Associations of interleukin-1 (IL-1) gene variation and IL-1 receptor antagonist phenotypes with immunological responses, metabolic dysregulation and type 2 diabetes

The upstream proinflammatory interleukin-1 (IL-1) cytokines, together with a naturally occurring IL-1 receptor antagonist (IL-1Ra), play a significant role in several diseases and physiologic conditions. The IL-1 proteins affect glucose homeostasis at multiple levels contributing to vascular injuries and metabolic dysregulations that precede diabetes. An association between IL-1 gene variations and IL-1Ra levels has been suggested, and genetic studies have reported associations with metabolic dysregulation and altered inflammatory responses. The principal aims of this study were to: 1) examine the associations of IL-1 gene variation and IL-1Ra expression in the development and persistence of thyroid antibodies in subacute thyroiditis; 2) investigate the associations of common variants in the IL-1 gene family with plasma glucose and insulin concentrations, glucose homeostasis measures and prevalent diabetes in a representative population sample; 3) investigate genetic and non-genetic determinants of IL-1Ra phenotypes in a cross-sectional setting in three independent study populations; 4) investigate in a prospective setting (a) whether variants of the IL-1 gene family are predictors for clinically incident diabetes in two population-based observational cohort studies; and (b) whether the IL-1Ra levels predict the progression of metabolic syndrome to overt diabetes during the median follow-up of 10.8 and 7.1 years. Results from on patients with subacte thyroiditis showed that the systemic IL-1Ra levels are elevated during a specific proinflammatory response and they correlated with C-reactive protein (CRP) levels. Genetic variation in the IL-1 family seemed to have an association with the appearance of thyroid peroxidase antibodies and persisting local autoimmune responses during the follow-up. Analysis of patients suffering from diabetes and metabolic traits suggested that genetic IL-1 variation and IL-1Ra play a role in glucose homeostasis and in the development of type 2 diabetes. The coding IL-1 beta SNP rs1143634 was associated with traits related to insulin resistance in cross-sectional analyses. Two haplotype variants of the IL-1 beta gene were associated with prevalent diabetes or incident diabetes in a prospective setting and both of these haplotypes were tagged by rs1143634. Three variants of the IL-1Ra gene and one of the IL-1 beta gene were consistently identified as significant, independent determinants of the IL-1Ra phenotype in two or three populations. The proportion of the phenotypic variation explained by the genetic factors was modest however, while obesity and other metabolic traits explained a larger part. Body mass index was the strongest predictor of systemic IL-1Ra concentration overall. Furthermore, the age-adjusted IL-1Ra concentrations were elevated in individuals with metabolic syndrome or diabetes when compared to those free of metabolic dysregulation. In prospective analyses the systemic IL-1Ra levels were found as independent predictors for the development of diabetes in people with metabolic syndrome even after adjustment for multiple other factors, including plasma glucose and CRP levels. The predictive power of IL-1Ra was better than that of CRP. The prospective results also provided some evidence for a role of common IL-1 alpha promoter SNP rs1800587 in the development of type 2 diabetes among men and suggested that the role may be gender specific. Likewise, common variations in the IL-1 beta coding region may have a gender specific association with diabetes development. Further research on the potential benefits of IL-1Ra measurements in identifying individuals at high risk for diabetes, who then could be targeted for specific treatment interventions, is warranted. It has been reported in the recent literature that IL-1Ra secreted from adipose tissue has beneficial effects on glucose homeostasis. Furthermore, treatment with recombinant human IL-1Ra has been shown to have a substantial therapeutic potential. The genetic results from the prospective analyses performed in this study remain inconclusive, but together with the cross-sectional analyses they suggest gender-specific effects of the IL-1 variants on the risk of diabetes. Larger studies with more extensive genotyping and resequencing may help to pinpoint the exact variants responsible and to further elucidate the biological mechanisms for the observed associations. This would improve our understanding of the pathways linking inflammation and obesity with glucose and insulin metabolism.

Tyrphostin-A47 inhibitable tyrosine phosphorylation of flagellar proteins is associated with distinct alteration of motility pattern in hamster spermatozoa

To acquire fertilizing potential, mammalian spermatozoa must undergo capacitation and acrosome reaction. Our earlier work showed that pentoxifylline (0.45 mM), a sperm motility stimulant, induced an early onset of hamster sperm capacitation associated with tyrosine phosphorylation of 45-80 kDa proteins, localized to the mid-piece of the sperm tail. To assess the role of protein tyrosine phosphorylation in sperm capacitation, we used tyrphostin-A47 (TP-47), a specific protein tyrosine kinase inhibitor. The dose-dependent (0.1-0.5 mM) inhibition of tyrosine phosphorylation by TP-47 was associated with inhibition of hyperactivated motility and 0.5 mM TP-47-treated spermatozoa exhibited a distinct circular motility pattern. This was accompanied by hypo-tyrosine phosphorylation of 45-60 kDa proteins, localized to the principal piece of the intact-sperm and the outer dense fiber-like structures in detergent treated-sperm. Sperm kinematic analysis (by CASA) of spermatozoa, exhibiting circular motility (at 1st hr), showed lower values of straight line velocity, curvilinear velocity and average path velocity, compared to untreated controls. Other TP-47 analogues, tyrphostin-AG1478 and -AG1296, had no effect either on kinematic parameters or sperm protein tyrosine phosphorylation. These studies indicate that TP-47-induced circular motility of spermatozoa is compound-specific and that the tyrosine phosphorylation status of 45-60 kDa flagellum-localized proteins could be key regulators of sperm flagellar bending pattern, associated with the hyperactivation of hamster spermatozoa.

The role of inflammation and matrix proteins in airway remodelling

Asthma is a chronic inflammatory disorder of the airways. Remodelling in asthma is defined as the structural changes seen in the airways of asthmatics in comparison to healthy controls. Progressive loss of lung function also seen in asthma might be caused by remodelling. The research aims of this thesis were to investigate inflammation and remodelling in the airways of different types of asthmatics and smokers. The association between inflammation and remodelling was also examined in a mouse model of allergic airway inflammation. Healthy smokers showed increased numbers of macrophages in the BAL with no changes in the inflammatory cells in biopsies. Macrophages seemed to be quite quiescent, since mRNA expression for a wide variety of inflammatory mediators, especially chemokines CCL3, CCL4, CCL5 and CCL20, secreted by macrophages was significantly lower than in healthy non-smokers. Attenuated macrophage activity in the airway lumen may render smokers more susceptible to airway infections and have an impact on the development of other airway pathology. Patients with diisocyanate-induced asthma (DIA) on inhaled corticosteroids (ICS) who still had non-specific bronchial hyperreactivity (NSBHR) at the end of the follow-up showed increased expression of TNF-α, IL-6 and IL-15 mRNA in BAL cells compared to those without NSBHR. In addition to being markers for poor prognosis and possible slight glucocorticoid resistance, these cytokines might aid in guiding the treatment of DIA. The increase in the thickness of tenascin-C layer in the bronchial basement membrane (BM) was much less than usually seen in other types of asthma, which might not make tenascin-C a good marker for DIA. OVA-induced tenascin-C expression in the lung was attenuated in STAT4-/- mice with impaired Th1-type immunity compared to WT mice. Interestingly, STAT6-/- mice with impaired Th2-type immunity showed tenascin-C expression levels similar to those of WT mice. The clearest difference between these two knockout strains in response to OVA was that STAT4-/- mice exhibited no upregulation of IFN-γ and TNF-α mRNA expression. Thus, tenascin-C expression was unexpectedly more related to Th1 type reactions. In vitro studies confirmed the results. Human fibroblasts stimulated by TNF-α and IFN-γ showed increased expression of tenascin-C. Patients with newly diagnosed asthma showed increased expression of laminin α2 in the bronchial BM in comparison to patients with asthma symptoms only and healthy controls. Both patients with asthma and those with only asthma symptoms showed increased expression of the laminin β2 chain in comparison to controls. Thus, laminin α2 expression differentiated patients with clinical asthma from patients with symptoms only. Furthermore, the expression of laminin α2 and β2 was associated with NSBHR, linking very specific remodelling events to clinical findings.

Prediction of protein-protein interactions in dengue virus coat proteins guided by low resolution cryoEM structures

Background: Dengue virus along with the other members of the flaviviridae family has reemerged as deadly human pathogens. Understanding the mechanistic details of these infections can be highly rewarding in developing effective antivirals. During maturation of the virus inside the host cell, the coat proteins E and M undergo conformational changes, altering the morphology of the viral coat. However, due to low resolution nature of the available 3-D structures of viral assemblies, the atomic details of these changes are still elusive. Results: In the present analysis, starting from C alpha positions of low resolution cryo electron microscopic structures the residue level details of protein-protein interaction interfaces of dengue virus coat proteins have been predicted. By comparing the preexisting structures of virus in different phases of life cycle, the changes taking place in these predicted protein-protein interaction interfaces were followed as a function of maturation process of the virus. Besides changing the current notion about the presence of only homodimers in the mature viral coat, the present analysis indicated presence of a proline-rich motif at the protein-protein interaction interface of the coat protein. Investigating the conservation status of these seemingly functionally crucial residues across other members of flaviviridae family enabled dissecting common mechanisms used for infections by these viruses. Conclusions: Thus, using computational approach the present analysis has provided better insights into the preexisting low resolution structures of virus assemblies, the findings of which can be made use of in designing effective antivirals against these deadly human pathogens.

A novel supersecondary structure in globular proteins comprising the collagen-like helix and ?-turn

A structure consisting of the polyproline-II or collagen-like helix immediately succeeded by a ?-turn is seen in several synthetic peptides and has been suggested to be the conformational requirement for proline hydroxylation in nascent procollagen. Using a simple algorithm for detecting secondary structures, we have analysed crystal structure data on 40 globular proteins and have found eight examples of the collagen-helix + ?-turn supersecondary structure in 15 proteins that contain the collagen-like helical segments.

A microcomputer program to analyze the CD spectrum of proteins and nucleic acids—Use of LOTUS 1-2-3 spread sheet

A user friendly interactive computer program, CIRDIC, is developed which calculates the molar ellipticity and molar circular dichroic absorption coefficients from the CD spectrum. This, in combination with LOTUS 1-2-3 spread sheet, will give the spectra of above parameters vs wavelength. The code is implemented in MicroSoft FORTRAN 77 which runs on any IBM compatible PC under MSDOS environment.

Over-expression and purification strategies for recombinant multi-protein oligomers: A case study of Mycobacterium tuberculosis sigma/anti-sigma factor protein complexes

The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the sigma factor/anti-sigma factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of sigma factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems. (C) 2010 Elsevier Inc. All rights reserved.

1-Anilino-8-naphthalene sulfonate (ANS) binding to proteins revisited: Investigation of dye binding to molten globule states by electrospray ionization mass spectrometry

The binding of 1-anilino-8-naphthalene-sulfonic acid to globular proteins at acidic pH has been investigated by electrospray ionization mass spectrometry (ESIMS). Mass spectra of apomyoglobin recorded in the pH range 2−7 establish that maximal ANS binding is observed at pH 4.0. As many as seven distinct species may be observed in the gas phase which correspond to protein molecules containing one to six molecules of bound ANS. At neutral pH only a single molecule of ANS is bound. In the case of cytochrome c, maximal binding is observed at pH 4.0, with five molecules being bound. Binding is suppressed at neutral pH. In both cases ESIMS demonstrates maximal ANS binding at pH values where the proteins have been reported to exist in molten globule states. ANS binding is not observed for lysozyme, which has a tightly folded structure over the entire pH range. Reduction of disulfide bonds in lysozyme leads to the detection of ANS-bound species at neutral pH. Binding is suppressed at low pH due to complete unfolding of the reduced protein. The results suggest that ESIMS may provide a convenient method of probing the stoichiometry and distribution of dye complexes with molten protein globules

A C-H…O hydrogen bond stabilized polypeptide chain reversal motif at the C-terminus helices in proteins.

The serendipitous observation of a C–Hcdots, three dots, centeredO hydrogen bond mediated polypeptide chain reversal in synthetic peptide helices has led to a search for the occurrence of a similar motif in protein structures. From a dataset of 634 proteins, 1304 helices terminating in a Schellman motif have been examined. The C–Hcdots, three dots, centeredO interaction between the T−4 CαH and T+1 C=O group (Ccdots, three dots, centeredO≤3.5 Å) becomes possible only when the T+1 residue adopts an extended β conformation (T is defined as the helix terminating residue adopting an αL conformation). In all, 111 examples of this chain reversal motif have been identified and the compositional and conformational preferences at positions T−4, T, and T+1 determined. A marked preference for residues like Ser, Glu and Gln is observed at T−4 position with the motif being further stabilized by the formation of a side-chain–backbone Ocdots, three dots, centeredH–N hydrogen bond involving the side-chain of residue T−4 and the N–H group of residue T+3. In as many as 57 examples, the segment following the helix was extended with three to four successive residues in β conformation. In a majority of these cases, the succeeding β strand lies approximately antiparallel with the helix, suggesting that the backbone C–Hcdots, three dots, centeredO interactions may provide a means of registering helices and strands in an antiparallel orientation. Two examples were identified in which extended registry was detected with two sets of C–Hcdots, three dots, centeredO hydrogen bonds between (T−4) CαHcdots, three dots, centeredC=O (T+1) and (T−8) CαHcdots, three dots, centeredC=O (T+3).

Mother-Infant Antibodies to Pneumococcal Polysaccharides and Proteins : Transfer and Persistence of Maternal Antibodies and Development of Vaccine-Induced and Naturally Acquired Antibodies in Infants

Symptomless nasopharyngeal carriage of Streptococcus pneumoniae (pneumococcus) is very common in young children. Occasionally the carriage proceeds into mild mucosal diseases, such as sinusitis or acute otitis media, or into serious life-threatening diseases, such as pneumonia, sepsis or meningitis. Each year, up to one million children less than five years of age worldwide die of invasive pneumococcal diseases (IPD). Especially in the low-income countries IPD is a leading health problem in infants; 75% of all IPD cases occur before one year of age. This stresses the need of increased protection against pneumococcus in infancy. Anti-pneumococcal antibodies form an important component in the defence against pneumococcal infection. Maternal immunisation and early infant immunisation are two possible ways by which potentially protective antibody concentrations against pneumococci could be achieved in early infancy. The aim of this thesis is to increase the knowledge of antibody mediated protection against pneumococcal disease in infants and young children. We investigated the transfer of maternal anti-pneumococcal antibodies from Filipino mothers to their infants, the persistence of the transferred antibodies in the infants, the immunogenicity of the 23-valent pneumococcal polysaccharide vaccine (PPV) in infants and the response of the children to a second dose of PPV at three years of age. We also investigated the development of antibodies to pneumococcal protein antigens in relation to culture-confirmed pneumococcal carriage in infants. Serum samples were collected from the mothers, the umbilical cords and from the infants at young age as well as at three years of age. The samples were used to determine the antibody concentrations to pneumococcal serotypes 1, 5, 6B, 14, 18C and 19F, as well as to the pneumococcal proteins PspA, PsaA, Ply, PspC, PhtD, PhtDC and LytC by the enzyme immunoassay. The findings of the present study confirm previously obtained results and add to the global knowledge of responses to PPV in young children. Immunising pregnant women with PPV provides the infants with increased concentrations of pneumococcal polysaccharide antibodies. Of the six serotypes examined, serotypes 1 and 5 were immunogenic already in infants. At three years of age, the children responded well to the second dose of PPV suggesting that maternal and early infant immunisations might not induce hyporesponsiveness to polysaccharide antigens after subsequent immunisations. The anti-protein antibody findings provide useful information for the development of pneumococcal protein vaccines. All six proteins studied were immunogenic in infancy and the development of anti-protein antibodies started early in life in relation to pneumococcal carriage.

Isolation of a phosphoryl choline-binding protein from the hemolymph of the snail, Achatina fulica

A phosphorylcholine-binding protein from the hemolymph of the snail Achatina fulica was purified to near homogeneity using a Sepharose phenylphosphorylcholine affinity column. The protein bound to the affinity column was eluted with 5 mM phosphorylcholine as a single symmetrical peak. The purified protein (400 Kda) contained 35–40% carbohydrate. On SDS-PAGE the protein separated into two bands of 20 and 24 Kda, and had a pI of 5.9. On immunodiffusion, antiserum to the snail phosphorylcholine binding protein did not cross-react against other phosphorylcholine binding proteins, like rat serum phosphorylcholine-binding protein (PCBP), limulus C-reactive protein (CRP), or human CRP. On pretreatment of the snail hemolymph with this antiserum, the hemagglutination titer of the hemolymph was markedly decreased. The purified snail phosphorylcholine binding protein agglutinated rabbit erythrocytes in the absence of divalent cation (Ca+2) but trace amount of Ca+2 increased its binding. The strongest inhibitor of the agglutination reaction was lactose, followed by melibiose and 2-deoxygalactose. The relationships of the snail phosphorylcholine binding protein to other hemolymph agglutinins and to CRPs are discussed in light of common phylogeny.

Nucleotide sequence of the 3terminal region of belladonna mottle virus (renamed Physalis mottle virus) RNA and analysis of the evolutionary relationships among tymoviral coat proteins

The 3' terminal 1255 nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3' terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addiition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.