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

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 non-specific 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 avidin-biotin 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.     

Age-related changes of glucose metabolism in rat cerebral cortex with reference to glucose-derived amino acids

The parietal cortical slices obtained from 8 week-old (young) and 78 week-old (middle-aged) male Wistar rats were incubated withd-[U-¹⁴C]glucose in oxygensaturated Gey's balanced salt solution. Subsequently, the radioactivities of liberated CO₂ and glucose-derived amino acids (alanine, aspartate, GABA, glutamate and glutamine) obtained from the slices were measured. In the middle-aged rats as compared to the young rats, the amount of radioactivity of CO₂ (P<0.01) and glutamate (P<0.05) showed a significant raduction with glutamine unchanged, while that of alanine (P<0.01), aspartate (P<0.05) and GABA (P<0.05) increased significantly. The results indicate that with advancing age the overall glucose oxidation in the cerebral cortex declines but the metabolic pathway to form amino acids is not uniformly suppressed. Therefore, the above characteristic glucose metabolism could be related to the development of heterogeneous enzyme activities associated with aging in the brain.     

The effects of PGD2 and 9-deoxy-Δ-PGD2 on colony formation of murine osteosarcoma cells

Effects of antineoplastic prostaglandins (PG), PGD₂ and 9-deoxy-Δ⁹-PGD₂, on colony formation of cloned Dunn osteosarcoma (TA 102), normal Swiss 3T3 and V-79 cell lines were evaluated. PGD₂ significantly inhibited the colony formation of TA 102 cells in a dose-dependent manner at concentrations between 0.5 and 5 ug/ml. The IC₅₀ value was calculated to be 0.72 ug/ml. A dose-dependent inhibition of TA 102 colony formation was also observed with 9-deoxy-Δ⁹-PGD₂ between 0.01 to 1 ug/ml, the IC₅₀ value being 0.22 ug/ml. These prostaglandins did not exert cytocidal effects in vitro on Swiss 3T3 cells at concentrations between 0.01 to 1 ug/ml. The two agents had no significant cytocidal effects on V-79 cells except for 9-deoxy-Δ⁹-PGD₂ at a concentration of 5 ug/ml. These results suggest that PGD₂ and 9-deoxy-Δ⁹-PGD₂ are considered to have cytocidal activity on Dunn osteosarcoma cells in dosages which do not affect non-malignant cells.     

Population dynamics of the japanese clouded apolloParnassius glacialis butler (Lepidoptera: Papilionidae). I. changes in population size and related population parameters for three successive generations

1. Population dynamics of a univoltine butterfly Parnassius glacialis (Lepidoptera: Papilionidae) was studied with mark-recapture methods for three successive generations in a hilly region in Kanazawa City, Ishikawa Prefecture, Japan in 1981–1983.
2. Jolly (1965) andSeber's (1973) method was applied to the mark-recapture data to estimate population parameters (daily survival rate, longevity, population size, sex ratio, etc.).
3. Sampling ratios were at least 50% and 30% for males and females, respectively.
4. Mean daily survival rate for males ranged 0.81–0.86 and that for females 0.80–0.84. Mean longevity was about 4–7 days for the males and about 5 days for the females. Spiders killed more males than females. Maximum longevity for an individual recorded during the study was 31 days for males and 18 days for females.
5. Emergence of the butterflies was later and less synchronous in 1981 than in 1982 and 1983. This was thought to be due to later extinction of heavier snow in 1981 than in the other years.
6. The population remained relatively stable for the three successive generations, with estimated total numbers of 914, 1277, and 869.
7. Estimated sex ratio (% females) was 30–40% at emergence

     

Proliferative Response of Mouse Submandibular Gland to Androgen But Not to Thyroid Hormone

Abstract. Hormone-induced differentiation and proliferation of cells were investigated in the submandibular gland of castrated female mice, by determining the esteroprotease activity and ³H-thymidine labelling index, respectively. Injections of 5α-dihydrotestosterone (4 μg/g body weight/day) or l -thyroxine (0.5 μg/g body weight/day) induced a significant increase in the activity of esteroprotease, which has been shown to be localized in the convoluted tubule cells of the submandibular gland. Injections of the above-mentioned dose of 5α-dihydrotestosterone for 3 days induced a 43-fold increase in the labelling index of the convoluted tubule cells, but injections of the above-mentioned dose of l -thyroxine for any duration did not induce a significant increase in the labelling index. the present result suggests that hormones which induce differentiation of cells in mouse submandibular gland do not necessarily induce cell proliferation.     

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.     

Medium-term <Emphasis Type="Italic">in vitro</Emphasis> conservation of virus-free parthenocarpic tomato plants

Infection of field-maintained parthenocarpic Solanum lycopersicum L. (tomato) plants with Tomato yellow leaf curl virus provided the motivation to preserve the germplasm by in vitro methods. In this study, a method for medium-term in vitro conservation of parthenocarpic tomato plants was established. As a preliminary study, the non-parthenocarpic tomato ‘Momotaro’ was used to obtain a number of uniform explants for vegetative propagation under aseptic conditions at 23°C. The modification of sucrose or mannitol concentrations in the medium alone was insufficient for the slow-growth storage of shoot cultures. In contrast, temperature had a considerable effect on the time of conservation. ‘Momotaro’ shoot cultures were pre-cultured with Murashige and Skoog (MS) medium supplemented with 2% (w/v) sucrose at 23°C for 6 d for rooting and were then stored at 10°C for further conservation. When maintained at 10°C, only 27% of the shoot cultures needed subculture even after 3 mo, whereas 100% of plants needed subculturing after approximately 2 wk., when conserved at 23°C. When the same method was used with parthenocarpic tomatoes, plants were successfully conserved at 10°C without subculture for approximately 9 mo. Moreover, field performance and genetic stability of the stored tomato plants were assessed. This newly developed method allows for easy and efficient medium-term in vitro conservation to maintain virus-free parthenocarpic tomato plants.     

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.