Photoaffinity labeling of scorpion toxin receptors associated with insect synaptosomal Na+ channels

Photoreactive and radioiodinated derivatives of several scorpion toxins acting on insect Na+ channels were prepared without loss of their pharmacological activities. Photoaffinity experiments were carried out on a synaptosomal fraction from the nerve cord of the cockroach Periplaneta americana: with all toxin derivatives, a single specifically labeled band was obtained with a molecular weight of 188,000 +/- 12,000 (n = 17). These results indicate for the first time the molecular weight of the scorpion toxin receptor from the insect nervous system which is probably associated with voltage sensitive Na+ channels. One of these toxins, toxin VII from Tityus serrulatus venom, has been previously shown to be active both in mammals and in insects, in rat brain synaptosomes this toxin labeled a Mr = 31,000 +/- 4,000 band in contrast, to observations in the insect preparation.     

Comparison of the Molecular Forms of the Cholinesterases in Tissues of Normal and Dystrophic Chickens

Abstract: The levels and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and pseudocholinesterase (ΦChE, EC 3.1.1.8) were examined in various skeletal muscles, cardiac muscles, and neural tissues from normal and dystrophic chickens. The relative amount of the heavy (H_c) form of AChE in mixed-fibre-type twitch muscles varies in proportion to the percentage of glycolytic fast-twitch fibres. Conversely, muscles with higher levels of oxidative fibres (i.e., slow-tonic, oxidative-glycolytic fast-twitch, or oxidative slow-twitch) have higher proportions of the light (L) form of AChE. The effects of dystrophy on AChE and ΦChE are more severe in muscles richer in glycolytic fast-twitch fibres (e.g., pectoral or posterior latissimus dorsi, PLD); there is no alteration of AChE or ΦChE in a slow-tonic muscle. In the pectoral or PLD muscles from older dystrophic chickens, however, the AChE forms revert to a normal distribution while the ΦChE pattern remains abnormal. Muscle ΦChE is sensitive to collagenase in a similar way as is AChE, thus apparently having a similar tailed structure. Unlike skeletal muscle, cardiac muscle has very high levels of ΦChE, present mainly as the L form; AChE is present mainly as the medium (M) form, with smaller amounts of L and H_c. The latter pattern of AChE forms resembles that seen in several neural tissues examined. No alterations in AChE or ΦChE were found in cardiac or neural tissues from dystrophic chickens.     

Size of vasopressin receptors from rat liver and kidney

Specific vasopressin receptors in rat liver membranes were recently characterized [Cantau et al., Journal of Receptors Research, in the press]. They differ from the receptors characterized earlier in kidney membranes as regards coupling with adenylate cyclase and specificity towards vasopressin structural analogues [Rajerison et al. (1974) J. Biol. Chem. 249, 6390-6400; Butlen et al. (1978) Mol. Pharmacol. 14, 1006-1017]. The object of the present work was to see whether these differences reflect a difference in the molecular size of liver and kidney vasopresin receptors. For this purpose, rat liver and kidney membranes were incubated with [3H]vasopressin and solubilized with Triton X-100 (0.3%). The properties of the macromolecular components of soluble extracts to which labelled vasopressin remained bound were observed to resemble those of the intact membrane receptors as regards binding reversibility at 30-37 degrees C and sensitivity to guanyl nucleotides. The hydrodynamic parameters of the soluble hormone-receptor complexes were estimated from Utrogel column filtration experiments and from sucrose density gradient ultracentrifugation experiments. The following values were obtained for liver and kidney receptors respectively: Stokes' radii: 5.4 and 5.6 nm; standard sedimentation coefficients: 3.7 and 3.7 S; partial specific volumes: 0.75 and 0.78 ml x g-1; molecular weight: 83000 and 80000. These results indicate that the marked functional differences between liver and kidney receptors are not accompanied by appreciable differences in molecular size.     

Binding of scorpion and sea anemone neurotoxins to a common site related to the action potential Na+ ionophore in neuroblastoma cells.

The protein neurotoxin II from the venom of the scorpion $\text{Androctonus}\text{australis}$ Hector was labeled with ¹²⁵I by the lactoperoxidase method to a specific radioactivity of about 100 μCi/μg without loss of biological activity. The labeled neurotoxin binds specifically to a single class of non intereacting binding sites of high affinity (K_D = 0.3 – 0.6 nM) and low capacity (4000 – 8000 sites/cell) to electrically excitable neuroblastoma cells. Relation of these sites to the action potential Na⁺ channel is derived from identical concentration dependence of scorpion toxin binding and increase in duration and amplitude of action potential. The protein neurotoxin II from the sea anemone $\text{Anemona sulcata}$ also affects the closing of the action potential Na⁺ ionophore in nerve axons. The unlabelled sea anemone toxin modifies ¹²⁵I-labeled scorpion toxin II binding to neuroblastoma cells by increasing the apparent K_D for labeled scorpion toxin without modification of the number of binding sites. It is concluded that both $\text{Androctonus}$ scorpion toxin II and $\text{Anemona}$ sea anemone toxin II interact competitively with a regulatory component of the action potential Na⁺ channel.     

Characterisation of structures in salivary secretion film formation. An experimental study with atomic force microscopy

The purpose of the present study was to characterise the structure dynamics of pure salivary secretions retained on controlled surfaces with different surface energies in the early stage of salivary film formation. Germanium prisms prepared to have either low surface energy or medium surface energy were incubated in fresh secretions of either human parotid saliva (HPS) or human submandibular/sublingual saliva (HSMSLS) for 15, 90, and 180 min. After controlled rinsing with distilled water, the surfaces were air dried and thereafter imaged with atomic force microscopy (AFM). The amount of adsorbed material and the size of the structures detected increased with increased saliva exposure time. The film thicknesses varied from 10 to 150 nm, and both HPS and HSMSLS films contained structures with diameters varying from 40 nm to 2 microm. Some of these were clustered into special formations. The HPS films exhibited a more granular morphology than the HSMSLS films. Furthermore, branched lines were detected on the low surface energy germanium prisms incubated in saliva. The results indicate that exposure time, surface energy, and type of salivary secretion all are factors affecting the adsorption characteristics of salivary films.     

Characterisation of mildew resistant wheat-rye substitution lines and identification of an inverted chromosome by fluorescent in situ hybridisation

Seven different mildew resistant wheat lines derived from crosses between triticale and bread wheat were examined by molecular cytogenetics and chromosome C-banding in order to determine their chromosomal composition. Genomic in situ hybridisation (GISH) showed the presence of rye germplasm in all the lines and identified three substitution lines, three double substitution lines and one addition-substitution line. C-banding identified rye chromosomes 1R and 4R in the addition-substitution line, rye chromosomes 1R and 6R in two substitution lines and 1R and 2R in the third line, and rye chromosome 1R in the three substitution lines. Two of the latter lines (7-102 and 7-169) contained a modified form of the chromosome; fluorescent in situ hybridisation (FISH) using five different repetitive DNA-probes showed a pericentric inversion of 1R in both lines. The breakpoints of the 1R inversion were between (1) the 5S rDNA site and the NOR-region on the satellite of the short arm, and (2) between two AAC(5) sites close to the centromere on the long arm. The role of the rye chromosomes in the mildew resistance, the utilisation of the inverted 1R and the significance of the lines in wheat breeding are discussed.     

Na(+) channel regulation by calmodulin kinase II in rat cerebellar granule cells

The effects of specific CaM kinase II inhibitors were investigated on Na(+) channels from rat cerebellar granule cells. A maximal effect of KN-62 was observed at 20 microM and consisted of an 80% reduction of the peak Na(+) current after only a 10-min application. A hyperpolarizing shift of 8 mV in the steady-state inactivation was also observed. KN-04 (20 microM), an inactive analog, had no detectable effect. KN-62 was however inactive on Na(+) currents recorded from Chinese hamster ovary cells expressing the type II A alpha subunit. We have also analyzed the inhibitory effects of CaM kinase II 296-311 and CaM kinase II 281-309 peptides. Both peptides (75 microM) induced a maximum peak Na(+) current reduction within 30 min. Under similar conditions, a truncated peptide CaM kinase II 284-302 was ineffective. These results demonstrate that CaM kinase II acts as a modulator of Na(+) channel activity in cerebellar granule cells.     

Interactions between brain endothelial cells and human T-cell leukemia virus type 1-infected lymphocytes: mechanisms of viral entry into the central nervous system

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a variety of clinical manifestations, including tropical spastic paraparesis or HTLV-1-associated myelopathy (TSP/HAM). Viral detection in the central nervous system (CNS) of TSP/HAM patients demonstrates the ability of HTLV-1 to cross the blood-brain barrier (BBB). To investigate viral entry into the CNS, rat brain capillary endothelial cells were exposed to human lymphocytes chronically infected by HTLV-1 (MT2), to lymphocytes isolated from a seropositive patient, or to a control lymphoblastoid cell line (CEM). An enhanced adhesion to and migration through brain endothelial cells in vitro was observed with HTLV-1-infected lymphocytes. HTLV-1-infected lymphocytes also induced a twofold increase in the paracellular permeability of the endothelial monolayer. These effects were associated with an increased production of tumor necrosis factor alpha by HTLV-1-infected lymphocytes in the presence of brain endothelial cells. Ultrastructural analysis showed that contact between endothelial cells and HTLV-1-infected lymphocytes resulted in a massive and rapid budding of virions from lymphocytes, followed by their internalization into vesicles by brain endothelial cells and apparent release onto the basolateral side, suggesting that viral particles may cross the BBB using the transcytotic pathway. Our study also demonstrates that cell-cell fusion occurs between HTLV-1-infected lymphocytes and brain endothelial cells, with the latter being susceptible to transient HTLV-1 infection. These aspects may help us to understand the pathogenic mechanisms associated with neurological diseases induced by HTLV-1 infection.     

Human blood-brain barrier disruption by retroviral-infected lymphocytes: role of myosin light chain kinase in endothelial tight-junction disorganization

The blood-brain barrier (BBB), which constitutes the interface between blood and cerebral parenchyma, has been shown to be disrupted during retroviral associated neuromyelopathies. Human T cell leukemia virus (HTLV-1)-associated myelopathy/tropical spastic paraparesis is a slowly progressive neurodegenerative disease, in which evidence of BBB breakdown has been demonstrated by the presence of lymphocytic infiltrates in the CNS and plasma protein leakage through cerebral endothelium. Using an in vitro human BBB model, we investigated the cellular and molecular mechanisms involved in endothelial changes induced by HTLV-1-infected lymphocytes. We demonstrate that coculture with infected lymphocytes induces an increase in paracellular endothelial permeability and transcellular migration, via IL-1alpha and TNF-alpha secretion. This disruption is associated with tight junction disorganization between endothelial cells, and alterations in the expression pattern of tight junction proteins such as zonula occludens 1. These changes could be prevented by inhibition of the NF-kappaB pathway or of myosin light chain kinase activity. Such disorganization was confirmed in histological sections of spinal cord from an HTLV-1-associated myelopathy/tropical spastic paraparesis patient. Based on this BBB model, the present data indicate that HTLV-1-infected lymphocytes can induce BBB breakdown and may be responsible for the CNS infiltration that occurs in the early steps of retroviral-associated neuromyelopathies.     

Characterization of the axon initial segment (AIS) of motor neurons and identification of a para-AIS and a juxtapara-AIS, organized by protein 4.1B

Background

The axon initial segment (AIS) plays a crucial role: it is the site where neurons initiate their electrical outputs. Its composition in terms of voltage-gated sodium (Nav) and voltage-gated potassium (Kv) channels, as well as its length and localization determine the neuron's spiking properties. Some neurons are able to modulate their AIS length or distance from the soma in order to adapt their excitability properties to their activity level. It is therefore crucial to characterize all these parameters and determine where the myelin sheath begins in order to assess a neuron's excitability properties and ability to display such plasticity mechanisms. If the myelin sheath starts immediately after the AIS, another question then arises as to how would the axon be organized at its first myelin attachment site; since AISs are different from nodes of Ranvier, would this particular axonal region resemble a hemi-node of Ranvier?

Results

We have characterized the AIS of mouse somatic motor neurons. In addition to constant determinants of excitability properties, we found heterogeneities, in terms of AIS localization and Nav composition. We also identified in all α motor neurons a hemi-node-type organization, with a contactin-associated protein (Caspr)⁺ paranode-type, as well as a Caspr2⁺ and Kv1⁺ juxtaparanode-type compartment, referred to as a para-AIS and a juxtapara (JXP)-AIS, adjacent to the AIS, where the myelin sheath begins. We found that Kv1 channels appear in the AIS, para-AIS and JXP-AIS concomitantly with myelination and are progressively excluded from the para-AIS. Their expression in the AIS and JXP-AIS is independent from transient axonal glycoprotein-1 (TAG-1)/Caspr2, in contrast to juxtaparanodes, and independent from PSD-93. Data from mice lacking the cytoskeletal linker protein 4.1B show that this protein is necessary to form the Caspr⁺ para-AIS barrier, ensuring the compartmentalization of Kv1 channels and the segregation of the AIS, para-AIS and JXP-AIS.

Conclusions

α Motor neurons have heterogeneous AISs, which underlie different spiking properties. However, they all have a para-AIS and a JXP-AIS contiguous to their AIS, where the myelin sheath begins, which might limit some AIS plasticity. Protein 4.1B plays a key role in ensuring the proper molecular compartmentalization of this hemi-node-type region.