Quantitative detection of changes in the leaf‐mesophyll tonoplast proteome in dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants

Although the vacuole is the most important final store for toxic heavy metals like cadmium (Cd²⁺), our knowledge on how they are transported into the vacuole is still insufficient. It has been suggested that Cd²⁺ can be transported as phytochelatin‐Cd²⁺ by an unknown ABC transporter or in exchange with protons by cation/proton exchanger (CAX) transporters. To unravel the contribution of vacuolar transporters to Cd²⁺ detoxification, a quantitative proteomics approach was performed. Highly purified vacuoles were isolated from barley plants grown under minus, low (20 μM), and high (200 μM) Cd²⁺ conditions and protein levels of the obtained tonoplast samples were analyzed using isobaric tag for relative and absolute quantitation (iTRAQ?). Although 56 vacuolar transporter proteins were identified, only a few were differentially expressed. Under low‐Cd²⁺ conditions, an inorganic pyrophosphatase and a γ‐tonoplast intrinsic protein (γ‐TIP) were up‐regulated, indicating changes in energization and water fluxes. In addition, the protein ratio of a CAX1a and a natural resistance‐associated macrophage protein (NRAMP), responsible for vacuolar Fe²⁺ export was increased. CAX1a might play a role in vacuolar Cd²⁺ transport. An increase in NRAMP activity leads to a higher cytosolic Fe²⁺ concentration, which may prevent the exchange of Fe²⁺ by toxic Cd²⁺. Additionally, an ABC transporter homolog to AtMRP3 showed up‐regulation. Under high Cd²⁺ conditions, the plant response was more specific. Only a protein homologous to AtMRP3 that showed already a response under low Cd²⁺ conditions, was up‐regulated. Interestingly, AtMRP3 is able to partially rescue a Cd²⁺‐sensitive yeast mutant. The identified transporters are good candidates for further investigation of their roles in Cd²⁺ detoxification.     

Periodontal therapy increases neutrophil extracellular trap degradation

In periodontitis, polymorphonuclear leucocytes (PMNs) are activated. They entrap and eliminate pathogens by releasing neutrophil extracellular traps (NETs). Abnormal NET degradation is part of a pro-inflammatory status, affecting co-morbidities such as cardiovascular disease. We aimed to investigate the ex vivo NET degradation capacity of plasma from periodontitis patients compared to controls (part 1) and to quantify NET degradation before and after periodontal therapy (part 2). Fresh NETs were obtained by stimulating blood-derived PMNs with phorbol 12-myristate 13-acetate. Plasma samples from untreated periodontitis patients and controls were incubated for 3 h onto freshly generated NETs (part 1). Similarly, for part 2, NET degradation was studied for 91 patients before and 3, 6 and 12 mo after non-surgical periodontal therapy with and without adjunctive systemic antibiotics. Finally, NET degradation was fluorospectrometrically quantified. NET degradation levels did not differ between periodontitis patients and controls, irrespective of subject-related background characteristics. NET degradation significantly increased from 65.6 ± 1.7% before periodontal treatment to 75.7 ± 1.2% at 3 mo post periodontal therapy, and this improvement was maintained at 6 and 12 mo, irrespective of systemic usage of antibiotics. Improved NET degradation after periodontitis treatment is another systemic biomarker reflecting a decreased pro-inflammatory status, which also contributes to an improved cardiovascular condition.     

Deficiency of terminal ADP‐ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

Adenosine diphosphate (ADP)‐ribosylation is a post‐translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP‐ribosylation reactions are the poly(ADP‐ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP‐ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP‐ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP‐ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP‐interacting protein that removes mono(ADP‐ribosyl)ation on glutamate amino acid residues in PARP‐modified proteins. X‐ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl‐(ADP‐ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP‐ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.     

Bax Exists in a Dynamic Equilibrium between the Cytosol and Mitochondria to Control Apoptotic Priming

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Highlights? Bax exists in equilibrium between the cytosol and mitochondria ? The retrotranslocation of Bax is independent of direct-activator BH3 proteins ? Changes in survival signaling modulate the dissociation rate of mitochondrial Bax ? Accumulation of Bax on mitochondria sensitizes cells to apoptosis     

Activity,synthesis, storage,and messenger RNA of cyclooxygenase in intrauterine tissues of guinea pigs near term and during labor

Whether the reported gestation-dependent increase in cyclooxygenase activity in gestational tissues is due to an accumulation of cyclooxygenase in vivo or an increasing capacity to synthesize cyclooxygenase in vitro is unknown. In this study in guinea pigs, COX activity was estimated from the net production rates of prostaglandins E(2) and F(2alpha) in the presence of optimal substrate concentrations. Cyclooxygenase activity in amnion increased between 45 days of gestation and labor in microsomes (150-fold in relation to PGF(2alpha) production and 116-fold in relation to PGE(2) production) and in tissue explants (42-fold in relation to PGF(2alpha) production). The capacity for de novo synthesis of cyclooxygenase after aspirin treatment increased nine-fold between 45 days of gestation and labor in amnion explants. Comparison of COX activity in amnion explants with or without prior aspirin treatment showed that COX activity is at least three-fold higher in controls than would be expected if the activity was due to de novo synthesis alone. Cyclooxygenase-2 mRNA predominated in amnion but neither cyclooxygenase-2 nor cyclooxygenase-1 mRNA levels (semi-quantitative RT-PCR) changed significantly. This suggests that the gestation-dependent increase in cyclooxygenase activity in guinea pig amnion is due in part to accumulation of cyclooxygenase in vivo, that COX-2 predominates, and that COX activity is not correlated with levels of COX mRNA.     

Structural model of the p14/SF3b155 · branch duplex complex

Human p14 (SF3b14), a component of the spliceosomal U2 snRNP, interacts directly with the pre-mRNA branch adenosine within the context of the bulged duplex formed between the pre-mRNA branch region and U2 snRNA. This association occurs early in spliceosome assembly and persists within the fully assembled spliceosome. Analysis of the crystal structure of a complex containing p14 and a peptide derived from p14-associated SF3b155 combined with the results of cross-linking studies has suggested that the branch nucleotide interacts with a pocket on a non-canonical RNA binding surface formed by the complex. Here we report a structural model of the p14 · bulged duplex interaction based on a combination of X-ray crystallography of an adenine p14/SF3b155 peptide complex, biochemical comparison of a panel of disulfide cross-linked protein-RNA complexes, and small-angle X-ray scattering (SAXS). These studies reveal specific recognition of the branch adenosine within the p14 pocket and establish the orientation of the bulged duplex RNA bound on the protein surface. The intimate association of one surface of the bulged duplex with the p14/SF3b155 peptide complex described by this model buries the branch nucleotide at the interface and suggests that p14 · duplex interaction must be disrupted before the first step of splicing.     

Serum after autologous transplantation stimulates proliferation and expansion of human hematopoietic progenitor cells

Regeneration after hematopoietic stem cell transplantation (HSCT) depends on enormous activation of the stem cell pool. So far, it is hardly understood how these cells are recruited into proliferation and self-renewal. In this study, we have addressed the question if systemically released factors are involved in activation of hematopoietic stem and progenitor cells (HPC) after autologous HSCT. Serum was taken from patients before chemotherapy, during neutropenia and after hematopoietic recovery. Subsequently, it was used as supplement for in vitro culture of CD34(+) cord blood HPC. Serum taken under hematopoietic stress (4 to 11 days after HSCT) significantly enhanced proliferation, maintained primitive immunophenotype (CD34(+), CD133(+), CD45(-)) for more cell divisions and increased colony forming units (CFU) as well as the number of cobblestone area-forming cells (CAFC). The stimulatory effect decays to normal levels after hematopoietic recovery (more than 2 weeks after HSCT). Chemokine profiling revealed a decline of several growth-factors during neutropenia, including platelet-derived growth factors PDGF-AA, PDGF-AB and PDGF-BB, whereas expression of monocyte chemotactic protein-1 (MCP-1) increased. These results demonstrate that systemically released factors play an important role for stimulation of hematopoietic regeneration after autologous HSCT. This feedback mechanism opens new perspectives for in vivo stimulation of the stem cell pool.     

A common VLDLR polymorphism interacts with APOE genotype in the prediction of carotid artery disease risk

The genetic factors associated with carotid artery disease (CAAD) are not fully known. Because of its role in lipid metabolism, we hypothesized that common genetic variation in the very low density lipoprotein receptor (VLDLR) gene is associated with severe CAAD (>80% stenosis), body mass index (BMI), and lipid traits in humans. VLDLR was resequenced for variation discovery in 92 subjects, and single nucleotide polymorphisms (tagSNPs) were chosen for genotyping in a larger cohort (n = 1,027). Of the 17 tagSNPs genotyped, one tagSNP (SNP 1226; rs1454626) located in the 5' flanking region of VLDLR was associated with CAAD, BMI, and LDL-associated apolipoprotein B (apoB). We also identified receptor-ligand genetic interactions between VLDLR 1226 and APOE genotype for predicting CAAD case status. These findings may further our understanding of VLDLR function, its ligand APOE, and ultimately the pathogenesis of CAAD in the general population.