Cabbage cryoprotectin is a member of the nonspecific plant lipid transfer protein gene family.

We have recently purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea) to electrophoretic homogeneity, which protects thylakoids isolated from the leaves of nonacclimated spinach (Spinacia oleracea) from freeze-thaw damage. Sequencing of cryoprotectin showed the presence of at least three isoforms of WAX9 proteins, which belong to the class of nonspecific lipid transfer proteins. Antibodies raised against two synthetic peptides derived from the WAX9 proteins recognized a band of approximately 10 kD in western blots of crude cryoprotectin preparations. This protein and the cryoprotective activity could be precipitated from solution by the antiserum. We show further that cryoprotectin is structurally and functionally different from WAX9 isolated from the surface wax of cabbage leaves. WAX9 has lipid transfer activity for phosphatidylcholine, but no cryoprotective activity. Cryoprotectin, on the other hand, has cryoprotective, but no lipid transfer activity. The cryoprotective activity of cryoprotectin was strictly dependent on Ca(2+) and Mn(2+) and could be inhibited by chelating agents, whereas the lipid transfer activity of WAX9 was higher in the presence of ethylenediaminetetraacetate than in the presence of Ca(2+) and Mn(2+).

Ca2+-induced Ca2+ release supports the relay mode of activity in thalamocortical cells.

Ca2+ ions play an important role during rhythmic bursting of thalamocortical neurons within sleep. The function of Ca2+ during the tonic relay mode of these neurons during wakefulness is less clear. Here, we report that tonic activity in thalamocortical cells results in an increase in the intracellular Ca2+ concentration and subsequent release of Ca2+ from intracellular stores mediated via ryanodine receptors (RyRs). Blockade of Ca2+ release shifted the regular firing of single action potentials toward the generation of spike clusters. Regular spike firing and intracellular Ca2+ release thus appear to be functionally coupled in a positive feedback manner, thereby supporting the relay mode of thalamocortical cells during wakefulness. Regulatory influences may be coupled to this system via the cyclic ADP ribose pathway.

Cellular reactivity to mechanical axonal injury in an organotypic in vitro model of neurotrauma.

An in vitro model of traumatic brain injury is described that is based on organotypic cocultures (OTCs) of rat neocortex and thalamus connected by reciprocal axonal projections. Localized mechanical compression of this projection was inflicted with a mechanical device, and the effects on cell viability, axonal morphology, and protein expression levels were analyzed. Within 24 h after insult, major cell damage occurred in infragranular cortical layers containing the corticothalamic projection neurons and in thalamic regions adjacent to the mechanical impact as was assessed through the use of the vital stain Syto 21, and propidium iodide labeling. A small, but significant number of calretinin-positive interneurons in cortical and thalamic areas displayed symptoms of injury. Axonal elements, as revealed by neurofilament (NF-H/M) immunohistochemistry, in the corticothalamic transition zone displayed pathomorphological changes, such as axonal bulbs and swellings, already 4 h after insult. Densitometric analysis revealed that MAP-2a,b expression was not significantly changed within 4 h after injury. A significant reduction in MAP-2a,b amount was evident at 20 h after injury in thalamus (by 31.6%) and cortex (by 30%) maintained for 12 days in vitro (DIV), but not in OTCs aged 20 DIV. The axonally localized form MAP-2c significantly increased in cortex of 12-DIV OTCs at 4 and 20 h after insult (65.6% and 33.4%, respectively). MAP-2c levels in cortex of 20 DIV initially increased by 47.7% and declined below control values 20 h after injury. Thalamic areas revealed a delay in MAP-2c reactivity, in that expression was significantly elevated only at 20 h after injury (by 84.4% in 12-DIV and by 39.6% in 20-DIV OTCs, respectively). These data may reflect the regenerative ability of juvenile, but not of older neurons in response to mechanical axonal injury.

Postnatal expression pattern of calcium-binding proteins in organotypic thalamic cultures and in the dorsal thalamus in vivo.

The present study describes the postnatal expression of calbindin, calretinin and parvalbumin and glutamic acid decarboxylase (GAD) and microtubule-associated protein 2 (MAP2) in organotypic monocultures of rat dorsal thalamus compared to the thalamus in vivo. Cultures were maintained for up to 7 weeks. Cortex-conditioned medium improved the survival of thalamic cultures. MAP2-immunoreactive material was present in somata and dendrites of small and large-sized neurons throughout the cultures. Parvalbumin immunoreactivity was present in larger multipolar or bitufted neurons along the edge of a culture. These neurons also displayed strong parvalbumin mRNA and GAD mRNA expression, and GABA immunoreactivity. They likely corresponded to cells of the nucleus reticularis thalami. Parvalbumin mRNA, but neither parvalbumin protein nor GAD mRNA, was expressed in neurons with large somata within the explant. They likely represented relay cells. GAD mRNA, but not parvalbumin mRNA, was expressed in small neurons within the explants. Small neurons also displayed calbindin- and calretinin-immunoreactivity. The small neurons likely represented local circuit neurons. The time course of expression of the calcium-binding proteins revealed that all were present at birth with the predicted molecular weights. A low, but constant parvalbumin expression was observed in vitro without the developmental increase seen in vivo, which most likely represented parvalbumin from afferent sources. In contrast, the explantation transiently downregulated the calretinin and calbindin expression, but the neurons recovered the expression after 14 and 21 days, respectively. In conclusion, thalamic monocultures older than three weeks represent a stable neuronal network containing well differentiated neurons of the nucleus reticularis thalami, relay cells and local circuit neurons.

Purification and Characterization of a Cryoprotective Protein (Cryoprotectin) from the Leaves of Cold-Acclimated Cabbage.

We have purified a protein (cryoprotectin) from the leaves of cold-acclimated cabbage (Brassica oleracea L.) that protects thylakoids from nonacclimated spinach (Spinacia oleracea L.) against freeze-thaw damage. The procedure involves precipitations by heat, ammonium sulfate, and the glycosaminoglycan heparin and column chromatography on Polyamide 6 and a C18 reverse-phase matrix. After reverse-phase chromatography we obtained a single band of an apparent molecular mass of 7 kD when fractions that showed cryoprotective activity were analyzed by sodium dodecyl sulfate gel electrophoresis and silver staining. Gel-filtration experiments confirmed that the active protein is a monomer of 7 kD native molecular mass. This 7-kD protein could be purified only from cold-acclimated cabbage, but not from plants grown under nonacclimating conditions. Using peroxidase-labeled lectins, we show that cryoprotectin is a glycoprotein and that the saccharide moiety contains [alpha]1-3-linked fucose.