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Biomedical subjects

Shigeo Ohta

Publications and source records attributed to Shigeo Ohta.

At least 19 recordsLinked to original sources
PubMed2007

Transduction of the anti-apoptotic PTD-FNK protein improves the efficiency of transplantation of bone marrow mononuclear cells.

Since most transplanted cells rapidly die in an ischemic environment by hypoxia and hyponutrition, it is crucial to know how to protect transplanted cells for improving transplantation efficiency. We examined whether the transduction of an artificial anti-cell death protein (PTD-FNK) into bone marrow mononuclear cells (BM-MNCs) prevents cell death and improves the transplantation efficiency of BM-MNCs in ischemic regions. Rat bone marrow cells were prepared from the femur and tibia and cultured on dishes precoated with human fibronectin in the absence of serum. BM-MNCs transduced with PTD-FNK survived better than those without the protein (P<0.008) and retained the potential to differentiate into endothelial progenitor cells (EPCs), as judged by the uptake of an acetylated low-density lipoprotein and the ability to bind lectin. Next, we used a co-culture system comprising human umbilical vein endothelial cells (HUVECs) and fibroblasts to examine angiogenic potential. HUVECs pretreated with PTD-FNK survived and formed a blood-vessel-like structure better than untreated cells (P<0.001). When BM-MNCs expressing EGFP were transplanted into ischemic areas of a male rat ischemic hindlimb model, the cells pretreated with PTD-FNK were incorporated into blood vessel with a higher efficiency than the untreated BM-MNCs (P=0.03). BM-MNCs protected through transduction of PTD-FNK maintained their angiogenic potential. Thus, PTD-FNK improves the transplantation efficiency of BM-MNCs into ischemic regions.

Amino Acid Motifs↗
PubMed2006

Dysfunction of mitochondria and oxidative stress in the pathogenesis of Alzheimer's disease: on defects in the cytochrome c oxidase complex and aldehyde detoxification.

The mitochondrion is an organelle that plays a central role in energy production. It, at the same time, generates reactive oxygen species as by-products. Large-scale epidemiological case-control studies suggest the involvements of dihydrolipoamide succinyltransferase (DLST) of the mitochondrial Krebs cycle and mitochondrial aldehyde dehydrogenase-2 (ALDH2) in Alzheimer's disease (AD). The DLST gene has two gene-products, one of which, a novel gene product MIRTD, mediates the molecular assembly of the cytochrome c oxidase complex whose defect has been a candidate of the causes of AD. Since levels of MIRTD mRNA in the brains of AD patients were significantly low, a decrease in MIRTD could affect energy production. ALDH2, a matrix enzyme, was found to act as a protector against oxidative stress through oxidizing toxic aldehydes, such as 4-hydroxy-2-nonenal, that are spontaneously produced from lipid peroxides. Hence, a decrease in ALDH2 activity is proposed to contribute to AD. Indeed, transgenic mice with low activity of ALDH2 exhibited an age-dependent neurodegeneration accompanying memory loss. Since amyloid beta peptide has been recently shown to be present in neuronal mitochondria to decline energy production and enhance ROS production, it has become possible to link AD more closely with roles of mitochondria in the pathogenesis.

Alcohol Dehydrogenase↗
PubMed2005

Association of alcohol dehydrogenase 2*1 allele with liver damage and insulin concentration in the Japanese.

The Japanese have a polymorphism in the alcohol dehydrogenase 2 gene (ADH2). The alleles of ADH2 (ADH2*1 and ADH2*2) encode more active and less active forms for ethanol metabolism, respectively. We examined whether liver damage and the insulin-glucose axis vary according to ADH2 genotype in the Japanese. The 2,232 subjects (1,126 men and 1,106 women) were recruited from a population-based prospective cohort study. Clinical evaluations including alcohol consumption, percentage of alcohol drinkers, plasma glucose, HbA1c, insulin, AST, ALT, gamma-GTP, and prevalence of diabetes were compared among the ADH2 genotypes. The percentage of drinkers, alcohol consumption, AST, ALT, and gamma-GTP were higher in group ADH2*1/1 than in group ADH2*1/2 or ADH2*2/2 (all P < 0.05). Hence, ADH2*1/1 is associated with excess alcohol intake and liver disorders. However, the prevalence of diabetes did not differ among the three groups. For the glucose-insulin axis, we examined subjects who did not receive insulin therapy or oral anti-diabetes medication. While amounts of alcohol consumed and glucose levels were nearly the same between ADH*1/2 and ADH2*2/2, insulin concentrations were lower in ADH2*2/1 than in ADH2*2/2 (P < 0.05 in men). This finding suggests that the ADH2*1 allele is associated with a lower insulin concentration when alcohol intake is light or moderate. It also suggests that the genetic effect of ADH2*1 plays an important role in alcohol drinking behavior and in the occurrence of liver injury, but the effect is so mild that it does not influence the glucose-insulin axis or prevalence of diabetes.

Alcohol Dehydrogenase↗
PubMed2005

MIDAS/GPP34, a nuclear gene product, regulates total mitochondrial mass in response to mitochondrial dysfunction.

To investigate the regulatory system in mitochondrial biogenesis involving crosstalk between the mitochondria and nucleus, we found a factor named MIDAS (mitochondrial DNA absence sensitive factor) whose expression was enhanced by the absence of mitochondrial DNA (mtDNA). In patients with mitochondrial diseases, MIDAS expression was increased only in dysfunctional muscle fibers. A majority of MIDAS localized to mitochondria with a small fraction in the Golgi apparatus in HeLa cells. To investigate the function of MIDAS, we stably transfected HeLa cells with an expression vector carrying MIDAS cDNA or siRNA. Cells expressing the MIDAS protein and the siRNA constitutively showed an increase and decrease in the total mass of mitochondria, respectively, accompanying the regulation of a mitochondria-specific phospholipid, cardiolipin. In contrast, amounts of the mitochondrial DNA, RNA and proteins did not depend upon MIDAS. Thus, MIDAS is involved in the regulation of mitochondrial lipids, leading to increases of total mitochondrial mass in response to mitochondrial dysfunction.

Cardiolipins↗
PubMed2005

Wobble modification deficiency in mutant tRNAs in patients with mitochondrial diseases.

Point mutations in mitochondrial (mt) tRNA genes are associated with a variety of human mitochondrial diseases. We have shown previously that mt tRNA(Leu(UUR)) with a MELAS A3243G mutation and mt tRNA(Lys) with a MERRF A8344G mutation derived from HeLa background cybrid cells are deficient in normal taurine-containing modifications [taum(5)(s(2))U; 5-taurinomethyl-(2-thio)uridine] at the anticodon wobble position in both cases. The wobble modification deficiency results in defective translation. We report here wobble modification deficiencies of mutant mt tRNAs from cybrid cells with different nuclear backgrounds, as well as from patient tissues. These findings demonstrate the generality of the wobble modification deficiency in mutant tRNAs in MELAS and MERRF.

Base Sequence↗
PubMed2005

The anti-cell death FNK protein protects cells from death induced by freezing and thawing.

The FNK protein, constructed from anti-apoptotic Bcl-xL with enhanced activity, was fused with the protein transduction domain (PTD) of the HIV/Tat protein to mediate the delivery of FNK into cells. The fusion protein PTD-FNK was introduced into chondrocytes in isolated articular cartilage-bone sections, cultured neurons, and isolated bone marrow mononuclear cells to evaluate its ability to prevent cell death induced by freezing and thawing. PTD-FNK protected the cells from freeze-thaw damage in a concentration-dependent manner. Addition of PTD-FNK with conventional cryoprotectants (dimethyl sulfoxide and hydroxyethyl starch) increased surviving cell numbers around 2-fold compared with controls treated only with the cryoprotectants. Notably, PTD-FNK allowed CD34+ cells among bone marrow mononuclear cells to survive more efficiently (12-fold more than the control cells) from two successive freeze-thaw cycles. Thus, PTD-FNK prevented cell death induced by freezing and thawing, suggesting that it provides for the successful cryopreservation of biological materials.

Animals↗
PubMed2005

Positive contribution of pathogenic mutations in the mitochondrial genome to the promotion of cancer by prevention from apoptosis.

The role of mitochondrial dysfunction in cancer has been a subject of great interest and much ongoing investigation. Although most cancer cells harbor somatic mutations in mitochondrial DNA (mtDNA), the question of whether such mutations contribute to the promotion of carcinomas remains unsolved. Here we used trans-mitochondrial hybrids (cybrids) containing a common HeLa nucleus and mtDNA of interest to compare the role of mtDNA against the common nuclear background. We constructed cybrids with or without a homoplasmic pathogenic point mutation at nucleotide position 8,993 or 9,176 in the mtDNA ATP synthase subunit 6 gene (MTATP6) derived from patients with mitochondrial encephalomyopathy. When the cybrids were transplanted into nude mice, the MTATP6 mutations conferred an advantage in the early stage of tumor growth. The mutant cybrids also increased faster than wild type in culture. To complement the mtDNA mutations, we transfected a wild-type nuclear version of MTATP, whose codons were converted to the universal genetic codes containing a mitochondrial target sequence, into the nucleus of cybrids carrying mutant MTATP6. The restoration of MTATP slowed down the growth of tumor in transplantation. Conversely, expression of a mutant nuclear version of MTATP6 in the wild-type cybrids declined respiration and accelerated the tumor growth. These findings showed that the advantage in tumor growth depended upon the MTATP6 function but was not due to secondary nuclear mutations caused by the mutant mitochondria. Because apoptosis occurred less frequently in the mutant versus wild-type cybrids in cultures and tumors, the pathogenic mtDNA mutations seem to promote tumors by preventing apoptosis.

Adenosine Triphosphatases↗
PubMed2005

Prognostic significance of the immunohistochemical index of survivin in glioma: a comparative study with the MIB-1 index.

OBJECTIVE: Survivin has been identified as a protein expressed in cancer cells and a member of the inhibitor-of-apoptosis protein family. Recent studies suggest that the expression of survivin increases during the G2/M phase of the cell cycle, and may be used in clinical prognosis. We examined whether survivin expression in human gliomas would be a correlative of prognosis. METHODS: We prepared polyclonal anti-survivin serum to establish a survivin index for stained sections, using an immunohistochemical procedure, according to the method used for scoring MIB-1 index, and then stained 29 paraffin-embedded sections from surgical specimens of 29 patients who were classified into three grades of World Health Organization with the mean age of low grade astocytoma (grade II) being 34.7; anaplastic astrocytoma (grade III), 48.8; and glioblastoma multiform (grade IV), 58.4. RESULTS: On staining with the anti-survivin antiserum, all specimens contained positive cells, but the survivin index was heterogeneous among grades. The mean percentage of immunoreactive cells in each specimen was 70.0 (SD 18.2) in grade II, 81.3 (16.5) in grade III, and 85.0 (13.6) in grade IV. Then we compared the survivin index to the MIB-1 index and found that in low-grade gliomas (grade II and III), the difference in survival times between the high and low survivin indexes was significant (P=0.007), whereas that between the high and low MIB-1 indexes was not significant (P=0.092). ONCLUSION: Survivin is more sensitive marker than MIB-1 for the evaluation of low-grade gliomas in that it helps to predict patient survival. Much larger glioma patient series are needed to validate the findings of our limited study.

Adult↗
PubMed2005

Inactive aldehyde dehydrogenase-2 increased the risk of pancreatic cancer among smokers in a Japanese male population.

OBJECTIVES: Most of the acetaldehyde, a recognized animal carcinogen, generated during alcohol metabolism is eliminated by liver mitochondrial aldehyde dehydrogenase 2 (ALDH2). More than 40% of Japanese have the inactive form of ALDH2, and inactive ALDH2 is a risk factor for multiple cancers of the esophagus as well as head and neck cancer. Possible associations between pancreatic cancer and ALDH2 gene polymorphism, in conjunction with smoking and/or drinking habits, were examined in a Japanese population. METHODS: We investigated 114 patients (70 male and 44 female) with pancreatic cancer and compared them with 2070 control subjects (1050 male and 1020 female). The drinking (5 g ethanol consumption/d) and/or smoking habits as well as ALDH2 gene polymorphism were examined. RESULTS: In male subjects, the frequency of the active form of ALDH2 (2*1/2*1) was lower in pancreatic cancer patients than in control subjects (P = 0.018). The frequency of subjects with both smoking and drinking habits was significantly higher in pancreatic cancer patients than in control subjects having ALDH2*1/2*1 and ALDH2*1/2*2. The frequency of smoking habit alone was significantly higher in pancreatic cancer patients compared with control subjects having inactive ALDH2. Drinking habit had no relation to pancreatic cancer. In female subjects, neither habit had a relation to pancreatic cancer. CONCLUSIONS: Smoking habit did increase the risk of pancreatic cancer, and this risk was further enhanced in subjects with inactive ALDH2 in a male population but not in a female population. There was no relationship between drinking habit and pancreatic cancer in either sex population.

Adult↗
PubMed2004

Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease.

Point mutations in the mitochondrial (mt) tRNA(Leu(UUR)) gene are responsible for mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), a subgroup of mitochondrial encephalomyopathic diseases. We previously showed that mt tRNA(Leu(UUR)) with an A3243G or T3271C mutation derived from patients with MELAS are deficient in a normal taurine-containing modification (taum5U; 5-taurinomethyluridine) at the anticodon wobble position. To examine decoding disorder of the mutant tRNA due to the wobble modification deficiency independent of the pathogenic point mutation itself, we used a molecular surgery technique to construct an mt tRNA(Leu(UUR)) molecule lacking the taurine modification but without the pathogenic mutation. This "operated" mt tRNA(Leu(UUR)) without the taurine modification showed severely reduced UUG translation but no decrease in UUA translation. We thus concluded that the UUG codon-specific translational defect of the mutant mt tRNAs(Leu(UUR)) is the primary cause of MELAS at the molecular level. This result could explain the complex I deficiency observed clinically in MELAS.

Base Sequence↗
PubMed2004

Green fluorescent protein causes mitochondria to aggregate in the presence of the Bcl-2 family proteins.

Green fluorescent protein (GFP) has been widely used in a variety of experiments in cell biology. When cells were co-transfected with the GFP gene and the bcl-2 family genes bcl-2, bcl-x(L), and bax, mitochondria appeared to aggregate at the periphery of the nucleus specifically where GFP was expressed. Little aggregation was seen in the presence of other members of the GFP family, EGFP (enhanced GFP), ECFP (enhanced cyan variant), and EYFP (enhanced yellow-green variant). GFP but not EGFP seemed to promote cell death induced by pro-apoptotic Bax. Thus, GFP specifically promotes the aggregation of mitochondria when co-expressed with a member of the Bcl-2 family in association with apoptosis.

Animals↗
PubMed2004

Transduction of anti-apoptotic proteins into chondrocytes in cartilage slice culture.

Peptides of the protein transduction domain (PTD) mediate the introduction of passenger proteins into cells in vitro and in vivo, where the domains are positively charged. This unusual ability can be exploited for medical applications in protein therapeutics. Chondrocytes are embedded in a dense extracellular matrix, whose components are highly negatively charged. We examined whether PTD mediates the delivery of functional proteins into chondrocytes through the matrix using the super anti-apoptotic protein FNK fused with Tat/PTD peptide (PTD-FNK), the FNK protein being constructed from anti-apoptotic Bcl-xL to enhance its activity. The PTD-FNK protein labeled with a fluorescent dye was incorporated into chondrocytes through the matrix and immunostaining confirmed the transduction into the cells. The PTD-FNK protein protected chondrocytes from cell death induced by Fas antibody and nitrogen oxide (NO). Thus, the PTD peptide has the ability to deliver passenger proteins into chondrocytes by penetrating the extracellular matrix of cartilage.

Apoptosis↗
PubMed2004

Accumulation of somatic mutation in mitochondrial DNA and atherosclerosis in diabetic patients.

A point mutation of mitochondrial DNA at nucleotide position 3243 A to G is responsible for the genetic cause of diabetes. Otherwise, this mutation is also reported to occur as a somatic mutation, possibility because of oxidative stress. Because diabetes may cause oxidative stress, we hypothesized that accumulation of the somatic A3243G mutation in mitochondrial DNA may be accelerated by diabetes. DNA was extracted from blood samples of 290 nondiabetic healthy subjects (aged 0-60 years) and from 383 type 2 diabetic patients (aged 18-80 years). Then, the extent of somatic A3243G mutation in total mitochondrial DNA was detected by real-time polymerase chain reaction (PCR) using the TaqMan probe. The genotyping of ACE I/D or p22phox C242T was done by PCR or PCR-restriction fragment length polymorphism. Although the level of the A3243G mutation was negligible in the newborn group, it increased in healthy subjects aged 20-29 and 41-60 years. In diabetic patients, the mutational rate increased along with age and the duration of diabetes. In the middle-aged group (41-60 years old), the A3243G mutation accumulates fourfold higher in the diabetic patients than in the healthy subjects. Moreover, multiple regression analysis revealed that the most critical factor associated with this mutation in diabetic patients was the duration of diabetes. Furthermore, the genotype of DD, DI-CC (ACE-p22phox) has the highest mutational rate and the thickest intima-media thickness of the carotid artery. In conclusion, diabetes accelerates the accumulation of the somatic A3243G mutation in mitochondrial DNA, and this somatic mutation may be a marker for the duration of diabetes and atherosclerosis.

Adolescent↗