Luminescence of thymine aqueous solutions at the room temperature

The distinguishing features of luminescence of aqueous thymine solutions at room temperature (a broadening of luminescence spectra as compared with low-temperature spectra and differences between excitation and absorption spectra) are discussed. It is shown that these features are due to bimolecular photochemical reactions that lead to the formation of photoproducts (at the first stages, photoadducts) with a comparatively high luminescence ability.     

On the hydrodynamic analysis of macromolecular conformation

Hydrodynamics provides a powerful complementary role to the traditional "high resolution" techniques for the investigation of macromolecular conformation, especially in dilute solution, conditions which are generally inaccessible to other structural techniques. This paper describes the state of art of hydrodynamic representations for macromolecular conformation, in terms of (1) simple but straightforward ellipsoid of revolution modelling; (2) general triaxial ellipsoid modelling; (3) hydrodynamic bead modelling; (4) the ability, especially for polydisperse macromolecular systems, to distinguish between various conformation types; (5) analysis of macromolecular flexibility.     

Direct measurement of osmotic pressure of glycosaminoglycan solutions by membrane osmometry at room temperature

Articular cartilage is a hydrated soft tissue composed of negatively charged proteoglycans fixed within a collagen matrix. This charge gradient causes the tissue to imbibe water and swell, creating a net osmotic pressure that enhances the tissue's ability to bear load. In this study we designed and utilized an apparatus for directly measuring the osmotic pressure of chondroitin sulfate, the primary glycosaminoglycan found in articular cartilage, in solution with varying bathing ionic strength (0.015 M, 0.15 M, 0.5 M, 1 M, and 2 M NaCl) at room temperature. The osmotic pressure (pi) was found to increase nonlinearly with increasing chondroitin sulfate concentration and decreasing NaCl ionic bath environment. Above 1 M NaCl, pi changes negligibly with further increases in salt concentration, suggesting that Donnan osmotic pressure is negligible above this threshold, and the resulting pressure is attributed to configurational entropy. Results of the current study were also used to estimate the contribution of osmotic pressure to the stiffness of cartilage based on theoretical and experimental considerations. Our findings indicate that the osmotic pressure resulting from configurational entropy is much smaller in cartilage (based on an earlier study on bovine articular cartilage) than in free solution. The rate of change of osmotic pressure with compressive strain is found to contribute approximately one-third of the compressive modulus (H(A)(eff)) of cartilage (Pi approximately H(A)(eff)/3), with the balance contributed by the intrinsic structural modulus of the solid matrix (i.e., H(A) approximately 2H(A)(eff)/3). A strong dependence of this intrinsic modulus on salt concentration was found; therefore, it appears that proteoglycans contribute structurally to the magnitude of H(A), in a manner independent of osmotic pressure.     

Chain and conformation stability of solid-state DNA: implications for room temperature storage

There is currently wide interest in room temperature storage of dehydrated DNA. However, there is insufficient knowledge about its chemical and structural stability. Here, we show that solid-state DNA degradation is greatly affected by atmospheric water and oxygen at room temperature. In these conditions DNA can even be lost by aggregation. These are major concerns since laboratory plastic ware is not airtight. Chain-breaking rates measured between 70°C and 140°C seemed to follow Arrhenius’ law. Extrapolation to 25°C gave a degradation rate of about 1–40 cuts/10⁵ nucleotides/century. However, these figures are to be taken as very tentative since they depend on the validity of the extrapolation and the positive or negative effect of contaminants, buffers or additives. Regarding the secondary structure, denaturation experiments showed that DNA secondary structure could be preserved or fully restored upon rehydration, except possibly for small fragments. Indeed, below about 500 bp, DNA fragments underwent a very slow evolution (almost suppressed in the presence of trehalose) which could end in an irreversible denaturation. Thus, this work validates using room temperature for storage of DNA if completely protected from water and oxygen.     

The active site of pepsin is formed in the intermediate conformation dominant at mildly acidic pH

Pepsin is an aspartic protease that acts in food digestion in the mammal stomach. An optimal pH of around 2 allows pepsin to operate in its natural acidic environment, while at neutral pH the protein is denatured. Although the pH dependence of pepsin activity has been widely investigated since the 40s, a renewed interest in this protein has been fueled by its homology to the HIV and other aspartic proteases. Recently, an inactive pepsin conformation has been identified that accumulates at mildly acidic pH, whose structure and properties are largely unknown. In this paper, we analyse the conformation of pepsin at different pHs by a combination of spectroscopic techniques, and obtain a detailed characterisation of the intermediate. Our analysis indicates that it is the dominant conformation from pH 4 to 6.5. Interestingly, its near UV circular dichroism spectrum is identical to that of the native conformation that appears at lower pH values. In addition, we show that the intermediate binds the active site inhibitor pepstatin with a strength similar to that of the native conformation. Pepsin thus adopts, in the 6.5-4.0 pH interval, a native-like although catalytically inactive conformation. The possible role of this intermediate during pepsin transportation to the stomach lumen is discussed.     

On the 710 nm fluorescence emitted by the diatom Phaeodactylum tricornutum at room temperature

The fluorescence emitted at 710 nm by Phaeodactylum tricornutum (F(710)) was characterized. Development of F(710) was found to be regulated by the quality of light needed for algal growth: weak red light absorbed mainly by Chl a induced its development, and weak blue-green light absorbed mainly by fucoxanthin and Chl c suppressed it. The difference spectra between cells grown under the two light conditions revealed two Chl a forms, absorption peaks of which were located at 692 nm (Chl a(692)) and at 703 nm (Chl a(703)), respectively, in red-light-grown cells. During cell growth under red light, the appearance and intensification of the emission correlated well with development of Chl a(692) and Chl a(703) suggesting that the two forms of Chl a are involved in the energy flow to F(710). A clear induction phenomenon characteristic of the PSII fluorescence was observed not only with the emission at 680 nm but also with F(710), indicating that F(710) is emitted by PSII Chl a. Development of F(710) under red light was sensitive to cycloheximide, indicating that the development of the energy flow to F(710) requires protein synthesis and that the emitter is installed in a protein encoded in the nuclear genome like the light-harvesting complex (LHC). Centrifugal fractionation of pigment-protein complexes revealed F(710) to be located at fractions slightly heavier than the major LHC. Development of F(710) was also found in red-light-grown cells of the diatom Nitzschia closterium.     

On the connection between bacterial growth and division

An expression for the variance in birth volumes during balanced growth of a cell population is derived. The requirement of this expression being positive and finite allows a discussion of some of the requirements imposed on the mechanisms of growth and division.     

Study of spectral and luminescent manifestations of the aggregation of thymine chromophores in aqueous solutions of polythymidylic acid at room temperature in connection with the task of photochemical record of information on thymine

Luminescence and excitation luminescence spectra of water solutions of polythymidylic acid at room temperature were studied. Three luminescence bands at different excitation wavelengths were observed: at 338 nm, which was known earlier, and two new bands, at 320 and 350 nm. The study of excitation luminescence spectra that have not been studied earlier led us to interpret the band at 320 nm as a band of chromophores that do not interact, the band at 338 nm as a band of photochemically most active densely packed stacking dimers (absorption band exciton splitting approximately 4000 cm(-1)), and the band at 350 nm as a band of photochemically inactive big stacking aggregates (n > or = 10, exciton splitting approximately 8000 cm(-1)). Changes in optical density at 270 nm of poly-T water solutions after consecutive irradiations with UV light at 297+302 and 248 nm were studied. The causes of incomplete reversibility are discussed.     

Electron paramagnetic resonance studies on spin-labelling of pepsin: effects of temperature, pH and urea on its conformation.

Pepsin was spin-labelled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidyl) bromoacetamide, possibly at the active site, at a beta-catboxyl group of a reactive aspartic acid. The spectrum of the spin-labelled pepsin showed that the spin probe was strongly immobilized (correlation time is greater than or equal to 10(-8) sec). Spin-labelled pepsin was thermally denatured at various temperatures and electron paramagnetic resonance (e.p.r.) spectra were taken at various times. Rates of denaturation estimated from the e.p.r. spectra at various temperatures showed that the enthalpy and entropy of thermal denaturation of spin-labelled pepsin at pH 3.5 were 48.0+/-4.9 kcal/mole and 214.7+/-14.5 e.u. respectively. Addition of conc. NaOH or 1 M acetate buffer at pH 6.0 sharpened e.p.r. spectra of the spin-labelled pepsin, indicating that the spin probe became mobilized by alkaline denaturation. Addition of urea caused unfolding of the protein which increased with the urea concentration, although only slight transition of conformational changes was observed in the e.p.r. spectra.     

Tryptophan phosphorescence of nascent and inactivated actin at the room temperature]

Millisecond internal dynamics of native and inactivated actin from rabbit skeletal muscle was examined using room temperature phosphorescence. Inactivated actin was prepared by incubation of G-actin at 70 degrees C, by treatment with 4 M urea or 1.5 M guanidinium hydrochloride, renaturation from fully unfolded state or by Ca2+ ion removal. It was shown that inactivation of actin, irrespective of the denaturation procedure applied, leads to a sharp decrease of millisecond fluctuations of the protein structure. Restriction of the slow intramolecular mobility in inactivated actin can result from changes of the protein conformation and/or specific association of macromolecules.     

Effect of temperature on the conformation of native DNA in aqueous solutions of various electrolytes

Effect of the temperature on the conformation of the native DNA molecule in solution of different electrolytes (LiCl, NaCl, KCl, CsCl, Gu-HCl) at ionic strengths mu = 5; 0.1; 0.01; 0.005 and temperatures ranging from 10 to 40 degrees C were studied by the methods of flow birefringence and viscometry. The experiments showed that the value of intrinsic viscosity [eta] of DNA increases at increase of temperatures in solutions of all the chlorides studied, excluding guanidine. The effect of temperature on the value of [eta] doesn't depend on the type of the cation at a fixed value of mu and is elevated when mu decreases. The observed alterations of the value of [eta] for DNA in water-salt solutions at different temperatures can be explained by an increase in the hydration of the alkaline ions at temperature increase. The experiments showed the specificity of the effect of different ions on the dimensions of the DNA molecule in solution. The data on optical anisotropy of the DNA molecule testify, that the thermodynamic rigidity of the latter doesn't depend on the temperature of solutions of different electrolytes in the temperature range studied.     

Biodiesel synthesis and conformation of lipase from Burkholderia cepacia in room temperature ionic liquids and organic solvents

Biodiesel synthesis and conformation of Burkholderia cepacia lipase (BCL) were studied in 19 different room temperature ionic liquids (RTLLs) with a range of cation and anion structures. Overall, anion selection had a greater influence on biodiesel conversion than cation choice. RTILs containing Tf2N- and PF6- anions were suitable reaction media, while RTIL of [OmPy][BF4] was the best reaction medium with a biodiesel yield of 82.2±1.2%. RTILs with strong water miscible properties showed very low biodiesel yields. Conformational analysis by FT-IR revealed that higher biodiesel conversion in RTILs was correlated with a low tendency in α-helix content of BCL. An ultrasound-assisted biocatalysis process in RTILs was used to improve mass transfer rate, leading to 83% reduction of the reaction time for biodiesel production.