Right time - right location - right move: TRPs find motors for common functions

TRP channels are localized at specialized sub-cellular compartments like filopodial tips, ciliary structures, growth cones and spines that have importance in the context of several sensory functions. Several motor proteins largely regulate these localizations. Recent studies indicate that both physical and genetic interactions exist between TRP channels with actin and microtubule-based motor proteins. These two groups of proteins share specialized and fine regulation underlying physiological functions. Indeed, mutations causing loss of these interactions and regulations result in development of pathophysiological disorders and syndromes. In this review we analyze the recent progress made in cell-biological, biochemical, electrophysiological and genetic studies and summarize the multi-dimensional crosstalk between TRP channels with different motor proteins.     

In situ FT-IR microscopic study on enzymatic treatment of poplar wood cross-sections

The feasibility of Fourier transform infrared (FT-IR) microscopy to monitor in situ the enzymatic degradation of wood was investigated. Cross-sections of poplar wood were treated with cellulase Onozuka RS within a custom-built fluidic cell. Light-optical micrographs and FT-IR spectra were acquired in situ from normal and tension wood fibers. Light-optical micrographs showed almost complete removal of the gelatinous (G) layer in tension wood. No structural and spectral changes were observed in the lignified cell walls. The accessibility of cellulose within the lignified cell wall was found to be the main limiting factor, whereas the depletion of the enzyme due to lignin adsorption could be ruled out. The fast, selective hydrolysis of the crystalline cellulose in the G-layer, even at room temperature, might be explained by the gel-like structure and the highly porous surface. Young plantation grown hardwood trees with a high proportion of G-fibers thus represent an interesting resource for bioconversion to fermentable sugars in the process to bioethanol.     

Arsenic co-exposure potentiates benzo[a]pyrene genotoxicity

The reactive industrial chemicals acrylamide (AA) and N-methylolacrylamide (MAA) are neurotoxic and carcinogenic in animals, MAA showing a lower potency than AA. The causative agent in AA-induced carcinogenesis is assumed to be the epoxy metabolite, glycidamide (GA), which in contrast to AA gives rise to stable adducts to DNA. The causative agent in MAA induced carcinogenesis is so far not studied. The two AAs were studied in mice and rats using analysis of hemoglobin (Hb) adducts as a measure of in vivo doses and the in vivo micronucleus (MN) assay as an end-point for chromosome damage. Male CBA mice were treated by intraperitoneal (i.p.) injection of three different doses and male Sprague-Dawley rats with one dose of each AA. Identical adducts were monitored from the two AAs [N-(2-carbamoylethyl)valine] and the respective epoxide metabolites [N-(2-carbamoyl-2-hydroxyethyl)valine]. Per unit of administered amount, AA gives rise to higher (three to six times) Hb adduct levels than MAA in mice and rats. Mice exhibit, compared with rats, higher in vivo doses of the epoxy metabolites, indicating that AAs were more efficiently metabolized in the mice. In mouse the two AAs induced dose-dependent increases in both Hb adduct level and MN frequency in peripheral erythrocytes. Per unit of administered dose MAA showed only half the potency for inducing micronuclei compared with AA, although the MN frequency per unit of in vivo dose of measured epoxy metabolite was three times higher for MAA than for AA. No increase in MN frequency was observed in rat bone marrow erythrocytes, after treatment with either AA. This is compatible with a lower sensitivity of the rat than of the mouse to the carcinogenic action of these compounds.     

Engineering E. coli for improved microaerobic pDNA production

Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA⁻, which, together with W3110recA⁻vgb⁺, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production.

     

A Transgenic Prox1-Cre-tdTomato Reporter Mouse for Lymphatic Vessel Research

The lymphatic vascular system plays an active role in immune cell trafficking, inflammation and cancer spread. In order to provide an in vivo tool to improve our understanding of lymphatic vessel function in physiological and pathological conditions, we generated and characterized a tdTomato reporter mouse and crossed it with a mouse line expressing Cre recombinase under the control of the lymphatic specific promoter Prox1 in an inducible fashion. We found that the tdTomato fluorescent signal recapitulates the expression pattern of Prox1 in lymphatic vessels and other known Prox1-expressing organs. Importantly, tdTomato co-localized with the lymphatic markers Prox1, LYVE-1 and podoplanin as assessed by whole-mount immunofluorescence and FACS analysis. The tdTomato reporter was brighter than a previously established red fluorescent reporter line. We confirmed the applicability of this animal model to intravital microscopy of dendritic cell migration into and within lymphatic vessels, and to fluorescence-activated single cell analysis of lymphatic endothelial cells. Additionally, we were able to describe the early morphological changes of the lymphatic vasculature upon induction of skin inflammation. The Prox1-Cre-tdTomato reporter mouse thus shows great potential for lymphatic research.     

Lipid components of aristolochia longa

The hexane extract (non-volatiles) of Aristolochia longa yielded fatty acids, methyl, ethyl, isobutyl and phytyl esters, and polyprenols.     

The effect of specific proline residues on the kinetic stability of the triosephosphate isomerases of two trypanosomes

The effect of specific residues on the kinetic stability of two closely related triosephosphate isomerases (from Trypanosoma cruzi, TcTIM and Trypanosoma brucei, TbTIM) has been studied. Based on a comparison of their β‐turn occurrence, we engineered two chimerical enzymes where their super secondary β‐loop‐α motifs 2 ((βα)₂) were swapped. Differential scanning calorimetry (DSC) experiments showed that the (βα)₂ motif of TcTIM inserted into TbTIM (2Tc) increases the kinetic stability. On the other hand, the presence of the (βα)₂ motif of TbTIM inserted into TcTIM (2Tb) gave a chimerical protein difficult to purify in soluble form and with a significantly reduced kinetic stability. The comparison of the contact maps of the (βα)₂ of TbTIM and TcTIM showed differences in the contact pattern of residues 43 and 49. In TcTIM these residues are prolines, located at the N‐terminal of loop‐2 and the C‐terminal of α‐helix‐2. Twelve mutants were engineered involving residues 43 and 49 to study the effect over the unfolding activation energy barrier (E_A). A systematic analysis of DSC data showed a large decrease on the E_A of TcTIM (ΔE_A ranging from 468 to 678 kJ/mol) when the single and double proline mutations are present. The relevance of Pro43 to the kinetic stability is also revealed by mutation S43P, which increased the free energy of the transition state of TbTIM by 17.7 kJ/mol. Overall, the results indicate that protein kinetic stability can be severely affected by punctual mutations, disturbing the complex network of interactions that, in concerted action, determine protein stability. Proteins 2017; 85:571–579. © 2016 Wiley Periodicals, Inc.     

Autumn migration of Montagu’s harriers Circus pygargus tracked by satellite telemetry

Although there is a general understanding of Montagu’s harriers migration routes and wintering areas, detailed information on the species’ migration is still lacking. However, improvements in satellite tracking technology in recent years, have enabled the study of medium-sized species by means of satellite telemetry. In 2006, ten adult Montagu’s harriers were fitted with satellite transmitters in northeastern Spain and tracked during their autumn migration to their wintering grounds in sub-Saharan Africa. The migration took between 10 and 30 days, and the end point was determined using breakpoint regressions. Whereas some birds had stopovers of more than a week, others stayed at the same site for only 1 or 2 days at the most. The tagged birds ultimately established at wintering grounds located along the border of Mauritania with Mali and Senegal, a distance of nearly 3000 km from the breeding sites. These sites are situated within a small range of latitudes (14° and 17°N), although distributed over a wider range of longitudes (−15°E and −4°E), with some birds occupying sites more than 1000 km apart. The distance covered in 1 day during the migration ranged between 93 and 219 km, with peaks of traveling speed of up to 65 km/h. Harriers were recorded traveling only during daytime, covering the longest distances in the late afternoon, suggesting that they are daytime migrants. Most of the distance was covered between 1500 and 2000 hours, and no traveling was recorded between 2000 and 0500 hours. During migration, harriers flew close to the ground (40–100 m on average). Improved knowledge of the harriers’ exact wintering sites may provide insights on the problems Montagu’s harriers face during the winter, highlighting the need to take into account what happens in both the breeding and wintering grounds to implement successful conservation measures.