Differential expression of two barley SNF1-related protein kinase genes

We have amplified and cloned DNA sequences derived from a gene encoding a SNF1 (sucrose-non-fermenting 1)-related protein kinase which differs from that previously reported from barley. Northern blot and polymerase chain reaction (PCR) analysis of RNA populations, using specific probes and oligonucleotide primers, indicated that the two SNF1-related genes are differentially regulated. One is expressed in all tissues, whereas the other is expressed at high levels in the seed endosperm and aleurone, but at levels undetectable by northern blot analysis in other tissues. Comparisons with other plant SNF1-related protein kinase genes suggest that the form which is expressed at greatly enhanced levels in the seed is less similar to the other plant homologues which have been reported and may be unique to cereals.     

l-trans-Pyrrolidine-2,4-Dicarboxylic Acid-Evoked Striatal Glutamate Levels Are Attenuated by Calcium Reduction, Tetrodotoxin, and Glutamate Receptor Blockade

Abstract: l - trans -Pyrrolidine-2,4-dicarboxylic acid ( l - trans -PDC) reverses plasma membrane glutamate transporters and elevates extracellular glutamate levels in vivo. We investigated the possibility that l - trans -PDC-stimulated glutamate levels are mediated partially by increases in transsynaptic activity. Therefore, the degree to which l - trans -PDC-evoked glutamate levels depend on calcium, sodium-channel activation, and glutamate-receptor activation was investigated by infusing via reverse microdialysis (a) 0.1 m M calcium, (b) 1 µ M tetrodotoxin, a selective blocker of voltage-dependent sodium channels, (c) R (−)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a selective NMDA-receptor antagonist, or (d) LY293558, a selective α-amino-3-hydroxy-5-methylisoxazole-4-propionate antagonist. In separate experimental groups, l - trans -PDC-evoked glutamate levels were reduced significantly by 55% in the presence of 0.1 m M calcium and by 46% in the presence of tetrodotoxin. Additionally, CPP and LY293558 significantly attenuated l - trans -PDC-evoked glutamate levels without altering basal glutamate levels. These data suggest that glutamate transporter reversal by l - trans -PDC initially elevates extracellular glutamate levels enough to stimulate postsynaptic glutamate receptors within the striatum. It is proposed that glutamate-receptor stimulation activates a positive feedback loop within the basal ganglia, leading to further glutamate release from corticostriatal and thalamostriatal afferents. Therefore, either extracellular striatal calcium reduction or tetrodotoxin perfusion leads to decreased action potential-dependent glutamate release from these terminals. In addition, blocking glutamate receptors directly reduces medium spiny neuronal firing and indirectly attenuates corticostriatal and thalamostriatal activity, resulting in an overall depression of l - trans -PDC-stimulated glutamate levels.     

Surfactant inhibition by plasma: gestational age and surfactant treatment effects in preterm lambs

Ikegami, Machiko, Celso M. Rebello, and Alan H. Jobe.Surfactant inhibition by plasma: gestational age and surfactant treatment effects in preterm lambs. J. Appl.Physiol. 81(6): 2517-2522, 1996.The preterminfant with respiratory distress syndrome has edematous lungs and smallamounts of surfactant that do not function normally. We reported thatsurfactant recovered from preterm lambs after surfactant treatment canhave decreased sensitivity to inhibition of surface tension by plasma.We asked whether this augmented resistance to inhibition was dependenton lung development (gestational age) by testing sensitivity to plasmainhibition of 1) endogenous surfactant from preterm lambs and 2)surfactant from preterm lambs after treatment with an organicsolvent-extracted natural sheep surfactant. Surfactant recovered aftersurfactant treatment of 121- or 128-days-gestation lambs had the samesensitivity to plasma inhibition as did the surfactant used to treatthe lambs. Surfactant recovered from 134-days-gestation lambs haddecreased sensitivity to inhibition. Lung maturation is a variableinfluencing surfactant inhibition by plasma.

     

Activated charcoal does not catalyze sucrose hydrolysis in tissue culture media during autoclaving

Tissue culture media or aqueous sucrose solutions containing activated charcoal buffered to pH 5.5 and autoclaved did not undergo appreciable sucrose hydrolysis as reported. Rather, the extent of sucrose hydrolysis in media containing activated charcoal was found to be directly proportional to the hydrogen ion concentration (pH). This finding is consistent with the known mechanism of acid-catalyzed hydrolysis of acetals such as sucrose. Several types of charcoal were identified that acidified culture media to the extent that considerable acid-catalyzed sucrose hydrolysis occurred under autoclave conditions, making it appear as though activated charcoal was responsible for catalyzing sucrose hydrolysis. A simple mathematical expression was empirically derived that can be used to predict the extent of sucrose hydrolysis based on the post-autoclave pH of the media. This revised version was published online in June 2006 with corrections to the Cover Date.     

Glutamate measured by 6-s resolution brain microdialysis: capillary electrophoretic and laser-induced fluorescence detection application

In the present experiment the combination of brain microdialysis and CZE–LIFD permitted the measurement of glutamate in 100 nl microdialysis samples collected every 5 or 6 s. Samples were collected every 6 s, in rats anesthetized with two different anesthetic agents (ketamine and sodium thiopental). A microdialysis probe was inserted in the cortex of an anesthetized rat in the territory irrigated by the middle cerebral artery. The artery was clamped for 30 s and then released. The samples were derivatized with fluorescein isothiocyanate I (FITC) by means of a continuous-flow reactor, collected and injected into a home-made CZE–LIFD instrument. Glutamate decreased immediately after clamping the artery in ketamine anesthetized rats and increased 1 min after the onset of the ischemia in sodium thiopental anesthetized rats. In another experiment a 60 mM KCl solution was injected through a microdialysis probe inserted in the hippocampus of an anesthetized rat. In the first 5 s after the KCl solution reached the tissue, glutamate increased but γ-aminobutytic acid and glutamine did not. The experiments show that time resolution of brain microdialysis can be reduced to a few seconds if the analytical technique is the proper one.     

Cells expressing preproenkephalin mRNA in the rat pineal gland are not serotonin-producing pinealocytes: Evidence usingin Situ hybridization combined with immunocytochemistry for serotonin

Summary 1. Preproenkephalin (PPEnk) mRNA expressing cells have been identified in rat pineal gland using radioactivein situ hybridization histochemistry. 2. Approximately 7% of the cells in the pineal gland (7.5±0.86, mean ± 95% CI) express PPEnk mRNA. These cells are distributed throughout the pineal as either scattered single cells or small groups of cells with large round or oval nuclei. 3. Usingin situ hybridization combined with ABC immunocytochemistry for serotonin (5-HT) in the same pineal sections, the PPEnk mRNA labeling cells are found not to be serotonin-immunoreactive cells. These data indicate that the PPEnk mRNA is expressed in a certain discrete subpopulation of cells in the rat pineal gland and these cells are not serotonin-producing pinealocytes. 4. The physiologic role of PPEnk-derived peptides in the pineal remains unknown. It is possible that these peptides either are synthesized and secreted as hormones or act as pineal paracrine signals.     

3,6 anhydrogalactose, sulfate and methoxyl contents of commercial agarophytes from different geographical origins

The chemical composition of agars extracted from six economically important species of Gracilaria from different geographical sources (Argentina, Brazil, Indonesia, China and Turkey) and one species of Gracilariopsis (G. lemaneiformis) collected from two different Japanese localities was investigated. Agar was extracted by pretreatment with various concentrations of NaOH (3%, 5%, 7%, 10%) for 2 h at 80°C. The sulfate, 3,6-anhyd, rogalactose and methoxyl contents of each agar extract were analyzed. High sulfate and 3,6 anhydrogalactose contents were found in non-alkali treated agar from Turkish Gracilaria gracilis (3.4%) and from Chilean G. chilensis (54.3%) after alkali treatment concentration of 5% NaOH, respectively. High methoxyl contents (4.9%) were obtained from non-alkali treated agar from Chinese G. tenuistipitata. This revised version was published online in August 2006 with corrections to the Cover Date.     

Human cystathionine β-synthase: gene organization and expression of different 5′ alternative splicing

The human cystathionine β-synthase (CBS) gene spans in excess of 30 kb and consists of 19 exons, with three different 5′ untranslated regions including three different exons 1 (exons 1 a, b, and c). Exon la and 1b are 390 bp apart from each other and are linked to exon 2 in cDNA « a » and cDNA « b ». Exon 1c, which linked to exon 5 in cDNA « c », is 7 kb apart from exon 1b. All splice sites conform to the GT/AG rule, including those from exon la or 1b to exon 2 and from exon 1c to exon 5. Upstream of exons la and 1b, we found two putative promoter sequences with high C + G nucleotide content, one CAAT box at —70 nucleotides (for exon lb), no TATA box, several Sp1 binding regulatory consensus sequences, and some other regulatory sequences. Human adult and fetal Northern blots hybridized with total cDNA containing exon 1b, or specific probes from exons 1 (b and c) showed mRNAs of 2.5 kb, 2.7 kb, and 3.7 kb. These results suggest that the mRNAs containing the different exons 1 are under the control of different promoters.     

Azide-sensitive thylakoid membrane insertion of chimeric cytochrome f polypeptides imported by isolated pea chloroplasts

Post-translational integration of cytochrome f into thylakoid membranes was observed after import by isolated pea chloroplasts of a chimeric protein consisting of the presequence of the small subunit of ribulose 1,5-bisphosphate carboxylase fused to the cytochrome f precursor. Import of a similar chimeric protein lacking the C-terminal 33 amino acid residues resulted in a soluble cytochrome f protein in the thylakoid lumen, indicating that the C-terminal region contains a stop-transfer sequence for membrane integration. Azide inhibited the insertion of cytochrome f into the thylakoid membrane, whereas the ionophores nigericin and valinomycin had little effect on membrane insertion. The precursor of the 33 kDa protein, but not the 23 kDa protein, of the photosystem II oxygen-evolving complex inhibited the thylakoid insertion of cytochrome f , suggesting competition for a component of the transport pathway. These experiments suggest that the post-translational insertion of cytochrome f into the thylakoid membrane uses a SecA-dependent pathway.     

Ca2+ effects on ethylene, carbon dioxide and 1-aminocyclopropane-1-carboxylic acid synthase activity

The response of pericarp disks from ripening tomato (Lycopersicon esculentum Mill. cv. Traveler‘76) to CaCl₂, additions was studied to determine the effect of Ca²⁺ on ethylene and CO₂ production. Application of 5 mM CaCl₂ resulted in a 2, 20, 33, 39, and 50% increase in ethylene production in disks obtained from preclimacteric minimum, climacteric rise, climacteric peak, one, and two days postclimacteric fruit, respectively. CaCl₂ concentrations of 10 and 50 mM gave no additional stimulation of ethylene production; CO₂ production at 5 mM CaCl₂ was not different from controls, but is increased at 10 and 50mM CaCl₂. CaCl₂ also increased ethylene production in disks treated with 1-aminocyclopropane-1-carboxylic acid (ACC) or aminoethoxy-vinylglycine. Chloride salts of K⁺, Na⁺, Mg²⁺, Sr²⁺ and La³⁺ did not stimulate ethylene production. SrCl₂ stimulated ethylene production to a lesser degree than CaCl₂. Disks from potato (Solanum tuberosum L. cv. Katahdin) tubers produced greater quantities of ethylene and ACC when 5 mM CaCl₂ was included in the incubation medium (K. B. Evensen, 1983. Physiol. Plant. 60:125–128). Ca²⁺-treated disks had more than three times as much ACC synthase activity as control disks after 18 to 24 h incubation, when ethylene and ACC were maximal. The apparent K_m for S-adenosylmethionine was 13 μM at 29°C, pH 8.0 in extracts from both Ca²⁺-treated and control disks. Inclusion of 1 to 50 mM CaCl₂ in the assay medium did not significantly affect enzyme activity. ACC synthase extracted from control and Ca²⁺-treated disks had a pH optimum of 8.5 and an apparent molecular weight of 72 kdalton, estimated by gel filtration. It is likely that the presence of Ca²⁺ in the buffer allows greater synthesis of ACC synthase as part of the wound-healing response in potato, while in tomato the predominant effect is on membrane stabilization.