Fluorosed Mouse Ameloblasts Have Increased SATB1 Retention and Gαq Activity

Dental fluorosis is characterized by subsurface hypomineralization and increased porosity of enamel, associated with a delay in the removal of enamel matrix proteins. To investigate the effects of fluoride on ameloblasts, A/J mice were given 50 ppm sodium fluoride in drinking water for four weeks, resulting serum fluoride levels of 4.5 µM, a four-fold increase over control mice with no fluoride added to drinking water. MicroCT analyses showed delayed and incomplete mineralization of fluorosed incisor enamel as compared to control enamel. A microarray analysis of secretory and maturation stage ameloblasts microdissected from control and fluorosed mouse incisors showed that genes clustered with Mmp20 appeared to be less downregulated in maturation stage ameloblasts of fluorosed incisors as compared to control maturation ameloblasts. One of these Mmp20 co-regulated genes was the global chromatin organizer, special AT-rich sequence-binding protein-1 (SATB1). Immunohistochemical analysis showed increased SATB1 protein present in fluorosed ameloblasts compared to controls. In vitro, exposure of human ameloblast-lineage cells to micromolar levels of both NaF and AlF₃ led to a significantly increase in SATB1 protein content, but not levels of Satb1 mRNA, suggesting a fluoride-induced mechanism protecting SABT1 from degradation. Consistent with this possibility, we used immunohistochemistry and Western blot to show that fluoride exposed ameloblasts had increased phosphorylated PKCα both in vivo and in vitro. This kinase is known to phosphorylate SATB1, and phosphorylation is known to protect SATB1 from degradation by caspase-6. In addition, production of cellular diacylglycerol (DAG) was significantly increased in fluorosed ameloblasts, suggesting that the increased phosphorylation of SATB1 may be related to an effect of fluoride to enhance Gαq activity of secretory ameloblasts.     

Social Orientation and Diabetes-Related Distress in Japanese and American Patients with Type 2 Diabetes

Objective

Recent evidence in cultural and social psychology suggests Eastern cultures'' emphasis on harmony and connection with others and Western cultures'' emphasis on self-direction and autonomy. In Eastern society, relational harmony is closely linked to people''s well-being. The impact of this cultural and social orientation on diabetes-related distress was investigated.

Research Design and Methods

Japanese and American patients with type 2 diabetes were surveyed by well-established questionnaire in Japan and in the United States, respectively. The association of personal values for interdependence, perceived emotional support, and the Problem Areas in Diabetes scale (PAID) were analyzed.

Results

A positive correlation between interdependence and PAID (r = 0.18; P = 0.025) and a negative correlation between perceived emotional support and PAID (r = − 0.24; P = 0.004) were observed after adjustments for other factors in Japanese data (n = 149), but not in American data (r = 0.00; P = 0.990, r = 0.02; P = 0.917, respectively, n = 50). In Japanese data, the three-factor structure of PAID (negative feelings about total life with diabetes, about living conditions with diabetes, and about treatment of diabetes) was identified, and interdependence showed significant positive correlations with the first and second factors and perceived emotional support showed significant negative correlations with all three factors of PAID.

Conclusions

These results suggest that personal values for interdependence may be linked to the level of diabetes-related distress and that the distress may be relieved by perception of emotional support, especially in an interdependent cultural context.     

The Unfolded Protein Response Transducer ATF6 Represents a Novel Transmembrane-type Endoplasmic Reticulum-associated Degradation Substrate Requiring Both Mannose Trimming and SEL1L Protein

Proteins misfolded in the endoplasmic reticulum (ER) are cleared by the ubiquitin-dependent proteasome system in the cytosol, a series of events collectively termed ER-associated degradation (ERAD). It was previously shown that SEL1L, a partner protein of the E3 ubiquitin ligase HRD1, is required for degradation of misfolded luminal proteins (ERAD-Ls substrates) but not misfolded transmembrane proteins (ERAD-Lm substrates) in both mammalian and chicken DT40 cells. Here, we analyzed ATF6, a type II transmembrane glycoprotein that serves as a sensor/transducer of the unfolded protein response, as a potential ERAD-Lm substrate in DT40 cells. Unexpectedly, degradation of endogenous ATF6 and exogenously expressed chicken and human ATF6 by the proteasome required SEL1L. Deletion analysis revealed that the luminal region of ATF6 is a determinant for SEL1L-dependent degradation. Chimeric analysis showed that the luminal region of ATF6 confers SEL1L dependence on type I transmembrane protein as well. In contrast, degradation of other known type I ERAD-Lm substrates (BACE457, T-cell receptor-α, CD3-δ, and CD147) did not require SEL1L. Thus, ATF6 represents a novel type of ERAD-Lm substrate requiring SEL1L for degradation despite its transmembrane nature. In addition, endogenous ATF6 was markedly stabilized in wild-type cells treated with kifunensine, an inhibitor of α1,2-mannosidase in the ER, indicating that degradation of ATF6 requires proper mannose trimming. Our further analyses revealed that the five ERAD-Lm substrates examined are classified into three subgroups based on their dependence on mannose trimming and SEL1L. Thus, ERAD-Lm substrates are degraded through much more diversified mechanisms in higher eukaryotes than previously thought.     

Epiblast integrity requires CLASP and Dystroglycan-mediated microtubule anchoring to the basal cortex

Amniote epiblast cells differentiate into mesoderm and endoderm lineages during gastrulation through a process called epithelial-to-mesenchymal transition (EMT). Molecular regulation of gastrulation EMT is poorly understood. Here we show that epiblast epithelial status was maintained by anchoring microtubules to the basal cortex via CLIP-associated protein (CLASP), a microtubule plus-end tracking protein, and Dystroglycan, a transmembrane protein that bridges the cytoskeleton and basement membrane (BM). Mesoderm formation required down-regulation of CLASP and Dystroglycan, and reducing CLASP activity in pregastrulation epiblast cells caused ectopic BM breakdown and disrupted epiblast integrity. These effects were mediated through the CLASP-binding partner LL5. Live-imaging using EB1–enhanced GFP (eGFP) revealed that reducing CLASP and LL5 levels in the epiblast destabilized basal microtubules. We further show that Dystroglycan is localized to basolateral membrane in epiblast cells. Basal but not lateral localization of Dystroglycan was regulated by CLASP. We propose that epiblast–BM interaction requires CLASP- and Dystroglycan-mediated cortical microtubule anchoring, the disruption of which initiates gastrulation EMT.     

Thiamine-responsive pyruvate dehydrogenase deficiency in two patients caused by a point mutation (F205L and L216F) within the thiamine pyrophosphate binding region

The human pyruvate dehydrogenase complex (PDHC) catalyzes the thiamine-dependent decarboxylation of pyruvate. Thiamine treatment is very effective for some patients with PDHC deficiency. Among these patients, five mutations of the pyruvate dehydrogenase (E1)alpha subunit have been reported previously: H44R, R88S, G89S, R263G, and V389fs. All five mutations are in a region outside the thiamine pyrophosphate (TPP)-binding region of the E1alpha subunit.We report the biochemical and molecular analysis of two patients with clinically thiamine-responsive lactic acidemia. The PDHC activity was assayed using two different concentrations of TPP. These two patients displayed very low PDHC activity in the presence of a low (1 x 10(-4) mM) TPP concentration, but their PDHC activity significantly increased at a high (0.4 mM) TPP concentration. Therefore, the PDHC deficiency in these two patients was due to a decreased affinity of PDHC for TPP. Treatment of both patients with thiamine resulted in a reduction in the serum lactate concentration and clinical improvement, suggesting that these two patients have a thiamine-responsive PDHC deficiency. The DNA sequence of these two male patients' X-linked E1alpha subunit revealed a point mutation (F205L and L216F) within the TPP-binding region in exon 7.     

Antihypertensive effects of onion on NO synthase inhibitor-induced hypertensive rats and spontaneously hypertensive rats

This study was designed to show the effects of onion on blood pressure in N(G)-nitro-L-arginine methyl ester (L-NAME) induced-hypertensive rats and stroke prone spontaneously hypertensive rats (SHRSP) using dried onion at 5% in their diets. For the experiment with L-NAME induced-hypertensive rats, male 6-weeks-old Sprague-Dawley rats were given tap water containing L-NAME to deliver 50 mg/kg BW/day. In this experiment, we found distinct antihypertensive effects of onion on the L-NAME induced-hypertensive rats and the SHRSP. Dietary onion decreased the thiobarbituric acid reactive substances (TBARS) in plasma in these hypertensive rats. Also, onion increased the nitrate/nitrite (products of nitric oxide (NO)) excreted in urine and the NO synthase (NOS) activity in the kidneys in SHRSP. These results suggested that the increased NO caused by the greater NOS activity, and additionally by the increased saving of NO by the antioxidative activity of onion, was one of the cause of the antihypertensive effect of onion in SHRSP. In the L-NAME induced hypertensive rats, onion did not significantly block the inhibition of NOS activity by L-NAME, and decreased nitrate/nitrite excretion in urine was not restored. The mechanism of the antihypertensive effect of onion probably involves increased saving of NO by antioxidative activity of onion in L-NAME induced-hypertensive rats.     

Redox-Dependent Domain Rearrangement of Protein Disulfide Isomerase Coupled with Exposure of Its Substrate-Binding Hydrophobic Surface

Protein disulfide isomerase (PDI) is a major protein in the endoplasmic reticulum, operating as an essential folding catalyst and molecular chaperone for disulfide-containing proteins by catalyzing the formation, rearrangement, and breakage of their disulfide bridges. This enzyme has a modular structure with four thioredoxin-like domains, a, b, b′, and a′, along with a C-terminal extension. The homologous a and a′ domains contain one cysteine pair in their active site directly involved in thiol-disulfide exchange reactions, while the b′ domain putatively provides a primary binding site for unstructured regions of the substrate polypeptides. Here, we report a redox-dependent intramolecular rearrangement of the b′ and a′ domains of PDI from Humicola insolens, a thermophilic fungus, elucidated by combined use of nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) methods. Our NMR data showed that the substrates bound to a hydrophobic surface spanning these two domains, which became more exposed to the solvent upon oxidation of the active site of the a′ domain. The hydrogen-deuterium exchange and relaxation data indicated that the redox state of the a′ domain influences the dynamic properties of the b′ domain. Moreover, the SAXS profiles revealed that oxidation of the a′ active site causes segregation of the two domains. On the basis of these data, we propose a mechanistic model of PDI action; the a′ domain transfers its own disulfide bond into the unfolded protein accommodated on the hydrophobic surface of the substrate-binding region, which consequently changes into a “closed” form releasing the oxidized substrate.     

Phylogenetic relationships of the hyphomycete generaChaetopsina andKionochaeta based on 18S rDNA sequences

A setiform dematiaceous hyphomycete,Kionochaeta spissa was newly collected and isolated from evergreen broad-leaved forests in the southern parts of Japan. Except for its dematiaceous nature, the species is morphologically similar to a nectriaceous hyphomycete,Chaetopsina fulva. The morphology and cultural properties of the Japanese isolates ofK. spissa were described, and the phylogenetic relationships betweenChaetopsina (C. fulva (type species)) andKionochaeta (K. ramifera (type species),K. spissa andK. ivoriensis) were inferred based on nuclear encoded small subunit (18S) rDNA sequences using the neighbor-joining method.Chaetopsina andKionochaeta were found to be phylogenetically related to the Hypocreales and Sordariales, respectively. Both should be maintained as separate genus for phylogenetic classification. The morphological resemblance especially betweenC. fulva andK. spissa is an example of the convergent evolution.