Voltage‐gated K+ channels play a role in cAMP‐stimulated neuritogenesis in mouse neuroblastoma N2A cells

Neuritogenesis is essential in establishing the neuronal circuitry. An important intracellular signal causing neuritogenesis is cAMP. In this report, we showed that an increase in intracellular cAMP stimulated neuritogenesis in neuroblastoma N2A cells via a PKA‐dependent pathway. Two voltage‐gated K⁺ (Kv) channel blockers, 4‐aminopyridine (4‐AP) and tetraethylammonium (TEA), inhibited cAMP‐stimulated neuritogenesis in N2A cells in a concentration‐dependent manner that remarkably matched their ability to inhibit Kv currents in these cells. Consistently, siRNA knock down of Kv1.1, Kv1.4, and Kv2.1 expression reduced Kv currents and inhibited cAMP‐stimulated neuritogenesis. Kv1.1, Kv1.4, and Kv2.1 channels were expressed in the cell bodies and neurites as shown by immunohistochemistry. Microfluorimetric imaging of intracellular [K⁺] demonstrated that [K⁺] in neurites was lower than that in the cell body. We also showed that cAMP‐stimulated neuritogenesis may not involve voltage‐gated Ca²⁺ or Na⁺ channels. Taken together, the results suggest a role of Kv channels and enhanced K⁺ efflux in cAMP/PKA‐stimulated neuritogenesis in N2A cells. J. Cell. Physiol. 226: 1090–1098, 2011. © 2010 Wiley‐Liss, Inc.     

Generation of mice with a novel conditional null allele of the <Emphasis Type="Italic">Sox9</Emphasis> gene

Sox9 is expressed in multiple tissues during mouse development and adulthood. Mutations in the Sox9 gene or changes in expression levels can be attributed to many congenital diseases. Heterozygous loss-of-function mutations in the human SOX9 gene cause Campomelic dysplasia, a semi-lethal skeletal malformation syndrome. Disruption of Sox9 by conventional gene targeting leads to perinatal lethality in heterozygous mice, hence hampering the feasibility to obtain the homozygous Sox9 null mice for in vivo functional studies. In this study, we generated a conditional allele of Sox9 (Sox9 ^tm4.Tlu ) by flanking exon 1 with loxP sites. Homozygous mice for the Sox9 ^tm4.Tlu allele (Sox9 ^flox/flox ) are viable, fertile and indistinguishable from wildtype (WT) mice, indicating that the Sox9 ^tm4.Tlu allele is a fully functional Sox9 allele. Furthermore, we demonstrated that Cre-mediated recombination using a Col2a1-Cre line resulted in specific ablation of Sox9 activity in cartilage tissues.     

The non-catalytic carboxyl-terminal domain of ARFGAP1 regulates actin cytoskeleton reorganization by antagonizing the activation of Rac1

Background

The regulation of the actin cytoskeleton and membrane trafficking is coordinated in mammalian cells. One of the regulators of membrane traffic, the small GTP-binding protein ARF1, also activates phosphatidylinositol kinases that in turn affect actin polymerization. ARFGAP1 is a GTPase activating protein (GAP) for ARF1 that is found on Golgi membranes. We present evidence that ARFGAP1 not only serves as a GAP for ARF1, but also can affect the actin cytoskeleton.

Principal Findings

As cells attach to a culture dish foci of actin appear prior to the cells flattening and spreading. We have observed that overexpression of a truncated ARFGAP1 that lacks catalytic activity for ARF, called GAP273, caused these foci to persist for much longer periods than non-transfected cells. This phenomenon was dependent on the level of GAP273 expression. Furthermore, cell spreading after re-plating or cell migration into a previously scraped area was inhibited in cells transfected with GAP273. Live cell imaging of such cells revealed that actin-rich membrane blebs formed that seldom made protrusions of actin spikes or membrane ruffles, suggesting that GAP273 interfered with the regulation of actin dynamics during cell spreading. The over-expression of constitutively active alleles of ARF6 and Rac1 suppressed the effect of GAP273 on actin. In addition, the activation of Rac1 by serum, but not that of RhoA or ARF6, was inhibited in cells over-expressing GAP273, suggesting that Rac1 is a likely downstream effector of ARFGAP1. The carboxyl terminal 65 residues of ARFGAP1 were sufficient to produce the effects on actin and cell spreading in transfected cells and co-localized with cortical actin foci.

Conclusions

ARFGAP1 functions as an inhibitor upstream of Rac1 in regulating actin cytoskeleton. In addition to its GAP catalytic domain and Golgi binding domain, it also has an actin regulation domain in the carboxyl-terminal portion of the protein.     

Hidden malignant cells within leukocyte aggregates: seeds for invasive and metastatic cancer?

Background: Our previous studies revealed that leukocyte infiltration into aged or injured myoepithelial cell layers is a key trigger for breast tumor invasion and metastasis. Our current study further assessed the possibility that leukocyte aggregates may harbor detached individual tumor cell or clusters of tumor cells. Materials and methods: Tissue sections from patients with pregnancy-associated breast cancer (PABC) and controls were subjected to morphological and immunohistochemical assessment with a panel of leukocyte and tumor cell related markers. Results: A total of 63 leukocyte aggregates were detected in the 20 PABC cases studied. Of these, 55 (87%) were distributed within normal or hyperplastic lobules adjacent to invasive lesions. Over 70% of these leukocyte aggregates harbored detached individual tumor cell or cell clusters with malignant properties, including strong p53 positivity, elevated proliferation, reduced cell surface adhesion molecules, and cytological resemblance to adjacent invasive cancer cells. A significant number of these tumor cells or condensed chromosomes of mitotic tumor cells were observed to conjoin with the plasma membrane of leukocytes. Similar alterations were seen in leukocyte aggregates within the inter-lobular space and in non-PABC with a lower frequency. Conclusions: These findings suggest that leukocyte infiltration may trigger dissemination of tumor cells from their primary site, and that leukocyte aggregates may serve as a reservoir for disseminated tumor cells that may be physically dragged to distant sites by leukocytes during their migration.     

Rice Big Grain 1 promotes cell division to enhance organ development,stress tolerance and grain yield

Grain/seed yield and plant stress tolerance are two major traits that determine the yield potential of many crops. In cereals, grain size is one of the key factors affecting grain yield. Here, we identify and characterize a newly discovered gene Rice Big Grain 1 (RBG1) that regulates grain and organ development, as well as abiotic stress tolerance. Ectopic expression of RBG1 leads to significant increases in the size of not only grains but also other major organs such as roots, shoots and panicles. Increased grain size is primarily due to elevated cell numbers rather than cell enlargement. RBG1 is preferentially expressed in meristematic and proliferating tissues. Ectopic expression of RBG1 promotes cell division, and RBG1 co‐localizes with microtubules known to be involved in cell division, which may account for the increase in organ size. Ectopic expression of RBG1 also increases auxin accumulation and sensitivity, which facilitates root development, particularly crown roots. Moreover, overexpression of RBG1 up‐regulated a large number of heat‐shock proteins, leading to enhanced tolerance to heat, osmotic and salt stresses, as well as rapid recovery from water‐deficit stress. Ectopic expression of RBG1 regulated by a specific constitutive promoter, GOS2, enhanced harvest index and grain yield in rice. Taken together, we have discovered that RBG1 regulates two distinct and important traits in rice, namely grain yield and stress tolerance, via its effects on cell division, auxin and stress protein induction.     

CFTR promotes malignant glioma development via up‐regulation of Akt/Bcl2‐mediated anti‐apoptosis pathway

Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP‐activated Cl^‐ channel, is extensively expressed in the epithelial cells of various tissues and organs. Accumulating evidence indicates that aberrant expression or mutation of CFTR is related to carcinoma development. Malignant gliomas are the most common and aggressive intracranial tumours; however, the role of CFTR in the development of malignant gliomas is unclear. Here, we report that CFTR is expressed in malignant glioma cell lines. Suppression of CFTR channel function or knockdown of CFTR suppresses glioma cell viability whereas overexpression of CFTR promotes it. Additionally, overexpression of CFTR suppresses apoptosis and promotes glioma progression in both subcutaneous and orthotopic xenograft models. Cystic fibrosis transmembrane conductance regulator activates Akt/Bcl2 pathway, and suppression of PI3K/Akt pathway abolishes CFTR overexpression–induced up‐regulation of Bcl2 (MK‐2206 and LY294002) and cell viability (MK‐2206). More importantly, the protein expression level of CFTR is significantly increased in glioblastoma patient samples. Altogether, our study has revealed a mechanism by which CFTR promotes glioma progression via up‐regulation of Akt/Bcl2‐mediated anti‐apoptotic pathway, which warrants future studies into the potential of using CFTR as a therapeutic target for glioma treatment.     

Staphylococcus aureus Keratitis: A Review of Hospital Cases

Background

Methicillin-resistant Staphylococcus aureus (MRSA) infection is an important public health issue. The study aimed to characterize the patient demographics, clinical features, antibiotic susceptibility, and clinical outcomes of keratitis caused by S. aureus, and to make a comparison between MRSA and methicillin-sensitive S. aureus (MSSA) isolates.

Methodology/Principal findings

Patients (n = 59) with culture-proven S. aureus keratitis treated in Chang Gung Memorial Hospital between January 1, 2006, and December 31, 2010, were included in our study. Patients'' demographic and clinical data were retrospectively reviewed. Twenty-six MRSA (44%) and 33 MSSA (56%) isolates were collected. The MRSA keratitis was significantly more common among the patients with healthcare exposure (P = 0.038), but 46.2% (12/26) of patients with MRSA keratitis were considered to have community-associated infections. All isolates were susceptible to vancomycin. MRSA isolates were significantly more resistant to clindamycin, erythromycin, and sulfamethoxazole/trimethoprim. Ocular surface disease was a significant risk factor for MRSA keratitis (P = 0.011). Visual outcome did not differ significantly between the MRSA and MSSA groups. However, age (B = 0.01, P = 0.035, 95% confidence interval [CI]: 0.001–0.019) and visual acuity at presentation (B = 0.749, P<0.001, 95% CI: 0.573–0.926) were significantly correlated with visual outcome.

Conclusions/Significance

Ocular surface disease is an important predisposing factor for S. aureus keratitis, especially for MRSA infections. Advanced age and poor visual acuity at presentation are important prognostic indicators for poor visual outcome in S. aureus keratitis. Oxacillin resistance may not be a significant prognostic indicator.     

Morphological and Molecular Characterization of Adult Midgut Compartmentalization in Drosophila

1. Download : Download full-size image

     

Irradiance,but not fluence,plays a crucial role in UVB‐induced immature pigment cell development: new insights for efficient UVB phototherapy

Light exposure modulates development of living organisms. In the field of medicine, light has frequently been used for regenerative purposes. Excimer light (308 nm) has demonstrated superior efficacy in treating vitiligo, a condition requiring development of melanoblasts and a model for studying nerve cell regeneration, as compared to narrow‐band ultraviolet B (NBUVB; 311 nm). Using mouse‐derived melanoblast cells to examine the pro‐differentiation effects of these two light sources, we demonstrated that at equivalent fluence, excimer light induces melanoblast differentiation, while NBUVB failed to so. Mechanistically, activation of aryl hydrocarbon receptor pathway and nuclear translocation of epidermal growth factor receptor are involved in pro‐differentiation effects of excimer light. Reduction in irradiance by filter abrogated the effects of excimer light in melanoblasts, even when equivalent fluence was delivered by the same light source. As ultraviolet B (UVB) irradiation is closely associated pigment cell development, future therapy employing UVB for pigmentation purposes should incorporate irradiance as a crucial specification.     

A Small Molecule Inhibitor of Src Family Kinases Promotes Simple Epithelial Differentiation of Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) provide unprecedented opportunities to study the earliest stages of human development in vitro and have the potential to provide unlimited new sources of cells for regenerative medicine. Although previous studies have reported cytokeratin 14+/p63+ keratinocyte generation from hPSCs, the multipotent progenitors of epithelial lineages have not been described and the developmental pathways regulating epithelial commitment remain largely unknown. Here we report membrane localization of β-catenin during retinoic acid (RA) – induced epithelial differentiation. In addition hPSC treatment with the Src family kinase inhibitor SU6656 modulated β-catenin localization and produced an enriched population of simple epithelial cells under defined culture conditions. SU6656 strongly upregulated expression of cytokeratins 18 and 8 (K18/K8), which are expressed in simple epithelial cells, while repressing expression of the pluripotency gene Oct4. This homogeneous population of K18+K8+Oct4− simple epithelial precursor cells can further differentiate into cells expressing keratinocyte or corneal-specific markers. These enriched hPSC-derived simple epithelial cells may provide a ready source for development and toxicology cell models and may serve as a progenitor for epithelial cell transplantation applications.