[Generation of mice with mitochondrial dysfunction by introducing mouse mtDNA carrying a deletion into zygotes].
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Animals↗
Biomedical subjects
K Isobe
Publications and source records attributed to K Isobe.
Interaction between DNA-damage protein GADD34 and a new member of the Hsp40 family of heat shock proteins that is induced by a DNA-damaging reagent.
GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest. To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with murine GADD34. As bait we used the product of the partial GADD34 cDNA, including the regions rich in proline, glutamic acid, serine and threonine (PEST) and gamma(1)34.5 regions. A cDNA clone, named GAHSP40, which is a mouse DnaJ family protein with a high similarity to human HLJ1 was cloned. The interaction between GADD34 and GAHSP40 in cultured cells was confirmed by a co-immunoprecipitation experiment and in NIH 3T3 cells by two-hybrid analysis in vivo. For binding of the two proteins, the gamma(1)34.5-similar region of GADD34 was necessary; however, the PEST region was also involved and the C-terminus of GAHSP40, but not the J-domain, was important. GAHSP40 was detected in all mouse tissues examined, but a different transcript was found in the testis. Both GADD34 mRNA and GAHSP40 mRNA were significantly elevated by treatment with methyl methanesulphonate, although the time courses were different. In addition, both GAHSP40 and GADD34 mRNA were induced by heat shock.
3T3 Cells↗
A patient with type 2 diabetes mellitus associated with mutations in calcium sensing receptor gene and mitochondrial DNA.
A 44-year-old female with familial hypocalciuric hypercalcemia (FHH) due to a homozygous missense mutation (Pro40Ala) in calcium sensing receptor (CaSR) gene has type 2 diabetes mellitus. The identical heterozygous mutation of CaSR gene was observed in consanguineous parents and all other family members examined except her two sisters. Many subjects with abnormal glucose tolerance were observed in this family, which is compatible with maternal inheritance. Mitochondrial function of complex I (NADH-coenzyme Q reductase) activity in cybrid cells between mitochondrial DNA (mtDNA)-deleted (rho(0)) HeLa cells and mtDNA from the proband was decreased by 35%. The proband has eight substitutions and among these 4833 A/G is a missense substitution in NADH dehydrogenase 2 gene and may probably be a major pathogenic mutation of impaired complex I activity. These results suggest that coexistence of nuclear gene and mtDNA mutations may have caused or modified the development of abnormal glucose tolerance in this family.
Adult↗
The diagnostic significance of left auricular thrombus in F344/N rats.
In the process of assessing the main cause of death in F344/N rats from the aging farm of our institute, we have often found left auricular thrombus in autopsy cases of moribund animals. In 319 autopsy cases, 45 were of left auricular thrombus and 44 were accompanied by hematopoietic neoplasms, including overt leukemia and a pre-leukemic condition of leukemia. In cases without splenomegaly, this lesion was found in 13 of 21 animals (61.9%) whereas, in cases with splenomegaly, 31 of 239 were positive for this lesion (13.0%). Thus, left auricular thrombus may be an important macroscopic diagnostic criteria of hematopoietic neoplasms, especially when splenomegaly is absent. Furthermore, this lesion tended to arise in aged animals despite the presence of splenomegaly. These results would therefore greatly contribute to aging science by confirming the health condition of experimental rats and the accuracy of subsequent results.
Journal Article↗
Target volume definition for upper abdominal irradiation using CT scans obtained during inhale and exhale phases.
PURPOSE: To evaluate the clinical utility of a treatment-planning technique involving the use of CT images obtained during both the static exhalation phase and static inhalation phase (two-phase planning). METHODS AND MATERIALS: Ten patients with pancreatic or liver tumors underwent CT scanning under static exhale and inhale conditions, after a period of mild ventilation. By setting image positions differently, we were able to treat the two-phase images as one dataset. Each gross tumor volume (GTV) was contoured separately and the mixed GTV was used for the two-phase treatment planning. Treatment plans were constructed to compare the two-phase plans with the plans constructed using static exhalation images. The shift of the center of the GTV and kidneys and the minimum dose of GTV were then calculated. RESULTS: The shift of the GTV ranged from 2.6 to 27. 3 mm and that of the kidneys from 2.2 to 24 mm. In some patients whose treatment was planned using exhalation planning, the minimum dose of GTV at inhalation was less than 90% of the isocenter dose. CONCLUSION: Two-phase planning is a simple technique that can visualize tumor and organ movement simultaneously using CT. It further defines adequate field margins around the tumor and prevents unexpected radiation exposure to critical organs. Routine use of this technique for upper abdominal irradiation is recommended.
Abdominal Neoplasms↗
Cancer incidence in old age.
The incidence of all cancer increases with age through most of the human life span, but its real incidence at very old ages has not been well elucidated to date. Clarification of the real incidence of cancer in old age, especially among centenarians, may well provide pivotal information to understand the characteristics of humankind. In this study, autopsy records of the Annual of the Pathological Autopsy Cases in Japan, 1991-1996, vols. 34-39 (Japanese Society of Pathology, Tokyo) were used. Cases over 90 years old were studied individually for accurate analysis. The incidence of cancer peaked in the 6th decade and that of multiple cases in the 8th decade. In groups over 90 years of age, the incidence at 5-year intervals did not show any significant decrement. Moreover, the metastatic rate and rate of death due to cancer among centenarians was about three-fourths and two-thirds, respectively, of that of cases aged 90-94 years. The decrease in the metastatic ratio and less mortality due to cancer occurring at the oldest ages are considered due to the nature of cancer itself. The fact that the incidence of cancer does not increase would suggest that certain people among those of advanced age have a special resistance to it.
Aged↗
Orexins suppress catecholamine synthesis and secretion in cultured PC12 cells.
New orexigenic peptides called orexin-A and -B have recently been described in neurons of the lateral hypothalamus and perifornical area. No orexins have been found in adipose tissues or visceral organs, including the adrenal gland. However, expression of the orexin-receptor 2 (OX2R) in the rat adrenal gland has been reported. To test the effects of orexins on peripheral organs, we investigated their effects on catecholamine synthesis and secretion in the rat pheochromocytoma cell line PC12. Orexin-A and -B (100 nM) significantly reduced basal and PACAP-induced tyrosine hydroxylase (TH) (the rate-limiting enzyme in the biosynthesis of catecholamines) mRNA levels. Orexin-A and -B (100 nM) also significantly inhibited the PACAP-induced increase in the cAMP level, suggesting that the suppressive effect on TH mRNA is mediated, at least in part, by the cAMP/protein kinase A pathway. Furthermore, orexin-A and -B (100 nM) significantly suppressed basal and PACAP-induced dopamine secretion from PC12 cells. Next, we examined whether orexin receptors (OX1R, OX2R) were present in the rat adrenal gland and PC12 cells. In the adrenal glands, OX2R was as strongly expressed as in the hypothalamus, but OX1R was not detected. On the other hand, neither OX1R nor OX2R was expressed in PC12 cells. However, binding assays showed equal binding of orexin-A and -B to PC12 cells, suggesting the existence in these cells of some receptors for orexins. These results indicate that orexins suppress catecholamine release and synthesis, and that the inhibitory effect is mediated by the cAMP/protein kinase A pathway.
Adrenal Gland Neoplasms↗
Tumor bulk as a prognostic factor for the management of localized aggressive non-Hodgkin's lymphoma: a survey of the Japan Lymphoma Radiation Therapy Group.
PURPOSE: To identify the prognostic factors that specifically predict survival rates of patients with localized aggressive non-Hodgkin's lymphoma (NHL). METHODS AND MATERIALS: The survey was carried out at 25 radiation oncology institutions in Japan in 1998. The 5-year event-free (EFS) and overall survival rates (OAS) were calculated, and univariate and multivariate analyses were done to identify which of the following factors, namely, gender, age, performance status (PS), serum lactate dehydrogenase (LDH) level, Stage (I vs. II), tumor bulk (maximum diameter), and treatment, were significant from the viewpoint of prognosis. RESULTS: A total of 1141 patients with Stage I and II NHL were treated by the Japanese Lymphoma Radiation Therapy Group between 1988 and 1992. Of them, 787 patients, who were treated using definitive radiotherapy with or without chemotherapy for intermediate- and high-grade lymphomas in working formulation, constituted the core of this study. Primary tumors arose mainly from extranodal organs (71%) in the head and neck (Waldeyer's ring: 36% and sinonasal cavities: 9%). The factors associated with poorer prognosis were age over 60 years old (p < 0. 0001), radiation therapy alone (p < 0.0001), PS = 2-4 (p = 0.0011), (sex male, p = 0.0078), a bulky tumor more than 6 cm in maximum diameter (p = 0.0088), elevated LDH (p = 0.0117), and stage II (p = 0.0642). A median dose of 42 Gy was delivered mainly to the involved fields. Short-course chemotherapy was provided in 549 (70%) patients. The 5-year OAS and EFS rates for all patients were 71% and 67%, respectively. According to the stage-modified International Prognostic Index, the 5-year EFS of the patients with risk factors from 0 to 1 was 76%, 61% for patients with two risk factors, and 26% for patients with three or more risk factors. CONCLUSION: Extranodal presentation, especially Waldeyer's ring and sinonasal cavities, is encountered more frequently in Japan than in Western countries. Tumor bulk is an important prognostic factor in patients with localized aggressive extranodal NHL. Short course chemotherapy followed by radiation therapy was associated with prolonged survival in patients with localized aggressive NHLs of extranodal origin and 0-1 risk factor.
Adult↗
Changes in electrophysiological properties of axon terminals of locus coeruleus neurons with age in F344 rat.
Age-dependent changes in electrophysiological properties of locus coeruleus (LC) neurons were studied in urethane anesthetized male F344 rats aged 8, 17 and 22 months. These properties, such as spontaneous firing rate, conduction time, and threshold currents were measured for individual LC neurons antidromically activated from terminals in the frontal cortex. We found no change in the electrophysiological properties with age except for an increased number of low-threshold LC neurons in 22-month-old rats compared to that of 8-month-old rats. These results suggested that the electrophysiological properties of cortical axon terminals of LC neurons changed with age.
Action Potentials↗
kappa-Opioid inhibits catecholamine biosynthesis in PC12 rat pheochromocytoma cell.
It was reported that nicotine-induced dopamine release in the rat pheochromocytoma cell line, PC12 cells, was inhibited by kappa-opioid. However, it is not known whether inhibition of catecholamine biosynthesis is involved in the inhibitory mechanisms of kappa-opioids in PC12 cells. U-69593 (a kappa-opioid agonist: >/=100 nM) significantly inhibited the nicotine-induced increase of tyrosine hydroxylase (TH, a rate-limiting enzyme in biosynthesis of catecholamine) enzyme activity and TH mRNA levels. These inhibitory effects were completely reversed by naloxone and nor-binaltorphimine dihydrochloride (nor-BNI), a specific kappa-antagonist, whereas pertussis toxin (PTX) only partially reversed this inhibitory effect. Also, U-69593 (>/=100 nM) significantly inhibited the nicotine-induced increase of cAMP production. This inhibitory effect was completely reversed by naloxone and nor-BNI, whilst only partially reversed by PTX. Moreover, U-69593 (>/=100 nM) significantly inhibited the nicotine-induced increase of both the TH protein level and intracellular catecholamine levels. These results indicate that the anti-cholinergic actions of kappa-opioid can be explained partially by its inhibition of both TH enzyme activity and TH synthesis, through suppression of the cAMP/protein kinase A pathway. It would also appear that the PTX-sensitive G-protein mediates the inhibitory effect of this pathway, at least in part.
Animals↗
Angiotensin-II subtype 2 receptor agonist (CGP-42112) inhibits catecholamine biosynthesis in cultured porcine adrenal medullary chromaffin cells.
Angiotensin II subtype 2 receptor (AT(2)-R) is abundantly expressed in adrenal medullary chromaffin cells. However, the physiological roles of AT(2)-R in chromaffin cells remain to be clarified. Therefore, we investigated the effects of CGP42112 (AT(2)-R agonist) on catecholamine biosynthesis in cultured porcine adrenal medullary cells. We initially confirmed AT(2)-R was predominantly expressed in porcine adrenal medullary cells by [(125)I]-Ang II binding studies. CGP42112 (>==1 nM) significantly inhibited cGMP production from the basal value. Tyrosine hydroxylase (TH) is a rate-limiting enzyme in the biosynthesis of catecholamine, and its activity is regulated by both TH-enzyme activity and TH-synthesis. CGP42112 (>==1 nM) significantly inhibited TH-enzyme activity from the basal value. These inhibitory effects of CGP42112 on TH-enzyme activity and-cGMP production were abolished by PD123319 (AT(2)-R antagonist) while CV-11974 (AT(1)-R antagonist) was ineffective. We also tested whether decrease of cGMP is involved in the inhibitory effect of CGP42112 on TH-enzyme activity. Pretreatment of 8-Br-cGMP (membrane-permeable cGMP analogue) prevented the inhibitory effect of CGP 42112 on TH-enzyme activity. Similar to that of TH-enzyme activity, CGP42112 (>==1 nM) significantly reduced TH-mRNA and TH-protein level from the basal value, and these inhibitory effects were abolished by PD123319 but not CV-11974. These findings demonstrate that CGP 42112 reduces both TH-enzyme activity and TH-synthesis and that these inhibitory effects could be mediated by decrease of cGMP production.
8-Bromo Cyclic Adenosine Monophosphate↗
Effects of natriuretic peptides (ANP, BNP, CNP) on catecholamine synthesis and TH mRNA levels in PC12 cells.
Atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are present in adrenal chromaffin cells, and are co-secreted with catecholamines suggesting that these natriuretic peptides (NPs) may modulate functions of chromaffin cells in an autocrine and/or paracrine manner. Therefore, we investigated the effects of NPs on tyrosine hydroxylase (TH: a rate-limiting enzyme in biosynthesis of catecholamine) mRNA in rat pheochromocytoma PC12 cells. It was also determined whether the cyclic GMP/cGMP-dependent protein kinase (cGMP/PKG) pathway was involved in theses effects. Finally, we examined the effects of NPs on intracellular catecholamine content to confirm increase of catecholamine synthesis following TH mRNA induction. NPs (0.1 microM) induced significant increases of the TH mRNA (ANP= BNP> CNP). Also, the effects of NPs on TH mRNA were mimicked by 8-bromo cyclic GMP (1mM), and were blocked by KT5823 (1 microM) (inhibitor PKG) or LY83583 (1 microM) (guanylate cyclase inhibitor). Moreover, NPs were shown to induce significant increases of intracellular catecholamine contents (ANP= BNP> CNP). These findings suggest that NPs induced increases of TH mRNA through cGMP/PKG dependent mechanisms, which, in turn, resulted in stimulation of catecholamine synthesis in PC12 cells.
Animals↗
PTRF (polymerase I and transcript-release factor) is tissue-specific and interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor which binds to the two mouse type-I collagen gene promoters.
We have used the yeast two-hybrid system to clone the protein that interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor that was cloned previously as the factor that binds to the two mouse proximal promoters of the type-I collagen genes. We utilized as bait the N-terminal domain of BFCOL1 that includes the zinc-finger DNA-binding domain. One cDNA contained a potential open reading frame for a polypeptide of 392 amino acids and was identical to PTRF (polymerase I and transcript-release factor), which is involved in transcription termination of the RNA polymerase I reaction. Northern-blot analysis revealed that the pattern of mRNA expression was similar to that of the type-I collagen gene. In addition, we detected the mRNA expression only in a fibroblast cell line and two bone cell lines, but not in other blood and neuronal cell lines. Recombinant protein was shown to enhance the binding of BFCOL1 to its binding site in the mouse proalpha2(I) collagen proximal promoter in vitro. The transient-transfection experiment showed that PTRF had a suppressive effect on the mouse proalpha2(I) collagen proximal promoter activity. We speculate that PTRF might play a role in the RNA polymerase II reaction as well as that of RNA polymerase I.
Animals↗
Vertebral decancellation for severe scoliosis.
STUDY DESIGN: The results of staged surgery including vertebral decancellation were reviewed retrospectively for 21 patients with severe scoliosis. OBJECTIVES: To evaluate the benefits and limitations of vertebral decancellation as new anterior surgical procedure. SUMMARY OF BACKGROUND DATA: The curvatures of severe scoliosis are often very rigid, and surgical correction using the anterior or posterior approach may not achieve the desired correction. Some studies reported neurologic complications might appear due to the aggressive approach or excessive correction force. METHODS: Twenty-one patients (average age, 17.0 years) with severe scoliosis, in whom Cobb angle was over 80 degrees (average angle, 107 degrees), underwent staged anterior and posterior spinal reconstruction. Vertebral decancellation was performed as anterior procedure, and until posterior instrumentation, halo traction was carried out. The transition of curvatures in coronal and sagittal planes was assessed in this series. RESULTS: The average correction rate of lateral curvature at the final follow-up was 46%. The average loss of correction was 2.5 degrees. Kyphosis, measured between T5 and T12, changed from 41 degrees to 36 degrees. Lordosis, measured between L1 and S1, changed from 56 degrees to 45 degrees. Transient neurologic deficit was seen in one case after vertebral decancellation. CONCLUSIONS: Staged surgery including vertebral decancellation is an effective surgical method for patients with severe scoliosis, where an inflexible rigid curve or the risk of occurrence of neurologic complications due to temporary correction may exist.
Adolescent↗
Interaction between GADD34 and kinesin superfamily, KIF3A.
GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest. To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with the murine GADD34. One cDNA clone was the C-terminal part of KIF3A gene including the tail domain. The interaction between GADD34 and KIF3A was confirmed in the NIH3T3 cells by in vivo two-hybrid analysis. We could detect that GADD34 was induced with methyl methanesulfonate; however, the mRNA induction of KIF3A was not detected.
3T3 Cells↗
p300 collaborates with Sp1 and Sp3 in p21(waf1/cip1) promoter activation induced by histone deacetylase inhibitor.
We have reported that histone acetylation induced by trichostatin A (TSA) promotes p21(waf1/cip1) (p21) expression, the GC-box located just upstream of TATA box was responsible for TSA-induced promoter activation, and both Sp1 and Sp3 were the working activator of this GC-box. To understand the molecular pathway from histone acetylation to this Sp1 family factors-mediated promoter activation, we investigated the function of p300, one of the histone acetyltransferase, in the present work. The evidence supporting the linkage between p300 and TSA-induced p21 promoter activation were realized from the following findings: 1) cotransfection of p300 elevated p21 promoter activity, and this elevation was dependent on TSA-responsive GC-box; 2) TSA-induced promoter activation was blocked by the introduction of p300 dominant-negative mutant into cells; and 3) Sp1- or Sp3-mediated activation was also suppressed by this p300 dominant-negative mutant. Our data also suggested that p300 collaborates with Sp1 in a way which is different from that when p300 collaborates with p53 in p21 transcription.
Animals↗
Cu/Zn- and Mn-superoxide dismutases are specifically up-regulated in neurons after focal brain injury.
In previous studies, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed cell death due to the cytotoxic effect of microglial nitric oxide (NO), and are finally eliminated by activated microglia. In contrast, neurons in a narrow surrounding region nearby this boundary area remain alive even though they may encounter cytotoxic NO. To investigate the mechanism by which neurons tolerate this oxidative stress, we examined the in vitro and in vivo expression levels of superoxide dismutase (SOD) under pathological conditions. Results from our in situ hybridization and immunohistochemical studies showed up-regulation of Cu/Zn-SOD only in neurons outside the boundary area, whereas up-regulation of Mn-SOD was detected in both neurons and glial cells in the same region. In vitro experiments using rat PC12 pheochromocytoma and C6 glioma cell lines showed that induction of both Cu/Zn- and Mn-SOD mRNA could only be detected in PC12 cells after treatment with NO donors, while a slight induction of Mn-SOD mRNA alone could be seen in C6 glioma cells. The mechanism of resistance toward oxidative stress therefore appears to be quite different between neuronal and glial cells. It is assumed that these two types of SOD might play a critical role in protecting neurons from NO cytotoxicity in vivo, and the inability of SOD induction in damaged neurons seems to cause their selective elimination after focal brain injury.
Animals↗
Treatment for pyothorax-associated lymphoma.
In eight patients with pyothorax-associated lymphoma (PAL), which resulted from artificial pneumothorax for the treatment of pulmonary tuberculosis, seven patients received radiotherapy and five showed no local recurrence. All four patients treated by primary chemotherapy had disease progression. Radiotherapy of 50 Gy with wide margins is recommended to treat PAL.
Aged↗