THE EFFECTS FROM COMBINING UREA AND AN ALCOHOL ON THE HEAT-INDUCED REVERSIBLE DENATURATION OF RIBONUCLEASE

The effects of a combination of an alcohol and urea on the transition temperature of bovine ribonuclease were investigated. The combined effects on the transition temperature of ribonuclease of a polyvalent alcohol and urea are about equal to the algebraic sum of the effects of each individual additive. The effects of a monovalent alcohol and urea are not cumulative, especially not at low temperatures (30°C). The presence of urea decreases the hydrophobic effect of a monovalent alcohol, strongly at low temperatures, to a lesser degree at high temperatures (60°C). Consequently, urea hinders the interhydrophobic interactions by affecting the water molecules.     

THE EFFECT OF POLYHYDRIC AND MONOHYDRIC ALCOHOLS ON THE HEAT-INDUCED REVERSIBLE DENATURATION OF LYSOZYME AND RIBONUCLEASE

The reversible thermal denaturation of lysozyme and ribonuclease was investigated in aqueous solutions of mono- and polyvalent alcohols. The polyvalent alcohols show a stabilizing influence whereas the monovalent alcohols destabilize the native conformation. The concentration of the monovalent alcohol proportionally lowers the transition temperature Tm with concentration. The increments of Tm of lysozyme and of ribonuclease, excerted by a polyvalent alcohol, are of the same magnitude. This was also found for chymotrypsinogen A (1). It is reasoned that these stabilizing effects may find their origin in the intensification of the intra-hydrophobic interactions of the protein. The effects of the monovalent alcohols on the Tm of lysozyme as well as on the Tm of ribonuclease are enhanced with the increase of the hydrophobic character of the alcohol. These increments of Tm for lysozyme are slightly larger than those obtained for ribonuclease at 50–60°C, as could be expected from their amino acid compositions.     

The influence of carbohydrates and polyhydric alcohols on the stability of cephalosporins in aqueous solution

The kinetics of the degradation of cephaloridine, cephalothin and cefazolin in aqueous solutions containing various carbohydrates (glucose, fructose, sucrose and dextrans) or polyhydric alcohols (sorbitol, mannitol and glycerol) was studied over the pH range 5–12 at 35°C. The degradation rate increased linearly with the hydroxy compound concentration up to 15% at constant pH for pH > 6.5. The rate-accelerating effect of the compounds was directly proportional to the hydroxide ion activity up to pH about 11 above which value the rate of reactions levelled off with increasing pH as demonstrated for glucose. Maximal stability of the cephalosporins in solutions containing carbohydrates or polyhydric alcohols is attained at pH 5–6.5. It is proposed that the rate-accelerating effect of the hydroxy compounds is due to a nucleophilic reaction mechanism involving opening of the cephalosporin β-lactam moiety by an alkoxide ion derived from proton ionization of one of the hydroxyl groups in the compounds.     

Protein–Solvent Interactions in Pharmaceutical Formulations

The stability of proteins is affected by a variety of solvent additives. Sugars, certain amino acids and salts, and polyhydric alcohols stabilize proteins in solution and during freeze-thawing. Urea and guanidine hydrochloride destabilize proteins under either condition. These effects can be explained from the preferential interactions of the cosolvents with the proteins; i.e., the protein stabilizers are preferentially excluded from the proteins, while the destabilizers bind to them. There is a class of compounds, such as polyethylene glycol and 2-methyl-2,4-pentanediol, that destabilize proteins at high temperature but stabilize them during freeze-thawing. Such effects can be accounted for by their preferential exclusion from the native proteins determined at room temperature and from their hydrophobic character, which depends on temperature. During freeze-drying, only a few sugars appear to be effective in protecting proteins from inactivation, as most other stabilizers cannot exert their action on proteins without water. The stabilization is due to hydrogen bonding between the sugars and the dried proteins, the sugars acting as water substitute. Understanding the mechanism of the effects of solvent additives on the protein stability should aid in the development of a suitable formulation for protein.     

MODIFICATION OF THE PROPERTIES OF BOVINE PANCREATIC RIBONUCLEASE A BY COVALENT ATTACHMENT OF D-GLUCONYL-GLYCINE RESIDUES*

Bovine pancreatic ribonuclease A was reacted with D-gluconyl-glycine azide in aqueous solution at pH 8.9, in absence of phosphates. Five out of 11 amino groups can be reproducibly modified and the penta D-gluconyl-glycinated ribonuclease A had greater than 70% of the enzymic activity of the unmodified enzyme toward cytidine 2′, 3′-cyclic phosphate as well as uridine 2′, 3′-cyclic phosphate and yeast RNA. The kinetic parameters K_m and k₂ of the modified enzyme were calculated from double-reciprocal Lineweaver-Burk plots, using cytidine 2, 3′-cyclic phosphate as the substrate. The native and chemically modified protein exhibited identity in their reversible thermal transitions at neutral pH, with midpoint at about 60°. Circular dichroism measurements indicated that the overall conformation of the D-gluconyl-glycinated enzyme is not significantly different from that of the unglycosylated parent enzyme. The modified protein was less sensitive than the native ribonuclease A to attack by chymotrypsin, pepsin and elastase, indicating a protecting effect of the D-gluconyl-glycine units. Similar properties are shown by the glycosylated bovine pancreatic ribonuclease B.     

Helix-enhancing propensity of fluoro and alkyl alcohols: influence of pH,temperature and cosolvent concentration on the helical conformation of peptides

We have analyzed the effects of trifluoroethanol (TFE) and three other alcohols(1-propanol, 2-propanol and hexafluoro-2-propanol) on S-peptide (residues 1–20) of ribonuclease A, an analog of S-peptide (QHM → AAA, Sa-peptide) and TC-peptide (residues 295–316) of thermolysin to assess the helix-enhancing propensity of fluoro and alkyl alcohols under different environmental conditions of cosolvent concentration, pH and temperature by circular dichroism (CD). The dependence of cosolvent concentration on helix-induction showed a plateauing effect in all cases. 1-Propanol and 2-propanol were as effective as TFE in all the three peptides. Hexafluoro-2-propanol (HFIP) was a better helix enhancer in all cases however, the relative effectiveness varied with the peptide sequence. The alcohol transitions were analyzed assuming a two-state transition. The free energy decreased linearly in the cosolvent concentration range of 0–5 m for all the three peptides. The m-value (constant of proportionality) varied between peptides but was similar for any given peptide for TFE, 1-propanol or 2-propanol. The m-values of HFIP for all three peptides was much higher compared to other cosolvents. The isothermal cosolvent helix-induction curves for the three peptides exhibited similar features of shape and character for 1-propanol, 2-propanol and TFE. The additivity of cosolvent-induced helix formation was observed for different blends of alkyl and/or fluoro cosolvents. The pH-dependence of helix formation was observed in both TFE and 1-propanol solutions for S-peptide and TC-peptide, respectively, while in Sa-peptide, which was designed to perturb the pH-effect, helix formation was unaffected. The overall results provide some insight into the mechanism of cosolvent-mediated helix-enhancement in protein segments and are likely to facilitate optimization of conditions for cosolvent usage in chemistry and biology.     

Pseudo-ternary Phase Diagrams of Lecithin-based Microemulsions: Influence of Monoalkylphosphates

The formation of macroscopically homogeneous, stable, fluid, optically transparent, isotropic solutions (microemulsions) was delineated, at 25°C, for systems containing water, soybean lecithin, sodium monoalkylphosphate (hexyl or octyl), alcohol and isopropyl myristate. Six straight or branched alcohols (1-butanol, 2-butanol, isobutanol, 1-pentanol, 2-pentanol, 3-pentanol) were investigated as co-surfactants. A constant lecithin/alcohol mixing ratio was used, while the aqueous phase consisted of a solution of alkylphosphates at different concentrations. An increase of the microemulsion domain was seen by increasing the concentration of the alkylphosphate. With 0·2 m hexylphosphate, as aqueous phase, the microemulsion domain consisted of a single, region, that, in the presence of butylic alcohols, spanded the greater portion of the phase diagram. In the presence of amyl alcohols the area of this region was much smaller. With 0·2 m octylphosphate the realm of existence of the microemulsions, except for 1-pentanol, consisted of two regions separated by a liquid-crystal region. With all the alcohols examined, the liquid-crystal phase solubilized a larger amount of oil in the presence of octylphosphate than in the presence of hexylphosphate. The stability ranges of microemulsions in systems containing soybean lecithin, alcohol, water, and isopropyl myristate can be greatly increased by using a second hydrophobic amphiphile, such as hexylphosphate, to adjust the hydrophilic-lipophilic balance or the spontaneous peaking properties of lecithin-alcohol systems.     

Alcohols and neurotransmitter gated ion channels: past, present and future

The neurotransmitter-gated ion channels form a superfamily of neurotransmitter receptors specialized for recognizing transmitters and rapidly gating ion channels that are contained within the same holoprotein complex. A large body of research indicates that alcohols alter the function of this class of receptor at concentrations relevant to the intoxicating and anesthetic effects of the alcohols. In addition, studies have implicated several types of neurotransmitter-gated channels in intoxicating and anesthetic alcohol actions. All of the neurotransmitter-gated ion channels contain conserved features such as N-terminal ligand binding domains, hydrophobic membrane spanning domains and charged pore-lining domains. However, at least two, and possibly three families of receptors have been identified within the superfamily, including the nicotinic ACh-like receptors, the ionotropic glutamate receptors and the ATP-gated ion channels. This review will begin with a brief overview of the structural features of the different receptors, with an emphasis on comparing and contrasting features of the different families of neurotransmitter-gated channels. The emphasis will be mostly on the nicotinic-like receptors and the iGluRs, since more is known about these receptors than about other types of ligand-activated cation channels. The remainder of the review focuses on the latest studies aimed at determining the mechanism of alcohol actions on this superfamily of receptor-channels as well as the relationship between the molecular structure of these channels and the effects of alcohols on channel function. In addition, emerging directions for future study of these effects of alcohols and possible regions of the protein that may be altered during alcohol exposure are discussed. Received: 21 January / Accepted: 21 May 1997     

THE EFFECT OF TRIFLUOPERAZINE ON THE GENOTOXICITY OF BLEOMYCIN IN CULTURED HUMAN LYMPHOCYTES

The effects of trifluoperazine on the toxicity and mutagenicity of bleomycin were examined in cultured human lymphocytes. Lymphocyte cultures were initiated from three adult healthy non-smoking male volunteers. Cultures were exposed to the drugs for either three or twenty hours prior to cell collection. The toxic and clastogenic effects of the different treatments were represented by the reduction in the mitotic indices and the induction of chromosomal aberrations (CA) respectively. Both TFP and BLM significantly increased CA frequencies and reduced the mitotic indices (MI) following all treatments. The reduction in the mitotic indices and the increase in CA frequencies induced by the combined administration of both BLM and TFP were highly significant (p ≤ 0.001), but they were not significantly different from the sum of those induced by the separate treatments with the two drugs. These combined treatments, however, potentiated the odds ratios compared to those of the separate drug treatments. Therefore, though the effect of TFP on the clastogenic and cytotoxic effects of BLM was additive, the observed potentiation of the odds ratios of the combined treatments compared to those of the separate treatments suggested a significant enhancement in the expected chemotherapeutic effects of BLM when administered with TFP.     

Defining maximum levels of higher alcohols in alcoholic beverages and surrogate alcohol products

Higher alcohols occur naturally in alcoholic beverages as by-products of alcoholic fermentation. Recently, concerns have been raised about the levels of higher alcohols in surrogate alcohol (i.e., illicit or home-produced alcoholic beverages) that might lead to an increased incidence of liver diseases in regions where there is a high consumption of such beverages. In contrast, higher alcohols are generally regarded as important flavour compounds, so that European legislation even demands minimum contents in certain spirits. In the current study we review the scientific literature on the toxicity of higher alcohols and estimate tolerable concentrations in alcoholic beverages. On the assumption that an adult consumes 4 x 25 ml of a drink containing 40% vol alcohol, the maximum tolerable concentrations of 1-propanol, 1-butanol, 2-butanol, isobutanol, isoamyl alcohol and 1-hexanol in such a drink would range between 228 and 3325 g/hl of pure alcohol. A reasonable preliminary guideline level would be 1000 g/hl of pure alcohol for the sum of all higher alcohols. This level is higher than the concentrations usually found in both legal alcoholic beverages and surrogate alcohols, so that we conclude that scientific data are lacking so far to consider higher alcohols as a likely cause for the adverse effects of surrogate alcohol. The limitations of our study include the inadequate toxicological data base leading to uncertainties during the extrapolation of toxicological data between the different alcohols, as well as unknown interactions between the different higher alcohols and ethanol.     

INFLUENCE OF LOWERED TEMPERATURE UPON THE RESPONSE OF PROSTATIC AND EPIDIDYMAL PORTION OF THE RAT ISOLATED VAS DEFERENS TO FIELD STIMULATION

This paper reports the effects of lowering (bath) temperature upon contractile responses of prostatic and epididymal ends of the rat isolated vas deferens to electrical field stimulation. Responses to field stimulation with single pulses at 60 s intervals were potentiated by lowering the bath temperature. This effect was maximal at temperatures of 25-27 degrees C and was reduced in preparations treated with phentolamine (5 mumol/l). Field stimulation with trains of 50 pulses at 5 Hz every 5 min produced biphasic contractions of preparations. The initial, twitch-like component, prominent at the prostatic end and resistant to blockade by phentolamine (5 mumol/l) was little affected by lowering the bath temperature from 37.5 degrees C to 19 degrees C. In contrast, the second, more prolonged, component of the response to pulse trains, which was prominent at the epididymal end of the tissue was decreased by lowering the bath temperature and by phentolamine. In the epididymal segment, responses to exogenous noradrenaline were enhanced by lowering the bath temperature. It is concluded that lowering the bath temperature enhances the contractility of the vas deferens in response to the release of neurotransmitters and to the application of exogenous noradrenaline via a postsynaptic action. In addition, via a presynaptic action, lowering the bath temperature depresses or delays the release of the noradrenergic transmitter from the sympathetic nerve terminals.     

Solvent Effects on the Solubility and Physical Stability of Human Insulin-Like Growth Factor I

Purpose. The solubility and physical stability of human Insulin-like Growth Factor I (hIGF-I) were studied in aqueous solutions with different excipients. Methods. The solubility of hIGF-I was determined by UV-absorption and quantification of light blocking particles. The physical stability of hIGF-I was studied with differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Results. Human IGF-I precipitated at low temperature in the presence of 140 mM benzyl alcohol and 145 mM sodium chloride. CD data showed that the tertiary structure of hIGF-I during these conditions was perturbed compared to that in 5 mM phosphate buffer. In the presence of benzyl alcohol 290 mM mannitol stabilized hIGF-I. Sodium chloride or mannitol by themselves had no effect on either the solubility or the tertiary structure. Benzyl alcohol was attracted to hIGF-I, whereas sodium chloride was preferentially excluded. The attraction of benzyl alcohol was reinforced by sodium chloride leading to salting-out of hIGF-I. The CD-data indicated interactions of benzyl alcohol with phenylalanine in hIGF-I. Thermal denaturation of hIGF-I occurred in all solutions with sodium chloride, whereas mannitol or benzyl alcohol had no effect on the thermal stability. The thermal stability of hlGF-I was thus decreased in 145 mM sodium chloride although it was excluded from hIGF-I. Conclusions. The self-association and thermal aggregation of hIGF-I is driven by hydrophobic interactions. Benzyl alcohol is attracted to hIGF-I and induces changes in the tertiary structure causing hydrophobic attraction of the protein at low temperatures.     

The effect of anesthetics and heart treatment on deformability and osmotic fragility of red blood cells.

The effect of a number of anesthetics on osmotic fragility and deformability of human red blood cells was studied. These anesthetics all protected erythrocytes against osmotic lysis. An indication of separate mechanisms of action of these anesthetics emerged from the effects on cell deformability. Chlorpromazine and the lower alcohols caused a clear decrease of cell deformability, whereas alcohols with a chain-length exceeding C = 4 had no effect on deformability. This indicates that these two effects are not interrelated.This conclusion was supported by the observation that heating of the cells to 48.8° for 4 min antagonized the effects of anesthetics on deformability, without modifying the protection against osmotic hemolysis.Heating of the cells to 48.8° caused an irreversible modification of some membrane proteins, in particular spectrin and bands 7 and 8 protein. These proteins exhibited an irreversible loss of extractability at low ionic strength. Thus it seems likely that the influence of anesthetics on cell deformability is mediated via a direct or indirect effect on one or more of the heat-affected proteins, whereas the protection against osmotic hemolysis is caused by a different drug-membrane interaction.     

Structural Characterization and Formulation Development of a Trivalent Equine Encephalitis Virus-Like Particle Vaccine Candidate

The zoonotic equine encephalitis viruses (EEVs) can cause debilitating and life-threatening disease, leading to ongoing vaccine development efforts for an effective virus-like particle (VLP) vaccine based on 3 strains of EEV (Eastern, Western, and Venezuelan or EEE, WEE and VEE VLPs, respectively). In this work, transmission electron microscopy and light scattering studies showed enveloped, spherical, and ～70 nm sized VLPs. Biophysical studies demonstrated optimal VLP physical stability in the pH range of 7.5-8.5 and at temperatures below ～50°C. Interestingly, the individual stability profiles differed notably between the 3 VLPs. Numerous pharmaceutical excipients were screened for their VLP stabilizing effects against thermal stress. Sucrose, sorbitol, sodium chloride, and pluronic F-68 were identified as promising stabilizers and the concentrations and combinations of these additives were optimized. Candidate monovalent VLP bulk formulations were incubated at temperatures ranging from ?80°C to 40°C to establish freeze-thaw, long-term (2°C-8°C) and accelerated stability trends. Good VLP stability profiles were observed at each storage temperature, except for a distinct instability observed at ?20°C. The interaction of monovalent and trivalent VLP formulations with aluminum adjuvants was examined, both in terms of antigen adsorption and desorption over time. The implications of these findings on future vaccine formulation development of EEV VLPs are discussed.     

Effect of estrogenic binary mixtures in the yeast estrogen screen (YES)

Endocrine disrupting compounds (EDCs) of natural or synthetic origin can interfere with the balance of the hormonal system, either by altering hormone production, secretion, transport, or their binding and consequently lead to an adverse outcome in intact animals. An important aspect is the prediction of effects of combined exposure to two or more EDCs at the same time. The yeast estrogen assay (YES) is a broadly used method to assess estrogenic potential of chemicals. Besides exhibiting good predictivity to identify compounds which interfere with the estrogen receptor, it is easy to handle, rapid and therefore allows screening of a large number of single compounds and varying mixtures. Herein, we applied the YES assay to determine the potential combination effects of binary mixtures of two estrogenic compounds, bisphenol A and genistein, as well as one classical androgen that in vitro also exhibits estrogenic activity, trenbolone. In addition to generating data from combined exposure, we fitted these to a four-parametric logistic dose–response model. As all compounds tested share the same mode of action dose additivity was expected. To assess this, the Loewe model was utilized. Deviations between the Loewe additivity model and the observed responses were always small and global tests based on the whole dose–response data set indicated in general a good fit of the Loewe additivity model. At low concentrations concentration additivity was observed, while at high concentrations, the observed effect was lower than additivity, most likely reflecting receptor saturation. In conclusion, our results suggest that binary combinations of genistein, bisphenol A and trenbolone in the YES assay do not deviate from expected additivity.     

CARDIAC ARREST TEMPERATURE: THE EFFECT OF ETHYL ALCOHOL AND CARBON DIOXIDE ON RATS, GUINEA-PIGS AND ISOLATED RAT HEARTS

1. Previous clinical and experimental reports have suggested that the systemic administration of ethyl alcohol permits lower body temperatures before the onset of hypothermic cardiac arrest. 2. In the investigations described here, the effect of ethyl alcohol on the temperature at which cardiac arrest occurred in rats, guinea-pigs and isolated rat hearts was studied. Comparison was also made of the difference between pulmonary ventilation with air and with 5% carbon dioxide in oxygen. 3. The results demonstrated that both the administration of ethyl alcohol and ventilation with 5% carbogen reduced the cardiac arrest temperature of rats. Ethyl alcohol did not influence the cardiac arrest temperature of guinea-pigs nor of isolated rat hearts.     

Temperature affects the toxicity of lead-contaminated food in Geophagus brasiliensis (QUOY & GAIMARD, 1824)

Lead is toxic to fish, and its toxicity can be aggravated by the water temperature. Geophagus brasiliensis populations are geographically widespread and thus live in areas with different temperatures. The objective of this work was to evaluate the effects of lead-contaminated feed in fish (Geophagus brasiliensis) exposed to different temperatures. A factorial experiment was performed with two temperatures (25 and 28°C), and two feeds (control and lead contaminated - 60 mg/kg) for a sum of four treatments (25°C, Control = 25/C; 25°C, 60 mg/kg = 25/60; 28°C, Control = 28/C and 28°C, 60 mg/kg = 28/60). Analyses of the lead accumulation, oxidative stress and genotoxic damage were performed. The gills and liver showed increased lead concentrations in fish receiving lead-contaminated food at both temperatures. The lead concentrations in the intestines and muscles of fish exposed to the 25/60 treatment was greater than it was in fish exposed to the 25/C treatment. The enzyme response in the gills and the micronuclei count increased in fish exposed to the 25/60 treatment. Higher temperatures can be a beneficial factor for Geophagus brasiliensis because they can hinder the absorption of lead, thereby reducing the damage caused to the organism.     

Physiological disturbances in rainbow trout, Salmo gairdneri (R.), exposed at two temperatures to effluents from a titanium dioxide industry

Rainbow trout (Salmo gairdneri R.) were exposed to the supernatant waste water (260 μl/l) from a TiO₂ producing plant in a continuous flow test (Diluent: brackish water 7‰) for 2 wk at two temperatures (7–8 and 13–15°C). Presedimentation chambers were used to separate the solid, precipitated fraction of the waste water from the soluble part.After 2 wk of exposure the fish were killed and blood samples for hematological and biochemical analyses were taken via caudal vessels. The effect pattern of the fish exposed at low temperature (7–8°C) differed from that of the fish exposed at 13–15°C. The exposure at 13–15°C significantly increased the blood hematocrit level and increased the plasma sodium and chloride concentrations. Moreover, increased plasma glucose and lactate contents were found in these fish. These parameters were unaffected in the fish exposed at 7–8°C. Instead this group suffered from a plasma hypocalcemia and a sex-dependent inhibition of erythrocytic delta-aminolevulinic dehydrates (ALA-D) enzyme activity.The effects obtained at 13–15°C were characterized as general stress symptoms. However, the effects obtained at 7–8°C indicated a more serious toxic response of the exposed fish. The reason behind this more pronounced effect at low temperature is assumed to be related to a higher concentration of metals present in the waste water at low temperature.     

Effect of ambient temperature on the hypometabolic and hypothermic effects of sulfolane in rats

Sulfolane toxicity may be partially due to its effects on body temperature. To examine the effects of sulfolane on thermoregulation, we measured metabolic rate (oxygen uptake) and body temperature in rats injected intraperitoneally with sulfolane at dosages of 0, 200, 400, and 800 mg/kg. At ambient temperatures (T_a) of 15 and 25° C sulfolane caused a dose-related inhibition of metabolic rate accompanied by hypothermia 60 min post-injection. At a T_a of 35° C, sulfolane had no effect on body temperature or metabolic rate. The hypometabolic and hypothermic effect of sulfolane at a T_a of 25° C lasted at least 2.5 h. Sulfolane had near identical effects on body temperature at T_as of 15 and 25° C, hence, the sulfolane-treated rat had some control over body temperature at a relatively low T_a.