Enzymatic synthesis of [14C]ceramide, [14C]glycosphingolipids, and omega-aminoceramide.
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Amidohydrolases↗
Biomedical subjects
K Kita
Publications and source records attributed to K Kita.
Increase in ultraviolet sensitivity by overexpression of calpastatin in ultraviolet-resistant UVr-1 cells derived from ultraviolet-sensitive human RSa cells.
Human RSa cells are highly sensitive to apoptotic-like cell death by ultraviolet irradiation (UV) while UVr-1 cells are their variant with an increased resistance to UV. Three days after UV at 10 J/m2, the viability of RSa cells was approximately 17% while that of UVr-1 cells was 65%. This different survival might reflect apoptotic cell death since apoptosis-specific DNA ladder was more clearly observed in RSa cells than in UVr-1 cells after UV. Addition of ALLN/calpain inhibitor I to the culture medium after UV resulted in similar survival (14 - 18%) between RSa and UVr-1 cells. Immunoblot analysis showed down-regulation of protein kinase CTheta, Src, Bax and mu-calpain after UV was more prominent in UVr-1 than in RSa cells. Activated mu-calpain appeared within 1 h post-UV only in UVr-1 cells. The expression of calpastatin, a specific endogenous inhibitor of calpain, was higher in RSa than in UVr-1 cells. To further examine the role of calpain in UV-induced cell death, cDNA of human calpastatin was transfected into UVr-1 cells. The results showed that overexpression of calpastatin suppressed down-regulation of Src, mu-calpain and Bax. Concomitantly, colony survival after UV was reduced in calpastatin-transfected cells as compared to vector control cells. Our results suggest that activation of calpain might account for, at least in part, the lower susceptibility to UV-induced cell death in UVr-1 cells.
Apoptosis↗
Geographic heterogeneity of cellular characteristics of acute myeloid leukemia: a comparative study of Australian and Japanese adult cases.
We assessed a large number of adults (368 from Australia and 494 from Japan) with de novo acute myeloid leukemia (AML) to define the biological differences between the two populations. In this study, AML was classified using the French-American-British (FAB) criteria into seven groups (M1-M7). M2 was more common in Japan than in Australia, whereas M4 occurred more frequently in Australia than in Japan. Other FAB subtypes were evenly distributed. Cytogenetically, Japanese M2 displayed a higher frequency of t(8;21) than Australian (33.1% vs 15.3%, P < 0.05). The t(15;17), inv/del(16), 11q23 aberrations and 5/7/8 abnormalities were seen at similar frequencies. Immunophenotypically, Japanese M4/M5 more frequently displayed CD13 and CD14 than Australian, whereas the stem cell markers, CD34 and HLA-DR were observed at a relatively higher rate in Australian M3 than in Japanese M3. The B cell antigen, CD19 was more frequently seen in Japanese M2 than in Australian M2, but found more often in Australian M5 than in Japanese M5. In both populations, a close relationship was observed between the expression of CD19 and t(8;21). These findings suggest different biological characteristics of AML between the two populations, the main differences being generated by a higher frequency of t(8;21) chromosomal abnormality in Japanese AML. Leukemia(2000) 14, 163-168.
Acute Disease↗
Heterogenous fusion transcripts involving the NUP98 gene and HOXD13 gene activation in a case of acute myeloid leukemia with the t(2;11)(q31;p15) translocation.
We report the characterization of a rare chromosomal translocation, a t(2;11)(q31;p15), which occurred in a patient with de novo acute myeloid leukemia (AML-M4). By 3'-RACE and RT-PCR analyses, two kinds of NUP98-HOXD13 fusion transcript were detected. In addition, we identified a novel fusion transcript, NUP98-FN1, in the same patient. Ectopic expression of the wild-type HOXD13 gene was also observed in the patient, suggesting that HOXD13 contributes to the development of this type of leukemia. The NUP98-HOXD13 fusion transcript was predicted to encode a 552 or 569-amino acid protein containing the Phe-Gly (FG) repeat region of NUP98 and the homeodomain of HOXD13. The NUP98-FN1 fusion transcript was predicted to encode a 482 or 499-amino acid protein consisting of the same N-terminal region of NUP98 and a C-terminal region of 12 amino acids derived from a previously unidentified sequence. We isolated and characterized the chromosomal breakpoints. The breakpoint at 11p15 is mapped within a LINE repetitive element in a 9 kb intron of NUP98, and more than 60% of the sequenced 3 kb region surrounding the breakpoint junction consists of repetitive elements. The other breakpoint at 2q31 is in an intron of FN1, which is located 7 kb upstream of HOXD13, and the repetitive sequence content of the breakpoint junction is low. Local sequence duplications at genomic breakpoints suggest that the t(2;11) translocation is mediated through staggered double-strand DNA breaks. These results throw light on the mechanisms responsible for the generation of t(2;11) translocation and on the processes leading to t(2;11) leukemia.
Acute Disease↗
Different responses of protein synthesis to refeeding in various muscles of fasted chicks.
1. The change in the rate of protein synthesis of different muscles, concentrations of plasma insulin, plasma insulin-like growth factor-I (IGF-I) and other plasma components were investigated after refeeding in fasted chicks. 5.2 g of the complete diet was refed. This was the maximum that could be force-fed with water. 2. The fractional synthesis rates (FSR) of breast (M. pectoralis major) and leg (M. gastrocnemius) muscles were measured after injection of L-[2, 6-(3)H]phenylalanine. Plasma insulin and IGF-I concentration were determined by radioimmunoassay. 3. In the breast muscle, FSR was significantly reduced by 2-d fasting. The FSR had recovered completely after 1 h of refeeding and was maintained until 6 h. The change in FSR after refeeding was associated with the change in ribosomal efficiency (K(RNA); absolute synthesis rate per unit RNA), while no change in ribosomal capacity (C(S); RNA: protein ratio) was observed. 4. In the leg muscle, FSR was decreased by 2-d fasting and increased gradually toward 6 h after refeeding but did not reach the level of the fed control. In contrast to the breast muscle, no significant changes in Cs and K(RNA) in the leg muscle were observed. 5. Plasma glucose concentration increased significantly at 1 h after refeeding but returned to the fasted level after 24 h. Plasma insulin concentration in chicks refed for 1 h was higher than in the fasted group. There was no significant change in plasma IGF-I concentration. 6. These results suggest that the FSR of breast muscle was more sensitive to refeeding than that of leg muscle which may be explained, in part, by differences in sensitivity to the change in circulating plasma insulin concentration after refeeding.
Animals↗
Is allantoin in serum and urine a useful indicator of exercise-induced oxidative stress in humans?
To assess whether allantoin levels in serum and urine are influenced by exhaustive and moderate exercise and whether allantoin is a useful indicator of exercise-induced oxidative stress in humans, we made subjects perform exhaustive and moderate (100% and 40% VO2max) cycling exercise and examined the levels of allantoin, thiobarbituric acid reactive substances (TBARS) and urate in serum and urine. Immediately after exercise at 100% VO2max, the serum allantoin/urate ratio was significantly elevated compared with the resting levels while the serum urate levels was significantly elevated 30 min after exercise. The serum TBARS levels did not increase significantly compared with the resting levels. Urinary allantoin excretion significantly increased during 60 min of recovery after exercise, however, urinary urate excretion decreased significantly during the same period. The urinary allantoin/urate ratio also rapidly increased during 60 min of recovery after exercise. Urinary TBARS excretion decreased during the first 60 min of the recovery period and thereafter significantly increased during the latter half of the recovery period. On the contrary, after 40% VO2max of exercise, no significant changes in the levels of urate, allantoin and TBARS in serum or urine were observed. These findings suggest that allantoin levels in serum and urine may reflect the extent of oxidative stress in vivo and that the allantoin which appeared following exercise may have originated not from urate formed as a result of exercise but from urate that previously existed in the body. Furthermore, these findings support the view that allantoin in serum and urine is a more sensitive and reliable indicator of in vivo oxidative stress than lipid peroxidation products measured as TBARS.
Adult↗
Various macronutrient intakes additively stimulate protein synthesis in liver and muscle of food-deprived chicks.
We investigated the influence of refeeding food-deprived chicks with either protein, carbohydrate, fat or combinations thereof on the rates of liver and muscle protein synthesis. After 2 d of food-deprivation, chicks were given individual or mixed protein, carbohydrate and fat. At 30 min after refeeding, the protein fractional synthesis rate (K(s)) was measured by a large dose injection of L-[2,6-(3)H]phenylalanine. When chicks were food-deprived for 2 d, liver K(s) was 67% lower and muscle fractional synthesis rate was half that of well-fed controls. Upon refeeding starved chicks a complete diet, K(s) in the liver and muscle returned to the level of fed controls within 30 min. When food-deprived chicks were refed protein alone or two of the three macronutrients, liver and muscle K(s) were significantly higher than those in the starved group. There was no effect of refeeding with carbohydrate or fat alone. Plasma glucose concentration was significantly greater than in fed or starved groups in chicks refed the complete diet, carbohydrate or carbohydrate mixed with either protein or fat. Refeeding chicks with the various macronutrients did not affect the plasma insulin or insulin-like growth factor-I concentrations. These results suggest that intakes of individual macronutrients additively increase liver and muscle protein synthesis and that the acute increase in muscle protein synthesis after refeeding chicks diets containing the three macronutrients was mainly regulated by the change in ribosomal efficiency.
Analysis of Variance↗
Urinary dysfunction and orthostatic hypotension in multiple system atrophy: which is the more common and earlier manifestation?
OBJECTIVES: Urinary dysfunction and orthostatic hypotension are the prominent autonomic features in multiple system atrophy (MSA). A detailed questionnaire was given and autonomic function tests were performed in 121 patients with MSA concerning both urinary and cardiovascular systems. METHODS: Replies to the questionnaire on autonomic symptoms were obtained from 121 patients including three clinical variants; olivopontocerebellar atrophy (OPCA) type in 48, striatonigral degeneration (SND) type in 17, and Shy-Drager type in 56. Urodynamic studies comprised measurement of postmicturition residuals, EMG cystometry, and bethanechol injection. Cardiovascular tests included head up tilt test, measurement of supine plasma noradrenaline (norepinephrine,NA), measurement of R-R variability (CV R-R), and intravenous infusions of NA and isoproterenol. RESULTS: Urinary symptoms (96%) were found to be more common than orthostatic symptoms (43%) (p<0.01) in patients with MSA, particularly with OPCA (p<0.01) and SND (p<0.01) types. In 53 patients with both urinary and orthostatic symptoms, patients who had urinary symptoms first (48%) were more common than those who had orthostatic symptoms first (29%), and there were patients who developed both symptoms simultaneously (23%). Post-micturition residuals were noted in 74% of the patients. EMG cystometry showed detrusor hyperreflexia in 56%, low compliance in 31%, atonic curve in 5%, detrusor-sphincter dyssynergia in 45%, and neurogenic sphincter EMG in 74%. The cystometric curve tended to change from hyperreflexia to low compliance, then atonic curve in repeated tests. Bethanechol injection showed denervation supersensitivity of the bladder in 19%. Cardiovascular tests showed orthostatic hypotension below -30 mm Hg in 41%, low CV R-R below 1.5 in 57%, supine plasma NA below 100 pg/ml in 28%, and denervation supersensitivity of the vessels (alpha in 73%; beta2 in 60%) and of the heart (beta1 in 62%). CONCLUSION: It is likely that urinary dysfunction is more common and often an earlier manifestation than orthostatic hypotension in patients with MSA, although subclinical cardiovascular abnormalities appear in the early stage of the disease. The responsible sites seem to be central and peripheral for both dysfunctions.
Adult↗
Phorbor myristate acetate induces NADPH oxidase activity of cytochalasin B-primed neutrophils through the protein kinase C-independent pathway.
We examined the effect of cytochalasin B (CB) or granulocyte colony stimulating factor (GCSF) on superoxide radical (O2-) production of neutrophils by phorbor myristate acetate (PMA)-stimulation. It was observed that O2- generation of intact and GCSF-treated neutrophils by PMA-stimulation showed a lag during the early stage, and was largely inhibited by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (200 microm) or GF109203X (GFX) (0.2 microM), but not by ethanol (1%) and wortmannin (100 nM). In contrast, O2- generation of CB-pretreated neutrophils by PMA-stimulation did not show a lag, but was less than that of intact cells, and was only minimally depressed by the above inhibitors, but was markedly depressed by the simultaneous addition of GFX and ethanol or GFX and wortmannin. Although translocation of p47phox and p67phox to the membrane fraction by PMA-stimulation of intact and GCSF-treated neutrophils occurred in parallel with O2- production, that of CB-treated neutrophils by PMA-stimulation was not always proportional to O2- production. These findings suggest that pretreatment of neutrophils with CB dramatically alters the PMA response of the cells; that is, the protein kinase C-dependent pathway is largely depressed, and a phospholipase D-dependent one for NADPH oxidase activation appears in CB-treated cells.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Curative resection of orthotopically implanted pancreatic cancer in Syrian golden hamsters.
BACKGROUND: Homologous orthotopic implantation models of pancreatic ductal cancer in Syrian golden hamsters can mimic the clinical features of the disease in humans. However, there have been no studies attempting a surgical approach to this animal model. Our aim was to clarify the possibility of curative resection in these tumor-bearing animals. METHODS: A nitrosamine-induced pancreatic ductal adenocarcinoma cell line, HaP-T1, was used for the experiments. Hamsters underwent two surgeries: first, the cells or the tissue previously grown subcutaneously were implanted into the pancreas, and second, pancreatectomy and splenectomy were performed within the following 4 wk after implantation. Animals were examined until death, when necropsies were performed. Surgically resected and necropsied specimens were studied histopathologically and at the molecular level by detection of K-ras point mutation at codon 12. RESULTS: At the second surgery, the success of implantation was 100%, and the curative resection rate was 75%. After observation for 400 d, 5 of 12 hamsters, which underwent second surgery within 15 d, were still alive. They were sacrificed and were found to be tumor-free. CONCLUSION: Orthotopic homologous implantation model of hamster pancreatic cancer combined with surgical resection may be useful for further research of adjuvant or neoadjuvant therapy for pancreatic carcinoma.
Animals↗
Effects of cyclosporin A on refractory B-cell chronic lymphocytic leukemia.
This report details the effects of cyclosporin A (Cs-A) on the clinical course of B-cell chronic lymphocytic leukemia (B-CLL) with an increase in circulating T cells that express CD3, CD8, CD25, CD45RO, and T-cell receptor alpha beta chain. Cs-A was used for treatment of a female patient with refractory B-CLL and was shown to increase dramatically the number of leukemic B cells in association with a simultaneous increase in T cells in vivo. To study this effect more precisely, CD19+ (B cells) and CD3+ (T cells) cells were isolated from the patient and cultured in the presence or absence of Cs-A. When Cs-A was added to the culture, no cell stimulation was seen. However, CD3+ cells from the patient underwent cell death, relative to those from healthy donors. On the other hand, CD19+ cells from the patient were more resistant to Cs-A than those from healthy donors. Because in vitro experiments cannot explain this phenomenon of Cs-A increasing leukemic cells (not only B-CLL cells but also T cells), care should be exercised in the clinical use of Cs-A in B-CLL patients.
Antigens, CD19↗
Expression of B cell-associated transcription factors in B-cell precursor acute lymphoblastic leukemia cells: association with PU.1 expression, phenotype, and immunogenotype.
B-lymphocyte development progresses through discrete stages characterized by regular DNA rearrangements of the immunoglobulin (Ig) loci that lead to the transcription of Ig genes and expression of B-cell antigen receptors. These developmental processes can also be distinguished by the expression of specific cell-surface markers. Therefore, rearrangement of the Ig, T-cell receptor (TCR) genes, and surface markers are generally considered as useful markers of the B- and T-cell lineage in lymphoproliferative disorders. However, concomitant rearrangement of Ig and TCR genes (double genotype) has been reported in the most immature lymphoid malignancies (lineage promiscuity), mainly in B-cell precursor acute lymphoblastic leukemia (pre-B ALL), but the mechanism is not fully understood. DNA rearrangements and specific cell-surface markers are regulated by several specific transcription factors. To better characterize the lineage promiscuity, we studied the relationship among the expression of lymphoid-associated transcription factors, phenotype, and immunogenotype. Rearrangement of the Ig light chain kappa gene was found in 37% of pre-B ALL samples and in all B-cell chronic lymphocytic leukemia (B-CLL) samples. Rearrangement of TCR gamma gene was shown in 40% of pre-B ALL samples but was not detected in any of the B-CLL samples. Among the tested B cell-associated transcription factors, Pax5 and E47 genes were expressed in all pre-B ALL and B-CLL samples. RAG-1 gene was expressed in all pre-B ALL samples but not in the B-CLL samples. Oct-2 gene was expressed in 82% of pre-B ALL and all B-CLL samples. The expression of PU.1 gene was shown in 56% of pre-B ALL but not in the B-CLL samples. Interestingly, the samples of pre-B ALL, which did not express the PU.1 gene, showed a significantly high frequency of TCR gamma gene rearrangement. This phenomenon was not found with Oct-2 gene expression. These findings suggest that absence of PU.1 expression may result in lineage promiscuity, such as the simultaneous rearrangements of Ig and TCR genes in pre-B ALL cells.
Adult↗
[Activities, which modulate mutability of cells, in human serum after parabolic flight (Report No. 2)].
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Base Sequence↗
Search for molecules that are biological indicators of gravity stress in the human body.
In this study, we analyzed levels of excretion of 8-OHdG into urine before and after exposure to gravitational changes including both microgravity and hypergravity by an enzyme-linked immunosorbent assay (ELISA).
8-Hydroxy-2'-Deoxyguanosine↗
Mutation modulating activity in human serum factors induced after parabolic flight.
We have reported that human interferon, one of cytokines present in serum, can confer hypomutability on various human cells. On the contrary, we have also reported that serum factors from cancer patients can enhance cell mutability. Therefore, it seems likely that cell mutability is changed by cytokine-like serum factors in our body. It is one of important space problems whether the mutability of human cells is regulated in response to microgravity and hypergravity (gravitational stress). However, there is little information about cell mutability during such stress. In this study, we investigated whether the mutability is changed by exposing cells to human serum factors after gravitational stress.
Adult↗
Search for gravity-responsive genes by PCR-based mRNA differential display in human cells.
Mechanisms of molecular responses of human cells to gravity change and/or space radiation are one of the most important physiological problems in space science. We have previously reported that expression levels of several genes are changed in cultured human cells after UVC irradiation, and a few of those genes are responsible for UVC sensitivity. In this study, to find candidates for genes that play roles in susceptibility of human cells to gravity stressors, including those responsible for genetic stability in humans, we analyzed genes expressed differentially after gravity stress in human cells, using a PCR-based mRNA differential display (D.D.) method. Cells used were RSa and its variant cell lines, with discrepant sensitivity to radiation cell-killing and mutagenicity [correction of mutagenecity].
Cell Line↗
Suppression of okadaic acid-induced apoptosis by overexpression of calpastatin in human UV(r)-1 cells.
Proteolytic systems have various involvements in apoptotic pathways. To understand the role of calpain in apoptosis, calpastatin, a specific inhibitor of calpain, was overexpressed in human UV(r)-1 fibroblasts by transfection of its cDNA. The elevated expression of calpastatin resulted in decreased survival in the presence of okadaic acid (OA) but in no apparent alteration in the sensitivity toward other drugs such as 5-fluorouracil, mitomycin C and methotrexate. After treatment with OA, a typical apoptotic DNA ladder was observed in control vector-transfected cells but not in calpastatin-transfected cells. This indicates that OA-induced apoptosis was suppressed by overexpression of calpastatin. Further immunoblot analysis showed that the OA-induced hyperphosphorylation of c-Jun was inhibited in calpastatin-transfected cells. This might be involved in the resistance to OA-induced cell death in calpastatin-overproducing cells.
Apoptosis↗
Characterization of the human SDHD gene encoding the small subunit of cytochrome b (cybS) in mitochondrial succinate-ubiquinone oxidoreductase.
We have mapped large (cybL) and small (cybS) subunits of cytochrome b in the succinate-ubiquinone oxidoreductase (complex II) of human mitochondria to chromosome 1q21 and 11q23, respectively (H. Hirawake et al., Cytogenet. Cell Genet. 79 (1997) 132-138). In the present study, the human SDHD gene encoding cybS was cloned and characterized. The gene comprises four exons and three introns extending over 19 kb. Sequence analysis of the 5' promoter region showed several motifs for the binding of transcription factors including nuclear respiratory factors NRF-1 and NRF-2 at positions -137 and -104, respectively. In addition to this gene, six pseudogenes of cybS were isolated and mapped on the chromosome.
Amino Acid Sequence↗