Effect of nutrients on the toxicity of pesticides carbofuran and hexachlorocyclohexane to blue-green alga Nostoc muscorum

The effects of various levels of nutrients like potassium phosphate (dibasic), calcium nitrate, and calcium chloride individually and in combinations were studied on the toxicity of the commonly used pesticides carbofuran and hexachlorocyclohexane (HCH) in growth medium to the N₂-fixing blue-green alga Nostoc muscorum. It was observed that all these chemicals had some effects on toxicity. The toxicity of both carbofuran and HCH was reduced to some extent in the presence of higher concentrations of the nutrients when compared to normal levels of the chemicals in the medium. The higher doses of nutrients in combinations antagonized the effect of individual treatment and enhanced the toxicity of pesticides.     

Alterations in the calcium homeostasis of skeletal muscle from postmyocardial infarcted rats

In chronic heart failure, skeletal muscles develop a weakness that is not associated to an impaired circulatory function but rather to alterations in the skeletal muscle fibers themselves. To understand these changes, the steps in excitation–contraction coupling of rats that underwent a left anterior coronary artery occlusion were studied. About 24 weeks after the myocardial infarction, neither the total amount nor the voltage dependence of intramembrane charge were altered. In contrast, calcium release from the sarcoplasmic reticulum was considerably suppressed, and its voltage dependence shifted toward more positive voltages. Elementary calcium-release events showed altered morphology as the relative proportion of embers increased. Calcium sparks were smaller in amplitude and had larger time-to-peak values. Isolated ryanodine receptors (RyR) displayed an unusual rectification with increased single-channel conductance at positive (cis vs trans) voltages. In addition, the bell-shaped calcium dependence of channel activity was broader, with a slight shift of activation to lower and a larger shift in inactivation to higher calcium concentrations. These data indicate that the number of channels that open during a calcium-release event is decreased and that RyR function is altered; thus, calcium-release is suppressed after a myocardial infarction. These observations give an explanation for the impaired skeletal muscle function in these animals.     

Use of batch assays to assess the toxicity of atrazine to some selected cyanobacteria. II. Effect of atrazine on heterocyst frequency,nitrogen and phosphorus metabolism of four heterocystous cyanobacteria

Atrazine added in different doses to the culture media, augmented with glucose, of Aulosira fertilissima, Tolypothrix tenuis, Anabaena oryzae and Nostoc muscorum enhanced heterocyst frequency and efficiency of nitrogen fixation. Variations in the amounts of fixed nitrogen between the four test organisms may be attributed to differences in the levels of the high energy substance ATP, and also to various effects on the permeability barrier of cells. Although atrazine is a metabolic inhibitor, it enhanced particularly the nitrogen and phosphorus metabolism, leading to more amino-N and protein-N accumulation, yielding active synthesis of organic phosphorus, total soluble and insoluble phosphorus contents.     

Regulatory role of calcium on histamine secretion

Calcium seems to have two opposing effects on histamine secretion from mast cells. A rise in the cytosol calcium concentration initiates the chain of reactions leading to histamine secretion. On the other hand, calcium appears to have a regulatory role, limiting the secretion. Removal of cell surface calcium enhances histamine secretion. The present work demonstrates an inhibitory effect of calcium in the medium, using low concentrations of compound 48/80 as the secretagogue. Histamine secretion in response to compound 48/80 primarily utilizes intracellular calcium. When low concentrations of compound 48/80 were used (usually 20–50 ng/ml), calcium (1 mM) inhibited the secretion, the inhibition being more pronounced as the pH was increased from 6.5 to 8.5. The higher pH conceivably promotes the binding of calcium to the phospholipids in the cell membrane. Calcium at this site seems to depress the efflux of calcium from the intracellular stores to the cytosol. The possibility that the removal of calcium from the cell surface causes increased sodium permeability was considered. However, the sodium channel blocker tetrodotoxin (10^–5 M) was equally ineffective in influencing histamine release in the presence and absence of calcium, indicating that a change of sodium permeability was not involved.Antigen-induced (anaphylactic) histamine secretion depends mainly on extracellular calcium, although some secretion occurs in a calcium-free medium. Addition of calcium alone to the medium caused only slight increase in the secretion, but when both phosphatidylserine and calcium were added histamine secretion was remarkably stimulated, apparently through the effect of phosphatidylserine on calcium transport across the plasma membrane.     

A comparative study of stimulation of erythropoiesis during renal anemia with the preparation of antibodies against erythropoietin in ultralow doses and Recormon

We compared the stimulatory effects of a preparation of antibodies against erythropoietin in ultralow doses (Poetam) and recombinant erythropoietin (Recormon) on erythropoiesis that was suppressed by carboplatin. Both drugs possessed high erythropoiesis-stimulating activity, which was manifested in an increase in the number of erythrokaryocytes and erythroid precursors in hemopoietic tissue and count of erythrokaryocytes and reticulocytes in the peripheral blood during postcytostatic recovery. Recormon produced a rapid and short-term stimulatory effect on the erythron. The effect of Poetam developed slowly, but persisted for a longer time. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 6, pp. 641–644, June, 2007     

Formation of fertile aerial mycelium within asporogenous streptomycetes as induced by fungal metabolites

Asporogenous streptomycetes, e. g. Streptomyces flavochromogenes and S. luteolutescens, become able to form fertile aerial mycelium when they are cultivated in close contact with fungi. This may be caused by the production and secretion of fungal metabolites which induce or stimulate the aerial mycelium formation. An attempt was made to characterize the stimulatory factor. Among the numerous fungi tested the most active ones were Cladosporium cladosporioides and Drechslera erythrospila.     

In vitro stimulation of penetration gland emptying by Trichobilharzia szidati and T. regenti (Schistosomatidae) cercariae. Quantitative collection and partial characterization of the products

Induction of penetration gland emptying by cercariae of the bird schistosomes Trichobilharzia szidati and T. regenti employing linoleic acid, linolenic acid, praziquantel and calcium ionophore A23187 showed that both postacetabular and circumacetabular cells released their content at chosen stimulant concentrations. The gland secretions consisted of soluble and insoluble parts. The former one adhering to the ground seemed to have different saccharide composition from the glands of Schistosoma mansoni. It bound labelled saccharides, thus exhibiting lectin-like activity. Protein profiles of the latter one were identical after stimulation by all four stimulants in T. szidati. The soluble secretions contained several proteolytic enzymes; 31 kDa and 33 kDa cysteine proteases were identified in E/S products of T. szidati and T. regenti, respectively. The circumacetabular glands contained a significant amount of calcium. Immunohistochemistry revealed that the origin of E/S products after in vitro stimulation is in both penetration glands and tegumental structures. No crossreactivity was observed between the bird schistosomes and a serum raised against S. mansoni elastase.The project was supported by the following grants: Grant Agency of the Czech Republic no. 524/04/P082 and 524/03/1263, Grant Agency of the Charles University no. 263/2004/B/Bio, the Wellcome Trust Collaborative Research Initiative Grant no. 072255 and the grant of the Ministry of Education of the Czech Republic no. 0021620828.     

Experimental extrinsic allergic alveolitis induced in the guinea-pig with Streptomyces olivaceus

Streptomyces olivaceus was isolated from the roof-thatch of New Guinea huts, and a soluble extract was prepared from it. Using sera from New Guinea natives as a source of precipitating antibody, the S. olivaceus antigen showed a reaction of partial identity with antigens in crude roof-thatch extracts. The inhalation of aerosols of S. olivaceus antigen by guinea-pigs, previously given parenteral doses of the same antigenic solution, induced extrinsic allergic alveolitis.     

A screening method for autoregulators of anthracycline-producing streptomycetes

A novel method was proposed for screening of autoregulators by anthracycline-producing strains of Streptomyces griseus. By means of a modified cosynthesis procedure, an indicator strain was detected capable of producing both leukaemomycin and aerial mycelium only in the presence yielding organisms and their mutants, but also by a streptomycin-producing strain of Streptomyces griseus as well as its non-differentiation derivative.     

Use of batch assays to assess the toxicity of atrazine to some selected cyanobacteria. I. Influence of atrazine on the growth,pigmentation and carbohydrate contents of Aulosira fertilissima,Anabaena oryzae,Nostoc muscorum and Tolypothrix tenuis

The data presented in this investigation revealed that the gain in the dry weights of all algae tested was suppressed in herbicide treatments, particularly, at higher concentrations. However, at the lowest dose of the herbicide, the growth inhibition was statistically significant for Anabaena and insignificant for the other organisms. The chlorophyll a content in Anabaena was slightly, however, insignificantly increased at lower concentrations; a phenomenon that was enhanced by increasing the herbicide dose. On the other hand, the pigment biosynthesis was suppressed in all treatments, in Tolypothrix and Aulosira, being more effective and statistically significant in the latter. The pigment accumulation in Nostoc was slightly and insignificantly promoted by all herbicide doses except at the highest concentration where a slight drop was noticed. The herbicide was most efficient at lower doses, in stimulating glucose absorption by either Nostoc and Anabaena up to 0.1 ppm. Applying higher doses, a remarkable drop was maintained. It induced significant retardation in the sugar uptake by Tolypothrix and Aulosira. This trend was confirmed by increasing the herbicide concentration. In all treatments with atrazine the total carbohydrate accumulation in all test organisms was suppressed and was more effective at the lowest concentration in Tolypothrix and Aulosira, however, of less significant effect in Anabaena and Nostoc. It may be concluded that the toxic effects of atrazine are partially due to its inhibitory effects on the synthesis of carbohydrates.     

Decolorisation of the polymeric dye Poly R by Streptomyces viridosporus T7A

The ability of a range of actinomycetes to decolorise the polymeric dye Poly R was investigated. Three well characterised lignocellulose-degrading actinomycetes, Streptomyces viridosporus, Streptomyces badius and Thermomonospora mesophila were found to be the most efficient at decolorising Poly R, with a maximum decolorisation rate of 0.10 unit per day. Extracellular fractions taken from S. viridosporus grown on a variety lignocellulose-related substrates were also capable of decolorising Poly R, indicating that dye decolorisation was not merely due to biomass uptake. The activity of extracellular fractions from straw-grown cultures of S. viridosporus was three to six times greater than those from other substrates examined. Purification of this dye-decolorising activity from S. viridosporus using anion exchange chromatography revealed the presence of a single active fraction. This was confirmed using gel permeation chromatography which estimated the molecular mass of the decolorising protein to be approximately 30000 Da.     

Effect of reduced chloride reabsorption on renin release in the isolated rat kidney

To investigate the relationship between tubular reabsorption of chloride and renal renin release in the isolated perfused rat kidney, perfusate renin activity was measured during substitution of either nitrate or thiocyanate for varying amounts of perfusate chloride but with maintained perfusate sodium concentration. Renin rose significantly as perfusate chloride fell; there was a sevenfold increase between perfusion with normal chloride and almost complete substitution of chloride by nitrate. With a normal perfusate chloride the addition of furosemide 10^–4 M to the perfusate also led to an increase in renin and a reduction in tubule chloride reabsorption. For all these experiments there was a significant negative correlation between renin and absolute tubular reabsorption of chloride (r=–0.68,P<0.001), but no such relationship with absolute sodium reabsorption. Renin release in a nonfiltering kidney, produced by elevating perfusate albumin concentration, increased approximately 40-fold. Thus increasing plasma oncotic pressure elevates renin by mechanisms additional to cessation of tubular chloride absorption. However, substitution of chloride in the perfusate by nitrate in this nonfiltering kidney did not further elevate renin release. We conclude that renin release is influenced by a signal dependent on, and inversely proportional to, chloride reabsorption in the thick ascending limb of the Loop of Henle.     

Non-specific effects of calcium entry antagonists in mast cells

Calcium entry in non-excitable cells occurs through calcium-selective currents activated secondarily to store depletion and/or through non-selective cation channels (e.g., receptor- or second-messenger-activated channels). The driving force for calcium influx can be modified by chloride or potassium channels, which set the membrane potential of cells. Together, these conductances determine the extent of calcium entry. Mast cells are an excellent model system for studying calcium influx, because calcium-release-activated calcium currents (I _CRAC), second-messenger-activated non-selective currents and chloride currents are present in these cells. Whole-cell patch-clamp recordings were used to test the Effects of the commonly used calcium entry blockers econazole and SK&F 96365, as well as the antiallergic and anti-inflammatory drugs tenidap, ketotifen and cromolyn on these channels. All tested drugs blocked the three different channel types with a similar order of magnitude (IC₅₀ values ranging from micromolar to millimolar). Hence, these drugs cannot be used to discriminate between different calcium entry mechanisms.     

Protease activity in extracellular products secreted in vitro by trophozoites of <Emphasis Type="Italic">Giardia duodenalis</Emphasis>

There are evidences that Giardia trophozoites contain and/or release proteolytic enzymes that may be implicated in pathogenesis of giardiasis. This report describes a preliminary characterization of the proteolytic activity in excretory/secretory (E/S) products of Giardia duodenalis trophozoites of an axenic Brazilian strain (BTU-11) and the reference strain Portland 1 (P1). The protease activity of E/S products in conditioned medium by trophozoites of each strain was analyzed using substrate (gelatin and collagen) impregnated SDS-PAGE and hemoglobin assay. The protease characterization was based on inhibition assays including synthetic inhibitors. Proteolytic products were detected in the conditioned medium by trophozoites of both assayed strains. In the gels containing copolymerized gelatin and collagen, E/S products promoted degradation of the substrates and the most evident proteolysis zones were distributed in the migration regions of 77 to 18 kDa and 145 to 18 kDa, respectively, in the patterns of gelatinolytic and collagenolytic activities. Degradation of hemoglobin was also observed, and the pattern of hydrolysis was similar in both E/S products assayed. Inhibition assays showed that the main proteolytic activity in both E/S products is due to cysteine proteases although the presence of serine proteases was also indicated, mainly in the hydrolysis of hemoglobin.     

Modified Alizarin Red S and Modified Silver Nitrate Methods for Calcium Demonstration

Abstract

A modified alizarin red S and a silver nitrate method for the demonstration of calcium are described. In the alizarin red S method, isopropyl alcohol is used to dehydrate the sections, thus minimizing the solubility of the calcium lake. The sensitive silver method, based on sensitization and physical development, demonstrates minute traces of calcium deposits mainly in the form of phosphates and is comparable to the alizarin red S. (J Histotechnol 12:225, 1989).     

New protease mutants in Aspergillus niger result in strongly reduced in vitro degradation of target proteins; genetical and biochemical characterization of seven complementation groups

Several mutants of Aspergillus niger, deficient in extracellular protease expression, have been isolated and characterized both genetically and biochemically. The mutant strains, obtained after in vivo UV-mutagenesis of conidiospores and selected by haloscreening on a new dual-substrate plate assay, belong to at least seven different complementation groups. These seven prt loci were assigned to linkage groups using master strains with marked chromosomes. One prt locus (prtC) could be assigned to linkage group I, three (prtB, prtE and prtG) to linkage group III, one (prtF) to linkage group V and the two remaining loci (prtA and prtD) to linkage group VIII. Extracellular proteolytic activities varied from 2 to 3% up to 80% of the protease activity of the parental strain. Assigning the different prt mutants to structural or regulatory genes is difficult since only one structural gene, pepA, has been mapped unambiguously on linkage group I but is not identical to prtC. All prt mutants except for prtC are likely to be regulatory mutants or else belong to a proteolytic cascade because residual activities showed that more proteolytic activities were affected simultaneously. Double mutants were constructed both by recombination and by a second round of mutagenesis. In both cases mutants with further reduced extracellular proteolytic activities were isolated. A sensitive in vitro degradation assay, based on the homologous pectin lyase B (PELB) protein to analyze proteolytic degradation in A. niger, was developed and used to show extremely reduced proteolytic PELB degradation in the culture media of some of these mutants.     

The effect of external anions on steady-state chloride exchange across ascites tumour cells

1. The rate of cell chloride exchange, or efflux coefficient, was measured after equilibration in media of different anionic composition.2. When sulphate substituted for chloride in the medium, the efflux coefficient was always higher than in control chloride solutions and varied inversely with external chloride concentration. In sulphate the chloride efflux coefficient varied from 19.6 to 100.7 hr(-1). The mean control efflux coefficient was 6.60 +/- 0.677 (S.E. of mean).3. In contrast, when external nitrate substituted for chloride, the efflux coefficient was independent of external chloride concentration and the same as in control chloride media. The mean value in nitrate was 6.42 +/- 0.603 (S.E. of mean). The results confirm findings of Hempling & Kromphardt (1965).4. Steady-state chloride flux varied in direct proportion to the external chloride concentration, which would be expected for passive chloride exchange. However, the slope of the line relating these variables was higher in sulphate than in nitrate and control media. Thus at any given external chloride concentration chloride flux was greater in sulphate than in nitrate and control solutions.5. It is suggested that the effect of sulphate to increase cell chloride exchange may be related to its greater tendency to bind water, relative to chloride and nitrate.     

Proteolytic activity of Aeromonas caviae

Aeromonas strains produce a variety of virulence factors including proteases. Studies on the kinetics of growth of Aeromonas caviae NRRL B-966 and its proteases suggest that the proteolytic activities are produced throughout the growth phase, with peak level occurring at stationary phase. A. caviae synthesize both intracellular and extracellular proteases with the latter account for major portion of the total activity. Optimum pH for the A. caviae proteolytic activity is at 7.0. A. caviae produces a thermoresistant protease, whose activity is dependent on Mg⁺⁺ and Ca⁺⁺ ions. Inhibition of proteolytic activity by phenyl methyl sulfonyl fluoride suggest the presence of a serine protease in A. caviae. Nitrogenous compounds enhance the proteolytic activity while carbohydrates tested in this study inhibit the activity.     

Immunomodulatory effects of clove (Syzygium aromaticum) constituents on macrophages: In vitro evaluations of aqueous and ethanolic components

Abstract

The present work sought to investigate potential suppressive effects on mouse macrophages by in vitro treatment with clove (Syzygium aromaticum) ethanolic extracted essential oil (containing eugenol) or its water-soluble extract. Using doses (ranging from 0.001–1000?µg/ml) of each material freshly prepared in the laboratory, cell survival and production of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12 by the treated cells (that in all cases also had received LPS stimulation) were measured. Results indicated that, except at doses ≥100?µg/ml, viability was unaffected in all groups. NO release by LPS-stimulated macrophages was generally significantly suppressed by either material; in contrast, low (i.e. 0.001–1?µg/ml) doses of either extract class appeared to enhance NO release by non-LPS (unstimulated)-treated macrophages. Among LPS-stimulated cells, TNFα release was also significantly affected by each extract; the ethanolic extract was suppressive at all doses tested, while the aqueous material was so up to 1?µg/ml and then became stimulatory. In contrast, nearly every dose of either extract appeared to stimulate IL-6 release from the LPS-treated cells. Effects on IL-12 production were overall inconsistent; in general, the ethanolic extract tended to be stimulatory of production by the LPS-treated cells. The data for the aqueous material showed no discernable pattern of effect. The results suggest that clove extracts do not have a distinct cytotoxic activity, but do impart potential anti- and pro-oxidant effects in cells, depending on their concentrations and on the activation state of the macrophages themselves at the time of exposure to the extracts. The impact of the extracts on macrophage cytokine release also displays a pattern of dose-relatedness.     

Regulation of nitrate uptake and nitrite efflux in the cyanobacterium Nostoc MAC

Nitrate uptake, nitrite efflux and their regulation have been studied in the cyanobacterium Nostoc MAC. Nitrate uptake as well as nitrite-assimilation-dependent nitrite efflux systems consisted of two distinct phases comprising an initial rapid phase followed by a slower one. Whereas, 3-(3,4-dichlorophenyl)-1-1 dimethyl urea (DCMU), an inhibitor of photosystem II inhibited both nitrate uptake and nitrite efflux significantly, exogenous supply of ATP, however, stimulated both the processes, suggesting that PS II-mediated energy generation plays vital role in regulating both nitrate uptake as well as nitrite efflux. The inhibition of both the processes by p-chloromercuribenzoate (pCMB)¹, N,N dicyclohexylcarbodiimide (DCCD) and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) indicated the involvement of -SH groups and ATP hydrolysis in the regulation of both the processes. Tungstate-treated cells having nonfunctional nitrate reductase although had a significant level of nitrate uptake, yet nitrite efflux by these cells was found to be negligible. These results suggest that (i) nitrate uptake and nitrite efflux processes in Nostoc MAC are energy-dependent; (ii) assimilation of nitrate via nitrate reductase is necessarily required for nitrite efflux to occur; (iii) nitrate uptake and nitrite efflux processes are regulated at different levels by different metabolic inhibitors; and (iv) cations of larger ionic radius facilitate the nitrate uptake and nitrite efflux processes.