The protein-tyrosine kinase Syk interacts with the C-terminal region of tensin2

Syk is a 72-kDa protein-tyrosine kinase that regulates signaling through multiple cell surface receptors including those for antigens, immunoglobulins and proteins of the extracellular matrix. As part of its function, Syk binds a variety of downstream effectors through interactions that are often mediated by motifs that recognize phosphotyrosines. In a search for novel Syk-interacting proteins by yeast two-hybrid analysis, we identified tensin2 as a Syk-binding protein. Syk interacts with a fragment of tensin2 located near the C-terminus that contains SH2 and PTB domains. In epithelial cells, tensin2 localizes both to focal adhesions and to large cytoplasmic puncta. It is within these punctuate structures that Syk and tensin2 are co-localized. The clustering of Syk within these structures leads to its phosphorylation on tyrosine.     

Examination of the quality of various force fields and solvation models for the equilibrium simulations of GA88 and GB88

Elucidating the relationship between sequence and conformation is essential for the understanding of functions of proteins. While sharing 88 % sequence identity and differing by only seven residues, GA88 and GB88 have completely different structures and serve as ideal systems for investigating the relationship between sequence and function. Benefiting from the continuous advancement of the computational ability of modern computers, molecular dynamics (MD) simulation is now playing an increasingly important role in the study of proteins. However, the reliability of MD simulations is limited by the accuracy of the force fields and solvent model approximations. In this work, several AMBER force fields (AMBER03, AMBER99SB, AMBER12SB, AMBER14SB, AMBER96) and solvent models (TIP3P, IGB5, IGB7, IGB8) have been employed in the simulations of GA88 and GB88. The statistical results from 19 simulations show that GA88 and GB88 both adopt more compact structures than the native structures. GB88 is more stable than GA88 regardless of the force fields and solvent models utilized. Most of the simulations overestimated the salt bridge interaction. The combination of AMBER14SB force field and IGB8 solvent model shows the best overall performance in the simulations of both GA88 and GB88. AMBER03 and AMBER12SB also yield reasonable results but only in the TIP3P explicit solvent model.     

Effluxes of solutes from developing seed coats of Phaseolus vulgaris L. and Vicia faba l.: locating the effect of turgor in a coupled chemiosmotic system

Cells lining the developing seed coats of legumes efflux photosynthates (mostly sucrose) and salts (mostly of potassium) into the apoplast for uptake by the developing embryo. These effluxes increase transiently in response to an increase in turgor in the effluxing cells. Detached coats of developing seed of P. haseolus vulgaris and Vicia faba were used to study the effects of turgor on the rates of efflux, on the membrane potential difference and on the membrane pH difference, using a number of inhibitors and agents which might affect signal cascades involving cytoplasmic calcium concentration. Effluxes were measured by measuring the concentrations of solutes of interest in solution samples placed in halves of detached seed coats, the paired halves serving as control and treated sample where appropriate. It is shown that a number of substances affect sucrose and potassium effluxes differently, and that hypo-osmotic shock depolarizes the efflux cells and acidifies the cytoplasm (in P. vulgaris). It is concluded that sucrose and potassium effluxes, although both are increased by an increase in turgor, are affected by different signal pathways. Further, it is also concluded that the signal that increases the rates of both sucrose efflux (via sucrose-proton antiport) and proton pump acts directly on the antiporter rather than on the pump. There are interesting parallels and contrasts between these processes and those in plants such as the charophyte Lamprothamnium after hypo-osmotic shock.     

High-level production of poly (β-<Emphasis Type="SmallCaps">l</Emphasis>-malic acid) with a new isolated <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> strain

Poly (β-l-malic acid) (PMLA) is a water-soluble polyester with many attractive properties in chemical industry and medicine development. However, the low titer of PMLA in the available producer strains limits further industrialization efforts and restricts its many potential applications. In order to solve this problem, a new strain with the distinguished high productivity of PMLA was isolated from fresh plants samples. It was characterized as the candidate of Aureobasidium pullulans based on the morphology and phylogenetic analyses of the internal transcribed spacer sequences. After the optimization of culture conditions, the highest PMLA concentration (62.27 g l⁻¹) could be achieved in the shake flask scale. In addition, the contribution of the carbon flux to exopolysaccharide (EPS) and PMLA could be regulated by the addition of CaCO₃ in the medium. This high-level fermentation process was further scaled up in the 10 l benchtop fermentor with a high PMLA concentration (57.2 g l⁻¹) and productivity (0.35 g l⁻¹ h⁻¹), which are the highest level in all the literature. Finally, the suitable acid hydrolysis conditions of PMLA were also investigated with regard to the production of l-malic acid, and the kinetics of PMLA acid hydrolysis was modeled to simulate the whole degradation process. The present work paved the road to produce this multifunctional biomaterial (PMLA) at industrial scale and promised one alternative method to produce l-malic acid in the future.     

Aldosterone induction of hepatic stellate cell contraction through activation of RhoA/ROCK-2 signaling pathway

The RhoA/ROCK-2 signaling pathway is necessary for activated hepatic stellate cell (HSC) contraction. HSC contraction plays an important role in the pathogenesis of cirrhosis and portal hypertension. This study investigated whether aldosterone contributes to HSC contraction by activation of the RhoA/ROCK-2 signaling pathway. Primary HSCs were isolated from Sprague-Dawley rats via in situ pronase/collagenase perfusion. We found that aldosterone enhanced the contraction of a collagen lattice seeded with HSCs. This induced contraction was suppressed by the mineralcorticoid receptor (MR) inhibitor spironolactone, the ROCK-2 inhibitor Y27632, and the angiotensin II type 1 receptor (AT(1)R) inhibitor irbesartan. Moreover, actin fiber staining showed that aldosterone significantly increased actin fiber formation in HSCs. Pre-incubating with spironolactone, Y27632, or irbesartan inhibited the aldosterone-induced actin fiber reorganization. Molecularly, the effect of aldosterone on activation of HSC contraction was mediated by phosphorylated myosin light chain (P-MLC) through the RhoA/ROCK-2 signaling pathway. All these inhibitors had the ability to block aldosterone-induced protein expressions in the RhoA/ROCK-2/P-MLC cascade in HSCs. Taken together, our current study suggests that aldosterone induces contraction of activated HSCs through the activation of the RhoA/ROCK-2 signaling pathway. This finding may provide a potential therapeutic target for control of cirrhosis and portal hypertension.     

Determinants of the interaction of the spinal muscular atrophy disease protein SMN with the dimethylarginine-modified box H/ACA small nucleolar ribonucleoprotein GAR1

Deletion or mutation of the SMN1 (survival of motor neurons) gene causes the common, fatal neuromuscular disease spinal muscular atrophy. The SMN protein is important in small nuclear ribonucleoprotein (snRNP) assembly and interacts with snRNP proteins via arginine/glycine-rich domains. Recently, SMN was also found to interact with core protein components of the two major families of small nucleolar RNPs, fibrillarin and GAR1, suggesting that SMN may also function in the assembly of small nucleolar RNPs. Here we present results that indicate that the interaction of SMN with GAR1 is mediated by the Tudor domain of SMN. Single point mutations within the Tudor domain, including a spinal muscular atrophy patient mutation, impair the interaction of SMN with GAR1. Furthermore, we find that either of the two arginine/glycine-rich domains of GAR1 can provide for interaction with SMN, but removal of both results in loss of the interaction. Finally, we have found that unlike the interaction of SMN with the Sm snRNP proteins, interaction with GAR1 and fibrillarin is not enhanced by arginine dimethylation. Our results argue against post-translational arginine dimethylation as a general requirement for SMN recognition of proteins bearing arginine/glycine-rich domains.     

Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor–modified effector cells

Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αβ T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)–engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting.