A high-throughput screen for single gene activities: isolation of apoptosis inducers

We describe a novel genetic screen that is performed by transfecting every individual clone of an expression library into a separate population of cells in a high-throughput mode. The screen allows one to achieve a hitherto unattained sensitivity in expression cloning which was exploited in a first read-out to clone apoptosis-inducing genes. This led to the isolation of several genes whose proteins induce distinct phenotypes of apoptosis in 293T cells. One of the isolated genes is the tumor suppressor cytochrome b(L) (cybL), a component of the respiratory chain complex II, that diminishes the activity of this complex for apoptosis induction. This gene is more efficient and specific for causing cell death than a drug with the same activity. These results suggest further applications, both of the isolated genes and the screen.     

Adenoviral infection decreases mortality from lipopolysaccharide-induced liver failure via induction of TNF-alpha tolerance

Effects of adenoviral infection on in vivo responses to LPS mediated by TNF-alpha were evaluated in a murine model. Adenovirus-infected mice showed decreased mortality from fulminant hepatitis induced by administration of LPS or staphylococcal enterotoxin B in the presence of D-galactosamine. Importantly, TNF-alpha resistance genes within adenoviral E3 region were not required, because E1,E3-deleted vectors showed similar effects. Adenovirus-infected mice exhibited higher TNF-alpha levels after LPS stimulation, no difference in TNFR1 expression, and similar mortality from Fas-induced fulminant hepatitis. Decreased production of IL-6 and KC in response to exogenous TNF-alpha, in addition to protection from TNF-alpha, suggested that adenoviral infection results in TNF-alpha tolerance.     

Chimeric ribonuclease as a source of human adapter protein for targeted drug delivery

Assembled modular complexes for targeted drug delivery can be based on strong non-covalent interactions between a cargo module containing an adapter protein and a docking tag fused to a targeting protein. We have recently constructed a completely humanized adapter/docking tag system based on interactions between 15 amino acid (Hu-tag) and 110 amino acid (HuS) fragments of human ribonuclease I (RNase I). Although recombinant HuS can be expressed and refolded into a functionally active form, the purification procedure is cumbersome and expensive, and more importantly, it yields a significant proportion of improperly folded proteins. Here we describe engineering, high-yield expression, and purification of a chimeric bovine/human RNase (BH-RNase) comprising 1-29 N-terminal amino acids of bovine ribonuclease A and 30-127 amino acids of human RNase I. Unlike RNase I, the chimeric BH-RNase can be cleaved by either subtilisin or proteinase K between A20 and S21, providing a functionally active HuS. The HuS obtained from chimeric BH-RNase differs from wild-type HuS by an N24T substitution; therefore, we have reverted this substitution by mutating N24 to T24 in BH-RNase. This BH-RNase mutant can also be cleaved by subtilisin or proteinase K yielding wild-type HuS. The affinity of HuS obtained from BH-RNase to Hu-tag is approximately five times higher than that for recombinant HuS, reflecting a higher percentage of properly folded proteins.     

Respiratory syncytial virus decreases p53 protein to prolong survival of airway epithelial cells

Respiratory syncytial virus (RSV) is a clinically important pathogen. It preferentially infects airway epithelial cells causing bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and life-threatening pneumonia in the immunosuppressed. The p53 protein is a tumor suppressor protein that promotes apoptosis and is tightly regulated for optimal cell growth and survival. A critical negative regulator of p53 is murine double minute 2 (Mdm2), an E3 ubiquitin ligase that targets p53 for proteasome degradation. Mdm2 is activated by phospho-Akt, and we previously showed that RSV activates Akt and delays apoptosis in primary human airway epithelial cells. In this study, we explore further the mechanism by which RSV regulates p53 to delay apoptosis but paradoxically enhance inflammation. We found that RSV activates Mdm2 1-6 h after infection resulting in a decrease in p53 6-24 h after infection. The p53 down-regulation correlates with increased airway epithelial cell longevity. Importantly, inhibition of the PI3K/Akt pathway blocks the activation of Mdm2 by RSV and preserves the p53 response. The effects of RSV infection are antagonized by Nutlin-3, a specific chemical inhibitor that prevents the Mdm2/p53 association. Nutlin-3 treatment increases endogenous p53 expression in RSV infected cells, causing earlier cell death. This same increase in p53 enhances viral replication and limits the inflammatory response as measured by IL-6 protein. These findings reveal that RSV decreases p53 by enhancing Akt/Mdm2-mediated p53 degradation, thereby delaying apoptosis and prolonging survival of airway epithelial cells.     

Ligand-dependent localization and intracellular stability of sigma-1 receptors in CHO-K1 cells

Background

Sigma-1 receptors are involved in regulation of neuronal activities presumably through regulation of the activity of ion channels. Sigma-1 receptors also play a role in growth and metastasis of cancerous cells. Intracellular distribution of sigma-1 receptors have been linked to sphingolipid-enriched domains.

Results

We report that in CHO-K1 cells sigma-1 receptors target to focal adhesion contacts (FAC) where they colocalize with Talin and Kv1.4 potassium channels. The levels of sigma-1 receptors in the FAC were significantly increased by application of sigma-1 receptor ligands and by filamentous actin (F-actin) polymerization with phalloidin. The total length of FAC (measured by the focal adhesion marker, talin) was concomitantly increased in the presence of sigma-1 receptors upon phalloidin treatment. Only sigma-1 receptor ligands, however, resulted in an increase of sigma-1 receptors in the FAC, independent of talin. Additionally, a novel approach was utilized to allow an assessment of the half life of endogenous sigma-1 receptors in CHO-K1 cells, which was measured to be at least 72 hours.

Conclusion

Ligand activated sigma-1 receptors translocate into FAC from a pool of receptors stored in ER lipid rafts presumably for inhibition of Kv1.4 channels. Stabilization of actin filaments is likely to be important for targeting sigma-1 receptors to Focal Adhesion Contacts in CHO-K1 cells.     

Estimation of stature in Turkish adults using knee height

Body height is an important clinical indicator to derive body mass index (BMI), which is a useful screening tool for both excess adiposity and malnutrition. Height measurement in the elderly may impose some difficulties and the reliability is doubtful. Stature estimation from knee height is one of the commonly used methods; nevertheless no study has been carried out so far on the Turkish population. A cross sectional anthropometric study was conducted to develop body height estimation equations by using knee height measurement for Turkish people. Measurements of height and knee height were taken according to the International Biological Programme procedures from 1422 adults (610 males, 812 females) aged 18-90 years from Ankara, the capital city of Turkey. Samples were randomly split into two sub-samples, training and validation (control group) sub-samples. Height estimation equations were developed from the knee height measurements by linear regression analysis according to age groups and sexes. Males were significantly taller and have higher knee height values than females in all age groups. Height and the knee height variables showed a gradual decrease (P 50) with aging in females and males. Evaluated knee height equations for stature estimation were tested through the validation sample and the results showed high accuracy. The study presents sex and age specific regression equations for height estimation by using the knee height measurement for Turkish adults and suggests facilitating the accurate usage of knee height.     

Neutrophil-dendritic cell interaction plays an important role in live attenuated Leishmania vaccine induced immunity

BackgroundNeutrophils are involved in the initial host responses to pathogens. Neutrophils can activate T cell responses either independently or through indirect involvement of Dendritic cells (DCs). Recently we have demonstrated direct neutrophil-T cell interactions that initiate adaptive immune responses following immunization with live attenuated Leishmania donovani centrin deleted parasite vaccine (LdCen^-/-). However, neutrophil-DC interactions in T cell priming in vaccine immunity in general are not known. In this study we evaluated the interaction between neutrophils and DCs during LdCen^-/- infection and compared with wild type parasite (LdWT) both in vitro and in vivo.Methodology/findingsLdCen^-/- parasite induced increased expression of CCL3 in neutrophils caused higher recruitment of DCs capable of inducing a strong proinflammatory response and elevated co-stimulatory molecule expression compared to LdWT infection. To further illustrate neutrophil-DCs interactions in vivo, we infected LYS-eGFP mice with red fluorescent LdWT/LdCen^-/- parasites and sort selected DCs that engulfed the neutrophil containing parasites or DCs that acquired the parasites directly in the ear draining lymph nodes (dLN) 5d post infection. The DCs predominantly acquired the parasites by phagocytosing infected neutrophils. Specifically, DCs containing LdCen^-/- parasitized neutrophils exhibited a proinflammatory phenotype, increased expression of costimulatory molecules and initiated higher CD4⁺T cell priming ex-vivo. Notably, potent DC activation occurred when LdCen^-/- parasites were acquired indirectly via engulfment of parasitized neutrophils compared to direct engulfment of LdCen^-/- parasites by DCs. Neutrophil depletion in LdCen^-/- infected mice significantly abrogated expression of CCL3 resulting in decreased DC recruitment in ear dLN. This event led to poor CD4⁺Th1 cell priming ex vivo that correlated with attenuated Tbet expression in ear dLN derived CD4⁺ T cells in vivo.ConclusionsCollectively, LdCen^-/- containing neutrophils phagocytized by DC markedly influence the phenotype and antigen presenting capacity of DCs early on and thus play an immune-regulatory role in shaping vaccine induced host protective response.     

PepBank - a database of peptides based on sequence text mining and public peptide data sources

Background  

Peptides are important molecules with diverse biological functions and biomedical uses. To date, there does not exist a single, searchable archive for peptide sequences or associated biological data. Rather, peptide sequences still have to be mined from abstracts and full-length articles, and/or obtained from the fragmented public sources.     

TM4SF10 and ADAP interaction in podocytes: role in Fyn activity and nephrin phosphorylation

TM4SF10 [transmembrane tetra(4)-span family 10] is a claudin-like cell junction protein that is transiently expressed during podocyte development where its expression is downregulated in differentiating podocytes coincident with the appearance of nephrin at the slit diaphragm. In a yeast two-hybrid screen, we identified adhesion and degranulation-promoting adaptor protein (ADAP), a well-known Fyn substrate and Fyn binding partner, as a TM4SF10 interacting protein in mouse kidney. Using coimmunoprecipitation and immunohistochemistry experiments in cultured human podocytes, we show that TM4SF10 colocalizes with Fyn and ADAP but does not form a stable complex with Fyn. Cytoskeletal changes and phosphorylation events mediated by Fyn activity were reversed by TM4SF10 overexpression, including a decrease in the activating tyrosine phosphorylation of Fyn (Y(421)), suggesting TM4SF10 may have a regulatory role in suppressing Fyn activity. In addition, TM4SF10 was reexpressed following podocyte injury by puromycin aminonucleoside treatment, and its expression enhanced the abundance of high-molecular-weight forms of nephrin indicating it may participate in a mechanism controlling nephrin's appearance at the plasma membrane. Therefore, these studies have identified ADAP as another Fyn adapter protein expressed in podocytes, and that TM4SF10, possibly through ADAP, may regulate Fyn activity. Since TM4SF10 expression is temporally regulated during kidney development, these studies may help define a mechanism by which the slit diaphragm matures as a highly specialized cell junction during podocyte differentiation.