Assessing physiological responses and oxidative stress effects in Rhamdia voulezi exposed to high temperatures.

Exposure to high temperatures induces changes in fish respiration, resulting in an increased production of reactive oxygen species. This, in turn, affects the enzymatic and non-enzymatic components of antioxidant defenses, which are essential for mitigating cellular stress. Rhamdia voulezi, an economically important fish species endemic to Brazil's Iguaçu River, served as the subject of our study. Our goal was to assess enzymatic antioxidant biomarkers (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, glucose-6-phosphate dehydrogenase), non-protein thiol levels (reduced glutathione), and markers of oxidative damage (lipoperoxidation and carbonylation) in the liver, gills, and kidneys of R. voulezi after acute exposure to high temperatures (31°C) for 2, 6, 12, 24, and 96 h. Control groups were maintained at 21°C. Our findings revealed that the liver exhibited increased superoxide dismutase levels up to 12 h and elevated glutathione S-transferase levels at 12 and 96 h at 31°C. In the gills, superoxide dismutase levels increased up to 24 h, along with increased lipoperoxidation at 2, 6, 12, and 96 h of exposure to high temperatures. The kidneys responded to heat stress at 12 h, with an increase in superoxide dismutase and catalase activity, and lipid peroxidation was observed at 2 and 6 h at 31°C. The three tissues evaluated responded differently to heat stress, with the liver demonstrating greater physiological adjustment to high temperatures. The intricate interplay of various antioxidant defense biomarkers and oxidative damage suggests the presence of oxidative stress in R. voulezi when exposed to high temperatures.

Metabolic responses in the gills of Yellowtail Lambari Astyanax lacustris under low- and high-temperature thermal stress.

OBJECTIVE: Ectothermic fish are directly affected by temperature changes in the environment. The aim of this study was to evaluate the metabolic responses in the gills of Yellowtail Lambari Astyanax lacustris under thermal stress. METHODS: To this end, we used spectrophotometry to evaluate the biomarkers of carbohydrate and protein metabolism, antioxidant defense, and oxidative damage in fish subjected to low (15°C) and high (31°C) temperatures, with control groups held at 23°C, for 2, 6, 12, 24, 48, and 96 h. RESULT: The results showed that cold thermal stress did not change the energy demand, and the antioxidant defense was reduced; therefore, the gills were vulnerable to the action of reactive oxygen species (ROS), presenting increased protein carbonylation at 12 h. With heat thermal stress, a higher energy demand was observed, which was verified by an increase in aerobic metabolism by glycolysis and the citric acid cycle. High-temperature stress also increased the antioxidant defenses, as verified by the increased activities of glutathione peroxidase, glutathione reductase, and glutathione S-transferase. However, the antioxidant defense system could not protect tissues from the action of ROS, as protein carbonylation increased at 6 and 24 h, indicating oxidative stress. CONCLUSION: The results showed that (1) temperature variations caused metabolic adjustments in the gills of Yellowtail Lambari, (2) the adaptive responses were different for winter and summer temperatures, and (3) Yellowtail Lambari recovered homeostasis when subjected to thermal stress, even with the occurrence of oxidative stress.

Effects of heat shock on energy metabolism and antioxidant defence in a tropical fish species Psalidodon bifasciatus.

Predictions about global warming have raised interest in assessing whether ectothermic organisms will be able to adapt to these changes. Understanding the physiological mechanisms and metabolic adjustment capacity of fish subjected to heat stress can provide subsidies that may contribute to decision-making in relation to ecosystems and organisms subjected to global climate change. This study investigated the antioxidant defence system and energy metabolism of carbohydrate and protein responses in the gill, liver and kidney tissues of Psalidodon bifasciatus (Garavello & Sampaio 2010), a Brazilian freshwater fish used in aquaculture and in biological studies, following exposure to heat shock at 31°C for 2, 6, 12, 24 and 48 h. The fish presented signs of stress in all tissues tested, as evidenced by increased lipid peroxidation concentration at 2 h and phosphofructokinase, hexokinase and malate dehydrogenase activity at 48 h in the gills; increased glutathione-S-transferase activity at 12 h, citrate synthase activity at 24 h and concentration of reduced glutathione (GSH) concentration at 12 and 48 h in the liver; and through increased activity of superoxide dismutase at 48 h, glutathione reductase at 24 h, glucose-6-phosphate dehydrogenase at 48 h and concentration of GSH at 24 h in the kidney. In the kidneys, changes in the antioxidant system were more prominent, whereas in the gills, there were greater changes in the carbohydrate metabolism. These results indicated the importance of glycolysis and aerobic metabolism in the gills, aerobic metabolism in the liver and pentose-phosphate pathway in the kidneys during homeostasis. The biomarker response was tissue specific, with the greatest number of biomarkers altered in the gills, followed by those in the kidneys and liver.

Gradual increase of temperature trigger metabolic and oxidative responses in plasma and body tissues in the Antarctic fish Notothenia rossii.

Antarctica is considered a thermally stable ecosystem; however, climate studies point to increases in water temperatures in this region. These thermal changes may affect the biological processes and promote metabolic changes in the adapted organisms that live in this region, rendering the animals more vulnerable to oxidative damage. This study assessed the effect of acclimation temperature on the levels of stress response markers in plasma, kidney, gill, liver, and brain tissues of Notothenia rossii subjected to gradual temperature changes of 0.5 °C/day until reaching temperatures of 2, 4, 6, and 8 °C. Under the effect of the 0.5 °C/day acclimation rate, gill tissue showed increased glutathione-S-transferase (GST) activity; kidney tissue showed increased H+-ATPase activity. In the liver, there was also an increase in GSH. In plasma, gradual decreases in the concentrations of total proteins and globulins were observed. These responses indicate a higher production of reactive oxygen species ROS, an imbalance in energy demand, and a lack in protein synthesis. Gradual increase in temperature may cause opposite responses to the thermal shock model in N. rossii.

Effect of heat stress on the antioxidant defense system and erythrocyte morphology of Antarctic fishes.

This study analyzed the effect of thermal stress on erythrocytes of Notothenia rossii and Notothenia coriiceps, abundant notothenioids in Admiralty Bay, Antarctic Peninsula. In both species, the antioxidant defense system enzymes, superoxide dismutase, catalase, glutathione peroxidase, glutathione-S transferase, glutathione reductase were punctually altered (8°C for 1, 3 and 6 days) in erythrocytes, indicating that these markers are not ideal for termal stress. However, under the influence of thermal stress, morphological changes in Notothenia coriiceps erythrocytes were observed at all exposure times (1, 3 and 6 days at 8°C), and in Notothenia rossii occurred in 6 days. These results suggest that Notothenia corriceps presents a lower tolerance to thermal stress at 8°C for up to 6 days, since the cellular and nuclear alterations recorded are pathological and may be deleterious to the cells. Among the morphological markers analyzed in this work, we believe that the shape change and nuclear bubble formation may be good stress biomarkers in erythrocytes of Notothenia rossii and Notothenia coriiceps.

Effect of long-term thermal challenge on the Antarctic notothenioid Notothenia rossii.

The thermal stability of the Antarctic Ocean raises questions concerning the metabolic plasticity of Antarctic notothenioids to changes in the environmental temperature. In this study, Notothenia rossii survived 90 days at 8 °C, and their condition factor level was maintained. However, their hepatosomatic (0.29×) index decreased, indicating a decrease in nutrient storage as a result of changes in the energy demands to support survival. At 8 °C, the plasma calcium, magnesium, cholesterol, and triglyceride concentrations decreased, whereas the glucose (1.91×) and albumin (1.26×) concentrations increased. The main energy substrate of the fish changed from lipids to glucose due to a marked increase in lactate dehydrogenase activity, as demonstrated by an increase in anaerobic metabolism. Moreover, malate dehydrogenase activity increased in all tissues, suggesting that fish acclimated at 8 °C exhibit enhanced gluconeogenesis. The aerobic demand increased only in the liver due to an increase (2.23×) in citrate synthase activity. Decreases in the activities of superoxide dismutase, catalase, and glutathione-S-transferase to levels that are most likely sufficient at 8 °C were observed, establishing a new physiological activity range for antioxidant defense. Our findings indicate that N. rossii has some compensatory mechanisms that enabled its long-term survival at 8 °C.

Antileishmanial Activity of a Calophyllum brasiliense Leaf Extract.

Calophyllum brasiliense (Clusiaceae) is a tree that grows mainly in the Atlantic Forest in Brazil. Its leaves possess antibacterial activity, cytotoxic activity against certain tumor cell lines, and antimicrobial activity in BALB/c mice infected with Leishmania (Leishmania) amazonensis.Aiming to identify ultrastructural changes and DNA fragmentation in Leishmania (Viannia) braziliensis, promastigotes were treated with a concentration of the dichloromethane extract and coumarin (-) mammea A/BB from C. brasiliense leaves that inhibited 50 % of the parasites (IC50), and were evaluated by transmission and scanning electron microscopy. Ultrastructural changes showed different levels of mitochondrial alterations, including mitochondrial swelling and a reduction in the density of the mitochondrial matrix. Induced DNA fragmentation, as observed by TUNEL, suggested that the extract and coumarin (-) mammea A/BB induced apoptosis-like cell death. These results suggest that the combination of C. brasiliense extract and coumarin (-) mammea A/BB can be considered a promising candidate for the development of new antiprotozoal agents, because of its significant leishmanicidal activity.

Evidence of metabolic microevolution of the limpet Nacella concinna to naturally high heavy metal levels in Antarctica.

The gastropod Nacella concinna is the most conspicuous macroinvertebrate of the intertidal zone of the Antarctic Peninsula and adjacent islands. Naturally high levels of copper and cadmium in coastal marine ecosystems are accumulated in N. concinna tissues. We aimed to study the effects of metal cations on N. concinna arginase in the context of possible adaptive microevolution. Gills and muscle had the highest argininolytic activity, which was concentrated in the cytosol in both tissues. Gills had the highest levels of arginase and may be involved in the systemic control of l-arginine levels. The relatively high argininolytic activity of the N. concinna muscular foot, with KM=25.3±3.4mmolL-1, may be involved in the control of l-arginine levels during phosphagen breakdown. N. concinna arginases showed the following preferences for metal cations: Ni2+>Mn2+>Co2+>Cu2+ in muscle and Mn2+>Cu2+ in gills. Cu2+ activation is a unique characteristic of N. concinna arginases, as copper is a potent arginase inhibitor. Cu2+ partly neutralized N. concinna arginase inhibition by Cd2+, worked synergistically in muscle arginase activation by Co2+ and neutralized muscle arginase activation by Ni2+. Mn2+ was able to activate muscle arginase in the presence of Fe3+ and Pb2+. The selection of arginases that are activated by Cu2+ and resistant to inhibition by Cd2+ in the presence of Cu2+ over evolutionary timescales may have favored N. concinna occupation of copper- and cadmium-rich niches.

Ultrastructural and morphological changes in Leishmania (Viannia) braziliensis treated with synthetic chalcones.

Cutaneous leishmaniasis has an estimated incidence of 1.5 million new cases per year and the treatment options available are old, expensive, toxic, and difficult to administer. Chalcones have shown good activity against several species of Leishmania. However few studies have discussed the mechanisms of action and drug target of this group of compounds in Leishmania. The synthetic chalcones that were evaluated in the present study were previously shown to exhibit activity against Leishmania (Viannia) braziliensis. The objective of the present study was to identify ultrastructural and morphological changes in L. (V.) braziliensis after treatment with three synthetic chalcones (1-3). Promastigotes were treated with chalcones 1-3 and evaluated by transmission and scanning electron microscopy. Cellular and nuclear morphology of the parasites, changes in membrane permeability, and DNA fragmentation in agarose electrophoresis gel were also investigated after exposure to synthetic chalcones. All three synthetic chalcones (1-3) induced ultrastructural alterations in mitochondria, intense vacuolization, two nuclei with rounding of parasites, and cellular and nuclear shrinkage. Chalcones 1-3 also induced no changes in membrane permeability, and presence of nucleosome-sized DNA fragments. Synthetic chalcones 1-3 induced ultrastructural and morphological changes, suggesting that chalcones 1-3 induce apoptosis-like cell death. Further studies should be conducted to elucidate other aspects of the action of these chalcones against Leishmania spp. and their use for the treatment of cutaneous leishmaniasis.

In vitro characterization of Leishmania (Viannia) braziliensis isolates from patients with different responses to Glucantime(®) treatment from Northwest Paraná, Brazil.

Leishmaniasis is a group of diseases that presents various clinical manifestations. Many studies have shown that the parasite plays an important role in the clinical manifestations and prognosis of this disease. The cutaneous and mucosal forms of American tegumentary leishmaniasis (ATL) are associated with Leishmania (Viannia) braziliensis, which exhibits intraspecific genetic polymorphisms and various clinical manifestations. The present study focused on four different L. braziliensis strains that were isolated from patients with distinct Glucantime(®) treatment responses. The isolates were described based on their molecular, biological, and infective characteristics. Growth patterns in culture medium and different grow phases were analyzed, MID-Logarithimic (Mid-LOG), Logarithimic (LOG) and Stationary (STAT) phases. Complement resistance was evaluated using guinea pig serum. Infection to murine peritoneal macrophages, cytokine and nitric oxide were analyzed. Ultrastructural features were determined by transmission electron microscopy, and molecular characteristics were determined based on random amplified polymorphic DNA (RAPD). All of the L. braziliensis isolates showed typical growth and similar complement sensitivity patterns. Markedly lower infectivity indexes were observed for all strains in the LOG phase, with different cytokine profiles. The ultrastructure analysis revealed distinct differences between the MID-LOG, LOG, and STAT phases. The RAPD results showed a divergence between the isolates of the L. braziliensis. The in vitro characterization of L. braziliensis isolates from humans with different treatment responses using various parameters enabled us to observe differences among the isolates. Molecular and in vivo characterizations are currently under study to improve understanding of the parasite-host interaction that can imply in the clinical manifestation differences.

Antileishmanial activity of essential oil and 6,7-dehydroroyleanone isolated from Tetradenia riparia.

Tetradenia riparia plant is used as a traditional medicine in Africa for the treatment of inflammatory and infectious diseases as like parasitic. Therapy for leishmaniasis caused by Leishmania (Leishmania) amazonensis specie often fails, and the conventional drugs are toxic, expensive, require a long period of treatment, and adverse effects are common. The alternative therapies using natural products are inexpensive and have few or any adverse reaction. These reasons are sufficient to investigate the new natural therapeutic for leishmaniasis. We evaluated the potential of the essential oil (TrEO) and 6,7-dehydroroyleanone (TrROY) isolated from T. riparia on L. (L.) amazonensis promastigote and amastigote forms, cytotoxicity on human erythrocytes and murine macrophages, nitric production and inducible nitric oxide synthase (iNOS) mRNA expression. TrEO was the most effective to promote the Leishmania promastigote death. After 72 h incubation, the lethal dose of TrEO and TrROY that promoted 50% Leishmania death (LD50) were 0.8 µg/mL and 3 µg/mL, respectively. TrEO and TrROY were not cytotoxic to human erythrocytes, but TrROY was toxic to murine macrophages resulting in a low selectivity index. The transmission electronic microscopy showed that TrEO (0.03 µg/mL) was able to modify the promastigote ultrastructures suggesting autophagy as chromatin condensation, blebbing, membranous profiles and nuclear fragmentation. Infected-macrophages treated with TrEO (0.03 µg/mL) or TrROY (10 µg/mL) had an infection index decreased in 65 and 48%. TrEO did not induce iNOS mRNA expression or nitrite production in macrophages infected with Leishmania. TrROY and mainly TrEO promoted the Leishmania death, and TrROY showed loss toxicity to erythrocytes cells. Other compounds derived from T. riparia and the essential oil could be explored to develop a new alternative treatment for leishmaniasis.

Cellular Structural Changes in Candida albicans Caused by the Hydroalcoholic Extract from Sapindus saponaria L.

Vulvovaginal candidiasis (VVC) is a disease caused by the abnormal growth of yeast-like fungi in the mucosa of the female genital tract. Candida albicans is the principal etiological agent involved in VVC, but reports have shown an increase in the prevalence of Candida non-C. albicans (CNCA) cases, which complicates VVC treatment because CNCA does not respond well to antifungal therapy. Our group has reported the in vitro antifungal activity of extracts from Sapindus saponaria L. The present study used scanning electron microscopy and transmission electron microscopy to further evaluate the antifungal activity of hydroalcoholic extract from S. saponaria (HE) against yeast obtained from VVC and structural changes induced by HE. We observed the antifungal activity of HE against 125 vaginal yeasts that belonged to four different species of the Candida genus and S. cerevisae. The results suggest that saponins that are present in HE act on the cell wall or membrane of yeast at the first moments after contact, causing damage to these structures and cell lysis.

Metabolic responses of the Antarctic fishes Notothenia rossii and Notothenia coriiceps to sewage pollution.

The present study aimed to assess the sewage effects of the Brazilian Antarctic Station Comandante Ferraz, Admiralty Bay, King George Island, on the hepatic metabolism (energetic, antioxidant, and arginase levels) and levels of plasma constituents of two Antarctic fish species Notothenia rossii and N. coriiceps. The bioassays were conducted under controlled temperature (0 °C) and salinity (35 psu), exposing the fish for 96 h, to sewage effluent diluted in seawater to 0.5 % (v/v). Liver homogenates were tested for the specific activities of the enzymes glucose-6-phosphatase (G6Pase), glycogen phosphorylase (GPase), hexokinase, citrate synthase, lactate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, superoxide dismutase, glutathione reductase, catalase, and arginase. Plasma levels of glucose, triacylglycerides, cholesterol, total protein, albumin, chloride, magnesium, calcium, and inorganic phosphate were also determined. In N. rossii, the decrease in citrate synthase and the increase in G6Pase and GPase suggested that the sewage effluent activated glycogenolysis and hepatic gluconeogenesis, whereas is N. coriiceps, only G6Pase levels were increased. In N. rossii, sewage effluent induced hypertriglyceridemia without modulating glucose plasma levels, in contrast to N. coriiceps, which developed hypoglycemia without elevating plasma triglyceride levels. The decrease in glutathione reductase levels in N. coriiceps and in superoxide dismutase and catalase in N. rossii suggest that these two species are susceptible to oxidative stress stemming from the production of reactive oxygen species. An increase in magnesium in N. rossii and a decrease in N. coriiceps showed that sewage effluent compromised the control of plasma levels of this cation. Although phylogenetically close, both species of Antarctic fish exhibited different metabolic responses to the sewage effluent, with N. coriiceps showing greater susceptibility to the toxic effects of the pollutants. The present study suggests that the biochemical responses of these two species are potential indicators of metabolic changes caused by sewage effluents.

Dual RNA-seq transcriptional analysis of wheat roots colonized by Azospirillum brasilense reveals up-regulation of nutrient acquisition and cell cycle genes.

BACKGROUND: The rapid growth of the world's population demands an increase in food production that no longer can be reached by increasing amounts of nitrogenous fertilizers. Plant growth promoting bacteria (PGPB) might be an alternative to increase nitrogenous use efficiency (NUE) in important crops such wheat. Azospirillum brasilense is one of the most promising PGPB and wheat roots colonized by A. brasilense is a good model to investigate the molecular basis of plant-PGPB interaction including improvement in plant-NUE promoted by PGPB. RESULTS: We performed a dual RNA-Seq transcriptional profiling of wheat roots colonized by A. brasilense strain FP2. cDNA libraries from biological replicates of colonized and non-inoculated wheat roots were sequenced and mapped to wheat and A. brasilense reference sequences. The unmapped reads were assembled de novo. Overall, we identified 23,215 wheat expressed ESTs and 702 A. brasilense expressed transcripts. Bacterial colonization caused changes in the expression of 776 wheat ESTs belonging to various functional categories, ranging from transport activity to biological regulation as well as defense mechanism, production of phytohormones and phytochemicals. In addition, genes encoding proteins related to bacterial chemotaxi, biofilm formation and nitrogen fixation were highly expressed in the sub-set of A. brasilense expressed genes. CONCLUSIONS: PGPB colonization enhanced the expression of plant genes related to nutrient up-take, nitrogen assimilation, DNA replication and regulation of cell division, which is consistent with a higher proportion of colonized root cells in the S-phase. Our data support the use of PGPB as an alternative to improve nutrient acquisition in important crops such as wheat, enhancing plant productivity and sustainability.

Physiological strategies of acute thermal conditions of Rhamdia voulezi collected in the Iguaçu river watershed, Paraná, Brazil: biochemical markers of metabolic and oxidative stress.

Thermal pollution creates substantial challenges that alter energy demand and produce reactive oxygen species that damage fish DNA, proteins, and lipids. Rhamdia voulezi is a species of fish native to the Iguaçu river, Paraná, Brazil, that does not have scientific records of minimum (CTmin) and maximum (CTmax) temperatures required for survival. As it is a top predator species in the food chain and lives at temperatures below 22 °C, the loss of the species can cause functional problems in controlling the ecosystem and energy flow. The study evaluated the tissue metabolism of the brain, heart, and muscle of R. voulezi (n = 72) subjected to acute thermal stress of 31 °C for 2, 6, 12, 24, and 96 h after acclimatization to 21 °C. The biochemical markers SOD, GPx, MDH, HK, and CK of the brain, PCO of the heart and CAT, glycogen, G6PDH, and ALT of muscle were significant. PCA, IBR, thermal sensitive, and condition factor suggested that R. voulezi has different physiological strategies for acclimatization to 31 °C to mobilize and sustain the metabolic needs of oxygenation and energy allocation/utilization for tissue ATP production.

Correction: Boia-Ferreira et al. TCTP from Loxosceles Intermedia (Brown Spider) Venom Contributes to the Allergic and Inflammatory Response of Cutaneous Loxoscelism. Cells 2019, 8, 1489.

In the original publication [...].

Influence of acute heat shock on antioxidant defense of tropical fish, Psalidodon bifasciatus.

Psalidodon bifasciatus is a fish species sensitive to physical and chemical changes in water. It serves as a good bioindicator of temperature variations and is utilized in environmental monitoring studies in Brazilian rivers. The objective of this study was to evaluate antioxidant defense biomarkers in the heart, brain, and muscle of P. bifasciatus exposed to a 10 °C thermal increase. P. bifasciatus were collected and divided into a control group (21 °C) and groups subjected to thermal shock (31 °C) for periods of 2, 6, 12, 24, and 48h. Two-way ANOVA indicated that a 10 °C temperature increase caused oxidative stress in P. bifasciatus. This was evidenced by altered levels of lipid peroxidation (LPO), carbonylated proteins (PCO), and glutathione peroxidase (GPx) in the heart, catalase (CAT) and LPO in the brain, and LPO in the muscle. Principal component analysis (PCA) and integrated biomarker response (IBR) analysis indicated that, compared to the heart and muscle, the brain exhibited a greater activation of the antioxidant response. Sensitivity analysis indicated that the muscle was the most sensitive organ, followed by the brain and heart. Our results indicate that the stress response is tissue-specific through the activation of distinct mechanisms. These responses may be associated with the tissue's function as well as its energy demand. As expected, P. bifasciatus showed changes in response to thermal stress, with the brain showing the greatest alteration in antioxidant defenses and the muscle being the most sensitive tissue.

Integrated biomarker response index as an ally in the observation of metabolic biomarkers in muscle of Astyanax lacustris exposed to thermal variation.

Temperature is an important factor that conditions the physiological responses of fish, generating a stressful condition when in non-ideal parameters. Thus, the objective was to evaluate metabolic aspects in the muscle of fish Astyanax lacustris submitted to thermal shock. The specimens were subjected to 2, 6, 12, 24, 48, and 96 h of exposure to 15 °C and 31 1 °C, with their respective controls 23 °C. At 15 °C there was a reduction in glycogenolysis in the initial periods indicated by changes in glycogen phosphorylase (GP), pyruvate and lactate. Subsequently, there was an increase in GP activity, pyruvate levels and hexokinase activity in the next time periods, suggesting an increase in energy demand. At 31 °C there was observed low need for the protein metabolism, indicated by reduction in the activity of aspartate aminotransferase and alanine aminotransferase. At 15 °C, initial periods show an increase in glutathione reductase activity and an increase in carbonylated protein levels, indicating induction of oxidative stress for muscle. At 31 °C, there was a punctual increase in reduced glutathione levels at 24 h. In addition, the integrated biomarker response index proved to be a good ally in the evaluation of a set of biomarkers, corroborating the results observed by the biomarkers individually. Thus, it is possible to conclude that the acute thermal shock affects the metabolism of A. lacustris muscle, which undergoes rearrangements to deal with temperature, where 15 °C is more stressful than 31 °C.

The metabolic response of Araucaria angustifolia embryogenic cells to heat stress is associated with their maturation potential.

Araucaria angustifolia is a critically endangered species and its distribution can be affected by an increase in temperature. In this study, we evaluated the effects of heat stress (30°C) on Araucaria angustifolia cell lines responsive (SE1) and non-responsive (SE6) to the development of somatic embryos. The viability of both cell lines was reduced by heat stress and mitochondria were the organelles most affected. Heat stress for 24h increased the reactive oxygen species (ROS) levels in SE1 cells, followed by a reduction at 48 and 72h. In SE6 cells, an increase occurred after 24 and 48h of stress, returning to control levels at 72h. H2 O2 levels were increased after 24h for both SE1 and SE6 cells, being higher for SE6. Interestingly, at 48 and 72h, H2 O2 levels decreased in SE1 cells, while in SE6, the values returned to the control levels. The respiration of SE6 cells in the presence of oxidisable substrates was inhibited by heat stress, in agreement with the high lipid peroxidation levels. The AaSERK1 gene was identified in both cultures, with greater expression in the SE1 line. Heat stress for 24 and 48h increased gene expression only in this cell line. The activity of peroxidase, superoxide dismutase and enzymes of the glutathione/ascorbate cycle was increased in both cell lines subjected to heat stress. Catalase activity was increased only in SE6 cells at 72h of exposure. These results show that responsive SE1 cells can modulate ROS levels more efficiently than SE6 when these cells are stressed by heat. This ability may be related to the maturation capacity of these cells.

Bax/Bcl-2 protein expression ratio and leukocyte function are related to reduction of Walker-256 tumor growth after ß-hydroxy-ß-methylbutyrate (HMB) administration in Wistar rats.

This study investigated the mechanisms by which ß-hydroxy-ß-methylbutyrate (HMB) administration in rats reduces Walker-256 tumor growth. Male Wistar rats were supplemented with HMB (76 mg/kg/day) (HW), or a placebo (W), during 8 wk by gavage. At the 6th wk, rats were inoculated with a suspension of Walker 256 tumor cells (3 × 10(7)/mL). Fifteen days after inoculation, the HW group showed higher glycemia (109.4 ± 5.53 vs. 89.87 ± 7.02 mg/dL, P < 0.05) and lower spleen (1.35 ± 0.05 vs. 1.65 ± 0.12 g, P < 0.05) and tumor weights (9.64 ± 1.07 vs. 13.55 ± 1.19 g, P < 0.05) compared to the W group. Tumor cells extracted from the HMB-treated rats displayed a 36.9% decrement in rates of proliferation ex vivo and a significant increase in the Bax/Bcl-2 protein expression ratio in comparison to those extracted from the placebo-treated rats (P < 0.05). Both phagocytic capacity and H(2)O(2) production rates were higher in polymorphnuclear cells that were obtained from the blood of the HW rats in comparison to those from the W rats (P < 0.05). Reduction of necrotic regions and an intense infiltration of leukocytes and activated granulocytes in HW were evident by transmission electron microscopy. Our findings suggest that HMB supplementation decreases tumor burden by modifying the inner environment of tumor cells and by interfering with blood leukocyte function.