State-dependent blocking actions of azimilide dihydrochlo-ride (NE-10064) on human cardiac Na(+) channels.

BACKGROUND: Azimilide reportedly blocks Na(+) channels, although its mechanism remains unclear. METHODS AND RESULTS: The kinetic properties of the azimilide block of the wild-type human Na(+) channels (WT: hH1) and mutant DeltaKPQ Na(+) channels (DeltaKPQ) expressed in COS7 cells were investigated using the whole-cell patch clamp technique and a Markovian state model. Azimilide induced tonic block of WT currents by shifting the h infinity curve in the hyperpolarizing direction and caused phasic block of WT currents with intermediate recovery time constant. The peak and steady-state DeltaKPQ currents were blocked by azimilide, although with only a slight shift in the h infinity curve. The phasic block of peak and steady-state DeltaKPQ currents by azimilide was significantly larger than the blocking of the peak WT current. The affinity of azimilide predicted by a Markovian state model was higher for both the activated state (Kd(A) =1.4 micromol/L), and the inactivated state (Kd(I) =1.4 micromol/L), of WT Na(+) channels than that for the resting state (Kd(R) =102.6 micromol/L). CONCLUSIONS: These experimental and simulation studies suggest that azimilide blocks the human cardiac Na(+) channel in both the activated and inactivated states.     

Anti-fibrillogenic and fibril-destabilizing activities of anti-Parkinsonian agents for alpha-synuclein fibrils in vitro

The aggregation of alpha-synuclein (alphaS) in the brain has been implicated as a critical step in the development of Lewy body diseases (LBD) and multiple system atrophy (MSA). Among the antioxidant strategies proposed, increasing evidence points to the possibility of achieving neuroprotection by dopamine agonists, as well as monoamine oxidase B inhibitors. We showed previously that the anti-Parkinsonian agents dose-dependently inhibited beta-amyloid fibrils (fAbeta)(1-40) and fAbeta(1-42) formation as well as destabilized preformed fAbetas. Using fluorescence spectroscopy with thioflavin S, electron microscopy, and atomic force microscopy, we examined the effects of anti-Parkinsonian agents, selegiline, dopamine, pergolide, bromocriptine, and trihexyphenidyl on the formation of alphaS fibrils (falphaS) and on preformed falphaS. All molecules except for trihexyphenidyl, dose-dependently inhibited the formation of falphaS. Moreover, these molecules dose-dependently destabilized preformed falphaS. The overall activity of the molecules examined was in the order of: selegiline = dopamine > pergolide > bromocriptine. These agents and other compounds related structurally could be key molecules for the development of therapeutics for LBD and MSA.     

Changes in deviation of absorbed dose to water among users by chamber calibration shift

Purpose

The JSMP01 dosimetry protocol had adopted the provisional ⁶⁰Co calibration coefficient \(N_{{{\text{D,w,Q}}_{ 0} }}^{{}}\), namely, the product of exposure calibration coefficient N _C and conversion coefficient k _D,X. After that, the absorbed dose to water D _w standard was established, and the JSMP12 protocol adopted the \(N_{{{\text{D,w,Q}}_{ 0} }}^{{}}\) calibration. In this study, the influence of the calibration shift on the measurement of D _w among users was analyzed.

Materials and methods

The intercomparison of the D _w using an ionization chamber was annually performed by visiting related hospitals. Intercomparison results before and after the calibration shift were analyzed, the deviation of D _w among users was re-evaluated, and the cause of deviation was estimated.

Results

As a result, the stability of LINAC, calibration of the thermometer and barometer, and collection method of ion recombination were confirmed. The statistical significance of standard deviation of D _w was not observed, but that of difference of D _w among users was observed between N _C and \(N_{{{\text{D,w,Q}}_{ 0} }}^{{}}\) calibration.

Conclusion

Uncertainty due to chamber-to-chamber variation was reduced by the calibration shift, consequently reducing the uncertainty among users regarding D _w. The result also pointed out uncertainty might be reduced by accurate and detailed instructions on the setup of an ionization chamber.

     

Relationship between plasma total homocysteine level and dietary caffeine and vitamin B6 intakes in pregnant women

A high total homocysteine (tHcy) level during pregnancy is a risk factor for adverse perinatal outcomes, such as fetal growth restriction and preeclampsia. Caffeine is assumed to increase tHcy levels by acting as a vitamin B₆ antagonist. The objective of this study was to examine a relationship between circulating tHcy levels and dietary caffeine and vitamin B₆ intakes in pregnant Japanese women. A total of 321 healthy women with singleton pregnancies were recruited in metropolitan Tokyo, from June to December 2008, resulting in the final number included in the study as 254. Dietary caffeine intakes did not correlate with plasma tHcy levels. When we analyzed the data according to caffeinated beverages, caffeinated tea consumption was positively associated with plasma tHcy levels only among the women with a high intake of vitamin B₆, after controlling for confounding factors (P = 0.029). No correlation between coffee consumption and plasma tHcy levels was found. Pregnant Japanese women might need to cut down the consumption of caffeinated tea as well as take sufficient vitamin B₆ in order to prevent the tHcy levels from increasing.     

Venous resistance increases during rat anaphylactic shock

Anaphylactic shock is a sudden, life-threatening allergic reaction associated with severe hypotension. The increased venous resistance accounts for the anaphylactic hypotension in anesthetized dogs. However, the change in peripheral vascular resistances during anaphylactic hypotension in other animals such as rats is not known. We measured the mean circulatory filling pressure using the mechanical occlusion method of inflation of the right atrial balloon along with systemic arterial pressure (Psa), central venous pressure, and portal venous pressure. Cardiac output was also measured with the thermodilution method. From these hemodynamic variables, we calculated the total peripheral and venous (Rv) resistances during anaphylactic hypotension in anesthetized rats. These hemodynamic variables were compared with those in the hemorrhagic shock. After an intravenous injection of 0.6 mg antigen ovalbumin in sensitized rats, Psa decreased from 119 +/- 4 to 43 +/- 2 mmHg, cardiac output decreased from 84.5 +/- 5.7 to 37.8 +/- 2.1 mL min, central venous pressure decreased from 0.9 +/- 0.1 to 0.1 +/- 0.1 mmHg, and mean circulatory filling pressure also decreased from 6.0 +/- 0.2 to 5.2 +/- 0.3 mmHg. Thus, the Rv increased from 0.06 +/- 0.05 to 0.15 +/- 0.02 mmHg mL(-1) min(-1), but total peripheral resistance did not significantly change. Portal venous pressure also increased from 5.6 +/- 0.5 to 21.5 +/- 0.9 mmHg. Hematocrit markedly increased from the baseline values of 43% +/- 1% to 55% +/- 1% at 15 min after antigen. During hemorrhagic shock, Psa decreased in the manner similar to anaphylactic shock; however, Rv did not significantly change, and portal venous pressure decreased. In conclusion, in rat anaphylactic shock, a substantial increase in Rv presumably due to hepatic venoconstriction may decrease venous return, resulting in systemic hypotension.     

Induction of cross‐protective immunity against influenza A virus H5N1 by an intranasal vaccine with extracts of mushroom mycelia

The identification of a safe and effective adjuvant that is able to enhance mucosal immune responses is necessary for the development of an efficient inactivated intranasal influenza vaccine. The present study demonstrated the effectiveness of extracts of mycelia derived from edible mushrooms as adjuvants for intranasal influenza vaccine. The adjuvant effect of extracts of mycelia was examined by intranasal co‐administration of the extracts and inactivated A/PR8 (H1N1) influenza virus hemagglutinin (HA) vaccine in BALB/c mice. The inactivated vaccine in combination with mycelial extracts induced a high anti‐A/PR8 HA‐specific IgA and IgG response in nasal washings and serum, respectively. Virus‐specific cytotoxic T‐lymphocyte responses were also induced by administration of the vaccine with extract of mycelia, resulting in protection against lethal lung infection with influenza virus A/PR8. In addition, intranasal administration of NIBRG14 vaccine derived from the influenza A/Vietnam/1194/2004 (H5N1) virus strain administered in conjunction with mycelial extracts from Phellinus linteus conferred cross‐protection against heterologous influenza A/Indonesia/6/2005 virus challenge in the nasal infection model. In addition, mycelial extracts induced proinflammatory cytokines and CD40 expression in bone marrow‐derived dendritic cells. These results suggest that mycelial extract‐adjuvanted vaccines can confer cross‐protection against variant H5N1 influenza viruses. The use of extracts of mycelia derived from edible mushrooms is proposed as a new safe and effective mucosal adjuvant for use for nasal vaccination against influenza virus infection. J. Med. Virol. 82:128–137, 2010. © 2009 Wiley‐Liss, Inc.     

Intranasal administration of adjuvant‐combined vaccine protects monkeys from challenge with the highly pathogenic influenza A H5N1 virus

The effectiveness in cynomolgus macaques of intranasal administration of an influenza A H5N1 pre‐pandemic vaccine combined with synthetic double‐stranded RNA (polyI/polyC12U) as an adjuvant was examined. The monkeys were immunized with the adjuvant‐combined vaccine on weeks 0, 3, and 5, and challenged with the homologous virus 2 weeks after the third immunization. After the second immunization, the immunization induced vaccine‐specific salivary IgA and serum IgG antibodies, as detected by ELISA. The serum IgG antibodies present 2 weeks after the third immunization not only had high neutralizing activity against the homologous virus, they also neutralized significantly heterologous influenza A H5N1 viruses. The vaccinated animals were protected completely from the challenge infection with the homologous virus. These results suggest that intranasal immunization with the Double stranded RNA‐combined influenza A H5N1 vaccine induce mucosal IgA and serum IgG antibodies which could protect humans from homologous influenza A H5N1 viruses which have a pandemic potential. J. Med. Virol. 82:1754–1761, 2010. © 2010 Wiley‐Liss, Inc.     

Analysis of linear B-cell epitopes of the nucleoprotein of ebola virus that distinguish ebola virus subtypes

Ebola virus consists of four genetically distinguishable subtypes. We developed monoclonal antibodies (MAbs) to the nucleoprotein (NP) of Ebola virus Zaire subtype and analyzed their cross-reactivities to the Reston and Sudan subtypes. We further determined the epitopes recognized by these MAbs. Three MAbs reacted with the three major subtypes and recognized a fragment containing 110 amino acids (aa) at the C-terminal extremity. They did not show specific reactivities to any 10-aa short peptides in Pepscan analyses, suggesting that these MAbs recognize conformational epitope(s) located within this region. Six MAbs recognized a fragment corresponding to aa 361 to 461 of the NP. Five of these six MAbs showed specific reactivities in Pepscan analyses, and the epitopes were identified in two regions, aa 424 to 430 and aa 451 to 455. Three MAbs that recognized the former epitope region cross-reacted with all three subtypes, and one that recognized the same epitope region was Zaire specific. One MAb, which recognized the latter epitope region, was reactive with Zaire and Sudan subtypes but not with the Reston subtype. These results suggest that Ebola virus NP has at least two linear epitope regions and that the recognition of the epitope by MAbs can vary even within the same epitope region. These MAbs showing different subtype specificities might be useful reagents for developing an immunological system to identify Ebola virus subtypes.     

Brain-derived neurotrophic factor-mediated retrograde signaling required for the induction of long-term potentiation at inhibitory synapses of visual cortical pyramidal neurons

High-frequency stimulation (HFS) induces long-term potentiation (LTP) at inhibitory synapses of layer 5 pyramidal neurons in developing rat visual cortex. This LTP requires postsynaptic Ca²⁺ rise for induction, while the maintenance mechanism is present at the presynaptic site, suggesting presynaptic LTP expression and the necessity of retrograde signaling. We investigated whether the supposed signal is mediated by brain-derived neurotrophic factor (BDNF), which is expressed in pyramidal neurons but not inhibitory interneurons. LTP did not occur when HFS was applied in the presence of the Trk receptor tyrosine kinase inhibitor K252a in the perfusion medium. HFS produced LTP when bath application of K252a was started after HFS or when K252a was loaded into postsynaptic cells. LTP did not occur in the presence of TrkB-IgG scavenging BDNF or function-blocking anti-BDNF antibody in the medium. In cells loaded with the Ca²⁺ chelator BAPTA, the addition of BDNF to the medium enabled HFS to induce LTP without affecting baseline synaptic transmission. These results suggest that BDNF released from postsynaptic cells activates presynaptic TrkB, leading to LTP. Because BDNF, expressed activity dependently, regulates the maturation of cortical inhibition, inhibitory LTP may contribute to this developmental process, and hence experience-dependent functional maturation of visual cortex.