Effects of the Actin‐Stabilizing Drug,Jasplakinolide, on Pigment Granule Motility in Isolated Retinal Pigment Epithelial (RPE) Cells of Green Sunfish,Lepomis cyanellus

The retinal pigment epithelium (RPE) of teleosts contains pigment granules that migrate in response to changes in light condition. Dissociated, cultured RPE cells in vitro can be triggered to aggregate or disperse pigment granules by the application of cAMP or dopamine, respectively. Previous research using the actin‐disrupting drug, cytochalasin D, suggested that pigment granule motility is actin dependent. To further examine the role of actin in pigment granule motility, we tested the effects of the actin‐stabilizing drug, jasplakinolide, on pigment granule motility. Pigment granules in previously dispersed RPE cells remained dispersed after jasplakinolide exposure (0.1–1 μM), but the drug halted movement of most pigment granules and stimulated rapid bi‐directional movements in a small subset of granules. Jasplakinolide also blocked net pigment granule aggregation and interfered with the maintenance of full aggregation. Although jasplakinolide did not block pigment granule dispersion, it did alter the motility of dispersing granules compared to control cells; rather than the normal saltatory, primarily centrifugal movements, granules of jasplakinolide‐treated cells demonstrated slow, creeping centrifugal movements and more rapid bi‐directional movements. Jasplakinolide also altered cell morphology; the length and thickness of apical projections increased, and enlarged, paddle‐like structures, which contained F‐actin appeared at the tips of projections. Actin antibody labeling of jasplakinolide‐treated cells revealed a more reticulated network of actin compared to antibody‐labeled control cells. These results indicate that jasplakinolide‐induced disruption of the actin network compromises normal pigment granule dispersion and aggregation in isolated RPE cells, thus providing further evidence that these movements are actin dependent.     

Differential Effect of Selected Phenoxy-acid Compounds on Oxidative Phosphorylation of Mitochondria from Vicia faba L.

The effect of six phenoxy-acid herbicides, 4-chloro-2-methylphenoxyaceticacid (MCPA), 4-(4-chloro-2-thylphenoxy)butyric acid (MCPB),2, 4-dichlorophenoxyacetic acid (2, 4-D), 4-(2, 4-dichlorophenoxy)butyricacid (2, 4-DB), 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T),and 4-(2, 4, 5-trichlorophenoxy)butyric acid (2, 4, 5-TB) onoxidative phosphorylation of mitochondria isolated from younghypocotyls of Vicia faba L. has been investigated. When NADHwas used as substrate all the test herbicides were found tostimulate state 4 respiration with the loss of phosphorylationand respiratory control in varying degrees. When malate andsuccinate were used separately as substrates, treatment with2, 4-DB, 2, 4, 5-T, and 2, 4, 5-TB at low concentration resultedin a marked stimulation of state 4 respiration; this effectwas not obtained with MCPA, MCPB, or 2, 4-D. At higher concentrationsall herbicides strongly inhibited respiration. These compoundsreleased oligomycin inhibition during NADH oxidation in varyingdegrees, stimulated mitochondrial adenosine-triphosphatase activity,and induced swelling of isolated mitochondria. In many respectsand in differing degrees they resemble 2, 4-dinitrophenol (DNP)in their action as uncouplers. Phenoxy-butyric acids were foundto be more toxic in vitro as uncouplers than their correspondingphenoxyacetic acids. Phenoxyacetic acids were very active as uncouplers in vivo whilephenoxybutyric acids had negligible effect. It is concludedthat in vivo, non-activity of phenoxybutyric acids is due totheir restricted entry into plants and that if available atthe site of action they would be inherently toxic.     

Diminished transmission of drug resistant HIV-1 variants with reduced replication capacity in a human transmission model

Background

Different patterns of drug resistance are observed in treated and therapy naïve HIV-1 infected populations. Especially the NRTI-related M184I/V variants, which are among the most frequently encountered mutations in treated patients, are underrepresented in the antiretroviral naïve population. M184I/V mutations are known to have a profound effect on viral replication and tend to revert over time in the new host. However it is debated whether a diminished transmission efficacy of HIV variants with a reduced replication capacity can also contribute to the observed discrepancy in genotypic patterns.As dendritic cells (DCs) play a pivotal role in HIV-1 transmission, we used a model containing primary human Langerhans cells (LCs) and DCs to compare the transmission efficacy M184 variants (HIV-M184V/I/T) to HIV wild type (HIV-WT). As control, we used HIV harboring the NNRTI mutation K103N (HIV-K103N) which has a minor effect on replication and is found at a similar prevalence in treated and untreated individuals.

Results

In comparison to HIV-WT, the HIV-M184 variants were less efficiently transmitted to CCR5⁺ Jurkat T cells by both LCs and DCs. The transmission rate of HIV-K103N was slightly reduced to HIV-WT in LCs and even higher than HIV-WT in DCs. Replication experiments in CCR5⁺ Jurkat T cells revealed no apparent differences in replication capacity between the mutant viruses and HIV-WT. However, viral replication in LCs and DCs was in concordance with the transmission results; replication by the HIV-M184 variants was lower than replication by HIV-WT, and the level of replication of HIV-K103N was intermediate for LCs and higher than HIV-WT for DCs.

Conclusions

Our data demonstrate that drug resistant M184-variants display a reduced replication capacity in LCs and DCs which directly impairs their transmission efficacy. As such, diminished transmission efficacy may contribute to the lower prevalence of drug resistant variants in therapy naive individuals.

     

Fluorescein-Conjugated Folate as an Indicator of Specific Folate Binding to Paramecium1

Normal Paramecium tetraurelia cells stained with fluorescein-conjugated folate show intense fluorescence that can be reduced to near background autofluorescence with excess K₂-folate, but not with excess KCl. Mutant d4–534, which is not attracted to folate and does not specifically bind ³H-folate, shows reduced fluorescence when stained. This method of monitoring specific folate binding to cells can be adapted to a microscale for rapid screening of clones since cells are routinely fixed and stained in microtiter wells.     

Coordinated approaches to quantify long‐term ecosystem dynamics in response to global change

Many serious ecosystem consequences of climate change will take decades or even centuries to emerge. Long‐term ecological responses to global change are strongly regulated by slow processes, such as changes in species composition, carbon dynamics in soil and by long‐lived plants, and accumulation of nutrient capitals. Understanding and predicting these processes require experiments on decadal time scales. But decadal experiments by themselves may not be adequate because many of the slow processes have characteristic time scales much longer than experiments can be maintained. This article promotes a coordinated approach that combines long‐term, large‐scale global change experiments with process studies and modeling. Long‐term global change manipulative experiments, especially in high‐priority ecosystems such as tropical forests and high‐latitude regions, are essential to maximize information gain concerning future states of the earth system. The long‐term experiments should be conducted in tandem with complementary process studies, such as those using model ecosystems, species replacements, laboratory incubations, isotope tracers, and greenhouse facilities. Models are essential to assimilate data from long‐term experiments and process studies together with information from long‐term observations, surveys, and space‐for‐time studies along environmental and biological gradients. Future research programs with coordinated long‐term experiments, process studies, and modeling have the potential to be the most effective strategy to gain the best information on long‐term ecosystem dynamics in response to global change.     

Ingestion, transmission, and persistence of Chino del tomate virus (CdTV), a New World begomovirus, by Old and New World biotypes of the whitefly vector Bemisia tabaci

Two whitefly biotypes of Bemisia tabaci, from either the Eastern or Western Hemisphere, respectively, were compared with respect to their competency to ingest and their efficiency to transmit the New World begomovirus, Chino del tomate virus (CdTV). The AZ A biotype of B.tabaci originates from the arid southwestern USA and northwestern Mexico, while the B biotype has an origin in the Middle East or Northern Africa. The ability of these two vector biotypes to ingest and subsequently to transmit CdTV were evaluated for an acquisition‐access period (AAP) that ranged from 0 to 72 h, followed by a 48 h inoculation‐access period (IAP). Individual adult whiteflies were monitored for CdTV ingestion using polymerase chain reaction (PCR) to detect the viral coat protein gene (AV1 ORF), and transmission efficiency (frequency) was determined by allowing potentially viruliferous whiteflies access to tomato seedlings following each experimental AAP. PCR results for individual adult whiteflies indicated that CdTV was ingested from infected tomato plants by both biotypes 93% of the time. Transmission frequencies by both vector biotypes increased with longer AAPs. However, the AZ A biotype transmitted CdTV 50% of the time, compared to only 27% for the B biotype. Evidence that virus was ingested with equal competency by the A and B biotypes confirmed that both vectors were capable of ingesting CdTV from tomato at the same frequency, even when the AAP was 0.5 h. Consequently, either the acquisition and/or transmission stages of the pathway, rather than ingestion competency, were responsible for differences in vector‐mediated transmissibility. Detection frequency of CdTV, after 48 h AAP, by PCR in single females of AZ B biotype was significantly higher than males.     

Lipid Metabolism in Fucus serratus as Modified by Environmental Factors

The effect of changes in the environment on lipid metabolismhas been studied in the brown alga, Fucus serratus L. Lightstimulated the incorporation of radioactivity from /{^I4C/}acetateinto oleic and, especially, into linoleic acid. The same effectwas caused by lowering the incubation temperature from 15 °Cto 4 °C. Incubations in the presence of Cd ^{+ +}, Pb ^{+ +} orZn^{+ +} had no effect on the total uptake of /{¹⁴C/}acetate intothe frond tip samples, but lowered the labelling of total lipidsrelative to aqueous-soluble components. However, pre-exposureof the algae to heavy metal cations caused changes in the uptakeof radioactivity but had less effect on the relative labellingof lipids than incubations in the presence of heavy metal cations.Algae collected from sites where the dissolved levels of heavymetals were elevated, showed a decrease in the relative labellingof lipids from /{¹⁴C/}acetate. Concentrations of Cd^{+ +}, Pb⁺⁺ or Zn^{+ +} at 10 x levels found at the collection site had littleeffect on the pattern of fatty acids made by Fucus serratus. Key words: Lipid metabolism, Fucus serratus L., Environmental changes     

Thirty‐two single nucleotide polymorphism markers for high‐throughput genotyping of sockeye salmon

We characterize 32 single nucleotide polymorphism genotyping assays for resolving genotypic variation in sockeye salmon Oncorhynchus nerka in the Pacific Rim. These assays are based on the 5′‐nuclease reaction and thus facilitate high‐throughput genotyping with minimal optimization time. Minor allele frequency differences (Δq) among collections were between 4.7% and 97.9%, resulting in per locus F_ST estimates of 0.02–0.71 with an average of 0.22.     

Methyl bromide emissions to the atmosphere from temperate woodland ecosystems

The environmental importance of methyl bromide (CH₃Br) arises from its contribution to stratospheric ozone loss processes and, as a consequence, its emissions from anthropogenic sources are subject to the Montreal Protocol. A better understanding of the natural budget of CH₃Br is required for assessing the benefit of anthropogenic emission reductions and for understanding any potential effects of environmental change on global CH₃Br concentrations. Measurements of CH₃Br flux in temperate woodland ecosystems, in particular, are very sparse, yet these cover a large fraction of terrestrial land surface. Results presented here from 18 months of field measurements of CH₃Br fluxes in four static flux chambers in a woodland in Scotland and from enclosures of rotting wood and deciduous and coniferous leaf litter suggest net emissions from temperate woodlands. Net CH₃Br fluxes in the woodland varied between the chambers, fluctuating between net uptake and net emissions (?73 to 279 ng m^?2 h^?1 across 161 individual measurements), and with no strong seasonality, but with time‐averaged net emission overall [27±57 (1 SD)] ng m^?2 h^?1]. This work demonstrates that scale‐up needs to be based on sufficient individual measurements to provide a reasonably constrained estimate of the long‐term mean. Mean (±1 SD) net CH₃Br emissions from deciduous and coniferous leaf litter were 43 (±33) ng kg^?1 (dry weight) h^?1 and 80 (±37) ng kg^?1 (dry weight) h^?1, respectively, and ～1–2 ng kg^?1 (fresh weight) h^?1 from rotting woody litter. Despite the intrinsic variability, data obtained here consistently point to the conclusion that the temperate forest soil/litter ecosystem is a net source of CH₃Br to the atmosphere.