Occurrence of l-Saccharopine and γ-L-Glutamylglycine in the Mushroom,Agaricus bisporus

Microfolicoumarin (1), a prenylcoumarin from Cedrelopsis microfoliata, was synthesized from 2,4,5-trimethoxybenzaldehyde in five steps. 1 did not show significant antioxidative activity, but the key intermediates, esculetin (3) and 5-prenylesculetin (6), exhibited strong antioxidative activity in both the superoxide-radical and 1,1-diphenyl-2-picrylhydrazyl radical-scavenging models.     

Responses of cattle to the combined exposure to diurnal temperature rhythm (−5 to 25°C) and to simulated high-altitude (4,000 m)

Eight 1/2-year old calves were exposed in a climatized altitude chamber to the following four conditions: 400 and 4,000 m at constant T_a (17°C), 400 and 4,000 m at alternating T_a (–5° to 25°C). Each exposure lasted for 24 h and for the rhythmic conditions included a cold night and warm midday hours, supplemented by infrared heaters. During exposure, hourly measurements were made of heart rate, respiratory rate, rectal and three skin temperatures. Every 3-h blood samples were collected for the determination of 10 blood variables. The following main results were obtained: (a) Altitude alone caused increases in respiratory rate, heart rate, erythrocyte number, haemoglobin, specific gravity of blood and plasma, LDH and all four body temperatures. (b) In the rhythmic exposures, high correlation coefficients were found between ambient temperature on the one hand and skin temperatures (0.88 to 0.94), rectal temperature (–0.43) and respiratory rate (0.49) on the other hand. A change in ambient temperature by 1°C lead, on average, to a change in ear temperature by 1.2°C. (c) in response to falling ambient temperature during the night, rectal temperature and heart rate increased. This was interpreted as indicating a compensatory elevation in meta bolic heat production. At the same time, there was haemoconcentration as shown by elevations in erythrocyte number, haematocrit and haemoglobin. This haemoconcentration might have reflected splenic discharge, possibly supplemented by some loss of water from the plasma. (d) The warm environmental conditions around midday produced mild heat responses in terms of elevated values for respiratory rate, heart rate and body temperatures. (e) It is concluded that the rhythmic temperature with alternating stress of cold and mild heat, especially in combination with high altitude, was a strain on the animals and that they were forced to expend extra energy for combatting altitude- and temperature stress, energy which no longer would be available for productive processes.Presented at the Eight International Congress of Biometeorology, 9–14 September 1979, Shefayim, Israel.     

Cryopreservation at −80°C of Agaricus blazei on rice grains

The preservation of Agaricus blazei is generally done by mycelial subculturing, but this technique may cause genetic degenerations. Despite this, there is not an efficient protocol established to preserve this fungus and cryopreservation could be an alternative. This study aimed to evaluate two freezing protocols for cryopreservation at −80°C of A. blazei strains. Five fungus strains grown on rice grains with husk and were transferred to glycerol (10%) in cryovials. Next, the cryovials were submitted to two freezing temperature protocols: (1) cryopreservation starting at 25°C, then at 8°C for 30 min and kept at −80°C; (2) cryopreservation starting at 25°C, then 8°C for 30 min, −196°C for 15 min and kept at −80°C. After 1 year of cryopreservation, the cryovials were thawed in a water bath at 30°C for 15 min and transferred to malt extract agar medium. It was concluded that the one-year cryopreservation process of A. blazei, grown on rice grains and cryopreserved at −80°C in glycerol 10%, is viable. The slow freezing, from 8 to −80°C, is effective whereas the fast freezing, from 8 to −196°C and then to −80°C, is ineffective. The different genetic characteristics among the strains of this fungus do not interfere in the cryopreservation process.     

Influence of pH and ionic strength on the lysis ofMicrococcus luteus cells by hen lysozyme at low (20°C) and high (physiological, 40°C) temperature

From isoactivity curves (showing activity as a function of pH and ionic strength) it was found that in the pH domain 6.7–8.6 frequently used in experiments involving hen lysozyme, the pH optimum of lysis ofMicrococcus luteus ceils at low ionic strength (0.02–0.05) by the high-temperature form (40°C physiological temperature) was one to two pH units lower than that by the low-temperature form (20°C).116th communication on lysozymes; 115th communication: Harada Y, Lifchitz A, Berthou J, & Jollès P (1981) Acta Cryst. in press.     

A comparison of the drying of chymotrypsin bound to bead cellulose by lyophilisation and in air at 25 to 70°C

Summary The effect was studied of the method of drying chymotrypsin attached to bead cellulose on its chemical and physical characteristics. These characteristics are not deteriorated when replacing the commonly used lyophilisation by fluid drying in the air stream. The preparations saved essentially the same proteolytic activity as well as stability even when increasing the drying air temperature to 70°C.     

Characterization of a glucose 3-dehydrogenase from the cultivated mushroom (Agaricus bisporus )

An extracellular enzyme with glucose dehydrogenase activity was purified from liquid cultures of the basidiomycete Agaricus bisporus after growth with d-cellobiose or d-glucose as carbon source. The molecular mass was measured as 57 kDa by gel filtration and 55 kDa by sodiumdodecyl sulphate/polyacrylamide gel electrophoresis, while the isoelectric point was at pH 3.6. By analysis of ¹H-NMR spectra in D₂O, the product of d-glucose oxidation was identified as 3-ketoglucose. The substrates oxidized included d-cellobiose, l-arabinose, d-xylose and sucrose, but the specificity parameter (k _cat/K _m) was highest for d-glucose. Two electron acceptors were identified, namely 2,6-dichloroindophenol and p-benzoquinone, but reduction of dioxygen, ferricyanide or cytochrome c was not detectable. The selective C-3 oxidation of d-glucose is well-characterized for Agrobacterium and Flavobacterium, but this is the first report for a fungus. Received: 19 June 1998 / Received revision: 15 September 1998 / Accepted: 17 September 1998     

Effects of Mild Cold Shock (25°C) Followed by Warming Up at 37°C on the Cellular Stress Response

Temperature variations in cells, tissues and organs may occur in a number of circumstances. We report here that reducing temperature of cells in culture to 25°C for 5 days followed by a rewarming to 37°C affects cell biology and induces a cellular stress response. Cell proliferation was almost arrested during mild hypothermia and not restored upon returning to 37°C. The expression of cold shock genes, CIRBP and RBM3, was increased at 25°C and returned to basal level upon rewarming while that of heat shock protein HSP70 was inversely regulated. An activation of pro-apoptotic pathways was evidenced by FACS analysis and increased Bax/Bcl2 and BclX_S/L ratios. Concomitant increased expression of the autophagosome-associated protein LC3II and AKT phosphorylation suggested a simultaneous activation of autophagy and pro-survival pathways. However, a large proportion of cells were dying 24 hours after rewarming. The occurrence of DNA damage was evidenced by the increased phosphorylation of p53 and H2AX, a hallmark of DNA breaks. The latter process, as well as apoptosis, was strongly reduced by the radical oxygen species (ROS) scavenger, N-acetylcysteine, indicating a causal relationship between ROS, DNA damage and cell death during mild cold shock and rewarming. These data bring new insights into the potential deleterious effects of mild hypothermia and rewarming used in various research and therapeutical fields.     

Break points in Arrhenius plots of the Hill reaction of spinach chloroplast fragments in the temperature range from -25 to 25?C

The relationship between temperature and 2,6-dichlorophenolindophenol(DPIP) photoreduction activity in chloroplast fragments isolatedfrom spinach grown at different temperatures was investigatedin the temperature range from –25 to 25?C. Two break points in the slope of the Arrhenius plot of the Hillreaction were observed at –9 and 11?C in chloroplast fragmentsisolated from spinach grown at low temperatures (0–10?C).These breaks were not affected by an uncoupler. In chloroplastfragments from spinach grown in a higher temperature range (10–30?C),only one break point was observed at –9?C. The other breakpoint at around 10?C appeared after treatment with detergent.Both break points were completely abolished by fixing the chloroplastfragments with glutaraldehyde. The break at around 10?C in chloroplast fragments isolated fromspinach grown at lower temperature was interpreted as due toacclimation rather than chilling sensitivity. (Received September 6, 1977; )     

CD measurement of aqueous protein solution at high temperature up to 180 °C —Thermodynamic analysis of thermophilic protein by pressure-proof cell compartment

Summary During investigation of thermal transitions of peptides and proteins, the transition temperature is occasionally too high to trace the whole transition profile. In order to solve this problem and perform conformational analysis at high temperature, we have recently developed a pressure-proof cell compartment for circular dichroism measurements. Here we demonstrate how well this system works to collect CD spectra, at high temperature up to 180°c in aqueous solution. Ribosome recycling factor (RRF), which consists of two domains; three stranded α-helix bundle domain (Domain I) and β/α/β domain (Domain II), was used as an example. We constructed models of isolated Domain I substituting Domain II with tripeptide (Gly-Gly-Gly) and compared these models from mesophilic and thermophilic bacteria. The melting profiles of these models revealed that thermal stability is enhanced by the increased enthalpy provided by hydrogen bonds and ionic pairing.     

Induced in vitro phagocytosis of the Antarctic starfish Odontaster validus (Koehler 1906) at 0°C

Phagocytosis was studied in vitro using coelomic fluid of the Antarctic starfish Odontaster validus at 0°C. The number of coelomocytes present was determined and the phagocytic activity of the phagocytic amoebocytes (PA) was quantified with yeast during incubations of 1 and 2 h. The percentage of PA phagocytosing increased significantly from 42.29 ± 10.50% (SD) at 1 h to 52.57 ± 13.96% at 2 h. Numbers of yeast per PA also rose significantly from 2.27 to 2.45 cells per amoebocyte, indicating that phagocytic activity was maintained. In vitro phagocytosis of an Antarctic invertebrate at 0°C is shown for the first time, and the types of amoebocytes involved identified. Rates of phagocytosis were similar to, or higher than, reported data for temperate starfish, although this conclusion must be treated cautiously because of scarcity of data and differences in methods used. However, the data suggest that phagocytosis in O. validus is well adapted to low temperature. Accepted: 27 September 1999     

Defining the response of a microorganism to temperatures that span its complete growth temperature range (-2°C to 28°C) using multiplex quantitative proteomics

The growth of all microorganisms is limited to a specific temperature range. However, it has not previously been determined to what extent global protein profiles change in response to temperatures that incrementally span the complete growth temperature range of a microorganism. As a result it has remained unclear to what extent cellular processes (inferred from protein abundance profiles) are affected by growth temperature and which, in particular, constrain growth at upper and lower temperature limits. To evaluate this, 8-plex iTRAQ proteomics was performed on the Antarctic microorganism, Methanococcoides burtonii. Methanococcoides burtonii was chosen due to its importance as a model psychrophilic (cold-adapted) member of the Archaea, and the fact that proteomic methods, including subcellular fractionation procedures, have been well developed. Differential abundance patterns were obtained for cells grown at seven different growth temperatures (-2°C, 1°C, 4°C, 10°C, 16°C, 23°C, 28°C) and a principal component analysis (PCA) was performed to identify trends in protein abundances. The multiplex analysis enabled three largely distinct physiological states to be described: cold stress (-2°C), cold adaptation (1°C, 4°C, 10°C and 16°C), and heat stress (23°C and 28°C). A particular feature of the thermal extremes was the synthesis of heat- and cold-specific stress proteins, reflecting the important, yet distinct ways in which temperature-induced stress manifests in the cell. This is the first quantitative proteomic investigation to simultaneously assess the response of a microorganism to numerous growth temperatures, including the upper and lower growth temperatures limits, and has revealed a new level of understanding about cellular adaptive responses.     

Permeability and reactivity of Thermotoga maritima in latex bimodal blend coatings at 80°C: a model high temperature biocatalytic coating

Thermostable polymers cast as thin, porous coatings or membranes may be useful for concentrating and stabilizing hyperthermophilic microorganisms as biocatalysts. Hydrogel matricies can be unstable above 65°C. Therefore a 55-m thick, two layer (cell coat + polymer top coat) bimodal, adhesive latex coating of partially coalesced polystyrene particles was investigated at 80°C using Thermotoga maritima as a model hyperthermophile. Coating permeability (pore structure) was critical for maintaining T. maritima viability. The permeability of bimodal coatings generated from 0.8 v/v of a suspension of non-film-forming 800 nm polystyrene particles with high glass transition temperature (T_g= 94°C, 26.9% total solids) blended with 0.2 v/v of a suspension of film-forming 158 nm polyacrylate/styrene particles (T_g –5°C, 40.9% total solids) with 0.3 g sucrose/g latex was measured in a KNO₃ diffusion cell. Diffusivity ratio remained above 0.04 (D_eff/D) when incubated at 80°C in artificial seawater (ASW) for 5 days. KNO₃ permeability was corroborated by cryogenic-SEM images of the pore structure. In contrast, the permeability of a mono-dispersed acrylate/vinyl acetate latex Rovace SF091 (T_g~10°C) rapidly decreased and became impermeable after 2 days incubation in ASW at 80°C. Thermotoga maritima were entrapped in these coatings at a cell density of 49 g cell wet weight/liter of coating volume, 25-fold higher than the density in liquid culture. Viable T. maritima were released from single-layer coatings at 80°C but accurate measurement of the percentage of viable entrapped cells by plate counting was not successful. Metabolic activity could be measured in bilayer coatings by utilization of glucose and maltose, which was identical for latex-entrapped and suspended cells. Starch was hydrolyzed for 200 h by latex-entrapped cells due to the slow diffusion of starch through the polymer top coat compared to only 24 h by suspended T. maritima. The observed reactivity and stability of these coatings was surprising since cryo-SEM images suggested that the smaller low T_g polyacrylate/styrene particles preferentially bound to the T. maritima toga-sheath during coat formation. This model system may be useful for concentrating, entrapment and stabilization of metabolically active hyperthermophiles at 80°C.     

The synthesis of oligosaccharides by the reversed hydrolysis reaction of β-glucosidase at high substrate concentration and at high temperature

Summary In the presence of -glucosidase from almond, a 90% glucose solution gave four kind of -linked glucose-disaccharides. The yield increased as the concentration of glucose was increased and as the reaction temperature was raised. The maximum yield of disaccharides from 90% glucose solution was 40% at 55°C.     

Adaptation of Methanogenic Communities to the Cofermentation of Cattle Excreta and Olive Mill Wastes at 37°C and 55°C

The acclimatization of methanogens to two-phase olive mill wastes (TPOMW) was investigated in pilot fermenters started up with cattle excreta (37°C) and after changing their feed to excreta plus TPOMW (37°C or 55°C) or TPOMW alone (37°C) until a steady state was reached (28 days). Methanogenic diversity was screened using a phylogenetic microarray (AnaeroChip), and positive targets were quantified by real-time PCR. Results revealed high phylogenetic richness, with representatives of three out of the four taxonomic orders found in digesters. Methanosarcina dominated in the starting excreta (>96% of total 16S rRNA gene copies; over 45 times more abundant than any other methanogen) at high acetate (0.21 g liter⁻¹) and ammonia N concentrations (1.3 g liter⁻¹). Codigestion at 37°C induced a 6-fold increase of Methanosarcina numbers, correlated with CH₄ production (r_Pearson = 0.94; P = 0.02). At 55°C, the rise in temperature and H₂ partial pressure induced a burst of Methanobacterium, Methanoculleus, Methanothermobacter, and a group of uncultured archaea. The digestion of excreta alone resulted in low but constant biogas production despite certain oscillations in the methanogenic biomass. Unsuccessful digestion of TPOMW alone was attributed to high Cu levels inducing inhibition of methanogenic activity. In conclusion, the versatile Methanosarcina immediately adapted to the shift from excreta to excreta plus TPOMW and was responsible for the stimulated CH₄ production at 37°C. Higher temperatures (55°C) fostered methanogenic diversity by promoting some H₂ scavengers while yielding the highest CH₄ production. Further testing is needed to find out whether there is a link between increased methanogenic diversity and reactor productivity.Turning residues into energy is a societal and scientific priority due to climate change, fossil fuel exhaustion, and waste accumulation. In 2006, in Europe (EU27), less than 3% of electricity production came from biomass and wastes (11). Biogas plants, which anaerobically treat organic wastes to produce energy, are increasingly promoted in Europe, but their distribution is highly biased (35). While thousands of full- and farm-scale biogas plants are spread over central and northern Europe, anaerobic digestion technology in Mediterranean countries—Portugal, Spain, Italy, Greece, and Turkey—is in its early stages (35). These nations and other circum-Mediterranean countries lead in the production of olive oil and thus in olive mill wastes and wastewaters, which have a huge biogas production potential due to their lipid composition (1). Spain alone generates one-third of the world''s oil production and millions of tons of two-phase olive mill wastes (TPOMW) per year. TPOMW are mostly burned or composted (28), hence releasing methane into the atmosphere. This compels a change in strategy: methane production from TPOMW should be optimized in engineered environments and transformed into energy.TPOMW is a humid residue containing the olive pulp and stone. Its anaerobic digestibility is hampered by its low pH, low ammonia N, and high content in antimicrobial substances (1). However, it has been successfully fermented under laboratory conditions by supplementing it with nutrients and increasing the reactor organic loading rate stepwise (2) or by codigesting it with residues with a high buffering capacity, e.g., cattle excreta (17). These approaches seem to facilitate the adaptation of the methane-producing anaerobic community to the environmental conditions that TPOMW impose.Methanogenic archaea—microbes clustered within five orders of the Euryarchaeota—constitute the last step in the trophic chain of decomposers degrading organic matter in oxygen-free environments (36). Methanogenesis is often the rate-limiting step of anaerobic digestion of organic wastes (3) due to the fast duplication times of bacteria, which generate all substrates for the slow-growing methane-producing archaea. It is also the most sensitive step in processing imbalances (4), likely due to the lack of functional redundancy among methanogens (8). High concentrations of volatile fatty acids, salts, ammonia, and heavy metals can be inhibitory for methanogens (5, 22) and are the most common reasons for reactor failure (3). Our objective was to understand the adaptation of methanogenic communities to TPOMW. We investigated methanogenic diversity and abundance in pilot digesters fed with cattle excreta and after changing their feed to TPOMW or TPOMW plus excreta. We expected that mixing both residues would allow a faster adaptation and more efficient performance of the methanogenic communities in digesting TPOMW. The cofermentation was evaluated at 37°C and 55°C. During an acclimatization period of 28 days, we screened the methanogenic diversity using an in-house-devised phylogenetic microarray, the AnaeroChip (13), and quantified dominant genera by real-time quantitative PCR (qPCR). We have taken primers from the literature, and we present four new sets of genus-specific primers and SYBR green I-optimized assays for quantifying methanogens in anaerobic environments.     

The coupling of electron flow to ATP synthesis in pea chloroplasts stored in the presence of glycerol at −70°C

A method is described for preserving coupling activity in isolated chloroplasts stored in the frozen state for periods up to 16 weeks. Pea chloroplasts stored in a high osmolarity medium supplemented with 50% (v/v) glycerol and 10 mg/ml bovine serum albumin show high rates of photophosphorylation, good coupling efficiencies, photosynthetic control, and modulation of the high energy state by adenine nucleotides.     

Effect of hypothermia (20–25°C) on mitosis in PtK1 cells

PtK₁ cells enter prophase and complete mitosis at 24–25°C but are inhibited from entering prophase at 20–21°C. Cells which have progressed up to midprophase at 24–37°C return to interphase when cooled to 20–21°C, but those in late prophase complete a normal, although prolonged mitosis. If prophase cells which have reverted to interphase at 20–21°C are incubated at 24–37°C they reenter prophase and complete mitosis. This temperature-induced prophase-interphase-prophase transition can be repeated several times on the same cell. At 24–25°C the process of spindle formation (i.e. prometaphase to the initiation of anaphase) encompasses approximately 75% of the total mitotic interval, with a duration of 8–12 h, compared to about 50% of the mitotic interval and a duration of 0.5 to 1.0 h at 37°C.     

Thyroliberin is rapidly transferred to the nucleus of GH3 pituitary cells at both 4°C and 37°C

The time-course of thyroliberin transfer to the nucleus of GH3/B6 rat pituitary prolactin cells was studied by both autoradiography and cell fractionation of intact cells exposed to [³H]thyroliberin at 4°C or 37°C. It was previously shown that thyroliberin is not degraded in these conditions. It is found by autoradiography that [³H]-thyroliberin is transferred to the nucleus of GH3/B6 cells within 5 min at least at both 37° C and 4°C. Consistent results are obtained by fractionation of cells exposed to [³H]thyroliberin at 37°C. However after binding at 4°C 50% of the cell radioactivity is extractible by glutaraldehyde and after fractionation the isolated nuclei retain only 1–1.5% of the cell radioactivity. This suggests the existence of both tightly bound and loosely bound internalized thyroliberin molecules.