A method for the rapid and efficient elution of native affinity-purified protein A tagged complexes

A problem faced in proteomics studies is the recovery of tagged protein complexes in their native and active form. Here we describe a peptide, Bio-Ox, that mimics the immunoglobulin G (IgG) binding interface of Staphylococcus aureus Protein A, and competitively displaces affinity-purified Protein A fusion proteins and protein complexes from IgG-Sepharose. We show that Bio-Ox elution is a robust method for the efficient and rapid recovery of native tagged proteins, and can be applied to a variety of structural genomics and proteomics studies.     

Exposure to 900 MHz electromagnetic field induces an unbalance between pro‐apoptotic and pro‐survival signals in T‐lymphoblastoid leukemia CCRF‐CEM cells

The original article to which this Erratum was published in J. Cell. Physiol. 198:324–332, 2004 It has been recently established that low‐frequency electromagnetic field (EMFs) exposure induces biological changes and could be associated with increased incidence of cancer, while the issue remains unresolved as to whether high‐frequency EMFs can have hazardous effect on health. Epidemiological studies on association between childhood cancers, particularly leukemia and brain cancer, and exposure to low‐ and high‐frequency EMF suggested an etiological role of EMFs in inducing adverse health effects. To investigate whether exposure to high‐frequency EMFs could affect in vitro cell survival, we cultured acute T‐lymphoblastoid leukemia cells (CCRF‐CEM) in the presence of unmodulated 900 MHz EMF, generated by a transverse electromagnetic (TEM) cell, at various exposure times. We evaluated the effects of high‐frequency EMF on cell growth rate and apoptosis induction, by cell viability (MTT) test, FACS analysis and DNA ladder, and we investigated pro‐apoptotic and pro‐survival signaling pathways possibly involved as a function of exposure time by Western blot analysis. At short exposure times (2–12 h), unmodulated 900 MHz EMF induced DNA breaks and early activation of both p53‐dependent and ‐independent apoptotic pathways while longer continuous exposure (24–48 h) determined silencing of pro‐apoptotic signals and activation of genes involved in both intracellular (Bcl‐2) and extracellular (Ras and Akt1) pro‐survival signaling. Overall our results indicate that exposure to 900 MHz continuous wave, after inducing an early self‐defense response triggered by DNA damage, could confer to the survivor CCRF‐CEM cells a further advantage to survive and proliferate. J. Cell. Physiol. 198: 324–332, 2004. © 2003 Wiley‐Liss, Inc.     

Distribution of α-synuclein in normal human jejunum and its relations with the chemosensory and neuroendocrine system

Alpha-synuclein (α-syn) is a presynaptic neuronal protein and its structural alterations play an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson’s disease (PD). It has been originally described in the brain and aggregated α-syn has also been found in the peripheral nerves including the enteric nervous system (ENS) of PD patients. ENS is a network of neurons and glia found in the gut wall which controls gastrointestinal function independently from the central nervous system. Moreover, two types of epithelial cells are crucial in the creation of an interface between the lumen and the ENS: they are the tuft cells and the enteroendocrine cells (EECs). In addition, the abundant enteric glial cells (EGCs) in the intestinal mucosa play a key role in controlling the intestinal epithelial barrier. Our aim was to localize and characterize the presence of α-syn in the normal human jejunal wall. Surgical specimens of proximal jejunum were collected from patients submitted to pancreaticoduodenectomy and intestinal sections underwent immunohistochemical procedure. Alpha-syn has been found both at the level of the ENS and the epithelial cells. To characterize α-syn immunoreactive epithelial cells, we used markers such as choline acetyltransferase (ChAT), useful for the identification of tuft cells. Then we evaluated the co-presence of α-syn with serotonin (5-HT), expressed in EECs. Finally, we used the low-affinity nerve growth factor receptor (p75NTR), to detect peripheral EGCs. The presence of α-syn has been demonstrated in EECs, but not in the tuft cells. Additionally, p75NTR has been highlighted in EECs of the mucosal layer and co-localized with α-syn in EECs but not with ChAT-positive cells. These findings suggest that α-syn could play a possible role in synaptic transmission of the ENS and may contribute to maintain the integrity of the epithelial barrier of the small intestine through EECs.Key words: Small intestine, jejunum, tuft cells, enteroendocrine cells (EECs), α-synuclein (α-syn), enteric     

Cisplatin resistance can be curtailed by blunting Bnip3-mediated mitochondrial autophagy

Cisplatin (CDDP) is commonly used to treat a multitude of tumors including sarcomas, ovarian and cervical cancers. Despite recent investigations allowed to improve chemotherapy effectiveness, the molecular mechanisms underlying the development of CDDP resistance remain a major goal in cancer research. Here, we show that mitochondrial morphology and autophagy are altered in different CDDP resistant cancer cell lines. In CDDP resistant osteosarcoma and ovarian carcinoma, mitochondria are fragmented and closely juxtaposed to the endoplasmic reticulum; rates of mitophagy are also increased. Specifically, levels of the mitophagy receptor BNIP3 are higher both in resistant cells and in ovarian cancer patient samples resistant to platinum-based treatments. Genetic BNIP3 silencing or pharmacological inhibition of autophagosome formation re-sensitizes these cells to CDDP. Our study identifies inhibition of BNIP3-driven mitophagy as a potential therapeutic strategy to counteract CDDP resistance in ovarian carcinoma and osteosarcoma.Subject terms: Cancer therapeutic resistance, Mitophagy     

I-MOVE multi-centre case control study 2010-11: overall and stratified estimates of influenza vaccine effectiveness in Europe

Background

In the third season of I-MOVE (Influenza Monitoring Vaccine Effectiveness in Europe), we undertook a multicentre case-control study based on sentinel practitioner surveillance networks in eight European Union (EU) member states to estimate 2010/11 influenza vaccine effectiveness (VE) against medically-attended influenza-like illness (ILI) laboratory-confirmed as influenza.

Methods

Using systematic sampling, practitioners swabbed ILI/ARI patients within seven days of symptom onset. We compared influenza-positive to influenza laboratory-negative patients among those meeting the EU ILI case definition. A valid vaccination corresponded to > 14 days between receiving a dose of vaccine and symptom onset. We used multiple imputation with chained equations to estimate missing values. Using logistic regression with study as fixed effect we calculated influenza VE adjusting for potential confounders. We estimated influenza VE overall, by influenza type, age group and among the target group for vaccination.

Results

We included 2019 cases and 2391 controls in the analysis. Adjusted VE was 52% (95% CI 30-67) overall (N = 4410), 55% (95% CI 29-72) against A(H1N1) and 50% (95% CI 14-71) against influenza B. Adjusted VE against all influenza subtypes was 66% (95% CI 15-86), 41% (95% CI -3-66) and 60% (95% CI 17-81) among those aged 0-14, 15-59 and ≥60 respectively. Among target groups for vaccination (N = 1004), VE was 56% (95% CI 34-71) overall, 59% (95% CI 32-75) against A(H1N1) and 63% (95% CI 31-81) against influenza B.

Conclusions

Results suggest moderate protection from 2010-11 trivalent influenza vaccines against medically-attended ILI laboratory-confirmed as influenza across Europe. Adjusted and stratified influenza VE estimates are possible with the large sample size of this multi-centre case-control. I-MOVE shows how a network can provide precise summary VE measures across Europe.     

Role of Palatine Tonsils as a Prion Entry Site in Classical and Atypical Experimental Sheep Scrapie

Atypical and classical scrapie-infected sheep brain tissue was monolaterally injected into the tonsils of lambs to investigate their role as a prion entry point. We first detected classical PrP^Sc within the inoculated tonsil and in the ipsilateral retropharyngeal lymph node at 3 months postinoculation (p.i.). At 7 months p.i., PrP^Sc colonized other lymphoid tissues bilaterally, including ileal Peyer''s patches. The earliest PrP^Sc deposition within the brain was ipsilaterally observed at 9 months p.i. in the substantia reticularis of the medulla oblongata. At 12 months p.i., PrP^Sc deposition was present bilaterally in the nucleus parasympathicus nervi vagi, as well as in the intermediolateral cell column of the thoracolumbar spinal cord. No PrP^Sc was detected in the lambs inoculated with atypical scrapie. These findings suggest that neuroinvasion may naturally occur from the tonsil after a widespread prion replication within the lymphoid tissues during classical scrapie only, thus mimicking the pathogenesis after oral ingestion.     

Peptidoglycan synthesis by Enterococcus faecalis penicillin binding protein 5

Low-affinity penicillin binding proteins (PBPs) are a particular class of proteins involved in β-lactam antibiotic resistance of enterococci. The activity of these PBPs is just sufficient to allow the cells to survive in the presence of high concentrations of β-lactams that cause saturation (and inhibition) of the other PBPs. For this reason, the low-affinity PBPs are thought to be multifunctional enzymes capable of catalyzing the entire peptidoglycan synthesis. To test the validity of this claim, we analyzed the muropeptide composition by reversed-phase high-performance liquid chromatography of the peptidoglycan synthesized by PBP5 (the low-affinity PBP) of Enterococcus faecalis, in comparison with the peptidoglycan produced normally by the concerted action of the usual PBPs (namely PBPs 1, 2, and 3). Cross-linked peptidoglycan was produced. The main difference consisted in the lack of oligomers higher than trimers, thus suggesting that this oligomer cannot be used as an acceptor/donor by the transpeptidase component of PBP5. The lack of higher oligomers had little impact on total cross-linking because of the increase observed in the dimer family. This increase was distributed among the various members of the dimer family with the result that minor dimer components figured among the prevalent ones in cells in which peptidoglycan was synthesized by PBP5. This also suggests that E. faecalis PBP5 is capable of catalyzing the synthesis of a peptidoglycan that is less precise and refined than usual, and for this reason PBP5 can be considered an enzyme endowed with poor specificity for substrates, as may be expected on the basis of its survival function. Received: 18 March 1998 / Accepted: 26 May 1998