Identification of beta-tubulin isoform V as an autoantigen in allergic rhinitis by a proteomic approach

Autoantibodies to IgE and beta2-adrenergic receptor have been reported in patients with allergic rhinitis. To investigate whether autoimmunity in allergic rhinitis is directed to such limited molecules or directed to a wide range of self proteins, we here attempted to survey autoantigens/autoantibodies comprehensively, using proteomics. Specifically, we separated proteins extracted from peripheral blood mononuclear cells by 2-dimensional electrophoresis and then detected autoantigens by subsequent western blotting with sera from patients with allergic rhinitis. As a result, we detected multiple autoantigens, some of which were further identified by mass fingerprinting. Next, we confirmed antigenicity of one of the identified autoantigens, beta-tubulin isoform V (beta-tubV), using a recombinant protein and then measured prevalence of the anti-beta-tubV autoantibodies. As a result, 52% of the tested patients with allergic rhinitis were found to possess anti-beta-tubV autoantibodies. Our study indicates that autoimmunity is a common phenomena and beta-tubV is one of the major autoantigens in allergic rhinitis.     

Synthesis and evaluation of 6-methylene-bridged uracil derivatives. Part 2: optimization of inhibitors of human thymidine phosphorylase and their selectivity with uridine phosphorylase

A series of novel 6-methylene-bridged uracil derivatives have been optimized for clinical use as the inhibitors of human thymidine phosphorylase (TP). We describe their synthesis and evaluation. Introduction of a guanidino or an amidino group enhanced the in vitro inhibitory activity of TP comparing with formerly reported inhibitor 1. Their selectivity for TP based on uridine phosphorylase inhibitory activity was also evaluated. Compound 2 (TPI) has been selected for clinical evaluation based on its strong TP inhibition and excellent modulation of 2'-deoxy-5-(trifluoromethyl)uridine (F(3)dThd) pharmacokinetics. As a result, TAS-102 (a combination of F(3)dThd and TPI) is currently in phase 1 clinical studies.     

Expression of death-associated protein kinase and recruitment to the tumor necrosis factor signaling pathway following brief seizures

Death-associated protein (DAP) kinase is calcium-regulated and known to function downstream of death receptors, prompting us to examine its role in the mechanism of seizure-induced neuronal death. Brief seizures were focally evoked in rats, eliciting neuronal death within the CA3 subfield of the hippocampus, and to a lesser extent, cortex. Western blotting confirmed expression of DAP kinase within hippocampus and cortex at the predicted weight of approximately 160 kDa. Immunohistochemistry revealed seizures triggered a significant increase in numbers of DAP kinase-expressing cells within CA3 and cortex, without affecting cell counts within seizure-resistant CA2 or the dentate gyrus. Numbers of DAP kinase-expressing cells were increased in relation to specific patterns of injury-causing seizure activity, electrographically defined. Seizures caused an early increase in DAP kinase binding to actin, and association with calmodulin. Co-immunoprecipitation studies also revealed seizures triggered binding of DAP kinase to the tumor necrosis factor receptor 1 and the Fas-associated death domain protein, commensurate with caspase-8 proteolysis. In contrast, within surviving fields of the hippocampus, DAP kinase interacted with the molecular chaperone 14-3-3. These data suggest DAP kinase is involved in the molecular pathways activated during seizure-induced neuronal death.     

Oxidative stress induces nuclear loss of DNA repair proteins Ku70 and Ku80 and apoptosis in pancreatic acinar AR42J cells

Cell death linked to oxidative DNA damage has been implicated in acute pancreatitis. The severe DNA damage, which is beyond the capacity of the DNA repair proteins, triggers apoptosis. It has been hypothesized that oxidative stress may induce a decrease in the Ku70 and Ku80 levels and apoptosis in pancreatic acinar cells. In this study, it was found that oxidative stress caused by glucose oxidase (GO) acting on beta-d-glucose, glucose/glucose oxidase (G/GO), induced slight changes in cytoplasmic Ku70 and Ku80 but drastically induced a decrease in nuclear Ku70 and Ku80 both time- and concentration-dependently in AR42J cells. G/GO induced apoptosis determined by poly(ADP-ribose) polymerase cleavage, an increase in expression of p53 and Bax, and a decrease in Bcl-2 expression. G/GO-induced apoptosis was in parallel with the loss of nuclear Ku proteins in AR42J cells. Caspase-3 inhibitor prevented G/GO-induced nuclear Ku loss and cell death. G/GO did not induce apoptosis in the cells transfected with either the Ku70 or Ku80 expression gene but increased apoptosis in those transfected with the Ku dominant negative mutant. Pulse and pulse-chase results show that G/GO induced Ku70 and Ku80 syntheses, even though Ku70 and Ku80 were degraded both in cytoplasm and nucleus. G/GO-induced decrease in Ku binding to importin alpha and importin beta reflects possible modification of nuclear import of Ku proteins. The importin beta level was not changed by G/GO. These results demonstrate that nuclear decrease in Ku70 and Ku80 may result from the decrease in Ku binding to nuclear transporter importins and the degradation of Ku proteins. The nuclear loss of Ku proteins may underlie the mechanism of apoptosis in pancreatic acinar cells after oxidative stress.     

Age and anesthetic effects on murine electrocardiography

Murine models offer potential insights regarding human cardiac disease, but efficient and reliable methods for phenotype evaluation are necessary. We employed non-invasive electrocardiography (ECG) in mice, investigating statistical reliability of these parameters with respect to anesthetic and animal age. Mice (C57BL/6, 8 or 48 weeks) were anesthetized by ketamine/xylazine (K/X, 80/10 mg/kg ip) or by inhalation anesthetic (halothane, HAL; sevoflurane, SEV) and 6 lead ECGs were recorded. P wave duration and QT interval was significantly prolonged with K/X compared to HAL and SEV, indicating slowed atrial and ventricular conduction. P-R interval (atrio-ventricular conduction) was significantly increased in aged mice under all anesthetics. Heart rate was inversely correlated to QT interval and P wave duration. We also detected significant age effects with respect to optimal approaches for QT interval corrections. Power analysis showed 4-fold higher number of mice/group, were required for K/X, to achieve identical statistical sensitivity. These data demonstrate the importance of anesthetic selection for relevant and reliable ECG analysis in mice and illustrate the selective influences of anesthetics and age on cardiac conductance in this species.     

An anhydrous polymorphic form of trehalose

An anhydrous polymorphic form of alpha,alpha-trehalose was prepared from trehalose dihydrate by two different drying methods: (1) heating under vacuum; and (2) heating in hot air. Preparation of this anhydrous form by vacuum heating showed good reproducibility. This form was characterized by X-ray powder diffraction analysis and differential scanning calorimetry. This anhydrous form was converted to an amorphous phase at 127 degrees C and was found to be hygroscopic. At 43% relative humidity at 25 degrees C, this form rapidly reverted to dihydrate, while the amorphous phase remained unchanged. When an amorphous phase coexisted with this form, the rate of water adsorption to the amorphous phase was slower than that to the amorphous phase alone. These properties of this anhydrous form of alpha,alpha-trehalose may explain the effects of trehalose in dehydration tolerance of plants and insects in the desert.     

Accumulation of inorganic polyphosphate in phoU mutants of Escherichia coli and Synechocystis sp. strain PCC6803

The biological process for phosphate (P(i)) removal is based on the use of bacteria capable of accumulating inorganic polyphosphate (polyP). We obtained Escherichia coli mutants which accumulate a large amount of polyP. The polyP accumulation in these mutants was ascribed to a mutation of the phoU gene that encodes a negative regulator of the P(i) regulon. Insertional inactivation of the phoU gene also elevated the intracellular level of polyP in Synechocystis sp. strain PCC6803. The mutant could remove fourfold more P(i) from the medium than the wild-type strain removed.     

Reduced expression of STAT4 and IFN-gamma in macrophages from BALB/c mice

BALB/c mice have been shown to easily induce Th2 type responses in several infection models. In this study, to examine the mechanisms of Th2 dominant responses in BALB/c mice, we assessed several macrophage functions using C3H/HeN, C57BL/6, and BALB/c mouse strains. Peritoneal macrophages from three strains of mice equally produced IL-12 by stimulation with LPS plus IFN-gamma. However, IFN-gamma production in response to IL-12 or IL-12 plus IL-18 was much lower in macrophages from BALB/c mice than other strains. IFN-gamma produced by activated macrophages induced IL-12R mRNA expression in T cells and macrophages themselves depending on their amount of IFN-gamma; namely, macrophages from BALB/c mice induced lower expression of IL-12R. Intracellular levels of STAT4 were much lower in macrophages from BALB/c mice. However, other STATs, such as STAT1 or STAT6, were expressed similarly in the three mouse strains. STAT4 and IFN-gamma production by other cell types such as T cells and B cells were equal in C3H/HeN and BALB/c mice. These results indicate that macrophages from Th2-dominant BALB/c mice have different functional characters compared with other mouse strains; that is, STAT4 expression and IFN-gamma production are reduced, which is one of the causes to shift to Th2-type responses.     

How Tlg2p/syntaxin 16 'snares' Vps45

Soluble N-ethylmaleimide sensitive factor-attachment protein receptors (SNAREs) and Sec1p/Munc18-homologs (SM proteins) play key roles in intracellular membrane fusion. The SNAREs form tight four-helix bundles (core complexes) that bring the membranes together, but it is unclear how this activity is coupled to SM protein function. Studies of the yeast trans-Golgi network (TGN)/endosomal SNARE complex, which includes the syntaxin-like SNARE Tlg2p, have suggested that its assembly requires activation by binding of the SM protein Vps45p to the cytoplasmic region of Tlg2p folded into a closed conformation. Nuclear magnetic resonance and biochemical experiments now show that Tlg2p and Pep12p, a late- endosomal syntaxin that interacts functionally but not directly with Vps45p, have a domain structure characteristic of syntaxins but do not adopt a closed conformation. Tlg2p binds tightly to Vps45p via a short N-terminal peptide motif that is absent in Pep12p. The Tlg2p/Vps45p binding mode is shared by the mammalian syntaxin 16, confirming that it is a Tlg2p homolog, and resembles the mode of interaction between the SM protein Sly1p and the syntaxins Ufe1p and Sed5p. Thus, this mechanism represents the most widespread mode of coupling between syntaxins and SM proteins.