Utility of Ethylene-Diamine-Tetraacetic Acid Buffer Solution With Boric Acid for Immunostaining of Specimens Stored for an Extended Period

Antigen modification and denaturation are recognized causes of false negatives in immunostaining. Specimens that have been stored for an extended period at room temperature show decreased immunoreactivity and may mislead the diagnosis. Studies of the molecular targeting of drugs often involve immunostaining of previous samples and, in some situations, only unstained specimens can be used. The present study aimed to develop an effective staining method to recover antigen activation in unstained specimens stored for an extended period by using ethylene-diamine-tetraacetic acid (EDTA) buffer solution with boric acid. We compared several commonly used antigen retrieval solutions and found that Tris-borate-EDTA (TBE) buffer solution with a pH ≥8.3 provided sufficient antigen retrieval. However, pH values higher than 8.3 (9.0, 10.0, and 11.0) frequently caused severe tissue damage. Thus, TBE with pH 8.3 was the most suitable antigen retrieval solution for recovering the antigenicity of specimens stored for an extended period. This procedure may allow useful immunohistochemical information, even from sections that have been stored for an extended period

PcpA, which is involved in the degradation of pentachlorophenol in Sphingomonas chlorophenolica ATCC39723, is a novel type of ring-cleavage dioxygenase

AbstractThe pentachlorophenol (PCP) mineralizing bacterium Sphingomonas chlorophenolica ATCC39723 degrades PCP via 2,6-dichlorohydroquinone (2,6-DCHQ). The pathway converting PCP to 2,6-DCHQ has been established previously; however, the pathway beyond 2,6-DCHQ is not clear, although it has been suggested that a PcpA plays a role in 2,6-DCHQ conversion. In this study, PcpA expressed in Escherichia coli was purified to homogeneity and shown to have novel ring-cleavage dioxygenase activity in conjunction with hydroquinone derivatives, and converting 2,6-DCHQ to 2-chloromaleylacetate

Steroid transhydrogenase activity of 3-ketosteroid-Δ1-dehydrogenase from Nocardia corallina

金沢大学自然科学研究科　　金沢大学理工研究域自然システム学系3-Ketosteroid-Δ1-dehydrogenase from Nocardia corallina catalyzes transhydrogenation of 3-keto-4-ene-steroid to 3-keto-1,4-diene-steroid e.g., progesterone to 1,4-androstadiene-3,17-dione. The reaction proceeded linearly at first and then soon slowed down owing to equilibration. The turnover number of this reaction was of the same magnitude as that of the dehydrogenation of 3-keto-4-ene-steroid. The pH optimum was 8.4, which is lower than that of the dehydrogenase reaction. The enzyme has a wide specificity for hydrogen acceptor steroids. The K(m)\u27 and K(max)\u27 values for these steroids and the values of the corresponding 3-keto-4-ene-steroids were compared. Kinetic studies of the steroid transhydrogenase reaction demonstrated a typical ping-pong mechanism. The enzyme oxidized 1,2-tritiated progesterone and transferred the tritium atoms to the reaction product, 4-androstene-3,17-dione, and water. Transhydrogenation in D2O resulted in the incorporation of a deuterium atom into the C2-position of 4-androstene-3,17-dione. The results indicate that the enzyme catalyzes C1,C2-trans axial abstraction of hydrogen atoms from progesterone, transfer of the 1α-hydrogen to the C1-position of 1,4-androstadiene-3,17-dione and release of the 2β-hydrogen to water. Reaction schemes based on the experimental results are proposed. The enzyme also catalyzes the reduction of 3-keto-1,4-diene-steroids with reduced benzoyl viologen

3-keto-5α-steroid-Δ4-dehydrogenase from Nocardia corallina: Purification and characterization

金沢大学自然科学研究科　　金沢大学理工研究域自然システム学系The inducible 3-keto-5α-steroid-Δ4-dehydrogenase of Nocardia corallina was purified to homogeneity using affinity chromatography on 19-nortestosterone-17-acetoxyaminoethyl Sepharose 4B. SDS-polyacrylamide gel electrophoresis, gel filtration and spectral analysis of flavin suggest that the purified dehydrogenase is a monomeric protein of M(r) 60,000 containing one flavin. It has a typical absorption spectrum of flavoprotein with maxima at 457, 375, and 277 nm. The values shifted to 470 and 395 nm on binding of 19-nortestosterone. The enzyme catalyzed the dehydrogenation of 3-keto-5α-steroid at the 4- and 5-position, e.g. the conversion of 5α-androst-1-ene-3,17-dione to 1,4-androstadiene-3,17-dione with the reduction of phenazine methosulfate. The substrate 3-ketosteroid has essentially the 5α-configuration. The enzyme did not reduce potassium ferricyanide but did reduce cytochrome c at a moderate rate, and exhibited only a weak steroid oxidase activity. Stereochemical study demonstrated that the enzyme abstracts the 4β, 5α-hydrogens of the substrate as a hydrogen ion through a protein-based reaction and as a hydride ion by transfer to FAD, respectively. The enzyme oxidizes a wide variety of 3-keto-5α-steroids but not 3β-hydroxysteroid. The dehydrogenase also catalyzed steroid transhydrogenation between 3-keto-5α-steroid and 3-keto-1,4-diene-steroid. The properties of this enzyme are compared with those of 3-keto-steroid-Δ1-dehydrogenase

Effect of Continuous Feeding of Ayu-Narezushi on Lipid Metabolism in a Mouse Model of Metabolic Syndrome

Ayu-narezushi, a traditional Japanese fermented food, comprises abundant levels of lactic acid bacteria (LAB) and free amino acids. This study aimed to examine the potential beneficial effects of ayu-narezushi and investigated whether ayu-narezushi led to improvements in the Tsumura Suzuki obese diabetes (TSOD) mice model of spontaneous metabolic syndrome because useful LAB are known as probiotics that regulate intestinal function. In the present study, the increased body weight of the TSOD mice was attenuated in those fed the ayu-narezushi-comprised chow (ayu-narezushi group) compared with those fed the normal rodent chow (control group). Serum triglyceride and cholesterol levels were significantly lower in the Ayu-narezushi group than in the control group at 24 weeks of age. Furthermore, hepatic mRNA levels of carnitine-palmitoyl transferase 1 and acyl-CoA oxidase, which related to fatty acid oxidation, were significantly increased in the ayu-narezushi group than in the control group at 24 weeks of age. In conclusion, these results suggested that continuous feeding with ayu-narezushi improved obesity and dyslipidemia in the TSOD mice and that the activation of fatty acid oxidation in the liver might contribute to these improvements

Fast and Slow Oscillation Electrooculography in Harada Disease

We assessed clinical utility of fast and slow oscillations (FO and SO) of the electrooculogram (EOG) in Harada disease. In 12 eyes of 4 female and 2 male subject patients aged 18 to 77 years (average: 41.8 years), FO and SO were recorded using an automated electrooculograph, the Nidek EOG-2, in the acute period before treatment and in the remission period under corticosteroid therapy. FO parameters, namely the RfFO [the average ratio in percentage of the maximum amplitude in the dark period (AD)/the minimum amplitude in the light period (AL) during FO measurement] and the dfFO (the average difference in ?V between AD and AL) were evaluated. The L/DSO (the light peak/dark trough ratio of the SO) was calculated as an SO parameter. The RfFO, dfFO and L/DSO showed low values in 7 (58.3%), 10 (83.3%) and 8 (66.7%) out of all 12 eyes in the acute period, respectively. In the remission period, values in the normal range were obtained in 12 (100%), 11 (91.7%) and 8 (66.7%) out of 12 eyes in the RfFO, dfFO and L/DSO, respectively. In mutual relation to each RfFO, dfFO and L/DSO in the acute and remission periods, all 12 eyes showed recovery values both in the RfFO and dfFO in the remission stage after systemic administration of corticosteroids, but 4 out of 12 eyes (33.3%) showed no recovery in the L/DSO. The FO may therefore well reflect the affected or ameliorated conditions in the outer layers of the retina and the choroid in Harada disease, in contrast to the SO. However, further observations are requested in more Harada disease patients

Assessment of Macular Function by Multifocal Electroretinography and Optical Coherence Tomography before and after Panretinal Photocoagulation in Diabetic Retinopathy

We evaluated macular function before and after panretinal photocoagulation (PRP) in diabetic retinopathy using a multifocal electroretinogram (mfERG) and optical coherence tomogram (OCT). In mfERGs, the 1st positive wave (P1) minus the 1st negative wave (N1) amplitude (P1 ? N1 amplitude), the P1 peak latency and the response density were measured in 7, 19, 37 and 103 hexagonal areas or elements (Areas 1, 2, 3 and 4) within a central radius of 5, 7, 10 and 20 degrees, respectively. The mean retinal thickness was estimated from 9 calculation points at the foveal region within 5 degrees; the central and each of the other 4 points at a distance of 250 ?m and 500 ?m from the central por tion on horizontal and vertical sections on OCT. The P1 peak latencies from the 4 areas were remarkably prolonged in 14 eyes of 9 patients with preproliferative or early proliferative diabetic retinopathy showing no clinically significant macular edema before PRP as compared with those in 15 normal control eyes, without a tendency of recovery throughout the course after PRP except for area 1. The P1-N1 amplitudes and the mean response density levels from the 4 areas were remarkably decreased in the diabetic eyes before PRP as compared with those in the control eyes, followed by a maximum decrease in both parameters at 3 months after PRP. However, remarkable recoveries were detected in both decreased parameters from the 4 areas at 6 months after PRP. The mean foveal retinal thickness on OCT was remarkably increased in the diabetic eyes before PRP as compared with the thickness in 16 normal control eyes. Most remarkably, a transient increase in thickness was detected in diabetic eyes 1 month after PRP, followed by a tendency of recovery 3 to 6 months after PRP. These results indicate that mfERG and OCT examinations are useful in the assessment of macular function before and after PRP in diabetic retinopathy, especially within 5 degrees of the central portion, and that the effects of PRP on macular function in this entity seem to be reversible at the foveal region, although we need to do further investigation in relation to the outcome of visual acuity

Universality, the QCD critical/tricritical point and the quark number susceptibility

The quark number susceptibility near the QCD critical end-point (CEP), the tricritical point (TCP) and the O(4) critical line at finite temperature and quark chemical potential is investigated. Based on the universality argument and numerical model calculations we propose a possibility that the hidden tricritical point strongly affects the critical phenomena around the critical end-point. We made a semi-quantitative study of the quark number susceptibility near CEP/TCP for several quark masses on the basis of the Cornwall-Jackiw-Tomboulis (CJT) potential for QCD in the improved-ladder approximation. The results show that the susceptibility is enhanced in a wide region around CEP inside which the critical exponent gradually changes from that of CEP to that of TCP, indicating a crossover of different universality classes.Comment: 18 pages, 10 figure