Zooplankton of Lake Peipsi-Pihkva in 1909–1987

164 taxa were identified in the net zooplankton of the pelagial of L. Peipsi-Pihkva in 1909–1987, including 3 species of protozoans, 74 species of rotifers, 58 species of cladocerans, 28 species of copepods and 1 mollusc. One rotifer species, Ploesoma peipsiense Mäemets et Kutikova, has been described as new for science here. The zooplankton of L. Peipsi-Pihkva is remarkably rich in species including rarities in Estonia: Limnosida frontosa, Drepanothrix dentata, Bythotrephes longimanus, B. cederstroemi etc. Due to its large surface area, L. Peipsi-Pihkva provides a large scale of biotopes of a diverse trophic state and humic content, which support species with different ecological requirements. Most of the aquatory of the lake has lately been mesotrophic, favouring the coexistence of indicators of oligo- and mesotrophic state and species preferring a higher trophic state. The occurrece of 10 species of the genus Bosmina including B. berolinensis, B. gibbera, B. lilljeborgi, B. thersites and B. crassicornis, sparse in Estonian lakes, is the most noteworthy feature of the zooplankton of L. Peipsi-Pihkva. The coexistence of B. coregoni and B. berolinensis, B. gibbera, B. lilljeborgi etc. which were earlier regarded as subspecies of B. c. coregoni proves that they are different species producing usually no hybrids. The species composition was subjected to certain changes during the years under consideration. Larvae of Dreissena were first found in zooplankton in 1962. The oligo-mesotrophic indicator Holopedium gibberum occurred in the lake in 1909–1964, but was lacking in later samples.     

Identification of mutations in the ALD-gene of 20 families with adrenoleukodystrophy/adrenomyeloneuropathy

Adrenoleukodystrophy (ALD), an X-linked inherited metabolic disorder, is the most frequent inborn peroxisomal disease. It leads to demyelination in the central and peripheral nervous system. Defective -oxidation of saturated very long chain fatty acids (VLCFAs; C22:0–C26:0) in peroxisomes has been shown to lead to an accumulation of VLCFAs in leukoid areas of the central nervous system, peripheral nerves, adrenal gland, and blood. The ALD gene has been recently identified and encodes a 745-amino-acid protein. We screened patients with adrenoleukodystrophy/adrenomyeloneuropathy (ALD/AMN) from 20 kindreds for mutations in the ALD gene. Eleven missense and two nonsense mutations, five deletions, and one insertion were detected by direct sequencing of eight reverse transcribed fragments of the ALD-gene mRNA. Four mutations could be shown to be de novo. All mutations could be confirmed in carriers by sequencing genomic DNA. No correlation between the type of mutation and the severity of the phenotype could be observed. The mutations were not detected in the ALD gene of 30 healthy persons.     

Water and solute transport along developing maize roots

Hydraulic and osmotic properties were measured along developing maize (Zea mays L.) roots at distances between 15 and 465 mm from the root tip to quantify the effects of changes in root structure on the radial and longitudinal movement of water and solutes (ions). Root development generated regions of different hydraulic and osmotic properties. Close to the root tip, passive solute permeability (root permeability coefficient, P_sr) was high and selectivity (root reflection coefficient, _sr) low, indicative of an imperfect semipermeable root structure. Within the apical 100–150 mm, P_sr decreased by an order of magnitude and _sr increased significantly. Root hydraulic conductivity (Lp_r) depended on the nature of the force (hydrostatic and osmotic). Osmotic Lp_r was smaller by an order of magnitude than hydrostatic Lp_r and decreased with increasing distance from the root tip. Throughout the root, responses in turgor of cortical cells and late metaxylem to step changes in xylem pressure applied to the base of excised roots were measured at high spatial resolution. The resulting profiles of radial and longitudinal propagation of pressure showed that the endodermis had become the major hydraulic barrier in older parts of the root, i.e. at distances from the apex ä 150 mm. Other than at the endodermis, no significant radial hydraulic resistance could be detected. The results permit a detailed analysis of the root's composite structure which is important for its function in collecting and translocating water and nutrients.Abbreviations and Symbols CPP cell pressure probe - IT root segments with intact tips; - Lp_r root hydraulic conductivity - Lp_rh hydrostatic hydraulic conductivity of root - Lp_ro osmotic hydraulic conductivity of root - P_app hydrostatic pressure applied to cut end of root - P_c cell turgor - P_{c, cor} turgor of cortical cell - P_c,xyl turgor of late metaxylem vessel - P_ro stationary root pressure - P_r0,seal stationary root pressure of sealed root segment - P_sr solute permeability coefficient of root - RPP root pressure probe - TR root segments with tip removed - _sr reflection coefficient of root Dedicated to Professor Andreas Sievers on the occasion of his retirement     

Characterization of devA, a gene required for the maturation of proheterocysts in the cyanobacterium Anabaena sp. strain PCC 7120.

Mutant M7, obtained by transposon mutagenesis of the cyanobacterium Anabaena sp. strain PCC 7120, is impaired in the development of mature heterocysts. Under aerobic conditions, the mutant is unable to fix N2 because of a deficiency of at least two components of the oxygen-protective mechanisms: a hemoprotein-coupled oxidative reaction and heterocyst-specific glycolipids. DNA contiguous with the inserted transposon was recovered from the mutant and sequenced. The transposon had inserted itself within a 732-bp open reading frame designated devA. The wild-type form of devA, obtained from a lambda-EMBL3 library of Anabaena sp. DNA, had the identical sequence. Directed mutagenesis of devA in the wild-type strain showed that the phenotype of the mutant was caused by insertion of the transposon. The wild-type form of devA on a shuttle vector complemented the mutation in M7. Expression of devA by whole filaments, monitored following nitrogen stepdown by using luxAB as the reporter, increased ca. eightfold during differentiation; the increase within differentiating cells was much greater. The deduced sequence of the DevA protein shows strong similarity to the ATP-binding subunit of binding protein-dependent transport systems. The product of devA may, therefore, be a component of a periplasmic permease that is required for the transition from a proheterocyst to a mature, nitrogen-fixing heterocyst.     

α2-Adrenoceptor-Mediated Inhibition of Electrically Evoked [3H]Noradrenaline Release from Chick Sympathetic Neurons: Role of Cyclic AMP

Abstract: This study explores the role of cyclic AMP in electrically evoked [³H]noradrenaline release and in the α₂-adrenergic modulation of this release in chick sympathetic neurons. Along with an increase in stimulation-evoked tritium overflow, applications of forskolin enhanced the formation of intracellular cyclic AMP. Both effects of forskolin were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. The forskolin-induced increase in overflow was abolished by the Rp-diastereomer of cyclic AMP-thioate, an antagonist at cyclic AMP-dependent protein kinases, and 1,9-dideoxy-forskolin, an inactive analogue at adenylyl cyclase, had no effect on the evoked overflow. A 24-h pretreatment with either cholera toxin or forskolin reduced the subsequent forskolin-induced accumulation of cyclic AMP and inhibited the stimulation-evoked release. Basal cyclic AMP production, however, remained unaltered after forskolin treatment and was enhanced after 24 h of cholera toxin exposure. The α₂-adrenergic agonist bromoxidine did not affect the formation of cyclic AMP stimulated by forskolin but reduced electrically evoked release. However, effects of bromoxidine on ³H overflow were attenuated by forskolin as well as by 8-bromo-cyclic AMP. Effects of bromoxidine on [³H]noradrenaline release were paralleled by an inhibition of voltage-activated Ca²⁺ currents, primarily through a delayed time course of current activation. This effect was abolished when either forskolin or 8-bromo-cyclic AMP was included in the pipette solution. Both substances, however, failed to affect Ca²⁺ currents in the absence of bromoxidine. These results suggest that the signaling cascade of the α₂-adrenergic inhibition of noradrenaline release involves voltage-activated Ca²⁺ channels but not cyclic AMP. Elevated levels of cyclic AMP, however, antagonize this α₂-adrenergic reduction, apparently through a disinhibition of Ca²⁺ channels.     

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5 end of the cDNA was amplified by a 5-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.     

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen