Characterisation and Regional Localisation of Pre- and Postsynaptic Glycoproteins of the Chick Forebrain Showing Changed Fucose Incorporation Following Passive Avoidance Training

To identify those glycoproteins whose synthesis or modification is necessary for memory formation, we have studied the uptake of radiolabelled fucose into synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) derived from two specific left and right forebrain loci, at two different times after training of 1-day-old chicks on a one-trial passive avoidance learning task. To increase the reliability of the comparison, a double-labelling method was used. Tissue samples from intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) were isolated at 6 and 24 h after training. At both times, training resulted in region-specific changes, both increases and decreases, in incorporated radioactivity into pre- and postsynaptic glycoproteins. After 6 h, there was a relative decline in incorporation into both SPMs and PSDs of the right IMHV of trained chicks, a decline that persisted in the PSDs until 24 h. A small decline in incorporation in SPMs from the right LPO of trained chicks at 6 h was reversed by 24 h, by which time there was a 64% increase in incorporation into SPMs and a 24% increase into PSDs of the left LPO. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of left and right hemisphere samples containing LPO revealed that 6 h after training the main effect was presynaptic, including a reduction of incorporation into high molecular mass glycoproteins, of 150-180 kDa, and an increase in a lower molecular mass (41 kDa) fraction. By 24 h after training, a left hemisphere presynaptic glycoprotein of molecular mass approximately 50 kDa showed the biggest increase in fucosylation. In addition, a wide group of postsynaptic glycoproteins of both hemispheres, in the ranges 150-180, 100-120, and 33 kDa now showed increases in incorporation. Some other fractions showed decreases. These results are in accord with previous data on incorporation obtained using the amnesic agent 2-deoxygalactose. They also support the hypothesis that memory formation involves the strengthening of connections between pre- and postsynaptic neurons of the LPO by growth or modulation of pre- and postsynaptic structures.     

On the computation of the tertiary structure of globular proteins. III. Inter-residue distances and computed structures

An analysis of geometrical models for computing the tertiary structure of globular proteins from the primary structure is presented. The roles of initial configuration, input information on inter-residue distances and the errors in this information are delineated. It is shown that for local information like that on secondary structure, the calculated structure is very sensitive to errors and to the initial configuration. Thus, such information is far from adequate for predicting the tertiary structure. On the other hand, global information on all the inter-residue distances is quite insensitive to errors. A semi-empirical method is presented to estimate these distances and the calculated structures are given for two proteins—pancreatic trypsin inhibitor and parvalbumin. These structures have good resemblances to those determined by X-ray diffraction. A strategy for further refinement of the method is indicated.     

The subcellular distribution and partial characterization of cholinesterase activities of canine platelets.

The multiple cholinesterase activities in canine platelets have been investigated. Platelets were homogenized by rapid decompression under nitrogen, glass tube/Teflon pestle, and glycerol lysis techniques. Rapid decompression under nitrogen technique was found to be the most efficient and gentle method for cell disruption. Homogenates were subfractionated using sodium diatrizoate density gradients. Marker enzyme assays and pulse labeling experiments with 5-hydroxyl[14C] tryptamine and [125I] thrombin on prepared subcellular fractions confirmed that the soluble, plasma membrane and the granule-1 fractions were all in reasonably pure form. Furthermore, labeling of the plasma membrane with [125I] thrombin is cited as the first successful attempt at attaining significantly bound marker for this structure. Cholinesterase activity distributions measured in these fractions indicated that about 30% of the activity was present in the plasma membrane, 50% in granule-1 and 5% in soluble fractions. Kinetic data of cholinesterase activities obtained from intact platelets, plasma membrane preparations and platelet release supernatants indicated that they are strikingly similar.     

Flexural and Dynamic Mechanical Properties of Alkali-Treated Coir/Pineapple Leaf Fibres Reinforced Polylactic Acid Hybrid Biocomposites

Polylactic acid(PLA)possesses good mechanical and biodegradability properties which make it a suitable material for polymer composites whereas brittleness and high costs limit its utilization in various applications.The reinforcement of natural fibres with biopolymers has been formed to be an efficient technique to develop composites having the ability to be fully biodegradable.This study concerns with the incorporation of various percentages of untreated and alkali-treated Coir Fibres(CF)and pineapple leaf fibres(PALF)in PLA biocomposites and characterizations of flexural,morphological and dynamic mechanical properties.Flexural properties showed that the treated C1P1 hybrid composites(C1P1A)displayed highest flexural strength(35.81 MPa)and modulus(5.28 GPa)among all hybrid biocomposites.Scanning Electron Micros-copy(SEM)revealed a behaviour of fibre-matrix adhesion in untreated treated biocomposites.SEM observation revealed good dispersion of the fillers in PLA.Dynamic mechanical analysis revealed that C1P1A showed highest glass transition temperature(Tg)and storage modulus(E')while untreated C3P7 displayed the least Tg and E'.Overall findings showed that alkali-treated hybrid biocomposites(CF/PALF/PLA)especially C1P1A have improved flexural properties,dynamic and morphological properties over untreated biocomposites.Success of these findings will provide attracting consideration of these hybrid biocomposites for various lightweight uses in a broad selection of industrial applications such as biomedical sectors,automobile,construction,electronics equipment,and hardware tools.     

A natural fusion of flavodiiron,rubredoxin, and rubredoxin oxidoreductase domains is a self-sufficient water-forming oxidase of Trichomonas vaginalis

Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H₂O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H₂O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD⁺ while simultaneously removing O₂ from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX–like enzyme and instead harbors three paralogous genes (FDPF1–3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a “stand-alone” FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.     

Assessment of T Cell Receptor Complex Expression Kinetics in Natural Killer Cells

Among the polypeptides that comprise the T cell receptor (TCR), only CD3ζ is found in Natural Killer (NK) cells, where it transmits signals from activating receptors such as CD16 and NKp46. NK cells are potent immune cells that recognize target cells through germline-encoded activating and inhibitory receptors. Genetic engineering of NK cells enables tumor-specific antigen recognition and, thus, has a significant promise in adoptive cell therapy. Ectopic expression of engineered TCR components in T cells leads to mispairing with the endogenous components, making a knockout of the endogenous TCR necessary. To circumvent the mispairing of TCRs or the need for knockout technologies, TCR complex expression has been studied in NK cells. In the current study, we explored the cellular processing of the TCR complex in NK cells. We observed that in the absence of CD3 subunits, the TCR was not expressed on the surface of NK cells and vice versa. Moreover, a progressive increase in surface expression of TCR between day three and day seven was observed after transduction. Interestingly, the TCR complex expression in NK92 cells was enhanced with a proteasome inhibitor (bortezomib) but not a lysosomal inhibitor (chloroquine). Additionally, we observed that the TCR complex was functional in NK92 cells as measured by estimating CD107a as a degranulation marker, IFNγ cytokine production, and killing assays. NK92 cells strongly degranulated when CD3ε was engaged in the presence of TCR, but not when only CD3 was overexpressed. Therefore, our findings encourage further investigation to unravel the mechanisms that prevent the surface expression of the TCR complex.     

Enhancement of keratinocyte growth factor potential in inducing adipose‐derived stem cells differentiation into keratinocytes by collagen‐targeting

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose‐derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen‐binding domain from Vibrio mimicus metalloprotease (vibrioCBD‐KGF). KGF and vibrioCBD‐KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK‐293 and MCF‐7 cells. vibrioCBD‐KGF demonstrated enhanced collagen‐binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD‐KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen‐coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin‐10 and Involucrin), and stem cell markers (Collagen‐I and Vimentin) by real‐time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD‐KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down‐regulation of Collagen‐I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD‐KGF. The present study showed that vibrioCBD‐KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen‐based biomaterials.     

Insights into plastome of Fagonia indica Burm.f. (Zygophyllaceae): organization,annotation and phylogeny

The enhanced understanding of chloroplast genomics would facilitate various biotechnology applications; however, the chloroplast (cp) genome / plastome characteristics of plants like Fagonia indica Burm.f. (family Zygophyllaceae), which have the capability to grow in extremely hot sand desert, have been rarely understood. The de novo genome sequence of F. indica using the Illumina high-throughput sequencing technology determined 128,379 bp long cp genome, encode 115 unique coding genes. The present study added the evidence of the loss of a copy of the IR in the cp genome of the taxa capable to grow in the hot sand desert. The maximum likelihood analysis revealed two distinct sub-clades i.e. Krameriaceae and Zygophyllaceae of the order Zygophyllales, nested within fabids.