Chloroform Treatment Research Articles

Chloroform toxicity in mice: Correlation of renal and hepatic necrosis with covalent binding of metabolites to tissue macromolecules

Chloroform (CHCl 3) treatment caused centrolobular hepatic necrosis in mice of both sexes whereas renal necrosis was observed only in male mice. Following administration of 14CHCl 3 to mice, substantial amounts (about 3 mmole/g) of radiolabeled material were covalently bound to proteins in the liver and kidney. The amount of convalent binding paralleled the extent of renal and hepatic necrosis both in normal animals and in male mice pretreated with either phenobarbital or piperonyl butoxide, agents which induce or block, respectively, microsomal drug metabolizing enzymes. These results suggest that the covalent binding is due to a metabolite of CHCl 3. Evidence that the covalent binding is causally related to the tissue necrosis was obtained from autoradiograms showing that the radioactivity is located mainly in the necrotic lesions.

Soil Degradation of Two Phenyl Pyridazinone Herbicides

Degradation of SAN 6706 [4-chloro-5-(dimethylamino)-2-(α,α,α-trifluoro-m-tolyl)-3(2H)-pyridazinone] and SAN 9789 [4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl)-3 (2H)-pyridazinone] was studied in a sandy loam soil at temperatures of 5, 20, and 35 C. After 210 days of incubation 10, 80, and 97% of SAN 6706 had been dissipated from soil at 5, 20, and 35 C., respectively. SAN 6706 was converted to SAN 9789 and a demethylated metabolite in the soil. SAN 9789 was converted to the demethylated metabolite. It was difficult to differentiate between first and second-order kinetics in the degradation of these compounds. SAN 6706, at 20 and 35 C, had a half life of 50 and 9 days respectively, and an Arrhenius activation energy of 15.8 Kcal/mole. SAN 9789, at 20 and 35 C, had a half life of 270 and 70 days, respectively, and an activation energy of 11.8 Kcal/mole. The degradation of SAN 6706 was inhibited by a chloroform treatment, and microbiological degradation is suggested.

Partial purification, structure and infectivity of complete maize rough dwarf virus particles

Maize rough dwarf virus, a reolike virus, was purified from roots of maize by clarification with the fluorocarbon 1,1,2-trifluoro-1,2,2-trichloroethane (Freon 113) or with carbon tetrachloride followed by sucrose density gradient centrifugation. The product consisted mostly of complete particles, but some subviral particles were present. The virus was spherical, varying from 63 to 70 nm in diameter under different staining conditions; it had a double capsid and typical reovirus structure with, probably, 92 morphological units in the outer capsid. In addition, each particle possessed 12 projecting spikes (A spikes) each some 11 nm long, one at each 5-fold symmetry axis. Various physical or chemical treatments stripped off the A spikes and part of the outer capsid to give a spherical 50–57 nm subviral particle possessing 12 previously hidden spikes (B spikes) each about 8 nm long, coaxial with the A spikes. Each B spike was implanted on a differentiated part of the inner capsid here called a baseplate. Treatment of whole virus or subviral particles with aqueous neutral potassium phosphotungstate produced smooth subviral particles without B spikes, and longer treatment with phosphotungstate or brief treatment with n-butanol also removed the nucleic acid to give ghosts exhibiting internal structures. Detached B spikes were composed of 5 morphological subunits surrounding a central hole. Infectivity of preparations was tested by injecting them into adult females of the planthopper vector Laodelphax striatellus, which were later screened for production duction of virus symptoms on maize and barley. Crude and purified preparations containing the large virus particles were infectious; Freon-treated preparations possessed the highest infectivity. Subviral preparations made by chloroform treatment were not or very slightly infectious and smooth subviral particles made by treatment with phosphotungstate were not infectious.

Inhibitors of the bone-marrow colony formation in sera of patients with leukemia.

AbstractAn increased variation of the bone‐marrow colony stimulating factor (CSF) was found in the sera of patients with acute leukemia as compared to normal sera. In sequential samples from the same patient, there was no apparent correlation between changes in the degree of colony stimulating activity and changes in the clinical and hematologic status. CSF levels as measured in untreated sera seemed to depend upon a variation in serum inhibitor levels. After chloroform treatment, sera from patients with acute lymphoid or myeloid leukemias had higher mean CSF activity than similarly treated normal sera, though the variation was considerable in both groups. The increase was most evident in sera which initially exhibited a normal or low degree of colony‐stimulating activity. In order to evaluate CSF in normal persons and in various diseases, it appears important to remove inhibitory substances efficiently, for instance by chloroform treatment.

Phage-host Relationships of Xanthomonas citri Compared with those of Other Xanthomonads

The temperate phages of Xanthomonas citri are completely inactivated by chloroform treatment, whereas the virulent phages are quite stable to the treatment. About 80 percent (but not all) of an extensive series of X. citri isolates is lysed by phage Cp1. Cp2, or both. Among 136 isolates of 43 Xanthomonas nomenspecies, only 9 isolates belonging to 7 nomenspecies [X. begoniae (XB8, XB111), X. blepharidis (XB115), X. cucurbitae (XC6), X. phaseoli (XP4, Xph), X. phaseoli var. sojense (XP21), X. marantae (XM126), and X. translucens f. sp. phleum-pratense (XT103)] showed susceptibility to any of the X. citri phages. One of the Stolp-Starr phages, ph25, showed lytic reactions on almost all isolates of X. citri, as well as on many other Xanthomonas nomenspecies. From the cross-susceptibility tests between Xanthomonas bacteria and various phages from different sources, it was concluded that, in general, the Xanthomonas phages which form tiny plaques of pinhead size could frequently be found in soil, and that they showed very broad host ranges, which extended not only over the different isolates of a homologous nomenspecies but also over strains of many heterologous nomenspecies. In contrast, the Xanthomonas phages which were isolated from diseased plant tissues usually showed very limited host ranges.

Adsorption of a ribonucleic acid bacteriophage of Pseudomonas aeruginosa.

The adsorption of a ribonucleic acid bacteriophage, PP7, of Pseudomonas aeruginosa was investigated using the following approaches: electron microscopic observation of the ultrastructure of phage-pili complexes, an adsorption technique employing chloroform and rapid dilution treatment which assays the number of phages remaining unadsorbed, the effect of shear treatment on the availability of the bacterial host's adsorption sites, and the effect of metabolic inhibition of the bacterial host on phage adsorption. The specific adsorption sites on the bacterial host were found to be the pili and only when attached to the bacterial cell. Adsorption is additionally dependent upon the metabolic integrity of the host cell. Variations in the morphology of P. aeruginosa pili were observed. The mechanism of adsorption of bacteriophage PP7 appears to be similar to that of the RNA phages of two other bacterial genera, Escherichia and Caulobacter.

Plant Mucilages. IV. Main Structural Features of the Mucous Polysaccharide isolated from Digenea simplex

The mucilage of Digenea simplex AGARDH has been isolated and subjected to partial methanolysis. Agarobiose dimethylacetal was obtained in high yield in addition to 3, 6-anhydro-L-galactose dimethylacetal, methyl D-galactoside, methyl D-xyloside and the other oligosaccharides. By acetylation of the mucilage followed by extraction with chloroform and alkali treatment, a pure polysaccharide was obtained. Partial methanolysis and methylation studies provided the evidence that the polysaccharide is mainly composed of alternately repeated units of 1, 3-linked D-galactose and 1, 4-linked 3, 6-anhydro-L-galactose, but the presences of 1, 4-linked D-xylose and sulfate ester were also found in part of it.

Biochemical and biophysical properties of vesicular exanthema of swine virus

Vesicular exanthema of swine virus (VESV) was purified and some of its biochemical and biophysical properties measured. VESV grew in the presence of 2 μg/ml actinomycin D, was ether resistant, and was not stabilized by cations. Its sedimentation rate was approximately 207 S, and its composition was approximately 20% RNA and 80% protein. The RNA, which was not readily obtained in intact form, appeared to be single stranded with a molecular weight of about 2 × 10 6. Heterogeneous distributions of VESV were observed upon centrifugation in CsCl density gradients. Crude virus, or virus concentrated by differential centrifugation or by ethanol precipitation, showed a major band at 1.36 g/ml, a secondary band at 1.38 g/ml, and some infectious virus of intermediate density. Sequential treatment with ethanol and chloroform produced a sharp peak at 1.38 g/ml. Chloroform treatment in conjunction with centrifugal concentration produced a sharp band of infectious virus at an intermediate density, 1.37 g/ml, and a band of low infectivity at 1.38 g/ml. The properties of VESV were different from those of the recognized groups of viruses, but most closely resembled properties of the picornaviruses, of which poliovirus and swine enteroviruses were employed for some direct comparisons in these studies.

Determination of Water Stress of Eucalypts in the Field

Abstract Two methods for determining plant hydrature in the field have been tested on Eucalyptus viminalis Labill. and E. dalrympleana Maiden one-year-old seedlings. The parameters selected are the physico-chemical properties of sap and the diffusion pressure deficit of leaves. Before extracting sap, leaves have been subjected to two different preliminary treatments to improve permeability of cellular membranes, viz. chloroform and liquid nitrogen treatments, the latter being chosen since it was quicker. Sap has been obtained by espressing at 272 Atm., then filtered and analyzed. Diffusion pressure deficit has been determined by equilibration with vapour. A series of samples has been placed in moist chambers with known water potential until equilibrium has been attained. DPD corresponds to the water potential of the chamber where the sample neither gains nor loses water.

Effect of some organic solvents on the cytoplasmic-polyhedrosis virus of the forest tent caterpillar, Malacosoma disstria

Effect of ether, chloroform, fluorocarbon, and butanol on the cytoplasmic-polyhedrosis virion of the forest tent caterpillar, Malacosoma disstria, labeled with uridine- 3H for viral RNA, has been investigated by zonal sucrose gradient centrifugagation. Free virions from the cytoplasm of midgut cells were not affected by ether treatment at room temperature (23°C), but about 50% were disrupted at higher temperature (37°C) releasing a light component, with the characteristics of viral RNA. Occluded virions, liberated from polyhedra were fairly stable to ether treatment at 37°C. It is suggested that this difference was due to the difference in maturity of free virions in the cytoplasm and occluded virions within the polyhedra. Free virions were not affected by chloroform treatment at 23°C, but at 37°C they were completely disrupted, releasing a 148 S component and viral RNA. Fluorocarbon caused only slight damage at 23°C but at 37°C two components were released, 175 S which is presumed to be part of the core, and viral RNA. Butanol disrupted intact virions completely at both 23°C and 37°C resulting in two distinct components similar to those obtained by fluorocarbon treatment at 37°C.

Location of Aryl Sulfatase in Conidia and Young Mycelia of Neurospora crassa

Aryl sulfatase (arylsulfate sulfohydrolase, EC 3.1.6.1) was found to have multiple locations in Neurospora conidia. Some enzyme activity remained in the supernatant when a spore suspension was centrifuged or filtered. Part of the cell-bound activity could be detected by adding the assay ingredients to a suspension of intact spores (patent enzyme), and additional activity was only detectable when the spores were first treated to destroy their permeability barriers (cryptic enzyme). Such treatments include: disruption with an X-press, brief rinsing with chloroform or acetone, incubation at 60 C for 5 min, and incubation with phenethyl alcohol, nystatin, or ascosin. Part of the patent aryl sulfatase was inactivated by briefly acid treating the intact spores (no loss of conidial viability). This enzyme was considered to have a cell surface location. Some enzyme was acid-resistant in intact spores, but all of the enzyme was acid-sensitive in spores whose permeability barriers had been disrupted. The pH dependence, kinetic properties, and p-nitrophenyl sulfate uptake were investigated in acid-treated conidia. No aryl sulfatase was detected in ascospores. Young mycelia contained more aryl sulfatase than did conidia, but little, if any, was secreted into the growth medium. Cryptic activity was demonstrated in young mycelia by brief chloroform treatment or by rinsing the cells with 0.1 m acetate buffer. Enzyme activity in young mycelia was completely labile to acid treatment, as was cell viability.

Complement fixation reaction for the diagnosis of type II avian (Marek's) leukosis.

The present study was designed to find a complement fixation (CF) reaction for the diagnosis of type II lymphoid leukosis, to learn some of the characteristics of the CF antigen, and to investigate the development of CF antibody response to this infection. JM virus-specific antigen was demonstrated in tumorous chicken tissue, in JM virus-infected chick embryo material, in JM virus-infected chicken kidney, and in duck embryo fibroblast tissue culture by using JM virus-immune rabbit serum. This CF antigen did not show cross-reactivity with Rous sarcoma virus or with RIF-type viruses. It was partially heat-labile. The CF activity was restored at -70 C for 10 months and was resistant to intermittent freeze-thaw treatment. The CF antigen may be denatured by ethyl alcohol, but no significant deleterious effects were noted after ether or chloroform treatment. JM virus-specific CF antibody could not be demonstrated by the direct complement dilution method or by the indirect or inhibition form of the CF test in infected or immunized chicken sera.

Recovery and Characterization of a Minute Virus of Canines

Four antigenically related transmissible agents were recovered from canine fecal specimens. The agents produced cytopathic effects in a continuous dog cell line developed in this laboratory. Increased antibody titers were demonstrated in three of the four dogs which provided the isolates. The virus did not produce cytopathic effects in primary canine kidney or thymus cell cultures, or in cell cultures of human, simian, porcine, bovine, feline, and murine origin. The agent is resistant to ether, chloroform, and heat treatment, and the growth of the virus is inhibited by 5-iodo-2-deoxyuridine. After acridine orange staining, green fluorescent intranuclear inclusions are seen in infected cell cultures. By electron microscopy, the virions measure approximately 20 to 21 nm in overall diameter and are present in the nuclei of infected cells. These properties are consistent with membership in the parvovirus or picodnavirus group. The agent hemagglutinates rhesus red blood cells at 5 C and by hemagglutination-inhibition tests could be readily distinguished from H-1, rat virus, and the minute virus of mice. Canine gamma globulin contains high titers of neutralizing antibody and neutralizing antibody was found in a high percentage of military dogs and in beagles of a breeding colony.

Complement Fixation Reaction for the Diagnosis of Type II Avian (Marek's) Leukosis 1

The present study was designed to find a complement fixation (CF) reaction for the diagnosis of type II lymphoid leukosis, to learn some of the characteristics of the CF antigen, and to investigate the development of CF antibody response to this infection. JM virus-specific antigen was demonstrated in tumorous chicken tissue, in JM virus-infected chick embryo material, in JM virus-infected chicken kidney, and in duck embryo fibroblast tissue culture by using JM virus-immune rabbit serum. This CF antigen did not show cross-reactivity with Rous sarcoma virus or with RIF-type viruses. It was partially heat-labile. The CF activity was restored at -70 C for 10 months and was resistant to intermittent freeze-thaw treatment. The CF antigen may be denatured by ethyl alcohol, but no significant deleterious effects were noted after ether or chloroform treatment. JM virus-specific CF antibody could not be demonstrated by the direct complement dilution method or by the indirect or inhibition form of the CF test in infected or immunized chicken sera.

Action of chloroform on the hemagglutinin of ECHO virus types 7 and 11.

Chloroform treatment of ECHO virus types 7 and 11 had no effect on either the infectivity or the hemagglutinin of these viruses. This is in contrast to reoviruses, the only other ether-resistant RNA virus group, where the hemagglutinin is destroyed but infectivity retained. The two ECHO viruses showed no morphological changes visible by electron microscopy after chloroform treatment, whereas capsomeres of reovirus particles break off.

Studies on the prekallikrein (kallikreinogen)--kallikrein enzyme system of human plasma. II. Evidence relating the kaolin-activated arginine esterase to plasma kallikrein.

Evidence is presented in this paper that the kaolin-activated arginine esterase of plasma is related to plasma kallikrein activity. Such a relationship is based on studies that (1) establish a constant ratio of esterase activity on various synthetic substrates for the kaolin-activated arginine esterase, purified kallikrein(s), and preparations obtained during the fractionation procedure; (2) exclude other known plasma and tissue arginine esterases; (3) confirm the requirement for factor XII in the activation of the enzyme precursor; and (4) show similarities in behavior between the plasma esterase and purified kallikrein(s) toward a variety of inhibitors. Based on this probable identification, evidence is provided that the concentration of active factor XII determines the rate of activation of plasma kallikreinogen, and that the activation may be blocked by polybrene. Once activated, plasma kallikrein is rapidly inactivated by the naturally occurring plasma inhibitor, but the inhibition is incomplete. Acid or chloroform treatment of plasma rapidly inactivates the plasma inhibitor without affecting the concentration of plasma kallikreinogen. Another plasma arginine esterase with properties suggestive of permeability factor is activated by factor XII in the presence of synthetic substrates, but only at low ionic strength. The data suggest that this enzyme is closely related to plasma kallikrein and that it arises from a common precursor.

Morphological and immunological studies on reovirus type 2 hemagglutinin.

1. Evidence is presented that reovirus hemagglutinin is an integral part of the structurally intact virus regardless of the presence or absence of infectivity. No hemagglutinin apart from that associated with the morphologically intact virus could be demonstrated. 2. Chloroform treatment of reovirus type 2 causes disintegration of the viral capsid with destruction of capsomeres. Furthermore, profound antigenic changes of the viral protein occur after chloroform treatment. Therefore chloroform seems to act on the protein of the virus destroying hemagglutinating activity without affecting infectivity and should therefore not be interchangeably used with ether to determine the presence or absence of “essential lipids” as it is commonly used in classifying viruses.

Rapid Method for the Extraction of Light Filth from Peanut Butter

Abstract A method has been developed for the extraction of light filth from peanut butter. The new method eliminates the petroleum ether and chloroform treatments in 36.021 and the pancreatin digestion in 36.022. Results are comparable to the official methods, and analysis time is reduced by one-half. The method is recommended for adoption as official, first action.

Preparation and properties of sea-urchin ribonuclease

1. 1. A method is described for the purification of a ribonuclease (polyribonucleotide 2-oligonucleotide transferase (cyclizing), EC 2.7.7.16) from mature, unfertilized eggs of the sea urchin Psammechinus miliaris. The enzyme was purified 19 000-fold with a yield of 48% with the aid of chloroform treatment, (NH 4) 2SO 4 fractionation, and by chromatography on Sephadex G-100, on SE-Sephadex C-50 and on CM-Sephadex C-50. 2. 2. The enzyme had a pH-optimum at 5.3–5.5. 3. 3. The enzyme was heat labile, loosing about 50% of its activity in 3 min at 60° and pH 5.3, 70% of its activity in 3 min at 60° and pH 2.1, and 80% of its activity in 3 min at 40° and pH 9.0. 4. 4. The enzyme hydrolyzed yeast RNA completely to 2′,3′-cyclic nucleotides, and finally hydrolyzed the purine cyclic nucleotides slowly to their corresponding 3′-isomers. 5. 5. The enzyme hydrolyzed guanosine 2′-3′-cyclic phosphate about three times faster than adenosine 2′,3′-cyclic phosphate. 6. 6. The purified enzyme was found to be free of deoxyribonuclease and non-specific phosphodiesterase activity. 7. 7. A molecular weight of about 37 000 was estimated for the enzyme by comparative gel filtration experiments.

STRAIN ALTERATION IN ENTERIC BACTERIA ISOLATED FROM RIVER WATER

Enteric bacteria recovered from the Cache la Poudre River and refrigerated for approximately 3 months were observed to form sectored colonies on MacConkey agar subculture. Organisms taken from each sector of the colonies were compared and a wide range of biochemical and serological variation was observed. Selected variant characteristics observed in the pure culture isolates from the sectored colonies were shown to be reproducible with membrane filter U tubes. Further investigation revealed that the agent responsible for the observed variations was submicrobial, survived chloroform and heat (56 C for 30 minutes) treatments, and produced occasional lysis in the non-lactose-fermenting strains. Additional cultures resembling the original variant organisms were recovered from the river but attempts to produce the sectoring phenomenon with these organisms were without success, although numerous sectoring isolates having similar variant characteristics were recovered from the stream.