Hypoxia-induced gene activity in disused oxidative muscle

Hypoxia is an important modulator of the skeletal muscle's oxidative phenotype. However, little is known regarding the molecular circuitry underlying the muscular hypoxia response and the interaction of hypoxia with other stimuli of muscle oxidative capacity. We hypothesized that exposure of mice to severe hypoxia would promote the expression of genes involved in capillary morphogenesis and glucose over fatty acid metabolism in active or disused soleus muscle of mice. Specifically, we tested whether the hypoxic response depends on oxygen sensing via the alpha-subunit of hypoxia-inducible factor-1 (HIF-1 alpha). Spontaneously active wildtype and HIF-1 alpha heterozygous deficient adult female C57B1/6 mice were subjected to hypoxia (PiO2 70 mmHg). In addition, animals were subjected to hypoxia after 7 days of muscle disuse provoked by hindlimb suspension. Soleus muscles were rapidly isolated and analyzed for transcript level alterations with custom-designed AtlasTM cDNA expression arrays (BD Biosciences) and cluster analysis of differentially expressed mRNAs. Multiple mRNA elevations of factors involved in dissolution and stabilization of blood vessels, glycolysis, and mitochondrial respiration were evident after 24 hours of hypoxia in soleus muscle. In parallel transcripts of fat metabolism were reduced. A comparable hypoxia-induced expression pattern involving complex alterations of the IGF-I axis was observed in reloaded muscle after disuse. This hypoxia response in spontaneously active animals was blunted in the HIF-1 alpha heterozygous deficient mice demonstrating 35% lower HIF-1 alpha mRNA levels. Our molecular observations support the concept that severe hypoxia provides HIF-1-dependent signals for remodeling of existing blood vessels, a shift towards glycolytic metabolism and altered myogenic regulation in oxidative mouse muscle and which is amplified by enhanced muscle use. These findings further imply differential mitochondrial turnover and a negative role of HIF-1 alpha for control of fatty acid oxidation in skeletal muscle exposed to one day of severe hypoxia.

Locomotor activity of two sympatric slugs: implication for the invasion success of terrestrial invertebrates.

A central focus of invasion biology is to identify the traits that predict which introduced species will become invasive. Behavioral traits related to locomotor activity most likely play a pivotal role in determining a species’invasion success but have rarely been studied, particularly in terrestrial invertebrates. Here, we experimentally investigated the small-scale locomotor activity of two slug species with divergent invasion success in Europe, the highly invasive slug, Arion lusitanicus, and the closely related, non-invasive and native slug, Arion rufus. To do so, we used a multi-state capture-mark-recapture approach, and hypothesized that the invasive slug has a higher moving rate (keeps on moving) and leaving rate (leaves more frequently known places). A total of 221 invasive and 241 non-invasive slugs were individually marked using magnetic transponders and released in three study sites differing in habitat type. The slugs were recaptured using shelter traps, and moving and leaving rates were estimated. Both rates were significantly higher for the invasive slug, demonstrating a higher locomotor activity which might partly explain its invasion success. Our results provide evidence for the recently suggested idea that locomotor activity might be an important trait underlying animal invasions using for the first time terrestrial invertebrates.

Theileria-induced constitutive IKK activation is independent of functional Hsp90

The intracellular parasite Theileria induces uncontrolled proliferation and host cell transformation. Parasite-induced transformation is accompanied by constitutive activation of IkappaB kinase (IKK), resulting in permanently high levels of activated nuclear factor (NF)-kappaB. IKK activation pathways normally require heat shock protein 90 (Hsp90), a chaperone that regulates the stability and activity of signalling molecules and can be blocked by the benzoquinone ansamycin compound geldanamycin (GA). In Theileria-transformed cells, IkappaBalpha and p65 phosphorylation, NF-kappaB nuclear translocation and DNA binding activity are largely resistant to GA and also NF-kappaB-dependent reporter gene expression is only partly affected. Our findings indicate that parasite-induced IKK activity does not require functional Hsp90.

Variation in human platelet glycoprotein VI content modulates glycoprotein VI-specific prothrombinase activity

- Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen that figures prominently in signal transduction. An addition to binding to type I and III collagens, GPVI is also bound specifically by collagen-related peptide and convulxin (CVX), a snake venom protein. We developed a quantitative assay of platelet GPVI in which biotin-conjugated CVX binds selectively to GPVI in separated total platelet proteins by a ligand blot procedure. Using this approach, we have documented a 5-fold range in platelet GPVI content among 23 normal healthy subjects. In addition, we have determined that CVX-induced or collagen-related peptide-induced prothrombinase activity is directly proportional to the platelet content of GPVI. A statistically significant correlation was observed at 2 CVX concentrations: 14.7 ng/mL (R(2)=0.854 and P<0.001, n=11) and 22 ng/mL (R(2)=0.776 and P<0.001, n=12). In previous studies, we established a similar range of expression of the integrin collagen receptor alpha(2)beta(1) on platelets of normal subjects. Among 15 donors, there is a direct correlation between platelet alpha(2)beta(1) density and GPVI content (R(2)=0.475 and P=0.004). In view of the well-documented association of GPVI with platelet procoagulant activity, this study suggests that the variation in GPVI content is a potential risk factor that may predispose individuals to hemorrhagic or thromboembolic disorders.

Locomotor velocity and striatal adaptive gene expression changes of the direct and indirect pathways in Parkinsonian rats

BACKGROUND In Parkinson's disease (PD), bradykinesia, or slowness of movement, only appears after a large striatal dopamine depletion. Compensatory mechanisms probably play a role in this delayed appearance of symptoms. OBJECTIVE Our hypothesis is that the striatal direct and indirect pathways participate in these compensatory mechanisms. METHODS We used the unilateral 6-hydroxydopamine (6-OHDA) rat model of PD and control animals. Four weeks after the lesion, the spontaneous locomotor activity of the rats was measured and then the animals were killed and their brain extracted. We quantified the mRNA expression of markers of the striatal direct and indirect pathways as well as the nigral expression of dopamine transporter (DAT) and tyrosine hydroxylase (TH) mRNA. We also carried out an immunohistochemistry for the striatal TH protein expression. RESULTS As expected, the unilateral 6-OHDA rats presented a tendency to an ipsilateral head turning and a low locomotor velocity. In 6-OHDA rats only, we observed a significant and positive correlation between locomotor velocity and both D1-class dopamine receptor (D1R) (direct pathway) and enkephalin (ENK) (indirect pathway) mRNA in the lesioned striatum, as well as between D1R and ENK mRNA. CONCLUSIONS Our results demonstrate a strong relationship between both direct and indirect pathways and spontaneous locomotor activity in the parkinsonian rat model. We suggest a synergy between both pathways which could play a role in compensatory mechanisms and may contribute to the delayed appearance of bradykinesia in PD.

CD40 activation induces NREM sleep and modulates genes associated with sleep homeostasis

The T-cell derived cytokine CD40 ligand is overexpressed in patients with autoimmune diseases. Through activation of its receptor, CD40 ligand leads to a tumor necrosis factor (TNF) receptor 1 (TNFR1) dependent impairment of locomotor activity in mice. Here we report that this effect is explained through a promotion of sleep, which was specific to non-rapid eye movement (NREM) sleep while REM sleep was suppressed. The increase in NREM sleep was accompanied by a decrease in EEG delta power during NREM sleep and by a decrease in the expression of transcripts in the cerebral cortex known to be associated with homeostatic sleep drive, such as Homer1a, Early growth response 2, Neuronal pentraxin 2, and Fos-like antigen 2. The effect of CD40 activation was mimicked by peripheral TNF injection and prevented by the TNF blocker etanercept. Our study indicates that sleep-wake dysregulation in autoimmune diseases may result from CD40 induced TNF:TNFR1 mediated alterations of molecular pathways, which regulate sleep-wake behavior.

Paleolimnological evidence for increased landslide activity due to forest clearing and land-use since 3600 cal BP in the western Swiss Alps

Schwarzsee is located in the western Swiss Alps, in a region that has been affected by numerous landslides during the Holocene, as evidenced by geological surveys. Lacustrine sediments were cored to a depth of 13 m. The vegetation history of the lake's catchment was reconstructed and investigated to identify possible impacts on slope stability. The pollen analyses record development of forest cover during the middle and late Holocene, and provide strong evidence for regional anthropogenic influence such as forest clearing and agricultural activity. Vegetation change is characterized by continuous landscape denudation that begins at ca. 4300 cal. yrs BP, with five distinct pulses of increased deforestation, at 3650, 2700, 1500, 900, and 450 cal. yrs BP. Each pulse can be attributed to increased human impact, recorded by the appearance or increase of specific anthropogenic indicator plant taxa. These periods of intensified deforestation also appear to be correlated with increased landslide activity in the lake's catchment and increased turbidite frequency in the sediment record. Therefore, this study gives new evidence for a strong influence of vegetation changes on slope stability during the middle and late Holocene in the western Swiss Alps, and may be used as a case study for anthropogenically induced landslide activity.

Effects of MASP-1 of the complement system on activation of coagulation factors and plasma clot formation

Background Numerous interactions between the coagulation and complement systems have been shown. Recently, links between coagulation and mannan-binding lectin-associated serine protease-1 (MASP-1) of the complement lectin pathway have been proposed. Our aim was to investigate MASP-1 activation of factor XIII (FXIII), fibrinogen, prothrombin, and thrombin-activatable fibrinolysis inhibitor (TAFI) in plasma-based systems, and to analyse effects of MASP-1 on plasma clot formation, structure and lysis. Methodology/Principal Findings We used a FXIII incorporation assay and specific assays to measure the activation products prothrombin fragment F1+2, fibrinopeptide A (FPA), and activated TAFI (TAFIa). Clot formation and lysis were assessed by turbidimetric assay. Clot structure was studied by scanning electron microscopy. MASP-1 activated FXIII and, contrary to thrombin, induced FXIII activity faster in the Val34 than the Leu34 variant. MASP-1-dependent generation of F1+2, FPA and TAFIa showed a dose-dependent response in normal citrated plasma (NCP), albeit MASP-1 was much less efficient than FXa or thrombin. MASP-1 activation of prothrombin and TAFI cleavage were confirmed in purified systems. No FPA generation was observed in prothrombin-depleted plasma. MASP-1 induced clot formation in NCP, affected clot structure, and prolonged clot lysis. Conclusions/Significance We show that MASP-1 interacts with plasma clot formation on different levels and influences fibrin structure. Although MASP-1-induced fibrin formation is thrombin-dependent, MASP-1 directly activates prothrombin, FXIII and TAFI. We suggest that MASP-1, in concerted action with other complement and coagulation proteins, may play a role in fibrin clot formation.

Antibacterial and antiinflammatory kinetics of curcumin as a potential antimucositis agent in cancer patients

The antiinflammatory agent curcumin (diferuloylmethane) has a potential to mitigate cancer therapy-induced mucositis. We assessed the in vitro extent of its bactericidal activity and determined the kinetics of its antiinflammatory effect on pharyngeal cells. Bactericidal activity was assessed using the LIVE/DEAD® Kit after 4 h of exposure to curcumin (50-200 μM) in 18 oropharyngeal species commonly associated with bacteremia in febrile neutropenia. Moraxella catarrhalis or its outer membrane vesicles were used to determine the inhibitory effect of curcumin on bacteria-induced proinflammatory activity as determined by cytokine release into the supernatant of Detroit 562 pharyngeal cells using the Luminex® xMAP® technology. Curcumin exerted a concentration-dependent bactericidal effect on all 18 species tested. After 4 h at 200 μM, 12 species tested were completely killed. Preincubation of Detroit cells with 200 μM curcumin for 5 to 60 min resulted in complete suppression of the release of tumor necrosis factor-α, interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1, granulocyte macrophage-colony stimulating factor, and vascular endothelial growth factor. Fibroblast growth factor-2 and interferon-γ were not affected. Repetitive exposure to curcumin resulted in repetitive suppression of cytokine/chemokine expression lasting from 4 to 6 h. Through reduction of oral microbial density as well as suppression of inflammation cascades curcumin may prevent cancer therapy-induced oral mucositis, e.g., when applied as multiple daily mouth washes.

Different migration of vascular smooth muscle cells from human coronary artery bypass vessels. Role of Rho/ROCK pathway

BACKGROUND: We examined whether vascular smooth muscle (VSMC) or endothelial cell (EC) migration from internal mammary artery (MA) differed from VSMC or EC migration from saphenous vein (SV). METHODS AND RESULTS: Migration to PDGF-BB (1-10 ng/ml) was lower in VSMC from MA than SV; however, attachment, movement without chemokine, and chemokinesis were identical. Unlike VSMC, migration of EC was similar in response to several mediators. Expression of PDGF receptor-beta was lower in VSMC from MA than SV, while alpha-receptor expression was higher. PDGF-BB-induced RhoA activity was lower in MA than SV, while basal activity was identical. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced migration of VSMC from MA and SV. Mevalonate and geranylgeranylpyrophosphate rescued inhibition by rosuvastatin. PDGF-BB induced less stress fiber formation in VSMC from MA than SV. A dominant negative RhoA mutant inhibited stress fiber formation to PDGF-BB, while a constitutively active mutant resulted in maximal stress fiber formation in MA and SV. Rosuvastatin and hydroxyfasudil impaired PDGF-BB-induced stress fiber formation in MA and SV. CONCLUSIONS: VSMC migration to PDGF-BB is lower in MA than SV, which is at least in part related to lower activity of the Rho/ROCK pathway.

Adenosine A2A receptor gene expression in the normal striatum and after 6-OH-dopamine lesion

Adenosine A2A receptors are present on enkephalinergic medium sized striatal neurons in the rat and have an important function in the modulation of striatal output. In order to establish more accurately whether adenosine transmission is a generalized phenomenon in mammalian striatum we compared the A2A R expression in the mouse, rat, cat and human striatum. Secondly we compared the modulation of enkephalin gene expression and A2A receptor gene expression in rat striatal neurons after 6-OH-dopamine lesion of the substantia nigra. Hybridization histochemistry was performed with a 35S-labelled radioactive oligonucleotide probe. The results showed high expression of A2A adenosine receptor genes only in the medium-sized cells of the striatum in all examined species. In the rat striatum, expression of A2A receptors was not significantly altered after lesion of the dopaminergic pathways with 6-OH-dopamine even though enkephalin gene expression was up-regulated. The absence of a change in A2A receptor gene expression after 6-OH-dopamine treatment speaks against a dependency on dopaminergic innervation. The maintained inhibitory function of A2A R on motor activity in spite of dopamine depletion could be partly responsible for the depression of locomotor activity observed in basal ganglia disorders such as Parkinson's disease.

Reducing Myofibroblast Arrhythmogeneicity by Pharmacological Targeting of Their Cytoskeleton

Introduction: Slow conduction and ectopic activity are key elements of cardiac arrhythmogenesis. Both anomalies can be caused by myofibroblasts (MFBs) following establishment of heterocellular gap junctional coupling with cardiomyocytes. Because MFBs are characterized by the expression of {alpha}-smooth muscle actin ({alpha}-SMA) containing stress fibers, we investigated whether pharmacological interference with stress fiber formation might affect myofibroblast arrhythmogenicity. Methods: Experiments were done with patterned growth strands of neonatal rat ventricular cardiomyocytes coated with cardiac MFBs. Impulse propagation characteristics were measured optically using voltage sensitive dyes. Electrophysiological characteristics of single MFBs were assessed using patch clamp techniques. Actin polymerization was inhibited by latrunculin B (LtB). Data are given as mean±S.D. (n=5 to 22). Results: As assessed by immunocytochemistry, exposure of MFBs to LtB (0.3–10 µmol/L) profoundly disrupted stress fiber formation. This led, within minutes, to a dramatic change in cell morphology with MFBs assuming an astrocyte-like shape. In pure cardiomyocyte preparations, LtB had negligible effects on impulse conduction velocity ({theta}) and maximal action potential upstroke velocities (dV/dtmax). In contrast, LtB applied to MFB coated cardiomyocyte strands substantially increased {theta} from 247±32 to 371±26 mm/s and dV/dtmax from 40±7 to 81±1 %APA/ms, i.e., to values similar to those of pure cardiomyocyte strands (342±13 mm/s; 82±1 %APA/ms). Moreover, LtB at 1 µmol/L completely abolished MFB induced ectopic activity. LtB induced normalization of electrophysiologic parameters can be explained by the finding that LtB hyperpolarized MFBs from –25 mV to –50 mV, thus limiting their depolarizing effect on cardiomyocytes which was shown before to cause slow conduction and ectopic activity. Conclusions: Pharmacological interference with the cytoskeleton of cardiac MFBs alters their electrophysiological phenotype to such an extent that detrimental effects on cardiomyocyte electrophysiology are completely abolished. This observation might form a basis for the development of therapeutic strategies aimed at limiting the arrhythmogenic potential of MFBs.

Antinociceptive effect of buprenorphine and evaluation of the nociceptive withdrawal reflex in foals.

OBJECTIVE To elicit and evaluate the NWR (nociceptive withdrawal reflex) in 2 and 11 day old foals, to investigate if buprenorphine causes antinociception and determine if the NWR response changes with increasing age. The effect of buprenorphine on behaviour was also evaluated. STUDY DESIGN Prospective, experimental cross-over trial. ANIMALS Nine Norwegian Fjord research foals. METHODS Buprenorphine, 10 μg kg(-1) was administered intramuscularly (IM) to the same foal at 2 days and at 11 days of age. The NWR and the effect of buprenorphine were evaluated by electromyograms recorded from the left deltoid muscle following electrical stimulation of the left lateral palmar nerve at the level of the pastern. Mentation, locomotor activity and respiratory rate were recorded before and after buprenorphine administration. RESULTS We were able to evoke the NWR and temporal summation in foals using this model. Buprenorphine decreased the root mean square amplitude following single electrical stimulation (p < 0.001) in both age groups, and increased the NWR threshold following single electrical stimulation in 2 day old foals (p = 0.0012). Repeated electrical stimulation at 2 Hz was more effective to elicit temporal summation compared to 5 Hz (p < 0.001). No effect of age upon the NWR threshold was found (p = 0.34). Sedation when left undisturbed (11 occasions), increased locomotor activity when handled (9 occasions) and tachypnea (13 occasions) were common side-effects of buprenorphine. CONCLUSION AND CLINICAL RELEVANCE These findings indicate that buprenorphine has antinociceptive effect in foals. Opioid side effects often recognized in adult horses also occur in foals.

Effects of induced parturition in goats on immunoglobulin G and chitotriosidase activity in colostrum and plasma and on plasma concentrations of prolactin

The effect of induction of parturition with a PGF(2)alpha analog on plasma concentration of prolactin (PRL) and its effects on colostrum concentration of IgG and chitotriosidase (ChT) activity were studied in 16 pregnant Majorera goats. Treated goats, those in which parturition was induced, had greater concentrations of PRL than control goats 24 h before parturition (P < 0.05) and 48 h after parturition (P < 0.05). Control goats had greater concentrations of PRL than treated goats 96 h after parturition (P < 0.05). Plasma concentration of IgG did not differ between groups during the experimental period, but colostrum concentrations of IgG were greater in control goats than in treated goats at parturition (P < 0.05). Plasma ChT activity decreased during the period 72 h before parturition to 24 h after parturition in control and treated goats. Time evolution after partum affected the colostrum ChT activity, being greater at parturition than after parturition in both groups (P < 0.05). In summary, concentration of IgG in colostrum is slightly diminished if parturition is induced. Induction of parturition causes an early increase in PRL, which is most likely responsible for preterm suppression of IgG transport into mammary secretions. (C) 2011 Elsevier Inc. All rights reserved.