Effects of VMAT2 inhibitors lobeline and GZ‐793A on methamphetamine‐induced changes in dopamine release,metabolism and synthesis in vivo

Vesicular monoamine transporter‐2 (VMAT2) inhibitors reduce methamphetamine (METH) reward in rats. The current study determined the effects of VMAT2 inhibitors lobeline (LOB; 1 or 3 mg/kg) and N‐(1,2R‐dihydroxylpropyl)‐2,6‐cis‐di(4‐methoxyphenethyl)piperidine hydrochloride (GZ‐793A; 15 or 30 mg/kg) on METH‐induced (0.5 mg/kg, SC) changes in extracellular dopamine (DA) and its metabolite dihydroxyphenylacetic acid (DOPAC) in the reward‐relevant nucleus accumbens (NAc) shell using in vivo microdialysis. The effect of GZ‐793A (15 mg/kg) on DA synthesis in tissue also was investigated in NAc, striatum, medial prefrontal cortex and orbitofrontal cortex. In NAc shell, METH produced a time‐dependent increase in extracellular DA and decrease in DOPAC. Neither LOB nor GZ‐793A alone altered extracellular DA; however, both drugs increased extracellular DOPAC. In combination with METH, LOB did not alter the effects of METH on DA; however, GZ‐793A, which has greater selectivity than LOB for inhibiting VMAT2, reduced the duration of the METH‐induced increase in extracellular DA. Both LOB and GZ‐793A enhanced the duration of the METH‐induced decrease in extracellular DOPAC. METH also increased tissue DA synthesis in NAc and striatum, whereas GZ‐793A decreased synthesis; no effect of METH or GZ‐793A on DA synthesis was found in medial prefrontal cortex or orbitofrontal cortex. These results suggest that selective inhibition of VMAT2 produces a time‐dependent decrease in DA release in NAc shell as a result of alterations in tyrosine hydroxylase activity, which may play a role in the ability of GZ‐793A to decrease METH reward.

     

Characterization and localization of Ac-SDKP receptor binding sites using 125I-labeled Hpp-Aca-SDKP in rat cardiac fibroblasts

We have shown that the tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) inhibited endothelin-1 (ET-1)-induced cell proliferation and collagen synthesis in cultured rat cardiac fibroblasts (CFs) and reduced left ventricle collagen deposition in rats with aldosterone (salt)- and ANG II-induced hypertension. However, it is not known whether these effects are mediated by receptor binding sites specific for Ac-SDKP. We hypothesized that Ac-SDKP exerts antifibrotic effects by binding to specific receptor sites in cultured rat CFs, which mediate the inhibitory effects of Ac-SDKP on ET-1-stimulated collagen synthesis. Ac-SDKP binding sites in rat CFs and hearts were characterized by a specific radioligand, (125)I-labeled 3-(p-hydroxyphenyl)-propionic acid (or desaminotyrosine) (Hpp)-Aca-SDKP, a biologically active analog of Ac-SDKP. (125)I-labeled Hpp-Aca-SDKP bound to rat CFs and fractionated membranes with similar affinities and specificity in a concentration- and time-dependent fashion. Scatchard plot analyses revealed a single class of high-affinity Hpp-Aca-SDKP binding sites (maximal binding: 1,704 +/- 198 fmol/mg protein; dissociation constant: 3.3 +/- 0.6 nM). (125)I-labeled Hpp-Aca-SDKP binding in CFs was displaced by unlabeled native peptide Ac-SDKP (inhibition constant: 0.69 +/- 0.15 nM) and the analog Hpp-Aca-SDKP (inhibition constant: 10.4 +/- 0.2 nM) but not the unrelated peptide ANG II or ET-1 (10 microM). In vitro, both Ac-SDKP and Hpp-Aca-SDKP inhibited ET-1-stimulated collagen synthesis in CFs in a dose-dependent fashion, reaching a maximal effect at 1 nM (control: 7.5 +/- 0.4, ET-1: 19.9 +/- 1.2, ET-1+SDKP: 7.7 +/- 0.4, ET-1+Hpp-Aca-SDKP: 9.7 +/- 0.1 microg/mg protein; P < 0.001). Ac-SDKP also significantly attenuated ET-1-induced increases in intracellular calcium and MAPK ERK1/2 phosphorylation in CFs. In the rat heart, in vitro autoradiography revealed specific (125)I-labeled Hpp-Aca-SDKP binding throughout the myocardium, primarily interstitially. We believe that these results demonstrate for the first time that Hpp-Aca-SDKP is a functional ligand specific for Ac-SDKP receptor binding sites and that both Ac-SDKP and Hpp-Aca-SDKP exert antifibrotic effects by binding to Ac-SDKP receptors in rat CFs.     

Induction of selective blood-tumor barrier permeability and macromolecular transport by a biostable kinin B1 receptor agonist in a glioma rat model

Treatment of malignant glioma with chemotherapy is limited mostly because of delivery impediment related to the blood-brain tumor barrier (BTB). B1 receptors (B1R), inducible prototypical G-protein coupled receptors (GPCR) can regulate permeability of vessels including possibly that of brain tumors. Here, we determine the extent of BTB permeability induced by the natural and synthetic peptide B1R agonists, LysdesArg(9)BK (LDBK) and SarLys[dPhe(8)]desArg(9)BK (NG29), in syngeneic F98 glioma-implanted Fischer rats. Ten days after tumor inoculation, we detected the presence of B1R on tumor cells and associated vasculature. NG29 infusion increased brain distribution volume and uptake profiles of paramagnetic probes (Magnevist and Gadomer) at tumoral sites (T(1)-weighted imaging). These effects were blocked by B1R antagonist and non-selective cyclooxygenase inhibitors, but not by B2R antagonist and non-selective nitric oxide synthase inhibitors. Consistent with MRI data, systemic co-administration of NG29 improved brain tumor delivery of Carboplatin chemotherapy (ICP-Mass spectrometry). We also detected elevated B1R expression in clinical samples of high-grade glioma. Our results documented a novel GPCR-signaling mechanism for promoting transient BTB disruption, involving activation of B1R and ensuing production of COX metabolites. They also underlined the potential value of synthetic biostable B1R agonists as selective BTB modulators for local delivery of different sized-therapeutics at (peri)tumoral sites.     

In vivo imaging of hepatic excretory function in the rat by fluorescence microscopy

Applying intravital fluorescence microscopy, we assessed sinusoidal delivery and biliary clearance of two different polymethine dyes. DY635, a benzopyrylium‐based hemocyanine dye with shorter excitation wavelength than indocyanine green (ICG), was validated for assessment of hepatic excretory function. Decrease of DY635 and ICG reflecting transcellular transport was 83 ± 4% (DY635) and 14 ± 2% (ICG; p < 0.05) over 35 minutes, respectively. In cholestasis, hepatobiliary excretion of DY635 was markedly impaired (control 3176 ± 148 pmol vs. cholestatic 1929 ± 179 pmol; p < 0.05). DY635 even enabled an analysis at high resolution suggesting 1.) hepatocyte uncoupling and 2.) failure of primarily the canalicular pole, allowing in vivo insights into molecular mechanisms of this critical facet of hepatobiliary function. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Efficient, chemoselective synthesis of immunomicelles using single-domain antibodies with a C-terminal thioester

Background  

Classical bioconjugation strategies for generating antibody-functionalized nanoparticles are non-specific and typically result in heterogeneous compounds that can be compromised in activity. Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester, providing a unique handle for site-specific conjugation using native chemical ligation (NCL). However, current methods to generate antibody fragments with C-terminal thioesters require cumbersome refolding procedures, effectively preventing application of NCL for antibody-mediated targeting and molecular imaging.     

Ultrasound enhanced prehospital thrombolysis using microbubbles infusion in patients with acute ST elevation myocardial infarction: Rationale and design of the Sonolysis study

Background

The CONSORT Statement provides recommendations for reporting randomized controlled trials. We assessed the extent to which leading medical journals that publish reports of randomized trials incorporate the CONSORT recommendations into their journal and editorial processes.

Methods

This article reports on two observational studies. Study 1: We examined the online version of 'Instructions to Authors' for 165 high impact factor medical journals and extracted all text mentioning the CONSORT Statement or CONSORT extension papers. Any mention of the International Committee of Medical Journal Editors (ICMJE) or clinical trial registration were also sought and extracted. Study 2: We surveyed the editor-in-chief, or editorial office, for each of the 165 journals about their journal's endorsement of CONSORT recommendations and its incorporation into their editorial and peer-review processes.

Results

Study 1: Thirty-eight percent (62/165) of journals mentioned the CONSORT Statement in their online 'Instructions to Authors'; of these 37% (23/62) stated this was a requirement, 63% (39/62) were less clear in their recommendations. Very few journals mentioned the CONSORT extension papers. Journals that referred to CONSORT were more likely to refer to ICMJE guidelines (RR 2.16; 95% CI 1.51 to 3.08) and clinical trial registration (RR 3.67; 95% CI 2.36 to 5.71) than those journals which did not. Study 2: Thirty-nine percent (64/165) of journals responded to the on-line survey, the majority were journal editors. Eighty-eight percent (50/57) of journals recommended authors comply with the CONSORT Statement; 62% (35/56) said they would require this. Forty-one percent (22/53) reported incorporating CONSORT into their peer-review process and 47% (25/53) into their editorial process. Eighty-one percent (47/58) reported including CONSORT in their 'Instructions to Authors' although there was some inconsistency when cross checking information on the journal's website. Sixty-nine percent (31/45) of journals recommended authors comply with the CONSORT extension for cluster trials, 60% (27/45) for harms and 42% (19/45) for non-inferiority and equivalence trials. Few journals mentioned these extensions in their 'Instructions to Authors'.

Conclusion

Journals should be more explicit in their recommendations and expectations of authors regarding the CONSORT Statement and related CONSORT extensions papers.     

End-of-life modelling in life cycle assessment—material or product-centred perspective?

Purpose

End-of-life (EoL) modelling in life cycle assessment has already been broadly discussed within several studies. However, no consensus has been achieved on how to model recycling in LCA, even though several approaches have been developed. Within this paper, results arising from the application of two new EoL formulas, the product environmental footprint (PEF) and the multi-recycling-approach (MRA) ones, are compared and discussed. Both formulas consider multiple EoL scenarios such as recycling, incineration and landfill.

Methods

The PEF formula has been developed within the PEF programme whose intent is to define a harmonized methodology to evaluate the environmental performance of products. The formula is based on a 50:50 allocation approach, as burdens and benefits associated with recycling are accounted for a 50% rate. The MRA formula has been developed to change focus from products to materials. Recycling cycles and material losses over time are considered with reference to material pools. Allocation between systems is no longer needed, as the actual number of potential life cycles for a certain material is included in the calculation. Both the approaches have been tested within two case studies.

Results and discussion

Methodological differences could thereof be determined, as well as applicability concerns, due to the type of data required for each formula. As far as the environmental performance is concerned, impacts delivered by MRA are lower than those delivered by PEF for aluminium, while the opposite happens for plastic and rubber due to the higher share of energy recovery accounted in PEF formula. Stainless steel impacts are almost the same.

Conclusions and recommendations

The application of the two formulas provides some inputs for the EoL dilemma in LCA. The use of a wider perspective, better reflecting material properties all over the material life cycle, is of substantial importance to properly represent recycling situations. In MRA, such properties are treated and less data are required compared to the PEF formula. On the contrary, the PEF model better accommodates the modelling of products whose materials, at end of life, can undertake the route of recycling or recovery (or landfill), depending on country-specific EoL management practices. However, its application requires more data.

     

Survival of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> in Wilted and Additive-Treated Grass Silage

Grass was field-dried to 3 different dry matter (DM) levels (200, 430 and 540 g/kg) and inoculated with 10⁶–10⁷ cfu/g of a Listeria monocytogenes strain sharing a phagovar occasionally involved in food-borne outbreaks of listeriosis. Formic acid (3 ml/kg) or lactic acid bacteria (8·10⁵/g) with cellulolytic enzymes were applied only to forages with low and intermediate DM levels. Forages were ensiled in laboratory silos (1700 ml) and were stored at 25°C for 30 or 90 days. After 90 days of storage, L. monocytogenes could not be detected in any silo, except one with the high dry matter grass without additive. After 30 days of storage, between 10² and 10⁶ cfu L. monocytogenes/g silage were isolated from the untreated silages. Increasing the DM content from 200 to 540 g/kg did not reduce listeria counts possibly because of the lower production of fermentation acids (higher pH). In silages treated with additives, counts of L. monocytogenes were always lower than in silages without additive. In wet silages (DM 200 g/kg) both additives were effective, but in the wilted silages (DM 430 g/kg) only the bacterial additive reduced listeria counts below detection level. Listeria counts were highly correlated to silage pH (r = 0.92), the concentration of lactic acid (r = -0.80) and the pooled amount of undissociated acids (r = -0.83).     

Light-flecks cause non-uniform stomatal opening – studies with special emphasis on Fagus sylvatica L.

The appearance of stomatal patchiness in response to rapid (seconds) changes in light has been studied in European beech, Fagus sylvatica L., and, by comparison, in a further 17 different woody species from the understorey of a European beech forest, using a simple water infiltration method. Water infiltrated areoles indicate open stomata. Since infiltration changes optical characteristics of a leaf section it can be analysed by photography, computer-aided image analysis and by weighing. For F. sylvatica clear differences were found between infiltration of cotyledons (no patchy pattern) and any other leaf type. Despite identical cultivation, leaves of the same type and age from different individual plants responded differently to application of 30 s of light after darkness. In contrast, the patchiness patterns were very similar for leaves of the same type originating from the same plant. Infiltration patterns after a light-fleck, observed on different leaves as a series of momentary clusters, probably indicate waves of opening stomata moving across the leaf blade. During and after a 30 s light-fleck infiltration increased and it continued to increase in the dark up to 10 min, indicating increasing stomatal opening over that period. In general, shade leaves became more infiltrated (by weight) than half-shade or sun leaves, due to larger intercellular air spaces. All species, without exception, showed patchy infiltration and, thus, non-uniform stomatal opening. Measuring leaf gas exchange (as ”quasi-steady states” using a fast responding system) during photosynthetic induction resulted in very similar CO₂ responses of net photosynthesis (A/c _i) as in the true steady state, proving that, in shade and half-shade leaves, the presence of stomatal patchiness does not necessarily affect the calculation of intercellular CO₂ concentrations. Causes and consequences of stomatal patchiness are discussed. Received: 18 November 1998 / Accepted: 1 July 1999