Structure and function study of prostaglandin H synthase

Prostaglandin H synthase (PGHS) is a key enzyme in biosynthesis of prostaglandins, thromboxane, and prostacyclin. It has two activities, cyclooxygenase and peroxidase. "PGHS" means PGHS-1. A current hypothesis considers the cyclooxygenase reaction to be a free radical chain reaction, initiated by interaction of the synthase peroxidase with hydroperoxides leading to the production of a tyrosyl free radical. According to this hypothesis, tyrosyl residue(s) may play a key role in the cyclooxygenase reaction. Tetranitromethane (TNM) can relatively selectively nitrate tyrosines at pH 8.0. The effect of TNM on both cyclooxygenase activity and peroxidase activity has been examined: reaction of the synthase holoenzyme with TNM at pH 8.0 led to inactivation of both activities, with the cyclooxygenase activity being lost rapidly and completely, while the peroxidase activity was lost more slowly. Indomethacin, a non-steroidal anti-inflammatory agent, can protect the synthase from the inactivation of TNM. Amino acid analyses indicated that a loss of tyrosine and formation of nitrotyrosine residues occurred during reaction with TNM, and that TNM-reacted holoenzyme with $<$10% residual cyclooxygenase activity had about 2.0 nitrotyrosine/subunit.^ PGH synthase is known to be an endoplasmic reticulum membrane-associated protein. Antibodies directed at particular PGHS peptide segments and indirect immunofluorescence have been used to characterize the membrane topology of crucial portions of PGHS. PGHS was expressed in COS-1 cells transfected with the appropriate cDNA. Stably-transfected human endothelial cells were also used for the topology study. The cells were treated with streptolysin-O, which selectively permeabilizes the plasma membrane, or with saponin to achieve general membrane disruption, before incubation with the antipeptide antibodies. Bound antipeptide antibody was stained by FITC-labelled secondary antibody and visualized by fluorescence microscopy. With the antipeptide antibodies against residues 51-66, 156-170 or 377-390, there was a significant reticular and perinuclear pattern of staining in cells permeabilized with saponin but not in cells permeabilized with SLO alone. Antibodies directed against the endogenous C-terminal peptide or against residues 271-284 produced staining in cells permeabilized with saponin, and also in a lower, but significant fraction of cells permeabilized with SLO. Similar results were obtained when COS-1 cells expressing recombinant PGHS with a viral reporter peptide inserted at the C-terminus were stained with antibody against the reporter epitope.^ The PGHS C-terminal sequence is similar to that of the consensus KDEL ER retention signal. The potential function of the PGHS C-terminus segment in ER retention was examined by mutating this segment and analyzing the subcellular distribution of the mutants expressed in COS-1 cells. None of the mutants had an altered subcellular distribution, although some had greatly diminished the enzyme activities. (Abstract shortened by UMI.) ^

Structure-function analyses of {\it PAX6\/} and the molecular bases of aniridia

PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. I dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. In both systems, the results show consistently that all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act cooperatively to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6 even though the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. I conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree, and that its full transactivation activity relies on the cooperative action of the four exon fragments.^ Most PAX6 mutations detected in patients with aniridia result in truncations of the protein. Some of the truncation mutations occur in the PST region of PAX6, resulting in mutant proteins that retain their DNA-binding ability but have no significant transactivation activity. It is not clear whether such mutants are true loss-of-function or dominant-negative mutants. I show that these mutants are dominant-negative if they are coexpressed with wild-type PAX6 in cultured cells and that the dominant-negative effects result from enhanced DNA-binding ability of these mutants due to removal of the PST domain. These mutants are able to repress the wild-type PAX6 activity not only at target genes with paired domain binding sites but also at target genes with homeodomain binding sites.^ Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis, and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, I performed a functional analysis of two missense mutations in the paired domain: the R26G mutation reported in a case of Peters' anomaly, and the I87R mutation identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. I found that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. My data support the haploinsufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele. ^

Nebulizing poractant alfa versus conventional instillation: Ultrastructural appearance and preservation of surface activity.

BACKGROUND Nebulized surfactant therapy has been proposed as an alternative method of surfactant administration. The use of a perforated vibrating membrane nebulizer provides a variety of advantages over conventional nebulizers. We investigated the molecular structure and integrity of poractant alfa pre- and post-nebulization. METHOD Curosurf® was nebulized using an Investigational eFlow® Nebulizer System. Non-nebulized surfactant ("NN"), recollected surfactant droplets from nebulization through an endotracheal tube ("NT") and nebulization of surfactant directly onto a surface ("ND") were investigated by transmission electron microscopy. Biophysical characteristics were assessed by the Langmuir-Wilhelmy balance and the Captive Bubble Surfactometer. RESULTS Volume densities of lamellar body-like forms (LBL) and multi-lamellar forms (ML) were high for "NN" and "NT" samples (38.8% vs. 47.7% for LBL and 58.2% vs. 47.8% for ML). In the "ND" sample, we found virtually no LBL's, ML's (72.6%) as well as uni-lamellar forms (16.4%) and a new structure, the "garland-like" forms (9.4%). Surface tension for "NN" and "NT" was 23.33 ± 0.29 and 25.77 ± 1.12 mN/m, respectively. Dynamic compression-expansion cycling minimum surface tensions were between 0.91 and 1.77 mN/m. CONCLUSION The similarity of surfactant characteristics of nebulized surfactant via a tube and the non-nebulized surfactant suggests that vibrating membrane nebulizers are suitable for surfactant nebulization. Alterations in surfactant morphology and characteristics after nebulization were transient. A new structural subtype of surfactant was identified. Pediatr Pulmonol. 2014; 49:348-356. © 2013 Wiley Periodicals, Inc.

Capture of activity-induced ultrastructural changes at synapses by high-pressure freezing of brain tissue.

Electron microscopy (EM) allows for the simultaneous visualization of all tissue components at high resolution. However, the extent to which conventional aldehyde fixation and ethanol dehydration of the tissue alter the fine structure of cells and organelles, thereby preventing detection of subtle structural changes induced by an experiment, has remained an issue. Attempts have been made to rapidly freeze tissue to preserve native ultrastructure. Shock-freezing of living tissue under high pressure (high-pressure freezing, HPF) followed by cryosubstitution of the tissue water avoids aldehyde fixation and dehydration in ethanol; the tissue water is immobilized in ∼50 ms, and a close-to-native fine structure of cells, organelles and molecules is preserved. Here we describe a protocol for HPF that is useful to monitor ultrastructural changes associated with functional changes at synapses in the brain but can be applied to many other tissues as well. The procedure requires a high-pressure freezer and takes a minimum of 7 d but can be paused at several points.

Chromatin structure and DNase I hypersensitivity in the transcriptionally active and inactive porcine tumor necrosis factor gene locus

We have analyzed the chromatin structure of the porcine tumor necrosis factor gene locus (TNF-alpha and TNF-beta). Nuclei from porcine peripheral blood mononuclear cells were digested with different nucleases. As assessed with micrococcal nuclease, the two TNF genes displayed slightly faster digestion kinetics than bulk DNA. Studies with DNaseI revealed distinct DNaseI hypersensitive sites (DH-sites) within the porcine TNF locus. Four DH-sites could be observed in the promoter and mRNA leader regions of the TNF-beta gene. Two DH-sites could be observed for the TNF-alpha gene, one located in the promoter region close to the TATA-box and the other site in intron 3. This pattern of DH-sites was present independently of the activation state of the cells. Interestingly in a porcine macrophage-like cell line, we found that the TNF-alpha promoter DH-site disappeared and another DH-site appeared in the region of intron 1. Additionally, the DH-site of intron 3 could be enhanced by PMA-stimulation in these cells. TNF-beta sites were not detected in this cell line. However, DH-sites were totally absent in fibroblasts (freshly isolated from testicles) and in porcine kidney cells (PK15 cell line) both of which do not transcribe the TNF genes. Therefore, the pattern of DH-sites corresponds to the transcriptional activity of analyzed cells.

Virus-like particle-mediated intracellular delivery of mRNA cap analog with in vivo activity against hepatocellular carcinoma.

UNLABELLED Adenovirus dodecahedron (Dd), a nanoparticulate proteinaceous biodegradable virus-like particle (VLP), was used as a vector for delivery of an oncogene inhibitor to hepatocellular carcinoma (HCC) rat orthotopic model. Initiation factor eIF4E is an oncogene with elevated expression in human cancers. Cell-impermeant eIF4E inhibitor, cap structure analog (cap) and anti-cancer antibiotic doxorubicin (Dox) were delivered as Dd conjugates. Dd-cap and Dd-dox inhibited cancer cell culture proliferation up to 50 and 84%, respectively, while with free Dox similar results could be obtained only at a 5 times higher concentration. In animal HCC model the combination treatment of Dd-cap/Dd-dox caused 40% inhibition of tumor growth. Importantly, the level of two pro-oncogenes, eIF4E and c-myc, was significantly diminished in tumor sections of treated rats. Attachment to Dd, a virus-like particle, permitted the first demonstration of cap analog intracellular delivery and resulted in improved doxorubicin delivery leading to statistically significant inhibition of HCC tumor growth. FROM THE CLINICAL EDITOR Adenovirus dodecahedron, a nanoparticulate proteinaceous biodegradable virus-like particle was used in this study as a vector for the concomitant delivery of cap structure analog and doxorubicine to hepatocellular carcinoma in a rat model, resulting in significant inhibition of tumor growth.

Altered structure of cortical sulci in gilles de la Tourette syndrome: Further support for abnormal brain development.

Gilles de la Tourette syndrome is a neurodevelopmental disorder characterized by the presence of motor and vocal tics. We hypothesized that patients with this syndrome would present an aberrant pattern of cortical formation, which could potentially reflect global alterations of brain development. Using 3 Tesla structural neuroimaging, we compared sulcal depth, opening, and length and thickness of sulcal gray matter in 52 adult patients and 52 matched controls. Cortical sulci were automatically reconstructed and identified over the whole brain, using BrainVisa software. We focused on frontal, parietal, and temporal cortical regions, in which abnormal structure and functional activity were identified in previous neuroimaging studies. Partial correlation analysis with age, sex, and treatment as covariables of noninterest was performed amongst relevant clinical and neuroimaging variables in patients. Patients with Gilles de la Tourette syndrome showed lower depth and reduced thickness of gray matter in the pre- and post-central as well as superior, inferior, and internal frontal sulci. In patients with associated obsessive-compulsive disorder, additional structural changes were found in temporal, insular, and olfactory sulci. Crucially, severity of tics and of obsessive-compulsive disorder measured by Yale Global Tic severity scale and Yale-Brown Obsessive-Compulsive scale, respectively, correlated with structural sulcal changes in sensorimotor, temporal, dorsolateral prefrontal, and middle cingulate cortical areas. Patients with Gilles de la Tourette syndrome displayed an abnormal structural pattern of cortical sulci, which correlated with severity of clinical symptoms. Our results provide further evidence of abnormal brain development in GTS. © 2015 International Parkinson and Movement Disorder Society.

Chiral Templating Activity of Tris(bipyridine)ruthenium(II) Cation in the Design of Three-Dimensional (3D) Optically Active Oxalate-Bridged {[Ru(bpy) 3 ][Cu 2 x Ni 2(1 - x ) (C 2 O 4 ) 3 ]} n (0 ≤ x ≤ 1; bpy = 2,2‘-bipyridine): Structural, Optical, and Magnetic Studies

Three-dimensional oxalate-based {[Ru(bpy)3][Cu2xNi2(1-x)(ox)3]}n (0≤ x ≤ 1, ox = C2O42-, bpy = 2,2‘bipyridine) were synthesized. The structure was determined for x = 1 by X-ray diffraction on single crystal. The compound crystallizes in the cubic space group P4132. It shows a three-dimensional 10-gon 3-connected (10,3) anionic network where copper(II) has an unusual tris(bischelated) environment. X-ray powder diffraction patterns and their Rietveld refinement show that all the compounds along the series are isostructural and single-phased. According to X-ray absorption spectroscopy, copper(II) and nickel(II) have an octahedral environment, respectively elongated and trigonally distorted. As shown by natural circular dichroism, the optically active forms of {[Ru(bpy)3][CuxNi2(1-x)(ox)3]}n are obtained starting from resolved Δ- or Λ-[Ru(bpy)3]2+. The Curie−Weiss temperatures range between −55 (x = 1) and −150 K (x = 0). The antiferromagnetic exchange interaction thus decreases when the copper contents increases in agreement with the crystallographic structure of the compounds and the electronic structure of the metal ions. At low temperature, the compounds exhibit complex long-range ordered magnetic behavior.

KLIKK proteases of Tannerella forsythia: putative virulence factors with a unique domain structure.

Comparative genomics of virulent Tannerella forsythia ATCC 43037 and a close health-associated relative, Tannerella BU063, revealed, in the latter, the absence of an entire array of genes encoding putative secretory proteases that possess a nearly identical C-terminal domain (CTD) that ends with a -Lys-Leu-Ile-Lys-Lys motif. This observation suggests that these proteins, referred to as KLIKK proteases, may function as virulence factors. Re-sequencing of the loci of the KLIKK proteases found only six genes grouped in two clusters. All six genes were expressed by T. forsythia in routine culture conditions, although at different levels. More importantly, a transcript of each gene was detected in gingival crevicular fluid (GCF) from periodontitis sites infected with T. forsythia indicating that the proteases are expressed in vivo. In each protein, a protease domain was flanked by a unique N-terminal profragment and a C-terminal extension ending with the CTD. Partially purified recombinant proteases showed variable levels of proteolytic activity in zymography gels and toward protein substrates, including collagen, gelatin, elastin, and casein. Taken together, these results indicate that the pathogenic strain of T. forsythia secretes active proteases capable of degrading an array of host proteins, which likely represents an important pathogenic feature of this bacterium.

Myelination of the right parahippocampal cingulum is associated with physical activity in young healthy adults.

Recent evidence suggests that individual differences in physical activity (PA) may be associated with individual differences in white matter microstructure and with grey matter volume of the hippocampus. Therefore, this study investigated the association between PA and white matter microstructure of pathways connecting to the hippocampus. A total of 33 young, healthy adults underwent magnetic resonance imaging (MRI). High angular resolution diffusion-weighted imaging and multi-component relaxometry MRI scans (multi-component driven equilibrium pulse observation of T1 and T2) were acquired for each participant. Activity levels (AL) of participants were calculated from 72-h actigraphy recordings. Tractography using the damped Richardson Lucy algorithm was used to reconstruct the fornix and bilateral parahippocampal cinguli (PHC). The mean fractional anisotropy (FA) and the myelin water fraction (MWF), a putative marker of myelination, were determined for each pathway. A positive correlation between both AL and FA and between AL and MWF were hypothesized for the three pathways. There was a selective positive correlation between AL and MWF in the right PHC (r = 0.482, p = 0.007). Thus, our results provide initial in vivo evidence for an association between myelination of the right PHC and PA in young healthy adults. Our results suggest that MWF may not only be more specific, but also more sensitive than FA to detect white matter microstructural alterations. If PA was to induce structural plasticity of the right PHC this may contribute to reverse structural alterations of the right PHC in neuropsychiatric disorder with hippocampal pathologies.

CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^

Purification and cloning of the novel phosphoprotein copine III and characterization of its associated kinase activity

The copines, named and first described by Creutz et al. (1998), comprise a two C2 domain-containing protein family that can aggregate phosphatidylserine membranes in a calcium-dependent manner. Although no enzymatic function has been attributed to copines, their carboxyl terminus shows homology to the A domain found in integrins that allows binding of magnesium ions. The secondary structure of A domains resembles a Rossmann fold, which can bind dinucleotides and is present in a number of intracellular enzymes. Due to a crossreacting activity of Mik b 1, an antibody to the IL-2R b chain, we were able to serendipitously clone human copine III (CIII). CIII is 65% identical to copine I (CI) and the 5 kb CIII transcript is expressed ubiquitously as determined by a multitissue Northern blot. A polyclonal antibody generated against the carboxyl terminus of CIII recognized CIII in immunoblots and immunoprecipitations. Phosphorylation of CIII was observed on serine and threonine residues, as determined by phosphoamino acid analysis. ^ Experiments were designed to determine whether or not any enzymatic activity, specifically kinase activity, was intrinsic to or associated with CIII. In vitro and in gel kinase assays were performed using transfected HA-tagged CI and CIII, immunoprecipitated endogenous CIII and purified endogenous CIII. The exogenous substrate MBP was phosphorylated in all in vitro kinase assays containing CIII protein purification and column chromatography expertise with me. ^

The characterization of mouse carboxypeptidase N small subunit gene structure and presence in developing embryos

Carboxypeptidase N (CPN) is a plasma zinc metalloprotease, which consists of two enzymatically active small subunits and two large subunits that protect the protein from degradation. CPN cleaves carboxy-terminal arginines and lysines from peptides found in the bloodstream such as complement anaphylatoxins, kinins, and creatine kinase MM. In this study, the mouse CPN small subunit (CPN1) coding region, gene structure, and chromosomal location were characterized and the expression of CPN1 was investigated in mouse embryos at different stages of development. The CPN1 gene, which was approximately 29 kb in length, contained nine exons and localized to mouse chromosome 19D2. The fifth and sixth exons of CPN1 encoded the amino acids necessary for substrate binding and catalytic activity. CPN1 RNA was expressed predominately in adult liver and contained a 1371 bp open reading frame encoding 457 amino acids. In the mouse embryo, CPN1 RNA was observed at 8.5 days post coitus (dpc), while its protein was detected at 10.5 dpc. In situ hybridization of the fetal liver detected CPN1 RNA in erythroid progenitor cells at 10.5, 13.5, and 16.5 dpc and in hepatocytes at 16.5 dpc. This was compared to the expression of the complement component C3, the parent molecule of complement anaphylatoxin C3a. Consistently throughout the experiments, CPN1 message and protein preceded the expression of C3. To obtain a better understanding of the biological significance of CPN1 in vivo, studies were initiated to produce a genetically engineered mouse in which the CPN1 gene was ablated. To facilitate this project a targeting vector was constructed by removing the functionally important fifth and sixth exons of the CPN1 gene. Collectively, these studies have: (1) provided important detailed information regarding the structure and organization of the murine CPN1 gene, (2) yielded insights into the developmental expression of mouse CPN1 in relationship to C3 expression, and (3) set the stage for the generation of a CPN1 “knock-out” mouse, which can be used to determine the biological significance of CPN1 in both normal and diseased conditions. ^

Aligning Library Strategy and Structure With the Campus Academic Plan: A Case Study

Colleges and universities’ missions are typically comprised of educating students, training professionals, engaging in scholarship and research, promoting creative activity, improving healthcare, and providing public service. Academic libraries exist to support these core functions, yet most academic libraries are organized based on library functions rather than the primary missions of their college or university. This paper describes one academic library’s attempt to align library strategy and structure with its university’s academic plan.

Identfying predictors of St. Louis encephalitis activity in Houston Texas

Generalized linear Poisson and logistic regression models were utilized to examine the relationship between temperature and precipitation and cases of Saint Louis encephalitis virus spread in the Houston metropolitan area. The models were investigated with and without repeated measures, with a first order autoregressive (AR1) correlation structure used for the repeated measures model. The two types of Poisson regression models, with and without correlation structure, showed that a unit increase in temperature measured in degrees Fahrenheit increases the occurrence of the virus 1.7 times and a unit increase in precipitation measured in inches increases the occurrence of the virus 1.5 times. Logistic regression did not show these covariates to be significant as predictors for encephalitis activity in Houston for either correlation structure. This discrepancy for the logistic model could be attributed to the small data set.^ Keywords: Saint Louis Encephalitis; Generalized Linear Model; Poisson; Logistic; First Order Autoregressive; Temperature; Precipitation. ^