Voltage-gated Ca2+ channels in accessory lobe neurons of the chick

Birds have ten pairs of protrusions, “accessory lobes”, on the lateral sides of the lumbosacral spinal cord. It has been proposed that accessory lobes act as a sensory organ of equilibrium and neurons in accessory lobes transmit sensory information to the motor center. We have reported that cells in chick accessory lobes express functional voltage-gated Na⁺ and K⁺ channels and generate action potentials. In this study, we examined properties of voltage-gated Ca²⁺ channels (VGCCs). The amplitude of voltage-gated Ca²⁺ channel currents carried by Ca²⁺ and Ba²⁺ increased gradually during 10 min rather than showing the usual run-down. The current–voltage relationship of Ba²⁺ currents was consistent with that of the high-voltage-activated Ca²⁺ channel. The proportion of Ba²⁺ currents inhibited by ω-conotoxin GVIA was larger than 80 %, indicating that the major subtype is N type. Amplitudes of tail currents of Ca²⁺ currents evoked by repetitive pulses at 50 Hz are stable for 1 s. If the major subtype of VGCCs at synaptic terminals is also N type, this property may contribute to the establishment of stable synaptic connections between accessory lobe neurons, which are reported to fire at frequencies higher than 15 Hz, and postsynaptic neurons in the spinal cord.     

Egg yolk phosvitin inhibits hydroxyl radical formation from the fenton reaction

Phosvitin, a phosphoprotein known as an iron-carrier in egg yolk, binds almost all the yolk iron. In this study, we investigated the effect of phosvitin on Fe(II)-catalyzed hydroxyl radical ((.-)OH) formation from H(2)O(2) in the Fenton reaction system. Using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) and deoxyribose degradation assays, we observed by both assays that phosvitin more effectively inhibited (.-)OH formation than iron-binding proteins such as ferritin and transferrin. The effectiveness of phosvitin was related to the iron concentration, indicating that phosvitin acts as an antioxidant by chelating iron ions. Phosvitin accelerates Fe(II) autoxidation and thus decreases the availability of Fe(II) for participation in the (.-)OH-generating Fenton reaction. Furthermore, using the plasmid DNA strand breakage assay, phosvitin protected DNA against oxidative damage induced by Fe(II) and H(2)O(2). These results provide insight into the mechanism of protection of the developing embryo against iron-dependent oxidative damage in ovo.     

Production of a recombinant cholera toxin B subunit-insulin B chain peptide hybrid protein by Brevibacillus choshinensis expression system as a nasal vaccine against autoimmune diabetes

Mucosally induced tolerance is an attractive strategy for preventing or reducing autoimmune diseases. Here, we produced a recombinant CTB fusion protein linked with autoantigen T cell epitope of insulin B chain peptide 9-23 (C19S) at levels up to 200 mg/L culture media in Brevibacillus choshinensis secretion-expression system. Receptor-competitive assay showed that the CTB-insulin peptide binds to GM1 receptor almost equivalent degree as the native form of CTB. Non-obese diabetes (NOD) mice that spontaneously develop an insulin-dependent diabetes were nasally immunized with CTB-insulin peptide (5 microg) for three times. The nasal treatment significantly reduced the development of insulin-dependent diabetes and peptide specific DTH responses after systemic immunization with the insulin peptide B 9-23(C19S) in CFA. Nasal administration of as high as 50 microg of the peptide alone demonstrated a similar level of the disease inhibition. In contrast, all mice given 5 microg of the insulin peptide alone or 5 microg of insulin peptide with 25 microg of the free form of CTB did not lead to the suppression of diabetes development and DTH responses. Because molecular weight of the insulin peptide is about one tenth of that of the CTB-insulin peptide, the results demonstrate that the recombinant hybrid of autoantigen and CTB increased its tolerogenic potential for nasal administration by up 100-fold on molar base of autoantigen peptide. Taken together, nasally-induced tolerance by administration of the recombinant B. choshinensis-derived hybrid protein of CTB and autoantigen T cell-epitope peptide could be useful mucosal immunetherapy for the control of T cell-mediated autoimmune diseases.     

Overexpression of chitinase 3-like 1/YKL-40 in lung-specific IL-18-transgenic mice, smokers and COPD

We analyzed the lung mRNA expression profiles of a murine model of COPD developed using a lung-specific IL-18-transgenic mouse. In this transgenic mouse, the expression of 608 genes was found to vary more than 2-fold in comparison with control WT mice, and was clustered into 4 groups. The expression of 140 genes was constitutively increased at all ages, 215 genes increased gradually with aging, 171 genes decreased gradually with aging, and 82 genes decreased temporarily at 9 weeks of age. Interestingly, the levels of mRNA for the chitinase-related genes chitinase 3-like 1 (Chi3l1), Chi3l3, and acidic mammalian chitinase (AMCase) were significantly higher in the lungs of transgenic mice than in control mice. The level of Chi3l1 protein increased significantly with aging in the lungs and sera of IL-18 transgenic, but not WT mice. Previous studies have suggested Chi3l3 and AMCase are IL-13-driven chitinase-like proteins. However, IL-13 gene deletion did not reduce the level of Chi3l1 protein in the lungs of IL-18 transgenic mice. Based on our murine model gene expression data, we analyzed the protein level of YKL-40, the human homolog of Chi3l1, in sera of smokers and COPD patients. Sixteen COPD patients had undergone high resolution computed tomography (HRCT) examination. Emphysema was assessed by using a density mask with a cutoff of -950 Hounsfield units to calculate the low-attenuation area percentage (LAA%). We observed significantly higher serum levels in samples from 28 smokers and 45 COPD patients compared to 30 non-smokers. In COPD patients, there was a significant negative correlation between serum level of YKL-40 and %FEV(1). Moreover, there was a significant positive correlation between the serum levels of YKL-40 and LAA% in COPD patients. Thus our results suggest that chitinase-related genes may play an important role in establishing pulmonary inflammation and emphysematous changes in smokers and COPD patients.     

Autism-associated gene expression in peripheral leucocytes commonly observed between subjects with autism and healthy women having autistic children

Autism spectrum disorder (ASD) is a severe neuropsychiatric disorder which has complex pathobiology with profound influences of genetic factors in its development. Although the numerous autism susceptible genes were identified, the etiology of autism is not fully explained. Using DNA microarray, we examined gene expression profiling in peripheral blood from 21 individuals in each of the four groups; young adults with ASD, age- and gender-matched healthy subjects (ASD control), healthy mothers having children with ASD (asdMO), and asdMO control. There was no blood relationship between ASD and asdMO. Comparing the ASD group with control, 19 genes were found to be significantly changed. These genes were mainly involved in cell morphology, cellular assembly and organization, and nerve system development and function. In addition, the asdMO group possessed a unique gene expression signature shown as significant alterations of protein synthesis despite of their nonautistic diagnostic status. Moreover, an ASD-associated gene expression signature was commonly observed in both individuals with ASD and asdMO. This unique gene expression profiling detected in peripheral leukocytes from affected subjects with ASD and unaffected mothers having ASD children suggest that a genetic predisposition to ASD may be detectable even in peripheral cells. Altered expression of several autism candidate genes such as FMR-1 and MECP2, could be detected in leukocytes. Taken together, these findings suggest that the ASD-associated genes identified in leukocytes are informative to explore the genetic, epigenetic, and environmental background of ASD and might become potential tools to assess the crucial factors related to the clinical onset of the disorder.     

Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.     

Kinematic analysis of the technique for elite male long-distance speed skaters in curving

The purpose of this study was to investigate technical factors for maintaining skating velocity by kinematic analysis of the skating motion for elite long-distance skaters during the curve phase in official championship races. Sixteen world-class elite male skaters who participated in the 5,000-m race were videotaped with two synchronized high-speed video cameras (250 Hz) in a curve lane by using a panning DLT technique. Three-dimensional coordinates of the body and blades during the first and second halves of the races were collected to calculate kinematic parameters. In the group that maintained greater skating velocity, the thigh angle during the gliding phase of the left stroke during the second half was greater than that during the first half, and the center of mass was located more forward during the second half. Thus, it was suggested that long-distance speed skaters should change the support leg position during the gliding phase in the left stroke of the curve phase under fatigued conditions so that they could extend the support leg with a forward rotation of the thigh and less shank backward rotation.     

Vesicular transport system in myotubes: ultrastructural study and signposting with vesicle-associated membrane proteins

Myofibers have characteristic membrane compartments in their cytoplasm and sarcolemma, such as the sarcoplasmic reticulum, T-tubules, neuromuscular junction, and myotendinous junction. Little is known about the vesicular transport that is believed to mediate the development of these membrane compartments. We determined the locations of organelles in differentiating myotubes. Electron microscopic observation of a whole myotube revealed the arrangement of Golgi apparatus, rough endoplasmic reticulum, autolysosomes, mitochondria, and smooth endoplasmic reticulum from the perinuclear region toward the end of myotubes and the existence of a large number of vesicles near the ends of myotubes. Vesicles in myotubes were further characterized using immunofluorescence microscopy to analyze expression and localization of vesicle-associated membrane proteins (VAMPs). VAMPs are a family of seven proteins that regulate post-Golgi vesicular transport via the fusion of vesicles to the target membranes. Myotubes express five VAMPs in total. Vesicles with VAMP2, VAMP3, or VAMP5 were found near the ends of the myotubes. Some of these vesicles are also positive for caveolin-3, suggesting their participation in the development of T-tubules. Our morphological analyses revealed the characteristic arrangement of organelles in myotubes and the existence of transport vesicles near the ends of the myotubes.     

Dose-dependent differential regulation of cytokine secretion from macrophages by fractalkine

Although expression of the fractalkine (CX3CL1, FKN) is enhanced in inflamed tissues, it is detected at steady state in various organs such as the intestine, and its receptor CX3CR1 is highly expressed in resident-type dendritic cells and macrophages. We hypothesized that FKN might regulate the inflammatory responses of these cells. Therefore, murine macrophages were pretreated with FKN and then stimulated with LPS. We found that macrophages pretreated with 0.03 nM FKN but not with 3 nM FKN secreted 50% less TNF-alpha than did cells treated with LPS alone. Cells treated with 0.03 nM FKN and LPS also showed reduced phosphorylation of ERK1/2 and reduced NF-kappaB p50 subunit. Interestingly, the p65 subunit of NF-kappaB was translocated to the nuclei but redistributed to the cytoplasm in the early phase by forming a complex with peroxisome proliferator-activated receptor (PPAR) gamma. Exogenous 15-deoxy-Delta(12,14)-prostaglandin J2, a natural ligand for PPAR-gamma, also induced redistribution of p65 with decreased TNF-alpha secretion after LPS challenge. Pretreatment with 0.03 nM but not 3 nM FKN increased the cellular levels of 15-deoxy-Delta(12,14)-prostaglandin J2 as well as mRNA of PPAR-gamma. Requirement of PPAR-gamma for the effect of 0.03 nM FKN was confirmed by small interfering RNA of PPAR-gamma. In contrast, pretreatment with 3 nM FKN induced higher levels of IL-23 compared with cells pretreated with 0.03 nM FKN and produced TNF-alpha in a CX3CR1-dependent manner. These dose-dependent differential effects of FKN establish its novel role in immune homeostasis and inflammation.