Counting emissions from potassium-42 in plant tissue with a liquid scintillation spectrometer

Summary A method has been devised to count emissions from K⁴² in plant tissue by liquid scintillation spectrometer with low quenching. The plant samples were ashed in aluminium dishes, folded into aluminium wafers and suspended in the liquid scintillator of counting vials. The samples were counted in a scintillation spectrometer at one to five minute intervals seven or more times and until the total exceeded 1000 counts. The efficiency of counting was approximately 70%.A computer program was written to calculate decay corrections for the short half-life isotope K⁴². The program computed the weighted mean of the counts, standard deviation of the mean, percentage standard deviation of the mean and Chi-square value for each sample. The measurements were precise enough for K-uptake studies.     

Synchronization of 9L rat brain tumor cells by centrifugal elutriation

Asynchronous 9L cells were separated into relatively homogeneously-sized populations using centrifugal elutriation with both a conventional collection method and a long collection method. A substantial increase in the homogeneity of the volume distributions and in the degree of synchrony of the separated fractions was obtained using the long collection method. Autoradiographic data indicated that fractions containing greater than or equal to 97% G1 cells, greater than or equal to 80% S cells, and 70-75% G2 cells could be routinely recovered with this procedure. Recovery in these fractions varied from 5 to 8% of the total number of cells elutriated. The colony forming efficiency (CFE) of cells from fractions representing each phase of the cell cycle was a constant 60-70%, which was comparable to the 60-80% usually found for asynchronous 9L cells. The percentage of cells in the G1, S, and G2 phases in the elutriated fractions was more accurately determined from the volume distribution than from computer fits of the DNA histogram obtained from flow cytometry. In genereal, the degree of synchrony was related to the coefficient of variation (CV) of the volume distributions of the elutriated fractions. The CV was about 14% for all elutriated fractions. When the greater than or equal to 97% G1 population was allowed to progress to S and G2, the CVs were about 17 and 20.2%, respectively. Thus, the best nonperturbing method for obtaining synchronous 9L cells in the S or G2 phases was direct elutriation with the long collection method.     

Indirect determination of nitric oxide production by reduction of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli MC1061

Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described. To obtain the crude bacterial lysate containing nitrate reductase activity, E. coli MC1061 was subjected to 16-20 freeze-thawing cycles, from -70 to 60 degrees C, until nitrite reductase activity was < or = 25%. Nitrate reductase activity was detected mainly in the crude preparation. To validate the nitrate reduction procedure, standard nitrate solutions (1.6-100 microM) were incubated with the nitrate reductase preparation for 3 h at 37 degrees C, and nitrite was estimated by the Griess reaction in a microassay. Nitrate solutions were reduced to nitrite in a range of 60-70%. Importantly, no cofactors were necessary to perform nitrate reduction. The biological samples were first reduced with the nitrate reductase preparation. After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified. The utility of the nitrate reductase preparation was assessed by nitrate+nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E. coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages. Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate+nitrite in biological fluids by spectrophotometric methods.     

Kinetic mechanisms of two NAD:arginine ADP-ribosyltransferases: the soluble, salt-stimulated transferase from turkey erythrocytes and choleragen, a toxin from Vibrio cholerae

A subunit of choleragen and an erythrocyte ADP-ribosyltransferase catalyze the transfer of ADP-ribose from NAD to proteins and low molecular weight guanidino compounds such as arginine. These enzymes also catalyze the hydrolysis of NAD to nicotinamide and ADP-ribose. The kinetic mechanism for both transferases was investigated in the presence and absence of the product inhibitor nicotinamide by using agmatine as the acceptor molecule. To obtain accurate estimates of kinetic parameters, the transferase and glycohydrolase reactions were monitored simultaneously by using [adenine-2,8-3H]NAD and [carbonyl-14C]NAD as tracer compounds. Under optimal conditions for the transferase assay, NAD hydrolysis occurred at less than 5% of the Vmax for ADP-ribosylation; at subsaturating agmatine concentrations, the ratio of NAD hydrolysis to ADP-ribosylation was significantly higher. Binding of either NAD or agmatine resulted in a greater than 70% decrease in affinity for the second substrate. All data were consistent with a rapid equilibrium random sequential mechanism for both enzymes.     

A simple methodological approach for counting and identifying culturable viruses adsorbed to cellulose nitrate membrane filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.     

Quench correction in Cerenkov counting: channels ration and external source channels ratio methods

Channels ratio and external source ratio methods which may be used for accurate quench correction in ?erenkov counting systems have been developed. In general, the channels ratio method will be more accurate for quench correction in high count rate samples where good ratio statistics are available. For low level samples, the external source channels ratio method will provide more accurate quench correction if efficiencies are greater that 10%.     

Effects of tritiated water on germ cells in medaka embryos

Embryos of medaka, Oryzias latipes, were exposed to tritiated water and 137Cs gamma rays continuously from the one-cell stage until hatching (10 days at 26 degrees C). Germ cells in the gonads of newly hatched fry were counted in histological sections and compared with controls. The accumulated dose for 50% survival of germ cells was 195 rad for tritium beta rays and 350 rad for 137Cs gamma rays. Female progeny were produced using Yamamoto's method. The 50% survival doses for female germ cells treated in a manner similar to that described above were 140 rad for beta rays and 305 rad for gamma rays. When embryos of medaka were irradiated with gamma rays below an accumulated dose of 475 rad or treated with tritiated water at a concentration of 0.2 mCi/ml or lower, the dose response of the germ cells showed an exponential relationship. It appeared that there was no threshold or significant dose-rate effect for either beta or gamma rays on germ cell survival, and that tritium beta rays were more effective than 137Cs gamma rays in germ cell killing.     

A Simple Methodological Approach for Counting and Identifying Culturable Viruses Adsorbed to Cellulose Nitrate Membrane Filters

We identified conditions under which Buffalo green monkey cells grew on the surfaces of cellulose nitrate membrane filters in such a way that they covered the entire surface of each filter and penetrated through the pores. When such conditions were used, poliovirus that had previously been adsorbed on the membranes infected the cells and replicated. A plaque assay method and a quantal method (most probable number of cytopathic units) were used to detect and count the viruses adsorbed on the membrane filters. Polioviruses in aqueous suspensions were then concentrated by adsorption to cellulose membrane filters and were subsequently counted without elution, a step which is necessary when the commonly used methods are employed. The pore size of the membrane filter, the sample contents, and the sample volume were optimized for tap water, seawater, and a 0.25 M glycine buffer solution. The numbers of viruses recovered under the optimized conditions were more than 50% greater than the numbers counted by the standard plaque assay. When ceftazidime was added to the assay medium in addition to the antibiotics which are typically used, the method could be used to study natural samples with low and intermediate levels of microbial pollution without decontamination of the samples. This methodological approach also allowed plaque hybridization either directly on cellulose nitrate membranes or on Hybond N+ membranes after the preparations were transferred.     

Pherogram normalization in capillary electrophoresis and micellar electrokinetic chromatography analyses in cases of sample matrix-induced migration time shifts

When analyzing bio-matrix samples using capillary electrophoresis (CE) or micellar electrokinetic chromatography (MEKC), unwanted shifts in the time axis are often observed, both between samples and standards and between samples, thus hampering identification. These shifts are caused by either or both of two sample matrix-induced effects: variations in stacking conditions (effective field strength or migration length) and variations in electroosmotic flow. Based on elementary CE principles and provided that any two peaks in the pherograms can be linked, these variations can be separately accounted and quantitatively corrected for, so that perfectly overlapping pherograms of standards and samples can be obtained after normalization. The method was validated using samples of a DNA ladder, separated in a sieving polymer. In addition, a number of data files from CE and MEKC analyses (steroids, opioids, beta-blockers, amines, and inorganic anions) previously published by other authors were successfully normalized. A freeware computer programme, CEqualizer, for normalizing ASCII files of detector signals using the method described, is available to the CE community from http: //www.ceyork.f2s.com.     

Cell-cycle duration and sister-chromatid exchange frequency in cultured human lymphocytes

Whole heparinized blood samples from normal human donors were grown in culture media containing 10 μg/ml of bromodeoxyuridine. Lymphocytes were harvested after 58, 70, 72 and 80 h and scored for sister-chromatid exchanges (SCEs) under a fluorescence microscope. SCEs which occured during the first and second cell cycles were counted in second or third generation cells selected on the basis of their chromosome fluorescence patterns. The results of a preliminary study showed the mean SCE frequency per cell at 72 h to be 9.0 for second generation cells and 7.8 in third generation cells (P < 0.01). A second study, using culture medium with heat-inactivated fetal-calf serum, gave similar results (9.4 vs. 7.8, P 0.001) at 70 h. Therefore, the difference in SCE frequency between second and third generation cells at 70 or 72 h cannot be attributed to heat-labile substances of serum origin. An additional finding in the second study was that SCE frequencies in third division cells at 70 and80 h were the samee as those of second division cells at 58 h but significantly less (P < 0.001) than the frequency in second division cells at 70 h. These data were interpreted as arising from at least two different lymphocyte populations; one group of cells that is either slower growing or slower in phytohemagglutinin stimulation, with a higher SCE frequency which does not reach second division until 70 or 80 h, and a more rapidly dividing (or more quickly stimulated by phytohemagglutinin) population with a lower SCE frequency which reaches second division at 58 h and third division by 70–80 h. Whether or not this hypothesis is correct, the data show that SCE frequency varies significantly with cell-cycle duration. Since some carcinogens have been shown to alter cell kinetics (Craig-Holmes and Shaw, Mutation Res. 46 (1977) 375), changes in SCE frequency which are caused by a change in cell kinetics must be considered a factor in determining the mutagenicity of an agent by its ability to increase SCE frequency.     

Direct comparison of kinematic data collected using an electromagnetic tracking system versus a digital optical system

The purpose of this study was to quantify the dynamic accuracy of kinematics measured by a digital optical motion analysis system in a gait analysis laboratory (capture volume approximately 20m(3)) compared to a standard range direct-current electromagnetic (EM) tracking device (capture volume approximately 1m(3)). This is a subset of a larger effort to establish an appropriate marker set for the optical system to quantify upperlimb kinematics simultaneously with gait, in comparison to previous studies of isolated upperlimb movements that have employed EM tracking devices. Rigid clusters of spherical reflective markers and EM sensors were attached to a mechanical articulator that mimicked three-dimensional joint rotations, similar to the elbow. As the articulator was moved through known ranges of motion (i.e. gold standard), kinematic data were collected simultaneously using both tracking systems. Both systems were tended to underestimate the range of motion; however, the application of post hoc smoothing and least-squares correction algorithms reduced these effects. When smoothing and correction algorithms were used, the magnitude of the mean difference between the gold standard and either the EM or optical system did not exceed 2 degrees for any of the compound motions performed. This level of agreement suggests that the measurements obtained from either system are clinically comparable, provided appropriate smoothing and correction algorithms are employed.     

The Benzidine Staining Method for Blood Vessels

Two modifications of the method are described: A. Living specimens of sabellid and serpluid polychaetes, earthworms, small tadpoles, or fish larvae are immersed in an approximately saturated solution of benzidine for 30 minutes and then 3% hydrogen peroxide is added until bubbles of gas appear. When the blood vessels appear dark blue, the specimens are fixed in acidified 70% alcohol, dehydrated, cleared and either mounted in Canada balsam as whole mounts, or embedded in paraffin, sectioned at 100 to 250µ and mounted. B. Material fixed in 10% formalin in sea-water, or in formalin hypertonic saline, is incubated at 37°C. for one hour in an aqueous mixture containing sodium nitroprusside, 0.1%; benzidine, acetic acid 0.5%, followed by a weak (0.01–0.02%) hydrogen peroxide solution for a further hour, embedded in paraffin, cut into thick sections and mounted.     

Detection of disease-specific augmentation of abnormal immunoglobulin G in sera of patients with rheumatoid arthritis

Galactose-free immunoglobulin G (IgG), which is known to be higher in the sera of patients with rheumatoid arthritis, was prepared from IgG of healthy volunteers using enzymes. Its reactivity to lectins was analyzed. The galactose-free IgG showed no reactivity to Ricinus communis agglutinin 120 but displayed greater reactivity to concanavalin A and Lens culinaris lectin than did intact human IgG. Then, IgG in serum samples was bound to protein A immobilized on a nitrocellulose membrane, and its reactivity to biotinylated concanavalin A was measured with streptavidin-conjugated horseradish peroxidase. When the reactivity to concanavalin A of IgG in sera from healthy individuals and patients with rheumatoid arthritis (RA), osteoarthritis, systemic lupus erythematosus, or hepatic disease was compared, higher levels were shown in patients with RA, notably in 60% of the seronegative patients and 80% of the early phase patients. Therefore, it was suggested that augmentation of the abnormal IgG in sera was highly specific to patients with RA and that this novel serum test could be very useful for an accurate diagnosis of this disease.     

The Benzidine Staining Method for Blood Vessels

Two modifications of the method are described: A. Living specimens of sabellid and serpluid polychaetes, earthworms, small tadpoles, or fish larvae are immersed in an approximately saturated solution of benzidine for 30 minutes and then 3% hydrogen peroxide is added until bubbles of gas appear. When the blood vessels appear dark blue, the specimens are fixed in acidified 70% alcohol, dehydrated, cleared and either mounted in Canada balsam as whole mounts, or embedded in paraffin, sectioned at 100 to 250µ and mounted. B. Material fixed in 10% formalin in sea-water, or in formalin hypertonic saline, is incubated at 37°C. for one hour in an aqueous mixture containing sodium nitroprusside, 0.1%; benzidine, acetic acid 0.5%, followed by a weak (0.01-0.02%) hydrogen peroxide solution for a further hour, embedded in paraffin, cut into thick sections and mounted.     

Effect of insemination timing on the fertilizing capacity of frozen/thawed equine spermatozoa

A breeding trial was conducted to evaluate the effect of insemination timing on the fertility of mares bred with frozen/thawed equine semen. One stallion and 60 reproductively sound, estrous-synchronized mares were included in the study. Mares were assigned to one of three groups (n = 20): 1) insemination with fresh semen every other day during estrus from detection of a 35-mm follicle until ovulation, 2) insemination with frozen/thawed semen every day during estrus from detection of a 35-mm follicle until ovulation or 3) insemination with frozen/thawed semen once, within 6 h after ovulation. Single-cycle 18-d pregnancy rates resulting from insemination with fresh semen (70%), preovulation insemination with frozen/thawed semen (60%) and postovulation insemination with frozen/thawed semen (55%) were not different (P > 0.05). Possibly, equivalent pregnancy rates could be achieved with frozen/thawed semen using either daily inseminations until ovulation occurs or frequent ovarian palpations with a single post-ovulation insemination. Further studies regarding the effect of insemination timing on stallion fertility are needed since the present investigation included only one stallion and a small number of mares.     

Genotype Imputation with Millions of Reference Samples

We present a genotype imputation method that scales to millions of reference samples. The imputation method, based on the Li and Stephens model and implemented in Beagle v.4.1, is parallelized and memory efficient, making it well suited to multi-core computer processors. It achieves fast, accurate, and memory-efficient genotype imputation by restricting the probability model to markers that are genotyped in the target samples and by performing linear interpolation to impute ungenotyped variants. We compare Beagle v.4.1 with Impute2 and Minimac3 by using 1000 Genomes Project data, UK10K Project data, and simulated data. All three methods have similar accuracy but different memory requirements and different computation times. When imputing 10 Mb of sequence data from 50,000 reference samples, Beagle’s throughput was more than 100× greater than Impute2’s throughput on our computer servers. When imputing 10 Mb of sequence data from 200,000 reference samples in VCF format, Minimac3 consumed 26× more memory per computational thread and 15× more CPU time than Beagle. We demonstrate that Beagle v.4.1 scales to much larger reference panels by performing imputation from a simulated reference panel having 5 million samples and a mean marker density of one marker per four base pairs.     

Survival of Campylobacter jejuni inoculated into ground beef.

Ground beef was inoculated with mixed cultures of Campylobacter jejuni, and the samples were subjected to various cooking and cold-storage temperatures. When samples were heated in an oven at either 190 or 218 degrees C, approximately 10(7) cells of C. jejuni per g were inactivated (less than 30 cells per g) in less than 10 min after the ground beef reached an internal temperature of 70 degrees C. When the samples were held at -15 degrees C over 14 days of storage, the numbers of C. jejuni declined by 3 log10. When inoculated samples were stored with an equal amount of Cary-Blair diluent at 4 degrees C, no changes in viability were observed over 14 days of storage. Twenty-five times as much C. jejuni was recovered from inoculated ground beef when either 10% glycerol or 10% dimethyl sulfoxide was added to an equal amount of ground beef before freezing as was recovered from peptone-diluted ground beef. Twice as much inoculated C. jejuni was recovered from ground beef plus Cary-Blair diluent as was recovered from ground beef plus peptone diluent.     

Estimating kinetic constants from single channel data.

The process underlying the opening and closing of ionic channels in biological or artificial lipid membranes can be modeled kinetically as a time-homogeneous Markov chain. The elements of the chain are kinetic states that can be either open or closed. A maximum likelihood procedure is described for estimating the transition rates between these states from single channel data. The method has been implemented for linear kinetic schemes of fewer than six states, and is suitable for nonstationary data in which one or more independent channels are functioning simultaneously. It also provides standard errors for all estimates of rate constants and permits testing of smoothly parameterized subhypotheses of a general model. We have illustrated our approach by analysis of single channel data simulated on a computer and have described a procedure for analysis of experimental data.