Induced expression of MMP-9 in C6 glioma cells is inhibited by PDGF via a PI 3-kinase-dependent pathway

The involvement of phosphatidylinositol 3 (PI 3)-kinase in the signalling pathways leading to MMP-9 expression in glioma cells remains unclear. Here, we report that PI 3-kinase inhibits MMP-9 expression induced by either IL-1 or TNF-alpha in rat C6 glioma cells. Using zymography and semi-quantitative RT-PCR analysis, we showed that treatment of C6 cells with wortmannin, an inhibitor of PI 3-kinase activity, potentiated the expression of MMP-9 induced by both cytokines. In contrast, platelet-derived growth factor (PDGF), an inducer of PI 3-kinase activity in C6 cells, inhibited IL-1- or TNF-alpha-induced MMP-9 secretion. Accordingly, this inhibition by PDGF was prevented by wortmannin. Furthermore, stable C6 clones over-expressing the dominant-negative form the regulatory subunit of PI 3-kinase potentiated the expression of MMP-9 induced by IL-1 or TNF-alpha. Taken together, these results suggest that PI 3-kinase may act as a negative regulator of MMP-9 expression in C6 glioma cells.     

Structural analysis of the epitopes recognized by monoclonal antibodies to angiotensin II

Six clones were obtained that secrete anti-angiotensin II antibodies after somatic cell fusions between splenocytes of immunized BALB/c or outbred OF1 mice and NS-1 myeloma cells. The dissociation constants for angiotensin II ranged from 0.3 to 2.9 nM. A panel of 20 structural analogs of the hormone were used as probes to analyze the specificity of binding. From the binding studies and the putative three-dimensional structures of the tested peptides, three families of antibodies could be distinguished that recognized overlapping epitopes; the conservation of the native conformation of the angiotensin II molecule in the analogs appeared essential for the preservation of a high affinity to the antibodies. With one antibody, the affinities of the angiotensin II analogs have been correlated with their intrinsic biologic activities (as measured by in vivo pressor tests), and not with their binding affinity to the membrane receptor. These results are interpreted as mimicry, by the antibody binding site, of the active conformation of the receptor site.     

Activation of the Voltage-Sensitive Sodium Channel by a β-Scorpion Toxin in Rat Brain Nerve-Ending Particles

Neurotoxins purified from scorpion venoms previously had been divided into two classes according to their binding properties in rat brain synaptosomes. However, the pharmacological action of beta-scorpion toxin (beta-ScTx) on this preparation has not yet been described. In this report we show that a beta-ScTx induced an increase in 22Na+ uptake through synaptosomal voltage-sensitive sodium channels since this stimulation was abolished by tetrodotoxin (TTX). The increase was smaller than with veratridine and no synergy was observed between beta-ScTx and veratridine, as is the case for alpha-scorpion toxin (alpha-ScTx) and veratridine. The effects of alpha- and beta-ScTx were additive and the concentration-effect curves for each type of toxin were not modified by the other, suggesting that these two types of toxins act through distinct and noninteracting receptor sites. This was confirmed by the absence of mutual modification of the equilibrium and kinetic binding properties. beta-ScTx was shown to inhibit the uptake and to stimulate the release of [3H]gamma-aminobutyric acid. These effects were blocked by TTX, and no synergy was observed with veratridine. It was concluded that all these effects are mediated by the activation of voltage-sensitive sodium channels induced by the binding of beta-ScTx to a receptor site (site 4) distinct from those for other neurotoxins acting on sodium channels.     

Marker-assisted breeding of <Emphasis Type="Italic">Musa balbisiana</Emphasis> genitors devoid of infectious endogenous Banana streak virus sequences

Breeding new interspecific banana hybrid varieties relies on the use of Musa acuminata and M. balbisiana parents. Unfortunately, infectious alleles of endogenous Banana streak virus (eBSV) sequences are present in the genome of Musa balbisiana genitors. Upon activation by biotic and abiotic stresses, these infectious eBSVs lead to spontaneous infections by several species of Banana streak virus in interspecific hybrids harboring both Musa acuminata and M. balbisiana genomes. Here we provide evidence that seedy M. balbisiana diploids display diverse eBSV allelic combinations and that some eBSVs differ structurally from those previously reported. We also show that segregation of infectious and non-infectious eBSV alleles can be achieved in seedy M. balbisiana diploids through self-pollination or chromosome doubling of haploid lines. We report on the successful breeding of M. balbisiana diploid genitors devoid of all infectious eBSV alleles following self-pollination and on the potential of breeding additional M. balbisiana diploid genitors free of infectious eBSVs by crossing parents displaying complementary eBSV patterns. Our work paves the way to the safe use of M. balbisiana genitors for breeding banana interspecific hybrid varieties with no risk of activation of infectious eBSVs.     

Binding of beta-scorpion toxin: a physicochemical study

The binding to rat brain synaptosomes of a beta-scorpion toxin, i.e., toxin II of Centruroides suffusus suffusus (Css II), was studied as a function of pH, temperature, and concentration of some monovalent and divalent cations. At 10 degrees C and pH 6.0, the specific binding of 125I-labeled Css II corresponds to a single class of noninteracting high-affinity binding sites (KD = 0.18 nM) with a capacity (4.2 pmol/mg of protein) that is almost identical with that generally accepted for saxitoxin. The equilibrium dissociation constant of beta-scorpion toxin is pH independent, but the maximum binding capacity is reduced with increasing pH. Li+, guanidinium, Ca2+, Mg2+, and Mn2+ modified the apparent KD of the 125I-labeled Css II toxin. The equilibrium dissociation constant varies markedly with the temperature. The van't Hoff plot of the data is curvilinear, corresponding to a standard free-energy change associated with an entropy-driven process. The association rate constant also varies considerably with the temperature whereas the Arrhenius plot is linear between 1 and 30 degrees C. The energy of activation determined from these data is 17.6 kcal/mol. These results support the hypothesis that a cluster of nonpolar amino acid residues present on one face of the molecule is involved in the toxin-receptor interaction.     

Acetylcholinesterase Molecular Forms in Chick Ciliary Ganglion: Pre- and Postsynaptic Distribution Derived from Denervation, Axotomy, and Double Section

Abstract: Four main molecular forms of acetylcholinesterase (AChE) characterized by their sedimentation coefficients (5S, 7.5S, 11.5S, and 20S), are found in chick ciliary ganglion. After transection of the preganglionic nerve (denervation), total AChE activity in the ganglion dropped by 35% in 2 days. By then, 11.5s and 20s forms had diminished by 60 and 75% respectively, where as 7.5s remained practically unchanged. Since presynaptic structures disappeared 2 days after denervation, we inferred that at most 35% of total ganglion AChE was presynaptic: 11.5s and 20s might be mainly presynaptic and 7.5S, postsynaptic. At later time intervals. total AChE continued to decline up to day 5, possibly as a result of orthograde transynaptic regulation of the enzyme activity. After transection of postganglionic nerves (axotomy), total ganglion activity showed little change; 11.5s and 20s decreased by 40 and 6076, respectively, in 5 days, but these drops were compensated for by an early increase in 7 5S, which started the day after axotomy. After simultaneous transection of both pre- and postganglionic nerves (double section), total ganglion AChE dropped rapidly by 35% in 1 day and remained at that level up to 21 days. The 11.5S diminished rapidly by 60% in 1 day. The early increase of the 7.5s form induced by axotomy alone did not occur. Since the effect resulting from double section was not the equivalent of the cumulative effects observed after denervation and axotomy, respectively, the level of AChE forms in the ganglion may be regulated by reciprocal interaction of pre- and postsynaptic elements. After denervation and double section but not after axotomy alone, the contralateral non-operated ganglion exhibited a fall in the 20s form. This suggests that a transynaptic effect is exerted on AChE by the contralateral preganglionic neuron. Taken together, these results indicate that the various AChE molecular forms in chick ciliary ganglion are preferentially but not exclusively distributed as follows: the pre- and postganglionic axons contain mainly the 11.5S form, whereas nerve endings and synaptic structures are enriched in 20S, and ganglion cell bodies, in 7.5s.     

Long-Lasting Cerebral Vasospasm,Microthrombosis, Apoptosis and Paravascular Alterations Associated with Neurological Deficits in a Mouse Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a devastating disease with high mortality and morbidity. Long-term cognitive and sensorimotor deficits are serious complications following SAH but still not well explained and described in mouse preclinical models. The aim of our study is to characterize a well-mastered SAH murine model and to establish developing pathological mechanisms leading to cognitive and motor deficits, allowing identification of specific targets involved in these long-term troubles. We hereby demonstrate that the double blood injection model of SAH induced long-lasting large cerebral artery vasospasm (CVS), microthrombosis formation and cerebral brain damage including defect in potential paravascular diffusion. These neurobiological alterations appear to be associated with sensorimotor and cognitive dysfunctions mainly detected 10 days after the bleeding episode. In conclusion, this characterized model of SAH in mice, stressing prolonged neurobiological pathological mechanisms and associated sensitivomotor deficits, will constitute a validated preclinical model to better decipher the link between CVS, long-term cerebral apoptosis and cognitive disorders occurring during SAH and to allow investigating novel therapeutic approaches in transgenic mice.     

BRCA2 is required for neurogenesis and suppression of medulloblastoma

Defective DNA damage responses in the nervous system can result in neurodegeneration or tumorigenesis. Despite the importance of DNA damage signalling, the neural function of many critical DNA repair factors is unclear. BRCA2 is necessary for homologous recombination repair of DNA and the prevention of diseases including Fanconi Anemia and cancer. We determined the role of BRCA2 during brain development by inactivating murine Brca2 throughout neural tissues. In striking contrast to early embryonic lethality after germ-line inactivation, Brca2(LoxP/LoxP);Nestin-cre mice were viable. However, Brca2 loss profoundly affected neurogenesis, particularly during embryonic and postnatal neural development. These neurological defects arose from DNA damage as Brca2(LoxP/LoxP);Nestin-cre mice showed extensive gammaH2AX in neural tissue and p53 deficiency restored brain histology but lead to rapid formation of medulloblastoma brain tumors. In contrast, loss of the Atm kinase did not markedly attenuate apoptosis after Brca2 loss, but did partially restore cerebellar morphology, supporting a genomic surveillance function for ATM during neurogenesis. These data illustrate the importance of Brca2 during nervous system development and underscore the tissue-specific requirements for DNA repair factors.