Heterocyst formation in cyanobacteria.

When deprived of combined nitrogen, many filamentous cyanobacteria develop a one-dimensional pattern of specialised nitrogen-fixing cells, known as heterocysts. Recent years have seen the identification and characterisation of some of the key genes and proteins involved in heterocyst development and spacing, including the positive regulator HetR and the diffusible inhibitor PatS.

Pyrolysis mass spectrometry analysis of free-living and symbiotic cyanobacteria.

The potential of pyrolysis mass spectrometry to distinguish closely related cyanobacterial strains was assessed by using the technique to compare symbiotic cyanobacteria isolated from the hornwort Phaeoceros laevis and free-living cyanobacterial strains at the same field site. The same strains had previously been compared using polymerase chain reaction-based DNA fingerprinting techniques (West & Adams 1997, Appl. Environ. Microbiol. 63: 4479-4484). Many of the strains were grouped identically by the two techniques, although there were some differences, possibly resulting from the ability of these cyanobacteria to develop a range of specialised cell types having different chemical compositions to the vegetative cells. Although growth conditions were chosen to suppress cellular differentiation, this may not always have been completely successful. With careful control of growth conditions pyrolysis mass spectrometry has considerable potential as an additional tool for the phenetic comparison of cyanobacterial strains. It has the advantage that analysis is directly derived from whole cells, and hence is simpler and cheaper than DNA-based methods, although it does require the growth of axenic strains. The technique may be particularly useful in the study of some of the more cryptic unicellular and non-heterocystous filamentous cyanobacterial groups, in which the lack of cellular differentiation should minimise any variability in the chemical composition of cells.

Fibrillar array in the cell wall of a gliding filamentous cyanobacterium.

The cell walls of a number of filamentous, gliding cyanobacteria of the genus Oscillatoria were examined by transmission electron microscopy of ultrathin sections, of freeze-etched replicas, and of whole cells crushed between glass slides and negatively stained. All three techniques revealed the presence of a highly ordered array of parallel fibrils, seen in transverse sections to be situated between the peptidoglycan and the outer membrane. Approximately 200 individual fibrils, each 25 to 30 nm in width, form a parallel, helical array that completely surrounds each cyanobacterial filament, running at an angle of 25 to 30 degrees to its long axis. This highly regular arrangement of the fibrillar layer may imply some underlying symmetry responsible for its organization. A possible source of such symmetry would be the peptidoglycan, and some form of interaction between this layer and the fibrils might provide the necessary scaffolding for the fibrillar array. In crushed, negatively stained samples of fresh cells, individual fibrils were seen outside the filament, released from the cell wall. These released fibrils were of the same width as those observed in situ but were in short lengths, mostly of 100 to 200 nm, and were invariably bent, sometimes even into U shapes, implying great flexibility. Negative staining of released fibrils showed no evidence that they were hollow tubes but did give some indication of a substructure, implying that they were composed of many subunits. The function of this fibrillar array is unknown, although its position in the cell wall, as well as the correspondence between the angle of the fibrils with respect to the long axis of the filament and the rotation of the filament during gliding, may imply an involvement in gliding motility.

Platelet-activating factor induction of activator protein-1 signaling in bronchial epithelial cells.

Platelet-activating factor (PAF) has been implicated in the pathogenesis of allergic and inflammatory events in the airway. In the present study, we sought to determine if PAF receptors are present on human bronchial epithelial cells and whether PAF binding to these receptors leads to activation of activator protein-1 (AP-1)-mediated transcription. Radioligand binding studies demonstrated specific binding sites for the PAF antagonist [3H]WEB 2086 (3-[4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f]-[1,2,4]triazolo[4,3- a][1,4]diazepine-2-yl]-1-(4-morpholinyl)-1-propanone) on primary bronchial epithelial cells with an equilibrium dissociation constant (Kd) = 9.8 nM and maximal density of binding sites (Bmax) = 42.4 fmol/mg of protein. The expression of PAF receptors in these cells was further confirmed by reverse transcriptase-polymerase chain reaction, which revealed amplification products derived from PAF receptor mRNA corresponding to transcripts 1 and 2. In the bronchial epithelial cell line BEAS-2B transfected with an expression plasmid for the human PAF receptor, PAF stimulation increased AP-1 DNA binding activity as determined by electrophoretic mobility shift assays. The Fos and Jun family proteins were identified as components of the DNA-protein complexes by anti-peptide antibodies in gel supershift assays. Additionally, PAF significantly induced AP-1 mediated transcription which was dependent on the expression of PAF receptors. The PAF antagonist WEB 2086 blocked the PAF effect but not that induced by 12-O-tetradecanoyl phorbol-13-acetate, indicating the specificity of the PAF response. These results indicate that activation of airway epithelial cells through stimulation of PAF receptors includes up-regulation of the nuclear transcription factor AP-1 and AP-1 transcriptional activity.

Phenotypic and genotypic comparison of symbiotic and free-living cyanobacteria from a single field site.

PCR amplification techniques were used to compare cyanobacterial symbionts from a cyanobacterium-bryophyte symbiosis and free-living cyanobacteria from the same field site. Thirty-one symbiotic cyanobacteria were isolated from the hornwort Phaeoceros sp. at several closely spaced locations, and 40 free-living cyanobacteria were isolated from the immediate vicinity of the same plants. One of the symbiotic isolates was a species of Calothrix, a genus not previously known to form bryophyte symbioses, and the remainder were Nostoc spp. Of the free-living strains, two were Calothrix spp., three were Chlorogloeopsis spp. and the rest were Nostoc spp. All of the symbiotic and all but one of the free-living strains were able to reconstitute the symbiosis with axenic cultures of both Phaeoceros and the liverwort Blasia sp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the regions flanking the 16S-23S rRNA internal transcribed spacer. With one exception, the two techniques produced complementary results and confirmed for the first time that a diversity of symbiotic cyanobacteria infect Phaeoceros in the field. Symbionts from adjacent colonies were different as often as they were the same, showing that the same thallus could be infected with many different cyanobacterial strains. Strains found to be identical by the techniques employed here were often found as symbionts in different thalli at the same locale but were never found free-living. Only one of the free-living strains, and none of the symbiotic strains, was found at more than one sample site, implying a highly localized distribution of strains.

Anabaena flos-aquae and other cyanobacteria possess diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) phosphorylase activity.

Diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) phosphorylase, previously only known in lower eukaryotes, has been detected in extracts of the cyanobacteria Anabaena flos-aquae, Anabaena variabilis and Synechococcus sp. The 32 kDa enzyme was partially purified from A. flos-aquae and separated from a 23 kDa hydrolytic activity. It had a pH optimum of 9.5 and required a bivalent cation for activity: Mg2+, Mn2+, Ca2+, Co2+ or Zn2+. Diadenosine tri-, tetra- and penta-phosphates were all effective substrates (relative rates 0.85, 1.00 and 0.27 respectively), while the hexaphosphate was a poor substrate and the diphosphate was inactive. ADP was always one of the products of phosphorolysis. Arsenate and vanadate could substitute for phosphate (relative rates 1.80, 2.25 and 1.00 respectively), but tungstate and sulphate could not. Chromate and molybdate were poor substrates. A search of the GenBank non-redundant database revealed a putative Ap4A phosphorylase gene in the cyanobacterium Synechocystis sp. The gene showed significant blocks of identity/similarity with yeast Ap4A phosphorylases I and II, particularly the latter.

Tryptophan Decarboxylase, Tryptamine, and Reproduction of the Whitefly.

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts tryptophan to the indole-alkaloid tryptamine. When the TDC gene was expressed in transgenic tobacco, the 55-kD TDC enzyme and tryptamine accumulated. Bemisia tabaci (sweetpotato whitefly) reproduction on transgenic plants decreased up to 97% relative to controls. Production of tryptamine, its derivatives, or other products resulting from TDC activity may discourage whitefly reproduction and provide a single-gene-based plant protection strategy.

Cloning and sequence of ftsZ and flanking regions from the cyanobacterium Anabaena PCC 7120.

Using degenerate oligodeoxyribonucleotide primers based on conserved regions of the cell-division protein FtsZ, a 220-bp fragment of DNA was amplified by the polymerase chain reaction from Anabaena PCC 7120 (Ana). This fragment, which showed significant homology with Escherichia coli ftsZ, was used as a probe to isolate a 15-kb genomic clone containing ftsZ from an Ana DNA library. Sequence analysis revealed an open reading frame (ORF) encoding a protein of 379 amino acids, with 49% identity with E. coli FtsZ. Upstream of Ana ftsZ is a small, unidentified ORF, transcribed in the same direction. An ORF lying downstream of the ftsZ coding region and transcribed in the opposite orientation, shows homology with bacterial glutathione synthetase-encoding genes. Single copies of ftsZ have been identified in Ana and two other cyanobacteria. Multiple transcripts hybridising to ftsZ were detected by Northern hybridisation.

Protease inhibitors of Manduca sexta expressed in transgenic cotton.

To explore the effectiveness of insect derived protease inhibitors in protecting plants against insect feeding, anti-trypsin, anti-chymotrypsin and anti-elastase protease inhibitor (PI) genes from Manduca sexta L. were expressed in transgenic cotton (Gossypium hirsutum L.). From 198 independent transformants, 35 elite lines were further analyzed. Under the control of the 35S promoter of CaMV, PI accumulated to approximately 0.1% of total protein, depending on the tissue analyzed. Using cell-flow cytometry, DNA content/ nuclei of transgenic and non-transformed cotton were identical. On cotton plants expressing PIs, fecundity of Bemisia tabaci (Genn.), the sweetpotato whitefly, was reduced compared to controls. Expression of these protease inhibitors may reduce the developmental rate of B. tabaci and other insects, and provide a strategy for cotton protection.

Sleep disorders in older patients. Conservative treatment is usually enough.

Sleep disorders in older patients can be caused by the changes of aging, physical disorders, psychological problems, certain drugs, or a combination of these. A complete physical examination and a thorough sleep history help in selecting appropriate treatment. Pharmacologic or surgical therapy may be needed, but one or more sleep-hygiene measures are adequate to improve most patients' quality of life when they are asleep--and awake.

Prophylaxis of cyanobacterial and mushroom cyclic peptide toxins.

The pathogenicity of virulent cyclic peptide toxins of the cyanobacterium, Microcystis aeruginosa, and the mushroom, Amanita phalloides, was prevented in mice by pretreatment with a variety of chemically unrelated agents including hydrocortisone, shellac, certain diazo and triazine dyes and cyclosporine. A. Despite the diverse nature of the protective agents, a feature commonly associated with protection was the ability to impair hepatic uptake of 51Cr-labeled sheep erythrocytes, a function of hepatic macrophages (Kupffer cells). In addition, several of the protective agents are known to affect other aspects of reticuloendothelial cell function. Therefore it seems likely that the hepatic macrophage is involved in the observed protection, although by what mechanism(s) is unknown. The most remarkable prophylaxis was seen with a single injection of Trypan red, which provided nonimmunologic protection against a lethal dose of a cyanobacterial toxin, cyanoginosin-LR, for periods up to 3 months.

Isolation and restriction analysis of DNA from heterocysts and vegetative cells of cyanobacteria.

A procedure was developed for the isolation of heterocysts from cyanobacterial filaments without recourse to mechanical disruption of the vegetative cells. DNA was then extracted from purified heterocysts by heating with 2% (w/v) SDS at 70 degrees C for 10 min. Following purification, this DNA was used for treatment with a range of restriction endonucleases and the results compared with DNA isolated from vegetative cells. Both heterocyst and vegetative DNAs from Anabaena PCC 7120 and Anabaena CA (ATCC 33047) were cut by XbaI, HindIII, EcoRI, ClaI, HpaII and MspI. However, none of the DNAs were cut by XhoI, SalI or MboI, indicating that the DNA from both organisms is methylated, but that no gross changes in methylation occur during heterocyst formation. Treatment of the DNAs with the former enzymes, followed by separation of the fragments by agarose gel electrophoresis, resulted in most cases in patterns of bands, which allowed a limited comparison of heterocyst and vegetative DNAs. No major differences were seen between the heterocyst and vegetative DNAs of either organism, implying that there are unlikely to be extensive rearrangements or major loss of DNA during heterocyst differentiation.

Effects of deuteration on hematopoiesis in the mouse.

Mice ingesting 30 to 50% D2O (heavy water, deuterium oxide) developed a dose-dependent depression of formed peripheral blood elements in 4 to 9 days. The principal mechanism of anemia and thrombocytopenia is impaired hematopoiesis. Despite pancytopenia in the peripheral blood, bone marrow cellularity and morphology remained normal. Upon replacement of D2O with tap water, platelet and neutrophil concentrations returned to normal within 48 to 72 hr. In contrast, blood lymphocyte concentrations remained low for several weeks. B-lymphocytes may be more affected by deuteration than other lymphocyte subsets. In vivo reticuloendothelial cell function, as assessed by 51Cr-labeled sheep erythrocyte clearance, was unaffected by D2O. Although a dose-dependent decrease in fluid intake occurred during deuteration, hematocytopenia was not a consequence of dehydration. In view of the known kinetics of D2O in biological systems, the rapid response of myeloid elements to deuteration must be due primarily to the solvent (nonmetabolic) isotope effect. Prolonged deuteration has proven toxic when included in regimens for treatment of neoplasia, including leukemia, in animal models. The present study shows that modulation of hematopoiesis by D2O is possible without invoking the toxicities associated with prolonged deuteration.

Pathophysiologic effects of a toxic peptide from Microcystis aeruginosa.

Toxin-LR, a hexapeptide produced by Microcystis aeruginosa, causes marked hepatic vascular congestion, thrombocytopenia, microscopic pulmonary thrombi and death in 50-70 min when injected into mice. Although it is considered an hepatotoxin, we report that sublethal hepatocellular damage produced by CCl4 given 24 hr prior to toxin-LR administration prevents the acute deaths. However, CCl4-treated mice surviving toxin-LR acute effects often died during the subsequent three days. Pretreatment of mice with the microsomal enzyme inhibitors SKF 525A or cobaltous chloride did not alter the acute lethality of toxin-LR, but pharmacologic doses of hydrocortisone prevented both the acute and delayed deaths. X irradiation-induced thrombocytopenia or thrombocytopenia and leukopenia did not significantly affect the toxin's lethality. In vitro platelet aggregation or lysis did not occur during incubation with toxin-LR, nor was a humoral aggregating factor detected in plasma from toxin-injected mice.

Heterocyst differentiation and cell division in the cyanobacterium Anabaena cylindrica: effect of high light intensity.

Heterocyst differentiation in the cyanobacterium Anabaena cylindrica is initiated by the removal of fixed nitrogen from the medium. These specialized cells occur singly at regular intervals within filaments of vegetative cells. Incubation of cultures for periods of up to 12 h immediately prior to or following removal of fixed nitrogen, at a light intensity (500 mi Einsteins cm-2 s-1) approximately 10-fold higher than that required for optimum growth, resulted in the differentiation of pairs of adjacent (double) heterocysts. The frequency of double heterocysts was proportional to the length of the period of high light intensity. During growth at normal light intensity approximately 5% of cell divisions were symmetrical, but this increased more than 3-fold during 10-h incubation at high light intensity. The frequency of dividing cells remained constant during this period, but increased rapidly on return to normal light. The frequency of double heterocysts was reduced if a period of incubation at normal light intensity was interposed between the 12-h period at high light intensity and transfer to nitrogen-free medium. A period of 8 h normal light was required to reduce the frequency of double heterocysts to control values, and this corresponded to the length of time needed for the frequency of symmetrical divisions to return to control levels following 12 h at high light intensity. We confirm that cell division in Anabaena cylindrica is asymmetrical and conclude that the presence of double heterocysts results from an increase in the symmetry of cell division during incubation at high light intensity. The results also support the finding of previous workers that a cell is only susceptible to differentiation during a short period following its formation. During the period of high light the rate of doubling of the absorbance of the culture at 750 mn increased from 24 h to approximately 10 h and decreased to more than 100 h on return to normal light. The very high rate could be explained by increases in the volume and granular content of cells during incubation at high light intensity and did not represent an equivalent increase in the rate of cell division.