Simultaneous inhibition of endogenous avidin-binding activity and peroxidase applicable for the avidin-biotin system using monoclonal antibodies

Summary The use of the avidin-biotin technique in immunoperoxidase staining provides a simple and highly sensitive method for detecting the localization of antigens defined by monoclonal antibodies. However, endogenous biotin, which is widely distributed in tissues, often causes nonspecific staining by binding to avidin [endogenous avidin-binding activity (EABA)]. Endogenous peroxidase activity (EPA) also makes the estimation of specific staining difficult. In the present study, several methods for the inhibition of EABA and/or EPA were examined using the avidinbiotin technique and monoclonal antibodies against murine Mac-1 and Ia antigen. Of these, the overnight incubation of sections in 40% methanol in phosphate-buffered saline containing 0.3% hydrogen peroxide gave the best result, as it inhibited EABA and EPA simultaneously without denaturating of the antigenic determinants recognized by the monoclonal antibodies.     

UDP-induced relaxation is enhanced in aorta from female obese Otsuka Long–Evans Tokushima Fatty rats

Uridine 5′-diphosphate (UDP) plays an important role in controlling vascular tone; however, UDP-mediated response in metabolic syndromes, including obesity and type 2 diabetes in females, remains unclear. In this study, we investigated UDP-mediated response in the aorta of female obese Otsuka Long–Evans Tokushima Fatty (OLETF) rats and control Long–Evans Tokushima Otsuka (LETO) rats. In OLETF rat aortas precontracted by phenylephrine (PE) (vs. LETO), (1) UDP-induced relaxation was increased, whereas acetylcholine (ACh)-induced relaxation was decreased; (2) no UDP- or ACh-induced relaxations were observed in endothelial denudation, whereas UDP-induced small contraction was observed; and (3) N^G-nitro-L-arginine [L-NNA, a nitric oxide (NO) synthase inhibitor] eliminated UDP-induced relaxation and small contraction, whereas caused contrasting responses by ACh, including slight relaxations (LETO) and contractions (OLETF). Indomethacin, a cyclooxygenase inhibitor, eliminated the difference in UDP- and ACh-induced relaxations between the groups by increased UDP-induced relaxation in the LETO group and increased ACh-induced relaxation in the OLETF group. MRS2578, a P2Y₆ receptor antagonist, eliminated the difference in UDP-induced relaxations between the groups by decreasing UDP-induced relaxation in the OLETF group. MRS2578 had no effect on UDP-induced contraction in endothelium-denuded aortas. Therefore, these findings demonstrate opposite trends of relaxations by UDP and ACh in OLETF and LETO rat aortas. These differences may be attributed to the imbalance between NO and vasoconstrictor prostanoids upon stimulations. Increased UDP-induced relaxation in OLETF rat aorta may be caused by the activation of endothelial MRS2578-sensitive P2Y₆ receptor.     

Detection of QTLs controlling alpha-amylase activity in a diversity panel of 343 barley accessions

The α–amylase activity of cultivated barley is critically important to the brewing industry. Here, we surveyed variation in malt α–amylase activity in 343 cultivated barley accessions from around the world. Population structure analysis based on genotype data at 1536 SNPs clustered these accessions into two groups, one comprising South-East Asian and Ethiopian accessions and one group containing the other accessions. A genome-wide association study identified significant quantitative trait loci (QTLs) for α–amylase activity on all seven chromosomes of barley. Accessions showing high and low α–amylase activity were crossed with the high-quality Japanese malting barley cv. Harun Nijo to develop F₂ mapping populations. We identified two QTLs on chromosome 6H in a cross between Haruna Nijo (high activity) × Weal (highest activity). Single QTLs were identified each on 3H, 4H, and 5H from a cross between Haruna Nijo (high activity) × VLB-1 (low activity), indicating that the high α–amylase activity in Haruna Nijo might be derived from loci on these chromosomes. The addition of the high α–amylase activity QTL alleles from chromosome 6H in cv. Weal further increased the α–amylase activity conferred by alleles of Haruna Nijo. These results demonstrate that a target haplotype can be successfully improved using a strategy comprising diversity analysis of ex situ collections followed by introducing effective new alleles.     

Comparative studies with EPR and MRI on the in vivo tissue redox status estimation using redox-sensitive nitroxyl probes: influence of the choice of the region of interest

In vivo decay rates of a nitroxyl contrast agent were estimated by a MR redox imaging (MRRI) technique and compared with the decay rates obtained by the electron paramagnetic resonance spectroscopy (EPRS) and imaging (EPRI). MRRI is a dynamic imaging technique employing T₁-weighted pulse sequence, which can visualise a nitroxyl-induced enhancement of signal intensity by T₁-weighted contrast. EPR techniques can directly measure the paramagnetic nitroxyl radical. Both the squamous cell carcinoma (SCC) tumour-bearing and normal legs of a female C3H mouse were scanned by T₁-weighted SPGR sequence at 4.7 T with the nitroxyl radical, carbamoyl-proxyl (CmP), as the contrast agent. Similarly, the time course of CmP in normal muscle and tumour tissues was obtained using a 700-MHz EPR spectrometer with a surface coil. The time course imaging of CmP was also performed by 300?MHz CW EPR imager. EPRS and EPRI gave slower decay rates of CmP compared to the MRRI. Relatively slow decay rate at peripheral region of the tumour tissues, which was found in the image obtained by MRRI, may contribute to the slower decay rates observed by EPRS and/or the EPRI measurements. To reliably determine the tissue redox status from the reduction rates of nitroxyls such as CmP, heterogenic structure in the tumour tissue must be considered. The high spatial and temporal resolution of T₁-weighted MRI and the T₁-enhancing capabilities of nitroxyls support the use of this method to map tissue redox status which can be a useful biomarker to guide appropriate treatments based on the tumour microenvironment.     

Integrative Analyses of De Novo Mutations Provide Deeper Biological Insights into Autism Spectrum Disorder

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Imaging of intracellular Ca waves induced by muscarinic receptor stimulation in rat parotid acinar cells

Changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) following muscarinic receptor stimulation were studied with digital imaging microscopy in small clusters of Fura-2 loaded rat parotid acinar cells. In the absence of extracellular Ca²⁺, the increase in [Ca²⁺]_i evoked by a high concentration (10 IM) of carbachol (CCh) was initiated in the apical pole of the acinar cells about 0.4 s after stimulation and then rapidly spread as a Ca²⁺ wave toward the basolateral region. The [Ca²⁺]_i reached the maximum high level throughout the cells 1–2 s after stimulation. As Ca²⁺ was eliminated from the extracellular medium, the Ca²⁺ wave was a result of Ca²⁺ release from intracellular stores. The magnitude and velocity of the Ca²⁺ wave decreased with decreasing concentration of CCh, and the increase in [Ca²⁺]_i induced by low CCh concentrations (≤ 0.5 μM) was always larger in the apical region of acinar cells than in the basal region. The Ca²⁺ wave was also observed in isolated single acinar cells, indicating that the maintenance of acinar structure is not essential for the development of the Ca²⁺ wave. Thapsigargin (ThG), an inhibitor of the endoplasmic reticulum Ca²⁺ pump, caused a slow and homogeneous increase in [Ca²⁺]_i throughout the cells. Addition of ThG after CCh, or addition of CCh after ThG, did not stimulate further increases in [Ca²⁺]_i suggesting that the inositol-1,4,5-trisphosphate (InsP₃) and ThG-sensitive Ca²⁺ stores overlap in parotid acinar cells. The present study supports the hypothesis that formation of InsP₃ is essential to trigger the Ca²⁺ wave and that the development of the Ca²⁺ wave may be attributed to regional differences in InsP₃ sensitivity of Ca²⁺ stores. The agonist-induced Ca²⁺ wave is probably a general phenomenon in exocrine acinar cells.