An in silico erythropoiesis model rationalizing synergism between stem cell factor and erythropoietin

Stem cell factor (SCF) and erythropoietin (EPO) are two most recognized growth factors that play in concert to control in vitro erythropoiesis. However, exact mechanisms underlying the interplay of these growth factors in vitro remain unclear. We developed a mathematical model to study co-signaling effects of SCF and EPO utilizing the ERK1/2 and GATA-1 pathways (activated by SCF and EPO) that drive the proliferation and differentiation of erythroid progenitors. The model was simplified and formulated based on three key features: synergistic contribution of SCF and EPO on ERK1/2 activation, positive feedback effects on proliferation and differentiation, and cross-inhibition effects of activated ERK1/2 and GATA-1. The model characteristics were developed to correspond with biological observations made known thus far. Our simulation suggested that activated GATA-1 has a more dominant cross-inhibition effect and stronger positive feedback response on differentiation than the proliferation pathway, while SCF contributed more to the activation of ERK1/2 than EPO. A sensitivity analysis performed to gauge the dynamics of the system was able to identify the most sensitive model parameters and illustrated a contribution of transient activity in EPO ligand to growth factor synergism. Based on theoretical arguments, we have successfully developed a model that can simulate growth factor synergism observed in vitro for erythropoiesis. This hypothesized model can be applied to further computational studies in biological systems where synergistic effects of two ligands are seen.     

Contribution of Orb2A Stability in Regulated Amyloid-Like Oligomerization of Drosophila Orb2

How learned experiences persist as memory for a long time is an important question. In Drosophila the persistence of memory is dependent upon amyloid-like oligomers of the Orb2 protein. However, it is not clear how the conversion of Orb2 to the amyloid-like oligomeric state is regulated. The Orb2 has two protein isoforms, and the rare Orb2A isoform is critical for oligomerization of the ubiquitous Orb2B isoform. Here, we report the discovery of a protein network comprised of protein phosphatase 2A (PP2A), Transducer of Erb-B2 (Tob), and Lim Kinase (LimK) that controls the abundance of Orb2A. PP2A maintains Orb2A in an unphosphorylated and unstable state, whereas Tob-LimK phosphorylates and stabilizes Orb2A. Mutation of LimK abolishes activity-dependent Orb2 oligomerization in the adult brain. Moreover, Tob-Orb2 association is modulated by neuronal activity and Tob activity in the mushroom body is required for stable memory formation. These observations suggest that the interplay between PP2A and Tob-LimK activity may dynamically regulate Orb2 amyloid-like oligomer formation and the stabilization of memories.     

IPRO: an iterative computational protein library redesign and optimization procedure

A number of computational approaches have been developed to reengineer promising chimeric proteins one at a time through targeted point mutations. In this article, we introduce the computational procedure IPRO (iterative protein redesign and optimization procedure) for the redesign of an entire combinatorial protein library in one step using energy-based scoring functions. IPRO relies on identifying mutations in the parental sequences, which when propagated downstream in the combinatorial library, improve the average quality of the library (e.g., stability, binding affinity, specific activity, etc.). Residue and rotamer design choices are driven by a globally convergent mixed-integer linear programming formulation. Unlike many of the available computational approaches, the procedure allows for backbone movement as well as redocking of the associated ligands after a prespecified number of design iterations. IPRO can also be used, as a limiting case, for the redesign of a single or handful of individual sequences. The application of IPRO is highlighted through the redesign of a 16-member library of Escherichia coli/Bacillus subtilis dihydrofolate reductase hybrids, both individually and through upstream parental sequence redesign, for improving the average binding energy. Computational results demonstrate that it is indeed feasible to improve the overall library quality as exemplified by binding energy scores through targeted mutations in the parental sequences.     

Cyclin F-mediated degradation of ribonucleotide reductase M2 controls genome integrity and DNA repair

F-box proteins are the substrate binding subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes. Using affinity purifications and mass spectrometry, we identified RRM2 (the ribonucleotide reductase family member 2) as an interactor of the F-box protein cyclin F. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides (dNTPs), which are necessary for both replicative and repair DNA synthesis. We?found that, during G2, following CDK-mediated phosphorylation of Thr33, RRM2 is degraded via SCF(cyclin F) to maintain balanced dNTP pools and genome stability. After DNA damage, cyclin F is downregulated in an ATR-dependent manner to allow accumulation of RRM2. Defective elimination of cyclin F delays DNA repair and sensitizes cells to DNA damage, a phenotype that is reverted by expressing a nondegradable RRM2 mutant. In summary, we have identified a biochemical pathway that controls the abundance of dNTPs and ensures efficient DNA repair in response to genotoxic stress.     

Mechanism of spasmolytic activity of a fraction of Sarcostemma brevistigma Wight

The effect of chloroform soluble fraction (F-A) of twigs of Sarcostemma brevistigma on contractions induced by KCl, histamine, and acetylcholine in the isolated guinea pig ileum and taenia coli smooth muscles has been evaluated. F-A (19.5 microg/ml) significantly inhibited the contraction induced by 40 mM KCl to the extent of 87.6% in the isolated guinea pig ileum. In the isolated guinea pig ileum, F-A (64.3 and 59.2 microg/ml) significantly inhibited the contractions induced by acetylcholine and histamine to the extent of 85 and 83% respectively. In the isolated guinea pig taenia coli, F-A (65.2 microg/ml) significantly inhibited the contraction induced by 40 mM KCl to the extent of 96.0%. The inhibitory effect of F-A (40 microg/ml) on the isolated guinea pig taenia coli was reduced by Bay K 8644 (10(-6) M) to the extent of 61.6 from 73.6%. These results suggest that the F-A may exhibit smooth muscle relaxant activity by blocking the Ca2+ channels.     

Effective attenuation of glyphosate‐induced oxidative stress and granulosa cell apoptosis by vitamins C and E in caprines

Pesticides are known to cause a wide range of reproductive problems that possess degenerative effects on mammalian fertility. Glyphosate (GLP), a broad‐spectrum organophosphate herbicide, is known to be a potent mammalian toxicant. The present study aims at assessing the GLP‐induced (0.1, 2.0, and 4.0 mg/ml) granulosa cells toxicity and evaluating the mitigating effects of vitamins C and E (0.5 mM and 1.0 mM) in healthy caprine antral follicles, cultured in vitro in a dose‐ and time‐dependent manner (24, 48, and 72 hr) and subjected to various cytotoxic and geno‐toxic analysis, namely, classic histology, EB/AO differential staining, oxidative stress parameters, and antioxidant enzymatic activity. The histomorphological analysis and EB/AO staining elucidated increase in the incidence of apoptotic attributes within granulosa cells with increasing dose and duration of the GLP treatment. The highest apoptotic frequency was observed at 4.0 mg/ml GLP after 72‐hr exposure duration in comparison with the control. GLP exposure also led to a significant decline in the antioxidant enzymes’ activity, namely, SOD, catalase, and GST along with enhanced lipid peroxidation and reduced FRAP activity in a dose‐ and time‐dependent manner. Vitamins C and E supplementation decreased oxidative stress‐mediated granulosa cells apoptosis, suggesting its efficiency to diminish GLP‐mediated GCs cytotoxicity and thereby, preventing associated fertility disorders.