Improved low pH bicelle system for orienting macromolecules over a wide temperature range

We have prepared and characterized a novel bicelle system composed of 1,2-di-O-dodecyl-sn-glycero-3-phos- phocholine (DIODPC) and 3-(chloramidopropyl)dimethylammonio-2-hydroxyl-1-propane sulfonate (CHAPSO). At the optimal DIODPC/CHAPSO molar ratio of 4.3:1, this medium becomes magnetically oriented from pH 6.5 down to pH 1.0. Unlike previously reported bicelle preparations, these bicelles are chemically stable at low pH and are capable of inducing protein alignment, as illustrated by the large residual dipolar couplings measured for rusticyanin from Thiobacillus ferrooxidans at pH 2.1. The DIODPC/CHAPSO system is particularly useful for measuring residual dipolar couplings of macromolecules that require very acidic conditions.     

Thermotaxis of human sperm cells in extraordinarily shallow temperature gradients over a wide range

On the basis of the finding that capacitated (ready to fertilize) rabbit and human spermatozoa swim towards warmer temperatures by directing their movement along a temperature gradient, sperm thermotaxis has been proposed to be one of the processes guiding these spermatozoa to the fertilization site. Although the molecular mechanism underlying sperm thermotaxis is gradually being revealed, basic questions related to this process are still open. Here, employing human spermatozoa, we addressed the questions of how wide the temperature range of thermotaxis is, whether this range includes an optimal temperature or whether spermatozoa generally prefer swimming towards warmer temperatures, whether or not they can sense and respond to descending temperature gradients, and what the minimal temperature gradient is to which they can thermotactically respond. We found that human spermatozoa can respond thermotactically within a wide temperature range (at least 29-41°C), that within this range they preferentially accumulate in warmer temperatures rather than at a single specific, preferred temperature, that they can respond to both ascending and descending temperature gradients, and that they can sense and thermotactically respond to temperature gradients as low as <0.014°C/mm. This temperature gradient is astonishingly low because it means that as a spermatozoon swims through its entire body length (46 μm) it can sense and respond to a temperature difference of <0.0006°C. The significance of this surprisingly high temperature sensitivity is discussed.     

Cholesterol sulfate and calcium affect stratum corneum lipid organization over a wide temperature range

The main diffusion barrier for drugs penetrating through the skin is located in the intercellular lipid matrix in the upper layer of the skin, the stratum corneum (SC). The main lipid classes in the SC are ceramides (CER), free fatty acids (FFA) and cholesterol (CHOL). The lipids in SC are organized into two lamellar phases with periodicities of approximately 13 and 6 nm, respectively. Similar lipid organization has been found with equimolar CHOL:CER:FFA mixtures in SAXD studies performed at room temperature. However, one may conclude that the phase behavior of the mixtures is similar to that in SC only when the lipid organization of the lipid mixtures resembles that in SC over a wide temperature range. Therefore, in the present study, the organization of the lipid mixtures has been studied in a temperature range between 20 degrees and 95 degrees C. From these experiments it appeared that at elevated temperatures in equimolar CHOL:CER:FFA mixtures a new prominent 4.3 nm phase is formed between 35;-55 degrees C, which is absent or only weakly formed in intact human and pig SC, respectively. As it has been suggested that gradients of pH and cholesterol sulfate exist in the SC and that Ca(2+) is present only in the lowest SC layers, the effect of pH, cholesterol sulfate, and Ca(2+) on the lipid phase behavior has been investigated with lipid mixtures. Both an increase in pH from 5 (pH at the skin surface) to 7.4 (pH at the SC;-stratum granulosum interface) and the presence of cholesterol sulfate promote the formation of the 13 nm lamellar phase. Furthermore, cholesterol sulfate reduces the amount of CHOL that is present in crystalline domains, causes a shift in the formation of the 4.3 nm phase to higher temperatures, and makes this phase less prominent at higher temperatures. The finding that Ca(2+) counteracts the effects of cholesterol sulfate indicates the importance of a proper balance of minor SC components for appropriate SC lipid organization. In addition, when the findings are extrapolated to the in vivo situation, it seems that cholesterol sulfate is required to dissolve cholesterol in the lamellar phases and to stabilize SC lipid organization. Therefore, a drop in cholesterol sulfate content in the superficial layers of the SC is expected to destabilize the lipid lamellar phases, which might facilitate the desquamation process.     

Improving Pb2+ detection using DNAzyme-based fluorescence sensors by pairing fluorescence donors with gold nanoparticles

For previously reported fluorescence Pb(2+) sensors, DNAzymes have lead to a significant increase in Pb(2+) detecting sensitivity and specificity. However, these sensors suffer from incomplete fluorescence quenching and require additional steps for annealing DNAzymes and substrates as well as for removing the uncoupled substrates. In this study, we successfully overcome these issues by immobilizing the substrate nucleic acids on gold nanoparticles through thiol linkages. The immobilization of the substrate molecules to the gold nanoparticles lead to almost-complete fluorescence quenching and fast Pb(2+) detection, without altering the Pb(2+) specificity of the DNAzymes. After optimizing the concentration of DNAzymes, reaction time and pH, we could detect Pb(2+) as low as 5 nM within 20 min without the preliminary and the post treatments. Considering the multi-color-fluorescence quenching capability of gold nanoparticles and the to-be-developed functional nucleic acids for other metal ions, this study could extend the application of DNAzymes to the detection of multiple heavy metal ions.     

An effective iodide formulation for killing Bacillus and Geobacillus spores over a wide temperature range

AIMS: To develop a sporicidal reagent which shows potent activity against bacterial spores not only at ambient temperatures but also at low temperatures. METHODS AND RESULTS: Suspension tests on spores of Bacillus and Geobacillus were conducted with the reagent based on a previously reported agent (N. Kida, Y. Mochizuki and F. Taguchi, Microbiology and Immunology 2003; 47: 279-283). The modified reagent (tentatively designated as the KMT reagent) was composed of 50 mmol l(-1) EDTA-2Na, 50 mmol l(-1) ferric chloride hexahydrate (FeCl(3).6H(2)O), 50 mmol l(-1) potassium iodide (KI) and 50% ethanol in 0.85% NaCl solution at pH 0.3. The KMT reagent showed significant sporicidal activity against three species of Bacillus and Geobacillus spores over a wide range of temperature. The KMT reagent had many practical advantages, i.e. activity was much less affected by organic substances than was sodium hypochlorite, it did not generate any harmful gas and it was stable for a long period at ambient temperatures. The mechanism(s) of sporicidal activity of the KMT reagent was considered to be based on active iodine species penetrating the spores with enhanced permeability of the spore cortex by a synergistic effect of acid, ethanol and generated active oxygen. CONCLUSIONS: The data suggest that the KMT reagent shows potent sporicidal activity over a wide range temperatures and possesses many advantages for practical applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate development of a highly applicable sporicidal reagent against Bacillus and Geobacillus spores.     

Enhanced dynamic range in a genetically encoded Ca2+ sensor

Genetically encoded fluorescence resonance energy transfer (FRET) indicators are powerful tools for real-time detection of second messenger molecules and activation of signal proteins. However, these fluorescent protein-based sensors typically display marginal FRET efficiency. To improve their FRET efficiency for optical imaging and screening, we developed a number of fluorescent protein mutants based on cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). To improve FRET ratios, which were initially within a narrow dynamic range, we used DNA shuffling to develop a new FRET pair called 3xCFP/Venus. The optimized 3xCFP/Venus pair exhibited higher FRET ratios than CyPet/YPet, which has one of the greatest dynamic ranges of protein-based FRET pairs. We converted this FRET pair to a Ca(2+) FRET indicators using circular permutation Venus (cpVenus) linked with 3xCFP to form 3xCFP/cpVenus, which displayed an ～11-fold change in dynamic range in response to Ca(2+) binding. The enhanced dynamic range for Ca(2+) concentration detection using 3xCFP/cpVenus was confirmed in PC12 cells using previously established indicators (TN-XXL, ECFP/cpCitrine). To our knowledge, this FRET pair displays the largest dynamic range so far among genetically-encoded sensors, and can be used for sensitive FRET detection.     

MD simulation of self-diffusion and structure in some n-alkanes over a wide temperature range at high pressures

Self-diffusion and structural properties of n-alkanes have been studied by molecular dynamics simulation in the temperature range between the melting pressure curve and 600 K at pressures up to 300 MPa. The simulated results of lower n-alkanes are in good agreement with the existing experimental data, and support the reliability of results of the simulations of self-diffusion coefficients obtained at the extreme conditions. We predict the self-diffusion coefficients for methane, ethane, propane and n-butane at the similar reduced temperatures and pressures to draw a comparison between them. Then the correlation between self-diffusion and structural properties are further investigated by calculating the coordination numbers. Moreover, we define four distances and their corresponding relative deviations to characterize the flexibility of long-chain n-alkanes. The simulated results show that the self-diffusion of n-alkane molecules is mainly affected by the close packing, and the flexibility has a strong impact on the self-diffusion of longer n-alkane molecules.

A sugar-aza-crown ether-based fluorescent sensor for Cu2+ and Hg2+ ions

A sugar-aza-crown ether (SAC)-based fluorescent sensor 4 was prepared. It contains a pyrene as the fluorophore and its fluoroionophoric properties toward transition metal ions were investigated. Chemosensor 4 exhibits highly selective recognition toward Cu(2+) and Hg(2+) ions among a series of tested metal ions in methanol solution. The association constants for 4*Cu(2+) and 4*Hg(2+) in methanol solution were calculated to be 7.4×10(1)M(-1) and 4.4×10(3)M(-1), respectively. Chemosensor 4 formed complexes with the Cu(2+) or Hg(2+) ion at a 1:1 ligand-to-metal ratio with a detection limit of 1.3×10(-4)M Cu(2+) and 1.26×10(-5)MHg(2+), respectively.     

A pyrenyl-appended triazole-based ribose as a fluorescent sensor for Hg2+ ion

For the efficient detection of toxic trace metal ions, two pyrenyl-appended triazole-based d-ribose fluorescent chemosensors 6 and 7 were prepared and their fluoroionophoric properties toward transition metal ions were investigated. Chemosensors 6 and 7 exhibit highly selective recognition toward Hg²⁺ ion among a series of tested metal ions in CH₂Cl₂/MeOH solution. The association constants of 6 and 7 are calculated to be 1.73 × 10⁵ M⁻¹ and 4.44 × 10⁵ M⁻¹, respectively. Both 6 and 7 formed complexes with the Hg²⁺ ion at a 1:1 ligand-to-metal ratio with a detection limit of 10-15 μM Hg²⁺. Computational analysis demonstrated that the Hg²⁺ ion occupied the coordination center of 6 with N² and N³ atoms in two triazole groups, thus separating and distorting the two parallel pyrenes away from each other.     

In Vitro measurement of tissue impedance over a wide frequency range

Summary A simple method is described to determine the loci of the specific impedance of biological materialsin vitro. A waveform generator and a x-y-oscilloscope were used. With this setup impedance loci from 1 kHz to 6.4 MHz could be obtained. The error caused by the final input impedance of the amplifier was corrected mathematically. The loci of the specific impedance of centrifuged human erythrocytes and of rabbit brain cortex, liver and kidney were determined.     

A novel system for expressing recombinant proteins over a wide temperature range from 4 to 35 degrees C

Escherichia coli cells are the most commonly used host cells for large-scale production of recombinant proteins, but some proteins are difficult to express in E. coli. Therefore, we tested the nocardioform actinomycete Rhodococcus erythropolis, which grows at temperatures ranging from 4 to 35 degrees C, as an expression host cell. We constructed inducible expression vectors, where the expression of the target genes could be controlled with the antibiotic thiostrepton. Using these expression vectors, several milligrams of reporter proteins could be isolated from 1 liter of culture of R. erythropolis cells grown at a temperature range from 4 to 35 degrees C. Moreover, we successfully purified serum amyloid A1, NADH dehydorogenase 1 alpha subcomplex 4, cytochrome b5-like protein, apolipoprotein A-V, cathepsin D, pancreatic Rnase, and HMG-1 that are all difficult to express in E. coli. In the case of kallikrein 6, mouse deoxyribonuclease I and Kid1, which are also difficult to express in E. coli, the expression level of each protein increased when proteins were expressed at low temperature (4 degrees C). Based on these results, we conclude that a recombinant protein expression system using R. erythropolis as the host cell is superior to respective E. coli systems.     

Circadian rhythms in the thermophilic cyanobacterium Thermosynechococcus elongatus: compensation of period length over a wide temperature range

Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical, and X-ray crystallographic studies. We developed an automated bioluminescence real-time monitoring system for the circadian clock in the thermophilic cyanobacterium T. elongatus BP-1 that uses a bacterial luciferase gene set (Xl luxAB) derived from Xenorhabdus luminescens as a bioluminescence reporter gene. A promoter region of the psbA1 gene of T. elongatus was fused to the Xl luxAB gene set and inserted into a specific targeting site in the genome of T. elongatus. The bioluminescence from the cells of the psbA1-reporting strain was measured by an automated monitoring apparatus with photomultiplier tubes. The strain exhibited the circadian rhythms of bioluminescence with a 25-h period length for at least 10 days in constant light and temperature. The rhythms were reset by light-dark cycle, and their period length was almost constant over a wide range of temperatures (30 to 60 degrees C). Theses results indicate that T. elongatus has the circadian clock that is widely temperature compensated.     

The freshwater red alga Batrachospermum turfosum (Florideophyceae) can acclimate to a wide range of light and temperature conditions

Batrachospermum turfosum Bory is one of the generalists among the few red algae that have adapted to freshwater habitats, occurring in a variety of primarily shaded, nutrient-poor micro-habitats with lotic (running) or lentic (standing) waters. Seasonal variations in water level and canopy cover can expose this sessile alga to widely fluctuating temperatures, solar irradiation and nutrient availability. Here we report on the ecophysiology of B. turfosum collected from an ultra-oligotrophic bog pool in the Austrian Alps. Photosynthesis as a function of photon fluence density (PFD) and temperature was studied by measuring oxygen evolution in combination with chlorophyll fluorescence. In addition, the effects of ultraviolet radiation (UVR) on photosynthetic pigments were analysed using HPLC and spectrophotometric methods, and cellular ultrastructure was studied using transmission electron microscopy. We found that B. turfosum is adapted to low light, with a light compensation point (I_c) and a light saturation point (I_k) of 8.4 and 29.7 µmol photons m^–2 s^–1, respectively, but also tolerates higher PFDs of ~1000 µmol photons m^–2 s^–1, and is capable of net photosynthesis at temperatures between 5°C and 35°C. Exposure to either UV-A or UV-AB for 102 h led to a strong transient drop in effective quantum yield (ΔF/F_M’), followed by an acclimation to about 70% of initial ΔF/F_M’ values. Ultrastructural changes included the accumulation of plastoglobules and dilated membranes after UVR treatment. Although all photosynthetic pigments strongly decreased upon UVR exposure and no UV-photoprotectants (e.g. mycosporine-like amino acids) could be detected, the alga was capable of recovering ΔF/F_M’ and phycobiliproteins after UVR treatment. In summary, B. turfosum tolerates a wide range of irradiation and temperature regimes, and these traits may be the basis for its successful adaptation to challenging environments.     

Synthesis of a ratiometric fluorescent peptide sensor for the highly selective detection of Cd2+

A novel ratiometric fluorescent peptidyl chemosensor (Dansyl-Cys-Pro-Gly-Cys-Trp-NH(2), D-P5) for metal ions detection has been synthesized via Fmoc solid-phase peptide synthesis. The chemosensor exhibited a high selectivity for Cd(2+) over other metal ions including competitive transition and Group I and II metal ions in neutral pH. The fluorescence emission intensity of D-P5 was significantly enhanced in the presence of Cd(2+) by fluorescent resonance energy transfer (FRET) and chelation enhanced fluorescence (CHEF) effects. The binding stoichiometry, detection limit, binding affinity, reversibility and pH sensitivity of the sensor for Cd(2+) were investigated.     

A generic geometric transformation that unifies a wide range of natural and abstract shapes

To study forms in plants and other living organisms, several mathematical tools are available, most of which are general tools that do not take into account valuable biological information. In this report I present a new geometrical approach for modeling and understanding various abstract, natural, and man-made shapes. Starting from the concept of the circle, I show that a large variety of shapes can be described by a single and simple geometrical equation, the Superformula. Modification of the parameters permits the generation of various natural polygons. For example, applying the equation to logarithmic or trigonometric functions modifies the metrics of these functions and all associated graphs. As a unifying framework, all these shapes are proven to be circles in their internal metrics, and the Superformula provides the precise mathematical relation between Euclidean measurements and the internal non-Euclidean metrics of shapes. Looking beyond Euclidean circles and Pythagorean measures reveals a novel and powerful way to study natural forms and phenomena.     

Adaptive advantages of myrmecochory: the predator-avoidance hypothesis tested over a wide geographic range

The predator‐avoidance hypothesis states that once released from the parent plant, myrmecochorous seeds are rapidly taken by ants to their nests, where they are protected from predators. Previous studies conducted to test this hypothesis have frequently neglected two major aspects necessary for its verification: 1) the influence of processes acting after the seed release and 2) the spatial evenness of such processes. Thus, large‐scale variations in the mechanisms acting beyond seed release, and possibly influencing seed escape from predators, remain poorly documented. Here, we present the results of a post‐dispersal seed‐removal experiment on the myrmecochorous herb Helleborus foetidus, aimed at verifing the predator‐avoidance hypothesis by considering two key post‐release aspects of seed fate: seed destination (dispersed or nondispersed) and seed burial (buried or not buried). Experiments were performed in four different regions in the Iberian Peninsula. After three days of exposure of seeds to the main predator (fieldmice Apodemus sylvaticus), ca 30% of the seeds were removed. Seed destination affected the proportion of seeds escaping predation, but the sign, magnitude and statistical significance of the effect varied among the geographical regions. In the southern region (Cazorla), seeds dispersed in ant nests or intermediate destinations suffered scarcely any predation, but seeds under reproductive‐age plants experienced losses ca 50%. Conversely, in the northern region (Caurel), seeds in nests suffered significantly greater losses than seeds under plants or intermediate destinations, suggesting that nests were especially unsafe destinations. Seed burial had a strong impact on seed escape from predators, and its effect was highly consistent among geographical regions. In view of the consistency of its effect at different spatial scales, seed burial was a more general mechanism for predation avoidance than seed relocation to ant nests, which was habitat‐ and/or ant‐species‐dependent. Our results thus only partially support the predator‐avoidance hypothesis for the evolution of myrmecochory.     

Electric signals during the bacteriorhodopsin photocycle, determined over a wide pH range.

From the electric signals measured after photoexcitation, the electrogenicity of the photocycle intermediates of bacteriorhodopsin were determined in a pH range of 4.5-9. Current measurements and absorption kinetic signals at five wavelengths were recorded in the time interval from 300 ns to 0.5 s. To fit the data, the model containing sequential intermediates connected by reversible first-order reactions was used. The electrogenicities were calculated from the integral of the current signal, by using the time-dependent concentrations of the intermediates, obtained from the fits. Almost all of the calculated electrogenicities were pH independent, suggesting that the charge motions occur inside the protein. Only the N intermediate exhibited pH-dependent electrogenicity, implying that the protonation of Asp96, from the intracellular part of the protein, is not from a well-determined proton donor. The calculated electrogenicities gave good approximations of all of the details of the measured electric signals.     

The gel-filtration behaviour of proteins related to their molecular weights over a wide range

1. Correlation between elution volume, V_e, and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (α-crystallin) on Sephadex G-200 columns at pH7·5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V_e–log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V_e–log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of `typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including γ-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10⁶. 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of γ-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.     

Quantification of gene expression over a wide range by the polymerase chain reaction.

We investigated the usefulness of the polymerase chain reaction (PCR) method for the relative quantification of gene expression using a simultaneously amplified sequence of beta-actin mRNA as an internal control for the target sequence of tax/rex mRNA of human T-cell leukemia virus type I. The PCR product of the internal control was reduced by delaying the addition of the primers for its sequence. The photostimulated luminescence of the bands was measured with a laser image analyzer, and the values were plotted against the cycle number. The cycle differences between the logarithmic phase of the curves for the target sequence and for beta-actin (delta cycle) showed a linear correlation with the initial concentration of the sample. This method is highly sensitive for evaluating gene expression over a wide range.     

Kinetic and thermodynamic study of the bacteriorhodopsin photocycle over a wide pH range.

The photocycle of bacteriorhodopsin and its thermodynamic parameters were studied in the pH range of 4.5-9. Measurements were performed at five different wavelengths (410, 500, 570, 610, and 650 nm), in the time interval 300 ns to 0.5 s, at six temperatures between 5 and 30 degreesC. Data were fitted to different photocycle models. The sequential model with reversible reactions gave a good fit, and the linear character of the Eyring plots was fulfilled. The parallel model with unidirectional reactions gave a poor fit, and the Eyring plot of the rate constants did not follow the expected linear behavior. When a parallel model with reversible reactions, which has twice as many free parameters as the sequential model, was considered, the quality of the fit did not improve and the Eyring plots were not linear. The sequential model was used to determine the thermodynamic activation parameters (activation enthalpy, entropy, and free energy) of the transitions and the free energy levels of the intermediates. pH dependence of the parameters revealed details of the transitions between the intermediates: the transitions M1 to M2 and N to O disclosed a large entropy increase, which could be interpreted as a loosening of the protein structure. The pH dependence of the energy levels explains the disappearance of intermediate O at high pH. A hypothesis is proposed to interpret the relation between the observed pKa of the photocycle energetics and the role of several amino acids in the protein.