Dynactin's pointed-end complex is a cargo-targeting module

Dynactin is an essential part of the cytoplasmic dynein motor that enhances motor processivity and serves as an adaptor that allows dynein to bind cargoes. Much is known about dynactin''s interaction with dynein and microtubules, but how it associates with its diverse complement of subcellular binding partners remains mysterious. It has been suggested that cargo specification involves a group of subunits referred to as the “pointed-end complex.” We used chemical cross-linking, RNA interference, and protein overexpression to characterize interactions within the pointed-end complex and explore how it contributes to dynactin''s interactions with endomembranes. The Arp11 subunit, which caps one end of dynactin''s Arp1 filament, and p62, which binds Arp11 and Arp1, are necessary for dynactin stability. These subunits also allow dynactin to bind the nuclear envelope prior to mitosis. p27 and p25, by contrast, are peripheral components that can be removed without any obvious impact on dynactin integrity. Dynactin lacking these subunits shows reduced membrane binding. Depletion of p27 and p25 results in impaired early and recycling endosome movement, but late endosome movement is unaffected, and mitotic spindles appear normal. We conclude that the pointed-end complex is a bipartite structural domain that stabilizes dynactin and supports its binding to different subcellular structures.     

Crystal Structures of a Piscine Betanodavirus: Mechanisms of Capsid Assembly and Viral Infection

Betanodaviruses cause massive mortality in marine fish species with viral nervous necrosis. The structure of a T = 3 Grouper nervous necrosis virus-like particle (GNNV-LP) is determined by the ab initio method with non-crystallographic symmetry averaging at 3.6 Å resolution. Each capsid protein (CP) shows three major domains: (i) the N-terminal arm, an inter-subunit extension at the inner surface; (ii) the shell domain (S-domain), a jelly-roll structure; and (iii) the protrusion domain (P-domain) formed by three-fold trimeric protrusions. In addition, we have determined structures of the T = 1 subviral particles (SVPs) of (i) the delta-P-domain mutant (residues 35−217) at 3.1 Å resolution; and (ii) the N-ARM deletion mutant (residues 35−338) at 7 Å resolution; and (iii) the structure of the individual P-domain (residues 214−338) at 1.2 Å resolution. The P-domain reveals a novel DxD motif asymmetrically coordinating two Ca²⁺ ions, and seems to play a prominent role in the calcium-mediated trimerization of the GNNV CPs during the initial capsid assembly process. The flexible N-ARM (N-terminal arginine-rich motif) appears to serve as a molecular switch for T = 1 or T = 3 assembly. Finally, we find that polyethylene glycol, which is incorporated into the P-domain during the crystallization process, enhances GNNV infection. The present structural studies together with the biological assays enhance our understanding of the role of the P-domain of GNNV in the capsid assembly and viral infection by this betanodavirus.     

EPR Oximetry Sensor—Developing a TAM Derivative for In Vivo Studies

Oxygenation is one of the most important physiological parameters of biological systems. Low oxygen concentration (hypoxia) is associated with various pathophysiological processes in different organs. Hypoxia is of special importance in tumor therapy, causing poor response to treatment. Triaryl methyl (TAM) derivative radicals are commonly used in electron paramagnetic resonance (EPR) as sensors for quantitative spatial tissue oxygen mapping. They are also known as magnetic resonance imaging (MRI) contrast agents and fluorescence imaging compounds. We report the properties of the TAM radical tris(2,3,5,6-tetrachloro-4-carboxy-phenyl)methyl, (PTMTC), a potential multimodal (EPR/fluorescence) marker. PTMTC was spectrally analyzed using EPR and characterized by estimation of its sensitivity to the oxygen in liquid environment suitable for intravenous injection (1?mM PBS, pH?=?7.4). Further, fluorescent emission of the radical was measured using the same solvent and its quantum yield was estimated. An in vitro cytotoxicity examination was conducted in two cancer cell lines, HT-29 (colorectal adenocarcinoma) and FaDu (squamous cell carcinoma) and followed by uptake studies. The stability of the radical in different solutions (PBS pH?=?7.4, cell media used for HT-29 and FaDu cells culturing and cytotoxicity procedure, full rat blood and blood plasma) was determined. Finally, a primary toxicity test of PTMTC was carried out in mice. Results of spectral studies confirmed the multimodal properties of PTMTC. PTMTC was demonstrated to be not absorbed by cancer cells and did not interfere with luciferin-luciferase based assays. Also in vitro and in vivo tests showed that it was non-toxic and can be freely administrated till doses of 250?mg/kg BW via both i.v. and i.p. injections. This work illustrated that PTMTC is a perfect candidate for multimodal (EPR/fluorescence) contrast agent in preclinical studies.     

Inhibition of mitochondrial complex I improves glucose metabolism independently of AMPK activation

Accumulating evidences showed metformin and berberine, well‐known glucose‐lowering agents, were able to inhibit mitochondrial electron transport chain at complex I. In this study, we aimed to explore the antihyperglycaemic effect of complex I inhibition. Rotenone, amobarbital and gene silence of NDUFA13 were used to inhibit complex I. Intraperitoneal glucose tolerance test and insulin tolerance test were performed in db/db mice. Lactate release and glucose consumption were measured to investigate glucose metabolism in HepG2 hepatocytes and C2C12 myotubes. Glucose output was measured in primary hepatocytes. Compound C and adenoviruses expressing dominant negative AMP‐activated protein kinase (AMPK) α1/2 were exploited to inactivate AMPK pathway. Cellular NAD⁺/NADH ratio was assayed to evaluate energy transforming and redox state. Rotenone ameliorated hyperglycaemia and insulin resistance in db/db mice. It induced glucose consumption and glycolysis and reduced hepatic glucose output. Rotenone also activated AMPK. Furthermore, it remained effective with AMPK inactivation. The enhanced glycolysis and repressed gluconeogenesis correlated with a reduction in cellular NAD⁺/NADH ratio, which resulted from complex I suppression. Amobarbital, another representative complex I inhibitor, stimulated glucose consumption and decreased hepatic glucose output in vitro, too. Similar changes were observed while expression of NDUFA13, a subunit of complex I, was knocked down with gene silencing. These findings reveal mitochondrial complex I emerges as a key drug target for diabetes treatment. Inhibition of complex I improves glucose homoeostasis via non‐AMPK pathway, which may relate to the suppression of the cellular NAD⁺/NADH ratio.     

Evaluation of hydrophobic properties of Yersinia enterocolitica strains isolated from humans and pigs]

The aim of the study was to evaluate the hydrophobic properties of Yersinia enterocolitica and to determine the influence of the culture conditions, such as: type of medium, temperature, and duration of the culture on the manifestation of these properties. The subject of the study were 117 of Y. enterocolitica strains isolated from humans and pigs. The ammonium sulphate salt aggregation test according to Lindahl modified method was used to evaluate the hydrophobic properties of Y. enterocolitica strains. Strains of Y. enterocolitica were cultured for 24 h at 25 degrees C on TSA (Difco) medium. During investigation of the influence of the culture conditions the chosen strains were incubated for 24 h and 48 h at 25 degrees C and 37 degrees C on TSA (Difco), LB (Difco), enrichment agar (Biomed), and enrichment agar with 5% sheep blood (Graso). A total of 44.5%, 17.9%, 9.4%, and 28.2% strains of Y. enterocolitica showed very strong hydrophobic properties, strong hydrophobic properties, some hydrophobic properties, and were non-hydrophobic, respectively when strains of Y. enterocolitica were cultured for 24 h at 25 degrees C on TSA medium. A total of 75.5% strains isolated from humans showed very strong hydrophobic properties and 13.5% strains were non-hydrophobic. Among strains isolated from pigs 30% showed very strong hydrophobic properties but 35% were non-hydrophobic. The hydrophobic properties of Y. enterocolitica depended on the temperature, duration of the culture and the type of media. The highest number of strains with very strong hydrophobic properties (89.6%) was obtained after 48 h of the incubation at 37 degrees C on the enrichment agar with 5% sheep blood. The highest number of non-hydrophobic strains of Y. enterocolitica (28.5%) was obtained after 24 h at 25 degrees C on TSA medium.     

Insulin-like growth factor I-dependent regulation of prolidase activity in cultured human skin fibroblasts

The objectives of this article are to: (i) discuss the origins and the nature of ischemic injury, (ii) identify factors influencing the evolution of injury, (iii) consider various cellular targets for ischemic injury, (iv) assess the overall importance of reperfusion injury, (v) discuss conceptual approaches to cardioprotection and (vi) to identify new ideas and approaches in the realm of myocardial protection. In the human heart, myocardial ischemia may take many forms, it may exist for periods as short as a few seconds or minutes, it may last for hours or it may persist for years. In terms of discussing interventions to combat myocardial ischemia, this article will focus on: (i) regional ischemia as occurs during evolving myocardial infarction and (ii) whole heart or global ischemia as occurs during cardiac surgery and transplantation.     

Activity Budgets of Captive Cape Fur Seals (Arctocephalus pusillus) Under a Training Regime

Ethograms and time budgets are crucial for the behavioral assessment of nonhuman animals in zoos, and they serve as references for welfare research. This study was conducted to obtain detailed time budgets of trained Cape fur seals (Arctocephalus pusillus) in captivity, to evaluate variations of these patterns, and to determine whether abnormal behaviors had been displayed. Behavioral data for 3 Cape fur seals in the Wroclaw Zoo were collected, and more than 300 observation hours (during a 12-month period) per individual were analyzed. The studied animals exhibited a diversified repertoire of natural behaviors with apparent seasonal and daily patterns, and they did not present stereotypic behaviors. Significant differences of interaction rates between individuals suggest more frequent affiliative interactions among related animals. The absence of stereotypic behaviors, good health of individuals, and the presence of diversified natural behaviors indicated relatively good welfare of Cape fur seals kept in the Wroclaw Zoo.