V-ATPase engagement in autophagic processes

The proton pumping activity of V-ATPase is responsible for acidification of the lysosome/vacuole. The low lumenal pH of this organelle stimulates the activity of a battery of resident hydrolases responsible for the degradation of various nonselective and selective cargos delivered by autophagic processes. However, the role of V-ATPase in membrane dynamics required for the uptake of autophagic cargo is far from fully understood. Consideration of the available data leads us to speculate that autophagic processes involving direct membrane-to-membrane contacts between the selected cargo and the vacuolar membrane require functional V-ATPase.     

The effects of selection for early and late reproduction on metabolite pools in Acanthoscelides obtectus Say

Abstract The present study was aimed at revealing the responses of metabolite pools to selection for alternative reproductive schedules in the seed beetle, Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae: Bruchinae). The levels of metabolites (free sugars, glycogen, lipids, soluble and hydrophobic proteins) that were determined in virgin females and males at three ages from adult eclosion onwards were compared among the base population (B) and two derived lines that were selected for either early (Y) or late (O) reproduction. The results showed differences in the accumulation of metabolites during pre‐adult development, as well as in the pattern of their changes during adult ageing. Generally, in comparison to the B population, the short‐lived beetles from the Y line showed increased protein content and reduced carbohydrate and lipid content, whereas the opposite was true for the long‐lived beetles from the O line. Females from the O line exhibited slower utilization of energy reserves and a slower increase in protein contents than females from the Y line. Females contained higher levels of free sugars, glycogen and hydrophobic proteins and lower levels of lipids and soluble proteins than males, although the sexual dimorphism was not evenly expressed among lines. Age‐specific changes in metabolite contents were slower in females than males. Our findings suggest that trade‐offs among capital resources are a physiological basis of early/late fitness trade‐offs and point to a conservation of resources that can be used for somatic maintenance.     

Dipeptidyl peptidase-IV inhibits glioma cell growth independent of its enzymatic activity

Malignant gliomas exhibit abnormal expression of proteolytic enzymes that may participate in the uncontrolled cell proliferation and aberrant interactions with the brain extracellular matrix. The multifunctional membrane bound serine aminopeptidase dipeptidyl peptidase (DPP)-IV has been linked to the development and progression of several malignancies, possibly both through the enzymatic and nonenzymatic mechanisms. In this report we demonstrate the expression of DPP-IV and homologous proteases fibroblast activation protein, DPP8 and DPP9 in primary cell cultures derived from high-grade gliomas, and show that the DPP-IV-like enzymatic activity is negatively associated with their in vitro growth. More importantly, the DPP-IV positive subpopulation isolated from the primary cell cultures using immunomagnetic separation exhibited slower proliferation. Forced expression of the wild as well as the enzymatically inactive mutant DPP-IV in glioma cell lines resulted in their reduced growth, migration and adhesion in vitro, as well as suppressed glioma growth in an orthotopic xenotransplantation mouse model. Microarray analysis of glioma cells with forced DPP-IV expression revealed differential expression of several candidate genes not linked to the tumor suppressive effects of DPP-IV in previous studies. Gene set enrichment analysis of the differentially expressed genes showed overrepresentation of gene ontology terms associated with cell proliferation, cell adhesion and migration. In conclusion, our data show that DPP-IV may interfere with several aspects of the malignant phenotype of glioma cells in great part independent of its enzymatic activity.     

Insights into the catalytic mechanism of tyrosine phenol-lyase from X-ray structures of quinonoid intermediates

Amino acid transformations catalyzed by a number of pyridoxal 5'-phosphate (PLP)-dependent enzymes involve abstraction of the Calpha proton from an external aldimine formed between a substrate and the cofactor leading to the formation of a quinonoid intermediate. Despite the key role played by the quinonoid intermediates in the catalysis by PLP-dependent enzymes, limited accurate information is available about their structures. We trapped the quinonoid intermediates of Citrobacter freundii tyrosine phenol-lyase with L-alanine and L-methionine in the crystalline state and determined their structures at 1.9- and 1.95-A resolution, respectively, by cryo-crystallography. The data reveal a network of protein-PLP-substrate interactions that stabilize the planar geometry of the quinonoid intermediate. In both structures the protein subunits are found in two conformations, open and closed, uncovering the mechanism by which binding of the substrate and restructuring of the active site during its closure protect the quinonoid intermediate from the solvent and bring catalytically important residues into positions suitable for the abstraction of phenol during the beta-elimination of L-tyrosine. In addition, the structural data indicate a mechanism for alanine racemization involving two bases, Lys-257 and a water molecule. These two bases are connected by a hydrogen bonding system allowing internal transfer of the Calpha proton.     

Short description of an alternative simplified method for screening recombinant clones within the "AdEasy-System" by Duplex-PCR

Background  

Recombinant adenoviral vectors are highly efficient for in vitro and in vivo gene delivery. They can easily be produced in large numbers, transduce a wide variety of cell types and generate high levels of transgene expression.     

Spectral Phase Shift of Surface Plasmon Resonance in the Kretschmann Configuration: Theory and Experiment

The spectral phase shift of surface plasmon resonance (SPR) in the Kretschmann configuration is modeled for aqueous solutions of NaCl (analytes) and an SPR structure consisting of gold and chromium layers deposited on an SF10 glass slide. Using the material dispersion of the SPR structure and the analyte, the SPR phase shift, its spectral derivative, and the spectral dependence of the ratio of the reflectances of p- and s-polarized waves are determined for aqueous solutions of NaCl when the concentration of NaCl in water and the refractive index range from 0 to 10 weight percent (wt%) and from 1.3334 to 1.3515 RIU, respectively. In addition, theoretical modeling is accompanied by experiment and the position of a sharp maximum in the measured spectral derivative of the SPR phase shift changes in a range from 596 to 626 nm. From the measurements, a sensitivity to concentration of 3.83 nm/wt% and a detection limit of 7.3 × 10^?7 RIU at a wavelength of 612.36 nm are obtained, and very good agreement between theory and experiment is confirmed.     

Red pigment-concentrating hormone is not limited to crustaceans

A peptide that was previously assumed to occur exclusively in crustaceans is found in the corpora cardiaca of the stinkbug, Nezara viridula. The sequence of the peptide was deduced from the multiple MS(N) electrospray mass data as that of an octapeptide: pGlu-Ile/Leu-Asn-Phe-Ser-Pro-Gly-Trp amide. This peptide with Leu at position 2 is known as crustacean red pigment-concentrating hormone and code-named Panbo-RPCH. The ambiguity about the amino acid at position 2, Leu or Ile, was solved by isolating the peptide in a single-step by reversed-phase HPLC and establishing co-elution with authentic Panbo-RPCH but not with the Ile(2)-analog. When injected into stinkbugs, synthetic Panbo-RPCH elicited an increase of lipids in the haemolymph. Thus, it is assumed that Panbo-RPCH functions in the stinkbug as a lipid-mobilizing hormone.