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A joint preparation of the two myosin light chains and actin from bovine cardiac muscle acetone powder is described. There is a significant improvement in the ease of purification, while the yield of the myosin light chains equals the best yields obtained from the use of established techniques. The actin yield greatly exceeds that obtained in an earlier published report.
The acetone precipitation of a partially purified tyrosine 3-monooxygenase (L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) resulted in the complete loss of enzymatic activity. The enzymatic activity was restored by incubation with iron and dithiothreitol. The restoration of the activity was a pH-, temperature- and time-dependent reaction. Since cobalt, nickel, copper, zinc, manganese, cadmium, magnesium calcium and barium ions were all ineffective in restoring activity, iron ion appeared to be specifically required in the restoration of the enzyme activity. Dithiothreitol could be partially replaced in the restoration step by glutathione, 2-mercaptoethanol or cysteine.
Monoclonal antibodies against human and bovine 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) were generated by fusing FOX-NY myeloma cells with spleen cells from RBF/Dn mice previously immunized with the purified brain antigens. The enzyme isolated from bovine brain was quite basic, with an isoelectric point of 9.71 and both the bovine and human enzymes consisted of a closely spaced doublet at approximately 44 and 46 kDa on SDS-PAGE. Six monoclonals were were identified as strongly recognizing the enzyme on both ELISA plates and on immunoblots of whole brain protein. Four monoclonals very weakly cross-reacted with guinea pig myelin basic protein. In contrast with two previous reports, some of our monoclonal antibodies did immunostain 2 or 3 protein bands in peripheral nerve, two bands closely corresponding to those immunostained in central nervous system (CNS) myelin, the Wolfgram protein fraction and in acetone powders of whole brain. Each of the 6 monoclonals reacting strongly on immunoblots recognized the enzyme in from 2 to 5 of the species examined (human, bovine, rat, mouse and rabbit). In addition, all 6 monoclonals that immunostained the enzyme in whole brain, myelin and Wolfgram protein immunoblots recognized both CNP1 (44 kDa) and CNP2 (46 kDa). The two closely spaced protein bands observed on SDS-PAGE and previously stained on immunoblots of CNS CNPase using polyvalent rabbit anti-bovine CNPase antisera, and now different monoclonal antibodies, appear to be immunologically related and to contain highly conserved sequences.
Potentiation of haloalkane hepatonecrosis by various ketones is a well-documented observation. The present study investigates the hepatobiliary effects of such treatments. Male Sprague-Dawley rats were pretreated with acetone (A), 2-butanone (MEK), 2-hexanone (MBK), 15 mmol/kg (po), or chlordecone (CD) and its nonketonic analog, mirex (M), 50 mg/kg (po). Following the pretreatment at various time intervals ranging from 10 to 96 hr, groups of animals received a challenging dosage of CHCl3 (0.5 ml/kg, po). In a collateral experiment, groups of animals were pretreated with vehicle and 18 hr later received either 0.50, 0.75, or 1.00 ml/kg CHCl3 (po). In each case hepatobiliary function was evaluated 24 hr later using bile flow rate and plasma bilirubin concentration. The results showed (1) that the ketones alone had no effect; mirex alone increased bile flow; (2) CHCl3 alone had no effect on bile flow but slightly increased plasma bilirubin; (3) all pretreatments potentiated the effect of CHCl3 on plasma bilirubin; (4) combinations of A, MBK, or CD plus CHCl3 were cholestatic within a restricted time frame. A study of biliary tree permeability by the segmented retrograde intrabiliary injection technique, using mannitol and inulin as marker compounds, suggested that cholestasis may result from potentiation of CHCl3-induced alterations in canalicular membrane permeability.
Thin-layer chromatographic (t.l.c.) analysis of the products formed from the incubation of an acetone-pentane powder of sheep vesicular gland microsomes with 7,10,13,16-docosatetraenoic acid (adrenic acid) revealed the presence of two products having Rf values identical to PGE2 and PGF2alpha. These products were purified by t.l.c., derivatized by treatment with methoxyamine, diazomethane, and N,O-bis-(trimethylsily1)-trifluoroacetamide, and these derivatives used for gas chromatography and gas chromatography-mass spectrometry. The results were consistent with 1a, 1b-dihomo-PGF2 and 1a, 1b-dihomo-PGF2alpha proposed structures. Formation of 1a, 1b-dihomo-PGF2 alpha could be increased, at the expense of 1a, 1b-dihomo-PGE2 by the addition of copper and reduced glutathione to the incubation mixture. Reduction of 1a, 1b-dihomo-PGE2 with NaBH4 in methanol resulted in total conversion to two products having chemical and physical properties consistent with 1a, 1b-dihomo-PGF2alpha and 1a, 1b-dihomo-PGF2beta proposed structures. The initial rate of adrenic acid-dependent oxygen uptake was determined to be 25% of that of arachidonic acid. The prostaglandin synthetase inhibitors, naproxen and 5,8,11,14-eicosatetraynoic acid (Ro 3-1428) inhibited adrenic acid-dependent oxygen uptake; Ro 3-1428 was shown to be a time-dependent inhibitor.
Single crystals of amylose complexed with isopropanol or acetone were prepared by adding these precipitants to a metastable aqueous solution of amylose. With both precipitants, similar micrometre sized platelet crystals were obtained. They gave indistinguishable electron diffraction diagrams which could be indexed in an orthorhombic unit cell, with a = 28.26 A, b = 29.30 A, c = 8.01 A and in a space group P2(1)2(1)2(1) or P2(1)2(1)2. Within the unit cell, the amylose chains are organized in antiparallel pairs of parallel 6(5) amylose helices occupying 70% of the cell content, the remaining 30% consisting of isopropanol/acetone and water, with an estimate of 10 isopropanol/acetone molecules for 52 water molecules per unit cell. If the crystals are suspended in pure isopropanol at various temperatures or in pure methanol at room temperature, they undergo a de-solvation process that ultimately converts them into VH amylose. De-solvation with isopropanol left the crystals intact whereas with methanol, they became cracked during the shrinkage. An explanation is proposed for such difference.
The frequency of recessive chlorophyll and embryonic lethals included by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Arabidopsis thaliana was markedly increased when exposure of the seeds to MNNG (3 h) was carried out in the presence of 4-12% acetone, 4-16% ethanol or 8-32% dimethylformamide. The enhancement of MNNG mutagenicity was proportional to the concentrations of these organic solvents. In contrast, neither of them, applied at the same conditions and doses, influenced the mutagenic activity of N-methyl-N-nitrosourea. The solvents without mutagens did not influence the spontaneous rate of mutations and revealed no or very weak toxic effect as measured by the seed germination.
The following studies examined the impact of the diabetic state on cisplatin nephrotoxicity. This study also investigated the potential mechanisms for diabetes mediated reduction of cisplatin toxicity. A diabetic state was induced in male Fischer 344 (F344) rats after intraperitoneal (i.p.) injection of 27-35 mg/kg STZ. Cisplatin (5 mg/kg, i.p.) nephrotoxicity was examined in normoglycemic and diabetic rats after 48 and 96 h. Cisplatin was nephrotoxic within 96 h to normoglycemic animals as indicated by an increased kidney weight, marked elevations in serum BUN levels as well as significant P less than 0.05) decreases in renal cortical slice accumulation of p-aminohippurate (PAH) and tetraethylammonium (TEA). Cisplatin failed to depress renal cortical slice accumulation of PAH and TEA in the diabetic rats. Cisplatin was also less effective in increasing BUN levels or kidney weight in diabetic rats. Further studies investigated the impact of glycosuric diuresis and ketone bodies on cisplatin nephrotoxicity. Dextrose diuresis of normoglycemic rats failed to reduce the effect of cisplatin on BUN levels, kidney weight and renal cortical slice uptake of PAH and TEA. Acetone pretreatment of normoglycemic rats also did not reduce cisplatin nephrotoxicity. These results indicate: (1) cisplatin nephrotoxicity is attenuated in the experimental diabetic state, (2) diabetes does not reduce cisplatin nephrotoxicity through glycosuric diuresis and (3) ketone body accumulation does not modulate cisplatin nephrotoxicity.
When the enol of isobutanal is added to polymorphonuclear cells, it undergoes an intracellular, myeloperoxidase-catalyzed aerobic oxidation with the formation of triplet acetone. The latter induces considerable damage if the enol concentration exceeds 2 mM. Cells which do not have myeloperoxidase are not damaged.
The nucleotide sequence of three open reading frames in the acetone-production locus of Clostridium acetobutylicum ATCC824 has been established. The three gene products, corresponding to acetoacetate decarboxylase (EC 4.1.1.4) and both subunits of the acetoacetyl-CoA:acetate/butyrate:CoA transferase (EC 2.8.3.9) are transcribed in two convergently arranged operons. The intervening DNA region separating the two transcripts is characterized by an inverted repeat which appears capable of forming a stem-loop structure functioning as a Rho-independent transcription terminator in both directions.
Acetone extracts from six plants were evaluated for mutagenic activity with the Salmonella/mammalian-microsome mutagenicity test (Ames) utilizing tester strains TA98 and TA100 and in the presence and absence of induced rat liver microsomes. Extracts from alfalfa (Medicago sativa), lettuce (Lactuca sativa) and thread-leaf groundsel (Senecio longilobus) produced only negative responses. Comfrey (Symphytum officinale) and tansy ragwort (Senecio jacobaea) extracts produced toxic responses that were abolished in the presence of the microsomal bioactivation system. Bracken (Pteridium aquilinum) and tansy ragwort extracts produced positive responses following bioactivation with the liver microsomal system. The results suggest that the Ames mutagenicity test may be of some value in initial evaluations for potential toxic effects of plants consumed by animals and man.
NADPH-dependent production of acetaminophen-glutathione conjugate has been partly characterized in rat liver microsomes. The reaction showed the characteristics of mixed function oxidase when glutathione concentration was higher than 0.2 mM. It is suggested that hydroxy radical and superoxide are not involved in this reaction. When the reaction was compared among rat, mouse and rabbit microsomes, mouse microsomes showed the highest activity; this was almost 4 times higher than the others, which may, at least in part, explain the susceptibility of mouse to acetaminophen-induced hepatotoxicity. There was also a species difference in the effects of acetone; this was enhanced in the rat microsomes, inhibited in the mouse, and minimally affected in the rabbit.
Formaldehyde (FA), acetaldehyde (ACT), malondialdehyde (MDA) and acetone (ACON) were simultaneously identified in urine, and their excretion quantitated in response to chemically induced oxidative stress. Urine samples of female Sprague-Dawley rats were collected over dry ice and derivatized with 2,4-dinitrophenylhydrazine. The hydrazones of the four lipid metabolic products were quantitated by high-performance liquid chromatography on a Waters 10-microns mu-Bondapak C18 column. The identities of FA, ACT, MDA and ACON in urine were confirmed by gas chromatography-mass spectrometry. An oxidative stress was induced by orally administering 100 micrograms/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin, 75 mg/kg paraquat, 6 mg/kg endrin or 2.5 ml/kg carbon tetrachloride to rats. Urinary excretion of FA, ACT, MDA and ACON increased relative to control animals 24 h after treatment with all xenobiotics. The system has wide-spread applicability to the investigation of altered lipid metabolism in disease states and exposure to environmental pollutants.
A multidisciplinary study to investigate 2,5-hexanedione (2,5-HD)-induced CNS-neurotoxicity and its proposed potentiation by acetone (AC) was conducted. Neurobehavioral testing, estimation of the brain-swelling reaction, neuropathological and morphometric measurements as well as synaptosomal 5-hydroxytryptamine (5-HT) uptake were evaluated. Male Wistar rats were exposed through their drinking water, for seven weeks, either to 0.5% 2,5-HD alone or to 0.5% 2,5-HD in combination with 0.5% AC. This 2,5-HD dose is known to cause neurotoxicity in the peripheral nervous system. Exposed animals were compared to a control group that received tap water. Acquisition but not performance of spatial learning, as measured in the radial 8-arm maze, was significantly inhibited by the coexposure to 2,5-HD and AC. Brain weights of rats exposed to 2,5-HD alone and to 2,5-HD and AC in combination, were slightly, albeit significantly, reduced. Synaptosomal high-affinity 5-HT uptake rate and uptake capacity were significantly reduced by 2,5-HD alone and in combination with AC. The morphometric results are reported in a companion paper. In conclusion, the present findings indicate that 2,5-HD is a CNS-neurotoxicant. The hypothesis about AC potentiation of 2,5-HD CNS-neurotoxicity was supported.
Direct asymmetric aldol reaction of acetone with aromatic aldehydes was achieved in good yields and high enantioselectivity using 5-amino-5-deoxy-beta-L-ido-(alpha-D-gluco)-heptofuranuronic acids as a new class of organocatalysts.
The high hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) greatly hamper their degradation in liquid media. The use of an organic solvent can assist the degradative action of ligninolytic enzymes from white rot fungi. The enzymatic action of the enzyme manganese peroxidase (MnP) in media containing a miscible organic solvent, acetone (36% v/v), was evaluated as a feasible system for the in vitro degradation of three PAHs: anthracene, dibenzothiophene and pyrene. These compounds were degraded to a large extent after a short period of time (7, 24 and 24h, respectively), at conditions maximizing the MnP-oxidative system. The initial amount of enzyme present in the reaction medium was determinant for the kinetics of the process. The order of degradability, in terms of degradation rates was as follows: anthracene>dibenzothiophene>pyrene. The intermediate compounds were determined using gas chromatography-mass spectrometry and the degradation mechanisms were proposed. Anthracene was degraded to phthalic acid. A ring cleavage product of the oxidation of dibenzothiophene, 4-methoxybenzoic acid, was also observed.
In this paper, we addressed the utilization of photochemical method as an innovative technology for the destruction and removal of two potent greenhouse gases, SF(6) and SF(5)CF(3). The destruction and removal efficiency (DRE) of the process was determined as a function of excitation wavelength, irradiation time, initial ratio of acetone to SF(5)X (X represented F or CF(3)), initial SF(5)X concentration, additive oxygen and water vapor concentration. A complete removal was achieved by a radiation period of 55min and 120min for SF(6)-CH(3)COCH(3) system and SF(5)CF(3)-CH(3)COCH(3) system respectively under 184.9nm irradiation. Extra addition of water vapor can enhance DRE by approximately 6% points in both systems. Further studies with GC/MS and FT-IR proved that no hazardous products such as S(2)F(10), SO(2)F(2), SOF(2), SOF(4) were generated in this process.
A continuous acetone-butanol-ethanol (ABE) production system with high cell density obtained by cell-recycling of Clostridium saccharoperbutylacetonicum N1-4 has been studied. In conventional continuous culture of ABE without cell-recycling, the cell concentration was below 5.2 g l(-1) and the maximum ABE productivity was only 1.85 g l(-1)h(-1) at a dilution rate of 0.20 h(-1). To obtain a high cell density at a faster rate, we concentrated the solventogenic cells of the broth 10 times by membrane filtration and were able to obtain approximately 20 g l(-1) of active cells after only 12h of cultivation. Continuous culture with cell-recycling was then started, and the cell concentration increased gradually through cultivation to a value greater than 100 g l(-1). The maximum ABE productivity of 11.0 gl(-1)h(-1) was obtained at a dilution rate of 0.85 h(-1). However, a cell concentration greater than 100 gl(-1) resulted in heavy bubbling and broth outflow, which made it impossible to carry out continuous culture. Therefore, to maintain a stable cell concentration, cell-bleeding was performed together with cell-recycling. At dilution rates of 0.11h(-1) and above for cell-bleeding, continuous culture with cell-recycling could be operated for more than 200 h without strain degeneration and the overall volumetric ABE productivity of 7.55 gl(-1)h(-1) was achieved at an ABE concentration of 8.58 gl(-1).