Polymorphism, Heteroplasmy, Mitochondrial Fusion and Diabetes

Mitochondrial DNA (mtDNA) is highly susceptible to mutations that result in polymorphisms and diseases including diabetes. We analyzed heteroplasmy, polymorphisms related to diabetes, and complementation by fusogenic proteins. Cytoplast fusion and microinjection allow, defects in mutated mtDNA inside a heteroplasmic cell to be complemented by fusing two mitochondria via human fusogenic proteins. We characterized three hfzos as well as two OPAls that prevent apoptosis. Two coiled coil domains and GTPase domains in these fusogenic proteins regulate membrane fusion. The hfzo genes were expressed mainly in the brain and in muscle that are postmitotic, but not in the pancreas. Under the influence of polymorphisms of mtDNA and nDNA, the vicious circle of reactive oxygen species and mutations in cell can be alleviated by mitochondrial fusion.     

Identification of genes specifically expressed in the accumulated visceral adipose tissue of OLETF rats

The Otsuka Long-Evans Tokushima fatty (OLETF) rat is an animal model of type 2 diabetes, characterized by abdominal obesity, insulin resistance, hypertension, and dyslipidemia. To elucidate the underlying molecular mechanism of obesity and its related complications, we used representational difference analysis and identified the genes more abundantly and specifically expressed in the visceral adipose tissue (VAT) of obese OLETF rats compared with the diabetes-resistant counterpart, that is, Long-Evans Tokushima Otsuka (LETO) rats. By Northern blot analysis, we confirmed the differential expression of 13 genes, including 3 novel genes. The upregulated expression of well-characterized lipid metabolic enzymes, such as lipoprotein lipase, phosphoenolpyruvate carboxykinase, and cholesterol esterase, were observed in VAT of OLETF rats. We demonstrated the differential expression of secreted proteins in VAT of OLETF rats, such as thrombospondin 1 and contrapsin-like protease inhibitor. In contrast to lipid enzymes, the secreted proteins revealed exclusive mRNA expression and they were not detected in VAT of LETO rats. Furthermore, the novel genes OL-16 and OL-64 were also expressed specifically in VAT of OLETF rats and were absent in that of LETO rats and other tissues, including subdermal and brown adipose tissues. The C-terminal partial amino acid sequence of OL-64 revealed that it showed approximately 40% homology with alpha(1)-antitrypsin and it seemed to be a new member of the serine proteinase inhibitor (SERPIN) gene family. VAT of OLEFT rats had a unique gene expression profile, and the accumulated VAT-specific known and novel secreted proteins may play a role(s) in the pathogenesis of obesity and its related complications.     

Responses of immunocompetent cells in the dental pulp to replantation during the regeneration process in rat molars

Responses of immunocompetent cells to tooth replantation during the regeneration process of the dental pulp in rat molars were investigated by immunocytochemistry using antibodies to class II major histocompatibility complex (MHC) molecules (OX6 antibody), monocyte/macrophage lineage cells (ED1 antibody) and protein gene product 9.5 (PGP 9.5), as well as by histochemical reaction for periodic acid-Schiff (PAS). Tooth replantation caused an increase in both the number of OX6- and ED1-positive cells and their immunointensity in the replanted pulp, but almost all PGP 9.5-immunoreactive nerves diminished in the initial stages. By postoperative day 3, many OX6- and ED -immunopositive cells had accumulated along the pulp-dentin border to extend their cytoplasmic processes into the dentinal tubules in successful cases. Once reparative dentin formation had begun after postoperative day 7, OX6- and ED1-immmunopositive cells became scattered in the odontoblast layer, while reinnervation was found in the coronal pulp. The temporal appearance of these immunocompetent cells at the pulp-dentin border suggests their participation in odontoblast differentiation as well as in initial defense reactions during the pulpal regeneration process. On postoperative day 14, the replanted pulp showed three regeneration patterns: (1) reparative dentin, (2) bone-like tissue formation, and (3) an intermediate form between these. In all cases, PAS-reactive cells such as polymorphonuclear leukocytes (PML) and mesenchymal cells occurred in the pulp space. However, the prolonged stagnation of inflammatory cells was also discernible in the latter two cases. Thus, the findings on PAS reaction suggest that the migration of the dental follicle-derived cells into the pulp space and the subsequent total death of the proper pulpal cells are decisive factors for eliciting bone-like tissue formation in the replanted pulp.     

Novel acyl-CoA synthetase in adrenoleukodystrophy target tissues

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder characterized by demyelination of white matter. The X-ALD gene product adrenoleukodystrophy protein (ALDP) is expressed broadly among various tissues. However, deficiency of functional ALDP exclusively impairs brain, adrenal gland, and testis. Thus, loss of ALDP function is assumed to involve inactivation of a putative mediating factor that functions in a tissue-specific manner. Here we cloned a mouse cDNA encoding a novel protein, Lipidosin, that possesses long-chain acyl-CoA synthetase (LCAS) activity. Lipidosin is expressed exclusively in mouse brain, adrenal gland, and testis, which are affected by X-ALD. LCAS activity of Lipidosin was diminished by mutation of conserved amino acids within the AMP-binding domain. Mutation of the Drosophila homologue of Lipidosin has been reported to cause neuronal degeneration. Thus, Lipidosin may mediate the link between ALDP dysfunction and the impairment of fatty acid metabolism in X-ALD.     

Characterization of chloroplast psbA transformants of Chlamydomonas reinhardtii with impaired processing of a precursor of a photosystem II reaction center protein,D1

One of the photosystem II reaction center proteins, D1, is encoded by the psbA gene and is synthesized as a precursor form with a carboxyl-terminal extension that is subsequently cleaved between Ala-344 and Ser-345. We have generated three psbA transformants of the green alga Chlamydomonas reinhardtii in which Ala-344 or Ser-345 have been substituted with Pro or Glu (A344P, S345E, and S345P) to understand the effects of the amino acid substitutions on the processing of the precursor D1. S345E grew photoautotrophically and showed PSII activity like the wild type. However, A344P and S345P were unable to grow photoautotrophically and were significantly photosensitive. A344P was deficient in the processing of precursor D1 and in oxygen-evolving activity, but assembled photosystem II complex capable of charge separation. In contrast, both precursor and mature forms of D1 accumulated in S345P cells from the logarithmic phase and the cells evolved oxygen at 18% of wild-type level. However, S345P cells from the stationary phase contained mostly the mature D1 and showed a twofold increase in oxygen-evolving activity. The rate of processing of the accumulated pD1 was estimated to be about 100 times slower than in the wild type. It is therefore concluded that the functional oxygen-evolving complex is assembled when the precursor D1 is processed, albeit at a very low rate. These results suggest the functional significance of the amino acid residues at the processing site of the precursor D1.     

Rapid Genotyping of Mice with Hemoglobinopathies and Globin Transgenes

The hematology of the laboratory mouse has beenwell characterized. Normal genetic differences at thealpha- and beta-globin gene loci serve as useful markersfor a wide variety of types of experimental studies. There are a number of naturallyoccurring or induced mutations that disrupt globinexpression and produce thalassemic phenotypes. Inaddition, much has been learned of the workings of theglobin locus control region from studies of transgenicmice, including those with mutations induced by targetedsite-specific modifications. After a new mutation ortransgene has been created, it must be maintained in living mice, and the genotypes of theoffspring must be ascertained. While it is possible todetermine genotypes by DNA analyses, such assays aretime consuming and relatively expensive. An osmoticchallenge test -- originally developed for thegenotyping of large-deletion alpha-thalassemia mutationsin mice -- has proven useful in detecting bothsevere and milder alpha- and beta-thalassemias, as wellas some transgenic genotypes in mice carrying human globin genes.Reliable genotyping can, in some cases, be completedwithin a few minutes with minimal expense.Quantification of red cell fragility for a variety ofthalassemic and transgenic mice is described here, alongwith a simplified test suitable for rapid, routinegenotyping. The osmotic challenge test is perfectlyreliable for distinguishing genotypes that causesignificantly decreased release of hemoglobin from the redcells, but it is also useful for some of the conditionsin which overall erythrocyte osmotic fragility isessentially normal.     

Abnormal Localization of STK17A in Bile Canaliculi in Liver Allografts: An Early Sign of Chronic Rejection

The biological significance of STK17A, a serine/threonine kinase, in the liver is not known. We analyzed STK17A expression in HepG2 cells and human liver tissue. Accordingly, we investigated whether STK17A could help in identifying earlier changes during the evolution of chronic rejection (CR) after liver transplantation. RT-PCR and immunofluorescence were used to analyze STK17A expression in HepG2 cells. Antibody microarray was performed using human liver samples from CR and healthy donors. Immunohistochemistry was used to verify the clinical utility of STK17A on sequential biopsies for the subsequent development of CR. A novel short isoform of STK17A was found in HepG2 cells. STK17A was localized in the nuclei and bile canaliculi in HepG2 cells and human livers. Microarray of STK17A revealed its decrease in failed liver allografts by CR. During the evolution of CR, the staining pattern of bile canalicular STK17A gradually changed from diffuse linear to focal intermittent. The focal intermittent staining pattern was observed before the definite diagnosis of CR. In conclusion, the present study was the first to find localization of STK17A in normal bile canaliculi. Abnormal expression and localization of STK17A were associated with CR of liver allografts since the early stage of the rejection process.     

Melatonin Inhibits Embryonic Salivary Gland Branching Morphogenesis by Regulating Both Epithelial Cell Adhesion and Morphology

Many organs, including salivary glands, lung, and kidney, are formed by epithelial branching during embryonic development. Branching morphogenesis occurs via either local outgrowths or the formation of clefts that subdivide epithelia into buds. This process is promoted by various factors, but the mechanism of branching morphogenesis is not fully understood. Here we have defined melatonin as a potential negative regulator or “brake” of branching morphogenesis, shown that the levels of it and its receptors decline when branching morphogenesis begins, and identified the process that it regulates. Melatonin has various physiological functions, including circadian rhythm regulation, free-radical scavenging, and gonadal development. Furthermore, melatonin is present in saliva and may have an important physiological role in the oral cavity. In this study, we found that the melatonin receptor is highly expressed on the acinar epithelium of the embryonic submandibular gland. We also found that exogenous melatonin reduces salivary gland size and inhibits branching morphogenesis. We suggest that this inhibition does not depend on changes in either proliferation or apoptosis, but rather relates to changes in epithelial cell adhesion and morphology. In summary, we have demonstrated a novel function of melatonin in organ formation during embryonic development.     

Inhibition of Excessive Cell Proliferation by Calcilytics in Idiopathic Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease of unknown pathogenesis. Vascular remodeling due to excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a critical pathogenic event that leads to early morbidity and mortality. The excessive cell proliferation is closely linked to the augmented Ca²⁺ signaling in PASMCs. More recently, we have shown by an siRNA knockdown method that the Ca²⁺-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, involved in the enhanced Ca²⁺ response and subsequent excessive cell proliferation. In this study, we examined whether pharmacological blockade of CaSR attenuated the excessive proliferation of PASMCs from IPAH patients by MTT assay. The proliferation rate of PASMCs from IPAH patients was much higher (~1.5-fold) than that of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Treatment with NPS2143, an antagonist of CaSR or calcilytic, clearly suppressed the cell proliferation in a concentration-dependent manner (IC₅₀ = 2.64 μM) in IPAH-PASMCs, but not in normal and CTEPH PASMCs. Another calcilytic, Calhex 231, which is structurally unrelated to NPS2143, also concentration-dependently inhibited the excessive proliferation of IPAH-PASMCs (IC₅₀ = 1.89 μM). In contrast, R568, an activator of CaSR or calcimimetic, significantly facilitated the proliferation of IPAH-PASMCs (EC₅₀ = 0.33 μM). Similar results were obtained by BrdU incorporation assay. These results reveal that the excessive PASMC proliferation was modulated by pharmacological tools of CaSR, showing us that calcilytics are useful for a novel therapeutic approach for pulmonary arterial hypertension.