More than a 100-fold increase in immunoblot signals of laser-microdissected inclusion bodies with an excessive aggregation property by oligomeric actin interacting protein 2/D-lactate dehydrogenase protein 2

We established a histobiochemical approach targeting micron-order inclusion bodies possessing extensive aggregation properties in situ by using a nonchemical denaturant (oligomeric actin interacting protein 2/d-lactate dehydrogenase protein 2 [Aip2p/Dld2p]) with the combinatorial method of laser-microdissection and immunoblot analysis. As a model, pick bodies were chosen and laser-microdissected from three different brain regions of two patients with Pick's disease. Initially, 500 to 2000 pick bodies were applied onto SDS-PAGE gels after boiling in Laemmli's sample buffer according to established immunoblotting procedures; however, only faint signals were obtained. Following negative results with chemical denaturants or detergent, including 6 M guanidine hydrochloride, 8 M urea, and 2% SDS, the laser-microdissected pick bodies were pretreated with oligomeric Aip2p/Dld2p, which possesses robust protein unfolding activity under biological conditions. Strikingly, only one pick body was sufficient to illustrate an immunoblot signal, indicating that pretreatment with oligomeric Aip2p/Dld2p enhanced the immunoblot sensitivity by more than 100-fold. Pretreatment with oligomeric Aip2p/Dld2p also allowed us to quantify the total protein content of pick bodies. Thus, use of oligomeric Aip2p/Dld2p significantly contributed toward the acquisition of information pertaining to the molecular profile of proteins possessing an extensive aggregation property, particularly in small amounts.     

An autosomal dominant cerebellar ataxia linked to chromosome 16q22.1 is associated with a single-nucleotide substitution in the 5' untranslated region of the gene encoding a protein with spectrin repeat and Rho guanine-nucleotide exchange-factor domains

Autosomal dominant cerebellar ataxia (ADCA) is a group of heterogeneous neurodegenerative disorders. By positional cloning, we have identified the gene strongly associated with a form of degenerative ataxia (chromosome 16q22.1-linked ADCA) that clinically shows progressive pure cerebellar ataxia. Detailed examination by use of audiogram suggested that sensorineural hearing impairment may be associated with ataxia in our families. After restricting the candidate region in chromosome 16q22.1 by haplotype analysis, we found that all patients from 52 unrelated Japanese families harbor a heterozygous C-->T single-nucleotide substitution, 16 nt upstream of the putative translation initiation site of the gene for a hypothetical protein DKFZP434I216, which we have called "puratrophin-1" (Purkinje cell atrophy associated protein-1). The full-length puratrophin-1 mRNA had an open reading frame of 3,576 nt, predicted to contain important domains, including the spectrin repeat and the guanine-nucleotide exchange factor (GEF) for Rho GTPases, followed by the Dbl-homologous domain, which indicates the role of puratrophin-1 in intracellular signaling and actin dynamics at the Golgi apparatus. Puratrophin-1--normally expressed in a wide range of cells, including epithelial hair cells in the cochlea--was aggregated in Purkinje cells of the chromosome 16q22.1-linked ADCA brains. Consistent with the protein prediction data of puratrophin-1, the Golgi-apparatus membrane protein and spectrin also formed aggregates in Purkinje cells. The present study highlights the importance of the 5' untranslated region (UTR) in identification of genes of human disease, suggests that a single-nucleotide substitution in the 5' UTR could be associated with protein aggregation, and indicates that the GEF protein is associated with cerebellar degeneration in humans.     

Transcytosis of calcium from bone by osteoclast-like cells evidenced by direct visualization of calcium in cells

'Transcytosis' of calcium (Ca) from bone by osteoclasts was identified by using a newly developed method that uses fixed or living osteoclast-like cells previously differentiated in vitro, a Ca-specific cell-membrane-impermeable fluorescent dye, and confocal laser scanning microscopy. This method, called the cell-membrane-impermeable dye method, revealed that in fixed osteoclast-like cells, a large quantity of Ca was confined within vacuoles and transported toward the apical cell membrane in the cells. These Ca-confined vacuoles were co-localized with marker proteins of both ruffled border and lysosome. The vacuoles were disrupted when treated with an inhibitor of ruffled border ATPase. In living osteoclast-like cells, Ca-confined vacuoles were again preferentially located at the central region and near the apical cell membrane. These results suggest actual transcytosis of Ca from bone by osteoclasts, and are the first direct evidence of the significant role of osteoclasts in the entire process of Ca metabolism in bone.     

Approach to a metallacycling polymerization — reactions of [(η-C5H5)Co(PPh3)2] with α,β-diynes and Me3SiCCC6H4C6H4CCSiMe3

Reactions of [CpCo(PPh₃)₂](Cp=η⁵-cyclopentadienyl) with conjugated diacetylenes were investigated in terms of the synthesis of π-conjugated organometallic polymers. The reaction of an α,β-diyne, PhCC---CCPh, gave three geometric isomers of dialkynylcobaltacyclopentadienes, 1a-c, and an insoluble polymeric product, 1d. A 2,4-dialkynyl complex, 2, and a 2,5-dialkynyl complex, 3, were obtained solely from Me₃SiCC---CCSiMe₃ and MeCC---CCMe, respectively. 1,1′-Bis(trimethylsilylethynyl)-4,4′-biphenyl afforded two isomers of 1,3-dialkynylcyclobutadiene complexes, 4a and 4b. The stability of the one-electron oxidized forms of the cobalacyclopentadiene and cyclobutadiene complexes was examined by cyclic voltammetry.     

A new type of 3‐D peripheral ultrastructure in Glaucocystis (Glaucocystales,Glaucophyta) as revealed by ultra‐high voltage electron microscopy

The coccoid glaucophyte genus Glaucocystis is characterized by having a thick cell wall, which has to date prohibited examination of the native ultrastructural features of the protoplast periphery. Recently, however, the three‐dimensional (3‐D) ultrastructure of the protoplast periphery was revealed in two divergent Glaucocystis species, with the world's most powerful ultra‐high voltage electron microscope (UHVEM). The two species exhibit morphological diversity in terms of their 3‐D ultrastructural features. However, these two types do not seem to encompass actual ultrastructural diversity in the genetically diverse genus Glaucocystis. Here, we report a new type of peripheral 3‐D ultrastructure resolved in “G. incrassata” SAG 229‐2 cells by 3‐D modeling based on UHVEM tomography using high‐pressure freezing and freeze‐substitution fixation. The plasma membrane and underlying flattened vesicles in “G. incrassata” SAG 229‐2 exhibited grooves at intervals of 200–600 nm, and the flattened vesicles often overlapped one another at the protoplast periphery. This 3‐D ultrastructure differs from those of the two types previously reported in other species of Glaucocystis. The possibility of classification of Glaucocystis species based on the 3‐D ultrastructure of the protoplast periphery is discussed.     

The endodermis and shoot gravitropism

Shoots and roots of higher plants exhibit negative and positive gravitropism, respectively. A variety of gravitropic mutants have recently been isolated from Arabidopsis, the characterization of which demonstrates that the molecular mechanisms of the gravitropic responses in roots, hypocotyls and inflorescence stems are different. The cytological and molecular analysis of two mutants, shoot gravitropism 1 (sgrl), which is allelic to scarecrow (scr), and sgr7, which is allelic to short-root(shr), indicate that the endodermis is the site of gravity perception in shoots. These data suggest a new model for shoot gravitropism.     

Regulatory dendritic cells pulsed with carbonic anhydrase I protect mice from colitis induced by CD4+CD25- T cells

Inflammatory bowel disease (IBD), which is characterized by a dysregulated intestinal immune response, is postulated to be controlled by intestinal self-antigens and bacterial Ags. Fecal extracts called cecal bacterial Ag (CBA) have been implicated in the pathogenesis of IBD. In this study, we identified a major protein of CBA related to the pathogenesis of IBD and established a therapeutic approach using Ag-pulsed regulatory dendritic cells (Reg-DCs). Using two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry, carbonic anhydrase I (CA I) was identified as a major protein of CBA. Next, we induced colitis by transfer of CD4(+)CD25(-) T cells obtained from BALB/c mice into SCID mice. Mice were treated with CBA- or CA I-pulsed Reg-DCs (Reg-DCs(CBA) or Reg-DCs(CA1)), which expressed CD200 receptor 3 and produced high levels of IL-10. Treatment with Reg-DCs(CBA) and Reg-DCs(CA1) ameliorated colitis. This effect was shown to be Ag-specific based on no clinical response of irrelevant Ag (keyhole limpet hemocyanin)-pulsed Reg-DCs. Foxp3 mRNA expression was higher but RORγt mRNA expression was lower in the mesenteric lymph nodes (MLNs) of the Reg-DCs(CA1)-treated mice compared with those in the MLNs of control mice. In the MLNs, Reg-DCs(CA1)-treated mice had higher mRNA expression of IL-10 and TGF-β1 and lower IL-17 mRNA expression and protein production compared with those of control mice. In addition, Reg-DCs(CBA)-treated mice had higher Foxp3(+)CD4(+)CD25(+) and IL-10-producing regulatory T cell frequencies in MLNs. In conclusion, Reg-DCs(CA1) protected progression of colitis induced by CD4(+)CD25(-) T cell transfer in an Ag-specific manner by inducing the differentiation of regulatory T cells.     

PHYLOGENETIC RELATIONSHIPS OF BRYDE'S WHALES IN THE WESTERN NORTH PACIFIC AND ADJACENT WATERS INFERRED FROM MITOCHONDRIAL DNA SEQUENCES1

To clarify phylogenetic relationships of Bryde's whales, we examined the nucleotide sequence of the mitochondrial control region and cytochrome b gene in 33 animals: 12 from offshore waters of the western North Pacific, five from off the Solomon Islands, and 16 from the East China Sea and coastal waters of Kochi in southwestern Japan. For reference purposes, homologous sequences from four Balaenoptera species including four Bryde's whales collected in the eastern Indian Ocean were added. We found whales from the three sampling areas to be genetically distinct. The control region sequences suggested that the whales from the three areas separate at higher than the populational level from one another. The cytochrome b data indicated that genetic differences between whales off the Solomon Islands and animals in the other two areas are equivalent to values found among recognized Balaenoptera species, although such a relationship was not observed between the other two areas. We conclude that whales in the East China Sea and coastal waters of Kochi separate from Bryde's whales in offshore waters of the western North Pacific at higher than the populational level but lower than the specific level (i. e., at the subspecific level) and that whales off the Solomon Islands do not belong genetically to the Bryde's whale as previously recognized.