Effects of high temperature and LPS injections on the hemocytes of the crab Neohelice granulata.

Temperature fluctuations, particularly elevated temperatures, can significantly affect immune responses. These fluctuations can influence the immune system and alter its response to infection signals, such as lipopolysaccharide (LPS). Therefore, this study was designed to investigate how high temperatures and LPS injections collectively influence the immune system of the crab Neohelice granulata. Two groups were exposed to 20 °C (control) or 33 °C for four days. Subsequently, half were injected with 10 µL of physiological crustacean (PS), while the rest received 10 µL of LPS [0.1 mg.kg-1]. After 30 min, the hemolymph samples were collected. Hemocytes were then isolated and assessed for various parameters using flow cytometry, including cell integrity, DNA fragmentation, total hemocyte count (THC), differential hemocyte count (DHC), reactive oxygen species (ROS) level, lipid peroxidation (LPO), and phagocytosis. Results showed lower cell viability at 20 °C, with more DNA damage in the same LPS-injected animals. There was no significant difference in THC, but DHC indicated a decrease in hyaline cells (HC) at 20 °C following LPS administration. In granular cells (GC), an increase was observed after both PS and LPS were injected at the same temperature. In semi-granular cells (SGC), there was a decrease at 20 °C with the injection of LPS, while at a temperature of 33 °C, the SGC there was a decrease only in SGC injected with LPS. Crabs injected with PS and LPS at 20 °C exhibited higher levels of ROS in GC and SGC, while at 33 °C, the increase was observed only in GC and SGC cells injected with LPS. A significant increase in LPO was observed only in SGC cells injected with PS and LPS at 20 °C and 33 °C. Phagocytosis decreased in animals at 20 °C with both injections and exposed to 33 °C only in those injected with LPS. These results suggest that elevated temperatures induce changes in immune system parameters and attenuate the immune responses triggered by LPS.

Can hypoosmotic shock and calcium influx lead to translocation of aquaporin-1 in shrimp muscle cells?

The physiological variations during the crustacean molting cycle have intrigued researchers for many years. Maintaining osmotic homeostasis in the face of hemolymph dilution and dealing with dynamic intracellular and extracellular calcium fluctuations are challenges these animals continuously confront. It has recently been shown that water channels present in the cell membrane (aquaporins) are essential for water uptake during premolt and postmolt. This study aims to investigate whether hypoosmotic shock and intracellular and extracellular calcium variations can lead to translocation of Aquaporin 1 (AQP-1) from the intracellular region to the plasma membrane during premolt and postmolt, thus allowing increased water flow in these stages. For this, we investigate in vitro the rapid change of AQP-1 positions in the abdominal muscle cells in the freshwater shrimp, Palaemon argentinus. Using cell volume analysis and immunohistochemistry, we show that hypoosmotic conditions and an elevation of the intracellular and extracellular calcium concentrations are concurrent with the translocation of AQP-1 to the plasma membrane. These results indicate that calcium flux and hypoosmotic shock may be regulators of AQP 1 in the translocation process.

Oxygen sensing in crustaceans: functions and mechanisms.

Animals that live in changing environments need to adjust their metabolism to maintain body functions, and sensing these changing conditions is essential for mediating the short- and long-term physiological and behavioral responses that make these adjustments. Previous research on nematodes and insects facing changing oxygen levels has shown that these animals rapidly respond using atypical soluble guanylyl cyclases (sGCs) as oxygen sensors connected to downstream cGMP pathways, and they respond more slowly using hypoxia-inducible transcription factors (HIFs) that are further modulated by oxygen-sensing prolyl hydroxylases (PHs). Crustaceans are known to respond in different ways to hypoxia, but the mechanisms responsible for sensing oxygen levels are more poorly understood than in nematodes and insects. Our paper reviews the functions of and mechanisms underlying oxygen sensing in crustaceans. Furthermore, using the oxygen sensing abilities of nematodes and insects as guides in analyzing available crustacean transcriptomes, we identified orthologues of atypical sGCs, HIFs, and PHs in crustaceans, including in their chemosensory organs and neurons. These molecules include atypical sGCs activated by hypoxia (Gyc-88E/GCY-31 and Gyc-89D/GCY-33) but not those activated by hyperoxia (GCY-35, GCY-36), as well as orthologues of HIF-α, HIF-ß, and PH. We offer possible directions for future research on oxygen sensing by crustaceans.

Synthesis of pyrazoline fatty chain derivatives and its effects on melanoma cells.

Skin cancer is the most common type of cancer in Brazil, representing 30% of all cases. Among these, melanoma represents only 3% of malignant neoplasms; however, it is the most serious and has a high capacity for metastasis. For this reason, it is extremely important to identify more efficient compounds and treatments that stop or decrease the proliferation of melanoma, even in its more advanced stages. This work reports the synthesis and biological evaluation of two homologous series of pyrazoline fatty chain derivatives as potent antitumoral agents in the melanoma B16F10 cell line. Cells were treated with pyrazoline fatty chain compounds (3, 30, 300, and 3000 µM) for 0, 24, 48, and 72 h. Decreased cell viability was observed when using most compounds at different concentrations and times. The structure-activity relationship (SAR) between antitumoral activity and the number of carbons and lipophilicity, as well as the oxygen-sulfur bioisosteric exchange, was evaluated. Among the tested derivatives, the lipophilic compounds 5-hydroxy-5-(trifluoromethyl)-3-undecyl-4,5-dihydro-1H-pyrazole-1-carboxamide (2d) and 5-hydroxy-5-(trifluoromethyl)-3-undecyl-4,5-dihydro-1H-pyrazole-1-thiocarboxamide (3d) showed the best results in the B16F10 cell line, as they produced the best cell viability decrease effects. The presence of fatty unbranched undecyl chain in the molecular structure appears to be important for its antimelanoma properties.

Emersion behavior of the semi-terrestrial crab Neohelice granulata during hypoxic conditions: Lactate as a trigger.

Climate changes affecting aquatic environments are increasing, and the resultant environmental challenges require animals to adopt alternative compensatory behavioral and physiological strategies. In particular, low levels of dissolved O2 are a regular problem for estuarine animals, leading to activation of a series of behavioral and physiological responses. This study on the semi-terrestrial crab Neohelice granulata examined patterns of emersion behavior under different levels of dissolved O2 availability and the role of lactate in this behavior. Emersion behavior was recorded for 4.5 h for crabs in water at four different levels of dissolved O2 (6, 3, 2, and 1 mg O2/L) and with free access to air. Oxygen consumption and hemolymphatic lactate levels were measured using the same experimental design. Emersion behavior was also recorded for 70 min in normoxic water after lactate or saline injections. Crabs increased their emersion behavior only in severe hypoxia (1 mg O2/L), and O2 consumption decreased under more severe hypoxic conditions. Despite the increase in emersion behavior, which leads to higher O2 availability, an increase in hemolymphatic lactate levels indicates that the animals still need to resort to anaerobic pathways to fulfill their metabolic demand. Furthermore, animals injected with lactate showed higher emersion behaviors than animals injected with a saline solution even in normoxia. These results suggest that the increase in hemolymphatic lactate can act directly or indirectly as a trigger for the increase in emersion behavior in the semi-terrestrial crab N. granulata.

High temperature acclimation alters the emersion behavior in the crab Neohelice granulata.

An increase in environmental temperature can deleteriously affect organisms. This study investigated whether the semiterrestrial estuarine crab Neohelice granulata uses emersion behavior as a resource to avoid thermal stress and survive higher aquatic temperatures. We also examined whether this behavior is modulated by exposure to high temperature; whether, during the period of emersion, the animal loses heat from the carapace to the medium; and whether this behavior is altered by the temperature at which the animal has been acclimated. The lethal temperature for 50% of the population (LT50) was determined through 96-h mortality curves in animals acclimated at 20 °C and 30 °C. The behavioral profile of N. granulata during thermal stress was based on monitoring crab movement in aerial, intermediary, and aquatic zones. Acclimation at a higher temperature and the possibility of emersion increased the thermotolerance of the crabs and the synergistic effect of acclimation temperature. The possibility of leaving the hot water further increased the resistance of these animals to thermal stress. We observed that when the crab was subjected to thermal stress conditions, it spent more time in the aerial environment, unlike under control conditions. Under the experimental conditions, it made small incursions into the aquatic environment and stayed in the aerial environment for a longer time in order to cool its body temperature. The animals acclimated at 20 °C and placed into water at 35 °C remained in the aerial zone. The animals acclimated and maintained at 30 °C (control) that were placed in water at 35 °C with the possibility of emerging into hot air transited more frequently between the aquatic and aerial zones than did the animals that were put in water at 35 °C with the possibility of emerging into a cooler air environment. We conclude that emergence behavior allows N. granulata to survive high temperatures and that this behavior is influenced by acclimation temperature.

Synthesis and antioxidant activity of new lipophilic dihydropyridines.

Dihydropyridines (DHPs) obtained from Hantzsch multicomponent reactions are an important pharmaceutical class of compounds marketed as antihypertensive (e.g., nifedipine, nitrendipine, and amlodipine) drugs. This study synthesized new symmetrical and unsymmetrical long-chain fatty DHPs using multicomponent reactions under metal-free conditions with sulfamic acid as a catalyst. The DHPs were tested for antioxidant activity using three different methods. The insertion of a long chain into the DHP core contributed to antioxidant potential, and compounds derived from nitro aldehydes have better antioxidant potential than the antihypertensive drug nifedipine. In addition, fatty analogs to nifedipine derived from palmitic and oleic chains showed similar antioxidant activity to the common standards butylated hydroxytoluene and vitamin E. These results showed that our new synthesized products may find novel applications as antioxidant additives or for tools for use in drug discovery.

Damage caused during hypoxia and reoxygenation in the locomotor muscle of the crab Neohelice granulata (Decapoda: Varunidae).

The aim of this work was to determine whether different durations of severe hypoxia (0.5 mg O2 L(-1)) followed by reoxygenation cause damage to the locomotor muscle of the crab Neohelice granulata. We evaluated reactive oxygen species (ROS), lipid peroxidation (LPO), mitochondrial membrane potential, and aerobic fiber area of the locomotor muscle after different periods of hypoxia (1, 4, or 10h) followed by 30 or 120 min of reoxygenation. Additionally, changes in cell volume, mitochondrial dysfunction, and infiltration of hemocytes were evaluated after hypoxia and a subsequent 2, 24, or 48 h of reoxygenation. After hypoxia, neither ROS nor LPO increased. However, mitochondrial membrane potential and aerobic fiber area decreased in a time-dependent manner. After reoxygenation, the ROS and LPO levels increased and mitochondrial membrane potential decreased, but these quickly recovered in crabs exposed to 4h of hypoxia. On the other hand, alterations of mitochondria resulted in morphological changes in aerobic fibers, which required more time to recover during reoxygenation after 10h of hypoxia. The locomotor muscles of the crab N. granulata suffer damage after hypoxia and reoxygenation. The intensity of this damage is dependent on the duration of hypoxia. In all experimental situations analyzed, the locomotor muscle of this crab was capable of recovery.

Melatonin as a signaling molecule for metabolism regulation in response to hypoxia in the crab Neohelice granulata.

Melatonin has been identified in a variety of crustacean species, but its function is not as well understood as in vertebrates. The present study investigates whether melatonin has an effect on crustacean hyperglycemic hormone (CHH) gene expression, oxygen consumption (VO2) and circulating glucose and lactate levels, in response to different dissolved-oxygen concentrations, in the crab Neohelice granulata, as well as whether these possible effects are eyestalk- or receptor-dependent. Melatonin decreased CHH expression in crabs exposed for 45 min to 6 (2, 200 or 20,000 pmol·crab-1) or 2 mgO2·L-1 (200 pmol·crab-1). Since luzindole (200 nmol·crab-1) did not significantly (p > 0.05) alter the melatonin effect, its action does not seem to be mediated by vertebrate-typical MT1 and MT2 receptors. Melatonin (200 pmol·crab-1) increased the levels of glucose and lactate in crabs exposed to 6 mgO2·L-1, and luzindole (200 nmol·crab-1) decreased this effect, indicating that melatonin receptors are involved in hyperglycemia and lactemia. Melatonin showed no effect on VO2. Interestingly, in vitro incubation of eyestalk ganglia for 45 min at 0.7 mgO2·L-1 significantly (p < 0.05) increased melatonin production in this organ. In addition, injections of melatonin significantly increased the levels of circulating melatonin in crabs exposed for 45 min to 6 (200 or 20,000 pmol·crab-1), 2 (200 and 20,000 pmol·crab-1) and 0.7 (200 or 20,000 pmol·crab-1) mgO2·L-1. Therefore, melatonin seems to have an effect on the metabolism of N. granulata. This molecule inhibited the gene expression of CHH and caused an eyestalk- and receptor-dependent hyperglycemia, which suggests that melatonin may have a signaling role in metabolic regulation in this crab.

Responses to ROS inducer agents in zebrafish cell line: differences between copper and UV-B radiation.

Fish are commonly exposed to environmental pollutants, which in turns could induce an oxidative stress. So, it is important to understand the effects and the responses elicited by these toxicants in fish species, being fish cell lines important tools for this purpose. Thus, the aim of the present study was to compare the effects of copper and UV-B radiation exposure on zebrafish hepatocytes (ZFL lineage) in terms of reactive oxygen species (ROS) levels, sulfhydril groups content and mRNA levels of important genes related to cellular response to toxic agents. Exposure of ZFL cells to UV-B radiation (23.3 mJ/cm(2)) significantly increased levels of intracellular ROS and mRNA of both superoxide dismutase isoforms (sod1 and sod2), three glutathione S-transferase isoforms (gstα, gstµ and gstπ) and a heat shock protein (hsp70). However, no changes in nonprotein sulfhydryl groups (NP-SH) content, as well as in the mRNA levels of genes related to glutathione (GSH) synthesis and recycling, were observed. Contrary to this, copper exposure (20 mg/L) diminished NP-SH content and increased the levels of mRNA of genes related to GSH synthesis (gclc and gs). Moreover, copper exposure increases the mRNA levels of some genes related to antioxidant defenses (gpx and gstπ), biotransformation reactions (cyp1a1) and protein repair (hsp70). In conclusion, these results demonstrated that both toxicants could increase ROS levels in ZFL cell line, but the responses are different, which could be related to activation of different signaling pathways.

Chronic immune challenge is detrimental to female survival, feeding behavior, and reproduction in the field cricket Gryllus assimilis (Fabricius, 1775).

Physiological trade-offs among expensive fitness-related traits, such as reproduction and immunity, are common in life histories of animals. An immune challenge can have different effects on female reproduction mediated by resource allocation and acquisition. In this study, employing a widely used method to challenge the insect immune system (nylon implant), we assessed the effects of mounting a chronic immune response simulating three successive immune assaults on survival and reproduction of mated females of Gryllus assimilis. We also verified feeding behavior following an implantation, which can be important in explaining trade-off dynamics in terms of energy acquisition. For this, three experimental groups were designed (Control, Sham, and Implant) with oviposition rates, egg morphometry, and nymph vigour observed over 3 weeks, at which ovarian mass and unlaid eggs were quantified from remaining individuals. The results showed that chronic implants were detrimental to female survival and reproduction throughout the experiments; Surgical Sham had no effect on survival compared to the control, but did on reproductive aspects such as oviposition rates and hatchling vigour. These negative effects on reproduction in Sham disappeared in the last experimental week, but still strong in the implanted females. Such immune challenge affected the feeding behavior of implanted females by reducing food consumption compared to control after infection, which is probably explained by illness-induced anorexia that takes place to maximize the immune system performance as a part of sickness behavior, exacerbating the adverse effects observed on reproduction (i.e., fewer and smaller eggs, and low vigour of nymphs) and survival.

Effects of seasonality and moult cycle on the proliferation of nerve cells and on the labelling of ecdysone receptors in an estuarine crab.

Decapod crustaceans show proliferation of the nerve cells in the olfactory lobe throughout their lives. However, the regulation of this process is still poorly understood, since it may vary with endogenous and exogenous factors. The objective of the present investigation was to quantify the proliferation of nerve cells and number of nerve cells with ecdysone receptors in the clusters of the central olfactory system in Neohelice granulata, according to moult stages and in different seasons (summer and winter). Three injections of bromodeoxyuridine (BrdU) were administered to the crabs. Brains were sectioned by microtome and fixed on slides for immunohistochemistry with anti-BrdU and anti-EcR antibodies. The proliferation of nerve cells was higher in winter than in summer, probably because in winter the crabs do not breed and the premoult and postmoult periods are longer. Crabs in postmoult exhibited more BrdU-labelled cells than crabs in premoult or intermoult in winter, because of a greater number of mitoses related to an increase in body size and addition of olfactory receptor neurons. The number of EcR-labelled cells was higher in premoult than in postmoult or intermoult in winter. The proliferation of nerve cells is regulated seasonally and according to moult stages.

Reproductive Status Modulates Mortality Rate, Lifespan and Egg Production, but Do Not the Physiological Aging in the Field Cricket Gryllus assimilis (Fabricius, 1775) (Orthoptera: Gryllidae).

The effects of reproduction are variable among insects, as well as the time of mate. In animals, a trade-off is usually observed between reproduction and self-maintenance, mostly in females because of egg producing costs. In the present investigation, manifestations of aging and senescence at population and cellular levels were studied associated with the mating opportunities. The main goal of this study was to evaluate the effect of mating on lifespan and also on the physiological aging in adult Jamaican field cricket females, Gryllus assimilis (Fabricius, 1775). Three experimental groups were set: virgin, normal-mated (14 days old), and late-mated (30 days old) insects. Lifespan, age-specific mortality rate, number of eggs laid, and in situ amount of the age-pigment neurolipofuscin were quantified. Results showed a trade-off between reproductive females and lifespan, with the strongest effects found in late-mated. Age-specific mortality showed a faster increase for late-mated females from 35 to 50 days old, followed by normal-mated females (35 to 60 days old). Virgins had the lowest age-specific mortality of all, increasing just from 65 to 73 days old. Normal-mated had the highest number of eggs laid, followed by late-mated and virgins, respectively. Neurolipofuscin accumulation rate was similar among groups, which was reflected in similar physiological rate of aging. Results indicate that reproductive status did affect the life-history of these field cricket females.

Effect of melatonin in the antioxidant defense system in the locomotor muscles of the estuarine crab Neohelice granulata (Decapoda, Brachyura).

In vertebrates, many studies verified different effects of melatonin in the antioxidant defense system (ADS). In crustaceans, few studies have been conducted to verify this possibility. We verified the melatonin effects in the crab Neohelice granulata using low (0.002 and 0.02 pmol/crab) and high (2.0 and 20.0 pmol/crab) melatonin dosages in short-term (0.5h) and long-term (9.5h) experiments. We analyzed the antioxidant capacity against peroxyl radicals (ACAP), reactive oxygen species (ROS) concentration, levels of by products of lipid peroxidation (LPO), oxygen consumption (VO(2)), the activity of glutamate cysteine ligase (gamma-GCL) and catalase (CAT) and glutathione content (GSH). Finally, the effects of exogenous melatonin were verified in terms of melatonin and N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) content in the muscles of N. granulata. In short-term experiment and low dosages, melatonin increased the VO(2), gamma-GCL activity and GSH content (p<0.05) and decreased melatonin content (p<0.05) without effects in ROS, ACAP and LPO (p>0.05). Possibly, melatonin is acting in the ADS increasing its efficiency and/or acting in mitochondrial activity and/or through signaling muscles to increase its consumption. AFMK was only detected in the eyestalk and cerebroid ganglia. In high dosages melatonin effects decreased, possibly by the desensitization of their receptors. In long-term experiment, melatonin decreased ACAP (p<0.05), and CAT activity (p<0.05) in low dosages. In high dosages melatonin reduced VO(2) (p<0.05) and increased ACAP (p<0.05), possibly stimulating others components of the ADS. In conclusion, melatonin in the locomotor muscles of N. granulata affects the antioxidant/pro-oxidant balance in a time and dosage dependent manner.

Effects of melatonin in connection with the antioxidant defense system in the gills of the estuarine crab Neohelice granulata.

Numerous studies have shown that melatonin exerts some influence on the antioxidant defense system (ADS) in vertebrates, but for crustaceans no such effect has been demonstrated till now. However, earlier reports did show a similar profile of daily variations in the ADS of the gills and the melatonin content of the eyestalk in the crab Neohelice granulata and, thus, the aim of this study was to take a closer look at the effects of melatonin in the gill ADS of N. granulata. Gill ADS is to a minor extent modulated by reactive oxygen species (ROS), because only the nonproteic sulfhydryl (NP-SH) content increases (p<0.05) in the presence of hydrogen peroxide (H(2)O(2)). No significant differences (p>0.05) were observed in the melatonin content of the hemolymph between intact and eyestalkless crabs. Gills from intact and eyestalkless crabs injected with physiological saline showed a daily variation in the total peroxyl radical scavenging capacity (TPRSC) (p<0.05) with two peaks, one at the photophase and another at the scotophase. However, in the gills of eyestalkless crabs injected with melatonin (2 x 10(-12)mol crab(-1)), the daily variation in TPRSC values was abolished (p>0.05). This molecule did not change the NP-SH content (p>0.05) in vitro, but decreased (p<0.05) the oxygen consumption in gills when incubated for 120 min. In the in vivo experiments melatonin also decreased (p<0.05) the oxygen consumption in eyestalkless crabs after 390 min. The results suggest that melatonin does not act directly on the ADS of the gills of N. granulata, but decreases the aerobic metabolism possibly involved in variations of tissue ADS.

Single and repeated low-dose UVB radiation exposures affect the visual system.

The visual system is an important biological indicator of effects induced by ultraviolet (UV) radiation. However, research has extensively investigated the effects of high-dose UV radiation in a single exposure, thus, the differential of this work was to investigate the effects of UVB radiation in low doses in single and repeated exposure. Therefore, we investigated the effects of repeated exposure to environmental UVB doses (0.09 J/cm2) on the retina and optic lobes of the crab Neohelice granulata. We evaluated the reactive oxygen species (ROS) concentration, antioxidant capacity against peroxyl radicals (ACAP) levels, catalase (CAT) and glutathione S-transferase (GST) activities and lipoperoxidation (LPO) levels and performed histological analysis. The crabs were exposed to UVB radiation for 1 or 60 days, while the control group was exposed to visible light. In the retina region, increases in ROS concentration and CAT and GST activities after the single exposure were observed. After 60 days of exposure, we observed an increase in ACAP levels. In the optic lobes, we observed an increase in GST activity and a decrease in LPO levels after the single exposure. However, we observed an increase in ROS concentration after 60 days of exposure. Moreover, after 60 days of exposure, infiltrating hemocytes in the retina and disorganization in neuron cell bodies of the external medulla were observed. In this sense, single and repeated exposure to low doses of UVB radiation induced changes in oxidative status and inflammatory process in the visual system of the crab Neohelice granulata.

Long-chain fatty dihydropyridines: Docking calcium channel studies and antihypertensive activity.

AIMS: From the synthesis of 43 lipophilic dihydropyridines, the aim of this study was to verify whether the new dihydropyridines have calcium channel affinity using coupling studies and to determine antihypertensive and antioxidant properties, as well as toxicology and toxicity nifedipine and three new compounds, were chosen from the previous results. MATERIALS AND METHODS: The animals were treated for 56 days, 28 days with N (ω) -nitro-L-arginine methyl ester to induce hypertension, and then treated for another 28 days with the new di- hydropyridine and the standard drug nifedipine. Throughout the treatment the animals had their blood pressure measured and their heart rate checked by pletysmography. After treatment the animals were euthanised, blood samples were collected for creatine kinase and urea analysis, and the brain, heart and liver were collected for oxidative status analysis (quantification of reactive oxygen species, total antioxidant capacity, and lipid peroxidation). KEY FINDINGS: Compounds 2c, and 9a, and nifedipine significantly reduced blood pressure to control group levels. The tachycardia caused by the induction of hypertension was reversed by 2c and 9a compounds. Regarding oxidative stress analyzes, the compounds that had the best performances were also 2c and 9a. Overall the results demonstrate that two of the three new dihydropyridines tested demonstrated performance equal to or superior to the standard drug nifedipine. SIGNIFICANCE: In this study, for the first time, docking was applied to analyse 43 fatty dihydropyridines regarding their calcium channel binding. Afterwards, three fatty dihydropyridines were chosen and their antihypertensive and antioxidant properties.

Infrared radiation influence on molt and regeneration of Neohelice granulata Dana, 1851 (Grapsidae, Sesarminae).

This paper analyzes the influence of infrared radiation (IR) on regeneration, after autotomy of limb buds of Neohelice granulata and consequently the time molt. Eyestalks were ablated to synchronize the start of molt. Afterward, animals were autotomized of five pereopods and divided into control and irradiated groups. The irradiated group was treated for 30 min daily until molt. Limb buds from five animals of days 4, 16 and 20 were collected and histological sections were made from them. These sections were photographed and chitin and epithelium content measured. Another group was made, and after 15 days limb buds were extracted to analyze mitochondrial enzymatic activity from complex I and II. The irradiated group showed a significant reduction in molt time (19.38+/-1.22 days) compared with the control group (32.69+/-1.57 days) and also a significant increase in mitochondrial complex I (388.9+/-27.94%) and II (175.63+/-7.66%) in the irradiated group when compared with the control group (100+/-17.90; 100+/-7.82, respectively). However, these effects were not accompanied by histological alterations in relation to chitin and epithelium. This way, it was possible to demonstrate that IR increases complex I and II activity, reduces the time molt and consequently increases the appendage regeneration rate.

Biochemical and physiological adaptations in the estuarine crab Neohelice granulata during salinity acclimation.

Neohelice granulata (Chasmagnathus granulatus) is an intertidal crab species living in salt marshes from estuaries and lagoons along the Atlantic coast of South America. It is a key species in these environments because it is responsible for energy transfer from producers to consumers. In order to deal with the extremely marked environmental salinity changes occurring in salt marshes, N. granulata shows important and interesting structural, biochemical, and physiological adaptations at the gills level. These adaptations characterize this crab as a euryhaline species, tolerating environmental salinities ranging from very diluted media to concentrated seawater. These characteristics had led to its use as an animal model to study estuarine adaptations in crustaceans. Therefore, the present review focuses on the influence of environmental salinity on N. granulata responses at the ecological, organismic and molecular levels. Aspects covered include salinity tolerance, osmo- and ionoregulatory patterns, morphological and structural adaptations at the gills, and mechanisms of ion transport and their regulation at the gills level during environmental salinity acclimation. Finally, this review compiles information on the effects of some environmental pollutants on iono- and osmoregulatory adaptations showed by N. granulata.

Protective role of the novel hybrid 3,5-dipalmitoyl-nifedipine in a cardiomyoblast culture subjected to simulated ischemia/reperfusion.

This work investigated the acute effects of the calcium channel blocker nifedipine and its new fatty hybrid derived from palmitic acid, 3,5-dipalmitoyl-nifedipine, compared to endocannabinoid anandamide during the process of inducing ischemia and reperfusion in cardiomyoblast H9c2 heart cells. The cardiomyoblasts were treated in 24 or 96-well plates (according to the test being performed) and maintaining the treatment until the end of hypoxia induction. The molecules were tested at concentrations of 10 and 100µM, cells were treated 24h after assembling the experimental plates and immediately before the I/R. Cell viability, apoptosis and necrosis, and generation of reactive oxygen species were evaluated. Nifedipine and 3,5-dipalmitoyl-nifedipine were used to assess radical scavenging potential and metal chelation. All tested molecules managed to reduce the levels of reactive oxygen species compared to the starvation+vehicle group. In in vitro assays, 3,5-dipalmitoyl-nifedipine showed more antioxidant activity than nifedipine. These results indicate the ability of this molecule to act as a powerful ROS scavenger. Cell viability was highest when cells were induced to I/R by both concentrations of anandamide and the higher concentration of DPN. These treatments also reduced cell death. Therefore, it was demonstrated that the process of hybridization of nifedipine with two palmitic acid chains assigns a greater cardioprotective effect to this molecule, thereby reducing the damage caused by hypoxia and reoxygenation in cardiomyoblast cultures.