Intraspecific variation in the venom of the vermivorous cone snail Conus vexillum

A combination of proteomic and biochemical assays was used to examine variations in the venom of Conus vexillum taken from two locations (Hurgada and Sharm El-Shaikh) in the Red Sea, Egypt. Using MALDI/TOF-MS, a remarkable degree of intra-species variation between venom samples from both locations was identified. To evaluate variability in the cytotoxic effects of Conus venom, mice were injected with the same dose from each location. The oxidative stress biomarkers [malondialdehyde (MDA), protein carbonyl content (PCC)], antioxidants [glutathione (GSH), superoxide dismutase (SOD), catalase (CAT)], total antioxidant capacity (TAC) and nitric oxide (NO), were measured 3, 6, 9 and 12 h post venom injection. The venoms induced a significant increase in the levels of PCC, MDA, NO, GSH and CAT. The venoms significantly inhibited the activity of SOD and reduced the TAC. Toxicological data showed that the venom obtained from Hurgada was more potent than that obtained from Sharm El-Shaikh. It can be concluded that: (1) the venom of the same Conus species from different regions is highly diversified (2) the venoms from different locations reflect clear differences in venom potency and (3) the cytotoxic effects of C. vexillum venom can be attributed to its ability to induce oxidative stress.     

The cytoskeleton of the neuron--an essay in celebration of Paul Letourneau's career

The neuronal cytoskeleton consists of microtubules, actin filaments, neurofilaments, and an array of accessory proteins that regulate and modify these three main filament systems. This essay celebrates the career of Paul Letourneau, a pioneer of the neuronal cytoskeleton, to whom the community owes a debt of gratitude.     

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans¹. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system^2,3. However, solid-state pulse lasers of modest costs (<$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (<1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers⁴). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.     

Biological activity of neurotrophins is dependent on recruitment of Rac1 to lipid rafts

The Rho family of small GTPases, key regulators of the actin cytoskeleton in eukaryotic cells, is implicated in the control of neuronal morphology. Here, we report that neurotrophin dependent cytoskeletal changes, characteristic of the phenotype of Rac1, in the hippocampal neurons or PC12 cells are inhibited by the disruption of lipid raft integrity. Activation of Rac1 induced by NGF is impaired in cholesterol-depleted PC12 cells. Pretreatment with gammaGTP shifted significant amount of Rac1, presumably in a GTP-bound form, from non-raft to raft fractions. Proper recruitment of activated Rac1 to lipid rafts, structures that represent specialized signaling organelles, is of fundamental importance in determining neurotrophins' bioactivity.     

New insights into the nanometer-scaled cell-surface interspace by cell-sensor measurements

The culture of adherent cells on solid surfaces is an established in vitro method, and the adhesion process of a cell is considered as an important trigger for many cellular processes (e.g., polarity and tumor genesis). However, not all of the eliciting biochemical or biophysical reactions are yet understood. Interestingly, there are not much experimental data about the impact that the interspace between an adherent cell and the (solid) substrate has on the cell's behavior. This interspace is mainly built by the basolateral side of epithelial cells and the substrate. This paper gives some new results of non-invasive and non-optical measurements in the interspace. The measurements were made with silicon cell-sensor hybrids. Measurements of acidification, adhesion, and respiration are analyzed in view of the situation in the interspace. The results show that, in general, the release of an ion or molecule on the basolateral side can have much more influence on the biophysical situation than a release of an ion or molecule on the apical side. In particular, the apical acidification (i.e., amount of extruded protons) of, e.g., epithelial tumor cells is several orders of magnitude higher than the basolateral acidification. These experimental results are a simple consequence of the fact that the basolateral volume of the interspace is several orders of magnitudes smaller than the apical volume. These results have the following consequences for the cell adhesion:     

Neurogenesis and neuronal communication on micropatterned neurochips

Neural networks are formed by accurate connectivity of neurons and glial cells in the brain. These networks employ a three-dimensional bio-surface that both assigns precise coordinates to cells during development and facilitates their connectivity and functionality throughout life. Using specific topographic and chemical features, we have taken steps towards the development of poly(dimethylsiloxane; PDMS) neurochips that can be used to generate and study synthetic neural networks. These neurochips have micropatterned structures that permit adequate cell positioning and support cell survival. Within days of plating, cells differentiate into neurons displaying excitability and communication, as evidenced by intracellular calcium oscillations and action potentials. The structural and functional capacities of such simple neural networks open up new opportunities to study synaptic communication and plasticity.     

Specific Proteolysis of Neuronal Protein GAP-43 by Calpain: Characterization,Regulation, and Physiological Role

The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of micro-calpain (PD151746), but it is completely blocked by micro- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by micro-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G(o); this results in growth cone turning in response to repulsive signals.     

Fyn promotes phosphorylation of collapsin response mediator protein 1 at tyrosine 504, a novel,isoform‐specific regulatory site

In vertebrates the collapsin response mediator proteins (CRMPs) are encoded by five highly related genes. CRMPs are cytosolic phosphoproteins abundantly expressed in developing and mature mammalian brains. CRMPs are best understood as effectors of Semaphorin 3A signaling regulating growth cone collapse in migratory neurons. Phosphorylation in the carboxyl‐terminal regulatory domain of CRMPs by several serine/threonine kinases has been described. These phoshorylation events appear to function, at least in part, to disrupt the interaction of CRMPs with tubulin heterodimers. In a large‐scale phosphoproteomic analysis of murine brain, we recently identified a number of in vivo tyrosine phosphorylation sites on CRMP isoforms. Using biochemical approaches and quantitative mass spectrometry we demonstrate that one of these sites, CRMP1 tyrosine 504 (Y504), is a primary target of the Src family of tyrosine kinases (SFKs), specifically Fyn. Y504 is adjacent to CDK5 and GSK‐3β sites that regulate the interaction of CRMPs with tubulin. Although Y504 is highly conserved among vertebrate CRMP1 orthologs, a residue corresponding to Y504 is absent in CRMP isoforms 2–5. This suggests an isoform‐specific regulatory role for CRMP1 Y504 phosphorylation and may help explain the observation that CRMP1‐deficient mice exhibit neuronal migration defects not compensated for by CRMPs 2–5. J. Cell. Biochem. 111: 20–28, 2010. © 2010 Wiley‐Liss, Inc.     

A mathematical model for photoreceptor interactions

The interactions between rods and cones in the retina have been the focus of innumerable experimental and theoretical biological studies in previous decades yet the understanding of these interactions is still incomplete primarily due to the lack of a unified concept of cone photoreceptor organization and its role in retinal diseases. The low abundance of cones in many of the non-primate mammalian models that have been studied make conclusions about the human retina difficult. A more complete knowledge of the human retina is crucial for counteracting the events that lead to certain degenerative diseases, in particular those associated with photoreceptor cell death (e.g., retinitis pigmentosa). In an attempt to gain important insight into the role and interactions of the rods and the cones we develop and analyze a set of mathematical equations that model a system of photoreceptors and incorporate a direct rod-cone interaction. Our results show that the system can exhibit stable oscillations, which correspond to the rhythmic renewal and shedding of the photoreceptors. In addition, our results show the mathematical necessity of this rod-cone direct interaction for survival of both and gives insight into this mechanism.