Molecular dynamics characterization of the C2 domain of protein kinase Cbeta.

Protein kinase C (PKC) isozymes comprise a family of related enzymes that play a central role in many intracellular eukaryotic signaling events. Isozyme specificity is mediated by association of each PKC isozyme with specific anchoring proteins, termed RACKs. The C2 domain of betaPKC contains at least part of the RACK-binding sites. Because the C2 domain contains also a RACK-like sequence (termed pseudo-RACK), it was proposed that this pseudo-RACK site mediates intramolecular interaction with one of the RACK-binding sites in the C2 domain itself, stabilizing the inactive conformation of betaPKC. BetaPKC depends on calcium for its activation, and the C2 domain contains the calcium-binding sites. The x-ray structure of the C2 domain of betaPKC shows that three Ca(2+) ions can be coordinated by two opposing loops at one end of the domain. Starting from this x-ray structure, we have performed molecular dynamics (MD) calculations on the C2 domain of betaPKC bound to three Ca(2+) ions, to two Ca(2+) ions, and in the Ca(2+)-free state, in order to analyze the effect of calcium on the RACK-binding sites and the pseudo-RACK sites, as well as on the loops that constitute the binding site for the Ca(2+) ions. The results show that calcium stabilizes the beta-sandwich structure of the C2 domain and thus affects two of the three RACK-binding sites within the C2 domain. Also, the interactions between the third RACK-binding site and the pseudo-RACK site are not notably modified by the removal of Ca(2+) ions. On that basis, we predict that the pseudo-RACK site within the C2 domain masks a RACK-binding site in another domain of betaPKC, possibly the V5 domain. Finally, the MD modeling shows that two Ca(2+) ions are able to interact with two molecules of O-phospho-l-serine. These data suggest that Ca(2+) ions may be directly involved in PKC binding to phosphatidylserine, an acidic lipid located exclusively on the cytoplasmic face of membranes, that is required for PKC activation.     

DNA methylation and chromatin accessibility predict age in the domestic dog

Across mammals, the epigenome is highly predictive of chronological age. These “epigenetic clocks,” most of which have been built using DNA methylation (DNAm) profiles, have gained traction as biomarkers of aging and organismal health. While the ability of DNAm to predict chronological age has been repeatedly demonstrated, the ability of other epigenetic features to predict age remains unclear. Here, we use two types of epigenetic information—DNAm, and chromatin accessibility as measured by ATAC-seq—to develop age predictors in peripheral blood mononuclear cells sampled from a population of domesticated dogs. We measured DNAm and ATAC-seq profiles for 71 dogs, building separate predictive clocks from each, as well as the combined dataset. We also use fluorescence-assisted cell sorting to quantify major lymphoid populations for each sample. We found that chromatin accessibility can accurately predict chronological age (R²_ATAC = 26%), though less accurately than the DNAm clock (R²_DNAm = 33%), and the clock built from the combined datasets was comparable to both (R²_combined = 29%). We also observed various populations of CD62L+ T cells significantly correlated with dog age. Finally, we found that all three clocks selected features that were in or near at least two protein-coding genes: BAIAP2 and SCARF2, both previously implicated in processes related to cognitive or neurological impairment. Taken together, these results highlight the potential of chromatin accessibility as a complementary epigenetic resource for modeling and investigating biologic age.     

Expression of double Vitreoscilla hemoglobin enhances growth and alters ribosome and tRNA levels in Escherichia coli

In several organisms, expression of a gene encoding dimeric hemoglobin (VHb) from the obligate aerobic bacterium Vitreoscilla stercoraria has been shown to increase microaerobic cell growth and enhance oxygen-dependent cell metabolism. In an attempt to further improve these effects of VHb, a gene encoding two vhb genes connected by a short linker of six base pairs was constructed and expressed in Escherichia coli(double VHb). Escherichia coli cells expressing double VHb reached a cell density 19% higher than that of cells expressing native VHb. The protein production per cell remained constant since the increase in cell growth was accompanied by an increase in protein content by 16%. Investigation of ribosome and tRNA content revealed that cells expressing double VHb reached their maximal capacity of protein synthesis later during cultivation than cells expressing native VHb, and furthermore they reached considerably higher levels of ribosome and tRNA compared to that of the VHb-expressing cells.     

Host use of a specialist lichen-feeder: dealing with lichen secondary metabolites

Host use by herbivores is largely determined by host properties such as nutrient content and chemical defence against foragers. The impacts of these attributes on a herbivore may largely depend on its life cycle stage. Lichen species are known to differ in nutritional quality and level of chemical defence and, consequently, vary as fodder for herbivores. The aim of this study was to explore the impact of several lichen species and the presence of their secondary metabolites on their use as hosts by a specialist lichen-feeder, Cleorodes lichenaria. This study also addressed, for the first time, how a specialist lichen-feeder deals with different lichen secondary metabolites. In the beginning of their development, larvae grew better on Xanthoria parietina than on the other host lichens, whereas older larvae grew best on Ramalina fraxinea. Lichen secondary chemicals in R. fraxinea and Parmelia sulcata hindered larval growth in the beginning but after 75 days lichen secondary chemicals had no impact on the mass of larvae. Physodic acids in Hypogymnia physodes were lethal to larvae. In general, larvae metabolized 70–95% of ingested lichen secondary chemicals and the rest of these were excreted in frass. Lichen secondary metabolites in P. sulcata restrict and in H. physodes prevent their use as a host for C. lichenaria larvae. Our main finding, the ability of larvae to metabolize several lichen secondary metabolites, indicates digestive adaptation to these chemicals. No signs of sequestration of these chemicals were found.     

Brain is an important source of GnRH in general circulation in the rat during prenatal and early postnatal ontogenesis

This study was aimed to test our hypothesis that, in contrast to adult rats, in fetuses and neonates, a large amount of the brain-derived GnRH is delivered to the general circulation. The GnRH concentration and content were estimated in general circulation and in the forebrain in rats on the 18th embryonic day (E18), E21, 3rd postnatal day (P3) and P30-36. Moreover, the GnRH concentration was measured in general circulation on E21 following microsurgical lesion on E18 of the forebrain containing most GnRH neurons. The concentration and content of GnRH in plasma on E18, E21 and P3 enormously exceeded those on P30-36. Reverse was true for the ontogenetic dynamics of the GnRH concentration in the forebrain. The lesion of the forebrain resulted in a drop of the GnRH concentration in plasma. The above data strongly suggest that the forebrain is the principal source of GnRH in general circulation in fetal and neonatal rats. Thus, the brain-derived GnRH is delivered to the general circulation in fetal and neonatal rats in amounts likely sufficient to influence the potential peripheral targets.     

Protection Against Ischemic Brain Injury by Inhibition of Mitochondrial Oxidative Stress

Mitochondria are both targets and sources of oxidative stress. This dual relationship is particularly evident in experimental paradigms modeling ischemic brain injury. One mitochondrial metabolic enzyme that is particularly sensitive to oxidative inactivation is pyruvate dehydrogenase. This reaction is extremely important in the adult CNS that relies very heavily on carbohydrate metabolism, as it represents the sole bridge between anaerobic and aerobic metabolism. Oxidative injury to this enzyme and to other metabolic enzymes proximal to the electron transport chain may be responsible for the oxidized shift in cellular redox state that is observed during approximately the first hour of cerebral reperfusion. In addition to impairing cerebral energy metabolism, oxidative stress is a potent activator of apoptosis. The mechanisms responsible for this activation are poorly understood but likely involve the expression of p53 and possibly direct effects of reactive oxygen species on mitochondrial membrane proteins and lipids. Mitochondria also normally generate reactive oxygen species and contribute significantly to the elevated net production of these destructive agents during reperfusion. Approaches to inhibiting pathologic mitochondrial generation of reactive oxygen species include mild uncoupling, pharmacologic inhibition of the membrane permeability transition, and simply lowering the concentration of inspired oxygen. Antideath mitochondrial proteins of the Bcl-2 family also confer cellular resistance to oxidative stress, paradoxically through stimulation of mitochondrial free radical generation and secondary upregulation of antioxidant gene expression.     

The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M(-1)s(-1), 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.     

The chemo enzymatic functionalization of chitosan zeolite particles provides antioxidant and antimicrobial properties

Silicate‐based microporous materials like zeolites are nano enabled particles and used for various applications including pharmaceutical formulations. This study reports on the chemo‐enzymatic functionalization of chitosan‐zeolite particles (CTS‐zeolites) with caffeic acid (CA) and glucose oxidase (GOX) to impart combined antioxidant and antimicrobial properties. CA was grafted on the chitosan moieties by using laccase generating stable particles (zeta potential –36.7 mV) of high antioxidant activity (44% DPPH inhibition). GOX was immobilized both on CTS‐zeolites and on CA modified CTS‐zeolites and creating a hydrogen peroxide generation system continuously and in‐situ producing this oxidative and antimicrobial agent. The system prevented bacterial growth of E. coli and S. aureus over 24 h whereby a steady‐state concentration of around 60 μM hydrogen peroxide in the culture medium was observed. CA and GOX functionalized CTS‐zeolite particles additionally showed combinatorial antioxidant and antimicrobial properties providing a powerful bioactive system for medical applications. These particles proved their suitability for incorporation in bioactive formulations which could be used, inter alia, for topical wound treatments.