Comprehensive mutational scanning of the p53 coding region by two- dimensional gene scanning

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long- distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li- Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.      

Association of granulocyte-macrophage colony-stimulating factor with the crystalloid granules of human eosinophils

We have previously shown that normal-density human peripheral blood eosinophils transcribe and translate mRNA for granulocyte-macrophage colony-stimulating factor (GM-CSF) and that the intracellular distribution was granular as assessed by light microscopy immunocytochemistry. The present study was conducted to confirm this apparent association between GM-CSF and the crystalloid granule using a subcellular fractionation method for human eosinophils and immunogold electron microscopy (EM). Highly purified (> 99%, by negative selection using anti-CD16 immunomagnetic microbeads) human peripheral blood eosinophils were obtained from four asthmatic subjects (not taking systemic medication), homogenized and density fractionated (5 x 10(7) cells/subject) on linear Nycodenz gradients. Twenty-four fractions were collected from each cell preparation and analyzed for marker enzyme activities as well as total protein. Dot blot analysis with specific monoclonal antibodies (MoAbs) was used to detect the eosinophil granule proteins major basic protein (MBP) and eosinophil cationic protein (ECP). An anti-CD9 MoAb was used as an eosinophil plasma membrane marker. Lactate dehydrogenase (LDH) was used as a cytosolic marker. Immunoreactivity for GM-CSF was detected by a specific enzyme-linked immunosorbent assay using a polyclonal antihuman GM-CSF antibody and confirmed by dot blot. GM-CSF coeluted with the cellular fractions containing granule markers (MBP, ECP, eosinophil peroxidase, hexosaminidase, and arylsulphatase), but not those containing cytoplasm (LDH+) or membrane (CD9+) markers. EM examination of pooled fractions associated with the peak of GM-CSF immunoreactivity confirmed that they contained crystalloid and small granules, but not plasma membrane. In addition, quantification, using immunogold labeling with an anti/GM-CSF MoAb, indicated preferential localization of gold particles over the eosinophil granule cores of intact cells. Thus, our results indicate that GM-CSF resides as a granule-associated, stored mediator in unstimulated human eosinophils.     

An unusual case of vaccine-associated paralytic poliomyelitis

The present article reports a case involving an immunocompetent, previously well child who, despite two previous doses of inactivated poliovirus vaccine, developed severe flaccid paralysis consistent with polio after receiving oral polio vaccine.     

Neuropeptide Y Attenuates Stress‐Induced Bone Loss Through Suppression of Noradrenaline Circuits

Chronic stress and depression have adverse consequences on many organ systems, including the skeleton, but the mechanisms underlying stress‐induced bone loss remain unclear. Here we demonstrate that neuropeptide Y (NPY), centrally and peripherally, plays a critical role in protecting against stress‐induced bone loss. Mice lacking the anxiolytic factor NPY exhibit more anxious behavior and elevated corticosterone levels. Additionally, following a 6‐week restraint, or cold‐stress protocol, Npy‐null mice exhibit three‐fold greater bone loss compared to wild‐type mice, owing to suppression of osteoblast activity. This stress‐protective NPY pathway acts specifically through Y2 receptors. Centrally, Y2 receptors suppress corticotropin‐releasing factor expression and inhibit activation of noradrenergic neurons in the paraventricular nucleus. In the periphery, they act to control noradrenaline release from sympathetic neurons. Specific deletion of arcuate Y2 receptors recapitulates the Npy‐null stress response, coincident with elevated serum noradrenaline. Importantly, specific reintroduction of NPY solely in noradrenergic neurons of otherwise Npy‐null mice blocks the increase in circulating noradrenaline and the stress‐induced bone loss. Thus, NPY protects against excessive stress‐induced bone loss, through Y2 receptor‐mediated modulation of central and peripheral noradrenergic neurons. © 2014 American Society for Bone and Mineral Research.     

Regenerative potential of silk conduits in repair of peripheral nerve injury in adult rats

Various attempts have been made to develop artificial conduits for nerve repair, but with limited success. We describe here conduits made from Bombyx mori regenerated silk protein, and containing luminal fibres of Spidrex^®, a silk-based biomaterial with properties similar to those of spider silk. Assessment in vitro demonstrated that Spidrex^® fibres support neurite outgrowth. For evaluation in vivo, silk conduits 10 mm in length and containing 0, 100, 200 or 300 luminal Spidrex^® fibres, were implanted to bridge an 8 mm gap in the rat sciatic nerve. At 4 weeks, conduits containing 200 luminal Spidrex^® fibres (PN200) supported 62% and 59% as much axon growth as autologous nerve graft controls at mid-conduit and distal nerve respectively. Furthermore, Spidrex^® conduits displayed similar Schwann cell support and macrophage response to controls. At 12 weeks, animals implanted with PN200 conduits showed similar numbers of myelinated axons (81%) to controls, similar gastrocnemius muscle innervation, and similar hindpaw stance assessed by Catwalk footprint analysis. Plantar skin innervation was 73% of that of controls. PN200 Spidrex^® conduits were also effective at bridging longer (11 and 13 mm) gaps. Our results show that Spidrex^® conduits promote excellent axonal regeneration and function recovery, and may have potential for clinical application.     

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.