Structural basis of human cytoglobin for ligand binding

Cytoglobin (Cgb), a newly discovered member of the vertebrate globin family, binds O(2) reversibly via its heme, as is the case for other mammalian globins (hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb)). While Cgb is expressed in various tissues, its physiological role is not clearly understood. Here, the X-ray crystal structure of wild type human Cgb in the ferric state at 2.4A resolution is reported. In the crystal structure, ferric Cgb is dimerized through two intermolecular disulfide bonds between Cys38(B2) and Cys83(E9), and the dimerization interface is similar to that of lamprey Hb and Ngb. The overall backbone structure of the Cgb monomer exhibits a traditional globin fold with a three-over-three alpha-helical sandwich, in which the arrangement of helices is basically the same among all globins studied to date. A detailed comparison reveals that the backbone structure of the CD corner to D helix region, the N terminus of the E-helix and the F-helix of Cgb resembles more closely those of pentacoordinated globins (Mb, lamprey Hb), rather than hexacoordinated globins (Ngb, rice Hb). However, the His81(E7) imidazole group coordinates directly to the heme iron as a sixth axial ligand to form a hexcoordinated heme, like Ngb and rice Hb. The position and orientation of the highly conserved residues in the heme pocket (Phe(CD1), Val(E11), distal His(E7) and proximal His(F8)) are similar to those of other globin proteins. Two alternative conformations of the Arg84(E10) guanidium group were observed, suggesting that it participates in ligand binding to Cgb, as is the case for Arg(E10) of Aplysia Mb and Lys(E10) of Ngb. The structural diversities and similarities among globin proteins are discussed with relevance to molecular evolutionary relationships.     

Insights into the CtrA regulon in development of stress resistance in obligatory intracellular pathogen Ehrlichia chaffeensis

Ehrlichia chaffeensis is an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Ehrlichiae have a biphasic developmental cycle consisting of dense-cored cells (DCs) and reticulate cells (RCs). Isolated DCs are more stress resistant and infectious than RCs. Here, we report that a response regulator, CtrA was upregulated in human monocytes at the late growth stage when DCs develop. E. chaffeensis CtrA bound to the promoters of late-stage transcribed genes: ctrA, ompA (peptidoglycan-associated lipoprotein), bolA (stress-induced morphogen) and surE (stationary-phase survival protein), which contain CtrA-binding motifs, and transactivated ompA, surE and bolA promoter-lacZ fusions in Escherichia coli. OmpA was predominantly expressed in DCs. E. chaffeensis binding to and subsequent infection of monocytes were inhibited by anti-OmpA IgG. E. chaffeensis BolA bound to the promoters of genes encoding outer surface proteins TRP120 and ECH_1038, which were expressed in DCs, and transactivated trp120 and ECH_1038 promoter-lacZ fusions. E. chaffeensis bolA complemented a stress-sensitive E. coli bolA mutant. E. coli expressing E. chaffeensis SurE exhibited increased resistance to osmotic stress. Our results suggest that E. chaffeensis CtrA plays a role in co-ordinating development of the stress resistance for passage from the present to the next host cells through its regulon.     

IgE-dependent mast cell activation potentiates airway responses in murine asthma models

We have studied murine models of asthma using FcepsilonRIalpha-chain-deficient (FcepsilonRIalpha(-/-)) mice to investigate the role of IgE-dependent mast cell activation in these models. When mice were either 1) immunized once with OVA in alum i.p. and then challenged with OVA intranasally, or 2) repeatedly immunized with OVA in the absence of adjuvant and subsequently challenged with nebulized OVA, FcepsilonRalpha(-/-) mice had significantly fewer eosinophils and lower IL-4 levels in their bronchoalveolar lavage fluid compared with wild-type mice. When mice were given anti-IL-5 antibody before OVA challenge in protocol 1, eosinophilic infiltration into the airways was significantly suppressed in both genotypes, but only FcepsilonRIalpha(-/-) mice showed significantly reduced airway hyperresponsiveness (AHR). In addition, when mice immunized and challenged with OVA also received a late OVA provocation at a higher concentration and were then exposed to methacholine, only wild-type mice developed a substantial increase in AHR. Since FcepsilonRI is expressed mainly on mast cells in mouse airways, we conclude that IgE-dependent activation of this cell type plays an important role in the development of allergic airway inflammation and AHR in mice. The models used may be of value for testing inhibitors of IgE or mast cells for development of therapeutic agents for human asthma.     

Identification of nuclear localization signals of Drosophila G9a histone H3 methyltransferase

G9a is one of the well-characterized histone methyltransferases. G9a regulates H3K9 mono- and dimethylation at euchromatic region and consequently plays important roles in euchromatic gene regulation. Mammalian G9a contains several distinct domains, such as GHD (G9a homology domain), ANK, preSET, SET and PostSET. These domains are highly conserved between mammals and Drosophila. Although mammalian G9a has nuclear localization signal (NLS) in its N-terminal region, the amino acid sequences of this region are not conserved in Drosophila. Here we have examined the subcellular localization of a series of truncated forms of Drosophila G9a (dG9a). The identified region (aa337-aa470) responsible for nuclear localization of dG9a contains four short stretches of positively charged basic amino acids (NLS1, aa334-aa345; NLS2, aa366-aa378; NLS3, aa407-aa419; NLS4, aa461-aa472). Each of NLS1, NLS3 and NLS4 is sufficient for the nuclear localization when they are fused with the enhanced green fluorescent protein. These NLSs of dG9a are distinct from those of mammalian G9a in their positions and amino acid sequences.     

Detection of the antibody to lymphocytic choriomeningitis virus in sera of laboratory rodents infected with viruses of laboratory and newly isolated strains by ELISA using purified recombinant nucleoprotein

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody against lymphocytic choriomeningitis virus (LCMV) in sera of laboratory animals. In this ELISA system, LCMV-nucleoprotein (NP) expressed by recombinant baculovirus and purified with high molar urea was used as the antigen. Sera from laboratory animals experimentally infected with the Armstrong strain or the newly isolated M1 strain of LCMV were examined to detect anti-LCMV antibody by the ELISA system, and the reactivity was compared with that of IFA test. Regardless of LCMV strain, all the sera of adult mice infected with LCMV were positive with very high optical density (OD). Also, the sera from mice neonatally infected with LCMV M1 strain were positive with slightly lower OD than adult mice. In contrast, all the sera of uninfected mice were negative to LCMV-NP antigen. Similarly, anti-LCMV antibodies were detected in all the sera of hamsters, mastomyses, and gerbils infected with the LCMV Armstrong strain. The results of the ELISA were in complete agreement with those of IFA, and indicate the high sensitivity and specificity of the ELISA system in the detection of anti-LCMV antibody. Because this ELISA system does not require handling infectious LCMV in the course of the antigen preparation and serological assay, there is no risk of contamination in the laboratory or nearby animal facility. In addition, by using negative control antigen in parallel with positive antigen in ELISA, we can exactly check the LCMV contamination in laboratory animals.     

Synthesis and biological activity of 1-methyl-tryptophan-tirapazamine hybrids as hypoxia-targeting indoleamine 2,3-dioxygenase inhibitors

We have designed and synthesized new hypoxic-neoplastic cells-targeted indoleamine 2,3-dioxygenase (IDO) inhibitors. 1-Methyl-tryptophan (1MT)-tirapazamine (TPZ, 3-amino-1,2,4-benzotriazine 1,4-dioxide) hybrid inhibitors including 1 (TX-2236), 2 (TX-2235), 3 (TX-2228), and 4 (TX-2234) were prepared. All of these compounds were uncompetitive IDO inhibitors. TPZ-monoxide hybrids 1 and 3 showed higher IDO inhibitory activities than TPZ hybrids 2 and 4. Among these hybrids, hybrid 1 was the most potent IDO inhibitor. TPZ hybrids 2 and 4 showed stronger hypoxia-selective cytotoxicity than TPZ to EMT6/KU cells. These data suggest that TPZ hybrids 2 and 4 may act through their dual biological functions: first, they function as hypoxic cytotoxins in hypoxic cells, and then are metabolized to their TPZ-monoxide (3-amino-1,2,4-benzotriazine 1-oxide) hybrids, which function as IDO inhibitors.     

High plasma norepinephrine attenuates the dynamic heart rate response to vagal stimulation

To better understand the pathophysiological significance of high plasma norepinephrine (NE) concentration in regulating heart rate (HR), we examined the interactions between high plasma NE and dynamic vagal control of HR. In anesthetized rabbits with sinoaortic denervation and vagotomy, using a binary white noise sequence (0-10 Hz) for 10 min, we stimulated the right vagus and estimated the transfer function from vagal stimulation to HR response. The transfer function approximated a first-order low-pass filter with pure delay. Infusion of NE (100 microg. kg(-1) x h(-1) iv) attenuated the dynamic gain from 6.2 +/- 0.8 to 3.9 +/- 1.2 beats x min(-1) x Hz(-1) (n = 7, P < 0.05) without affecting the corner frequency or pure delay. Simultaneous intravenous administration of phentolamine (1 mg x kg(-1) x h(-1)) and NE (100 microg x kg(-1) x h(-1)) abolished the inhibitory effect of NE on the dynamic gain (6.3 +/- 0.8 vs. 6.4 +/- 1.3 beats x min(-1) x Hz(-1), not significant, n = 7). The inhibitory effect of NE at infusion rates of 10, 50, and 100 microg x kg(-1) x h(-1) on dynamic vagal control of HR was dose-dependent (n = 5). In conclusion, high plasma NE attenuated the dynamic HR response to vagal stimulation, probably via activation of alpha-adrenergic receptors on the preganglionic and/or postganglionic cardiac vagal nerve terminals.     

Purification and characterization of an alpha-haloketone-resistant formate dehydrogenase from Thiobacillus sp. strain KNK65MA, and cloning of the gene

Thiobacillus sp. strain KNK65MA, which produced an NAD-dependent formate dehydrogenase (FDH) highly resistant to alpha-haloketones, was newly isolated, i.e., the enzyme showed no loss of activity after a 5-h incubation with alpha-haloketones, such as ethyl 4-chloro-3-oxobutanoate. The enzyme was also resistant to SH reagents. The enzyme, purified to homogeneity, was a dimer composed of identical subunits. The specific activity was 7.6 u/mg, and the apparent Km values for formate and NAD+ were 1.6 and 0.048 mM, respectively. The cloned gene of FDH contained one open reading frame (ORF) of 1206 base pairs, predicted to encode a polypeptide of 401 amino acids, with a calculated molecular weight of 44,021; this gene was highly expressed in E. coli cells. The deduced amino acid sequence of this FDH had high identity to other bacterial FDHs.     

A novel framework of circulatory equilibrium

A novel framework of circulatory equilibrium was developed by extending Guyton's original concept. In this framework, venous return (CO(V)) for a given stressed volume (V) was characterized by a flat surface as a function of right atrial pressure (P(RA)) and left atrial pressure (P(LA)) as follows: CO(V) = V/W - G(S)P(RA) - G(P)P(LA), where W, G(S), and G(P) denote linear parameters. In seven dogs under total heart bypass, CO(V), P(RA), P(LA), and V were varied to determine the three parameters in each animal with use of multivariate analysis. The coefficient of determination (r(2) = 0.92-0.99) indicated the flatness of the venous return surface. The averaged surface was CO(V) = V/0.129 - 19.61P(RA) - 3.49P(LA). To examine the invariability of the surface parameters among animals, we predicted the circulatory equilibrium in response to changes in stressed volume in another 12 dogs under normal and heart failure conditions. This was achieved by equating the standard surface with the individually measured cardiac output (CO) curve. In this way, we could predict CO [y = 0.90x + 5.6, r(2) = 0.95, standard error of the estimate (SEE) = 8.7 ml.min(-1).kg(-1)], P(RA) (y = 0.96x, r(2) = 0.98, SEE = 0.2 mmHg), and P(LA) (y = 0.89x + 0.5, r(2) = 0.98, SEE = 0.8 mmHg) reasonably well. We conclude that the venous return surface accurately represents the venous return properties of the systemic and pulmonary circulations. The characteristics of the venous return surface are invariable enough among animals, making it possible to predict circulatory equilibrium, even if those characteristics are unknown in individual animals.