X-ray absorption near edge structure and first-principles spectral investigations of cationic disorder in MgAl2O4 induced by swift heavy ions.

Cationic disorder in the MgAl2O4 spinel induced by swift heavy ions was investigated using the X-ray absorption near edge structure. With changes in the irradiation fluences of 200 MeV Xe ions, the Mg K-edge and Al K-edge spectra were synchronously changed. The calculated spectra based on density function theory indicate that the change in the experimental spectra was due to cationic disorder between Mg in tetrahedral sites and Al in octahedral sites. These results suggest a high inversion degree to an extent that the completely random configuration is achieved in MgAl2O4 induced by the high density electronic excitation under swift heavy ion irradiation.

Phase I/II study of divided-dose docetaxel, cisplatin and fluorouracil for patients with recurrent or metastatic squamous cell carcinoma of the esophagus.

Squamous cell carcinoma of the esophagus (SCCE) has a poor prognosis compared with other gastrointestinal cancers. Many patients present with locoregional unresectable or metastatic disease at the time of diagnosis. For these patients with metastatic esophageal cancer, chemotherapy is generally indicated. The aim of this phase I/II study was to evaluate the efficacy and safety of the combined use of docetaxel, cisplatin (CDDP) and 5-fluorouracil (5-FU)(DCF) in patients with recurrent/metastatic SCCE. This study adopted divided doses of docetaxel and CDDP in order to reduce the toxicities of the treatment. The dose of docetaxel was escalated using the following protocol in the phase I stage: level 1, 30 mg/m2; level 2, 35 mg/m2 and level 3, 40 mg/m2, which was intravenously infused for 2 hours on days 1 and 8. CDDP was administered at a dose of 12 mg/m2 infused for 4 hours on days 1-5. The 5-FU was administered at a dose of 600 mg/m2 continuously infused from day 1 to 5. This regimen was repeated every 4 weeks. The study subjects were nine patients (phase I) and 48 patients (phase II). The recommended dose was determined as level 3 in phase I. In the phase II stage, the overall response rate was 62.5%, with a complete response rate of 12.5%. The median progression-free survival was 6 months, and the median overall survival was 13 months. Grade 3/4 toxicities of leukopenia, neutropenia and febrile neutropenia occurred in 64.6%, 68.8% and 14.6% of the patients, while grade 3/4 non-hematological toxicities were relatively rare. No treatment-related death was recorded. This modified DCF regimen with divided doses can be a tolerable and useful regimen of definitive chemotherapy for unresectable SCCE because of its high efficacy, although adequate care for severe neutropenia must be administered.

Characterization of mesenchymal progenitor cell populations from non-epithelial oral mucosa.

OBJECTIVES: The characteristics of cell populations extracted from oral mucosal non-epithelial tissues and their ability to differentiate were evaluated in vitro as a potential source of cells for mandibular and corneal regeneration. MATERIALS AND METHODS: Oral mucosal non-epithelial cells (OMNECs) were extracted from tissue samples and were studied by flow cytometry and RT-PCR. Cells differentiating into osteoblasts, adipocytes, chondrocytes, neurocytes, or keratocytes were characterized by RT-PCR and cell staining. RESULTS: OMNECs expressed CD44, CD90, CD105, CD166, and STRO-1 antigens, which are markers for mesenchymal stem cells. In addition, Oct3/4, c-Myc, Nanog, KLF4, and Rex, which are expressed by embryonic or pluripotent stem cells, were detected by RT-PCR. Expression of CD49d, CD56, and PDGFRα, proteins closely associated with the neural crest, was observed in OMNECs, as was expression of Twist1, Sox9, Snail1 and Snail2, which are early neural crest and neural markers. Specific differentiation markers were expressed in OMNECs after differentiation into osteoblasts, adipocytes, chondrocytes, or keratocytes. CONCLUSIONS: Populations of OMNECs may contain both mesenchymal stem cells and neural crest origin cells and are a potential cell source for autologous regeneration of mandibular or corneal stroma.

Characterization of 83-kilodalton nonmuscle caldesmon from cultured rat cells: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin.

Nonmuscle caldesmon purified from cultured rat cells shows a molecular weight of 83,000 on SDS gels, Stokes radius of 60.5 A, and sedimentation coefficient (S20,w) of 3.5 in the presence of reducing agents. These values give a native molecular weight of 87,000 and a frictional ratio of 2.04, suggesting that the molecule is a monomeric, asymmetric protein. In the absence of reducing agents, the protein is self-associated, through disulfide bonds, into oligomers with a molecular weight of 230,000 on SDS gels. These S-S oligomers appear to be responsible for the actin-bundling activity of nonmuscle caldesmon in the absence of reducing agents. Actin binding is saturated at a molar ratio of one 83-kD protein to six actins with an apparent binding constant of 5 X 10(6) M-1. Because of 83-kD nonmuscle caldesmon and tropomyosin are colocalized in stress fibers of cultured cells, we have examined effects of 83-kD protein on the actin binding of cultured cell tropomyosin. Of five isoforms of cultured rat cell tropomyosin, tropomyosin isoforms with high molecular weight values (40,000 and 36,500) show higher affinity to actin than do tropomyosin isoforms with low molecular weight values (32,400 and 32,000) (Matsumura, F., and S. Yamashiro-Matsumura. 1986. J. Biol. Chem. 260:13851-13859). At physiological concentration of KCl (100 mM), 83-kD nonmuscle caldesmon stimulates binding of low molecular weight tropomyosins to actin and increases the apparent binding constant (Ka from 4.4 X 10(5) to 1.5 X 10(6) M-1. In contrast, 83-kD protein has slight stimulation of actin binding of high molecular weight tropomyosins because high molecular weight tropomyosins bind to actin strongly in this condition. As the binding of 83-kD protein to actin is regulated by calcium/calmodulin, 83-kD protein regulates the binding of low molecular weight tropomyosins to actin in a calcium/calmodulin-dependent way. Using monoclonal antibodies to visualize nonmuscle caldesmon along microfilaments or actin filaments reconstituted with purified 83-kD protein, we demonstrate that 83-kD nonmuscle caldesmon is localized periodically along microfilaments or actin filaments with similar periodicity (36 +/- 4 nm) as tropomyosin. These results suggest that 83-kD protein plays an important role in the organization of microfilaments, as well as the control of the motility, through the regulation of the binding of tropomyosin to actin.

Intracellular localization of the 55-kD actin-bundling protein in cultured cells: spatial relationships with actin, alpha-actinin, tropomyosin, and fimbrin.

The 55-kD protein is a new actin-bundling protein purified from HeLa cells (Yamashiro-Matsumura, S., and F. Matsumura, 1985, J. Biol. Chem., 260:5087-5097). We have prepared monoclonal antibodies against the 55-kD protein and examined its intracellular localization, as well as its spatial relationships with other components of microfilaments in cultured cells by double-label immunofluorescence. The localization of the 55-kD protein is similar to that of actin. The antibody to the 55-kD protein stained strongly both microspikes and stress fibers. The 55-kD protein was found from the basal portions to the extremities of microspikes while alpha-actinin was localized only in the basal portions. In stress fibers, the 55-kD protein was found rather continuously in comparison to the periodic localizations of alpha-actinin and tropomyosin. Although fimbrin is located in microspikes and ruffling membranes, fimbrin is hardly found in stress fibers unlike the 55-kD protein. These observations coupled with the actin-bundling activity of the 55-kD protein imply that the 55-kD protein is involved in the formation of microfilament bundles in both microspikes and stress fibers.

Tropomyosin-enriched and alpha-actinin-enriched microfilaments isolated from chicken embryo fibroblasts by monoclonal antibodies.

Antitropomyosin and anti-alpha-actinin monoclonal antibodies have been used to isolate two classes of microfilaments, i.e., tropomyosin-enriched and alpha-actinin-enriched microfilaments, respectively, from cultured chicken embryo fibroblasts. Electron microscopic studies of the isolated tropomyosin-enriched microfilaments showed periodic localization of tropomyosin along the microfilaments, with a 35-nm repeat. On the contrary, the isolated alpha-actinin-enriched microfilaments showed no obvious periodicity. Many individual alpha-actinin-enriched microfilaments with length greater than 1 micron (ranging from 1 to 10 microns) were aggregated by anti-alpha-actinin monoclonal antibodies. Both of the isolated microfilaments had the ability to activate the Mg2+-ATPase activity of skeletal muscle myosin, although different extents of activation were observed. These two classes of microfilaments also differed in their protein composition. Molar ratios of major identifiable proteins in the isolated microfilaments were alpha-actinin(dimer):actin(monomer):tropomyosin(dimer) = less than 0.02:8.06:1.00 for tropomyosin-enriched microfilaments and 0.44:13.91:1.00 for alpha-actinin-enriched microfilaments. By two-dimensional gel analysis of the isolated microfilaments, we have found seven spots which possess typical tropomyosin properties including pI 4.5, immunological cross-reaction, lack of proline and tryptophan, and heat stability. Pulse-chase experiments suggested that the assembly of microfilament-associated proteins, at least for alpha-actinin and tropomyosins, was coordinately regulated by the assembly of actin into microfilaments.

Reg IV is a serum biomarker for gastric cancer patients and predicts response to 5-fluorouracil-based chemotherapy.

Regenerating gene family, member 4 (Reg IV), a secreted protein, is overexpressed in several cancers, including gastric cancer (GC). In the present study, we measured Reg IV levels in sera from patients with GC by enzyme-linked immunosorbent assay. We also examined the effect of forced Reg IV expression on the apoptotic susceptibility to 5-fluorouracil (5-FU). Forced expression of Reg IV inhibited 5-FU-induced apoptosis. Induction of Bcl-2 and dihydropyrimidine dehydrogenase was involved in inhibition of apoptosis. Among 36 GC patients treated with a combination chemotherapy of low-dose 5-FU and cisplatin, all 14 Reg IV-positive patients showed no change or disease progression. The serum Reg IV concentration was similar between healthy individuals (mean+/-s.e., 0.52+/-0.05 ng/ml) and patients with chronic-active gastritis (0.36+/-0.09 ng/ml). However, the serum Reg IV concentration in presurgical GC patients was significantly elevated (1.96+/-0.17 ng/ml), even at stage I. The diagnostic sensitivity of serum Reg IV (36.1%) was superior to that of serum carcinoembryonic antigen (11.5%) or carbohydrate antigen 19-9 (13.1%). These results indicate that expression of Reg IV is a marker for prediction of resistance to 5-FU-based chemotherapy in patients with GC. Serum Reg IV represents a novel biomarker for GC.

Involvement of stem cell factor and its receptor tyrosine kinase c-kit in pain regulation.

The c-kit receptor tyrosine kinase is expressed in a subpopulation of small- and medium-sized neurons of the dorsal root ganglia (DRG) and in the superficial layer of the spinal cord. Stem cell factor (SCF), a ligand of the c-kit receptor, induces neurite outgrowth from DRG and supports the survival of c-kit-expressing neurons. To clarify the possible function of the SCF/c-kit receptor system in the adult animal, we investigated the expression of c-kit receptor in the spinal cord and DRG in relation to pain by using H2C7, a newly developed anti-c-kit monoclonal antibody. S.c. and intrathecal injection of SCF markedly reduced the paw withdrawal threshold to mechanical stimuli and intrathecal SCF at 10 pg maximally induced mechanical allodynia in conscious mice. Intrathecal SCF also reduced the paw withdrawal latency to heat stimuli significantly but transiently. The c-kit receptor was co-expressed in 58.4% of calcitonin gene-related peptide (CGRP) -positive, but only 5.1% of isolectin B4-positive, DRG neurons. In the spinal cord, the c-kit receptor was detected in the superficial layer of the dorsal horn and co-localized there with CGRP in central terminals of DRG neurons. Selective elimination of unmyelinated C-fibers by neonatal capsaicin treatment resulted in marked reduction of the c-kit receptor and CGRP expression in the superficial layer of the spinal cord. Cell-size profiles showed that c-kit receptor expression was significantly up-regulated and down-regulated in medium-sized DRG neurons after neonatal capsaicin treatment and nerve injury, respectively. These results suggest that the c-kit receptor is mainly expressed in peptidergic small-sized DRG neurons and may be involved in pain regulation both peripherally and centrally.

Omega filter installed in the 1MV microscope of Kyushu University.

A 1.25MV high-voltage electron microscope with a B-type omega filter has been successfully installed at Kyushu University. An image detection chamber has been set inside a concrete block below the ground level without changing the frame structure for anti-vibration. Nearly the same design as that for the 200kV microscope has been kept for the present omega filter except for its size. A new pre- and post-filter lens system with rotation-free imaging has been designed. Energy resolution, beam shape and stability of the filter have been measured. Some application data have been obtained to demonstrate the performance of the filter.

Kinetics of B2- and D0(3)-type ordering and formation of domain structures in Fe-Al alloys.

Time-dependent Ginzburg-Landau (TDGL) formulation has been developed for the ordering processes of B2 and D0(3) types in binary alloy systems. In the formulation, three order parameters are defined in order to describe the state of order. Equivalent variants of B2 and D0(3) structures are distinguished using these order parameters. The mean-field free energy is defined in the form of a Landau-type expansion using the order parameters and a composition parameter. Interface energies due to local variations in the degrees of order and concentration are given with a gradient square approximation. Kinetic equations are derived from the Ginzburg-Landau-type potential in order to describe the time-evolutions of the order parameters and the concentration. Numerical simulations of the kinetic equations have been performed for B2- and D0(3)-type ordering as well as concurrent ordering and phase separation to disordered A2+D0(3). The simulated results provide a good reproduction of the formation processes of B2 and D0(3) ordered domains in an Fe(3)Al alloy.

Comparative Study on Variation of Quality of Life of Patients of Preemptive Kidney Transplantation and Nonpreemptive Kidney Transplantation.

BACKGROUND: There have been few studies that have reported the influence of kidney transplantation on the quality of life (QOL) of patients of preemptive kidney transplantation (PKT) and nonpreemptive kidney transplantation (NPKT). MATERIAL AND METHODS: Fifty patients of PKT and 49 patients of NPKT were employed as study subjects. A questionnaire survey using Short Form 36 and Kidney Disease QOL on patients' physical and psychological QOL was performed for these patients prior to transplantation and 1 month, 3 months, and 1 year after transplantation. RESULTS: The analysis of results has revealed that transplantation clearly has improved the physical and psychological QOL in patients with end-stage renal disease. For the items regarding physical burdens incurred by the transplantation, patient QOL deteriorated on a single occasion 1 month after the transplantation while it was improved 1 year after the transplantation. For the items regarding psychological burdens, the mental condition of the patients was improved overall without deterioration over time. Concerning the "Effect of Kidney Disease" and "Burden of Kidney Disease," QOL was significantly better in PKT than NPKT at baseline before transplantation, although the significant difference gradually decreased 1 month and 3 months after the transplantation and disappeared after 1 year. CONCLUSION: Transplantation certainly improved the QOL of patients with end-stage renal disease. Before transplantation, PKT was clearly better than NPKT in the QOL items associated with "Burden of Kidney Disease." This indicated that patients of PKT have improved QOL compared to patients of NPKT, and that the overall awareness of kidney disease is decreased. A postoperative gap in mental and bodies was observed especially in PKT, however, could be overcome by nursing interventions.

Azathioprine-induced Agranulocytosis and Severe Alopecia After Kidney Transplantation Associated With a NUDT15 Polymorphism: A Case Report.

BACKGROUND: Azathioprine (AZA) is the drug recommended for the continuation of immunosuppressive treatment after renal transplant in women during pregnancy. CASE REPORT: A 37-year-old Japanese female developed agranulocytosis and severe alopecia after initiation of AZA (50 mg), used as an alternative to mycophenolate mofetil (MMF, 1000 mg) therapy in anticipation of a planned pregnancy. Within 4 days of the initiation of AZA therapy, the patient developed a high fever, leucopenia, and cranial alopecia. Genetic testing revealed a homozygous polymorphism of NUDT15 (rs116855232, NM_018283.3:c.415C>T: p.Arg139Cys), which has previously been identified as a risk factor for AZA-related complications in patients with inflammatory bowel disease. CONCLUSION: Genetic screening for NUDT15 could contribute to the prevention of serious adverse reactions to AZA and provide the opportunity for personalized medicine. Identification of a safe alternative to MMF during pregnancy after a renal transplant is a problem to be resolved in the future.

Increase in transforming growth factor-beta in the brain during infection is related to fever, not depression of spontaneous motor activity.

When viral infection occurs, this information is transmitted to the brain, and symptoms such as fever and tiredness are induced. One of the causes of these symptoms is the secretion of proinflammatory cytokines in blood and the brain. In this study, the i.p. administration of polyinosinic:polycytidylic acid (poly I:C), a synthetic double-stranded RNA, to rats was used as an infection model. Poly I:C decreased spontaneous motor activity (SMA) 2 h after i.p. administration, and this decrease was maintained thereafter. The concentration of active transforming growth factor-beta (TGF-beta) in cerebrospinal fluid (CSF) increased 1 h after the administration. This increase occurred earlier than those in the concentrations of other proinflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), in serum. The intracisternal administration of an anti-TGF-beta antibody partially inhibited fever induced by poly I:C administration; however, this treatment did not affect the decrease in SMA. Furthermore, intracisternal administration of TGF-beta raised the body temperature. These results indicate that TGF-beta in the brain, which was increased by poly I:C administration, is associated with fever but not with a decrease in SMA.

IGF-1 Mediates EphrinB1 Activation in Regulating Tertiary Dentin Formation.

Eph receptors belong to a subfamily of receptor tyrosine kinases that are activated by membrane-spanning ligands called ephrins. Previously, we demonstrated that the ephrinB1-EphB2 interaction regulates odontogenic/osteogenic differentiation from dental pulp cells (DPCs) in vitro. The goal of this study was to identify the molecular mechanisms regulated by the EphB2/ephrinB1 system that govern tertiary dentin formation in vitro and in vivo. During tooth development, ephrinB1, and EphB2 were expressed in preodontoblast and odontoblasts at postnatal day 4. EphrinB1 was continuously expressed in odontoblasts and odontoblastic processes until the completion of tooth eruption. In addition, ephrinB1 was expressed in odontoblastic processes 2 wk following tooth injury without pulp exposure, whereas EphB2 was expressed in the center of pulp niches but not odontoblasts. In a model of tooth injury with pulp exposure, ephrinB1 was strongly expressed in odontoblasts 4 wk postinjury. In vitro studies with human and mouse DPCs treated with calcium hydroxide (CH) or mineral trioxide aggregate (MTA) showed an increased expression of insulin-like growth factor 1 (IGF-1). Experiments using several inhibitors of IGF-1 receptor signaling revealed that inhibiting the Ras/Raf-1/MAPK pathway inhibited EphB2 expression, and inhibiting the PI3K/Akt/mTOR pathway specifically inhibited ephrinB1 gene expression. Tooth injury in mice with odontoblast-specific IGF-1 receptor ablation exhibited a reduced tertiary dentin volume, mineral density, and ephrinB1 expression 4 wk following injury. We conclude that the IGF-1/ephrinB1 axis plays significant roles in the early stages of tooth injury. Further research is needed to fully understand the potential of targeting ephrinB1 as a regenerative pulp therapy.

Partial purification and characterization of a triacyglycerol lipase from rat liver cytosol.

A triacyglycerol lipase (EC 3.1.1.3) was purifiec about 60-fold from rat liver cytosol by delipidation with acetone and ethyl ether, hydroxyapatitie and Sephadex G-100 column chromatographies and isoelectrofocusing electrophoresis. The partially purified enzyme had a molecular weight of approximately 42 000 and an isolectric point of 7.2. The Km for trioleylglycerol was 0.33 mM and the pH optimum was around 8.0. The activity of the enzyme was not dependent on serum lipoproteins, but was stimulated about 2-fold by several proteins such as serum albumin, lipoproteins, gamma-globulin and ovalbumin. The lipase hydrolyzed trioleyglycerol to oleic acid and glycerol. NaCl had no effect on the enzymatic activity. Some physical and kinetic properties of the partially purified lipid-free lipase were different from those of crude non-delipidated lipase and also from those of a neutral triacylglycerol lipase which was recently purified partially from pig liver cytosol (Ledford, J.H. and Alaupovic, P. (1975) Biochim. Biophys. Acta 398, 132-148).

Comparative studies on the extractability of collagen from aortas of stroke-prone spontaneously hypertensive and normotensive rats.

The molecular states of collagen in the aortas of age-matched stroke-prone spontaneously hypertensive (SHRSP) and normotensive Wistar Kyoto rats (WKY) were studied by analyzing its extractability under defined conditions. The monomeric and oligomeric collagen extractable with 0.5 M acetic acid/6 M urea from aortic homogenates of 9-month-old SHRSP and WKY comprised approx. 0.6 and 2.0%, respectively, of the total collagen. On incubation of the acetic acid/urea-extracted residues with pepsin at 4 degrees C, the levels of the collagen alpha 1(I) and alpha 2(I) chains solubilized from the SHRSP residues were both less than 50% of those from the WKY residues. When the residues were incubated with pepsin at 15 or 25 degrees C, the differences became smaller. When the acetic acid/urea residues were hydrolyzed with cyanogen bromide, nearly identical peptide maps were obtained for SHRSP and WKY. The aortas from 2-month-old SHRSP and WKY contained much larger proportions of acid/urea-extractable collagen than those of the older rats (8.2 and 13% of the respective total collagen). The levels of the alpha 1(I) and alpha 2(I) chains solubilizable from the respective residues by pepsin at 4 degrees C were similar to each other. These results indicate that aortic collagen fibrils in SHRSP are stiffened more prominently than those in WKY.

Tissue-specific and non-tissue-specific heavy-chain isoforms of myosin in the brain as revealed by monoclonal antibodies.

Four types of monoclonal antibody (BM-1, BM-2, BM-3 and BM-4) each having distinctive tissue specificity were obtained by immunizing mice with purified bovine cerebrum myosin. Both BM-1 and BM-2 reacted most efficiently with cerebrum myosin and less efficiently with myosins from other limited nonmuscle tissues, the tissue specificity of BM-1 being much narrower than that of BM-2. BM-3 reacted more efficiently with several other nonmuscle myosins than with cerebellar or cerebral myosin. BM-4 recognized various nonmuscle and smooth muscle myosins with a nearly equal efficiency. Cerebral myosin as well a cerebellar myosin contained two or more electrophoretic variants of the heavy chains. BM-1 and BM-3 as well as BM-2 and BM-3 were found to recognize selectively these distinct heavy-chain isoforms. The antigenic sites of the three tissue-specific antibodies (BM-1, BM-2 and BM-3) were all localized near the head/tail junction of the myosin molecules, while that of non-tissue-specific antibody BM-4 was near the center of the tail. These and additional results indicate that mammalian brain tissues as well as several other nonmuscle tissues contain multiple heavy-chain isoforms of myosin, the levels of which differed considerably from one tissue to another.

Involvement of Rho-kinase in inflammatory and neuropathic pain through phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS).

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a major in vivo substrate for protein kinase C in the brain and has been implicated in cellular processes associated with cytoskeletal restructuring such as synaptic trafficking and neurotransmitter release. A phosphorylation-site specific antibody against Ser159-phospho-MARCKS (pS159-Mar-Ab) revealed that MARCKS is phosphorylated at Ser159 by Rho-kinase and that its phosphorylation is inhibited by the Rho-kinase specific inhibitor H-1152. Since the function of MARCKS is regulated by phosphorylation at multiple sites, here we examined the involvement of Rho-kinase in relation to phosphorylation of MARCKS at Ser159 in inflammatory and neuropathic pain by H-1152. When intrathecally administered 10 min before s.c. injection of formalin, H-1152 at 10 and 100 ng attenuated the second-phase, but not the first-phase, pain-like behaviors in the formalin test. Neuropathic pain induced by selective L5 spinal nerve transection was also relieved by intrathecal injection of H-1152. Nitric oxide synthase activity visualized by NADPH diaphorase histochemistry increased in the superficial layer of the spinal cord 30 min after formalin injection and 7 days after nerve transection, which were blocked by H-1152. Phosphorylation of MARCKS at Ser159 was detected in the spinal cord by pS159-Mar-Ab and the level of phosphorylation increased in the superficial layer after nerve transection. In contrast, immunoreactivities of neuronal nitric oxide synthase and MARCKS did not change significantly in the spinal cord before and after nerve transection. Taken together, the present study demonstrates that Rho-kinase is involved in inflammatory pain and the maintenance of neuropathic pain through phosphorylation of MARCKS at Ser159.

The Piccolo Intronic Single Nucleotide Polymorphism rs13438494 Regulates Dopamine and Serotonin Uptake and Shows Associations with Dependence-Like Behavior in Genomic Association Study.

Piccolo (PCLO) inhibits methamphetamine-induced neuropharmacological effects via modulation of dopamine (DA) uptake and regulation of the transport of synaptic vesicles in neuronal cells. Clinical studies have recently suggested that the single nucleotide polymorphism (SNP) rs13438494 in the intron 24 of the PCLO gene is associated with psychiatric disorder, in the meta-analysis of GWAS. Therefore, in this study, we attempted to evaluate the possible role of the PCLO SNP in the mechanisms of uptake of monoamines. To characterize rs13438494 in the PCLO gene, we constructed plasmids carrying either the C or A allele of the SNP and transiently transfected them into SH-SY5Y cells to analyze genetic effects on the splicing of PCLO mRNA. The C and A allele constructs produced different composition of the transcripts, indicating that the intronic SNP does affect the splicing pattern. We also transfected DA and serotonin (5-hydroxytryptamine; 5- HT) transporters into cells and analyzed their uptakes to elucidate the association to psychiatric disorders. In the cells transfected with the C allele, both the DA and 5-HT uptake were enhanced compared to the A allele. We also conducted a clinical study, in order to clarify the genetic associations. PCLO rs13438494 exhibits a relationship with the symptoms of drug dependence or related parameters, such as the age of first exposure to methamphetamine, eating disorders, tobacco dependence and fentanyl requirement. Our findings suggest that rs13438494 is associated with drug abuse and contributes to the pathogenesis of psychiatric disorders via modulation of neurotransmitter turnover.