Automatic mapping of viable microbial cells distributed in the surface layer of cotton fabrics

Viable microbial cells distributed in a 130 microim thick surface layer of cotton fabrics were stained with a fluorescent glucose, 2- [N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] -2-deoxy-D-glucose (2-NBDG), and automatically mapped with an ultra-deep focusing range microscope (UDF) system. The software of the UDF system was upgraded and the number of Candida albicans cells could be counted at a higher precision than before. Bacterial cells of Pseudomonas fluorescens, Serratia marcescens, and Citrobacter freundii, which were smaller than 1-2 microm, were successfully mapped for the first time. These results indicate the practical importance of the present method in the evaluation of the antibacterial properties of fabrics and the efficacy of washing.     

Conditional Hypovascularization and Hypoxia in Islets Do Not Overtly Influence Adult β-Cell Mass or Function

It is generally accepted that vascularization and oxygenation of pancreatic islets are essential for the maintenance of an optimal β-cell mass and function and that signaling by vascular endothelial growth factor (VEGF) is crucial for pancreas development, insulin gene expression/secretion, and (compensatory) β-cell proliferation. A novel mouse model was designed to allow conditional production of human sFlt1 by β-cells in order to trap VEGF and study the effect of time-dependent inhibition of VEGF signaling on adult β-cell fate and metabolism. Secretion of sFlt1 by adult β-cells resulted in a rapid regression of blood vessels and hypoxia within the islets. Besides blunted insulin release, β-cells displayed a remarkable capacity for coping with these presumed unfavorable conditions: even after prolonged periods of blood vessel ablation, basal and stimulated blood glucose levels were only slightly increased, while β-cell proliferation and mass remained unaffected. Moreover, ablation of blood vessels did not prevent β-cell generation after severe pancreas injury by partial pancreatic duct ligation or partial pancreatectomy. Our data thus argue against a major role of blood vessels to preserve adult β-cell generation and function, restricting their importance to facilitating rapid and adequate insulin delivery.Tissue recombination and misexpression experiments revealed that endothelial cells are indispensable for ontogeny of the endocrine pancreas (1). Mice devoid of Kdr, the vascular endothelial growth factor (VEGF) receptor type 2, lack endothelial cells and show impaired expression of Ptf1a, a functional marker of pancreas progenitor cells, and absence of insulin and glucagon gene expression (2). Moreover, vascular density near the branching pancreatic epithelium appears crucial for pancreatic cell type specification during development (3–5). During development, endothelial cell signaling induces VEGF-A production by β-cells, thereby further increasing islet blood vessel density and permeability (6–9). In addition, islet blood vessels provide adult β-cells with a basement membrane that is essential for β-cell proliferation as well as glucose responsiveness of insulin production and secretion (8,10).Pdx1Cre/Vegf^fl/fl mice show decreased β-cell mass with a reduced density of insulin granules and impaired insulin gene expression and secretion, resulting in impaired glycemic control (8,11,12). RIPCre/Vegf^fl/fl mice possess normal β-cell mass but show a retarded glucose clearance and decreased glucose-induced insulin release (7,9), while insulin secretion from perifused transgenic islets was accelerated compared with wild-type islets (9). The above models unfortunately lack temporal control and can therefore not distinguish between the effects of VEGF signaling on β-cell mass and function in adult pancreas from those provoked in the developing pancreas. The current study describes a novel, conditional transgenic model to induce islet vessel regression to investigate the genuine role of the islet vasculature of adult mice with regard to β-cell mass and function.     

A case of Schneiderian middle-ear and sinonasal papilloma with intracranial complications

Unlike Schneiderian papilloma, a widespread benign epithelial neoplasm arising in the sinonasal tract mucosa of the nasal cavity, paranasal sinus, trachea, and larynx, middle-ear Schneiderian papilloma is extremely rare. We report a case of recurrent Schneiderian papilloma spreading to the bilateral middle ear and right paranasal sinus, and eventually causing cerebellar complications. A-52-year old woman seen for episodes of inarticulateness was first, found to have middle right ear and right ethmoid papilloma, that occurred thereafter is the middle left ear. This bilateral middle-ear papilloma is, to our knowledge, the only case reported thus far, and fell into a low-risk malignant formation group based on HPV-DNA testing. Given previous cases, we concluded that ours warranted meticulous follow-up because recurrence and malignancy are more common in multiple-site middle-ear papilloma as in our case rather than papilloma of the middle ear alone.     

Tracing phenotypic reversibility of pancreatic β‐cells in vitro

Aims/Introduction: Studies have suggested that pancreatic β‐cells undergo dedifferentiation during proliferation in vitro. However, due to limitations of the methodologies used, the question remains whether such dedifferentiated cells can redifferentiate into β‐cells. Materials and Methods: We have established a method for cell tracing in combination with fluorescence‐activated cell sorter (FACS). Using this method, mouse pancreatic β‐cells labeled with green fluorescent protein (GFP) under the control of the insulin promoter are collected by FACS. These β‐cells can be traced and characterized throughout the culture process, even when insulin becomes undetectable, because the cells are also marked with monomeric red fluorescent protein (mRFP) driven by the CAG promoter. Results: When cultured with fetal mouse pancreatic cells, FACS sorted β‐cells lost GFP expression, but retained mRFP expression. The cells also lost expressions of genes characteristic of the β‐cell phenotype, such as Pdx1 and glucokinase, indicating dedifferentiation. More than 30% of such dedifferentiated pancreatic β‐cells were detected in S or G2/M phase. Furthermore, these dedifferentiated cells redifferentiated into insulin‐expressing cells on cultivation with a MEK1/2 inhibitor. Conclusions: Our data provide direct evidence that pre‐existing β‐cells can undergo dedifferentiation and redifferentiation in vitro, their phenotype is reversible and that dedifferentiation in β‐cells is associated with progression of the cell cycle. (J Diabetes Invest, doi: 10.1111/j.2040‐1124.2010.00051.x, 2010)     

Up‐regulation of PSF1 promotes the growth of breast cancer cells

PSF1 is a subunit of the GINS complex that functions along with the MCM2‐7 complex and Cdc45 in eukaryotic DNA replication. Although mammalian PSF1 is predominantly expressed in highly proliferating cells and organs, little is known about the roles of PSF1 in mature cells or cancer cells. We found that PSF1 was expressed at relatively high levels in breast tumor cells, but at low levels in normal breast cells. Knockdown of PSF1 expression using small interfering RNA (siRNA) slowed the growth of breast cancer cell lines by delaying DNA replication but did not affect proliferation of normal human mammary epithelial cells. Reduced PSF1 expression also inhibited anchorage‐independent growth in breast cancer cell lines. These results suggest that PSF1 over‐expression is specifically involved in breast cancer cell growth. Therefore, PSF1 inhibition might provide new therapeutic approaches for breast cancer.     

Comparison of T cell subsets between somatic cloned and normal cow

PROBLEM: Somatic cloning technology is beneficial for genetically producing excellent animals. However, many developmental problems of somatically cloned animals have been described. Some of them may cause disorders of the immune system, resulting in the fluctuation of the proportion of white blood cells (WBC), different from that of normal animals in peripheral blood. METHOD OF STUDY: In Holstein- cloned and normal cows, the fluctuation of granulocytes, monocytes, B cells and T cells, and further T cell subsets (CD4+, CD8+, gammadelta, CD8+gammadelta and WC1+gammadelta T cell) in peripheral blood were analyzed in early lactation stage (ELS) and mid to late lactation stage (MLS) by flow cytometry using specific monoclonal antibodies for cell surface markers. RESULTS: In both ELS and MLS, there were no significant differences in the proportions of granulocytes, monocytes, B cells and T cells between cloned and normal cows. In T cell subsets, gammadelta and WC1+gammadelta T cells in cloned cows were significantly less frequent than in normal cows in ELS. The decreased proportions of gammadelta and WC1+gammadelta T cells recovered to the level of normal cows in MLS. CONCLUSIONS: The population of granulocytes, monocytes, B cells and T cells, and T cell subsets except for gammadelta and WC1+gammadelta T cells in cloned cows fluctuated in a manner similar to those of normal cows during lactation. In ELS, the proportions of gammadelta and WC1+gammadelta T cells temporarily declined in cloned cows, suggesting that cloned cows may fall into an immunosuppressive state in ELS.     

Two new indole alkaloids, 2-(3,3-dimethylprop-1-ene)-costaclavine and 2-(3,3-dimethylprop-1-ene)-epicostaclavine,from the marine-derived fungus Aspergillus fumigatus

Two new indole alkaloids, 2-(3,3-dimethylprop-1-ene)-costaclavine (1) and 2-(3,3-dimethylprop-1-ene)-epicostaclavine (2), together with the known compounds costaclavine (3), fumgaclavine A (4) and C (5), were isolated from the marine-derived fungus Aspergillus fumigatus. The planar structures of the two new compounds were elucidated on the basis of chemical and physicochemical evidence including MS, UV, IR and NMR spectra. Their stereochemistry was studied by NOESY, ¹H–¹H coupling constant and CD spectra. The compounds 1, 2, 3 and 5 showed weak cytotoxicity against a mouse leukemia cell line (P388).     

A CLCN1 mutation in dominant myotonia congenita impairs the increment of chloride conductance during repetitive depolarization

Myotonia congenita is caused by mutation of the CLCN1 gene, which encodes the human skeletal muscle chloride channel (ClC-1). The ClC-1 protein is a dimer comprised of two identical subunits each incorporating its own separate pore. However, the precise pathophysiological mechanism underlying the abnormal ClC-1 channel gating in some mutants is not fully understood. We characterized a ClC-1 mutation, Pro-480-Thr (P480T) identified in dominant myotonia congenita, by using whole-cell recording. P480T ClC-1 revealed significantly slowed activation kinetics and a slight depolarizing shift in the voltage-dependence of the channel gating. Wild-type/mutant heterodimers exhibited similar kinetic properties and voltage-dependency to mutant homodimers. Simulating myotonic discharge with the voltage clamp protocol of a 50 Hz train pulse, the increment of chloride conductance was impaired in both wild-type/mutant heterodimers and mutant homodimers, clearly indicating a dominant-negative effect. Our data showed that slow activation gating of P480T ClC-1 impaired the increment of chloride conductance during repetitive depolarization, thereby accentuating the chloride conductance reduction caused by a slight depolarizing shift in the voltage-dependence of the channel gating. This pathophysiology may explain the clinical features of myotonia congenita.