Conjugal Transfer of Plasmid-Borne Multiple Antibiotic Resistance in Streptococcus faecalis var. zymogenes

A strain of Streptococcus faecalis var. zymogenes, designated JH1, had high-level resistance to the antibiotics streptomycin, kanamycin, neomycin, erythromycin, and tetracycline. These resistances were lost en bloc from approximately 0.1% of cells grown in nutrient broth at 45 C. The frequency of resistance loss was not increased by growth in the presence of the "curing" agents acriflavine or acridine orange, but after prolonged storage in nutrient agar 17% of cells became antibiotic sensitive. Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated from the parental strain and from antibiotic-sensitive segregants by using cesium chloride-ethidium bromide gradients. DNA molecular species were identified by using neutral sucrose gradients. Strain JH1 contained two covalently closed circular DNA species of molecular weights 50 x 10(6) and 38 x 10(6). An antibiotic-sensitive segregant, strain JH1-9, had lost the larger molecular species. A second sensitive segregant, strain JH1-5, had also lost the larger molecular species but a new molecular species of approximate molecular weight 6 x 10(6) was present. The antibiotic resistances that were curable from the parental strain were transferred to antibiotic-sensitive strains of S. faecalis and to strain JH1-9, during mixed incubation in nutrient broth at 37 C. Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism. Analysis of the plasmid species in recipient clones showed that all had received the plasmid of molecular weight 50 x 10(6). Strain JH1-5 was not a good recipient. Analysis of one successful recipient clone of JH1-5 revealed that it had gained the 50 x 10(6) molecular weight plasmid but lost the 6 x 10(6) molecular weight species. These data are interpreted to mean that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 10(6) molecular weight, and that in clone JH1-5 this plasmid suffered a large deletion leaving only a 6 x 10(6) remnant which was incompatible with the complete replicon.     

Oestradiol uptake and retention, and high-affinity binding sites in cultured rabbit uterus

Oestradiol uptake and turnover was examined in rabbit uterus maintained in organ culture for up to 3 days. Serum decreased the uptake of [(3)H]oestradiol, whereas insulin had no significant effect. During the first 24h of culture unoccupied high-affinity receptors for oestradiol were markedly depleted in the cytosol. Nuclear binding sites remained high during the first day of culture, and were still present after 3 days. The stability of nuclear-bound oestradiol was confirmed by examining the turnover of radioactivity during culture of uteri of rabbits injected with [(3)H]oestradiol 6h before death. Over half of the radioactivity was retained for as long as 3 days in tissue cultured in the absence of oestrogen. In tissue cultured for 24h with unlabelled oestrogen, there was a progressive increase in the displacement of [(3)H]oestradiol as the concentration of unlabelled hormone in the medium was increased from 0.1 to 5nm. Higher concentrations of oestradiol had little additional effect. The oestradiol involved in this displacement reaction was associated with macromolecules, characterized by Sephadex G-25 chromatography and sucrose-density-gradient ultracentrifugation of the 0.4m-KCl extract of the nuclear pellet.     

Studies on the lipid composition of the rat liver endoplasmic reticulum after induction with phenobarbitone and 20-methylcholanthrene

1. The cholesterol content, proportions of different phospholipids and fatty acid components of phosphatidylcholine and phosphatidylethanolamine were studied in rat liver endoplasmic-reticulum membrane, after a single injection of 20-methylcholanthrene or injections of phenobarbitone for 5 days. 2. A marked decrease in the proportion of cholesterol occurred 5 days after injection of 20-methylcholanthrene or phenobarbitone. 3. The proportion of phosphatidylcholine was increased by injection of phenobarbitone and minor changes occurred in other phospholipids. 4. Phenobarbitone caused the proportion of linoleic acid in phosphatidylcholine and phosphatidylethanolamine to increase to 120-125% of the control and the proportion of oleic acid, arachidonic acid and docosahexaenoic acid to decrease. 5. 20-Methylcholanthrene caused an increase in the proportion of oleic acid in phosphatidylcholine and ethanolamine to 125-140% of the control, 1 day after injection. 6. The increased proportion of linoleic acid in phosphatidylcholine after phenobarbitone injection occurs simultaneously with the increase of cytochrome P-450 concentration, the rate of oxidative demethylation of aminopyrine and the rate of hydroxylation of biphenyl. It is therefore considered that distinct species of phosphatidylcholine or phosphatidylethanolamine containing linoleic acid in the beta position are essential in the endoplasmic-reticulum membrane for optimal activity of oxidative demethylation.     

Adenylate cyclase in sea anemone implication for chemoreception

Adenylate cyclase of the sea anemoneAnthopleura elegantissima was found to be associated with the heavy particulate fraction of the cell and to be activated by NaF and 2-mercaptoethanol. Reduced glutathione, which elicits the ciliary swallowing response during feeding, also activated adenylate cyclase in particles from the oral disc and pharynx. The GSH effect was dependent on homogenization procedure, whereas the NaF and 2-mercaptoethanol activation was not. The activation of adenylate cyclase from the oral disc and pharynx by GSH was correlated with increased Ca²⁺ binding to the particulate fraction. When activation by GSH was abolished by mechanical homogenization, no increasea in Ca²⁺ binding was observed in the presence of GSH. It is suggested that chemoreception for the swallowing response of this organism is mediated by cyclic AMP control of Ca²⁺ distribution in the cell.     

Comparison of Undefined Medium and Its Dialyzable Fraction for Growth of Mycoplasma

In examining the medium used in cultivation of Mycoplasma for deoxyribonucleic acid isolation, it was found that an aggregate was present which sedimented with the organisms and which was ethyl alcohol-precipitated during deoxyribonucleic acid purification. To eliminate the contaminating material, a method was devised to obtain only the dialyzable constituents of the medium. This report describes the preparation of a dialysate of soy peptone-yeast extract. The medium, obtained by immersion of the encased dehydrated ingredients in sodium chloride solution for 5.5 hr at approximately 80 C, has been employed as the basal medium for cultivation of a number of Mycoplasma species. Comparative growth curves of two saprophytic strains and two parasitic species indicated that multiplication in dialysate, with suitable supplement, followed the pattern typical of the common eubacteria. Thus, by elimination of the sediment which occurred in nondialyzed medium, Mycoplasma could be concentrated without concomitant accumulation of contaminating macromolecules.     

Mycobacterial Polysaccharides II. Comparison of Polysaccharides from Strains of Four Species of Mycobacteria

Evidence from chemical and serological studies indicates that a cellular heteropolysaccharide, also found in lipid extracts and culture filtrate, is present as a group antigen in Mycobacterium tuberculosis H37Ra and in other strains of mycobacteria representing M. kansasii, scotochromogenic and Battey strains. Polysaccharides from the four strains contain the same main sugars, arabinose, and galactose, as revealed by thin-layer chromatography and spectrophotometric studies. In Ouchterlony gel diffusions, bands of identity are produced between the polysaccharides by using rabbit antiserum prepared against any of the four mycobacteria. Immune adsorption studies also confirm the presence of identical antigenic determinant groups. In skin tests with tuberculopolysaccharide I, a skin reaction of about equal size was elicited in guinea pigs sensitized with either M. tuberculosis H37Ra or heterologous mycobacterial antigens in Freund's incomplete adjuvant. In animals sensitized with M. tuberculosis H37Ra, skin tests with both homologous and heterologous polysaccharides elicited similar responses.     

Specificity of the Induced Bactericidin of the West Indian Spiny Lobster, Panulirus argus

To investigate the specificity of an inducible hemolymph bactericidin, West Indian spiny lobsters were immunized with a variety of antigens. Bactericidal assays were conducted with strain EMB-1, a gram-negative bacillus isolated from normal lobster intestine. Animals given saline solution with or without 0.3% Formalin exhibited only a slight response similar to sham controls. Injections of type 2 pneumococci or bovine albumin produced slightly higher responses. These were much less pronounced than the homologous response to EMB-1. Formalin exhibited an adjuvant-like effect with EMB-1.     

Methacarn (methanol-Carnoy) fixation

Summary According to chemical data, methanol raises the shrinkage temperature of collagen significantly more than ethanol (86° C versus 70° C). Since increase of shrinkage temperature appears desirable in tissues to be embedded in paraffin, methanol was substituted for ethanol in Carnoy's fluid. This methanol-Carnoy mixture is referred to as methacarn solution. The fixation-embedding procedure was similar to that described in the study of Carnoy fixation. Methacarn-fixed sections showed little or no shrinkage and compared well with material fixed in Carnoy's or Zenker's fluid. Myofibrils, especially in endothelial and epithelial cells, were more prominent in methacarn- than in Carnoy-fixed tissues.A review of the chemical literature showed that methanol, ethanol and chloroform stabilize or even enhance helical conformations of proteins, presumably by strengthening of hydrogen bonds. Interference with hydrophobic bonds causes unfolding and/or structural rearrangements in globular proteins. The twin-helical structure of DNA collapses in alcoholic solutions. Hence, methacarn fixation can be expected to preserve the helical proteins in myofibrils and collagen, but the conformations of globular proteins and DNA will be significantly altered. Literature on conformational effects produced by fixatives used in electron microscopy was also reviewed. Glutaraldehyde and OsO₄ cause considerable loss of helix (22–29% and 39–66% respectively). KMnO₄ and glutaraldehyde followed by OsO₄ produce extensive transitions from helical to random-coil conformations similar to those seen in powerful denaturants such as 8 M urea. Evidently these fixatives are unsuitable for studies of helical proteins. In contrast ethylene glycol preserves helical conformations.