Streptococcal protein G-gold complex: comparison with staphylococcal protein A-gold complex for spot blotting and immunolabeling

Protein G, a cell wall protein isolated from human group G streptococci strain G148, binds in a similar manner as protein A from Staphylococcus aureus to the Fc portion of IgG molecules. Indeed, protein G has been proposed as a superior Fc binding protein due to its broader species reactivity. Thus, we have prepared a complex of protein G with particles of colloidal gold and determined its applicability for spot-blot analysis and postembedding immunolabeling by comparing it with protein A-gold complex. By spot-blot analysis no difference in binding of protein G-gold or protein A-gold to IgG molecules from a whole spectrum of animal species was observed. Moreover, using rabbit, sheep, or goat anti-rat albumin antibodies to detect nitrocellulose-immobilized rat albumin or antigenic sites in paraffin and Lowicryl K4M thin sections from rat liver, no difference was found with protein G-gold or protein A-gold. Similarly, no difference in binding to protein G-gold or protein A-gold was observed with a battery of monoclonal antibodies. However, in contrast to expectations, protein A-gold reacted well with both sheep and goat IgG molecules; indeed, for the light and electron microscopic localization of albumin with sheep or goat antibodies it was as efficient as protein G-gold. These results demonstrate, therefore, that both protein G-gold and protein A-gold are useful second step reagents for immunolabeling and that protein G-gold was not a superior probe in the systems tested.     

Determination of “active” cytochrome P-450 from relaxation kinetics of product formation

We estimate the active part of cytochrome P-450, which is involved in a special substrate transformation, by measuring the initial change of the production rate as a function of the relaxation transitions between two different steady states of the reaction cycle of cytochrome P-450 using the light-reversibility of the carbon monoxide inhibition. The kinetic data of such relaxations are interpreted within a model cycle, which reduces the reaction cycle to three steps. The estimation of the rate constant of the first reduction step, derived from model simulation of the production rate, is confirmed by independent experimental study of the reduction kinetics.An application of our model to the O-deethylation of 7-ethoxycoumarin reveals that — in a time average — 10%–15% of the spectroscopically detectable cytochrome P-450 is involved in that transformation.Abbreviations Cyt. P-450 microsomal cytochrome P-450 - 7-EC 7-ethoxycoumarin     

Effects of cultural conditions on the production of phenylalanine from a plasmid-harboring E. coli strain

Summary Phenylalanine production from E. coli KA 197/pJN6 (plasmid harboring genes for aro F, phe A^FBR, Amp^R and Tc^R) was studied under varying nutritional conditions in batch and continuous cultures. In batch culture experiments where growth was deliberately interrupted by limiting concentrations of sulphate and phosphate the phenylalanine production continued from the non-growing cells. However, the depletion of phosphate resulted in an immediate cessation of phenylalanine production but thereafter a low specific rate of phenylalanine formation resumed, while the decrease in specific rate of product formation was less after sulphate depletion. In the chemostat experiments, however, phosphate limitation was the only case where the specific rate of phenylalanine formation remained constant, while at the corresponding time in sulphate and glucose limited chemostats it was declining respectively had ceased.     

Effects of cultivation techniques and media on yields and morphology of the basidiomycete Armillaria mellea

Summary The yield of cell mass and the morphology of Armillaria mellea, strain ATCC 11114, was studied using a variety of cultivation methods: solid media, standing liquid culture, shake flasks, tower reactors and impeller-stirred reactors. Two different media, malt extract broth and a glucose/asparagine/peptone-medium, and the corresponding agar media, were used. Yields were higher in the malt extract media than in the glucose media. Generally the highest yields were obtained on solid media while agitated cultures gave the lowest yields. Morphological characteristics such as pellet formation, adhesion to surfaces and pigment production were significantly affected by culture conditions.     

The population dynamics of Cheilosia fasciata (Diptera,Syrphidae): significance of environmental factors and behavioural adaptations in a phytophagous insect

Summary A population of Cheilosia fasciata Schin. & Egg., 1853 was studied in a beech forest (Melico-Fagetum allietosum) near Göttingen (FRG). This syrphid species is closely associated with ramson (Allium ursinum L.), as the larvae mine the plant's leaves. Adult abundance was determined using emergence traps (1981–1986); egg and larval abundance was determined by examining ramson leaves: puparia were extracted from soil samples (1984 and 1986). Cheilosia fasciata is a univoltine species; the imagines emerge in late April/early May. Larval development took five (1986) to six (1984) weeks. In late June/early July the larvae enter the soil for pupariation. A linear relationship was found between the area of the mines and larval biomass (dry wt.). It was concluded that one larva had to mine 51.90 cm² of leaf area in order to gain the mean maximum dry weight (11.15 mg). Furthermore, it was shown that ovipositing females, laying just one egg per leaf, tended to select large leaves providing this minimum amount of food. It is hypothesized that females probably follow straight-lined routes on their oviposition flights, rather than ovipositing on leaves chosen at random. By applying such a strategy, females can almost completely avoid competition for food among their own offspring. Furthermore, competition among the progeny of the whole population is also reduced, particularly in years when adult abundance is very low. The significance of certain factors influencing the population dynamics of Cheilosia fasciata was evaluated by applying a rank correlation analysis. It was shown that high precipitation rates in April reduced the time available for mating and oviposition resulting in low emergence abundance in the next spring. Larval survivorship was enhanced by high precipitation rates in May and June, indicating that drought may be unfavourable to larvae and reduce food availability. Predation by a parasitoid Phygadeuon ursini Horstmann, 1986 (Hymenoptera, Ichneumonidae), appears to be just an imperfect (Milne 1984) density-dependent control mechanism; in fact, it was shown that food limitation is the only significant mechanism of density-dependent population control.     

Natural and Chernobyl-caused radioactivity in mushrooms,mosses and soil-samples of defined biotops in SW Bavaria

Summary Different mushrooms, mosses and corresponding soil samples have been collected mainly from two sites in the alpine region of southwestern Bavaria. At the end of the growthseason, September 1986, gamma spectroscopic analysis showed that the moss-, mould, and needle-layer contained considerably more ¹³⁴Cs and ¹³⁷Cs activity per unit fresh weight than eight different species of mushroom. These two isotopes were carried into the biotop mainly as a consequence of the Chernobyl accident. ¹³¹J could not be found any more in the samples ca. 5–6 months after the catastrophe. The activity of the cesium isotopes decreased with increasing soil depth. In the mushrooms the activity was relatively high in Xerocomus badius and surprisingly low in Boletus edulis; samples of the latter and of Cantharellus cibarius collected in September 1985 (before the accident) and kept deep frozen contained almost identical amounts of ¹³⁷Cs as those collected from August to October 1986. Mushrooms contained considerably more of the natural isotope ⁴⁰K than the needlelayers and the soil samples in the neighbourhood. In all mushrooms except Xerocomus badius the activity of ⁴⁰K was generally higher than the ¹³⁷Cs activity. The results indicate that except Xerocomus badius the analyzed mushrooms do not actively take up Cs from the soil, in contrast to K.     

Numerical responses by populations of red fox and mountain hare during an outbreak of sarcoptic mange

Summary During a severe outbreak of sarcoptic mange (Sarcoptes scabiei vulpes) starting among red foxes (Vulpes vulpes) in Sweden in the 1970s, we studied: 1) the establishment and spread of the disease in northernmost Sweden (by inquiries), and 2) the 1970–84 bag records for foxes and mountain hares (Lepus timidus) (an alternative prey to the fox's main prey, voles). Since the first case of sarcoptic mange in 1975 the disease spread rapidly, with >50% of the hunting organizations having reported the disease in 1981 and >75% in 1983. Also the disease became more abundant within the areas affected. In areas with a low mange infection rate (index) the number of foxes killed in the 1980s did not deviate markedly from the average level in the 1970s. However, there was a slight tendency towards a decline in areas with a medium index and numbers declined markedly where the index was high. Hare harvests initially were low (after a tularemia epidemic) in the 1970s. In that decade harvests increased dramatically and stabilized, increased gradually or changed little, respectively, where mange infection rates were low, medium or high in the early 1980s. In areas with a low mange index hare harvests remained cyclical and at the same level in the 1980s as in most of the 1970s. However, in areas with a medium index harvests increased and seemed to begin to lose their cyclicity, and where the index was high the low and relatively stable hare harvests increased annually. A predator-prey hypothesis, assuming predators to synchronize alternative prey declines to those of the cyclic main prey, predicts that a predator reduction would cause a gradual disappearance of the cyclicity and increasing numbers among alternative prey. Our hare data are partially consistent with this prediction.     

Carbohydrate formation in rewetted terrestrial cyanobacteria

Summary In the terrestrial cyanobacterium Nostoc commune Vauch. formation of carbohydrate polymers was measured upon rewetting the mats in a light-dark regime. To discriminate between carbohydrates of different physiological function, total carbohydrate was determined as anthrone-reactive material (ARM) and storage carbohydrate (glycogen) assayed by an enzymic test. In the dry thalli glycogen was found to represent less than one tenth of the ARM. After rewetting an increase of total carbohydrate was observed in illuminated samples. Only glycogen, however, showed a regular pattern of synthesis and degradation during a 12:12 h light-dark cycle. This indicates that most carbohydrates detected by anthrone belong to the metabolically inert sheath material.When illuminated colonies were kept submerged after rewetting glycogen was hydrolyzed indicative of being used in the rapid recovery of cellular functions as observed in rewetted colonies. Apparently, photosynthesis allowed for net glycogen synthesis only, provided the mats were sufficiently aerated. These findings give evidence that the (carbohydrate) sheath plays an important role in water retention in an organism bound to a terrestrial habitat.     

Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield of O2 evolution in leaves of higher plants

High-light treatments (1750–2000 mol photons m^–2 · s^–1) of leaves from a number of higher-plant species invariably resulted in quenching of the maximum 77K chlorophyll fluorescence at both 692 and 734 nm (F _M, 692 and F _M, 734). The response of instantaneous fluorescence at 692 nm (F _O, 692) was complex. In leaves of some species F _O, 692 increased dramatically in others it was quenched, and in others yet it showed no marked, consistent change. Regardless of the response of F _O, 692 an apparently linear relationship was obtained between the ratio of variable to maximum fluorescence (F _V/F _M, 692) and the photon yield of O₂ evolution, indicating that photoinhibition affects these two variables to approximately the same extent. Treatment of leaves in a CO₂–free gas stream containing 2% O₂ and 98% N₂ under weak light (100 mol · m^–2 · s^–1) resulted in a general and fully reversible quenching of 77K fluorescence at 692 and 734 nm. In this case both F _O, 692 and F _M, 692 were invariably quenched, indicating that the quenching was caused by an increased non-radiative energy dissipation in the pigment bed. We propose that high-light treatments can have at least two different, concurrent effects on 77K fluorescence in leaves. One results from damage to the photosystem II (PSII) reaction-center complex and leads to a rise in F _O, 692; the other results from an increased non-radiative energy dissipation and leads to quenching of both F _O, 692 and F _M, 692 This general quenching had a much longer relaxation time than reported for pH-dependent quenching in algae and chloroplasts. Sun leaves, whose F _V/F _M, 692 ratios were little affected by high-light exposure in normal air, suffered pronounced photoinhibition when the exposure was made under conditions that prevent photosynthetic gas exchange (2% O₂, 0% CO₂). However, they were still less susceptible than shade leaves, indicating that the higher capacity for energy dissipation via photosynthesis is not the only cause of their lower susceptibility. The rate constant for recovery from photoinhibition was much higher in mature sun leaves than in mature shade leaves, indicating that differences in the capacity for continuous repair may in part account for the difference in their susceptibility to photoinhibition.Abbreviations and symbols kDa kilodalton - LHC-II light-harvesting chlorophyll-protein complex - PFD photon flux density (photon fluence rate) - PSI, PSII photosystem I, II - F _O, F _M, F _V instantaneous, maximum, variable fluorescence emission - absorptance - _a photon yield of O₂ evolution (absorbed light) C.I.W.-D.P.B. Publication No. 925     

Characteristics of aSerratia plymuthica isolate from plant rhizospheres

An isolate (G15) of a bacterium, frequently isolated from roots of various plant species, was identified asSerratia plymuthica. At low temperature (viz. 2–8°C), the studied isolated readily produced a red pigment which proved useful in recognizing the bacteria on reisolation. In laboratory tests it exhibited strong antagonism againstBotrytis cinerea andGerlachia nivalis and moderate antagonism againstRhizoctonia solani, Fusarium culmorum andPythium sp. The bacterium significantly increased growth of lettuce plants when applied to the roots under non-sterile conditions in greenhouse tests. Various strains ofSerratia plymuthica are supposed to be common as rhizosphere bacteria under Swedish conditions.