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At least 19 recordsLinked to original sources
PubMed2025

A universal, high-quality, and high-yield DNA purification method for mycobacteria, including Mycobacterium tuberculosis: large-scale assessment of the chloroform-bead method.

UNLABELLED: Genomic analysis of mycobacteria has become increasingly crucial for understanding drug-resistance mechanisms, molecular epidemiology, and pathogenesis. However, efficient extraction of high-molecular-weight genomic DNA from these organisms remains challenging because of their thick mycolic acid-rich cell walls. In this study, we report the chloroform-bead method, a universal DNA extraction protocol that combines chemical and mechanical disruptions to overcome these challenges. Multi-laboratory evaluation (16 sites) demonstrated the chloroform-bead method's superiority over conventional methods for Mycobacterium tuberculosis (DNA yield: 17.9 vs 1.9 &#xb5;g, purity A260/A230: 1.86 vs 1.22, both P < 0.001). Single-facility assessment extended these findings to >32 nontuberculous mycobacterial species (n = 1,058), showing performance comparable to M. tuberculosis (n = 1,000), with both achieving median yields of 22.2 &#xb5;g DNA and consistent quality metrics. The chloroform-bead method significantly reduced the processing time from 2 to 3 days to 2 h while ensuring complete sample sterilization, eliminating the need for species-specific optimization. This streamlined and universally applicable protocol represents a practical advancement in mycobacterial DNA extraction methodology, ideal for high-throughput genomic studies and routine clinical diagnostics. IMPORTANCE: Mycobacterial genomics is crucial for understanding pathogenesis and drug resistance; however, DNA extraction remains a significant challenge because of its unique cell wall. Traditional methods rely on enzymatic treatments, resulting in complex and time-consuming protocols with variable results. The chloroform-bead method introduces a paradigm shift by chemically and mechanically disrupting the mycolic acid layer and eliminating the need for enzymatic treatment. This standardized approach ensures consistent, high-quality DNA extraction across diverse mycobacterial species, thereby enhancing research capabilities and clinical applications.

Chloroform↗
PubMed1978

Affinity chromatography of H+-translocating adenosine triphosphatase isolated by chloroform extraction of Rhodospirillum rubrum chromatophores. Modification of binding affinity by divalent cations and activating anions.

1. ATPase isolated from Rhodospirillum rubrum by chloroform extraction and purified by gel filtration or affinity chromatography shows three bands (alpha, beta and gamma) upon electrophoresis in sodium dodecyl sulphate. 2. Ca2+-ATPase activity of the preparation is inhibited by aurovertin and efrapeptin but not by oligomycin. Activity may be inhibited by treatment with 4-chloro-7-nitrobenzofurazan and subsequently restored by dithiothreitol. 3. The enzyme fails to reconstitute photophosphorylation in chromatophores depleted of ATPase by sonic irradiation. 4. Most of the active protein from the crude chloroform extract binds to an affinity chromatography column bearing an immobilised ADP analogue but not to a column bearing immobilised pyrophosphate. 5. In the absence of divalent cations, a component with a very high specific activity for Ca2+-ATPase is eluted from the column by 1.6 mM ATP. This protein migrates asa single band on 5% polyacrylamide gel electrophoresis and only possesses three subunits. At 12 mM ATP an inactive protein is eluted which does not run on acid or alkali polyacrylamide gels and shows a complex subunit structure. 6. ATPase preparations prepared by acetone extraction or by sonic irradiation of chromatophores may also be purified 10-fold by affinity chromatography. 7. The inclusion of 5 mM MgCl2 or CaCl2 during affinity chromatography of chloroform ATPase increases the capacity of the column for the enzyme and demands a higher eluting concentration of ATP. 8. When the enzyme is more than 90% inhibited by efrapeptin or 4-chloro-7-nitrobenzofurazan, the binding characteristics of the enzyme are not affected. 9. 10 mM Na2SO3, which greatly stimulates the Ca2+- and Mg2+-dependent ATPase activity of the enzyme and increases Ki (ADP) for Ca2+-ATPase from 50 to 850 micron, prevents binding to the affinity column. Binding may be restored by the addition of divalent cations. 10. Na2SO3 increases the rate of ATP hydrolysis, ATP-driven H+ translocation and ATP-driven transhydrogenase in chromatophores. 11. It is proposed that anions such as sulphite convert the chromatophore ATPase into a form which is a more efficient energy transducer.

Adenosine Triphosphatases↗
PubMed2026

Stable chloroform emissions in southeastern China: insights from recent observations.

Chloroform (CHCl3) is a short-lived ozone-depleting substance not currently regulated under the Montreal Protocol. Due to the unique meteorological conditions in East Asia, CHCl3 emitted in this region has a greater potential to reach the stratosphere and contribute to ozone depletion. As an essential component of national CHCl3 emissions, southeastern China has attracted increasing attention. However, long-term observational data in this region remain relatively scarce, with no updates since 2020. In this study, we continuously measured atmospheric CHCl3 concentrations at a remote monitoring station in southeastern China from 1 June 2023 to 31 May 2025. Frequent concentration enhancements were observed during the monitoring period, with mixing ratios ranging from 9.3 to 134.1 ppt and an average value of 36.8 &#xb1; 19.7 ppt. Back-trajectory analysis indicated that air masses associated with elevated CHCl3 levels primarily originated from coastal industrial provinces in eastern China. Using the Potential Source Contribution Function and Concentration Weighted Trajectory methods, we identified the Yangtze River Delta (Jiangsu, Anhui, and Zhejiang Provinces) and Jiangxi Province as dominant source regions. Emissions of CHCl3 in southeastern China were estimated using the Tracer Ratio Method to be approximately 30.1 &#xb1; 5.3 Gg/yr from 2023-06-01 to 2025-05-31, indicating overall stability relative to earlier estimates and no apparent upward trend. These findings provide updated insights into the current status of CHCl3 emissions in southeastern China and highlight the need for continued monitoring and emission assessment of CHCl3 in East Asia, given its unregulated status and implications for ozone layer recovery.

Air Pollutants↗
PubMed1977

Reduction of nitro-BT by some components of the chloroform-methanol extract of the grey matter of the rat brain.

An attempt was made to extract the substance present in the nerve fibres of the cerebral cortex and basal ganglia which reduced Nitro-BT in alkaline medium pH 9-5 [4,5]. The investigations were performed on 40 white Wistar rats of both sexes and ca. 200 g of body weight. The brains taken for studies were fixed in Baker's formalin for 1,5 hr, thereafter grinded and homogenized in chloroform-methanol solution supplemented up to 100 ml of final volume after homogenisation. Thus prepared solution was left for 24 hr at 4 degrees C temperature and than centrifuged 2000 g/min. The extract was evaporated and the sediment further investigated. The following fraction were received: 1. fraction soluble in aceton, 2. fraction soluble in ethylalcohol, 3. fraction soluble in ether, 4. fraction soluble in water and 5. the sediment. Each fraction was incubated with a standard medium containing Nitro-BT. After incubation the amount of reduced Nitro-BT in each of the incubation medium was spectrophotometrically measured. The most intense reduction was found in the incubation medium with the water fraction. The spectrophotometrically determined reduction of Nitro-BT of the water fraction compared with that of the gangliosides solution were similar. The addition into the incubation media biogenic amines and N,N-diethyl-p-phenylenediamine enhances the reduction of Nitro-BT significantly. According to the results obtained an interference of gangliosides and of the investigated amines in the reduction of Nitro-BT is suggested.

Animals↗
PubMed1975

A comparative study on the irreversible binding of labeled halothane trichlorofluoromethane, chloroform, and carbon tetrachloride to hepatic protein and lipids in vitro and in vivo.

1) After intraperitoneal injection of labeled CCl4, CHCl3, and halothane in mice, 14C is preferentially bound to liver endoplasmic protein and lipid. A considerable activity is also associated with mitochondrial constituents. Maximal protein binding (nmol/mg): CCl4: 2.8 (0.5 hrs); CHCl3: 11.5 (6 hrs); halothane: 5 (6 hrs). Lipid binding: CCl4: 6.4 (5 min); CHCl3: 8 (4 hrs); halothane: 13.5 (2 hrs). The form of the binding curves in microsomal and mitochondrial protein and lipid differed with the individual haloalkanes. 2) The irreversible (covalent) binding of 14C from labeled haloalkanes in anaerobic suspensions of isolated rabbit liver microsomes and NADPH after 30 min was for protein (lipid) (nmol/mg): CCl4: 15 (58); CHCl3: 3.4 (3.2); halothane: 2.3 (10); trichlorofluoromethane: 6.5 (30). Anerobic incubation favored dehalogenation, but CHCl3 metabolism and irreversible binding requires oxygen. The greatest differences in the in vitro "covalent" binding rates were observed with CHCl3 in rat, mouse, and rabbit. 3) Altered microsomal cytochrome P-450 concentrations in newborn animals, or produced by pretreatment of rats with phenobarbital, 3-methylcholanthrene (MC), or CoCl2 effected similar, but not proportional changes in the rates of irreversible protein and lipid binding. Upon addition of CCl4 the difference of light absorption of reduced liver microsomes from MC-pretreated rats containing cytochrome P-448 appeared at 452 nm. The irreversible binding rate in these microsomes was also increased. The small accleration in irreversible binding in liver microsomes from rats pretreated with isopropanol is not proportional to the high increase of CCl4 toxicity. 4) Practically no binding to added, soluble albumin or RNA was observed in microsomal incubates. However, 14C is bound to the nicotine-adenine dinucleotides of the NADPH system. All haloalkanes produced a similar increase of NADPH oxidation in incubates of rabbit liver microsomes and NADPH.

1-Propanol↗
PubMed1976

Unusual behaviour of a protein: reversible quantitative precipitation of staphylococcal delta-haemolysin by low concentrations of some organic solvents.

Staphylococcal delta-haemolysin is precipitated from watery solutions by low concentrations of chloroform. The effect on this precipitation of pH, kind and concentration of salts, and concentration of, and time of exposure to, chloroform have been investigated. Some but not all of the other solvents tested cause a similar precipitation. The haemolytic activity is not destroyed and under appropriate conditions precipitation is quantitative. The dry lysin will take up nearly double its weight of chloroform when exposed to the vapour. Some is bound strongly.

Ammonium Sulfate↗
PubMed1976

pH, temperature, humidity and the dynamic force-area curve of dipalmitoyl lecithin.

Both high pH at 25 degrees C and humidity at 37 degrees C prevent DPL films from attaining zero surface tension, whereas humidity at 25 degrees C and high pH at 37 degrees C do not. At 37 degrees C DPL lowered surface tension to zero when spread from organic solvent or when absorbed from aqueous 0.15 M NaCl in the surface balance in which the surface film was exposed to the room air (dry film). Upon saturation of the atmosphere with water vapor in equilibrium with the aqueous phase at 37 degrees C in a closed chamber, DPL lost the ability to produce zero surface tension, and the gamma min of the DPL film increased from zero to 22 dyne/cm. Addition of DPL in chloroform to distilled water before dispersion by sonication did not prevent the effect of the humidity. However, when the chloroform solution of DPL was added to 0.15 M NaCl before sonication, the adsorbed film produced immediately a stable gamma min of zero in a saturated atmosphere, 37 degrees C. In the absence of chloroform, with DPL adsorbed from either distilled water or 0.15 M NaCl, the effect of humidity was reversed either by removing the chamber and returning the wet film to room air or by introducing small quantities of dispersing agents such as cholesteryl palmitate. However, whereas the effect of humidifying the air was reversible indefinitely, the effect of cholesteryl palmitate (zero surface tension, wet or dry film) was irreversible. This means that there are substances or conditions that can assist DPL films in maintaining zero surface tension when such films are exposed to humidity-saturated air at 37 degrees C.

Humidity↗
PubMed1976

Biotransformation, sex hormones, and toxicity of two volatile anesthetics in mice.

Male and female DBA 11 mice recovered from 1 hr of anesthesia with chloroform of fluoroxene apparently unharmed. However, many of the animals died within 24-48 hr after anesthesia. Pretreatment with phenobarbital increased, while pretreatment with a small dose of carbon tetrachloride decreased, this toxicity. Relatively more males than females died. Pretreatment with estradiol in males and testosterone in females reversed this ratio. We conclude that the murine toxicity of chloroform and fluoxene is dependent on biotransformation by hepatic microsomal enzymes and that the testosterone enhances postanesthetic toxicity of these agents.

Animals↗
PubMed1976

[Physico-chemical properties of swine vesicular disease virus].

Titration of SVDV on primary pig kidney cell cultures revealed a plating efficiency of less than or equal to 0,9 X 10(-3). Concentration and purification of the SVD-Virus propagated on pig kidney cell cultures were done by chloroform treatment, adsorption, differential- and density gradient centrifugation. The following physical parameters were found: SVDV is an isometrical RNA-virus having a diameter of 25,1 +/- 1,0 nm. It is resistent to the action of chloroform, ether and pH. The virus has a sedimentation coefficient of 156 +/- 3S and a bouyant density in CsCl of 1,33 +/- 0,01 g/ml. Within the family of picornaviruses the SVDV belongs to the subgroup of enteroviruses and can be distinguished from the foot-and-mouth disease virus by the difference in pH-sensitivity and bouyant density in CsCl.

Animals↗
PubMedDate not supplied

Mechanisms of chronotropic effects of volatile inhalation anesthetics.

The chronotropic effects of all currently available volatile anesthetics were investigated in isolated rat atrial preparations. Anesthetic ethers, diethyl ether, methoxyflurane, and enflurane elicited a dose-dependent positive chronotropic effect. Fluroxene produced a slight depression at low concentrations. The halogenated hydrocarbon anesthetics, halothane, chloroform, and trichloroethylene, did not show a uniform pattern. Halothane's effect was small and biphasic. Chloroform caused a dose-dependent decrease in heart rate, and trichloroethylene caused a marked positive chronotropic effect. The dose-response curves in all anesthetics remained unaltered in the presence of either 3 x 10(-7) M dl-propranolol or 1 x 10(-6) M atropine. It is concluded that volatile anesthetics elicit significant direct chronotropic actions on rat atrial preparations. The mechanism of their actions does not involve stimulation of beta-adrenergic or cholinergic receptors.

Anesthesia, Inhalation↗
PubMed1978

[Extraction and polarimetric method of determining phenacetyl-D(-)-alpha-aminophenylacetic acid].

Equilibrium distribution of phenacetyl-D-(--)-alpha-aminophenylacetic acid obtained on fermentative hydrolysis of D,L-aminophenylacetic acid in a two-phase system (chloroform-water) was studied within a wide range of the substance concentration in the organic phase. It was shown than in the organic phase the distributing substance formed associates. The empirical equation: y = A + Bx describing the dependence of the effective distribution of the non-dissociated form of phenylacetyl-D-(--)-alpha-aminophenylacetic acid on its concentration in the organic phase was suggested. Coefficients A and B of the equation were determined and an equation for evaluating equilibrium concentrations in the equeous phase was suggested. On the basis of the studies an extraction-polarometric method for quantitative determination of phenacetyl-D-(--)-alpha-aminophenylacetic acid concentration was developed. The method consists of extraction of the non-dissociated form of phenylacetyl-D-(--)-alpha-aminophenylacetic acid with chloroform, determination of the equilibrium concentration of the distributing substance in the organic phase by the polarimetric method and subsequent estimation of the equilibrium and initial concentrations of the electrolyte in the aqueous phase.

Chloroform↗
PubMed1979

The effect of exposure to halogenated anaesthetics on liver glutathione levels in rats. An index of hepatotoxicity.

Having studied anaesthetic drug interactions in rats, we report the effects of halogenated anaesthetics on the liver glutathione levels and histology, as well as the results of the enhancement of these effects by microsomal enzyme induction. The anaesthetic agents studied included methoxyflurane, halothane, ethrane, chloroform and fluroxene. While exposure of rats to methoxyflurane, helothane and ethrane produced no significant changes in hepatic glutathione levels, or in liver histology, exposure to chloroform and fluroxene produced marked depression of liver glutathione, especially after microsomal enzyme induction. Furthermore, rats exposed to thses agents after enzyme induction developed gross centrilobular necrosis and died. It is suggested that the study of the effects of any new anaesthetic agent on liver glutathione levels could be a valuable screening test of its hepatotoxic potential, before its clinical trial.

Anesthetics↗
PubMed1976

Spectrophotometric determination of diphenhydramine hydrochloride using dipicrylamine.

A spectrophotometric procedure for the determination of diphenhydramine hydrochloride based on the reaction with dipicrylamine was developed. A yellow complex forms and is easily extractable by chloroform at pH 5. The mole ratio of diphenydramine hydrochloride to dipicrylamine in the complex is 1:3. The absorbance of the complex obeys Beer's law over the concentration range of 3-10 mug of diphenhydramine hydrochloride per ml of chloroform. This procedure can be carried out in the presence of other compounds without interference.

Aniline Compounds↗
PubMed1976

Simple and rapid determination of a new pharmaceutically active amine hydrochloride.

The quantitative analysis of a new pharmaceutically active amine hydrochloride is described. Samples are extracted with chloroform. A yellow amine-dye complex is formed by buffering a sample-bromthymol blue solution at pH 8.5 +/- 0.1 and subsequently extracted with chloroform. The complex is treated with 0.01 N NaOH to convert it back to the sodium salt of bromthymol blue, which is then measured at 615 nm in the aqueous layer. The amount of complex extracted is linearly related to the amount of amine present, from 0.020 to 0.20 mg/ml. Under the selected conditions, the compound can be determined in the presence of degradation products and there is no interference from common pharmaceutical excipients. The method is suitable for stability studies.

Benzopyrans↗
PubMed1978

Substance P: binding to lipids in the brain.

1. Substance P (SP) could be extracted from brain homogenates with chloroform-methanol by a method which extracts all lipids. 2. SP could be transferred form this total lipid extract (TLE) into an aqueous solution at low pH values (2.0--3.0). 3. At higher pH values (5.5) SP could be transferred from an aqueous phase into an organic phase (chloroform:methanol, 2:1) and recombined with TLE (which was previously freed from endogenous SP) contained in this phase. The binding capacity of TLE for SP exceeded by far the amount of endogenous SP bound originally in the brain extracts. 4. Among the lipids present in TLE, phosphatidylserine was able to bind and release SP in a pH dependent manner. 5. It is suggested that SP bound to phosphatidylserine is the storage form of SP in the brain. The mechanisms by which it is released are still unknown. The possibility that the SP-receptor is also a phospholipid is considered.

Animals↗