Changes in gill histology of fathead minnows and yellow perch transferred to soft water or acidified soft water with particular reference to chloride cells

Summary Fathead minnows, Pimephales promelas, and yellow perch, Perca flavescens, were transferred from moderately soft Lake Superior water (hardness 45mg/l as CaCO₃) to very soft diluted Lake Superior water (hardness 4.5mg/l). Sulfuric acid was added in some treatments by means of a multichannel diluter. In very soft water, chloride cells proliferated in the gills, especially in the epithelium of the secondary lamellae. When exposed to acid, chloride cells were damaged and less abundant in the secondary lamellae, and blood osmolality was reduced at pH 5.0 (x = 188 mOsm/kg, 9 days exposure; normal 280 mOsm/kg) for the minnows and pH 4.1 (x = 218 mOsm/kg, 58 days exposure; normal 329 mOsm/kg) for the perch. Certain chloride cells which form gland-like clusters in the primary lamellae of perch gills showed little damage even at pH 4.1. The present study supports the view that chloride cells proliferate in very soft fresh water to help maintain ionic balances, and that damage to these cells in acidified soft water may be related to diminished ionoregulatory capacity. The greater acid tolerance of chloride cells of, and the higher blood osmolality maintained by, perch could help to explain the greater tolerance of this species to low pH. In some cases, a species' ability to acclimate to very soft water and acidified soft water may depend upon the number, distribution, and physiology of its chloride cells.     

Glycerol production in relation to the ATP pool and heat production rate of the yeasts Debaryomyces hansenii and Saccharomyces cerevisiae during salt stress

Changes in glycerol production and two parameters related to energy metabolism i. e. the heat production rate and the ATP pool, were assayed during growth of Saccharomyces cerevisiae and Debaryomyces hansenii in 4 mM and 1.35 M NaCl media. For both of the yeasts, the specific ATP pool changed during the growth cycle and reached maximum values around 10 nmol per mg dry weight in both types of media. The levels of glycerol were markedly enhanced by high salinity. In the presence of 1.35 M NaCl, D. hansenii retained most of its glycerol produced intracellularly, while S. cerevisiae extruded most of the glycerol to the environment. The intracellular glycerol level of S. cerevisiae equalled or exceeded that of D. hansenii, however, with values never lower than 3 mol per mg dry weight at all phases of growth. When D. hansenii was grown at this high salinity the intracellular level of glycerol was found to correlate with the specific heat production rate. No such correlation was found for S. cerevisiae. We concluded that during salt stress, D. hansenii possesses the capacity to regulate the metabolism of glycerol to optimize growth, while S. cerevisiae may not be able to regulate when exposed to different demands on the glycerol metabolism.     

Molecular cloning and nucleotide sequencing of oxyR, the positive regulatory gene of a regulon for an adaptive response to oxidative stress in Escherichia coli: homologies between OxyR protein and a family of bacterial activator proteins

Summary Treatment of Escherichia coli and Salmonella typhimurium cells with a low dose of hydrogen peroxide induces expression of a large number of genes, and confers resistance to oxidative stresses. The oxyR gene encodes a positive regulatory protein for a subset of these genes involved in the defense against oxidative damage. We cloned a DNA fragment that contains the E. coli oxyR region on a plasmid vector, and analyzed the nucleotide sequence of the gene. The amino acid sequence of OxyR protein, deduced from the nucleotide sequence, shows a high degree of homology to the sequences of a number of bacterial activator proteins including LysR, cysB, IlvY, MetR and NodD. The product of the oxyR gene identified by the maxicell procedure was a 34 kDa protein, which agrees with the size predicted from the nucleotide sequence of the gene.     

Polygalacturonase activity and variation in ripening of papaya fruit with tissue depth and heat treatment

Effects of tissue position (viz. outer vs inner mesocarp) and heat treatment (48°C, 20 min) on variations in polygalacturonase (EC 3.2.1.15 and EC 3.2.1.67) activity and ripening of fruits of Carica papaya L. cv. Backcross Solo were investigated. Polygalacturonase activity increased during ripening concomitantly with an increase in tissue softness and soluble polyuronide level. Throughout ripening, inner mesocarp tissue was softer and contained higher polygalacturonase activity than outer mesocarp tissue. Titratable acidity as well as ß-galactosidase (EC 3.2.1.23) activity also increased during ripening; however, unlike polygalacturonase, their level or activity was lower in inner than in outer mesocarp. Ascorbic acid could partially account for the increase in titratable acidity during ripening but contributed very little to the differences in titratable acid levels between outer and inner mesocarp. Heat treatment had no effect on either fruit softness or titratable acidity, but it markedly reduced the increase in ascorbic acid and polygalacturonase activity during ripening. Ripening, as reflected by changes in tissue softness and polygalacturonase activity, progressed outwardly from the interior towards the exterior of the fruit. The effect of heat treatment in suppressing polygalacturonase activity was relatively greater in inner than in outer mesocarp, suggesting that sensitivity of the enzyme to heat treatment may vary with stage of ripeness of the tissue.     

Effect of osmotic stress on protein turnover in Lemna minor fronds

Evidence is presented that although many proteins from the fronds of Lemna minor L. undergo enhanced degradation during osmotic stress, ribulose-1,5-bisphosphate carboxylase (RuBPCase) is not degraded. Instead RuBPCase is converted in a series of steps to a very high-molecular-weight form. The first step involves the induction of an oxidase system which after 24 h of stress converts RuBPCase to an acidic and catalytically inactive form. Subsequently, the oxidised RuBPCase protein is gradually polymerized to a number of very large aggregates (molecular weight of several million).The conversion of RuBPCase to a high-molecular-weight form appears to be correlated with (i) a reduction in the number of-SH residues and (ii) the susceptibility to in-vitro proteolysis. Indeed, the number of-SH groups per RuBPCase molecule decreases from 89 in the native enzyme to 54 and 22 in the oxidised and polymerized forms, respectively. On the other hand, the oxidised enzyme is more susceptible to in-vitro proteolysis than the native form. However, it is the polymerized form of RuBPCase which is particularly susceptible to in-vitro proteolysis.Western-blotting experiments and anti-ubiquitin antibodies were used to detect the presence of ubiquitin conjugates in extracts from osmotically stressed Lemna fronds. The possible involvement of ubiquitin in the formation of the aggregates is discussed.Abbreviations DTT dithiothreitol - EDTA ethylenediamine-tetraacetic acid - FPLC fast protein liquid chromatography - kDa kilodaltons - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulphonyl fluoride - RuBPCase ribulose bisphosphate carboxylase - SDS sodium dodecyl sulphate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons

Members of the Chenopodiaceae can accumulate high levels (>100 mol·(g DW)^-1) of glycine betaine (betaine) in leaves when salinized. Chenopodiaceae synthesize betaine by a two-step oxidation of choline (cholinebetaine aldehyde betaine), with the second step catalyzed by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). High betaine levels have also been reported in leaves of species from several distantly-related families of dicotyledons, raising the question of whether the same betaine-synthesis pathway is used in all cases.Fast atom bombardment mass spectrometry showed that betaine levels of >100 mol·(g DW)^-1 are present in Lycium ferocissimum Miers (Solanaceae), Helianthus annuus L. (Asteraceae), Convolvulus arvensis L. (Convolvulaceae), and Amaranthus caudatus L. (Amaranthaceae), that salinization promotes betaine accumulation in these plants, and that they can convert supplied choline to betaine aldehyde and betaine. Nicotiana tabacum L. and Lycopersicon lycopersicum (L.) Karst. ex Farw. (Solanaceae), Lactuca sativa L. (Asteraceae) and Ipomoea purpurea L. (Convolvulaceae) also contained betaine, but at a low level (0.1–0.5 mol·(g DW)^-1. Betaine aldehyde dehydrogenase activity assays, immunotitration and immunoblotting demonstrated that the betaine-accumulating species have a BADH enzyme recognized by antibodies raised against BADH from Spinacia oleracea L. (Chenopodiaceae), and that the M_r of the BADH monomer is in all cases close to 63 000. These data indicate that the cholinebetaine aldehydebetaine pathway may have evolved by vertical descent from an early angiosperm ancestor, and might be widespread (albeit not always strongly expressed) among flowering plants. Consistent with these suggestions, Magnolia x soulangiana was found to have a low level of betaine, and to express a protein of M_r 63 000 which cross-reacted with antibodies to BADH from Spinacia oleracea.Abbreviations BADH Betaine aldehyde dehydrogenase - DCIMS desorption chemical ionization mass spectrometry - FABMS fast atom bombardment mass spectrometry - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TLC thin-layer chromatography     

Osmoregulation in Dunaliella tertiolecta: effects of salt stress,and the external pH on the internal pH

The intracellular pH of the halotolerant green algae Dunaliella tertiolecta, was determined by the distribution of 5,5-dimethyl-2(¹⁴C)-oxalolidine-2,5-dione (DMO) between the cell and the surrounding medium. 5,5-dimethyl-2(¹⁴C)oxalolidine-2,4-dione was not metabolized by the algal cells. The intracellular pH of Dunaliella tertiolecta was 6.8 in the dark and 7.4 in the light. During a salt stress, after two hours, the intracellular pH was increased by 0.2 pH units in both light and dark. The salt stressed cells maintained a constant pH of about 7.5 over the pH range of 6.5 to 8.5. Because of the relatively low permeability coefficient of the plasma membrane for DMO, this technique does not permit rapid pH determinations during the induction period after a salt stress. The magnitude of the salt induced pH changes measured 2 h after the salt stress implies a minor importance of this alkalization in this time range, but does not exclude a larger importance of pH changes for osmoregulation during the induction period.Abbreviations Chl chlorophyll - DMO 5,5-dimethyl-2(¹⁴C)oxalolidine-2,4-dione - PCV packed cell volume - SDS sodium dodecyl sulfate     

Effect of acetaminophen on hepatic content and biliary efflux of glutathione disulfide in mice

The increased expiration of ethane and pentane by mice treated with hepatotoxic doses of acetaminophen suggests the possibility of oxidant mechanisms associated with the necrosis. However, studies in rats are not consistent with oxidant stress mechanisms causing the damage, because acetaminophen given to rats does not increase GSSG efflux, a sensitive index of intrahepatic oxidant stress. To compare the extent of oxidant stress generated by acetaminophen in mice versus rats, hepatic content and biliary efflux of GSSG and GSH in mice have been examined. Bile was collected from anesthetized male ICR mice before and after intraperitoneal administration of acetaminophen (325 mg/kg, 2.15 mmol/kg), t-butyl hydroperoxide (TBHP) (1.5 mmol/kg), diethyl maleate (400 mg/kg, 2.33 mmol/kg, in corn oil) or saline (control) and GSH and GSSG were measured by the enzymatic recycling method of Tietze. An increase in biliary GSSG efflux was produced by t-butyl hydroperoxide, but not by the other agents. Biliary GSH/GSSG ratios decreased in acetaminophen-treated animals, presumably reflecting the marked depletion of hepatic GSH, since a similar decrease was observed with non-hepatotoxic doses of diethyl maleate. The failure of acetaminophen to increase the hepatic content or biliary efflux of GSSG in ICR mice is not consistent with the view that oxidant stress mechanisms cause the damage, despite the increases in alkanes expired after acetaminophen administration in this specific animal model.     

Is the ‘microbial loop’ an early warning indicator of anthropogenic stress?

Various components of the Microbial loop such as bacteria, heterotrophic nanoflagellates and autotrophic picoplankton were analyzed, for the first time across the Great Lakes, during a cruise in the summer of 1988. In addition, the size fractionated primary productivity using carbon-14 techniques was also determined. The statistical analysis indicated that bacteria, autotrophic picoplankton and ultraplankton/picoplankton productivity were significantly higher in Lakes Ontario and Erie than Lakes Huron and Michigan. The autotrophic picoplankton and ultraplankton/picoplankton productivity was higher in Lake Erie compared to Lake Ontario.The autotrophic picoplankton showed sensitivity to nutrients and contaminants in various types of environments. A dramatic decrease of autotrophic picoplankton in eutrophic-contaminated areas, such as Ashbridges Bay, Hamilton Harbour and western Lake Erie was observed. Conversely, in Saginaw Bay, another eutrophic environment, the autotrophic picoplankton were significantly higher than in Lake Huron. The sensitivity of autotrophic picoplankton to nutrients/contaminants might have implications to trophic interactions. Our results suggest that structural and functional characteristics of the microbial loop may be operating differently in stressed versus unstressed ecosystems. The possibility of using autotrophic picoplankton as an early warning indicator of environmental perturbation is proposed.     

Acid shock proteins of Escherichia coli

Synthesis of total cellular proteins of Escherichia coli was studied after transfer of cultures from pH 6.9 to pH 4.3. Proteins induced by such an external pH shift down were identified by mono- and bi-dimensional electrophoresis. 30 to 45 min after an acid shift, a group of at least sixteen polypeptides was markedly induced. Four of these polypeptides corresponded to the well known heat shock proteins GroEL, DnaK, HtpG and HtpM. Their pH induction was RpoH-dependent. Three other pH-induced proteins were previously identified as stress proteins induced either by osmolarity or aerobiosis or low temperature (proteins 32 (defined in this paper), C70.0 and C62.7). Seven other proteins were specifically induced after an acid shift and were called acid shock proteins (ASP). The induction of one of these proteins was RpoH-dependent, whereas that of others was RpoH-independent.