Covalent attachment of osmium complexes to glucose oxidase and the application of the resulting modified enzyme in an enzyme switch responsive to glucose.

Pyridine-based osmium complexes bearing either a carboxylate or aldehyde group were covalently attached to glucose oxidase and were shown to work as mediators for the reoxidation of the enzyme. For the complex containing the carboxylate group, the binding was made through carbodiimide coupling to the amine residues in the protein. For the complex containing the aldehyde group, the reductive coupling was carried out by condensation with the amino groups on the protein in the presence of sodium cyanoborohydride.

Cytokines that signal through the leukemia inhibitory factor receptor-beta complex in the nervous system.

Cytokines that signal through the leukemia inhibitory factor (LIF) receptor, such as LIF and ciliary neuronotrophic factor, have a wide range of roles within both the developing and mature nervous system. They play a vital role in the differentiation of neural precursor cells into astrocytes and can prevent or promote neuronal differentiation. One of the conundrums regarding signalling through the LIF receptor is how it can have multiple, often conflicting roles in different cell types, such as enhancing the differentiation of astrocytes while inhibiting the differentiation of some neuronal cells.

Endogenous IGF-1 regulates the neuronal differentiation of adult stem cells.

Stem cells from the adult forebrain of mice were stimulated to form clones in vitro using fibroblast growth factor-2 (FGF-2). At concentrations above 10 ng/ml of FGF-2, very few clones gave rise to neurons; however, if FGF-2 was removed after 5 days, 20-30% of clones subsequently gave rise to neurons. The number of neuron-containing clones and the number of neurons per clone was significantly enhanced, if insulin-like growth factor (IGF)-1 or heparin were added subsequent to FGF-2 removal.

An instrument for simultaneous EQCM impedance and SECM measurements.

A novel combination of an electrochemical quartz crystal microbalance (EQCM) and a scanning electrochemical microscope (SECM) has been built. Unlike conventional EQCMs, the instrument described here allows rapid in situ measurement of the modulus of the quartz crystal's transfer function. Data analysis in the complex plane for the Butterworth-Van Dyke (BVD) equivalent circuit yields the real and the imaginary components R (damping resistance) and XL (reactive inductance) of the crystal's electroacoustic impedance around its resonant frequency of 10 MHz.

Precursor cells in the subventricular zone of the adult mouse are actively inhibited from differentiating into neurons.

The adult mouse subventricular zone (SVZ) contains precursor cells capable of generating new neurons which populate the olfactory bulb. The SVZ precursors, however, appear to be restricted in their capacity to generate neurons in other regions of the brain indicating a tight regulation of their differentiation. We demonstrate in vitro that explants of SVZ are unable to generate neurons from dividing precursors, even though precursors are present and dividing within explant cultures.

Signaling of neuronal cell death by the p75NTR neurotrophin receptor.

The neurotrophin receptor (p75NTR) is best known for mediating tropic support by participating in the formation of high-affinity nerve growth factor (NGF) receptor complexes with trkA, however, p75NTR more recently has been shown to act as a bona fide death-signaling receptor, which can signal independently of trkA. This article discusses the evidence for an active role of p75NTR in neuronal cell death and the mechanisms controlling this process, including roles for Bcl-2 family members, the c-jun stress kinase JNK, the transcription factor nuclear factor kappa B (NFkappaB), and caspases.

The neurotrophins act synergistically with LIF and members of the TGF-beta superfamily to promote the survival of spiral ganglia neurons in vitro.

A number of growth factor families have been implicated in normal inner ear development, auditory neuron survival and protection. Several growth factors, including transforming growth factor-beta5 (TGF-beta5) and TGF-beta3, neurotrophin-3 (NT-3), brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) were tested for their ability, individually or in combination, to promote auditory neuron survival in dissociated cell cultures of early rat post-natal spiral ganglion cells (SGCs). The results indicate that at discrete concentrations all growth factors act in an additive fashion and some in synergy when promoting neuronal survival.

Regulation of spinal motoneuron differentiation by the combined action of Sonic hedgehog and neurotrophin 3.

1. The development of ventral cell types in the spinal cord, including motor neurons, requires the growth factor Sonic hedgehog (Shh). However, it is still unknown whether Shh acts directly on precursors to induce these cell types and whether additional factors are required for induction.

Differential loss of spinal sensory but not motor neurons in the p75NTR knockout mouse.

Sensory neurons in the dorsal root ganglia (DRG) and motor neurons in the spinal cord express the 75 kDa low-affinity neurotrophin receptor (p75NTR) during prenatal development. The p75NTR gene knockout mouse provides a unique opportunity to assess the role of p75NTR during this period. Quantitative analysis of the p75NTR knockout mouse revealed a significant developmental loss of sensory neurons.

Pax-3 regulates neurogenesis in neural crest-derived precursor cells.

The peripheral nervous system consists of multiple neural lineages derived from the neural crest (NC). Pax-3 is expressed in the NC and when mutated in the splotch mouse (Sp) results in the loss of derivatives from this precursor cell population. We have investigated the role of Pax-3 in regulating the generation of neurons from NC-derived precursor cells in vitro.

Nerve growth factor signaling through p75 induces apoptosis in Schwann cells via a Bcl-2-independent pathway.

Apoptosis is involved in the regulation of Schwann cell numbers during normal development and after axonal damage, but the molecular regulation of Schwann cell death remains unknown. We have used stably transfected rat Schwann cell lines to study the potential roles of nerve growth factor (NGF), the antiapoptotic protein Bcl-2 and the cytokine response modifier A (CrmA) in modulating Schwann cell death in vitro. Bcl-2 inhibited Schwann cell apoptosis induced by survival factor withdrawal, whereas CrmA did not.

p75 neurotrophin receptor-mediated neuronal death is promoted by Bcl-2 and prevented by Bcl-xL.

The p75 neurotrophin receptor (p75NTR) has been shown to mediate neuronal death through an unknown pathway. We microinjected p75NTR expression plasmids into sensory neurons in the presence of growth factors and assessed the effect of the expressed proteins on cell survival. We show that, unlike other members of the TNFR family, p75NTR signals death through a unique caspase-dependent death pathway that does not involve the "death domain" and is differentially regulated by Bcl-2 family members: the anti-apoptotic molecule Bcl-2 both promoted, and was required for, p75NTR killing, whereas killing was inhibited by its homologue Bcl-xL.

Nerve growth factor modulates myelin-associated glycoprotein binding to sensory neurons.

Myelin-associated glycoprotein (MAG) is a molecule expressed by myelinating cells at the myelin/axon interface, which binds to an as yet unidentified molecule on neurons. We have used a MAG-immunoglobulin Fc fusion protein to examine the expression and regulation of the MAG binding molecule on sensory neurons in culture. Binding of the MAG-Fc reached a maximum at 24-48 h and was higher on neurons which expressed high levels of neurofilament.

Cellular distribution and developmental expression of AMP-activated protein kinase isoforms in mouse central nervous system.

The mammalian AMP-activated protein kinase is a heterotrimeric serine/threonine protein kinase with multiple isoforms for each subunit (alpha, beta, and gamma) and is activated under conditions of metabolic stress. It is widely expressed in many tissues, including the brain, although its expression pattern throughout the CNS is unknown. We show that brain mRNA levels for the alpha2 and beta2 subunits were increased between embryonic days 10 and 14, whereas expression of alpha1, beta1, and gamma1 subunits was consistent at all ages examined.

Sonic hedgehog promotes neuronal differentiation of murine spinal cord precursors and collaborates with neurotrophin 3 to induce Islet-1.

Sonic hedgehog (Shh) is strongly implicated in the development of ventral structures in the nervous system. Addition of Sonic hedgehog protein to chick spinal cord explants induces floor plate and motoneuron development. Whether Shh acts directly to induce these cell types or whether their induction is mediated by additional factors is unknown.

Short echo time single-voxel 1H magnetic resonance spectroscopy in magnetic resonance imaging-negative temporal lobe epilepsy: different biochemical profile compared with hippocampal sclerosis.

Single-voxel proton magnetic resonance spectroscopy (1H MRS) has shown abnormalities in patients with temporal lobe epilepsy (TLE) and hippocampal sclerosis (HS). Many TLE patients, however, do not have HS or other lesions on quantitative magnetic resonance imaging (MRI) (MRI-negative). Fifteen control subjects, 15 patients with unilateral HS, and 15 MRI-negative TLE patients underwent 1H MRS at an echo time of 30 msec on a 1.

Enhanced downregulation of the p75 nerve growth factor receptor by cholesteryl and bis-cholesteryl antisense oligonucleotides.

The effects of conjugating cholesterol to either or both ends of a phosphorothioate (PS) oligonucleotide were analyzed in terms of cellular uptake and antisense efficacy. The oligo sequence was directed against the p75 nerve growth factor receptor (p75), and was tested in differentiated PC12 cells, which express high levels of this protein. The addition of a single cholesteryl group to the 5'-end significantly increased cellular uptake and improved p75 mRNA downregulation compared with the unmodified PS oligo.

Lowering the entropic barrier for binding conformationally flexible inhibitors to enzymes.

The design of inhibitors with enhanced potency against proteolytic enzymes has many applications for the treatment of human diseases. In addition to the optimization of chemical interactions between the enzyme and inhibitor, the binding affinity can be increased by constraining the inhibitor to the conformation that is recognized by the enzyme, thus lowering the entropic barrier to complex formation. We have structurally characterized the complexes of a macrocyclic pentapeptide inhibitor and its acyclic analogue with penicillopepsin, an aspartic proteinase, to study the effect of conformational constraint on the binding affinity.

Almost linear VC-dimension bounds for piecewise polynomial networks.

We compute upper and lower bounds on the VC dimension and pseudo-dimension of feedforward neural networks composed of piecewise polynomial activation functions. We show that if the number of layers is fixed, then the VC dimension and pseudo-dimension grow as WlogW, where W is the number of parameters in the network. This result stands in opposition to the case where the number of layers is unbounded, in which case the VC dimension and pseudo-dimension grow as W2.

Regulation of neural stem cell differentiation in the forebrain.

In the developing forebrain, mounting evidence suggests that neural stem cell proliferation and differentiation is regulated by growth factors. In vitro in the presence of serum, stem cell proliferation is predominantly mediated by fibroblast growth factor-2 (FGF-2) whereas neuronal differentiation can be triggered by FGF-1 in association with a specific heparan sulphate proteoglycan. On the other hand, astrocyte differentiation in vivo and in vitro appears to be dependent on signalling through the leukaemia inhibitory factor receptor (LIFR).

EphA4 (Sek1) receptor tyrosine kinase is required for the development of the corticospinal tract.

Members of the Eph family of tyrosine kinase receptors have been implicated in the regulation of developmental processes and, in particular, axon guidance in the developing nervous system. The function of the EphA4 (Sek1) receptor was explored through creation of a null mutant mouse. Mice with a null mutation in the EphA4 gene are viable and fertile but have a gross motor dysfunction, which is evidenced by a loss of coordination of limb movement and a resultant hopping, kangaroo-like gait.