An engineered folded PLP-bound monomer of Treponema denticola cystalysin reveals the effect of the dimeric structure on the catalytic properties of the enzyme

Cystalysin, a dimeric pyridoxal 5'-phosphate (PLP)-dependent lyase, is a virulence factor of the human oral pathogen Treponema denticola. Guided by bioinformatic analysis, two interfacial residues (Leu57 and Leu62) and an active site residue (Tyr64*), hydrogen-bonded with the PLP phosphate group of the neighboring subunit, have been mutated. The wild-type and the L57A, L62A, Y64*A, L57A/L62A, L57A/Y64*A, L57A/L62A/Y64*A mutants, all having a C-terminal histidine tag, have been constructed, expressed, and purified. The impact of these mutations on the dimeric state of cystalysin in the apo- and holo-form has been analyzed by size-exclusion chromatography. The results demonstrate that (i) Leu57 is more critical than Leu62 for apodimer formation, (ii) Tyr64*, more than Leu62, interferes with dimerization of holocystalysin without affecting that of apoenzyme, (iii) while each single mutation is inadequate in significantly altering the extent of monomerization of both apo- and holo-cystalysin, their combination leads to species which remain in a folded monomeric state at a reasonably high concentration in both the apo- and holo-forms. Although L57A/L62A or L57A/Y64*A, even to a different extent, are stimulated to dimer formation in the presence of either unproductive or productive ligands, L57A/L62A/Y64*A remains prevalently monomer at a concentration up to 50 microM. Kinetic analyses show that in this monomeric species the alpha,beta-eliminase, alanine racemase, and D-alanine half-transaminase activities are almost abolished, while the L-alanine half-transaminase activity is slightly enhanced when compared with that of wild-type. The structural basis of the stereospecific transaminase activity displayed by the engineered folded PLP-bound monomer has been analyzed and discussed.     

Induced pluripotent stem cells for therapy personalization in pediatric patients:Focus on drug-induced adverse events

Adverse drug reactions(ADRs) are major clinical problems, particularly in special populations such as pediatric patients. Indeed, ADRs may be caused by a plethora of different drugs leading, in some cases, to hospitalization, disability or even death. In addition, pediatric patients may respond differently to drugs with respect to adults and may be prone to developing different kinds of ADRs,leading, in some cases, to more severe consequences. To improve the comprehension, and thus the prevention, of ADRs, the set-up of sensitive and personalized assays is urgently needed. Important progress is represented by the possibility of setting up groundbreaking patient-specific assays. This goal has been powerfully achieved using induced pluripotent stem cells(iPSCs). Due to their genetic and physiological species-specific differences and their ability to be differentiated ideally into all tissues of the human body, this model may be accurate in predicting drug toxicity, especially when this toxicity is related to individual genetic differences. This review is an up-to-date summary of the employment of iPSCs as a model to study ADRs, with particular attention to drugs used in the pediatric field. We especially focused on the intestinal, hepatic,pancreatic, renal, cardiac, and neuronal levels, also discussing progress in organoids creation. The latter are three-dimensional in vitro culture systems derived from pluripotent or adult stem cells simulating the architecture and functionality of native organs such as the intestine, liver, pancreas, kidney, heart,and brain. Based on the existing knowledge, these models are powerful and promising tools in multiple clinical applications including toxicity screening,disease modeling, personalized and regenerative medicine.     

Fungi,dendritic cells and receptors: a host perspective of fungal virulence

An association between morphogenesis and virulence has long been presumed for dimorphic fungi that are pathogenic to humans, as one morphotype exists in the environment or during commensalism, and another within the host during the disease process. For Candida albicans, putative virulence factors include the ability to switch between saprophytic yeast and pathogenic, filamentous forms of the fungus. Dendritic cells sense either form in a specific way, resulting in distinct, T-helper-cell-dependent protective and non-protective immunities. Recent evidence suggests that the use of distinct recognition receptors contributes to the disparate patterns of reactivity observed locally in response to challenge with C. albicans. These findings offer new interpretive clues to the mechanisms of fungal virulence: rather than dimorphism per se, the engagement of different recognition receptors on dendritic cells might select the mode of fungal internalization and antigen presentation, condition the nature of the T-helper response and, ultimately, favor saprophytism or infection.     

The making of a professional secretory cell: architectural and functional changes in the ER during B lymphocyte plasma cell differentiation

B lymphocytes are small cells that express antigen receptors and secrete little if any IgM. Upon encounter with antigen, they differentiate into short-lived plasma cells, which secrete large amounts of polymeric IgM. Plasma cell differentiation entails a massive development of the endoplasmic reticulum to sustain high levels of Ig production. Recent findings suggest a role for the unfolded protein response in orchestrating the architectural and functional changes during terminal plasma cell differentiation.     

An Italian survey on “palliative intent” radiotherapy

BackgroundThe aim of this paper is to provide a comprehensive overview of the scenario on radiotherapy (RT) delivered with palliative intent in Italy.Materials and methodsA structured online questionnaire was submitted to Italian radiation oncologists in order to explore the clinical practice in different areas of palliation, namely: bone, lung, brain, liver, and emergencies suitable to RT.Results209 radiation oncologists took part in the study. Stereotactic body irradiation was found to be the preferred technique in lung and liver metastases, whereas 3D conformal RT was registered as the technique of choice for bone and brain metastases. The majority (98%) of participants stated to treat mainly radiotherapy emergencies with 3D conformal RT at doses ranging from 25 to 50 Gy. Re-irradiation is delivered by the majority of respondents, whereas post-treatment follow-up is done only by 51.4% of them.ConclusionsThis nationwide study highlights some heterogeneity among Italian radiation oncologists regarding treatment and follow-up of metastatic cancer patients.     

Induction of cell cycle arrest and DNA damage by the HDAC inhibitor panobinostat (LBH589) and the lipid peroxidation end product 4-hydroxynonenal in prostate cancer cells

Histone deacetylase inhibitors (HDACIs) are promising antineoplastic agents for the treatment of cancer. Here we report that the lipid peroxidation end product 4-hydroxynonenal (HNE) significantly potentiates the anti-tumor effects of the HDAC inhibitor panobinostat (LBH589) in the PC3 prostate cancer cell model. Panobinostat and HNE inhibited proliferation of PC3 cells and the combination of the two agents resulted in a significant combined effect. Cell cycle analysis revealed that both single agents and, to a greater extent, their combined treatment induced G2/M arrest, but cell death occurred in the combined treatment only. Furthermore, HNE and, to a greater extent, the combined treatment induced dephosphorylation of Cdc2 leading to progression into mitosis as confirmed by α-tubulin/DAPI staining and phospho-histone H3 (Ser10) analysis. To evaluate possible induction of DNA damage we utilized the marker phosphorylated histone H2A.X. Results showed that the combination of panobinostat and HNE induced significant DNA damage concomitant with the mitotic arrest. Then, by using androgen receptor (AR)-expressing PC3 cells we observed that the responsiveness to HNE and panobinostat was independent of the expression of functional AR. Taken together, our data suggest that HNE potentiates the antitumoral effect of the HDACI panobinostat in prostate cancer cells.     

Human wild-type alanine:glyoxylate aminotransferase and its naturally occurring G82E variant: functional properties and physiological implications

Human hepatic peroxisomal AGT (alanine:glyoxylate aminotransferase) is a PLP (pyridoxal 5'-phosphate)-dependent enzyme whose deficiency causes primary hyperoxaluria Type I, a rare autosomal recessive disorder. To acquire experimental evidence for the physiological function of AGT, the K(eq),(overall) of the reaction, the steady-state kinetic parameters of the forward and reverse reactions, and the pre-steady-state kinetics of the half-reactions of the PLP form of AGT with L-alanine or glycine and the PMP (pyridoxamine 5'-phosphate) form with pyruvate or glyoxylate have been measured. The results indicate that the enzyme is highly specific for catalysing glyoxylate to glycine processing, thereby playing a key role in glyoxylate detoxification. Analysis of the reaction course also reveals that PMP remains bound to the enzyme during the catalytic cycle and that the AGT-PMP complex displays a reactivity towards oxo acids higher than that of apoAGT in the presence of PMP. These findings are tentatively related to possible subtle rearrangements at the active site also indicated by the putative binding mode of catalytic intermediates. Additionally, the catalytic and spectroscopic features of the naturally occurring G82E variant have been analysed. Although, like the wild-type, the G82E variant is able to bind 2 mol PLP/dimer, it exhibits a significant reduced affinity for PLP and even more for PMP compared with wild-type, and an altered conformational state of the bound PLP. The striking molecular defect of the mutant, consisting in the dramatic decrease of the overall catalytic activity (approximately 0.1% of that of normal AGT), appears to be related to the inability to undergo an efficient transaldimination of the PLP form of the enzyme with amino acids as well as an efficient conversion of AGT-PMP into AGT-PLP. Overall, careful biochemical analyses have allowed elucidation of the mechanism of action of AGT and the way in which the disease causing G82E mutation affects it.     

Decreasing negative impacts of harvesting over insect communities using variable retention in southern Patagonian forests

Variable retention is an alternative silvicultural approach to timber forest management, which consist in a regeneration treatment with different degrees and patterns of stand retention. It has been proposed to mitigate harmful effects of harvesting, but effectiveness in insect conservation remains unknown in southern Patagonian Nothofagus pumilio forests. Here, the objectives were to: (1) define a baseline of insect diversity in old-growth forests along a site quality gradient (high, medium and low, associated to the forest productivity of each site); (2) evaluate stands with different retention treatments [aggregated (AR) surrounded by dispersed (DR) retention, and aggregated retention surrounded by clear-cut (CC)] and to compare with old-growth unmanaged forests (OGF); and (3) assess temporal changes during the first 4 years after harvesting (YAH). In a long term forest research plot, mobile epigean insect richness and relative abundance were characterized and classified in seven response type groups, using a wide spectrum sampling set. Data analyses included parametric and permutational ANOVAs, multivariate classification and ordinations. There were found 79 species before harvesting, and that richness was not related to site quality. After harvesting, 84 new species were added considering all treatments along the first four sampled YAH, of which 65 % were added to OGF, while in harvested sites richness and abundance directly diminished with retention degree (OGF > AR > DR > CC) due to incoming species cannot compensate the lost of them. However, fluctuations in diversity were observed along the YAH. Therefore, harvesting reduces insect richness in N. pumilio forests independently of the treatment, but the original insect assemblage significantly changes due to loss of sensitive species and introduction of others from surrounding environments. Despite this, inclusion of aggregates greatly diminished harvesting impacts because insect assemblage is favoured when structural complexity is preserved, conserving richness and abundance at similar levels than in old-growth forests. However, more studies are necessary to evaluate effects of different aggregate size, shape and distribution into harvested forests, as well as their fragmentation and connectivity at landscape level.