PcoB is a defense outer membrane protein that facilitates cellular uptake of copper

Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid‐encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical β‐barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion‐conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco‐harboring pathogens.     

Replication study of 34 common SNPs associated with prostate cancer in the Romanian population

Prostate cancer is the third‐most common form of cancer in men in Romania. The Romanian unscreened population represents a good sample to study common genetic risk variants. However, a comprehensive analysis has not been conducted yet. Here, we report our replication efforts in a Romanian population of 979 cases and 1027 controls, for potential association of 34 literature‐reported single nucleotide polymorphisms (SNPs) with prostate cancer. We also examined whether any SNP was differentially associated with tumour grade or stage at diagnosis, with disease aggressiveness, and with the levels of PSA (prostate specific antigen). In the allelic analysis, we replicated the previously reported risk for 19 loci on 4q24, 6q25.3, 7p15.2, 8q24.21, 10q11.23, 10q26.13, 11p15.5, 11q13.2, 11q13.3. Statistically significant associations were replicated for other six SNPs only with a particular disease phenotype: low‐grade tumour and low PSA levels (rs1512268), high PSA levels (rs401681 and rs11649743), less aggressive cancers (rs1465618, rs721048, rs17021918). The strongest association of our tested SNP's with PSA in controls was for rs2735839, with 29% increase for each copy of the major allele G, consistent with previous results. Our results suggest that rs4962416, previously associated only with prostate cancer, is also associated with PSA levels, with 12% increase for each copy of the minor allele C. The study enabled the replication of the effect for the majority of previously reported genetic variants in a set of clinically relevant prostate cancers. This is the first replication study on these loci, known to associate with prostate cancer, in a Romanian population.     

Developing outcome measures for pediatric mitochondrial disorders: Which complaints and limitations are most burdensome to patients and their parents?

Since some drug intervention effects are only experienced by the patient, organizations such as the Food and Drug Administration prefer clinically meaningful outcome measures. Here, we evaluated which symptoms and limitations in daily life are most burdensome to pediatric patients with mitochondrial disorders and their parents, using two questionnaires. In a study of 78 patients, the most burdensome complaints included fatigue, behavior and speech disturbances, epilepsy and muscle weakness and a high degree of limitations in daily activities was found. Importantly, there was a discrepancy between what symptoms metabolic pediatricians estimated would be most burdensome compared to the patients'/caretakers' opinion. To include feasible and relevant outcome measures in intervention studies, the experience and opinions of patients and caretakers should therefore be heard.     

Nuclear localization of TRK-A in liver cells

The liver represents a site of expression of neurotrophins and their receptors. We have characterized the expression and intracellular localization of the nerve growth factor (NGF) receptor, Trk-A, in liver cells in vivo and in vitro. In both normal and fibrotic liver tissue, Trk-A immunostaining was present in different cell types, including parenchymal cells and cells of the inflammatory infiltrate. In hepatocytes and activated stellate cells (HSC), Trk-A showed a predominant nuclear localization, both in the presence and absence of injury. In cultured HSC, Trk-A was found to be functional, because exposure of the cells to recombinant NGF resulted in stimulation of cell migration and activation of intracellular signaling pathways, including Ras-ERK and PI3K/Akt. Remarkably, in cultured HSC, Trk-A staining was found constitutively in the nucleus. In these cells, Trk-A could be stained only by antibodies directed against the intracellular domain but not by those recognizing the extracellular portion of Trk-A suggesting that the intracellular portion of the receptor is the major determinant of nuclear Trk-A staining. In contrast to HSC, freshly isolated hepatocytes did not show any nuclear localization of the intracellular portion of Trk-A. In pheocromocytoma cells, nuclear staining for Trk-A was not present in conditions of serum deprivation, but could be induced by exposure to NGF or to a mixture of soluble mediators. We conclude that nuclear localization of the intracellular domain of Trk-A is observed constitutively in liver cells such as HSC, while in other cell types it could be induced in response to soluble factors.     

The Mammalian Cell Cycle Regulates Parvovirus Nuclear Capsid Assembly

It is unknown whether the mammalian cell cycle could impact the assembly of viruses maturing in the nucleus. We addressed this question using MVM, a reference member of the icosahedral ssDNA nuclear parvoviruses, which requires cell proliferation to infect by mechanisms partly understood. Constitutively expressed MVM capsid subunits (VPs) accumulated in the cytoplasm of mouse and human fibroblasts synchronized at G0, G1, and G1/S transition. Upon arrest release, VPs translocated to the nucleus as cells entered S phase, at efficiencies relying on cell origin and arrest method, and immediately assembled into capsids. In synchronously infected cells, the consecutive virus life cycle steps (gene expression, proteins nuclear translocation, capsid assembly, genome replication and encapsidation) proceeded tightly coupled to cell cycle progression from G0/G1 through S into G2 phase. However, a DNA synthesis stress caused by thymidine irreversibly disrupted virus life cycle, as VPs became increasingly retained in the cytoplasm hours post-stress, forming empty capsids in mouse fibroblasts, thereby impairing encapsidation of the nuclear viral DNA replicative intermediates. Synchronously infected cells subjected to density-arrest signals while traversing early S phase also blocked VPs transport, resulting in a similar misplaced cytoplasmic capsid assembly in mouse fibroblasts. In contrast, thymidine and density arrest signals deregulating virus assembly neither perturbed nuclear translocation of the NS1 protein nor viral genome replication occurring under S/G2 cycle arrest. An underlying mechanism of cell cycle control was identified in the nuclear translocation of phosphorylated VPs trimeric assembly intermediates, which accessed a non-conserved route distinct from the importin α2/β1 and transportin pathways. The exquisite cell cycle-dependence of parvovirus nuclear capsid assembly conforms a novel paradigm of time and functional coupling between cellular and virus life cycles. This junction may determine the characteristic parvovirus tropism for proliferative and cancer cells, and its disturbance could critically contribute to persistence in host tissues.     

Phosphorus saturation and pH differentially regulate the efficiency of organic acid anion-mediated P solubilization mechanisms in soil

Exudation of organic acid anions by plants as well as root-induced changes in rhizosphere pH can potentially improve phosphate (P_i) availability in the rhizosphere and are frequently found to occur simultaneously. In non-calcareous soils, a major proportion of P_i is strongly sorbed to metal oxi(hydr)oxides of mainly iron (Fe) and aluminium (Al) and organic anions are known to compete with P_i for the same sorption sites (ligand exchange) or solubilize P_i via ligand-promoted mineral dissolution. Root-induced co-acidification may also further promote P_i release from soil. The relative efficiency of these different solubilization mechanisms, however, is poorly understood. The aims of this study were to gain a better mechanistic understanding of the solubilizing mechanisms of four carboxylates (citrate, malate, oxalate, malonate) in five soils with high and low P surface site saturation. Results indicate that at a lower P saturation of solid phase sorption sites, ligand-promoted mineral dissolution was the main P_i solubilization mechanism, while ligand exchange became more important at higher soil P concentrations. Co-acidification generally increased P_i solubility in the presence of carboxylates; however the relative solubilizing effect of carboxylates compared to the background electrolyte (KCl) control decreased by 20–50%. In soils with high amounts of exchangeable calcium (Ca), the proton-induced Ca solubilization reduced soluble P_i, presumably due to ionic-strength-driven changes in the electric surface potential favoring a higher P_i retention. Across a wider soil pH range (pH 3–8), P_i solubility increased with increasing alkalinity, as a result of both, more negatively charged sorption sites, as well as DOC-driven changes in Fe and Al solubility, which were further enhanced by the presence of citrate. Overall, the relative efficiency of carboxylates in solubilizing P_i was greatest in soils with medium to high amounts of anionic binding sites (mainly Fe- and Al-oxy(hydr)oxides) and a medium P sorption site coverage, with citrate being most effective in solubilizing P_i.     

Hydrolysis of polyethyleneterephthalate by p‐nitrobenzylesterase from Bacillus subtilis

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p‐nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET‐hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2‐hydroxyethyl benzoate (HEB), and mono‐(2‐hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET‐hydrolase was isolated from non‐denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC‐MS/MS analysis. BsEstB was expressed in Escherichia coli with C‐terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p‐nitrophenyl acetate and 108 U/mg on p‐nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half‐life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2‐hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The k_cat values decreased with increasing complexity of the substrate from 6 and 8 (s?1) for p‐nitrophenyl‐acetate (4NPA) and p‐nitrophenyl‐butyrate (4NPB), respectively, to 0.14 (s?1) for bis(2‐hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water‐contact angle (WCA) from 68.2° ± 1.7° to 62.6° ± 1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2‐(bromomethyl)naphthalene. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Generation of useful insertionally blocked sterol degradation pathway mutants of fast-growing mycobacteria and cloning, characterization, and expression of the terminal oxygenase of the 3-ketosteroid 9alpha-hydroxylase in Mycobacterium smegmatis mc(2)155

Integration of the pCG79 temperature-sensitive plasmid carrying Tn611 was used to generate libraries of mutants with blocked sterol-transforming ability of the sterol-utilizing strains Mycobacterium smegmatis mc(2)155 and Mycobacterium phlei M51-Ept. Of the 10,000 insertional mutants screened from each library, 4 strains with altered activity of the sterol-degrading enzymes were identified. A blocked 4-androstene-3,17-dione-producing M. phlei mutant transformed sitosterol to 23,24-dinorcholane derivatives that are useful starting materials for corticosteroid syntheses. A recombinant plasmid, pFJ92, was constructed from the genomic DNA of one of the insertional mutants of M. smegmatis, 10A12, which was blocked in 3-ketosteroid 9alpha-hydroxylation and carrying the transposon insertion and flanking DNA sequences, and used to isolate a chromosomal fragment encoding the 9alpha-hydroxylase. The open reading frame encodes the 383-amino-acid terminal oxygenase of 3-ketosteroid 9alpha-hydroxylase in M. smegmatis mc(2)155 and has domains typically conserved in class IA terminal oxygenases. Escherichia coli containing the gene could hydroxylate the steroid ring at the 9alpha position.