G2/M population in t(8;21)AML was larger than in the other types of AML. In t(8;21)AML, spontaneous apoptosis after a 12-h liquid culture was prominent, and the autonomous DNA synthesis after a 72-h liquid culture was low. G-CSF and IL-5 promoted the colony formation of t(8;21)AML cells. The data suggest that, in vivo, the low BCL-2 in t(8;21)AML induced entry of cells from the G0/G1 phase to S phase, but the cells easily die by apoptosis, in vitro. The low BCL-2 expression and the supportive effects of G-CSF and IL-5 in t(8;21)AML is thought to be a key phenomenon which might be related to the formation of the in vivo blood picture, such as prominent neutrophilic differentiation and eosinophilia. Cellular extracts from t(8;21)AML cell line Kasumi-1 bound to both the AML1 and CRE binding sites in the bcl-2 promoter, but none of the cellular extracts from de novo t(8;21)AML bound to either of these sites. The DNA binding activity of transactivators in de novo t(8;21)AML is different from that in Kasumi-1 cells probably due to the phosphorylation status.

Acute Disease↗

PubMed1999

Expressions of four major protein Ser/Thr phosphatases in human primary leukemic cells.

Activity and expression of four major protein serine/threonine (Ser/Thr) phosphatases, protein phosphatase type 1 (PP1), protein phosphatase type 2A (PP2A), protein phosphatase type 2B (PP2B) and protein phosphatase type 2C (PP2C) were evaluated in normal peripheral leukocytes, and in various leukemic cells from patients with acute myelogenous leukemia (AML), common acute lymphocytic leukemia (cALL), or chronic lymphocytic leukemia (CLL). PP1 was the most abundant phosphatase in blood cells, and relative abundance of each phosphatase was: PP1 > PP2A > PP2B approximately = PP2C. PP1 activity and its expressions were higher in blasts of AML-M4 and -M5 than in cells of AML-M1, cALL and CLL. PP2A activity and its expression were higher in blasts of AML-M3, -M4 and -M5 than in cells of AML-M1, cALL and CLL. Activity and expression of both PP1 and PP2A in normal monocytes were highest, and PP2A activity in normal neutrophils was lowest among normal leukocytes. PP2B activity and its expression were higher in blasts of AML-M2, -M3 and normal lymphocytes. PP2C activity and its expression were relatively constant in various leukemic cell types. Activities of PP1 and PP2A of AML blasts correlated positively with the expression of CD11b, whereas activities of PP1 and PP2B correlated negatively with the expression of CD7. Thus, each phosphatase was ubiquitously but differently expressed in various leukemic cell types and in normal leukocytes. These data also suggest that expressions of PP1, PP2A and PP2B are relatively low in leukemic blasts arresting at the stage of early pluripotent stem cells, and are differently modulated during the course of myelomonocytic commitment and maturation.

Acute Disease↗

PubMed1999

Survival of human leukaemic B-cell precursors is supported by stromal cells and cytokines: association with the expression of bcl-2 protein.

We searched for cytokines with the potential to support the survival of human B-cell precursor acute lymphoblastic leukaemia (pre-B ALL) cells. 47 patients with pre-B ALL were classified into four stages: stage I, CD19+CD10-CD20-; stage II, CD19+CD10+CD20-; stage III, CD19+CD10+CD20+cytoplasmic mu-heavy chain (cmu)-; stage IV, CD19+CD10+CD20+cmu. Interleukin (IL)-3 receptor alpha chain (IL-3Ralpha) was expressed in all stages, whereas the expressions of IL-7Ralpha and IL-2Ralpha were pronounced in stages IV and II, respectively. Neither IL-3, IL-7 nor IL-2 supported the survival of pre-B ALL cells. When pre-B ALL cells were layered on stromal, MS-10, cells, viability of the pre-B ALL cells increased. Addition of IL-3 to culture containing MS-10 cells enhanced the survival of pre-B ALL cells in all cases, whereas addition of IL-7 augmented the survival of pre-B ALL cells of some cases of stage III and all cases of stage IV. The survival of pre-B ALL cells was also supported by the conditioned media of MS-10 cells. Stromal-cell-derived factor 1 (SDF-1) supported the survival of pre-B ALL cells. Effects of the conditioned media of MS-10 cells were abrogated by an anti-SDF-1 neutralizing antibody. The extent of survival of pre-B ALL cells supported by stromal cells and IL-3 and IL-7, correlated with the expression level of bcl-2 protein. The effects of stromal cells may be in part related to SDF-1.

Adult↗

PubMed1999

Myeloid differentiation antigen and cytokine receptor expression on acute myelocytic leukaemia cells with t(16;21)(p11;q22): frequent expression of CD56 and interleukin-2 receptor alpha chain.

We report the cellular characteristics of cells from three patients with de novo acute myelocytic leukaemia (AML) with t(16;21)(p11;q22), two M4 and one M5a according to the FAB classification, and two permanent cell lines with t(16;21)(p11;q22), TSU1621MT and YNH-1. The FUS/ERG fusion mRNA was demonstrated in all cases by reverse transcriptase-polymerase chain reaction (RT-PCR). The immunophenotypes of the AML cells, and YNH-1 and TSU1621MT cell lines with t(16;21) were characterized as CD34+CD33+CD13+CD11b+CD18+CD56+ HLA-DR-/+. Cells from all samples strongly expressed c-kit, granulocyte colony-stimulating factor receptor (G-CSFR), c-fms (macrophage colony-stimulating factor receptor), interleukin-3 receptor alpha chain (IL-3Ralpha), and granulocyte macrophage colony-stimulating factor receptor alpha chain (GM-CSFRalpha), and these data corresponded well to the growth responsiveness to the cytokines. IL-2Ralpha expression was also found in all t(16;21) samples, but IL-2 did not act on the proliferation of the leukaemic cells in in vitro cultures. G-CSF distinctly promoted the proliferation of leukaemic cells of t(16;21) AML, but did not enhance the expression of MPO and neutrophil differentiation of these cells. Our findings indicate that AML cells with t(16;21) preserve stem cell properties such as CD34 and c-kit expression, and suggest that they have the potential to differentiate into a monocytic lineage. The relationship between the unique cellular characteristics (especially CD56 and IL-2Ralpha expression) and FUS/ERG protein remains undetermined.

Adolescent↗

PubMed1999

De novo CD5-positive diffuse large B-cell lymphoma: clinical characteristics and therapeutic outcome.

To determine the clinical significance of CD5 expression in diffuse large B-cell lymphoma (DLBL) without a clinical history of low-grade B-cell lymphoma, we have reviewed the clinical features and therapeutic outcome of 25 patients with de novo CD5-positive DLBL, and compared the results with those of 87 patients with CD5-negative DLBL and 22 patients with mantle cell lymphoma (MCL). The patients with de novo CD5-positive DLBL had clinical characteristics of elderly onset (median age 63, range 37-91), and female predominance (male/female 10/15). 21 (84%) of these patients had extranodal involvement at presentation, with great variation in the sites. In comparison with the patients with CD5-negative DLBL, the treatment outcome for the patients with de novo CD5-positive DLBL was very poor with frequent relapse. The failure-free survival curve was almost identical to that of patients with MCL, showing that standard chemotherapy for DLBL was not effective for most of the patients with de novo CD5-positive DLBL. These findings suggest that de novo CD5-positive DLBL forms a distinct subgroup of DLBL.

Adult↗

PubMed1999

Preparation of fluorescence-labeled GM1 and sphingomyelin by the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase as substrates for assay of sphingolipid-degrading enzymes and for detection of sphingolipid-binding proteins.

Sphingolipid ceramide N-deacylase is an enzyme capable of hydrolyzing the N-acyl linkages of ceramides of various sphingolipids. Recently, it was found that the enzyme catalyzes the reverse hydrolysis reaction in which free fatty acids are condensed to lyso-sphingolipids to produce sphingolipids. This paper describes a simple method for the synthesis of fluorescence-labeled sphingolipids utilizing the condensation reaction of the enzyme. N-TFAc-aminododecanoic acids were efficiently condensed by the enzyme to the lyso-forms of GM1 and sphingomyelin in glycine buffer (pH 10). The reaction products, N-TFAc-amino-GM1 and sphingomyelin, were obtained with overall yields of 60%. The purified products were identified to be omega-amino-GM1 and omega-amino-sphingomyelin, respectively, by TLC and FAB-MS or ESI-LC/MS analysis after removal of the N-TFAc by mild alkaline treatment. NBD-labeled GM1 and sphingomyelin were prepared from omega-amino-GM1 and omega-amino-sphingomyelin by coupling with 4-fluoro-NBD. These fluorescence-labeled substrates, C12-NBD-GM1 and C12-NBD-sphingomyelin, were hydrolyzed by endoglycoceramidase and sphingomyelinase, respectively, to produce NBD-dodecanoylsphingosines, but were resistant to hydrolysis by sphingolipid ceramide N-deacylase. C12-NBD-sphingomyelin was found to be a better substrate than the commercially available C6-NBD-sphingomyelin for the assay of sphingomyelinase from various sources. We also describe a new method to detect GM1-binding proteins using fluorescence-labeled GM1.

Amidohydrolases↗

PubMed1999

Cloning, overexpression, and mutagenesis of the Sporobolomyces salmonicolor AKU4429 gene encoding a new aldehyde reductase, which catalyzes the stereoselective reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (S)-4-chloro-3-hydroxybutanoate.

We cloned and sequenced the gene encoding an NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolor AKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3beta-hydroxysteroid dehydrogenase-plant dihydroflavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism (K. Kita, K. Matsuzaki, T. Hashimoto, H. Yanase, N. Kato, M. C.-M. Chung, M. Kataoka, and S. Shimizu, Appl. Environ. Microbiol. 62:2303-2310, 1996). The ARII protein was overproduced in Escherichia coli about 2, 000-fold compared to the production in the original yeast cells. The enzyme expressed in E. coli was purified to homogeneity and had the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif G(19)-X-X-G(22)-X-X-A(25), which is located in the N-terminal region, during ARII catalysis, we replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G(19)-->A and G(22)-->A mutant enzymes by 4-COBE did not occur. The A(25)-->G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH.

Acetoacetates↗

PubMed1999

Evidence of horizontal transfer of the EcoO109I restriction-modification gene to Escherichia coli chromosomal DNA.

A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5'-(A/G)GGNCC(C/T)-3', was cloned from the chromosomal DNA of Escherichia coli H709c. The EcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. The sid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.

Amino Acid Sequence↗

PubMed1999

Enhancement of XPG mRNA transcription by human interferon-beta in Cockayne syndrome cells with complementation group B.

Human interferon-beta (HuIFN-beta) confers UV-refractoriness in association with increased DNA repair capacity to human cells. We examined the modulation of XPG gene expression by HuIFN-beta in UV-sensitive cells from Cockayne syndrome complementation B (CSB), xeroderma pigmentosum complementation A (XPA) and normal control cells. Northern blot analysis revealed that XPG mRNA was more extensively transcribed in CSB cells treated with HuIFN-beta than in those without HuIFN-beta treatment. XPG mRNA from XPA cells and normal control cells was not markedly transcribed by HuIFN-beta treatment compared to that from CSB cells. The findings suggested that different mechanisms of UV-refractoriness by HuIFN-beta exist between CS and XP.

Blotting, Northern↗

PubMed1999

Protease activity induced by nicotine in human cells.

Nicotine has a wide range of biological effects, and proteases have been extensively studied for their biological roles in living creatures. The aim of this study is to determine whether nicotine can induce proteolytic protease activity in cultures of various human cell lines. Plasminogen activator-like fibrinolytic protease activity, using 125I-fibrin as substrate in the presence of plasminogen, was estimated in cells with and without nicotine treatment. Among 16 cell lines tested, APr-1 cells were found to have the highest induced protease activity. Partial purification of the proteases was carried out by high performance liquid chromatography gel filtration on TSKG2000SW. Protease inhibitor tests indicated that the proteases induced by nicotine are serine proteases.

Cell Line↗