While K-ras is essential for mouse development, expression of the K-ras 4A splice variant is dispensable

In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras(tmDelta4A/+) mice produced viable K-ras(tmDelta4A/tmDelta4A) offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras(tmDelta4A/tmDelta4A) mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola x C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B.     

Evolution of the CD4 family: teleost fish possess two divergent forms of CD4 in addition to lymphocyte activation gene-3

The T cell coreceptor CD4 is a transmembrane glycoprotein belonging to the Ig superfamily and is essential for cell-mediated immunity. Two different genes were identified in rainbow trout that resemble mammalian CD4. One (trout CD4) encodes four extracellular Ig domains reminiscent of mammalian CD4, whereas the other (CD4REL) codes for two Ig domains. Structural motifs within the amino acid sequences suggest that the two Ig domains of CD4REL duplicated to generate the four-domain molecule of CD4 and the related gene, lymphocyte activation gene-3. Here we present evidence that both of these molecules in trout are homologous to mammalian CD4 and that teleosts encode an additional CD4 family member, lymphocyte activation gene-3, which is a marker for activated T cells. The syntenic relationships of similar genes in other teleost and non-fish genomes provide evidence for the likely evolution of CD4-related molecules in vertebrates, with CD4REL likely representing the primordial form in fish. Expression of both CD4 genes is highest in the thymus and spleen, and mRNA expression of these genes is limited to surface IgM- lymphocytes. consistent with a role for T cell functionality. Finally, the intracellular regions of both CD4 and CD4REL possess the canonical CXC motif involved in the interaction of CD4 with p56LCK, implying that similar mechanisms for CD4+ T cell activation are present in all vertebrates. Our results therefore raise new questions about T cell development and functionality in lower vertebrates that cannot be answered by current mammalian models and, thus, is of fundamental importance for understanding the evolution of cell-mediated immunity in gnathosomes.     

Thermal adaptations to extreme freeze–thaw cycles in the high tropical Andes

Temperature plays a key role in the biology of ectotherms, including anurans, which are found at higher elevations in the tropics than anywhere in the temperate zone. High elevation tropical environments are characterized by extreme daily thermal fluctuation including high daily maxima and nightly freezing. Our study investigated the contrasting operative temperatures of the anurans Telmatobius marmoratus and Pleurodema marmoratum in different environmental contexts at the same elevation and biome above 5,200 m. Telmatobius marmoratus avoids extremes of daily temperature fluctuation by utilizing thermally buffered aquatic habitat at all life stages, with minimal operative temperature variation (range: 4.6–8.0°C). Pleurodema marmoratum, in contrast, experienced operative temperatures from ?3.5 to 44°C and has one of the widest thermal breadths reported for any tropical frog, from >32°C (critical thermal maximum) to surviving freezing periods of 1 and 6 hr down to ?3.0°C. Our findings expand experimental evidence of frost tolerance in amphibians to the widespread Neotropical family Leptodactylidae, the first such evidence of frost tolerance in a tropical amphibian. Our study identifies three strategies (wide thermal tolerance breadth, use of buffered microhabitats, and behavioral thermoregulation), which allow these tropical frogs to withstand the current wide daily thermal fluctuation above 5,000 m.a.s.l. and which may help them adapt to future climatic changes. Abstract in Spanish is available with online material     

Life-cycle assessment and techno-economic analysis of biochar produced from forest residues using portable systems

The International Journal of Life Cycle Assessment - Producing biochar from forest residues can help resolve environmental issues by reducing forest fires and mitigating climate change. However,...     

Cell elongation in Arabidopsis hypocotyls involves dynamic changes in cell wall thickness

Field-emission scanning electron microscopy was used to measure wall thicknesses of different cell types in freeze-fractured hypocotyls of Arabidopsis thaliana. Measurements of uronic acid content, wall mass, and wall volume suggest that cell wall biosynthesis in this organ does not always keep pace with, and is not always tightly coupled to, elongation. In light-grown hypocotyls, walls thicken, maintain a constant thickness, or become thinner during elongation, depending upon the cell type and the stage of growth. In light-grown hypocotyls, exogenous gibberellic acid represses the extent of thickening and promotes cell elongation by both wall thinning and increased anisotropy during the early stages of hypocotyl elongation, and by increased wall deposition in the latter stages. Dark-grown hypocotyls, in the 48 h period between cold imbibition and seedling emergence, deposit very thick walls that subsequently thin in a narrow developmental window as the hypocotyl rapidly elongates. The rate of wall deposition is then maintained and keeps pace with cell elongation. The outer epidermal wall is always the thickest ( approximately 1 mum) whereas the thinnest walls, about 50 nm, are found in inner cell layers. It is concluded that control of wall thickness in different cell types is tightly regulated during hypocotyl development, and that wall deposition and cell elongation are not invariably coupled.     

Poly(ethylene glycol)-containing hydrogel surfaces for antifouling applications in marine and freshwater environments

This work describes the fabrication, characterization, and biological evaluation of a thin protein-resistant poly(ethylene glycol) (PEG)-based hydrogel coating for antifouling applications. The coating was fabricated by free-radical polymerization on silanized glass and silicon and on polystyrene-covered silicon and gold. The physicochemical properties of the coating were characterized by infrared spectroscopy, ellipsometry, and contact angle measurements. In particular, the chemical stability of the coating in artificial seawater was evaluated over a six-month period. These measurements indicated that the degradation process was slow under the test conditions chosen, with the coating thickness and composition changing only marginally over the period. The settlement behavior of a broad and diverse group of marine and freshwater fouling organisms was evaluated. The tested organisms were barnacle larvae (Balanus amphitrite), algal zoospores (Ulva linza), diatoms (Navicula perminuta), and three bacteria species (Cobetia marina, Marinobacter hydrocarbonoclasticus, and Pseudomonas fluorescens). The biological results showed that the hydrogel coating exhibited excellent antifouling properties with respect to settlement and removal.     

Differential sensitivity of fungi to lithium chloride in culture media

Forty species of fungi, representing a range of ecological and taxonomic groups, were tested for their ability to grow on agar media amended with lithium chloride (LiCl) at 1.5, 3 and 6 g l⁻¹. Species of Trichoderma varied considerably in their sensitivity to LiCl; at one week on 6 g l⁻¹ LiCl medium, the growth of seven species of Trichoderma was considerably inhibited; however, by three weeks at this level, four of the species tested were able to attain ≥30 % of control growth. Of the seven species tested, an isolate of T. viride was the most sensitive to LiCl in agar. Eleven other imperfect fungi also showed a range of ability to grow on agar amended with LiCl, from total inhibition to complete lack of inhibition. Six ascomycete fungi were greatly inhibited by LiCl at all levels; however, an isolate of Chaetomium globosum was highly tolerant of LiCl. Seven basidiomycete wood-decay fungi were quite sensitive to LiCl in agar, showing total to nearly total inhibition even at the lowest level; however, after three weeks, an isolate of Postia placenta was nearly uninhibited except at 6 g l⁻¹. Five ectomycorrhizal basidiomycete fungi were totally inhibited by all levels of LiCl; however, one ectomycorrhizal imperfect fungus (Cenococcum graniforme) was able to grow at 3 g l⁻¹ and was uninhibited at 1.5 g l⁻¹. Four zygomycete fungus isolates were nearly unaffected in their growth by all levels of LiCl.     

Extracellular Norepinephrine Clearance by the Norepinephrine Transporter Is Required for Skeletal Homeostasis

Changes in bone remodeling induced by pharmacological and genetic manipulation of β-adrenergic receptor (βAR) signaling in osteoblasts support a role of sympathetic nerves in the regulation of bone remodeling. However, the contribution of endogenous sympathetic outflow and nerve-derived norepinephrine (NE) to bone remodeling under pathophysiological conditions remains unclear. We show here that differentiated osteoblasts, like neurons, express the norepinephrine transporter (NET), exhibit specific NE uptake activity via NET and can catabolize, but not generate, NE. Pharmacological blockade of NE transport by reboxetine induced bone loss in WT mice. Similarly, lack of NE reuptake in norepinephrine transporter (Net)-deficient mice led to reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and mechanical properties associated with low sympathetic outflow and high plasma NE levels. Last, daily sympathetic activation induced by mild chronic stress was unable to induce bone loss, unless NET activity was blocked. These findings indicate that the control of endogenous NE release and reuptake by presynaptic neurons and osteoblasts is an important component of the complex homeostatic machinery by which the sympathetic nervous system controls bone remodeling. These findings also suggest that drugs antagonizing NET activity, used for the treatment of hyperactivity disorders, may have deleterious effects on bone accrual.     

A selective, slow binding inhibitor of factor VIIa binds to a nonstandard active site conformation and attenuates thrombus formation in vivo

The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.     

Statistical convenience vs biological insight: consequences of data transformation for the analysis of fitness variation in heterogeneous environments

In plants, more favourable environmental conditions can lead to dramatic increases in both mean fitness and variance in fitness. This results in data that violate the equality-of-variance assumption of anova, a problem that most empiricists would address by log-transforming fitness values. Using heuristic data sets and simple simulations, we show that anova on log-transformed fitness consistently fails to match the outcome of selection in a heterogeneous environment or its sensitivity to environmental frequency. Only anova based on relative fitness within environments accurately predicts the sensitivity of genotype selection to the frequency of alternative environments. Parallel analyses of variance based on absolute fitness and relative fitness can bracket the expected success of alternative genotypes under hard and soft selection, respectively. For example, for Sinapis arvensis growing in full sun and partial shade treatments, families achieving high fitness in the best environment are favoured under hard selection, whereas soft selection favours different families that achieve consistently good performance across environments. Based on these findings, we recommend that log-transformation of fitness should no longer be standard practice in ecological genetics studies. Weighted anova is a preferable method for dealing with unequal variances, and investigators should also make greater use of techniques such as quantile regression or resampling to describe and evaluate fitness variation across heterogeneous environments.