Biochemistry and Regulation of a Novel Escherichia coli K-12 Porin Protein, OmpG, Which Produces Unusually Large Channels

A novel porin, OmpG, is produced in response to a chromosomal mutation termed cog-192. Molecular characterization of cog-192 revealed that it is a large chromosomal deletion extending from the 3′ end of pspA through to the 5′ end of an open reading frame located immediately upstream of ompG. As a result of this 13.1-kb deletion, the expression of ompG was placed under the control of the pspA promoter. Characterization of OmpG revealed that it is quite different from other porins. Proteoliposome swelling assays showed that OmpG channels were much larger than those of the OmpF and OmpC porins, with an estimated limited diameter of about 2 nm. The channel lacked any obvious solute specificity. The folding model of OmpG suggests that it is the first 16-stranded β-barrel porin that lacks the large external loop, L3, which constricts the channels of other nonspecific and specific porins. Consistent with the folding model, circular dichroism showed that OmpG contains largely a β-sheet structure. In contrast to other Escherichia coli porins, there is no evidence that OmpG exists as stable oligomers. Although ompG DNA was present in all E. coli strains examined so far, its expression under laboratory conditions was seen only due to rare chromosomal mutations. Curiously, OmpG was constitutively expressed, albeit at low levels, in Salmonella, Shigella, and Pseudomonas species.     

Effect of Heat Shock on Intracellular Calcium Mobilization in Neuroblastoma × Glioma Hybrid Cells

Abstract: The effect of heat shock on agonist-stimulated intracellular Ca²⁺ mobilization and the expression of heat shock protein 72 (hsp72) in neuroblastoma × glioma hybrid cells (NG 108–15 cells) were examined. Hsp72 was expressed at 6 h after heat shock (42.5°C, 2 h), reached a maximum at 12 h, and decreased thereafter. Bradykinin-induced [Ca²⁺], rise was attenuated to 28% of control by heat shock at 2 h after heat shock, and reversion to the control level was seen 12 h later. When the cells were treated with quercetin or antisense oligodeoxyribonucleotide against hsp72 cDNA, the synthesis of hsp72 was not induced by heat shock, whereas bradykinin-induced [Ca²⁺]_i rise was abolished and the [Ca²⁺]_i rise was not restored. Recovery from this stressed condition was evident when cells were stimulated by the Ca²⁺-ATPase inhibitor thapsigargin, even in the presence of either quercetin or antisense oligodeoxyribonucleotide. Inositol 1,4,5-trisphosphate (IP₃) production was not altered by heat shock at 12 h after heat shock, whereas IP₃ receptor binding activity was reduced to 45.3%. In the presence of quercetin or antisense oligodeoxyribonucleotide, IP₃ receptor binding activity decreased and reached 27.2% of the control 12 h after heat shock. Our working thesis is that heat shock transiently suppresses the IPs-mediated intracellular Ca²⁺ signal transduction system and that hsp72 is involved in the recovery of bradykinin-induced [Ca²⁺]_i rise.     

Small amount of concanavalin A modifies radiation-induced alteration in cell-surface charge depending on its binding condition

Cell electrophoretic mobility of cultured melanoma cells or rat erythrocytes decreased with time after X-irradiation. Addition of tetravalent concanavalin A or divalent succinyl-concanavalin A before (not after) irradiation, completely blocked the mobility reduction in greater concentrations than 5 μg/l.At 5 μg/1 only 3.7 · 10³ concanavalin A molecules bound to receptors per cell, while 4.18 · 10⁷ molecules/cell bound at saturating concentrations. Preincubation with concanavalin A at 37°C was effective even when the cells were treated with α-methylmannoside immediately after irradiation. At low temperature, however, concanavalin A was not effective despite a sufficient amount of bound ¹²⁵I-labelled concanavalin A. Treatment with α-methylmannoside following the binding of concanavalin A at 37°C before irradiation inhibited the concanavalin A effect depending on temperature. The residual amount of bound lectin could not account for the temperature dependence. The amount of sialic acid (the main charged substance) was not altered by X-irradiation with or without the lectin. Divalent succinyl-concanavalin A was also effective in blocking the radiation effect on electrophoretic mobility. These results seem to suggest that binding of a very small amount of concanavalin A without causing cell agglutination or clustering of its receptors, induces some alteration in the conformation of receptor glycoprotein, which blocks the internalization of acidic sugar residues by subsequent irradiation.     

Genome-wide association mapping in Arabidopsis identifies previously known flowering time and pathogen resistance genes

There is currently tremendous interest in the possibility of using genome-wide association mapping to identify genes responsible for natural variation, particularly for human disease susceptibility. The model plant Arabidopsis thaliana is in many ways an ideal candidate for such studies, because it is a highly selfing hermaphrodite. As a result, the species largely exists as a collection of naturally occurring inbred lines, or accessions, which can be genotyped once and phenotyped repeatedly. Furthermore, linkage disequilibrium in such a species will be much more extensive than in a comparable outcrossing species. We tested the feasibility of genome-wide association mapping in A. thaliana by searching for associations with flowering time and pathogen resistance in a sample of 95 accessions for which genome-wide polymorphism data were available. In spite of an extremely high rate of false positives due to population structure, we were able to identify known major genes for all phenotypes tested, thus demonstrating the potential of genome-wide association mapping in A. thaliana and other species with similar patterns of variation. The rate of false positives differed strongly between traits, with more clinal traits showing the highest rate. However, the false positive rates were always substantial regardless of the trait, highlighting the necessity of an appropriate genomic control in association studies.     

Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 regulates the migration of Langerhans cells from the epidermis to draining lymph nodes

Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1) is a member of the signal regulatory protein family in which the extracellular region interacts with its ligand, CD47. Recent studies have demonstrated that SHPS-1 plays an important role in cell migration and cell adhesion. We demonstrate in this study, using immunohistochemical and flow cytometric analyses, that murine Langerhans cells (LCs) express SHPS-1. Treatment of mice ears with 2,4-dinitro-1-fluorobenzene significantly reduced the number of epidermal LCs, and that reduction could be reversed by pretreatment with mAb to SHPS-1 or the CD47-Fc fusion protein. Treatment with the SHPS-1 mAb in vivo reduced the number of FITC-bearing cells in the lesional lymph nodes after the application of FITC to the skin. The SHPS-1 mAb inhibited the in vivo TNF-alpha-induced migration of LCs. The emigration of dendritic cells expressing I-A(b+) from skin explants to the medium was also reduced by the SHPS-1 mAb. We further demonstrate that the chemotaxis of a murine dendritic cell line, XS52, by macrophage inflammatory protein-3beta was significantly inhibited by treatment with the SHPS-1 mAb or CD47-Fc recombinant protein. Finally, we show that migration of LCs was attenuated in mutant mice that lack the intracellular domain of SHPS-1. These observations show that the ligation of SHPS-1 with the SHPS-1 mAb or with CD47-Fc abrogates the migration of LCs in vivo and in vitro, which suggests that the SHPS-1-CD47 interaction may negatively regulate LC migration.     

Demonstration of functional role of TECK/CCL25 in T lymphocyte-endothelium interaction in inflamed and uninflamed intestinal mucosa

It has recently been suggested that C-C chemokines may play a role in the organ-specific homing of lymphocytes, but there is not enough in vivo evidence in intestinal mucosa. The aim of this study was to examine whether thymus-expressed chemokine (TECK)/CCL25 and its ligand CCR9 are involved in T-lymphocyte interaction with microvessels of murine intestinal mucosa. T lymphocytes from the small intestine were fluorescence labeled, and their adhesion to mucosal microvessels was observed by intravital microscopy. Lamina proprial lymphocytes (LPL) and intraepithelial lymphocytes (IEL) adhered to both the small intestine and colon, and desensitization of CCR9 with TECK/CCL25 or anti-TECK/CCL25 antibody significantly inhibited these adhesions only in small intestine. At both sites, TNF-alpha significantly increased LPL adhesion but not IEL adhesion. Desensitization of CCR9 or anti-TECK/CCL25 antibody also attenuated the TNF-alpha-induced LPL adhesion in the small intestine. Increased expression of TECK/CCL25 by TNF-alpha was observed in the lamina propria of small intestine. TECK/CCL25 may thus play an important role in the adherence of mucosal lymphocytes to the microvessels of the small intestine but not the colon under uninflamed as well as inflamed conditions.     

Effects of different anti-tau antibodies on tau fibrillogenesis: RTA-1 and RTA-2 counteract tau aggregation

Tau is the major antigenic component of neurofibrillary pathology in tauopathy, including Alzheimer's disease. Although conversion of soluble tau to an insoluble polymerized fibrillar form is a key factor in the pathogenesis of tauopathy, the mechanism of the change is unclear and no inhibitors of fibril formation are available. Monoclonal antibodies against the 1st or 2nd repeat of the microtubule binding domain, but not the C-terminal 16 residues, completely inhibited tau aggregation into PHF. Furthermore, they did not inhibit tau-induced tubulin assembly. Thus, they are useful to investigate tau protein conversion and will be useful therapeutic lead materials.     

Xrs2p regulates Mre11p translocation to the nucleus and plays a role in telomere elongation and meiotic recombination

The Mre11-Rad50-Xrs2 (MRX) protein complex plays pivotal roles in meiotic recombination, repair of damaged DNA, telomere elongation, and cell cycle checkpoint control. Xrs2p is known to be essential for all the functions of the complex, but its role in the complex has not been clearly elucidated. A 32-amino acid region near the C terminus of Xrs2p was identified as an Mre11p-binding site. No more function of Xrs2p than translocation of Mre11p from the cytoplasm to the nucleus is necessary for response to DNA damage. However, domains in Xrs2p located both 49 amino acids upstream and 104 amino acids downstream of the Mre11p binding site are required for meiotic recombination and telomere elongation, respectively, in addition to the 32-amino acid region. These findings demonstrate that Xrs2p acts as a specificity factor that allows the MRX complex to function in meiotic recombination and in telomere elongation.     

Behavior and function of paternally inherited centrioles in brown algal zygotes

In brown algal cells, the centrosome, consisting of a pair of centrioles and the pericentriolar material, is primarily involved in the organization of microtubules (MTs) throughout the cell cycle. In motile cells, the centrioles participate in the formation of flagellar axoneme as flagellar basal bodies, and in somatic cells they play a crucial role in many cellular activities as a part of the centrosome. With respect to the role of the centrosome as a microtubule organizing center (MTOC), brown algal cells resemble animal cells. In most animal fertilization processes, the sperm cell introduces centrioles, the core of the centrosome, into the egg cytoplasm. In this study, the behavior of centrioles from gametogenesis and fertilization to the first cell division of the zygote was examined in the three sexual reproduction patterns occurring in brown algae, i.e., oogamy, anisogamy and isogamy, by electron- and immunofluorescence-microscopy. The pair of centrioles contained in somatic cells was shown to be derived from the male gamete, irrespective of the sexual reproductive pattern. The paternally derived centrioles were duplicated before mitosis and were involved in spindle pole formation. Moreover, MTs from the centrosome play a crucial role in the process of cytokinesis, as the position of centrosomes accompanying daughter nuclei seems to determine the cytokinetic plane. A new approach to clarifying the mode of cytokinesis in brown algae is presented in this study.Chikako Nagasato was the recipient of the Botanical Society of Japan Award for Young Scientist, 2004.     

Protection against malaria by anti-erythropoietin antibody due to suppression of erythropoiesis in the liver and at other sites

We have previously reported that erythropoiesis commences in the liver and spleen after malarial infection, and that newly generated erythrocytes in the liver are essential for infection of malarial parasites as well as continuation of infection. At this time, erythropoietin (EPO) is elevated in the serum. In the present study, we administered EPO or anti-EPO antibody into C57BL/6 (B6) mice to modulate the serum level of EPO. When mice were infected with a non-lethal strain (17NXL) of Plasmodium yoelii (blood-stage infection of 10(4) parasitized erythrocytes per mouse), parasitemia continued for 1 month, showing a peak at day 17. Daily injection of EPO (200 IU/day per mouse) from day five to day 14 prolonged parasitemia, whereas injection of anti-EPO antibody (1.5 mg/day per mouse) every second day from day five to day 28 decreased it. Erythropoiesis was confirmed in the liver, spleen and bone marrow by the appearance of nucleated erythrocytes (TER119+). When anti-EPO antibody was injected by the same protocol into mice infected with a lethal strain (17XL) of P. yoelii, all mice showed decreased parasitemia and recovered from the infection. These results suggest that the use of anti-EPO antibody after malarial infection may be of therapeutic value in severe cases of malaria.