Basic Fibroblast Growth Factor Selectively Increases AMPA-Receptor Subunit GluR1 Protein Level and Differentially Modulates Ca2+ Responses to AMPA and NMDA in Hippocampal Neurons

Abstract: The excitatory neurotransmitter glutamate is believed to play important roles in development, synaptic plasticity, and neurodegenerative conditions. Recent studies have shown that neurotrophic factors can modulate neuronal excitability and survival and neurite outgrowth responses to glutamate, but the mechanisms are unknown. The present study tested the hypothesis that neurotrophic factors modulate responses to glutamate by affecting the expression of specific glutamate-receptor proteins. Exposure of cultured embryonic rat hippocampal cells to basic fibroblast growth factor (bFGF) resulted in a concentration-dependent increase in levels of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor subunit GluR1 protein as determined by western blot, dot-blot, and immunocytochemical analyses. In contrast, bFGF did not alter levels of GluP2/3, GluR4, or the NMDA-receptor subunit NR1. Nerve growth factor did not affect GluR1 levels. Calcium-imaging studies revealed that elevation of [Ca²⁺]_i, resulting from selective AMPA-receptor activation, was enhanced in bFGF-pretreated neurons. On the other hand, [Ca²⁺]_i responses to NMDA-receptor activation were suppressed in bFGF-treated neurons, consistent with previous studies showing that bFGF can protect neurons against NMDA toxicity. Moreover, neurons pretreated with bFGF were relatively resistant to the toxicities of glutamate and AMPA, both of which were shown to be mediated by NMDA receptors. These data suggest that differential regulation of the expression of specific glutamate-receptor subunits may be an important mechanism whereby neurotrophic factors modulate activity-dependent neuronal plasticity and vulnerability to excitotoxicity.     

Immune effects of mesenchymal stem cells: implications for Charcot-Marie-Tooth disease

Mesenchymal stem cell (MSC) therapy is the most clinically advanced form of cell therapy, second to hematopoietic stem cell transplants. To date, MSC have been used for immune modulation in conditions such as Graft Versus Host Disease (GVHD) and Crohn's Disease, for which Phase III clinical trials are currently in progress. Here, we review the immunological properties of MSC and make a case for their use in treatment of Charcot-Marie-Tooth disease type 1 (CMT1), a group of inherited peripheral neuropathies. CMT1 is characterized by demyelination and aberrant immune activation making this condition an ideal target for exploration of MSC therapy, given the ability of these cells to promote sheath regeneration as well as suppress inflammation. Studies supporting this hypothesis will be presented and placed into the context of other cell-based approaches that are theoretically feasible. Given that MSCs selectively home to areas of inflammation, as well as exert effects in an allogeneic manner, the possibility of an "off the shelf" therapy for CMT1 will be discussed.     

Limited proteolysis of streptokinase and properties of some fragments.

Limited proteolysis of streptokinase (Sk) by trypsin and thermolysin was performed under various incubation conditions and analysed by polyacrylamide gel electrophoresis. Several fragments (Sk1, Tr27, Tr17, Th26, and Th16) were isolated and characterized further. The N-terminal sequences of Tr27, Tr17, Th26, Th16 and the C-terminal sequences of Tr27 and Th26 were determined by partial sequencing. The evidence available allows the positioning of these fragments within the Sk sequence. Fragment Sk1 is obtained by carefully standardized tryptic digestion of Sk and gel chromatography under non-denaturing conditions. Sk1 is formed by a large polypeptide Ser60-Lys293 and non-covalently bonded smaller polypeptides composed of amino acids from the N-terminal region Ile1-Lys59 of Sk. Fragment Tr27 consists of the large polypeptide Ser60-Lys293 of Sk1, and can be obtained from Sk1 by removal of the smaller N-terminal polypeptides under denaturing conditions. Fragment Th26 is composed of amino acids Phe63-His291. The N-termini of fragments Tr17 and Th16 start with Glu148 and Ile151. From their electrophoretically-determined sizes it can be concluded that they most probably have the same C-terminal amino acids, Lys293 and His291, as fragments Tr27 and Th26, respectively. Secondary structure elements of similar composition were found in all the fragments studied using circular dichroism (c.d.) and infrared (i.r.) measurements. Differential scanning calorimetric (d.s.c.) measurements were performed in order to correlate the sequence regions of Sk to energetic folding units of the protein. Fragments Sk1, Tr27, Th26, Tr17, and Th16 show one melting peak in the temperature range from 42.8 to 46.1 degrees C (thermal unfolding stage). For fragment Sk1, this melting peak can be separated by deconvolution into two transitions at T1 = 46.1 degree C and T2 = 47.3 degrees C with delta H1 = 450 kJ/mol and delta H2 = 219 kJ/mol, respectively. Fragments Tr17 and Th16 show one two-state transition at T = 42.8 degrees C with delta H = 326 kJ/mol.     

Effects of functional constraints and opportunism on the functional structure of a vertebrate predator assemblage

1. Within mainstream ecological literature, functional structure has been viewed as resulting from the interplay of species interactions, resource levels and environmental variability. Classical models state that interspecific competition generates species segregation and guild formation in stable saturated environments, whereas opportunism causes species aggregation on abundant resources in variable unsaturated situations. 2. Nevertheless, intrinsic functional constraints may result in species-specific differences in resource-use capabilities. This could force some degree of functional structure without assuming other putative causes. However, the influence of such constraints has rarely been tested, and their relative contribution to observed patterns has not been quantified. 3. We used a multiple null-model approach to quantify the magnitude and direction (non-random aggregation or divergence) of the functional structure of a vertebrate predator assemblage exposed to variable prey abundance over an 18-year period. Observed trends were contrasted with predictions from null-models designed in an orthogonal fashion to account independently for the effects of functional constraints and opportunism. Subsequently, the unexplained variation was regressed against environmental variables to search for evidence of interspecific competition. 4. Overall, null-models accounting for functional constraints showed the best fit to the observed data, and suggested an effect of this factor in modulating predator opportunistic responses. However, regression models on residual variation indicated that such an effect was dependent on both total and relative abundance of principal (small mammals) and alternative (arthropods, birds, reptiles) prey categories. 5. In addition, no clear evidence for interspecific competition was found, but differential delays in predator functional responses could explain some of the unaccounted variation. Thus, we call for caution when interpreting empirical data in the context of classical models assuming synchronous responses of consumers to resource levels.     

Turbo-mixing in microplates

How to effectively mix small volumes of liquids within microplate wells is a still underestimated and often neglected challenge. The method the authors introduce here relies on violent turbulent motion within a liquid caused by spotting an organic solvent drop onto its surface. The amount needed, less than 1 to 3 microL, is generally small enough not to alter bioactive molecules. Moreover, a solvent may be selected for its compatibility with assay components. The method was tested with layers of aqueous liquids that differ in pH and concentration of a pH-dependent dye, allowing mixing to be monitored optically. Rapid mixing was caused by spotting drops of alcohols, acetone, acetonitrile, and aqueous solutions of these, as long as the difference of surface tension between the drop and the uppermost layer of the bulk liquid surpassed 30 dynes/cm. Along with this difference, position and velocity of spotting, as well as viscosity and geometry of the bulk liquid volume, may influence the turbulence evoked. No significant difference was found for the activity of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase when measured after mixing by shaking and after mixing by spotting 1 microL of methanol onto assays within 96-well microplates.     

Free-air exposure systems to scale up ozone research to mature trees

Because seedlings and mature trees do not necessarily respond similarly to O(3) stress, it is critically important that exposure systems be developed that allow exposure of seedlings through to mature trees. Here we describe three different O(3) Free-Air Exposure Systems that have been used successfully for exposure at all growth stages. These systems of spatially uniform O(3) release have been shown to provide reliable O(3) exposure with minimal, if any, impact on the microclimate. This methodology offers a welcome alternative to chamber studies which had severe space constraints precluding stand or community-level studies and substantial chamber effects on the microclimate and, hence physiological tree performance.     

Generation of mice deficient for Lbx2, a gene expressed in the urogenital system, nervous system, and Pax3 dependent tissues

Lbx2 is a member of the ladybird family of homeobox genes. The first murine ortholog identified, Lbx1, is required for hypaxial musculature and dorsal spinal cord neuron development. The second murine ortholog, Lbx2, is expressed in the developing urogenital and nervous systems. To elucidate the function of Lbx2, we generated a gene-targeted allele of Lbx2 in mice. Lbx2 deficiency did not impair mouse development, and Lbx2 null mice appeared healthy and fertile. Replacement of Lbx2 by the lacZ gene provides a valuable histological marker for Lbx2-expressing cells. Given the important role of Pax3 in neural crest, we intercrossed our Lbx2 deficient mice with Splotch Pax3 mutant mice to determine if Pax3 affects Lbx2 expression. There was reduced Lbx2 expression in dorsal root ganglia and cranial nerve ganglia with Pax3 deficiency, but not in the genital tubercle. This suggested that Pax3 is required for Lbx2 expression in affected neural crest-derived tissues.