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EXPLANATION FOR LIMITATION OF POPULATIONS OF ESCHERICHIA COLI IN BROTH CULTURES.

Freter, Rolf (Jefferson Medical College, Philadelphia, Pa.), and Atsushi Ozawa. Explanation for limitation of populations of Escherichia coli in broth cultures. J. Bacteriol. 86:904-910. 1963.-Veal Infusion Broth surrounding a cellophane bag containing a 24-hr culture of Escherichia coli was inhibitory to an inoculum of the same strain when kept under an atmosphere of nitrogen plus CO(2). The inhibition could be abolished by addition of glucose or by aeration. When a small inoculum of a dulcitol-positive E. coli strain was introduced into a fully grown (24 hr) static Veal Infusion Broth culture of a dulcitol-negative E. coli, no multiplication occurred. However, the inoculum did grow in the presence of a carbon source (dulcitol) which could not be utilized by the static population. The logarithmic growth rate attained by the dulcitol-positive inoculum under these conditions was independent of the dulcitol concentration. In contrast, the maximal population size was a function of the amount of dulcitol present. Similar results were obtained when sucrose or salicin was substituted for the dulcitol. All strains grew well in filtrates of 24-hr broth cultures with growth rates and maximal populations approximating those attained in fresh broth. It was thus concluded that populations of E. coli in broth cultures were limited by exhaustion of all those carbon and energy sources which could be utilized under the prevailing highly reduced conditions. No evidence of inhibitory metabolic end products was found except in broth supplemented with 1% glucose. Partial inhibition of multiplication was noted in the latter medium even when the pH was maintained between 6.9 and 7.2.

Culture Media↗

FACTORS THAT INFLUENCE TOXIGENICITY OF PSEUDOMONAS AERUGINOSA.

Liu, Pinghui V. (University of Louisville, Louisville, Ky.). Factors that influence toxigenicity of Pseudomonas aeruginosa. J. Bacteriol. 88:1421-1427. 1964.-Hemolysin, lecithinase, and protease of Pseudomonas aeruginosa were produced with cellophane-covered plates and completely synthetic agar of simple composition containing alpha-alanine, aspartic acid, glutamic acid, glucose, and salts. The most critical factor in the production of these extracellular toxins appeared to be the concentration of phosphate, which had to be in the range of 0.003% for the lecithinase, 0.005 to 0.01% for the hemolysin, and 0.01 to 0.2% for the protease. alpha-Alanine and glutamic acid favored the production of the hemolysin and lecithinase, and aspartic acid supported the production of protease. The role of glucose in the production of these extracellular toxins appeared to be more important than just an energy source. It is likely that products of anaerobic metabolism of glucose are needed in the production of these toxins. In the absence of glucose, the production of protease was enhanced by lactic acid, pyruvic acid, or alpha-ketoglutaric acid. Possible implications of these findings in the pathogenesis of P. aeruginosa are discussed.

Alanine↗

Experimental Salmonellosis VI. In Vitro Transfer of Cellular Immunity of Mouse Mononuclear Phagocytes.

Saito, Kazuko (Gunma University, Maebashi, Japan), and Susumu Mitsuhashi. Experimental salmonellosis. VI. In vitro transfer of cellular immunity of mouse mononuclear phagocytes. J. Bacteriol. 90:629-634. 1965.-A culture medium of the mononuclear phagocytes (monocytes) of mice immunized with live vaccine of Salmonella enteritidis contains the transfer agent (TA) of cellular immunity from immune to non-immune monocytes. The TA is of ribonucleic acid nature, is nondialyzable through cellophane, and maintains its active state for 3 months in a frozen state (-10 C) and for 24 hr at 37 C.

Journal Article↗

New approach to the genetics of Streptomyces coelicolor.

Sermonti, G. (Istituto Superiore di Sanità, Rome, Italy), Milena Bandiera, and Isabella Spada-Sermonti. New approach to the genetics of Streptomyces coelicolor. J. Bacteriol. 91:384-392. 1966.-Mixed cultures of complementary auxotrophic strains of Streptomyces coelicolor A3(2) were preincubated on discs of cellophane on complete medium and were then transferred onto selective media. When one strain was streptomycin-resistant and the other was streptomycin-sensitive, and the transfer medium contained streptomycin, distinct minute tufts of aerial mycelium appeared on the background growth of the mixed culture. They turned out to be heterozygous clones (heteroclones) in which the streptomycin-sensitive allele was, as a rule, missing. The pattern of marker contribution of the streptomycin-sensitive parent to the zygotes was indicative of a continuous structure carrying the hereditary material. A gradual transfer of the donor genome during conjugation was suggested by the progressive completion of the zygotes obtained by increasing preincubation time.

Genetics↗

Replication of bacteriophage T4 DNA in vitro. I. Basic properties of the system.

A new in vitro system for T4 DNA replication was developed by concentrating cell lysates on cellophane disks. The time course of [3H]dTTP incorporation into DNA by the system was separated into two phases: one was a very rapid incorporation which was terminated within 2 min (phase I reaction), and the other was a slow but continuous incorporation thereafter (phase II reaction). More than half of the phase I reaction product was Escherichia coli DNA, but the phase II reaction was mostly T4 DNA. Phase II reaction required four deoxyribonucleoside triphosphates, ATP, Mg2+, and KCl. 5-Hydroxymethyldeoxycytidine triphosphate was essential for the reaction and not substitutable by dCTP. The presence of KCN or NaN3 in the reaction mixture did not interfere with [3H]dTTP incorporation, but the addition of deoxyribonuclease completely degraded the system. Alkaline sucrose sedimentation analysis of phage II reaction product revealed that phase II reaction proceeded by the discontinuous mode of DNA replication as in vivo. After T4 infection, the activity for phase II reaction appeared in parallel with the activity of T4 phage DNA replication in vivo.

Adenosine Triphosphate↗

Effect of intravenous prostaglandin E 2 on platelet function, coagulation, and fibrinolysis.

Prostaglandin E(2) (PGE(2)) was infused intravenously to eight women for the termination of pregnancy and tests of platelet function: coagulation and fibrinolysis were studied before and during the infusion.Platelet adhesiveness, as measured by a cellophane membrane test-cell system, was significantly diminished by PGE(2), a change which was not noted by the glass-bead column technique. The administration of PGE(2) caused more rapid platelet disaggregation following ADP-induced aggregation but had no effect on the platelet count, collagen-induced aggregation, or platelet factor 3 activity. An increase in plasma antithrombin concentration and euglobulin lysis activity was also noted. These results support the concept that prostaglandin E(2) might have a role in the prevention of thrombosis.

Abortion, Induced↗

Staphylococcal protein A; its preparation and an application to rubella serology.

Good yields of staphylococcal protein A are obtained by growing the staphylococcus Cowan type 1 on cellophane agar. The activity of these preparations in removing immunoglobulin G (IgG) from human serum can be readily measured by the Mancini radial-diffusion technique and the correct in-use dilution determined. Treatment with protein A of sera from women with a history of rubella may help in the identification of those having specific antibody in the IgM and IgA fractions. This relatively simple procedure may have worthwhile application in the diagnosis of rubella.

Antibodies, Viral↗

Plasma atrial natriuretic factor and atrial wall stress in hypertensive and normotensive rabbits after pacing and volume expansion.

Atrial natriuretic factor (ANF) is present in high concentration in atria but in very low concentration in the ventricles. Under conditions of haemodynamic overload ventricular gene expression may become activated, but it is not clear if ventricular ANF can be released through a regulated or constitutive pathway. The purpose of this study was to determine whether basal and stimulated release of ANF are increased in perinephritic rabbits with mild hypertension. Six rabbits were rendered hypertensive by wrapping both kidneys in cellophane, and six sham-operated rabbits were used as controls. Eight weeks after renal wrapping, mean arterial pressure was approximately 20 mmHg higher in the experimental group. After anaesthesia, the renal-wrapped group had a higher vascular resistance. Right and left atrial wall stress was measured using sonomicrometry. Volume expansion by 30% of blood volume, using donor blood, caused a small increase in right and left atrial diastolic and systolic wall stress but did not significantly increase plasma ANF. Pacing the heart at 6 Hz caused increases in systolic but not diastolic wall stress and caused a significant increase in plasma ANF; the increase was larger after volume expansion. There were no significant differences between the responses of the experimental and control groups. It is concluded that mild hypertension, in the rabbit, does not lead to changes in atrial wall stress or either basal or stimulated release of ANF.

Anesthesia↗

Development of chronic perinephritic hypertension in dogs without volume expansion.

The theory of whole body autoregulation to explain the pathogenesis of experimental renal hypertension states that hypertension is initiated in response to an early increase in salt and water retention and a subsequent elevation of the cardiac output. This hypothesis was evaluated in the present study. Dogs (n,5) were made hypertensive by wrapping the left kidney in cellophane and removing the contralateral kidney 3 wk later. One week prior to right nephrectomy, the dogs were volume depleted by placing them on a low sodium intake (less than 3 meq of sodium/day) and giving them a mercurial diuretic for the first 3 days of the diet. This superimposed sodium depletion (negative sodium balance of 137 +/- 17 meq) increased plasma renin activity 3-5 times but did not change arterial pressure or heart rate. Within 2 days after nephrectomy, the mean arterial pressure increased from the control level of 105 +/- 1 to 135 +/- 6 mmHg (P less than 0.005) and pressure remained elevated throughout an additional 4-wk period in which volume depletion was enforced. The present study suggests, therefore, that initial blood volume expansion with such possible consequences as elevated cardiac output are not essential to the pathogenesis of experimental renal hypertension.

Animals↗

Gas flux through human skin: effect of temperature, stripping, and inspired tension.

The flux of He and O2 through intact adult human skin was measured at various inspired concentrations and skin temperatures. The skin surface was then stripped with cellophane tape to alter the diffusional conductance of the stratum corneum. He flux for stripped skin was used to estimate skin perfusion as a function of local temperature, and diffusional conductance for O2 was estimated from O2 flux and perfusion. The flux of He or O2 at constant skin temperature can be related to inspired concentration by a simple linear model. Increasing surface temperature in the range 33-43 degrees C produced a much larger increase in O2 flux than in He flux for intact skin. Skin stripping greatly increased skin O2 flux. Estimated skin conductance for O2 showed a more marked temperature dependence than estimated skin perfusion. The results suggest that raising skin temperature in the range 38-43 degrees C has only a modest effect on skin perfusion and that stratum corneum conductance may have a major role in the large increase of O2 flux with temperature.

Biological Transport↗

Microdialysis probe for transcutaneous monitoring of ethanol and glucose in humans.

A heated (42 degrees C) microdialysis probe and its application for continuous transcutaneous sampling of ethanol and glucose through cellophane-stripped forearm skin are described. Ethanol and glucose concentration in the dialysate were measured on-line with continuous-flow analysis and compared with blood values in human volunteers after ethanol consumption (n = 4) and oral glucose testing (n = 5), respectively. For ethanol and glucose, the dialysate and blood concentrations were linearly related in each subject (r > or = 0.91, P < 0.005), although the dialysate-to-blood ratio varied among subjects. The recovery in vivo was 22.4 +/- 22.7 and 4.7 +/- 2.3% (SD) of the recovery in vitro for ethanol and glucose, respectively. The dialysate glucose concentration was independent of blood flow. When the probe temperature was increased from 32 to 42 degrees C, the dialysate-to-blood glucose ratio increased, with 2.4 +/- 1.4%/degrees C (SD) in fasting subjects (n = 4), which was similar to an increase of 2.1 +/- 0.045%/degree C in dialysate-to-medium ratio in vitro. The present approach for transcutaneous sampling may possibly be used for other substances of low molecular weight as well.

Adult↗

Gordon Murray: heparin, hemodialysis and hubris.

Gordon Murray (1894-1976), a brilliant and innovative surgeon who spent the majority of his professional career at the University of Toronto, Ont., Canada, is properly credited with having performed the first successful hemodialyses in humans in North America. Neither he nor Kolff, working in the Netherlands, were aware of each other's work during the middle 1940s when wartime hampered communication. Murray's extensive investigations and experience in the use of heparin in vascular surgery laid the groundwork for the use of this anticoagulant with the artificial kidney. He first designed a coil dialyzer in which cellophane tubing was wound about a steel frame. His second-generation apparatus was a plate dialyzer. In all, he performed dialysis on 11 patients with presumed acute renal failure, 50% of whom survived. Those who died succumbed to sepsis or irreversible chronic renal failure. Not much has changed in 50 years.

Canada↗

Action potential generation in reaggregates of rat aortic smooth muscle cells in primary culture.

Cultured vascular smooth muscle cells were prepared from adult rat aortas. The cells were dispersed by collagenase/elastase, and allowed to reaggregate into small spheres (50-200 micron in diameter) by plating on to cellophane. These primary cultures were incubated for 5-14 days, and then impaled with microelectrodes. The mean resting potential was -55 mV, and the mean input resistance was 9.0 M omega. The cells were quiescent, electrically and mechanically, and electrical stimulation usually did not elicit responses. However, addition of Ba++ (1 mM) or tetraehtylammonium ion (TEA; 5-15 mM) induced excitability (with accompanying contractions), either as spontaneous action potentials or by allowing responses to electrical stimulation. The cells became partially depolarized (e.g., to -36 mV) by these agents, and the input resistance increased. The frequency of spontaneous firing of the Ba++-induced spikes was affected by polarizing current pulses, as expected for pacemaker behavior. Elevation of Ca++ in the bathing solution increased the amplitude (overshoot) of the action potentials, and the spikes were blocked by verapamil (10(-5) M). Electron microscopy showed that the reaggregates consisted of a tight packing of elongated small-diameter cells, some of which exhibited thick and thin myofilaments and 'dense bodies'; many cells possessed surface caveolae. The results of this study demonstrate that reaggregates of arterial smooth muscle in primary culture can maintain functional and morphological characteristics of intact arterial smooth muscle, and therefore provide a useful preparation for the study of vascular smooth muscle function and control.

Action Potentials↗

Serum beta2-microglobulin in hemodialyzed patients.

beta2-Microglobulin (beta2-M) concentrations were measured by radioimmunoassay in 63 patients treated with maintenance hemodialysis. Serum levels ranged from 12.5 to 92 microgram/ml and were found stable upon sequential measurements over a 6-month period. Excretion of beta2-M was achieved through urinary loss (up to 150 mg/day) and to a lesser extent by ultrafiltration across acrylopolynitrile but not cellophane dialysis membranes. Proximal tubular cells being the major site of beta2-M catabolism, stable levels in anephric patients indicated a defective production of beta2-M in uremic patients. Serum beta2-M levels were not correlated with predialysis urea, creatinine, hematocrit or middle molecule material. In vitro experiments do not support a possible toxicity of purified beta2-M but do not rule out the contribution of other low molecular weight proteins to uremic toxicity.

Adolescent↗

In vivo studies of dialysis-related endotoxemia and bacteremia.

The transfer of bacteria and endotoxin and the development of fever was monitored during hemodialysis of dogs. Bacterial and endotoxin levels in dialysis fluids exceeded those reportedly associated with pyrogenic reactions, and bacteria-free dialysate filtrates administered intravenously were shown to be pyrogenic in 2 of 2 dogs. During 18 hollow-fiber dialyses, neither bacteria nor endotoxin, as measured in the Limulus lysate assay (LLA), was detected in venous and arterial blood samples. Body temperatures did not increase during or within 1/2 h after dialysis. In experiments with parallel dialyzers, contaminated dialysate was simultaneously ultrafiltered into the second blood compartment. Ultrafiltrates were unreactive in the LLA and the absence of pyrogens was confirmed with the pyrogen test, USP, in five of five experiments. This investigation suggests that the intact cellophane membrane of the hollow-fiber dialyzer is an effective barrier to endotoxin and bacteria.

Animals↗

Plasma fibronectin levels in patients with chronic uremia.

Plasma fibronectin (FN) concentration was measured in patients with idiopathic glomerulonephritis (GN) with or without impaired renal function, in uremic patients undergoing periodic hemodialysis and in renal transplant patients before and after an acute rejection crisis. Results show normal FN levels in idiopathic GN and in renal transplant patients with normal renal function, while significantly lower levels were found in GN with severe renal damage, in uremia before and after dialysis, and in renal transplant patients during acute and chronic graft rejection. Significant correlations between high serum creatinine values and low plasma FN levels were found in renal transplant patients. These findings suggest that the kidney may influence FN levels in the blood since acute (rejection crisis) and chronic renal failure (uremia) cause low concentrations of this protein, while levels tend to return to normal values in patients with uremia after renal transplantation. We hypothesize that the normal kidney removes or perhaps degrades some substances or hormones that may control the release or synthesis of FN. These substances are not dialyzed by cellophane membranes since low plasma FN levels persist after periodic hemodialysis. Only the renal graft provokes an increase of FN in the blood stream.

Adult↗

Mebendazole (R 17635) in enterobiasis. A clinical trial in mental retardates.

The efficacy of mebendazole against enterobiasis was tested on 150 institutionalized mental retardates (range 5-25 years). At the start of the trial all patients swallowed on tablet containing 100 mg mebendazole. Another tablet was administered to all patients 2 weeks and 1, 2 and 3 months later. 19 patients were further treated with one tablet 4 and 5 months after the start of the trial. The presence of E. vermicularis was checked with cellophane adhesive tapes from the anal skin and from the right-hand finger-tips before the trial and after 1, 3 and 6 months. 94 subjects were positive before the trial. At the first post-treatment examination, all patients were found negative. However, 12% were reinfected. No side effects were observed or reported.

Adolescent↗

Bactericidal and antineoplastic effect of combination of gentamicin and adriamycin.

Gentamicin and adriamycin were tested against eight strains of Proteus mirabilis: using the cellophane transfer technique, combinations of these antibiotics were screened for bactericidal activity. On mice bearing Ehrlich ascites carcinoma, the antitumoral effect of a combination of gentamicin and adriamycin was determined. Antagonism occurred with the combination in bactericidal activity and not in antitumoral activity.

Animals↗