Cell density-dependent changes in the insulin action pathway: evidence for involvement of protein-tyrosine phosphatases

Three gas chromatographic procedures for the determination of ethanol in postmortem blood using alternative internal standards to n-propanol are presented: a direct injection procedure using t-butanol, and two headspace methods using t-butanol and methyl ethyl ketone. t-Butanol and methyl ethyl ketone were well resolved from ethanol, acetone, methanol and other commonly observed putrefactive volatiles using direct injection or headspace analysis. CVs for the direct injection method were below 5% for ethanol and below 10% for the other volatiles. The lower limits of detection (LOD) were 25-50 mg/L. The CVs for the headspace methods were below 5% for ethanol and below 6% for the other volatiles. The LODs were 10 mg/L using either t-butanol or methyl ethyl ketone as internal standards. The use of t-butanol or methyl ethyl ketone as alternatives to n-propanol avoids the possibility of error in the quantitation of ethanol due to the presence of n-propanol and allows for the identification of other volatiles that may aid in distinguishing antemortem ingestion from postmortem production of ethanol.     

Locomotor activity responses to ethanol, other alcohols and GABA-A acting compounds in forward- and reverse-selected FAST and SLOW mouse lines.

Mice selectively bred for high (FAST) or low (SLOW) locomotor stimulant response to ethanol have been found to differ in response to drugs with gamma-aminobutyric acid (GABA)-ergic actions. Reverse selection produced lines that are similar in sensitivity to ethanol stimulation (r-FAST and r-SLOW) and provided a unique model for testing hypotheses about shared genetic influence on sensitivity to ethanol and GABAergic drugs. FAST mice were more stimulated than SLOW mice by all drugs tested: ethanol, methanol, n-propanol, t-butanol, pentobarbital, diazepam, and allopregnanolone. In contrast, r-FAST and r-SLOW mice differed in sensitivity to only a few isolated drug doses. Locomotor responses of each reverse-selected line were significantly different from the responses of their respective forward-selected line for all drugs. Results support an effect of selection for ethanol sensitivity on allosteric modulation of the GABA-A receptor. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Comparative drug adsorption by activated charcoal

Comparative in vitro studies were carried out to determine the adsorption characteristics of 12 drugs on activated charcoal. At pH 1.3 and 37 degrees, the adsorption capacity of activated charcoal (milligrams per gram of charcoal) was: aspirin, 262; glutethimide, 252; methaqualone, 179; chlordiazepoxide, 157; propoxyphene napsylate, 137; diazepam, 136; amitriptyline, 133; propoxyphene hydrochloride, 127; secobarbital, 124, pentobarbital, 103; phenobarbital, 70; and amobarbital, 51. The adsorption of the weak acids was most markedly decreased at pH 10.8. In patients, actual drug adsorption probably is lower than these maxima because of the presence of mucus, bile salts, and other drugs. In patients investing large amounts of poorly adsorbed drugs, activated charcoal would not be helpful.     

Thermodynamic and fluorescence studies of the underlying factors in benzyl alcohol-induced lipid interdigitated phase

To further investigate factors contributing to the action of alcohol in the solute-induced lipid interdigitation phase, thermodynamic and fluorescence polarization measurements were carried out to study the interaction of benzyl alcohol with dipalmitoyl phosphatidylcholine bilayer vesicles. The obtained results were compared with those previously reported for ethanol and cyclohexanol (L. G. Roth and C-H. Chen, Arch. Biochem. Biophys. 296, 207, 1992). Similar to ethanol, benzyl alcohol was found to exhibit a biphasic effect on the enthalpy (delta Hm) and the temperature (tm) of the lipid-phase transition and the steady-state fluorescence polarization (P) monitored by 1,6-diphenyl-1,3,5-hexatriene. At a total concentration of benzyl alcohol < 30 mg/ml (the alcohol concentration in lipid phase < 21 mg/ml), benzyl alcohol was found to exhibit large increases in delta Hm and P, which were correlated with the formation of a lipid interdigitated phase, as evidenced by reported X-ray diffraction data. Combining the results with benzyl alcohol and ethanol suggested that simultaneously large changes in delta Hm and P can be used as an indication of the occurrence of a solute-induced lipid interdigitated phase. The overall interacting force in the formation of this lipid phase, as derived from the interactions of the hydroxyl portion of an alcohol with the lipid phosphate head group and the hydrophobic portion of an alcohol with the lipid hydrocarbon chains, may or may not be dominated by hydrophobic interaction. Although lipid/water partition coefficients and the contribution of hydrophobic interaction to the overall interacting force were comparable between benzyl alcohol and cyclohexanol, benzyl alcohol induced lipid interdigitated phase, but not for cyclohexanol. This was due to the ability of benzyl alcohol to be more effective than cyclohexanol in simultaneously interacting with the phosphate head group and the hydrocarbon chains of lipid.     

TGF-alpha sustains clonal expansion by promoter-dependent, chemically initiated rat hepatocytes

A series of promoting and non-promoting barbiturates and hydantoins were examined for their ability to sustain the growth of a phenobarbital (PB)-dependent hepatocyte line in cell culture. The effective liver tumor promoters, pentobarbital, allobarbital and 5-ethyl-5-phenylhydantoin, replaced PB and supported 6/27C1 hepatocyte colony formation in vitro at 52-87% of the level induced by PB. The weak promoters secobarbital and amobarbital supported colony formation at only 11-19% of the PB control. A significant correlation was observed for in vivo and in vitro promotion activities of barbiturates and hydantoins, indicating that clonal expansion by 6/27C1 hepatocytes was promoter-dependent. Cell density also appeared to influence hepatocyte growth in vitro. Hepatocyte colonies acquired the ability to grow in the absence of PB, such that after 10 days incubation with PB, approximately 50% of colonies continued to grow in the absence of promoter. This phenomenon of clone-size-dependent hepatocyte growth suggested the operation of an autocrine growth factor pathway. Addition of the hepatocyte mitogen and autocrine growth factor, transforming growth factor-alpha (TGF-alpha), to culture medium lacking PB induced a dose-dependent increase in 6/27C1 hepatocyte colony formation. At the optimal concentration of 3 ng/ml, TGF-alpha sustained hepatocyte clonal expansion at 84% of the level induced by 2 mM PB. Individual 6/27C1 colonies that grew from single cells in the presence of TGF-alpha were tested for promoter-dependent colony formation. Either PB or TGF-alpha supported colony formation by these cells at similar levels and when combined at optimal concentrations, the response appeared to be saturated. When these factors were tested in combination at suboptimal concentrations, the two compounds were additive for supporting colony formation by the parental 6/27C1 line. The ability of TGF-alpha to replace PB and sustain hepatocyte clonal expansion was confirmed with the tumorigenic 6/15 hepatocyte line. These results suggest that TGF-alpha and PB may promote hepatocarcinogenesis by stimulating a common signal transduction pathway.     

Inhibition of gap junctional intercellular communication by barbiturates in long-term primary cultured rat hepatocytes is correlated with liver tumour promoting activity

Rodent liver tumor formation can be promoted by certain barbiturates and this may involve their ability to inhibit hepatocyte gap junctional intercellular communication (GJIC). In order to address the mechanisms and specificity of action of barbiturates on hepatocyte gap junctions, we have compared the effects of liver tumor-promoting barbiturates (phenobarbital, sodium barbital and amobarbital: PB, SB and AB, respectively) and a non-liver tumor-promoting barbiturate (barbituric acid: BA) on primary cultured rat hepatocyte GJIC and connexin32 (Cx32) expression after short (1-24 h) and long (2-14 days) treatment. GJIC was evaluated by fluorescent dye microinjection (dye-coupling); Cx32 expression was monitored by Northern blot, Western blot and immunohistochemistry. Both parameters were maintained at high levels over 14 days by coculture of the cells with WB-F344 rat liver epithelial cells in the presence of dexamethasone. Treatment with PB (2 mM) for 1 h sharply reduced dye-coupling from approximately 90-30%, but the cells fully recovered by 24 h. No inhibition was seen with the other barbiturates over this 1-day treatment period. Longer treatments (2-14 days) with the promoters PB, SB and AB, however, gradually reduced hepatocyte dye-coupling to approximately 30-50%. The non-promoter, BA, did not affect hepatocyte GJIC. These decreases in hepatocyte dye-coupling occurred without changes in Cx32 or gap junction expression. Dye-coupling of WB-F344 cells and expression of their predominant gap junction protein, connexin43 (Cx43), were also not affected. Thus, the inhibition of GJIC was specific to liver tumor promoting barbiturates in hepatocytes, was time-dependent and was not due to altered Cx32 expression.     

Effect of chain length on interactions of aliphatic alcohols with suspended human serum albumin

Enthalpy changes on the immersion of human serum albumin (HSA) into n-butanol, n-propanol, ethanol and methanol containing different amounts of water have been measured calorimetrically at 25 degrees C. Water sorption isotherms on HSA were also determined in water-n-butanol and water-ethanol mixtures. From comparison of the calorimetric and sorption data, it was concluded that there is a significant enthalpy change on the HSA immersion into methanol and ethanol even under conditions where there is no change in the quantity of adsorbed water. No similar contribution was found in the n-butanol based suspensions. Water monolayer capacity evaluated from the Langmuir model decreases also significantly when going from ethanol to n-butanol. Considering this non water sorption contribution, values of the monolayer capacity and the shape of the experimental dependences, it was inferred that a relatively small change of the solvent molecule structure (from n-propanol to ethanol) affects strongly the interactions of the protein with the solvent.     

Supercritical fluid extraction and negative ion electrospray liquid chromatography tandem mass spectrometry analysis of phenobarbital, butalbital, pentobarbital and thiopental in human serum

Four commonly used barbiturates (phenobarbital, butalbital, pentobarbital and thiopental) were analyzed in human serum using supercritical fluid extraction (SFE) and negative ionization LC/ESI-MS/MS. Barbital was used as the internal standard. Carbon dioxide SFE was performed at 40 degrees C and 500 atm, with a total extraction time of 35 min. The analytes were collected off-line in a liquid trap containing absolute methanol. Samples were then concentrated by vacuum centrifugation. The high performance liquid chromatography separation utilized gradient elution with a total analysis time of 21 min. The precursor and major product ions for the four barbiturates were monitored on a triple quadrupole mass spectrometer with negative ion electrospray ionization (ESI) in the multiple reaction monitoring mode as follows: (1) thiopental (m/z 241.20-->58.00), (2) phenobarbital (m/z 231.10-->188.0), (3) pentobarbital (m/z 225.10-->181.90) and (4) butalbital (m/z 222.80-->179.90). In the case of phenobarbital, pentobarbital and butalbital, the most abundant product ion arises from the loss of 43 u (HCNO loss). However, in the case of thiopental, the most abundant product ion was observed at m/z 58.0 (the [M-183]-ion, or NCS-). Mechanisms for the formation of the collision induced dissociation reaction products of these barbiturates are proposed.     

Methodology of the survey]

To evaluate the effects of various barbiturates on lidocaine metabolism by cytochrome P-450 (P-450), enzyme kinetics were analyzed in an in vitro study using rat hepatic microsomes. Phenobarbital, amobarbital, hexobarbital, pentobarbital, and thiamylal showed the mixed type inhibition of lidocaine metabolism with inhibition constants being 4.89, 1.08, 2.76, 0.77 and 0.65 mM, respectively. Same as lidocaine, all barbiturates used in the present study, corresponding to binding with P-450, induced the I type of spectral change of P-450. Since these did not affect cytochrome C reductase activity, it was suggested that this inhibition of lidocaine metabolism in hepatic microsomes may have been caused by the reduction of activity on P-450 by the barbiturates.     

Gas chromatographic properties of 1,3-dialkyl barbiturate derivatives

The gas chromatographic properties of 1,3-dialkyl barbiturates have been studied. The retention times for 56 different 1,3-dialkyl derivatives of both aprobarbital and phenobarbital, and for 65 different 1,3-dialkyl derivatives of secobarbital were determined by gas-liquid chromatography (GLC) using a 3% SE-30 column. Retention times were found to increase with the number of carbons added. A plot of the log of the retention time versus the number of carbons in the alkyl group added for 3-alkyl derivatives of 1-alkylbarbiturates was made for each of the three barbiturates studied. A linear relationship was found to exist for derivatives having butyl or greater groups attached to the second nitrogen of the 1-alkylbarbiturates. The slopes of the graphs for the 3-alkyl derivatives of 10 different 1-alkylbarbiturates for each barbiturate studied were all found to be identical with a similar plot for straight chain hydrocarbons. This linear relationship allowed calculation of the retention times of large N,N-dialkyl barbiturates, such as N,N-eicosanylsecobarbital, which are not readily synthesized or chromatographed.     

Modulation of GABAA receptor function by alcohols: effects of subunit composition and differential effects of ethanol

We sought to test the hypotheses that closely related alcohols would have effects on GABAA receptor function that were not predicted by differences in lipid solubility, and that the subunit structure of the GABAA receptor would significantly affect the actions of different alcohols. Cloned subunits of human GABAA receptors were expressed in Xenopus oocytes, and two-electrode voltage-clamp recording was used to quantify the membrane current response to GABA in the presence and absence of different alcohols. 1-Butanol and 2-butanol differentially potentiated the response to 20 microM GABA in oocytes expressing the alpha 1 beta 2 gamma 2L and alpha 2 beta 2 gamma 2L receptor isoforms. In the alpha 1 beta 2 gamma 2L receptor construct, 1-butanol was more potent than 2-butanol to potentiate GABAA receptor function, but 2-butanol had a greater efficacy. In the alpha 2 beta 2 gamma 2L receptor construct, 1-butanol and 2-butanol were equipotent, but 2-butanol again had a greater efficacy. In the alpha 2 beta 2 receptor construct, both 1-butanol and 2-butanol produced large potentiations of the current response to 3 microM GABA. The efficacy for butanol potentiation of GABA responses in the absence of a gamma 2L subunit was greater, but the potency was greatly reduced. Low concentrations (20 mM) of ethanol potentiated GABA responses in the alpha 1 beta 2 gamma 2L receptor construct. Ethanol potentiation of GABAA receptor function was completely blocked by the benzodiazepine receptor partial inverse agonist RO15-4513 at a concentration (0.5 microM) that did not alter the control GABA response. In contrast, RO15-4513 did not block potentiation of GABAA receptor activity induced by n-propanol, 1-butanol, 2-butanol, 1-heptanol, or propofol (2,6-diisopropylphenol). These results suggest that alcohols have specific interactions with GABAA receptors, and that ethanol may have unique effects not shared by other longer chain alcohols.     

Intensification of treatment for adults with acute lymphoblastic leukaemia: results of U.K. Medical Research Council randomized trial UKALL XA. Medical Research Council Working Party on Leukaemia in Adults

The effects of short-chain alcohols (methanol, ethanol and n-propanol) on the fast-inactivating, A-type, potassium current of Lymnaea neurons were examined using macroscopic recording techniques. Alcohols produced a blockade of the current and modified its inactivation mechanism. The extracellular concentrations of methanol, ethanol and n-propanol causing 50% suppression of the current were 2970, 830 and 230 mM, respectively. The main effects of alcohols on inactivation were a decrease in the amplitude of the fast component and a simultaneous increase in the amplitude of the slow component of inactivation. In a model, the suppression of the fast component could be reproduced by an increase of the backward rate constant related to the dissociation of the inactivation particle from its binding site. The blockade and modification of inactivation reveal similar dependences on ethanol concentration, indicating that the same type of interaction of ethanol with the channel underlies both of these events. Ethanol was effective only in extracellular applications. The data support an action of alcohols at a hydrophobic site near the extracellular portion of the channel.     

Development of rapid tolerance to pentobarbital and cross-tolerance to ethanol on a motor performance test with intoxicated practice

Male Wistar rats given a single moderate dose (1.7 mg/kg, IP) of pentobarbital (PB), followed by six trials on the moving belt apparatus during the next hour, showed tolerance to the motor-impairing effects of a second dose of 17 mg/kg given 24 h later. A control group that received saline before the first test showed the usual initial sensitivity when tested with PB 24 h later. Three weeks later, the first group showed cross-tolerance to the effects of ethanol (1.7 g/kg, IP) on the same test, while the second group did not. These findings support the suggestion that rapid tolerance is closely similar to chronic tolerance and that the contribution of intoxicated practice results in a long-lasting component that applies to cross-tolerance to ethanol on the same test.     

Effects of chronic ethanol consumption and withdrawal on the neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one in male and female rats

Prolonged alcohol (ethanol) consumption leads to the development of alcohol tolerance and cross-tolerance to some benzodiazepines and barbiturates. In contrast, rats undergoing alcohol withdrawal are sensitized to the anticonvulsant effects of the endogenous GABA(A) receptor modulator, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-THP). Alterations in endogenous, cerebral cortical levels of 3alpha,5alpha-THP during alcohol withdrawal could contribute to the observed sensitization to 3alpha,5alpha-THP. Therefore, this study investigated plasma and brain levels of 3alpha,5alpha-THP, progesterone, and corticosterone during alcohol dependence and withdrawal in the rat. Plasma corticosterone, progesterone (a precursor of 3alpha,5alpha-THP) and 3alpha,5alpha-THP levels were unchanged in alcohol-dependent animals. Cerebral cortical levels of 3alpha,5alpha-THP decreased in dependent male animals, but not in dependent female rats. During alcohol withdrawal, plasma corticosterone and progesterone levels increased in male, but not female rats. However, neither plasma nor cerebral cortical 3alpha,5alpha-THP levels were altered from control levels in male or female rats during alcohol withdrawal. Plasma and brain levels of 3alpha,5alpha-THP were markedly higher in female compared with male rats. Cerebral cortical levels of 3alpha,5alpha-THP during the diestrus phase of the estrus cycle were approximately 4 to 6 ng/g, a concentration that may approach physiological relevance. These findings suggest that sensitization to 3alpha,5alpha-THP during alcohol withdrawal is not mediated by elevations in brain levels of endogenous 3alpha,5alpha-THP in male or female rats. However, elevations in circulating corticosterone and progesterone levels during ethanol withdrawal in male rats may underlie gender differences in allopregnanolone sensitivity during ethanol withdrawal.     

Body temperature in mice: a quantitative measure of alcohol tolerance and physical dependence

Mice undergoing withdrawal after chronic ethanol consumption were found to be hypothermic if kept at room temperature. The extent of the hypothermia correlated well with the behavioral withdrawal symptoms and could be used as a quantitative measure of the severity and time course of the withdrawal syndrome. Placing mice in a cold environment (4 degrees C) exacerbated the hypothermia whereas placing animals at 34 degrees C reversed the hypothermia and produced hyperthermia. It was concluded that the temperature set point mechanism and the ability to regulate around this set point was disturbed in animals physically dependent on alcohol. During consumption of the ethanol-containing diets, mice exhibited tolerance to the hypothermic effects of an acutely administered dose od ethanol. Tolerance to the hypothermic effects of ethanol mirrored the development of behavioral tolerance as measured by performance on a tilting plane. Temperature and behavioral tolerance were both shown to extend well beyond the period of the withdrawal syndrome. Ethanol-treated mice were found to be cross-tolerant to the hypothermic effects of barbiturates but not to the hypothermia produced by the monoamine oxidase inhibitor, pargyline.     

有机磷一元酸四面体钴配合物加合醇反应中的取代基效应

用光度法测算了醇与有机磷一元酸四面体钴配合物加合反应的平衡常数。试验的有机磷酸是苯乙烯膦酸的单正辛基酯，单2－乙基己基酯和单1－甲基庚基酯，以及2－乙基己基膦酸单2－乙基己基酸和二（2－乙基己基）磷酸。研究的醇的烃基分别为正辛基、2－乙基己基、1－甲基庚基、叔丁基和苯甲基。试验结果表明，酸和醇的烃基的位阻效应与电子效应影响醇与四面体钴配合物的加合趋势，并且酸的取代基效应更重要。     

Corticosterone increases severity of acute withdrawal from ethanol, pentobarbital, and diazepam in mice

It has been suggested that withdrawal from several subclasses of central nervous system (CNS) depressants involves common underlying mechanisms. For example, mice genetically selected for severe ethanol withdrawal convulsions (Withdrawal Seizure Prone or WSP) have also been found to express severe withdrawal following treatment with barbiturates and benzodiazepines. Corticosteroids appear to modulate severity of withdrawal from CNS depressants. Therefore, it was hypothesized that corticosterone would enhance withdrawal convulsions following acute ethanol, pentobarbital, and diazepam in WSP mice. Corticosterone (20 mg/kg) administered following each of these drugs significantly increased severity of handling-induced convulsions during withdrawal. Corticosterone did not affect pre-withdrawal convulsion scores or handling-induced convulsions of drug-naive mice. These results suggest that withdrawal convulsions following acute ethanol, pentobarbital, and diazepam are sensitive to modulation by corticosterone and they support the hypothesis that stress may increase drug withdrawal severity.     

Effect of alcohols on lactose solubility

In tests of effects of high (70 to 90% volume/volume) and low (2 and 5% volume/volume) alcohol concentrations, solubility of lactose decreased with increased alcohol concentration and decreased as alcohol chain length increased. Since lactose has lower solubility in alcohol, crystallization would be expected to be speeded by increased supersaturation. Composition of precipitates formed by the action of ethanol changed with time; alpha-lactose precipitated more rapidly at first, then beta-lactose. Time of crystallization was related directly to the percentage of beta-lactose (therefore, the relation was inverse for total alpha). However, the percentage of alpha-hydrate increased with time and with water content. Agitation during crystallization increased production of beta-lactose. Composition of the lactose precipitate varied greatly with concentration of alcohol (ethanol). When ethanol concentration was low, only alpha-hydrate was precipitated whereas at higher concentrations stable anhydrous alpha lactose also was precipitated, with the percentage of total alpha decreasing while the percentage of beta-lactose increased. Crystal shape changed from prisms initially to partially or fully developed tomahawks as time went by or as the percentage of ethanol decreased, and crystal color increased with crystallization time and as ethanol percentage decreased. Choosing long-chain alcohols and controlling suitable parameters enabled recovery of greater amounts and more desirable forms of lactose.     

Mycotic sinusitis

Previous drug discrimination studies have elucidated the importance of gamma-aminobutyric acidA (GABAA), N-methyl-D-aspartate (NMDA) glutamate, and serotonin (5-HT) receptor systems in mediating the discriminative stimulus effects of ethanol. The present study used a three-choice operant drug discrimination procedure in an attempt to determine if salient GABAergic effects could be separated from other stimulus effects of 2.0 g/kg ethanol. Adult male Long-Evans rats (n = 7) were trained to discriminate pentobarbital (10.0 mg/kg; intragastrically (i.g.) from ethanol (2.0 g/kg; i.g.) from water (4.7 ml; i.g.) using food reinforcement. Stimulus substitution tests were conducted following the administration of allopregnanolone (1.0-17.0 mg/kg; intraperitoneally (i.p.)), diazepam (0.1-7.3 mg/kg; i.p.), midazolam (0.0056-17.0 mg/kg; i.p.), dizocilpine (0.01-0.56 mg/kg; i.p.), phencyclidine (1.0-5.6 mg/kg; i.p.), CGS 12066B (3-30 mg/kg; i.p.), RU 24969 (0.1-5.6 mg/kg; i.p.) and morphine (1 or 3.0 mg/kg; i.p.). Within the group, allopregnanolone and midazolam completely substituted (> 80%), and diazepam partly substituted (67%) for the discriminative stimulus effects of pentobarbital. Dizocilpine and phencyclidine partly substituted (58 and 57%, respectively) for ethanol without substantial pentobarbital-appropriate responding. RU 24969, CGS 12066B and morphine did not result in complete substitution for either ethanol or pentobarbital, although RU 24969 resulted in partial (68%) pentobarbital substitution. The ability to train the present three-choice discrimination in rats indicates that the discriminative stimulus effects of 10.0 mg/kg pentobarbital were separable from those of 2.0 g/kg ethanol. The results suggest that the pharmacological effects of ethanol, which can control behavior, may seemingly be modified by training conditions (two-versus three-choice discrimination procedures), to the extent that a receptor system prominently linked to the behavioral activity of ethanol (i.e. GABAA) appears no longer to be involved in the interoceptive effects of the drug.     

Distinct promoter sequences of two precursor genes for salmon gonadotropin-releasing hormone in masu salmon

Three phase partitioning (TPP) uses t-butanol and ammonium sulfate to precipitate enzymes and proteins from aqueous solutions. The method is useful both upstream with crude samples and downstream where a scaleable simple step is needed. About 25 enzymes and proteins have been isolated by various laboratories using TPP-t-butanol. The relation of t-butanol used in TPP, with n-butanol used as an extraction agent from Morton's work, is reviewed. Some t-butanol appears bound to TPP-precipitated proteins which are actually protein-t-butanol coprecipitates. They float above denser aqueous salts because bound t-butanol increases their buoyancy, similar to the behavior of many lipoproteins. On redissolving TPP-precipitated enzymes, total and specific activities usually are regained and sometimes increased. Sulfate ion-in large concentrations-likely exerts itself through its kosmotropic action as in conventional salting out. t-Butanol likewise appears to be a kosmotrope and crowding agent at room temperature or above, whereas C1 and C2 cosolvents (e.g., ethanol) do not so behave except at near or below zero temperatures. However, kosmotropy is not the entire origin of TPP, nor probably of conventional salting out. Electrostatic forces, capacity to force protein conformation tightening and protein hydration shifts, also contribute. Electrostatic forces, and the tendency for salt ions to bind and tighten protein molecule conformation, are indicated by the sharp pH dependency of both conventional salting out and TPP, around pH regions where proteins undergo conformation changes. Sulfate anion is densely-perhaps extraordinarily-hydrated, adding much to its effective size, and therefore it has a tendency to crowd or exclude proteins, when sulfate concentrations are in the 0.5 to 3 M range.