Relationship between thymidine transport and phosphorylation in Novikoff rat hepatoma cells as analyzed by a rapid sampling technique

Incorporation of thymidine into Novikoff rat hepatoma cells was analyzed with a rapid sampling technique which allowed collection of 12 time points in 20 sec. Transport was studied in the absence of metabolism by using either ATP-depleted cells or a thymidine kinase negative subline. Transport was a rapid, saturable, nonconcentrative process with a K_m of about 85 μM. The intracellular thymidine pool was also rapidly labeled in cells which phosphorylated thymidine, so that a group translocation process involving thymidine kinase can be ruled out. Under all conditions examined, phosphorylation, not the transport, of thymidine was the rate-determining step in its incorporation into the acid-soluble pool. Estimation of transport rates from total incorporation into cells which phosphorylate the substrate is invalid in this cell system and must be questioned in all instances.     

The Interactions of P-Glycoprotein with Antimalarial Drugs,Including Substrate Affinity,Inhibition and Regulation

The combination of passive drug permeability, affinity for uptake and efflux transporters as well as gastrointestinal metabolism defines net drug absorption. Efflux mechanisms are often overlooked when examining the absorption phase of drug bioavailability. Knowing the affinity of antimalarials for efflux transporters such as P-glycoprotein (P-gp) may assist in the determination of drug absorption and pharmacokinetic drug interactions during oral absorption in drug combination therapies. Concurrent administration of P-gp inhibitors and P-gp substrate drugs may also result in alterations in the bioavailability of some antimalarials. In-vitro Caco-2 cell monolayers were used here as a model for potential drug absorption related problems and P-gp mediated transport of drugs. Artemisone had the highest permeability at around 50 x 10⁻⁶ cm/sec, followed by amodiaquine around 20 x 10⁻⁶ cm/sec; both mefloquine and artesunate were around 10 x 10⁻⁶ cm/sec. Methylene blue was between 2 and 6 x 10⁻⁶ cm/sec depending on the direction of transport. This 3 fold difference was able to be halved by use of P-gp inhibition. MRP inhibition also assisted the consolidation of the methylene blue transport. Mefloquine was shown to be a P-gp inhibitor affecting our P-gp substrate, Rhodamine 123, although none of the other drugs impacted upon rhodamine123 transport rates. In conclusion, mefloquine is a P-gp inhibitor and methylene blue is a partial substrate; methylene blue may have increased absorption if co-administered with such P-gp inhibitors. An upregulation of P-gp was observed when artemisone and dihydroartemisinin were co-incubated with mefloquine and amodiaquine.     

Perfluoroalkyl Acid Concentrations in Blood Samples Subjected to Transportation and Processing Delay

Background

In studies of perfluoroalkyl acids, the validity and comparability of measured concentrations may be affected by differences in the handling of biospecimens. We aimed to investigate whether measured plasma levels of perfluoroalkyl acids differed between blood samples subjected to delay and transportation prior to processing and samples with immediate processing and freezing.

Methods

Pregnant women recruited at Aarhus University Hospital, Denmark, (n = 88) provided paired blood samples. For each pair of samples, one was immediately processed and plasma was frozen, and the other was delayed and transported as whole blood before processing and freezing of plasma (similar to the Danish National Birth Cohort). We measured 12 perfluoroalkyl acids and present results for compounds with more than 50% of samples above the lower limit of quantification.

Results

For samples taken in the winter, relative differences between the paired samples ranged between -77 and +38% for individual perfluoroalkyl acids. In most cases concentrations were lower in the delayed and transported samples, e.g. the relative difference was -29% (95% confidence interval -30; -27) for perfluorooctane sulfonate. For perfluorooctanoate there was no difference between the two setups [corresponding estimate 1% (0, 3)]. Differences were negligible in the summer for all compounds.

Conclusions

Transport of blood samples and processing delay, similar to conditions applied in some large, population-based studies, may affect measured perfluoroalkyl acid concentrations, mainly when outdoor temperatures are low. Attention to processing conditions is needed in studies of perfluoroalkyl acid exposure in humans.     

Determining specific biomass activity in anaerobic wastewater treatment processes

An experimental method for the measurement of specific gas production rate was developed and tested with biomass samples taken from anaerobic fluidized bed reactors, operating with a variety of carriers with molasses, condensate from cellulose production and brewery wastewater as feeds. The method is based on reactor sampling and offline gas volume measurement during a known time interval. Important factors are biomass and liquid sampling under oxygen-free conditions, using the liquid from the reactor as substrate, providing sufficient mixing and maintaining the physical integrity of the biomass. The method was developed in such a way that small samples (20 ml) were taken under anaerobic conditions (poising agent) for short-term (2–3 min.) gas rate measurements in a small fluidized bed (25 ml) batch reactor with U-tube. Biomass content was measured by an instrumental nitrogen method (Dumas), followed by weight determination of the carrier. The gas rates measured with the test system, and their dependence on substrate concentration, were in good agreement with those directly measured from the continuous fluidized bed reactor. Additions of molasses and acetate to the sample proved that the influence of concentration on the biomass activity can be obtained only by operating the continuous reactor at the concentration levels of interest. Comparison between the reactors showed large differences in the specific activity and the total reactor activity. It was found when comparing two reactors, that the values of the specific and the total activities permitted the calculation of the relative biomass quantities. In this way the influence of the carrier-type could be evaluated.     

Analysis of kinetic data in transport studies: New insights from kinetic studies of Na+-d-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling,rapid filtration apparatus

Summary Using the fast sampling, rapid filtration apparatus (FSRFA) recently developed in our laboratory (Berteloot et al., 1991.J. Membrane Biol. 122:111–125), we have studied the kinetic characteristics of Na⁺-d-glucose cotransport in brush-border membrane vesicles isolated from normal adult human jejunum. True initial rates of transport have been determined at both 20 and 35°C using a dynamic approach which involves linearregression analysis over nine time points equally spaced over 4.5 or 2.7 sec, respectively. When the tracer rate of transport was studied as a function of unlabeled substrate concentrations added to the incubation medium, a displacement curve was generated which can be analyzed by nonlinear regression using equations which take into account the competitive inhibition of tracer flux by unlabeled substrate. This approach was made imperative since at 20°C, in the presence of high substrate concentrations or 1mm phlorizin, no measurable diffusion was found and the resultant zero slope values cannot be expressed into a classicalv versus S plot. All together, our results support the existence of a single Na⁺-d-glucose cotransport system in these membranes for which Na⁺ is mandatory for uptake. This conclusion is at variance with that of a recent report using the same preparation (Harig et al., 1989.Am J. Physiol. 256:8618–8623). Since the discrepancy seems difficult to resolve on the consideration of experimental conditions alone, we have determined the kinetic parameters ofd-glucose transport using one time point measurements and linear transformations of the Michaelis-Menten equation, in order to investigate the potential problems of such a widely used procedure. Comparing these approaches, we conclude that: (i) the dynamic uptake measurements give a better understanding of the different uptake components involved; (ii) it does not matter whether a dynamic or a one time point approach is chosen to generate the uptake data provided that a nonlinear-regression analysis with proper weighting of the data points is performed; (iii) analytical procedures which rely on linearization of Michaelian process(es) are endowed with a number of difficulties which make them unsuitable to resolve multicomponent systems in transport studies. A more general procedure which uses a nonlinear-regression analysis and a displacement curve is proposed since we demonstrate that it is far superior in terms of rapidity, data interpretation, and visual information.     

Analysing the performance of a micro soil solution sampling device in a laboratory examination and a field experiment

The performance of a micro soil solution sampling device was tested in a laboratory examination and in a field experiment. The instrument allows detection of temporal and spatial changes in soil solution chemistry at a spatially high resolution. The flexible tube of the suction cell is made of a porous polymer with a diameter of 2.3 mm. To achieve more stability and to minimize disturbance of the instrument during field installation, the original device was modified by embedding the suction cell in a stainless steel and pressure absorbing corpus. During a laboratory test the new sampling system was compared to ceramic P-80 suction cells. Solution samples taken with the new device adapted more quickly to the given concentrations compared to the ceramic suction cells. In a field test, micro samplers were implanted in an existing soil solution monitoring plot, equipped with standard ceramic samplers. Bi-weekly sampling using the micro cells indicated high temporal and spatial variation, and in June 1995 it was possible, to identify a distinct nitrification. However, in a statistical comparison of the entire sampling period and respective sub-sampling areas the two sampler types indicated identical concentration ranges for nitrate. It is concluded that the new micro samplers can help to identify processes in soils which may cause short-term changes in the soil solution chemistry, whereas the standard sampling technique with ceramic cells seems to be still a suitable tool if long-term mean soil solution concentrations are to be measured.     

Sampling dust from human dwellings to estimate the prevalence ofDermatophagoides mite and cat allergens

Summary Quantification of specific allergens in household dust samples may provide important information for selecting appropriate allergen control methods, and monitoring efficacy and compliance. The purpose of this study was to investigate the source of variation in mite and cat allergen measurements associated with dust sample collection. Discrete and composite dust samples were collected on a filter using a special vacuum sampling device. Aqueous extracts of the dust samples were prepared thenDer p I,Der f I, andFel d I were quantitated by enzyme immunoassays (EIA). Mite and cat allergens were frequently detected in dust samples from human dwellings, and the amounts of these allergens varied significantly (p<0.01) among dwellings. The differences of allergen measurements among duplicate samples taken immediately and up to three weeks later appear to be much smaller than differences among houses and between rooms. Variation among dust samples taken from living rooms and bedrooms of the same dwelling suggest differences in allergen reservoirs. Composite samples formed by sampling specific objects within a room may provide a reliable estimate of allergen exposure in that room. Dust samples from discrete objects are useful to find and monitor specific reservoirs of mite and cat allergens.     

Short-term variability of chlorophyll a concentrations in Lake Ontario

Research on aquatic ecosystems has shown the presence of diel (24 hour) variations of chlorophylla. In order to investigate this phenomenon in the Great Lakes, an IFYGL program was set up in which 8 cruises were made roughly monthly between April and October 1972. Two Lake Ontario Stations were chosen, one inshore near Oshawa, Ontario and the other mid-lake. Chlorophylla samples were taken approximately every two hours at depths of 1, 5 and 1o m at the inshore station and 1, 10 and 20 m at the mid-lake station.Chlorophylla concentrations at the 1 m depth were reduced during periods of high light intensities, but this phenomenon was observed only during the late June and July cruises at the near-shore station. Apparently, during most cruises, light intensities were too low to produce this effect. Coefficients of variation of chlorophylla concentrations as great as too% occurred at the deeper sampling depth during periods when the thermocline was well developed. This variability was associated with a thermocline motion in which the fixed depths sampled moved from the epilimnion into the thermocline or the hypolimnion and back again over the sampling period. When the lake was not thermally stratified, variability of chlorophylla concentrations was also observed with coefficients of variations between 20–3o%. Horizontal advection by currents, vertical turbulence, and presence of Langmuir cells were possible mechanisms causing such chlorophylla variability.In light of these observations, caution must be used in interpreting spatial and temporal distributions of chlorophylla in lakes. Special care must be taken when sampling at depths near the thermocline due to movements that take place. For this reason, water samples taken from the epilimnion during periods of thermal stratification are recommended as opposed to fixed depth or integrated samples which could be affected by thermocline movements.This study was undertaken as part of the International Field Year for the Great Lakes, a joint Canadian-U.S. contribution to the International Hydrological Decade Program     

Some factors influencing the microdistribution of a population of spined loach,Cobitis taenia (L.)

Monthly observations (October 1972–October 1974) of a natural population of spined loach, Cobitis taenia (L.), in the River Great Ouse at Newport Pagnell indicated a patchy microdistribution, which varied seasonally. The distribution was clearly linked to the areas of fine substrate, which altered seasonally in position.Flow rates measured at monthly intervals in areas where Cobitis taenia (L.) were present were found to be approximately half those of areas in which Cobitis taenia L. were not found (0.148 m/sec and 0.293 m/sec). Substrate samples from areas where Cobitis taenia L. were present had a fine organic component, in contrast to the remainder of the river bed, which consisted of hard-packed gravel.In the laboratory, choice chamber experiments demonstrated a positive selection for fine organic deposits.     

Single Versus Multiple Sampler Comparisons

Experiments comparing spore concentrations obtained by a single versus multiple Rotorod samplers in a 2 m×2 m×2 m grid were conducted within similar ecological areas and environmental conditions. Mean spore concentrations obtained by samplers at the same height above ground level were not significantly different at the p=0.05 level in all tests. However, significant differences in spore concentrations were measured among samplers operating at different heights above ground level when wind velocities were greater than 5 m/sec and no free moisture was on the spore bearing tissue. Samplers operating near a spore source measured significant differences in spore concentrations at various heights above ground but not among samplers at the same height. A single volumetric sampler will adequately measure spore concentrations for powering simulation models, but care must be taken to determine the location of the sampling unit in relationship to spore source and ecological or environmental conditions.     

Comparison between two methods of assaying relative microbial activity in marine environments.

Two methods for determining relative microbial activity in the marine environment were compared. In one method, a single concentration of a labeled substrate was used to calculate rates of substrate utilization; in the other, multiple concentrations of the same substrate (heterotrophic activity method) were used to calculate maximum potential substrate utilization rates. These studies were made on 232 seawater and 79 sediment samples taken from a variety of marine environments. The highest correlations between these two methods were seen in the sediment samples tested. The lowest correlation coerfficient seen in the sediment samples was 0.90, and the highest was 0.98. In seawater samples (six studies), the lowest correlation coefficient was 0.77 and the highest was 0.95. The correlation between these two methods was also substrate concentration dependent. Higher correlation coefficients were observed when higher substrate concentrations were used. Under certain conditions, these two methods appear to be comparable for estimating relative levels of microbial activity in the marine environment.     

Comparison between two methods of assaying relative microbial activity in marine environments.

Two methods for determining relative microbial activity in the marine environment were compared. In one method, a single concentration of a labeled substrate was used to calculate rates of substrate utilization; in the other, multiple concentrations of the same substrate (heterotrophic activity method) were used to calculate maximum potential substrate utilization rates. These studies were made on 232 seawater and 79 sediment samples taken from a variety of marine environments. The highest correlations between these two methods were seen in the sediment samples tested. The lowest correlation coerfficient seen in the sediment samples was 0.90, and the highest was 0.98. In seawater samples (six studies), the lowest correlation coefficient was 0.77 and the highest was 0.95. The correlation between these two methods was also substrate concentration dependent. Higher correlation coefficients were observed when higher substrate concentrations were used. Under certain conditions, these two methods appear to be comparable for estimating relative levels of microbial activity in the marine environment.     

Microbial Decomposition in Aquatic Environments: Combined Process of Extracellular Enzyme Activity and Substrate Uptake

The aim of this study was to define a model for the coupling between extracellular enzyme activity and substrate uptake by bacterial populations in natural waters. The balance between uptake of leucine and extracellular hydrolytic production of leucine from a peptide model substrate was investigated in a combined fluorescence-radiotracer experiment with [H]leucine as a marker for the leucine pool and l-leucine-4 methyl-7-coumarinylamide (Leu-MCA) as a marker for the pool of dissolved peptide substrates. Results show that at low concentrations of the model substrate the input and uptake processes of leucine are nearly balanced, whereas at high concentrations of the model substrate much more leucine is liberated than taken up. In addition, samples from one polluted and one less polluted station in the Kiel Fjord were investigated for their extracellular enzymatic and uptake properties in an annual cycle. It was found that turnover rates of leucine (T(r), percent per hour) and hydrolysis rates of Leu-MCA (H(r), percent per hour), as well as the quotient T(r)/H(r), reflect the impact of environmental conditions on decomposition processes at both sampling sites. The quotient T(r)/H(r) is interpreted as an indirect measurement of the pool size ratio (polymers/monomers), which may serve as an index of hydrolysis-uptake coupling in bacterial utilization of dissolved protein. Calculated on an annual average basis, turnover rates are ca. nine times higher than hydrolysis rates at the polluted station and ca. five times higher at the less polluted station. From the described model, this would mean that the relative fraction of polymers within the total dissolved organic carbon pool (with regard to the substrate combination dissolved protein-leucine) is about twice that at the polluted than at the less polluted station.     

Inshore migration,seasonal distribution and sizes of larval bonefish,Albula, in the Gulf of California

Synopsis The leptocephalous larval stages of the bonefish (Albulidae: Albula) are abundant in the coastal regions and hypersaline lagoons (esteros) throughout the Gulf of California during winter and spring. Inshore movements of leptocephali were studied by sampling the entrance to Estero Miramar, at Guaymas, Sonora, Mexico, from February to May, 1981. Thirty-three samples were taken at various times during flood tide. All but four of the samples were taken after sunset. Leptocephali were collected in all months except May. The results suggest that leptocephali enter the estero only during the first few hours of flood tide. Large numbers of larvae were observed during the day at ebb tide maintaining their near-shore position in the entrance channel. These larvae did not appear to be migrating into the estero. This, together with the absence of larvae in daytime samples, suggests that the migration is nocturnal. Most of the 667 leptocephali collected during the sampling period had completed larval growth or were just beginning the metamorphic interval. The median standard length (SL) was 61 mm and the mode was 62 mm. The largest leptocephali measured 71 mm SL. Since metamorphosis is accompanied by a decrease in length, it was concluded that the maximum size attained during larval growth is about 71 mm SL.     

Taking the stress out of blood collection: comparison of field blood‐sampling techniques for analysis of baseline corticosterone

Many ecological studies use stress hormones to assess the condition, health or disturbance levels of wild organisms. Common blood sampling protocols for this research involve trapping individuals and taking blood within three minutes to obtain a “baseline” for analysis of stress hormones (“conventional method”). In some situations it may be difficult to get an accurate measure of baseline values; therefore, alternative sampling techniques may be preferable. We compared corticosterone levels in samples taken via a newly developed, minimally invasive blood sampling technique with corticosterone levels in blood taken via the conventional method. We collected samples from incubating adult common terns Sterna hirundo via blood sucking bugs (Heteroptera, Triatominae) contained in “dummy eggs” (“bug method”) and compared measured corticosterone concentrations to concentrations in blood taken from the same birds using the conventional method. We found no significant differences in mean or variance of baseline corticosterone levels between samples collected via the different methods. This suggests that the bug method offers a viable alternative for hormone sampling.     

Effects on transport of rapidly penetrating,competing substrates: Activation and inhibition of the choline carrier in erythrocytes by imidazole

Summary The properties of the choline transport system are fundamentally altered in saline solution containing 5mm imidazole buffer instead of 5mm phosphate: (i) The system no longer exhibits accelerated exchange. (ii) Choline in the external compartment fails to increase the rate of inactivation of the carrier by N-ethylmaleimide. (iii) Depending on the relative concentrations of choline and imidazole, transport may be activated or inhibited. The maximum rates are increased more than fivefold by imidazole, but at moderate substrate concentrations activation is observed with low concentrations of imidazole and inhibition with high concentrations. (iv) The imidazole effect is asymmetric, there being a greater tendency to activate exit than entry. All this behavior is predicted by the carrier model if imidazole is a substrate of the choline carrier having a high maximum transport rate but a relatively low affinity, and if imidazole rapidly enters the cell by simple diffusion, so that it can add to carrier sites on both sides of the membrane. Addition at thecis side inhibits, and at thetrans side activates. According to the carrier model, asymmetry is a necessary consequence of the potassium ion gradient in red cells, potassium ion being another substrate of the choline system.     

Effects of time and watershed characteristics on the concentration of Cryptosporidium oocysts in river water.

Water samples were collected from four locations on two rivers in Washington State and analyzed by membrane filtration-immunofluorescence assay to establish Cryptosporidium oocyst concentrations. Sampling locations were selected to evaluate effects of watershed character, from pristine mountain to downstream agricultural, on oocyst concentrations. Samples were collected at six biweekly intervals from late June to early September, with two additional sets of five samples taken on separate days (one set taken at bihourly intervals and one set taken simultaneously). Cryptosporidium oocysts were found in 34 of 35 samples at concentrations ranging from about 0.2 to 65 oocysts per liter. Oocyst concentrations were highest early in the sampling period, when they were influenced by postrainfall runoff, and decreased through the summer months. Oocyst concentrations found in ten samples collected on two days (5 samples per day) showed no short-term variations. Oocyst concentrations and oocyst production per square mile (ca. 2.6 km2) of watershed found in water draining a controlled public water supply watershed were the lowest observed. The concentrations and production rates for drainage from an adjacent, comparable, but uncontrolled watershed were nearly 10 times higher. The concentration and production rates of the downstream area influenced by dairy farming were nearly 10 times higher than rates at the upstream stations. The data showed clearly that oocyst concentrations were consistently observed above the detection limit of the analytical method, about 0.1 oocysts per liter; that oocyst concentrations were continuous as opposed to intermittent; and that watershed character and management affected surface water oocyst concentrations significantly.     

Effects of time and watershed characteristics on the concentration of Cryptosporidium oocysts in river water.

Water samples were collected from four locations on two rivers in Washington State and analyzed by membrane filtration-immunofluorescence assay to establish Cryptosporidium oocyst concentrations. Sampling locations were selected to evaluate effects of watershed character, from pristine mountain to downstream agricultural, on oocyst concentrations. Samples were collected at six biweekly intervals from late June to early September, with two additional sets of five samples taken on separate days (one set taken at bihourly intervals and one set taken simultaneously). Cryptosporidium oocysts were found in 34 of 35 samples at concentrations ranging from about 0.2 to 65 oocysts per liter. Oocyst concentrations were highest early in the sampling period, when they were influenced by postrainfall runoff, and decreased through the summer months. Oocyst concentrations found in ten samples collected on two days (5 samples per day) showed no short-term variations. Oocyst concentrations and oocyst production per square mile (ca. 2.6 km2) of watershed found in water draining a controlled public water supply watershed were the lowest observed. The concentrations and production rates for drainage from an adjacent, comparable, but uncontrolled watershed were nearly 10 times higher. The concentration and production rates of the downstream area influenced by dairy farming were nearly 10 times higher than rates at the upstream stations. The data showed clearly that oocyst concentrations were consistently observed above the detection limit of the analytical method, about 0.1 oocysts per liter; that oocyst concentrations were continuous as opposed to intermittent; and that watershed character and management affected surface water oocyst concentrations significantly.     

SKIN SWABBING FOR GENETIC ANALYSIS: APPLICATION TO DUSKY DOLPHINS (LAGENORHYNCHUS OBSCURUS)

Exfoliated skin was collected from bowriding dolphins with the use of a sterilized nylon scrub pad affixed to a wooden dowel. Initial tests of the effectiveness of the technique and dolphin behavioral responses were conducted on dusky dolphins off Kaikoura, New Zealand. During 14 sampling days, 128 contacts using this procedure were made with bowriding dolphins, of which 114 showed behavioral response. Responses during sampling were mild, with 11% of contacted individuals showing no visible response, and 66% of individuals which could be monitored for 30 sec after contact returning to bowride within 30 sec. Mean return time was 10 sec postcontact and did not vary significantly for groups of different sizes. Behavioral controls suggest that a proportion of responses could be explained by typical dolphin behavior in the presence of boats. Seventy-eight percent of contacts resulted in successful collection of tissue samples. Sample time was three minutes on average. Size of group and behavioral state did not appear to influence sample time. Preliminary genetic analyses revealed that tissue collected by this technique was suitable for amplification and sequencing of mitochondrial DNA (mtDNA) via PCR. Comparisons of mtDNA control region sequences with those from known L. obscurus and other delphinid samples verified that this technique was robust against contamination from elements in sea water.     

Inhibition of thymidine uptake in asynchronous HeLa cells by nitrobenzylthioinosine

Nitrobenzylthioinosine (NBMPR), an inhibitor of nucleoside transport by human erythrocytes, was found to be a potent inhibitor of thymidine uptake by asynchronous monolayer cultures of HeLa cells. Rates of thymidine uptake by the cultures at 20 °C were constant between 10 and 40 sec after thymidine addition and were assayed during this interval; TTP was the principal metabolite of thymidine and the thymidine phosphates accumulated at constant rates which extrapolated through time zero. The lack of an effect of NBMPR on thymidine kinase activity, or on the relative proportions of thymidine metabolites in cell extracts, indicated that NBMPR inhibited thymidine transport. When mediated entry (transport) was eliminated by 2 μM NBMPR, a significant diffusional component of thymidine entry was apparent. The mediated component of thymidine uptake exhibited Michaelis-Menten kinetics and apparent K_m and V_max values of 0.5 μM and 10–21 pmoles/min/10⁶ cells were obtained. When NBMPR-treated cells were transferred to NBMPR-free medium, inhibition of thymidine uptake persisted, suggesting that NBMPR was firmly bound to the transport inhibitory sites.