Ovariectomy enhances renal cortical expression and function of cyclooxygenase-2

Ovariectomy enhances renal cortical expression and function of cyclooxygenase-2.BackgroundCyclooxygenase-2 (COX-2) inhibitors are used as analgesics in postmenopausal women, who develop edema and require a salt-restricted diet. This study was performed to determine the renal expression of COX-2 and on COX-2–dependent regulation of renal blood flow (RBF) in ovariectomized rats.MethodsSprague-Dawley rats were divided into 4 groups: sham-operated rats fed a normal-salt diet (Sh+NS) or a low-salt diet (Sh+LS), and bilaterally ovariectomized rats fed a normal-salt diet (Ox+NS) or a low-salt diet (Ox+LS) (N = 6 in each group). Estrogen replacement therapy was performed on other ovariectomized rats. A renal clearance study was performed in anesthetized animals.ResultsOvariectomy increased renal cortical COX-2 expression independently of dietary salt intake (Sh+NS <Ox+N; Sh+LS <Ox+LS). Inhibition of COX-2 by NS398 reduced the urinary excretion of 6-keto-prostaglandin F1α in all 4 groups, although the reduction was greater in the Ox+LS group than in the Ox+NS and Sh+LS groups, which in turn had a greater reduction than the Sh+NS group. RBF significantly decreased in every group except the Sh+NS group, but no effect on blood pressure, inulin clearance, or urinary sodium excretion was seen. The decrease in RBF was significantly greater in the Ox+LS group than in the Sh+LS and Ox+NS group. The decrease in RBF was dependent on cortical RBF in the Sh+LS and Ox+NS groups, and on both cortical and medullary RBF in the Ox+LS group. Estrogen replacement therapy reversed the ovariectomy-induced changes.ConclusionEstrogen-dependent COX-2 expression plays an important role in the RBF regulation in female rats

Fish mesonephric model of polycystic kidney disease in medaka (Oryzias latipes) pc mutant

Fish mesonephric model of polycystic kidney disease in medaka (Oryzias latipes) pc mutant.BackgroundPolycystic kidney disease (PKD) is a common hereditary disease. A number of murine and zebrafish mutants have been generated and used for the study of PKD as metanephric and pronephric models, respectively. Here, we report a medaka (Oryzias latipes) mutant that develops numerous cysts in the kidney in adulthood fish in an autosomal-recessive manner as a mesonephric model of PKD.MethodsThe phenotypes of the medaka pc mutant were described in terms of morphologic, histologic, and ultrastructural features. The pc see-through stock was produced by crossing a pc mutant and a fish from the see-through stock and used for observing the kidney through the transparent body wall of a live fish.ResultsThe mutant developed bilateral massive enlargement of the kidney in adulthood. They sexually matured normally within 2 months of age and died within 6 months of age. The affected kidney was occupied by numerous, fluid-filled cysts, which were lined by attenuated squamous epithelial cells. Developmentally, cystic formation began in the pronephros in 10-day-old fry and in the mesonephros in 20-day-old fry at the microscopic level. The pc see-through stock was useful in observing disease progression in live fish.ConclusionThe kidney disorder that develops in the medaka pc mutant is a mesonephric counterpart of PKD, particularly an autosomal-dominant PKD, based on its morphologic, histologic, and ultrastructural features, and slow progression

A new cancer diagnostic system based on a CDK profiling technology

AbstractA series of molecular pathological investigations of the molecules that stimulate the cyclin dependent kinases (CDK1, 2, 4, and 6) have led to enormous accumulation of knowledge of the clinical significance of these molecules for cancer diagnosis. However, the molecules have yet to be applied to clinical cancer diagnosis, as there is no available technology for application of the knowledge in a clinical setting. We hypothesized that the direct measurement of CDK activities and expressions (CDK profiling) might produce clinically relevant values for the diagnosis. This study investigated the clinical relevance of CDK profiling in gastrointestinal carcinoma tissues by using originally developed expression and activity analysis methods. We have established novel methods and an apparatus for analyzing the expression and activities of the CDK molecules in lysate of tumor tissue in a clinical setting, and examined 30 surgically dissected gastrointestinal carcinomas and corresponding normal mucosal specimens. We demonstrate here that remarkably elevated CDK2 activity is evident in more than 70% of carcinoma tissues. Moreover, a G1-CDK activity profiling accurately mirrored the differences in proliferation between tumor and normal colonic tissues. Our results suggest that CDK profiling is a potent molecular–clinical approach to complement the conventional pathological diagnosis, and to further assist in the individualized medications

Pillar[6]arene acts as a biosensor for quantitative detection of a vitamin metabolite in crude biological samples

ビタミン代謝物を迅速定量できる超分子バイオセンサーを開発. 京都大学プレスリリース. 2020-12-09.Metabolic syndrome is associated with obesity, hypertension, and dyslipidemia, and increased cardiovascular risk. Therefore, quick and accurate measurements of specific metabolites are critical for diagnosis; however, detection methods are limited. Here we describe the synthesis of pillar[n]arenes to target 1-methylnicotinamide (1-MNA), which is one metabolite of vitamin B3 (nicotinamide) produced by the cancer-associated nicotinamide N-methyltransferase (NNMT). We found that water-soluble pillar[5]arene (P5A) forms host–guest complexes with both 1-MNA and nicotinamide, and water-soluble pillar[6]arene (P6A) selectively binds to 1-MNA at the micromolar level. P6A can be used as a “turn-off sensor” by photoinduced electron transfer (detection limit is 4.38 × 10−6 M). In our cell-free reaction, P6A is used to quantitatively monitor the activity of NNMT. Moreover, studies using NNMT-deficient mice reveal that P6A exclusively binds to 1-MNA in crude urinary samples. Our findings demonstrate that P6A can be used as a biosensor to quantify 1-MNA in crude biological samples

Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases

Aims The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them. Methods and Results We performed WES of 23 probands diagnosed with early-onset (&amp;lt;65 years) CCSD and analyzed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency &amp;lt; 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as “pathogenic” by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that 2 variants in KCNH2 and SCN5A, 4 variants in SCN10A, and 1 variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from “Uncertain significance” to “Likely pathogenic” in 6 probands. Conclusions Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD. Translational Perspective Whole-exome sequencing (WES) may be helpful in determining the causes of cardiac conduction system disease (CCSD), however, the identification of pathogenic variants remains a challenge. We performed WES of 23 probands diagnosed with early-onset CCSD, and identified 12 pathogenic or likely pathogenic variants in 11 of these probands (48%) according to the 2015 ACMG standards and guidelines. In this context, functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants, and SCN10A may be one of the major development factors in CCSD

Effect of extracted garlic powder ingestion for two months on exercise-induced immunological responses

Introduction. Exhaustive exercise is associated with an increased risk of upper respiratory tract infection. Previously, allicin supplementation has been reported to reduce the incidence of common cold symptoms and production of exercise-induced interleukin (IL)-6. However, it is not clear if daily ingestion of the edible portion of whole garlic (Allium sativum) alters the exercise-induced immunological response. The present study investigated the effects of extracted garlic powder ingestion for 2 months on immune cell counts, natural killer cell activity (NKCA), as well as changes in cytokines, cortisol, and lactic acid in response to high-intensity cycling exercise. Methods. The present study employed a before-after study design. Six sedentary male participants (age, 22.0±0.3 years) consumed extracted garlic powder for 2 months, and underwent 45 minutes of cycling exercise at 80% of the heart rate reserve once before and once after the supplementation period. A thousand milligrams of extracted garlic powder, comparable to 6 g (1 clove) of raw garlic, was ingested every day. Blood samples were obtained at the following five time points: before exercise, 0 min, 30 min, 60 min, and 120 min after exercise. We measured NKCA, leukocyte counts, neutrophil counts, lymphocyte counts, as well as levels of serum IL-6, IL-10, cortisol, and lactic acid. Repeated measures ANOVA was used for statistical analyses. When interaction effects were significant, measurement values at the various time points were compared between pre- and post-supplementation period using the paired t-test. Changes were deemed statistically significant when p\u3c0.05. Results. We observed no significant difference in pre-exercise measurements between pre- and post-supplementation periods. In addition, we found no significant interaction effect for leukocytes, neutrophils, NKCA, IL-10, and cortisol. However, we did identify a significant interaction effect for lymphocytes, IL-6, and lactic acid (p=0.033, p=0.030, and p\u3c0.001, respectively). Lymphocyte counts were significantly lower post-supplementation relative to pre-supplementation immediately after exercise (p=0.014). In addition, IL-6 was significantly lower post-supplementation relative to pre-supplementation immediately and 30 minutes after exercise (p=0.015 and p=0.018, respectively). Lactic acid levels were significantly lower post-supplementation relative to pre-supplementation immediately after exercise (p=0.018). Conclusions. The extracted garlic powder did not significantly influence exercise-induced responses by leukocytes, neutrophils, NKCA, IL-10, or cortisol. However, exercise-induced responses by lymphocytes, IL-6, and lactic acid were suppressed after ingestion of extracted garlic powder. Thus, daily ingestion of the edible portion of whole garlic may suppress exercise-induced immunological responses and lactic acid levels