An Amino Acid Substitution at Position 740 in σ70 of Ralstonia solanacearum Strain OE1-1 Affects Its In Planta Growth

Growth of Ralstonia solanacearum strain OE1-1 in roots after invasion is required for virulence. An Arg740Cys substitution in σ⁷⁰ of OE1-1 resulted in loss of in planta growth and virulence. The negative dominance of mutant σ⁷⁰ over the wild-type protein suggested that the amino acid substitution may affect the in planta growth of OE1-1, leading to a lack of virulence.     

Diazepam-induced sedative effects follow the pattern of enhanced heat loss in human

Sleep and Biological Rhythms - Although sleepiness and cognitive decline frequently appear concurrently after benzodiazepine administration, their functional linkage and the underlying...     

A new family of D-2-hydroxyacid dehydrogenases that comprises D-mandelate dehydrogenases and 2-ketopantoate reductases

The gene for the D-mandelate dehydrogenase (D-ManDH) of Enterococcus faecalis IAM10071 was isolated by means of an activity staining procedure and PCR and expressed in Escherichia coli cells. The recombinant enzyme exhibited high catalytic activity toward various 2-ketoacid substrates with bulky hydrophobic side chains, particularly C3-branched substrates such as benzoylformate and 2-ketoisovalerate, and strict coenzyme specificity for NADH and NAD(+). It showed marked sequence similarity with known NADP-dependent 2-ketopantoate reductases (KPR). These results indicate that together with KPR, D-ManDH constitutes a new family of D-2-hydroxyacid dehydrogenases that act on C3-branched 2-ketoacid substrates with various specificities for coenzymes and substrates.     

Clostridium botulinum serotype D neurotoxin and toxin complex bind to bovine aortic endothelial cells via sialic acid

Botulinum neurotoxin (BoNT) is produced as a large toxin complex (L-TC) associated with nontoxic nonhemagglutinin (NTNHA) and three hemagglutinin subcomponents (HA-70, -33 and -17). The binding properties of BoNT to neurons and L-TC to intestinal epithelial cells are well documented, while those to other tissues are largely unknown. Here, to obtain novel insights into the pathogenesis of foodborne botulism, we examine whether botulinum toxins bind to vascular endothelial cells. BoNT and 750 kDa L-TC (a complex of BoNT, NTNHA and HAs) of Clostridium botulinum serotype D were incubated with bovine aortic endothelial cells (BAECs), and binding to the cells was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot. Both BoNT and L-TC bound to BAECs, with L-TC showing stronger binding. Binding of BoNT and L-TC to BAECs was significantly inhibited by N-acetyl neuraminic acid in the cell culture medium or by treatment of the cells with neuraminidase. However, galactose, lactose or N-acetyl galactosamine did not significantly inhibit toxin binding to the cells. This is the first report demonstrating that BoNT and L-TC bind to BAECs via sialic acid, and this mechanism may be important in the trafficking pathway of BoNT in foodborne botulism.     

Seasonal changes in the microbial population of the water column and sediments of the Ongagawa River,northern Kyushu,Japan

The total viable count, population density of Escherichia coli and coliform bacteria, and nitrogen in the microbiomass (microbiomass-N) in sediments were monitored monthly at 12 points in the Ongagawa River basin from June 2002 to May 2006. The measurement of the sediment microbiomass-N was used for evaluation of the sediment’s microbial population in the river ecosystem. An extraordinarily high population of E. coli was observed during the season when there was stagnant water in the basin, with a high population and an insufficient drain diffusion system, and, thus hydrological water control is indispensable to prevent rapid E. coli growth. Microbiomass-N in sediments showed a negative correlation or independent fluctuation in relation to the bacterial population in the water column of the river. Seasonal changes in extracted nitrogen (N) in river sediments did not show correspondence with microbiomass-N in sediments. The microbiomass-N in sediments changed independently of the bacterial population in the river water, indicating that the high population of bacteria in the water does not lead to a high microbial population in river sediments. Ordination of the microbial parameters by canonical correspondence analysis (CCA) showed that microbiomass-N in sediments was quite different from other parameters. Relatively higher H⁺ (lower pH), PO₄ ³⁻ concentration and dissolved oxygen (DO) were the determinant parameters of higher microbiomass-N in sediments. A relative microbial abundance between the water column and sediments as well as each of the microbial populations in the water column and sediments could be a quantitative parameter for evaluating the biochemical processes of stream water.     

Peripheral nerve pericytes originating from the blood-nerve barrier expresses tight junctional molecules and transporters as barrier-forming cells

The objective of this study was to establish pure blood-nerve barrier (BNB)-derived peripheral nerve pericyte cell lines and to investigate their unique properties as barrier-forming cells. We isolated peripheral nerve, brain, and lung pericytes from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines expressed several pericyte markers such as alpha-smooth muscle actin, NG2, osteopontin, and desmin, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, these cell lines expressed several tight junction molecules such as occludin, claudin-12, ZO-1, and ZO-2. In particular, the expression of occludin was detected in peripheral nerve and brain pericytes, although it was not detected in lung pericytes by a Western blot analysis. An immunocytochemical analysis confirmed that occludin and ZO-1 were localized at the cell-cell boundaries among the pericytes. Brain and peripheral nerve pericytes also showed significantly higher trans-pericyte electrical resistance values and lower inulin clearances than lung pericytes. We considered that occludin localized at the cell-cell boundaries among the pericytes might mechanically stabilize the microvessels of the BNB and the blood-brain barrier. Furthermore, we also showed that these cell lines expressed many barrier-related transporters. ABCG2, p-gp, MRP-1, and Glut-1 were detected by a Western blot analysis and were observed in the cytoplasm and outer membrane by an immunocytochemical analysis. These transporters on pericytes might facilitate the peripheral nerve-to-blood efflux and blood-to-peripheral nerve influx transport of substrates in cooperation with those on endothelial cells in order to maintain peripheral nerve homeostasis.     

TRIM-directed selective autophagy regulates immune activation

Selectivity of autophagy is achieved by target recognition; however, the number of autophagy receptors identified so far is limited. In this study we demonstrate that a subset of tripartite motif (TRIM) proteins mediate selective autophagy of key regulators of inflammatory signaling. MEFV/TRIM20, and TRIM21 act as autophagic receptors recognizing their cognate targets and delivering them for autophagic degradation. MEFV recognizes the inflammasome components NLRP3, CASP1 and NLRP1, whereas TRIM21 specifically recognizes the activated, dimeric from of IRF3 inducing type I interferon gene expression. MEFV and TRIM21 have a second activity, whereby they act not only as receptors but also recruit and organize key components of autophagic machinery consisting of ULK1, BECN1, ATG16L1, and mammalian homologs of Atg8, with a preference for GABARAP. MEFV capacity to organize the autophagy apparatus is affected by common mutations causing familial Mediterranean fever. These findings reveal a general mode of action of TRIMs as autophagic receptor-regulators performing a highly-selective type of autophagy (precision autophagy), with MEFV specializing in the suppression of inflammasome and CASP1 activation engendering IL1B/interleukin-1β production and implicated in the form of cell death termed pyroptosis, whereas TRIM21 dampens type I interferon responses.     

Building-block architecture of botulinum toxin complex: Conformational changes provide insights into the hemagglutination ability of the complex

Clostridium botulinum produces the botulinum neurotoxin (BoNT). Previously, we provided evidence for the “building-block” model of botulinum toxin complex (TC). In this model, a single BoNT is associated with a single nontoxic nonhemagglutinin (NTNHA), yielding M-TC; three HA-70 molecules are attached and form M-TC/HA-70, and one to three “arms” of the HA-33/HA-17 trimer (two HA-33 and one HA-17) further bind to M-TC/HA-70 via HA-17 and HA-70 binding, yielding one-, two-, and three-arm L-TC. Of all TCs, only the three-arm L-TC caused hemagglutination. In this study, we determined the solution structures for the botulinum TCs using small-angle X-ray scattering (SAXS). The mature three-arm L-TC exhibited the shape of a “bird spreading its wings”, in contrast to the model having three “arms”, as revealed by transmission electron microscopy. SAXS images indicated that one of the three arms of the HA-33/HA-17 trimer bound to both HA-70 and BoNT. Taken together, these findings regarding the conformational changes in the building-block architecture of TC may explain why only three-arm L-TC exhibited hemagglutination.     

Comparative study of the N-terminal hydrophilic region in Streptomyces lividans and E. coli FtsY

Streptomyces lividans FtsY (SlFtsY) was cloned and overexpressed in Escherichia coli. Analysis of the amino acid (aa) sequence showed a concentration of hydrophilic aa's in the N-terminal half region of SlFtsY as observed in that of E. coli FtsY (EcFtsY). However, the length of the hydrophilic region was shorter in SlFtsY than in EcFtsY. Overexpression of SlFtsY in E. coli resulted in growth suppression as in the case of the overexpression of EcFtsY, while growth suppression as a result of the overexpression of the C-terminal half region of SlFtsY was limited. This result suggests that the N-terminal hydrophilic region of SlFtsY, regardless of its short length, would behave like its counterpart region of EcFtsY in E. coli.     

Fertilized eggs obtained from transplantation of frozen ovaries and parthenogenesis in combination with artificial insemination of frozen semen of the silkworm,Bombyx mori

A reliable method is reported for the long-term preservation of ovaries and spermatozoa of the silkworm (Bombyx mori). Three studies are presented. In the first, ovaries were removed from larvae at either 3rd, 4th, or 5th instar, cryopreserved, and stored in liquid nitrogen. Thawed ovaries were transplanted to surgically castrated female larvae at the same or a different developmental stage. The highest percentage of recipient females producing eggs resulted into either 3rd or 4th instar larvae (respectively, 22.1 and 8.7%). Similarly, the highest levels of other measurements of successful cryopreservation and transplanted ovary, and number of eggs laid, occurred with the same combination of donor and recipient developmental stages. Other combinations of ovary/recipient developmental stages yielded lower results. In the second experiment, semen was collected from male moths, cryopreserved, and then thawed semen was diluted with trypsin solution and artificially inseminated into females obtained from the best conditions of first experiment. A small percentage of inseminated moths laid eggs (8-10.3%) compared to that of controls (100%). In addition, the fertility of eggs from experimental moths was lower than that of control females (respectively, 40.3-88% and 97.5%). In the third experiment, eggs were surgically removed from ovarian tubules of moth following transplantation of thawed ovaries and subjected to parthenogenetic activation and artificial hatching. As expected, all resulting moths were female and, following natural mating or artificial insemination with thawed semen, yielded normal offspring at high rates.