Knockout mouse production assisted by Blm knockdown

Production of knockout mice using targeted embryonic stem cells (ESCs) is a powerful approach for investigating the function of specific genes in vivo. Although the protocol for gene targeting via homologous recombination (HR) in ESCs is already well established, the targeting efficiency varies at different target loci and is sometimes too low. It is known that knockdown of the Bloom syndrome gene, BLM, enhances HR-mediated gene targeting efficiencies in various cell lines. However, it has not yet been investigated whether this approach in ESCs is applicable for successful knockout mouse production. Therefore, we attempted to answer this question. Consistent with previous reports, Blm knockdown enhanced gene targeting efficiencies for three gene loci that we examined by 2.3–4.1-fold. Furthermore, the targeted ESC clones generated good chimeras and were successful in germline transmission. These data suggest that Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production.     

Habitat use and movement of hatchery-reared F2 Mekong giant catfish in the Mae Peum reservoir, Thailand, studied by acoustic telemetry

The horizontal and vertical movements of eight immature hatchery-reared (F2) Mekong giant catfish Pangasianodon gigas were monitored using acoustic telemetry in the Mae Peum reservoir, Thailand, from September to December 2005. All tagged fish were successfully monitored throughout the study period. All fish moved throughout the reservoir for approximately 1 month after release. Subsequently, their utilized areas became small, and the fish utilized deep areas of the reservoir. The fish displayed diel spatial movement patterns between deep areas in the day and shallow areas in the night. The vertical movements of the fish were related to the environment declination such as existence of hypoxic water and thermocline. Our results suggest that the establishment of a protected area in addition to conventional fisheries regulations may sustain the fish population in this reservoir.     

Effects of a meal rich in 1,3-diacylglycerol on postprandial cardiovascular risk factors and the glucose-dependent insulinotropic polypeptide in subjects with high fasting triacylglycerol concentrations

It was previously reported that compared to triacylglycerol (TAG) oil, diacylglycerol (DAG) oil improves postprandial lipid response. However, the effects of DAG oil on postprandial hyperglycemia and incretin response have not yet been determined. In this study, the effects of DAG oil on both postprandial hyperlipidemia and hyperglycemia and the response to the glucose-dependent insulinotropic polypeptide (GIP) were studied. This randomized, double-blind, crossover study analyzed data for 41 individuals with high fasting triacylglycerol concentrations. The subjects ingested test meals (30.3 g of protein, 18.6 g of fat, and 50.1 g of carbohydrate) containing 10 g of DAG oil (DAG meal) or TAG oil (TAG meal) after fasting for at least 12 h. Blood samples were collected prior to and 0.5, 2, 3, 4, and 6 h after ingestion of the test meal. Postprandial TAG concentrations were significantly lower after the DAG meal compared with the TAG meal. Postprandial TAG, insulin, and GIP concentrations were significantly lower after the DAG meal compared with the TAG meal in 26 subjects with fasting serum TAG levels between 1.36 and 2.83 mmol/L. DAG-oil-based meals, as a replacement for TAG oil, may provide cardiovascular benefits in high-risk individuals by limiting lipid and insulin excursions.     

Expression and localization of alpha-tubulin N-acetyltransferase 1 in the reproductive system of male mice

The structure of microtubules is essential for the fertilizing ability of spermatozoa. Acetylation of α-tubulin plays an important role in flagellar elongation and spermatozoa motility. Previous reports have suggested that alpha-tubulin N-acetyltransferase 1 (ATAT1) is the main acetyltransferase involved in the acetylation of α-tubulin. Although ATAT1 is reported to express in the testis, no information is available regarding its expression in elongated spermatids, epididymis, and mature spermatozoa. Hence, it remains unclear whether ATAT1 is involved in spermatozoa maturation and capacitation. Therefore, we evaluated the expression of ATAT1 in the mouse male reproductive system using immunostaining and western blotting. Our results showed that ATAT1 was expressed in spermatids during spermiogenesis in mouse testes, but its expression varied according to the seminiferous tubule stage. We observed ATAT1 in the cytoplasm of round spermatids, the flagella of elongated spermatids, and in the cytoplasm of step 16 spermatids, just before its release into the lumen. In addition, ATAT1 was expressed in epithelial cells of the epididymis. In spermatozoa of the cauda epididymis, ATAT1 expression was primarily observed in the midpiece of the spermatozoa. The localization of ATAT1 protein in the male germline was observed during spermiogenesis as well as during spermatozoa maturation. Our results suggest that ATAT1 may be involved in the formation of flagella and in the acetylation process, which has attracted attention in recent years regarding male infertility.     

PDE4 and PDE5 regulate cyclic nucleotide contents and relaxing effects on carbachol-induced contraction in the bovine abomasum

The effects of various selective phosphodiesterase (PDE) inhibitors on carbachol (CCh)-induced contraction in the bovine abomasum were investigated. Various selective PDE inhibitors, vinpocetine (type 1), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA, type 2), milrinone (type 3), Ro20-1724 (type 4), vardenafil (type 5), BRL-50481 (type 7) and BAY73-6691 (type 9), inhibited CCh-induced contractions in a concentration-dependent manner. Among the PDE inhibitors, Ro20-1724 and vardenafil induced more relaxation than the other inhibitors based on the data for the IC₅₀ or maximum relaxation. In smooth muscle of the bovine abomasum, we showed the expression of PDE4B, 4C, 4D and 5 by RT-PCR analysis. In the presence of CCh, Ro20-1724 increased the cAMP content, but not the cGMP content. By contrast, vardenafil increased the cGMP content, but not the cAMP content. These results suggest that Ro20-1724-induced relaxation was correlated with cAMP and that vardenafil-induced relaxation was correlated with cGMP in the bovine abomasum. In conclusion, PDE4 and PDE5 are the enzymes involved in regulation of the relaxation associated with cAMP and cGMP, respectively, in the bovine abomasum.     

Central somatostatin-somatostatin receptor 2 signaling mediates lactational suppression of luteinizing hormone release via the inhibition of glutamatergic interneurons during late lactation in rats

Reproductive function is suppressed during lactation owing to the suckling-induced suppression of the kisspeptin gene (Kiss1) expression in the arcuate nucleus (ARC) and subsequent suppression of luteinizing hormone (LH) release. Our previous study revealed that somatostatin (SST) neurons mediate suckling-induced suppression of LH release via SST receptor 2 (SSTR2) in ovariectomized lactating rats during early lactation. This study examined whether central SST-SSTR2 signaling mediates the inhibition of ARC Kiss1 expression and LH release in lactating rats during late lactation and whether the inhibition of glutamatergic neurons, stimulators of LH release, is involved in the suppression of LH release mediated by central SST-SSTR2 signaling in lactating rats. A central injection of the SSTR2 antagonist CYN154806 (CYN) significantly increased ARC Kiss1 expression in lactating rats on day 16 of lactation. Dual in situ hybridization revealed that few ARC Kiss1-positive cells co-expressed Sstr2, and some of the ARC Slc17a6 (a glutamatergic neuronal marker)-positive cells co-expressed Sstr2. Furthermore, almost all ARC Kiss1-positive cells co-expressed Grin1, a subunit of N-methyl-D-aspartate (NMDA) receptors. The numbers of Slc17a6/Sstr2 double-labeled and Slc17a6 single-labeled cells were significantly lower in lactating dams than in non-lactating rats whose pups had been removed after parturition. A central injection of an NMDA antagonist reversed the CYN-induced increase in LH release in lactating rats. Overall, these results suggest that central SST-SSTR2 signaling, at least partly, mediates the suppression of ARC Kiss1 expression and LH release by inhibiting ARC glutamatergic interneurons in lactating rats.     

NASAL SALT GLAND: INDEPENDENCE OF SALT AND WATER TRANSPORT

The nasal salt-secreting gland of the domestic duck can produce a fluid with a sodium chloride concentration about three times that in blood plasma. To study the cellular mechanism responsible for the formation of the highly concentrated fluid, the gland was poisoned by retrograde injection of mercuric chloride into the lumen, decreasing the salt concentration to that in plasma while the volume of secretion was unchanged. Carbonic anhydrase inhibitor (acetazoleamide) caused a moderate decrease in salt concentrations, again with maintenance of volume of secretion. The results suggest that concentration and volume of the secreted fluid depend on two different cellular mechanisms.     

Evaluation of the heat‐killed and dried cell preparation of Enterococcus faecalis against villous atrophy in early‐weaned mice and pigs

Early weaning induces villous atrophy in the small intestine (SI) of piglets. Oral administration of live lactic acid bacteria (LAB) can improve villous shortening. In this study, we evaluated the oral administration of a heat‐killed and dried cell preparation of Enterococcus faecalis (a LAB) strain EC‐12 against villous atrophy in early‐weaned mice (Experiment 1) and pigs (Experiments 2 and 3). Twelve 16‐days‐old mice were divided into two groups in Experiment 1: gavage of EC‐12 (10 mg/kg body weight (BW)/day), or control. On day 21, SI was collected. Eighteen 21‐day‐old pigs were divided into two groups in Experiment 2: gavage of EC‐12 (10 mg/kg BW/day), or control. After 10 days, the villous height of jejunum was measured. Six 21‐day‐old pigs were divided into two groups in Experiment 3: the basal diet supplemented with EC‐12 at 0.05%‐fed group, or the basal diet‐fed group. After 10 days, the villous height of jejunum was measured. The villous heights in SI were significantly higher by EC‐12 administration in all experiments. EC‐12 successfully improved the villous atrophy in the early‐weaned mice and pigs when EC‐12 was administered orally.     

Mixed germ cell-sex cord-stromal tumor with a concurrent interstitial cell tumor in a ferret

A 5-year-old male ferret presented with an enlarged canalicular testis in the left inguinal region. Microscopically, the enlarged testis consisted of a diffuse intimately admixed proliferation of c-kit-positive germ cell-like and Wilms tumor-1 protein-positive Sertoli cell-like components, but no Call-Exner body was detected. In addition, the compact proliferation of steroidogenic acute regulatory protein-intense positive interstitial cells was identified in a separate peripheral area of the mass. Based on histopathological and immunohistochemical findings, the tumor was diagnosed as a mixed germ cell-sex cord-stromal tumor with a concurrent interstitial cell tumor.