De novo transcriptome analysis of the centrohelid Raphidocystis contractilis to identify genes involved in microtubule-based motility.

The centrohelid heliozoan Raphidocystis contractilis has many radiating axopodia, each containing axopodial microtubules. The axopodia show rapid contraction at nearly a video rate (30 frames per second) in response to mechanical stimuli. The axopodial contraction is accompanied by cytoskeletal microtubule depolymerization, but the molecular mechanism of this phenomenon has not been elucidated. In this study, we performed de novo transcriptome sequencing of R. contractilis to identify genes involved in microtubule dynamics such as the rapid axopodial contraction. The transcriptome sequencing generated 7.15-Gbp clean reads in total, which were assembled as 31,771 unigenes. Using the obtained gene sets, we identified several microtubule-severing proteins which might be involved in the rapid axopodial contraction, and kinesin-like genes that occur in gene duplication. On the other hand, some genes for microtubule motor proteins involved in the formation and motility of flagella were not found in R. contractilis, suggesting that the gene repertoire of R. contractilis reflected the morphological features of nonflagellated protists. Our transcriptome analysis provides basic information for the analysis of the molecular mechanism underlying microtubule dynamics in R. contractilis.

Bone Marrow Sarcoidosis with Pancytopenia and Renal Failure Presenting as Fever of Unknown Origin: The Pivotal Role of 18F-FDG PET/CT in Lesion Detection.

We describe a case of fever of unknown origin (FUO), renal failure, and pancytopenia. Initially, lymph proliferative disorder was suspected; therefore, bone marrow biopsy and 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) were performed. Bronchoscopy and lung biopsy were performed because of abnormal FDG uptake in both lung fields. Imaging data and laboratory and histological results confirmed sarcoidosis with bone marrow invasion. The patient was discharged after favorable response to corticosteroid therapy. Sarcoidosis may present as FUO without typical specific presentations in the skin or lungs. Combined 18F-FDP PET/CT helped identify the biopsy site and confirmed the sarcoidosis diagnosis.

Influence of Stent Flexibility on Artery Wall Stress and Wall Shear Stress in Bifurcation Lesions.

PURPOSE: Stent flexibility can influence clinical outcome, especially in bifurcation lesions. For instance, an overly rigid stent can impose mechanical stress on the artery at the stent edges and alter both arterial geometry and blood flow dynamics in bifurcations. This study investigated the influence of stent flexibility on vessel geometry, histology, wall stress, and blood flow dynamics in arterial bifurcations. MATERIALS AND METHODS: We compared arterial angulation, stenosis, histopathology, simulated wall shear stress (WSS), and simulated blood flow velocity distribution in swine coronary artery bifurcations following placement of the less flexible Multi-link 8 or more flexible Kaname stent (4.1 ± 0.5 vs 1.5 ± 0.1 mN, p < 0.05, t-test). Stents were implanted into six coronary artery bifurcations each using the single-stent crossover technique without side branch strut dilatation. Outcomes were examined after 28 days. RESULTS: Implantation of both stents significantly increased site angulation (Multi-link 8: 148° ± 8° to 172° ± 2°, p < 0.05, paired t-test; Kaname: 152° ± 5° to 164° ± 4°, p < 0.05, paired t-test), but the change tended to be greater after Multi-link 8 stent implantation (24° ± 15° vs 11° ± 7°, p = 0.1, t-test), suggesting greater straightening of the bifurcation. The Multi-link 8 stent induced greater neointimal thickness than the Kaname stent (0.53 ± 0.3 mm vs 0.26 ± 0.1 mm, p < 0.05, t-test). The distribution of neointimal hyperplasia following stent implantation as revealed by longitudinal histopathology matched the distribution of WSS simulated using computational fluid dynamics (CFD). The endothelium at low WSS areas exhibited aberrant cell morphology and leukocyte adhesion. A CFD model of a curved bifurcation suggested that the region of low WSS is expanded by artery straightening. CONCLUSION: In bifurcated lesions, stent flexibility influences not only mechanical stress on the artery but also WSS, which may induce local neointimal hyperplasia.

Aquaporin 4 in the chicken oviduct during a pause in laying induced by food deprivation.

In the present study we hypothesize that aquaporin 4 (AQP4) expression in the chicken oviduct would change during a pause in egg laying that was induced by fasting. Accordingly, the aim of this investigation was to examine the AQP4 mRNA and protein expression, and immunolocalization in the chicken oviduct during the course of regression. The experiment was carried out on laying hens subjected to a pause in laying that was induced by food deprivation for 5 days. Control hens were fed ad libitum. The birds were sacrificed on day 6 of the experiment and all segments of the oviduct were isolated, including the infundibulum, magnum, isthmus, shell gland, and vagina. Subsequently, the gene and protein expressions of AQP4 in the tissues were tested by real-time PCR and Western blot, respectively. The relative mRNA expression of AQP4 was the highest in the infundibulum and vagina and the lowest, and least detectable, in the magnum. The level of AQP4 protein was the highest in the infundibulum and the lowest in the magnum. Fasting resulted in a decrease of the AQP4 mRNA expression (P<0.001) in the infundibulum, a decrease in protein abundance (P<0.01) in the shell gland, and an increase in protein level (P<0.001) in the vagina. Immunohistochemistry demonstrated tissue- and cell-dependent localization of AQP4 protein in the oviductal wall. The intensity of staining was as follows: the infundibulum > shell gland > vagina ≥ isthmus ≫ magnum. In the control hens, the immunoreactivity for AQP4 in the vagina was similar, whereas in other oviductal segments, the immunoreactivity was stronger when compared with the chickens subjected to a pause in laying. In summary, these findings suggest that the AQP4 is an essential protein involved in the regulation of water transport required to create a proper microenvironment for fertilization and egg formation in the hen oviduct.

Dans la présente étude, nous posons l'hypothèse que l'expression de l'aquaporine 4 (AQP4) dans l'oviducte de poule changerait pendant une pause lors de la ponte induite par un jeûne. Ainsi, le but de notre expérimentation était de déterminer l'expression de l'ARNm et de la protéine AQP4 ainsi que son immunolocalisation dans l'oviducte de poule au cours de la régression. L'expérience a été réalisée sur des poules pondeuses soumises à une pause de ponte induite par une privation alimentaire pendant 5 jours. Les poules témoins ont été nourries ad libitum. Les oiseaux ont été sacrifiés au jour 6 de l'expérience et tous les segments de l'oviducte ont été isolés, à savoir l'infundibulum, le magnum, l'isthme, la glande coquillière, et le vagin. Les expressions géniques et protéiques d'AQP4 dans ces tissus ont été testées respectivement par PCR en temps réel et Western blot. L'expression relative d'ARNm d'AQP4 était la plus élevée dans l'infundibulum et le vagin et la plus faible et la moins détectable dans le magnum. Le niveau de la protéine AQP4 était le plus élevé dans l'infundibulum et le plus bas dans le magnum. Le jeûne a entraîné une diminution de l'expression de l'ARNm AQP4 (P<0,001) dans l'infundibulum, une diminution de l'abondance des protéines (P<0,01) dans la glande coquillière et une augmentation du niveau de protéines (P<0,001) dans le vagin. L'immunohistochimie a démontré une localisation dépendante des tissus et des cellules de la protéine AQP4 dans la paroi oviductale. L'intensité de la coloration était la suivante : infundibulum > glande coquillière > vagin ≥ isthme ≫ magnum. Chez les poules témoins, l'immunoréactivité de l'AQP4 dans le vagin était similaire, tandis que dans d'autres segments oviductaux, l'immunoréactivité était plus forte par rapport aux poulets soumis à une pause de ponte. En résumé, ces résultats suggèrent que l'AQP4 est une protéine essentielle impliquée dans la régulation du transport de l'eau nécessaire pour créer un micro-environnement approprié pour la fécondation et la formation d'œufs dans l'oviducte de poule.

Expression of aquaporin 4 in the chicken oviduct following tamoxifen treatment.

This study was designed to examine whether aquaporin 4 (AQP4) is present in the chicken oviduct, and if so, whether its expression changes during pause in laying induced by tamoxifen (TMX; oestrogen receptor modulator) treatment. The control chickens were injected with a vehicle (ethanol) and the experimental ones with TMX at a dose of 6 mg/kg of body weight. Birds were treated daily until complete cessation of egg laying. The oviductal parts, that is the infundibulum, magnum, isthmus, shell gland and vagina were isolated from hens on day 8 of the experiment, and subsequently, the gene and protein expressions of AQP4 in tissues were examined by real-time PCR and Western blot, respectively. Immunohistochemical localization of AQP4 in the wall of the chicken oviduct was also investigated. Both mRNA and protein of AQP4 were found in all segments of the chicken oviduct. The relative expression [RQ] of AQP4 was the highest in the infundibulum and the vagina and the lowest, less detectable, in the magnum and isthmus. The pattern of AQP4 protein expression was similar to that of mRNA. Treatment of hens with TMX decreased the mRNA and protein levels of AQP4 in the oviduct. Immunohistochemistry demonstrated tissue and cell-dependent localization of AQP4 protein in the oviductal wall. The intensity of the immunopositive reaction was as follows: the infundibulum > vagina > shell gland ≥ isthmus >˃ magnum. In the control chickens, the immunoreactivity for AQP4 in all oviductal segments was stronger compared with the TMX-treated hens. The results obtained indicate that AQP4 takes part in the regulation of water transport required for the formation of egg in the chicken oviduct. Moreover, a relationship between oestrogen action and AQP4 gene and protein expression is suggested.

Large patternable metal nanoparticle sheets by photo/e-beam lithography.

Techniques for micro/nano-scale patterning of large metal nanoparticle sheets can potentially be used to realize high-performance photoelectronic devices because the sheets provide greatly enhanced electrical fields around the nanoparticles due to localized surface plasmon resonances. However, no single metal nanoparticle sheet currently exists with sufficient durability for conventional lithographical processes. Here, we report large photo and/or e-beam lithographic patternable metal nanoparticle sheets with improved durability by incorporating molecular cross-linked structures between nanoparticles. The cross-linked structures were easily formed by a one-step chemical reaction; immersing a single nanoparticle sheet consisting of core metals, to which capping molecules ionically bond, in a dithiol ethanol solution. The ligand exchange reaction processes were discussed in detail, and we demonstrated 20 µm wide line and space patterns, and a 170 nm wide line of the silver nanoparticle sheets.

Female-biased sex difference in vasotocin-immunoreactive neural structures in the developing quail brain.

The bed nucleus of the stria terminalis pars medialis (BSTM), medial preoptic nucleus (POM), and lateral septal region (LS) exhibit more vasotocin-immunoreactive (VT-ir) neural structures in male than in female adult quail. VT-ir cells and fibers in these regions are sensitive to gonadal steroids only in males. The insensitivity of adult female VT-ir neural structures to sex steroids is attributed to estradiol exposure during a critical period in embryonic life. Although the VT-ir system has been intensively examined in adult quail, information is limited in embryos and juveniles. Therefore, we herein investigated the development of VT-immunoreactive neural structures from embryonic day (E) 9 to adulthood with a particular focus on the BSTM, POM and LS of both sexes. VT-ir neural structures were more evident in female than in male embryos from E9 (BSTM and POM) and E11 (LS). This sex difference disappeared between E15 and post-hatch day 1 in the BSTM and POM, and during the first week of life in the LS. Male-biased sex differences in VT-ir structures appeared at puberty. Female-biased sexual dimorphism in the density of the VT-ir structures of BSTM was reflected by the stronger expression of VT mRNA in females than in males. However, the density of VT mRNA somata was comparable in the two sexes. The exposure of male embryos to estradiol resulted in the feminization of VT-ir neural structures in the BSTM, but not in the POM or LS at E11. Collectively, these results suggest that sex differences in VT-ir neural structures changes drastically throughout quail life. In embryos, endogenous estradiol may stimulate the expression of VT in females, resulting in a robust sex difference in VT-ir cells and fibers in favor of this sex.

Drug diffusion and biological responses of arteries using a drug-eluting stent with nonuniform coating.

The purpose of this study was to determine the effect of a nonuniform coating, abluminal-gradient coating (AGC), which leaves the abluminal surface of the curves and links parts of the stent free from the drug coating, on the diffusion direction of the drug and the biological responses of the artery to drug-eluting stent (DES) by comparing the AGC-sirolimus stent and the conventional full-surface coating (CFC) sirolimus stent. The study aimed to verify whether the AGC approach was appropriate for the development of a safer DES, minimizing the risks of stent thrombosis due to delayed endothelialization by the drug and distal embolization due to cracking of the coating layer on the hinge parts of the DES on stent expansion. In the in vitro local drug diffusion study, we used rhodamine B as a model drug, and rhodamine B released from the AGC stent diffused predominantly into the abluminal side of the alginate artery model. Conversely, rhodamine B released from the CFC stent quickly spread to the luminal side of the artery model, where endothelial cell regeneration is required. In the biological responses study, the luminal surface of the iliac artery implanted with the AGC-sirolimus stent in a rabbit iliac artery for 2 weeks was completely covered with endothelial-like cells. On the other hand, the luminal surface of the iliac artery implanted with the CFC-sirolimus stent for 2 weeks only showed partial coverage with endothelial-like cells. While thrombosis was observed in two of the three CFC-sirolimus stents, it was observed in only one of the three AGC-sirolimus stents. Taken together, these findings indicate that the designed nonuniform coating (AGC) is an appropriate approach to ensure a safer DES. However, the number of studies is limited and a larger study should be conducted to reach a statistically significant conclusion.

Clinical significance of hepatocyte growth factor/c-Met expression in the assessment of gastric cancer progression.

Among the mechanisms that control cancer progression, cell mobility is a significant factor required for cellular liberation from the primary focus and infiltration. Hepatocyte growth factor (HGF) has been shown to facilitate cell mobility. In the present study, the clinical significance of the HGF/c­Met pathway in the assessment of gastric cancer progression was evaluated. From a cohort of patients with gastric cancer who underwent surgical resection between April 1999 and March 2003, 110 subjects were randomly selected. Preoperative serum HGF levels were measured and various pathological factors were analyzed. Furthermore, 50 subjects were randomly selected from within this group and immunohistochemical staining of tissue preparations for HGF and its receptor c­Met were performed. In the infiltrative growth pattern [(INF)α,ß vs. INFγ], advanced progression was associated with elevated preoperative serum HGF levels (P<0.001). No correlation was identified between serum HGF levels and immunostaining for HGF or c­Met in the tissue preparations. Immunostaining revealed a significant correlation between c­Met expression and lymphatic vessel invasion (ly0.1 vs. 2.3; P=0.0416), lymph node metastasis (n0.1 vs. 2; P=0.0184) and maximum tumor diameter (≤50 mm vs. >50 mm; P=0.0469). Furthermore, c­Met­positivity was associated with a significant difference in overall survival (P=0.0342), despite stage I and II cases accounting for 82% of the total cohort (41 of 50 cases). These results suggested that the expression of the HGF/c­Met pathway in gastric cancer may be a potential predictive factor for disease progression.

Clinical significance of fibroblast growth factor (FGF) expression in colorectal cancer.

In this study, we serologically and pathologically examined the clinical significance of fibroblast growth factor (FGF) expression in patients with colorectal cancer. Serum basic FGF (bFGF) levels in 92 surgical colorectal cancer patients and 31 controls were measured, and the relationship between those levels and clinicopathological factors were examined. Immunohistochemical study was also conducted on specimens from 51 cancer patients, and the association between bFGF staining and serum levels were investigated. An examination of clinicopathological factors revealed significant differences in bFGF levels between stage 0-IIIb and stage IV cancers (P = 0.013). Lymphatic invasion was one factor that differed significantly. Patients with a tumor 30 mm or smaller had a bFGF level of 7.65 ± 1.11 pg/ml while patients with a tumor 31 mm or larger had a bFGF level of 8.53 ± 3.22 pg/ml; significant differences in these bFGF levels were noted (P < 0.05). Patients with a tumor that had no lymphatic invasion (ly0) had a bFGF level of 7.25 ± 0.66 pg/ml, those with a tumor that had minimal lymphatic invasion (ly1) had a bFGF level of 7.99 ± 1.68 pg/ml, and those with a tumor that had moderate lymphatic invasion (ly2) had a bFGF level of 9.17 ± 4.23 pg/ml. bFGF levels differed significantly for tumors with no/minimal lymphatic invasion (ly0-ly1) and those with moderate lymphatic invasion (ly2) (P < 0.0001). Serological examination of bFGF levels during the proliferation of colorectal cancer revealed that moderate lymphatic invasion can be readily distinguished.

Reflection and feedback in ambulatory education.

BACKGROUND: It has been argued that the best method of acquiring clinical reasoning is through seeing new out-patients. The purpose of this interventional study was to establish a clinical clerkship course for Japanese medical students in an out-patient care setting, with multiple opportunities for reflective practice and improving the clinical reasoning abilities of the students. The effectiveness of the course was also examined. METHODS: Students performed examinations of new patients and made diagnostic decisions in 20 minutes. They presented their case using the SNAPPS (Summarize the case, Narrow the differential, Analyze the differential, Probe the preceptor, Plan management, and Select an issue for self directed learning) method, and this was followed by feedback from faculty members using the 1-minute preceptor method and a mini clinical evaluation exercise (mini-CEX). Students' clinical reasoning abilities were assessed by the objective structured clinical examination (OSCE) and the script concordance test (SCT). Students' written comments and responses to an interview about the course were also analysed. Cross-sectional data were examined by comparing individual OSCE and SCT scores, and the multiple-choice question examination (MCQ) completed by students who did and did not participate in this project. RESULTS: Students in the programme had higher scores on the mini-CEX in all areas. The SCT and OSCE scores were also significantly higher than the scores for the control group. Students' comments about the course, which provided an opportunity for daily reflection, were positive. DISCUSSION: Students rapidly acquired clinical reasoning skills through reflective practice. Students also demonstrated motivation to learn through the examination of new patients. The clinical clerkship programme with multiple opportunities for reflective practice in an out-patient care setting substantially improved the clinical reasoning abilities of medical students.

Clinical significance of keratinocyte growth factor and K-sam gene expression in gastric cancer.

Although gastric cancer is increasingly being detected at an early stage of development, diffuse growth­type malignant tumors, such as scirrhous gastric cancer, are usually at an advanced stage at the time of diagnosis, resulting in poor treatment outcomes. The aim of this study was to determine whether the K-sam gene and keratinocyte growth factor (KGF) expression may be used to identify malignant tumors with a poor prognosis. K-sam and KGF expression was retrospectively evaluated in samples from 86 patients with early and advanced gastric cancer according to type, by examining serum levels and using immunohistochemical staining. The associations with clinicopathological characteristics and survival were also examined. The mean serum KGF levels were 11.191±3.808 pg/ml in early stage and 10.715±3.4991 pg/ml in advanced gastric cancer patients. KGF levels were significantly higher in types 4 and 5 (14.498±3.812 pg/ml, n=6) compared with types 1, 2 and 3 (10.747±3.571 pg/ml, n=80; P=0.028). Stage classification was identified as the only significant factor which determined overall survival. Patients with KGF-positive tumors had significantly higher serum KGF levels compared with those who had KGF-negative tumors. Patients with K-sam­positive tumors had significantly higher KGF levels compared with those who had K-sam-negative tumors. Pathological KGF expression was not significantly correlated with the degree of differentiation; however, there was a positive correlation between high K-sam expression in scirrhous gastric tumors and serum KGF levels. The present study revealed that high serum KGF levels are a risk factor for diffuse infiltrative gastric cancer and may provide a simple method of identifying patients with a poor prognosis among previously diagnosed preoperative gastric cancer patients.

The mechanism underlying the central glucagon-induced hyperglycemia and anorexia in chicks.

We investigated the mechanism underlying central glucagon-induced hyperglycemia and anorexia in chicks. Male 8-day-old chicks (Gallus gallus) were used in all experiments. Intracerebroventricular administration of glucagon in chicks induced hyperglycemia and anorexia from 30 min after administration. However, the plasma insulin level did not increase until 90 min after glucagon administration, suggesting that glucose-stimulated insulin secretion from pancreatic beta cells may be suppressed by central glucagon. The plasma corticosterone concentration significantly increased from 30 min to 120 min after administration, suggesting that central glucagon activates the hypothalamic pituitary adrenal (HPA) axis in chicks. However, central administration of corticotropin-releasing factor (CRF), which activates the HPA axis in chicken hypothalamus, significantly reduced not only food intake but also plasma glucose concentration, suggesting that CRF and the activation of the HPA axis are related to the glucagon-induced anorexia but not hyperglycemia in chicks. Phentolamine, an α-adrenergic receptor antagonist, significantly attenuated the glucagon-induced hyperglycemia, suggesting that glucagon induced hyperglycemia at least partly via α-adrenergic neural pathway. Co-administration of phentolamine and α-helical CRF, a CRF receptor antagonist, significantly attenuated glucagon-induced hyperglycemia and anorexia. It is therefore likely that central administration of glucagon suppresses food intake at least partly via CRF-induced anorexigenic pathway in chicks.

Inheritance and developmental pattern of cerebral hernia in the crested Polish chicken.

A number of crested chicken strains, such as the Polish chicken, exhibit a bony protuberance in the anterodorsal region of their skulls. The shape of their brain shows the anatomical peculiarity that is characterized by the upthrusting of cerebral hemispheres, called "cerebral hernia." Some early works suggested that this phenotype may be caused by a genetic factor and any modifiers influencing the development of brain and/or skull. However, the causative gene and its formation mechanism are still unclear. The present study is aimed to analyze the inheritance and ontogenic process of cerebral hernia in the crested Polish chicken. Firstly, we constructed the resource family with the Polish chicken and PNP inbred strain. Genetic analysis of this family revealed that cerebral hernia is controlled by a single autosomal recessive gene and is closely associated with crest formation. Furthermore, our morphological analysis of brain structures in the progenies suggested that the significant enlargement of brain cavity at later stages of embryos, particularly after 15 days of incubation, may be the main cause of cerebral hernia.

[Influence of impaired renal function and magnesium oxide administration on serum magnesium levels in elderly inpatients].

AIM: This study aimed to clarify whether serum magnesium (Mg) levels increased in elderly inpatients with impaired renal function receiving magnesium oxide (MgO) administration. METHODS: We recruited a total of 1,282 inpatients (505 men, 777 women, mean age 79.6 years) in this study. Fasting blood samples were obtained early in the morning. Serum Mg was measured using xylidyl blue method. Estimated glomerular filtration rate (eGFR) levels were calculated according to the formula for ethnic Japanese, inserting sex, age and serum creatinine (cr) levels into the formula. Inpatients were divided into 5 groups according to eGFR levels (ml/min/1.73 m(2)): <30 eGFR (group 1), ≥ 30 but <60 (group 2), ≥ 60 but <90 (group 3), ≥ 90 but <130 (group 4), and ≥ 130 (group 5). Division into a further 4 groups was also carried out, into the same groups (1-3) as described above and ≥ 90 (group 4). In these subgroups we investigated how serum Mg levels changed according to different eGFR levels, or after being given MgO. RESULTS: In 552 inpatients not given MgO and 372 given MgO, the percentages of subjects with ≥ 2.7 mg/dl of serum Mg were 38.5% in those not given MgO and 78.5% in those given MgO in group 1, 28.1% and 49%, respectively, in group 2, 0% and 23.1% to 29.6% in groups 3 to 5; the percentage of patients with < 2.4 mg/dl of serum Mg was higher in groups 1 to 5 in those not given MgO than in those given MgO. These findings suggest an increase in serum Mg levels after initiation of MgO administration. At an average of 6.9 months in 22 men and 6.4 months in 39 women, both groups not receiving MgO serum Mg increased significantly, while eGFR reduced considerably. At an average of 6.4 months in 18 men and 10 months in 30 women who received MgO, serum Mg increased considerably, although eGFR did not show any significant change. In 4 cases spanning 4 to 14 months, seesawing alterrations between eGFR and serum Mg were often noted. We measured subjects from the 4 subgroups (divided according to eGFR), comprising 88 inpatients not given MgO, 116 who were given daily doses of 0.5 g to 1.5 g MgO, and 118 who were given daily doses of 2 g to 3 g MgO. In those without MgO serum Mg was markedly higher in group 1 than in groups 3 and 4. In all 4 groups, serum Mg was markedly higher in those given MgO than in those not given MgO. In group 1 only, serum Mg was markedly higher in those given daily doses of 2 g to 3 g than in those given 0.5 g to 1.5 g MgO. In 23 subjects with serum Mg levels of over 3.8 mg/dl (normal range: 1.7 mg/dl to 2.6 mg/dl), 7 not given MgO had markedly lower eGFR levels than 16 given MgO, and the mean levels of serum Mg were similar among these. The highest levels of serum Mg were 5.2 mg/dl in those not given MgO and 5.9 mg/dl in those given MgO. CONCLUSION: The important factors associated with elevated serum Mg levels noted in this study were: a reduction in eGFR to below 30 ml/min/1.73 m(2), and MgO administration for treatment of chronic constipation and the simultaneous occurrence of the above two factors.

Immunolocalization of aquaporin-4 in the brain, kidney, skeletal muscle, and gastro-intestinal tract of chicken.

Aquaporin-4 (AQP4), a member of the water channel family, has high water permeability and multi-functional potentiality. Although an avian AQP4 homolog has recently been identified, its overall localization is still largely unknown. This study demonstrates the presence of AQP4 in several organs of chicken by using a specific chicken AQP4 antibody. Western blot analysis has revealed two bands of chicken AQP4 (30 and 32 kDa) in the brain, proventriculus, pectoral muscle, kidney, and ureter. The brain is the primary expression site of AQP4 in chicken. Immunohistochemical analysis of the brain has shown the highest AQP4 immunoreactivity around the cerebral ventricles, blood vessels, and the Purkinje cells. In peripheral organs, AQP4-immunoreactive elements have been observed in the ureter, glandular cells of the proventriculus, sarcolemma of the pectoral muscle, and the epithelium of the ceca and the rectum. Moreover, a heavily stained network of AQP4-immunoreactive fibers has been detected within the enteric plexuses.

Novel method of gene transfer in birds: intracytoplasmic sperm injection for green fluorescent protein expression in quail blastoderms.

This study was conducted to establish a new method of avian transgenesis by intracytoplasmic sperm injection (ICSI). First, we evaluated the fertilization ability of quail oocytes after microinjection of Triton X-100 (TX-100)-treated quail sperm with PLCZ cRNA. The quail oocytes were cultured for 24 h, and blastoderm development was examined by histological observation. The TX-100 treatment induced damage to the quail sperm membrane and interfered with fertilization of oocytes injected with sperm. On the other hand, when quail oocytes were injected with TX-100-treated sperm and PLCZ cRNA simultaneously, 43.5% (10/23) of the oocytes developed into blastoderms. This rate of development was comparable to that for oocytes injected with sperm without TX-100 treatment but with PLCZ cRNA (6 [42.9%] of 14). Second, we evaluated the rate of transduction of the enhanced green fluorescent protein (EGFP) gene in quail oocytes injected with TX-100-treated sperm and PLCZ cRNA. The EGFP expression was assessed by histological observation of fluorescence emission in the embryos. The intracytoplasmic injection of sperm without TX-100 treatment but with PLCZ cRNA and EGFP vector induced blastoderm development in 40% (4/10) of the oocytes, but those oocytes showed no fluorescence emission. In contrast, the intracytoplasmic injection of TX-100-treated sperm and PLCZ cRNA induced blastoderm development in 43.8% (7/16) of the oocytes, and, importantly, 85.7% (6/7) of oocytes showed fluorescence emission. In addition, PCR analysis detected GFP fragments in 50% (3/6) of GFP-expressing blastoderms. These results indicate that this ICSI method with additional treatments described herein may be the first step toward the production of transgenic birds.

Distribution and sex differences in aromatase-producing neurons in the brain of Japanese quail embryos.

The biochemical properties, neuroanatomical location, and function of aromatase (ARO), the enzyme that converts testosterone to 17beta-estradiol, have been studied extensively in the adult quail brain. Conversely, very little is known about ARO in quail embryos. This study investigated the distribution of ARO in quail prosencephalon at embryonic days (E) 9, 11, and 15 by immunocytochemistry. ARO-immunoreactive cells were observed within the walls of the cerebral ventricles, the ventral striatum, medial preoptic nucleus (POM), medial part of the bed nucleus of the stria terminalis (BSTM), lateral part of the BST, and in the tuberal region. The BSTM and to a lesser extent the POM showed transient, female-biased sex-differences. In the BSTM, the number of the ARO-immunoreactive cells, the fractional area covered by ARO-immunoreactive structures, and the overall extension of ARO-immunoreactivity were greater in females at E9 and E11, but these differences largely disappeared at E15 and post-hatch day 1. The sex differences were confirmed at the transcriptional level by in situ hybridization. In the lateral part of the POM, females showed slightly more ARO-immunoreactivity than males at E11. Treatment of E9 male embryos with estradiol completely feminized ARO-immunoreactivity at E11. The origins and the functional significance of these sex differences remain unknown.

TonEBP regulates hyperosmolality-induced arginine vasotocin gene expression in the chick (Gallus domesticus).

Arginine vasotocin (AVT) is expressed mainly in the paraventircular and supraoptic nuclei of the hypothalamus in chicken. This peptide is known to act as an antidiuretic hormone and its gene expression is stimulated by hyperosmolality. However, the transcription factors that regulate the AVT gene expression induced by hyperosmolality are still unknown. In this study, we examined the role of hyper-tonicity enhancer binding protein (TonEBP) in the transcriptional regulation of AVT gene in chicken. TonEBP mRNA expression levels increased at 1h after salt-loading treatment in the hypothalamus. This increase preceded that in AVT and c-fos mRNA expression. Intracerebroventricular injections of TonEBP antisense oligonucleotides, before the salt-loading treatment, prevented the increase in AVT gene expression. These results, all together, suggest that the transcription factor TonEBP may be involved in the regulation of AVT genes expression in response to a hyperosmotic environment in chicken.

Phospholipase Czeta mRNA expression and its potency during spermatogenesis for activation of quail oocyte as a sperm factor.

This study was conducted to investigate the role of a sperm-borne compound in oocyte activation in special reference to the time when oocyte activation is required by testicular cells during spermatogenesis in quail. First, effects of a microinjection of quail sperm extract (SE) and quail phospholipase Czeta (PLCzeta) cRNA into quail oocytes were assessed by observation of pronuclear formation and cytoplasmic segmentation, respectively. Secondly, the effects of a microinjection of round spermatids with or without PLCzeta cRNA into quail oocytes were studied by observation of development. When the oocytes were injected with SE at 0.13 mg protein/ml, both pronuclear formation and cytoplasmic segmentation were optimally induced. However, pronuclear formation was blocked when SE was pretreated with heat or when the oocyte was pretreated with BAPTA (a Ca(2+) chelator) before SE injection. On the other hand, when the oocytes were injected with PLCzeta cRNA at 60 microg/ml, not only pronuclear formation but also cytoplasmic segmentation were optimally induced. However, PLCzeta cRNA-induced pronuclear formation was blocked by pretreatment with cycloheximide (an inhibitor of protein synthesis) or with BAPTA. Most interestingly, round spermatids alone cannot induce blastodermal development but microinjection of a round spermatid with PLCzeta cRNA can induce development. In addition, RT-PCR revealed that PLCzeta mRNA is expressed in elongated spermatids and testicular sperm but not in round spermatids. It is concluded that PLCzeta is a functional sperm factor for oocyte activation to initiate resumption of meiotic division in quail and its potency is acquired after elongated spermatid formation during the spermatogenesis.