Human pluripotent stem cell-derived cells endogenously expressing follicle-stimulating hormone receptors: modeling the function of an inactivating receptor mutation.

Follicle-stimulating hormone (FSH) is crucial in the development and regulation of reproductive functions. The actions of human FSH and its receptor (FSHR) and mutations therein have mainly been studied using in vivo models, primary cells, cancer cells and cell lines ectopically expressing the FSHR. To allow studies of endogenous FSHR function in vitro, we differentiated FSHR-expressing cells from human pluripotent stem cells. FSH stimulation of the wild-type (WT), but not the inactivating Finnish founder mutant (A189V) receptor, activated the canonical cyclic adenosine monophosphate (cAMP)-dependent signaling pathway and downstream mediators. To investigate protein-protein interaction partners of FSHR at resting state and upon FSH stimulation, we expressed FSHR in HEK293 cells followed by affinity purification mass spectrometry analyses. We found 19 specific high-confidence interacting proteins for WT FSHR and 14 for A189V FSHR, several of which have been linked to infertility. Interestingly, while only WT FSHR interacted with FSH, insulin-like growth factor 1 receptor (IGF1R), for example, interacted with both WT and A189V FSHR upon FSH stimulation. In conclusion, our protocol allows detailed studies of FSH action and disease modeling in human cells endogenously expressing FSHR.

Filaggrin mutations in relation to skin barrier and atopic dermatitis in early infancy.

BACKGROUND: Loss-of-function mutations in the skin barrier gene filaggrin (FLG) increase the risk of atopic dermatitis (AD), but their role in skin barrier function, dry skin and eczema in infancy is unclear. OBJECTIVES: To determine the role of FLG mutations in impaired skin barrier function, dry skin, eczema and AD at 3 months of age and throughout infancy. METHODS: FLG mutations were analysed in 1836 infants in the Scandinavian population-based PreventADALL study. Transepidermal water loss (TEWL), dry skin, eczema and AD were assessed at 3, 6 and 12 months of age. RESULTS: FLG mutations were observed in 166 (9%) infants. At 3 months, carrying FLG mutations was not associated with impaired skin barrier function (TEWL > 11·3 g m-2  h-1 ) or dry skin, but was associated with eczema [odds ratio (OR) 2·89, 95% confidence interval (CI) 1·95-4·28; P < 0·001]. At 6 months, mutation carriers had significantly higher TEWL than nonmutation carriers [mean 9·68 (95% CI 8·69-10·68) vs. 8·24 (95% CI 7·97-8·15), P < 0·01], and at 3 and 6 months mutation carriers had an increased risk of dry skin on the trunk (OR 1·87, 95% CI 1·25-2·80; P = 0·002 and OR 2·44, 95% CI 1·51-3·95; P < 0·001) or extensor limb surfaces (OR 1·52, 95% CI 1·04-2·22; P = 0·028 and OR 1·74, 95% CI 1·17-2·57; P = 0·005). FLG mutations were associated with eczema and AD in infancy. CONCLUSIONS: FLG mutations were not associated with impaired skin barrier function or dry skin in general at 3 months of age, but increased the risk for eczema, and for dry skin on the trunk and extensor limb surfaces at 3 and 6 months.

Simultaneous integrated boost concepts in definitive radiation therapy for esophageal cancer: outcomes and toxicity.

BACKGROUND: Radiation therapy and chemoradiation therapy play a major role in the definitive management of esophageal cancer. Survival in esophageal cancer patients is still relatively poor, mostly due to high rates of local recurrence and distant metastases. It is hypothesized that dose escalation in radiotherapy could improve outcomes. Therefore, this retrospective analysis aimed to investigate the outcomes and toxicity in patients treated with local dose escalation by means of using simultaneous integrated boost concepts. METHODS: Between 2012 and 2018, 101 patients with esophageal carcinoma were analyzed in this monocentric, retrospective study. All patients received definitive chemoradiation or radiation therapy alone as intensity modulated radiotherapy. The prescribed dose was 50.4 Gy in 28 fractions to the primary tumor and the elective lymph nodes as well as a simultaneous integrated boost (SIB) with 58.8 Gy to macroscopic tumor and lymph node metastases. Endpoints were overall survival (OS), progression free survival (PFS), local control rate (LCR) and toxicity. RESULTS: 60 patients (59.4%) received chemoradiation, 41 patients (40.6%) radiotherapy alone. The median follow up was 17 months (range 0-75 months). OS, PFS and LCR were at 63.9%, 53.9% and 59.9% after 1 year and 37.6%, 34.5% and 36.1%, respectively after 3 years. 16 patients (15.8%) in total developed a locoregional recurrence within the field of radiation. In 48 patients (47.5%) at least one grade III° (CTCAE) toxicity was documented during radiotherapy, mostly dysphagia (36 pat., 75%). One patient suffered from a grade IV° pneumonia. CONCLUSION: This retrospective analysis demonstrates that a SIB concept in definitive (chemo)radiation therapy is safe and feasible, showing acceptable outcomes in this patient cohort. Considering that this cohort mainly consists of elderly patients not eligible for chemotherapy in many cases, we emphasize the aspect of SIB radiation therapy as potential partial compensation for omitted simultaneous chemotherapy. Prospective studies are needed for validation.

Production and characterization of chicken blood hydrolysate with antihypertensive properties.

Chicken blood has limited utilization despite its high protein content. Production of a blood hydrolysate exhibiting angiotensin I-converting enzyme (ACE)-inhibitory activity would be means of valorizing chicken blood. The optimized conditions used to produce chicken blood corpuscle hydrolysate (BCH) by Alcalase were 51.1°C, 4% enzyme, and pH 9.6 for 6 h, resulting in a 35.8% degree of hydrolysis and 37.7% ACE inhibition at a peptide concentration of 0.2 mg/mL. The permeate of a 1-kDa membrane, BCH-III, showed a 2.5-fold increase in ACE inhibition compared with that of BCH. BCH-III was resistant to in vitro gastrointestinal digestion, whereas the BCH digesta exhibited an increased ACE-inhibitory activity after digestion. Both BCH and BCH-III were rich in hydrophobic amino acids. A single administration of BCH and BCH-III to spontaneously hypertensive rats at concentrations of 600 and 100 mg/kg, respectively, lowered the systolic blood pressure by -57.7 and -70.9 mmHg, respectively, 6 h after oral administration compared with the control group. The blood pressure-lowering effect of the 600 mg/kg BCH dose was comparable with that of the 100 mg/kg BCH-III dose after 4 wk of oral administration. Both BCH and BCH-III could be developed for use as nutraceutical products with antihypertensive effects.

Severe hypoglycaemia is a major predictor of incident diabetic retinopathy in Japanese patients with type 2 diabetes.

AIM: Hypoglycaemia is a common complication in diabetes patients. However, its relationship with retinopathy has not been well documented in patients with type 2 diabetes (T2D). This study aimed to investigate the associations between hypoglycaemia and the incidence and progression of diabetic retinopathy (DR). METHODS: In this longitudinal cohort study, which was part of the Japan Diabetes Complications Study (JDCS), adult patients with T2D were recruited at 59 diabetes clinics across Japan. Their history of hypoglycaemia was assessed by standardized self-reported questionnaires. Severe hypoglycaemia was defined as having at least one episode with coma requiring an outpatients visit or hospitalization. Adjusted hazard ratios (HRs) for incidence and progression of DR over 8 years of follow-up were determined. RESULTS: Of 1221 patients without DR, 127 (10.4%) had experienced non-severe hypoglycaemia within the previous year, whereas 10 (0.8%) reported severe hypoglycaemia episodes. During the 8-year follow-up involving 8492 person-years, 329 patients developed DR. In 410 patients with prevalent DR, the adjusted HRs for incident DR were 4.35 (95% CI: 1.98-9.56; P<0.01) and, for progression of DR, 2.29 (95% CI: 0.45-11.78; P=0.32) with severe hypoglycaemia. CONCLUSION: Having a history of severe hypoglycaemia was one of the strongest predictors of incident DR in patients with T2D, with a fourfold increased risk. Identifying patients with greater risks of DR based on their history of hypoglycaemia may help to personalize risk evaluation in patients with diabetes.

Factors predicting successful discontinuation of continuous renal replacement therapy.

This multicentre, retrospective observational study was conducted from January 2010 to December 2010 to determine the optimal time for discontinuing continuous renal replacement therapy (CRRT) by evaluating factors predictive of successful discontinuation in patients with acute kidney injury. Analysis was performed for patients after CRRT was discontinued because of renal function recovery. Patients were divided into two groups according to the success or failure of CRRT discontinuation. In multivariate logistic regression analysis, urine output at discontinuation, creatinine level and CRRT duration were found to be significant variables (area under the receiver operating characteristic curve for urine output, 0.814). In conclusion, we found that higher urine output, lower creatinine and shorter CRRT duration were significant factors to predict successful discontinuation of CRRT.

Single-cell transcriptome analysis of endometrial tissue.

STUDY QUESTION: How can we study the full transcriptome of endometrial stromal and epithelial cells at the single-cell level? SUMMARY ANSWER: By compiling and developing novel analytical tools for biopsy, tissue cryopreservation and disaggregation, single-cell sorting, library preparation, RNA sequencing (RNA-seq) and statistical data analysis. WHAT IS KNOWN ALREADY: Although single-cell transcriptome analyses from various biopsied tissues have been published recently, corresponding protocols for human endometrium have not been described. STUDY DESIGN, SIZE, DURATION: The frozen-thawed endometrial biopsies were fluorescence-activated cell sorted (FACS) to distinguish CD13-positive stromal and CD9-positive epithelial cells and single-cell transcriptome analysis performed from biopsied tissues without culturing the cells. We studied gene transcription, applying a modern and efficient RNA-seq protocol. In parallel, endometrial stromal cells were cultured and global expression profiles were compared with uncultured cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: For method validation, we used two endometrial biopsies, one from mid-secretory phase (Day 21, LH+8) and another from late-secretory phase (Day 25). The samples underwent single-cell FACS sorting, single-cell RNA-seq library preparation and Illumina sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: Here we present a complete pipeline for single-cell gene-expression studies, from clinical sampling to statistical data analysis. Tissue manipulation, starting from disaggregation and cell-type-specific labelling and ending with single-cell automated sorting, is managed within 90 min at low temperature to minimize changes in the gene expression profile. The single living stromal and epithelial cells were sorted using CD13- and CD9-specific antibodies, respectively. Of the 8622 detected genes, 2661 were more active in cultured stromal cells than in biopsy cells. In the comparison of biopsy versus cultured cells, 5603 commonly expressed genes were detected, with 241 significantly differentially expressed genes. Of these, 231 genes were up- and 10 down-regulated in cultured cells, respectively. In addition, we performed a gene ontology analysis of the differentially expressed genes and found that these genes are mainly related to cell cycle, translational processes and metabolism. LIMITATIONS, REASONS FOR CAUTION: Although CD9-positive single epithelial cells sorting was successfully established in our laboratory, the amount of transcriptome data per individual epithelial cell was low, complicating further analysis. This step most likely failed due to the high dose of RNases that are released by the cells' natural processes, or due to rapid turnaround time or the apoptotic conditions in freezing- or single-cell solutions. Since only the cells from the late-secretory phase were subject to more focused analysis, further studies including larger sample size from the different time-points of the natural menstrual cycle are needed. The methodology also needs further optimization to examine different cell types at high quality. WIDER IMPLICATIONS OF THE FINDINGS: The symbiosis between clinical biopsy and the sophisticated laboratory and bioinformatic protocols described here brings together clinical diagnostic needs and modern laboratory and bioinformatic solutions, enabling us to implement a precise analytical toolbox for studying the endometrial tissue even at the single-cell level.

Changes in mouse gastrointestinal microbial ecology with ingestion of kale.

Kale, a cultivar of Brassica oleracea, has attracted a great deal of attention because of its health-promoting effects, which are thought to be exerted through modulation of the intestinal microbiota. The present study was performed to investigate the effects of kale ingestion on the gastrointestinal microbial ecology of mice. 21 male C57BL/6J mice were divided into three groups and housed in a specific pathogen-free facility. The animals were fed either a control diet or experimental diets supplemented with different commercial kale products for 12 weeks. Contents of the caecum and colon of the mice were processed for the determination of active bacterial populations by a bacterial rRNA-based quantification method and short-chain fatty acids by HPLC. rRNAs of Bacteroides-Prevotella, the Clostridium coccoides-Eubacterium rectale group, and Clostridium leptum subgroup constituted the major fraction of microbiota regardless of the composition of the diet. The ratio of Firmicutes to Bacteroidetes was higher in the colon samples of one of the kale diet groups than in the control. The colonic butyrate level was also higher with the kale-supplemented diet. Overall, the ingestion of kale tended to either increase or decrease the activity of specific bacterial groups in the mouse gastrointestinal tract, however, the effect might vary depending on the nutritional composition.

Identification and functional analysis of fructosyl amino acid-binding protein from Gram-positive bacterium Arthrobacter sp.

AIM: Fructosyl amino acid-binding protein (FABP) is a substrate-binding protein (SBP), which recognizes fructosyl amino acids (FAs) as its ligands. Although FABP has been shown as a molecular recognition tool of biosensing for glycated proteins, the availability of FABP is still limited and no FABP was reported from Gram-positive bacteria. In this study, a novel FABP from Gram-positive bacteria, Arthrobacter spp., was reported. METHOD AND RESULTS: BLAST analysis revealed that FABP homologues exist in some of Arthrobacter species genomes. An FABP homologue cloned from Arthrobacter sp. FV1-1, FvcA, contained a putative lipoprotein signal sequence, suggesting that it is a lipoprotein anchored to the bacterial cytoplasmic membrane, which is a typical characteristic for SBPs from Gram-positive bacteria. In contrast, FvcA also exhibits high amino acid sequence similarity to a known Gram-negative bacterial FABP, which exists as a free periplasmic protein. FvcA, without the N-terminal anchoring region, was then recombinantly produced as soluble protein and was found to exhibit Nα-FA-specific binding activity by intrinsic fluorescent measurement. CONCLUSION: This study identified a novel FABP from a Gram-positive bacterium, Arthrobacter sp., which exhibited Nα-FA-specific binding ability. This is the first report concerning an FABP from a Gram-positive bacterium, suggesting that FABP-dependent FA catabolism system is also present in Gram-positive bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel FABP exhibits the ability to specifically bind to Nα-FA with a high affinity. This selectivity is beneficial for applying FABP in HbA1c sensing. The successful preparation of water-soluble, functionally expressed Gram-negative bacterial FABP may make way for future applications for a variety of SBPs from Gram-positive bacteria employing the same expression strategy. The results obtained here enhance our understanding of bacterial FA catabolism and contribute to the improved development of FABP as Nα-FA-sensing molecules.

Epidermal growth factor receptor localized to exosome membranes as a possible biomarker for lung cancer diagnosis.

Detection of drug-target proteins and biomarkers that are expressed in cancer tissue has significant potential for both diagnosis and treatment of cancer. However, current immuno-histochemical and cytogenetic analyses of biopsy specimens for pre-operational diagnosis are highly invasive and often difficult to apply to lung cancer patients. The purpose of this study was to evaluate the possible utility of determining epidermal growth factor receptor (EGFR) expression on exosomal membranes using a targeted ELISA with an anti-CD81 antibody as a capture antibody for lung cancer diagnosis. While soluble EGFR (sEGFR) levels in plasma were not remarkably different between lung cancer patients and normal controls, significantly higher exosomal EGFR expression levels were observed in 5/9 cancer cases compared to normal controls. These results suggest that measurement of exosomal protein levels could be useful for in vitro diagnosis, and that exosomal EGFR is a possible biomarker for characterization of lung cancer.

Prevalence and characterization of Shiga toxin-producing Escherichia coli O157 and O26 in beef farms.

Rectal content grab samples were collected from 2436 beef cattle reared on 406 beef farms in Japan between November 2007 and March 2008. STEC strains O157 and O26 were isolated from 110 (27.1%) and 7 (1.7%) farms, respectively. Farms that tested positive for STEC O157 were located in 35 out of all 47 Japanese prefectures. This indicates that STEC O157 strains are widespread on beef farms nationwide. Of the 2436 tested beef cattle, 218 (8.9%) and 10 (0.4%) had STEC strains O157 and O26 in the rectal content, respectively. The most common Shiga toxin genes detected in the isolated STEC O157 strains were: stx(2c) alone (32.1%), stx(2)/stx(2c) (27.2%), and stx(1)/stx(2) (21.8%). Almost all of the STEC O157 and STEC O26 strains expressed Shiga toxins (Stx). Most of the STEC O157 and STEC O26 strains possessed eaeA and EHEC-hlyA. These results strongly suggest that STEC strains O157 and O26 from beef cattle would be pathogenic to humans. Therefore, it is important to reduce STEC strains O157 and O26 in beef cattle in order to prevent foodborne disease caused by STEC. The presence of dogs and/or cats on a farm was significantly (P=0.02) associated with the prevalence of STEC O157. More research is needed to clarify the role of dogs and cats.

Low transition rate from normo- and low microalbuminuria to proteinuria in Japanese type 2 diabetic individuals: the Japan Diabetes Complications Study (JDCS).

AIMS/HYPOTHESIS: The aim of the study was to determine the transition rate and factors associated with the progression of normo- and low microalbuminuria to diabetic nephropathy (overt proteinuria). METHODS: For 8 years we prospectively observed 1,558 Japanese patients with type 2 diabetes mellitus whose basal urinary albumin:creatinine ratio (UACR) had been measured as <17.0 mg/mmol at entry. The incidence of nephropathy (UACR >33.9 mg/mmol) was determined by measuring UACR twice a year. RESULTS: Progression to nephropathy occurred in 74 patients. The annual transition rate was 0.67%, and was substantially higher for the low-microalbuminuric group than for the normoalbuminuric group (1.85% and 0.23%, respectively; hazard ratio for the low-microalbuminuric group 8.45, p < 0.01). The hazard ratio for an HbA(1c) of 7-9% or ≥9% was 2.72 (p < 0.01) or 5.81 (p < 0.01) relative to HbA(1c) <7.0%, respectively. In comparison with individuals with a systolic blood pressure (SBP) of <120 mmHg, the hazard ratios for patients with an SBP of 120-140 mmHg or ≥140 mmHg were 2.31 (p = 0.06) and 3.54 (p < 0.01), respectively. Smoking also affected progression to proteinuria (hazard ratio 1.99, p < 0.01). In contrast, 30.3% of the low-microalbuminuric group returned to normoalbuminuria (i.e. were in remission). CONCLUSIONS/INTERPRETATION: These results suggest that if patients with type 2 diabetes mellitus are receiving treatment from diabetologists for hyperglycaemia and hypertension when they are in the early stages of nephropathy (i.e. normo- or low microalbuminuria), their rate of transition to proteinuria is considerably lowered, and that differentiating patients with low microalbuminuria from those with high microalbuminuria might be clinically useful. TRIAL REGISTRATION: UMIN Clinical Trials Registry C000000222.

Office and ambulatory pulse pressure--association with clinical characteristics and cardiovascular risk factors in normoalbuminuric patients with type 2 diabetes (ROADMAP study).

To investigate the association of office and ambulatory 24-h pulse pressure (PP) with clinical characteristics and cardiovascular risk factors in normoalbuminuric type 2 diabetic patients enrolled to the Randomised Olmesartan and Diabetes Microalbuminuria Prevention study, 4449 patients (2054 male and 2395 female; mean age 57.7±8.7 years) with type 2 diabetes, normoalbuminuria and at least one additional cardiovascular risk factor were included into the analysis. After adjustment by age, there were significant correlations between office PP and presence of hypertension (r=0.24; P<0.001), presence of cardiac and vascular disorders (r=0.17; P<0.001), metabolic syndrome (r=0.10; P<0.001), duration of diabetes (r=0.09; P<0.001), fasting blood glucose (r=0.08; P<0.001), albumin/creatinine ratio (r=0.07; P<0.001), insulin treatment, glycosylated haemoglobin (HbA1c), male gender and current smoking. In the subgroup of 1234 patients with ambulatory blood pressure measurement performed, ambulatory PP adjusted for office PP correlated with fasting blood glucose (r=0.16; P<0.001), metabolic syndrome (r=0.14; P<0.001), albumin/creatinine ratio (r=0.11; P<0.001) and indices of glycemic control (HbA1c: r=0.11; P<0.001). In this group of normoalbuminuric type 2 diabetic patients, office and ambulatory PP were associated with duration of diabetes, indices of glycemic control and cardiovascular risk factors. There was relationship between office and ambulatory PP and albuminuria even within normal albuminuria range.

Risk factors for Campylobacter colonization in broiler flocks in Japan.

The present study was conducted to estimate the prevalence of and to identify the risk factors for Campylobacter colonization in broiler flocks in Japan. Campylobacter colonization status in flock was evaluated by culturing pooled caecal excrement from 124 broiler flocks. Potential exposure to risk factors was evaluated with a questionnaire for the broiler producers. Odds ratios (ORs) were calculated using a multivariable logistic regression analysis. The prevalence of Campylobacter-positive flocks was 43.5% (upper and lower limits of 95% confidence interval (CI(95%) ): 34.8, 52.3). Multivariable logistic regression model identified two variables as risk factors for Campylobacter colonization. The ORs of Campylobacter colonization were higher in flocks in western Japan (OR=2.68; CI(95%) : 1.04, 6.91) than in eastern Japan, and in flocks supplied with undisinfected drinking water (OR=7.41; CI(95%) : 3.11, 17.66) than in those supplied with disinfected drinking water. These findings indicate that water may play an important role in Campylobacter colonization in broiler flocks in Japan and the use of disinfected water may reduce the risk of Campylobacter colonization.

Integration of cap analysis of gene expression and chromatin immunoprecipitation analysis on array reveals genome-wide androgen receptor signaling in prostate cancer cells.

The androgen receptor (AR) is a critical transcriptional factor that contributes to the development and the progression of prostate cancer (PCa) by regulating the transcription of various target genes. Genome-wide screening of androgen target genes provides useful information to understand a global view of AR-mediated gene network in PCa. In this study, we performed 5'-cap analysis of gene expression (CAGE) to determine androgen-regulated transcription start sites (TSSs) and chromatin immunoprecipitation (ChIP) on array (ChIP-chip) analysis to identify AR binding sites (ARBSs) and histone H3 acetylated (AcH3) sites in the human genome. CAGE determined 13 110 distinct, androgen-regulated TSSs (P<0.01), and ChIP-chip analysis identified 2872 androgen-dependent ARBSs (P<1e-5) and 25 945 AcH3 sites (P<1e-4). Both androgen-regulated coding genes and noncoding RNAs, including microRNAs (miRNAs) were determined as androgen target genes. Besides prototypic androgen-regulated TSSs in annotated gene promoter regions, there are many androgen-dependent TSSs that are widely distributed throughout the genome, including those in antisense (AS) direction of RefSeq genes. Several pairs of sense/antisense promoters were newly identified within single RefSeq gene regions. The integration of CAGE and ChIP-chip analyses successfully identified a cluster of androgen-inducible miRNAs, as exemplified by the miR-125b-2 cluster on chromosome 21. Notably, the number of androgen-upregulated genes was larger in LNCaP cells treated with R1881 for 24 h than for 6 h, and the percentage of androgen-upregulated genes accompanied with adjacent ARBSs was also much higher in cells treated with R1881 for 24 h than 6 h. On the basis of the Oncomine database, the majority of androgen-upregulated genes containing adjacent ARBSs and CAGE tag clusters in our study were previously confirmed as androgen target genes in PCa. The integrated high-throughput genome analyses of CAGE and ChIP-chip provide useful information for elucidating the AR-mediated transcriptional network that contributes to the development and progression of PCa.

Salmonella prevalence in commercial raw shell eggs in Japan: a survey.

We examined 20 300 raw shell chicken eggs sold at retail stores in Japan for Salmonella outside and inside eggs. The eggs were purchased at 220 retail stores throughout Japan between August 2007 and January 2008. Of 2030 pooled egg samples (10 eggs/sample), Salmonella was isolated from five shell samples (0.25%), but not from any of egg-content samples. The serovars of the isolates were Salmonella Enteritidis (2), S. Derby, S. Livingstone and S. Cerro. The samples positive for Salmonella originated from five different egg grading and packaging (GP) centres. All the GP centres washed their egg shells according to government guidelines for hygienic practice in GP centres. Thus, practical control measures at GP centres need to be reviewed and implemented to diminish Salmonella prevalence of egg shells because Salmonella contamination on eggs is a potential hazard for foodborne salmonellosis in Japan.

miR-148a is an androgen-responsive microRNA that promotes LNCaP prostate cell growth by repressing its target CAND1 expression.

Recent advances in cancer biology reveal that microRNAs (miRNAs) are involved in the regulation of cancer-related genes, or they function as tumor suppressors or oncogenes. In prostate cancer, evidence has accumulated for the contribution of the androgen-dependent gene network to tumor growth, although the precise functions of miRNAs in prostate cancer remain to be investigated. Here, we identified androgen-responsive miRNAs by the short RNA sequencing analysis in LNCaP prostate cancer cells. Among 10 miRNAs with known sequences, we have determined that miR-148a reduces the expression of cullin-associated and neddylation-dissociated 1 (CAND1), a negative regulator of SKP1-Cullin1-F-box (SCF) ubiquitin ligases, by binding to the 3'-untranslated region of CAND1 mRNA. CAND1 knockdown by small interfering RNA promoted the proliferation of LNCaP cells. Our study indicates the potential contribution of miR-148a to the growth of human prostate cancer.

Intracerebroventricular injection of orexin-A, but not orexin-B, induces arousal of layer-type neonatal chicks.

In order to determine if orexins affect arousal in neonatal chicks, we intracerebroventricularly (ICV) injected either orexin-A or orexin-B to layer and broiler type chicks (Gallus gallus) and measured their behaviors and food intake following injection. Layer chicks treated with orexin-A at 0.2 and 2.0 nmol had increased arousal but their food intake was not affected. However, arousal was not affected in broiler chicks treated with orexin-A, but they spent less time feeding. When orexin-B was administered to layer and broiler chicks, neither had altered arousal and their food intake was not affected. Therefore, the orexin peptides may differentially affect arousal in the two stocks tested; orexin-A causes a stock dependant increase whereas orexin-B does not affect either.

Molecular cloning and localization of the luteinizing hormone beta subunit and glycoprotein hormone alpha subunit from Japanese anchovy Engraulis japonicus.

Although Clupeiformes contain many economically important species, there is limited information on their reproductive physiology. To obtain more insight into reproductive mechanisms in clupeiform fishes, molecular cloning of the Japanese anchovy Engraulis japonicus luteinizing hormone beta (LHbeta) and glycoprotein hormone alpha (GPHalpha) subunits, and immunocytochemistry of gonadotrophs in the pituitary using antisera raised against the synthetic peptides for both subunits were carried out. The cDNAs for LHbeta and GPHalpha subunits consisted of 963 and 535 nucleotides encoding 141 and 122 amino acids, respectively. The deduced amino acid sequences of the E. japonicus LHbeta subunit showed a 60% similarity to the Pacific herring Clupea pallasii LHbeta subunit and 24-31% similarities to FSHbeta subunits of other fish species. The E. japonicus GPHalpha subunit showed 52-57% similarities to anguilliform and cypriniform GPHalpha subunits. Both the subunits have typical structural characteristics of each subunit such as N-linked glycosylation sites, conserved cysteine residues and highly conserved short amino acid sequences. These results indicate that cDNAs cloned in this study encode the E. japonicus LHbeta and GPHalpha subunits. Reverse-transcription polymerase chain reaction (RT-PCR) revealed that both the LHbeta and GPHalpha subunit genes were abundantly expressed in the pituitary, and the GPHalpha subunit was observed to be weakly expressed in the extrapituitary tissues. Immunocytochemistry of the E. japonicus pituitary showed that cells that immunoreacted with antiserum against the LHbeta subunit were distributed in the peripheral regions of proximal pars distalis, and these cells were also immunoreactive to antiserum against the GPHalpha subunit. An abundant number of both LHbeta and GPHalpha cells in the pituitary of matured fish were observed, in comparison with immature fish. These results indicate that the E. japonicus LH is involved in the final reproductive maturation as well as those of other teleosts.