Indian medicinal plants as antiradicals and DNA cleavage protectors

Celastrus paniculatus L. (Celastraceae) (CP), Picrorhiza kurroa L. (Scrophulariaceae) (PK) and Withania somnifera L. (Solanaceae) (WS) are Indian medicinal plants having a remarkable reputation, as a factor of health care, among the indigenous medical practitioners. The plants exhibit varying degrees of therapeutic value some of which useful in the treatment of cognitive dysfunction, epilepsy, insomnia, rheumatism, gout, dyspepsia. In this work, we have investigated the free radical scavenging capacity of methanolic extracts from CP, PK, WS and the effect on DNA cleavage induced by H2O2 UV-photholysis. In addition, we investigated whether these plant extracts are capable of reducing the hydrogen peroxide-induced cytotoxicity and DNA damage in human non-immortalized fibroblasts. These extracts showed a dose-dependent free radical scavenging capacity and a protective effect on DNA cleavage; methanolic extracts from PK was more active than extracts from CP and WS. These results were confirmed by a significant protective effect on H2O2-induced cytoxicity and DNA damage in human non-immortalized fibroblasts. These antioxidant effects of active principle of CP, PK and WS may explain, at least in part, the reported anti-stress, immunomodulatory, cognition-facilitating, anti-inflammatory and antiaging effects produced by them in experimental animal and in clinical situations and may justify the further investigation of their other beneficial biological properties.     

Effect of the excretory-secretory products of some marine invertebrates on byssus production of the blue mussel <Emphasis Type="Italic">Mytilus edulis</Emphasis> (Bivalvia: Mytilidae)

Under laboratory conditions, we investigated byssus production in the blue mussel Mytilus edulis, as affected by the excretory-secretory products (ESPs) of the mussel itself and some marine invertebrates: the predatory starfish Asterias rubens, and organisms competing with mussels in White Sea fouling communities—a bivalve Hiatella arctica, the solitary ascidians Styela rustica and Molgula citrine, and a sponge Halichondria panicea. The number of attachment disks produced by a mussel per day and the thickness of byssal threads were estimated. Excretory-secretory products of H. arctica and M. citrine had no effect on the number of attachment disks, while ESPs of S. rustica, H. panacea and A. rubens stimulated mussels to produce attachment plaques. The activity of the mussel was slightly increased at low levels of its own ESPs in seawater. The thickness of byssal threads decreased with an increase in the ESPs of mussels in seawater, but it increased in experiments with the ESPs of any other species tested.     

Conversion of phenoloxidase and peroxidase indicators in individual haemocytes of Mytilus edulis specimens and isolation of phenoloxidase from haemocyte extract

Haemocytes oxidized 3-amino-9-ethylcarbazole and other peroxidase indicators such as 3,3-diaminobenzidine·4HCl, 3,3,5,5 tetramethylbenzidine·2HCl and 4-chloro-1-naphthol without addition of H₂O₂ indicating that the reaction was possibly not caused by a peroxidase. As these chromogens were also converted by a mushroom phenoloxidase in the absence of H₂O₂, cell smears were incubated with known substrates of phenoloxidases. One of these, l-dopa, caused strong melanin formation in several haemocytes and the reaction could be blocked by a variety of inhibitors including KCN, NaF, 1-phenyl-2-thiourea, cysteine, glutathione, ascorbic acid and HgCl₂. The enzymatic activity was isolated using a concanavalin A column and separated into two fractions with an ion-exchange cartridge. The molecular weights of the glycoproteins were estimated to be 381±13.7 kDa and 316±11.1 kDa. After isoelectric focusing of a haemocyte extract and the two ion-exchange peaks, seven enzyme bands were detected with isoelectric points between pH 5.0 and 5.5. The isolated enzyme fractions both converted 3,3,5,5-tetramethylbenzidine·2HCl best at pH 5–6 and l-dopa at pH 7.0 without addition of H₂O₂. Heat-treated cells lost their enzymatic activity; however, a group of haemocytes still bound preoxidized 3-amino-9-ethylcarbazole (= AECox). Also, some of the phenoloxidase inhibitors mentioned above blocked this non-enzymatic staining reaction. About 30–57% of haemocytes from individual mussels were AECox-positive, whereas Mytilus specimens without phenoloxidase-containing cells often occurred. Haemocytes containing this enzyme exhibited a high mobility and a large percentage of them belonged to a cytotoxic cell population.Abbreviations AEC 3-amino-9-ethylcarbazole - AECox preoxidized AEC - BSA bovine serum albumin - 4CN 4-chloro-1-naphthol - DAB 3,3-diaminobenzidine·4HCl - DMFA dimethylformamide - EDTA ethylenediaminetetra-acetic acid - LGT Iow gelling temperature - MW molecular weight - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulphonylfluoride - PTU 1-phenyl-2-thiourea - RBC red blood cells - TCA trichloroacetic acid - TMB 3,3,5,5-tetramethylbenzidine·2 HCl     

A comparative study on the influence of manganese on the bactericidal response of marine invertebrates

Manganese, Mn, is a naturally abundant metal in marine sediments. During hypoxic conditions the metal converts into a bioavailable state and can reach levels that have been shown immunotoxic to the crustacean Nephrops norvegicus. For this species it has previously been shown that exposure to 15 mg L⁻¹ of Mn decreased the number of circulating haemocytes while it for the echinoderm Asterias rubens increased the number of coelomocytes. Here, we compared if five days of exposure to the same concentration of Mn affects the bactericidal capacity of these two species and the mollusc Mytilus edulis when inoculated with the bacterium Vibrio parahaemolyticus. Viable counts of the bacteria were investigated at a time-course post-injection in the blood and the digestive glands of Mn-exposed and un-exposed (controls) animals. Accumulation of Mn was also analyzed in these tissues. When exposed to Mn the haemocyte numbers were significantly reduced in M. edulis and it was shown that the bactericidal capacity was impaired in the mussels as well as in N. norvegicus. This was most obvious in the digestive glands. These two species also showed the highest accumulation of the metal. In A. rubens the bactericidal capacity was not affected and the metal concentration was similar to the exposure concentration. After a recovery period of three days the concentration of Mn was significantly reduced in all three species. However, in M. edulis and N. norvegicus it was still double that of A. rubens which could explain the remaining bactericidal suppression observed in N. norvegicus. This study pointed out that exposure to such Mn-levels that are realistic to find in nature could have effects on the whole organism level, in terms of susceptibility to infections. The effect seemed associated to the accumulated concentration of Mn which differed on species level.     

Reattachment of certain species of mytilid bivalves to various substrates

Reattachment to various substrates (concrete, wood, and iron) was studied in three species of Mytilidae in the laboratory. Each of the investigated species exhibited substrate selectivity. The most successful substrate colonization occurred on concrete, which, owing to its physicochemical properties, proved to be more appropriate for rapid and firm anchorage of the mollusks. Mytilid response to any specific substrate, judged from the reattachment rate and substrate preference, may indirectly indicate the possibile existence of natural communities of organisms associated with a corresponding biotope.     

Cytochemical demonstration of latency of lysosomal hydrolases in digestive cells of the common mussel,Mytilus edulis,and changes induced by thermal stress

Latent beta-glucuronidase and glucosaminidase activities have been demonstrated in small cytoplasmic particles, which may possibly be primary lysosomes, as well as some larger granules of the digestive cells of the common mussel. Latency was indicated by increased staining of these structures following incubation in buffer at pH 4.5 at 37 degrees C. The exposure of mussels to temperatures of 25-28 degrees C over a period of four days induced a significant decrease in the latency of lysosomal glucosaminidase. Thermal death produced labilization of lysosomes although selective release of hydrolase activity was indicated by the differential latency of glucosaminidase and glucuronidase. The injection of hydrocortisone induced a significant increase in latency in stressed animals, indicating that the stress response involved changes in structure and function of membranes.     

Apical surface of the epithelial cells in the gallbladder of the rainbow trout and the tench

Summary The aluminium-formaldehyde (ALFA) histofluorescence method was used to study the innervation of the gill of the marine bivalve mollusc Mytilus edulis and the results were contrasted with those obtained with the standard formaldehyde-induced-fluorescence (FIF) method. The ALFA method produced more fluorescing structures than the FIF method, thus revealing fine branches of the branchial nerve running beneath the gill epithelium which previously remained undetected. This study demonstrates the usefulness of the ALFA histofluorescence method in the study of marine invertebrates.This study was supported in part by Grants 1506RR08171 from NIMH and 5T32GM07641 from the MARC Program of NIGMS. I wish to thank E. Aiello for thoughtful discussions of the work     

Sodiumd-glucose cotransport in the gill of marine mussels: Studies with intact tissue and brush-border membrane vesicles

Summary Glucose transport was studied in marine mussels of the genusMytilus. Initial observations, with intact animals and isolated gills, indicated that net uptake of glucose occurred in mussels by a carrier-mediated, Na⁺-sensitive process. Subsequent studies included use of brush-border membrane vesicles (BBMV) in order to characterize this transport in greater detail. The highest activity of Na⁺-dependent glucose transport was found in the brush-border membrane fractions used in this study, while basal-lateral membrane fractions contained the highest specific binding of ouabain. Glucose uptake into BBMV showed specificity for Na⁺, and concentrative glucose transport was observed in the presence of an inwardly directed Na⁺ gradient. There was a single saturable pathway for glucose uptake, with an apparentK _t of 3 m in BBMV and 9 m in intact gills. The kinetics of Na⁺ activation of glucose uptake were sigmoidal, with apparent Hill coefficients of 1.5 in BBMV and 1.2 in isolated gills, indicating that more than one Na⁺ may be involved in the transport of each glucose. Harmaline inhibited glucose transport in mussel BBMV with aK _i of 44 m. The uptake of glucose was electrogenic and stimulated by an inside-negative membrane potential. The substrate specificity in intact gills and BBMV resembled that of Na⁺-glucose cotransporters in other systems;d-glucose and -methyl glucopyranoside were the most effective inhibitors of Na⁺-glucose transport,d-galactose was intermediate in its inhibition, and there was little or no effect ofl-glucose,d-fructose, 2-deoxy-glucose, or 3-O-methyl glucose. Phlorizin was an effective inhibitor of Na⁺-glucose uptake, with an apparentK _i of 154nm in BBMV and 21nm in intact gills. While the qualitative characteristics of glucose transport in the mussel gill were similar to those in other epithelia, the quantitative characteristics of this process reflect adaptation to the seawater environment of this animal.     

The application of combined tissue residue chemistry and physiological measurements of mussels (Mytilus edulis) for the assessment of environmental pollution

The rationale for the use of combined tissue residue chemistry and physiological energetics measurements of Mytilus edulis in the assessment and monitoring of environmental pollution is outlined. Laboratory derived relationships between the concentration of toxicants in tissues and sublethal responses (eg. feeding, respiration and growth rate) provide a toxicological database for the interpretation of physiological responses measured in the field. The role of quantitative structure-activity relationships (QSAR's) in establishing tissue concentration-effect relationships for organic contaminants is discussed. The application of this approach is illustrated with reference to two field studies, a monitoring programme in the Shetlands and a practical biological effects workshop in Oslo.     

Protection against suspended sand: the function of the branchial membrane in the blue mussel Mytilus edulis

Blue mussels (Mytilus edulis) living in estuaries have to cope with varying concentrations of suspended sand. Sand flowing through the inhalant siphons comes into the infrabranchial chamber. The inhalant siphon can be partially closed by the branchial membrane. As a result the inward flow decreases, and suspended sand sinks and can be eliminated. Experiments with mussels from three ecologically different locations showed about the same response of the branchial membrane on contact with suspended sand. The presence and function of the branchial membrane appears to be an adaptation of mussels to their estuarine environment.     

Turnover rate of metallothionein and cadmium in Mytilus edulis

The results demonstrate the first attempt to determine metallothionein turnover in the whole soft tissues of mussels Mytilus edulis exposed to cadmium. Half-lives for metallothionein and cadmium are 25 and 300 days, respectively. As metallothionein degrades the released cadmium induces further synthesis of the protein, to which the metal becomes resequestered. The slow metallothionein turnover rates (compared with mammals) and the lack of significant cadmium excretion testify to the relatively stable nature of the cadmium-metallothionein complex in these invertebrates and supports the view of a detoxifying role for metallothionein in the mussels.     

Oxidative stress in Perna perna and other bivalves as indicators of environmental stress in the Brazilian marine environment: antioxidants, lipid peroxidation and DNA damage

Oxidative stress can take place in marine bivalves under a series of environmental adverse conditions. The study of different systems related to oxidative stress in these organisms can give important information about their physiological status and also about environmental health. Bivalves have been proposed as good sentinel organisms in pollution monitoring studies through the analysis of biochemical biomarkers, and most of the biomarkers analyzed are those related to oxidative stress. However, it is very important to know how other environmental factors not associated to the presence of pollutants might affect these parameters. We have studied a series of mechanisms related to oxidative stress in mussels which inhabit the Brazilian coast, especially in Perna perna species, subjected to different stress conditions, such as the exposure to different contaminants in the laboratory and in the field, the exposure of mussels to air and re-submersion, simulating the tidal oscillations, and in mussels collected at different seasons. Both oxidative damage levels and antioxidant defense systems were strongly affected by the different environmental stress. This review summarizes the data obtained in some studies carried out in bivalves from the Brazilian coast.     

Cytochemical and immunofluorescence investigations on insulin-like producing cells in the intestine of Mytilus edulis L. (Bivalvia)

Summary Insulin-like immunoreactivity can be localized to cells of the intestine in the-area of the hepatopancreas of Mytilus edulis L. No cross-reactivity can be obtained with anti-glucagon, anti-gastrin, anti-pentagastrin or anticaerulin. The cells containing the substance immunoreactive to mammalian anti-insulin, can be restained with paraldehyde-fuchsin.I thank Prof. A.G.E. Pearse for the opportunity to carry out this work in the Department of Histochemistry, Royal Postgraduate Medical School, London, England. The work was carried out with the aid of a grant from the Studienstiftung des Deutschen Volkes e.V., Germany (FRG).Dedicated to Prof. Dr. Drs. h.c. W. Bargmann at his 70th birthday.     

Prediction of heavy-metal concentrations in mature plants by chemical analysis of seedlings

Five plant species were cultivated on a soil from the Neckar alluvial fan near Heidelberg (FRG) polluted by the emissions of a cement plant. Thallium, cadmium and lead concentrations in seedlings and mature plants were determined by atomic absorption analysis. AdditionallyBrassica napus L.napus was grown on soils containing 5 different concentrations of heavy metals, achieved by mixing two similar soils, from the same area but with different metal concentrations. Thallium and cadmium were shown to be taken up by roots whilst lead which was also absorbed, was deposited mainly on the plant surface. However during cultivation in the winter months, a remarkable deposit of lead via the roots was found. Thallium in the soil from a anthrorogen source was more available to plants than thallium of geological origin. During the lifetime of a plant concentrations of thallium and cadmium were always highest in the seedling. The decrease in metal concentration with maturity depended on the plant species and the element, but was not a function of the metal concentration in the soil.     

Differential binding of lectins to haemocytes of the mussel Mytilus edulis

Summary Pre- and post-embedding techniques were used to investigate the ultrastructural binding of a range of lectins to the haemocytes of the mussel Mytilus edulis. Direct and indirect labelling procedures were employed using colloidal gold and ferritin-labelled lectins, or biotinylated lectins followed by gold-labelled streptavidin. Cell surface receptors were present for lectins from Helix pomatia (HPA), Helix aspersa (HAA), Triticum vulgaris (WGA) and Tetragonolobus purpureas (TPA). Double labelling of haemocytes with HPA and WGA demonstrated binding sites for both lectins on the plasma membrane of the majority of haemocytes. Endocytosis of colloidal gold-labelled HPA was observed for unfixed haemocytes. Three classes of haemocyte were identified by use of morphological criteria: hyalinocytes; granulocytes containing small granules; and granulocytes containing large granules. Lectin binding showed the small granules of the granulocytes to be HPA-positive and the large granules of the granulocytes to be WGA-positive. The WGA-positive granules demonstrated a differential pattern of binding according to granule size. Binding sites for the lectin from Arachis hypogaea (PNA) were not demonstrated on the cell surface, but did show an affinity for the heterochromatin region of the nucleus in post-embedding protocols.     

Reduction of cyanobacterial toxins through coprophagy in Mytilus edulis

An experiment was conducted to follow the fate of the cyanobacterial toxin, nodularin, produced by Nodularia spumigena through ingestion by Mytilus edulis and re-ingestion of faecal material (coprophagy). Mussels were fed with cultures of N. spumigena, and the faeces that were produced were fed to other mussels not previously exposed to N. spumigena. Concentrations of nodularin were measured in the food (N. spumigena), the mussels and in the faeces in order to make a toxin budget. High concentrations of nodularin were found in the mussels and their faeces after 48 h incubation with N. spumigena. When the toxic faeces were fed to new mussels, the toxin content of faeces was reduced from 95 μg nod g⁻¹ dry weight (DW) to 1 μg nod g⁻¹ DW through the process of coprophagy. Hence, when toxic faeces were fed to mussels, the nodularin concentration of the resulting faecal material was reduced by 99%. Pseudofaeces were produced when the mussels were grazing on N. spumigena, but not when grazing on faeces. The pseudofaeces contained high concentrations of nodularin and apparently intact N. spumigena cells. However, these cells were growth-inhibited and their potential contribution to seeding a bloom is probably limited. Our data indicate that a large fraction of ingested nodularin in M. edulis is egested with the faeces, and that the concentration of nodularin in the faeces is reduced when faeces are re-ingested.     

A new approach for the assessment of stochastic variation: analysis of behavioural response in blue mussel (Mytilus edulis L.)

One of the most effective techniques for evaluating stress is the analysis of developmental stability, measured by stochastic variation based particularly on fluctuating asymmetry, i.e. a variance in random deviations from perfect bilateral symmetry. However, the application of morphological methods is only possible when an organism lives under testing conditions during a significant part of its ontogenesis. Contrary to morphological characters, behavior can change very fast. Consequently, methods based on behavioural characters may have advantages over more traditional approaches. In this study we describe the technique of assessing stochastic variation, using not morphological, but behavioural characters. To measure stochastic variation of behavioural response, we assessed the stability of the isolation reaction of blue musselMytilus edulis at regular changes of salinity. With increasing temperature from +12°C to +20°C stochastic variation of the isolation reaction increased, which is a common response to change of environmental conditions. In this way, we have developed a method of assessing stochastic variation of behavioural response in molluscs. This method may find a great range of applications, because its usage does not require keeping animals in tested conditions for a long time.     

Effect of nutrient supply on growth of blue mussels (Mytilus edulis) in a landlocked bay

The growth of blue mussels (Mytilus edulis) was studied in the landlocked bay Hopavågen in central Norway for 3 years, of which in 2 years (1998 and 1999) nutrients were added to increase the primary production. Nutrients (N:Si:P) were added daily from May to October in 1998 (molar ratio 15:5:4.1) and 1999 (molar ratio 16:8:1). The doses of nutrients correspond to 0.4 and 0.8 g P l^–1 day^–1 in 1998 and 1999 respectively. The growth of blue mussels (Mytilus edulis) in 1997 was followed at four depths (1, 2, 4 and 7 m). In 1998 and 1999 growth was followed at 2 and 10 m depths at four locations in Hopavågen and at a control station outside on the coast. The nutrient supply in 1998 only slightly increased the algal biomass (chlorophyll a), whereas mean daily primary production during the summer remained at the same level as the previous year. The increased nutrient supply in 1999 caused a nearly 50 and 100% increase in mean summer biomass and daily primary production, respectively. The growth of blue mussels in Hopavågen in 1997 and 1998 was within the same size range during the summer. In 1999 the shell length of blue mussels kept at 2 m depth was significantly higher than in the previous year at end of the growth season. The recorded growth was also significant higher than for mussels at 2 m depth at the control station. No difference in shell length was observed on mussels grown at 10 m depth in Hopavågen and in the control stations in 1998 and 1999. A higher tissue content was found in blue mussels grown at 2 m depth in Hopavågen, both in 1998 and 1999 when compared to the control groups. At 10 m depth no differences were recorded.     

Density dependence,spatial scale and patterning in sessile biota

Sessile biota can compete with or facilitate each other, and the interaction of facilitation and competition at different spatial scales is key to developing spatial patchiness and patterning. We examined density and scale dependence in a patterned, soft sediment mussel bed. We followed mussel growth and density at two spatial scales separated by four orders of magnitude. In summer, competition was important at both scales. In winter, there was net facilitation at the small scale with no evidence of density dependence at the large scale. The mechanism for facilitation is probably density dependent protection from wave dislodgement. Intraspecific interactions in soft sediment mussel beds thus vary both temporally and spatially. Our data support the idea that pattern formation in ecological systems arises from competition at large scales and facilitation at smaller scales, so far only shown in vegetation systems. The data, and a simple, heuristic model, also suggest that facilitative interactions in sessile biota are mediated by physical stress, and that interactions change in strength and sign along a spatial or temporal gradient of physical stress.