Distinct subcellular localization of three isoforms of insulinoma-associated protein 2β in neuroendocrine tissues

AimsInsulinoma-associated protein 2β (IA-2β) is considered to play a significant role in regulated secretion. Recent studies have shown that the mouse brain expresses three major isoforms of IA-2β, named IA-2β60, IA-2β64, and IA-2β71. In this study, we analyzed the tissue-, cell- and organelle-specific distributions of IA-2β isoforms in mice.Main methodsTo localize IA-2β expression in mouse tissues and cells, western blot and immunohistochemical analyses were carried out. The subcellular distribution of IA-2β isoforms was assessed by sedimentation of mouse brain homogenates in a discontinuous sucrose density gradient.Key findingsIA-2β60 was abundant in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, and pituitary, and in the muscular and mucosal layers of the digestive organs. In contrast, the expression of IA-2β64 and IA-2β71 was restricted to the cerebrum, cerebellum, medulla oblongata, and pituitary, and the muscular layers of the digestive organs. Immunohistochemical analysis of mouse pancreatic islets revealed that pancreatic beta cells expressed IA-2β60 exclusively, whereas alpha and delta cells expressed all three isoforms. By the sedimentation of mouse brain homogenates, it was shown that IA-2β64 and IA-2β71 were co-localized with IA-2 on secretory granules, but were absent from synaptic vesicles (SVs). On the other hand, IA-2β60 was co-localized with synaptophisin on SVs, but was absent from secretory granules.SignificanceThe tissue-, cell- and organelle-specific distributions of IA-2β isoforms suggest that IA-2β60 has a role in secretion from SVs, whereas IA-2β64 and IA-2β71 are involved in secretion from secretory granules.     

A novel saccharification method of starch using microwave irradiation with addition of activated carbon

Activated carbons were investigated for their heat catalytic effects to improve saccharification of starch by autohydrolysis in water under microwave electromagnetic field, and the results were compared with graphite and carbon nanotubes. The activated carbons with low adsorptive capacity of maltose showed high saccharification rate, while those with high adsorptive capacity exhibited low saccharification. In addition, the former activated carbons decreased the saccharification temperature by 10-30 °C. Maltooligosaccharides produced in the presence of the latter activated carbons were recovered by desorption with 50% aqueous ethanol. The results indicated that both adsorptive capacities of maltooligosaccharides and catalytic effects of hot spots arisen from the uneven surface structure of activated carbons might contribute to the improvement in starch saccharification.     

Peculiar hyporheic habitat of some <Emphasis Type="Italic">Rhyacophila</Emphasis> species (Trichoptera; Rhyacophilidae) in Japanese mountain streams

We investigated the distribution patterns of Rhyacophila larvae at 2 habitat scales in mountain streams in central Honshu. One was at a rapid glide within a stream reach and the other was in microvertical layers within each rapid and glide. Cobbles were predominant in rapids, whereas sand and gravels were predominant in glides. Mean sediment particle size was smaller in glides, but evenness of sediment size and porosity were greater. A total of 9 species of Rhyacophila larvae were collected from rapids. On the other hand, in glides, only 4 species, R. nipponica, R. shikotsuensis, R. kawamurae, and R. transquilla, were collected, but they were more abundant than in rapids. The larvae of R. transquilla were found mainly in upper layers of the stream bed, whereas the other 3 species, R. nipponica, R. shikotsuensis, and R. kawamurae, occurred both in upper and lower layers. The latter 3 species belong to R. nigrocephala species group, the larvae of which share several common morphological and behavioral characters adapted to hyporheic habitat, such as slender body shape and vermicular movement. It is suggested that higher porosity in glides provides more microhabitat for them, and small-sized sediment particles are appropriate for their burrowing and creeping movement in hyporheic habitat.     

Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis

We investigated the direct constitution of membrane proteins into giant liposomes in cell-free (in vitro) protein synthesis. Giant liposomes were present in a translation reaction cocktail of a wheat germ cell-free protein translation system. Apo cytochrome b(5) (b5) and its fusion proteins were synthesized and directly localized in the liposomes. After the translation reaction, the proteo-liposomes were isolated by simplified discontinuous density-gradient centrifugation. Apo cytochrome b(5) conjugated dihydrofolate reductase (DHFR) was synthesized in the same procedure and the protein was directly displayed on the liposome surface. b5 acts as a "hydrophobic tag" for recruitment to the liposome surface.     

Impairment of superoxide dismutase activation by N-terminally truncated prion protein (PrP) in PrP-deficient neuronal cell line

Previous studies have reported a neuroprotective role for cellular prion protein (PrP(C)) against apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line, but the mechanisms remain unclear. In this study, to investigate the mechanisms by which PrP(C) prevents apoptosis, the authors compared apoptosis of Prnp(-/-) cells with that of Prnp(-/-) cells expressing the wild-type PrP(C) or PrP(C) lacking N-terminal octapeptide repeat region under serum-free conditions. Re-introduction of Prnp rescued cells from apoptosis, upregulated superoxide dismutase (SOD) activity, enhanced superoxide anion elimination, and inhibited caspase-3/9 activation. On the other hand, N-terminally truncated PrP(C) enhanced apoptosis accompanied by potentiation of superoxide production and caspase-3/9 activation due to inhibition of SOD. These results suggest that PrP(C) protects Prnp(-/-) cells from apoptosis via superoxide- and caspase-3/9-dependent pathways by upregulating SOD activity. Furthermore, the octapeptide repeat region of PrP(C) plays an essential role in regulating apoptosis and SOD activity.     

Differential expression and distribution of Kir5.1 and Kir4.1 inwardly rectifying K+ channels in retina

Kir5.1 is an inwardly rectifying K⁺ channel subunit whose functional role has not been fully elucidated. Expression and distribution of Kir5.1 in retina were examined with a specific polyclonal antibody. Kir5.1 immunoreactivity was detected in glial Müller cells and in some retinal neurons. In the Kir5.1-positive neurons the expression of glutamic acid decarboxylase (GAD₆₅) was detected, suggesting that they may be GABAergic-amacrine cells. In Müller cells, spots of Kir5.1 immunoreactivity distributed diffusely at the cell body and in the distal portions, where Kir4.1 immunoreactivity largely overlapped. In addition, Kir4.1 immunoreactivity without Kir5.1 was strongly concentrated at the endfoot of Müller cells facing the vitreous surface or in the processes surrounding vessels. The immunoprecipitant obtained from retina with anti-Kir4.1 antibody contained Kir5.1. These results suggest that heterotetrameric Kir4.1/Kir5.1 channels may exist in the cell body and distal portion of Müller cells, whereas homomeric Kir4.1 channels are clustered in the endfeet and surrounding vessels. It is possible that homomeric Kir4.1 and heteromeric Kir4.1/Kir5.1 channels play different functional roles in the K⁺-buffering action of Müller cells. inwardly rectifying potassium channel; heteromerization; glial Müller cells; amacrine cells; potassium siphoning     

Inward rectifier K+ channel Kir2.3 is localized at the postsynaptic membrane of excitatory synapses

Classical inwardly rectifyingK⁺ channels (Kir2.0) are responsible for maintaining theresting membrane potential near the K⁺ equilibriumpotential in various cells, including neurons. Although Kir2.3 is knownto be expressed abundantly in the forebrain, its precise localizationhas not been identified. Using an antibody specific to Kir2.3, weexamined the subcellular localization of Kir2.3 in mouse brain. Kir2.3immunoreactivity was detected in a granular pattern in restricted areasof the brain, including the olfactory bulb (OB). Immunoelectronmicroscopy of the OB revealed that Kir2.3 immunoreactivity wasspecifically clustered on the postsynaptic membrane of asymmetricsynapses between granule cells and mitral/tufted cells. Theimmunoprecipitants for Kir2.3 obtained from brain contained PSD-95 andchapsyn-110, PDZ domain-containing anchoring proteins. In vitro bindingassay further revealed that the COOH-terminal end of Kir2.3 isresponsible for the association with these anchoring proteins.Therefore, the Kir channel may be involved in formation of the restingmembrane potential of the spines and, thus, would affect the responseof N-methyl-D-aspartic acid receptor channels atthe excitatory postsynaptic membrane.

     

Analysis of T-cell receptor Vbeta gene from infiltrating T cells in insulitis and myocarditis in encephalomyocarditis virus-infected BALB/C mice

Encephalomyocarditis (EMC) virus induces insulin-dependent diabetes and myocarditis in several strains of mice. The T-cell receptor (TCR) Vbeta genes of infiltrating T cells in the pancreas and myocardium of BALB/C mice infected with EMC virus D-variant (EMC-D virus) were analyzed. Using a nested two-step polymerase chain reaction (PCR), TCR Vbeta cDNAs were cloned and sequenced. Two and four kinds of TCR Vbeta clones were obtained from T cells infiltrating into the pancreas and myocardium of BALB/C mice infected with EMC-D virus, respectively. The infiltrating lymphocytes in the diabetic mice expressed Vbeta 8.1, 8.2, and 8.3 genes predominantly. Previously, the use of Vbeta 8.2 has been reported in autoimmune diseases such as murine experimental allergic encephalomyelitis (EAE) and non-obese diabetic (NOD) mouse. This study suggests that mice infected with EMC virus are a useful animal model for autoimmune diseases such as insulin-dependent diabetes.