Mutually exclusive interactions of EHD1 with GS32 and Syndapin II

GS32/SNAP-29 is a SNAP-25-like SNARE and has been shown to interact with syntaxin 6. Using immobilized recombinant GS32, we have recovered EHD1 as a major GS32-interacting protein from total HeLa cell extracts. This interaction is mediated by the EH domain of EHD1 and the N-terminal NPF-containing 17-residue region of GS32. Co-immunoprecipitation suggests that GS32 could also interact with EHD1 in intact cells. When immobilized GST-EHD1 was used to fish out interacting proteins from total brain extracts, syndapin II was identified as a major interacting partner. Similar to the GS32-EHD1 interaction, syndapin II also interacts with the EH domain of EHD1 via its NPF repeat region. Interaction of endogenous EHD1 and syndapin II was also established by co-immunoprecipitation. Furthermore, interaction of GS32 and syndapin II with EHD1 was shown to be mutually exclusive, suggesting that EHD1 may regulate/participate in the functional pathways of both GS32 and syndapin II in a mutual exclusive manner. Opposing roles of GS32 and syndapin II in regulating the surface level of transferrin receptor (TfR) were observed.     

A TAXONOMIC STUDY OF THE GENUS PADINA (DICTYOTALES,PHAEOPHYCEAE) INCLUDING THE DESCRIPTIONS OF FOUR NEW SPECIES FROM JAPAN,HAWAII, AND THE ANDAMAN SEA1

A taxonomic study of the genus Padina from Japan, Southeast Asia, and Hawaii based on morphology and gene sequence data (rbcL and cox3) resulted in the recognition of four new species, that is, Padina macrophylla and Padina ishigakiensis from Ryukyu Islands, Japan; Padina maroensis from Hawaii; and Padina usoehtunii from Myanmar and Thailand. All species are bistratose and morphologically different from one another as well as from any known taxa by a combination of characters relating to degree of calcification; the structure, position, and arrangement of hairlines (HLs) and reproductive sori; and the presence or absence of rhizoid‐like groups of hairs and an indusium. Molecular phylogenetic analyses demonstrated a close relationship between P. ishigakiensis, P. macrophylla, P. maroensis, and Padina australis Hauck. The position of P. usoehtunii, however, was not fully resolved, being either sister to a clade comprising the other three new species and P. australis in the rbcL tree or more closely related to a clade comprising several other recently described species in the cox3 tree. The finding of the four new species demonstrates high species diversity particularly in southern Japan. The following characters were first recognized here to be useful for species delimitation: the presence or absence of small rhizoid‐like groups of hairs on the thallus surface, structure and arrangement of HLs on both surfaces either alternate or irregular, and arrangement of the alternating HLs between both surfaces in equal or unequal distance. The evolutionary trajectory of these and six other morphological characters used in species delineation was traced on the phylogenetic tree.     

Does the combination of optimal substitutions at the C²-, N⁵- and N⁸-positions of the pyrazolo-triazolo-pyrimidine scaffold guarantee selective modulation of the human A₃ adenosine receptors?

In an attempt to study the optimal combination of a phenyl ring at the C(2)-position and different substituents at the N(5)- and N(8)-positions towards the selective modulation of human A(3) adenosine receptors (hA(3)AR), we synthesized a new series of 2-para-(un)substituted-phenyl-pyrazolo-triazolo-pyrimidines bearing either a methyl or phenylethyl at N(8) and chains of variable length at N(5). Through biological evaluation, it was found that the majority of the compounds had good affinities towards the hA(3)AR in the low nanomolar range. Compound 16 possessed the best hA(3)AR affinity and selectivity profile (K(i)hA(3)=1.33 nM; hA(1)/hA(3)=4880; hA(2A)/hA(3)=1100) in the present series of 2-(substituted)phenyl-pyrazolo-triazolo-pyrimidine derivatives. In addition to pharmacological characterization, a molecular modeling investigation on these compounds further elucidated the effect of different substituents at the pyrazolo-triazolo-pyrimidine scaffold on affinity and selectivity to hA(3)AR.     

Experimental Characterization of the Chronic Constriction Injury-Induced Neuropathic Pain Model in Mice

Number of ligations made in the chronic constriction injury (CCI) neuropathic pain model has raised serious concerns. We compared behavioural responses, nerve morphology and expression of pain marker, c-fos among CCI models developed with one, two, three and four ligations. The numbers of ligation(s) on sciatic nerve shows no significant difference in displaying mechanical and cold allodynia, and mechanical and thermal hyperalgesia throughout 84 days. All groups underwent similar levels of nerve degeneration post-surgery. Similar c-fos level in brain cingulate cortex, parafascicular nuclei and amygdala were observed in all CCI models compared to sham-operated group. Therefore, number of ligations does not impact intensity of pain symptoms, pathogenesis and neuronal activation. A single ligation is sufficient to develop neuropathic pain, in contrast to the established model of four ligations. This study dissects and characterises the CCI model, ascertaining a more uniform animal model to surrogate actual neuropathic pain condition.

     

Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system

The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display.     

Lipidomic analysis of human tear fluid reveals structure-specific lipid alterations in dry eye syndrome

As current diagnostic markers for dry eye syndrome (DES) are lacking in both sensitivity and specificity, a pressing concern exists to develop activity markers that closely align with the principal axes of disease progression. In this study, a comprehensive lipidomic platform designated for analysis of the human tear lipidome was employed to characterize changes in tear lipid compositions from a cohort of 93 subjects of different clinical subgroups classified based on the presence of dry eye symptoms and signs. Positive correlations were observed between the tear levels of cholesteryl sulfates and glycosphingolipids with physiological secretion of tears, which indicated the possible lacrimal (instead of meibomian) origin of these lipids. Notably, we found wax esters of low molecular masses and those containing saturated fatty acyl moieties were specifically reduced with disease and significantly correlated with various DES clinical parameters such as ocular surface disease index, tear breakup time, and Schirmer''s I test (i.e., both symptoms and signs). These structure-specific changes in tear components with DES could potentially serve as unifying indicators of disease symptoms and signs. In addition, the structurally-specific aberrations in tear lipids reported here were found in patients with or without aqueous deficiency, suggesting a common pathology for both DES subtypes.     

Assembly and stability of Salmonella enterica ser. Typhi TolC protein in POPE and DMPE

In this work we assessed the suitability of two different lipid membranes for the simulation of a TolC protein from Salmonella enterica serovar Typhi. The TolC protein family is found in many pathogenic Gram-negative bacteria including Vibrio cholera and Pseudomonas aeruginosa and acts as an outer membrane channel for expulsion of drug and toxin from the cell. In S. typhi, the causative agent for typhoid fever, the TolC outer membrane protein is an antigen for the pathogen. The lipid environment is an important modulator of membrane protein structure and function. We evaluated the conformation of the TolC protein in the presence of DMPE and POPE bilayers using molecular dynamics simulation. The S. typhi TolC protein exhibited similar conformational dynamics to TolC and its homologues. Conformational flexibility of the protein is seen in the C-terminal, extracellular loops, and α-helical region. Despite differences in the two lipids, significant similarities in the motion of the protein in POPE and DMPE were observed, including the rotational motion of the C-terminal residues and the partially open extracellular loops. However, analysis of the trajectories demonstrated effects of hydrophobic matching of the TolC protein in the membrane, particularly in the lengthening of the lipids and subtle movements of the protein’s β-barrel towards the lower leaflet in DMPE. The study exhibited the use of molecular dynamics simulation in revealing the differential effect of membrane proteins and lipids on each other. In this study, POPE is potentially a more suitable model for future simulation of the S. typhi TolC protein.