The kinetics of ribosomal peptidyl transfer revisited

The speed of protein synthesis determines the growth rate of bacteria. Current biochemical estimates of the rate of protein elongation are small and incompatible with the rate of protein elongation in the living cell. With a cell-free system for protein synthesis, optimized for speed and accuracy, we have estimated the rate of peptidyl transfer from a peptidyl-tRNA in P site to a cognate aminoacyl-tRNA in A site at various temperatures. We have found these rates to be much larger than previously measured and fully compatible with the speed of protein elongation for E. coli cells growing in rich medium. We have found large activation enthalpy and small activation entropy for peptidyl transfer, similar to experimental estimates of these parameters for A site analogs of aminoacyl-tRNA. Our work has opened a useful kinetic window for biochemical studies of protein synthesis, bridging the gap between in vitro and in vivo data on ribosome function.     

Differential effects of Parkin and its mutants on protein aggregation, the ubiquitin-proteasome system, and neuronal cell death in human neuroblastoma cells

Mutations in Parkin, an E3 ligase, which participates in the ubiquitin-proteasome system (UPS), cause juvenile onset Parkinson's disease (PD). Some mutants aggregate upon over-expression, but the effects of such aggregation on the UPS and neuronal survival have not been characterized. We show in this study that transient over-expression of wild type (WT) Parkin or various mutants in human neuroblastoma cells leads to localized accumulation of green fluorescent protein (GFP(u)), an artificial proteasomal substrate, indicative of UPS dysfunction. Parkin mutants, but not WT, aggregated, and GFP(u) and ubiquitin accumulated within such aggregates. Apoptotic death occurred only with mutant Parkin over-expression, and correlated with aggregation, but not GFP(u) accumulation. Enzymatic proteasomal activity was slightly increased with WT Parkin and decreased with mutant Parkin over-expression. This decrease was, at least in part, due to caspase activation. We conclude that mutant forms of Parkin can exert toxic effects on neuronal cells, possibly through their propensity to aggregate. Both WT and mutant forms can induce localized UPS dysfunction, likely through different mechanisms. This raises a note of caution regarding forced over-expression of Parkin as a neuroprotective strategy in PD or other neurodegenerative conditions and suggests a possible toxic gain of function for certain mutant forms of Parkin.     

High Platelet Reactivity in Patients with Acute Coronary Syndromes Undergoing Percutaneous Coronary Intervention: Randomised Controlled Trial Comparing Prasugrel and Clopidogrel

Background

Prasugrel is more effective than clopidogrel in reducing platelet aggregation in acute coronary syndromes. Data available on prasugrel reloading in clopidogrel treated patients with high residual platelet reactivity (HRPR) i.e. poor responders, is limited.

Objectives

To determine the effects of prasugrel loading on platelet function in patients on clopidogrel and high platelet reactivity undergoing percutaneous coronary intervention for acute coronary syndrome (ACS).

Patients

Patients with ACS on clopidogrel who were scheduled for PCI found to have a platelet reactivity ≥40 AUC with the Multiplate Analyzer, i.e. “poor responders” were randomised to prasugrel (60 mg loading and 10 mg maintenance dose) or clopidogrel (600 mg reloading and 150 mg maintenance dose). The primary outcome measure was proportion of patients with platelet reactivity <40 AUC 4 hours after loading with study medication, and also at one hour (secondary outcome). 44 patients were enrolled and the study was terminated early as clopidogrel use decreased sharply due to introduction of newer P2Y12 inhibitors.

Results

At 4 hours after study medication 100% of patients treated with prasugrel compared to 91% of those treated with clopidogrel had platelet reactivity <40 AUC (p = 0.49), while at 1 hour the proportions were 95% and 64% respectively (p = 0.02). Mean platelet reactivity at 4 and 1 hours after study medication in prasugrel and clopidogrel groups respectively were 12 versus 22 (p = 0.005) and 19 versus 34 (p = 0.01) respectively.

Conclusions

Routine platelet function testing identifies patients with high residual platelet reactivity (“poor responders”) on clopidogrel. A strategy of prasugrel rather than clopidogrel reloading results in earlier and more sustained suppression of platelet reactivity. Future trials need to identify if this translates into clinical benefit.

Trial Registration

ClinicalTrials.gov NCT01339026     

Assignment of red antenna states in photosystem I from Thermosynechococcus elongatus by single-molecule spectroscopy

Single-molecule spectroscopy at cryogenic temperatures was used to elucidate spectral properties, heterogeneities, and dynamics of the chlorophyll a (Chla) molecules responsible for the fluorescence in photosystem I (PSI) from the cyanobacteria Thermosynechococcus elongatus. Absorption and hole burning data suggest the presence of three pools absorbing at wavelengths greater than 700 nm with their absorption maxima at 708, 715, and 719 nm. The responsible Chla molecules are termed C708, C715, and C719. In the emission spectra of single PSI complexes, zero-phonon lines (ZPLs) were observed over the whole red emission range of PSI. The spectral region of the C708 pool is dominated by intense ZPLs; on the other hand, the broad emission of C715/C719 is unstructured and ZPLs are seen in this region much less frequently. Spectral jumps of ZPLs were observed. The dynamics as well as the spectral range covered by such jumps differ for C708 and C715/C719. This heterogeneity is likely caused by differences in the close environment of the chromophores. A tentative assignment of C708 and C715/C719 to Chla dimers and a Chla trimer is discussed, which is based on the remarkable structural differences in the environment of the most probable candidates for the red-most fluorescence.     

Cellular response to DNA damage is enhanced by the pR plasmid in mouse cells and in Escherichia coli.

The pR plasmid, which enhances the survival of Escherichia coli C600 exposed to UV light by induction of the SOS regulatory mechanism, showed the same effect when it transformed mouse LTA cells (tk-, aprt-). With Tn5 insertion mutagenesis which inactivates UV functions in the pR plasmid, we recognized two different regions of the plasmid, uvp1 and uvp2. These pR UVR- mutants exhibited the same effect in LTA transformed cells, demonstrating that resistance to UV light, carried by the pR plasmid, was really due to the expression of these two regions, which were also in the mouse cells. Statistical analysis showed that the expression of the uvp1 and uvp2 regions significantly increased (P less than 0.01) the survival upon exposure to UV light in mouse cells and bacteria. These results might suggest the presence of an inducible repair response to DNA damage in mouse LTA cells.     

Differential biofilm formation and chemical disinfection resistance of sessile cells of listeria monocytogenes strains under monospecies and dual-species (with Salmonella enterica) conditions

This study aimed to investigate the possible influence of bacterial intra- and interspecies interactions on the ability of Listeria monocytogenes and Salmonella enterica to develop mixed-culture biofilms on an abiotic substratum, as well as on the subsequent resistance of sessile cells to chemical disinfection. Initially, three strains from each species were selected and left to attach and form biofilms on stainless steel (SS) coupons incubated at 15°C for 144 h, in periodically renewable tryptone soy broth (TSB), under either monoculture or mixed-culture (mono-/dual-species) conditions. Following biofilm formation, mixed-culture sessile communities were subjected to 6-min disinfection treatments with (i) benzalkonium chloride (50 ppm), (ii) sodium hypochlorite (10 ppm), (iii) peracetic acid (10 ppm), and (iv) a mixture of hydrogen peroxide (5 ppm) and peracetic acid (5 ppm). Results revealed that both species reached similar biofilm counts (ca. 10(5) CFU cm(-2)) and that, in general, interspecies interactions did not have any significant effect either on the biofilm-forming ability (as this was assessed by agar plating enumeration of the mechanically detached biofilm bacteria) or on the antimicrobial resistance of each individual species. Interestingly, pulsed-field gel electrophoresis (PFGE) analysis clearly showed that the three L. monocytogenes strains did not contribute at the same level either to the formation of mixed-culture sessile communities (mono-/dual species) or to their antimicrobial recalcitrance. Additionally, the simultaneous existence inside the biofilm structure of S. enterica cells seemed to influence the occurrence and resistance pattern of L. monocytogenes strains. In sum, this study highlights the impact of microbial interactions taking place inside a mixed-culture sessile community on both its population dynamics and disinfection resistance.     

Mutational analysis of TRAF6 reveals a conserved functional role of the RING dimerization interface and a potentially necessary but insufficient role of RING-dependent TRAF6 polyubiquitination towards NF-κB activation

TRAF6 is an E3 ubiquitin ligase that plays a pivotal role in the activation of NF-κB by innate and adaptive immunity stimuli. TRAF6 consists of a highly conserved carboxyl terminal TRAF-C domain which is preceded by a coiled coil domain and an amino terminal region that contains a RING domain and a series of putative zinc-finger motifs. The TRAF-C domain contributes to TRAF6 oligomerization and mediates the interaction of TRAF6 with upstream signaling molecules whereas the RING domain comprises the core of the ubiquitin ligase catalytic domain. In order to identify structural elements that are important for TRAF6-induced NF-κB activation, mutational analysis of the TRAF-C and RING domains was performed. Alterations of highly conserved residues of the TRAF-C domain of TRAF6 did not affect significantly the ability of the protein to activate NF-κB. On the other hand a number of functionally important residues (L77, Q82, R88, F118, N121 and E126) for the activation of NF-κB were identified within the RING domain of TRAF6. Interestingly, several homologues of these residues in TRAF2 were shown to have a conserved functional role in TRAF2-induced NF-κB activation and lie at the dimerization interface of the RING domain. Finally, whereas alteration of Q82, R88 and F118 compromised both the K63-linked polyubiquitination of TRAF6 and its ability to activate NF-κB, alteration of L77, N121 and E126 diminished the NF-κB activating function of TRAF6 without affecting TRAF6 K63-linked polyubiquitination. Our results support a conserved functional role of the TRAF RING domain dimerization interface and a potentially necessary but insufficient role for RING-dependent TRAF6 K63-linked polyubiquitination towards NF-κB activation in cells.     

Changes in Protein Expression in Two Cholangiocarcinoma Cell Lines Undergoing Formation of Multicellular Tumor Spheroids In Vitro

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and resistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutaminase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The arising protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conversion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the carbohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen.