Spontaneous calcium transients regulate neuronal plasticity in developing neurons

Calcium ions play critical roles in neuronal differentiation. We have recorded transient, repeated elevations of calcium in embryonic Xenopus spinal neurons over periods of 1 h in vitro and in vivo, confocally imaging fluo 3-loaded cells at 5 s intervals. Calcium spikes and calcium waves are found both in neurons in culture and in the intact spinal cord. Spikes rise rapidly to approximately 400% of baseline fluorescence and have a double exponential decay, whereas waves rise slowly to approximately 200% of baseline fluorescence and decay slowly as well. Imaging of fura 2-loaded neurons indicates that intracellular calcium increases from 50 to 500 nM during spikes. Both spikes and waves are abolished by removal of extracellular calcium. Developmentally, the incidence and frequency of spikes decrease, whereas the incidence and frequency of waves are constant. Spikes are generated by spontaneous calcium-dependent action potentials and also utilize intracellular calcium stores. Waves are produced by a mechanism that does not involve classic voltage-dependent calcium channels. Spikes are required for expression of the transmitter GABA and for potassium channel modulation. Waves in growth cones are likely to regulate neurite extension. The results demonstrate the roles of a novel signaling system in regulating neuronal plasticity, that operates on a time scale 10⁴ times slower than that of action potentials. © 1995 John Wiley & Sons, Inc.     

Galectin-3 mRNA level depends on transformation phenotype in ras-transformed NIH 3T3 cells

Summary— The increase in galectin-3 lectin content observed in tumours or in in vitro transformed cells suggests that this lectin is important in the transformation process. In the present study, we investigated the mRNA expression level of the galectin-3, galectin-I and macrophage mannose receptor in normal and ras-transformed NIH 3T3 cells in relation to their transformation state. The galectin3 mRNA content in ras-transformed cells is increased in fully transformed cells, with a maximum in ras-transformed cells that have lost their growth anchorage-dependence. Under the same conditions, the galectin-1 mRNA level which was high in normal cells, increased slightly in transformed cells. The mRNA for the macrophage mannose receptor was not detected in 3T3 cells or in their ras-transformed counterparts.     

Theoretical Basis of Protocols for Seed Storage III. Optimum Moisture Contents for Pea Seeds Stored at Different Temperatures

In previous work, we demonstrated that there was an optimummoisture level for seed storage at a given temperature (Vertucciand Roos, 1990), and suggested, using thermodynamic considerations,that the optimum moisture content increased as the storage temperaturedecreased (Vertucci and Roos, 1993b). In this paper, we presentdata from a two year study of aging rates in pea (Pisum sativum)seeds supporting the hypothesis that the optimum moisture contentfor storage varies with temperature. Seed viability and vigourwere monitored during storage under dark or lighted conditionsat relative humidities between 1 and 90%, and temperatures between-5 and 65°C. The optimum moisture content varied from 0·015g H₂O g^-1 d.wt at 65°C to 0·101 g H₂O g^-1 d.wt at15°C under dark conditions and from 0·057 at 35°Cto 0·092 g H₂O g^-1 d.wt at -5°C under lighted conditions.Our results suggest that optimum moisture contents cannot beconsidered independently of temperature. This conclusion hasimportant implications for 'ultra-dry' and cryopreservationtechnologies.Copyright 1994, 1999 Academic Press Seed storage, seed aging, seed longevity, water content, temperature, glass, desiccation damage, ultradry, Pisum sativum L., pea, cryopreservation     

Cloning of Multiple Forms of Goldfish Vimentin: Differential Expression in CNS

Abstract: In efforts to determine the primary structure of intermediate filament proteins in the goldfish visual pathway, we isolated clones from a retinal λgt11 cDNA expression library that represent goldfish vimentin. We show that there are at least two forms of goldfish vimentin, designated as vimentin α and vimentin β. RNase protection assays indicate that vimentin α mRNA is expressed in low amounts in retina, optic nerve, and brain and in higher amounts in spinal cord. In contrast, vimentin β mRNA is expressed in low amounts in retina, optic nerve, brain, and spinal cord and in very high amounts in eye lens. Immunohistochemical studies show that in the optic nerve, vimentin α is mainly restricted to blood vessels, meninges, and septa. Light staining is observed with this antibody in an astrocytic glial pattern throughout the optic nerve. Two-dimensional gel analysis shows that all of these goldfish vimentins are low abundant components of optic nerve cytoskeletal preparations.     

Cloning and Expression of a 5-Hydroxytryptamine7 Receptor Positively Coupled to Adenylyl Cyclase

Abstract: In a number of different cell types, phosphorylation of a 63-kDa protein has been shown to increase rapidly in response to stimuli that lead to an increase in intracellular calcium. Here, a stimulus-sensitive protein at this molecular weight is identified in PC12 cells and rat cortical synaptosomes as phosphoglucomutase. In addition, the added phosphate is shown to be in an oligosaccharide terminating in phosphodiester-linked glucose. In synaptosomes, incorporated radioactivity, following incubation with [¹⁴C]glucose or the [β-³⁵S]phosphorothioate analogue of UDP-glucose, was found to increase within 5 s of stimulation and return to baseline within 25 s. Despite the many pathways utilizing glucose, this was the only detectable protein glycosylation observed in synaptosomes. These results indicate that cytoplasmic glycosylation is reversible and rapidly regulated, and suggest that phosphoglucomutase undergoes an alteration in function and/or topography in response to increases in intracellular calcium.     

Mutations in somePolycomb group genes ofDrosophila interfere with regulation of segmentation genes

Mutations in severalPolycomb (Pc) group genes cause maternal-effect or zygotic segmentation defects, suggesting thatPc group genes may regulate the segmentation genes ofDrosophila. We show that individuals doubly heterozygous for mutations inpolyhomeotic and six otherPc group genes show gap, pair rule, and segment polarity segmentation defects. We examined double heterozygous combinations ofPc group and segmentation mutations for enhancement of adult and embryonic segmentation defects.Posterior sex combs andpolyhomeotic interact withKrüppel ² and enhance embryonic phenotypes ofhunchback andknirps, andpolyhomeotic enhanceseven-skipped. Surprisingly, flies carrying duplications ofextra sex combs (esc), that were heterozygous for mutations ofeven-skipped (eve), were extremely subvital. Embryos and surviving adults of this genotype showed strong segmentation defects in even-numbered segments. Antibody studies confirm that expression ofeve is suppressed by duplications ofesc. However,esc duplications have no effect on other gap or pair rule genes tested. To our knowledge, this is only the second triplo-abnormal phenotype associated withPc group genes. Duplications of nine otherPc group genes have no detectable effect oneve. Expression ofengrailed (en) was abnormal in the central nervous systems of mostPc group mutants. These results support a role forPc genes in regulation of some segmentation genes, and suggest thatesc may act differently from otherPc group genes.     

Genetic analysis of the minimal replicon of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid

Using a combination of mutagenesis with the transposon and polymerase chain reaction subcloning, the essential elements of the replication region of the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid have been identified. An open reading frame, coding for a protein with homology to Rep proteins from other Lactococcus plasmids, is essential. This protein is trans-acting and could not be replaced by the Rep protein from another Lactococcus plasmid. A second open reading frame immediately downstream from the first could be removed or inactivated with no apparent effect on plasmid replication. A region containing two 10 by direct repeats and three tandem repeats of a 22 by sequence, immediately upstream of the essential open reading frame, is also essential and probably includes the origin of replication. A 181-bp DNA fragment containing this region was sufficient to allow replication in Lactococcus if the trans-acting protein was provided on another replicon. Single-stranded replication intermediates could not be detected, suggesting that the citrate plasmid uses theta replication rather than rolling-circle replication.