Protein synthesis and proteolysis in immobilized cells of the cyanobacterium Nostoc commune UTEX 584 exposed to matric water stress.

Cells of the cyanobacterium Nostoc commune UTEX 584 in exponential growth were subjected to acute water stress by immobilizing them on solid supports and drying them at a matric water potential (psi m) of -99.5 MPa. Cells which had been grown in the presence of Na235SO4 before immobilization and rapid drying continued to incorporate 35S into protein for 90 min. This incorporation was inhibited by chloramphenicol. No unique proteins appeared to be synthesized during this time. Upon further drying, the level of incorporation of 35S in protein began to decrease. In contrast, there was an apparent increase in the level of certain phycobiliprotein subunits in solubilized protein extracts of these cells. Extensive proteolysis was detected after prolonged desiccation (17 days) of the cells in the light, although they still remained intact. Phycobilisomes became dissociated in both light- and dark-stored desiccated material.     

Sensitivity of Nostoc commune UTEX 584 (Cyanobacteria) to water stress

Cells of Nostoc commune UTEX 584 from liquid cultures expressed an upshift in nitrogenase activity when immobilised on inert supports and exposed to matric water potentials between -1.10 and -99.5 MPa. Cells incubated at 0.10 MPa (a_w=c 1.0) maintained increased activity for at least 48 h following immobilization. At water potentials below -23.1 MPa (a_w=0.85), the upshift was transitory. Nitrogenase activity decreased rapidly when immobilised cells were incubated at lower values of _m.Desiccated cells stored at -99.5 MPa (a_w=0.50) underwent an upshift in nitrogenase activity, and in the size of the intracellular ATP pool, when rewetted with either distilled water or liquid MB_o medium (_o =-0.18 MPa). The upshift in nitrogenase activity was chloramphenicol-sensitive and was preceeded by a lag. The duration of the lag depended on the time taken to equilibrate cells to-99.5 MPa, the time desiccated, and the conditions of storage and rewetting. Cells that had no, or very low, nitrogenase activity when rewetted in air, showed a marked stimulation of nitrogenase activity in the presence of 5% v/v CO₂ under both aerobic and anerobic conditions.When rewetted in the presence of 1% w/v glucose (_o =-0.14 MPa), vegetative cells remained intact, but heterocysts underwent autolysis and nitrogenase activity was not detected, even in the presence of 5% v/v CO₂.Abbreviations TTC 2,3,5-triphenyl-2-tetrazolium chloride - _m matric water potential - _o osmotic water potential - a_w water activity     

The protein index ofNostoc commune UTEX 584 (cyanobacteria): changes induced in immobilized cells by water stress

Two-dimensional gel electrophoresis was used to analyze the effects of water stress (immobilization and rapid drying, desiccation, rewetting) on the protein index of the desiccation-tolerant cyanobacteriumNostoc commune UTEX 584. Five major landmark protein constellations were detected in the protein index of control cells (in liquid culture) and were designated A (1 protein), B (7 proteins), C (8 proteins), D (3 proteins) and E (2 proteins). These included proteins which showed different sensitivities to water stress. Upon immobilization and rapid drying of the cells at a water potential ({ie87-1}) of -99.5 MPa (a_w=0.5) for 30 min, few changes took place in the index. Four conspicuous proteins and the majority of proteins in the size range 18 to 97 K diminished in abundance while most proteins of constellations A, B and C were detected in fluorographs with the same intensity as in the control. Although protein synthesis continued during this time of drying, no novel class of proteins was detected. The level of incorporation of³⁵S in protein increased rapidly during the first 60 min of rehydration, and then decreased gradually for a further 2.5 h. Extant proteins that were hardly detectable after 24 h of drying, reappeared and increased in abundance upon rewetting of cells for 60 min while a number of proteins which disappeared after drying did not appear during this time. No novel class of proteins appeared upon rewetting. During further rehydration, extensive proteolysis was observed.ThenifH product (Fe protein of nitrogenase) was detected on Western blots — through cross-reaction with antibody — as an acidic polypeptide with a molecular mass of 33.8 K. Fe-protein was detected in immobilized cells after 30 min of drying, in desiccated material, and in rehydrated cells.Abbreviations PMSF Phenylmethylsulfonyl fluoride - IEF Isoelectric focussing     

Desiccation independence of terrestrialNostoc commune ecotypes (cyanobacteria)

The subspeciesNostoc commune var.flagelliforme andN. commune var.commune are found in China (Ningxia Province, Inner Mongolia) as two morphologically different ecotypes of the desiccation-independent cyanobacteriumN. commune. The first ecotype, but not the second, colonizes arid areas. Various biochemical parameters and water dependence of photosynthesis and nitrogen fixation were compared for both ecotypes. Different patterns of water stress proteins were found in the two ecotypes. Repeated desiccation resulted in an enhanced desiccation independence for photosynthesis and, in the case of the ecotypecommune, for nitrogen fixation. The different response of nitrogenase of both ecotypes towards repeated cycles of rewetting and desiccation under conditions simulating the natural environment is discussed in terms of the energy balance of the colonies that are adapted to different environmental conditions.     

Peroxide reduction by a metal-dependent catalase in Nostoc punctiforme (cyanobacteria)

This study investigated the role of a novel metal-dependent catalase (Npun_R4582) that reduces hydrogen peroxide in the cyanobacterium Nostoc punctiforme. Quantitative real-time PCR showed that npun_R4582 relative mRNA levels were upregulated by over 16-fold in cells treated with either 2 μM added Co, 0.5 μM added Cu, 500 μM Mn, 1 μM Ni, or 18 μM Zn. For cells treated with 60 μM H₂O₂, no significant alteration in Npun_R4582 relative mRNA levels was detected, while in cells treated with Co, Cu, Mn, Ni, or Zn and 60 μM peroxide, relative mRNA levels were generally above control or peroxide only treated cells. Disruption or overexpression of npun_R4582 altered sensitivity to cells exposed to 60 μM H₂O₂ and metals for treatments beyond the highest viable concentrations, or in a mixed metal solution for Npun_R4582⁻ cells. Moreover, overexpression of npun_R4582 increased cellular peroxidase activity in comparison with wild-type and Npun_R4582⁻ cells, and reduced peroxide levels by over 50%. The addition of cobalt, manganese, nickel, and zinc increased the capacity of Npun_R4582 to reduce the rate or total levels of peroxide produced by cells growing under photooxidative conditions. The work presented confirms the function of NpunR4582 as a catalase and provides insights as to how cells reduce potentially lethal peroxide levels produced by photosynthesis. The findings also show how trace elements play crucial roles as enzymatic cofactors and how the role of Npun_R4582 in hydrogen peroxide breakdown is dependent on the type of metal and the level available to cells.

     

Induction of water-stress proteins in cyanobacteria exposed to matric- or osmotic-water stress

Abstract: Specific stress polypeptides were detected in three drought-resistant cyanobacteria ( Phormidium autumnale , LPP₄ and Chroococcidiopsis sp.) subjected to matric- and osmotic-water stress. Drought stress caused the induction of at least 2–3 new polypeptides of apparent molecular masses of 30 kDa and 40–45 kDa. The polypeptide of 30 kDa was located in the thylakoid membranes, and the 45-kDa polypeptide in the cytoplasm. When these cyanobacteria were exposed to salt stress polypeptides of similar size appeared.     

Mechanisms to avoid photoinhibition in a desiccation-tolerant cyanobacterium, Nostoc commune

A desiccation-tolerant cyanobacterium, Nostoc commune, showsunique responses to dehydration. These responses are: (i) lossof PSII activity in parallel with the loss of photosynthesis;(ii) loss of PSI activity; and (iii) dissipation of light energyabsorbed by pigment–protein complexes. In this study,the deactivation of PSII is shown to be important in avoidingphotoinhibition when the Calvin–Benson cycle is repressedby dehydration. Furthermore, our evidence suggests that dissipationof light energy absorbed by PSII blocks photoinhibition understrong light in dehydrated states.     

Carbon turnover in peatland mesocosms exposed to different water table levels

Changes of water table position influence carbon cycling in peatlands, but effects on the sources and sinks of carbon are difficult to isolate and quantify in field investigations due to seasonal dynamics and covariance of variables. We thus investigated carbon fluxes and dissolved carbon production in peatland mesocosms from two acidic and oligotrophic peatlands under steady state conditions at two different water table positions. Exchange rates and CO₂, CH₄ and DOC production rates were simultaneously determined in the peat from diffusive-advective mass-balances of dissolved CO₂, CH₄ and DOC in the pore water. Incubation experiments were used to quantify potential CO₂, CH₄, and DOC production rates. The carbon turnover in the saturated peat was dominated by the production of DOC (10–15 mmol m^–2 d^–1) with lower rates of DIC (6.1–8.5 mmol m^–2 d^–1) and CH₄ (2.2–4.2 mmol m^–2 d^–1) production. All production rates strongly decreased with depth indicating the importance of fresh plant tissue for dissolved C release. A lower water table decreased area based rates of photosynthesis (24–42%), CH₄ production (factor 2.5–3.5) and emission, increased rates of soil respiration and microbial biomass C, and did not change DOC release. Due to the changes in process rates the C net balance of the mesocosms shifted by 36 mmol m^–2 d^–1. According to our estimates the change in C mineralization contributed most to this change. Anaerobic rates of CO₂ production rates deeper in the peat increased significantly by a factor of 2–3.5 (DOC), 2.9–3.9 (CO₂), and 3–14 (CH₄) when the water table was lowered by 30 cm. This phenomenon might have been caused by easing an inhibiting effect by the accumulation of CO₂ and CH₄ when the water table was at the moss surface.     

Regulation of gene expression in Dictyostelium discoideum cells exposed to immobilized carbohydrates

When amoebae of Dictyostelium discoideum develop on gels of polyacrylamide that are derivatized with glucosides, they become capable of aggregation at the same time as cells not exposed to glucosides. However, the aggregation centers and streams of adherent cells formed on immobilized glucosides suddenly disintegrate. The cells repeatedly re-aggregate, but never form tight aggregates as they do on other substrata. Tight aggregates formed in the absence of glucosides disperse after their transfer to glucoside gels, and the cells undergo aggregation-disaggregation cycles. The formation of tight aggregates is correlated with the expression of specific post-aggregative poly(A)⁺ RNAs. These RNAs are not expressed in cells developing on glucoside gels, and the dispersal of tight aggregates on such gels is accompanied by the almost complete loss of these RNAs. A developmentally regulated membrane glycoprotein called contact site A, which is a marker of aggregation-competent cells, is normally expressed on glucoside gels. Cyclic AMP is also produced, indicating that the strong increase of adenylate cyclase activity during the preaggregation phase is not affected. In conclusion, cell contact with immobilized glucosides specifically inhibits postaggregative gene expression and arrests development at the aggregation stage.     

Reproductive allocation in Daphnia exposed to toxic cyanobacteria

We investigated experimentally how resources were allocated to reproduction in Daphnia pulex and Daphnia longispina when varying levels of toxic Microcystis were added to higher quality food. We used multiple regression models to estimate mean offspring size and clutch size in relation to maternal size and clutch number, and analysed effects of treatments on residuals from the models. We also measured variation in per offspring investment. At a high cyanobacterial level, D.pulex was virtually unable to reproduce. At a lower level, D.pulex produced small clutches. However, the regression model residuals indicated that the presence of cyanobacteria increased the portion of available resources allocated to reproduction. The observed allocation may be a means to maximize reproduction under diminished longevity. Effects on mean offspring size were marginal in D.pulex but variation in per offspring investment sometimes decreased in cyanobacterial exposures. Daphnia longispina was affected by a higher cyanobacterial level only, where offspring sized was reduced. Deviations from the regression model indicated that effects on maternal size alone do not explain this effect. Clutch size residuals and per offspring investment were unaffected by treatments in D.longispina. The observed responses differ from theoretical models on reproductive allocation under food imitation.      

Soluble guanylyl cyclase activation by HMR-1766 (ataciguat) in cells exposed to oxidative stress

Many vascular diseases are characterized by increased levels of ROS that destroy the biological activity of nitric oxide and limit cGMP formation. In the present study, we investigated the cGMP-forming ability of HMR-1766 in cells exposed to oxidative stress. Pretreatment of smooth muscle cells with H(2)O(2) reduced cGMP production stimulated by sodium nitroprusside (SNP) or BAY 41-2272. However, pretreatment with H(2)O(2) significantly increased HMR-1766 responses. Similar results were obtained with SIN-1, menadione, and rotenone. In addition, HMR-1766 was more effective in stimulating heme-free sGC compared with the wild-type enzyme. Interestingly, in cells expressing heme-free sGC, H(2)O(2) inhibited instead of potentiated HMR-1766 responses, suggesting that the ROS-induced enhancement of cGMP formation was heme dependent. Moreover, using truncated forms of sGC, we observed that the NH(2)-terminus of the beta(1)-subunit is required for the action of HMR-1766. Finally, to study tolerance development to HMR-1766, cells were pretreated with this sGC activator and reexposed to HMR-1766 or SNP. Results from these experiments demonstrated lack of tolerance development to HMR-1766 as well as lack of cross-tolerance with SNP. We conclude that HMR-1766 is an improved sGC activator as it has the ability to activate oxidized/heme-free sGC and is resistant to the development of tolerance; these observations make HMR-1766 a promising agent for treating diseases associated with increased vascular tone combined with enhanced ROS production.     

青海高原地木耳中氨基酸特征与分析

地木耳在青海民间视为山珍 ,能清热解毒、凉血明目 ,是一种天然的营养保健食品。文中采用美国 6 0 0E氨基酸分析仪 ,日本岛津RF5 4 0荧光仪测定了青海高原地木耳中 1 8种氨基酸的含量 ,为科学地开发利用青海高原地木耳食物资源提供一些基础资料和科学依据     

Ultrastructural analysis of the rehydration of desiccatedNostoc commune HUN (cyanobacteria) with particular reference to the immunolabelling of NifH

Summary Vegetative cells and heterocysts of the filamentous desiccation-tolerant cyanobacteriumNostoc commune HUN retain their ultrastructural organisation and the integrity of their intra- and extracellular membranes after two years of desiccation and subsequent rehydration. Immunogold-labelling of thin sections demonstrated the presence of NifH (Fe protein of nitrogenase) in vegetative cells and heterocysts within five minutes of the rehydration of dried colonies. Immunogold label accumulated in discrete areas vegetative cells within 5 minutes of the rewetting of cells, and after 30 minutes a conspicuous association of NifH protein with heterocyst ribosomes was detected. After longer periods of rehydration, the deposition of gold particles became more random within both cell types but occurred with a greater frequency in heterocysts. Up to 24 hours after the rewetting of cells, two morphologically-distinct forms of heterocyst could be discerned. NifH protein was detected through Western blotting (subunit M_r=33,800) in protein extracts from samples ofNostoc commune, collected in different parts of the world and including some which had been desiccated for periods of up to 10 years. The results are discussed in relation to the sequential recovery of metabolic functions, particularly nitrogen fixation, which occurs upon the rehydration of cells after their prolonged storage in the air-dry state.     

Response to stress of mussels (Mytilus edulis) exposed in Dutch tidal waters.

1. Mussels were exposed in the Dutch coastal zone and the Western Scheldt estuary. After six weeks of exposure, trace metals, PCBs and PAHs were measured in the soft tissue. 2. Tissue concentrations of contaminants are high in the Western Scheldt and intermediate near the Rhine outflow, except for the lower PCB congeners. 3. Results show that the survival in air is significantly lower at higher tissue concentrations, in particular of the lower PCB congeners. 4. The clearance rate is reduced at the highest tissue concentrations.