Piezo1 forms a slowly-inactivating mechanosensory channel in mouse embryonic stem cells

Piezo1 is a mechanosensitive (MS) ion channel with characteristic fast-inactivation kinetics. We found a slowly-inactivating MS current in mouse embryonic stem (mES) cells and characterized it throughout their differentiation into motor-neurons to investigate its components. MS currents were large and slowly-inactivating in the stem-cell stage, and became smaller and faster-inactivating throughout the differentiation. We found that Piezo1 is expressed in mES cells, and its knockout abolishes MS currents, indicating that the slowly-inactivating current in mES cells is carried by Piezo1. To further investigate its slow inactivation in these cells, we cloned Piezo1 cDNA from mES cells and found that it displays fast-inactivation kinetics in heterologous expression, indicating that sources of modulation other than the aminoacid sequence determine its slow kinetics in mES cells. Finally, we report that Piezo1 knockout ES cells showed a reduced rate of proliferation but no significant differences in other markers of pluripotency and differentiation.

Unravelling the 2D self-assembly of Fmoc-dipeptides at fluid interfaces

Fmoc-dipeptides are self-assembled at the air/liquid interface as a function of their aminoacid sequence.

The nucleoid as a scaffold for the assembly of bacterial signaling complexes

ABSTRACTThe FrzCD chemoreceptor from the gliding bacterium Myxococcus xanthus forms cytoplasmic clusters that occupy a large central region of the cell body also occupied by the nucleoid. In this work, we show that FrzCD directly binds to the nucleoid with its N-terminal positively charged tail and recruits active signaling complexes at this location. The FrzCD binding to the nucleoid occur in a DNA-sequence independent manner and leads to the formation of multiple distributed clusters that explore constrained areas. This organization might be required for cooperative interactions between clustered receptors as observed in membrane-bound chemosensory arrays.AUTHOR SUMMARYIn this work, we show that the cytoplasmic chemoreceptor of the Frz chemosensory system, FrzCD, does not bind the cytoplasmic membrane like most MCPs but bind the bacterial nucleoid directly, thus forming distributed protein clusters also containing the Frz kinase. In vitro and in vivo experiments show that DNA-binding is not sequence-specific and is mediated by a basic aminoacid sequence of the FrzCD N-terminal domain. The deletion of this motif abolishes FrzCD DNA-binding and cooperativity in the response to signals. This work shows the importance of the nucleoid in the organization and functioning of cytoplasmic signaling systems in bacteria.

CLONING, EXPRESSION, BIOCHEMICAL PARTIAL CHARATERIZATION AND MODELING OF ESTERASE FROM ACINETOBACTER CALCOACETICUS SP DSM587

The aim of this paper has been to clone, express, purify and characterization the EstB from<br />Acinetobacter calcoaceticus encoded by the gen X8895. The esterase was cloned in Pet28a and<br />partially purified. The molecular mass of the purified enzyme was 36 kDa (by SDS) and 68.9<br />KDa (by gel filtration chromatography). The EstB showed a maximum activity at 35ºC and pH 8<br />and towards shorter acyl chain lengths (PNPA, PNPB, PNPC) and showed activity about Smethyltiobutanoate,<br />too. The catalytic triad has been predicted by aminoacid sequence<br />alignment and the structure modeling was performed used esterase 1QoR as template.

Difference of hemaglutinins between wild-type and vaccine measles virus in Indonesia

Background Hemaglutinin (H) protein of measles virus is veryimportant in the process of host cell infection. H protein is alsoable to induce specific antibodies which can neutralize measlesvirus and block the cell infection.Objective This study aimed to explore the nucleotide and aminoacid sequence differences between wild-type measles virus (G2,G3 and D9) with CAM-70, Schwarz and Edmonston-wt vaccinevirus.Methods The exctration and amplification of the gene wereconducted in the laboratory using biomolecular technology. Thegene and protein analysis were conducted using the bioinformatictechnology.Results The results showed that the differences in nucleotidesequences were highest between wild-type virus and CAM-70vaccine virus (76-77 nucleotides), followed by Schwarz (61-64nucleotides) and Edmonston (60-63 nucleotides). The differencesin amino acid sequences were highest between wild-type virusand CAM-70 (24-29 residues), followed by Schwarz (13-20residues) and Edmonston (12-19 residues).Conclusion The Indonesian wild-type measles virus was geneticallycloser to Schwarz vaccine virus than CAM-70 vaccine virus,hence the neutralizing antibodies generated by Schwarz vaccinewere more specific against Indonesian wild-type virus comparedto CAM-70 vaccine.

Periostin Is a Previously Uncharacterised Vitamin K Dependent γ-Carboxyglutamic Acid (Gla) Containing Protein Expressed by Marrow-Derived Mesenchymal Stromal Cells.

The modification of glutamic acid residues to g-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent γ-glutamylcarboxylase enzyme. Despite ubiquitous expression of the γ-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-proteins after the liver, we sought to identify Gla proteins secreted by bone-marrow derived mesenchymal stromal cells (MSCs), a precursor to all non-hematopoietic cells in bones. We used a proteomics approach to screen the secretome of MSCs with a combination of 2D gel electrophoresis and tandem mass spectrometry. The most abundant Gla-protein secreted by MSCs was identified as periostin, a previously unrecognized γ-carboxylated protein. In silico aminoacid sequence analysis of periostin demonstrated the presence of four consensus γ-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation using anti-Gla antibodies and could be inhibited by warfarin in MSCs. In conclusion, periostin is a novel vitamin K-dependent γ-carboxylated protein distinguished from other Gla-proteins by the presence of multiple γ-carboxylase recognition sites and MSCs are an abundant source of periostin including its γ-carboxylated variant.

ADAM15 is an adhesion receptor for platelet GPIIb-IIIa and induces platelet activation

SummaryCell adhesion and proteolytic matrix degradation are central processes in atherosclerosis. Being a member of the family of ADAMs (“a disintegrin and metalloproteinase”), metargidin (ADAM15) combines a metalloproteinase domain and an RGD aminoacid sequence. We studied the potential role of ADAM15 as an adhesion receptor on endothelial cells and interactions between platelets and ADAM15 with respect to platelet adhesion, activation and thrombus formation. ADAM15 was found to be expressed on cultured endothelial cells (HUVEC). Platelet adhesion to immobilized recombinantADAM15 was effectively enhanced under both static and high shear rate conditions reaching the maximum level of adhesion to fibrinogen. Consistently, platelet adhesion onto ADAM15 overexpressing endothelial cells was significantly increased. Adhesion to ADAM15 was reduced by blockade of GPIIb-IIIa using neutralizing anti-αIIbβ3 mAbs (7E3, 2G12), but not by anti-α v β3 (LM609). Soluble ADAM15 binds to activated but not to resting GPIIb-IIIa. Moreover, platelets adherent to ADAM15 additionally attracted platelets under high shear rates indicating an initial role of platelet- ADAM15 interactions for thrombus formation. Furthermore, incubation of platelets with solubleADAM15 showed a dose-dependent increase in secretion of CD62P and CD40L. ADAM15 is expressed on endothelial cells and can serve as an adhesion receptor for platelets via GPIIb-IIIa binding. Platelet adhesion to ADAM15 leads to platelet activation, secretion and promotes thrombus formation. Thus, ADAM15 may represent a novel target for antithrombotic strategies in cardiovascular pathologies.

Overview of the most commonly used methods in allergen characterization

The characterization of an allergen is a troublesome and difficult process, as it requires both the precise biochemical characterization of a (glyco)protein molecule and the establishment of its susceptibility to IgE antibodies, as they are the main link to histamine release in some hypersensitivity states (type I allergies). As the characterization of an allergen includes molecular weight determination of the allergenic molecule, its structure determination, physicochemical properties, IgE binding properties of the allergen molecule, and its allergenicity, an overall review of which biochemical and immunochemical methods are used in achieving this goal are presented in this paper. The information on the molecular level on the structures of allergens indicates that allergens are considerably heterogeneous protein structures, and that there is no particular aminoacid sequence which is responsible for the allergenicity. Therefore, information gained from detailed structural, functional and immunochemical studies of these intriguing molecules, which nowadays modulate a variety of pathophysiological conditions, would greatly improve our understanding of the underlying disease mechanisms, and the way to handle them.