Transcription of Human Resistin Gene Involves an Interaction of Sp1 with Peroxisome Proliferator-Activating Receptor Gamma (PPAR gamma)

Background: Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.Methodology/Principal Finding: We show that the minimal promoter of human resistin lies within similar to 80 bp sequence upstream of the transcriptional start site (-240) whereas binding sites for cRel, CCAAT enhancer binding protein alpha (C/EBP-alpha), activating transcription factor 2 (ATF-2) and activator protein 1 (AP-1) transcription factors, important for induced expression, are present within sequences up to -619. Specificity Protein 1(Sp1) binding site (-276 to -295) is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPAR gamma) is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPAR gamma in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA) upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPAR gamma on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPAR gamma interaction. Chromatin immunoprecipitation (ChIP) assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPAR gamma, chromatin modifier histone deacetylase 1 (HDAC1) and the acetylated form of histone H3 but not cRel, C/EBP-alpha and phospho c-Jun during resistingene transcription.Conclusion: Our findings suggest a complex interplay of Sp1 and PPAR gamma along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.

Appendiceal sonography in children: A retrospective analysis as a platform for potential implementation of diagnostic categories

- Background Sonography is an important diagnostic tool in children with suspected appendicitis. Reported accuracy and appendiceal visualisation rates vary significantly, as does the management of equivocal ultrasound findings. The aim of this study was to audit appendiceal sonography at a tertiary children's hospital, and provide baseline data for a future prospective study. - Summary of work Records of children who underwent ultrasound studies for possible appendicitis between January 2008 and December 2010 were reviewed. Variables included patient demographics, sonographic appendix characteristics, and secondary signs. Descriptive statistics and analysis using ANOVA, Mann-Whitney U test, and ROC curves were performed. Mater Human Research Ethic Committee approval was granted. - Summary of results There were 457 eligible children. Using a dichotomous diagnostic model (including equivocal results), sensitivity was 89.6%, specificity 91.6%, and diagnostic yield of 40.7%. ROC curve analysis of a 6mm diameter cut-off was 0.88 AUC (95% CI 0.80 to 0.95). - Discussion and conclusions Sonography is an accurate test for acute appendicitis in children, with a high sensitivity and negative predictive value. A diameter of 6mm as an absolute cut-off in a binary model can lead to false findings. Results were compared with available literature. Recent publications propose categorising diameter1 and integrating secondary signs2 to improve accuracy and provide more meaningful results to clinicians. This study will be a benchmark for future studies with multiple diagnostic categorisation.

Salt in the desert: A comparison of clinical versus serum-calculated osmolality determination of dehydration

Background: Clinicians frequently use their own judgement to assess patient’s hydration status although there is limited evidence for the diagnostic utility of any individual clinical symptom. Hence, the aim of this study was to compare the diagnostic accuracy of clinically assessed dehydration in older hospital patients (using multiple symptoms), versus dehydration measured using serum-calculated osmolality (CO) as the reference standard. Method: Participants were 44 hospital patients aged ≥ 60 years. Dehydration was assessed clinically and pathologically (CO) within 24 hours of admission and at study exit. Indicators of diagnostic accuracy were calculated. Results: Clinicians identified 27% of patients as dehydrated at admission, and 19% at exit, compared to 19% and 16% using CO. Agreement between the measures was fair at admission and poor at exit. Clinical assessment showed poor sensitivity for predicting dehydration with reasonable specificity. Conclusions: Compared to the use of CO, clinical assessment of dehydration in older patients was poor. Given that failure to identify dehydration in this population may have serious consequences, we recommend that clinicians do not rely upon their own assessments without also using the reference standard.

Novel peptides of therapeutic promise from Indian Conidae

Highly structured small peptides are the major toxic constituents of the venom of cone snails, a family of widely distributed predatory marine molluscs. These animals use the venom for rapid prey immobilization. The peptide components in the venom target a wide variety of membrane-bound ion channels and receptors. Many have been found to be highly selective for a diverse range of mammalian ion channels and receptors associated with pain-signaling pathways. Their small size, structural stability, and target specificity make them attractive pharmacologic agents. A select number of laboratories mainly from the United States, Europe, Australia, Israel, and China have been engaged in intense drug discovery programs based on peptides from a few snail species. Coastal India has an estimated 20-30% of the known cone species; however, few serious studies have been reported so far. We have begun a comprehensive program for the identification and characterization of peptides from cone snails found in Indian Coastal waters. This presentation reviews our progress over the last 2 years. As expected from the evolutionary history of these venom components, our search has yielded novel peptides of therapeutic promise from the new species that we have studied.

Analysis of the impact of a uracil DNA glycosylase attenuated in AP-DNA binding in maintenance of the genomic integrity in Escherichia coli

Uracil DNA glycosylase (Ung)initiates the uracil excision repair pathway. We have earlier characterized the Y66W and Y66H mutants of Ung and shown that they are compromised by similar to 7- and similar to 170-fold, respectively in their uracil excision activities. In this study, fluorescence anisotropy measurements show that compared with the wild-type, the Y66W protein is moderately compromised and attenuated in binding to AP-DNA. Allelic exchange of ung in Escherichia coli with ung::kan, ungY66H:amp or ungY66W:amp alleles showed similar to 5-, similar to 3.0- and similar to 2.0-fold, respectively increase in mutation frequencies. Analysis of mutations in the rifampicin resistance determining region of rpoB revealed that the Y66W allele resulted in an increase in A to G (or T to C) mutations. However, the increase in A to G mutations was mitigated upon expression of wild-type Ung from a plasmid borne gene. Biochemical and computational analyses showed that the Y66W mutant maintains strict specificity for uracil excision from DNA. Interestingly, a strain deficient in AP-endonucleases also showed an increase in A to G mutations. We discuss these findings in the context of a proposal that the residency of DNA glycosylase(s) onto the AP-sites they generate shields them until recruitment of AP-endonucleases for further repair.

Exploring the interaction energies for the binding of hydroxydiphenyl ethers to enoyl-acyl carrier protein reductases

It is now well established that the potent anti-microbial compound, triclosan, interrupts the type II fatty acid synthesis by inhibiting the enzyme enoyl-ACP reductase in a number of organisms. Existence of a high degree of similarity between the recently discovered enoyl-ACP reductase from R falciparum and B. napus enzyme permitted building of a satisfactory model for the former enzyme that explained some of the key aspects of the enzyme such as its specificity for binding to the cofactor and the inhibitor. We now report the interaction energies between triclosan and other hydroxydiphenyl ethers with the enzymes from B. napus, E. coli and R falciparum. Examination of the triclosan-enzyme interactions revealed that subtle differences exist in the ligand binding sites of the enzymes from different sources i.e., B. napus, E. coli and P falciparum. A comparison of their binding propensities thus determined should aid in the design of effective inhibitors for the respective enzymes.

Kinetics and the Mechanism of Interaction of the Endoplasmic Reticulum Chaperone, Calreticulin, with Monoglucosylated (Glc1Man9GlcNAc2) Substrate

Calreticulin is a lectin-like molecular chaperone of the endoplasmic reticulum in eukaryotes. Its interaction with N-glycosylated polypeptides is mediated by the glycan, Glc(1)Man(9)GlcNAc(2), present on the target glycoproteins. In this work, binding of monoglucosyl IgG (chicken) substrate to calreticulin has been studied using real time association kinetics of the interaction with the biosensor based on surface plasmon resonance (SPR). By SPR, accurate association and dissociation rate constants were determined, and these yielded a micromolar association constant. The nature of reaction was unaffected by immobilization of either of the reactants. The Scatchard analysis values for K-a agreed web crith the one obtained by the ratio k(1)/k(-1). The interaction was completely inhibited by free oligosaccharide, Glc(1)Man(9)GlcNAc(2), whereas Man(9)GlcNAc(2) did not bind to the calreticulin-substrate complex, attesting to the exquisite specificity of this interaction. The binding of calreticulin to IgG was used for the development of immunoassay and the relative affinity of the lectin-substrate association was indirectly measured. The values are in agreement with those obtained with SPR. Although the reactions are several orders of magnitude slower than the diffusion controlled processes, the data are qualitatively and quantitatively consistent with single-step bimolecular association and dissociation reaction. Analyses of the activation parameters indicate that reaction is enthalpically driven and does not involve a highly ordered transition state. Based on these data, the mechanism of its chaperone activity is briefly discussed.

Microcalorimetric indications for ligand binding as a function of the protein for galactoside-specific plant and avian lectins

The process cascade leading to the final accommodation of the carbohydrate ligand in the lectin’s binding site comprises enthalpic and entropic contributions of the binding partners and solvent molecules. With emphasis on lactose, N-acetyllactosamine, and thiodigalactoside as potent inhibitors of binding of galactoside-specific lectins, the question was addressed to what extent these parameters are affected as a function of the protein. The microcalorimetric study of carbohydrate association to the galectin from chicken liver (CG-16) and the agglutinin from Viscum album (VAA) revealed enthalpy–entropy compensation with evident protein type-dependent changes for N-acetyllactosamine. Reduction of the entropic penalty by differential flexibility of loops or side chains and/or solvation properties of the protein will have to be reckoned with to assign a molecular cause to protein type-dependent changes in thermodynamic parameters for lectins sharing the same monosaccharide specificity.

Cloning by genomic PCR and production of peanut agglutinin in Escherichia coli

Using the polymerase chain reaction, the coding sequence for peanut agglutinin (PNA) was cloned and expressed in Escherichia coli. Amplified PNA is identical to previously reported cDNA, suggesting the absence of any introns in PNA gene. Recombinant (re-) PNA forms inclusion bodies in E. coli. Production of PNA was confirmed by probing Western blots with polyclonal anti-PNA immunoglobulin G. Inclusion bodies were solubilized with 6 M guanidine-HCl and renatured by rapid dilution in the presence of metal ions. The renatured lectin was then purified by affinity chromatography. The re-lectin shows carbohydrate-binding properties similar to the natural PNA. This expression system provides a model for future mutagenesis studies of the carbohydrate-binding site and thus facilitates ongoing efforts to explore the molecular basis for the specificity of lectin-carbohydrate interaction.

Thermodynamics of monosaccharide binding to concanavalin A, pea (Pisum sativum) lectin, and lentil (Lens culinaris) lectin

Titration calorimetry measurements of the binding of methyl alpha-D-mannopyranoside (Me alpha Man), D-mannopyranoside (Man), methyl alpha-D-glucopyranoside (Me alpha Glu), and D-glucopyranoside (Glu) to concanavalin A (Con A), pea lectin, and lentil lectin were performed at 281 and 292 K in 0.01 M dimethylglutaric acid-NaOH buffer (pH 6.9) containing 0.15 M NaCl and Mn+2 and Ca+2 ions. The site binding enthalpies, delta H, are the same at both temperatures and range from -28.4 +/- 0.9 (Me alpha Man) to -16.6 +/- 0.5 kJ mol-1 (Glu) for Con A, from -26.2 +/- 1.1 (Me alpha Man) to -12.8 +/- 0.4 kJ mol-1 (Me alpha Glu) for pea lectin, and from -16.6 +/- 0.7 (Me alpha Man) to -8.0 +/- 0.2 kJ mol-1 (Me alpha Glu) for lentil lectin. The site binding constants range from 17 +/- 1 x 10(3) M-1 (Me alpha Man to Con A at 281.2 K) to 230 +/- 20 M-1 (Glu to lentil lectin at 292.6 K) and exhibit high specificity for Con A where they are in the Me alpha Man:Man:Me alpha Glu:Glu ratio of 21:4:5:1, while the corresponding ratio is 5:2:1.5:1 for pea lectin and 4:2:2:1 for lentil lectin. The higher specificity for Con A indicates more interactions between the amino acid residues at the binding site and the carbohydrate ligand than for the pea and lentil lectin-carbohydrate complexes. The carbohydrate-lectin binding results exhibit enthalpy-entropy compensation in that delta Hb (kJ mol-1) = -1.67 +/- 0.06 x 10(4) + (1.30 +/- 0.12)T(K) delta Sb (J mol-1K-1). Differential scanning calorimetry measurements on the thermal denaturation of the lectins and their carbohydrate complexes show that the Con A tetramer dissociates into monomers, while the pea and lentil lectin dimers dissociate into two submonomer fragments. At the denaturation temperature, one carbohydrate binds to each monomer of Con A and the pea and lentil lectins. Complexation with the carbohydrate increases the denaturation temperature of the lectin and the magnitude of the increases yield binding constants in agreement with the determinations from titration calorimetry.

Topography of the combining region of a Thomsen-Friedenreich-antigen-specific lectin jacalin (Artocarpus integrifolia agglutinin). A thermodynamic and circular-dichroism spectroscopic study

Thermodynamic analysis of carbohydrate binding by Artocarpus integrifolia (jackfruit) agglutinin (jacalin) shows that, among monosaccharides, Me alpha GalNAc (methyl-alpha-N-acetylgalactosamine) is the strongest binding ligand. Despite its strong affinity for Me alpha GalNAc and Me alpha Gal, the lectin binds very poorly when Gal and GalNAc are in alpha-linkage with other sugars such as in A- and B-blood-group trisaccharides, Gal alpha 1-3Gal and Gal alpha 1-4Gal. These binding properties are explained by considering the thermodynamic parameters in conjunction with the minimum energy conformations of these sugars. It binds to Gal beta 1-3GalNAc alpha Me with 2800-fold stronger affinity over Gal beta 1-3GalNAc beta Me. It does not bind to asialo-GM1 (monosialoganglioside) oligosaccharide. Moreover, it binds to Gal beta 1-3GalNAc alpha Ser, the authentic T (Thomsen-Friedenreich)-antigen, with about 2.5-fold greater affinity as compared with Gal beta 1-3GalNAc. Asialoglycophorin A was found to be about 169,333 times stronger an inhibitor than Gal beta 1-3GalNAc. The present study thus reveals the exquisite specificity of A. integrifolia lectin for the T-antigen. Appreciable binding of disaccharides Glc beta 1-3GalNAc and GlcNAc beta 1-3Gal and the very poor binding of beta-linked disaccharides, which instead of Gal and GalNAc contain other sugars at the reducing end, underscore the important contribution made by Gal and GalNAc at the reducing end for recognition by the lectin. The ligand-structure-dependent alterations of the c.d. spectrum in the tertiary structural region of the protein allows the placement of various sugar units in the combining region of the lectin. These studies suggest that the primary subsite (subsite A) can accommodate only Gal or GalNAc or alpha-linked Gal or GalNAc, whereas the secondary subsite (subsite B) can associate either with GalNAc beta Me or Gal beta Me. Considering these factors a likely arrangement for various disaccharides in the binding site of the lectin is proposed. Its exquisite specificity for the authentic T-antigen, Gal beta 1-3GalNAc alpha Ser, together with its virtual non-binding to A- and B-blood-group antigens, Gal beta 1-3GalNAc beta Me and asialo-GM1 should make A. integrifolia lectin a valuable probe for monitoring the expression of T-antigen on cell surfaces.

Erythrocyte-binding studies on an acidic lectin from winged bean (Psophocarpus tetragonolobus)

n acidic lectin (WBA II) was isolated to homogeneity from the crude seed extract of the winged bean (Psophocarpus tetragonolobus) by affinity chromatography on lactosylaminoethyl-Bio-Gel. Binding of WBA II to human erythrocytes of type-A, -B and -O blood groups showed the presence of 10(5) receptors/cell, with high association constants (10(6)-10(8) M-1). Competitive binding studies with blood-group-specific lectins reveal that WBA II binds to H- and T-antigenic determinants on human erythrocytes. Affinity-chromatographic studies using A-, B-, H- and T-antigenic determinants coupled to an insoluble matrix confirm the specificity of WBA II towards H- and T-antigenic determinants. Inhibition of the binding of WBA II by various sugars show that N-acetylgalactosamine and T-antigenic disaccharide (Thomsen-Friedenreich antigen, Gal beta 1-3GalNAc) are the most potent mono- and di-saccharide inhibitors respectively. In addition, inhibition of the binding of WBA II to erythrocytes by dog intestine H-fucolipid prove that the lectin binds to H-antigenic determinant.

A simple and rapid method for quantification of hapten specific antibodies

A sensitive and simple method for quantification of antibodies against small molecules is described using DNP-lysozyme as the enzyme conjugate. The anti-DNP antiserum was raised against DNP-bovin serum albumin conjugate. Anti-DNP antibody or its monovalent fragment (Fab) reduced the enzyme activity of DNP-lysozyme conjugate in a concentration-dependent manner. The inhibition of enzyme activity is a specific measure of the antibody and Fab content of the sample. The specificity of the reaction was assessed by reduction of antibody-induced inhibition by DNP-lysine. The ability of DNP-lysine to reduce the antibody-induced inhibition of DNP-lysozyme activity also makes possible a sensitive assay for DNP-lysine.

On the conformations of isomeric trimethyl -methylcyclohexane-1,2,3-tricarboxylates

A study has been made of the stereochemistry of three of the four possible configurational isomers of trimethyl 1-methylcyclohexane-1,2,3-tricarboxylate. Two of the isomers undergo highly stereoselective methylation at the 3-position; the third cannot be methylated under similar conditions. Conformations have been suggested for these three isomers on the basis of n.m.r. results. It is thought that axial ester groups at the 1-position in the first two solvate the axial protons at the 3-position and facilitate their removal by trityl anion, while in the third, which has an axial methyl at the 1-position, the effect is not possible and the anion is not formed. The role of A(1.3) strain in causing the high stereoselectivity and position-specificity in the two cases where alkylation does take place and the reasons for slow inversion at the anion centre at position 3 in one of them are discussed.

Alppihiihdon alkutaival : Pujottelu- ja tunturihiihto Suomessa 1920-luvulta 1960-luvulle

The object of study in this thesis is Finnish skiing culture and Alpine skiing in particular from the point of view of ethnology. The objective is to clarify how, when, why and by what routes Alpine skiing found its way to Finland. What other phenomena did it bring forth? The objective is essentially linked to the diffusion of modern sports culture to Finland. The introduction of Alpine skiing to Finland took place at a time when skiing culture was changing: flat terrain skiing was abandoned in favour of cross-country skiing in the early decades of the 20th century, and new techniques and equipment made skiing a much more versatile sport. The time span of the study starts from the late 19th century and ends in the mid-20th century. The spatial focus is in Finland. People and communities formed through their actions are core elements in the study of sports and physical activity. Organizations tend to raise themselves into influential actors in the field of physical culture even if active individuals work in their background. Original archive documents and publications of sports organizations are central source material for this thesis, complemented by newspapers and sports magazines as well as photographs and films on early Alpine skiing in Finland. Ever since their beginning in the late 19th century skiing races in Finland had mostly taken place on flat terrain or sea ice. Skiing in broken cross-country terrain made its breakthrough in the 1920 s, at a time when modern skiing techniques were introduced in instruction manuals. In the late 1920 s the Finnish Women s Physical Education Association (SNLL) developed unconventional forms of pedagogical skiing instruction. They abandoned traditional Finnish flat terrain skiing and boldly looked for influences abroad, which caused friction between the leaders of the women s sports movement and the (male) leaders of the central skiing organization. SNLL was instrumental in launching winter tourism in Finnish Lapland in 1933. The Finnish Tourism Society, the State Railways and sports organizations worked in close co-operation to instigate a boom in tourism, which culminated in the inauguration of a tourist hotel at Pallastunturi hill in the winter of 1938. Following a Swedish model, fell-skiing was developed as a domestic counterpart to Alpine skiing as practiced in Central Europe. The first Finnish skiing resorts were built at sites of major cross-country skiing races. Inspired by the slope at Bad Grankulla health spa, the first slalom skiing races and fell-skiing, slalom enthusiasts began to look for purpose-built sites to practice turn technique. At first they would train in natural slopes but in the late 1930 s new slopes were cleared for slalom races and recreational skiing. The building of slopes and ski lifts and the emergence of organized slalom racing competitions gradually separated Alpine skiing from the old fell-skiing. After the Second World War fell-skiing was transformed into ski trekking on marked courses. At the same time Alpine skiing also parted ways with cross-country skiing to become a sport of its own. In the 1940 s and 1950 s Finnish Alpine skiing was almost exclusively a competitive sport. The specificity of Alpine skiing was enhanced by rapid development of equipment: the new skis, bindings and shoes could only be used going downhill.