Functional dissection of hnRNP D suggests that nuclear import is required before hnRNP D can modulate mRNA turnover in the cytoplasm

Many shuttling proteins not only function in the nucleus but also control mRNA fates in the cytoplasm. We test whether a link exists between their nuclear association with mRNPs and their cytoplasmic functions using the p37 isoform of hnRNP D, which inhibits the rapid cytoplasmic mRNA decay in NIH3T3 cells. We showed that p37 shuttles between nucleus and cytoplasm, and narrowed down the nuclear import signal to a 50-amino-acid C-terminal domain. A p37 mutant missing this domain, still capable of associating with target mRNAs in vitro, was confined to the cytoplasm, where it was unable to block cytoplasmic mRNA turnover. Introducing heterologous shuttling domains to this mutant, thereby restoring its ability to enter the nucleus, concomitantly restored its cytoplasmic function. Association of p37 with its target mRNAs can only be detected when it can enter the nucleus. Our results suggest that nuclear import of hnRNP D is a prerequisite for it to exert its cytoplasmic function. This study provides a useful model system to elucidate the mechanisms by which "nuclear history" affects cytoplasmic mRNA fates.     

The study of gelation kinetics and chain-relaxation properties of glutaraldehyde-cross-linked chitosan gel and their effects on microspheres preparation and drug release

The effects of gelation kinetics and chain-relaxation properties of glutaraldehyde-cross-linked chitosan gel on microspheres preparation or drug release were studied. The rate of gelation is zero order corresponding to the chitosan concentration but non-zero order corresponding to the glutaraldehyde concentration. It was suggested that the cross-linking reaction was mainly dominated by the concentration of small molecule reactant, glutaraldehyde. The relaxation of an entangled polymer chain in a gel network as a result of the swelling of cross-linked chitosan hydrogel was investigated by the stress–strain determination. The higher the cross-linking density of chitosan hydrogel, the lower the swelling ability of chitosan hydrogel due to the slower relaxation rate of polymer chain, which then results in the decreased drug-release rate.     

Mechanical Stretch Induces Apoptosis Regulator TRB3 in Cultured Cardiomyocytes and Volume-Overloaded Heart

The expression of TRB3 (tribbles 3), an apoptosis regulated gene, increases during endoplasmic reticulum (ER) stress. How mechanical stress affects the regulation of TRB3 in cardiomyocytes during apoptosis is not fully understood. An in vivo model of aorta-caval shunt in adult rats demonstrated the increased TRB3 protein expression in the myocardium. The tumor necrosis factor-alpha (TNF-α) antagonist etanercept reversed the TRB3 protein expression and cardiomyocyte apoptosis induced by AV shunt. An in vitro model of cyclic stretch in neonatal rats was also used to investigate TRB3 expression. We hypothesized that cardiomyocyte apoptosis induced by cyclic stretch is TRB3 dependent. Neonatal rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. Cyclic stretch significantly increased TRB3 protein and mRNA expression. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125, TNF-α antibody and etanercept 30 min before stretch reversed the induction of TRB3 protein induced by stretch. Cyclic stretch induced the DNA-binding activity of growth arrest and DNA damaged inducible gene-153 (GADD153) by electrophoretic mobility shift assay. SP600125, JNK siRNA, TNF-α antibody and etanercept abolished the binding activity induced by stretch. TRB3 promoter activity was enhanced by stretch and TRB3-mut plasmid, SP600125, TNF-α antibody and etanercept attenuated TRB3 promoter activity induced by stretch. Exogenous administration of TNF-α recombinant protein to the non-stretched cardiomyocytes increased TRB3 protein expression similar to that seen after stretch. Cyclic stretch induced cardiomyocyte apoptosis is inhibited by TRB3 siRNA and etanercept. The stretch-induced TRB3 is mediated by TNF-α、JNK and GADD153 pathway. These results indicate that TRB3 plays an important role in stretch-induced cardiomyocyte apoptosis.     

Calmodulin antagonists inhibit germination of Bacillus cereus T spores

M. RACINE, J. DUMONT, C.P. CHAMPAGNE AND A. MORIN. 1991. The effects of lactose, ammonium and phosphate on the production of extracellular polysaccharide from Propionibacterium acidi-propionici VM-25 were studied in whey-based media. The polysaccharide was composed of a water-soluble fraction (15% w/w), a water-insoluble fraction (27% w/w) and ca 65% (w/w) of ash. Up to 15 g/l of polysaccharide was produced during growth on partially deproteinated whey, supplemented with lactose, NH₄ Cl and KH₂ PO₄, after incubation at pH 7.0 and 25.C for 90 h. The final viscosity of the medium remained under 20 centipoises at the end of the fermentation (100–140 h). The fermentation of whey enabled a reduction of the lactose content up to 50%. The polysaccharide-containing fractions were composed of glucose, galactose, mannose, rhamnose and fucose and had M, < 5800. The polysaccharide may have applications as a low viscosity stabilizing agent.     

Induction of Tolerogenic Dendritic Cells by a PEGylated TLR7 Ligand for Treatment of Type 1 Diabetes

Autoimmune diabetes mellitus (DM) results from the destruction of pancreatic islet cells by activated T lymphocytes, which have been primed by activated dendritic cells (DC). Individualized therapy with ex vivo DC manipulation and reinfusion has been proposed as a treatment for DM, but this treatment is limited by cost, and requires specialized facilities. A means of in situ modulation of the DC phenotype in the host would be more accessible. Here we report a novel innate immune modulator, 1Z1, generated by conjugating a TLR7 ligand to six units of polyethylene glycol (PEG), which skews DC phenotype in vivo. 1Z1 was less potent in inducing cytokine production by DC than the parent ligand in vitro and in vivo. In addition, this drug only modestly increased DC surface expression of activation markers such as MHC class II, CD80, and CD86; however, the expression of negative regulatory molecules, such as programmed death ligand 1 (PD-L1), and interleukin-1 receptor-associated kinase M (IRAK-M) were markedly increased. In vivo transfer of 1Z1 treated DC into prediabetic NOD mice delayed pancreatic insulitis. Daily administration of 1Z1 effectively prevented the clinical onset of hyperglycemia and reduced histologic islet inflammation. Daily treatment with 1Z1 increased PD-L1 expression in the CD11c⁺ population in peri-pancreatic lymph nodes; however, it did not induce an increase in regulatory T cells. Pharmaceutical modulation of DC maturation and function in situ, thus represents an opportunity to treat autoimmune disease.     

3D-Assisted Quantitative Assessment of Orbital Volume Using an Open-Source Software Platform in a Taiwanese Population

Orbital volume evaluation is an important part of pre-operative assessments in orbital trauma and congenital deformity patients. The availability of the affordable, open-source software, OsiriX, as a tool for preoperative planning increased the popularity of radiological assessments by the surgeon. A volume calculation method based on 3D volume rendering-assisted region-of-interest computation was used to determine the normal orbital volume in Taiwanese patients after reorientation to the Frankfurt plane. Method one utilized 3D points for intuitive orbital rim outlining. The mean normal orbital volume for left and right orbits was 24.3±1.51 ml and 24.7±1.17 ml in male and 21.0±1.21 ml and 21.1±1.30 ml in female subjects. Another method (method two) based on the bilateral orbital lateral rim was also used to calculate orbital volume and compared with method one. The mean normal orbital volume for left and right orbits was 19.0±1.68 ml and 19.1±1.45 ml in male and 16.0±1.01 ml and 16.1±0.92 ml in female subjects. The inter-rater reliability and intra-rater measurement accuracy between users for both methods was found to be acceptable for orbital volume calculations. 3D-assisted quantification of orbital volume is a feasible technique for orbital volume assessment. The normal orbital volume can be used as controls in cases of unilateral orbital reconstruction with a mean size discrepancy of less than 3.1±2.03% in females and 2.7±1.32% in males. The OsiriX software can be used reliably by the individual surgeon as a comprehensive preoperative planning and imaging tool for orbital volume measurement and computed tomography reorientation.     

Determining Effects of Non-synonymous SNPs on Protein-Protein Interactions using Supervised and Semi-supervised Learning

Single nucleotide polymorphisms (SNPs) are among the most common types of genetic variation in complex genetic disorders. A growing number of studies link the functional role of SNPs with the networks and pathways mediated by the disease-associated genes. For example, many non-synonymous missense SNPs (nsSNPs) have been found near or inside the protein-protein interaction (PPI) interfaces. Determining whether such nsSNP will disrupt or preserve a PPI is a challenging task to address, both experimentally and computationally. Here, we present this task as three related classification problems, and develop a new computational method, called the SNP-IN tool (non-synonymous SNP INteraction effect predictor). Our method predicts the effects of nsSNPs on PPIs, given the interaction''s structure. It leverages supervised and semi-supervised feature-based classifiers, including our new Random Forest self-learning protocol. The classifiers are trained based on a dataset of comprehensive mutagenesis studies for 151 PPI complexes, with experimentally determined binding affinities of the mutant and wild-type interactions. Three classification problems were considered: (1) a 2-class problem (strengthening/weakening PPI mutations), (2) another 2-class problem (mutations that disrupt/preserve a PPI), and (3) a 3-class classification (detrimental/neutral/beneficial mutation effects). In total, 11 different supervised and semi-supervised classifiers were trained and assessed resulting in a promising performance, with the weighted f-measure ranging from 0.87 for Problem 1 to 0.70 for the most challenging Problem 3. By integrating prediction results of the 2-class classifiers into the 3-class classifier, we further improved its performance for Problem 3. To demonstrate the utility of SNP-IN tool, it was applied to study the nsSNP-induced rewiring of two disease-centered networks. The accurate and balanced performance of SNP-IN tool makes it readily available to study the rewiring of large-scale protein-protein interaction networks, and can be useful for functional annotation of disease-associated SNPs. SNIP-IN tool is freely accessible as a web-server at http://korkinlab.org/snpintool/.     

A rheumatoid factor paradox: inhibition of rituximab effector function

Introduction

Rituximab (RTX) therapy of rheumatoid arthritis (RA) exhibits enhanced effectiveness in seropositive patients. Using patient sera, we tested if this improved efficacy was associated with enhanced RTX mediated complement-dependent cytotoxicity (RTX-CDC).

Methods

We developed an in vitro assay for RTX-CDC using patient sera and the Daudi human B cell line. Using propidium iodide uptake and flow cytometry, we compared RTX-CDC with rheumatoid factor (RF)+ sera relative to normal volunteer, non-RA and RF- sera. Additional studies examined mixing studies of RF+ and RF- sera, as well as the effect of monoclonal IgA or IgM RF. Finally, the effect of RF on RTX mediated trogocytosis of normal B cells was evaluated.

Results

Using human sera, addition of RTX resulted in rapid and profound (> 50%) Daudi cell death that was complement dependent. Surprisingly, RF+ patient sera exhibited reduced RTX-CDC relative to RF- sera, with an inverse relationship of RTX-CDC and RF titer. Mixing studies indicated the presence of an inhibitor of RTX-CDC in RF+ sera. The addition of monoclonal IgM or IgA RF to RF- sera markedly inhibited RTX-CDC. This effect was specific for RF binding to the Fc portion of RTX as it was not apparent with the F(ab)'' domains of RTX engineered onto IgG3 heavy chain. RF also modestly inhibited RTX mediated trogocytosis.

Conclusions

Contrary to expectations, RF+ sera exhibits reduced RTX-CDC due to the presence of RF. The enhanced efficacy of RTX in seropositive RA patients cannot be attributed to improved B cell depletion through CDC. This result indicates that high RF levels may potentially modulate the efficacy of any therapeutic monoclonal antibody dependent on Fc effector function.