Vascular permeability enhancing activity of Porphyromonas gingivalis protease in guinea pigs

Abstract The complete nucleotide sequence of the Borrelia burgdorferi dnaA gene (encoding the initiator protein of chromosome replication) and its flanking regions was determined. The putative DnaA polypeptide exhibited 29–42% identity with those of other eubacteria. The gene order in the dnaA region at the centre of the B. burgdorferi linear chromosome is rnpA-rpmH-dnaN-dnaA-gyrB-gyrA in contrast to the consensus eubacterial order of rnpA-rpmH-dnaN-recF-gyrB , suggesting a rearrangement during the evolution of the Borrelia chromosome. We did not detect the multiple 9-nucleotide repeats known as DnaA boxes, which characterise origin of replications, in the dnaA-gyrB and dnaA-dnaN intergenic regions. In addition B. burgdorferi DnaA protein differs considerably from those of other eubacteria in a normally highly conserved region at the C-terminus of the polypeptide which may be involved in DNA binding.     

Inactivation of photosynthetic oxygen evolution by o-phenanthroline and LiClO4 in Photosystem 2 of the pea

We examined the effects of o-phenanthroline and LiClO₄ on oxygen evolution and electron transport in the Photosystem 2 complex of the pea. Treatment of Photosystem 2 particles with a combination of 3.0 mM o-phenanthroline and 1.0 M LiClO₄ for 30–40 min at 0°C decreased the oxygen-evolving activity with the electron acceptor (either phenyl-p-benzoquinone or 2,6-dichlorophenol indophenol) to less than 5% of the original level. However with the same treatment, the electron-transport activity from an artificial electron donor, 1,5-diphenylcarbohydrazide, to 2,6-dichlorophenol indophenol remained at 60% of the original activity. The amount of manganese in the Photosystem 2 complex decreased in parallel with the loss of oxygen evolution following treatment. These observations suggest that the treatment of the Photosystem 2 complex with o-phenanthroline and LiClO₄ inhibits electron transport on the oxygen-evolving side much more significantly than on the electron-acceptor side.Abbreviations Chl chlorophyll - DCPIP 2,6-dichlorophenol indophenol - DPC 1,5-diphenylcarbo hydrazide - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 4-morpholineethanesulfonic acid - PBQ phenyl-p-benzoquinone - PS 2 Photosystem 2     

Cleavage of hexokinase II to two domains by trypsin without significant change in catalytic activity

Hexokinase II prepared from Ehrlich-Lettre hyperdiploid tumor cells (ELD cells) was subjected to a limited digestion by trypsin. After 60 min digestion, hexokinase II (100 kDa) was completely cleaved to two fragments with the molecular weight of about 60 kDa and 40 kDa as manifested in SDS-PAGE. It was noteworthy that the enzyme activity was observed even at the time when the native enzyme molecule was no more detectable. These fragments were separated by SDS-PAGE irrespective of the presence of a reducing agent, but neither by native PAGE nor by cellulose acetate membrane electrophoresis under the nondenaturing conditions. Neither kinetic parameters such as Km values for ATP and glucose nor an ability of binding to mitochondria were changed significantly by the tryptic digestion. These results indicate that an essential conformation of hexokinase II can be restored by the self-association of two fragments produced as a result of the cleavage by trypsin at the middle of the molecule. Affinity labeling with 2-3-dialdehyde ATP followed by the trypsin digestion showed that ATP binding site resided in the 40 kDa fragment. Furthermore, the mode of the response in the incorporation of this ATP analog to hexose phosphate, moreover, was similar to that in the catalytic activity.Abbreviations SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis - EDTA Ethylenediamine Tetraacetic Acid - CBB Coomassie Brilliant Blue - PMSF Phenylmethylsulfonyl Fluoride - TPCK N-tosyl-Lphenylalanyl Chloromethyl Ketone - ELD cells Ehrlich-Lettre Hyperdiploid Tumor Cells     

A PP6-ASK3 Module Coordinates the Bidirectional Cell Volume Regulation under Osmotic Stress

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Prevalence of antibodies against Borna disease virus proteins in Japanese children with autism spectrum disorder

Bornavirus infection occurs in many animals, including humans. However, the epidemiology of bornavirus in humans, especially children, is as yet unclear. Here, antibodies against bornaviruses in Japanese children with autism spectrum disorder (ASD) were evaluated using immunofluorescence analysis, western blotting and radio ligand assay. The prevalence of antibodies against bornavirus‐specific speckles, nucleoprotein and phosphoprotein were 22%, 48%, and 33%, respectively, in children with ASD. According to our criteria, the prevalence of antibodies against bornaviruses was 7.4% in children with ASD. This is the first report of the serological prevalence of bornavirus in Japanese children. Our results provide valuable baseline‐data for future studies regarding bornavirus epidemiology in children.

     

Effects of Tenascin-C Knockout on Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage in Mice

A matricellular protein tenascin-C (TNC) has been suggested to play a role in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH), but the direct evidence remains lacking. In this study, we examined effects of TNC knockout (TNKO) on cerebral vasospasm after experimental SAH in mice. C57BL/6 wild-type (WT) or TNKO mice were subjected to SAH by endovascular puncture. Ten WT and ten TNKO mice were randomized to WT sham (n = 4), TNKO sham (n = 4), WT SAH (n = 6), and TNKO SAH (n = 6) groups. In addition to neurobehavioral impairments and severity of SAH, cerebral vasospasm was assessed by morphometric measurements of the left internal carotid artery (ICA). Infiltration of inflammatory cells in the subarachnoid periarterial space was also assessed, and expressions of TNC and mitogen-activated protein kinases (MAPKs) in the ICA were immunohistochemically evaluated at 24 h post-surgery. TNC was induced in the smooth muscle cell layers and the adventitia in the spastic ICAs as well as the periarterial inflammatory cells in WT SAH mice. Compared with WT SAH mice, TNKO SAH mice showed better neurological scores and less severe cerebral vasospasm, as well as fewer inflammatory cell infiltration in the periarterial space. Post-SAH activation of MAPKs in the smooth muscle cell layers of the ICAs was also prevented in TNKO SAH mice. The findings in the present study suggest that TNC causes the development of cerebral vasospasm via pro-inflammatory effects and activation of MAPKs.     

Microtopographic specialization and flexibility in tropical peat swamp forest tree species

Tropical tree species distributions are determined by a wide range of biotic and abiotic factors, including topography and hydrology. Tropical peat swamp forests (TPSFs) are characterized in part by small‐scale variations in topography (‘hummocks’ and ‘hollows’) that create distinct microhabitats and thus may contribute to niche diversification among TPSF tree species. Using tree elevations calibrated to daily peat water levels collected using a data logger and a permutation test, we evaluated topographical microhabitat preferences for 21 tree species in a relatively undisturbed TPSF in Central Kalimantan, Indonesia, to determine whether these species show preferential association with hummocks or hollows and to quantify the prevalence of microhabitat specialization among them. Only one species, Tetractomia tetrandrum, emerged as a hollow specialist, with no hummock specialists among the species tested. The remaining 20 species, including Psydrax dicoccos, which had the lowest mean observed elevation, and Maasia hypoleuca, which had the highest mean observed elevation, showed no clear microtopographic preference. This suggests that many TPSF species may be resilient to the natural hydrologic variations that occur in relatively intact peat swamp forests. Such studies of microtopographic preferences of tree species in TPSF and other wetland forest ecosystems can inform selection of tree species for reforestation projects, and potentially also provide information on how future climate change may impact these habitats and their resident tree species.     

Drive-selection equilibrium: Homopolymer evolution in the Drosophila gene mastermind

Interspecific sequence comparison of the highly repetitive Drosophila gene mastermind (mam) reveals extensive length variation in homopolymer domains. The length variation in homopolymers is due to nucleotide misalignment in the underlying triplet repeats, which can lead to slippage mutations during DNA replication or repair. In mam, the length variation in repetitive regions appears to be balanced by natural selection acting to maintain the distance between two highly conserved charge clusters. Here we report a statistical test of the null hypothesis that the similarity in the amino acid distance between the charge clusters of each species arose by chance. The results suggest that at mam there is a juxtaposition of length variability due to molecular drive and length conservation maintained by natural selection. The analysis of mam allows the extension of current theories of drive-selection interaction to encompass homopolymers. Our model of drive-selection equilibrium suggests that the physical flexibility, length variability, and abundance of homopolymer domains provide an important source of genetic variation for natural populations.Correspondence to: S.J. Newfeld 1072     

Apoptosis signal-regulating kinase (ASK) 2 functions as a mitogen-activated protein kinase kinase kinase in a heteromeric complex with ASK1

Apoptosis signal-regulating kinase (ASK) 1 is a mitogen-activated protein kinase kinase kinase (MAP3K) in the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways that play multiple important roles in cytokine and stress responses. Here we show that ASK2, a highly related serine/threonine kinase to ASK1, also functions as a MAP3K only in a heteromeric complex with ASK1. We found that endogenous ASK2 was constitutively degraded in ASK1-deficient cells, suggesting that ASK1 is required for the stability of ASK2. ASK2 in a heteromeric complex with a kinase-negative mutant of ASK1 (ASK1-KN) effectively activated MAP2K and was more competent to respond to oxidative stress than ASK2 alone. Knockdown of ASK2 revealed that ASK2 was required for oxidative stress-induced JNK activation. These results suggest that ASK2 forms a functional MAP3K complex with ASK1, in which ASK1 supports the stability and the active configuration of ASK2. Moreover, ASK2 was found to activate ASK1 by direct phosphorylation, suggesting that ASK1 and ASK2 in a heteromeric complex facilitate their activities to each other by distinct mechanisms. Such a formation of functional heteromeric complex between different MAP3Ks may be advantageous for cells to cope with a wide variety of stimuli by fine regulation of cellular responses.     

A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments

The central channel Tom40 of the preprotein translocase of outer membrane (TOM) complex is thought to be responsible for the import of virtually all preproteins synthesized outside the mitochondria. In this study, we analyze the topogenesis of the peripheral benzodiazepine receptor (PBR), which integrates into the mitochondrial outer membrane (MOM) through five hydrophobic transmembrane segments (TMSs) and functions in cholesterol import into the inner membrane. Analyses of in vitro and in vivo import into TOM component–depleted mitochondria reveal that PBR import (1) depends on the import receptor Tom70 but requires neither the Tom20 and Tom22 import receptors nor the import channel Tom40, (2) shares the post-Tom70 pathway with the C-tail–anchored proteins, and (3) requires factors of the mitochondrial intermembrane space. Furthermore, membrane integration of mitofusins and mitochondrial ubiquitin ligase, the MOM proteins with two and four TMSs, respectively, proceeds through the same initial pathway. These findings reveal a previously unidentified pathway of the membrane integration of MOM proteins with multiple TMSs.