Experimental Determination and Theoretical Calculation of the Eutectic Composition of Cefuroxime Axetil Diastereomers

Cefuroxime axetil (CFA), an ester prodrug of cefuroxime exists as a pair of diastereoemers, namely isomer A and isomer B. To enable phase diagram construction, crystallization of the diastereomers of CFA from the commercially available amorphous drug substance was carried out. Isomer A was separated with a purity approaching 100% whereas the maximum purity of isomer B was 85% as confirmed by solution state proton NMR spectroscopy. The crystalline forms of isomer A and isomer B were confirmed as forms AI and BI, respectively, based on differential scanning calorimetry (DSC) analysis and powder X-ray diffraction. DSC analysis was used to observe the melting behavior of different diastereomer mixture compositions. The binary solid-liquid phase diagram for mixture compositions ranging from 0 to 85% w/w isomer B indicated the formation of a eutectic mixture having a melting temperature of 124.7?±?0.4°C and a composition of 75% w/w (+/?5% wt.) isomer B. The eutectic composition was calculated using an index based on the van’t Hoff equation for melting point depression and was found to be 75% isomer B and 25% isomer A. As CFA is present in commercial preparations as a mixture of diastereomers, the formation of a eutectic mixture between the diastereomers may impact the solubility and stability of the commercial product. Eutectic formation can be explained on the basis of the chemical similarity of diastereomers that favor miscibility in the liquid state.     

Calorimetric,density and circular dichroism studies of the reversible structural transition in Pf1 filamentous bacterial virus

The macromolecular structural transition of Pf1 filamentous bacterial virus detected by X-ray diffraction analysis has been studied in virus solutions by density, circular dichroism, and microcalorimetric measurements. The reversible structural change occurring between 5 °C and 25 °C has a calorimetrically determined transition enthalpy ΔH_t,cal of 14·5 ± 1.5 kJ (mol protein)^?1. The transition curves resulting from the density, circular dichroism, and calorimetric measurements have been analysed in terms of a two-state process to extract the van't Hoff enthalpy. Comparison of the effective transition enthalpy and the calorimetric ΔH_t,cal values gives about 26 protein subunits as the size of the co-operative unit. Parallel heat capacity and density measurements on fd virus show no such transition, in agreement with X-ray diffraction studies.     

A comparison of van't Hoff and calorimetric heats of binding to DNA using an ethidium selective electrode

A liquid membrane electrode selective for ethidium ion was used to measure free ethidium in mixtures with calf thymus DNA. Electrode response was unaffected by variation in ionic strength from 1 mm to 0.5 m, and was not degraded over the temperature range studied. DNA-ethidium binding isotherms obtained with the electrode at 17.4, 25.4, 30.1, and 40.6 °C were fitted to a single class of excluded sites model for $\text{}\text{?}$gn ranging from 0.01 to 0.16. van't Hoff analysis of these data yielded ΔH = ?8300 cal/mol ethidium bound (in 0.5 m KCl, 10 mm Tris buffer, pH 10, 1 mm EDTA). Direct calorimetric measurements of the heat of complex formation led to a value of ?7600 cal/mol at 25 °C in the same medium; the two results were not significantly different at the 95% confidence level. The agreement supports the validity of the ethidium selective electrode, and illustrates its utility in the study of ligand binding to nucleic acids and related materials.     

The Effect of Tripalmitin Crystallization on the Thermomechanical Properties of Candelilla Wax Organogels

The effect of tripalmitin (TP) crystallization on the thermomechanical properties of organogels developed with candelilla wax (CW) was investigated using safflower oil high in triolein (HOSFO) as the liquid phase. Factorial combinations of CW (i.e., 0–3%) and TP (i.e., 0–1%) in the HOSFO were used to develop organogels at three different temperatures (T _set). The onset of crystallization (T _g) during the cooling stage (10 °C/min), the melting temperature (T _M), and the corresponding heat of melting (ΔH _M) of the organogels were determined by differential scanning calorimetry. Results showed that, without CW, the crystallization of TP in the HOSFO at the concentrations and T _set investigated (i.e., −10 °C to 25 °C) did not develop a three-dimensional network that provided significant viscoelasticity (i.e., solid-like behavior) to the HOSFO. The CW developed organogels in the HOSFO with T _M’s that increased from ≈30.5 °C up to ≈42.5 °C as a function of CW concentration. In contrast, in the CW–1% TP system, the co-crystallization of TP and CW resulted in organogels with T_M’s that varied just between 36 °C and 38 °C, independent of the CW concentration. Higher elastic modulus (G′) and yield stress (σ*) were obtained with 3% CW–1.0% TP organogels than with organogels developed just by CW, particularly at T _set’s of −5 °C and 15 °C. This research showed that co-crystallization of TP and CW, occurring at different extent as a function of T _set, resulted in organogels with thermomechanical properties different from the ones showed by CW organogels. The results showed that co-crystallization of triacylglycerides with CW might be a useful alternative to tailor particular physicochemical properties associated to a specific functionality (i.e., melting profile and texture). Organogelation of vegetable oil might be used to develop trans-free vegetable-oil-based spreads and coatings and also novel food products with new textural perceptions for the consumers.     

A Promising Evaluation Method for Dead Leaves of Posidonia oceanica (L.) in the Adsorption of Methyl Violet

Posidonia oceanica (Linnaeus) Delile is an endemic species in the Mediterranean Sea. In the present study, dead leaves of P. oceanica (L.) which are accumulated on the beach seasonally were used as an alternative low cost biosorbent to remove methyl violet (MV) from aqueous solution. In order to explain the adsorption kinetics, pseudo first-order and pseudo second-order kinetic models were investigated. It was obtained that the pseudo second-order model was well in line with our experimental results. Equilibrium data were analyzed according to Langmuir, Freundlich, and Dubinin–Radushkevich equations. Langmuir isotherm model corresponded well with equilibrium data than the others, and the maximum adsorption capacity of the biomass was found to be 119.05 mg g⁻¹ at 45°C. Thermodynamic parameters, ΔG°, ΔH°, and ΔS°, were calculated according to van’t Hoff equation. Negative values of Gibbs free energy imply that the process is spontaneous. Consequently, dried biomass of this species can be an alternative and low cost material for the removal of MV from wastewaters. Moreover, since the remarkable adsorption capacity of these dead leaves compared to other low cost adsorbents has been observed, this beach waste could provide an economic contribution to the countries in the Mediterranean region.     

Accounting for apparent deviations between calorimetric and van't Hoff enthalpies

Background

In theory, binding enthalpies directly obtained from calorimetry (such as ITC) and the temperature dependence of the binding free energy (van't Hoff method) should agree. However, previous studies have often found them to be discrepant.

A non-ideal replacement for the Boyle van't Hoff equation

A non-ideal osmotic equilibrium equation is proposed as a replacement for the Boyle van’t Hoff equation to describe the equilibrium volume of a living cell as a function of external osmolality. Contrary to common understanding, the Boyle van’t Hoff equation is only thermodynamically correct for ideal, dilute solutions. However, the Boyle van’t Hoff equation is commonly used to determine the osmotically inactive fraction of the cell. This involves extrapolating to infinite osmolality, which violates the ideal, dilute solution constraint. It has been noted that the osmotically inactive fractions obtained from the Boyle van’t Hoff equation for human erythrocytes are markedly larger than measured values of the dry volume fraction of the cell. Using the new osmotic equilibrium equation to analyze experimental osmotic equilibrium data reduces the inferred osmotically inactive fraction of human erythrocytes by approximately 20%.     

Cooperative unit size in the gel-liquid crytalline phase transition of dipalmitoyl phosphatidylcholine-water multilayers: An estimate from raman spectroscopy

The temperature dependence of the Raman spectral transitions assigned to the acyl chain C-C stretching modes of dipalmitoyl phosphatidylcholine was determined for the gel, phase transition and liquid crystalline states of the lipid multilayers. The van't Hoff enthalpy differences $\text{ΔH}{}_{\mathrm{<mtext>VH</mtext>}}$ between trans and gauche rotational isomers were obtained from the Raman spectral data for the temperature region characteristics of each bilayer state. An average size for the cooperative unit undergoing the chain melting process during the phase transition was estimated from the ratio of the appropriate van't Hoff enthalpy to an adjusted calorimetric enthalpy.     

Cyto B dependent and ouabain insensitive Regulatory Volume Decrease in bicellular mouse embryo

Mouse single-cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to determine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicellular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three-dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco’s experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van’ t Hoff law defines swelling phase with the effective hydraulic conductivity of 0.3 micron · min⁻¹ · atm⁻¹. Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment.     

A conformational change in concanavalin A detected by a calorimetric study of the binding of methyl α-D-mannopyranoside

The binding of methyl α-D-mannopyranoside to the lectin concanavalin A was studied by means of calorimetry. An apparent enthalpy of binding was also calculated from the variation of the equilibrium constant with temperature (van't Hoff ΔH). The ΔH measured directly was ?30 to ?38 kJ/mole indicating that the binding is driven by the ΔH change. In contrast, the van't Hoff ΔH was substantially smaller, about zero at pH 5.2. The difference in the ΔH measured directly and the van't Hoff ΔH implies that the conformation of concanavalin A undergoes a temperature dependent change at both pH's but most predominantly at pH 5.2. The existence of this conformational change was verified by difference absorption spectroscopy.     

A climate response function explaining most of the variation of the forest floor needle mass and the needle decomposition in pine forests across Europe

The forest floor needle mass and the decomposition rates of pine needle litter in a European climate transect were studied in order to estimate the impact of climate change on forest soil carbon sequestration. Eight pine forests preserved from fire were selected along a climatic latitudinal gradient from 40° to 60° N, from Spain and Portugal to Sweden. The forest floor (Oi and Oe layers) was sorted into five categories of increasing decomposition level according to morphological criteria. The needle mass loss in each category was determined using a linear mass density method. The needle decomposition rate was calculated from the needle fall (NF), the mass of each category and its mass loss. For each site, the remaining mass vs. the calculated time was best fitted by an asymptotic model which indicates that the organic matter should be made up of two fractions: a decomposable one and a recalcitrant one. NF was correlated with actual evapotranspiration (AET) whereas the decomposition parameters (decomposition rate of the decomposable fraction, first year mass loss, forest floor needle mass, age of the most-decomposed category) were related to a combined response function to climate (CRF) based on the van’t Hoff law for temperature and the water deficit (DEF) for moisture. Scenarios with temperature increases, without and with DEF increases, were applied to predict forest floor needle mass changes. C would be lost from the forest floor if only temperature increases and this loss would increase from south to north. If more droughts occur, the forest floor would then tend to sequester C according to the level of the DEF and the latitude of the site. For example, a site in Portugal which is presently the most active site of the transect in terms of decomposition because of its present favourable warm Atlantic climate would react with a large range of responses, losing carbon under an unchanged precipitation regime and sequestering up to 3 times its present stock of carbon under drier conditions.Section Editor: C. Neill     

The effects of day and night temperatures during early growth of winter oilseed rape (Brassica napus L. var. oleifera cv. Górczański) seedlings on their morphology and cold acclimation responses

Studies on the effects of temperature during the early stage of growth on frost resistance of winter rape seedlings under controlled conditions were performed. It was found that cold acclimation responses of plants were affected to a great extent by the conditions of the seedlings early growth. During this period, when the day temperatures were reduced to the range from +10 °C to +15 °C, a process termed “prehardening” was observed. During prehardening plants formed leaf rosettes. Their ability to develop frost resistance during acclimation at +2 °C also increased. Frost resistance of these plants was comparable with the resistance of plants growing in autumn under field conditions.     

Effects of short-term heat stress on oxidative damage and responses of antioxidant system in Lilium longiflorum

This paper aims to determine the changes in reactive oxygen species (ROS) and the responses of the lily (Lilium longiflorum L.) antioxidant system to short-term high temperatures. Plants were exposed to three levels of heat stress (37°C, 42°C, 47°C) for 10 h when hydrogen peroxide (H₂O₂) and superoxide (O₂⁻) production rate along with membrane injury indexes, and changes in antioxidants were measured. Compared with the control (20°C), electrolyte leakage and MDA concentration varied slightly after 10 h at 37°C and 42°C, while increased significantly at 47°C. During 10 h at 37°C and 42°C, antioxidant enzyme activities, such as SOD, POD, CAT, APX and GR, were stimulated and antioxidants (AsA and GSH concentrations) maintained high levels, which resulted in low levels of O₂⁻ and H₂O₂ concentration. However, after 10 h at 47°C, SOD, APX, GR activities and GSH concentration were similar to the controls, while POD, CAT activities and AsA concentration decreased significantly as compared with the control, concomitant with significant increase in O₂⁻ and H₂O₂ concentrations. In addition, such heat-induced effects on antioxidant enzymes were also confirmed by SOD and POD isoform, as Cu/ZnSOD maintained high stability under heat stress and the intensity of POD isoforms reduced with the duration of heat stress, especially at 47°C. It is concluded that in lily plants, the oxidative damage induced by heat stress was related to the changes in antioxidant enzyme activities and antioxidants.     

Effects of heat treatment on the protein secondary structure and pigment microenvironment in photosystem 1 complex

The protein secondary structure and pigments' microenvironment in photosystem 1 (PS1) complexes were studied in the temperature range of 25–80 °C using Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy, respectively. Quantitative analysis of the component bands of the amide I band (1 700–1 600 cm⁻¹) showed no significant change below 50 °C. However, apparent conformational changes occurred at 60 °C and further continued at 70 and 80 °C accompanied with transitions of secondary structure mainly from α-helix to the β-sheet structures. CD analysis demonstrated that the regular arrangement, viz. protein microenvironment of pigments of PS1 complexes, was destroyed by heat treatment which might come from the changes of protein secondary structure of PS1. The CD signals at 645 nm contributed by chlorophyll (Chl) b of light-harvesting complex 1 (LHC1) were easily destroyed at the beginning of heat treatment (25–60 °C). When temperature reached 70 and 80 °C, the CD signals at 478 nm contributed mainly by Chl b of LHC1 and 498 nm contributed by carotenoids decreased most rapidly, indicating that LHC1 was more sensitive to high temperature than core complexes. In addition, the oxygen uptake rate decreased by 90.81 % at 70 °C and was lost completely at 80 °C showing that heat treatment damaged the regular function of PS1 complexes. This may be attributed to heat-induced changes of pigment microenvironment and protein secondary structure, especially transmembrane α-helix located in PsaA/B of PS1.     

Differential scanning calorimetric study of the thermal unfolding of myosin rod,light meromyosin,and subfragment 2

The thermal unfolding of myosin rod, light meromyosin (LMM), and myosin subfragment 2 (S-2) was studied by differential scanning calorimetry (DSC) over the pH range of 6.5–9.0 in 0.5M KCl and either 0.20M sodium phosphate or 0.15M sodium pyrophosphate. Two rod samples were examined: one was purified by Sephadex G-200 without prior denaturation (native rod), and the other was purified by a cycle of denaturation-renaturation followed by Sephacryl S-200 chromatography (renatured rod). There were clearly distinguishable differences in the calorimetric behavior of these two samples. At pH 7.0 in phosphate the DSC curves of native rod were deconvoluted into six endothermic two-state transitions with melting temperatures in the range of 46–67°C and a total enthalpy of 4346 kJ/mol. Under identical conditions the melting profile of LMM was resolved into five endothermic peaks with transition temperatures in the range of 45–66°C, and the thermal profile of long S-2 was resolved into two endotherms, 46 and 57°C. Transition 4 observed with native rod was present in the deconvoluted DSC curve for long S-2, but absent in the DSC curve for LMM. This transition was identified with the high-temperature transition detected with long S-2 and attributed to the melting of the coiled-coil α-helical segment of subfragment 2 (short S-2). The low-temperature transition of long S-2 was attributed to the unfolding of the hinge region. The smallest transition temperatures observed for all three fragments were 45–46°C. It is suggested that the most unstable domain in rod (domain 1) responsible for the 46°C transition includes both the hinge region, which is the C-terminal segment of long S-2, and a short N-terminal segment of LMM. This domain, accounting for 21% of the rod structure, contains the S-2/LMM junction, and upon proteolytic cleavage yields the C-terminal and N-terminal ends of long S-2 and LMM, respectively. Over the pH range of 6.5–7.5, the observed specific heat of denaturation of rod was approximately equal to the sum of the specific heats of LMM and S-2. This finding provides an additional argument for the existence of independent domains in myosin rod.     

Heat shock on <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> inoculum increases glycerol production in wine fermentation

A heat shock was applied to Saccharomyces cerevisiae: a change from 18°C to 45°C over 5 min and then maintenance at later temperature for 20 min followed by cooling to 18°C. Such a treated inoculum, when used in an alcoholic fermentation of Welsch Riesling grape must at 18°C, gave up to 12 g glycerol l⁻¹ This is a new and easy method for high glycerol production in large scale wine production.     

‘Safe sites’ for the seed germination ofRhus javanica: A characterization by responses to temperature and light

Germination responses ofRhus javanica L. seeds to temperature and light were investigated with special reference to their gap-detecting mechanisms in germination, i.e., responses to elevated and/or fluctuating temperatures and sensitivity to leaf-canopy transmitted light. The seeds, which have water-impermeable coats to prevent imbibition, were shown to become permeable and germinable after exposure to higher temperatures of 48–74°C for a brief period depending on the temperature. Once the coat impermeability had been removed by such heat treatment, the seeds became readily germinable over a wide range of temperature and light conditions. The lower and higher temperature limits for germination were around 8° and 36°C, respectively, with an optimal temperature of around 25°C. Simple linear relationships were observed between the temperature and germination rates, i.e., the reciprocals of the time taken by the seed subpopulations to show 10–70% germination in the sub-optimal temperature range, where the required ‘thermal time’ for germination was 2300–3600 Kh. The presence or absence of light or a simulated ‘canopy light’ had little effect on the germination of this species. It was concluded that the seeds ofR. javanica are furnished with a gap-detecting mechanism in the form of a heat requirement for the breakage of water-impermeable seed dormancy, which may be fulfilled by either daytime elevation of the surface temperature of exposed soil, or more effectively by fire.     

Unfolding properties of recombinant human serum albumin products are due to bioprocessing steps

We have used differential scanning calorimetry (DSC) to determine the unfolding properties of commercial products of human serum albumin (HSA) prepared from pooled human blood, transgenic yeast, and transgenic rice. The initial melting temperatures (T_m1) for the unfolding transitions of the HSA products varied from 62°C to 75°C. We characterized the samples for purity, fatty acid content, and molecular weight. The effects of adding fatty acids, heat pasteurization, and a low pH defatting technique on the transition temperatures were measured. Defatted HSA has a structure with the lowest stability (T_m of ～62°C). When fatty acids are bound to HSA, the structure is stabilized (T_m of ～64–72°C), and prolonged heating (pasteurization at 60°C) results in a heat‐stabilized structural form containing fatty acids (T_m of ～75–80°C). This process was shown to be reversible by a low pH defatting step. This study shows that the fatty acid composition and bioprocessing history of the HSA commercial products results in the large differences in the thermal stability. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:62–69, 2015     

Behavior of Freezable Bound Water in the Bacterial Cellulose Produced by Acetobacter xylinum: An Approach Using Thermoporosimetry

The aim of the study is to examine thermal behavior of water within reticulated structure of bacterial cellulose (BC) films by sub-ambient differential scanning calorimetry (DSC). BC films with different carbon source, either manitol (BC (a)) or glycerol (BC (b)), were produced by Acetobacter xylinum using Hestrin and Shramm culture medium under static condition at 30 ± 0.2°C for 3 days. BC samples were characterized by electron scanning microscopy and X-ray diffraction spectroscopy. The pore analysis was done by B.H.J. nitrogen adsorption. The pre-treated with 100% relative humidity, at 30.0 ± 0.2°C for 7 days samples were subjected to a between 25 and −150°C-cooling–heating cycle of DSC at 5.00°C/min rate. The pre-treated samples were also hydrated by adding 1 μl of water and thermally run with identical conditions. It is observed that cellulose fibrils of BC (a) were thinner and reticulated to form slightly smaller porosity than those of BC (b). They exhibited slightly but non-significantly different crystalline features. The freezable bound water behaved as a water confinement within pores rather than a solvent of polymer which is possible to use thermoporosimetry based on Gibb–Thomson equation to approach pore structure of BC. In comparison with nitrogen adsorption, it was found that thermoporosimetry underestimated the BC porosity, i.e., the mean diameters of 23.0 nm vs. 27.8 nm and 27.9 nm vs. 33.9 nm for BC (a) and BC (b), respectively, by thermoporosimetry vs. B.H.J. nitrogen adsorption. It may be due to large non-freezable water fraction interacting with cellulose, and the validity of pore range based on thermodynamic assumptions of Gibb–Thomson theory.     

Combined NMR-observation of cold denaturation in supercooled water and heat denaturation enables accurate measurement of ΔC p of protein unfolding

Cold and heat denaturation of the double mutant Arg 3→Glu/Leu 66→Glu of cold shock protein Csp of Bacillus caldolyticus was monitored using 1D ¹H NMR spectroscopy in the temperature range from −12°C in supercooled water up to +70°C. The fraction of unfolded protein, f _u, was determined as a function of the temperature. The data characterizing the unfolding transitions could be consistently interpreted in the framework of two-state models: cold and heat denaturation temperatures were determined to be −11°C and 39°C, respectively. A joint fit to both cold and heat transition data enabled the accurate spectroscopic determination of the heat capacity difference between native and denatured state, ΔC _p of unfolding. The approach described in this letter, or a variant thereof, is generally applicable and promises to be of value for routine studies of protein folding.