Electrophoretic separation of large proteoglycans in large-pore polyacrylamide gradient gels (1.32-10.0% T) and a one-step procedure for simultaneous staining of proteins and proteoglycans.

A procedure is described for the preparation of 1.32-10% polyacrylamide gradient gels. Loose polyacrylamide gel on the top side of the gradient was stabilized with a layer of 0.4% agarose gel which also formed sample wells. The upper limit of separation achieved in these gels was estimated to be approximately 2 X 10(6) using globular protein standards. However, large aggregating proteoglycans from cartilage which have a molecular weight range of 1-4 X 10(6) penetrate and separate in these gels. A simple one-step procedure is also described for simultaneous staining of proteins and large proteoglycans in polyacrylamide gels.     

New immunofluorescent method for the rapid determination of microbial antibiotic sensitivity

A new procedure for rapid determination of the levels of antibiotic sensitivity in pathogenic microorganisms with the use of fluorescent antibodies is described. The procedure was developed with the use of a model of the vaccinal strains of Bacillus anthracis. It is based on determination of the microbial antibiotic resistance with the method of serial dilutions on solid media. Still, the medium with an antibiotic is inoculated instead of the pathogen with the native material subject to the analysis. The antibiotic effect on the microorganism is estimated with the method of fluorescent antibodies. The replica preparations obtained as a result of the pathogen growth in a mixed culture on nutrient media containing definite concentrations of the antibiotic are examined with the method of luminescence microscopy. The modification of the immunofluorescent procedure for rapid determination of the microbial sensitivity to antibiotics does not require obligatory isolation of the pathogen as a pure culture. This makes the procedure more economic with respect to the time necessary for the analysis. The following conditions for performing rapid analysis with respect to Bacillus anthracis are required: the minimal concentration of the pathogen in the specimen (2.10(5) spores/ml), preliminary thermal treatment of the specimen for destroying the spore microflora, additional cultivation for 6-8 hours at 37 degrees C. The presence of the accompanying sporulating microflora, i.e. common microorganisms present in the atmosphere, soil and open water bodies does not prevent the performance of the analysis.     

The turnover of hamster fibroblast lysosomal beta-D-glucuronidase.

The half-life of hamster fibroblast beta-D-glucuronidase (EC 3.2.1.31) was estimated to be 4-5 days by measuring the decay with time of the radioactivity in beta-D-glucuronidase isolated from cells grown in the presence of 14C-labelled amino acids. A new affinity-chromatographic procedure for the purification of beta-D-glucuronidase is described.     

Biochemical markers of apoptosis in different brain regions after training

Activity of caspase-3 and content of DNA fragments with sizes 0.2-0.6 kbp and more than 4.0 kbp in the worm of the cerebellum, hippocampus and prefrontal cortex of the adult rat brain were estimated in 4 and 24 hours after the procedure of acoustic startle habituation and fear conditioning. Heterochronic changes in apoptotic markers in the examined brain structures were observed after training. Caspase-3 activity was decreased in the worm of the cerebellum and hippocampus, and DNA fragmentation was suppressed in the hippocampus and brain cortex. At the same time, both caspase activity and DNA fragmentation were increased in the hypothalamus. These results provide evidence for the involvement of apoptosis in the mechanisms of learning and memory in adult brain.     

Rapid Embedding with Hot Low-Viscosity Nitrocellulose

A rapid method for embedding with low-viscosity nitrocellulose is described. Advantage is taken of the greater penetrating power of low-viscosity nitrocellulose than that of common varieties of celloidin. Fixation, dehydration and infiltration are carried out in screw-topped jars in the incubator at 56° C. The whole procedure from fixing to sectioning can be finished in 15-30 hours, and sections as thin as 6 to 10 μ may be cut without difficulty.     

Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts

Mitochondria participate in key metabolic reactions of the cell and regulate crucial signaling pathways including apoptosis. Although several approaches are available to study mitochondrial function in situ are available, investigating functional mitochondria that have been isolated from different tissues and from cultured cells offers still more unmatched advantages. This protocol illustrates a step-by-step procedure to obtain functional mitochondria with high yield from cells grown in culture, liver and muscle. The isolation procedures described here require 1-2 hours, depending on the source of the organelles. The polarographic analysis can be completed in 1 hour.     

The Germination and Staining of Basidia in Gymnosporangium

A procedure is described for germinating and staining rust teliospores on the slide. The spores are germinated on slides in a damp chamber, about 3 hours being required for the production of sporidia. The material is killed by inverting slides over osmic acid fumes for a few minutes. Germinated spores are then allowed to dry on the slide, thus becoming fixed to the slide in a gelatin produced by the breaking down of their own stalks during germination. No other fixative is required. Material must be thoroly dehydrated in the alcohols (one or more hours in each of the higher alcohols); returned to water; mordanted for 2-3 hours in 4% iron alum; stained for 2-3 hours in 0.5% aqueous solution of Heidenhain's hematoxylin; destained in 2% iron alum. The material is passed back thru the alcohols and mixtures of xylol and absolute alcohol (1:2, 1:1, 2:1) to xylol and mounted in balsam. The method is particularly satisfactory for the Gymnosporanghim rusts, which have telia very readily gelatinized. The details of germination are preserved intact, as in nature, and many details of nuclear division are excellent.     

Improved enrichment of insect gap junctions by filtration and sonication of NaOH-extracted subcellular fractions

The purification of gap junctions from insects has been hampered by low yields when starting with dissected tissues or by contamination with non-junctional structures when starting with intact insects. A method is described here involving filtration and sonication of NaOH-extracted crude membrane fractions from larvae of the lepidopteran Heliothis virescens which yields fractions containing approximately 50% gap junctions and approximately 7 microg total protein per g of larvae and represents an estimated 10-100 fold increase in gap junction enrichment compared with a previously described procedure (Cell Tissue Res. 274: 393-403, 1993). The remaining structures in the fractions consist of non-junctional membrane, septate junctions, lamellate bodies and amorphous material.     

The 48 kDa Ca2+-binding protein of bovine brain.

The integrated rate equation for reactions with stoichiometry A----P + Q is: e0t = -Cf . ln(1-delta P/A0) + C1 delta P + 1/2C2(delta P)2 where the coefficients C are linear or quadratic functions of the kinetic constants and the initial substrate and product concentrations. I have used the 21 progress curves described in the accompanying paper [Cox & Boeker (1987) Biochem. J. 245, 59-65] to develop computer-based analytical and statistical techniques for extracting kinetic constants by fitting this equation. The coefficients C were calculated by an unweighted non-linear regression: first approximations were obtained from a multiple regression of t on delta P and were refined by the Gauss-Newton method. The procedure converged in six iterations or less. The bias in the coefficients C was estimated by four methods and did not appear to be significant. The residuals in the progress curves appear to be normally distributed and do not correlate with the amount of product produced. Variances for Cf, C1 and C2 were estimated by four resampling procedures, which gave essentially identical results, and by matrix inversion, which came close to the others. The reliability of C2 can also be estimated by using an analysis-of-variance method that does not require resampling. The final kinetic constants were calculated by standard multiple regression, weighting each coefficient according to its variance. The weighted residuals from this procedure were normally distributed.     

Age validation and growth of bluenose Hyperoglyphe antarctica using the bomb chronometer method of radiocarbon ageing

Age validation of bluenose Hyperoglyphe antarctica was sought using the independent bomb chronometer procedure. Radiocarbon ((14) C) levels were measured in core micro-samples from 12 otoliths that had been aged using a zone count method. The core (14) C measurement for each fish was compared with the value on a surface water reference curve for the calculated birth year of the fish. There was good agreement, indicating that the line-count ageing method described here is not substantially biased. A second micro-sample was also taken near the edge of nine of the otolith cross-sections to help define a bomb-carbon curve for waters deeper than 200-300 m. There appears to be a 10 to 15 year lag in the time it takes the (14) C to reach the waters where adult H. antarctica are concentrated. The maximum estimated age of this species was 76 years, and females grow significantly larger than males. Von Bertalanffy growth curves were estimated, and although they fit the available data reasonably well, the lack of aged juvenile fish results in the K and t(0) parameters being biologically meaningless. Consequently, curves that are likely to better represent population growth were estimated by forcing t(0) to be -0·5.     

Quantitative determination of starch, amylose, and amylopectin in plant tissues using glass fiber paper

Methods for accurate and rapid determination of starch, amylose, and amylopectin in plant tissues are described. They are based on simplified extraction of starch with 32% perchloric acid and selective retention of the starch-iodine complex on a glass fiber disk (Whatman GF/A). The starch on the disk is dissolved in 0.75 M sulfuric acid and estimated with phenol. For amylose and amylopectin determination the starch on the disk is dissolved in perchloric acid, precipitated with ethanol, and retained on a 10-cm glass fiber strip. Both polysaccharides are separated by a chromatographic procedure involving development of the strip in a mixture of ethanol and dimethyl sulfoxide and in dimethyl sulfoxide. The strip is washed in ethanol and stained with iodine or used for polysaccharide quantitation. As little as 5 micrograms of starch or its components present in different amounts of plant material can be estimated.     

Heterogeneity of purified mouse interferons.

A procedure for obtaining highly purified stable interferon from mouse L cells is described. Interferon with a specific activity of 2.5 times 10-8 reference units/mg of protein is composed of 10 to 11 polypeptides. They differ in their molecular size as determined by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The molecular weight range is estimated to be from the smallest at 20, 000 to the largest at 32, 000. At least six of the polypeptides are glycoproteins and each of the polypeptides can be obtained as an apparent homogeneous entity and each has interferon activity.     

Nucleotide sequence-directed mapping of the nucleosomes

The concept of sequence-dependent deformational anisotropy of DNA proposed earlier is further elaborated and a computational procedure is developed for the sequence-directed mapping of the nucleosomes along chromatin DNA nucleotide sequences. The deformational anisotropy is found to be nonuniform along the molecule of the nucleosomal DNA, suggesting that the DNA superhelix in the nucleosome is slightly oval rather than circular in projection. The number of superhelical turns in the nucleosome core particle is estimated to be 2.0 +/- 0.2. Preliminary mapping of the nucleosomes in various chromatin DNA sequences yields the distribution of linker lengths which shows several minima separated by about 10 base-pairs. This is explained by sterical exclusion effects due to overlapping of the nucleosomes in space when some specific linker lengths are chosen. The mapping procedure described is tested by comparing its results with all the most accurate experimental mapping data reported so far. The comparison demonstrates that the exact positions of all the nucleosomes appear to be determined exclusively by the nucleotide sequences.     

Rabbit hindlimb glucose uptake assessed with positron-emitting fluorodeoxyglucose

The feasibility of estimating skeletal muscle glucose uptake in vivo was examined by using the glucose analogue 2-[18F]deoxy-2-fluoro-D-glucose (2-[18F]FDG) in the rabbit hindlimb. A pair of collimated coincidence gamma photon detectors was used to monitor the accumulation of tracer in the tissue after 2-[18F]FDG injection. Time-activity curves were generated on a second-by-second basis under control conditions, during increased contractile activity, or hyperinsulinemia. The arterial input of 2-[18F]FDG, plasma glucose, lactate, free fatty acids, and insulin were determined. A graphical (Patlak plot) procedure was used to determine the fractional rate of tracer phosphorylation and therefore trapping in the muscle. From the graphical analysis, the estimated rate of glucose phosphorylation (R) in the unperturbed state was calculated to be 0.037 mumol.min-1.ml-1 of tissue. During perturbation by electrical stimulation, an increase in the rate of tracer phosphorylation (K) was observed. No change in the rate of tracer phosphorylation was observed during hyperinsulinemia. The results support the use of 2-[18F]FDG and the graphical procedure for the noninvasive assessment of glucose uptake by skeletal muscle in vivo. The method described is sensitive to changes in the rate of tracer uptake with respect to time and physiological interventions.     

Purification of oat and rye phytochrome

A purification procedure employing normal chromatographic techniques is outlined for isolating phytochrome from etiolated oat (Avena sativa L.) seedlings. Yields in excess of 20% (25 milligrams or more) of phytochrome in crude extract were obtained from 10- to 15-kilograms lots. The purified oat phytochrome had an absorbance ratio (A₂₈₀ nm/A₆₆₅ nm) of 0.78 to 0.85, comparable to reported values, and gave a single major band with an estimated molecular weight of 62,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. A modification of the oat isolation procedure was used to isolate phytochrome from etiolated rye Secale cereale cv. Balbo) seedlings. During isolation rye phytochrome exhibited chromatographic profiles differing from oat phytochrome on diethylaminoethyl cellulose and on molecular sieve gels. It eluted at a higher salt concentration on diethylaminoethyl cellulose and nearer the void volume on molecular sieve gels. Yields of 5 to 10% (7.5-10 milligrams) of phytochrome in crude extract were obtained from 10- to 12-kilogram seedling lots. The purified rye phytochrome had an absorbance ratio of 1.25 to 1.37, significantly lower than values in the literature and gave a single major band with an estimated molecular weight of 120,000 on electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. It is suggested that the absorbance ratio and electrophoretic behavior of rye phytochrome are indices of purified native phytochrome, and that oat phytochrome as it has been described is an artifact which arises as a result of endogenous proteolysis during isolation. A rationale is provided for further modifications of the purification procedure to alleviate presumed protease contaminants.     

Electronic thrombocyte count from total blood]

Platelets were simultaneously counted in 52 persons by the conventional method of Cronkite and Brecher and with a new electronic cell volume analyser. The platelet counts estimated by the electronic analyser were 10% lower than that measured with the conventional method. This difference decreased to 1.5% when the same diluent was used for both methods. The described electronic counting method of platelets in whole blood is a simple procedure, which gives well reproducible and exact results.     

The Preparation of Stem Sections of Woody Herbarium Specimens

A procedure is described for sectioning the stems of woody herbarium specimens by the paraffin method. The specimens are cut into convenient sizes; boiled for one-half to one hour in order to exclude air and soak up the cell walls and membranes; cooled and placed in 5% NaOH for 24 hours in order to expand the collapsed cells and remove excessive coloring matter; washed in running water for a few hours; placed in hydrofluoric acid until sufficiently softened to cut easily with a razor blade; washed in running water for 24 hours to remove the acid; dehydrated and embedded in paraffin of high melting point (56-58°C.) according to the n-butyl alcohol method; sectioned with the rotary microtome and completed by the ordinary method. Soaking the paraffin blocks in water for a period of several hours to a day or more before sectioning greatly improves the cutting and reduces electrification of the paraffin ribbon. The method proves satisfactory not only for herbarium material but for seeds and specimens of old bark and wood varying in hardness from balsa to ebony. For seeds or specimens containing only xylem, the NaOH treatment should be omitted.     

Reexamination of spectroscopic properties of ceruloplasmin freshly isolated with a novel very rapid single-step procedure

A novel, single-step, chromatographic procedure for the purification of ceruloplasmin is described. The procedure consists of a single chromatographic step, leading in approximately 5 hours from 2 liters of plasma to an electrophoretically homogeneous ceruloplasmin preparation which exhibits different spectroscopic properties with respect to the protein obtained with more lengthy, currently available procedures. The method is suitable for either small or large scale purification from various vertebrate plasma.     

Thermodynamics of carp metmyoglobin unfolding

The conformational free energy of carp lateral muscle metmyoglobin at 25 degrees C pH 8 is 9.0 +/- 0.5 kcal/mol as estimated from isothermal unfolding by both urea and guanidinium chloride. A novel procedure for the simultaneous analysis of acid and guanidinium chloride unfolding data is described. Acid denaturation data suggest that binding of five protons to histidyl residues occurs on unfolding. Correlation of sequences and conformational stabilities of several myoglobins according to the tertiary structure of sperm whale myoglobin suggests an evolutionary turnover of side chain-side chain interactions.