Fixation,leachability, and decay resistance of wood treated with some commercial extracts and wood preservative salts

Beech (Fagus orientalis) and Scots pine (Pinus sylvestris) wood blocks were treated with some commercial extracts as well as water-based wood preservative salts at various concentrations to increase retention and fixation. The penetration, fixation, and antifungal properties of different treatment solutions were compared with statistical analysis. Retention levels of solutions were generally higher for Scots pine wood than beech wood. The highest retention levels were seen in wood treated with sumac leaf extract and oak valonia extract. Leaching tests indicated that both wood types treated with sumac extracts showed higher retention levels than wood treated with the other fruit and bark extract solutions. Adding 1% water-based wood preservative salts to valonia and sumac leaf extracts increased the retention levels. Concentrations of more than 1% did not contribute to retention either individually or with salt additions. Three percent and higher concentrations of wood-preserving salts accelerated and increased the amount of leaching. The results showed that the extract alone was resistant to leaching. Mycological tests showed that all extractives were significantly effective against wood decay.     

Amphetamine and Methamphetamine Differentially Affect Dopamine Transporters in Vitro and in Vivo

The psychostimulants d-amphetamine (AMPH) and methamphetamine (METH) release excess dopamine (DA) into the synaptic clefts of dopaminergic neurons. Abnormal DA release is thought to occur by reverse transport through the DA transporter (DAT), and it is believed to underlie the severe behavioral effects of these drugs. Here we compare structurally similar AMPH and METH on DAT function in a heterologous expression system and in an animal model. In the in vitro expression system, DAT-mediated whole-cell currents were greater for METH stimulation than for AMPH. At the same voltage and concentration, METH released five times more DA than AMPH and did so at physiological membrane potentials. At maximally effective concentrations, METH released twice as much [Ca²⁺]_i from internal stores compared with AMPH. [Ca²⁺]_i responses to both drugs were independent of membrane voltage but inhibited by DAT antagonists. Intact phosphorylation sites in the N-terminal domain of DAT were required for the AMPH- and METH-induced increase in [Ca²⁺]_i and for the enhanced effects of METH on [Ca²⁺]_i elevation. Calmodulin-dependent protein kinase II and protein kinase C inhibitors alone or in combination also blocked AMPH- or METH-induced Ca²⁺ responses. Finally, in the rat nucleus accumbens, in vivo voltammetry showed that systemic application of METH inhibited DAT-mediated DA clearance more efficiently than AMPH, resulting in excess external DA. Together these data demonstrate that METH has a stronger effect on DAT-mediated cell physiology than AMPH, which may contribute to the euphoric and addictive properties of METH compared with AMPH.The dopamine transporter (DAT)³ is a main target for psychostimulants, such as d-amphetamine (AMPH), methamphetamine (METH), cocaine (COC), and methylphenidate (Ritalin®). DAT is the major clearance mechanism for synaptic dopamine (DA) (1) and thereby regulates the strength and duration of dopaminergic signaling. AMPH and METH are substrates for DAT and competitively inhibit DA uptake (2, 3) and release DA through reverse transport (4–9). AMPH- and METH-induced elevations in extracellular DA result in complex neurochemical changes and profound psychiatric effects (2, 10–16). Despite their structural and pharmacokinetic similarities, a recent National Institute on Drug Abuse report describes METH as a more potent stimulant than AMPH with longer lasting effects at comparable doses (17). Although the route of METH administration and its availability must contribute to the almost four times higher lifetime nonmedical use of METH compared with AMPH (18), there may also be differences in the mechanisms that underlie the actions of these two drugs on the dopamine transporter.Recent studies by Joyce et al. (19) have shown that compared with d-AMPH alone, the combination of d- and l-AMPH in Adderall® significantly prolonged the time course of extracellular DA in vivo. These experiments demonstrate that subtle structural features of AMPH, such as chirality, can affect its action on dopamine transporters. Here we investigate whether METH, a more lipophilic analog of AMPH, affects DAT differently than AMPH, particularly in regard to stimulated DA efflux.METH and AMPH have been reported as equally effective in increasing extracellular DA levels in rodent dorsal striatum (dSTR), nucleus accumbens (NAc) (10, 14, 20), striatal synaptosomes, and DAT-expressing cells in vitro (3, 6). John and Jones (21), however, have recently shown in mouse striatal and substantia nigra slices, that AMPH is a more potent inhibitor of DA uptake than METH. On the other hand, in synaptosomes METH inhibits DA uptake three times more effectively than AMPH (14), and in DAT-expressing COS-7 cells, METH releases DA more potently than AMPH (EC₅₀ = 0.2 μm for METH versus EC₅₀ = 1.7 μm for AMPH) (5). However, these differences do not hold up under all conditions. For example, in a study utilizing C6 cells, the disparity between AMPH and METH was not found (12).The variations in AMPH and METH data extend to animal models. AMPH- and METH-mediated behavior has been reported as similar (22), lower (20), or higher (23) for AMPH compared with METH. Furthermore, although the maximal locomotor activation response was less for METH than for AMPH at a lower dose (2 mg/kg, intraperitoneal), both drugs decreased locomotor activity at a higher dose (4 mg/kg) (20). In contrast, in the presence of a salient stimuli, METH is more potent in increasing the overall magnitude of locomotor activity in rats yet is equipotent with AMPH in the absence of these stimuli (23).The simultaneous regulation of DA uptake and efflux by DAT substrates such as AMPH and METH, as well as the voltage dependence of DAT (24), may confound the interpretation of existing data describing the action of these drugs. Our biophysical approaches allowed us to significantly decrease the contribution of DA uptake and more accurately determine DAT-mediated DA efflux with millisecond time resolution. We have thus exploited time-resolved, whole-cell voltage clamp in combination with in vitro and in vivo microamperometry and Ca²⁺ imaging to compare the impact of METH and AMPH on DAT function and determine the consequence of these interactions on cell physiology.We find that near the resting potential, METH is more effective than AMPH in stimulating DAT to release DA. In addition, at efficacious concentrations METH generates more current, greater DA efflux, and higher Ca²⁺ release from internal stores than AMPH. Both METH-induced or the lesser AMPH-induced increase in intracellular Ca²⁺ are independent of membrane potential. The additional Ca²⁺ response induced by METH requires intact phosphorylation sites in the N-terminal domain of DAT. Finally, our in vivo voltammetry data indicate that METH inhibits clearance of locally applied DA more effectively than AMPH in the rat nucleus accumbens, which plays an important role in reward and addiction, but not in the dorsal striatum, which is involved in a variety of cognitive functions. Taken together these data imply that AMPH and METH have distinguishable effects on DAT that can be shown both at the molecular level and in vivo, and are likely to be implicated in the relative euphoric and addictive properties of these two psychostimulants.     

LFA-1 Affinity Regulation Is Necessary for the Activation and Proliferation of Naive T Cells

The activation of LFA-1 (lymphocyte function-associated antigen) is a critical event for T cell co-stimulation. The mechanism of LFA-1 activation involves both affinity and avidity regulation, but the role of each in T cell activation remains unclear. We have identified antibodies that recognize and block different affinity states of the mouse LFA-1 I-domain. Monoclonal antibody 2D7 preferentially binds to the low affinity conformation, and this specific binding is abolished when LFA-1 is locked in the high affinity conformation. In contrast, M17/4 can bind both the locked high and low affinity forms of LFA-1. Although both 2D7 and M17/4 are blocking antibodies, 2D7 is significantly less potent than M17/4 in blocking LFA-1-mediated adhesion; thus, blocking high affinity LFA-1 is critical for preventing LFA-1-mediated adhesion. Using these reagents, we investigated whether LFA-1 affinity regulation affects T cell activation. We found that blocking high affinity LFA-1 prevents interleukin-2 production and T cell proliferation, demonstrated by TCR cross-linking and antigen-specific stimulation. Furthermore, there is a differential requirement of high affinity LFA-1 in the activation of CD4⁺ and CD8⁺ T cells. Although CD4⁺ T cell activation depends on both high and low affinity LFA-1, only high affinity LFA-1 provides co-stimulation for CD8⁺ T cell activation. Together, our data demonstrated that the I-domain of LFA-1 changes to the high affinity state in primary T cells, and high affinity LFA-1 is critical for facilitating T cell activation. This implicates LFA-1 activation as a novel regulatory mechanism for the modulation of T cell activation and proliferation.LFA-1 (lymphocyte function-associated antigen), an integrin family member, is important in regulating leukocyte adhesion and T cell activation (1, 2). LFA-1 consists of the α_L (CD11a) and β₂ (CD18) heterodimer. The ligands for LFA-1, including intercellular adhesion molecule ICAM³-1, ICAM-2, and ICAM-3, are expressed on antigen-presenting cells (APCs), endothelial cells, and lymphocytes (1). Mice that are deficient in LFA-1 have defects in leukocyte adhesion, lymphocyte proliferation, and tumor rejection (3–5). Blocking LFA-1 with antibodies can prevent inflammation, autoimmunity, organ graft rejection, and graft versus host disease in human and murine models (6–10).LFA-1 is constitutively expressed on the surface of leukocytes in an inactive state. Activation of LFA-1 is mediated by inside-out signals from the cytoplasm (1, 11). Subsequently, activated LFA-1 binds to the ligands and transduces outside-in signals back into the cytoplasm that result in cell adhesion and activation (12, 13). The activation of LFA-1 is a critical event in the formation of the immunological synapse, which is important for T cell activation (2, 14, 15). The active state of LFA-1 is regulated by chemokines and the T cell receptor (TCR) through Rap1 signaling (16). LFA-1 ligation lowers the activation threshold and affects polarization in CD4⁺ T cells (17). Moreover, productive LFA-1 engagement facilitates efficient activation of cytotoxic T lymphocytes and initiates a distinct signal essential for the effector function (18–20). Thus, LFA-1 activation is essential for the optimal activation of T cells.The mechanism of LFA-1 activation involves both affinity (conformational changes within the molecule) and avidity (receptor clustering) regulation (21–23). The I-domain of the LFA-1 α_L subunit is the primary ligand-binding site and has been proposed to change conformation, leading to an increased affinity for ligands (24–26). The structural basis of the conformational changes in the I-domain of LFA-1 has been extensively characterized (27). Previously, we have demonstrated that the conformation of the LFA-1 I-domain changes from the low affinity to the high affinity state upon activation. By introducing disulfide bonds into the I-domain, LFA-1 can be locked in either the closed or open conformation, which represents the “low affinity” or “high affinity” state, respectively (28, 29). In addition, we identified antibodies that are sensitive to the affinity changes in the I-domain of human LFA-1 and showed that the activation-dependent epitopes are exposed upon activation (30). This study supports the presence of the high affinity conformation upon LFA-1 activation in cell lines. It has been demonstrated recently that therapeutic antagonists, such as statins, inhibit LFA-1 activation and immune responses by locking LFA-1 in the low affinity state (31–34). Furthermore, high affinity LFA-1 has been shown to be important for mediating the adhesion of human T cells (35, 36). Thus, the affinity regulation is a critical step in LFA-1 activation.LFA-1 is a molecule of great importance in the immune system, and its activation state influences the outcome of T cell activation. Our previous data using the activating LFA-1 I-domain-specific antibody MEM83 indicate that avidity and affinity of the integrin can be coupled during activation (37). However, whether affinity or avidity regulation of LFA-1 contributes to T cell activation remains controversial (23, 38, 39). Despite the recent progress suggesting that conformational changes represent a key step in the activation of LFA-1, there are considerable gaps to be filled. When LFA-1 is activated, the subsequent outside-in signaling contributes to T cell activation via immunological synapse and LFA-1-dependent signaling. It is critical to determine whether high affinity LFA-1 participates in the outside-in signaling and affects the cellular activation of T cells. Nevertheless, the rapid and dynamic process of LFA-1 activation has hampered further understanding of the role of high affinity LFA-1 in primary T cell activation. The affinity of LFA-1 for ICAM-1 increases up to 10,000-fold within seconds and involves multiple reversible steps (23). In addition, the activation of LFA-1 regulates both adhesion and activation of T cells, two separate yet closely associated cellular functions. When LFA-1 is constitutively expressed in the active state in mice, immune responses are broadly impaired rather than hyperactivated, suggesting the complexity of affinity regulation (40). Therefore, it is difficult to dissect the mechanisms by which high affinity LFA-1 regulates stepwise activation of T cells in the whole animal system.In the present study, we identified antibodies recognizing and blocking different affinity states of mouse LFA-1. These reagents allowed us to determine the role of affinity regulation in T cell activation. We found that blocking high affinity LFA-1 inhibited IL-2 production and proliferation in T cells. Furthermore, there is a differential requirement of high affinity LFA-1 in antigen-specific activation of CD4⁺ and CD8⁺ T cells. The activation of CD4⁺ T cells depends on both high and low affinity LFA-1. For CD8⁺ T cell activation, only high affinity LFA-1 provides co-stimulation. Thus, affinity regulation of LFA-1 is critical for the activation and proliferation of naive T cells.     

Structural basis for specific recognition of Lys 63‐linked polyubiquitin chains by NZF domains of TAB2 and TAB3

TAB2 and TAB3 activate the Jun N‐terminal kinase and nuclear factor‐κB pathways through the specific recognition of Lys 63‐linked polyubiquitin chains by its Npl4 zinc‐finger (NZF) domain. Here we report crystal structures of the TAB2 and TAB3 NZF domains in complex with Lys 63‐linked diubiquitin at 1.18 and 1.40 Å resolutions, respectively. Both NZF domains bind to the distal ubiquitin through a conserved Thr‐Phe dipeptide that has been shown to be important for the interaction of the NZF domain of Npl4 with monoubiquitin. In contrast, a surface specific to TAB2 and TAB3 binds the proximal ubiquitin. Both the distal and proximal binding sites of the TAB2 and TAB3 NZF domains recognize the Ile 44‐centred hydrophobic patch on ubiquitin but do not interact with the Lys 63‐linked isopeptide bond. Mutagenesis experiments show that both binding sites are required to enable binding of Lys 63‐linked diubiquitin. We therefore propose a mechanism for the recognition of Lys 63‐linked polyubiquitin chains by TAB2 and TAB3 NZF domains in which diubiquitin units are specifically recognized by a single NZF domain.     

Structural and magnetic diversity in metal-dicyanamido polymer moieties: Paramagnetic and antiferromagnetic 1D chain compound and weakly ferromagnetic 2D motif

The reaction of M(NO₃)₂·xH₂O (M = Cu, Ni and Co; x = 3 for Cu and x = 6 for Co/Ni), imidazole (Im) and sodium dicyanamide (dca) afforded the complexes [M(Im)₂(dca)₂] (where M = Cu for 1, M = Ni for 2, and M = Co for 3). All of them have been characterized structurally by single crystal X-ray diffraction measurements. X-ray analysis reveals that the dicyanamido ligand features the μ_1,3 bridging mode that led to the formation of two-dimensional structure of complex 1 while complexes 2 and 3 attribute an infinite one-dimensional chain like structure to generate the fascinating molecular assemblies. The {N(CN)₂}⁻ ligands present in the complexes 2 and 3 are coordinated in end-to-end (μ_1,5) fashion. All the complexes have distorted octahedral geometry around the central metal ion and coordinated by two amine nitrogen atoms from imidazole ligands and four nitrogen atoms from dca ligands. The variable temperature (2-300 K) magnetic susceptibility measurements showed that the magnetic interaction between the metal centers in the complex 1 is dominantly ferromagnetic while the metal ions in complex 3 are antiferromagnetically coupled. On the contrary, complex 2 is a simple paramagnet. The results of magnetic model are in good agreement with the experimental data.     

Substrate dependent production of extracellular biosurfactant by a marine bacterium

The potential of a marine microorganism to utilize different carbon substrates for the production of an extracellular biosurfactant was evaluated. Among the several carbon substrates tested for this purpose, production of the crude biosurfactant was found to be highest with glycerol (2.9+/-0.11 g L(-1)) followed by starch (2.5+/-0.11 g L(-1)), glucose (1.16+/-0.11 g L(-1)) and sucrose (0.94+/-0.07 g L(-1)). The crude biosurfactant obtained from glycerol, starch and sucrose media had significantly higher antimicrobial action than those obtained from glucose containing medium. RP-HPLC resolved the crude biosurfactants into several fractions one of which had significant antimicrobial action. The antimicrobial fraction was found in higher concentrations in biosurfactant obtained using glycerol, starch and sucrose as compared to the biosurfactants from glucose medium, thereby explaining higher antimicrobial activity. The carbon substrate was thus found to affect biosurfactant production both in a qualitative and quantitative manner.     

Attitudes towards forest,biomass and certification – A case study approach to integrate public opinion in Japan

The purpose of this study was to contribute to filling the knowledge gap in public opinion and knowledge about forest and its certification in Japan, as well as to identify key elements and the possible role of public opinion within integrated bottom-up policies, bridging the sectors of forest, environment and energy. For the study 1930 questionnaires were disseminated in a small town in early 2007. Results from the statistical analysis indicated that forest was perceived as an ecosystem with a protective function against e.g. soil erosion or flooding, rather than a place that might serve for wood production and providing jobs. Forest certification and bioenergy from forest were identified as key elements for future integrated bottom-up policies that need to concentrate on facilitating the linkage between forestry and renewable energy as well as on promoting environmentally sound management and forest certification.