Multiple presentation of Scfv800E6 on silica nanospheres enhances targeting efficiency toward HER-2 receptor in breast cancer cells

Spherical silica nanoparticles (SNP) have been synthesized and functionalized with anti-HER-2 scFv800E6 antibody by both localized histidine-tag recognition, leading to an oriented protein ligation, and glutaraldehyde cross-linking, exploiting a statistical reactivity of lysine amine groups in the primary sequence of the molecule. The targeting efficiency of nanocomplexes in comparison with free scFv was evaluated by flow cytometry using a HER-2 antigen-positive MCF-7 breast cancer cell line, exhibiting a 4-fold increase in scFv binding efficacy, close to the affinity of intact anti-HER-2 monoclonal antibody, which suggests the effectiveness of presenting multiple scFv molecules on nanoparticles in improving antigen recognition. Unexpectedly, the conjugation method did not affect the binding efficacy of scFv, suggesting a structural role of lysines in the scFv molecule. Confocal laser scanning microscopy confirmed the binding of nanocomplexes to HER-2 and also provided evidence of their localization at the cell surface.     

Importing statistical measures into Artemis enhances gene identification in the <Emphasis Type="Italic">Leishmania</Emphasis> genome project

Background

Seattle Biomedical Research Institute (SBRI) as part of the Leishmania Genome Network (LGN) is sequencing chromosomes of the trypanosomatid protozoan species Leishmania major. At SBRI, chromosomal sequence is annotated using a combination of trained and untrained non-consensus gene-prediction algorithms with ARTEMIS, an annotation platform with rich and user-friendly interfaces.

Results

Here we describe a methodology used to import results from three different protein-coding gene-prediction algorithms (GLIMMER, TESTCODE and GENESCAN) into the ARTEMIS sequence viewer and annotation tool. Comparison of these methods, along with the CODON USAGE algorithm built into ARTEMIS, shows the importance of combining methods to more accurately annotate the L. major genomic sequence.

Conclusion

An improvised and powerful tool for gene prediction has been developed by importing data from widely-used algorithms into an existing annotation platform. This approach is especially fruitful in the Leishmania genome project where there is large proportion of novel genes requiring manual annotation.

     

Cross talk between vestibular neurons and Schwann cells mediates BDNF release and neuronal regeneration

It is now well-established that an active cross-talk occurs between neurons and glial cells, in the adult as well as in the developing and regenerating nervous systems. These functional interactions not only actively modulate synaptic transmission, but also support neuronal growth and differentiation. We have investigated the possible existence of a reciprocal interaction between inner ear vestibular neurons and Schwann cells maintained in primary cultures. We show that ATP released by the extending vestibular axons elevates intracellular calcium levels within Schwann cells. Purinergic activation of the Schwann P2X₇ receptor induces the release of neurotrophin BDNF, which occurs via a regulated, tetanus-toxin sensitive, vesicular pathway. BDNF, in turn, is required by the vestibular neuron to support its own survival and growth. Given the massive release of ATP during tissue damage, cross-talk between vestibular neurons and Schwann cells could play a primary role during regeneration.     

Novel interactions of TG2 with heparan sulfate proteoglycans: reflection on physiological implications

This mini-review brings together information from publications and recent conference proceedings that have shed light on the biological interaction between transglutaminase-2 and heparan sulphate proteoglycans. We subsequently draw hypotheses of possible implications in the wound healing process. There is a substantial overlap in the action of transglutaminase-2 and the heparan sulphate proteoglycan syndecan-4 in normal and abnormal wound repair. Our latest findings have identified syndecan-4 as a possible binding and signalling partner of fibronectin-bound TG2 and support the idea that transglutaminase-2 and syndecan-4 act in synergy.     

Regulated delivery of AMPA receptor subunits to the presynaptic membrane

In recent years, a role for AMPA receptors as modulators of presynaptic functions has emerged. We have investigated the presence of AMPA receptor subunits and the possible dynamic control of their surface exposure at the presynaptic membrane. We demonstrate that the AMPA receptor subunits GluR1 and GluR2 are expressed and organized in functional receptors in axonal growth cones of hippocampal neurons. AMPA receptors are actively internalized upon activation and recruited to the surface upon depolarization. Pretreatment of cultures with botulinum toxin E or tetanus toxin prevents the receptor insertion into the plasma membrane, whereas treatment with alpha-latrotoxin enhances the surface exposure of GluR2, both in growth cones of cultured neurons and in brain synaptosomes. Purification of small synaptic vesicles through controlled-pore glass chromatography, revealed that both GluR2 and GluR1, but not the GluR2 interacting protein GRIP, copurify with synaptic vesicles. These data indicate that, at steady state, a major pool of AMPA receptor subunits reside in synaptic vesicle membranes and can be recruited to the presynaptic membrane as functional receptors in response to depolarization.     

Vesicle turnover in developing neurons: how to build a presynaptic terminal

Over the past decade, evidence has accumulated indicating that, during development, the construction of synapses--the sites of communication between neurons--might rely on the utilization of preassembled sets of synaptic proteins, which have already accumulated in the axon and are highly mobile, before getting recruited to the sites of contact with the postsynaptic neuron. In this review, we discuss evidence from most recent publications pointing to the existence of active vesicle traffic and turnover in developing neurons, which lead to the construction of new synaptic sites.     

Different localizations and functions of L-type and N-type calcium channels during development of hippocampal neurons

Using immunocytochemical assays and patch-clamp and calcium-imaging recordings, we demonstrate that L-type and N-type calcium channels have distinct patterns of expression and distribution and play different functional roles during hippocampal neuron differentiation. L-type channels, which support the depolarization-induced calcium influx in neurons from the very early developmental stages, are functionally restricted to the somatodendritic compartment throughout neuronal development and play a crucial role in supporting neurite outgrowth at early developmental stages. N-type channels, which start contributing at later neuronal differentiation stages (3-4 DIV), are also functionally expressed in the axons of immature neurons. At this developmental stage preceding synaptogenesis, N-type (but not L-type) channels are involved in controlling synaptic vesicle recycling. It is only at later developmental stages (10-12 DIV), when the neurons have established a clear axodendritic polarity and form synaptic contacts, that N-type channels are progressively excluded from the axon. Electrophysiological recordings of single neurons growing in microislands revealed that synaptic maturation coincides with a progressive increase in N-type channels in the somatodendritic region and a progressive decrease in the N-type channels supporting glutamate release from the presynaptic terminal. These results indicate that L-type and N-type calcium channels undergo dynamic, developmentally regulated rearrangements in regional distribution and function and also suggest that different mechanisms may be involved in the sorting and/or stabilization of these two types of channels in different plasma membrane domains during neuronal differentiation.