5-Hydroxydecanoate Abolishes Cardioprotective Effects of a Structural Analogue of Apelin-12 in Ischemia/Reperfusion Injury

Chemically modified peptide apelin-12 (MA) with enhanced resistance to degradation by proteolytic enzymes is able to protect the heart against myocardial ischemia and reperfusion. This study was aimed to explore the role of mitochondrial ATP-sensitive K⁺-channels (mitoKATP) in effects of MA on myocardial energy state and membrane integrity in ischemia/reperfusion (I/R) injury. Isolated perfused working rat hearts were used to simulate global ischemia and reperfusion. Acute myocardial infarction was induced by coronary artery occlusion followed by restoration of coronary blood flow in anesthetized rats. Myocardial infarct size and cardiac dysfunction were used as indices of I/R injury at the end of reperfusion. Co-infusion of 5-hydroxydecanoate (5HD), the mitoKATP blocker, along with MA before ischemia significantly decreased functional recovery of isolated hearts as compared to administration of MA alone. These effects were accompanied by increased LDH release in the myocardial effluent, reduced restoration of myocardial ATP, AN, Cr, adenylate energy charge (AEC), and lactate accumulation. Coadministration of 5HD and MA at the onset of reperfusion substantially reduced infarct-limiting effect of the peptide in rats in vivo and increased the plasma LDH and CK-MB activity compared with MA treatment. Additionally, 5HD abolished MA influence on the metabolic state of the area at risk (AAR) at the end of reperfusion. In this case, the contents of metabolites and AEC in the AAR did not differ significantly from the values in control. Therefore, restoration of myocardial energy metabolism and sarcolemma integrity via activation of mitoKATP may be of critical importance for MA-induced protection against I/R injury.     

Morphogenetic status of somatic embryos of <Emphasis Type="Italic">Citrus sinensis</Emphasis> from mature polyembryonic seeds and those produced in vitro

In tissue culture of sweet orange (Citrus sinensis (L.) Osbeck, cv. Tarocco), we obtained mass regeneration of somatic embryos with two morphologically distinct cotyledons about 3 mm in length, their numbers amounting to 110–150 embryos per petri dish and 60 to 80% of the population. The morphogenetic state of somatic embryos was compared using the embryos with the cotyledons of different size (from 3 to 10 mm) from mature polyembryonic seeds as a test system and the cell number, size, and ultrastructural organization, and the number of protein bodies in the cotyledon cells as morphological and biochemical criteria. Cell number in the cotyledons of different size was related to the content of protein bodies therein. Typical protein bodies where 33 kD polypeptide of storage proteins was identified were detected only in the cotyledons, which size was identical to that of embryonic cotyledons from monoembryonic seeds of citrus plants. In the cells of smaller cotyledons, we detected protein-accumulating vacuoles with electron-dense inclusions that irrespective of their size, shape and structure accumulated the gold label. The number of the cells with protein depositions in vacuoles decreased when the cotyledons became smaller. Irrespective of the origin of embryos (in vivo or in vitro), lipids were the major storage metabolites in the cells of 3-mm cotyledons. As the cotyledon-forming cells became smaller and less numerous, their metabolic activity tended to decrease in line with the fragmentation of endoplasmic reticulum, the absence of polysomic complexes, and indistinct inner organization of mitochondria and plastids. It was concluded that somatic embryos developing in vivo and in vitro were physiological dwarfs, that is, the structures with diminutive storage organ with characteristically incomplete cell differentiation. Apparently such forms emerged due to the suppression of cotyledon growth at the initial stages of their organogenesis; as a result, the cell population could not properly realize both organo- and histogenesis.     

Automated Blood Sampling in a Canine Telemetry Cardiovascular Model

Successful implementation of automated blood sampling (ABS) into a telemetry instrumented canine cardiovascular model provides simultaneous cardiovascular assessment of novel compounds while collecting multiple blood samples for analysis of drug level, cytokines, and biomarkers. Purpose-bred male Beagle dogs (n = 36) were instrumented with a dual-pressure telemetry transmitter and vascular access port. Modifications to acclimation practices, surgical procedures, and housing were required for implementation of ABS in our established cardiovascular canine telemetry colony. These modifications have increased the use and reproducibility of the model by combining early pharmacokinetic and cardiovascular studies, thus achieving both refinement and reduction from a 3R perspective. In addition, the modified model can shorten timelines and reduce the compound requirement in early stages of drug development. This telemetry–ABS model provides an efficient means to quickly identify potential effects on key cardiovascular parameters in a large animal species and to obtain a more complete pharmacokinetic–pharmacodynamic profile for discovery compounds.

Safety pharmacology is an important facet of drug discovery and development. Prior to the first in-human studies, safety pharmacology outcomes are used to predict clinical risk profiles of potential new drugs. Dogs that are chronically instrumented for telemetry are a well-characterized in vivo model for safety pharmacology studies and for screening new compounds for cardiovascular physiologic effects.² Relatively few blood collections are performed during conscious telemetry studies due to the potential disturbance of cardiovascular data.¹⁰ Personnel entering the room cause excitement in dogs, which can mask drug-induced changes in physiologic parameters, such as heart rate and blood pressure.¹⁰ The ‘work around’ for the excitation artifact involves obtaining blood samples from a different group of dogs (known as a satellite group) while recording physiologic changes in the first group or repeating the pharmacokinetic study in the telemetry-instrumented dogs. Pharmacokinetic properties of drugs vary due to individual dog variability and can vary across dosing events (for example, batch-to-batch variability). If adverse effects are noted on either study, determining a correlation of occurrences can be difficult especially when involving a single animal. These approaches do not compensate for individual variability, dosing variations between animals, or changing environmental conditions.⁹Heart rate and circulating cortisol concentration increase during manual blood sampling as compared with automated blood sampling in macaques¹⁷ and swine.¹² Automated blood sampling (ABS) is designed to collect blood painlessly from awake and freely moving animals.¹⁶ The use of ABS reduces stress from handling and eliminates the repeated venipunctures that are required for manual blood sampling.⁶ The implementation of ABS allowed us to achieve a complete pharmacokinetic–pharmacodynamic drug assessment in cardiovascular safety telemetry dog studies with no collection-related disturbance of physiologic parameters. Combining the pharmacokinetic and cardiovascular studies by using this method also reduces the need to synthesize more compound and shortens drug development timelines. Another advantage of this approach is the reduction of animal use by eliminating satellite animal groups.¹⁹Our incorporation of ABS into cardiovascular safety telemetry dog studies required changes to the dog acclimation procedure, addition of vascular access ports (VAP) to the surgical protocol, and modifications to the canine housing. This report is an overview of how we successfully incorporated ABS into a colony of telemetry-implanted dogs and the modifications necessary for continued success.     

Comparative proteomic characteristic of mycoplasmas (Mollicutes)

Using one- and two-dimensional gel electrophoresis, various types of mass-spectrometry, and combinations of these two methods, saturating identification of mycoplasm Acholeplasma laidlawii proteome and study of proteins carrying posttranslational modifications were performed. We compared our data with earlier identified proteome of Mycoplasma gallisepticum. It was shown that M. gallisepticum and A. laidlawii expressed 61 and 58% of the proteins of the annotated open reading frames, respectively. All subunits of DNA-polymerase III were identified for A. laidlawii during our study, which indicates that our techniques enable detection of single copies of mollicutes proteins per cell. Metabolic pathways of respective mycoplasmas is compared in this research.     

Production of nitric oxide as related to cardiomyocyte injury upon regional myocardial ischemia and reperfusion in rats

Changes in nitric oxide concentration in the rat myocardium in situ during temporary occlusion of the anterior descending coronary artery and subsequent reperfusion were monitored by microdialysis in the risk zone and a normal zone, using an NO trap (complex of ferrous ions with N-methyl-D-glucamine dithiocarbamate, Fe³⁺-MGD). The amplitude of the EPR signal of the reduced adduct NO-Fe²⁺-MGD in the samples from the risk zone increased during the 40-min occlusion and remained higher than the initial or the current control values during 60-min postischemic reperfusion, indicating substantial NO production. By the end of reperfusion, the infarct size was 47 ± 3% of the risk area. The contents of ATP, creatine phosphate, and total creatine in the risk zone decreased to respectively 44 ± 4, 51 ± 5, and 60 ± 3% of the initial values, whereas the level of lactate was six times the initial. The normal zone of the left ventricle showed no changes in NO or energy metabolite levels throughout the experiment. Thus, intense nitric oxide production in acute regional ischemia and reperfusion is associated with disturbance of energy metabolism, cell membrane damage, and death of cardiomyocytes.     

Pathogenic Huntingtin Repeat Expansions in Patients with Frontotemporal Dementia and Amyotrophic Lateral Sclerosis

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Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells

Background  

Interactions of cells with the extracellular matrix (ECM) are critical for the establishment and maintenance of stem cell self-renewal and differentiation. However, the ECM is a complex mixture of matrix molecules; little is known about the role of ECM components in human embryonic stem cell (hESC) differentiation into neural progenitors and neurons.     

Expression of catalytic antibodies in eukaryotic systems

Expression of recombinant antibodies in mammalian cells is one of the key problems in immuno-biotechnology. Alternatively, expression of a broad panel of antibodies and of their fragments may be effectively performed in yeast cells. We obtained expression strains of the methylotrophic yeast Pichia pastoris producing single-chain human catalytic antibody A17 (A.17scFv), Fab-fragment (A.17Fab), and full-size light chain (A.17Lch). These antibodies were characterized in terms of functional activity. The capacity to specifically bind and transform organophosphorus compounds has been demonstrated for A.17scFv and A.17Fab. The loss of activity of the antibody light chain when expressed alone indicates that the active site is formed by both heavy and light chains of the antibody. We determined the reversible constant K _d and the first order constant (k ₂) of the reaction of the covalent modification of A.17scFv and A.17Fab by irreversible inhibitor of the serine proteases p-nitrophenyl 8-methyl-8-azobicyclo[3.2.1]phosphonate (phosphonate X). Calculated values indicate that activity of the antibodies expressed in yeast is similar to the full-size antibody A17 and to the single-chain antibody A.17 expressed in CHO and E. coli cells, respectively.