Hypothalamic neuronal histamine modulates febrile response but not anorexia induced by lipopolysaccharide

This study examined the contribution of hypothalamic neuronal histamine (HA) to the anorectic and febrile responses induced by lipopolysaccharide (LPS), an exogenous pyrogen, and the endogenous pyrogens interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha). Intraperitoneal (ip) injection of LPS, IL-1beta, or TNF-alpha suppressed 24-hr cumulative food intake and increased rectal temperature in rats.To analyze the histaminergic contribution, rats were pretreated with intracerebroventricular (icv) injection of 2.44 mmol/kg or ip injection of 244 mmol/kg of alpha-fluoromethylhistidine (FMH), a suicide inhibitor of histidine decarboxylase (HDC), to deplete neural HA. The depletion of neural HA augmented the febrile response to ip injection of LPS and IL-1beta and alleviated the anorectic response to ip injection of IL-1beta. However, the depletion of neural HA did not modify the LPS-induced anorectic response or TNF-alpha-induced febrile and anorectic responses. Consistent with these results, the rate of hypothalamic HA turnover, assessed by the accumulation of tele-methylhistamine (t-MH), was elevated with ip injections of LPS and IL-1beta, but unaffected by TNF-alpha at equivalent doses. This suggests that (i) LPS and IL-1beta activate hypothalamic neural HA turnover; (ii) hypothalamic neural HA suppresses the LPS- and IL-1beta-induced febrile responses and accelerates the IL-1beta-induced anorectic response; and (iii) TNF-alpha modulates the febrile and anorectic responses via a neural HA-independent pathway. Therefore, hypothalamic neural HA is involved in the IL-1beta-dominant pathway, rather than the TNF-alpha-dominant pathway, preceding the systemic inflammatory response induced by exogenous pyrogens, such as LPS. Further research on this is needed.     

Autoregulation and homodimerization are involved in the activation of the plant steroid receptor BRI1

The leucine-rich-repeat receptor serine/threonine kinase, BRI1, is a cell-surface receptor for brassinosteroids (BRs), the steroid hormones of plants, yet its activation mechanism is unknown. Here, we report a unique autoregulatory mechanism of BRI1 activation. Removal of BRI1's C terminus leads to a hypersensitive receptor, indicated by suppression of dwarfism of BR-deficient and BR-perception mutants and by enhanced BR signaling as a result of elevated phosphorylation of BRI1. Several sites in the C-terminal region can be phosphorylated in vitro, and transgenic Arabidopsis expressing BRI1 mutated at these sites demonstrates an essential role of phosphorylation in BRI1 activation. BRI1 is a ligand-independent homo-oligomer, as evidenced by the transphosphorylation of BRI1 kinase in vitro, the dominant-negative effect of a kinase-inactive BRI1 in transgenic Arabidopsis, and coimmunoprecipitation experiments. Our results support a BRI1-activation model that involves inhibition of kinase activity by its C-terminal domain, which is relieved upon ligand binding to the extracellular domain.     

Protein expressed by the ho2 gene of the cyanobacterium Synechocystis sp. PCC 6803 is a true heme oxygenase. Properties of the heme and enzyme complex

Two isoforms of a heme oxygenase gene, ho1 and ho2, with 51% identity in amino acid sequence have been identified in the cyanobacterium Synechocystis sp. PCC 6803. Isoform-1, Syn HO-1, has been characterized, while isoform-2, Syn HO-2, has not. In this study, a full-length ho2 gene was cloned using synthetic DNA and Syn HO-2 was demonstrated to be highly expressed in Escherichia coli as a soluble, catalytically active protein. Like Syn HO-1, the purified Syn HO-2 bound hemin stoichiometrically to form a heme-enzyme complex and degraded heme to biliverdin IXalpha, CO and iron in the presence of reducing systems such as NADPH/ferredoxin reductase/ferredoxin and sodium ascorbate. The activity of Syn HO-2 was found to be comparable to that of Syn HO-1 by measuring the amount of bilirubin formed. In the reaction with hydrogen peroxide, Syn HO-2 converted heme to verdoheme. This shows that during the conversion of hemin to alpha-meso-hydroxyhemin, hydroperoxo species is the activated oxygen species as in other heme oxygenase reactions. The absorption spectrum of the hemin-Syn HO-2 complex at neutral pH showed a Soret band at 412 nm and two peaks at 540 nm and 575 nm, features observed in the hemin-Syn HO-1 complex at alkaline pH, suggesting that the major species of iron(III) heme iron at neutral pH is a hexa-coordinate low spin species. Electron paramagnetic resonance (EPR) revealed that the iron(III) complex was in dynamic equilibrium between low spin and high spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components. These observations suggest that the structure of the heme pocket of the Syn HO-2 is different from that of Syn HO-1.     

Hydrogen production from glucose by anaerobes

Various anaerobes were cultivated in media containing glucose. When 100 mL of thioglycollate medium containing 2.0% (w/v) glucose was used, Clostridium butyricum ATCC 859, NBRC 3315, and NBRC 13949 evolved 227-243 mL of biogas containing about 180 mL of hydrogen in 1 day. Although some strains had some resistance against oxygen, C. butyricum ATCC 859 and 860 did not have it. C. butyricum NBRC 3315 and Enterobacter aerogenes NBRC 13534 produced hydrogen in the presence of glucose or pyruvic acid, and E. aerogenes NBRC 13534 produced hydrogen by not only glucose and pyruvic acid but also dextrin, sucrose, maltose, galactose, fructose, mannose, and mannitol. When a medium containing 0.5% (w/v) yeast extract and 2.0% (w/v) glucose was used, E. aerogenes NBRC 13534 evolved more biogas and hydrogen than C. butyricum NBRC 3315 in the absence of reducing agent.     

Involvement of calpain in osteoclastic bone resorption

There is increasing evidence that calpain contributes to the reorganization of the cytoskeleton in the integrin-mediated signaling pathway. Osteoclastic bone resorption requires cell-matrix contact, an event mediated by integrin alphavbeta3, and subsequent cytoskeletal reorganization to form characteristic membrane domains such as the sealing zone and ruffled border. In this study, therefore, we investigated whether calpain is involved in osteoclastic bone resorption. Membrane-permeable calpain inhibitors suppress the resorption activity of human osteoclasts, but an impermeable inhibitor does not. Upon the attachment of osteoclasts to bone, micro-calpain is translocated from the cytosolic to the cytoskeletal fraction and is autolytically activated. Both the activation of micro-calpain and the formation of actin-rings, the cytoskeletal structures essential for bone resorption, are inhibited by membrane-permeable calpain inhibitors. The activated micro-calpain in osteoclasts selectively cleaves talin, which links the matrix-recognizing integrin to the actin cytoskeleton. These findings suggest that calpain is a regulator of the bone resorption activity of osteoclasts through reorganization of the cytoskeleton related to actin-ring formation.     

Effects of Y132H and F145L substitutions on the activity, azole resistance and spectral properties of Candida albicans sterol 14-demethylase P450 (CYP51): a live example showing the selection of altered P450 through interaction with environmental compounds

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds.     

Role of 14-3-3gamma in FE65-dependent gene transactivation mediated by the amyloid beta-protein precursor cytoplasmic fragment

The amyloid beta-protein precursor intracellular domain fragment (AICD) is generated from amyloid beta-protein precursor by consecutive cleavages. AICD is thought to activate FE65-dependent gene expression, but the molecular mechanism remains under consideration. We found that dimeric 14-3-3gamma bound both AICD and FE65 simultaneously, and this binding facilitated FE65-dependent gene transactivation by enhancing the association of AICD with FE65. 14-3-3gamma bound to the 667VTPEER672 motif of AICD and, most interestingly, the phosphorylation of AICD at Thr-668 in this motif inhibited the interaction with 14-3-3gamma and blocked gene transactivation. 14-3-3gamma required a sequence between the WW domain and the first phosphotyrosine interaction domain of FE65 for association with FE65. Deletion of this region blocked 14-3-3gamma binding to FE65 and suppressed AICD-mediated FE65-dependent gene transactivation, although the deletion mutant FE65 was still able to bind Tip60, a histone acetyltransferase that forms a complex with FE65 in the nucleus. Taken together, these data demonstrate that 14-3-3gamma facilitates FE65-dependent gene transactivation by forming a complex containing AICD and FE65, and phosphorylation of AICD down-regulates FE65-dependent gene transactivation through the dissociation of 14-3-3gamma and/or FE65 from AICD. Our findings suggest that multiple interactions of AICD with FE65 and 14-3-3gamma modulate FE65-dependent gene transactivation.     

ATP binding to nucleotide binding domain (NBD)1 of the ClpB chaperone induces motion of the long coiled-coil, stabilizes the hexamer, and activates NBD2

The molecular chaperone ClpB can rescue the heat-damaged proteins from an aggregated state in cooperation with other chaperones. It has two nucleotide binding domains (NBD1 and NBD2) and forms a hexamer ring in a manner dependent on ATP binding to NBD1. In the crystal structure of ClpB with both NBDs filled by nucleotides, the linker between two NBDs forms an 85-A-long coiled-coil that extends on the outside of the hexamer and leans to NBD1. To probe the possible motion of the coiled-coil, we tested the accessibility of a labeling reagent, fluorescence change of a labeled dye, and cross-linking between the coiled-coil and NBD1 by using the mutants with defective NBD1 or NBD2. The results suggest that the coiled-coil is more or less parallel to the main body of ClpB in the absence of nucleotide and that ATP binding to NBD1 brings it to the leaning position as seen in the crystal structure. This motion results in stabilization of the hexamer form of ClpB and promotion of ATP hydrolysis at NBD2.