Ethylene effects on cambial activity and cell wall formation in hypocotyls of Picea abies seedlings

Ethylene regulation of cell division in the vascular cambium and cell wall formation was studied in hypocotyls of Norway spruce ( Picea abies [L.] Karst.) seedlings. Cuttings from 6-week-old seedlings were placed in water culture to which compounds affecting the synthesis and action of ethylene were added. After a 3-week treatment period, growth, ethylene production, morphology and cell wall composition of the hypocotyls were determined. Addition of high concentrations of the potent ethylene releasing agent 2-chloroethylphosphonic acid (ethrel), which increased ethylene emission by more than twice compared to control plants, inhibited the expansion of xylem cells while stimulating the incorporation of cell wall material, especially cellulose. Addition of small amounts of ethrel, which slightly stimulated ethylene emission, led to increases in the size of xylem cells, the amount of phloem tissue and the number of intercellular spaces in the cortex, and thus to increased hypocotyl diameter. However, no significant change in cell wall composition was detected. When ethylene production was decreased by adding Co²⁺ to the nutrient solution, differentiation of new xylem was disturbed, but the rate of cell division was not affected. Although the incorporation of cell wall material was inhibited, the proportions of lignin and cellulose in the wall appeared to remain unchanged. Silver ions stimulated the expansion of both xylem and cortex cells, but had no significant effect on cell wall formation. We conclude that ethylene has a role in regulating the incorporation of wall carbohydrates.     

A p50 surface antigen restricted to human urinary bladder carcinomas and B lymphocytes

Summary We have previously described the derivation of a monoclonal antibody, S2C6, to a novel 50 Kdalton antigen associated with human urinary bladder carcinoma. No reactions were obtained with carcinomas of unrelated origin or with normal urothelial cells. However, the antibody also reacted with a similar antigen on some cell lines of B lymphocyte origin. Using large panels of target cells we have now shown that this reactivity was entirely restricted to cells of the B lineage within the haematopoietic system. As opposed to its apparent restriction to malignant cells of the urothelium, the S2C6 antigen was expressed by normal B lymphocytes as well as by many malignant B cells (chronic lymphocytic leukaemia, hairy cell leukaemia and immunocytoma). Pre-B cells derived from acute lymphocytic leukaemia and plasma cells from multiple myeloma lacked the antigen. Expression was significantly enhanced on cultured B cells from Burkitt lymphomas and on Epstein-Barr virus-transformed lymphoblastoid cell lines including those of the pre-B phenotype derived from fetal bone marrow. As judged from the molecular size and the distribution pattern displayed by the S2C6 antigen it appears to be distinct from other B cell antigens previously described. A possible relation of the S2C6 antigen to a receptor for B cell growth factors is discussed.     

Immunoaffinity column clean-up for the high-performance liquid chromatographic determination of aflatoxins B1, B2, G1, G2, M1 and Q1 in urine

A method for the determination of aflatoxins B₁, B₂, G₁, G₂, M₁ and Q₁ in human urine has been developed. The 10-ml urine samples were automatically cleaned up on immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC), including post-column derivatization with bromine and fluorescence detection. Average aflatoxin recoveries were: B₁ 103%, B₂ 106%, G₁ 98% and G₂ 96% in the range 6.8–73 pg/ml of urine and M₁ 103% and Q₁ 100% in the range 18–97 pg/ml of urine. The relative standard deviations were all between 1% and 21%. The determination limits of aflatoxins in urine were 6.8 pg/ml for B₁, B₂, G₁ and G₂ and 18 pg/ml for M₁ and Q₁.     

Concentrations of Amino Acids in Extracellular Fluid After Opening of the Blood-Brain Barrier by Intracarotid Infusion of Protamine Sulfate

Abstract: This article evaluates the influence of an opening of the blood-brain barrier (BBB) on compounds in brain extracellular fluid. The concentrations of amino acids and some other primary amines were determined in dialysates sampled from the right parietal cortex of rats before and after an intracarotid infusion of protamine sulfate. Extravasated plasma proteins were visualized by Evans blue/albumin and immunohistochemistry. CSF albumin— an indicator of blood-CSF barrier opening—was quantified with immunoelectrophoresis. The brains were macroscopically edematous after 10 mg but not after 5 mg of protamine sulfate. The higher dose led to a 50% death rate. The concentrations of amino acids did not change 10 min after the BBB opening. No significant alterations in the amino acid concentrations were observed after the lower dose. The concentrations of glutamate, aspartate, GABA, glycine, taurine, and phosphoethanolamine increased significantly within 50–80 min after the infusion of 10 mg of protamine sulfate. CSF albumin levels were significantly increased 1 h after infusion. We conclude that a dysfunction of the BBB, of a degree known to induce brain edema (10 mg of protamine sulfate), significantly increases the extracellular concentration of excitatory amino acids, GABA, taurine, and phosphoethanolamine in the extracellular space.     

Characterization of mutations on the rare duplicated C4/CYP21 haplotype in steroid 21-hydroxylase deficiency

We have defined the mutations causing congenital adrenal hyperplasia in three Swedish patients carrying a rare haplotype containing two mutated steroid 21-hydroxylase genes (CYP21) in addition to one pseudogene (CYP21P). The presence of such haplotypes complicates genetic diagnosis and screening of mutations in 21-hydroxylase deficiency, and we show how these genotypes can be resolved by amplification and analysis of each gene separately. In all cases, the rare haplotype carried the same combination of disease-causing mutations; one of the genes had the splice mutation at base 659 in intron 2, and the other had the nonsense mutation at base 1999 in exon 8 (CAG to TAG). We have thus characterized the most common haplotype containing duplicated CYP21 genes. The frequency of this haplotype is low, and if additional such haplotypes are present, they are rare in this population.     

Ethylene effects on peroxidases and cell growth patterns in Picea abies hypocotyl cuttings

The effect of 2-chloroethylphosphonic acid (ethrel) on cell growth patterns and per-oxidase activity (EC 1.11.1.7) and location in young Norway spruce cuttings ( Picea abies [L.] Karst.) was investigated. The peroxidase activity in a fraction containing soluble and membrane bound enzymes show a diurnal variation, with decreased activity during the light period and a corresponding increase during the following dark period. The decrease during the day could to some extent be counteracted by treatment with ethrel. It appears that ethrel affects only peroxidases in the isolated membrane fraction, since peroxidases bound to the cell wall were not affected by ethrel. In vitro experiments indicated that the hydrophobicity of soluble peroxidases was increased by treatment with ethylene. Cytochemical localization of peroxidase activity in differentiating tracheids revealed a clear ethrel-induced increase in the tonoplast. It appears that ethylene affects soluble peroxidases in vivo in such a way that they are directed to a more hydrophobic environment, like the tonoplast. Treatment with ethrel also changed the appearance of the rough endoplasmic reticulum (ER) and Golgi apparatus. Dilated ER cisternae were observed on electron micrographs, as a result of treatment with ethrel. The number of vesicles produced by the Golgi apparatus and also the amount of vesicles fusing with the plasma membrane in secondary-wall-forming tracheids increased considerably. The results clearly indicate that the stimulatory effect of ethylene in spruce seedlings on lignification and cell wall formation, is due to a general stimulation on both synthesis, transport and secretion of cell wall material and not on a stimulation of peroxidase activity as reported for other species.     

Molecular Phylogeny and Evolution of the Neurotrophins from Monotremes and Marsupials

We have investigated the phylogenetic relationships of monotremes and marsupials using nucleotide sequence data from the neurotrophins; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). The study included species representing monotremes, Australasian marsupials and placentals, as well as species representing birds, reptiles, and fish. PCR was used to amplify fragments encoding parts of the neurotrophin genes from echidna, platypus, and eight marsupials from four different orders. Phylogenetic trees were generated using parsimony analysis, and support for the different tree structures was evaluated by bootstrapping. The analysis was performed with NGF, BDNF, or NT-3 sequence data used individually as well as with the three neurotrophins in a combined matrix, thereby simultaneously considering phylogenetic information from three separate genes. The results showed that the monotreme neurotrophin sequences associate to either therian or bird neurotrophin sequences and suggests that the monotremes are not necessarily related closer to therians than to birds. Furthermore, the results confirmed the present classification of four Australasian marsupial orders based on morphological characters, and suggested a phylogenetic relationship where Dasyuromorphia is related closest to Peramelemorphia followed by Notoryctemorphia and Diprotodontia. These studies show that sequence data from neurotrophins are well suited for phylogenetic analysis of mammals and that neurotrophins can resolve basal relationships in the evolutionary tree. Received: 27 January 1997 / Accepted: 20 March 1997     

Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone

Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.     

Improvement of anther culture technique: Activated charcoal bound in agar medium in combination with liquid medium and elevated CO2 concentration

Anthers of different species of the genera Anemone, Clematis, Papaver and Nicotiana were cultured by floating on a liquid medium which overlay an agarified charcoal medium . This technique proved to be superior to conventional methods i.e. culture on either solid or liquid media. Cold treatment of Anemone anthers for 7 days after inoculation on the double layer medium gave about the same frequency of embryos per anther as corresponding cultures cold treated before inoculation. An elevation of the CO₂ concentration to 2% stimulated embryogenesis in anther cultures of Anemone canadensis, Anemone vitifolia, Papaver setigerum and Papaver radicatum . Cold treatment of cultures of Anemone canadensis inhibited embryogenesis if the ensuing culture was performed in 2% CO_2. On the other hand, cold treatment was stimulating, with an optimum of about 20 days, if the cultures were maintained in normal air. Chemical analysis of untreated anthers of Anemone canadensis showed the presence of abscisic acid (2.2 × 10⁻⁶ g/g anthers). Cold treatment reduced the concentration of abscisic acid to 0.6 × 10⁻⁶ g/g anthers. By use of assays with Lemna gibba as test organism, activated charcoal was shown to adsorb abscisic acid that was added to the medium. Medium treated with charcoal before inoculation of anthers of Anemone canadensis provided to inhibit embryo production.     

A COMPARISON BETWEEN FLUOROMETRIC AND MASS FRAGMENTOGRAPHIC DETERMINATIONS OF HOMOVANILLIC ACID AND 5-HYDROXYINDOLEACETIC ACID IN HUMAN CEREBROSPINAL FLUID

HVA and 5-HIAA in human cerebrospinal fluid were quantitatively determined by both fluorometry and mass fragmentography. The homovanillic acid values obtained by fluorometry were significantly lower than those obtained by mass fragmentography (P < 0.05). The correlation coefficient between values for HVA obtained by the two methods was high, r= 0.90. For 5-HIAA the concentrations obtained by the two methods were not significantly different while the correlation coefficient was lower, r= 0.55.