Rate of disappearance of isethionic acid in the rat central nervous system

The rates of disappearance of tritiated isethionic acid (2-hydroxyethanesulfonic acid) in eight regions of the rat central nervous system were studied. By utilizing the technique of graphical analysis (curve peeling), it was determined that seven areas (striatum, diencephalon, pons-medulla, midbrain, hippocampus, spinal cord, and cortex) exhibited triphasic (fast, intermediate, and slow) disappearance rates while only the cerebellum displayed a biphasic (fast, slow) rate. The half-lives for the fast component in the different regions of the central nervous system varied from 0.5 hr (midbrain and cortex) to 1.5 hr (diencephalon, cerebellum, and spinal cord); the half-lives for the intermediate component of the triphasic rate varied from 3.5 hr in the midbrain and spinal cord to 5.5 hr in the hippocampus. Half-lives estimated for the slow component of the multiphasic rate of disappearance of tritiated isethionic acid ([³H]ISA) varied from 28 hr (cerebellum) to 90 hr (spinal cord).     

Isolation of monovalent sexual binding components from Chlamydomonas eugametos flagellar membranes

Several treatments were tested to extract the sexual binding site from membrane vesicles derived from the flagellar surface of Chlamydomonas eugametos. Extraction with detergents, chaotropic and hydrogen bond-disrupting agents, as well as sonication, was effective in reducing the isoagglutination activity of these membrane vesicles. Complementary with this reduction, a sex-specific biological activity related to isoagglutination, called twitch activity appeared in the extract. This was only observed with vesicles derived from minus mating type (mt^-) gametes. After fractionation of the extract, one high-molecular weight glycoprotein fraction appeared to be responsible for this activity. When extracts were treated with cross-linking agents, a pelletable fraction was obtained with isoagglutinative activity. We conclude that the mt^- factor, responsible for twitch activity, causes isoagglutination when it is rendered multivalent.     

Monoclonal antibodies to surface glycoconjugates in Chlamydomonas eugametos recognize strain-specific O-methyl sugars

Monoclonal antibodies are described that are directed against cell surface components of the unicellular green alga Chlamydomonas eugametos. These antibodies recognize strain-specific epitopes which occur at the surface of vegetative and gametic cells. Two different groups of epitopes are distinguished that are never detectable together in one clonal cell culture. Evidence is presented showing that the antigenicity of cell surface molecules is a consequence of the presence of particular O-methylated sugars. Monoclonal antibodies reacting with one group of epitopes were studied in more detail, and immunoprecipitation and Western-blot studies showed that these epitopes can be arranged into four classes. The use of these monoclonal antibodies as strain-specific markers in light- and electron-microscopical techniques is illustrated.Abbreviations ELISA enzyme-linked immunosorbent assay - mt ^+/- mating type plus or minus - PAS periodic acid Schiff - Mab monoclonal antibody - PBS phosphate-buffered saline     

Differential Effects of Collagen Prolyl 3-Hydroxylation on Skeletal Tissues

Mutations in the genes encoding cartilage associated protein (CRTAP) and prolyl 3-hydroxylase 1 (P3H1 encoded by LEPRE1) were the first identified causes of recessive Osteogenesis Imperfecta (OI). These proteins, together with cyclophilin B (encoded by PPIB), form a complex that 3-hydroxylates a single proline residue on the α1(I) chain (Pro986) and has cis/trans isomerase (PPIase) activity essential for proper collagen folding. Recent data suggest that prolyl 3-hydroxylation of Pro986 is not required for the structural stability of collagen; however, the absence of this post-translational modification may disrupt protein-protein interactions integral for proper collagen folding and lead to collagen over-modification. P3H1 and CRTAP stabilize each other and absence of one results in degradation of the other. Hence, hypomorphic or loss of function mutations of either gene cause loss of the whole complex and its associated functions. The relative contribution of losing this complex''s 3-hydroxylation versus PPIase and collagen chaperone activities to the phenotype of recessive OI is unknown. To distinguish between these functions, we generated knock-in mice carrying a single amino acid substitution in the catalytic site of P3h1 (Lepre1^H662A). This substitution abolished P3h1 activity but retained ability to form a complex with Crtap and thus the collagen chaperone function. Knock-in mice showed absence of prolyl 3-hydroxylation at Pro986 of the α1(I) and α1(II) collagen chains but no significant over-modification at other collagen residues. They were normal in appearance, had no growth defects and normal cartilage growth plate histology but showed decreased trabecular bone mass. This new mouse model recapitulates elements of the bone phenotype of OI but not the cartilage and growth phenotypes caused by loss of the prolyl 3-hydroxylation complex. Our observations suggest differential tissue consequences due to selective inactivation of P3H1 hydroxylase activity versus complete ablation of the prolyl 3-hydroxylation complex.     

Multilayer fluorescence optically encoded beads for protein detection

An easy preparation method of multilayer fluorescence optically encoded beads for protein detection is presented. The beads, which consist of multicolored layers, are made from amino polyethylene glycol grafted polystyrene (PS-g-PEG) beads by using several fluorescent dyes such as fluorescein isothiocyanate (FITC) and rhodamine via controlling diffusion of an Fmoc-protecting group after HCl solution swelling. A biotin, glutathione S-transferase (GST) antibody, and an RNA aptamer that specifically recognize streptavidin, GST antigen, and hepatitis C virus (HCV) helicase are introduced to the optically encoded beads and monitored for their binding activity to the target molecules. After binding, the ligands are identified easily by their color codes.     

Establishment of a genotyping scheme for Coxiella burnetii

Coxiella burnetii is the causative agent of Q fever. The bacterium is highly infectious and is classified as a category B biological weapon. The tools of molecular biology are of utmost importance in a rapid and unambiguous identification of C. burnetii in naturally occurring Q fever outbreaks, or in cases of a deliberate release of the infectious agent. In this work, development of a multiple locus variable number tandem repeats (VNTR) analysis (MLVA) for the characterization of C. burnetii is described. Sixteen C. burnetii isolates and five passage history/laboratory variants were characterized. The VNTR markers revealed many polymorphisms resulting in nine unique MLVA types that cluster into five different clusters. This proves that the MLVA system is highly discriminatory. The selected VNTR markers were stable. The MLVA method developed in this report is a promising tool for the characterization of C. burnetii isolates and their epidemiological study.     

Apolipoprotein A-II-mediated conformational changes of apolipoprotein A-I in discoidal high density lipoproteins

It is well accepted that HDL has the ability to reduce risks for several chronic diseases. To gain insights into the functional properties of HDL, it is critical to understand the HDL structure in detail. To understand interactions between the two major apolipoproteins (apos), apoA-I and apoA-II in HDL, we generated highly defined benchmark discoidal HDL particles. These particles were reconstituted using a physiologically relevant phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) incorporating two molecules of apoA-I and one homodimer of apoA-II per particle. We utilized two independent mass spectrometry techniques to study these particles. The techniques are both sensitive to protein conformation and interactions and are namely: 1) hydrogen deuterium exchange combined with mass spectrometry and 2) partial acetylation of lysine residues combined with MS. Comparison of mixed particles with apoA-I only particles of similar diameter revealed that the changes in apoA-I conformation in the presence of apoA-II are confined to apoA-I helices 3-4 and 7-9. We discuss these findings with respect to the relative reactivity of these two particle types toward a major plasma enzyme, lecithin:cholesterol acyltransferase responsible for the HDL maturation process.     

Identification of novel inhibitors for a low molecular weight protein tyrosine phosphatase via virtual screening

The human cytoplasmic protein tyrosine phosphatase (HCPTP) has been identified as a potential target for inhibition in order to downregulate metastatic transformation in several human epithelial cancers such as breast, prostate and colon cancer. Docking with two scoring functions on both isoforms of HCPTP was employed as an initial virtual screen to identify potential inhibitors. Compounds identified as potential inhibitors via this in silico screen were subjected to kinetic analysis in order to validate their selection as improved inhibitors. Eleven compounds with IC₅₀’s of less than 100 μM were identified in a single concentration screen. Five of these compounds were determined to have an IC₅₀ of less than 10 μM; however, all but one of these compounds inhibited via non-specific aggregation. The validated effective inhibitor, which is based on a naphthyl sulfonic acid, strongly resembles a previously synthesized rationally designed azaindole phosphonic acid. This similarity suggests subsequent inhibitor optimization based on this scaffold may generate effective inhibitors of HCPTP. The structural elements of the computationally identified inhibitors are discussed to analyze the combined use of rational design and virtual screening to reduce false negatives in the identification of multiple strong inhibitors of HCPTP.