The Large Isoform of Myelin-Associated Glycoprotein Is Scarcely Expressed in the Quaking Mouse Brain

Two polypeptide isoforms of myelin-associated glycoprotein (MAG) with molecular masses of 72 and 67 kDa are produced by alternative splicing of the exon 12 portion. Our previous work has demonstrated that in the quaking mouse brain this alternative splicing is lacking and that the mRNA coding the large MAG isoform (L-MAG) is scarcely expressed, whereas that of small MAG isoform (S-MAG) is overexpressed. In the present study, we prepared antisera specific to the S-MAG and L-MAG amino acid residues, respectively. Immunoblots showed that the L-MAG band was scarcely detectable in the quaking mouse brain, whereas the S-MAG band had an apparently higher molecular mass than in the normal control. Our immunohistochemical study also showed that L-MAG was scarcely stained in the quaking mouse brain. These results seemed to reflect a reduction in content of L-MAG mRNA and abnormal glycosylation in the quaking mouse brain.     

Differential Expression of mRNA and Protein Encoding Retinal and Pineal S-Antigen During the Light/Dark Cycle

S-Antigen is a soluble cell protein unique to the retina and pineal gland. In the former, it is a well-characterized molecule that participates in light-induced signal transduction in photoreceptor cells. In the latter, the functional role is presently not known. The expression of S-antigen and its mRNA was examined in the rat retina and pineal gland throughout the diurnal cycle and with light interruption of the dark cycle. A cDNA for rat S-antigen was isolated from a pineal gland library to examine the mRNAs. A 1.7-kb mRNA for S-antigen was observed in both the pineal gland and the retina. Retinal S-antigen mRNA was expressed throughout the diurnal cycle and increased with light interruption of the dark cycle. In contrast, pineal gland S-antigen mRNA levels were detectable only during the dark and were absent preceding and during light. The phenotypic expression of immunoreactive S-antigen, identified with two S-antigen monoclonal antibodies (MAbs), MAb A9C6 and MAb C10C10, was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel (PAGE) and isoelectric focusing (IEF) electrophoresis. Immunoblot analysis of gels after SDS-PAGE revealed a single 46-kDa protein in retina. In contrast, two bands of approximately 43 and 46 kDa were identified in the pineal gland. Immunoblots of the retinal extracts separated by IEF electrophoresis revealed five S-antigen isomers, which vary quantitatively throughout the diurnal cycle and when light interrupted the dark cycle. Immunoblots of the pineal gland samples separated by IEF electrophoresis indicated that the pineal gland possesses four pineal gland-specific forms of S-antigen in addition to the five forms present in the retina. The differences observed in the mRNA and protein analyses suggest tissue-specific structural components for S-antigen in the retina and pineal gland that are not regulated in the same manner.     

Short- and Long-Term Alterations of Gene Expression in Limbic Structures by Repeated Electroconvulsive-Induced Seizures

Rats were submitted to a series of 10 daily electroconvulsive shocks (ECS). A first group of animals was killed 1 day after the last seizure and a second group 30 days later. Tyrosine hydroxylase (TH) activity was measured using an in vitro assay in the nucleus caudatus, anterior cortex, amygdala, substantia nigra, ventral tegmental area, and locus ceruleus. The mRNA corresponding to this enzyme (TH-mRNA) was evaluated using a cDNA probe at the cellular level in the ventral tegmental area, substantia nigra, and locus ceruleus. Met-enkephalin (MET)-immunoreactivity and the mRNA coding for the preproenkephalin (PPE-mRNA) were assayed in striatum and the central nucleus of the amygdala. The day after the last ECS an increase of TH activity was observed in the ventral tegmental area, locus ceruleus, and substantia nigra in parallel with a similar increase in the amygdala and striatum; in the anterior cortex TH activity remained unchanged. TH-mRNA was increased in the locus ceruleus, evidencing the presence in this structure of a genomic activation. The amounts of MET and PPE-mRNA were unaffected in the striatum but increased in the amygdala. Thirty days after the last ECS we observed a decrease of TH activity in the amygdala and of TH-mRNA amount in the ventral tegmental area. In the locus ceruleus TH-mRNA remained higher in treated animals than in controls whereas TH activity returned to control levels. These results demonstrate that a series of ECS induces an initial increase of the activity of mesoamygdaloid catecholaminergic neurons followed by a sustained decrease through alterations of TH gene expression which could mediate the clinical effect of the treatment.     

Induction of c-fos mRNA Expression by Afterdischarge in the Hippocampus of Naive and Kindled Rats

Periodic induction of focal electrical seizure [afterdischarge (AD)] is an absolute prerequisite for the development of kindling, an animal model of complex partial epilepsy. Once established, it is a permanent condition. The mechanism(s) that translate ADs, which last tens of seconds, into life-long alterations in the CNS is unclear. Cellular immediate-early genes have been implicated in the conversion of short-term stimuli to long-term alterations in cellular phenotypes by regulating target gene expression. We have investigated the contribution of one such early gene, c-fos, to this process. The relationship between ADs and expression of c-fos gene in the rat hippocampus, a key structure in kindling development, was studied by analysis of mRNA levels. The low constitutive expression of c-fos mRNA in the hippocampus was not altered by kindling. There was an "all-or-none" relationship between induction of c-fos and the duration of AD. The threshold for induction was approximately 30 s of AD. Above-threshold ADs induced c-fos in both naive and kindled animals to the same extent and with identical temporal profiles. Although the expression of c-fos is unchanged with kindling, c-fos may nonetheless contribute to many long-term changes of kindling, both adaptive and epileptogenic.     

吗啡耐受大鼠心房心钠素的释放及其基因转录

本工作应用心钠素放射免疫测定和分子杂交技术首次发现,吗啡耐受大鼠血浆心钠素水平显著降低,心房内心钠素含量明显升高,同时心房内心钠素特异性mRNA水平也相应提高,提示在吗啡耐受时大鼠心房内心钠素的合成和贮存增加,释放减少。     

Freezing pattern in apple seeds as affected by the temperature of fruit storage

Storage of apple fruits ( Pyrus malus L. cv. Golden Delicious) at different temperatures (0, 12 and 35°C) markedly altered the pattern of water freezing in the seeds. Higher temperatures of storage brought about a shift from the sequential to the discontinuous type of freezing in seeds, the low temperature exotherm (LTE) being much more pronounced than the high temperature one (HTE). The occurrence of LTE was highly correlated with embryo death. Fruit storage at higher temperatures also caused a decrease in the threshold super cooling temperature of seeds and of naked embryos, removed from the seed coat and endosperm. The decrease was less pronounced in naked embryos than in intact seeds. The results presented show that the frost resistance of apple seeds relies mainly on the supercooling ability of the embryos and is increased by the presence of seed coat and endosperm. The broad peak of the LTE indicates that, in contrast with other seeds and many super cooling organs, massive ice nucleation in apple seeds occurs within the embryo tissues and that extra organ freezing seems to be of less importance. Therefore, the increased super cooling ability of apple seeds, isolated from fruits stored at higher temperatures, seems to rely on those seed properties that protect embryo cells against heterogeneous ice nucleation.     

Elicitor-induced metabolic changes in cell cultures of chickpea (Cicer arietinum L.) cultivars resistant and susceptible to Ascochyta rabiei

Cell-suspension cultures of two chickpea (Cicer arietinum L.) cultivars, resistant (ILC 3279) and susceptible (ILC 1929) to the fungus Ascochyta rabiei (Pass.) Lab., showed differential accumulation of the phytoalexins medicarpin and maackiain, and transient induction of related enzyme activities after application of an A. rabiei-derived elicitor. The chalcone-synthase (CHS) activity (EC 2.3.1.74) which is involved in the first part of phytoalexin biosynthesis exhibited a maximum 8–12 h after elicitation in the cells of both cultivars. Concomitant with the fivefold-higher phytoalexin accumulation, CHS activity increased twofold in the cells of the resistant cultivar. The maximum of the elicitor-induced CHS-mRNA activity was determined 4 h after onset of induction in the cultures of both cultivars, although in cells of cultivar ILC 3279 this mRNA activity was induced at a level twofold higher than that in cells of the susceptible race ILC 1929. Investigations of CHS isoenzymes by two-dimensional gel electrophoresis of immunoprecipitated in-vitro-translated protein indicated the presence of five proteins. In the cells of both cultivars only two of the isoenzymes were induced after elicitor treatment. Analysis of the total in-vitro-translated proteins by two-dimensional gel electrophoresis showed that the constitutively expressed patterns of mRNA activities in the cell cultures of the two cultivars were identical. After elicitation, considerably more translatable mRNAs were induced in the cells of cultivar ILC 3279. The few induced proteins, and their respective mRNA activities, which could be detected in the cells of the susceptible cultivar, all existed in the cells of the resistant cultivar, too. One highly induced protein (M_r 18 kDa) found in the cells of cultivar ILC 3279 reached its maximum mRNA activity 6 h after elicitor application. The amount of this protein was hardly increased in the cells of the susceptible cultivar. This protein appears to be excreted from the cells into the growth medium.Abbreviations CHS chalcone synthase - IEF isoelectric focussing - ILC international legume chickpea - PR-protein pathogenesis-related protein - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. The authors thank Dr. K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for provision of antisera and the International Centre for Agricultural Research in the Dry Areas (Aleppo, Syria) for plant material.     

Down-regulation of phytochrome mRNA abundance by red light and benzyladenine in etiolated cucumber cotyledons

Northern blot analysis revealed that a single 4.2 kb phytochrome mRNA species was detectable in cotyledons excised from five-day-old etiolated cucumber seedlings. Intact etiolated five-day-old cucumber seedlings were given a red light or benzyladenine treatment, and cotyledons were harvested at various times following treatment. The abundance of phytochrome mRNA in the cotyledons was quantitated using ³²P-labeled RNA probes and slot blot analysis. By 2 h after irradiation the phytochrome mRNA level was reduced to 40% of the initial abundance and reaccumulation began by 3 h after irradiation. Reaccumulation of phytochrome mRNA to the time-zero dark control level was achieved by 10 h after treatment. A decrease in phytochrome mRNA abundance was evident by 2 h after benzyladenine treatment, and a maximal reduction to 45% of the time-zero dark control was attained by 4 h after treatment. No recovery of the phytochrome mRNA level was evident by 8 h after benzyladenine treatment. The abundance of actin mRNA was unaffected by benzyladenine treatment.     

Direct analysis of root zone data in a microculture system

Disks were isolated from young leaves of winter rape plants and grown in vitro at ambient (15°C) or low (2°C) temperatures for two weeks. In the control disks the growth cessation and beginning of chlorophyll degradation were observed after 1 week of culture. In the low-temperature treated disks the expansion of cells was slower than that in the control material but it continued for two weeks and was accompanied by a marked accumulation of dry matter. Practically, no chlorophyll degradation was observed. The low temperature treatment brought about the decrease in the frost killing temperature of the tissue which was associated with its increased capacity to subcool water. A short (18 h) exposure of the cold-grown leaf disks to slight frost (–5°C) increased further their resistance to freezing, despite the fact that the subcooling capacity of disks decreased in result of the treatment. Therefore, the two stages of hardening, observed previously for the whole plants, can also be detected in the isolated material. In the cold-grown disks, a transient accumulation of reducing sugars but a steady decrease in ATP and water-soluble protein contents were observed. These observations indicate that tissue isolation might affect processes involved in the functional adaptation of cells to cold.Abbreviations DTA differential thermal analysis - T_k50 frost killing temperature - T_in ice nucleation temperature