Self-organization of oligopeptides obtained on dissolution of feather keratins in superheated water

Keratins are self-organized proteins that are abundantly available in wool, feather, human hair, etc., making them a potential cheap feedstock for the modification of amino acids. This paper explores the hydrolysis of keratin in water under specific pressure-temperature conditions where the hydrolysis through scission of the protein chain yields oligopeptides. Here we report for the first time that, under appropriate conditions, these oligopeptides self-assemble into a hierarchical architecture, the process being followed in time by optical microscopy. Birefringent needle-like crystals are observed which tend to nucleate heterogeneously. When given sufficient time, these needles become tens of microns in length and act as further nuclei, developing a highly repetitive structure of several hundreds of microns in size. Micro-focus X-ray diffraction studies supported by in situ microscopy reveal that these needles have a crystal structure similar to that of the native protein, although better organized along the ab-plane. Spectroscopic studies on these structures show crystalline bands that disappear above 150 degrees C, coinciding with an endothermic peak in DSC. Amino acid analysis shows that the self-assembled birefringent entities are indeed oligopeptides, consisting of sequences of approximately 40 amino acids. The proposed ecofriendly route provides an effective route for obtaining oligopeptides that can be used as important building blocks for the synthesis of a range of novel polymers. The oligopeptides obtained from the sustainable source can be used as important building blocks for the synthesis of a range of novel polymers.     

Correlation of XMAP and ELISA cytokine profiles; development and validation for immunotoxicological studies in vitro

There is an emerging trend in immunotoxicological studies to use the multiplex technologies for testing the safety and the efficacy of new pharmaceuticals by using cytokines profiling as biomarker. The Luminex 200 xMAP (multi-analyte profiling) technology provides simultaneous measurement of multiple cytokines in small sample volumes, expressing rapidly the differences between various test compounds. The aim is to develop and validate the Luminex 200 multiplex immunoassays by correlation with ELISA (enzyme-linked immunosorbent assays) for implementation in evaluating cytokine profiling in immunotoxicological studies in vitro. METHODS: Human peripheral whole blood from healthy subject diluted 1+4 with RPMI 1640 was cultured 48 hours in 28 experimental variants: control, in presence of mitogens, bioflavonoid extracts (from Crataegus monogyna and Echinacea purpurea) as cytoprotectors and with a toxic compound [Pb(NO3)2]), separately or variously combined. IL-1beta and IL-2 were comparatively performed by xMAP and ELISA immunoassays from the same sample to initialize validation of multiplex cytokine panel: IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-alpha, IFN-gamma, usually performed by Luminex 200 system in our immunotoxicological studies. The results indicate similarly typed trends of cytokine values obtained by both methods, with comparable relative changes in presence of mitogens, bioflavonoids and toxic, respectively. Although xMAP absolute cytokine values were higher than ELISA values, the correlation between multiplexed assay and ELISA was good for IL-1beta and IL-2 with positive correlation coefficients near to 1. Conclusions. Quantitative differences between absolute values for IL-1beta and IL-2 obtained by xMAP and ELISA assays are found, but the relative values are comparable and the two methods keep similar trends in similar exposure conditions. The performance parameters of the xMAP assay and the good correlation coefficients with the "gold standard" ELISA recommend to validate the multiplex assay for analyzing cytokine profiles in immunotoxicological studies in vitro.     

Cardiac telocytes: serial dynamic images in cell culture

Telocytes (TC) are interstitial cells with telopodes (Tp). These prolongations (Tp) are quite unique: very long (several tens of micrometres) and very thin (≤0.5 μm), with moniliform aspect: thin segments (podomeres) alternating with dilations (podoms). To avoid any confusion, TC were previously named interstitial Cajal-like cells (ICLC). Myocardial TC were repeatedly documented by electron microscopy, immunohistochemistry and immunofluorescence. TC form a network by their Tp, either in situ or in vitro. Cardiac TC are (completely) different of 'classic' fibroblasts or fibrocytes. We hereby present a synopsis of monitoring, by time-lapse videomicroscopy, of Tp network development in cell culture. We used a protocol that favoured interstitial cell selection from adult mouse myocardium. Videomicroscopy showed dynamic interactions of neighbour TC during the network formation. During their movement, TC leave behind distal segments (podomeres) of their Tp as guiding marks for the neighbouring cells to follow during network rearrangement.     

Palmitoyl-protein thioesterase gene expression in the developing mouse brain and retina: implications for early loss of vision in infantile neuronal ceroid lipofuscinosis

Mutations in the palmitoyl-protein thioesterase (PPT) gene cause infantile neuronal ceroid lipofuscinosis (INCL), the clinical manifestations of which include the early loss of vision followed by deterioration of brain functions. To gain insight into the temporal onset of these clinical manifestations, we isolated and characterized a murine PPT (mPPT)-cDNA, mapped the gene on distal chromosome 4, and studied its expression in the eye and in the brain during development. Our results show that both cDNA and protein sequences of the murine and human PPTs are virtually identical and that the mPPT expression in the retina and in the brain is temporally regulated during development. Furthermore, the retinal expression of mPPT occurs much earlier and at a higher level than in the brain at all developmental stages investigated. Since many retinal and brain proteins are highly palmitoylated and depalmitoylation by PPT is essential for their effective recycling in the lysosomes, our results raise the possibility that inactivating mutations of the PPT gene, as occur in INCL, are likely to cause cellular accumulation of lipid-modified proteins in the retina earlier than in the brain. Consequently, the loss of vision occurs before the deterioration of brain functions in this disease.     

Molecular recognition between glyconectins as an adhesion self-assembly pathway to multicellularity

The appearance of multicellular forms of life has been tightly coupled to the ability of an organism to retain its own anatomical integrity and to distinguish self from non-self. Large glycoconjugates, which make up the outermost cell surface layer of all Metazoans, are the primary candidates for the primordial adhesion and recognition functions in biological self-assembly systems. Atomic force microscopy experiments demonstrated that the binding strength between a single pair of Porifera cell surface glyconectin 1 glycoconjugates from Microciona prolifera can hold the weight of 1600 cells, proving their adhesion functions. Here, measurement of molecular self-recognition of glyconectins (GNs) purified from three Porifera species was used as an experimental model for primordial xenogeneic self/non-self discrimination. Physicochemical and biochemical characterization of the three glyconectins, their glycans, and peptides using gel electrophoresis, ultracentrifugation, NMR, mass spectrometry, glycosaminoglycan-degrading enzyme treatment, amino acid and carbohydrate analyses, and peptide mapping showed that GNs define a new family of proteoglycan-like molecules exhibiting species-specific structures with complex and repetitive acidic carbohydrate motives different from the classical proteoglycans and mucins. In functional self-assembly color-coded bead, cell, and blotting assays, glyconectins displayed species-specific recognition and adhesion. Affinity-purified monospecific polyclonal antibodies prepared against GN1, -2, and -3 glycans selectively inhibited cell adhesion of the respective sponge species. These results together with species-specific coaggregation of GN carbohydrate-coated beads with cells showed that GN glycans are functional in cell recognition and adhesion. The specificity of carbohydrate-mediated homophilic GN interactions in Porifera approaches the binding selectivity of the evolutionarily advanced immunoglobulin superfamily. Xenoselectivity of primordial glyconectin to glyconectin recognition may be a new paradigm in the self-assembly and non-self discrimination pathway of cellular adhesion leading to multicellularity.     

Synthesis and Antiviral Activity of Carbocyclic 5-Substituted Uridines and Cytidines

Abstract

Some carbocyclic uridines and cytidines have been prepared in a palladium-catalyzed reaction between 5-substituted uracils and cytosines and diacetoxymethylcyclopentene, prepared in a Prins reaction. The antiviral activity of the nucleoside analogues have been tested.     

Taste Masking of Griseofulvin and Caffeine Anhydrous Using Kleptose Linecaps DE17 by Hot Melt Extrusion

The objective of this project was to investigate the potential of Kleptose Linecaps DE17 (KLD) in masking the unpleasant/bitter taste of therapeutic agents by hot melt extrusion (HME). Griseofulvin (GRI) and caffeine anhydrous (CA) were used as a bitter active pharmaceutical ingredient (API) model drugs. Thermogravimetric studies confirmed the stability of GRI, CA, and KLD at the employed extrusion temperatures. The differential scanning calorimetry (DSC) studies revealed a characteristic melting endotherm of GRI at 218–220°C and CA at 230–232°C in the physical mixtures as well as in all extrudates over the period of study, indicating the crystalline nature of drug. HME of KLD was achieved only in the presence of plasticizer. Among the several plasticizers investigated, xylitol showed improved processability of KLD at 15% w/w concentration. Dissolution studies of HME extrudates using simulated salivary medium exhibited ～threefold less release compared to physical mixture at the end of 5 min (the lesser drug release, better the taste masking efficiency). Furthermore, the results from the sensory evaluation of products in human panel demonstrated strong bitter taste in the case of physical mixture compared to the HME formulation, suggesting the potential of Kleptose Linecaps DE17 as taste masking polymer in melt extruded form.     

Water exchange through erythrocyte membranes: Nuclear magnetic resonance studies on the effects of inhibitors and of chemical modification of human membranes

The changes in water diffusion across human erythrocyte membranes following exposure to various inhibitors and proteolytic enzymes have been studied on isolated erythrocytes suspended in isotonic buffered solutions. An important issue was to investigate whether the sulfhydryl reacting reagents that have been applied in osmotic experiments showed similar effects on diffusional permeability. It was found that mercurials, including mersalyl, were the only sulfhydryl reacting reagents that were efficient inhibitors. Under optimal conditions a similar degree of inhibition (around 45%) was found with all mercury-containing sulfhydryl reagents. Other reagents, including the sulfhydryl reagent DTNB, phloretin, or H2DIDS, the specific inhibitor of the anion transport system in erythrocyte membrane, did not appear to inhibit significantly the diffusional permeability. No changes in water diffusion were noticed after exposure to erythrocytes to trypsin and chymotrypsin. A new kind of experiments was that in which the effects of exposure of erythrocytes to two or more agents were studied. It was found that none of the chemical manipulations of membranes that did not affect water diffusion hampered the inhibitory action of mercurials. These findings show that the SH groups involved in water diffusion across erythrocyte membrane do not react with any of the other SH reagents aside from mercurials and that the molecular mechanism of water transport is not affected by chymotryptic cleavage of band 3 protein into the 60 and 35 kD fragments. The NMR method appears as a useful tool for studying changes in water diffusion in erythrocyte membranes following various chemical manipulations of the membranes with the aim of locating the water channel.