A delta-conotoxin from Conus ermineus venom inhibits inactivation in vertebrate neuronal Na+ channels but not in skeletal and cardiac muscles

We have isolated delta-conotoxin EVIA (delta-EVIA), a conopeptide in Conus ermineus venom that contains 32 amino acid residues and a six-cysteine/four-loop framework similar to that of previously described omega-, delta-, microO-, and kappa-conotoxins. However, it displays low sequence homology with the latter conotoxins. delta-EVIA inhibits Na+ channel inactivation with unique tissue specificity upon binding to receptor site 6 of neuronal Na+ channels. Using amphibian myelinated axons and spinal neurons, we showed that delta-EVIA increases the duration of action potentials by inhibiting Na+ channel inactivation. delta-EVIA considerably enhanced nerve terminal excitability and synaptic efficacy at the frog neuromuscular junction but did not affect directly elicited muscle action potentials. The neuronally selective property of delta-EVIA was confirmed by showing that a fluorescent derivative of delta-EVIA labeled motor nerve endings but not skeletal muscle fibers. In a heterologous expression system, delta-EVIA inhibited inactivation of rat neuronal Na+ channel subtypes (rNaV1.2a, rNaV1.3, and rNaV1.6) but did not affect rat skeletal (rNaV1.4) and human cardiac muscle (hNaV1.5) Na+ channel subtypes. delta-EVIA, in the range of concentrations used, is the first conotoxin found to affect neuronal Na+ channels without acting on Na+ channels of skeletal and cardiac muscle. Therefore, it is a unique tool for discriminating voltage-sensitive Na+ channel subtypes and for studying the distribution and modulation mechanisms of neuronal Na+ channels, and it may serve as a lead to design new drugs adapted to treat diseases characterized by defective nerve conduction.     

Heparanase uptake is mediated by cell membrane heparan sulfate proteoglycans

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of, heparanase. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinase-treated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is approximately 30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase.     

PLA2G6 mutation underlies infantile neuroaxonal dystrophy

Infantile neuroaxonal dystrophy (INAD) is an autosomal recessive progressive neurodegenerative disease that presents within the first 2 years of life and culminates in death by age 10 years. Affected individuals from two unrelated Bedouin Israeli kindreds were studied. Brain imaging demonstrated diffuse cerebellar atrophy and abnormal iron deposition in the medial and lateral globus pallidum. Progressive white-matter disease and reduction of the N-acetyl aspartate : chromium ratio were evident on magnetic resonance spectroscopy, suggesting loss of myelination. The clinical and radiological diagnosis of INAD was verified by sural nerve biopsy. The disease gene was mapped to a 1.17-Mb locus on chromosome 22q13.1 (LOD score 4.7 at recombination fraction 0 for SNP rs139897), and an underlying mutation common to both affected families was identified in PLA2G6, the gene encoding phospholipase A2 group VI (cytosolic, calcium-independent). These findings highlight a role of phospholipase in neurodegenerative disorders.     

Generalization and discrimination of positive and negative acoustic stimuli in the common carp (Cyprinus carpio)

Groups of common carp (Cyprinus carpio) were trained for 33 days to discriminate between two acoustic signals differing in frequency and temporal pattern. One signal (positive stimulus) was reinforced by food, while the other (negative stimulus) was not rewarded. When exposed to 3 positive and 3 negative stimuli per day (training Paradigm 1) the fish responded similarly to the two signals. When daily negative stimuli outnumbered positive stimuli (3 positive and 33 negative, training Paradigm 2) the fish responded similarly to the two signals for the first 2 weeks and then began to discriminate between them. Discriminatory response was statistically significant from Day 21 of training. After 33 days, training was switched from Paradigm 2 to Paradigm 1 and the fish maintained their discriminatory capability. The results are interpreted in terms of anticipatory frustration and risk management.     

Single-beat estimation of end-diastolic pressure-volume relationship: a novel method with potential for noninvasive application

Whereas end-systolic and end-diastolic pressure-volume relations (ESPVR, EDPVR) characterize left ventricular (LV) pump properties, clinical utility of these relations has been hampered by the need for invasive measurements over a range of pressure and volumes. We propose a single-beat approach to estimate the whole EDPVR from one measured volume-pressure (Vm and Pm) point. Ex vivo EDPVRs were measured from 80 human hearts of different etiologies (normal, congestive heart failure, left ventricular assist device support). Independent of etiology, when EDPVRs were normalized (EDPVRn) by appropriate scaling of LV volumes, EDPVRns were nearly identical and were optimally described by the relation EDP = An.EDV (Bn), with An = 28.2 mmHg and Bn = 2.79. V0 (the volume at the pressure of approximately 0 mmHg) was predicted by using the relation V0 = Vm.(0.6 - 0.006.Pm) and V30 by V30 = V0 + (Vm,n - V0)/(Pm/An) (1/Bn). The entire EDPVR of an individual heart was then predicted by forcing the curve through Vm, Pm, and the predicted V0 and V30. This technique was applied prospectively to the ex vivo human EDPVRs not used in determining optimal An and Bn values and to 36 in vivo human, 12 acute and 14 chronic canine, and 80 in vivo and ex vivo rat studies. The root-mean-square error (RMSE) in pressure between measured and predicted EDPVRs over the range of 0-40 mmHg was < 3 mmHg of measured EDPVR in all settings, indicating a good predictive value of this approach. Volume-normalized EDPVRs have a common shape, despite different etiology and species. This allows the entire curve to be predicted by a new method with a potential for noninvasive application. The results are most accurate when applied to groups of hearts rather than to individual hearts.     

Direct evidence that receptor site-4 of sodium channel gating modifiers is not dipped in the phospholipid bilayer of neuronal membranes

In a recent note to Nature, R. MacKinnon has raised the possibility that potassium channel gating modifiers are able to partition in the phospholipid bilayer of neuronal membranes and that by increasing their partial concentration adjacent to their receptor, they affect channel function with apparent high affinity (Lee and MacKinnon (2004) Nature 430, 232-235). This suggestion was adopted by Smith et al. (Smith, J. J., Alphy, S., Seibert, A. L., and Blumenthal, K. M. (2005) J. Biol. Chem. 280, 11127-11133), who analyzed the partitioning of sodium channel modifiers in liposomes. They found that certain modifiers were able to partition in these artificial membranes, and on this basis, they have extrapolated that scorpion beta-toxins interact with their channel receptor in a similar mechanism as that proposed by MacKinnon. Since this hypothesis has actually raised a new conception, we examined it in binding assays using a number of pharmacologically distinct scorpion beta-toxins and insect and mammalian neuronal membrane preparations, as well as by analyzing the rate by which the toxin effect on gating of Drosophila DmNa(v)1 and rat brain rNa(v)1.2a develops. We show that in general, scorpion beta-toxins do not partition in neuronal membranes and that in the case in which a depressant beta-toxin partitions in insect neuronal membranes, this partitioning is unrelated to its interaction with the receptor site and the effect on the gating properties of the sodium channel. These results negate the hypothesis that the high affinity of beta-toxins for sodium channels is gained by their ability to partition in the phospholipid bilayer and clearly indicate that the receptor site for scorpion beta-toxins is accessible to the extracellular solvent.     

Magic-angle spinning NMR studies of cell wall bound aromatic-aliphatic biopolyesters associated with strengthening of intercellular adhesion in potato (Solanum tuberosum L.) tuber parenchyma

Intercellular adhesion strengthening, a phenomenon that compromises the texture and the edible quality of potatoes (Solanum tuberosum L.), has been induced reproducibly by exposure to low-pH acetic acid solutions under tissue culture conditions. The resulting parenchyma tissues have been examined by solid-state nuclear magnetic resonance (NMR) in order to characterize the biopolymer(s) thought to be associated with this syndrome. Cross polarization-magic angle spinning (CPMAS) (13)C NMR has been used to establish the presence of a polyphenol-suberin-like aromatic-aliphatic polyester within an abundant cell wall polysaccharide matrix in potato tubers that exhibit hardening due to strengthened intercellular adhesion. Dipolar dephasing and CP chemical shift anisotropy experiments suggest that the aromatic domain is composed primarily of guaiacyl and sinapyl groups. Two-dimensional wide-line separation experiments show that the biopolymer associated with parenchyma hardening contains rigid polysaccharide cell walls and mobile aliphatic long-chain fatty acids; (1)H spin diffusion experiments show that these flexible aliphatic chains are proximal to both the phenolics and a subpopulation of the cell wall polysaccharides. Finally, high-resolution MAS NMR of parenchyma samples swelled in DMSO in conjunction with two-dimensional through-bond and through-space NMR spectroscopy provides evidence for covalent linkages among the polysaccharide, phenolic, and aliphatic domains of the intercellular adhesion-strengthening biopolymer in potato parenchyma tissue.