A novel approach for assessing protein synthesis in channel catfish, Ictalurus punctatus

A comprehensive understanding of animal growth requires adequate knowledge of protein synthesis (PS), which in fish, has traditionally been determined by the flooding dose method. However, this procedure is limited to short-term assessments and may not accurately describe fish growth over extended periods of time. Since deuterium oxide (²H₂O) has been used to non-invasively quantify PS in mammals over short- and long-term periods, we aimed at determining if ²H₂O could also be used to measure PS in channel catfish. Fish were stocked in a 40-L aquarium with ~ 4% ²H₂O and sampled at 4, 8 and 24 h (n = 6 at each time period) to determine ²H-labeling of body water (plasma), as well as protein-free and protein-bound ²H-labeled alanine. The labeling of body water reflected that of aquarium water and the labeling of protein-free alanine remained constant over 24 h and was ~ 3.8 times greater than that of body water. By measuring ²H-labeled alanine incorporation after 24 h of ²H₂O exposure we were able to calculate a rate of PS: 0.04 ± 0.01% h^− 1. These results demonstrate that PS in fish can be effectively measured using ²H₂O and, because this method yields integrative measures of PS, is relatively inexpensive and accounts for perturbations such as feeding, it is a novel and practical assessment option.     

Preferential solvation and association constants of 2-exo and 2-endo norbornyl chlorides in water–acetone mixtures

Molecular dynamics simulations of 2-exo and 2-endo norbornyl chlorides are performed in water–acetone mixtures to investigate the role of preferential solvation on the dissociation process of norbornyl chlorides. Increase in the association constants between carbon (C2) and chloride (Cl) of norbornyl chlorides with an increase in mole fraction of acetone supports the experimental data for the corresponding decrease in the rates of solvolysis for both exo and endo norbornyl chlorides. When the mole fraction of water is increased in water–acetone mixtures, acetone molecules are replaced by water molecules around C2 as well as Cl of norbornyl chloride. Preferential solvation by water is analysed using running coordination numbers and excess coordination numbers around C2 and Cl.     

Headspace analyses of H labeling of acetone: Enabling studies of fatty acid oxidation in vivo

We demonstrate that one can measure low levels of ²H labeling (e.g., <0.025% excess ²H) by exchanging hydrogen (deuterium) in water with acetone and subjecting samples to gas chromatography–pyrolysis–isotope ratio mass spectrometry. This analytical method circumvents the need to use typical off-line reduction methods that convert water to hydrogen gas prior to isotope ratio mass spectrometry or the need to purchase extra peripheral devices that would permit the direct analysis of water labeling. This method enables routine measurements of fatty acid oxidation in rodents; that is, one administers a ²H-labeled fatty acid(s) and then quantifies the production of ²H-labeled water.     

Sequence analysis of small amounts of nonradioactive oligoribonucleotides containing ribose-methylated nucleosides by a combination of 3H- and 32P-labeling techniques

The oligonucleotides A-G-A-Cm-U and Gm-A-A-Y-A-ψ were used as model compounds to demonstrate how the complete nucleotide sequence of small amounts of nonradioactive oligoribonucleotides (0.2–0.3 nmol) can be derived by a combination of ³H-labeling procedures previously published and a new method for the characterization of 2′-O-methylated nucleosides based on enzymatic ³²P labeling. The newly developed method for the identification of ribose-methylated nucleosides entails ³²P labeling by [γ-³²P]ATP/polynucleotide kinase of the 5′-terminus of a ribonuclease T₂-stable 2′-O-methylated dinucleotide derived from the polyribonucleotide, conversion of the labeled dinucleotide to the ³²P-labeled 2′-O-methylated nucleoside 5′-monophosphate, and identification of the monophosphate by its chromatographic properties on a polyethyleneimine-cellulose thin layer. The novel method is simple, fast, and sensitive and, at present, represents the only way by which ribose-methylated nucleosides can be analyzed in small amounts (0.01 nmol) of nonradioactive oligonculeotides or RNA.     

Net glucose production from acetone in isolated murine hepatocytes. The effect of different pretreatments of mice

• 1.1. To evaluate the condition under which net glucose production from acetone, added as sole substrate, occurs different pretreatments of mice, in combination with starvation, were used; (i) acetone pretreatment (acetone is a known inducer of cytochrome P-450 isozymes involved in this pathway), (ii) fructose pretreatment (to induce NADPH + H⁺ generating enzymes) or (iii) their combination.
• 2.2. There was net glucose formation from acetone only in that case, when the cells were prepared from 48 hr fasted animals pretreated with both acetone and fructose. However, using 2-¹⁴C-acetone, incorporation of ¹⁴C-carbon into glucose could be detected in all the cases and, at the same time, acetone was without any effect on protein synthesis.
• 3.3. The addition of acetone increased gluconeogenesis from alanine in almost all the cases. The only exception from this general rule was that the case, when hepatocytes were prepared from acetone pretreated 48 hr starved mice where, instead of the elevation of glucose formation, a decrease of that was caused by acetone.
• 4.4. Acetone decreased ¹⁴C-carbon incorporation into glucose from ¹⁴C-(U)-alanine added at saturating concentration in hepatocytes prepared from starved mice.
• 5.5. Similarly to acetone there was no net glucose formation from acetol either when added alone, however, it enhanced gluconeogenesis from alanine at non-saturating concentrations of the amino acid.
• 6.6. Methylglyoxal proved gluconeogenic in all the cases.
• 7.7. It is concluded that net glucose formation from acetone as sole substrate occurs only under those conditions which are far from a physiological situation, however, when gluconeogenesis from another substrate takes place, acetone can contribute to net glucose formation in hepatocytes prepared from fasted mice.

     

Anaerobic degradation of acetone by Desulfococcus biacutus spec. nov.

From anaerobic digestor sludge of a waste water treatment plant, a gram-negative, strictly anaerobic sulfate-reducing bacterium was isolated with acetone as sole organic substrate. The bacterium was characterized as a new species, Desulfococcus biacutus. The strain grew with acetone with doubling times of 72 h to 120 h; the growth yield was 12.0 (±2.1) g · [mol acetone]^-1. Acetone was oxidized completely, and no isopropanol was formed. In labelling studies with ¹⁴CO₂, cell lipids (including approx. 50% PHB) of acetone-grown cells became labelled 7 times as high as those of 3-hydroxy-buyrate-grown cells. Enzyme studies indicated that acetone was degraded via acetoacetyl-CoA, and that acetone was channeled into the intermediary metabolism after condensation with carbon dioxide to a C₄-compound, possibly free acetoacetate. Acetoacetyl-CoA is cleaved by a thiolase reaction to acetyl-CoA which is completely oxidized through the carbon monoxide dehydrogenase pathway. Strain KMRActS was deposited with the Deutsche Sammlung von Mikroorganismen, Braunschweig, under the number DSM 5651.     

Feasibility of using water-immiscible organic solvents in biological waste-gas treatment

In order to conclude about the feasibility of using water-immiscible organic solvents in biological waste-gas treatment, a theoretical study was done in which different types of organic-solvent-containing systems are compared with systems where the pollutant is transferred directly to the water phase. For each system the total equipment volume needed to remove 99% of a pollutant from a waste-gas stream is calculated. Three different pollutants with a different solubility in water are considered: Hexane (m _gw =71), dichloromethane (m _gw =0.1) and acetone (m _gw =0.0016), withm _gw the partition coefficient (kg/m³ gas/kg/m³ water) of the pollutant between the gas and the water phase. From the results it is concluded that the use of organic solvents is only advantageous in case the specific area for mass transfer between solvent and water is large enough to compensate for the additional transport resistance introduced by the solvent, and secondly if the solvent shows a sufficiently high affinity for the pollutants.     

Exhaust gas purification using biocatalysts (fixed bacteria monocultures) — the influence of biofilm diffusion rate (O2) on the overall reaction rate

Summary This paper presents results of experiments on the influence of O₂ and substrate (pollutant) concentration on the overall reaction rate of a trickle-bed reactor used for biological waste gas purification. The biocatalyst was a pollutant-specific bacterial monoculture fixed on porous glass carriers. The conversion of acetone and propionaldehyde, as model pollutants that are easily soluble in water, was measured. Under constant hydrodynamic conditions (gas and liquid flow rates) the inlet pollutant concentration was varied. The O₂ partial pressure in the model gas was increased to investigate the influence of O₂ supply on pollutant conversion. At higher pollutant concentrations (>117 mg acetone.m^-3 gas and > 150 mg propionaldehyde.m^-3 gas) higher concentrations of dissolved O₂ led to a significant rise in the maximum degradation capacity of the reactor. This maximum reaction rate was independent of the pollutant mass flow. It seems that the diffusion of O₂ in the biofilm is rate-determining. The reaction rate at lower inlet concentrations was not affected by the improved O₂ supply. Here the external mass transfer through the liquid film limits the reaction rate and the maximum separation efficiency of about 80% at a residence time of 1.2s (space velocity 3000h^-1) is achieved.     

A novel radiochemical method for the microdetermination of total and individual ketone bodies in biological materials

A novel radiochemical method has been developed for ultramicrodetermination of acetone based on the principle that ¹²⁵I-labeled iodoform is produced by iodination of acetone with ¹²⁵ICl. [¹²⁵I]Iodoform is readily counted as a measure of acetone after separation from unreacted iodide ions. Quantitative conversion of 3-hydroxybutyrate to acetoacetate takes place when NAD-dependent oxidation of 3-hydroxybutyrate by 3-hydroxybutyrate dehydrogenase is coupled with NADH-dependent reduction of pyruvate (or 2-oxoglutarate) by lactic dehydrogenase (or glutamic dehydrogenase). Acetoacetate thus formed produces acetone spontaneously when the acidified (deproteinized) reaction mixture is maintained at 50°C for 2 hr. Thus, total and individual ketone bodies in plasma are determined conveniently by combining the radiochemical determination of acetone with these conversion procedures.     

Elucidation of the Origin of NOEs And ROEs that Show the Hydration in the Minor and Major Grooves of DNA Duplex with ATTAAT Tract by a Combination of NOESY and ROESY Experiments

Abstract

A combination of NOESY and ROESY experiments (using ammonia as a catalyst across the pH range of 5 to 8.6) has given us a clear understanding regarding the origin of nOes that are attributed to the stereochemical location and the residence time of water in the major and the minor grooves of d⁵'(¹C²C³A⁴T⁵T⁶A⁷A⁸T⁹G¹⁰G)₂ ³' duplex Our conclusions are the following: (i) In the major groove, the presence of ammonia in the buffer does not influence on the process of exchange between bound and bulk water, (ii) It has been found that the observation of the bound water in the minor groove is a result of straight dipole-dipole effect at the physiological pH. (iii) The residence time of water near H2 of adenine (H2A) in the minor groove has been estimated to be in the range of 0.3–0.5ns, which is closer to the residence time of the bound water found on the surface of protein, (iv) The hydration pattern in the minor groove in the physiological pH, under our NMR measurement condition, is similar to the ones found in the X-ray structure, (v) It has been shown that at pH > 8.0 the nOe/rOe intensities of the water-H2A crosspeaks dramatically increase due to dipole-dipole and/or relayed magnetization transfer from H2A to water through ammonia catalyst.     

Notes of Technic

The leaching of water-soluble and exchangeable calcium in histoautoradiog-raphy of oat tissue can be prevented by using acetone as the dehydration fluid (freeze substitution technique) and by keeping the tissue sections, while stretching on water, embedded in the methacrylate matrix. Ca⁴⁵ was either added to the mineral solution on which the oat plants were grown (75 μc), or applied on the leaf surface (8 μc). After freezing in melting isopentane, specimens of 1-2 mm dimensions are fixed for 24 hr in an acetone-OsO₄ (1%) solution at—80 C. Dehydration is obtained by transferring the material every day for 6 successive days to a fresh acetone solution at—80 C. The material is infiltrated by a three-time renewed monomer methacrylate mixture (methylmethacrylate I, butylmethacrylate 4) at—50 C. The specimens are embedded in the polymerizing methacrylate mixture at room temperature. Sections of 4-8 μ are easily cut with a rotating microtome. If the methacrylate is not removed from the sections, they can be stretched on water without leaching of calcium. The presence of methacrylate in no way hinders microscopic observation nor effective histoautoradiography.     

Kinetics of oxidation of ferrocene by tris-1,10-phenanthrolinecobalt(III) in t-butyl alcoholwater and acetonewater mixtures

Rate constants and activation parameters (ΔH^≠ and ΔS^≠)are reported for the oxidation of ferrocene by the tris-1,10-phenanthrolinecobalt(III) cation in t-butyl alcoholwater and in acetonewater solvent mixtures. Solvent effects on reactivity trends for these systems, for this same reaction in methanolwater mixtures, and for cobalt(II)-catalysed racemisation of Co(phen)_3³⁺ in t-butyl alcoholwater solvent mixtures are analysed into initial state and transition state contributions. The dependences of solubilities on solvent composition for ferrocene and for [Co(phen)₃](ClO₄)₃ in methanol, t-butyl alcohol, and acetonewater mixtures are also reported; these results are needed in order to establish solvent effects on the initial states of the reactions studied.     

Molecular Dynamics Simulation Studies of the Limiting Conductances of MgCl2 and CaCl2 in Supercritical Water Using SPC/E Model for Water

We report results of molecular dynamics (MD) simulations of the limiting conductances of MgCl² and CaCl² in supercritical water as a function of water density using the SPC/E model for water. The limiting conductances of Mg²⁺, Ca²⁺ and Cl^- over the whole range of water density considered exhibits a linear dependence of the limiting conductance on the water density. In the cases of Mg²⁺ and Ca²⁺, a solventberg picture for the behavior of small divalent cation emerges from our studies. From the view of the solventberg picture, the ion and its shell moving together as an entity interacts with the second hydration shell water molecules, and its mobility is restricted mostly by the number of the second hydration shell water which is proportional to the water density of the whole system. In the case of Cl^-, the range of water density considered in this study belongs to the higher-density region (above 0.45?g/cm³) in which the effect of the number of hydration water molecules around ions dominated. As the water density increases, the water molecules of the first hydration shell restrict the mobility of Cl^- and the limiting conductance of Cl^- decreases nearly linearly. Significant different dependence on the water density is observed between the calculated limiting conductances of MgCl² and CaCl² at 673?K and the experimental results over the water density of 0.60–0.90?g/cm³. Possible limitation of the extended simple point charge (SPC/E) model with regard to this difference should be pointed out and the use of a more precise model like the revised polarizable (RPOL) model is indispensable for a further MD study to gain a complete picture of the chemical circumstance around the ions.     

Bioconversion of isopropanol by a solvent tolerant Sphingobacterium mizutae strain

The bioconversion of high concentration isopropanol (2-propanol, IPA) was investigated by a solvent tolerant strain of bacteria, which was identified as Sphingobacterium mizutae ST2 by partial 16S rDNA gene sequencing. This strain of bacteria exhibited the ability to utilise high concentration isopropanol as the sole carbon source, with mineralization occurring via an acetone intermediate into central metabolism. The biodegradative performance of this strain for IPA was examined over a 2–38 g l⁻¹ concentration range, using specific growth rate (μ) and conversion rate analysis. Maximum specific growth rates (μ_max) of 0.0045 h⁻¹ were routinely obtainable on IPA. In addition, the highest specific IPA degradation rate was obtained at a concentration of 7.5 g l⁻¹ with a corresponding value of 0.045 g IPA g cells⁻¹ h⁻¹. While the highest acetone yield reached its maximum value of 0.940 g acetone g IPA⁻¹ at 7.5 g IPA l⁻¹. This is the first report on bioconversion of isopropanol at such high concentration by this solvent tolerant strain of S. mizutae and may allow its application in novel biocatalytic processes for effective biological conversion in two-phase solvent systems.     

Acetone production in solventogenic <Emphasis Type="Italic">Clostridium</Emphasis> species: new insights from non-enzymatic decarboxylation of acetoacetate

Development of a butanologenic strain with high selectivity for butanol production is often proposed as a possible route for improving the economics of biobutanol production by solventogenic Clostridium species. The acetoacetate decarboxylase (aadc) gene encoding acetoacetate decarboxylase (AADC), which catalyzes the decarboxylation of acetoacetate into acetone and CO₂, was successfully disrupted by homologous recombination in solventogenic Clostridium beijerinckii NCIMB 8052 to generate an aadc ⁻ mutant. Our fermentation studies revealed that this mutant produces a maximum acetone concentration of 3 g/L (in P2 medium), a value comparable to that produced by wild-type C. beijerinckii 8052. Therefore, we postulated that AADC-catalyzed decarboxylation of acetoacetate is not the sole means for acetone generation. Our subsequent finding that non-enzymatic decarboxylation of acetoacetate in vitro, under conditions similar to in vivo acetone–butanol–ethanol (ABE) fermentation, produces 1.3 to 5.2 g/L acetone between pH 6.5 and 4 helps rationalize why various knock-out and knock-down strategies designed to disrupt aadc in solventogenic Clostridium species did not eliminate acetone production during ABE fermentation. Based on these results, we discuss alternatives to enhance selectivity for butanol production.     

An efficient method for the application of PHA‐poor solvents to extract polyhydroxybutyrate from Cupriavidus necator

There are many published studies presenting ethanol and acetone as PHAs‐poor solvents, where these two solvents are shown to dissolve <2% (w/v) of PHAs at low temperatures. In this study, the suitability of ethanol and acetone for the recovery of PHB at different temperatures (from room temperature to near boiling point) in Cupriavidus necator was investigated. Experiments were performed using response surface methodology to examine the effects of different temperatures and heating incubation times on recovery percentage using the two solvents. The highest recovery percentage (92.3%) and product purity (up to 99%) were obtained with ethanol‐assisted extraction at 76°C for 32 min of incubation time. Under these conditions the extracted PHB exhibited a molecular mass of 1.2 × 10⁶. The present strategy showed that at temperatures near its boiling point, ethanol, as a nonhalogenated solvent, represents a good alternative to halogenated solvents, like chloroform, when PHB recovery is concerned. DSC analysis showed good thermal properties for ethanol‐ and acetone‐extracted biopolymers. GC and ¹H NMR analysis confirmed the extracted biopolymer to be polyhydroxybutyrate of good purity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1480–1486, 2016     

Boron nitride nanotube based nanosensor for acetone adsorption: a DFT simulation

We have investigated the adsorption properties of acetone on zigzag single-walled BNNTs using density functional theory (DFT) calculations. The results obtained show that acetone is strongly bound to the outer surface of a (5,0) BNNT on the top site directly above the boron atom, with a binding energy of ?96.16 kJ?mol^?1 and a B–O binding distance of 1.654 Å. Our first-principles calculations also predict that the ability of zigzag BNNTs to adsorb acetone is significantly stronger than the corresponding ability of zigzag CNTs. A comparative investigation of BNNTs with different diameters indicated that the ability of the side walls of the tubes to adsorb acetone decreases significantly for nanotubes with larger diameters. Furthermore, the stability of the most stable acetone/BNNT complex was tested using ab initio molecular dynamics simulation at room temperature.

Optimization of Two Immunofluorescent Antibodies for the Detection of <Emphasis Type="Italic">Escherichia coli</Emphasis> Using Immunofluorescent Microscopy and Flow Cytometry

Two commercially available fluorescein isothiocyanate (FITC) -conjugated anti-Escherichia coli antibodies, tested for immunofluorescence were assessed for their suitability in screening E. coli using flow cytometry. Staining efficacy was initially tested using immunofluorescent microscopy; and further optimization was carried out using flow cytometry. Initially, an acetone fixation step was utilized; however, it was determined statistically that the step could be omitted without impacting the assay and thus reduce the time involved. There was no statistical difference between the staining proficiency of the two antibodies employed. The percentage staining was quite low, approximately 10% for the two antibodies, which indicated that both were equally sensitive but ultimately, more specific antibodies are required for the detection of E. coli. Known proportions of target-E. coli (10⁵, 10⁶, and 10⁷ cells/ml) were mixed with large quantities of non-target bacteria; there was a significant correlation among all the antibodies at the different bacterial cell concentrations. Therefore, despite the low staining percentage achieved on the bacterial cultures, there is a representative and comparative level of staining occurring, between samples and between bacterial strains.     

Wilson and Wilson's comprehensive analytical chemistry: Vol. XII,Thermal Analysis,Part A,Simultaneous thermoanalytical examinations by means of the Derivatograph. By J. Paulik and F. Paulik,Elsevier, Amsterdam/New York, 1981. 278 pp., $83.00

The reductive methylation procedure of G.E. Means and R. E. Feeney (1968)Biochemistry7, 2192–2201) was adapted for ³H-labeling of membrane proteins using pigeon erythrocyte membrane. Usably high ³H incorporation into protein was obtained, e.g., 28 μCi/mg protein with 83 nmol (input) H₂CO/mg protein, B³H₄^? at 10 Ci/mmol, and a B³H₄^?/H₂CO ratio of 0.34. With this low H₂CO/protein ratio, methylation did not perturb ATP-dependent ⁴⁵Ca²⁺ uptake, Na⁺-dependent [¹⁴C]glycine uptake, membrane vesicle sealing, or isoelectric focusing patterns of methylated membrane proteins. The labeled membrane proteins were shown to be good tracers for the unlabeled proteins by using two-dimensional isoelectric focusing x sodium dodecyl sulfate gel electrophoresis.     

Short-chained oxygenated VOC emissions in <Emphasis Type="Italic">Pinus halepensis</Emphasis> in response to changes in water availability

Short-chained oxygenated VOC (oxVOCs) emissions from Pinus halepensis saplings were monitored in response to changes in water availability. Online measurements were made with a proton transfer reaction—mass spectrometer under controlled conditions, together with CO₂ and H₂O exchange measurements. Masses corresponding to methanol and acetone were the most emitted oxVOCs. All the oxVOC exchanges, except that of acetone (M59), were significantly related to stomatal conductance and transpiration. Acetaldehyde (M45) emission showed, moreover, a strong dependence on the concentration of acetaldehyde in the ambient: stomatal opening (stomatal conductance above 75 mmol m⁻² s⁻¹) only allowed increased emissions when external concentration were below 6 ppb. Acetone (M59) presented an important peak of emission following light and stomatal opening in the morning when plants were water stressed. Thus, the alterations in oxVOC emissions in P. halepensis caused by the water deficit seem to be mainly driven by water stress effect on stomatal closure and oxVOC air concentrations.