Expression, phosphorylation, and glycosylation of CNS proteins in aversive operant conditioning associated memory in Lymnaea stagnalis.

Long-term memory formation requires "de novo" expression and post-translational modification of many proteins. Understanding the temporal and spatial regulatory pattern of these proteins is fundamental to decoding the molecular basis of learning and memory. We characterized changes in expression, phosphorylation, and glycosylation of CNS proteins after operant conditioning in pond snail Lymnaea stagnalis. The phosphorylation and the glycosylation levels of proteins, measured by the ratio of Pro-Q Diamond (phosphoproteins) or Pro-Q Emerald (glycoproteins) vs. SYPRO-Ruby (total proteins) signals, increased during memory formation. Proteins whose modulation of phosphorylation might be involved in learning and memory were identified by mass spectrometry (MS) and are associated with cytoskeleton, glutamine cycle, energy metabolism, G-protein signaling, neurotransmitter release regulation, iron transport, protein synthesis, and cell division. Phosphorylation of actin increased during memory formation. To identify proteins whose expression levels changed in long-term memory formation we used two-dimensional difference gel electrophoresis followed by MS. The up-regulated proteins are mostly associated with lipoprotein and cholesterol metabolism, protein synthesis and degradation, cytoskeleton, nucleic acid synthesis, and energy supply. The down-regulated proteins are enzymes of aspartic acid metabolism involved in regulation of protein synthesis. Our proteomic analyses have revealed a number of candidate proteins associated with memory formation. These findings provide new directions for further investigation into the signaling networks required for memory formation and consolidation.

Neural phosphoproteomics of a chronic hypoxia model--Lymnaea stagnalis.

Chronic hypoxia is a common clinical event that induces adaptive responses and can result in behavioral deterioration. The reduction of metabolic rate during hypoxia may limit overall protein phosphorylation owing to the lack of high energy phosphate. However, the hypoxia-induced regulation of phosphoproteins is poorly understood. Here, we characterized the CNS phosphoproteome of Lymnaea stagnalis, a freshwater snail that has been used as a model to study chronic hypoxia-induced neural depression. After hypoxia treatment for 4 days, the motor behavior of the snail was suppressed. Electrophysiological measurements from Pedal A (PeA) interneurons showed that hypoxia increased the frequency of spontaneous postsynaptic excitatory potentials (sEPSPs), but reduced the firing frequency, the amplitude, and the half-width duration (APD(50)) of spontaneous action potentials. Imaging with a fluorescent phosphate label, Pro-Q Diamond, revealed that the neuronal phosphoprotein level was reduced after the hypoxia treatment. The hypoxia-induced changes in the phosphoproteome of the central ganglia were quantified using one-dimensional gel-electrophoresis by comparing the fluorescence intensity ratio of phospholabeled phosphoproteins versus total proteins between the hypoxia and control groups. We analyzed 16 protein bands: eight showed decreased phosphorylation levels after hypoxia treatment, and eight did not change. Using mass spectrometry analysis and protein database matching we found three phosphoproteins that may be associated with chronic hypoxia-induced neuronal adaptive response of the snail. This is the first proteomic screening for neural phosphoproteins in chronic hypoxia.

Regulation of the tip-high [Ca2+] gradient in growing hyphae of the fungus Neurospora crassa.

Previous work has shown that hyphal elongation in the fungus Neurospora crassa requires a tip-high cytosolic Ca2+ gradient. The source of the Ca2+ appears to be intracellular stores as there is no net transplasma membrane Ca2+ flux at the elongating hyphal tip and modification of ion fluxes across the plasma membrane using voltage clamp is without effect on tip growth. To decode the internal mechanisms which generate and maintain the tip-high Ca2+ gradient we first identified calcium regulators which affect hyphal growth and morphology, then determined how they modify cytosolic [Ca2+] and the actin cytoskeleton using fluorescent dyes and confocal microscopy. Cyclopiazonic acid (a known inhibitor of the endoplasmic reticulum calcium ATPase) inhibits growth and increases cytoplasmic [Ca2+] in the basal region 10-25 microm behind the hyphal tip. 2-APB (2-aminoethoxydiphenyl borate, an inhibitor of IP3-induced Ca2+ release) inhibits hyphal elongation and dissipates the tip-high Ca2 gradient 0-10 microm from the tip. Microinjections of the IP3 receptor agonists adenophostin A and IP3 (but not control microinjections of the biologically inactive L-IP3) transiently inhibited growth and induced subapical branches. IP3 microinjections, but not L-IP3, lowered tip-localized [Ca2+] and increased basal [Ca2+]. Even though their effect on [Ca2+] gradients was different, both cyclopiazonic acid and 2-APB disrupted similarly the normal actin pattern at the hyphal apex. Conversely, disruption of actin with latrunculin B dissipated tip-localized Ca2+. We conclude that the tip-high Ca2+ gradient is generated internally by Ca2+ sequestration into endoplasmic reticulum behind the tip and Ca2+ release via an IP3 receptor from tip-localized vesicles whose location is maintained by the actin cytoskeleton.

Structure and function analysis of the calcium-related gene spray in Neurospora crassa.

The spray gene was cloned, and wildtype and mutant alleles were sequenced. Spray(+) has a 3452-bp open reading frame plus seven introns. The spray mutant had a T --> G transversion close to the carboxyl end, creating a stop codon (TGA). The sequence shows no match to genes of known function, but the carboxyl end shows seven transmembrane domains and matches putative membrane proteins of yeast. The most abundant transcript detected was 4.4 kb in size. Repeat-induced point mutagenesis produced the mutant spray phenotype. Electrophysiological analysis showed that ion fluxes in the spray plasma membrane are normal; furthermore, whereas the spray mutant was known to have no organelle-based calcium fluorescence, the cytosol shows a tip-high calcium gradient. The spray mutant is sensitive to calcineurin inhibitors. The results suggest that the SPRAY protein is located in an organellar membrane, regulating the distribution of Ca(2+) via calcineurin.

A particular structure associated to the nucleoid of cyanobacteria.

Regressive staining as well as beta-radiations or trypsin treatment on Synechocystis PCC6803 and Spirulina platensis (Gom,-Geilteri.) whole cells or permeaplasts, respectively, have demonstrated the presence of a particular structure associated to the nucleoid of cyanobacteria. This structure with a tridimensional network aspect has been called scaffold-like. We presume that it represents the cellular-molecular support for the supercoiling of the nucleoid of cyanobacteria.

Biochemical and cytogenetical study of the mycoplasmal antigen and of the cyclophosphamide action in mammalians, in vivo. The action of some immunomodulatory antioxidants.

It was proved spectrophotometrically that Mycoplasma agalactiae antigen inoculated in vivo in sheep modifies the corresponding erythrocyte lysates reactivity toward methylene blue and neutral red and induces several types of chromosomal rearrangements. The treatment in vivo of sheep with an original preparation obtained from the Phaseolus vulgaris pods restores the erythrocyte lysates reactivity toward the two redox dyes and reduces the chromosomal abnormalities frequency induced by the mycoplasmal antigen. It was also demonstrated by optical and electronical microscopy that the Smise line mouse meiocytes exhibit chromosomal abnormalities induced by the cyclophosphamide treatment in vivo. In the case of concomitant treatment with the cyclophosphamide and C vitamin the same frequency of abnormalities was recorded as in the simple treatment with the drug.