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Volume 48,
Issue 3,
1967
Volume 48, Issue 3, 1967
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Purification and Characterization of the Lipase of Pseudomonas fragi
More LessSUMMARY: The lipase of Pseudomonas fragi (nrrl b-25) was purified 75-to 100-fold with an overall recovery of ca. 20%. The enzyme was found to exist in heavy and light forms exhibiting the same position specificity for triglycerides, Michaelis constants (Km), and apparent pH and temperature optima. The light form of the enzyme appeared to be present in the heavy one, probably in a complexed state. The purified lipase was found to hydrolyze only glycerol esters of fatty acids; it required a water-fat interface and exhibited a 1,3-position specificity for triglycerides. The optimum pH for the purified enzyme with purified tributyrin as substrate was calculated to be 8.6 to 8.7 from initial velocity measurements with a pH-stat at 25°. The Km for the purified lipase with tributyrin was found to be 0.9 x 10−3 m at 25°and pH 7.2. Exposure of the purified enzyme to 40°for 10 min. caused a complete loss of activity. A 50% loss of activity-occurred after 10 min. exposure to about 35°. When exposed to pH values ranging from 5.3 to 9.5 for 1 hr at 2°and then assayed at pH 7.0 and 35°, the purified enzyme was found to be stable in the pH range 6.6-7.8.
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Long-distance Spore Transport: Methods of Measurement, Vertical Spore Profiles and the Detection of Immigrant Spores
More LessSUMMARY: Airborne spores can be carried long distances, but little is known about the atmospheric transport processes involved or the rates at which spore clouds are depleted. Aircraft sampling is expensive and inevitably intermittent, and surface traps reveal only some of the processes involved. The best compromise is to combine surface and aircraft observations and to support both with detailed meteorological interpretation. Gravity slide traps exposed for 1 day indicate the arrival of spores less precisely than moving-slide impactors, which therefore provide a more accurate starting time for estimating the past track of spores from air trajectories. Catches of Puccinia graminis uredospores from continental European sources illustrated how immigration depends on the movement of atmospheric pressure systems and the gradients within them and suggested that in addition to surface air movement winds at the 700 and 500 mb. levels were important. Aircraft of the Meteorological Research Flight, using suction impactors which operated approximately isokinetically, sampled air in the lower troposphere, both to ascertain vertical spore profiles over land and to intercept immigrant Puccinia graminis uredospores over the English Channel. The vertical distribution of spores seemed to be determined in the same way as that of other aerosol particles; atmospheric turbulence was a major factor and there were indications that wind shear, precipitation and surface deposition might be important. However, most spores are liberated periodically and so encounter different degrees of atmospheric turbulence depending on the diurnal periodicity of their concentration near the ground. Concentrations of 104 spores/m.3 occurred at heights up to 1000 m. and hundreds/m.3 at 3000 m. In unstable air spore concentrations often declined roughly logarithmically with height, but layers of stable air were often associated with abrupt changes of concentration. Details of vertical spore profiles also depended on the history of both the temperature profile and the spore cloud. Such factors tended to affect all spore types similarly: but occasionally some components, e.g. P. graminis uredospores, showed unique vertical profiles. One such profile, characterized by preferential ‘erosion’ of the spore cloud from air near the surface, may indicate travel remote from sources. Spores of plant pathogenic fungi were frequent in samples of air moving northward over the English Channel but their viability was not tested.
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Long-distance Spore Transport: Vertical Sections of Spore Clouds over the Sea
More LessSUMMARY: To measure the rate at which spore clouds were depleted over the sea, spores were collected with isokinetic suction impactors mounted in aircraft of the Meteorological Research Flight, Farnborough. Remote from sources able to replenish spore clouds, preferential deposition from the lower layers caused ‘erosion’ of the base of vertical profiles of spore concentration leaving maxima at heights between 500 and 1500 m. To determine vertical spore distributions throughout the largest possible distance downwind of the English coast, a saw-tooth flight plan of alternating ascent and descent was used. Of four flights, two encountered favourable weather, rain interfered with sampling on one and unexpected winds across the track converted another into an unintended but interesting cross-wind section of spore distribution. Pollens and Cladosporium spores were counted as examples of large and small spores liberated typically by day, and a composite group of spores liberated in damp air was chosen as an indicator of spores liberated mostly at night. In two flights in fine weather maximum spore concentrations occurred hundreds of miles off-shore. Diagrams showing height, distance from the coast and lines of equal spore concentration (‘isospores’) demonstrated discrete clouds of each marker spore type. Pollen and Cladosporium clouds were centred at approximately the same distances from the coast but with the pollen about 500 m. lower, probably because the pollen grains sedimented faster. Maximum concentrations of the damp-air group sometimes coincided and sometimes alternated with the day-liberated groups. Known periodicities of these spores over land, surface air trajectories and previous weather, suggested that the spore clouds which the aircraft overtook over the North Sea, were the residue of those produced from the British Isles on previous days or nights. Interpretation of the results was limited by meteorological uncertainties, the geographical complexity of probable source areas, and perhaps most by changes in the number of spores crossing the coast at different times of day, which prevented accurate measurement of rates of spore deposition. Spores of many species were recognized over the North Sea. The plant pathogens included established distant migrants such as uredospores of Puccinia graminis, which apparently originated east of the Baltic. The viability of the spores was not tested, but it seems safe to assume that distant transport is both frequent and extensive and probably important in temperate latitudes in summer.
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The Action of Light on Nitrate and Nitrite Assimilation by the Marine Chlorophyte, Dunaliella tertiolecta (Butcher)
More LessSUMMARY: Light caused up to a 20-fold increase in the rate of nitrate and nitrite assimilation by the marine chlorophyte Dunaliella tertiolecta. While higher rates of oxygen evolution were observed during both nitrate and nitrite assimilation, the extra oxygen released was not related to the amounts of nitrate or nitrite assimilated. Carbon dioxide was required for light to increase nitrate or nitrite assimilation; the addition of glucose did not overcome this requirement. The light intensities at which nitrate and nitrite assimilation reached their maximum rates were 300 and 1000 ft. candles respectively, while oxygen evolution and carbon fixation required 1800 ft. candles. In the presence of 3-(4-chlorophenyl)-1,1-dimethylurea (10−4 m), nitrate and nitrite assimilation were still increased by 30% in light, although oxygen evolution decreased to 2% of the control rate. Assimilation of nitrate and nitrite in light was decreased to 50% by sodium azide or potassium cyanide at 1-5 x 10−5 m; oxygen evolution was less sensitive, requiring more than 3 x 10−4 m to decrease the evolution to 50%. Azide and cyanide both blocked dark nitrate and nitrite assimilation but at slightly higher concentrations than those required in the light. Iodoacetate (10−3 m) decreased dark nitrate and nitrite assimilation to 2% and 38% of control values, respectively; but, in the light, nitrate assimilation was not affected and nitrite assimilation was decreased by only 35% at this concentration. It is concluded that the action of light in stimulating nitrate and nitrite assimilation cannot be explained on the basis of a single site of action as has been proposed for spinach chloroplasts and that two relatively independent sites exist in the living D. tertiolecta cell.
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Effects of Organic Cations on the Gram-negative Cell Wall and Their Bactericidal Activity with Ethylenediaminetetra-acetate and Surface Active Agents
More LessSUMMARY: When Gram-negative cell walls were damaged by lysozyme and ethylene-diaminetetra-acetic acid (EDTA) in tris buffer, tris played an active role through its action as an organic cation. Other organic cations such as aliphatic amines and quaternary ammonium compounds were more effective than tris in modifying the cell wall and making it permeable to other solutes. The most effective organic cations had one C12 to C16 alkyl chain. At pH10, EDTA 100/°mUg./ml and N,N-dimethyldodecylamine (DDA) 2.5 °mUg./ml. modified the cell wall of Escherichia coli so as to permit a more than 99.99% kill by 20 °mU/g./ml. of a zwitterionic surface active agent, 2-hydroxy-3-(dimethylhexadecylammonio) propane-1-sulphonate (HAPS), in 10 min. at 37°dG. At these concentrations, the individual compounds were bactericidally ineffective. Four other Gram-negative species were shown to be similarly susceptible to killing by this system. Together, EDTA and DDA appeared to remove surface components of the Gram-negative cell wall. Alone, DDA and other organic cations removed somatic antigens from the cell wall.
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The Production and Characterization of Lipases from a Micrococcus and a Pseudomonad
More LessSUMMARY: Extracellular lipase production was a constitutive property of the micrococcus and pseudomonad studied but was considerably influenced by nutritional and physical conditions. The lipase in culture supernatant fluids of the micrococcus was markedly heat resistant but became increasingly more ther-molabile with the degree of purification obtained. The hydrolytic activity of partially purified extracellular lipase preparations from each organism was due to a single protein which was identical with a hydrolytic enzyme also found in cell-free extracts of each organism. The lipases from both organisms had general specificity towards ester linkages although the lipase from the micrococcus was markedly more active towards esters containing short chain fatty acids and comparatively less active towards trigylcerides containing long chain acids than was the pseudomonal lipase. The activity of both lipases showed an optimum for all substrates at pH 8.0-8.5 and did not decrease at higher pH values, indicating the involvement of an acidic group in the enzyme/substrate binding. The results of inhibition studies were consistent with the view that both lipases possess a serine-imidazole active centre and are therefore similar to esterolytic enzymes in mammalian systems.
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The Preparation and Biochemical Properties of Mitochondria from Neurospora crassa
More LessSUMMARY: Mitochondria were prepared in large quantities from Neurospora crassa by grinding hyphae with glass beads in an Eppenbach Micro-mill. Observation in the electron microscope indicated that some of the isolated mitochondria were probably damaged during preparation. Nevertheless, the crude mitochondrial fraction was capable of coupling phosphorylation to the oxidation of seven different substrates tested. The P: O ratio obtained with succinate ranged from 0.7 to 1.3 in different experiments with different mitochondrial preparations, but only low respiratory control ratios were observed. The effects of common inhibitors of oxidative phosphorylation were similar to those reported with mammalian mitochondria. In addition, ATPase and ATP-Pi exchange activities, as well as ion accumulation, were measured in the Neurospora mitochondria. The use of a ‘preparation’ medium containing sucrose, 0.25 m; EDTA, 0.005 m (pH 7.0); bovine serum albumin (0.15%, w/v) was necessary to obtain these results.
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Leuconostoc oenos sp.nov.
More LessSUMMARY: Nineteen strains of Gram-positive cocci isolated from wine and belonging to the genus Leuconostoc were examined and are considered to form a new species which is named Leuconostoc oenos. These strains differ from other leuconostocs because they grow better in a medium containing tomato juice than in yeast glucose citrate broth normally used in this laboratory for the genus. Furthermore, they grow well in media in which the pH value is too acid for other Leuconostoc species to initiate growth. The cultures of L. oenos do not form dextran from sucrose. They form acid from fructose, glucose, trehalose and aesculin, generally from melibiose and salicin. They may form acid from arabinose, xylose, galactose, mannose and cellobiose, but not from lactose, maltose, sucrose, raffinose, dextrin, glycerol, mannitol or sorbitol.
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The Growth Factor and Amino Acid Requirements of Species of the Genus Leuconostoc, including Leuconostoc paramesenteroides (sp.nov.) and Leuconostoc oenos
More LessSUMMARY: The vitamin requirements of 63 strains and the amino acid requirements of 40 strains belonging to the genus Leuconostoc have been determined. The amino acid requirements separate the genus into dextran and non-dextran formers and in particular indicated that non-dextran formers should be excluded from Leuconostoc mesenteroides. The growth factor requirements support the division of the leuconostocs by other methods into six species: Leuconostoc mesenteroides, L. dextranicum, L. paramesenteroides, L. lactis, L. cremoris and L. oenos.
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The Action of Cephaloridine with Cloxacillin or Methicillin against °bT-Lactamase-Producing Gram-negative Bacteria
More LessSUMMARY: Cephaloridine has a broad spectrum of antibacterial activity, but certain Gram-negative organisms (e.g. Aerobacter aerogenes, Proteus morgani) are resistant by reason of their ability to destroy the antibiotic. Methicillin and cloxacillin do not inhibit Gram-negative organisms but are resistant to the βbT-lactamases which some strains produce and can protect cephaloridine from destruction by such enzymes. This protection effect towards cephaloridine has been demonstrated in vitro and on experimentally infected mice. In vitro, a relatively small proportion of the penicillin exerted a maximal protecting effect towards cephalosporin and enhanced its bactericidal action against the Gram-negative organisms which produce °bT-lactamase. The clinical use of a combination of cloxacillin or methicillin with cephaloridine for the treatment of intractable systemic infections with °bT-lactamase-producing Gram-negative organisms is suggested, even though laboratory tests may show the organisms to be resistant to both cephaloridine and the penicillins when they are tested separately.
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