Rapid reverse phase-HPLC assay of HMG-CoA reductase activity

Radioisotope-based and mass spectrometry coupled to chromatographic techniques are the conventional methods for monitoring HMG-CoA reductase (HMGR) activity. Irrespective of offering adequate sensitivity, these methods are often cumbersome and time-consuming, requiring the handling of radiolabeled chemicals or elaborate ad-hoc derivatizing procedures. We propose a rapid and versatile reverse phase-HPLC method for assaying HMGR activity capable of monitoring the levels of both substrates (HMG-CoA and NADPH) and products (CoA, mevalonate, and NADP⁺) in a single 20 min run with no pretreatment required. The linear dynamic range was 10–26 pmol for HMG-CoA, 7–27 nmol for NADPH, 0.5–40 pmol for CoA and mevalonate, and 2–27 nmol for NADP⁺, and limit of detection values were 2.67 pmol, 2.77 nmol, 0.27 pmol, and 1.3 nmol, respectively.HMG-CoA reductase (HMGR) is the enzyme that catalyze the four-electron reductive deacylation of HMG-CoA to CoA and mevalonate (Fig. 1) (1). This reaction is the controlling step in the biosynthesis of sterols and isoprenoids (2, 3); hence, a large number of studies on the modulation of HMGR activity are continuously performed in the effort of developing new drugs in the treatment of hypercholesterolemic disorders (1).Open in a separate windowFig. 1.Schematic representation of HMGR enzymatic reaction.HMGR activity is conventionally assayed using elaborate radiochemical assay (4–9), chromatographic techniques coupled with mass spectrometry (10–15), or spectrophotometrically by monitoring the decrease in the absorbance of cofactor NADPH at 340 nm (16).Herein, as an alternative for laboratories with no access to the expensive LC/MS equipment, we propose a rapid and adequately sensitive HPLC-based method capable of monitoring both the levels of all the species involved in the equilibrium in a single analysis and the kinetics of HMGR-catalyzed reactions.     

Evaluation of six methods for extraction and purification of viral DNA from urine and serum samples

The sensitivity and reliability of PCR for diagnostic and research purposes require efficient unbiased procedures of extraction and purification of nucleic acids. One of the major limitations of PCR-based tests is the inhibition of the amplification process by substances present in clinical samples. This study used specimens spiked with a known amount of plasmid pBKV (ATCC 33-1) to compare six methods for extraction and purification of viral DNA from urine and serum samples based on recovery efficiency in terms of yield of DNA and percentage of plasmid pBKV recovered, purity of extracted DNA, and percentage of inhibition. The most effective extraction methods were the phenol/chloroform technique and the silica gel extraction procedure for urine and serum samples, respectively. Considering DNA purity, the silica gel extraction procedure and the phenol/chloroform method produced the most satisfactory results in urine and serum samples, respectively. The presence of inhibitors was overcome by all DNA extraction techniques in urine samples, as evidenced by semiquantitative PCR amplification. In serum samples, the lysis method and the proteinase K procedure did not completely overcome the presence of inhibitors.     

Molecular analysis of lichen-associated bacterial communities

The bacterial communities associated with 11 different lichen samples (belonging to eight different species) from different habitats were investigated. The culturable aerobic-heterotrophic fraction of the bacterial communities was isolated from nine lichen samples on protein-rich and sugar-rich/N-free media. Thirty-four bacterial isolates were purified and pooled into groups (phylotypes) by analysis of the ribosomal internal transcribed spacer polymorphism. Twenty five phylotypes were identified, each comprising between one and three isolates. One isolate of each phylotype was partially sequenced and the resulting 16S rRNA gene sequences were compared in a phylogenetic analysis. Three genera of Firmicutes, four of Actinobacteria and three of Proteobacteria were identified. Two phylotypes, belonging to the phyla Actinobacteria and Proteobacteria, respectively, were not identified at genus level. Some bacterial taxa were retrieved frequently in different lichen species sampled in the same or different sites. Paenibacillus and Burkholderia phylotypes seem to be common in lichens. Luteibactor rhizovicina was found in three different lichens of two different regions. In a cultivation-independent approach, total DNA was extracted from 11 lichen samples. Molecular fingerprints of the bacterial communities were obtained by PCR-amplification of the internal transcribed spacer region, and sequencing of selected bands indicated the presence of additional bacteria.     

CRTAP is required for prolyl 3- hydroxylation and mutations cause recessive osteogenesis imperfecta

Prolyl hydroxylation is a critical posttranslational modification that affects structure, function, and turnover of target proteins. Prolyl 3-hydroxylation occurs at only one position in the triple-helical domain of fibrillar collagen chains, and its biological significance is unknown. CRTAP shares homology with a family of putative prolyl 3-hydroxylases (P3Hs), but it does not contain their common dioxygenase domain. Loss of Crtap in mice causes an osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production. CRTAP can form a complex with P3H1 and cyclophilin B (CYPB), and Crtap-/- bone and cartilage collagens show decreased prolyl 3-hydroxylation. Moreover, mutant collagen shows evidence of overmodification, and collagen fibrils in mutant skin have increased diameter consistent with altered fibrillogenesis. In humans, CRTAP mutations are associated with the clinical spectrum of recessive osteogenesis imperfecta, including the type II and VII forms. Hence, dysregulation of prolyl 3-hydroxylation is a mechanism for connective tissue disease.     

Backbone dynamics of human parathyroid hormone (1-34): flexibility of the central region under different environmental conditions

The presence of a stable tertiary structure in the bioactive N-terminal portion of parathyroid hormone (PTH), a major hormone in the maintenance of extracellular calcium homeostasis, is still debated. In this work, 15N relaxation parameters of the 33 backbone amides of human PTH(1-34) were determined in phosphate-buffered saline solution (PBS) and in the presence of dodecylphosphocholine (DPC) micelles. The relaxation parameters were analyzed using both the model-free formalism (G. Lipari and A. Szabo, Journal of the American Chemical Society, 1982, Vol. 104, pp. 4546-4549) and the reduced spectral density functions approach (J.-F. Lefevre, K. T. Dayie, J. W. Peng, and G. Wagner, Biochemistry, 1996, Vol. 35, pp. 2674-2686). In PBS, the region around Gly12 possesses a high degree of flexibility and the C-terminal helix is less flexible than the N-terminal one. In the presence of DPC micelles, the mobility of the entire molecule is reduced, but the stability of the N-terminal helix increases relative to the C-terminal one. A point of relatively higher mobility at residue Gly12 is still present and a new site of local mobility at residues 16-17 is generated. These results justify the lack of experimental nuclear Overhauser effect (NOE) restraints with lack of tertiary structure and support the hypothesis that, in the absence of the receptor, the relative spatial orientation of the two N- and C-terminal helices is undefined. The flexibility in the midregion of PTH(1-34), maintained in the presence of the membrane-mimetic environment, may enable the correct relative disposition of the two helices, favoring a productive interaction with the receptor.     

CpG oligodeoxynucleotides induce Ca2+-dependent phospholipase D activity leading to phagolysosome maturation and intracellular mycobacterial growth inhibition in monocytes

Synthetic oligodeoxynucleotides containing CpG motifs (CpG ODN) have been reported to induce antimycobacterial activity both in vitro and in vivo. The present study analyzes the signals leading to CpG ODN-induced antimicrobial activity in monocytes. In this context, CpG, but not GpC, ODN induced cytosolic Ca2+ influx of extracellular origin which, in turn, activated host phospholipase D (PLD). The production of CpG-induced PLD-dependent phosphatidic acid induced the maturation of phagolysosomes and intracellular mycobacterial growth inhibition. These results show the presence of an antimicrobial pathway in monocytes, mediated by Ca2+-dependent PLD which can be useful for the exploitation of novel anti-tuberculosis immunotherapy approaches.     

The molecular basis of filamin binding to integrins and competition with talin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.     

Temperature preferences of bacteria isolated from seawater collected in Kandalaksha Bay,White Sea,Russia

Fifty-two bacteria were isolated from seawater collected in Kandalaksha Bay, White Sea, Russia, and classified by 16S rDNA sequencing. Most of the strains belonged to ubiquitous microorganisms. Pseudomonas was the most abundant genus (21 strains), including species of P. fluorescens, P. putida and P. syringae. Serratia was also common (10 strains) with species S. plymuthica and S. proteamaculans. Sphingobacterium, Flavobacterium and Pantoea were less represented (5, 3 and 2 strains, respectively). The only typical bacterium of marine Arctic regions was Shewanella baltica. The strains were tested for their optimal growth temperature in the range 0–45°C. The majority appeared to be psychrotolerant (42%) or mesophilic-psychrotolerant (40%). In addition, one strain (Bacillus pumilus) showed a rather narrow mesophilic profile. No true psychrophilic bacteria were found. Most of the strains showed a classical curve with fast growth decrease above the optimum; some others displayed uncommon flat curves with scarce differences between maximum and minimum of growth in a wide range of temperatures. Moreover, few strains presented an unusual profile being, in relation to the optimum, more tolerant to high rather than low temperatures. Preferences of the Kandalaksha Bay strains are generally different from those reported in literature for the same species: optima were at lower temperatures and, sometimes, ranges were broader showing increased eurythermism. This could indicate adaptation to the wide temperature variations recorded in this peculiar environment.     

High Selectivity of the γ-Aminobutyric Acid Transporter 2 (GAT-2, SLC6A13) Revealed by Structure-based Approach

The solute carrier 6 (SLC6) is a family of ion-dependent transporters that mediate uptake into the cell of osmolytes such as neurotransmitters and amino acids. Four SLC6 members transport GABA, a key neurotransmitter that triggers inhibitory signaling pathways via various receptors (e.g., GABA_A). The GABA transporters (GATs) regulate the concentration of GABA available for signaling and are thus targeted by a variety of anticonvulsant and relaxant drugs. Here, we characterize GAT-2, a transporter that plays a role in peripheral GABAergic mechanisms, by constructing comparative structural models based on crystallographic structures of the leucine transporter LeuT. Models of GAT-2 in two different conformations were constructed and experimentally validated, using site-directed mutagenesis. Computational screening of 594,166 compounds including drugs, metabolites, and fragment-like molecules from the ZINC database revealed distinct ligands for the two GAT-2 models. 31 small molecules, including high scoring compounds and molecules chemically related to known and predicted GAT-2 ligands, were experimentally tested in inhibition assays. Twelve ligands were found, six of which were chemically novel (e.g., homotaurine). Our results suggest that GAT-2 is a high selectivity/low affinity transporter that is resistant to inhibition by typical GABAergic inhibitors. Finally, we compared the binding site of GAT-2 with those of other SLC6 members, including the norepinephrine transporter and other GATs, to identify ligand specificity determinants for this family. Our combined approach may be useful for characterizing interactions between small molecules and other membrane proteins, as well as for describing substrate specificities in other protein families.