Measure Of Cell Damage Research Articles

Priming with a Seaweed Extract Strongly Improves Drought Tolerance in Arabidopsis.

Drought represents a major threat to plants in natural ecosystems and agricultural settings. The biostimulant Super Fifty (SF), produced from the brown alga Ascophyllum nodosum, enables ecologically friendly stress mitigation. We investigated the physiological and whole-genome transcriptome responses of Arabidopsis thaliana to drought stress after a treatment with SF. SF strongly decreased drought-induced damage. Accumulation of reactive oxygen species (ROS), which typically stifle plant growth during drought, was reduced in SF-primed plants. Relative water content remained high in SF-treated plants, whilst ion leakage, a measure of cell damage, was reduced compared to controls. Plant growth requires a functional shoot apical meristem (SAM). Expression of a stress-responsive negative growth regulator, RESPONSIVE TO DESICCATION 26 (RD26), was repressed by SF treatment at the SAM, consistent with the model that SF priming maintains the function of the SAM during drought stress. Accordingly, expression of the cell cycle marker gene HISTONE H4 (HIS4) was maintained at the SAMs of SF-primed plants, revealing active cell cycle progression after SF priming during drought. In accordance with this, CYCP2;1, which promotes meristem cell division, was repressed by drought but enhanced by SF. SF also positively affected stomatal behavior to support the tolerance to drought stress. Collectively, our data show that SF priming mitigates multiple cellular processes that otherwise impair plant growth under drought stress, thereby providing a knowledge basis for future research on crops.

Tolerance of subzero winter cold in kudzu (Pueraria montana var. lobata).

The use of species distribution as a climate proxy for ecological forecasting is thought to be acceptable for invasive species. Kudzu (Pueraria montana var. lobata) is an important invasive whose northern distribution appears to be limited by winter survival; however, kudzu's cold tolerance thresholds are uncertain. Here, we used biogeographic evidence to hypothesize that exposure to -20°C is lethal for kudzu and thus determines its northern distribution limit. We evaluated this hypothesis using survival tests and electrolyte leakage to determine relative conductivity, a measure of cell damage, on 14 populations from eastern North America. Relative conductivity above 36% was lethal. Temperatures causing this damage averaged -19.6°C for northern and -14.4°C for southern populations, indicating kudzu acclimates to winter cold. To assess this, we measured relative conductivity of above- and belowground stems, and roots collected throughout the winter at a kudzu population in southern Ontario, Canada. Consistent with acclimation, the cold tolerance threshold of aboveground stems at the coldest time of year was -26°C, while stems insulated from cold extremes survived to -17°C-colder than the survival limits indicated by kudzu's biogeographic distribution. While these results do not rule out alternative cold limitations, they indicate kudzu can survive wintersnorth of its current distribution. For kudzu, biogeography is not a proxy for climatic tolerance and continued northward migration is possible. Efforts to limit its spread are therefore prudent. These results demonstrate that physiological constraints inform predictions of climate-related changes in species distribution and should be considered where possible.

Redox Profiling Reveals Clear Differences between Molecular Patterns of Wound Fluids from Acute and Chronic Wounds.

Wound healing is a complex multiphase process which can be hampered by many factors including impaired local circulation, hypoxia, infection, malnutrition, immunosuppression, and metabolic dysregulation in diabetes. Redox dysregulation is a common feature of many skin diseases demonstrated by virtually all cell types in the skin with overproduction of reactive oxygen and nitrogen species. The objective of this study was to characterize the redox environment in wound fluids and sera from patients suffering from chronic leg ulcers (n = 19) and acute wounds (bulla fluids from second degree burns; n = 11) with serum data also compared to those from healthy volunteers (n = 7). Significantly higher concentrations of TNF-α, interleukine-8, vascular endothelial growth factor, and lactate dehydrogenase (measure of cell damage) were found in fluids from chronic wounds compared to acute ones. The extent of protein carbonylation (measure of protein oxidation), lipid peroxidation, and tyrosine nitration (indicator of peroxynitrite production) was similar in acute and chronic wound fluids, while radical scavenging activity and glutathione (GSH) levels were elevated in chronic wound fluids compared to acute wounds. Sera were also assessed for the same set of parameters with no significant differences detected. Nitrotyrosine (the footprint of the potent oxidant peroxynitrite) and poly(ADP-ribose) (the product of the DNA damage sensor enzyme PARP-1) could be detected in wound biopsies. Our data identify multiple signs of redox stress in chronic wounds with notable differences. In chronic wounds, elevations in antioxidant levels/activities may indicate compensatory mechanisms against inflammation. The presence of nitrotyrosine and poly(ADP-ribose) in tissues from venous leg ulcers indicate peroxynitrite production and PARP activation in chronic wounds.

Effect of photobiomodulation on endothelial cell exposed to Bothrops jararaca venom.

Bleeding is a common feature in envenoming caused by Bothrops snake venom due to extensive damage to capillaries and venules, producing alterations in capillary endothelial cell morphology. It has been demonstrated, in vivo, that photobiomodulation (PBM) decreases hemorrhage after venom inoculation; however, the mechanism is unknown. Thus, the objective was to investigate the effects of PBM on a murine endothelial cell line (tEnd) exposed to Bothrops jararaca venom (BjV). Cells were exposed to BjV and irradiated once with either 660- or 780-nm wavelength laser light at energy densities of 4 and 5J/cm(2), respectively, and irradiation time of 10s. Cell integrity was analyzed by crystal violet and cell viability/mitochondrial metabolism by MTT assay. The release of lactic dehydrogenase (LDH) was quantified as a measure of cell damage. In addition, cytokine IL1-β levels were measured in the supernatant. PBM at 660 and 780nm wavelength was able to increase cellular viability and decrease the release of LDH and the loss of cellular integrity. In addition, the concentration of pro-inflammatory cytokine IL1-β was reduced after PBM by both wavelengths. The data reported herein indicates that irradiation with red or near-infrared laser resulted in protection on endothelial cells after exposure to Bothrops venom and could be, at least in part, a reasonable explanation by the beneficial effects of PBM inhibiting the local effects induced by Bothrops venoms, in vivo.

Photobiomodulation Protects and Promotes Differentiation of C2C12 Myoblast Cells Exposed to Snake Venom.

BackgroundSnakebites is a neglected disease and in Brazil is considered a serious health problem, with the majority of the snakebites caused by the genus Bothrops. Antivenom therapy and other first-aid treatments do not reverse local myonecrose which is the main sequel caused by the envenomation. Several studies have shown the effectiveness of low level laser (LLL) therapy in reducing local myonecrosis induced by Bothropic venoms, however the mechanism involved in this effect is unknown. In this in vitro study, we aimed to analyze the effect of LLL irradiation against cytotoxicity induced by Bothrops jararacussu venom on myoblast C2C12 cells.MethodologyC2C12 were utilized as a model target and were incubated with B. jararacussu venom (12.5 μg/mL) and immediately irradiated with LLL at wavelength of red 685 nm or infrared 830 nm with energy density of 2.0, 4.6 and 7.0 J/cm2. Effects of LLL on cellular responses of venom-induced cytotoxicity were examined, including cell viability, measurement of cell damage and intra and extracellular ATP levels, expression of myogenic regulatory factors, as well as cellular differentiation.ResultsIn non-irradiated cells, the venom caused a decrease in cell viability and a massive release of LDH and CK levels indicating myonecrosis. Infrared and red laser at all energy densities were able to considerably decrease venom-induced cytotoxicity. Laser irradiation induced myoblasts to differentiate into myotubes and this effect was accompanied by up regulation of MyoD and specially myogenin. Moreover, LLL was able to reduce the extracellular while increased the intracellular ATP content after venom exposure. In addition, no difference in the intensity of cytotoxicity was shown by non-irradiated and irradiated venom.ConclusionLLL irradiation caused a protective effect on C2C12 cells against the cytotoxicity caused by B. jararacussu venom and promotes differentiation of these cells by up regulation of myogenic factors. A modulatory effect of ATP synthesis may be suggested as a possible mechanism mediating cytoprotection observed under laser irradiation.

Exposure of Carcinus maenas to waterborne fluoranthene: Accumulation and multibiomarker responses

Fluoranthene (FLU) is a priority polycyclic aromatic hydrocarbon (PAH) commonly detected in estuarine sediments, water and biota. Despite this, information on FLU detection, accumulation and effects on marine crustaceans is scarce. This work investigated the accumulation of FLU in Carcinus maenas and the responses of several early-warning biomarkers after a 7-day laboratory exposure to five FLU concentrations (2.56 to 100μgL−1). After exposure to FLU, sub-samples of the crabs' digestive gland and muscle were collected for biomarker determinations. The remaining digestive gland and muscle, together with the rest of the whole-body soft tissues, were analysed for FLU residues by gas chromatography–mass spectrometry (GC–MS). The biomarkers assessed were: i) the quantification of FLU-type compounds by fixed wavelength fluorescence (FF); ii) the activities of glutathione S-transferases (GST) and glutathione reductase (GR), and the levels of total glutathione (GT) and lipid peroxidation (LPO) for oxidative stress; iii) the activity of acetylcholinesterase (AChE) for neurotoxicity; iv) the activities of isocitrate dehydrogenase (IDH) and lactate dehydrogenase (LDH) enzymes, and total protein, glycogen and lipids as indicators of changes in energy metabolism and storage; and v) the lysosomal membrane stability (LMS) as a measure of cell damage. The results showed strong (R2>0.95) concentration-dependent accumulation of FLU residues (as measured by GC–MS) in the remaining whole-body soft tissues and of FLU-type compounds (as measured by FF) in the digestive gland and muscle. A strong positive linear relationship (R2=0.91) between FLU residues and FLU-type compounds was also found. Comparing to controls, activities of GST and GR were significantly higher in crabs exposed to ≥16 and ≥40μgL−1 FLU, respectively. TG levels and IDH activity showed a significant trend to increase with FLU concentrations whereas AChE activity exhibited the opposite trend. FF measurements in the digestive gland and muscle proved to be an expeditious cost-effective method to assess the uptake and availability of FLU and its metabolites in C. maenas. The results suggest that under continuous environmental exposure, FLU may enhance detoxification and anti-oxidant defences, and cause alterations in the aerobic energy pathway, as well as neuromuscular toxic effects that may increase C. maenas risk of predation.

Desiccation Tolerance of Green Ash and Sugar Maple Fine Roots

Abstract Exposed fine roots are subject to desiccation, which may affect their survival as well as new root growth following bare root transplanting. Fine roots of dormant 1-year-old green ash (Fraxinus pennsylvanica) and sugar maple (Acer saccharum) seedlings, subjected to desiccation treatments of 0, 1, 2, or 3 hours in December and March, lost up to 82 percent of their water. Root electrolyte leakage, a measure of cell damage, tripled after three hours of desiccation. The increase was moderately, but significantly, greater in March for both species. Desiccation treatments had no effect on fine root survival. Growth of new roots (RGP) was also unaffected by desiccation treatments. RGP of maple was greater in March than December, but not ash.

Comparative study of the cytolytic activity of myotoxic phospholipases A 2 on mouse endothelial (tEnd) and skeletal muscle (C2C12) cells in vitro

A rapid in vitro cytolytic effect of some myotoxic phospholipases A 2 (PLA 2s) isolated from the venoms of Viperidae snakes has been previously described. This study was undertaken to investigate if cytolytic activity is a common property of the myotoxic proteins from this group. Murine endothelial cells (tEnd) and skeletal muscle myotubes (C2C12) were utilized as targets. The release of lactic dehydrogenase was quantified as a measure of cell damage, 3 h after exposure of cells to the different PLA 2s, including representatives from the genera Bothrops, Agkistrodon, Trimeresurus, Crotalus (family Viperidae), and Notechis (family Elapidae). All of the group II myotoxic PLA 2s tested displayed rapid cytolytic activity when tested in the micromolar range of concentrations (8–32 μM). In contrast, the group I myotoxic PLA 2 notexin was devoid of this activity. Aspartate-49 and lysine-49 PLA 2 group II variants showed a comparable cytolytic effect. Skeletal muscle myotubes, obtained after fusion and differentiation of C2C12 myoblasts, were significantly more susceptible to the cytolytic action of myotoxins than endothelial cells, previously reported to be more susceptible than undifferentiated myoblasts under the same assay conditions. Cytolytic activity appears to be a common characteristic of group II myotoxic PLA 2s of the Viperidae. Bee venom PLA 2, a group III enzyme of known myotoxicity, also displayed cytotoxic activity on C2C12 myotubes, being devoid of activity on endothelial cells. These results suggest that in vitro differentiated skeletal muscle myotubes may represent a suitable model target for the study of myotoxic PLA 2s of the structural group II found in snake venoms.

Effects of the homoepathic remedy Arnica D30 on marathon runners: a randomized, double-blind study during the 1995 Oslo Marathon

Objective: To examine whether the homoeopathic remedy Arnica D30 has an effect on cell damage and muscle soreness after long-distance running. Design: Randomized, double-blind placebo-controlled trial. Interventions: Five pills of Arnica D30 or placebo were given morning and evening. Treatment started the evening before the marathon, continued the running day and for three days following. Main outcome measures: The sense of muscular soreness was assessed by the runners on a visual analogue scale (VAS). Measurement of cell damage was assessed by serological enzyme tests. Results: Muscle soreness was significantly lower in the Arnica group than in the placebo group immediately after the marathon (p = 0.017). Cell damage measured by enzymes was essentially the same whether the runners were treated with Arnica D30 or placebo. Conclusions: In this study Arnica D30 had a positive effect on muscle soreness after marathon running, but not on cell damage as measured by enzymes.

A cell culture model of cerebral ischemia as a convenient system to screen for neuroprotective drugs.

Aggregation cultures of rat brain were exposed to a combination of anoxia and hypoglycaemia for 30 minutes. Thereafter, the release of lactate dehydrogenase into the cell culture medium was monitored up to 4 days as a measure of cell damage after the ischemic insult. Some cultures were treated with different concentrations of deprenyl or tolcapone, selective inhibitors of monoamine oxidase B and catechol-O-methyltransferase, respectively. After 1 day in culture, the release of lactate dehydrogenase was significantly reduced in cultures treated with deprenyl (at 1 nM. 100 nM, and 10 microM), as well as in cultures treated with 1 nM or 100 nM tolcapone; 10 microM of tolcapone, on the other hand, resulted in a toxic effect on the cell aggregates. No differences in the release of lactate dehydrogenase into the medium was observed in the aggregates treated with drugs as compared with the control cultures after 2 or 4 days post-ischemia.

Role of milk fractions, serum, and divalent cations in protection of mammary epithelial cells of cows against damage by Staphylococcus aureus toxins

Abstract Objective To determine the effect of milk and blood serum constituents on cytotoxicity of Staphylococcus aureus on mammary epithelial cells. Design In vitro incubation of cells with cytotoxic agents and milk and serum constituents. Sample Population Mammary cells, milk, and blood obtained from 3 cows. Procedure Staphylococcal α-toxin and culture supernatants from S aureus M60 and an α-toxin-negative mutant of M60 were incubated with bovine mammary epithelial cells in the presence of milk fractions, serum, and divalent cations. Propidium iodide fluorescence was used as a measure of cell damage. Results Skim milk and milk whey inhibited S aureus cytotoxic agents. Skim milk protected against α-toxin damage to a greater extent than milk whey. Serum from an adult animal was more protective than was fetal serum. Milk fat and serum albumin had no protective effect. Divalent calcium and Mg2+ were more effective inhibitors of mammary epithelial cell damage caused by α-toxin than of damage attributable to M60 culture supernatant. Divalent calcium and Mg2+ at concentrations similar to those of free Ca2+ and Mg2+ in normal bovine milk decreased cytotoxic damage attributable to α-toxin. However, concentrations similar to those of total Ca2+ and Mg2+ in normal milk were required to decrease cell damage caused by M60 culture supernatant. The α-toxin-negative mutant was less cytotoxic than the M60 parent strain. Conclusions Casein, as well as Ca2+ and Mg2+ in bovine milk, inhibit the cytotoxic effect of S aureus on mammary epithelial cells.(Am J Vet Res 1996;57:308-312)

Energetic determinants of stunning and cell damage following reoxygenation of rabbit myocardium

The energetic conditions in anoxia which lead to stunning and cell damage upon reoxygenation of the myocardium are studied in isolated papillary muscles of rabbit contracting isometrically at 20°C. Before anoxia and thereafter the muscles are stimulated at 0.2 Hz, while during anoxia stimulation frequency is varied. Total creatine ( Cr T) content is taken as a measure of cell damage. The degree of stunning is estimated from the difference in force before anoxia and at the end of the recovery period when no creatine is lost. In anoxic rabbit papillary muscles glycogen is the substrate for lactate formation. The ‘ATP reserve’ of the muscle can then be calculated from: 2 ATP+PCr+ADP+3 (glycosyl units). In control muscles it equals 482.2 μmol.g −1 dw of ATP. By varying stimulus frequency (0, 0.2, 0.5, or 1 Hz) during and the length of the anoxic period (20 to 80 min), the fraction of the ATP reserve used in anoxia is varied. It is found that the degree of stunning depends on the degree of ATP reserve depletion. When the ATP reserve becomes virtually zero force recovers to only 67% of the preanoxic value. When anoxia continues after the ATP reserve is virtually zero, creatine in the muscle decreases during reoxygenation, indicating cell damage. Apparently a hierarchy of cellular ATPases exists in the myocardium when energy is short preserving cell integrity as long as possible.

Increased elastase activity in nasal mucus associated with nasal colonization by Pasteurella haemolytica in infectious bovine rhinotracheitis virus-infected calves

Summary Four healthy calves were inoculated with Pasteurella haemolytica serotype 1 by instillation of a broth culture into the middle nasal meatus of the left nostril. Four weeks later, calves were exposed to infectious bovine rhinotracheitis virus by aerosol into both nostrils. All calves became ill, from approximately day 3 through day 10 after virus exposure, and shed increased amounts of nasal mucus. Two calves were induced to shed P haemolytica by the virus infection, and 2 calves required reinoculation with P haemolytica for nasal passages to become actively colonized. Elastase activity in nasal mucus increased about 15-fold within 3 days and peaked about 60-fold over baseline by 7 days after virus exposure. Activity of N-acetyl-β-d-glucosaminidase, a measure of cell damage and serum leakage, increased slightly by day 3 and reached plateau on day 5, almost threefold over baseline activity. Protein and carbohydrate content increased at a rate similar to that of N-acetyl-β-d-glucosaminidase activity with about 12-fold and sixfold increases, respectively. None of the variables returned to baseline by 19 days after virus exposure. Increased elastase activity preceded colonization by P haemolytica and decreasing elastase activity preceded decreasing P haemolytica concentration in the nasal secretions. A causal relation between elastase activity and P haemolytica colonization could be mediated by cleavage of epithelial cell surface fibronectin and exposure of receptors.