Overexpression of constitutively activated glutamate dehydrogenase induces insulin secretion through enhanced glutamate oxidation

Glutamate dehydrogenase (GDH) catalyzes reversible oxidative deamination of l-glutamate to alpha-ketoglutarate. Enzyme activity is regulated by several allosteric effectors. Recognition of a new form of hyperinsulinemic hypoglycemia, hyperinsulinism/hyperammonemia (HI/HA) syndrome, which is caused by gain-of-function mutations in GDH, highlighted the importance of GDH in glucose homeostasis. GDH266C is a constitutively activated mutant enzyme we identified in a patient with HI/HA syndrome. By overexpressing GDH266C in MIN6 mouse insulinoma cells, we previously demonstrated unregulated elevation of GDH activity to render the cells responsive to glutamine in insulin secretion. Interestingly, at low glucose concentrations, basal insulin secretion was exaggerated in such cells. Herein, to clarify the role of GDH in the regulation of insulin secretion, we studied cellular glutamate metabolism using MIN6 cells overexpressing GDH266C (MIN6-GDH266C). Glutamine-stimulated insulin secretion was associated with increased glutamine oxidation and decreased intracellular glutamate content. Similarly, at 5 mmol/l glucose without glutamine, glutamine oxidation also increased, and glutamate content decreased with exaggerated insulin secretion. Glucose oxidation was not altered. Insulin secretion profiles from GDH266C-overexpressing isolated rat pancreatic islets were similar to those from MIN6-GDH266C, suggesting observation in MIN6 cells to be relevant in native beta-cells. These results demonstrate that, upon activation, GDH oxidizes glutamate to alpha-ketoglutarate, thereby stimulating insulin secretion by providing the TCA cycle with a substrate. No evidence was obtained supporting the hypothesis that activated GDH produced glutamate, a recently proposed second messenger of insulin secretion, by the reverse reaction, to stimulate insulin secretion.     

PKC mediates cyclic stretch-induced cardiac hypertrophy through Rho family GTPases and mitogen-activated protein kinases in cardiomyocytes

Signaling events, including Rho GTPases and protein kinase C (PKC), are involved in cardiac hypertrophy. However, the mechanisms by which these pathways cooperate during the hypertrophic process remain unclear. Using an in vitro cyclic stretch model with neonatal rat cardiomyocytes, we demonstrated that stretch-induced activation of RhoA, Rac1/Cdc42, and phosphorylation of Rho-guanine nucleotide dissociation inhibitor (GDI) were prevented by inhibition or depletion of PKC, using chelerythrine and phorbol 12-myristate 13-acetate, indicating that phorbol ester-sensitive PKC isozymes may be upstream regulators of Rho GTPases. Using adenoviral-mediated gene transfer of wild-type (WT) and dominant-negative (DN) mutants of PKCalpha and delta, we found that stretch-induced activation of Rho GTPases and phosphorylation of Rho-GDI were mainly regulated by PKCalpha. PKCdelta was involved in regulation of the activation of Rac1. Stretch-induced increases in [(3)H]-leucine incorporation, myofibrillar reorganization and cell size, were blocked by inhibition of Rho GTPases, or overexpression of DN PKCalpha and delta, suggesting that PKCalpha and delta are both required in stretch-induced hypertrophy, through Rho GTPases-mediated signaling pathways. The mechanism, whereby PKC and Rho GTPases regulate hypertrophy, was associated with mitogen-activated protein (MAP) kinases. Stretch-stimulated phosphorylation of MEK1/ERK1/2 and MKK4/JNK was inhibited by overexpression of DN PKCalpha and delta, and that of MKK3/p38 inhibited by DN PKCdelta. The phosphorylation of ERK and JNK induced by overexpression of WT PKCalpha, and the phosphorylation of p38 induced by WT PKCdelta, were regulated by Rho GTPases. This study represents the first evidence that PKCalpha and delta are important regulators in mediating activation of Rho GTPases and MAP kinases, in the cyclic stretch-induced hypertrophic process.     

Various Effects of Sandblasting of Dental Restorative Materials

Background

Sandblasting particles which remain on the surfaces of dental restorations are removed prior to cementation. It is probable that adhesive strength between luting material and sandblasting particle remnants might exceed that with restorative material. If that being the case, blasting particles adhere to sandblasted material surface could be instrumental to increasing adhesive strength like underlying bonding mechanism between luting material and silanized particles of tribochemical silica coating-treated surface. We hypothesize that ultrasonic cleaning of bonding surfaces, which were pretreated with sandblasting, may affect adhesive strength of a resin luting material to dental restorative materials.

Methods

We therefore observed adhesive strength of resin luting material to aluminum oxide was greater than those to zirconia ceramic and cobalt-chromium alloy beforehand. To measure the shear bond strengths of resin luting material to zirconia ceramic and cobalt-chromium alloy, forty specimens of each restorative material were prepared. Bonding surfaces were polished with silicon abrasive paper and then treated with sandblasting. For each restorative material, 40 sandblasted specimens were equally divided into two groups: ultrasonic cleaning (USC) group and non-ultrasonic cleaning (NUSC) group. After resin luting material was polymerized on bonding surface, shear test was performed to evaluate effect of ultrasonic cleaning of bonding surfaces pretreated with sandblasting on bond strength.

Results

For both zirconia ceramic and cobalt-chromium alloy, NUSC group showed significantly higher shear bond strength than USC group.

Conclusions

Ultrasonic cleaning of dental restorations after sandblasting should be avoided to retain improved bonding between these materials.     

Distribution and composition of colony founding associations of a subterranean termite, Reticulitermes kanmonensis

We investigated distribution and sexual composition of founding associations of Reticulitermes kanmonensis, the Japanese subterranean termite, which occurs only in the Kanmon area. These properties are discussed in relation to body size and mitochondrial genotype of the dealates. The founding colonies showed a highly aggregated distribution with a ‘hot spot’ of colony founding; however, mitochondrial haplotypes of the dealates suggested random mating. Monogamous colonies were predominant, but solitary colonies and colonies with two females and/or males also occurred. Paired dealates tended to be larger than solitary founders, suggesting that both sexes were under sexual selection related to body size.     

Identification and Characterization of Ambroxol as an Enzyme Enhancement Agent for Gaucher Disease

Gaucher disease (GD), the most prevalent lysosomal storage disease, is caused by a deficiency of glucocerebrosidase (GCase). The identification of small molecules acting as agents for enzyme enhancement therapy is an attractive approach for treating different forms of GD. A thermal denaturation assay utilizing wild type GCase was developed to screen a library of 1,040 Food and Drug Administration-approved drugs. Ambroxol (ABX), a drug used to treat airway mucus hypersecretion and hyaline membrane disease in newborns, was identified and found to be a pH-dependent, mixed-type inhibitor of GCase. Its inhibitory activity was maximal at neutral pH, found in the endoplasmic reticulum, and undetectable at the acidic pH of lysosomes. The pH dependence of ABX to bind and stabilize the enzyme was confirmed by monitoring the rate of hydrogen/deuterium exchange at increasing guanidine hydrochloride concentrations. ABX treatment significantly increased N370S and F213I mutant GCase activity and protein levels in GD fibroblasts. These increases were primarily confined to the lysosome-enriched fraction of treated cells, a finding confirmed by confocal immunofluorescence microscopy. Additionally, enhancement of GCase activity and a reduction in glucosylceramide storage was verified in ABX-treated GD lymphoblasts (N370S/N370S). Hydrogen/deuterium exchange mass spectrometry revealed that upon binding of ABX, amino acid segments 243–249, 310–312, and 386–400 near the active site of GCase are stabilized. Consistent with its mixed-type inhibition of GCase, modeling studies indicated that ABX interacts with both active and non-active site residues. Thus, ABX has the biochemical characteristics of a safe and effective enzyme enhancement therapy agent for the treatment of patients with the most common GD genotypes.Gaucher disease (GD),³ caused by deficiency of a lysosomal enzyme glucocerebrosidase (GCase), an acidic β-glucosidase (EC 3.2.1.45), encompasses a continuum of clinical spectrum from a perinatal lethal disorder to an asymptomatic form. Three major clinical subtypes (1–3) have been broadly defined, primarily based on the degree of neurological involvement. These are useful in determining prognosis and management (1, 2). Type 1 (GD-1), known as the non-neuronopathic form, is characterized by the presence of bone disease, hepatosplenomegaly, anemia, and thrombocytopenia. Type 2 and 3 (GD-2 and GD-3), also known as neuronopathic forms, are characterized by the presence of primary neurological disease. In the past, GD-2 and -3 were distinguished by age of onset and rate of disease progression, but these distinctions are not absolute (3). Generally, patients with GD-2 have an age of onset from days to months with a rapidly progressive neurological course. Individuals with GD-3 may have onset before reaching 2 years of age, often have a more slowly progressive neurological course, and may live into the 3rd or 4th decade. Interestingly, carriers of GD are also considered to have an increased risk of developing Parkinson disease (4–6).Skin fibroblasts from GD-1 patients contain 10–25% of residual GCase activity. The level of 25–30% is believed to represent the “critical threshold” of GCase activity, under which symptoms associated with GD are observed (7). Over 250 mutations have been reported in the GBA gene (encoding GCase) to cause GD. Of these, 203 are missense mutations (8). The most prevalent mutations found are N370S in GD-1 patients and L444P in GD-3 patients (9). However, L444P can also be found in GD-2 patients (10). Most of these missense mutations are believed to affect the proper folding of GCase in the endoplasmic reticulum (ER), re-directing the misfolded protein to the ER-associated degradation pathway. The mutations that have been demonstrated to affect the early folding of GCase are G202R, N370S, and F213I (11–14). In GD patient fibroblasts, particularly those with an N370S or F213I allele, growth in media containing a sub-inhibitory concentration of N-nonyl-deoxynojirimycin, N-octyl-β-valienamine, or the iminosugar isofagomine (IFG), specific competitive inhibitors of GCase, has been demonstrated to increase levels of lysosomal GCase activity 2–4-fold (7, 11, 13, 15, 16). These compounds function as pharmacological chaperones (PCs). PCs act by stabilizing the native conformation of the mutant GCase in the ER, allowing more functional molecules to form and evade the ER-associated degradation pathway by instead being passed onto the ER transport machinery (17), ultimately resulting in increased amounts in the lysosome. It is believed that once the complex enters the lysosomes of cells containing stored substrate, the inhibitors will be displaced allowing GCase to hydrolyze the substrate (18). However, the ideal PC for any lysosomal storage disease (LSD) should bind maximally at the neutral pH of the ER and minimally at the acidic pH of lysosomes.The current enzyme replacement therapy (ERT) for GD is limited to the treatment of non-neurological symptoms, because of the inability of the enzyme to cross the blood-brain barrier (19). This therapeutic approach is also very expensive (∼$200,000/year/patient) and must be administered intravenously. Small molecules are more likely to cross the blood-brain barrier, are less expensive to manufacture, and can be taken orally. Most of the small molecules, currently identified as PCs for GCase, are chemical compounds that have never been tested in humans and therefore are not approved by the drug regulation authorities. The screen of chemical libraries of Food and Drug Administration (FDA)-approved compounds has already proven to be a practical and successful strategy to identify small molecules that can behave as EET agents for specific lysosomal enzymes deficient in LSDs (20). To date, all reported approaches to library screening have utilized an assay aimed at identifying novel inhibitors of the target enzyme (20–23). In this context, we describe here the identification of ambroxol (ABX) as an EET agent for GCase using a more general thermal denaturation assay to screen the NINDS, National Institutes of Health, library of 1,040 drugs that have been previously used to treat humans. We then characterize the biochemical effects of ABX treatment on three GD patient cell lines, L444P/L444P, N370S/N370S, and F213I/L444P. Only the latter two cell lines responded with enhanced lysosomal GCase activity and protein levels. Additionally, substrate storage was reduced as demonstrated by liquid chromatography-electrospray ionization-tandem mass spectrometry after 15 days of treatment of an N370S/N370S lymphoblast line with ABX.     

Primary Gut Symbiont and Secondary,Sodalis-Allied Symbiont of the Scutellerid Stinkbug Cantao ocellatus

Symbiotic associations with midgut bacteria have been commonly found in diverse phytophagous heteropteran groups, where microbiological characterization of the symbiotic bacteria has been restricted to the stinkbug families Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae. Here we investigated the midgut bacterial symbiont of Cantao ocellatus, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified from the insects of different geographic origins. The bacterium was detected in all 116 insects collected from 9 natural host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the Gammaproteobacteria, not closely related to gut symbionts of other stinkbugs. Diagnostic PCR and in situ hybridization demonstrated that the bacterium is extracellularly located in the midgut 4th section with crypts. Electron microscopy of the crypts revealed a peculiar histological configuration at the host-symbiont interface. Egg sterilization experiments confirmed that the bacterium is vertically transmitted to stinkbug nymphs via egg surface contamination. In addition to the gut symbiont, some individuals of C. ocellatus harbored another bacterial symbiont in their gonads, which was closely related to Sodalis glossinidius, the secondary endosymbiont of tsetse flies. Biological aspects of the primary gut symbiont and the secondary Sodalis-allied symbiont are discussed.Insects are among the largest animal groups on the earth, embracing 750,000 to several millions of species (37, 52). Diverse insects are symbiotically associated with microorganisms, especially bacteria (5-7). In some insects, symbiotic bacteria are harbored in specialized host cells called bacteriocytes (or mycetocytes), constituting obligate mutualistic associations. For example, Buchnera aphidicola is harbored within bacteriocytes in the abdominal body cavity of almost all aphids and provides essential amino acids that are lacking in the phloem sap diet of the insects (9, 47). Wigglesworthia glossinidia is localized in a midgut-associated bacteriome of tsetse flies and plays pivotal roles in biosynthesis of B vitamins that are deficient in the vertebrate blood diet of the insects (2, 34). These obligate endocellular symbionts are often collectively referred to as “primary symbionts.”In contrast, there are facultative endosymbiotic microorganisms not essential for their host insects, often collectively called “secondary symbionts.” For example, many aphids are known to harbor various facultative symbionts, which belong to distinct lineages in the Gamma- and Alphaproteobacteria (33, 43) and the Mollicutes (10). While the majority of those facultative bacteria are either parasitic or commensalistic for their hosts, some of them affect the host fitness beneficially in particular ecological contexts (29, 32, 36, 44, 51). In addition to the obligate primary symbiont Wigglesworthia, tsetse flies harbor the facultative secondary symbiont Sodalis glossinidius, whose biological function for the hosts is currently elusive (3, 8).Members of the suborder Heteroptera, known as true bugs and consisting of over 38,000 described species, are characterized by their sucking mouthparts, half-membranous forewings, and incomplete metamorphosis (46). In the Heteroptera, symbiotic associations with bacteria are mainly found in phytophagous groups, especially in stinkbugs of the infraorder Pentatomomorpha. These stinkbugs generally possess many sacs or tubular outgrowths, called crypts or ceca, in a posterior region of the midgut, whose lumen is densely populated by a specific bacterial symbiont (7, 16). In some cases, experimental elimination of the symbiotic bacteria resulted in retarded growth and high mortality of the host insects (1, 13, 21, 26, 27, 39), indicating that these gut symbionts play important biological roles. Most of the gut symbionts are vertically transmitted through host generations by such mechanisms as egg surface contamination in the families Pentatomidae and Acanthosomatidae (1, 27, 39, 40, 42), coprophagy in the Cydnidae and Coreidae (22, 45), and capsule transmission in the Plataspidae (20), whereas a case of environmental acquisition has been reported from the Alydidae (26). Thus far, gut symbiotic bacteria of some members of the Acanthosomatidae, Plataspidae, Pentatomidae, Alydidae, and Pyrrhocoridae have been characterized using molecular techniques (21, 23, 25, 27, 38), while phylogenetic and biological aspects of gut symbiotic bacteria have been untouched in many other stinkbug groups.These gut symbiotic bacteria are, despite their extracellular localization, regarded as “primary symbionts” of the stinkbugs. On the other hand, some stinkbugs may, in addition to the gut symbiotic bacteria, also be associated with facultative “secondary symbionts.” For example, Wolbachia infections have been detected from diverse stinkbugs, most of which are probably of parasitic or commensalistic nature (24). Besides Wolbachia, there has been no report on facultative, secondary symbionts from stinkbugs.Members of the family Scutelleridae, often referred to as jewel bugs or shield-backed bugs, are stinkbugs characterized by their greatly enlarged convex scutellum that usually covers the entire abdomen. Some tropical species are also known for their vivid and beautiful body coloration (46). The family contains approximately 80 genera and 450 species, and in Japan, at least 7 genera and 9 species have been recorded (50). In the early 20th century, the presence of symbiotic bacteria was histologically described in midgut crypts of several scutellerid species (16, 31, 42). Since these pioneer works, however, no studies have been conducted on the symbiotic bacteria of scutellerid stinkbugs.Here we investigated the midgut symbiont of Cantao ocellatus, a scutellerid stinkbug widely distributed in Asian countries, including Japan, and known to guard their eggs and newborn nymphs (Fig. ​(Fig.1A)1A) (50). In addition to the gut symbiont, we also identified a Sodalis-allied facultative secondary symbiont from gonads of the insect.Open in a separate windowFIG. 1.(A) Adult female of Cantao ocellatus, guarding hatchlings under her body. (B) Dissected midgut from an adult female of C. ocellatus. 1st, midgut 1st section; 2nd, midgut 2nd section; 3rd, midgut 3rd section; 4th, midgut 4th section with crypts; hg, hindgut. (C) Enlarged image of the midgut 4th section with crypts. Arrowheads indicate three rows of crypts, while a fourth row is hidden behind. Glandular crypts (gc) are developed in adult females specifically, which may be involved in egg surface contamination with the symbiont. (D) An in situ hybridization image of the midgut 4th section, in which red and green signals indicate the gut symbiont and the host nucleus, respectively. Each arrow shows a crypt. (E) An enlarged image of the symbiotic bacteria in the crypts.     

Strict host-symbiont cospeciation and reductive genome evolution in insect gut bacteria

Host-symbiont cospeciation and reductive genome evolution have been identified in obligate endocellular insect symbionts, but no such example has been identified from extracellular ones. Here we first report such a case in stinkbugs of the family Plataspidae, wherein a specific gut bacterium is vertically transmitted via “symbiont capsule.” In all of the plataspid species, females produced symbiont capsules upon oviposition and their gut exhibited specialized traits for capsule production. Phylogenetic analysis showed that the plataspid symbionts constituted a distinct group in the γ-Proteobacteria, whose sister group was the aphid obligate endocellular symbionts Buchnera. Removal of the symbionts resulted in retarded growth, mortality, and sterility of the insects. The host phylogeny perfectly agreed with the symbiont phylogeny, indicating strict host-symbiont cospeciation despite the extracellular association. The symbionts exhibited AT-biased nucleotide composition, accelerated molecular evolution, and reduced genome size, as has been observed in obligate endocellular insect symbionts. These findings suggest that not the endocellular conditions themselves but the population genetic attributes of the vertically transmitted symbionts are probably responsible for the peculiar genetic traits of these insect symbionts. We proposed the designation “Candidatus Ishikawaella capsulata” for the plataspid symbionts. The plataspid stinkbugs, wherein the host-symbiont associations can be easily manipulated, provide a novel system that enables experimental approaches to previously untouched aspects of the insect-microbe mutualism. Furthermore, comparative analyses of the sister groups, the endocellular Buchnera and the extracellular Ishikawaella, would lead to insights into how the different symbiotic lifestyles have affected their genomic evolution.     

A sensitive immunochromatographic assay using gold nanoparticles for the semiquantitative detection of prostate-specific antigen in serum

In prostate cancer screening, prostate-specific antigen (PSA) has been utilized as a valuable biomarker. There are routinely used procedures based on enzyme-linked immunosorbent assay (ELISA) for PSA detection. The procedures based on ELISA, however, are time consuming, complicated, and costly. We have developed a rapid, very simple, cost effective and sensitive immunochromatographic assay using gold nanoparticles and evaluated its applications for first screening of prostate cancer in serum samples. The sensitive immunochromatographic assay requires only 40 μL of the serum sample. The assay used is rapid and simple, that it totally takes approx 15 min to complete. The method for sensitive immunochromatographic assay has the other advantage of decreasing the antibody concentration that is used for the test line. In this study, we show the advantage to decrease the antibody concentration and the evaluation of our sensitive immunochromatographic assay for the semiquantitative detection of PSA in serum. The results obtained from 163 serum samples using sensitive immunochromatographic assay are compared with the results obtained using the chemiluminescent enzyme immunoassay (CLEIA) and normal immunochromatographic assay. The results obtained in the sensitive immunochromatographic assay correlated well with the values obtained in CLEIA. We concluded that our sensitive immunochromatographic assay is applicable to the first screening test for the diagnosis of prostate cancer. Our developed sensitive immunochromatographic assay is a promising candidate for diagnosis or research use, which may become commercially available in the near future.