Undiluted Extracts Research Articles

Interferences, limitations and an improvement in the extraction and assessment of cellulase activity in soil

A practical modification of areducing-sugar method to estimate soil (carboxymethyl) cellulase activity is described and used to compare three non-sterile soils. The original method involves the formation of a ferric-ferrocyanide complex in soil extracts prepared and exposed to substrate in 2 M acetate buffer. Because high acetate concentrations compromise this reaction, such extracts require considerable (e.g. 20–30-fold) dilution prior to activity determinations. For soils with low organic matter, such as one used in this study, undetectable activity often results. We used 67 mM acetate buffer and were able to detect and compare cellulase activities in three diverse soils using undiluted extracts. The values obtained were more reproducible, but in a different range from those of the original method. Our modification yielded activities that appeared to be unchanged over the extraction buffer concentration range of 11–140 mM acetate. The use of undiluted soil extracts prepared in less concentrated buffer simplifies and extends the practicality of the assay by: (a) reducing the importance of using matrix-matched standard curves; (b) requiring less sample manipulation and glassware, and (c) preventing the dilution to extinction of enzyme activity. It also improves the usefulness of the method as an indicator of ecological effects (biomass turnover) due to the introduction of nonindigenous microorganisms and chemicals of environmental concern. These factors make the method more competitive with other reducing-sugar assays used to measure cellulase activity in soil.

Fluorescence spectroscopy of aqueous pine litter extracts: effects of humification and aluminium complexation

SUMMARYFluorescence excitation, emission, and synchronous‐scan spectra were obtained for aqueous extracts of needle and litter layer (O‐horizon) samples from ponderosa pine collected at a plantation site. The spectral lineshapes differed markedly between the needle and litter samples, and showed an increasing overall intensity with increasing extent of humification (increasing depth in the litter layer). At a dissolved organic C (DOC) concentration of 100 g m−3, these effects were accompanied by a general shift in spectral density from lower to higher wavelength such that, in the excitation spectra, there was increasing prominence of a peak at 390 nm. When the DOC concentration was decreased from 100 to 25 g m−3, the overall spectral intensity decreased and the peak at 390 nm in the excitation spectra of the litter samples gave way to a rising peak at 340 nm. Changes in pH from 4 to 5, characteristic of the undiluted litter extracts, produced little effect on the spectra. The addition of A1 at 40 mmol m−3 generally produced enhancement of the fluorescence intensity in all three kinds of spectra for the needle and upper litter‐layer extracts at 100 g C m−3 and pH 4. Spectral density below 320 nm in the excitation and synchronous‐scan spectra of the needle solution (possibly attributable to gentisic acid on the basis of model experiments) was, however, diminished by Al addition. For the lower litter‐layer extracts, A1 addition decreased the fluorescence intensity in excitation and emission spectra, whereas it increased the intensity in synchronous‐scan spectra. These trends indicated that the water‐soluble fluorophores in the litter layer differed significantly from those in the fresh needles, and changed with the extent of humification. The 390‐nm peak in the excitation spectrum, particularly its behaviour in the presence of added Al, may be useful as a spectral signature of products formed by litter humification processes.

Determination of coplanar polychlorinated biphenyl (CB) congeners in seal tissues by chromatography on active carbon, dual-column high resolution GC/ECD and high resolution GC/high resolution MS

A method for measurement of toxic coplanar chlorinated biphenyl (CB) congeners in seal tissues is described. Chromatography on Carbopack C efficiently separates the non-ortho substituted coplanar CB congeners CB-37/81/77/126 and CB-169 from the other (mono- and di-ortho substituted) coplanar CB congeners. In total 31 specific CB congeners are determined by this analytical method. Samples are fractionated, and analyzed by either high resolution dual-column electron capture gas chromatography (HR-GC/ECD) or high resolution gas chromatography/high resolution mass spectrometry (HR-GC/HR-MS) with isotope dilution technique. The method is validated through standard addition experiments, and by use of recovery surrogate standards. The recovery is (85 ± 16%; N=78) for the ECD analyses. Corresponding figures for the isotope dilution GC/MS analyses are (79 ± 14 (CB-77); 80 ± 15 (CB-126); 83 ± 17 (CB-169); N=63). Detection limits were 0,1 ng/g for the HR-GC/ECD analysis (undiluted extracts), and 0,005 ng/g by the isotope dilution GC/MS analysis.

Allelopathic potential ofnuphar lutea (L.) Sibth. & Sm. (Nymphaeaceae)

Aqueous extracts ofNuphar lutea (L.) Sibth. & Sm. leaves (blades plus petioles) and roots plus rhizomes were tested for allelopathic activity using lettuce seedling andLemna minor L. assay systems. The 12.5, 25, 125, and 250 parts per thousand (ppt) treatments of both extracts killed the lettuce seedlings. At 2.5 ppt of extract, radicle growth of lettuce was 29% of the control for leaves and 31% of the control for roots plus rhizomes.Lemna minor frond number was reduced to 34% of the control by the 25 ppt leaf extract and to 43% of the control by the 25 ppt roots plus rhizomes extract.L. minor was killed by concentrations of 125 ppt and above of both plant part extracts. As expected, the frond number and total chlorophyll content measured by theL. minor assay were highly correlated. Osmotic potentials below 143 mOsmol/kg had no influence onL. minor growth. Neither the osmotic potential nor the pH of the undiluted extracts ofN. lutea were in the range known to influence the growth of either lettuce seedlings orL. minor. Nuphar lutea extracts were many times more inhibitory than 16 other hydrophytes we previously examined.

Quantitative studies of cutaneous hypersensitivity: The prevalence of epicutaneous flare reactions to allergenic pollen extracts

The flare reactions produced by epicutaneous tests with 68 undiluted allergenic pollen extracts were measured in 550 allergic patients. Skin test reactions ≥2, ≥5, ≥10, ≥20, and ≥30 mm in diameter, respectively, were detected in approximately 67%, 22%, 10%, 3%, and 1% of the 34,700 skin tests. With the Kolmogorov-Smirnov difference test, the cumulative frequency of reaction diameters and log c-transformed diameters of all reactions and reactions to individual allergenic extracts differed significantly (p ≤ 0.01) from a normal distribution. The ability to identify specific differences between reactions to closely related pollen extracts was evaluated. Specific reactions could be reliably identified with ≥10 mm diameter flares. This arbitrary conservative threshold was used to estimate the relative prevalence of positive reactions to each allergenic extract. Seven allergenic extracts elicited the first quartile of all positive reactions. Thirteen, 18, and 30 allergenic extracts, respectively, were needed to elicit the second, third, and fourth quartiles of all positive reactions. Reactions to amphiphilous, as well as anemophilous, pollens were detected. Skin test reactions to grasses were more prevalent than reactions to weeds and trees. The most informative allergenic extracts for the detection of patients who exhibited a positive reaction to any extract were from red fescue-grass pollens, mesquite, short ragweed, red clover, and timothy-grass pollens.

Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity.

Undiluted extracts from eggs or oocytes of Xenopus laevis support the assembly of chromatin with physiologically spaced nucleosomes. Micrococcal nuclease and DNase I digestion experiments show that nucleosome formation as well as supercoiling of circular DNA concomitant to assembly do not require ATP or Mg2+. However these factors are essential for the stability and the physiological spacing of the assembled chromatin. gamma-S-ATP can substitute for ATP in this process. With topoisomers of defined linking number topological interconversions proceed by steps of unity, both in vitro as well as in vivo, indicating that topoisomerase I is dominantly acting in this process. Novobiocin sensitivity occurred only with diluted extracts and was unrelated to an inhibition of topoisomerase II. Finally, nucleosome assembly occurs efficiently on linear DNA although the assembled DNA is less stable than with circular DNA. From these results we propose that mature chromatin is formed in a two-step reaction. In the first step, nucleosome deposition occurs independently of ATP and Mg2+. Thus, nucleosome formation can be uncoupled from their spacing. In this step, topoisomerase activity is involved in the relaxation of the topological constraints generated by chromatin assembly rather than in the process of assembly itself. The second step, requiring ATP and Mg2+, generates properly spaced chromatin.

A simple kit for rapid field diagnosis of potato virus Y by latex serological test

A simple kit for rapid detection of potato virus Y by latex serological test was developed. The test is carried out on a white cardboard sheet and the results can be read by naked eye in two minutes. A test card of 10 x 6 cm holds latex sensitized antibodies, buffers and other necessary ingredients as dry blue colored formulate on the ringed areas of the card. A test card includes space for six tests and positive and negative controls. The kit also includes disposable plastic sticks for mixing the samples with test reagents and a hand press with disposable plastic tips. For testing, dried reagents are dissolved in drops of sample and mixed. After gentle rotation, samples containing virus appear clearly granulated while samples from healthy plants remain unagglutinated. The testing of undiluted extracts of evenly developed tuber sprouts resulted in over 91 % identity with the results obtained with ELISA that was used as a control method. Testing of diluted leaf extracts reached the same reliability but undiluted leaf extracts from glasshouse grown potatoes were not well suitable as test samples because of their dark green color. No such problems occurred with field grown material and a complete identity with the ELISA readings was true when the samples included secondarily infected potato plants. No reaction to other potato viruses than PVY was obtained by the test kit.

Activity of extracts from Euphorbia species on the germination of Striga species

SummaryAqueous extracts of fifteen Euphorbia species induced germination of Striga hermonthica (Del.) Benth. seeds to varying extents. Extracts from E. supina Raf., E. pilulifera Jacq., E. acalyphoides Hochst. ex. Boiss, E. prunifolia (Jacq.) Hort. Schoent., E. hirta L., E. aegyptiaca Boiss, E. splendens Boj., and E. granulata Forsk, were the most active. Extracts from E. acalyphoides and E. pilulifera were inhibitory at high concentrations. Undiluted extracts from E. pilulifera, E. aegyptiaca and E. hirta induced haustorial initiation. The stimulatory substance(s) were widely distributed in plants of E. aegyptiaca. Extracts from E. aegyptiaca also stimulated seed germination and haustorial initiation in a range of Striga species and strains.

Competition and Allelopathy Between Soybeans and Weeds1

AbstractThis research was initiated to determine how soybean [Glycine max (L.) Merr.] cultivars differ in competitive ability and also whether allelopathy functions to inhibit surrounding weed growth. Initially 280 soybean cultivars were screened in the field for competitive ability based on emergence, seedling vigor, quickness of canopy closure, and ability to inhibit growth of velvetleaf (Abutilon theophrasti Medic.) and foxtail millet [Setaria italica (L.) Beauv.]. Experiments were conducted at Lincoln, Nebr. on a Sharpsburg silty clay loam (fine, montmorillonitic, mesic Typic Argiudolls) during 1981 and 1982. Soybean cultivars of later maturity tended to compete more effectively with weeds. Competitive cultivars emerged quickly, rapidly formed a canopy, and were able to slow the growth of competing weed species. Twenty soybean cultivars of varying competitive ability were selected and grown in the greenhouse during 1981 and 1982 to determine the importance of allelopathy in competing with weeds. Exudates from roots of soybean cultivars grown in sand reduced the dry weight of 4‐week‐old velvetleaf plants an average of 15%, but foxtail millet was not inhibited. Incorporation of 1% ground soybean dry matter into Sharpsburg silty clay loam inhibited germination and dry weight of greenhouse grown velvetleaf an average of 46% each. Foxtail millet germination and dry weights were reduced an average of 82 and 65%, respectively. Undiluted soybean plant extracts of all cultivars tested slowed the germination and dry weight accumulation of 6‐day‐old velvetleaf and foxtail millet, but dilution of the extracts caused quite variable responses. Osmotic potential of the extract solution appeared to be the main cause of inhibition, however, extracts of some cultivars, diluted to decrease the osmotic potential, still inhibited growth. Allelopathy appears to be one mechanism for competition among soybeans and weeds. Soybean cultivars possessing the ability to chemically inhibit competing weed growth would be a great benefit to the soybean producer.

Sunflowers (Helianthus annuus) are Allelopathic to Weeds

Laboratory, greenhouse, and field studies were conducted to determine the allelopathic potential of sunflower (Helianthus annuusL.) cultivars to suppress weed germination and growth. Germination of wild mustard [Brassica kaber(DC.) L.C. Wheeler var.pinnatifida(Stokes) L.C. Wheeler] seeds at 25 C in undiluted aqueous extracts of sunflower leaf tissue was inhibited 75%, but was stimulated by up to 150% at 10- and 100-fold dilutions. Stem-tissue extracts at all concentrations stimulated wild mustard seed germination. The germination response of other weed species varied with the sunflower cultivar and concentration of tissue extract. In sand culture, leachates of dried sunflower leaf and stem tissue inhibited broadleaf - weed seedling growth, but had little or no effect on the growth of grass weeds. Sunflower root exudates inhibited seedling growth, but were less effective than leaf and stem tissue leachates. Germination of weed seeds was unaffected by root exudates. Over a 5 -yr period, weed density and percent ground cover increased less in field plots of sunflower than in control plots.

Cell-free extracts of Salmonella inhibit protein synthesis and cause cytotoxicity in eukaryotic cells

Cell-free, unconcentrated sonic extracts of several serotypes of Salmonella caused extensive detachment of intact Vero cells. Undiluted sonic extracts of these strains exhibited cell detachment in the range 20 – 50%. Upon dilution, the extract preparations caused a linear, dose-related cytotoxic effect on Vero cell monolayers. The heat-lability (100°C for 30 min) of much of the cytotoxic activity in the extract ruled out the possible involvement of endotoxin in this toxic effect for eukaryotic cells and suggested that this toxic factor is probably a protein. It was demonstrated that these sonic extracts also inhibited the incorporation of 3H-leucine by Vero cells and that the inhibitory events occurred 1 – 2 hr after exposure. When subjected to heating at 100°C for 30 min, the ability of Salmonella extracts to inhibit protein synthesis of Vero cells was significantly but only partially destroyed. Because the Salmonella extract-treated Vero cells did not release 3H-uridine until 24 – 48 hr after addition of sonic extracts, cell lysis was considered to be a secondary event resulting from the early shutdown of protein syntheis, rather than a direct effect of the toxic factor on membrane integrity. Further studies are needed to determine if these two biological activities of Salmonella sonic extracts are due to a single toxic molecule or result from two distinct toxin molecules.

Distribution and Interference of Common Milkweed (Asclepias syriaca) in Nebraska

Infestations of common milkweed (Asclepias syriacaL.), in descending order of incidence in Nebraska, occurred on areas occupied by soybean [Glycine max(L.) Merr.], oats (Avena sativaL.), sorghum [Sorghum bicolor(L.) Moench.], railroad rights-of-way, fallow land, roadsides, corn (Zea maysL.), winter wheat (Triticum aestivumL.), pasture, and alfalfa (Medicago sativaL.). Average crop yield reductions, within common milkweed communities of 11100 to 45 200 plants/ha, ranged from 2 to 10% for corn, 4 to 29% for sorghum, and 12 to 19% for soybeans. Reductions in crop yields tended to increase with increasing common milkweed populations. Germination percentage of common milkweed and sorghum were not significantly affected by growing medium conductivities below 7 and 13 mmho/cm2, respectively. Growth media at pH 2 significantly reduced germination percentage, hypocotyl length, and radicle length of both common milkweed and sorghum as compared with pH 4 to 10. Undiluted aqueous extracts of common milkweed shoot and root material significantly reduced germination percentage, hypocotyl length, and radicle length of sorghum.

ANTIVIRAL ACTIVITY OF FRUIT EXTRACTS

ABSTRACTInactivation of poliovirus with strawberry extract was time and concentration dependent and could be prevented or reduced but not reversed with serum or gelatin. Strawberry extract inactivated several enteric viruses and herpes simplex virus, and extracts of several fruits inactivated poliovirus; the degree of activity varied with the virus type and the fruit species showing a minimum two log reduction with undiluted fruit extracts, adjusted to pH 7.0. Step‐wise ultrafiltration of strawberry extract indicated that activity was found in fractions of molecular weight <500. 500‐2000. 10.000‐30.000 and >300.000. Several compoinds of plant origin were’ found to be antiviral; tannic acid was especially potent. Treatment of HEp2 cells with strawberry extract or tannic acid reduced subsequent infection with virus, almost a log; inhibition was prevented and reversed completely with serum or gelatin. The antiviral components of various fruits and plants may act both on viruses and host cells to prevent infection.

Der einflu� der temperatur auf thermostabilit�t, isoenzym-muster und reaktionskinetik der laktat-dehydrogenase aus fischen

Methodological problems complicate investigations on thermostability of lactate dehydrogenase (LDH). It is difficult to demonstrate a correlation between adaptation-temperature (AT) and LDH thermostability. Heat-inactivation characteristics change completely if diluted or undiluted tissue extracts are heated. In purified LDH (purchased from Boehringer, Mannheim, FRG), additions such as casein, bovine-serum albumin, NADH and pyruvate — even in small concentrations — can alter considerably the degree of heat resistance. It LDH activity is measured as a function of experimental temperature (ET) according to the composition of the actual test mixture (e.g. altered pyruvate concentration), a different temperature optimum is found. If tissue extracts containing unpurified enzymes are used, the accompanying substances act on the enzyme and modify its properties. Thus, possible influence of AT on enzyme properties can be concealed (suppressed, over-emphasized). In Idus idus acclimated to 10° or 20°C, brain, gill, gut and white dorsal muscle reveal identical LDH-isoenzyme patterns. However, liver-LDH shows a pattern dependent on the AT. A total of 11 bands with LDH activity were found. In 10°C fishes, the Isoenzymes 1, 3, 6 and 7 are especially active. However, 20°C fishes show marked activity of Isoenzymes 5 and 8, and a reduced activity of Isoenzyme 7. According to their electrophoretic mobility, the particular isoenzymes of LDH of white dorsal muscle of I. idus or Rhodeus amarus can be clearly distinguished. The ATs 10° or 20°C do not influence the dependence of reaction order on ET: this is not true for the velocity constant.

The effect of steroids and ammonium chloride acidosis on phosphoenolpyruvate carboxykinase in rat kidney cortex. I. Differentiation of the inductive process and characterization of enzyme activities.

The behaviour of rat kidney cortex phosphoenolpyruvate carboxykinase has been investigated under conditions of triamcinolone administration and ammonium chloride acidosis. The concentration of phosphoenolpyruvate carboxykinase as measured by enzyme activity and immunotitration was elevated under both conditions. The mechanism of induction is different in the two cases. At doses which produce maximum stimulation, the effects of steroid and ammonium chloride were additive; only the increment in enzyme activity produced by steroid was blocked by actinomycin D. PHOSPHOENOLPYRUVATE CARBOXYKINASE ACTIVITIES IN ALL CONDITIONS INVESTIGATED SHOW SIMILAR BEHAVIOR IN DILUTE EXTRACTS: these experiments involved antibody titration, stability studies, and molecular weight determinations on sucrose gradients. The molecular weight of phosphoenolpyruvate carboxykinase was also studied in undiluted extracts prepared by high-speed centrifugation; values were determined from sedimentation data obtained with a moving-partition cell as described by Yphantis and Waugh. Under these conditions, the apparent molecular weight of phosphoenolpyruvate carboxykinase was increased from 83,000 to 128,000 by ammonium chloride acidosis. These results are discussed and a hypothesis regarding the mechanism of phosphoenolpyruvate carboxykinase regulation in kidney cortex is presented.

Determination of free gossypol in chemically treated cottonseed meals containing dianilinogossypol

SummaryA method is proposed for the determination of free gossypol in chemically treated cottonseed meals containing dianilinogossypol. The procedure includes a rapid qualitative test for detecting the presence of dianilinogossypol in cottonseed meals.Investigation of the properties of dianilinogossypol showed that it was appreciably soluble in water‐free solvents, such as acetone, methyl ethyl ketone, or chloroform, but only slightly soluble in 70% acetone, an efficient solvent for the extraction of free gossypol.Both 70% acetone extracts of chemically treated meals and pure dianilinogossypol in the same solvent exhibited significant changes in optical density due to slow hydrolysis of dianilinogossypol. Dilution of aqueous acetone extracts with 80% isopropyl alcohol produced changes in optical density even greater than those occurring in undiluted extracts, introducing serious errors in the free gossypol determination.The addition of a small amount of aniline to the aqueous acetone for extraction stabilized the extract against changes in optical density and yielded constant values for free gossypol over a period of several hours. Use of aqueous acetone without aniline gave values which increased with the time interval between extraction and analysis. Extrapolation of these values to zero time gave results in good agreement with those obtained by the use of aqueous acetone containing aniline.The intense background color in extracts of chemically treated meals introduces a source of error when photoelectric colorimeters yielding non‐linear calibration curves are used for analysis. A modification in the customary method of calculation eliminates this source of error.

Chromosome Fragmentation inAllium CepaInduced by Seed Extracts ofPhaseolus Vulgaris

SUMMARYThe authors examined the influence of aqueous extracts of Phaseolus vulgaris on the root tip mitoses in Allium Cepa. Undiluted extracts of seeds, stored one year, induced an almost complete inhibition of mitotic activity after an 8 hrs'. treatment. After 60 hrs. recovery in tap water, mitotic activity was regained, but the new mitoses showed various chromosome aberrations, mainly fragmentations, The dilutions 1:2 and 1:4 induced similar effects in a lesser degree. Similar extracts of a variety of Phaseolus vulgaris seeds, which had been stored for 5 to 6 years exerted an even stronger activity. The undiluted extract killed the roots after an 8 hrs'. treatment, while the dilution 1:2 inhibited mitosis completely after 8 hrs.; after 60 hrs. recovery in tap water 21,3% abnormal anaphases could be counted. The authors discuss the possible implications of their findings for an explanation of the origin of spontaneous chromosome aberrations in the primary roots of aged seeds.

Field Spraying with undiluted Paraffin Extracts of Pyrethrum againstAntestiaandLyguson Coffee in Kenya

The application of an undiluted paraffin extract of pyrethrum for controllingAntestiaandLygus simonyion coffee trees is described.(1) The spray is applied with a small hand “ atomiser,” using 20 cc. of extract to a tree, and spraying under a cloth cover.(2) Tables are given with detailed figures in which the different methods of application may be compared. The kill ofAntestiabrought down by the most favourable method is 99%.(3) The proportion of insects brought down by the spray is in the neighbourhood of 96%. It appears that in practice a kill not much below 95% should be possible.(4) Of theLyguson trees sprayed, 99% were brought down and the kill of these was complete.(5) The method is a dual control forAntestiaandLygus, and examples are given of trees sprayed when both these insects were present in serious numbers.(6) An experimental field spraying againstLygusis described in detail. It was shown that the numbers ofLyguscould be kept below two to a tree by this spraying, and the proportion of fruit set on the sprayed block to that on the control was 3 : 2, indicating an increase of about 8 cwt. an acre.(7) The technique of the experimental sprayings, by which the detailed figures in the tables were obtained, is described. It is believed that these methods will be of more extended value in the experimental testing of sprays in the field.