Effect of pre-treatment and extraction conditions on the antioxidant properties of persimmon (Diospyros kaki) leaves

Persimmon is a very delicious fruit and the leaves of this tree are used as a traditional drug. This study aimed to investigate the effects of drying method (hot air and freeze-drying), extraction temperature (80, 90 and 100 °C) and extraction time (10, 30, 60 and 120 min) and harvest stage (flowering and fruiting) on the antioxidant contents and antioxidant activity of persimmon leaves. The results showed that the highest antioxidants were obtained in both methods of drying. Also, 100 °C for 120 min of extraction gave the highest antioxidant contents, but with no significant difference compared to 90 °C for 60 min of extraction. Persimmon leaves collected during flowering stage had the maximum amount of antioxidants compared to the fruiting stage. Finally, it can be said that persimmon leaves harvested during flowering stage and treated by hot air drying with these extraction conditions (90 °C for 60 min) are richer in bioactive compounds.     

Pivotal role of reactive oxygen species as intracellular mediators of hyperthermia-induced apoptosis

The effects of cellular antioxidant capacity on hyperthermia (HT)-induced apoptosis and production of antiapoptotic heat shock proteins (HSPs) were investigated in HL-60 cells and in HL-60AR cells that are characterized by an elevated endogenous catalase activity. Exposure of both cell lines to 43 degrees C for 1 h initiated apoptosis. Apoptosis peaked at 3-6 h after heat exposure in the HL-60 cells. Whereas HL-60AR cells were partially protected against HT-induced apoptosis at these early time points, maximal levels of apoptosis were detected later, i.e. 12-18 h after heat exposure. This differential induction of apoptosis was directly correlated to the induction of the antiapoptotic HSP27 and HSP70. In particular, in the HL-60 cells HSP27 was significantly induced at 12-18 h after exposure to 43 degrees C when apoptosis dropped. In contrast, coinciding with the late onset of apoptosis in HL-60AR cells at that time HL-60AR cells lacked a similar HSP response. In line with the higher antioxidant capacity HL-60AR cells accumulated reactive oxygen species to a lesser degree than HL-60 cells after heat treatment. Protection from HT-induced apoptosis as well as diminished heat-induced HSP27 expression was also observed after cotreatment of HL-60 cells with 43 degrees C and catalase but not with superoxide dismutase. These data emphasize the pivotal role of reactive oxygen species for HT induced pro- and antiapoptotic pathways.     

Anticancer efficacies of doxorubicin,verapamil and quercetin on FM3A cells under hyperthermic temperature

Hyperthermia (HT) in combination with anticancer drugs (ACDs) had proven to more efficacious in various cancers, although efficacies vary according to chemotherapeutic compounds and cancer types. Presently there are few data that compares anticancer efficacies among ACDs under hyperthermic conditions. Therefore, we selected three commonly used ACDs (quercetin, verapamil and doxorubicin) and compared their antitumor effects when each was treated with 43°C HT exposure. Firstly, FM3A, a murine breast cancer cell line, was treated with each ACD for 1 h followed by 43°C exposure for additional 1 h, and examined the effects of: 1) each drug, 2) 43°C HT exposure, and 3) the combination of each drug and 43°C HT exposure for 1, 6 and 24 h. The determined overall effects on FM3A cells were arrested cell proliferation, clonogenic efficiency and apoptosis. Pre-treatment of FM3A cells to each ACD followed by 43°C HT exposure produced greater antitumor effects including suppressed cell proliferation, reduced clonogenic efficiency and increased apoptotic cell death, compared to ACD treatment or HT exposure alone. Apoptotic cell death occurred in a time-dependent manner. Among the ACDs, antitumor efficacies varied in the order of doxorubicin > verapamil > quercetin. It was concluded that heat exposure during ACD treatment of caner cells may be an important factor to get a better antitumor benefit, even though this benefit may differ from one drug to another.     

Inhibition of protein synthesis sensitizes thermotolerant cells to heat shock induced apoptosis

Hyperthermia is a potent inducer of apoptosis in many cell lines. A brief exposure to mildly elevated temperatures elicits a transient state of augmented resistance to subsequent thermal stress. Here we show that a hyperthermic treatment of 43°C for 1 h is sufficient to induce apoptosis in the cell line HL-60. This observation is based on morphologic evaluation and on comet assay results (an extremely sensitive method of detecting and quantifying apoptotic DNA fragmentation in individual cells). The thermotolerance phenomenon was also verified in the same manner by giving the cells a brief 30 min sub-lethal heat conditioning treatment at 43°C followed by a 6 h incubation time prior to the administration of a lethal heat load (43°C for 1 h). We observed a dramatic decrease in resultant apoptoses in the thermotolerized cells in comparison to unconditioned cells. We assessed the necessity of de novo protein synthesis in the protective phenomenon. When the conditioned cells were given a cycloheximide treatment prior to heat conditioning we saw a sensitization of the conditioned cells to secondary thermal injury. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of heat stress on skeletal muscle contractile properties

An elevated heat-shock protein (HSP) content protects cells and tissues, including skeletal muscles, from certain stressors. We determined if heat stress and the elevated HSP content that results is correlated with protection of contractile characteristics of isolated fast and slow skeletal muscles when contracting at elevated temperatures. To elevate muscle HSP content, one hindlimb of Sprague–Dawley rats (21–28 days old, 70–90 g) was subjected to a 15 min 42 °C heat-stress. Twenty-four hours later, both extensor digitorum longus (EDL) and soleus muscles were removed, mounted in either 20 °C or 42 °C Krebs-Ringer solution, and electrically stimulated. Controls consisted of the same muscles from the contra-lateral (non-stressed) hindlimbs as well as muscles from other (unstressed) animals. Isolated muscles were twitched and brought to tetanus every 5 min for 30 min. As expected, HSP content was elevated in muscles from the heat-stressed limbs when compared with controls. Regardless of prior treatment, both EDL and soleus twitch tensions were lower at 42 °C when compared with 20 °C. In addition, when incubated at 42 °C, both muscles showed a drop in twitch tension between 5 and 30 min. For tetanic tension, both muscles also showed an increase in tension between 5 and 30 min when stimulated at 20 °C regardless of treatment but when stimulated at 42 °C no change was observed. No protective effect of an elevated HSP content was observed for either muscle. In conclusion, although heat stress caused an elevation in HSP content, no protective effects were conferred to isolated contracting muscles.     

Flavonoids in the Flowers of Gnaphalium affine D. Don

Thermonsenstivie division mutants were derived from Bacillus subtilis Marburg 168 thy trp₂ by means of membrane filtration after nitrosoguanidine mutagenesis. Among them, ts42 requiring uracil for normal growth at 48°C was investigated.

In the absence of uracil, the mutant cells grew normally at 37°C and stopped dividing after temperature shift to 48°C resulting in filaments of two to four times length of normal rods. The total cell number after temperature shift from 37 to 48°C, increased two to three fold in 90 min and remained constant thereafter. The viable count after the temperature shift to 48°C, increased 1.5 to 2 fold in initial 60 min and then decreased exponentially. A rapid restoration of colony forming ability was shown when the mutant cells were shifted back to the permissive temperature after 120 to 180 min of incubation at 48°C or when uracil was introduced to the culture at 48°C. This recovery of viability was partly observed even in the presence of chloramphenicol. The synthesis of RNA of this mutant was shown to decline 20 min after the temperature shift to 48°C whereas the syntheses of DNA and protein proceeded for more than 80 min at that temperature.

No newly isolated uracil requiring mutants formed filaments in the medium lacking uracil or showed growth pattern like ts42.     

Effect of heat stress on development in vitro and in vivo and on synthesis of heat shock proteins in porcine embryos

The present study was conducted (1) to examine the effect of an acute increase in ambient temperature on the development of porcine day 6 embryos in culture and after transfer to recipient gilts, and (2) to analyze intracellular production of heat shock proteins (hsps). The viability of porcine day 6 embryos following a temporary acute elevation in ambient temperature (at 42°–45.5°C and for 10–180 min) was examined. Synthesis of 70 kDa hsp (hsp 70) and 90 kDa hsp (hsp90) was determined by SDS-PAGE and Western blot analysis in porcine day 6 embryos subjected to heat stresses. Nonheat-stressed embryos were considered as control. Significantly higher numbers of viable nuclei were observed in treatment groups of 42°C-10 min (236.6 ± 71.4; P < 0.05) and 43°C-30 min (276.8 ± 89.4; P < 0.005) compared to control (173.9 ± 53.9). The 42°C-180 min group (158.0 ± 27.1 μm) had a greater increase in diameter after 24 hr in culture following heat stress compared to control (82.5 ± 47.3 μm), while heat stress with 43°C for ≧60 min, 44°–44.5°C for ≧30 min, or 45°-45.5°C for ≧10 min impaired their survival, as assessed by differences in number of viable nuclei. The embryos subjected to heat stresses under the conditions of 42°C-180 min, 43°C-10 min, 43°C-30 min, 44°C-10 min, or 45°C-10 min developed to normal piglets after transfer to recipient gilts. Overall pregnancy rate was 75% (6/8), and farrowing rate 62.5% (5/8). Of heat-stressed embryos transferred, 59% (36/61) developed to normal piglets. Heat-stress conditions of 42°C for 180 min, 43°C for 30 min, 44°C for 10 min, and 45°C for 10 min were determined as critical with respect to the in vitro and in vivo survival of porcine embryos. Porcine day 6 embryos constitutively synthesized hsp70 even without heat stress, while hsp90 was detected only at trace level. Neither hsp70 nor hsp90 levels increased in the embryos subjected to heat stresses. In conclusion, porcine day 6 embryos could continue to develop in vivo or during in vitro culture after exposure to acute and temporary rise in temperature. However, no increase of hsp70 and hsp90 was observed in the heat-stressed porcine embryos, while hsp70 was detected in the nonheat-stressed porcine embryos. The precise mechanism of the thermotolerance was unclear. © 1996 Wiley-Liss, Inc.     

Effects of high temperature on the chlorophyll a fluorescence of Alhagi sparsifolia at the southern Taklamakan Desert

Climate change is expected to result in an increase in the frequency and magnitude of extreme weather events. Alhagi sparsifolia is an important factor for wind prevention and sand fixation in the forelands of the Taklamakan Desert. The effects of high temperature on desert plants remain widely unknown. In this work, chlorophyll a fluorescence induction kinetics were investigated at different time stresses of 5, 20, 40, and 60 min at temperature gradients of 38–44 °C at 2 °C intervals. A pronounced K-step was found, and the values of the maximum quantum yield for primary photochemistry, the quantum yield of electron transport, the density of reaction centers and the performance index on absorption basis were lowest after 60 min at 44 °C, thus indicating that the oxygen-evolving complex was damaged, the inactivated reaction centers increased, and the activity of the photosystem II (PSII) reaction center in leaves was seriously limited. Therefore, we suggest that under normal temperature (below 42 °C), the PSII of A. sparsifolia would be unaffected. When such temperature is maintained for 40 min, the activity of PSII would be limited, and when retained for 60 min, PSII may be severely damaged.     

Time-of-day effects of exposure to solar radiation on thermoregulation during outdoor exercise in the heat

High solar radiation has been recognised as a contributing factor to exertional heat-related illness in individuals exercising outdoors in the heat. Although solar radiation intensity has been known to have similar time-of-day variation as body temperature, the relationship between fluctuations in solar radiation associated with diurnal change in the angle of sunlight and thermoregulatory responses in individuals exercising outdoors in a hot environment remains largely unknown. The present study therefore investigated the time-of-day effects of variations in solar radiation associated with changing solar elevation angle on thermoregulatory responses during moderate-intensity outdoor exercise in the heat of summer. Eight healthy, high school baseball players, heat-acclimatised male volunteers completed a 3-h outdoor baseball trainings under the clear sky in the heat. The trainings were commenced at 0900 h in AM trial and at 1600 h in PM trial each on a separate day. Solar radiation and solar elevation angle during exercise continued to increase in AM (672–1107 W/m² and 44–69°) and decrease in PM (717–0 W/m² and 34–0°) and were higher on AM than on PM (both P < 0.001). Although ambient temperature (AM 32–36°C, PM 36–30°C) and wet-bulb globe temperature (AM 31–33°C, PM 34–27°C) also continued to increase in AM and decrease in PM, there were no differences between trials in these (both P > 0.05). Tympanic temperature measured by an infrared tympanic thermometer and mean skin temperature were higher in AM than PM at 120 and 180 min (P < 0.05). Skin temperature was higher in AM than PM at the upper arm and thigh at 120 min (P < 0.05) and at the calf at 120 and 180 min (both P < 0.05). Body heat gain from the sun was greater during exercise in AM than PM (P < 0.0001), at 0–60 min in PM than AM (P < 0.0001) and at 120–180 min in AM than PM (P < 0.0001). Dry heat loss during exercise was greater at 0–60 min (P < 0.0001), and lower at 60–120 min (P < 0.05) and 120–180 min (P < 0.0001) in AM than PM. Evaporative heat loss during exercise was greater in PM than AM at 120–180 min (P < 0.0001). Total (dry + evaporation) heat loss at the skin was greater during exercise in PM than AM (P < 0.0001), at 0–60 min in AM than PM (P < 0.0001) and at 60–120 and 120–180 min in PM than AM (P < 0.05 and 0.0001). Heart rate at 120–150 min was also higher in AM than PM (P < 0.05). Neither perceived thermal sensation nor rating of perceived exertion was different between trials (both P > 0.05). The current study demonstrates a greater thermoregulatory strain in the morning than in the afternoon resulting from a higher body temperature and heart rate in relation to an increase in environmental heat stress with rising solar radiation and solar elevation angle during moderate-intensity outdoor exercise in the heat. This response is associated with a lesser net heat loss at the skin and a greater body heat gain from the sun in the morning compared with the afternoon.     

Effects of different heat–moisture treatments on the physicochemical properties of brown rice flour

We evaluated the effect of heat–moisture treatment (HMT) on the main chemical components, physical properties, and enzyme activities of two types of brown rice flour: high-amylose Koshinokaori and normal-quality Koshiibuki. Five different HMTs using brown rice (moisture content was 12.0%) were assessed: 0.1 MPa/120 °C for 5 or 10 min, 0.2 MPa/134 °C for 5 or 10 min and 0.3 MPa/144 °C for 10 min. HMT, decreased the α-amylase and lipase activities, and fat acidity, and slightly increased the dietary fiber and resistant starch levels. After 2 months’ storage at 35 °C, rice samples that were treated with 0.2 MPa/134 °C or 0.3 MPa/144 °C for 10 min had a lower fat acidity than untreated samples, which would be useful for long-term storage and export of rice flour. And HMT exhibited inhibition of retrogradation in the pasting and physical properties, which is profitable to promote the qualities of the rice products.     

Effects of heat treatment on oil-binding ability of rice flour

Heat-treated (120 °C for 120 min) rice flour showed high affinity to oil (oil-binding ability). This oil-binding ability could be observed by shaking the heat-treated rice flour (2.0 g), oil (4.0 mL), and water (20 mL) vigorously in a test tube, and the oil bound to the rice flour sank into the water. To examine the time-dependent levels of the oil-binding ability, rice flour was heat-treated at 120 °C for 10, 20, 40, 60, and 120 min, and the precipitated volume of oil/rice flour complex increased with an increase of the heating time. The oil-binding ability of the rice flour was not affected by the treatments with diethyl ether or boiled chloroform/methanol (2:1) solutions, which suggested no relationship to the oil in the rice flour, but was lost upon alkali (0.2% NaOH solution) or pepsin treatment, which suggested its relationship to the rice proteins.     

Thermotolerance induced by heat shock in Chlorella

Thermotolerance in cultures of Chlorella zofingiensis was induced by heat shock treatment at supraoptimal temperatures (40and 45 °C for 30 min). Thermotolerance was assayed by two methods: the survival of the cells at 70 °C and the growth of diluted cultures at 35 and 45 °C. A culture without heat shock treatment was unable to grow at 45 °C. According to eletrophoretic analyses, the synthesis of proteins of 95, 73, 60, 43 and 27 kDa was induced by heat shock treatment. The large molecular weight proteins (95, 73, 60 and43 kDa) were present in non-heat treated cells, but the heat shock treatment increased their quantity in cells. The synthesis of a low molecular weight protein (27 kDa) was induced by heat shock treatment. The induced thermotolerance could be inhibited by the presence of an 80S ribosomal translation inhibitor, cycloheximide(CHI). The first 12 amino acid residues from the N-terminus of the27 kDa heat shock induced protein are Val-Glu-Trp-Try-Gly-Pro-Asn-Arg-Ala-Lys-Phe-Leu. This revised version was published online in August 2006 with corrections to the Cover Date.     

Thermotolerance of IVM‐derived bovine oocytes and embryos after short‐term heat shock

A series of experiments were designed to study the effect of elevated temperatures on developmental competence of bovine oocytes and embryos produced in vitro. In experiment 1, the effect of heat shock (HS) by a mild elevated temperature (40.5°C) for 0, 30, or 60 min on the viability of in vitro matured (IVM) oocytes was tested following in vitro fertilization (IVF) and culture. No significant difference was observed between the control (39°C) and the heat‐treated groups in cleavage, blastocyst formation, or hatching (P > 0.05). In experiment 2, when the HS temperature was increased to 41.5°C, neither the cleavage rate nor blastocyst development was affected by treatment. However, the rate of blastocyst hatching appeared lower in the HS groups (13% in control group vs. 3.9% and 5.6% in 30 min and 60 min, respectively; P < 0.05). When IVM oocytes were treated at 43°C prior to IVF (experiment 3), no difference was detected in blastocyst and expanded blastocyst development following heat treatment for 0, 15, or 30 min, but heat treatment of oocytes for 45 or 60 min significantly reduced blastocyst and expanded blastocyst formation (P < 0.05). In experiment 4, the thermotolerance of day 3 and day 4 bovine IVF embryos were compared. When embryos were pre‐treated with a mild elevated temperature (40.5°C) for 1 hr, and then with a higher temperature (43°C) for 1 hr, no improvement in thermotolerance of the embryos was observed as compared to those treated at 43°C alone. However, a higher thermotolerance was observed in day 4 than day 3 embryos. In conclusion, treatment at 43°C, but not 40.5°C or 41.5°C significantly reduced oocyte developmental competence. An increase in thermotolerance was observed from day 3 to day 4 of in vitro embryonic development, which corresponds to the maternal to zygotic transition of gene expression in bovine embryos. Mol. Reprod. Dev. 53:336–340, 1999. © 1999 Wiley‐Liss, Inc.     

Thermal inhibition of repair of methylmethane sulfonate-damaged DNA in chick embryo fibroblasts

Chick embryo fibroblasts were treated with the monofunctional alkylating agent methylmethane sulfonate at various concentrations for 1 h at 42°C, rinsed and then incubated post-treatment at various temperatures at which the kinetics of alkali-labile bond disappearance was followed. Growth experiments showed that these cells grew similarly at temperatures of either 37°C or 42°C. Repair as assessed by removal of alkali-labile bond was also similar for postincubation in the temperature range 37–42°C for damage due to methylmethane sulfonate treatment at concentrations less than 1.5 mM. When the postincubation temperature was raised higher than 42.5–43°C, this type of repair was stopped. The normal internal body temperature of adult chickens is about 41.6°C. Hence the present finding indicates that chick cells are much more severely restricted in DNA repair at temperatures above normal than are mammalian cells, which can function in this respect for several deg. C above 37°C.     

Hyperthermia induced NFκB mediated apoptosis in normal human monocytes

Conceptual approaches of heat-induced cytotoxic effects against tumor cells must address factors affecting therapeutic index, i.e., the relative toxicity for neoplastic versus normal tissues. Accordingly, we investigated the effect of hyperthermia treatment (HT) on the induction of DNA fragmentation, apoptosis, cell-cycle distribution, NFκB mRNA expression, DNA-binding activity, and phosphorylation of IκBα in the normal human Mono Mac 6 (MM6) cells. For HT, cells were exposed to 43°C. FACS analysis showed a 48.5% increase in apoptosis, increased S-phase fraction, and reduced G2 phase fraction after 43°C treatments. EMSA analysis showed a dose-dependent inhibition of NFκB DNA-binding activity after HT. This HT-mediated inhibition of NFκB was persistent even after 48 h. Immunoblotting analysis revealed dose-dependent inhibition of IκBα phosphorylation. Similarly, RPA analysis showed that HT persistently inhibits NFκB mRNA. These results demonstrate that apoptosis upon HT exposure of MM6 cells is regulated by IκBα phosphorylation mediated suppression of NFκB.     

Denaturation and in Vitro Gastric Digestion of Heat-Treated Quinoa Protein Isolates Obtained at Various Extraction pH

The aim of this study was to determine the influence of heat processing on denaturation and digestibility properties of protein isolates obtained from sweet quinoa (Chenopodium quinoa Willd) at various extraction pH values (8, 9, 10 and 11). Pretreatment of suspensions of protein isolates at 60, 90 and 120 °C for 30 min led to protein denaturation and aggregation, which was enhanced at higher treatment temperatures. The in vitro gastric digestibility measured during 6 h was lower for protein extracts pre-treated at 90 and 120 °C compared to 60 °C. The digestibility decreased with increasing extraction pH, which could be ascribed to protein aggregation. Protein digestibility of the quinoa protein isolates was higher compared to wholemeal quinoa flour. We conclude that an interactive effect of processing temperature and extraction pH on in vitro gastric digestibility of quinoa protein isolates obtained at various extraction pH is observed. This gives a first indication of how the nutritional value of quinoa protein could be influenced by heat processing, protein extraction conditions and other grain components.     

Effect of hyperthermia on the pH of lysosomes of HeLa cells in culture

HeLa cells in the logarithmic phase of growth were heated up to 42-43 degrees C in the SHF field for 10 min. Then, after 30, 60 and 120 min pH of lysosomes was determined by neutral red. In 30 min, pH of lysosomes of heated cells decreased down to 5.51 +/- 0.1 against 5.87 +/- 0.07 in intact cells; in 120 min it reached the initial level.     

A simple method for cryopreservation of MDBK cells using trehalose and storage at −80°C

Madin Darby bovine kidney cells were stored at ?80°C using trehalose. Trehalose was loaded into the cells by fluid-phase endocytosis that was facilitated by heat shock at 40°C for 1 h. Loaded cells were gradually frozen and stored at ?80°C. Revival of cells was done by quick thawing and immediately seeded in the tissue culture flasks. The membrane integrity of cells was measured at different times post-storage by trypan blue dye exclusion method. It was estimated to be 96.23, 73.84, 57.33, 54.36, 25.47, 50.53 and 46.86% at 0, 7, 60, 90, 120, 160 and 180-day post-storage, respectively. Cryostorage of cells at ?80°C may help to reduce the use of liquid nitrogen.     

Heat shock increases levels of reactive oxygen species,autophagy and apoptosis

Hyperthermia is a promising anticancer treatment used in combination with radiotherapy and chemotherapy. Temperatures above 41.5 °C are cytotoxic and hyperthermia treatments can target a localized area of the body that has been invaded by a tumor. However, non-lethal temperatures (39–41 °C) can increase cellular defenses, such as heat shock proteins. This adaptive survival response, thermotolerance, can protect cells against subsequent cytotoxic stress such as anticancer treatments and heat shock (>41.5 °C). Autophagy is another survival process that is activated by stress. This study aims to determine whether autophagy can be activated by heat shock at 42 °C, and if this response is mediated by reactive oxygen species (ROS). Autophagy was increased during shorter heating times (<60 min) at 42 °C in cells. Levels of acidic vesicular organelles (AVO) and autophagy proteins Beclin-1, LC3-II/LC-3I, Atg7 and Atg12-Atg5 were increased. Heat shock at 42 °C increased levels of ROS. Increased levels of LC3 and AVOs at 42 °C were inhibited by antioxidants. Therefore, increased autophagy during heat shock at 42 °C (<60 min) was mediated by ROS. Conversely, heat shock at 42 °C for longer times (1?3 h) caused apoptosis and activation of caspases in the mitochondrial, death receptor and endoplasmic reticulum (ER) pathways. Thermotolerant cells, which were developed at 40 °C, were resistant to activation of apoptosis at 42 °C. Autophagy inhibitors 3-methyladenine and bafilomycin sensitized cells to activation of apoptosis by heat shock (42 °C). Improved understanding of autophagy in cellular responses to heat shock could be useful for optimizing the efficacy of hyperthermia in the clinic.