γ-Irradiation of malic acid in aqueous solutions

The -irradiation of malic acid in aqueous solutions was studied under initially oxygenated and oxygen-free conditions in an attempt to determine the possible interconversion of malic acid into other carboxylic acids, specifically those associated with Krebs cycle. The effect of dose on product formation of the system was investigated. Gas-liquid chromatography combined with mass spectrometry was used as the principal means of identification of the non-volatile products. Thin layer chromotography and direct probe mass spectroscopy were also employed.The findings show that a variety of carboxylic acids are formed, with malonic and succinic acids in greatest abundance. These products have all been identified as being formed in the -irradiation of acetic acid, suggesting a common intermediary. Since these molecules fit into a metabolic cycle, it is strongly suggestive that prebiotic pathways provided the basis for biological systems.     

Thermodynamics of denaturation of α-chymotrypsinogen A in aqueous urea and alkylurea solutions

The effects of pH, urea, and alkylureas on the thermal stability ofα-chymotrypsinogen A (α-ctg A) have been investigated by differential scanning calorimetry (DSC) and UV spectroscopy. Heat capacity changes and enthalpies of transition ofα-ctg A in the presence of urea and alkylureas were measured at the transition temperature. Using these data, the corresponding Gibbs free energies, enthalpies, and entropies of denaturation at 25°C were calculated. Comparison of these values shows that at 25°C denaturation with urea is characterized by a significantly smaller enthalpy and entropy of denaturation. At all denaturant concentrations the enthalpy term slightly dominates the entropy term in the Gibbs free energy function. The most obvious effect of alkylureas was lowering of the temperature of transition, which was increasing with alkylurea concentration and the size of alkyl chain. Destabilization of the folded protein in the presence of alkylureas appears to be primarily the result of the weakening of hydrophobic interactions due to diminished solvent ordering around the protein molecules. At pH lower than 2.0,α-ctg A still exists in a very stable form, probably the acid-denatured form (A-form).     

Ribonucleic acid–deoxyribonucleic acid hybridization in aqueous solutions and in solutions containing formamide

Hybridization in 6xSSC (SSC, 0.15m-sodium chloride-0.015m-sodium citrate) at 66 degrees C was compared with hybridization in formamide-6xSSC (1:1, v/v) at 35 degrees C. As expected, the RNA hybridization potential was labile in the former system and stable in the latter. DNA retention by filters was poor in the formamide system, but could be improved. Several other properties of the hybridization reaction were explored and it was concluded that the formamide system is generally superior.     

α-Chymotrypsin superactivity in aqueous solutions of cationic surfactants

α-Chymotrypsin (α-CT) activity was tested in aqueous media with the following cetyltrialkylammonium bromide surfactants in the series methyl, ethyl, propyl and butyl, different in the head group size, and for the sake of comparison also with the anionic sodium n-dodecyl sulfate and the zwitterionic myristyldimethylammonium propanesulfonate. N-glutaryl-l-phenylalanine p-nitroanilide hydrolysis rate was monitored at surfactant concentration above the critical micellar one. Only some cationic surfactants gave superactivity and the head group size had a major weight. The highest superactivity was measured in the presence of cetyltributylammonium bromide. The effect of both nature and concentration of three different buffers was also investigated. There is a dependence of enzyme superactivity on buffer type. Michaelis–Menten kinetics were found. The binding constants of substrate with micellar aggregates were determined in the used buffers and the effective improvement of reaction rate (at the same free substrate concentration in the medium) was calculated. k_cat significantly increased while K_m was little changed after correction to free substrate concentration. The ratio of k_cat to K_m was between 12 and 35 times higher than in pure buffer, depending on buffer and surfactant concentrations. The increase of α-CT activity (30%) was less important in the presence of 1×10⁻² M tetrabutylammonium bromide, a very hydrophobic salt, unable to micellise. Fluorescence spectra showed differences of enzyme conformation in the presence of various surfactants.     

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular β-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular β-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.     

High-temperature solvolysis of 2′-deoxyribonucleosides in aqueous amine solutions

Abstract

Degradation of 2′-deoxyribonucleosides in 0.5?M aqueous pyrrolidine at 110?°C proceeds at different rates, ordered as deoxyuridine?>?deoxyadenosine?>?deoxycytidine?>?deoxyguanosine ??deoxythymidine. Deoxyadenosine degradation produces the free base, adenine, while deoxycytidine by deamination produces deoxyuridine, and then uracil. The solvolysis of deoxyadenosine has an activation energy of 23.3?kcal/mol. Ammonolysis is slower than pyrrolidinolysis for deoxyadenosine, but faster for deoxyguanosine. In pyrrolidinolysis of the trinucleotides, d-TGT and d-TAT, the guanine moiety reacts faster than the adenine moiety. These trends are interpreted in terms of the ionization of the guanine moieties under basic conditions, rendering them less susceptible to nucleophilic attack.     

Oxidation of oat β-glucan in aqueous solutions during processing

The study investigated carbonyl group formation along the chain and the chain cleavage of cereal β-glucan during heat treatments, high pressure homogenisation, cold storage and ascorbic acid treatment of aqueous solutions of this soluble dietary fibre. The carbonyl group content and its distribution along the chain were simultaneously determined with the chain cleavage using a HPSEC/labelling method, originally developed for water-insoluble cellulose. Ascorbic acid treatment resulted in a relatively high degree of carbonyl content and extensive degradation of β-glucan, even in concentrations typically found in foods. The thermal oxidation of the β-glucan was considerable at 120 °C in a β-glucan solution with co-extracted compounds from oat ingredient, and in the highly purified solutions in presence of ferrous ions. Oxidation also probably contributed to the molecular properties during high pressure homogenisation, even thou the main degradation mechanism is the hydrolysis caused by mechanical energy. In addition to the cleavage of the β-glucan chain, the formation of compact, high molar mass species or molecule clusters were obtained in the study after ascorbic acid, heat (120 °C) and homogenisation treatments.     

Induction of lymphokine-activated killer activity in mice by prothymosin α

We have recently demonstrated that prothymosin (ProT) when administered intraperitoneally (i.p.) protects DBA/2 mice against the growth of syngeneic leukemic L1210 cells through the induction of tumoricidal peritoneal cells producing high levels of tumor necrosis factor (TNF) [Papanastasiou et al. (1992) Cancer Immunol Immunother 35: 145]. In this report we tested further immunological alterations that may be caused by the administration of ProT in vivo. We demonstrate that i.p. injections of ProT enhance natural killer (NK) cell activity and induce lymphokine-activated (LAK) activity in vivo. Thus, splenocytes from ProT-treated DBA/2 animals exhibited significantly higher cytotoxic activity (up to threefold) against the NK-sensitive YAC cell line and the NK-resistant P815 and L1210 syngeneic tumor cells, as compared to splenocytes from syngeneic control mice. The enhancement of the cytotoxic profile of DBA/2 splenocytes was associated with increased percentages of CD8⁺ cells, NK cells and activated CD3⁺ cells. The ProT-induced effect persisted for 30 days after the end of the ProT treatment period and returned to normal levels 20 days later. SPlenocytes from non-treated DBA/2 animals generated high NK and LAK activities in response to ProT in vitro. The ProT-induced NK an LAK activities reached 84% and 75% respectively of what was obtained with interleukin-2 (IL-2). High concentrations of TNF and IL-2 were generated in response to ProT in LAK cultures. These findings suggest that ProT may provide an overall protective effect against tumor growth in vivo through induction of NK and LAK activities possibly indirectly via the production of IL-2 and TNF in the spleen, peritoneal cavity and probably other lymphoid organs.This work was supported by a CEC grant to M. Papamichail     

Excess enthalpies of aqueous solutions of mono- and oligo-saccharide at 25°

The heats of dilution in water of d-xylose, d-fructose, d-galactose, d-mannose, lactose, and raffinose have been determined calorimetrically at 25°. The calorimetric data, expressed in terms of excess enthalpy, lead to an evaluation of pair- and triplet-interaction coefficients. Osmotic data, where known, permit the analogous coefficients of the excess free energy to be obtained and thence those of the excess entropy. Analysis of the excess functions and comparison with spectroscopic properties permits some qualitative hypotheses to be formulated on the molecular interactions occurring in these solutions.     

Verification of a decrease in the rigidity of the phage λ DNA polymeric chain in low ionic strength aqueous solutions by testing the polymer-polymer interlink interactions

Changes in the rigidity of the polymeric chain of phage λ double-strand DNA have been studied by laser correlation spectroscopy. It was shown that, as the ionic strength increases, the effect of the screening of the hydrodynamic interaction of the links of the polymeric chain specific for polymeric coils arises in a DNA solution. It is assumed that the screening occurs when the threshold of the overlapping of DNA coils is achieved. The overlapping of coils is the result of a previously observed significant rise of DNA coil size from abnormally small DNA coils in low ionic strength buffers (about 10⁻² M Na⁺ or less) to maximum possible large coils in the 5SSC and 5SSC-like buffers. Further analysis of the far interlink interactions in linear lambda phage DNA coils in similar buffers at pH 7 and 4 confirms the earlier proposal about the role of H+ ions in the appearance of abnormally small DNA coils. The abnormal decrease in the DNA coil size in low ionic strength buffers is not a specific feature of λ phage DNA only.     

Highly sensitive and selective photoelectrochemical DNA sensor for the detection of Hg²⁺ in aqueous solutions

A "turn-on" photoelectrochemical sensor for Hg(2+) detection based on thymine-Hg(2+)-thymine interaction is presented by using a thymine-rich oligonucleotide film and a double-strand DNA intercalator, Ru(bpy)(2)(dppz)(2+) (bpy=2,2'-bipyridine, dppz=dipyrido[3,2-a:2',3'-c]phenazine) as the photocurrent signal reporter. The presence of Hg(2+) induces the formation of a double helical DNA structure which provides binding sites for Ru(bpy)(2)(dppz)(2+). The double helical structure was confirmed by circular dichroism and fluorescence measurements. Under the optimized conditions, a linear relationship between photocurrent and Hg(2+) concentration was obtained over the range of 0.1 nM to 10 nM Hg(2+), with a detection limit of 20 pM. Interference by 10 other metal ions was negligible. Analytical results of Hg(2+) spiked into tap water and lake water by the sensor were in good agreement with mass spectrometry data. With the advantages of high sensitivity and selectivity, simple sensor construction, low instrument cost and low sample volume, this method is potentially suitable for the on-site monitoring of Hg(2+) contamination.     

Molecular association of betaine and betaine hydrochloride in aqueous solutions – a study by Raman spectroscopy

Raman vibrational spectroscopy, at 298 K, has been used to study the hydration of betaine hydrochloride and betaine in the concentration range 0.5–2 M. The observed changes in the internal vibrations of the solutes, namely, in the CO, COO⁻ and C–H stretchings, and in the components of the O–H stretching band are consonant with anionic water–betaine and betaine hydrochloride dimeric species involving simultaneously hydrogen-bonding between two solute and water molecules. In both cases, betaine hydrochloride and ‘zwitterionic’ betaine behave like structure-makers promoting a larger association in the ‘bulk’ liquid water.     

Induction of prophage λ by daunorubicin and derivatives correlation with antineoplastic activity

The antineoplastic drug daunorubicin and 15 other anthracyclines were tested for their ability to induce prophage λ in Escherichia coli K12. Prophage λ induction by daunorubicin was obtained in excision-repair deficient uvr⁻ bacteria at doses about 3-fold lower than in excision-repair proficient uvr⁺ cells; this suggests that some of the lesions produced in DNA by daunorubicin are subject to excision repair and may be adducts. Daunorubicin seems to be converted to active species capable of causing prophage inducing lesions in DNA by bacterial enzymes. The antineoplastic and prophage inducing potencies of the anthracyclines were compared in a blind test. These two parameters were correlated for two thirds of the compounds. Such a correlation supports the idea that the antineoplastic activity of the anthracyclines is a consequence of their capacity to damage DNA.     

Radiolysis of aqueous solutions of hydrogen cyanide (pH∼6): Compounds of interest in chemical evolution studies

Summary Oxygen-free aqueous solutions of hydrogen cyanide, 0.1 M and pH∼6, were exposed to gamma rays from a⁶⁰Co source, the mixture of nonvolatile products was fractionated and the fractions were analysed. It has been found that the complex mixture contains oligomers and polymers with molecular weights up to 20,000 daltons, mainly polyamides with urea and peptidic fragments. Among the constituents are carbamyl glycinonitrile and carbamyl glycinamide that represent 6.4% and 3.1% of the total of unfractionated material respectively. Urea content is 2.6%, but the derivatives of urea are more abundant. Acid hydrolysis releases several amino acids. Glycine is the most abundant (75% or more of total amino acid content), and its concentration considerably increases in some fractions when the hydrolysis is carried out at 130°C. The role of free radicals in reactions leading to the formations of radiolytic products is considered. Some comparisons are made between findings in the present work, at initial pH∼6, and an earlier study of ammonium cyanide at pH 9.     

Immunomodulatory and cytoprotective role of RP-1 in γ-irradiated mice

RP-1 has been reported to provide protection against lethal -irradiation in mice. The present study was undertaken to understand its mechanism of action, especially with respect to modulation of radiation-induced changes in immune cell function, plasma antioxidant potential, cell cycle perturbations, apoptosis in mouse bone marrow cells, and micronuclei frequency in mice reticulocytes. 2 Gy reduced mitogenic response of splenic lymphocytes significantly at 48 h. Pre-irradiation RP-1 treatment significantly countered the radiation-induced loss of splenocyte proliferation. RP-1 treatment, with or without radiation, suppressed macrophage activation as compared to control. Irradiation decreased plasma antioxidant status significantly (p < 0.05) at 1 and 2 h (4.8 ± 0.224 and 4.9 ± 0.057 mM Fe²⁺) as compared to control (6.29 ± 0.733 mM Fe²⁺) that was countered by RP-1 pre-treatment significantly (p < 0.05). RP-1 and irradiation individually caused G₂ delay in bone marrow cells. RP-1 pre-treatment augmented radiation-induced G₂ delay and elicited significant (p < 0.05) recovery in S phase fraction at 48 h in comparison to irradiated group. Radiation-induced apoptosis (3%) was significantly higher than the control. RP-1 pre-treatment further enhanced apoptosis frequency (7.2%) in bone marrow cells. RP-1 pre-treatment significantly (p < 0.05) reduced (1.23%) the radiation-induced MN frequency (2.9%) observed at 48 h post-irradiation interval. Since the radioprotective manifestation of RP-1 is mediated through multiple mechanisms, needs further investigation.     

Induction of catalytic activity of plasminogen by monoclonal antibody IV-Ic in the presence of divalent metal cations and α2-antiplasmin

Investigation of the influence of divalent metal cations on the induction of plasminogen catalytic activity by monoclonal antibody IV-Ic showed that the presence of metal cations in the reaction medium changes the induction by slowing down or accelerating the process. Ions of Zn²⁺, Mn²⁺, and Cu²⁺ completely inhibit activation. Ions of Co²⁺ and Ni²⁺ decrease the rate of the first and second phases of the reaction more than 2 times. Ca²⁺ ions do not have any effect on the activation rate. Ions of Mg²⁺, Ba²⁺, and Sr²⁺ increase the rate of the first phase of the reaction by 1.5, 2.0, and 2.0 times and the rate of the second phase by 2.0, 3.8, and 4.7 times, correspondingly. Sr²⁺ ions have the strongest stimulating effect on plasminogen activation by monoclonal antibody IV-Ic. Investigation of the dose dependent effect of Sr²⁺ on the rate of plasminogen activation by monoclonal antibody IV-Ic showed stimulating effect of Sr²⁺ at concentrations from 0.1 to 1.0 mM with half maximum at 0.6 mM. However, Sr²⁺ ions do not affect amidolytic activity of plasmin and activation of plasminogen by streptokinase. Sr²⁺ ions also do not affect monoclonal antibody IV-Ic binding to plasminogen. The effect of Sr²⁺ is specific and mediated by the IV-Ic component. The presence of metal cations affects conformational changes in the process of active site formation. Metal cations also affect structure of the plasminogen molecule active site in the complex with monoclonal antibody IV-Ic and enzyme-substrate interaction. The effect of α₂-antiplasmin on the induction of plasminogen catalytic activity by monoclonal antibody IV-Ic in range of concentrations from 5 to 30 nM has been studied. α₂-Antiplasmin at concentration 30 nM almost completely inhibits induction of plasminogen catalytic activity by monoclonal antibody IV-Ic at the ratio plasminogen/α₂-antiplasmin of 3:1. This can be explained by competition of α₂-antiplasmin and monoclonal antibody IV-Ic for the lysine-binding sites of plasminogen and inhibition of the active center in activated complex plasminogen*—mAB IV-Ic. Divalent metal cations and α₂-antiplasmin are important factors in induction of plasminogen catalytic activity by monoclonal antibody IV-Ic. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 6, pp. 778–785.     

Mechanistic aspects of radiation-induced free radical formation in frozen aqueous solutions of DNA constituents: consequences for DNA?

Frozen aqueous solutions of 1 M thymidine-5'-monophosphate were X-irradiated 77 K. The free radicals formed were analyzed by electron spin resonance spectroscopy between 77 K and about 260 K and were shown to result nearly exclusively from electron reaction at 77 K forming the thymine base anion, which converts into the well known 5-thymyl radical upon annealing. Primary oxidation of the substrate was not detectable. A minority species denoted TOH., which appeared at about 200 K, was suggested to result from OH. addition to carbon C6 of the base, perhaps via intermediate oxidation involving H2O2 or from direct reaction of OH. with the base. Another minority species at 77 K up to about 150 K, which was strongly enhanced by H2O2, was shown to be the allyl radical formed by reaction of the OH. with the methyl group. Support for this was given from experiments using BeF2 glasses. The possible spectral features for the cation of dTMP were extracted from aqueous pastes of the Ca2+ salt at 77 K. The mechanistic aspects derived from the results are in conflict with previous assumptions and are discussed for DNA model compounds and DNA.     

Saponins can perturb biologic membranes and reduce the surface tension of aqueous solutions: A correlation?

Saponins are secondary plant compounds. They have a triterpenoid or steroidal backbone. Sugars are attached to one or more points of this structure, forming chains that can be branched. This appearance leads to amphiphilic properties giving saponins the ability to interact with both lipophilic and hydrophilic structures. The surfactant behavior lets them lower the surface tension in aqueous solutions and form micelles when reaching the critical micelle concentration (cmc). It also lets them interact with biologic membrane layers that usually consist of phospholipids and cholesterol. This action may perturb the membrane and its function leading to membrane perforation or complete lysis. Thus saponins are also known for their cytotoxicity and membranolytic, respectively hemolytic features. In our studies we wanted to answer the question if there is a correlation between the unspecific detergent behavior when lowering the surface tension and the ability to perforate cell membranes and to act cytotoxic. Do saponins showing a considerable reduction in the surface tension also reveal an evident cytotoxicity or/and a marked cell membrane perforation?We tested a variety of saponins with distinct structures. The reduction in the surface tension and the cmc were analyzed on a tensiometer using the Wilhelmy plate method. The general cytotoxicity was determined in a cell model by DNA quantification. The cell membrane toxicity or membrane perforation was explored in a cell model by quantification of the leakage of the intracellular enzyme lactate dehydrogenase (LDH).The experiments revealed a correlation between the membrane toxicity and the reduction in surface tension.     

Synthesis of β-D-glucopyranuronosylamine in aqueous solution: kinetic study and synthetic potential

A systematic study of the synthesis of β-D-glucopyranuronosylamine in water is reported. When sodium D-glucuronate was reacted with ammonia and/or volatile ammonium salts in water a mixture of β-D-glucopyranuronosylamine and ammonium N-β-D-glucopyranuronosyl carbamate was obtained at a rate that strongly depended on the experimental conditions. In general higher ammonia and/or ammonium salt concentrations led to a faster conversion of the starting sugar into intermediate species and of the latter into the final products. Yet, some interesting trends and exceptions were observed. The use of saturated ammonium carbamate led to the fastest rates and the highest final yields of β-D-glucopyranuronosylamine/carbamate. With the exception of 1 M ammonia and 0.6 M ammonium salt, after 24 h of reaction all tested protocols led to higher yields of β-glycosylamine/carbamate than concentrated commercial ammonia alone. The mole fraction of α-D-glucopyranuronosylamine/carbamate at equilibrium was found to be 7-8% in water at 30°C. Concerning bis(β-D-glucopyranuronosyl)amine, less than 3% of it is formed in all cases, with a minimum value of 0.5% in the case of saturated ammonium carbamate. Surprisingly, the reaction was consistently faster in the case of sodium D-glucuronate than in the case of D-glucose (4-8 times faster). Finally, the synthetic usefulness of our approach was demonstrated by the synthesis of three N-acyl-β-D-glucopyranuronosylamines and one N-alkylcarbamoyl-β-D-glucopyranuronosylamine directly in aqueous-organic solution without resorting to protective group chemistry.