Cold activity of Beauveria and Metarhizium, and thermotolerance of Beauveria

Heat and cold are environmental abiotic factors that restrict the use of entomopathogenic fungi as agents for biological control of insects. The thermotolerance and cold activity of 60 entomopathogenic fungal isolates, including five species of Beauveria and one isolate of Engyodontium albus (=Beauveria alba) were examined as to tolerance of temperatures that might be encountered during field use. In addition, cold activity of eight Metarhizium spp. isolates was evaluated. The isolates were from various geographic regions, arthropod hosts or substrates. High variability in conidial thermotolerance was found among the Beauveria spp. isolates after exposure to 45 °C for 2 h, as evidenced by low (0-20%), medium (20-60%), or high germination (60-80%). The thermal death point (0% germination) for three rather thermotolerant B. bassiana isolates (CG 138, GHA and ARSEF 252) was 46 °C for 6 h. At low temperatures (5 °C), with few exceptions (e.g. CG 66, UFPE 479, CG 227, CG 02), most of the B. bassiana isolates germinated well (ca. 100%). On the other hand, only one isolate of Metarhizium sp. was cold-active (i.e. ARSEF 4343 from Macquarie Island, 54.4°S, Australia). This probably is a M. frigidum isolate. The E. albus isolate (UFPE 3138) was the most susceptible isolate to both heat and cold stress. Isolates ARSEF 252 and GHA of B. bassiana, on the other hand, presented exceptionally high thermotolerance and cold activity. Some isolates with high cold activity, however, were thermosensitive (e.g. ARSEF 1682) and others with high thermotolerance had low cold activity (e.g. CG 227). An attempt to correlate the latitude of origin with thermotolerance or cold activity indicated that B. bassiana isolates from higher latitudes were more cold-active than isolates from nearer the equator, but there was not a similar correlation for heat.     

Effects of rapamycin on cell proliferation and phosphorylation of mTOR and p70 in HepG2 and HepG2 cells overexpressing constitutively active Akt/PKB

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that plays an important role in the regulation of cell proliferation and protein synthesis through the activation of its downstream target ribosomal p70 S6 kinase (p70^S6K). The levels of p-mTOR are regulated by the protein kinase B (Akt/PKB). Therefore, the effects of insulin and rapamycin (an inhibitor of mTOR) on the phosphorylation of mTOR (Ser 2448) and p70^S6K (Thr 389) as well as on cell proliferation in parental HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB (HepG2-CA-Akt/PKB) were studied. Insulin increased the levels of phosphorylated mTOR and p70^S6K in both the cell lines. Rapamycin treatment partially decreased the phosphorylation of mTOR but completely abolished the phosphorylation of p70^S6K in the absence as well as presence of insulin in both cell lines. The effect of insulin and rapamycin on the cell proliferation in both cell lines was further studied. In the presence of serum, parental HepG2 cells and HepG2-CA-Akt/PKB showed an increase in cell proliferation until 120 and 168 h respectively. Rapamycin inhibited cell proliferation under all experimental conditions more evident under serum deprived conditions. Parental HepG2 cells showed decline in the cell proliferation after 48 h and the presence of insulin prolonged cell survival until 120 h and this effect were also inhibited by rapamycin under serum deprived conditions. On the contrary, HepG2-CA-Akt/PKB cells continued proliferation until 192 h. The effects of insulin on cell proliferation were more pronounced in parental HepG2 cells as compared to HepG2-CA-Akt/PKB cells. Long term effects of rapamcyin significantly decreased the levels of p-mTOR (Ser 2448) both in the presence and absence of insulin in these cells. A positive correlation between the levels of p-mTOR (Ser2448) and cell proliferation was observed (99% confidence interval, r² = 0.525, p < 0.0001). These results suggest that rapamycin causes a decline in the cell growth through the inhibition of mTOR.     

Cytotoxic triterpenoid saponins acylated with monoterpenic acids from fruits of Gleditsia caspica Desf.

Four bisdesmosidic triterpenoid saponins named caspicaosides A-D, were isolated from the fruits of Gleditsia caspica Desf. Their structures were determined by NMR spectroscopy including HOHAHA, ¹H-¹H COSY, ROE, HMQC, HMBC experiments and HRFAB-MS as well as acid hydrolysis. The four 3,28-O-bisdesmosidic triterpenoid saponins comprised echinocystic acid as the aglycone and common oligosaccharide moieties at C3 and C28. The saccharide moiety at C-3 was identified as β-d-xylopyranosyl-(1 → 2)-α-l-arabinopyranosyl-(1 → 6)-β-d-glucopyranosyl while that at C-28 was determined as β-d-xylopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-[α-l-rhamnopyranosyl-(1 → 6)-]β-d-glucopyranosyl. The pentasaccharide moiety linked to C-28 was acylated with monoterpenic acid and or monoterpene-arabinoside moieties at C-2 or C-2 and C-3 of the terminal rhamnose unit. The isolated saponins were assayed for their in vitro cytotoxicities against the three human tumor cell lines HepG2, A549 and HT29 using MTT method. The results showed that caspicaosides B and C bearing two and three monoterpene units, respectively, exhibited significant cytotoxic activities against the used cell lines with IC₅₀ values 1.5-6.5 μM. Caspicaosides A and D with one monoterpene unit exhibited significant cytotoxic activities on HepG2 cell line with IC₅₀ values equal to 4.5 and 5.4 μM, respectively, and IC₅₀ values >10 μM against the other two cell lines. The number of monoterpene units seems to play a main role in determining the activity.     

Intraspecific tolerance of Metarhizium anisopliae conidia to the upper thermal limits of summer with a description of a quantitative assay system

Conidial tolerance to the upper thermal limits of summer is important for fungal biocontrol agents, whose conidia are formulated into mycoinsecticides for field application. To develop an efficient assay system, aerial conidia of eight Metarhizium anisopliae, four M. anisopliae var. anisopliae, and six M. anisopliae var. acridum isolates with different host and geographic origins were wet-stressed for ≤180 min at 48 °C or incubated for 14 d colony growths at 10-35 °C. The survival ratios (relative to unstressed conidia) of each isolate, examined at 15-min intervals, fit a logistic equation (r² ≥ 0.975), yielding median lethal times (LT₅₀s) of 14.3-150.3 min for the 18 isolates stressed at 48 °C. Seven grasshopper isolates from Africa had a mean LT₅₀ of 110 (73-150) min, but could not grow at 10 or 15 °C. The mean LT₅₀ of five non-grasshopper isolates capable of growing at 10-35 °C was 16 (10-26) min only. Three isolates with typically low (type I), medium (type II), and high (type III) levels of tolerance to 48 °C were further assayed for ≤4-d tolerance of their conidia to the wet stress at 38, 40, 42, or 45 °C. The resultant LT₅₀s decreased to 20, 53 and 167 min at 48 °C from 507, 1612, and 8256 min at 38 °C for types I, II and III, respectively. For the distinguished types, the logarithms of the LT₅₀s were significantly correlated to the temperatures of 38-48 °C with an inverse linearity (r² ≥ 0.88). The method developed to assay quantitatively fungal thermotolerance would be useful for screening of fungal candidates for improved pest control in summer.     

Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae)

In order to preserve key activities or improve survival, insects facing variable and unfavourable thermal environments may employ physiological adjustments on a daily basis. Here, we investigate the survival of laboratory-reared adult Cydia pomonella at high or low temperatures and their responses to pre-treatments at sub-lethal temperatures over short time-scales. We also determined critical thermal limits (CTLs) of activity of C. pomonella and the effect of different rates of cooling or heating on CTLs to complement the survival assays. Temperature and duration of exposure significantly affected adult C. pomonella survival with more extreme temperatures and/or longer durations proving to be more lethal. Lethal temperatures, explored between −20 °C to −5 °C and 32 °C to 47 °C over 0.5, 1, 2, 3 and 4 h exposures, for 50% of the population of adult C. pomonella were −12 °C for 2 h and 44 °C for 2 h. Investigation of rapid thermal responses (i.e. hardening) found limited low temperature responses but more pronounced high temperature responses. For example, C. pomonella pre-treated for 2 h at 5 °C improved survival at −9 °C for 2 h from 50% to 90% (p < 0.001). At high temperatures, pre-treatment at 37 °C for 1 h markedly improved survival at 43 °C for 2 h from 20% to 90% (p < 0.0001). We also examined cross-tolerance of thermal stressors. Here, low temperature pre-treatments did not improve high temperature survival, while high temperature pre-treatment (37 °C for 1 h) significantly improved low temperature survival (−9 °C for 2 h). Inducible cross-tolerance implicates a heat shock protein response. Critical thermal minima (CTmin) were not significantly affected by cooling at rates of 0.06, 0.12 and 0.25 °C min⁻¹ (CTmin range: 0.3-1.3 °C). By contrast, critical thermal maxima (CTmax) were significantly affected by heating at these rates and ranged from 42.5 to 44.9 °C. In sum, these results suggest pronounced plasticity of acute high temperature tolerance in adult C. pomonella, but limited acute low temperature responses. We discuss these results in the context of local agroecosystem microclimate recordings. These responses are significant to pest control programmes presently underway and have implications for understanding the evolution of thermal tolerance in these and other insects.     

Mild thermotolerance induced at 40 °C increases antioxidants and protects HeLa cells against mitochondrial apoptosis induced by hydrogen peroxide: Role of p53

Exposure of cells to mild temperatures (40 °C) induces thermotolerance, which renders cells resistant to subsequent toxic insults. Thermotolerance is usually associated with accumulation of heat shock proteins. This study determines whether mild thermotolerance (40 °C, 3 h) can induce other defense proteins (e.g. antioxidants, anti-apoptosis proteins), and protect HeLa cells against apoptosis triggered by H₂O₂. Protein expression and enzymatic activity of MnSOD and catalase were increased in thermotolerant cells, as well as intracellular glutathione levels and γ-glutamylcysteine synthetase expression. Furthermore, levels of reactive oxygen species (ROS) were increased in thermotolerant cells, which caused mitochondrial membrane hyperpolarisation. Mild thermotolerance inhibited activation of the mitochondrial cascade of apoptosis by H₂O₂. This entailed inhibition of mitochondrial Bax translocation, mitochondrial membrane depolarisation, cytochrome c release, activation of caspases-9/-3 and chromatin condensation. Thermotolerance inhibited H₂O₂-induced caspase-independent apoptosis involving apoptosis-inducing factor, and activation of p53 and increased expression of its target protein PUMA. Thermotolerance induced at mild physiological temperatures protects cells against both caspase-dependent and caspase-independent apoptosis triggered by oxidative stress.     

Thermotolerance and HSP70 expression in the Mediterranean fruit fly Ceratitis capitata

The relationship between Hsp70 expression and thermotolerance has been well documented in Drosophila melanogaster. However, there is limited information on this relationship in other insect species. In this report we describe the Hsp70-thermotolerance relationship in one of the major fruit fly pests, Ceratitis capitata (medfly). Hsp70 expression and thermotolerance were assayed at a range of temperatures in several stages of medfly development. The most thermotolerant stage was found to be the late larval stage (100% survival at 41 °C) followed by adult flies and late embryos (100% survival at 39 °C). These three stages showed a positive relationship between Hsp70 expression and thermotolerance. Mid-larval and mid-embryonic stages were found less thermotolerant and the Hsp70-thermotolerance relationship was not evident. Early embryos did not express Hsp70 at any temperature and exhibited the lowest thermotolerance. The relationship between Hsp70 and inducible thermotolerance was also studied in late larvae. A pretreatment at 37-39 °C increased thermotolerance at higher temperatures by approximately 1 °C. In parallel, the pretreatment increased Hsp70 expression suggesting a close link between Hsp70 expression and inducible thermotolerance. The increased Hsp70 levels after pretreatment were found to be due to the increased levels of the hsp70 RNA.     

Application of screen-printed microband biosensors to end-point measurements of glucose and cell numbers in HepG2 cell culture

Microband glucose biosensors were produced by insulating and sectioning through a screen-printed, water-based carbon electrode containing cobalt phthalocyanine redox mediator and glucose oxidase enzyme. Under quiescent conditions at 37 °C, at an operating potential of +0.4 V, they produced an amperometric response to glucose in buffer solutions with a sensitivity of 26.4 nA/mM and a linear range of 0.45 to 9.0 mM. An optimal pH value of 8.5 was obtained under these conditions, and a value for activation energy of 40.55 kJ mol⁻¹ was calculated. In culture medium (pH 7.3), a sensitivity of 13 nA/mM was obtained and the response was linear up to 5 mM with a detection limit of 0.5 mM. The working concentration was up to 20 mM glucose with a precision of 11.3% for replicate biosensors (n = 4). The microband biosensors were applied to determine end-point glucose concentrations in culture medium by monitoring steady-state current responses 400 s after transfer of the biosensors into different sample solutions. In conjunction with cultures of HepG2 (human Caucasian hepatocyte carcinoma) cells, current responses obtained in 24-h supernatants showed an inverse correlation (R² = 0.98) with cell number, indicating that the biosensors were applicable for monitoring glucose metabolism by cells and of quantifying cell number. Glucose concentrations determined using the biosensor assay were in good agreement, for concentrations up to 20 mM, with those determined spectrophotometrically (R² = 0.99). This method of end-point glucose determination was used to provide an estimated rate of glucose uptake for HepG2 cells of 7.9 nmol/(10⁶ cells min) based on a 24-h period in culture.     

Heat inactivation of Escherichia coli K12 MG1655: Effect of microbial metabolites and acids in spent medium

Aim

The effect of spent medium, obtained after different time-temperature pre-histories, on the heat inactivation of Escherichia coli K12 MG1655 is studied.

Methods and results

Stationary E. coli cells were heated in BHI broth (initial pH 7.5) at different time-temperature scenarios, i.e., (1) 30 °C to 55 °C at 0.14 °C/min, (2) 30 °C to 42 °C at 0.14 °C/min and (3) 30 °C to 42 °C at 0.8 °C/min. After the heat treatment, spent medium was filter-sterilized, non-stressed cells were added and inactivation experiments took place at 54 °C and 58 °C. In all scenarios, increased resistance was observed. The main characteristics of the spent medium - compared to the unmodified BHI broth - are (1) the presence of proteins (proven via SDS-PAGE) and (2) a lower pH of approximately 6. Possibly, the increased resistance is due to these proteins and/or the lower pH. Further experiments revealed that each factor separately may lead to an increased heat resistance.

Conclusions

It can be concluded that this increased heat resistance resulted from both the presence of the heat shock proteins in the spent medium and the lowered pH. Experiments, which separate both effects, showed that mainly the lower pH resulted in the increased thermotolerance.

Significance and impact of study

This study may lead to a better understanding and control of the heat stress adaptation phenomenon as displayed by E. coli at lethal temperatures. Therefore, it contributes to an improved assessment of the effect of temperature during thermal processes in the food industry.     

Thermal preference and tolerance of Megastrea (Lithopoma) undosa (Wood, 1828; Gastropoda: Turbinidae)

The thermoregulatory behavior of the wavy turban snail Megastrea (Lithopoma) undosa was determined in a horizontal thermal gradient and was 16.31 in day cycle and 14.4 °C in night cycle. Displacement velocity of adults was 29.3±4.2 cm h⁻¹ during the light phase and 26.1±3.2 cm h⁻¹ during the dark phase. The critical thermal maxima of the wavy turban snail were determined. As a measure of thermal tolerance, snails were subjected to increasing water temperatures at a rate of 1 °C every 30 min until they were detached from the substrate. The critical thermal maximum at 50% was 29.7 °C.     

Heat tolerance, behavioural temperature selection and temperature-dependent respiration in larval Octopus huttoni

This study reports temperature effects on paralarvae from a benthic octopus species, Octopus huttoni, found throughout New Zealand and temperate Australia. We quantified the thermal tolerance, thermal preference and temperature-dependent respiration rates in 1-5 days old paralarvae. Thermal stress (1 °C increase h⁻¹) and thermal selection (∼10-24 °C vertical gradient) experiments were conducted with paralarvae reared for 4 days at 16 °C. In addition, measurement of oxygen consumption at 10, 15, 20 and 25 °C was made for paralarvae aged 1, 4 and 5 days using microrespirometry. Onset of spasms, rigour (CT_max) and mortality (upper lethal limit) occurred for 50% of experimental animals at, respectively, 26.0±0.2 °C, 27.8±0.2 °C and 31.4±0.1 °C. The upper, 23.1±0.2 °C, and lower, 15.0±1.7 °C, temperatures actively avoided by paralarvae correspond with the temperature range over which normal behaviours were observed in the thermal stress experiments. Over the temperature range of 10 °C-25 °C, respiration rates, standardized for an individual larva, increased with age, from 54.0 to 165.2 nmol larvae⁻¹ h⁻¹ in one-day old larvae to 40.1-99.4 nmol h⁻¹ at five days. Older larvae showed a lesser response to increased temperature: the effect of increasing temperature from 20 to 25 °C (Q₁₀) on 5 days old larvae (Q₁₀=1.35) was lower when compared with the 1 day old larvae (Q₁₀=1.68). The lower Q₁₀ in older larvae may reflect age-related changes in metabolic processes or a greater scope of older larvae to respond to thermal stress such as by reducing activity. Collectively, our data indicate that temperatures >25 °C may be a critical temperature. Further studies on the population-level variation in thermal tolerance in this species are warranted to predict how continued increases in ocean temperature will limit O. huttoni at early larval stages across the range of this species.     

Thermal dependence of cardiac SR Ca-ATPase from fish and mammals

The thermal sensitivity of metabolic performance in vertebrates requires a better understanding of the temperature sensitivity of cardiac function. The cardiac sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2) is vital for excitation–contraction (E–C) coupling and intracellular Ca²⁺ homeostasis in heart cells. To better understand the thermal dependency of cardiac output in vertebrates, we present comparative analyses of the thermal kinetics properties of SERCA2 from ectothermic and endothermic vertebrates. We directly compare SR ventricular microsomal preparations using similar experimental conditions from sarcoplasmic reticulum isolated from cardiac tissues of mammals and fish. The experiments were designed to delineate the thermal sensitivity of SERCA2 and its role in thermal sensitivity Ca²⁺ uptake and E–C coupling. Ca²⁺ transport in the microsomal SR fractions from rabbit and bigeye tuna (Thunnus obesus) ventricles were temperature dependent. In contrast, ventricular SR preparations from coho salmon (Onchorhychus kisutch) were less temperature dependent and cold tolerant, displaying Ca²⁺ uptake as low as 5 °C. As a consequence, the Q₁₀ values in coho salmon were low over a range of different temperature intervals. Maximal Ca²⁺ transport activity for each species occurred in a different temperature range, indicating species-specific thermal preferences for SERCA2 activity. The mammalian enzyme displayed maximal Ca²⁺ uptake activity at 35 °C, whereas the fish (tuna and salmon) had maximal activity at 30 °C. At 35 °C, the rate of Ca²⁺ uptake catalyzed by the bigeye tuna SERCA2 decreased, but not the rate of ATP hydrolysis. In contrast, the salmon SERCA2 enzyme lost its activity at 35 °C, and ATP hydrolysis was also impaired. We hypothesize that SERCA2 catalysis is optimized for species-specific temperatures experienced in natural habitats and that cardiac aerobic scope is limited when excitation–contraction coupling is impaired at low or high temperatures due to loss of SERCA2 enzymatic function.     

Determination of thermal properties of composting bulking materials

Thermal properties of compost bulking materials affect temperature and biodegradation during the composting process. Well determined thermal properties of compost feedstocks will therefore contribute to practical thermodynamic approaches. Thermal conductivity, thermal diffusivity, and volumetric heat capacity of 12 compost bulking materials were determined in this study. Thermal properties were determined at varying bulk densities (1, 1.3, 1.7, 2.5, and 5 times uncompacted bulk density), particle sizes (ground and bulk), and water contents (0, 20, 50, 80% of water holding capacity and saturated condition). For the water content at 80% of water holding capacity, saw dust, soil compost blend, beef manure, and turkey litter showed the highest thermal conductivity (K) and volumetric heat capacity (C) (K: 0.12–0.81 W/m °C and C: 1.36–4.08 MJ/m³ °C). Silage showed medium values at the same water content (K: 0.09–0.47 W/m °C and C: 0.93–3.09 MJ/m³ °C). Wheat straw, oat straw, soybean straw, cornstalks, alfalfa hay, and wood shavings produced the lowest K and C values (K: 0.03–0.30 W/m °C and C: 0.26–3.45 MJ/m³ °C). Thermal conductivity and volumetric heat capacity showed a linear relationship with moisture content and bulk density, while thermal diffusivity showed a nonlinear relationship. Since the water, air, and solid materials have their own specific thermal property values, thermal properties of compost bulking materials vary with the rate of those three components by changing water content, bulk density, and particle size. The degree of saturation was used to represent the interaction between volumes of water, air, and solids under the various combinations of moisture content, bulk density, and particle size. The first order regression models developed in this paper represent the relationship between degree of saturation and volumetric heat capacity (r = 0.95–0.99) and thermal conductivity (r = 0.84–0.99) well. Improved knowledge of the thermal properties of compost bulking materials can contribute to improved thermodynamic modeling and heat management of composting processes.     

Thermal tolerance of Litopenaeus vannamei (Crustacea: Penaeidae) acclimated to four temperatures

Critical thermal minima (CTMin) and maxima (CTMax) values were determined for the Pacific white shrimp Litopenaeus vannamei post-larvae and juveniles at four different acclimation temperatures (15, 20, 25, and 30 °C). The CTMin of shrimp at these acclimation temperatures were 7.82, 8.95, 9.80, and 10.96 °C for post-larvae and 7.50, 8.20, 10.20, and 10.80 °C for juveniles, respectively, at 1 °C h⁻¹ cooling rate. The CTMax values were 35.65, 38.13, 39.91, and 42.00 °C for post-larvae and 35.94, 38.65, 40.30, and 42.20 °C for juveniles at the respective acclimation temperatures. Both acclimation temperature and size of the shrimp affected CTMin values of L. vannamei (P<0.01). Overall, juveniles displayed significantly lower CTMin values than the post-larvae (P<0.0001). However, the CTMax response by post-larvae and juveniles were not significantly different from each other and no interaction was determined between the acclimation temperature and development stage (P>0.01). The area of the thermal tolerance polygon over four acclimation temperatures (15, 20, 25, and 30 °C) for the post-larvae of L. vannamei was calculated to be 434.94 °C². The acclimation response ratio (ARR) values were high ranging from 0.35 to 0.44 for both post-larvae and juveniles. L. vannamei appears to be more sensitive to low temperatures than other penaeid species and its cold tolerance zone ranged from 7.5 to 11 °C. In successful aquaculture temperature must never fall below 12 °C to prevent mortalities. Upper thermal tolerance is less of a problem as in most subtropical regions maximum water temperature rarely exceeds 34 °C, but care should be given if shallow ponds with low water renewal rate are being used.     

Phenylpropanoids from Juglans mandshurica exhibit cytotoxicities on liver cancer cell lines through apoptosis induction

Three new phenylpropanoids (1–3) together with six known congeners (4–9) were isolated from the bark of Juglans mandshurica Maxim using anti-hepatoma activity as a guide. Their structures were determined by comprehensive NMR and HRESIMS spectroscopic data analyses. All the isolated compounds were evaluated for their growth inhibitory activities against two kinds of liver cancer cell lines (HepG2 and Hep3B). Among them, compound 4 showed moderate cytotoxic activities against HepG2 and Hep3B cell lines with IC₅₀ values of 58.58 and 69.87 μM. Compound 5 exhibited 50% cell death rate in HepG2 and Hep3B cell lines at 63.70 and 46.45 μM, respectively. Further observation of morphological changes and Western blot demonstrated that compounds 4 and 5 exhibited their cytotoxic activities through the induction of apoptosis. A structure-activity relationship study suggested that an α, β-unsaturated aldehyde might be the most important functional group.     

Characteristics of the killing mechanism of human natural killer cells against hepatocellular carcinoma cell lines HepG2 and Hep3B

Purpose Unlike normal hepatocytes, most hepatocellular carcinomas (HCCs) are quite resistant to death receptor-mediated apoptosis when the cell surface death receptor is cross linked with either agonistic antibodies or soluble death ligand proteins in vitro. The resistance might play an essential role in the escape from the host immune surveillance; however, it has not been directly demonstrated that HCCs are actually resistant to natural killer (NK) cell-mediated death. Therefore, this study investigated the molecular mechanism of NK cell-mediated cytotoxicity against the HCCs, HepG2, and Hep3B, using two distinct cytotoxic assays: a 4-h ⁵¹Cr-release assay and a 2-h [³H] thymidine release assay which selectively measures the extent of necrotic and apoptotic target cell death, respectively.Methods Most of the target cells exhibited marked morphologic changes when they were co-incubated with the NK cells, and the NK cytotoxicity against these HCCs was comparable to that against K562, a NK-sensitive leukemia cell line, when the cytotoxicity was assessed by a 4-h ⁵¹Cr release assay.Results The NK cells also induced significant apoptotic cell death in the Hep3B targets, but not in the HepG2 targets, when the cytotoxicity was assessed by a 2-h [³H]-thymidine release assay. In agreement with these results, procaspase-3 was activated in the Hep3B targets, but not in the HepG2 targets. Interestingly, mildly fixed NK cells had no detectable activity in the 4-h ⁵¹Cr release assay against both HepG2 and Hep3B targets, while they were similarly effective as the untreated NK cells in the 2-h [³H]-thymidine release assay, suggesting that the level of apoptotic cell death of the Hep3B targets is granule independent and might be primarily mediated by the death ligands of the NK cells.Conclusion This study found that a tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/TRAIL receptor interaction is involved in the NK cell-mediated apoptotic death of the Hep3B targets, but a Fas/Fas ligand (FasL) interaction is not.     

Interaction of 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate with mimetic membranes and cytotoxic effect on leukemic cells

10-(Octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC) is an alkylphospholipid that can interact with cell membranes because of its amphiphilic character. We describe here the interaction of ODPC with liposomes and its toxicity to leukemic cells with an ED-50 of 5.4, 5.6 and 2.9 μM for 72 h of treatment for inhibition of proliferation of NB4, U937 and K562 cell lines, respectively, and lack of toxicity to normal hematopoietic progenitor cells at concentrations up to 25 μM. The ED-50 for the non-malignant HEK-293 and primary human umbilical vein endothelial cells (HUVEC) was 63.4 and 60.7 μM, respectively. The critical micellar concentration (CMC) of ODPC was 200 μM. Dynamic light scattering indicated that dipalmitoylphosphatidylcholine (DPPC) liposome size was affected only above the CMC of ODPC. Differential calorimetric scanning (DCS) of liposomes indicated a critical transition temperature (T_c) of 41.5 °C and an enthalpy (?H) variation of 7.3 kcal mol⁻¹. The presence of 25 μM ODPC decreased T_c and ?H to 39.3 °C and 4.7 kcal mol⁻¹, respectively. ODPC at 250 μM destabilized the liposomes (36.3 °C, 0.46 kcal mol⁻¹). Kinetics of 5(6)-carboxyfluorescein (CF) leakage from different liposome systems indicated that the rate and extent of CF release depended on liposome composition and ODPC concentration and that above the CMC it was instantaneous. Overall, the data indicate that ODPC acts on in vitro membrane systems and leukemia cell lines at concentrations below its CMC, suggesting that it does not act as a detergent and that this effect is dependent on membrane composition.     

Cytotoxic clerodane diterpenoids from Croton crassifolius

Hepatocellular carcinoma (HCC) is the most common type of liver cancer, and treatment options for HCC are limited. In addition, the discovery of new natural compounds with anti-hepatocarcinoma activity is attracting increasing attention. For this reason, phytochemical investigation of Croton crassifolius led to the isolation of 17 diterpenoids, including three new clerodane diterpenoids, named crassifolius A-C (1–3), along with 14 known ones (4–17). Their structures were established by 1D, 2D NMR, HR-ESI-MS, detailed calculated electronic circular dichroism (ECD) spectra and the assistance of quantum chemical predictions (QCP) of ¹³C NMR chemical shifts. The cytotoxicities of all these compounds against human liver cancer lines (HepG2 and Hep3B) were determined. Among them, compound 1 exhibited good cytotoxicity with IC₅₀ value of 17.91 μM against human liver tumor cells Hep3B. Following further studies of the anti-tumor mechanism of compound 1-induced cell growth inhibition, we found that compound 1 caused apoptotic cell death in Hep3B cells by detecting morphologic changes and Western blotting analysis.     

Decolorization of indigo carmine by laccase displayed on Bacillus subtilis spores

Blue multicopper oxidases, laccases displayed on the surface of Bacillus spores were used to decolorize a widely used textile dyestuff, indigo carmine. The laccase-encoding gene of Bacillus subtilis, cotA, was cloned and expressed in B. subtilis DB104, and the expressed enzyme was spontaneously localized on Bacillus spores. B. subtilis spores expressing laccase exhibited maximal activity for the oxidation of 2,2′-azino-bis (3-ethylthiazoline-6-sulfonate) (ABTS) at pH 4.0 and 80 °C, and for the decolorization of indigo carmine at pH 8.0 and 60 °C. The displayed enzyme retained 80% of its original activity after pre-treatment with organic solvents such as 50% acetonitrile and n-hexane for 2 h at 37 °C. The apparent K_m of the enzyme displayed on spores was 443 ± 124 μM for ABTS with a V_max of 150 ± 16 U/mg spores. Notably, 1 mg of spores displaying B. subtilis laccase (3.4 × 10² U for ABTS as a substrate) decolorized 44.6 μg indigo carmine in 2 h. The spore reactor (0.5 g of spores corresponding to 1.7 × 10⁵ U in 50 mL) in a consecutive batch recycling mode decolorized 223 mg indigo carmine/L to completion within 42 h at pH 8.0 and 60 °C. These results suggest that laccase displayed on B. subtilis spores can serve as a powerful environmental tool for the treatment of textile dye effluent.