Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (Arctium lappa L.)

<p>Abstract</p> <p>Background</p> <p>We have previously reported on the variation of total fructooligosaccharides (FOS), total inulooligosaccharides (IOS) and inulin in the roots of burdock stored at different temperatures. During storage at 0°C, an increase of FOS as a result of the hydrolysis of inulin was observed. Moreover, we suggested that an increase of IOS would likely be due to the synthesis of the IOS by fructosyltransfer from 1-kestose to accumulated fructose and elongated fructose oligomers which can act as acceptors for fructan:fructan 1-fructosyltransferase (1-FFT). However, enzymes such as inulinase or fructan 1-exohydorolase (1-FEH) involved in inulin degradation in burdock roots are still not known. Here, we report the isolation and functional analysis of a gene encoding burdock 1-FEH.</p> <p>Results</p> <p>A cDNA, named <it>aleh1</it>, was obtained by the RACE method following PCR with degenerate primers designed based on amino-acid sequences of FEHs from other plants. The <it>aleh1 </it>encoded a polypeptide of 581 amino acids. The relative molecular mass and isoelectric point (<it>pI</it>) of the deduced polypeptide were calculated to be 65,666 and 4.86. A recombinant protein of <it>aleh1 </it>was produced in <it>Pichia pastoris</it>, and was purified by ion exchange chromatography with DEAE-Sepharose CL-6B, hydrophobic chromatography with Toyopearl HW55S and gel filtration chromatography with Toyopearl HW55S. Purified recombinant protein showed hydrolyzing activity against β-2, 1 type fructans such as 1-kestose, nystose, fructosylnystose and inulin. On the other hand, sucrose, neokestose, 6-kestose and high DP levan were poor substrates.</p> <p>The purified recombinant protein released fructose from sugars extracted from burdock roots. These results indicated that <it>aleh1 </it>encoded 1-FEH.</p

Tissue distribution and differential expression of melanocortin 1 receptor, a malignant melanoma marker

The melanocortin 1 receptor is a G-protein-coupled receptor, described to be expressed on melanomas and melanocytes. Subsequent RT–PCR studies demonstrated the presence of melanocortin 1 receptor mRNA in other tissues such as pituitary gland and testis. Previously, we have demonstrated that three HLA-A2 binding nonamer peptides derived from melanocortin 1 receptor can elicit peptide-specific CTL which can recognize target cells transfected with the melanocortin 1 receptor gene and MHC class I matched melanoma lines. The potential of targeting melanocortin 1 receptor in therapy and diagnosis will depend on a preferential expression of this receptor in the majority of primary and metastatic melanomas vs normal tissues. We tested a panel of melanomas, carcinomas and other cell lines for the presence of melanocortin 1 receptor, using two monoclonal antibodies. The receptor was detected in 83% of the tested melanoma cell lines but not in other carcinoma lines. Immunohistochemistry revealed a strong expression of melanocortin 1 receptor in all tested primary and metastatic melanomas, but also demonstrated low levels of expression in adrenal medulla, cerebellum, liver and keratinocytes. Flow cytometry studies showed that melanocortin 1 receptor was expressed in in vitro activated monocytes/macrophages and in the THP-1 monocytic leukaemia line at levels of about 1 in 3 to 1 in 5 of that found in melanomas. Peripheral blood-derived dendritic cells, also express melanocortin 1 receptor in vitro. This extensive analysis of melanocortin 1 receptor tissue distribution may be of relevance not only for melanoma immunology, but also for research on the pathogenicity of inflammatory conditions in the skin and neurologic tissues. It remains to be seen if the over-expression of melanocortin 1 receptor in melanomas is sufficiently high to allow a ‘therapeutic window’ to be exploited in cancer immunotherapy

Multiple barriers against successful care provision for depressed patients in general internal medicine in a Japanese rural hospital: a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>A general internist has an important role in primary care, especially for the elderly in rural areas of Japan. Although effective intervention models for depressed patients in general practice and primary care settings have been developed in the US and UK medical systems, there is little information regarding even the recognition rate and prescription rate of psychotropic medication by general internists in Japan. The present study surveyed these data cross-sectionally in a general internal medicine outpatient clinic of a Japanese rural hospital.</p> <p>Methods</p> <p>Patients were consecutively recruited and evaluated for major depressive disorder or any mood disorder using the Patient Health Questionnaire (PHQ). Physicians who were blinded to the results of the PHQ were asked to diagnose whether the patients had any mental disorders, and if so, whether they had mood disorders or not. Data regarding prescription of psychotropic medicines were collected from medical records.</p> <p>Results</p> <p>Among 312 patients, 27 (8.7%) and 52 (16.7%) were identified with major depressive disorder and any mood disorder using the PHQ, respectively. Among those with major depressive disorder, 21 (77.8%) were recognized by physicians as having a mental disorder, but only three (11.1%) were diagnosed as having a mood disorder.</p> <p>Only two patients with major depressive disorder (7.4%) had been prescribed antidepressants. Even among those (n = 15) whom physicians diagnosed with a mood disorder irrespective of the PHQ results, only four (26.7%) were prescribed an antidepressant.</p> <p>Conclusions</p> <p>Despite a high prevalence of depression, physicians did not often recognize depression in patients. In addition, most patients who were diagnosed by physicians as having a mood disorder were not prescribed antidepressants. Multiple barriers to providing appropriate care for depressed patients exist, such as recognizing depression, prescribing appropriate medications, and appropriately referring patients to mental health specialists.</p

TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors.

The genomic regulatory programmes that underlie human organogenesis are poorly understood. Pancreas development, in particular, has pivotal implications for pancreatic regeneration, cancer and diabetes. We have now characterized the regulatory landscape of embryonic multipotent progenitor cells that give rise to all pancreatic epithelial lineages. Using human embryonic pancreas and embryonic-stem-cell-derived progenitors we identify stage-specific transcripts and associated enhancers, many of which are co-occupied by transcription factors that are essential for pancreas development. We further show that TEAD1, a Hippo signalling effector, is an integral component of the transcription factor combinatorial code of pancreatic progenitor enhancers. TEAD and its coactivator YAP activate key pancreatic signalling mediators and transcription factors, and regulate the expansion of pancreatic progenitors. This work therefore uncovers a central role for TEAD and YAP as signal-responsive regulators of multipotent pancreatic progenitors, and provides a resource for the study of embryonic development of the human pancreas

Multiple domains in the Crumbs Homolog 2a (Crb2a) protein are required for regulating rod photoreceptor size

Background Vertebrate retinal photoreceptors are morphologically complex cells that have two apical regions, the inner segment and the outer segment. The outer segment is a modified cilium and is continuously regenerated throughout life. The molecular and cellular mechanisms that underlie vertebrate photoreceptor morphogenesis and the maintenance of the outer segment are largely unknown. The Crumbs (Crb) complex is a key regulator of apical membrane identity and size in epithelia and in Drosophila photoreceptors. Mutations in the human gene CRUMBS HOMOLOG 1 (CRB1) are associated with early and severe vision loss. Drosophila Crumbs and vertebrate Crb1 and Crumbs homolog 2 (Crb2) proteins are structurally similar, all are single pass transmembrane proteins with a large extracellular domain containing multiple laminin- and EGF-like repeats and a small intracellular domain containing a FERM-binding domain and a PDZ-binding domain. In order to begin to understand the role of the Crb family of proteins in vertebrate photoreceptors we generated stable transgenic zebrafish in which rod photoreceptors overexpress full-length Crb2a protein and several other Crb2a constructs engineered to lack specific domains. Results We examined the localization of Crb2a constructs and their effects on rod morphology. We found that only the full-length Crb2a protein approximated the normal localization of Crb2a protein apical to adherens junctions in the photoreceptor inner segment. Several Crb2a construct proteins localized abnormally to the outer segment and one construct localized abnormally to the cell body. Overexpression of full-length Crb2a greatly increased inner segment size while expression of several other constructs increased outer segment size. Conclusions Our observations suggest that particular domains in Crb2a regulate its localization and thus may regulate its regionalized function. Our results also suggest that the PDZ-binding domain in Crb2a might bring a protein(s) into the Crb complex that alters the function of the FERM-binding domain

Influence of socioeconomic factors on medically unnecessary ambulance calls

<p>Abstract</p> <p>Background</p> <p>Unnecessary ambulance use has become a socioeconomic problem in Japan. We investigated the possible relations between socioeconomic factors and medically unnecessary ambulance calls, and we estimated the incremental demand for unnecessary ambulance use produced by socioeconomic factors.</p> <p>Methods</p> <p>We conducted a self-administered questionnaire-based survey targeting residents of Yokohama, Japan. The questionnaire included questions pertaining to socioeconomic characteristics, dichotomous choice method questions pertaining to ambulance calls in hypothetical nonemergency situations, and questions on the city's emergency medical system. The probit model was used to analyze the data.</p> <p>Results</p> <p>A total of 2,029 out of 3,363 targeted recipients completed the questionnaire (response rate, 60.3%). Probit regression analyses showed that several demographic and socioeconomic factors influence the decision to call an ambulance. Male respondents were more apt than female respondents to state that they would call an ambulance in nonemergency situations (p < 0.05). Age was an important factor influencing the hypothetical decision to call an ambulance (p < 0.05); elderly persons were more apt than younger persons to state that they would call an ambulance. Possession of a car and hesitation to use an ambulance negatively influenced the hypothetical decision to call an ambulance (p < 0.05). Persons who do not have a car were more likely than those with a car to state that they would call an ambulance in unnecessary situations.</p> <p>Conclusion</p> <p>Results of the study suggest that several socioeconomic factors, i.e., age, gender, household income, and possession of a car, influence a person's decision to call an ambulance in nonemergency situations. Hesitation to use an ambulance and knowledge of the city's primary emergency medical center are likely to be important factors limiting ambulance overuse. It was estimated that unnecessary ambulance use is increased approximately 10% to 20% by socioeconomic factors.</p

Fluorescent Transgenic Zebrafish Tg(nkx2.2a:mEGFP) Provides a Highly Sensitive Monitoring Tool for Neurotoxins

10.1371/journal.pone.0055474PLoS ONE82

Diagnostic Dilemma in a Patient with Jaundice: How to Differentiate between Autoimmune Pancreatitis, Primary Sclerosing Cholangitis and Pancreas Carcinoma

A 68-year-old male patient was referred to our institution in May 2011 for a suspected tumor in the pancreatic head with consecutive jaundice. Using magnetic resonance imaging, further differentiation between chronic inflammation and a malignant process was not possible with certainty. Apart from cholestasis, laboratory studies showed increased values for CA 19-9 to 532 U/ml (normal <37 U/ml) and hypergammaglobulinemia (immunoglobulin G, IgG) of 19.3% (normal 8.0–15.8%) with an elevation of the IgG4 subtype to 2,350 mg/l (normal 52–1,250 mg/l). Endoscopic retrograde cholangiopancreatography revealed a prominent stenosis of the distal ductus hepaticus communis caused by pancreatic head swelling and also a bihilar stenosis of the main hepatic bile ducts. Cytology demonstrated inflammatory cells without evidence of malignancy. Under suspicion of autoimmune pancreatitis with IgG4-associated cholangitis, immunosuppressive therapy with steroids and azathioprine was started. Follow-up endoscopic retrograde cholangiopancreatography after 3 months displayed regressive development of the diverse stenoses. Jaundice had disappeared and blood values had returned to normal ranges. Moreover, no tumor of the pancreatic head was present in the magnetic resonance control images. Due to clinical and radiological similarities but a consecutive completely different prognosis and therapy, it is of fundamental importance to differentiate between pancreatic cancer and autoimmune pancreatitis. Especially, determination of serum IgG4 levels and associated bile duct lesions induced by inflammation should clarify the diagnosis of autoimmune pancreatitis and legitimate immunosuppressive therapy

Functional Analysis of Conserved Motifs in Influenza Virus PB1 Protein

The influenza virus RNA polymerase complex is a heterotrimer composed of the PB1, PB2, and PA subunits. PB1, the catalytic core and structural backbone of the polymerase, possesses four highly conserved amino acid motifs that are present among all viral RNA-dependent RNA polymerases. A previous study demonstrated the importance of several of these conserved amino acids in PB1 for influenza polymerase activity through mutational analysis. However, a small number of viruses isolated in nature possesses non-consensus amino acids in one of the four motifs, most of which have not been tested for their replicative ability. Here, we assessed the transcription/replication activities of 25 selected PB1 mutations found in natural isolates by using minireplicon assays in human and avian cells. Most of the mutations tested significantly reduced polymerase activity. One exception was mutation K480R, observed in several pandemic (H1N1) 2009 viruses, which slightly increased polymerase activity relative to wild-type. However, in the background of the pandemic A/California/04/2009 (H1N1) virus, this mutation did not affect virus titers in cell culture. Our results further demonstrate the functional importance of the four conserved PB1 motifs in influenza virus transcription/replication. The finding of natural isolates with non-consensus PB1 motifs that are nonfunctional in minireplicon assays suggests compensatory mutations and/or mixed infections which may have ‘rescued’ the inactive PB1 protein