Activity of the natural algicide, cyanobacterin, on eukaryotic microorganisms

Abstract The natural product cyanobacterin has been shown to be toxic to most cyanobacteria at a concentration of approx. 5 μM. We demonstrate here that cyanobacterin will also inhibit the growth of most eukaryotic algae at a similar concentration. Some algae, such as Euglena gracilis , are resistant because they are able to maintain themselves by heterotrophic nutrition. Others, such as Chlamydomonas reinhardtii , can apparently induce a detoxification mechanism to maintain photosynthesis in the presence of low concentrations of the inhibitor. Non-photosynthetic microorganisms are not affected by cyanobacterin.     

Can zoosporic true fungi grow or survive in extreme or stressful environments?

Zoosporic true fungi are thought to be ubiquitous in many ecosystems, especially in cool, moist soils and freshwater habitats which are rich in organic matter. However, some of the habitats where these fungi are found may periodically experience extreme conditions, such as soils in extremely dry, hot and cold climates, acidic and alkaline soils, polluted rivers, anaerobic soil and water, saline soil and water, periglacial soils, oligotrophic soils, tree canopies and hydrothermal vents. It is clear that many ecotypes of zoosporic true fungi have indeed adapted to extreme or stressful environmental conditions. This conclusion is supported by studies in both the field and in the laboratory. Therefore, in our opinion, at least some true zoosporic fungi can be considered to be extremophiles.     

Cloning of a negative transcription factor that binds to the upstream conserved region of Moloney murine leukemia virus.

The long terminal repeat of Moloney murine leukemia virus (MuLV) contains the upstream conserved region (UCR). The UCR core sequence, CGCCATTTT, binds a ubiquitous nuclear factor and mediates negative regulation of MuLV promoter activity. We have isolated murine cDNA clones encoding a protein, referred to as UCRBP, that binds specifically to the UCR core sequence. Gel mobility shift assays demonstrate that the UCRBP fusion protein expressed in bacteria binds the UCR core with specificity identical to that of the UCR-binding factor in the nucleus of murine and human cells. Analysis of full-length UCRBP cDNA reveals that it has a putative zinc finger domain composed of four C2H2 zinc fingers of the GLI subgroup and an N-terminal region containing alternating charges, including a stretch of 12 histidine residues. The 2.4-kb UCRBP message is expressed in all cell lines examined (teratocarcinoma, B- and T-cell, macrophage, fibroblast, and myocyte), consistent with the ubiquitous expression of the UCR-binding factor. Transient transfection of an expressible UCRBP cDNA into fibroblasts results in down-regulation of MuLV promoter activity, in agreement with previous functional analysis of the UCR. Recently three groups have independently isolated human and mouse UCRBP. These studies show that UCRBP binds to various target motifs that are distinct from the UCR motif: the adeno-associated virus P5 promoter and elements in the immunoglobulin light- and heavy-chain genes, as well as elements in ribosomal protein genes. These results indicate that UCRBP has unusually diverse DNA-binding specificity and as such is likely to regulate expression of many different genes.     

Activity of the natural algicide, cyanobacterin, on angiosperms

Cyanobacterin is a secondary metabolite produced by the cyanobacterium (blue-green alga) Scytonema hofmanni. The compound had previously been isolated and chemically characterized. It was shown to inhibit the growth of algae at a concentration of approximately 5 micromolar. Cyanobacterin also inhibited the growth of angiosperms, including the aquatic, Lemna, and terrestrial species such as corn and peas. In isolated pea chloroplasts, cyanobacterin inhibited the Hill reaction when p-benzoquinone, K₃Fe(CN)₆, dichlorophenolindophenol, or silicomolybdate were used as electron acceptors. The concentration needed to inhibit the Hill reaction in photosystem II was generally lower than the concentration of the known photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea. Cyanobacterin had no effect on electron transport in photosystem I. The data indicate that cyanobacterin inhibits O₂ evolving photosynthetic electron transport in all plants and that the most probable site of action is in photosystem II.     

Cloning, expression, and characterization of the Anabaena thioredoxin gene in Escherichia coli.

The gene encoding thioredoxin in Anabaena sp. strain PCC 7119 was cloned in Escherichia coli based on the strategy that similarity between the two thioredoxins would be reflected both in the gene sequence and in functional cross-reactivity. DNA restriction fragments containing the Anabaena thioredoxin gene were identified by heterologous hybridization to the E. coli thioredoxin gene following Southern transfer, ligated with pUC13, and used to transform an E. coli strain lacking functional thioredoxin. Transformants that complemented the trxA mutation in E. coli were identified by increased colony size and confirmed by enzyme assay. Expression of the cloned Anabaena thioredoxin gene in E. coli was substantiated by subsequent purification and characterization of the algal protein from E. coli. The amino acid sequence derived from the DNA sequence of the Anabaena gene was identical to the known amino acid sequence of Anabaena thioredoxin. The E. coli strains which expressed Anabaena thioredoxin complemented the TrxA- phenotype in every respect except that they did not support bacteriophage T7 growth and had somewhat decreased ability to support bacteriophages M13 and f1.     

Synthesis and LTD4-antagonist activity of desamino-2-nor-leukotriene analogs

A series of desamino-2-nor-leukotriene analogs has been prepared by the reaction of various thiols with several methyl trans-4,5-epoxy-6Z-alkenoates, followed by deprotection. The products were assessed for their ability to antagonize the LTD4-induced contraction of the isolated guinea pig trachea. Several compounds displayed potent leukotriene antagonist activity, i.e., KB values in the sub-micromolar range, while only minimally affecting basal airway tone. The most potent analog, 4-hydroxy-5-(2-carboxyethylthio)-6Z-nonadecenoic acid, antagonized both LTD4- and LTE4-induced contractions of the trachea in an apparently competitive fashion. These agents possess increased potency relative to SK&F 101132, the first leukotriene analog identified as having LT-antagonist activity. Thus, these results demonstrate that deletion of the peptide amino group can produce leukotriene analogs which have minimal intrinsic contractile activity on the isolated guinea pig trachea, yet possess potent leukotriene-antagonistic effects.     

Lactate uptake by the fetal sheep liver

Lactate is produced by the sheep placenta and is an important metabolic substrate for fetal sheep. However, lactate uptake and release by the fetal liver have not been assessed directly. We measured lactate flux across the liver in 16 fetal sheep at 129 (120-138) days gestation that had catheters chronically maintained in the fetal descending aorta, inferior vena cava, right or left hepatic vein, and umbilical vein. Lactate and hemoglobin concentrations and oxygen saturation were measured in blood drawn from all vessels. Umbilical venous, portal venous, and hepatic blood flow were measured by injecting radionuclide-labeled microspheres into the umbilical vein while obtaining a reference sample from the descending aorta. We found net hepatic uptake of lactate (5.0 +/- 4.4 mg/min per 100 g liver). A large quantity of lactate was delivered to the liver (94.2 +/- 78.1 mg/min per 100 g), so that the hepatic extraction of lactate was only 7.7 +/- 6.5%. Hepatic oxygen consumption was 3.18 +/- 3.3 ml/min per 100 g, and the hepatic lactate/oxygen quotient was 2.07 +/- 1.54. There was no significant correlation between hepatic lactate uptake and hepatic lactate or glucose delivery, hepatic oxygen consumption, hepatic blood flow, hepatic glucose flux, total body oxygen consumption, arterial pH, oxygen content, or oxygen saturation. There was, however, a significant correlation between hepatic lactate uptake and umbilical lactate uptake (r = 0.74, P less than 0.005) such that net hepatic lactate uptake was nearly equivalent to that produced across the umbilical-placental circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sediment-related growth limitation of Elodea nuttallii as indicated by a fertilization experiment

1. A fertilization experiment was performed to identify the limiting nutrient for the growth of submerged vegetation in ditches of a peat-grassland system in the Netherlands, in which restoration measures involved ceasing fertilization, exporting nutrients by removal of above-ground plant mass and large-scale introduction of calcium-rich, nutrient-poor artesian water.
2. Growth of Elodea was significantly enhanced by enrichment with nitrogen alone, and by fertilization with nitrogen in combination with phosphorus, and by nitrogen in combination with phosphorus and potassium.
3. Plant tissue nutrient concentrations increased significantly, for nitrogen by enrichment with nitrogen alone, and with nitrogen in combination with phosphorus and potassium; for phosphorus by enrichment with phosphorus alone and with phosphorus in combination with nitrogen and potassium; tissue concentrations of potassium were not enhanced by any treatment.
4. The elemental ratios of treated plants indicated that nitrogen, rather than phosphorus, was limiting in all treatments, except in those involving nitrogen and NK enrichment (when phosphorus was limiting).
5. The efficiency with which plants used nutrients declined with increased supply of nitrogen and phosphorus, but was unchanged when potassium was increased. Efficiencies were similar to those of other aquatic macrophytes.     

Sediment-related growth limitation of Elodea nuttallii as indicated by a fertilization experiment

1. A fertilization experiment was performed to identify the limiting nutrient for the growth of submerged vegetation in ditches of a peat-grassland system in the Netherlands, in which restoration measures involved ceasing fertilization, exporting nutrients by removal of above-ground plant mass and large-scale introduction of calcium-rich, nutrient-poor artesian water.
2. Growth of Elodea was significantly enhanced by enrichment with nitrogen alone, and by fertilization with nitrogen in combination with phosphorus, and by nitrogen in combination with phosphorus and potassium.
3. Plant tissue nutrient concentrations increased significantly, for nitrogen by enrichment with nitrogen alone, and with nitrogen in combination with phosphorus and potassium; for phosphorus by enrichment with phosphorus alone and with phosphorus in combination with nitrogen and potassium; tissue concentrations of potassium were not enhanced by any treatment.
4. The elemental ratios of treated plants indicated that nitrogen, rather than phosphorus, was limiting in all treatments, except in those involving nitrogen and NK enrichment (when phosphorus was limiting).
5. The efficiency with which plants used nutrients declined with increased supply of nitrogen and phosphorus, but was unchanged when potassium was increased. Efficiencies were similar to those of other aquatic macrophytes.     

Contraction of isolated airway smooth muscle by synthetic leukotrienes C4 and D4

The pharmacology of leukotrienes (LT) C4 and D4 in isolated airway smooth muscle was investigated. In rat trachea, neither LTC4 or D4 elicited a response. In contrast, LTC4 was a potent contractile agonist in guinea-pig trachea, bronchus and parenchymal lung strip. Similar effects were obtained with LTD4 in trachea and parenchyma. In trachea and bronchus, the concentration-response curve to LTC4 was biphasic: indomethacin converted the biphasic response curve to a simple sigmoidal shape and enhanced the maximum contractile response. The SRS-A antagonist FPL 55712 antagonized the effect of LTD4 in both trachea and parenchyma. As regards LTC4-induced contraction of trachea and bronchus, FPL 55712, depending on concentration, either antagonized, or antagonized and enhanced the maximum contractile response. The enhancement of the maximum contractile response by FPL 55712 was not apparent when indomethacin was present. FPL 55712 failed to antagonize the effect of LTC4 in parenchyma.