Ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein 70

Hsp70 is often overexpressed in cancer cells, and the selective cellular survival advantage that it confers may contribute to the process of tumour formation. Thus, the pharmacological manipulation of Hsp70 levels in cancer cells may be an effective means of preventing the progression of tumours. We found that the downregulation of Hsp70 by ibuprofen in vitro enhances the antitumoural activity of cisplatin in lung cancer. Ibuprofen prominently suppressed the expression of Hsp70 in A549 cells derived from lung adenocarcinoma and sensitized them to cisplatin in association with an increase in the mitochondrial apoptotic cascade, whereas ibuprofen alone did not induce cell death. The cisplatin-dependent events occurring up- and downstream of mitochondrial disruption were accelerated by treatment with ibuprofen. The increase in cisplatin-induced apoptosis caused by the depletion of Hsp70 by RNA interference is evidence that the increased apoptosis by ibuprofen is mediated by its effect on Hsp70. Our observations indicate that the suppression of Hsp70 by ibuprofen mediates the sensitivity to cisplatin by enhancing apoptosis at several stages of the mitochondrial cascade. Ibuprofen, therefore, is a potential therapeutic agent that might allow lowering the doses of cisplatin and limiting the many challenge associated with its toxicity and development of drug resistance.     

Predation on invasive zebra mussel, Dreissena polymorpha, by pumpkinseed sunfish, rusty crayfish, and round goby

The enemy release hypothesis states that invasive species are successful in their new environment because native species are not adapted to utilize the invasive. If true for predators, native predators should have lower feeding rates on the invasive species than a predator from the native range of the invasive species. We tested this hypothesis for zebra mussel (Dreissena polymorpha) by comparing handling time and predation rate on zebra mussels in the laboratory by two North American species (pumpkinseed, Lepomis gibbosus, and rusty crayfish, Orconectes rusticus) and one predator with a long evolutionary history with zebra mussels (round goby, Neogobius melanostomus). Handling time per mussel (7 mm shell length) ranged from 25 to >70 s for the three predator species. Feeding rates on attached zebra mussels were higher for round goby than the two native predators. Medium and large gobies consumed 50–67 zebra mussels attached to stones in 24 h, whereas pumpkinseed and rusty crayfish consumed <11. This supports the hypothesis that the rapid spread of zebra mussels in North America was facilitated by low predation rates from the existing native predators. At these predation rates and realistic goby abundance estimates, round goby could affect zebra mussel abundance in some lakes.     

The effect of environmental salinity on the level of heat shock proteins in gill epithelium of Mytilus edilis L. mussel

The composition and the level of heat shock proteins in the gill epithelium cells of mussels Mytilus edulis L. from the White Sea under different levels of environmental salinity were studied by the method of immunoblotting. In mussels maintained under normal salinity (26%), constitutive Hsp70 and protein of about 40 kDa were revealed. After long-term (11?C14 days) acclimation to 14 and 35?? of the level Hsp70 in gill epithelium cells increased. Hsp70 induction was also observed in cells of isolated gills after salinity shock at 14% for 3 and 24 h.     

Space as a limiting resource in freshwater systems: competition between zebra mussels (Dreissena polymorpha) and freshwater sponges (Porifera)

Species interactions between two types of sessile benthic invertebrates, the zebra mussel (Dreissena polymorpha) and freshwater sponges (Porifera), were evaluated in Michigan City IN Harbor in southern Lake Michigan during 1996. The study objective was to define whether competition plays a role in structuring benthic communities using experimental techniques commonly employed in marine systems. Sponges were uninhibited by zebra mussel presence and overgrew zebra mussel shells on hard vertical substrata. In contrast, zebra mussels did not overgrow sponge colonies, but did show an ability to re-capture hard substrata if relinquished by the sponge. The negative affect of sponges on zebra mussels through overgrowth and recruitment suggests interactions that could eventually displace zebra mussels from these benthic communities. However, seasonal reduction of sponge biomass from autumn through winter appears to allow the zebra mussel a periodic respite from overgrowth, preventing exclusion of zebra mussels from the community and allowing these two taxa to co-exist.     

Induction of hsp70, alterations in oxidative stress markers and apoptosis against dichlorvos exposure in transgenic Drosophila melanogaster: Modulation by reactive oxygen species

We examined a hypothesis that reactive oxygen species (ROS) generated by organophosphate compound dichlorvos modulates Hsp70 expression and anti-oxidant defense enzymes and acts as a signaling molecule for apoptosis in the exposed organism. Dichlorvos (0.015–15.0 ppb) without or with inhibitors of Hsp70, superoxide dismutase (SOD) and catalase (CAT) were fed to the third instar larvae of Drosophila melanogaster transgenic for hsp70 (hsp70-lacZ) Bg⁹ to examine Hsp70 expression, oxidative stress and apoptotic markers. A concentration- and time-dependent significant increase in ROS generation accompanied by a significant upregulation of Hsp70 preceded changes in antioxidant defense enzyme activities and contents of glutathione, malondialdehyde and protein carbonyl in the treated organisms. An inhibitory effect on SOD and CAT activities significantly upregulated ROS generation and Hsp70 expression in the exposed organism while inhibition of Hsp70 significantly affected oxidative stress markers induced by the test chemical. A comparison made among ROS generation, Hsp70 expression and apoptotic markers showed that ROS generation is positively correlated with Hsp70 expression and apoptotic cell death end points indicating involvement of ROS in the overall adversity caused by the test chemical to the organism. The study suggests that (a) Hsp70 and anti-oxidant enzymes work together for cellular defense against xenobiotic hazard in D. melanogaster and (b) free radicals may modulate Hsp70 expression and apoptosis in the exposed organism.     

CD95-mediated alteration in Hsp70 levels is dependent on protein stabilization

Engagement of death receptors induces caspase activation and apoptosis. A recent study reported altered protein expression, including increased Hsp70 levels during CD95-mediated apoptosis. Here, we examined the mechanism underlying increased Hsp70 levels in cells challenged with a monoclonal antibody directed against the CD95 receptor. Levels of Hsp70 were found to increase in a dose-dependent manner, occurring independently of either heat shock factor 1 activation or the accumulation of Hsp70 messenger ribonucleic acid (mRNA), suggesting the involvement of posttranslational modifications. Inhibition of translation and de novo protein synthesis by cycloheximide resulted in Hsp70 protein levels diminishing over time in control cells, whereas its level remained constant during CD95 signaling. In addition, death receptor activation through exposure of cells to tumor necrosis factor-related apoptosis-inducing ligand did not alter Hsp70 levels. These findings demonstrate that receptor-specific signaling through the CD95 increases the stability of Hsp70 protein, rather than mRNA, when compared with control cells. The results describe a novel mechanism of heat shock protein accumulation, where increased protein stability and reduced turnover, is the mechanism by which Hsp70 accumulates in cells during CD95-mediated apoptosis.     

Hsp70 Regulates Immune Response in Experimental Autoimmune Encephalomyelitis

Heat shock protein (Hsp)70 is one of the most important stress-inducible proteins. Intracellular Hsp70 not only mediates chaperone-cytoprotective functions but can also block multiple steps in the apoptosis pathway. In addition, Hsp70 is actively released into the extracellular milieu, thereby promoting innate and adaptive immune responses. Thus, Hsp70 may be a critical molecule in multiple sclerosis (MS) pathogenesis and a potential target in this disease due to its immunological and cytoprotective functions. To investigate the role of Hsp70 in MS pathogenesis, we examined its immune and cytoprotective roles using both in vitro and in vivo experimental procedures. We found that Hsp70.1-deficient mice were more resistant to developing experimental autoimmune encephalomyelitis (EAE) compared with their wild-type (WT) littermates, suggesting that Hsp70.1 plays a critical role in promoting an effective myelin oligodendrocyte glycoprotein (MOG)-specific T cell response. Conversely, Hsp70.1-deficient mice that developed EAE showed an increased level of autoreactive T cells to achieve the same production of cytokines compared with the WT mice. Although a neuroprotective role of HSP70 has been suggested, Hsp70.1-deficient mice that developed EAE did not exhibit increased demyelination compared with the control mice. Accordingly, Hsp70 deficiency did not influence the vulnerability to apoptosis of oligodendrocyte precursor cells (OPCs) in culture. Thus, the immunological role of Hsp70 may be relevant in EAE, and specific therapies down-regulating Hsp70 expression may be a promising approach to reduce the early autoimmune response in MS patients.     

Seasonal effects of zebra mussels on littoral nitrogen transformation rates in Gull Lake, Michigan, U.S.A.

• 1 Zebra mussels (Dreissena polymorpha) are successful colonisers of lake littoral habitats and they interact strongly with littoral benthos. Previous research suggests that localised areas colonised by zebra mussels may be hotspots of nitrogen (N) cycling.
• 2 The effects of zebra mussels on nitrification and denitrification rates were examined approximately every other month for 1 year in Gull Lake, Michigan, U.S.A. Littoral sediment was collected from an area free of zebra mussels and distributed into shallow trays; rocks colonised with zebra mussels were placed in half of the trays, while uncolonised rocks were placed in the remaining trays. After an incubation period of 6–8 weeks in the lake, sediment and zebra mussels were collected from the trays, replaced with new sediment and zebra mussels, and placed in the lake for the next interval. In the laboratory, sediment nitrification and denitrification rates were measured for each tray.
• 3 Sediment nitrification rates did not increase in the presence of zebra mussels; instead nitrification rates were sensitive to changes in water temperature and increased with increasing exchangeable sediment ammonium. In contrast, denitrification rates increased in sediment trays with zebra mussels in the winter when nitrate (NO₃⁻) availability was high and when Chara did not grow in the trays.
• 4 Sediment denitrification was NO₃⁻‐limited in all seasons, regardless of zebra mussel treatment. However, sediment in the presence of zebra mussels responded less to NO₃⁻ addition, suggesting that NO₃⁻ limitation of denitrification can be reduced by zebra mussel activity. Zebra mussels have a seasonally variable impact on sediment denitrification rates, and this may translate into altered seasonal patterns of N cycling in localised areas of lakes where they are particularly abundant.

     

Non-Lethal Heat Shock of the Asian Green Mussel,Perna viridis,Promotes Hsp70 Synthesis,Induces Thermotolerance and Protects Against Vibrio Infection

Mild heat stress promotes thermotolerance and protection against several different stresses in aquatic animals, consequences correlated with the accumulation of heat shock protein 70 (Hsp70). The purpose of this study was to determine if non-lethal heat shock (NLHS) of the Asian green mussel, Perna viridis, an aquatic species of commercial value, promoted the production of Hsp70 and enhanced its resistance to stresses. Initially, the LT₅₀ and LHT for P. viridis were determined to be 42°C and 44°C, respectively, with no heat shock induced death of mussels at 40°C or less. Immunoprobing of western blots revealed augmentation of constitutive (PvHsp70-1) and inducible (PvHsp70-2) Hsp70 in tissue from adductor muscle, foot, gill and mantel of P. viridis exposed to 38°C for 30 min followed by 6 h recovery, NLHS conditions for this organism. Characterization by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that PvHsp70-1 and PvHsp70-2 respectively corresponded most closely to Hsp70 from P. viridis and Mytilus galloprovincialis. Priming of adult mussels with NLHS promoted thermotolerance and increased resistance to V. alginolyticus. The induction of Hsp70 in parallel with enhanced thermotolerance and improved protection against V. alginolyticus, suggests Hsp70 functions in P. viridis as a molecular chaperone and as a stimulator of the immune system.     

Factors affecting trematode infection rates in freshwater mussels

Mussels are intermediate hosts of digenean trematodes, but determinants of these infections remain unknown. To address this problem, we collected duck mussels Anodonta anatina in eighteen lakes from northeastern Poland and examined how mussel age, sex, and the encrustation with zebra mussels Dreissena polymorpha and environmental conditions in lakes influenced infection rates. We also assessed parasitic preferences to host gonads and hepatopancreas and the impact of parasites on female fertility. Mussels were infected with Rhipidocotyle campanula and Phyllodistomum sp. Infection rates were higher in older and female mussels but were unrelated to the biomass of encrusting D. polymorpha and the trophy, thermal conditions, and Ca²⁺ availability. Parasites occupied gonads more often than hepatopancreas. Infected females were less likely to carry glochidia and incubated fewer glochidia. We suggest that the risk of infection by digenean trematodes increases with the amount of water processed by filter-feeding hosts and/or that parasites actively seek hosts which can provide them with abundant resources. This mechanism explains why parasites more often occupied older and female mussels and targeted their gonads. Future research on trematode-mussel interactions should integrate knowledge on different elements of the complex trematode life cycles, including effects of higher-order hosts such as fish.     

Significant fish predation on zebra mussels Dreissena polymorpha in Lake Champlain,U.S.A.

The objectives of this study were to: (1) determine which fishes were consistently eating zebra mussels Dreissena polymorpha in Lake Champlain and document their feeding behaviour and (2) quantify the diet composition of the fish predators that were found to consume zebra mussels. From 2002 to 2005, freshwater drum Aplodinotus grunniens, pumpkinseed Lepomis gibbosus, yellow perch Perca flavescens and rock bass Ambloplites rupestris all consumed zebra mussels at varying frequencies and amounts. Aplodinotusgrunniens and L. gibbosus chewed clumps of zebra mussels, expelling shells, whereas P. flavescens and A. rupestris swallowed small individuals whole. Lepomis gibbosus consumed zebra mussels at the highest frequency (65–89% of prey consumed) and zebra mussels comprised a large part of this fish’s diet (up to 40% by dry mass). Zebra mussels were also an important component of the diet of A. grunniens (up to 59% of the diet by dry mass, 40–63% frequency of consumption). The percentage of the diet comprising zebra mussels in P. flavescens and A. rupestris varied significantly from year to year but never exceeded 10%. Because A. grunniens and L. gibbosus crushed zebra mussels, the nutritional return from consuming zebra mussels would be similar to other prey; for P. flavescens and A. rupestris zebra mussels were only partially digested and the nutritional return would probably be low. As predation on zebra mussels is widespread and significant, it is possible that fish predators could contribute to regulating the population of zebra mussels in Lake Champlain.     

Reciprocal Effects between Burying Behavior of a Larval Dragonfly (Odonata: Macromia illinoiensis) and Zebra Mussel Colonization

Invasive zebra mussels (Dreissena polymorpha) often colonize dragonfly larvae, especially spawling species whose survivorship to emergence as terrestrial predators is consequently reduced. Using individuals of the sprawler, Macromia illinoiensis, as their own controls, we compared the burying behavior of penultimate instar larvae before (i.e. baseline) and after their colonization by zebra mussels under ambient conditions. Individuals that took longer to bury themselves when mussel-free had a higher rate of colonization by mussels over a five-day period compared to those that buried faster. In contrast, the depth at which individuals buried when mussel-free was not predictive of subsequent colonization rate. Although mean bury time did not differ between baseline and when an individual carried one or more mussels, colonized larvae buried more shallowly than when mussel-free. Moreover, attached mussels increased the risk of subsequent colonization by zebra mussels. After naturally losing all of their attached mussels, bury time and depth of individuals did not differ from their baseline behavior, indicating that the changes in the behavior of colonized individuals were due to mussel loads and not their time in captivity. Under natural conditions, the positive feed-back between mussel attachment and increasing vulnerability to colonization helps explain how mussel loads, which are lost at molting, can accumulate quickly over the duration of the final larval stadium. Because zebra mussel attachment decreases the crypsis that that a M. illinoiensis gains from burying, the invasive mussel may also make dragonfly larvae more detectable to visual predators.     

Activation of spawning in zebra mussels by algae-, cryptomonad-, and gamete-associated factors

In zebra mussels (Dreissena polymorpha) simultaneous release of gametes and peaks in larval densities at particular locations suggest that spawning is triggered by synchronizing stimuli. Furthermore, spawning tends to occur only after an adequate environmental temperature is reached. To test the hypothesis that phytoplankton and gamete-associated chemicals initiate spawning in zebra mussels and that the responsiveness to such chemicals is affected by ambient temperature, the spawning response of zebra mussels to extracts from algae, a cryptomonad, and a cyanobacterium and to water associated with released gametes was assayed in animals acclimated to 12 ^C and 17 ^C. For animals held at 12 ^C, only serotonin, a known activator of bivalve spawning used as a positive control, stimulated spawning. However, for animals acclimated to 17 ^C, extracts made from a diatom (Phaeodactylum), a brown alga (Fucus), and a cryptomonad (Rhodomonas) stimulated spawning in both sexes; extracts from green algae (Platymonas and Dunaliella) and a cyanobacterium (Oscillatoria) did not cause spawning. Water associated with either released sperm or eggs elicited spawning in both females and males. Positive controls, stimulated with serotonin, spawned at a high (>90%) rate, whereas no negative control spawned. Thus, phytoplankton chemicals and gamete-associated factors may have a role in synchronizing spawning in zebra mussels once adequate ambient temperature is reached.     

Phosphorus recycling by zebra mussels in relation to density and food resource availability

Using flow-through microcosms, we examined phosphorus (P) recycling by zebra mussels under conditions of nearly constant food resource supply and varying zebra mussel population densities (600–5200 ind./m²). At all density levels, zebra mussels filtered substantial algae, measured as chlorophyll biomass. Because chlorophyll biomass inputs were low throughout the study, zebra mussel biomass-specific rates of chlorophyll filtration declined with increasing density, suggesting food resource limitation at the higher densities. We observed net total P export and high zebra mussel biomass-specific rates of P recycling over time in microcosms at high zebra mussel densities. In systems with a low zebra mussel density, net total P export did not occur over time. Our results suggest the occurrence of P remineralization by zebra mussels and net loss associated with emaciation during periods of temporary starvation. These findings have implications for P dynamics since zebra mussels can be subjected to periods of starvation over seasonal and annual time scales.     

Effects of zebra mussels on cladoceran communities under eutrophic conditions

The purpose of this study was to determine how zebra mussels affected cladoceran community structure under eutrophic conditions. We conducted a mesocosm study where we manipulated the presence of zebra mussels and the presence of large-bodied Daphnia (Daphnia magna and Daphnia pulicaria). We also conducted a complimentary life-table experiment to determine how water from the zebra mussel treatment affected the life history characteristics of the cladoceran species. We anticipated that small- and large-bodied cladoceran species would respond differently to changes in algal quality and quantity under the effects of zebra mussels. Large-bodied Daphnia successfully established in the zebra mussel treatment but failed to grow in the control. We did not observe positive relationships between food concentrations and cladoceran abundances. However, the phosphorus content in the seston indicated that food quality was below the threshold level for large-bodied cladocerans at the beginning of the experiment. We believe that zebra mussels quickly enhanced the phosphorus content in the seston due to the excretion of inorganic phosphorus, thus facilitating the development of large-bodied Daphnia. In conclusion, our results suggest that zebra mussels can alter the phosphorus content of seston in lakes and this can affect the dynamics of crustacean zooplankton.     

Elevated female fecundity as a possible compensatory mechanism in response to trematode infestation in populations of Littorina saxatilis (Olivi)

Co-evolution between parasites and their hosts may lead to changes in the life-history traits of the host that promote sustainability of their populations despite parasite pressure. Such changes are expected to be especially pronounced in the host-parasite systems where parasites cause complete castration of their hosts. We have studied populations of the rough periwinkle, Littorina saxatilis, infested by castrating trematode species, in order to determine whether high infestation levels are associated with a compensatory increase in host fecundity. To test this hypothesis, we determined female fecundity in populations with trematode prevalence spanning from <1% to 30-75%, and followed long-term changes in female fecundity and trematode infestation in two heavily infested populations of L. saxatilis. The broad-scale geographic analysis of populations with different trematode burdens showed that fecundity of uninfected females is significantly higher in highly infested L. saxatilis populations than in those with low trematode burdens. This is also supported by a comparison of fecundity in two pairs of geographically adjacent populations with contrasting trematode levels, revealing higher fecundity of uninfected females in heavily infested populations. Higher fecundity could be explained by the larger size of uninfected females in some heavily infested populations but not in others. Long-term (15-20 years) intra-population analysis performed in two heavily infested L. saxatilis populations showed that female fecundity increased in parallel with a long-term increase in trematode prevalence from 20% to >75% in one population, but remained high and relatively stable in the second population, reflecting its consistently high trematode prevalence (40-65%). These data support the hypothesis that an increase in female fecundity may be a population compensation mechanism in response to heavy trematode infestation in L. saxatilis and suggest the possible involvement of both natural selection and fast (physiological) regulation mechanisms.     

Altered trophic pathway and parasitism in a native predator (Lepomis gibbosus) feeding on introduced prey (Dreissena polymorpha)

Populations of invasive species tend to have fewer parasites in their introduced ranges than in their native ranges and are also thought to have fewer parasites than native prey. This ‘release’ from parasites has unstudied implications for native predators feeding on exotic prey. In particular, shifts from native to exotic prey should reduce levels of trophically transmitted parasites. We tested this hypothesis in native populations of pumpkinseed sunfish (Lepomis gibbosus) in Lake Opinicon, where fish stomach contents were studied intensively in the 1970s, prior to the appearance of exotic zebra mussels (Dreissena polymorpha) in the mid-1990s. Zebra mussels were common in stomachs of present-day pumpkinseeds, and stable isotopes of carbon and nitrogen confirmed their importance in long-term diets. Because historical parasite data were not available in Lake Opinicon, we also surveyed stomach contents and parasites in pumpkinseed in both Lake Opinicon and an ecologically similar, neighboring lake where zebra mussels were absent. Stomach contents of pumpkinseed in the companion lake did not differ from those of pre-invasion fish from Lake Opinicon. The companion lake, therefore, served as a surrogate “pre-invasion” reference to assess effects of zebra mussel consumption on parasites in pumpkinseed. Trophically transmitted parasites were less species-rich and abundant in Lake Opinicon, where fish fed on zebra mussels, although factors other than zebra mussel consumption may contribute to these differences. Predation on zebra mussels has clearly contributed to a novel trophic coupling between littoral and pelagic food webs in Lake Opinicon.     

Growth patterns of the pan‐European freshwater mussel,Anodonta anatina (Linnaeus, 1758) (Bivalvia: Unionidae), vary with sex and mortality in populations

1. Post‐maturation growth leading to indeterminate growth patterns is widespread in nature. However, its adaptive value is unclear. Life history theory suggests this allocation strategy may be favored by temporal pulses in the intensity of mortality and/or the capacity to produce new tissues.
2. Addressing the origin of indeterminate growth and the variability of growth patterns, we studied the growth of duck mussels, Anodonta anatina, a pan‐European unionid, in 18 Polish lakes. For each population, the sex, size, and age of collected mussels were measured to estimate Bertalanffy''s growth curve parameters. We integrated information on A. anatina mortality rates, lake trophy, biofouling by zebra mussels, Dreissena polymorpha, and the prevalence of parasitic trematode larvae to identify selective conditions in lakes.
3. We found two sources of mortality in A. anatina populations, pertaining to adverse effects of zebra mussel biofouling and trophy state on mussel survival. Additionally, populations with heavier biofouling presented a smaller abundance of parasites, indicative of a relationship between filtering intensity and contraction of water‐borne trematode larvae by filtering A. anatina.
4. Consistently for each sex, populations with a greater trophy‐related mortality were characterized in A. anatina by a smaller asymptotic size L_max, indicative of a life history response to mortality risk involving early maturation at a smaller body size. In all populations, females featured higher mortality and larger asymptotic size versus males.
5. Our findings support a theoretical view that adaptive responses to selection involve adjustments in the lifetime resource allocation patterns. These adjustments should be considered drivers of the origin of indeterminate growth strategy in species taking parental care by offspring brooding in body cavities.