Nucleocytoplasmic shuttling: a novel in vivo property of antisense phosphorothioate oligodeoxynucleotides

Phosphorothioate oligodeoxynucleotides (P=S ODNs) are frequently used as antisense agents to specifically interfere with the expression of cellular target genes. However, the cell biological properties of P=S ODNs are poorly understood. Here we show that P=S ODNs were able to continuously shuttle between the nucleus and the cytoplasm and that shuttling P=S ODNs retained their ability to act as antisense agents. The shuttling process shares characteristics with active transport since it was inhibited by chilling and ATP depletion in vivo. Transport was carrier-mediated as it was saturable, and nuclear pore complex-mediated as it was sensitive to treatment with wheatgerm agglutinin. Oligonucleotides without a P=S backbone chemistry were only weakly restricted in their migration by chilling, ATP depletion and wheatgerm agglutinin and thus moved by diffusion. P=S ODN shuttling was only moderately affected by disruption of the Ran/RCC1 system. We propose that P=S ODNs shuttle through their binding to yet unidentified cellular molecules that undergo nucleocytoplasmic transport via a pathway that is not as strongly dependent on the Ran/RCC1 system as nuclear export signal-mediated protein export, U-snRNA, tRNA and mRNA export. The shuttling property of P=S ODNs must be taken into account when considering the mode and site of action of these antisense agents.     

TGF-beta1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages

Transforming growth factor-beta1 (TGF-beta1), a key cytokine for control of cell growth, extracellular matrix formation, and inflammation control, is secreted by many cells present in the arteriosclerotic plaque. Lipid accumulation in the vessel wall is regarded as an early step in atherogenesis and depends on uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors and their transformation into foam cells. Prominent members of the scavenger receptor family are the class A type I and II receptors (ScR-A), the class B receptor CD36, and the recently detected lectin-like oxidized LDL receptor-1 (LOX-1), which, unlike the native LDL receptor (LDL-R), are not feedback controlled. CD36 is responsible for >50% of modified LDL uptake into human monocyte-derived macrophages. We therefore studied whether TGF-beta1 influences expression and function of ScR-A, CD36, and LOX-1 in monocytes using RT-PCR and flow cytometry. Total uptake of oxidized LDL by monocytoid cells, reflecting the combined function of all scavenger receptors, was significantly reduced by TGF-beta1. At initially low picomolar concentrations, TGF-beta1 decreased CD36 mRNA and protein surface expression and ScR-A mRNA levels in the human monocytic cell line THP-1 and in freshly isolated and cultivated human monocytes, whereas LOX-1 mRNA was increased. Expression of LDL-R and beta-actin was not affected by TGF-beta1. In conclusion, depression of scavenger receptor function in monocytes by TGF-beta1 in low concentrations reduces foam cell formation. Together with matrix control by TGF-beta1, this may be important for atherogenesis and plaque stabilization.     

Physiological Characterization of a Bacterial Consortium Reductively Dechlorinating 1,2,3- and 1,2,4-Trichlorobenzene

A bacterial mixed culture reductively dechlorinating trichlorobenzenes was established in a defined, synthetic mineral medium without any complex additions and with pyruvate as the carbon and energy source. The culture was maintained over 39 consecutive transfers of small inocula into fresh media, enriching the dechlorinating activity. In situ probing with fluorescence-labeled rRNA-targeted oligonucleotide probes revealed that two major subpopulations within the microbial consortium were phylogenetically affiliated with a sublineage within the Desulfovibrionaceae and the gamma subclass of Proteobacteria. The bacterial consortium grew by fermentation of pyruvate, forming acetate, propionate, CO₂, formate, and hydrogen. Acetate and propionate supported neither the reduction of trichlorobenzenes nor the reduction of sulfate when sulfate was present. Hydrogen and formate were used for sulfate reduction to sulfide. Sulfate strongly inhibited the reductive dechlorination of trichlorobenzenes. However, when sulfate was depleted in the medium due to sulfate reduction, dechlorination of trichlorobenzenes started. Similar results were obtained when sulfite was present in the cultures. Molybdate at a concentration of 1 mM strongly inhibited the dechlorination of trichlorobenzenes. Cultures supplied with molybdate plus sulfate did not reduce sulfate, but dechlorination of trichlorobenzenes occurred. Supplementation of electron-depleted cultures with various electron sources demonstrated that formate was used as a direct electron donor for reductive dechlorination, whereas hydrogen was not.Chlorobenzenes are widespread pollutants and accumulate in the food chain due to their hydrophobicity and strong persistence against chemical and microbial degradation (34). Anaerobic reductive dechlorination of chlorinated benzenes was demonstrated for enrichment cultures from biofilm reactors, sewage sludge, river sediment, and soil (3, 4, 15, 16, 22, 31, 37). Dechlorination pathways for all multiply chlorinated benzenes were elucidated (4, 15). Some dechlorination patterns can be rationalized by thermodynamic considerations (3, 13), but little is known about the microorganisms participating in chlorobenzene dechlorination.Anaerobic bacteria transforming chlorobenzoates and/or chlorophenols have been isolated in pure cultures (5, 7, 18, 27, 39, 40, 45, 48). Desulfomonile tiedjei (12), strain 2CP-1 (7), Desulfitobacterium chlororespirans (39), and Desulfitobacterium sp. strain PCE1 (18) grow anaerobically by chlororespiration. So far, it has not been possible to evaluate whether the anaerobic dechlorination of chlorobenzenes proceeds via a similar mechanism, since pure cultures are not available.While the effect of oxygen and nitrate on the dechlorination of chloroaromatics is reported to be negative for most cultures (32), the effect of sulfur oxyanions is controversial. Some reports stated an inhibitory role of sulfate in the reductive dehalogenation of various chlorinated or fluorinated aromatics (17, 19, 25, 26); other studies found only slight inhibition (24), no inhibition (14), or even a stimulated rate of dechlorination (17, 23). For one mixed culture, the mineralization of chlorophenols was concomitantly coupled to the reduction of sulfur oxyanions (20, 21). With pure cultures of D. tiedjei, it could be shown that sulfite and thiosulfate inhibited the dechlorination of 3-chlorobenzoate in growing cells, nongrowing cells, and cell extracts, while sulfate inhibited dechlorination only in growing cells (46).The high toxicity (22) and the low solubility of chlorobenzenes in water prevented the successful isolation of bacteria with chlorobenzenes as electron acceptors. It is therefore essential to study alternative electron acceptors that could be used by chlorobenzene-dechlorinating bacteria and that could substitute for chlorobenzenes during enrichment and isolation. Information about reductive dechlorination of chlorobenzenes in the presence of other electron acceptors is also needed for the evaluation of dechlorination processes at natural sites and for in situ remediation projects. To our knowledge, detailed studies of the effects of alternative electron acceptors on the dechlorination of chlorobenzenes have not been reported so far.The aim of the present study was to describe the physiological properties of a mixed culture effectively dechlorinating trichlorobenzenes and to determine the effects of various specific inhibitors and alternative electron acceptors. For these experiments, we used a stable, sediment-free mixed consortium growing in a defined, synthetic mineral medium. This consortium has been established in our laboratory from a fluidized bed bioreactor (1, 33) and reductively dechlorinates 1,2,3-trichlorobenzene to 1,3-dichlorobenzene and 1,2,4-trichlorobenzene to 1,4- and 1,3-dichlorobenzene. By inhibiting the activity of methanogenic bacteria using the specific inhibitor bromoethanesulfonate (BES), we showed that dechlorination occurs independently from methanogenic bacteria (1), as has also been shown for other enrichment cultures dechlorinating chlorobenzenes (22, 31).     

Use of polymerase chain reaction and electroporation of Escherichia coli to monitor the persistence of extracellular plasmid DNA introduced into natural soils.

A modified protocol for DNA amplification by polymerase chain reaction (PCR) coupled with laser densitometric determination of the amount of PCR products, which allowed quantitation of target sequence numbers in soil extracts, was developed. The method was applied to monitor target loss during incubation of purified plasmid DNA in natural nonsterile soils. It revealed soil-specific kinetics of target loss. After 60 days, 0.2, 0.05, and 0.01% of the initially added nahA genes on plasmids were detectable by PCR in a loamy sand soil, a clay soil, and a silty clay soil, respectively. Electroporation of Escherichia coli was used in parallel to quantitate plasmid molecules in soil extracts by their transforming activity. It was found that transformation by electroporation was about 20 times more efficient and much less inhibited by constituents of soil extracts than transformation of Ca(2+)-treated cells (G. Romanowski, M.G. Lorenz, G. Sayler, and W. Wackernagel, Appl. Environ. Microbiol. 58:3012-3019, 1992). By electroporation, greater than 10,000-fold plasmid loss was monitored in nonsterile soils. Transforming activity was found up to 60 days after inoculation of the soils. The studies indicate that PCR and electroporation are sensitive methods for monitoring the persistence of extracellular plasmid DNA in soil. It is proposed that plasmid transformation by electroporation can be used for the monitoring in soil and other environments of genetically engineered organisms with recombinant plasmids. The data suggest that genetic material may persist in soil for weeks and even for months after its release from cells.     

Physiological role of the alpha1- and alpha2-isoforms of the Na+-K+-ATPase and biological significance of their cardiac glycoside binding site

An interesting feature of Na+-K+-ATPase is that it contains four isoforms of the catalytic alpha-subunit, each with a tissue-specific distribution. Our laboratory has used gene targeting to define the functional role of the alpha1- and alpha2-isoforms. While knockout mice demonstrated the importance of the alpha1- and alpha2-isoforms for survival, the knockin mice, in which each isoform can be individually inhibited by ouabain and its function determined, demonstrated that both isoforms are regulators of cardiac muscle contractility. Another intriguing aspect of the Na+-K+-ATPase is that it contains a binding site for cardiac glycosides, such as digoxin. Conservation of this site suggests that it may have an in vivo role and that a natural ligand must exist to interact with this site. In fact, cardiac glycoside-like compounds have been observed in mammals. Our recent study demonstrates that the cardiac glycoside binding site of the Na+-K+-ATPase plays a role in the regulation of blood pressure and that it mediates both ouabain-induced and ACTH-induced hypertension in mice. Whereas chronic administration of ouabain or ACTH caused hypertension in wild-type mice, it had no effect on blood pressure in mice with a ouabain-resistant alpha2-isoform of Na+-K+-ATPase. Interestingly, animals with the ouabain-sensitive alpha1-isoform and a ouabain-resistant alpha2-isoform develop ACTH-induced hypertension to a greater extent than wild-type animals. Taken together, these results demonstrate that the cardiac glycoside binding of the Na+-K+-ATPase has a physiological role and suggests a function for a naturally occurring ligand that is stimulated by administration of ACTH.     

Differences between single and paired heifers in residency in functional areas, length of travel path, and area used throughout days 1–6 after integration into a free stall dairy herd

The integration of heifers into free stall dairy herds is a frequent management procedure, but little systematic research has been conducted on its effect on cow behavior. Previous studies mainly focused on aggressive interactions, but it is also of interest how integration affects the spatial distribution of both the cows in the herd and the integrated heifers. In the present study we integrated a single and a pair of heifers on each of six Swiss working farms in a balanced order. Using an automatic tracking system, we recorded the positions of all the cows and of the integrated heifers at 1 min intervals for six continuous 24 h periods. From these data we calculated the proportion of time the animals spent in the activity area, at the feed rack and in the lying cubicles, their average path length and the area of the barn that they used. We then compared the behavior of the integrated heifers with that of the cows in the introductory weeks. We also compared the behavior of the cows recorded in the control weeks directly preceding the integration and in the introductory weeks. For evaluation we used linear mixed-effects models. Singly integrated heifers spent a higher proportion of time in the activity area (0.29 vs. 0.14; P < 0.001) and a lower proportion of time in the lying area (0.40 vs. 0.53; interaction with day, P = 0.011) than the cows, whereas the heifers of the pairs mainly spent a lower proportion of time in the feeding area than the cows (0.23 vs. 0.32; interaction with day, P = 0.044). Average path length was longer for the integrated heifers soon after introduction but approached the values of the cows later on (interaction with day, P = 0.012). The total barn area used by a given animal was largest in the cows and was reduced in heifers integrated singly or in pairs (cows: 341/373 m², pairs: 306 m², single heifer: 333 m²; P = 0.055). Cows were little influenced in their space use by the integration of a single or pair of heifers. In summary, the behavior of the singly integrated heifers differed more markedly from that of the cows than the behavior of the heifers introduced in pairs during the introductory week. We would therefore recommend integrating pairs rather than single heifers into herds of dehorned dairy cows to ease their integration.     

Complete ammonia oxidization in agricultural soils: High ammonia fertilizer loss but low N2O production

The contribution of agriculture to the sustainable development goals requires climate-smart and profitable farm innovations. Increasing the ammonia fertilizer applications to meet the global food demands results in high agricultural costs, environmental quality deterioration, and global warming, without a significant increase in crop yield. Here, we reported that a third microbial ammonia oxidation process, complete ammonia oxidation (comammox), is contributing to a significant ammonia fertilizer loss (41.9 ± 4.8%) at the rate of 3.53 ± 0.55 mg N kg⁻¹ day⁻¹ in agricultural soils around the world. The contribution of comammox to ammonia fertilizer loss, occurring mainly in surface agricultural soil profiles (0–0.2 m), was equivalent to that of bacterial ammonia oxidation (48.6 ± 4.5%); both processes were significantly more important than archaeal ammonia oxidation (9.5 ± 3.6%). In contrast, comammox produced less N₂O (0.98 ± 0.44 μg N kg⁻¹ day⁻¹, 11.7 ± 3.1%), comparable to that produced by archaeal ammonia oxidation (16.4 ± 4.4%) but significantly lower than that of bacterial ammonia oxidation (72.0 ± 5.1%). The efficiency of ammonia conversion to N₂O by comammox (0.02 ± 0.01%) was evidently lower than that of bacterial (0.24 ± 0.06%) and archaeal (0.16 ± 0.04%) ammonia oxidation. The comammox rate increased with increasing soil pH values, which is the only physicochemical characteristic that significantly influenced both comammox bacterial abundance and rates. Ammonia fertilizer loss, dominated by comammox and bacterial ammonia oxidation, was more intense in soils with pH >6.5 than in soils with pH <6.5. Our results revealed that comammox plays a vital role in ammonia fertilizer loss and sustainable development in agroecosystems that have been previously overlooked for a long term.     

How will climate novelty influence ecological forecasts? Using the Quaternary to assess future reliability

Future climates are projected to be highly novel relative to recent climates. Climate novelty challenges models that correlate ecological patterns to climate variables and then use these relationships to forecast ecological responses to future climate change. Here, we quantify the magnitude and ecological significance of future climate novelty by comparing it to novel climates over the past 21,000 years in North America. We then use relationships between model performance and climate novelty derived from the fossil pollen record from eastern North America to estimate the expected decrease in predictive skill of ecological forecasting models as future climate novelty increases. We show that, in the high emissions scenario (RCP 8.5) and by late 21st century, future climate novelty is similar to or higher than peak levels of climate novelty over the last 21,000 years. The accuracy of ecological forecasting models is projected to decline steadily over the coming decades in response to increasing climate novelty, although models that incorporate co‐occurrences among species may retain somewhat higher predictive skill. In addition to quantifying future climate novelty in the context of late Quaternary climate change, this work underscores the challenges of making reliable forecasts to an increasingly novel future, while highlighting the need to assess potential avenues for improvement, such as increased reliance on geological analogs for future novel climates and improving existing models by pooling data through time and incorporating assemblage‐level information.     

Blood-derived small Dot cells reduce scar in wound healing

Wounds in fetal skin heal without scar, however the mechanism is unknown. We identified a novel group of E-cadherin positive cells in the blood of fetal and adult mice and named them "Dot cells". The percentage of Dot cells in E16.5 fetal mice blood is more than twenty times higher compared to adult blood. Dot cells also express integrin beta1, CD184, CD34, CD13low and Sca1low, but not CD45, CD44, and CD117. Dot cells have a tiny dot shape between 1 and 7 microm diameters with fast proliferation in vitro. Most of the Dot cells remain positive for E-cadherin and integrin beta1 after one month in culture. Transplantation of Dot cells to adult mice heals skin wounds with less scar due to reduced smooth muscle actin and collagen expression in the repair tissue. Tracking GFP-positive Dot cells demonstrates that Dot cells migrate to wounds and differentiate into dermal cells, which also express strongly to FGF-2, and later lose their GFP expression. Our results indicate that Dot cells are a group of previously unidentified cells that have strong wound healing effect. The mechanism of scarless wound healing in fetal skin is due to the presence of a large number of Dot cells.     

Substance P and secretoneurin in vitreous aspirates of patients with various vitreoretinal diseases

By means of highly sensitive radioimmunoassays, the levels of substance P (SP) and secretoneurin (SN) were detected in vitreous aspirates of patients with macular holes which served as controls, in patients with nonproliferative diabetic retinopathy (DR), active proliferative diabetic retinopathy (active PDR), inactive PDR, rhegmatogenous retinal detachment and proliferative vitreoretinopathy (PVR). Furthermore, SN-like immunoreactivities were characterized by reversed phase-HPLC. The concentration of SN was more than 20-fold higher in macular holes when compared with SP and reversed phase HPLC revealed evidence that the vitreous levels of SN represent authentic SN. SN was significantly decreased in patients with nonproliferative DR, active PDR and inactive PDR by more than 70% which seems to result from a reduced expression and/or secretion from the cilary epithelium and a reduced release from the retina both due to diabetes mellitus. By contrast SP was increased in rhegmatogenous retinal detachment most obviously due to an enhanced outflow of the peptide through retinal breaks. Despite their proangiogenic activities, SP and SN are unlikely to be involved in the pathogenesis of neovascularizations in DR because of their unchanged and reduced levels, respectively, but the low levels of both peptides may facilitate the regression of vasoproliferations following laser photocoagulation.