Placenta growth factor expression is regulated by hydrogen peroxide in vascular smooth muscle cells

When supply arteries become occluded, blood is diverted through preexisting collateral vessels. Shear stress arising from this increase in blood flow provides the initial physiological stimulus for expansion of the collateral circulation, a process termed arteriogenesis. Endothelial cells (EC) respond to increased shear stress by releasing a variety of mediators that can act on underlying smooth muscle cells (SMC). Placenta growth factor (PLGF) is known to mediate certain aspects of arteriogenesis, such as recruitment of monocytes to the vessel wall. Therefore, we tested whether SMC PLGF expression is influenced by mediators released by EC. We used A10 SMC cultured with medium that had been conditioned by EOMA EC for 4 days as a model. We found that EC-conditioned medium is able to upregulate PLGF gene expression in A10 SMC. Further experiments identified hydrogen peroxide (H(2)O(2)) as a key mediator of this response. We confirmed the physiological relevance of this mechanism in primary human coronary artery SMCs by demonstrating that exogenous H(2)O(2) specifically upregulates PLGF gene and protein expression. We also demonstrated that the physiological stimulus of shear stress raises endogenous H(2)O(2) levels in media into the range found to increase PLGF expression. In this study, we demonstrate that EC-released H(2)O(2) acts as a positive regulator of PLGF gene and protein expression in vascular SMC. To our knowledge, this is the first study to describe H(2)O(2) as a regulator of PLGF expression and therefore an upstream mediator of PLGF-driven arteriogenesis.     

Genetic diversity in clinical isolates of Mycobacterium avium complex from Guinea-Bissau, West Africa

Isolates of Mycobacterium avium complex (MAC) were cultured from sputum samples obtained from patients in Guinea-Bissau, West Africa. Twenty-eight isolates hybridising with MAC probe (AccuProbe) were further characterised by different molecular techniques: hybridisation with species-specific probes (AccuProbe) for M. avium and M. intracellulare, partial sequencing of 16S rRNA gene and PCR detection of the DT1-DT6 sequences and the macrophage-induced gene (mig). Only one of the 28 isolates reacted with the M. avium probe and four with the M. intracellulare probe. Two isolates expressed the DT1 sequence, and three the DT6. The mig was detected in 18 (64%) of the isolates. Sequencing of 16S rRNA had the greatest discriminative power of the typing methods applied, without strong correlation with any other technique. Clinical MAC isolates from Guinea-Bissau demonstrated a wide genetic diversity among the members of M. avium complex that might reflect on biotope variation.     

PECAM-directed immunotargeting of catalase: specific,rapid and transient protection against hydrogen peroxide

Vascular immunotargeting to Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM) facilitates drug delivery to endothelium. We used human PECAM-transfected REN cells (REN/PECAM) as a model to compare targeting of antioxidant enzyme catalase conjugated with PECAM antibody (anti-PECAM/catalase) with adenoviral catalase delivery. Anti-PECAM/(125)I-catalase bound to REN/PECAM, but not to REN cells (70 vs. 1 ng/well vs. < 2 ng/well of unmodified catalase). At a virus-to-cell ratio of 1, elevated levels of catalase protein were detected by immunoblotting after adenoviral transfection of REN/PECAM and REN cells alike; H(2)O(2)-degrading activity of cell lysates was elevated at ratios of 10 and higher. REN/PECAM cells internalize 66% of cell-bound anti-PECAM/(125)I-catalase. Confocal microscopy localized anti-PECAM/catalase to intracellular vesicles, while catalase expressed by adenovirus was distributed in vesicles and throughout the cytosol. Within 15 min of delivery, anti-PECAM/catalase augmented H(2)O(2)-degrading activity and survival of H(2)O(2)-exposed REN/PECAM cells. The effects of conjugate delivery reached a plateau within 1 h and declined to the basal level within 12 h. In contrast, adenoviral delivery required several hours for transduction and development of the effects, but permitted much longer duration of protection (at least 48 h). Simultaneous exposure of REN/PECAM cells to anti-PECAM/catalase and catalase-encoding adenovirus afforded protection against H(2)O(2) with a rapid onset and a prolonged duration. Therefore, PECAM-directed immunotargeting provides a specific, antigen-directed intracellular delivery of catalase that affords a rapid but transient protection against H(2)O(2) and may complement gene delivery strategies for antioxidant protection.     

Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae

We report here the effect of adding different types of carbohydrate-binding modules (CBM) to a single-module GH7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (TeCel7A). Both bacterial and fungal CBMs derived from families 1, 2 and 3, all reported to bind to crystalline cellulose, were used. Chimeric cellobiohydrolases with an additional S–S bridge in the catalytic module of TeCel7A were also made. All the fusion proteins were secreted in active form and in good yields by Saccharomyces cerevisiae. The purified chimeric enzymes bound to cellulose clearly better than the catalytic module alone and demonstrated high thermal stability, having unfolding temperatures (T _m) ranging from 72 °C to 77 °C. The highest activity enhancement on microcrystalline cellulose could be gained by a fusion with a bacterial CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA. The two CBM3 fusion enzymes tested were more active than the reference enzyme Trichoderma reesei Cel7A both at moderate (45 °C and 55 °C) and at high temperatures (60 °C and 65 °C), the hydrolysis yields being two- to three-fold better at 60 °C, and six- to seven-fold better at 65 °C. The best enzyme variant was also tested on a lignocellulosic feedstock hydrolysis, which demonstrated its potency in biomass hydrolysis even at 70 °C.     

Growth environment determines light sensitivity of shoot hydraulic conductance

Acclimation of light sensitivity of hydraulic conductance of shoots of silver birch (Betula pendula) and hybrid aspen (Populus × wettsteinii) to growth environments with three different air humidities was studied. Hydraulic conductance of shoots kept for 1–2 h in darkness (D) or in light (L) was measured by the pressure chamber method, and light sensitivity was defined as a significant difference between D and L shoots. Light sensitivity of shoots grown in three different air humidities was found to vary. Amongst shoots grown in current natural air, only the hydraulic conductance of the whole shoot and that of the leaf blades of birch upper foliage were significantly light sensitive. Amongst shoots grown in decreased air humidity, hydraulic conductance of the whole shoot, the leaf blades, and the stem and petioles of birch upper foliage, the conductance of the whole shoot and the leaf blades of birch lower foliage, and the conductance of the whole shoot of aspen upper foliage were light sensitive. None of the shoots grown in increased air humidity were significantly light sensitive. We predict that light sensitivity will become more widespread among species in regions where air humidity decreases as a result of global climate change, and vice versa. Low white light always caused the same increase in hydraulic conductance as high white light, and blue and white light always caused an increase in conductance about two times greater than red light, indicating that growth environment did not markedly modify the mechanism of light sensitivity.     

The first report of autochthonous non-vector-borne transmission of canine leishmaniosis in the Nordic countries

Background

Leishmania spp. are zoonotic protozoans that infect humans and other mammals such as dogs. The most significant causative species in dogs is L. infantum. In dogs, leishmaniosis is a potentially progressive, chronic disease with varying clinical outcomes. Autochthonous cases of canine leishmaniosis have not previously been reported in the Nordic countries.

Results

In this report we describe the first diagnosed autochthonous cases of canine leishmaniosis in Finland, in which transmission via a suitable arthropod vector was absent. Two Finnish boxers that had never been in endemic areas of Leishmania spp., had never received blood transfusions, nor were infested by ectoparasites were diagnosed with leishmaniosis. Another dog was found with elevated Leishmania antibodies. A fourth boxer dog that had been in Spain was considered to be the source of these infections. Transmission occurred through biting wounds and semen, however, transplacental infection in one of the dogs could not be ruled out.Two of the infected dogs developed a serious disease and were euthanized and sent for necropsy. The first one suffered from membranoproliferative glomerulonephritis and the second one had a chronic systemic disease. Leishmania sp. was detected from tissues by PCR and/or IHC in both dogs. The third infected dog was serologically positive for Leishmania sp. but remained free of clinical signs.

Conclusions

This case report shows that imported Leishmania-infected dogs may pose a risk for domestic dogs, even without suitable local arthropod vectors.

     

The effects of short-term pH decrease on the reproductive output of the copepod Acartia bifilosa – a laboratory study

This laboratory study reports some reproductive responses of the copepod Acartia bifilosa to rapid variations in pH. The imposed changes mimic those that copepods could experience due to coastal upwelling, changed mixing conditions or vertical migration. We measured effects of low pH on egg production, hatching and early nauplii development (H₀: no effects on response variables between low and ambient pH). On treatment with low pH, we found positive effects on egg production rate and nauplii development time. The positive response to low pH could be an initial stress response or show that A. bifilosa is tolerant to the experimental pH values. The result suggests that A. bifilosa is adapted to pH changes as it performs daily migrations between the depths with differing pH. It could also be advantageous for population development if eggs hatch at high speed and so reduce the possibility that they will sink into anoxic and low pH waters.     

Single-molecule Imaging Analysis of Elementary Reaction Steps of Trichoderma reesei Cellobiohydrolase I (Cel7A) Hydrolyzing Crystalline Cellulose Iα and IIII

Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose I_α and III_I were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose I_α and III_I are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose I_α and III_I. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose I_α and III_I. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose I_α and III_I and similar fractions of productive binding on cellulose I_α and III_I. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose I_α and III_I with similar translational rates. With structural models of cellulose I_α and III_I, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes.