Unzipping the cuticle of the human hair shaft to obtain micron/nano keratin filaments

An attempt has been made to obtain intact individual keratin filaments of various levels from micron cortical, micron macrofibril to nano intermediate filament and polypeptide alpha-helix from the human hair shaft. The feasibility of this initiative has been largely demonstrated by finding that there is a longitudinal seam/zipper on the cuticle of the human hair shaft, which can be unzipped by certain solvents such as performic acid and urea, allowing one to use an anatomical approach to separate intact individual micron/nano filaments. Micron cortical and macrofibril filaments have been obtained. It is also found that the cortical filaments are twisted together to form a yarn, giving rise to the strength for the hair shaft; and that individual cortical filaments are often 2-2 paired in a similar structure to the double helix.     

Involvement of ERK1/2 and p38 in Mg2+ accumulation in liver cells

Activation of PKC signaling induces Mg²⁺ accumulation in liver cells. To test the hypothesis that PKC induces Mg²⁺ accumulation via MAPKs activation, hepatocytes were incubated in the presence of PD98059 and SB202190 as specific inhibitors of ERK1/2 and p38, respectively, and stimulated for Mg²⁺ accumulation by addition of PMA or OAG. Accumulation of Mg²⁺ within the cells was measured by atomic absorbance spectrophotometry in the acid extract of cell pellet. The presence of either inhibitor completely abolished Mg²⁺ accumulation irrespective of the dose of agonist utilized while having no discernible effect on β -adrenoceptor mediated Mg²⁺ extrusion. A partial inhibition on α ₁-adrenoceptor mediated Mg²⁺ extrusion was observed only in cells treated with PD98059. To confirm the inhibitory effect of PD98509 and SB202190, total and basolateral liver plasma membrane vesicles were purified in the presence of either MAPK inhibitor during the isolation procedure. Consistent with the data obtained in intact cells, liver plasma membrane vesicles purified in the presence of PD98509 or SB202190 lost the ability to accumulate Mg²⁺in exchange for intra-vesicular entrapped Na⁺ while retaining the ability to extrude entrapped Mg²⁺ in exchange for extra-vesicular Na⁺. These data indicate that ERK1/2 and p38 are involved in mediating Mg²⁺ accumulation in liver cells following activation of PKC signaling. The absence of a detectable effect of either inhibitor on β -adrenoceptor induced, Na⁺-dependent Mg²⁺ extrusion in intact cells and in purified plasma membrane vesicles further support the hypothesis that Mg²⁺ extrusion and accumulation occur through distinct and differently regulated transport mechanisms.     

Comparative analysis of the chemical treatments used in keratin extraction from red sheep’s hair and the cell viability evaluations of this keratin for tissue engineering applications

Hair waste is one of the solid substances rejected by the leather industry. This waste finds its way into the surroundings causing serious environmental pollution. This hair waste may be utilized for effective extraction of keratin, thereby generating value-added products with numerous applications. Thus we focusing on utilizing red sheep’s hair waste for extracting keratin by the application of different chemical treatments such as sodium hydroxide, sodium sulfide, mercaptoethanol, cysteine, sodium metabisulfite with urea (SMB), and SMB with SDS (SMBS). CD spectrum and FTIR results of the keratin samples indicated a predominance of the helical conformation along with β sheets. SDS-PAGE confirmed the molecular weight of the keratin samples to be in the range of 40–60 kDa. DSC and TGA analysis exhibited the extracted keratin to have a higher denaturation temperature (>200 °C) and thermal stability. The keratin samples obtained using varied chemical treatments were compared in terms of yield, protein content, and cost-effectiveness, and the sample obtained using SMBS was preferred for in vitro studies. It is indicated that keratin extracted using SMBS effectively involved for fibroblast cell growth. Thus, we suggest that these keratin could produce biomaterials that can serve as a valuable material for biomedical applications.     

Molecular dynamics simulation approach for the prediction of transmembrane helix–helix heterodimers assembly

Computational methods are useful to identify favorable structures of transmembrane (TM) helix oligomers when experimental data are not available or when they cannot help to interpret helix-helix association. We report here a global search method using molecular dynamics (MD) simulations to predict the structures of transmembrane homo and heterodimers. The present approach is based only on sequence information without any experimental data and is first applied to glycophorin A to validate the protocol and to the HER2-HER3 heterodimer receptor. The method successfully reproduces the experimental structures of the TM domain of glycophorin A (GpA(TM)) with a root mean square deviation of 1.5 A. The search protocol identifies three energetically stable models of the TM domain of HER2-HER3 receptor with favorable helix-helix arrangement, including right-handed and left-handed coiled-coils. The predicted TM structures exhibit the GxxxG-like motif at the dimer interface which is presumed to drive receptor oligomerization. We demonstrate that native structures of TM domain can be predicted without quantitative experimental data. This search protocol could help to predict structures of the TM domain of HER heterodimer family.     

Calculation of pKas in RNA: on the structural origins and functional roles of protonated nucleotides

pK(a) calculations based on the Poisson-Boltzmann equation have been widely used to study proteins and, more recently, DNA. However, much less attention has been paid to the calculation of pK(a) shifts in RNA. There is accumulating evidence that protonated nucleotides can stabilize RNA structure and participate in enzyme catalysis within ribozymes. Here, we calculate the pK(a) shifts of nucleotides in RNA structures using numerical solutions to the Poisson-Boltzmann equation. We find that significant shifts are predicted for several nucleotides in two catalytic RNAs, the hairpin ribozyme and the hepatitis delta virus ribozyme, and that the shifts are likely to be related to their functions. We explore how different structural environments shift the pK(a)s of nucleotides from their solution values. RNA structures appear to use two basic strategies to shift pK(a)s: (a) the formation of compact structural motifs with structurally-conserved, electrostatic interactions; and (b) the arrangement of the phosphodiester backbone to focus negative electrostatic potential in specific regions.     

A large conformational change couples the ATP binding site of SecA to the SecY protein channel

In bacteria, the SecYEG protein translocation complex employs the cytosolic ATPase SecA to couple the energy of ATP binding and hydrolysis to the mechanical force required to push polypeptides through the membrane. The molecular basis of this energy transducing reaction is not well understood. A peptide-binding array has been employed to identify sites on SecYEG that interact with SecA. These results along with fluorescence spectroscopy have been exploited to characterise a long-distance conformational change that connects the nucleotide-binding fold of SecA to the transmembrane polypeptide channel in SecY. These movements are driven by binding of non-hydrolysable ATP analogues to a monomer of SecA in association with the SecYEG complex. We also determine that interaction with SecYEG simultaneously decreases the affinity of SecA for ATP and inhibitory magnesium, favouring a previously identified active state of the ATPase. Mutants of SecA capable of binding but not hydrolysing ATP do not elicit this conformationally active state, implicating residues of the Walker B motif in the early chain of events that couple ATP binding to the mobility of the channel.     

Cuticular waxes from ivy leaves (Hedera helix L.): analysis of high-molecular-weight esters

Soluble waxes were extracted from the cuticle of ivy (Hedera helix L.) leaves with dichloromethane in a yield of ca. 13%. The cuticular waxes were directly analysed by GC-MS, high-temperature GC-MS and ESI-MS/MS. The GC-MS analysis showed mostly n-alkanols (45.3%), monoacids (18.8%), triterpenes (9.7%), n-aldehydes (8.7%) and n-alkanes (7.7%). The high-temperature GC-MS and the ESI-MS/MS analyses showed the presence of ester waxes, namely alkyl alkanoates and alkyl coumarates. Alkyl alkanoates comprised esters of the hexadecanoic acid with n-alkanols ranging from C16 to C34. Alkyl coumarates included esters of coumaric acid with n-alkanols ranging from C16 to C32. The cuticular waxes were hydrolysed and the resulting organic and aqueous phases analysed by GC-MS. The hydrolysate showed a major increase in the quantities of n-alkanols, hexadecanoic acid and coumaric acid derived from the alkyl and acyl moieties from the ester waxes. A content of ester waxes of 38% was estimated based on the results from the GC-MS analysis of the non-hydrolysed and hydrolysed cuticular waxes. Alkyl alkanoates were analysed by ESI-MS/MS as [M + Li]+ adduct ions and the alkyl coumarates as [M - H]- deprotonated ions. The ESI-MS/MS analysis allowed the detection of a wider range of ester waxes than high-temperature GC-MS, and was shown to be a useful technique for the qualitative analysis of ester waxes from plant cuticles.     

The CD44 standard/ezrin complex regulates Fas-mediated apoptosis in Jurkat cells

The transmembrane receptor CD44 conveys important signals from the extracellular microenvironment to the cytoplasm, a phenomena known as “outside-in” signaling. CD44 exists as several isoforms that result from alternative splicing, which differ only in the extracellular domain but yet exhibit different activities. CD44 is a binding partner for the membrane-cytoskeleton cross-linker protein ezrin. In this study, we demonstrate that only CD44 standard (CD44s) colocalizes and interacts with the actin cross-linkers ezrin and moesin using well-characterized cell lines engineered to express different CD44 isoforms. Importantly, we also show that the association CD44s-ezrin-actin is an important modulator of Fas-mediated apoptosis. The results highlight a mechanism by which signals from the extracellular milieu regulate intracellular signaling activities involved in programmed cell death. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Control of osteopontin signaling and function by post-translational phosphorylation and protein folding

Osteopontin (OPN) plays roles in a variety of cellular processes from bone resorption and extracellular matrix (ECM) remodeling to immune cell activation and inhibition of apoptosis. Because it binds receptors (integrins, CD44 variants) typically engaged by ECM molecules, OPN acts as a "soluble" ECM molecule. A persistent theme throughout the characterization of how OPN functions has been the importance of phosphorylation. The source of the OPN used in specific experiments and the location of modified sites is an increasingly important consideration for OPN research. We review briefly some of the ways OPN impacts on the biology of mammalian systems with an emphasis on the importance of serine phosphorylation in modulating its signaling ability. We describe experiments that support the hypothesis that differences in the post-translational phosphorylation of OPN expressed by different cell types regulate how it impacts on target cells. Analyses of OPN's potential secondary structure reveal a possible beta-sheet conformation that offers an interpretation of certain experimental observations, specifically the effect of thrombin cleavage; it is consistent with an interaction between the C-terminal region of the protein and the central integrin-binding RGD sequence.     

Drug block of the hERG potassium channel: insight from modeling

Many commonly used, structurally diverse, drugs block the human ether-a-go-go-related gene (hERG) K(+) channel to cause acquired long QT syndrome, which can lead to sudden death via lethal cardiac arrhythmias. This undesirable side effect is a major hurdle in the development of safe drugs. To gain insight about the structure of hERG and the nature of drug block we have produced structural models of the channel pore domain, into each of which we have docked a set of 20 hERG blockers. In the absence of an experimentally determined three-dimensional structure of hERG, each of the models was validated against site-directed mutagenesis data. First, hERG models were produced of the open and closed channel states, based on homology with the prokaryotic K(+) channel crystal structures. The modeled complexes were in partial agreement with the mutagenesis data. To improve agreement with mutagenesis data, a KcsA-based model was refined by rotating the four copies of the S6 transmembrane helix half a residue position toward the C-terminus, so as to place all residues known to be involved in drug binding in positions lining the central cavity. This model produces complexes that are consistent with mutagenesis data for smaller, but not larger, ligands. Larger ligands could be accommodated following refinement of this model by enlarging the cavity using the inherent flexibility about the glycine hinge (Gly648) in S6, to produce results consistent with the experimental data for the majority of ligands tested.