Gram scale purification and preparation of rabbit liver zinc metallothionein.

A simplified procedure for purifying gram quantities of rabbit liver metallothionein (MT) using gel filtration and anion exchange chromatography is presented. The MT purification made use of anion exchange batch elution chromatography which greatly shortened the procedure. Quantitation techniques for use with crude and purified MT are discussed. This paper also describes the preparation of large amounts of ZnMT from Cd,ZnMT.     

Kinematics of biophysically asymmetric limbs within rate of velocity development

The purpose of this study was to investigate the movement speed characteristics of 2 intrinsically different limbs. Twenty subjects volunteered to participate (10 men and 10 women). Each subject performed 5 repetitions of concentric knee and elbow extension and flexion movements at 60 through 500 d.s(-1) on an isokinetic dynamometer. Kinematic data were collected at 1,000 Hz and separated into rate of velocity development (RVD) and peak torque. Results demonstrated a significant (p < 0.05) main effect for sex for RVD and peak torque. Significant (p < 0.05) differences were also demonstrated between knee and elbow RVD and between knee and elbow peak torque at every speed tested. Neither knee and elbow RVD nor peak torque demonstrated any significant Pearson correlations at any speed tested (r = -0.17-0.41). These results collectively point to the specificity of limb speed and torque as a result of biophysical differences such as length and mass. Therefore, strength and speed may be modulated by neuromotor patterns that differ based on individual limbs.     

A persistent, productive, and seasonally dynamic vibriophage population within Pacific oysters (Crassostrea gigas)

In an effort to understand the relationship between Vibrio and vibriophage populations, abundances of Vibrio spp. and viruses infecting Vibrio parahaemolyticus (VpVs) were monitored for a year in Pacific oysters and water collected from Ladysmith Harbor, British Columbia, Canada. Bacterial abundances were highly seasonal, whereas high titers of VpVs (0.5 x 10(4) to 11 x 10(4) viruses cm(-3)) occurred year round in oysters, even when V. parahaemolyticus was undetectable (< 3 cells cm(-3)). Viruses were not detected (<10 ml(-1)) in the water column. Host-range studies demonstrated that 13 VpV strains could infect 62% of the V. parahaemolyticus strains from oysters (91 pairings) and 74% of the strains from sediments (65 pairings) but only 30% of the water-column strains (91 pairings). Ten viruses also infected more than one species among V. alginolyticus, V. natriegens, and V. vulnificus. As winter approached and potential hosts disappeared, the proportion of host strains that the viruses could infect decreased by approximately 50% and, in the middle of winter, only 14% of the VpV community could be plated on summer host strains. Estimates of virus-induced mortality on V. parahaemolyticus indicated that other host species were required to sustain viral production during winter when the putative host species was undetectable. The present study shows that oysters are likely one of the major sources of viruses infecting V. parahaemolyticus in oysters and in the water column. Furthermore, seasonal shifts in patterns of host range provide strong evidence that the composition of the virus community changes during winter.     

Accuracy and dynamic range of spatial image correlation and cross-correlation spectroscopy

We present a comprehensive study of the accuracy and dynamic range of spatial image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS). We use simulations to model laser scanning microscopy imaging of static subdiffraction limit fluorescent proteins or protein clusters in a cell membrane. The simulation programs allow us to control the spatial imaging sampling variables and the particle population densities and interactions and introduce and vary background and counting noise typical of what is encountered in digital optical microscopy. We systematically calculate how the accuracy of both image correlation methods depends on practical experimental collection parameters and characteristics of the sample. The results of this study provide a guide to appropriately plan spatial image correlation measurements on proteins in biological membranes in real cells. The data presented map regimes where the spatial ICS and ICCS provide accurate results as well as clearly showing the conditions where they systematically deviate from acceptable accuracy. Finally, we compare the simulated data with standard confocal microscopy using live CHO cells expressing the epidermal growth factor receptor fused with green fluorescent protein (GFP/EGFR) to obtain typical values for the experimental variables that were investigated in our study. We used our simulation results to estimate a relative precision of 20% for the ICS measured receptor density of 64 microm(-2) within a 121 x 98 pixel subregion of a single cell.     

Similar controls on calcification under ocean acidification across unrelated coral reef taxa

Ocean acidification (OA) is a major threat to marine ecosystems, particularly coral reefs which are heavily reliant on calcareous species. OA decreases seawater pH and calcium carbonate saturation state (Ω), and increases the concentration of dissolved inorganic carbon (DIC). Intense scientific effort has attempted to determine the mechanisms via which ocean acidification (OA) influences calcification, led by early hypotheses that calcium carbonate saturation state (Ω) is the main driver. We grew corals and coralline algae for 8–21 weeks, under treatments where the seawater parameters Ω, pH, and DIC were manipulated to examine their differential effects on calcification rates and calcifying fluid chemistry (Ω_cf, pH_cf, and DIC_cf). Here, using long duration experiments, we provide geochemical evidence that differing physiological controls on carbonate chemistry at the site of calcification, rather than seawater Ω, are the main determinants of calcification. We found that changes in seawater pH and DIC rather than Ω had the greatest effects on calcification and calcifying fluid chemistry, though the effects of seawater carbonate chemistry were limited. Our results demonstrate the capacity of organisms from taxa with vastly different calcification mechanisms to regulate their internal chemistry under extreme chemical conditions. These findings provide an explanation for the resistance of some species to OA, while also demonstrating how changes in seawater DIC and pH under OA influence calcification of key coral reef taxa.     

A cost effective base-matching assay with low backgrounds.

Base-matching or so-called mini-sequencing is a powerful technique for genotyping and mutation identification. However, its application is often hampered by high background and high cost. We have decreased the background by approximately 5-fold by incorporating an end-blocking step and using only 1/10 of the usual nucleotide concentrations.     

Bioremediation of pentachlorophenol-contaminated soil by bioaugmentation using activated soil

The use of an indigenous microbial consortium, pollutant-acclimated and attached to soil particles (activated soil), was studied as a bioaugmentation method for the aerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil. A 125-l completely mixed soil slurry (10% soil) bioreactor was used to produce the activated soil biomass. Results showed that the bioreactor was very effective in producing a PCP-acclimated biomass. Within 30 days, PCP-degrading bacteria increased from 10⁵ cfu/g to 10⁸ cfu/g soil. Mineralization of the PCP added to the reactor was demonstrated by chloride accumulation in solution. The soil-attached consortium produced in the reactor was inhibited by PCP concentrations exceeding 250 mg/l. This high level of tolerance was attributed to the beneficial effect of the soil particles. Once produced, the activated soil biomass remained active for 5 weeks at 20 °C and for up to 3 months when kept at 4 °C. The activated attached soil biomass produced in the completely mixed soil slurry bioreactor, as well as a PCP-acclimated flocculent biomass obtained from an air-lift immobilized-soil bioreactor, were used to stimulate the bioremediation of a PCP-impacted sandy soil, which had no indigenous PCP-degrading microorganisms. Bioaugmentation of this soil by the acclimated biomass resulted in a 99% reduction (from 400 mg/kg to 5 mg/kg in 130 days) in PCP concentration. The PCP degradation rates obtained with the activated soil biomass, produced either as a biomass attached to soil particles or as a flocculent biomass, were similar. Received: 31 March 1997 / Received revision: 22 July 1997 / Accepted: 25 August 1997     

Somatic embryogenesis and plant regeneration in Triticum aestivum x Leymus angustus F1 hybrids and the parental lines

Somatic embryos and plants were produced from cultured inflorescence and leaf segments of Triticum aestivum X Leymus anaustus F₁ hybrids and the parental lines. Inflorescences showed a better capacity for somatic embryogenesis and plant regeneration than leaves. Leymus anaustus produced the highest number of embryogenic calli, while the hybrids were intermediate between this species and Triticum aestivum. Presence of 2,4-D was shown to be essential for induction and maintenance of somatic embryogenesis. Addition of five amino acids (glutamine, proline, asparagine, aspartic acid and glutamic acid) did not have any marked effect when they were used in the callus induction medium. The regenerated plants had the same morphology as the original plants. No cytological modification was observed in the examined plants.     

Extraction from natural planktonic microorganisms of DNA suitable for molecular biological studies

We developed a simple technique for the high-yield extraction of purified DNA from mixed populations of natural planktonic marine microbes (primarily bacteria). This is a necessary step for several molecular biological approaches to the study of microbial communities in nature. The microorganisms from near-shore marine and brackish water samples, ranging in volume from 8 to 40 liters, were collected on 0.22-mum-pore-size fluorocarbon-based filters, after prefiltration through glass fiber filters, to remove most of the eucaryotes. DNA was extracted directly from the filters in 1% sodium dodecyl sulfate that was heated to 95 to 100 degrees C for 1.5 to 2 min. This procedure lysed essentially all the bacteria and did not significantly denature the DNA. The DNA was purified by phenol extraction, and precautions were taken to minimize shearing. Agarose gel electrophoresis showed that most of the final preparation had a large molecular size (>23 kilobase pairs). The DNA was sufficiently pure to allow complete digestion by the restriction endonuclease Sau3AI and ligation to vector DNA. In a sample in which the extracted DNA was quantified by binding to the dye Hoechst H33258, DNA was quantitatively extracted, and 45% of the initially extracted DNA was recovered after purification. Final yields were a few micrograms of DNA per liter of seawater and were roughly 25 to 50% of the total bacterial DNA in the sample. Alternatives to the initial harvest by filtration method, including continuous-flow centrifugation and thin-channel or hollow-fiber concentration followed by centrifugation, were less efficient than filtration in terms of both time and yield, largely because of the difficulty of centrifuging the very small bacteria typical of marine plankton. These methods were judged to be less appropriate for studies of natural populations as they impose a strong selection for the larger bacteria.     

Role of inoculum preparation and density on the bioremediation of 2,4-D-contaminated soil by bioaugmentation

The effect of inoculum preparation and density on the efficiency of remediation of 2,4-dichlorophenoxyacetic acid (2,4-D) by bioaugmentation was studied in non-sterile soil. A 2,4-D-degrading Pseudomonas cepacia strain (designated BRI6001) was used initially in liquid culture to determine the effects of pre-growth induction and of inoculum density. The time for complete 2,4-D degradation was reduced by 0.5 day for each log increase of inoculum density. In mixed (BRI6001 and soil bacteria) liquid cultures, a competition effect for 2,4-D became apparent at low inoculum levels (less than 10 10⁵ cfu/ml BRI6001 for 10⁸ cfu/ml soil bacteria) but only when the soil bacteria included indigenous 2,4-D degraders. In static non-sterile soil, the effect of inoculum density on 2,4-D degradation was comparable to that in liquid culture but only at high inoculation levels. At lower levels, a biological effect for 2,4-D degradation became apparent, as was observed in mixed liquid cultures, whereas at intermediate levels, a combination of biological, physical and chemical factors decreased the efficiency of bioaugmentation. The acclimation period for 2,4-D degradation in soil bioaugmented with BRI6001 reflected mainly the time required for cell induction and, presumably, for overcoming the physical limitation of diffusion of both 2,4-D and added bacteria in the soil matrix. Correspondence to: R. SamsonISSUED AS NRCC 33848