On the non-respect of the thermodynamic cycle by DsbA variants

The mechanism of the disulfide-bond forming enzyme DsbA depends on the very low pKa of a cysteine residue in its active-site and on the relative instability of the oxidized enzyme compared to the reduced one. A thermodynamic cycle has been used to correlate its redox properties to the difference in the free energies of folding (deltadeltaGred/ox) of the oxidized and reduced forms. However, the relation was proved unsatisfied for a number of DsbA variants. In this study, we investigate the thermodynamic and redox properties of a highly destabilized variant DsbA(P151A) (substitution of cis-Pro151 by an alanine) by the means of intrinsic tryptophan fluorescence and by high-sensitivity differential scanning calorimetry (HS-DSC). When the value of deltadeltaGred/ox obtained fluorimetrically for DsbA(P151A) does not correlate with the value expected from its redox potential, the value of deltadeltaGred/ox provided by HS-DSC are in perfect agreement with the predicted thermodynamic cycle for both wild-type and variant. HS-DSC data indicate that oxidized wild-type enzyme and the reduced forms of both wild-type and variant unfold according to a two-state mechanism. Oxidized DsbA(P151A) shows a deviation from two-state behavior that implies the loss of interdomain cooperativity in DsbA caused by Pro151 substitution. The presence of chaotrope in fluorimetric measurements could facilitate domain uncoupling so that the fluorescence probe (Trp76) does not reflect the whole unfolding process of DsbA(P151A) anymore. Thus, theoretical thermodynamic cycle is respected when an appropriate method is applied to DsbA unfolding under conditions in which protein domains still conserve their cooperativity.     

Lipopolysaccharide enhances TGF‐β1 signalling pathway and rat pancreatic fibrosis

Pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during alcoholic chronic pancreatitis (ACP). Transforming growth factor‐beta1 (TGF‐β1) is a key regulator of extracellular matrix production and PSC activation. Endotoxin lipopolysaccharide (LPS) has been recognized as a trigger factor in the pathogenesis of ACP. This study aimed to investigate the mechanisms by which LPS modulates TGF‐β1 signalling and pancreatic fibrosis. Sprague‐Dawley rats fed with a Lieber‐DeCarli alcohol (ALC) liquid diet for 10 weeks with or without LPS challenge during the last 3 weeks. In vitro studies were performed using rat macrophages (Mφs) and PSCs (RP‐2 cell line). The results showed that repeated LPS challenge resulted in significantly more collagen production and PSC activation compared to rats fed with ALC alone. LPS administration caused overexpression of pancreatic TLR4 or TGF‐β1 which was paralleled by an increased number of TLR4‐positive or TGF‐β1‐positive Mφs or PSCs in ALC‐fed rats. In vitro, TLR4 or TGF‐β1 production in Mφs or RP‐2 cells was up‐regulated by LPS. LPS alone or in combination with TGF‐β1 significantly increased type I collagen and α‐SMA production and Smad2 and 3 phosphorylation in serum‐starved RP‐2 cells. TGF‐β pseudoreceptor BAMBI production was repressed by LPS, which was antagonized by Si‐TLR4 RNA or by inhibitors of MyD88/NF‐kB. Additionally, knockdown of Bambi with Si‐Bambi RNA significantly increased TGF‐β1 signalling in RP‐2 cells. These findings indicate that LPS increases TGF‐β1 production through paracrine and autocrine mechanisms and that LPS enhances TGF‐β1 signalling in PSCs by repressing BAMBI via TLR4/MyD88/NF‐kB activation.     

Survival Impact of Delaying Postoperative Radiotherapy in Patients with Esophageal Cancer

The purpose of the current study was to retrospectively assess the effect of postoperative radiotherapy (RT) delay on survival for patients with esophageal cancer. From 2008 to 2011, patients with esophageal cancer who had undergone postoperative RT in five different hospitals in China were reviewed. Clinical data, including time interval between surgery to RT, were prospectively collected. Kaplan-Meier method was conducted to estimate the effect of each variable on progression-free survival (PFS) and overall survival (OS), with differences assessed by log-rank test. Univariate Cox proportional-hazards models were performed for both PFS and OS for all assumed predictor variables. Statistically significant predictor variables (P < .05) on univariate analysis were then included in multivariate Cox proportional-hazards models, which were performed to compare the effects of RT delay on PFS and OS. A total of 316 patients were finally enrolled in this prospectively multicentric study. Time to RT after surgery varied from 12 days to over 60 days (median, 26 days). Multivariate analysis showed that delay to RT longer than the median does not appear to be a survival cost. There was also no statistically difference in PFS (P = .513) or OS (P = .236) between patients stratified by quartiles (≤21 days vs ≧35 days). However, patients with particularly long delays (≧42 days) demonstrated a detrimental impact on OS (P = .021) but not PFS (P = .580). Delaying postoperative RT of esophageal cancer does not impact PFS, but results in a significant reduction on OS if delaying longer than 6 weeks.     

Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons

Pyruvate recycling was studied in primary cultures of mouse cerebrocortical astrocytes, GABAergic cerebrocortical interneurons, and co-cultures consisting of both cell types by measuring production of [4-¹³C]glutamate from [3-¹³C]glutamate by aid of nuclear magnetic resonance spectroscopy. This change in the position of the label can only occur by entry of [3-¹³C]glutamate into the tricarboxylic acid (TCA) cycle, conversion of labeled -ketoglutarate to malate or oxaloacetate, malic enzyme-mediated decarboxylation of malate to pyruvate or phosphoenolpyruvate carboxykinase-mediated conversion of oxaloacetate to phosphoenolpyruvate and subsequent hydrolysis of the latter to pyruvate, and introduction of the labeled pyruvate into the TCA cycle, i.e., after exit of the carbon skeleton of pyruvate from the TCA cycle followed by re-entry of the same pyruvate molecules via acetyl CoA. In agreement with earlier observations, pyruvate recycling was demonstrated in astrocytes, indicating the ability of these cells to undertake complete oxidative degradation of glutamate. The recycled [4-¹³C]glutamate was not further converted to glutamine, showing compartmentation of astrocytic metabolism. Thus, absence of recycling into glutamine in the brain in vivo cannot be taken as indication that pyruvate recycling is absent in astrocytes. No recycling could be demonstrated in the cerebrocortical neurons. This is consistent with a previously demonstrated lack of incorporation of label from glutamate into lactate, and it also indicates that mitochondrial malic enzyme is not operational. Nor was there any indication of pyruvate recycling in the co-cultures. Although this may partly be due to more rapid depletion of glutamate in the co-cultures, this observation at the very least indicates that pyruvate recycling is not up-regulated in the neuronal-astrocytic co-cultures.     

Characterization of the tunicamycin gene cluster unveiling unique steps involved in its biosynthesis

Tunicamycin, a potent reversible translocase I inhibitor, is produced by several Actinomycetes species. The tunicamycin structure is highly unusual, and contains an 11-carbon dialdose sugar and an α, β-1″,11′-glycosidic linkage. Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression (HHE) strategy combined with a bioassay. Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains, demonstrating the role of the genes for the biosynthesis of tunicamycins. Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes (tunA– tunL). Amongst these is a putative radical SAM enzyme (Tun B) with a potentially unique role in biosynthetic carbon-carbon bond formation. Hence, a seven-step novel pathway is proposed for tunicamycin biosynthesis. Moreover, two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827. These data provide clarification of the novel mechanisms for tunicamycin biosynthesis, and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.     

Arabidopsis Villins Promote Actin Turnover at Pollen Tube Tips and Facilitate the Construction of Actin Collars

Apical actin filaments are crucial for pollen tube tip growth. However, the specific dynamic changes and regulatory mechanisms associated with actin filaments in the apical region remain largely unknown. Here, we have investigated the quantitative dynamic parameters that underlie actin filament growth and disappearance in the apical regions of pollen tubes and identified villin as the major player that drives rapid turnover of actin filaments in this region. Downregulation of Arabidopsis thaliana VILLIN2 (VLN2) and VLN5 led to accumulation of actin filaments at the pollen tube apex. Careful analysis of single filament dynamics showed that the severing frequency significantly decreased, and the lifetime significantly increased in vln2 vln5 pollen tubes. These results indicate that villin-mediated severing is critical for turnover and departure of actin filaments originating in the apical region. Consequently, the construction of actin collars was affected in vln2 vln5 pollen tubes. In addition to the decrease in severing frequency, actin filaments also became wavy and buckled in the apical cytoplasm of vln2 vln5 pollen tubes. These results suggest that villin confers rigidity upon actin filaments. Furthermore, an observed decrease in skewness of actin filaments in the subapical region of vln2 vln5 pollen tubes suggests that villin-mediated bundling activity may also play a role in the construction of actin collars. Thus, our data suggest that villins promote actin turnover at pollen tube tips and facilitate the construction of actin collars.     

Pharmacokinetic,metabolic stability,plasma protein binding and CYP450s inhibition/induction assessment studies of N-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5-pyrimidine-carboxamide as potential type 5 phosphodiesterase inhibitors

N-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5-pyrimidine-carboxamide (NHPPC) is a new potential of type 5 phosphodiesterase (PDE5) inhibitors, synthesized from the avanafil analogue for the treatment of erectile dysfunction. The targets of this article were to assess plasma protein binding, liver microsomal metabolic stability, inhibition and induction on cytochrome P450 isozymes and the pharmacokinetics of NHPPC. Equilibrium dialysis technique was applied to determine Plasma protein binding (PPB) and NHPPC was evaluated in male Sprague–Dawley rats and Beagle dogs in vivo pharmacokinetic. The NHPPC was highly bound to plasma proteins in rats, dogs and human tested and the mean values for PPB rate were 96.2%, 99.6% and 99.4%, respectively. After in vitro liver microsomes incubated for 60?min, the percent remaining of NHPPC was 42.8%, 0.8% and 42.0% in rats, dogs and human, respectively. In vitro intrinsic clearance was found to be 0.0233, 0.1204 and 0.0214 mL/min/mg protein in rat, dog and human liver microsomes of NHPPC, respectively. NHPPC showed no significant inhibitory effects on major CYP450 enzymes, and had no significant induction potential on CYP1A2 and CYP3A4. Following oral administration in rats and dogs, t_max was 6 and 0.5?h, respectively. The clearance for NHPPC was 1.19 and 1.46?L/h/kg in rats and dogs, respectively. And absolute bioavailability in rat and dog were approximately 34.5% and 53.1%, respectively. These results showed that NHPPC has a good development prospect.     

Novel Reassortant Highly Pathogenic H5N2 Avian Influenza Viruses in Poultry in China

There has been multiple evidence that domestic poultry may act as a vessel for the generation of novel influenza A viruses. In this study, we have analyzed the evolution and pathogenicity of 4 H5N2 avian influenza viruses isolated from apparently healthy poultry from H5N1 virus endemic areas in China. Phylogenetic analysis revealed that two of these viruses, A/duck/Eastern China/1111/2011 (DK/EC/1111/11) and A/goose/Eastern China/1112/2011 (GS/EC/1112/11) were derived from reassortment events in which clade 2.3.4 highly pathogenic avian influenza (HPAI) H5N1 viruses acquired novel neuraminidase and nonstructural protein genes. Another two isolates, A/chicken/Hebei/1102/2010 (CK/HB/1102/10) and A/duck/Hebei/0908/2009 (DK/HB/0908/09), possess hemagglutinin (HA) gene belong to clade 7 H5 viruses and other genes from endemic H9N2 viruses, or from viruses of various subtypes of the natural gene pool. All of these H5N2 isolates bear characteristic sequences of HPAI virus at the cleavage site of HA, and animal experiments indicated that all of these viruses but DK/HB/0908/09 is highly pathogenic to chickens. In particular, DK/EC/1111/11 and GS/EC/1112/11 are also highly pathogenic to ducks and moderately pathogenic to mice. All of these 4 viruses were able to replicate in domestic ducks and mice without prior adaptation. The emergence of these novel H5N2 viruses adds more evidence for the active evolution of H5 viruses in Asia. The maintenance of the highly pathogenic phenotype of some of these viruses even after reassortment with a new NA subtypes, their ability to replicate and transmit in domestic poultry, and the pathogenicity in the mammalian mouse model, highlight the potential threat posed by these viruses to both veterinary and public health.