Calorie restriction reduces biomarkers of cellular senescence in humans

Calorie restriction (CR) with adequate nutrient intake is a potential geroprotective intervention. To advance this concept in humans, we tested the hypothesis that moderate CR in healthy young-to-middle-aged individuals would reduce circulating biomarkers of cellular senescence, a fundamental mechanism of aging and aging-related conditions. Using plasma specimens from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE™) phase 2 study, we found that CR significantly reduced the concentrations of several senescence biomarkers at 12 and 24 months compared to an ad libitum diet. Using machine learning, changes in biomarker concentrations emerged as important predictors of the change in HOMA-IR and insulin sensitivity index at 12 and 24 months, and the change in resting metabolic rate residual at 12 months. Finally, using adipose tissue RNA-sequencing data from a subset of participants, we observed a significant reduction in a senescence-focused gene set in response to CR at both 12 and 24 months compared to baseline. Our results advance the understanding of the effects of CR in humans and further support a link between cellular senescence and metabolic health.     

AmyA, an α-Amylase with β-Cyclodextrin-Forming Activity, and AmyB from the Thermoalkaliphilic Organism Anaerobranca gottschalkii: Two α-Amylases Adapted to Their Different Cellular Localizations

Two α-amylase genes from the thermophilic alkaliphile Anaerobranca gottschalkii were cloned, and the corresponding enzymes, AmyA and AmyB, were investigated after purification of the recombinant proteins. Based on their amino acid sequences, AmyA is proposed to be a lipoprotein with extracellular localization and thus is exposed to the alkaline milieu, while AmyB apparently represents a cytoplasmic enzyme. The amino acid sequences of both enzymes bear high similarity to those of GHF13 proteins. The different cellular localizations of AmyA and AmyB are reflected in their physicochemical properties. The alkaline pH optimum (pH 8), as well as the broad pH range, of AmyA activity (more than 50% activity between pH 6 and pH 9.5) mirrors the conditions that are encountered by an extracellular enzyme exposed to the medium of A. gottschalkii, which grows between pH 6 and pH 10.5. AmyB, on the other hand, has a narrow pH range with a slightly acidic pH optimum at 6 to 6.5, which is presumably close to the pH in the cytoplasm. Also, the intracellular AmyB is less tolerant of high temperatures than the extracellular AmyA. While AmyA has a half-life of 48 h at 70°C, AmyB has a half-life of only about 10 min at that temperature, perhaps due to the lack of stabilizing constituents of the cytoplasm. AmyA and AmyB were very similar with respect to their substrate specificity profiles, clearly preferring amylose over amylopectin, pullulan, and glycogen. Both enzymes also hydrolyzed α-, β-, and γ-cyclodextrin. Very interestingly, AmyA, but not AmyB, displayed high transglycosylation activity on maltooligosaccharides and also had significant β-cyclodextrin glycosyltransferase (CGTase) activity. CGTase activity has not been reported for typical α-amylases before. The mechanism of cyclodextrin formation by AmyA is unknown.     

Par-4 Is an Essential Downstream Target of DAP-like Kinase (Dlk) in Dlk/Par-4–mediated Apoptosis

Prostate apoptosis response-4 (Par-4) was initially identified as a gene product up-regulated in prostate cancer cells undergoing apoptosis. In rat fibroblasts, coexpression of Par-4 and its interaction partner DAP-like kinase (Dlk, which is also known as zipper-interacting protein kinase [ZIPK]) induces relocation of the kinase from the nucleus to the actin filament system, followed by extensive myosin light chain (MLC) phosphorylation and induction of apoptosis. Our analyses show that the synergistic proapoptotic effect of Dlk/Par-4 complexes is abrogated when either Dlk/Par-4 interaction or Dlk kinase activity is impaired. In vitro phosphorylation assays employing Dlk and Par-4 phosphorylation mutants carrying alanine substitutions for residues S154, T155, S220, or S249, respectively, identified T155 as the major Par-4 phosphorylation site of Dlk. Coexpression experiments in REF52.2 cells revealed that phosphorylation of Par-4 at T155 by Dlk was essential for apoptosis induction in vivo. In the presence of the Par-4 T155A mutant Dlk was partially recruited to actin filaments but resided mainly in the nucleus. Consequently, apoptosis was not induced in Dlk/Par-4 T155A–expressing cells. In vivo phosphorylation of Par-4 at T155 was demonstrated with a phospho-specific Par-4 antibody. Our results demonstrate that Dlk-mediated phosphorylation of Par-4 at T155 is a crucial event in Dlk/Par-4-induced apoptosis.     

Lophodermium pini-mugonis sp. nov. on needles of Pinus mugo from the Alps based on morphological and molecular data

Lophodermium pini-mugonis, collected on needles of Pinus mugo from German Alps, is described as a species new to science. It is characterized by subcuticular ascomata with a wrinkled surface and a somewhat untidy outline, a complex structure of lip cells, and ellipsoidal conidia. An analysis of the internal transcribed spaces of rDNA showed that Lophodermium pini-mugonis is, sister to Lophodermium autumnale and distantly related to other Lophodermium species on pines. The hypothesis of cospeciation of Lophodermium species with members of the Pinaceae is discussed.     

Crystal structure of CbpF,a bifunctional choline‐binding protein and autolysis regulator from Streptococcus pneumoniae

Phosphorylcholine, a crucial component of the pneumococcal cell wall, is essential in bacterial physiology and in human pathogenesis because it binds to serum components of the immune system and acts as a docking station for the family of surface choline‐binding proteins. The three‐dimensional structure of choline‐binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, has been solved in complex with choline. CbpF shows a new modular structure composed both of consensus and non‐consensus choline‐binding repeats, distributed along its length, which markedly alter its shape, charge distribution and binding ability, and organizing the protein into two well‐defined modules. The carboxy‐terminal module is involved in cell wall binding and the amino‐terminal module is crucial for inhibition of the autolytic LytC muramidase, providing a regulatory function for pneumococcal autolysis.     

Regulation and function of specifier proteins in plants

Specifier proteins are responsible for the diversification of biologically active products formed upon myrosinase-catalyzed glucosinolate hydrolysis and are therefore assumed to have an impact on the defensive function of the glucosinolate–myrosinase system. Among glucosinolate hydrolysis products, the generation of epithionitriles and organic thiocyanates requires the presence of epithiospecifier protein (ESP) and thiocyanate-forming protein (TFP), respectively, while myrosinase alone is sufficient for the production of isothiocyanates. Both ESP and TFP also promote the formation of simple nitriles upon myrosinase-catalyzed glucosinolate hydrolysis. Only little is known about the biological effects of epithionitriles and thiocyanates. Moreover, simple nitriles have repeatedly been reported to be less toxic to plant pathogens and herbivorous insects than the correponding isothiocyanates. Thus, it has remained an open question how plants benefit from the presence of specifier proteins. In this review, we survey the biological effects of different types of glucosinolate hydrolysis products on insects and pathogens as well as the current knowlegde on the developmental, organ specific and stimuli-mediated regulation of specifier proteins. Integrating these findings can help us to better understand the ecological functions of plant specifier proteins as well as the co-evolution of glucosinolate-containing plants and their insect herbivores.     

Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts

Background

Mutations in Parkin are the most common cause of autosomal recessive Parkinson disease (PD). The mitochondrially localized E3 ubiquitin-protein ligase Parkin has been reported to be involved in respiratory chain function and mitochondrial dynamics. More recent publications also described a link between Parkin and mitophagy.

Methodology/Principal Findings

In this study, we investigated the impact of Parkin mutations on mitochondrial function and morphology in a human cellular model. Fibroblasts were obtained from three members of an Italian PD family with two mutations in Parkin (homozygous c.1072delT, homozygous delEx7, compound-heterozygous c.1072delT/delEx7), as well as from two relatives without mutations. Furthermore, three unrelated compound-heterozygous patients (delEx3-4/duplEx7-12, delEx4/c.924C>T and delEx1/c.924C>T) and three unrelated age-matched controls were included. Fibroblasts were cultured under basal or paraquat-induced oxidative stress conditions. ATP synthesis rates and cellular levels were detected luminometrically. Activities of complexes I-IV and citrate synthase were measured spectrophotometrically in mitochondrial preparations or cell lysates. The mitochondrial membrane potential was measured with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide. Oxidative stress levels were investigated with the OxyBlot technique. The mitochondrial network was investigated immunocytochemically and the degree of branching was determined with image processing methods. We observed a decrease in the production and overall concentration of ATP coinciding with increased mitochondrial mass in Parkin-mutant fibroblasts. After an oxidative insult, the membrane potential decreased in patient cells but not in controls. We further determined higher levels of oxidized proteins in the mutants both under basal and stress conditions. The degree of mitochondrial network branching was comparable in mutants and controls under basal conditions and decreased to a similar extent under paraquat-induced stress.

Conclusions

Our results indicate that Parkin mutations cause abnormal mitochondrial function and morphology in non-neuronal human cells.