Depressive disorder due to mitochondrial transfer RNALeu(UUR) mutation.
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Adult↗
Biomedical subjects
K Inoue
Publications and source records attributed to K Inoue.
Tumor necrosis factor receptors in the pituitary cells.
To clarify the site and mode of action of tumor necrosis factor (TNF) in the pituitary, we studied the effects, binding sites of TNF and its receptor mRNA in the two types of mouse pituitary-derived cell lines, AtT-20, ACTH-producing cells and TtT/GF, folliculo-stellate (FS)-like cells. First, we examined the expression of TNF receptor mRNA in these cells. Using Northern blot analyses with radiolabeled cDNA to murine TNF receptor p60 and p80 mRNAs as probes, we identified both types of mRNA in the poly(A)-containing RNA prepared from AtT-20 cells and p60 TNF receptor mRNA from TtT/GF. The identified mRNA were compatible in size with those detected in the immune-competent cells. Next, we studied the TNF-binding sites on these cells. Scatchard plot analysis of the significant binding of [125I]TNF revealed a single type of binding site with a Kd (dissociation constant) of 210 pM and 131 binding sites/cell on AtT-20. Similarly on TtT/GF, [125I]TNF showed 353 binding sites/cell with a Kd of 900 pM. [125I]TNF binding on both types of cells competed with TNF and lymphotoxin (TNF beta) in an equimolar fashion. Third, TNF stimulates ACTH synthesis in AtT-20 cells, while TNF increases immunoreactive interleukin (IL)-6 release from TtT/GF cells. These findings demonstrate that AtT-20 and TtT/GF cells are equipped with fully functional TNF receptor system, and suggest that ligand of the receptor, TNF alpha and/or TNF beta, can modulate ACTH synthesis and release as a direct hormonal effector on corticotrophs or indirect modulator through another paracrine mediator, such as IL-6 from FS cells.
Animals↗
The glycoproteins that occur in the colloids of senescent porcine pituitary glands are clusterin and glycosylated albumin fragments.
Periodic acid-Schiff-positive colloids occur in the pituitary glands of many vertebrates. These colloids are surrounded by folliculo-stellate (FS) cells and increase dramatically in number and size with age. The characterization of these colloidal substances is necessary for the establishment of FS cell function. In a previous study we showed that these colloids in the senescent pituitary contain a number of glycoproteins; however, the characterization of these glycoproteins has not yet been carried out. To characterize the pituitary colloid, we purified the colloids by centrifugation and Percoll gradient. The isolated colloids were unable to be solubilized in a regular buffer since they were very dense. However, solubilization was accomplished in sodium dodecyl sulfate (SDS) lysis buffer following treatment with exoglycosidase. Analysis using SDS-polyacrylamide gel electrophoresis showed that the porcine pituitary colloids contain four proteins, CP26, CP40, CP48 and CP60, whose estimated molecular weights were 26 kD, 36-43 kD, 48 kD, and 60 kD, respectively. Staining with concanavalin A (ConA) showed that both CP26 and CP40 were glycoproteins. Moreover, N-terminal amino acid sequencing and Western blot analysis revealed CP26, CP48 and CP60 to be porcine albumin while CP40, the major protein in the colloids, proved to be clusterin. Therefore we report here that the glycoproteins that occur in senescent porcine pituitary colloids are of two types, namely, albumin fragments and clusterin.
Aging↗
Identification of inheritance modes of mitochondrial diseases by introduction of pure nuclei from mtDNA-less HeLa cells to patient-derived fibroblasts.
A nuclear genome delivery system was developed to deduce the modes of inheritance of the clinical phenotypes observed in patients with mitochondrial diseases by transfer of pure nuclei from normal cells to fibroblasts from the patients. The problem of possible contamination of the nuclei with a small amount of mtDNA was overcome by using mtDNA-less (rho0) human cells as nuclear donors. In this study, intercellular transfer of pure nuclei was carried out by simple fusion of rho0 HeLa cells with 533 fibroblasts from a patient with a fatal mitochondrial disease, which were deficient in cytochrome c oxidase and succinate dehydrogenase activities. The results showed that the cytochrome c oxidase and succinate dehydrogenase activities were restored by the introduction of pure HeLa nuclei, suggesting that the observed phenotypes of mitochondrial dysfunction were not due to mtDNA mutations but to nuclear, recessive mutations. Thus, our nuclear transfer system is effective for determining whether a mitochondrial or nuclear genome of a patient is responsible for a disease and whether deficiency of mitochondrial enzymes, including enzymes exclusively encoded by nuclear genomes, is transmitted in a nuclear recessive or nuclear dominant way, providing the parents of the patients with valuable information for genetic counseling on the risk of mitochondrial diseases in their next babies.
Adenosine Triphosphatases↗
Functional asymmetry of cortical motor control in left-handed subjects.
We used positron emission tomography (PET) to investigate which cortical motor areas are active in relation to unilateral dominant or non-dominant finger movements in left-handed subjects. The right premotor area was activated by both contralateral and ipsilateral finger movements, in contrast to the primary motor, the left premotor and supplementary motor areas in which activation was only by contralateral movements. Bilateral finger movements activated all of these areas. We conclude that there exists cortical asymmetry for motor control in left-handers which is different from that apparent in right-handers.
Adolescent↗
Exposure of phosphatidylethanolamine on the surface of apoptotic cells.
In the early stages of apoptosis, phosphatidylserine (PS) is translocated from the inner side of the plasma membrane to the outer layer, which allows phagocytes to recognize and engulf the apoptotic cells. In this study we have analyzed the cell surface exposure of phosphatidylethanolamine (PE) in apoptotic CTLL-2 cells, a cytotoxic T cell line, using a tetracyclic polypeptide of 19 amino acids (Ro09-0198) which specifically recognizes the structure of PE and forms a tight equimolar complex with the phospholipid. Fluorescence microscopic analysis showed that the peptide, conjugated with fluorescence-labeled streptavidin (FL-SA-Ro), bound effectively to the cell surface of cells undergoing apoptosis in response to withdrawal of interleukin-2 from the culture media, but not to nonapoptotic cells. The binding of FL-SA-Ro to apoptotic cells was not uniform and the intense staining was observed on surface blebs of apoptotic cells. The FL-SA-Ro binding was inhibited specifically by liposomes containing PE, suggesting that PE is mainly exposed on the surface blebs of apoptotic cells. The specific binding of FL-SA-Ro to the apoptotic cells was also confirmed using a flourescence-activated cell sorter and the time-dependent cell surface exposure of PE correlated well with the exposure of PS, as detected by the binding of annexin V. This study provides the first direct evidence that PE as well as PS is exposed on the cell surface during the early stages of apoptosis, resulting in the total loss of asymmetric distribution of aminophospholipids in the plasma membrane bilayer.
Animals↗
Potent inhibition by trivalent cations of ATP-gated channels.
The effects of La3+ and other trivalent cations on ATP-gated channels (P2X purinoceptor/channels) were investigated using rat pheochromocytoma PC12 cells and Xenopus oocytes expressing these channels. La3+, Gd3+, Ce3+ and Nd3+ (30-300 microM) inhibited an inward current activated by 30 microM ATP in PC12 cells. The concentration-response curve for the ATP-activated current was shifted by La3+ or Gd3+ toward a higher concentration range, and the slope of the curve became steeper, suggesting the inhibition is non-competitive. La3+ or Gd3+ did not affect the current component that was slowly activated upon hyperpolarization, and selectively inhibited the remaining 'voltage-independent' component. La3+ and Gd3+ also inhibited currents mediated through P2X1 and P2X2 purinoceptors expressed in Xenopus oocytes. The results suggest that La3+ and other trivalent cations inhibit P2X purinoceptors at low concentrations. The inhibition may at least partly be attributed to an allosteric inhibition.
Adenosine Triphosphate↗
cDNA cloning and expression of chicken aminopeptidase H, possessing endopeptidase as well as aminopeptidase activity.
Chicken aminopeptidase H is a cysteine protease possessing endopeptidase as well as aminopeptidase activity [Rhyu, M. R., Nishimura, T., Kato, Y., Okitani, A. & Kato, H. (1992) Eur. J. Biochem. 208, 53-59]. This enzyme exhibits molecular masses of 400 kDa on gel filtration and 52 kDa on SDS/PAGE, indicating that it consists of eight subunits with the same molecular mass. In the current study, we cloned the cDNA for the catalytic subunit of chicken aminopeptidase H. The open reading frame of the aminopeptidase H gene consists of 1362 base pairs encoding a 52-kDa protein consistent with the molecular mass determined on SDS/PAGE; the deduced amino acid sequence contains all the partial sequences determined for the purified enzyme. The sequence is similar to that of the bleomycin hydrolase of rabbit lung, which has been partially determined. The recombinant 52-kDa protein expressed in COS7 cells exhibited both aminopeptidase and endopeptidase activities, which were inhibited by monoiodoacetic acid. Furthermore, the expression of aminopeptidase H in COS7 cells was also recognized on immunoblotting. This gene is the first one for aminopeptidase H in an animal tissue whose sequence has been completely determined.
Amino Acid Sequence↗
Genomic structure and expression analyses of serine acetyltransferase gene in Citrullus vulgaris (watermelon).
The genomic clones of Sat gene encoding serine acetyltransferase (SATase), a key enzyme in cysteine biosynthesis in plants, were isolated from the genomic library of Citrullus vulgaris (watermelon). The determination of nucleotide sequence of 5.7 kilobase pair (kbp) length revealed the presence of two introns of 1939 basepair (bp) and 515 bp length in the gene. The transcription start point was determined by primer extension experiments. Southern blot analysis indicated the presence of a single copy of the Sat gene and a couple of additional related sequences in the genome of C. vulgaris. The expression of Sat was analyzed in watermelon plants growth under sulfur- and/or nitrogen-starved conditions and in the presence of pyrazole, O-acetylserine and N-acetylserine. Only slight increment (ca. 1.5-2-fold) of Sat gene expression was observed upon sulfur starvation for 48 h. Interestingly, the addition of pyrazole, which is a precursor of beta-pyrazolealanine (beta-PA) synthesized by SATase and cysteine/beta-PA synthase, enhanced the expression of Sat by ca. 2-fold.
Acetyltransferases↗
Differential tissue distribution of the beta- and gamma-subunits of human cytosolic platelet-activating factor acetylhydrolase (isoform I).
The cDNA for human platelet-activating factor acetylhydrolase (PAF-AH) beta-subunit was cloned. The complete amino acid sequence deduced from cDNA contains 229 amino acids and is completely identical with that of the bovine subunit. Moreover, the sequence of the human beta-subunit protein shows 62.4% identity with the human gamma-subunit at the amino acid level. Southern blot analysis suggested the presence of multiple genes and we indeed found another closely related pseudo gene. Northern blot analysis showed that the 4.0 kb transcript was expressed in all tissues tested, suggesting the ubiquitous distribution of the subunit protein. Differential distribution of beta- and gamma-subunits might suggest that the oligomeric structure of PAF-AH is different from tissue to tissue.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
Pyloroduodenal duplication cyst: case report
A 6-day-old female presented with vomiting and an abdominal mass. At laparotomy, a pyloroduodenal duplication cyst was enucleated from the pyloric region. Of the diagnostic studies performed, IV cholangiography with spiral computed tomography and an upper gastrointestinal barium study were useful in the preoperative differential diagnosis.
Journal Article↗
The interorganellar interaction between distinct human mitochondria with deletion mutant mtDNA from a patient with mitochondrial disease and with HeLa mtDNA.
For the examination of possible intermitochondrial interaction of human mitochondria from different cells, cybrids were constructed by introducing HeLa mitochondria into cells with respiration-deficient (rho-) mitochondria. Respiration deficiency was due to the predominance of mutant mtDNA with a 5,196-base pair deletion including five tRNA genes (DeltamtDNA5196). The HeLa mtDNA and DeltamtDNA5196 encoded chloramphenicol-resistant (CAPr) and chloramphenicol-sensitive (CAPs) 16 S rRNA, respectively. The first evidence for the interaction was that polypeptides exclusively encoded by DeltamtDNA5196 were translated on the introduction of HeLa mitochondria, suggesting supplementation of the missing tRNAs by rho- mitochondria from HeLa mitochondria. Second, the exchange of mitochondrial rRNAs was observed; even in the presence of CAP, CAPs DeltamtDNA5196-specific polypeptides as well as those encoded by CAPr HeLa mtDNA were translated in the cybrids. These phenomena can be explained assuming that the translation in rho- mitochondria was restored by tRNAs and CAPr 16 S rRNA supplied from HeLa mitochondria, unambiguously indicating interorganellar interaction. These observations introduce a new concept of the dynamics of the mitochondrial genetic system and help in understanding the relationship among mtDNA mutations and expression of human mitochondrial diseases and aging.
Chloramphenicol Resistance↗
Aberrant overexpression of the Wilms tumor gene (WT1) in human leukemia.
To clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34- cell populations, and the CD34+ cells into nine subsets (CD34+ CD33-, CD34+ CD33+, CD34+ CD38-, CD34+ CD38+, CD34+ HLA-DR-, CD34+ HLA-DR+, CD34+ c-kit(high), CD34+ c-kit(low), and CD34+ c-kit-) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+ CD33-, CD34+ CD33+, CD34- CD33+, and CD34- CD33-). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34- cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 x 10(-2)) or undetectable (< 10(-2)) levels (the WT1 expression level of K562 cells was defined as 1.0), whereas the average levels of WT1 expression in FACS-sorted and -unsorted leukemic cells were 2.4 to 9.3 x 10(-1). Thus, the WT1 expression levels in normal hematopoietic progenitor cells were at least 10 times less than those in leukemic cells. Therefore, we could not find any normal counterparts of BM or CB that expressed the WT1 at levels comparable with those in leukemic cells. These results indicate an aberrant overexpression of the WT1 gene in leukemic cells and imply the involvement of this gene in human leukemogenesis.
Acute Disease↗
Serine phospholipid-specific phospholipase A that is secreted from activated platelets. A new member of the lipase family.
Rat platelets secrete two types of phospholipases upon stimulation; one is type II phospholipase A2 and the other is serine-phospholipid-selective phospholipase A. In the current study we purified serine-phospholipid-selective phospholipase A and cloned its cDNA. The final preparation, purified from extracellular medium of activated rat platelets, gave a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. [3H]Diisopropyl fluorophosphate, an inhibitor of the enzyme, labeled the 55-kDa protein, suggesting that this polypeptide possesses active serine residues. The cDNA for the enzyme was cloned from a rat megakaryocyte cDNA library. The predicted 456-amino acid sequence contains a putative short N-terminal signal sequence and a GXSXG sequence, which is a motif of an active serine residue of serine esterase. Amino acid sequence homology analysis revealed that the enzyme shares about 30% homology with mammalian lipases (lipoprotein lipase, hepatic lipase, and pancreatic lipase). Regions surrounding the putative active serine, histidine, and aspartic acid, which may form a "lipase triad," were highly conserved among these enzymes. The recombinant protein, which we expressed in Sf9 insect cells using the baculovirus system, hydrolyzed a fatty acyl residue at the sn-1 position of lysophosphatidylserine and phosphatidylserine, but did not appreciably hydrolyze phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidic acid, and triglyceride. The present enzyme, named phosphatidylserine-phospholipase A1, is the first phospholipase that exclusively hydrolyses the sn-1 position and has a strict head group specificity for the substrate.
Amino Acid Sequence↗
Vascular endothelial growth factor expression in primary esophageal squamous cell carcinoma. Association with angiogenesis and tumor progression.
BACKGROUND: Angiogenesis is essential for solid tumor growth and metastasis. Vascular endothelial growth factor (VEGF), a recently identified growth factor with significant angiogenic properties, may be a major tumor angiogenesis regulator in vivo. Conversely, there have been few studies of the association between angiogenic factor expression and angiogenesis in esophageal carcinoma. The authors examined VEGF expression and microvessel density in esophageal squamous cell carcinomas to clarify the association of VEGF expression with the clinicopathologic features of the disease. METHODS: Surgical specimens from 75 primary esophageal squamous cell carcinomas were examined for VEGF expression and microvessel density by immunocytochemical staining. Original anti-VEGF polyclonal antibody was used to determine VEGF expression, and antifactor VIII antibody was used to determine microvessel density. The isoforms of VEGF mRNA were determined by reverse transcriptase-polymerase chain reaction. RESULTS: Thirty-five (46.7%) of the 75 esophageal carcinomas were positive for VEGF protein. There was a close correlation between microvessel density and VEGF positivity (P = 0.0002). High VEGF levels were significantly associated with well differentiated tumors, advanced stage (depth of invasion and blood vessel invasion), high incidence of distant metastases after surgery, and poorer prognosis. CONCLUSIONS: The results of this study suggest that VEGF expression is associated with tumor progression and poor prognosis by stimulating angiogenesis in esophageal squamous cell carcinoma.
Adult↗
Identification of NAP-22 and GAP-43 (neuromodulin) as major protein components in a Triton insoluble low density fraction of rat brain.
NAP-22 is a membrane-localized brain enriched acidic protein having a Ca(2+)-dependent calmodulin binding activity. Further fractionation of the NAP-22 containing membrane showed the localization of NAP-22 in a Triton insoluble fraction of low density. Besides NAP-22, this fraction was found to contain GAP-43 (neuromodulin), trimeric G proteins, and some GPI-anchored proteins such as Thy-1 and N-CAM-120. Presence of some protein tyrosine kinases, such as src and fyn, was also shown.
Amino Acid Sequence↗
Brain acetylhydrolase that inactivates platelet-activating factor is a G-protein-like trimer.
The platelet-activating factor PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid first messenger active in general cell activation, fertilization, inflammatory and allergic reactions, asthma, HIV pathogenesis, carcinogenesis, and apoptosis. There is substantial evidence that PAF is involved in intracellular signalling, but the pathways are poorly understood. Inactivation of PAF is carried out by specific intra- and extracellular acetylhydrolases (PAF-AHs), a subfamily of phospholipases A2 that remove the sn-2 acetyl group. Mammalian brain contains at least three intracellular isoforms, of which PAF-AH(Ib) is the best characterized. This isoform contains a heterodimer of two homologous catalytic subunits alpha1 and alpha2, each of relative molecular mass 26K, and a non-catalytic 45K beta-subunit, a homologue of the beta-subunit of trimeric G proteins. We now report the crystal structure of the bovine alpha1 subunit of PAF-AH(Ib) at 1.7 A resolution in complex with a reaction product, acetate. The tertiary fold of this protein is closely reminiscent of that found in p21(ras) and other GTPases. The active site is made up of a trypsin-like triad of Ser 47, His 195 and Asp 192. Thus, the intact PAF-AH(Ib) molecule is an unusual G-protein-like (alpha1/alpha2)beta trimer.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
Major histocompatibility complex restriction between hematopoietic stem cells and stromal cells in vivo.
Graft failure is a mortal complication in allogeneic bone marrow transplantation (BMT); T cells and natural killer cells are responsible for graft rejection. However, we have recently demonstrated that the recruitment of donor-derived stromal cells prevents graft failure in allogeneic BMT. This finding prompted us to examine whether a major histocompatibility complex (MHC) restriction exists between hematopoietic stem cells (HSCs) and stromal cells. We transplanted bone marrow cells (BMCs) and bones obtained from various mouse strains and analyzed the cells that accumulated in the engrafted bones. Statistically significant cell accumulation was found in the engrafted bone, which had the same H-2 phenotype as that of the BMCs, whereas only few cells were detected in the engrafted bones of the third-party H-2 phenotypes during the 4 to 6 weeks after BMT. Moreover, the BMCs obtained from the MHC-compatible bone showed significant numbers of both colony-forming units in culture (CFU-C) and spleen colony-forming units (CFU-S). These findings strongly suggest that an MHC restriction exists between HSCs and stromal cells.
Animals↗