Heat Activation and Heat-induced Dormancy of Bacillus stearothermophilus Spores

Heat-induced dormancy was observed when spores of two strains of Bacillus stearothermophilus were heated in distilled water at 80, 90, and 100 C. At temperatures above 100 C, true activation occurred; however, maximal activation was not achieved until temperatures of 110 to 115 C were employed. A heat treatment of 115 C for 3 min was required to induce maximal activation in one suspension of strain 1518 spores, whereas a heat treatment of 110 C for 7 to 10 min was adequate for the other suspension of strain 1518 spores. Spores from both strain M suspensions required heat treatments of 110 C for 9 to 15 min for maximal activation. The degree to which the spores could be activated was strain dependent and variable among spore suspensions of the same strain.     

The role of cryoprotective agents as hydroxyl radical scavengers

The classic cryoprotective agents dimethylsulfoxide and glycerol are hydroxyl radical scavengers. In addition the cryoprotective agents tetramethylurea, dimethylformide, dimethylurea and monomethylurea act as hydroxyl radical scavengers as shown by the inhibition of ethylene production from methional and the inhibition of methane production from dimethylsulfoxide. Both cryoprotection and scavenger efficiency decrease in the same order within a homologous series: tetramethylurea > dimethylurea > monomethylurea. Urea does not act as a hydroxyl radical scavenger and urea is not a cryoprotective agent. Thus cryoprotection may involve scavengers in the prevention of membrane damage by hydroxyl radicals.     

Activation and killing of Dictyostelium discoideum spores with urea.

The optimal conditions for activation of Dictyostellium discoideum spores are an 8 M urea treatment for 30 min. The lag between activation and swelling is 45 min. Lower concentrations of urea do not activate entire spore populations. Incubating spores in 8 M urea for 60 min or treatment with 10 M urea for 30 min results in a lengthening of the post-activation lag and a decrease in the final percentage of germination. Urea-activated spores can be deactivated by azide, cyanide, osmotic pressure, and low-temperature incubation. Activated spores do not germinate if incubated in 1 M urea for 24 h but will complete germination upon resuspension in urea-free buffer. Shocking spores at 45 degrees C in 8 M urea or incubating spores in 4-8 M urea for 10 h at 23.5 degrees C causes inactivation. When suspended in urea-free buffer, a larger percentage of these dead spores release spheroplasts through a longitudinal split in the spore case. Sequential enzyme treatment of spheroplasts with cellulase and pronase causes them to release lysable protoplasts. The data of these experiments suggest that shedding of the outer and middle wall layers during physiological spore swelling may be a physical process rather than an enzymatic one.     

Ab Initio and Molecular Mechanics Calculations of Various Substituted Ureas - Rotational Barriers and a New Parametrization for Ureas

High-level ab initio calculations have been performed on urea, methylurea, tetramethylurea and three isomers of dimethylurea to obtain accurate rotational barriers. Results of MP2(fc)/6-31 G(d) calculations are compared to those with lower basis sets and semiempirical calculations. The MM2(87) force field has been parameterized.     

Effect of temperature on the dose–response curves for auxin-induced elongation growth in maize coleoptile segments

The dose–response curves for IAA-induced growth in maize coleoptile segments were studied as a function of time and temperature. In addition, the kinetics of growth rate responses at some auxin concentrations and temperatures was also compared. It was found that the dose–response curves for IAA-induced elongation growth were, independently of time and temperature, bell-shaped with an optimal concentration at 10⁻⁵ M IAA. The kinetics of IAA-induced growth rate responses depended on IAA concentration and temperature, and could be separated into two phases (biphasic reaction). The first phase (very rapid) was followed by a long lasting one (second phase), which began about 30 min after auxin addition. For coleoptile segments incubated at 30°C, the amplitudes of the first and second phase were significantly higher, when compared with 25°C, at all IAA concentrations studied. However, when coleoptile segments were incubated at 20°C, the elongation growth of coleoptile segments treated with suboptimal IAA concentrations was diminished, mainly as a result of both phases reduction. In conclusion, we propose that the shape of the dose–response curves for IAA-induced growth in maize coleoptile segments is connected with biphasic kinetic of growth rate response.     

Spores of microorganisms

Calcium-dipicolinate (Ca-DPA)-rich and Ca-DPA-deficientBacillus cereus spores were incubated in a synthetic medium with germination stimulants and in bactopeptone medium with a fairly high calcium ion concentration. In the complex medium the germination of Ca-DPA-rich spores was completely blocked at a concentration of 0.5m CaCl₂, whereas the complete blockage of germination in the synthetic medium required higher concentrations (0.6–0.8m) of calcium chloride. Ca-DPA-deficient spores germinated more slowly and less completely in the synthetic medium than in the bactopeptone medium. The germination of these spores took place, however, even at higher calcium ion concentrations (0.6–0.8m). On the contrary, lower calcium chloride concentrations (0.1–0.4m) accelerated the germination of these spores in the synthetic medium and the final percentage of phase-dark and stainable spores was higher. “H-forms” of the Ca-DPA-rich and Ca-DPA-deficient spores prepared by acid titration germinated in both media. The germination of the latter spores being slower and proceeding less completely. “H-forms” germinated completely or partially in media with a high concentration of calcium chloride. The percentage of germinated spores, however, was strongly influenced by the concentration of this cation, especially the “H-forms” of Ca-DPA-deficient spores. Moreover, the germination of Ca-DPA-deficient spores in this medium was affected by the length of previous storage and, in the case of “H-forms” by the pH at which they were titrated. It was assumed that the increased permeability of calcium into the calciumundersaturated spore periphery in Ca-DPA-deficient and in “H-forms” of spores of both types co-determines (in the presence of germinants) the germinability of bacterial spores.     

Failure to decontaminate Glomus clarum NT4 spores is due to spore wall-associated bacteria

 Exposure of spores of Glomus clarum NT4 to solutions of chloramine-T (2.5–10% w/v) for 10–120 min failed to fully decontaminate all spores. Scanning electron microscopy did not show the presence of contaminants on treated spores, but transmission electron microscopy revealed bacterial cells embedded within the outer spore wall layer. Bacteria that remained protected within the spore walls were detected only when the spores were placed on appropriate media. Nutrient agar and tryptic soy agar supported relatively high levels of contaminant growth and were regarded as good media for assessing contamination, whereas the detection of contaminant growth on water agar required prolonged incubation. Contamination and germination of G. clarum NT4 spores following decontamination treatments were dependent on spore age. Generally, lower concentrations of chloramine-T and shorter incubation periods were required to reduce contamination of freshly harvested spores than of mature spores. Exposure to 10% chloramine-T for 120 min was required to reduce the levels of contamination of mature spores to ≤10%. Unfortunately, spore germination was compromised by rigorous decontamination treatments, thus the success of any decontamination procedure should be evaluated prior to its routine use. Moreover, if the interpretation of experimental results rests on the assumption of true surface sterility of VAMF spores, we suggest that the axenic condition of spores be confirmed prior to experimentation on a medium that encourages contaminant growth. Accepted: 12 July 1995     

Plasmolysis shape in relation to freeze-hardening of cabbage plants and to the effect of penetrating solutes*

Abstract In conformity with earlier results, low-temperature hardening of cabbage seedlings lowered the osmotic potential and increased the permeability to thiourea of the petiole cells. It also decreased the time required for rounding-up of the protoplasts in cells plasmolysed in 1. 5 × isotonic (or higher) glucose or CaCl₂ solutions. Solutions of dimethylsulphoxide (DMSO), thiourea, urea, and glycerol each accelerated the rate of rounding-up of protoplasts in plasmolysed cells, compared to the rate in glucose solution of the same hypertonicity. Each also penetrated the cell membranes as indicated by deplasmolysis. Only in the case of DMSO, in which there was very rapid deplasmolysis (5–6 min), was this rounding-up due to protoplast expansion. In the case of thiourea (deplasmolysis within 30–60 min) rounding-up occurred almost immediately (less than 2 min), before protoplast expansion was sufficient to induce it. It was concluded that the accelerated rounding-up was due to a rapid osmotic adjustment in the protoplasm by the penetrating solution, which increased its water content and decreased its viscosity.     

Inhibition of glucagon-induced glycogenolysis in isolated rat hepatocytes by the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate

The ability of the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp cAMPS) to inhibit glucagon-induced glycogenolysis was studied in hepatocytes isolated from fed rats. Preincubation of the cells for 20 min with progressively higher concentrations of Rp cAMPS followed by a 1 X 10(-9) M glucagon challenge resulted in a 50% inhibition of glucose production over a 30-min period at 2-3 X 10(-6) M Rp cAMPS. A maximal inhibition of 50-74% was achieved, the actual value depending upon the length of preincubation with Rp cAMPS. The inhibitory effect did not increase when the concentration of Rp cAMPS was increased from 3 X 10(-6) to 3 X 10(-4) M. Addition of 1 X 10(-5) M Rp cAMPS to the cells followed by 10(-11) to 10(-6) M glucagon shifted the glucagon concentration required for half-maximal glucose production measured at 10 min to 6-fold higher glucagon concentrations and the concentration of glucagon required for apparent maximal glucose production measured at 10 min to greater than 10-fold higher glucagon concentrations. The cAMP-dependent protein kinase activation curve was similarly shifted to higher concentrations of glucagon. These data show that Rp cAMPS acts as a cAMP antagonist capable of opposing the glucagon-induced activation of cAMP-dependent protein kinase and the concomitant activation of the glycogenolytic cascade.     

Effects of urea and o-phenanthroline on F-695 emission in chloroplasts

To clarify the nature of the chlorophyll species which fluorescesat about 695 nm in vivo (F-695), effects of the addition ofurea and related compounds and of urea plus o-phenanthrolineon the emission spectra of spinach chloroplast fragments, at77°K, were examined. F-695 emission was partially decreased by the presence of alow concentration (0.1 M) of urea, thiourea, guanidine hydrochloride,methylguanidine hydrochloride, acetamide, N-methylurea, or dimethylurea. The concurrent addition of o-phenanthroline with the reagents(0.1–1 M) caused a marked increase in F-695 emission anda decrease in F-685 emission. Methyl-substituted ureas and acetamide,however, were less effective. The effect was largely dependenton the pH of the sample. These two effects, the decrease and increase in F-695 emission,were reversible and were inhibited by the presence of ethanol. The phenomena are probably due to specific interactions betweenadded reagents and the component(s) in chloroplasts which havean intimate connection with a postulated energy trap; an invivo species of chlorophyll responsible for the fluorescenceemission near 695 nm. (Received August 17, 1971; )     

An Evaluation of the Critical Parameters for Abiotic Peptide Synthesis in Submarine Hydrothermal Systems

It has been proposed that oligopeptides may be formed in submarine hydrothermal systems (SHSs). Oligopeptides have been synthesized previously under simulated SHS conditions which are likely geochemically implausible. We have herein investigated the oligomerization of glycine under SHS–like conditions with respect to the limitations imposed by starting amino acid concentration, heating time, and temperature. When 10⁻¹ M glycine solutions were heated at 250°C for < 20 min glycine oligomers up to tetramers and diketopiperazine (DKP) were detectable. At 200°C, less oligomerization was noted. Peptides beyond glycylglycine (gly₂) and DKP were not detected below 150°C. At 10⁻² M initial glycine concentration and below, only gly₂, DKP, and gly₃ were detected, and then only above 200°C at < 20 min reaction time. Gly₃ was undetectable at longer reaction times. The major parameters limiting peptide synthesis in SHSs appear to be concentration, time, and temperature. Given the expected low concentrations of amino acids, the long residence times and range of temperatures in SHSs, it is unlikely that SHS environments were robust sources of even simple peptides. Possible unexplored solutions to the problems presented here are also discussed.     

Effect of abscisic acid on betacyanin leakage from plant tissues

Effect of abscisic acid on cell permeability in leaves ofIresine u allisi hort. and roots ofBeta vulgaris L. were examined. An increase of betacyanin leakage from leaf cells was shown by ABA at 10⁻⁴, 10⁻⁷ or 10⁻⁹ M concentrations in water solution at 25 °C. The efflux of batacyanin from tissues did not change during the joint action of ABA and PEG 1000. ABA could lower the betacyanin leakage fromIresine leaves and beet-root slices under severe osmotic stress, as was found by deplasmolysis. The results suggest that ABA elicits some alteration in density of tonoplast membranes under dehydration. Presented at the International Symposium “Plant Growth Regulators” held on June 18–22, 1984 at Liblice, Czechoslovakia.     

Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique

 Sugar beet shoot tips from cold-acclimated plants were successfully cryopreserved using a vitrification technique. Dissected shoot tips were precultured for 1 day at 5  °C on solidified DGJ0 medium with 0.3 M sucrose. After loading for 20 min with a mixture of 2 M glycerol and 0.4 M sucrose (20  °C), shoot tips were dehydrated with PVS2 (0  °C) for 20 min prior to immersion in liquid nitrogen. Both cold acclimation and loading enhanced the dehydration tolerance of shoot tips to PVS2. After thawing, shoot tips were deloaded for 15 min in liquid DGJ0 medium with 1.2 M sucrose (20  °C). The optimal exposure time to both loading solution and PVS2 depended on the in vitro morphology of the clone. With tetraploid clones a higher sucrose concentration during cold acclimation and preculture further enhanced survival after cryopreservation. Survival rates ranged between 60% and 100% depending on the clone. Since only 10–50% of the surviving shoot tips developed into non-hyperhydric shoots, regrowth was optimized. Received: 13 September 1999 / Revision received: 2 March 2000 / Accepted: 16 March 2000     

Studies on the control and properties of ornithine transcarbamylase in germinated spores of Geotrichum candidum

The effects of arginine and urea on the levels of ornithinetranscarbamylase (OTC) were investigated in relation to thephysiological functions of this enzyme in Geotrichum candidum.OTC was repressed in germinated spores to half of its initiallevel when exogenous arginine exceeded 12 mM. The repressionof OTC could not be correlated with intracellular arginine concentration.The addition of urea at the final concentration of 0.035 M increasedthe specific activity of OTC by 5.5 and 2.5 fold as comparedto enzyme levels in arginine-repressed spores and control sporesrespectively. Simultaneous addition of urea and arginine duringgermination prevented either arginine repression or urea inductionof OTC. The enzyme was partially purified from germinated sporesand isolated as a single protein band after disc electrophoresis.Two distinct pH optima for the forward reaction (pH 8.8–9)and backward reaction (pH 7.8) were found. Km values for ornithineand carbamyl phosphate were 5 x 10^–3 M and 6.8 x 10^–4so respectively. The Km for citrulline in the catabolic directionwas 1 x 10^–2 M. Enzymes obtained from cell-free extractsof germinated spores could synthetize ATP from citrulline andADP under physiological conditions by coupling the phosphorolysisof citrulline with carbamate kinase activity. The initial rateof germination was stimulated in the presence of citrullineas the sole nitrogen source, as compared to arginine, glutamineor yeast extract. These observations suggest that citrullinemay be catabolized during germination by means of OTC ratherthan via the energy-consuming urea cycle. (Received June 26, 1971; )     

A simple method for the freeze-preservation of zoospores of the green macroalga Enteromorpha intestinalis

Settled zoospores of the green macroalga Enteromorpha intestinalis were subjected to several different freezing and storing treatments at both cryogenic and non-cryogenic temperatures after which their viability was assessed using a spore germination bioassay. Three different cooling rates were tested: slow cooling at –1°C min⁻¹ and –0.5°C min⁻¹ to end temperatures in the range –20°C to –40°C, and a two-step procedure whereby the spores were frozen to –30°C at a rate of –1°C min⁻¹ prior to immersion in liquid nitrogen at –196°C. Spore viability was also investigated using the cryoprotectants glycerol and dimethyl suphoxide (DMSO), a reduced saline medium and various storage times. In the majority of experiments, the use of a cryoprotectant during the freezing process significantly increased the viability of the spores, with DMSO affording slightly greater protection than glycerol. All treatments produced high viabilities (ranging from 75.3–100.0%) after 5-min storage at the different end temperatures. However, progressively longer storage up to 7 days generally resulted in a marked reduction in viability. This was with the exception of spores frozen in a reduced saline medium; a medium of 75% seawater and either 5 or 10% DMSO greatly increased spore viability, with values of > 40% recorded for spores stored at –20°C for up to 5 weeks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of naturally occurring polyols and urea on mitochondrial F0F1ATPase

We show that urea inhibits the ATPase activity of MgATP submitochondrial particles (MgATP-SMP) with Ki = 0.7 M, probably as a result of direct interaction with the structure of F0F1-ATPase. Counteracting compounds (sorbitol, mannitol or inositol), despite slightly (10-20%) inhibiting the ATPase activity, also protect the F0F1-ATPase against denaturation by urea. However, this protection was only observed at low urea concentrations (less than 1.5 M), and in the presence of three polyols, the Ki for urea shift from 0.7 M to 1.2 M. Urea also increases the initial activation rate of latent MgATP-SMP in a dose-dependent-manner. However, when the particles (0.5 mg/ml) were preincubated in the presence of 1 M, 2 M or 3 M urea, a decrease in the activation level occurred after 1 h, 30 and 10 min, respectively. At high MgATP-SMP concentration (3 mg/ml) a decrease in activation was observed after 2 h, 1 h and 20 min, respectively. These data indicate that the effect of urea on the activation of MgATP-SMP depends on time, urea and protein concentrations. It was also observed that polyols suppress the activation of latent MgATP-SMP in a dose-dependent manner, and protect the particles against urea denaturation during activation. We suppose that a decrease in membrane mobility promoted by interactions of polyols with phospholipids around the F0F1-ATPase may also increase the compactation of protein structure, explaining the inhibition of natural inhibitor protein of ATPase (IF1) release and the activation of the enzyme.     

Optimization of adenosine 5′-triphosphate extraction for the measurement of␣acidogenic biomass utilizing whey wastewater

A set of experiments was carried out to maximize adenosine 5′-triphosphate (ATP) extraction efficiency from acidogenic culture using whey wastewater. ATP concentrations at different microbial concentrations increased linearly as microbial concentration decreased. More than 50% of ATP was extracted from the sample of 39 mg volatile suspended solids (VSS)/l compared to the sample of 2.8 g VSS/l. The ATP concentrations of the corresponding samples were 0.74±0.06 and 0.49±0.05 mg/l, respectively. For low VSS concentrations ranging from 39 to 92 mg/l, the extracted ATP concentration did not vary significantly at 0.73±0.01 mg ATP/l. Response surface methodology with a central composite in cube design for the experiments was used to locate the optimum for maximal ATP extraction with respect to boiling and bead beating treatments. The overall designed intervals were from 0 to 15 min and from 0 to 3 min for boiling and bead beating, respectively. The extracted ATP concentration ranged from 0.01 to 0.74 mg/l within the design boundary. The following is a partial cubic model where η is the concentration of ATP and x _k is the corresponding variable term (k=boiling time and bead beating time in order): η=0.629+0.035x ₁–0.818x ₂–0.002x ₁ x ₂–0.003x ₁ ² +0.254x ₂ ² +0.002x ₁ ² x ₂. This model successfully approximates the response of ATP concentration with respect to the boiling- and bead beating-time. The condition for maximal ATP extraction was 5.6 min boiling without bead beating. The maximal ATP concentration using the model was 0.74 mg/l, which was identical to the experimental value at optimum condition for ATP extraction.     

Purification and characterization of a β-glucosidase fromAspergillus niger

The high-molar mass from of β-glucosidase fromAspergillus niger strain NIAB280 was purified to homogeneity with a 46-fold increase in purification by a combination of ammonium sulfate precipitation, hydrophobic interaction, ion-exchange and gel-filtration chromatography. The native and subunit molar mass was 330 and 110 kDa, respectively. The pH and temperature optima were 4.6–5.3 and 70°C, respectively. TheK _m andk _cat for 4-nitrophenyl β-d-glucopyranoside at 40°C and pH 5 were 1.11 mmol/L and 4000/min, respectively. The enzyme was activated by low and inhibited by high concentrations of NaCl. Ammonium sulfate inhibited the enzyme. Thermolysin periodically inhibited and activated the enzyme during the course of reaction and after 150 min of proteinase treatment only 10% activity was lost with concomitant degradation of the enzyme into ten low-molar-mass active bands. When subjected to 0–9 mol/L transverse urea-gradient-PAGE for 105 min at 12°C, the nonpurified β-glucosidase showed two major bands which denatured at 4 and 8 mol/L urea, respectively, with half-lives of 73 min.     

Regulation of UT-A1-mediated transepithelial urea flux in MDCK cells

Transepithelial [¹⁴C]urea fluxes were measured across cultured Madin-Darby canine kidney (MDCK) cells permanently transfected to express the urea transport protein UT-A1. The urea fluxes were typically increased from a basal rate of 2 to 10 and 25 nmol·cm^–2·min^–1 in the presence of vasopressin and forskolin, respectively. Flux activation consisted of a rapid-onset component of small amplitude that leveled off within 10 min and at times even decreased again, followed by a delayed, strong increase over the next 30–40 min. Forskolin activated urea transport through activation of adenylyl cyclase; dideoxyforskolin was inactive. Vasopressin activated urea transport only from the basolateral side and was blocked by OPC-31260, indicating that its action was mediated by basolateral V₂ receptors. In the presence of the phosphodiesterase inhibitor IBMX, vasopressin activated as strongly as forskolin. By itself, IBMX caused a slow increase over 50 min to 5 nmol·cm^–2·min^–1. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP; 300 µM) activated urea flux only when added basolaterally. IBMX augmented the activation by basolateral 8-BrcAMP. Urea flux activation by vasopressin and forskolin were only partially blocked by the protein kinase A inhibitor H-89. Even at concentrations >10 µM, urea flux after 60 min of stimulation was reduced by <50%. The rapid-onset component appeared unaffected by the presence of H-89. These data suggest that activation of transepithelial urea transport across MDCK-UT-A1 cells by forskolin and vasopressin involves cAMP as a second messenger and that it is mediated by one or more signaling pathways separate from and in addition to protein kinase A. urea transporter; Madin-Darby canine kidney cells     

Initial protein concentration and residual denaturant concentration strongly affect the batch refolding of hen egg white lysozyme

The effects of several variables on the refolding of hen egg white lysozyme have been studied. Lysozyme was denatured in both urea, and guanidine hydrochloride (GuHCl), and batch refolded by dilution (100 to 1000 fold) into 0.1M Tris-HCl, pH 8.2, 1 mM EDTA, 3 mM reduced glutathione and 0.3 mM oxidised glutathione. Refolding was found to be sensitive to temperature, with the highest refolding yield obtained at 50°C. The apparent activation energy for lysozyme refolding was found to be 56 kJ/mol. Refolding by dilution results in low concentrations of both denaturant and reducing agent species. It was found that the residual concentrations obtained during dilution (100-fold dilution: [GuHCl]=0.06 mM, [DTT]=0.15 mM) were significant and could inhibit lysozyme refolding. This study has also shown that the initial protein concentration (1–10 mg/mL) that is refolded is an important parameter. In the presence of residual GuHCl and DTT, higher refolding yields were obtained when starting from higher initial lysozyme concentrations. This trend was reversed when residual denaturant components were removed from the refolding buffer.