The regulation of proenkephalin expression in a distinct population of glial cells.

The expression of opioid genes was examined in isolated populations of glial cells in primary culture. Northern blot analysis of purified type I astrocytes, oligodendrocytes and mixed oligodendrocyte-type-2-astrocyte lineage cells derived from cerebral cortex demonstrated robust expression of proenkephalin mRNA exclusively in type I astrocytes. The expression of proenkephalin mRNA was stimulated by the beta-adrenergic agonist isoproterenol, and 8-(4-chlorophenyl thio)adenosine 3'-5'-cyclic monophosphate (cpt-cAMP). Both of these compounds regulated a proenkephalin-chloramphenicol acetyltransferase fusion gene transiently transfected into type I astrocytes. HPLC and immunoassay of the cell culture media revealed significant levels of unprocessed proenkephalin secreted by the cell and this secretion was stimulated by isoproterenol and cpt-cAMP. The relatively high levels of proenkephalin expressed suggest that enhanced expression in astrocytes may be important during neural development, in trauma-induced gliosis and in neuroimmune interactions.     

An abscisic acid analog inhibits abscisic acid-induced freezing tolerance and protein accumulation, but not abscisic acid-induced sucrose uptake in a bromegrass (Bromus inermis Leyss) cell culture

The application of abscisic acid (ABA), either as a racemic mixture or as optically resolved isomers, increases freezing tolerance in a bromegrass (Bromus inermis Leyss) cell culture and induces the accumulation of several heat-stable proteins. Two stereoisomers of an ABA analog, 23 dihydroacetylenic abscisyl alcohol (DHA), were used to study the role of ABA-induced processes in the acquisition of freezing tolerance in these cells. Freezing tolerance was unchanged in the presence of (–) DHA (LT₅₀ -9°C), and no increase in heat-stable protein accumulation was detected; however, the (+) enantiomer increased the freezing tolerance (LT₅₀ -13°C) and induced the accumulation of these polypeptides. All three forms of ABA increased freezing tolerance in the bromegrass cells, although (–) ABA was less effective than either (+) or (±) ABA when added at equal concentrations. Cells pretreated with 20 or 50 M (–) DHA displayed lower levels of freezing tolerance following the addition of 2.5, 7.5 or 25 M (±) ABA. Full freezing tolerance could be restored by increasing the concentration of (±) ABA to > 25 M. Pretreatment of cells with (–) DHA (20 or 50 M) had no effect on freezing tolerance when 25 M (+) ABA was added. The induction of freezing tolerance by 25 M (–) ABA was completely inhibited by the presence of 20 M (–) DHA. The accumulation of ABA-responsive heat-stable proteins was inhibited by pretreatment with 20 M (–) DHA in cells treated with 2.5 or 7.5M (+) ABA, and in cells treated with 25 M (–) ABA. The accumulation of these polypeptides was restored when (±) or (+) ABA was added at a concentration of 25 M. The analysis of proteins which cross-reacted with a dehydrin antibody revealed a similar inhibitory pattern as seen with the other ABA-responsive proteins. The effects of the various isomers of ABA and DHA on cell osmolarity and sucrose uptake was also investigated. In both cases, (±) and (+) ABA had pronounced effects on the parameters measured, whereas (–) ABA treated cells gave substantially different results. In both sucrose uptake and cell osmolarity, DHA had no significant effect on the results obtained following (±) or (+) ABA treatment. Maximum freezing tolerance was only observed in cells when both heat-stable protein accumulation and sucrose uptake were observed.Abbreviations ABA abscisic acid - DHA 2,3 dihydroacetylenicabscisyl alcohols - DMSO dimethyl sulfoxide - LT₅₀ temperature at which 50% of cells are killed The authors would like to acknowledge the technical assistance of Angela Bollman, Bruce Ewan and Angela Shaw. This work was supported by grants from the Natural Science and Engineering Research Council of Canada to L.V.G. and N.H.L., and a grant from the University of Saskatchewan to R.W.W.     

Analysis of nonmethylated GATC sites in the Escherichia coli chromosome and identification of sites that are differentially methylated in response to environmental stimuli.

Seven GATC sites that are nonmethylated in logarithmic growth phase cells using glycerol as a carbon source were isolated from the Escherichia coli chromosome. Three of these GATC sites are located upstream of the operons gut, mtl, and ppiA, whereas DNA sequences adjacent to three other nonmethylated GATC sites are not homologous to previously identified genes. The seventh nonmethylated GATC site is located downstream of uspA. The protection of this site from DNA methylation requires leucine-responsive regulatory protein and is leucine responsive. The carbon source and the growth phase influenced the protection of the GATC site 5' of the ppiA gene. The other five sites were protected under all the environmental conditions examined.     

Cholinergic innervation of the retrosplenial cortex via the fornix pathway as determined by high affinity choline uptake,choline acetyltransferase activity,and muscarinic receptor binding in the rat

The cholinergic projections from basal forebrain nuclei to the retrosplenial cortex (RSC) have previously been studied using a variety of histological approaches. Studies using acetylcholinesterase (AChE) histochemistry and choline acetyltransferase (ChAT) immunocytochemistry have demonstrated that this projection travels via the cingulum on route to the RSC. Preliminary studies from our laboratory, however, have shown that the fornix may also be involved in this projection. The present study uses the combination of pathway lesions, and the analysis of cholinergic neurochemical markers in the RSC to determine the role of the fornix in the cholinergic projection to the RSC. High affinity choline uptake (HACU) and ChAT activity were measured in the RSC of control rats, animals with cingulate lesions, and animals with fornix plus cingulate lesions. Fornix plus cingulate lesions resulted in significant deceases in HACU and ChAT activity in comparison to cingulate lesions alone. Muscarinic receptor binding was also evaluated in combination with the various lesions, and a significant increase in retrosplenial receptor binding was noted following fornix lesions. Together, these results support the concept of a fornix-mediated cholinergic pathway to the RSC.     

Studies on the striatal dopamine uptake system of weaver mutant mice and effects of ventral mesencephalic grafts

The dopamine (DA) uptake system was investigated in the mesostriatal system of normal and weaver mutant mice, which lose mesencephalic DA neurons, as well as in weaver mutants with ventral mesencephalic grafts to the striatum. Assays of [³H]DA uptake in striatal synaptosomal fractions in vitro and autoradiography of [³H]mazindol binding in brain sections were carried out in wild-type mice (+/+) and in the two hemispheres of homozygous weaver mutants (wv/wv) that had received unilateral grafts of mesencephalic cell suspensions to the right side. Net [³H]DA uptake, expressed as pmol/mg-protein/2-min, was on the average 50.6 in the striatum of wild-type mice, 7.9 in the non-grafted, and 10.1 in the transplanted striatum of weaver mutants. [³]DA uptake in wild-type mice differed significantly from both the grafted and non-grafted weaver striata (P<0.001). Paired comparisons for [³H]DA uptake between right and left sides of recipient weaver mice showed a significant side effect (P<0.02), the right side being 28–38% higher than the left side [mean of all individual (R-L)/L values]. The results of amphetamine-induced turning behavior tests were compared with the biochemical findings. Mice with grafts to the right side rotated an average of 22 turns to the left and 7 turns to the right during the five one-minute sessions; the mean value L/(L+R) was 64%. A plot of (L-R) rotations against (R-L) [³H]DA uptake gave a correlation coefficient of 0.552 (P<0.05), indicating that animals with a strong rotational bias to the left tended to have higher [³H]DA on the right. Similarly, the animals that were used for [³H]mazindol binding autoradiographic studies displayed on the average 72% rotations to the left side. In the [³H]mazindol binding data, non-grafted weaver mutants showed the severest depletion relative to wild-type in the dorsomedial and dorsolateral caudate-putamen (86% and 87%, respectively). Mice with unilateral grafts to the right side showed an increase in [³H]mazindol binding signal in the transplanted side of 40–64% (depending on dorsoventral topography) over the contralateral, non-grafted side. These findings attest to the functional effects of the grafts at the anatomical, biochemical, and behavioral levels. The parallel measurements of motor performance and DA uptake in the same animals offers an index of behavioral recovery as a function of transmitter-related activity. Furthermore, by conducting measurements of the synaptosomal DA uptake in vitro and of the binding characteristics of mazindol in brain slices by autoradiography, one has the advantage of combining the anatomical resolution of uptake site visualization with a dynamic indicator of function for DA uptake in the nerve terminal.Special issue dedicated to Professor Sidney Ochs     

The structure of feeding behavior in the colorado potato beetle,Leptinotarsa decemlineata (Coleoptera: Chrysomelidae)

Feeding behavior, in an ad libitum situation on potato plants in the laboratory, was continuously observed for approximately 7 h/day on 2 successive days for 18 adult femaleLeptinotarsa decemlineata. Additional behaviors were also recorded including resting, walking, biting, local movements, grooming, defecating, and regurgitating. These data were used to calculate a time budget for the various behaviors. The feeding data were analyzed to describe the structure of feeding for young adult females on their normal host plant. The criterion for a meal (minimum intermeal interval) was determined to be 286 s. This criterion was used to distinguish between intra- and intermeal interruptions in feeding for all subsequent analyses. Meals taken from leaves that were young, medium aged, or old did not differ, but on average beetles took 60% of their meals from young leaves. Meal size and meal duration were equally good predictors of when a meal would end. Feeding from stems was a prominent feature for most beetles. The stem meals had much longer durations than leaf meals, but stem feeding did not affect subsequent leaf feeding. The structure of feeding by these beetles is compared with that found in other insects, especiallyLocusta migratoria.     

Productivity of outdoor algal cultures in unstable weather conditions

In the outdoor cyclic fed batch cultures of Chlorella pyrenoidosa, some typical growth kinetics patterns in unstable weather conditions were observed. On cloudy days, the biomass output rate (R) was low, but the bioenergetic growth yield (Y) was generally high. In the cloudy morning-sunny afternoon condition, the values of Y were low, especially in the afternoon. In the sunny morning-cloudy afternoon condition, both R and Y were high. A few very high short-term Y values were measured during the cloudy the cloudy afternoon. (c) 1993 Wiley & Sons, Inc.     

Total chemical synthesis and NMR characterization of the glycopeptide tx5a, a heavily post-translationally modified conotoxin, reveals that the glycan structure is alpha-D-Gal-(1-->3)-alpha-D-GalNAc.

The 13-amino acid glycopeptide tx5a (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* = 6-bromotryptophan and Thr* = Gal-GalNAc-threonine), isolated from Conus textile, causes hyperactivity and spasticity when injected intracerebral ventricularly into mice. It contains nine post-translationally modified residues: four cysteine residues, two gamma-carboxyglutamic acid residues, and one residue each of 6-bromotryptophan, 4-trans-hydroxyproline and glycosylated threonine. The chemical nature of each of these has been determined with the exception of the glycan linkage pattern on threonine and the stereochemistry of the 6-bromotryptophan residue. Previous investigations have demonstrated that tx5a contains a disaccharide composed of N-acetylgalactosamine (GalNAc) and galactose (Gal), but the interresidue linkage was not characterized. We hypothesized that tx5a contained the T-antigen, beta-D-Gal-(1-->3)-alpha-D-GalNAc, one of the most common O-linked glycan structures, identified previously in another Conus glycopeptide, contalukin-G. We therefore utilized the peracetylated form of this glycan attached to Fmoc-threonine in an attempted synthesis. While the result-ing synthetic peptide (Gla-Cys-Cys-Gla-Asp-Gly-Trp*-Cys-Cys-Thr*-Ala-Ala-Hyp-OH, where Trp* =6-bromotryptophan and Thr* = beta-D-Gal-(1-->3)-alpha-D-GalNAc-threonine) and the native peptide had almost identical mass spectra, a comparison of their RP-HPLC chromatograms suggested that the two forms were not identical. Two-dimensional 1H homonuclear and 13C-1H heteronuclear NMR spectroscopy of native tx5a isolated from Conus textile was then used to determine that the glycan present on tx5a indeed is not the aforementioned T-antigen, but rather alpha-D-Gal-(1-->3)-alpha-D-GalNAc.