Germline recombination in a novel Cre transgenic line,Prl3b1‐Cre mouse

Spermatogenesis is a complex and highly regulated process by which spermatogonial stem cells differentiate into spermatozoa. To better understand the molecular mechanisms of the process, the Cre/loxP system has been widely utilized for conditional gene knockout in mice. In this study, we generated a transgenic mouse line that expresses Cre recombinase under the control of the 2.5 kbp of the Prolactin family 3, subfamily b, member 1 (Prl3b1) gene promoter (Prl3b1‐cre). Prl3b1 was initially reported to code for placental lactogen 2 (PL‐2) protein in placenta along with increased expression toward the end of pregnancy. PL‐2 was found to be expressed in germ cells in the testis, especially in spermatocytes. To analyze the specificity and efficiency of Cre recombinase activity in Prl3b1‐cre mice, the mice were mated with reporter R26GRR mice, which express GFP ubiquitously before and tdsRed exclusively after Cre recombination. The systemic examination of Prl3b1‐cre;R26GRR mice revealed that tdsRed‐positive cells were detected only in the testis and epididymis. Fluorescence imaging of Prl3b1‐cre;R26GRR testes suggested that Cre‐mediated recombination took place in the germ cells with approximately 74% efficiency determined by in vitro fertilization. In conclusion, our results suggest that the Prl3b1‐cre mice line provides a unique resource to understand testicular germ‐cell development. genesis 54:389–397, 2016. © 2016 Wiley Periodicals, Inc.     

Hypermethylation of the <Emphasis Type="Italic">CaSR</Emphasis> and <Emphasis Type="Italic">VDR</Emphasis> genes in the parathyroid glands in chronic kidney disease rats with high-phosphate diet

Chronic kidney disease (CKD) disrupts mineral homeostasis and its representative pathosis is defined as secondary hyperparathyroidism (SHPT). SHPT occurs during the early course of progressive renal insufficiency, and is associated with mortality and cardiovascular events. SHPT results in reduction of calcium-sensing receptor (CaSR) and vitamin D receptor (VDR) in the parathyroid glands during CKD. However, the precise mechanism of CaSR and VDR reduction is largely unknown. CKD was induced through two-step 5/6 nephrectomy, and then CKD rats and sham-operated rats were maintained for 8 weeks on diets containing 0.7 % phosphorus (normal phosphate) or 1.2 % phosphorus (high phosphate). In gene expression analysis, TaqMan probes were used for quantitative real-time polymerase chain reaction. Finally, CaSR and VDR protein expressions were analyzed using immunohistochemistry. DNA methylation analysis was performed using a restriction digestion and quantitative PCR. CaSR and VDR mRNA were reduced only in CKD rats fed the high-phosphorus diets (CKD HP), then CaSR and VDR immunohistochemical expressions were compatible with gene expression assay. SHPT was then confirmed only in CKD HP rats. Furthermore, sole CKD HP rats showed the hypermethylation in CaSR and VDR genes; however, the percentage methylation of both genes was low. Although CaSR and VDR hypermethylation was demonstrated in PTGs of CKD HP rats, the extent of hypermethylation was insufficient to support the relevance between hypermethylation and down-regulation of gene expression because of the low percentage of methylation. Consequently, our data suggest that mechanisms, other than DNA hypermethylation, were responsible for the reduction in mRNA and protein levels of CaSR and VDR in PTGs of CKD HP rats.     

The life cycle of Hymenoscyphus fraxineus on Manchurian ash,Fraxinus mandshurica,in Japan

Hymenoscyphus fraxineus causes a lethal disease known as “ash dieback” in the common ash, Fraxinus excelsior, in Europe. It is hypothesized that the fungus originated from East Asia. This fungus is found on the leaf litter of the Manchurian ash, Fraxinus mandshurica, in Japan and is reported to produce apothecia on pseudosclerotial plates formed mainly on decomposing rachises. However, dieback disease has not been reported in Japan, and little is known about the life cycle of H. fraxineus. This study was conducted to explore the behavior and life cycle of this fungus. It was revealed that, after infection by ascospores, H. fraxineus endophytically inhabits the living leaves of F. mandshurica. On fallen leaves, the fungus behaves saprophytically, producing apothecia on pseudosclerotial plates formed mainly on the decomposing rachises. Analysis by real-time quantitative polymerase chain reaction (qPCR) revealed that the amount of H. fraxineus DNA sharply increased in rachises, while such sharp increase of DNA was not found in leaflets.     

Essential structure of orexin 1 receptor antagonist YNT-707, part III: Role of the 14-hydroxy and the 3-methoxy groups in antagonistic activity toward the orexin 1 receptor in YNT-707 derivatives lacking the 4,5-epoxy ring

Morphinan derivatives lacking the 4,5-epoxy ring were synthesized to examine the participation of the 14-OH group, the 3-OMe group, and the aromaticity of the A-ring in the activity and selectivity for the orexin 1 receptor (OX₁R). The assay results and the conformational analyses of the 14-dehydrated and 14-H derivatives suggested that the orientations of the 6-amide side chain and the 17-benzenesulfonyl group would play important roles in the activity for OX₁R. In the 6β-derivatives, removal of the 3-OMe group and the reduction of the A-ring significantly decreased the activity toward the OX₁R, but these changes did not affect the 6α-derivatives. These results indicate that the 3-OMe group and the A-ring would be essential structural moieties for the 6β-derivatives.     

Affinity Improvement of a Cancer-Targeted Antibody through Alanine-Induced Adjustment of Antigen-Antibody Interface

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Interactions between peptides containing nucleobase amino acids and T7 phages displaying S. cerevisiae proteins

The importance of high-throughput analyses of protein abundances and functions is interestingly increasing in genomic/proteomic studies. In such postgenome sequencing era, a protein-detecting chip, in which a large number of molecules specifically capturing target proteins (capturing agents) such as antibodies, recombinant proteins, and small molecules are arrayed onto solid, wet, or semi-wet substrates, enables comprehensive analysis of proteomes by a single experiment. However, whole proteomes are generally complicated for comprehensive analyses so that alternative approaches to subproteome analysis categorized by protein functions and binding properties (focused proteome) would be effective. Approaching the goal of development of designed peptide chip for protein analysis, diversity increases in peptide structures and validation of target proteins are needed. We herein describe design and synthesis of nucleobase amino acid (NBA)-containing peptides, selection of nucleic acid-related proteins derived from S. cerevisiae, and detection of interactions between NBA-containing peptides and T7 phages displaying proteins by both enzyme-linked immunosorbent assays (ELISA) and label-free anomalous reflection of gold (AR) measurements. Twenty-eight phage clones were obtained by the phage-display method and sequenced. Ten of 28 clones were expected to be nucleic acid-related proteins including initiation factor, TYB protein, ribosomal proteins, elongation factor, ATP synthase subunit, GTP-binding protein, and ribonuclease. Other phage clones encoded several classes of enzymes such as reductase, oxidase, aldolase, metalloprotease, and hexokinase. Both ELISA and AR measurements suggested that the methodology of in vitro selection for recognition of the NBA-containing peptide presented in this study was successfully established. Such a combination of NBA and phage display technologies would be potential to efficiently confirm valuable target proteins binding specifically to capturing agents, to be arrayed onto solid surfaces to develop the designed peptide chip.     

Non-genomic effects of aldosterone on intracellular ion regulation and cell volume in rat ventricular myocytes

Aldosterone has non-genomic effects that express within minutes and modulate intracellular ion milieu and cellular function. However, it is still undefined whether aldosterone actually alters intracellular ion concentrations or cellular contractility. To clarify the non-genomic effects of aldosterone, we measured [Na+]i, Ca2+ transient (CaT), and cell volume in dye-loaded rat ventricular myocytes, and we also evaluated myocardial contractility. We found the following: (i) aldosterone increased [Na+]i at the concentrations of 100 nmol/L to 10 micromol/L; (ii) aldosterone (up to 10 micromol/L) did not alter CaT and cell shortening in isolated myocytes, developed tension in papillary muscles, or left ventricular developed pressure in Langendorff-perfused hearts; (iii) aldosterone (100 nmol/L) increased the cell volume from 47.5 +/- 3.6 pL to 49.8 +/- 3.7 pL (n=8, p<0.05); (iv) both the increases in [Na+]i and cell volume were blocked by a Na+-K+-2Cl- co-transporter (NKCCl) inhibitor, bumetanide, or by a Na+/H+ exchange (NHE) inhibitor, 5-(N-ethyl-N-isopropyl) amiloride; and (v) spironolactone by itself increased in [Na+]i and cell volume. In conclusion, aldosterone rapidly increased [Na+]i and cell volume via NKCC1 and NHE, whereas there were no changes in CaT or myocardial contractility. Hence the non-genomic effects of aldosterone may be related to cell swelling rather than the increase in contractility.