Molecular Identification and Expression Analysis of Filaggrin-2, a Member of the S100 Fused-Type Protein Family

Genes of the S100 fused-type protein (SFTP) family are clustered within the epidermal differentiation complex and encode essential components that maintain epithelial homeostasis and barrier functions. Recent genetic studies have shown that mutations within the gene encoding the SFTP filaggrin cause ichthyosis vulgaris and are major predisposing factors for atopic dermatitis. As a vital component of healthy skin, filaggrin is also a precursor of natural moisturizing factors. Here we present the discovery of a member of this family, designated as filaggrin-2 (FLG2) that is expressed in human skin. The FLG2 gene encodes a histidine- and glutamine-rich protein of approximately 248 kDa, which shares common structural features with other SFTP members, in particular filaggrin. We found that FLG2 transcripts are present in skin, thymus, tonsils, stomach, testis and placenta. In cultured primary keratinocytes, FLG2 mRNA expression displayed almost the same kinetics as that of filaggrin following Ca2+ stimulation, suggesting an important role in molecular regulation of epidermal terminal differentiation. We provide evidences that like filaggrin, FLG2 is initially expressed by upper granular cells, proteolytically processed and deposited in the stratum granulosum and stratum corneum (SC) layers of normal epidermis. Thus, FLG2 and filaggrin may have overlapping and perhaps synergistic roles in the formation of the epidermal barrier, protecting the skin from environmental insults and the escape of moisture by offering precursors of natural moisturizing factors

Targeted sensors to monitor oxidative stress in the endoplasmic reticulum

No abstract available

Purification and characterization of TbHsp70.c, a novel Hsp70 from Trypanosoma brucei

One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c

Właściwości i zastosowanie polimerów polietylenoiminowych modyfikowanych i niemodyfikowanych tyrozyną jako nośników siRNA

RNA interference (RNAi)-based therapy is a promising treatment option for many diseases, including cancer, neurodegenerative disorders and auto-immune diseases. RNAi may target a selected gene, allowing to avoid adverse effects related to standard treatment options. Due to its specificity, RNAi technology may be useful for the treatment of so-called ‘undruggable’ genes. Strategies based on RNAi have been broadly explored. Currently, increasing numbers of studies focus on the improvement of nanomedicines. Among many available nanocarriers for siRNA, cationic polymers gain special attention, due to efficient and non-toxic delivery mechanisms. As positively charged particles, they show improved interaction with negatively charged cell membranes and enhanced cellular uptake. The aim of the current work was to describe the properties of polyethyleneimines (PEIs) and their tyrosine modified counterparts. The first step focused on the evaluation of the biophysical properties of PEIs, their toxicity and ability to form complexes with siRNA. Linear and branched, tyrosine modified polymers complexed siRNA at favorable mass ratios which allowed significant gene expression knockdown with a limited cytotoxicity. Tyrosine modified PEIs were investigated in the further steps. The second task included a better understanding of complex formation, toxicity mechanisms and gene knockdown efficacy. The third task aimed to describe biophysical properties of tyrosine modified polymer: human serum albumin interactions. Summarizing, tyrosine modification significantly affected the properties of polyethyleneimines, changing their toxicity profile, siRNA binding capacity and interaction with human serum albumin. Tyrosine modified polymers performed better as siRNA carriers, exhibiting acceptable toxic effects and a very promising knockdown efficiency

Hydrolases

This book gives a current review of the links between the structure and function of hydrolases and ligases, as well as ideas for better using these critical enzymes. The book is split into two sections: “Cleavage” and “Ligases.” These enzymes are the biggest and most varied family of enzymes, allowing researchers to investigate the structural variety that underpins their different biological roles. In light of recent scientific advances, there is a desire to examine and update our knowledge of these enzymes’ functional and structural changes

Carbohydrate-Active Enzymes

Carbohydrate-active enzymes are responsible for both biosynthesis and the breakdown of carbohydrates and glycoconjugates. They are involved in many metabolic pathways; in the biosynthesis and degradation of various biomolecules, such as bacterial exopolysaccharides, starch, cellulose and lignin; and in the glycosylation of proteins and lipids. Carbohydrate-active enzymes are classified into glycoside hydrolases, glycosyltransferases, polysaccharide lyases, carbohydrate esterases, and enzymes with auxiliary activities (CAZy database, www.cazy.org). Glycosyltransferases synthesize a huge variety of complex carbohydrates with different degrees of polymerization, moieties and branching. On the other hand, complex carbohydrate breakdown is carried out by glycoside hydrolases, polysaccharide lyases and carbohydrate esterases. Their interesting reactions have attracted the attention of researchers across scientific fields, ranging from basic research to biotechnology. Interest in carbohydrate-active enzymes is due not only to their ability to build and degrade biopolymers—which is highly relevant in biotechnology—but also because they are involved in bacterial biofilm formation, and in glycosylation of proteins and lipids, with important health implications. This book gathers new research results and reviews to broaden our understanding of carbohydrate-active enzymes, their mutants and their reaction products at the molecular level