Serum amyloid-A (SAA) and haptoglobin (Hp) plasma concentrations in newborn calves

The concentrations of 2 major bovine acute-phase proteins, haptoglobin (Hp) and serum amyloid-A (SAA), were measured in plasma obtained shortly after birth from 22 healthy calves. In a separate group of diseased calves (n = 8), Hp and SAA concentrations were measured to determine whether newborn calves (up to 4 d old) are able to produce SAA and Hp. In blood samples taken directly after birth, the Hp plasma concentrations were all below the limit of detection. The mean SAA concentration was independent of weight (r = 0.063), degree of acidosis (r = -0.125), sex (p > 0.05), and were not different in calves born after different types of obstetrical help (p > 0.05). In the group of diseased calves, an increased Hp concentration was measured in only 2 of 8 animals, whereas the mean SAA concentration was significantly higher (p < 0.05) than in the healthy newborn calves. These data suggest that prenatal stress due to parturition does not form a stimulus for the production of acute-phase proteins in the fetal calf. The low Hp plasma concentrations might indicate that either it takes a few days to establish a detectable concentration of this protein, or that Hp production is not fully developed in newborn calves.     

Abnormal changes of plasma acute phase proteins in schizophrenia and the relation between schizophrenia and haptoglobin (Hp) gene

Summary. In this study we focused on detecting schizophrenia related changes of plasma proteins using proteomic technology and examining the relation between schizophrenia and haptoglobin (Hp) genotype. We investigated plasma proteins from schizophrenic subjects (n = 42) and healthy controls (n = 46) by two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry. To further reveal the genetic relationship between acute phase proteins (APPs) and schizophrenia disease, we tested Hp α1/Hp α2 (Hp 1/2) polymorphism and two single nucleotide polymorphisms (SNPs) of Hp, rs2070937 and rs5473, for associations with schizophrenia in the Chinese Han population. With the relatively high number of samples for 2-DE work, we found that four proteins in the family of positive APPs were all up-regulated in patients. In genetic association study, we found significant associations existing between schizophrenia and Hp polymorphisms, Hp 1/2 and rs2070937 variants. Schizophrenia is accompanied by both an altered expression of Hp protein and a different genotype distribution of Hp gene, demonstrating that Hp is associated with schizophrenia. The results from proteomic and genomic aspects both indicate that acute phase reaction is likely to be an aetiological agent in the pathophysiology of schizophrenia, but not just an accompanying symptom. The positive APPs are schizophrenic related proteins, with the highly concordant results on four positive APPs. The first two authors contributed equally.     

Evolution of haptoglobin: comparison of complementary DNA encoding Hp alpha 1S and Hp alpha 2FS.

Haptoglobin is a transport glycoprotein which removes free hemoglobin from the circulation of vertebrates. In human populations haptoglobin is polymorphic due to three alleles, Hp alpha 1F, Hp alpha 1S and Hp alpha 2. The Hp alpha 2 allele is roughly twice the length of the Hp alpha 1 alleles and is the product of a partial gene duplication possibly resulting from an unequal crossover event in a heterozygous genotype Hp alpha 1F/Hp alpha 1S. In the study described here we compare the cDNA encoding Hp alpha 1S to that encoding Hp alpha 2FS . Both have a leader sequence followed by the genotypic alpha chain sequence, a beta sequence and an untranslated sequence in the 3' end. The cDNA encoding Hp alpha 2FS is composed of alpha 1F and alpha 1S domains differing by four nucleotide replacements. Hp alpha 1S cDNA contains the same replacement site mutations found in the alpha 1S domain of Hp alpha 2FS , indicating that this coding region has sustained few, if any, mutations since its incorporation into the Hp alpha 2FS gene.     

Channel-forming (Porin) activity in Herpetosiphon aurantiacus Hp a2

Detergent extracts of cell envelopes of the gliding bacterium Herpetosiphon aurantiacus formed channels in lipid bilayers. Fast protein liquid chromatography across a HiTrap-Q cation-exchange column demonstrated that a 45-kDa protein forms the channel. The observation of a channel-forming protein suggests that Herpetosiphon aurantiacus Hp a2 has a permeability barrier on its surface.     

Products of trypsin digestion of haptoglobin beta (heavy) chain

Trypsin digestion of haptoglobin beta (heavy) chain resulted in five glycopeptides. The glycopeptides were characterized by carbohydrate and sulphydryl groups content; their molecular mass was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. None glycopeptide possessed hemoglobin-binding capacity. glycopeptide I did not form any precipitate with antihaptoglobin serum but was shown to inhibit strongly the reaction of haptoglobin or beta chain with the antiserum. Glycopeptide II showed dominant antigenic determinants in relation to native haptoglobin and to beta chain. Reaction of this glycopeptide with concanavalin A was almost twice higher than the corresponding reaction of haptoglobin. Glycopeptides IV and V were inactive in the reaction with the lectin. Glycopeptide III exhibited relatively the strongest cross-reactivity with the specific antihaptoglobin serum while its inhibitory activity in the immunoreaction was the lowest.     

Personal dose equivalent Hp(0.07) during 68Ga-DOTA-TATE production procedures

This work presents the exposure of hands of the personnel of a nuclear medicine department who prepare and administer ⁶⁸Ga-DOTA-TATE. Dosimetry measurements were performed during three 1-week sessions, for nine production procedures. A total of 360 measurements were made by using high-sensitivity MCP-N thermoluminescent detectors. Annealed detectors were and vacuum-packed in foil and then placed on each fingertip of both hands of five radiochemists and four nurses (one detector for one fingertip). The greatest exposure to ionizing radiation was found on the non-dominant left hand of radiochemists and nurses. A maximum H_p(0.07)/A value of 49.36?±?4.95 mSv/GBq was registered for radiochemists during the ⁶⁸ Ga-DOTA-DATE activity dispensing procedure. For nurses performing the radiopharmaceutical injection procedure, a corresponding maximum value of 1.28?±?0.13 mSv/GBq was measured, while the mean value for all the nurses was 0.38 mSv/GBq. The dispensing procedure accounted for approximately 60% of the total exposure of radiochemists' fingertips. Based on the results obtained it is recommended that a ring dosimeter should be routinely placed on the middle finger of the non-dominant hand of radiochemists and nurses. Furthermore, it is proposed to systematically train workers in handling open sources of ionizing radiation, with the aim of reducing the required handling time.

     

Kinetics of the reaction between oxygen and haemoglobin bound to haptoglobin (Short Communication)

The kinetics of the reaction between O(2) and haemoglobin bound to haptoglobin 1-1 were investigated by the stopped-flow and temperature-jump techniques. The reaction, which follows second-order kinetics in the lower concentration range, becomes independent of O(2) concentration above about 150mum-O(2).     

Helicobacter pylori outer membrane protein 18 (Hp1125) induces dendritic cell maturation and function

BACKGROUND: Dendritic cells (DCs) are potent antigen-presenting cells that initiate T-cell responses. A robust adaptive Th1 immune response is crucial to an adaptive (Th2) immune response necessary for vaccine-induced protective immunity against Helicobacter pylori. It has been shown that several outer membrane proteins (Omps) induce a robust antibody response. However, it is also known that the antibodies generated are not protective. Moreover there is great variation in the recognition of high molecular weight H. pylori proteins by sera from infected patients. In contrast to the high molecular weight proteins, serologic responses to small molecular weight proteins provide assessment of current infection with H. pylori and also of its eradication. AIM: The goal of the study was to analyze the activation of the immune response by a specific low molecular weight Omp that is universally expressed by all H. pylori strains. Therefore, we studied interaction of H. pylori Omp18 with DCs. METHODS: Activation of murine bone marrow-derived DCs and production of cytokines by Omp18 was assessed by fluorescence-activated cell sorter (FACS) for costimulatory markers and ELISA, respectively. The ability of Omp18 stimulated DCs to induce lymphocyte proliferation was measured in a mixed leukocyte reaction. RESULTS: Omp18 induced higher expression of the B7 (CD80 and CD86) costimulatory molecule after 18 hours indicating processing and presentation of the antigen on the surface by bone marrow-derived DCs. The maturing DCs also secreted significant levels of IL-12, but was 4-fold less than that stimulated by whole bacteria. Omp18-primed DCs induced proliferation and release of IFNgamma by syngeneic splenocytes. CONCLUSION: We concluded that Omp18 is capable of activating DCs initiating a Th1 immune response.     

Targeted Identification of Glycosylated Proteins in the Gastric Pathogen Helicobacter pylori (Hp)

Virulence of the gastric pathogen Helicobacter pylori (Hp) is directly linked to the pathogen''s ability to glycosylate proteins; for example, Hp flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, and this modification is absolutely essential for Hp to synthesize functional flagella and colonize the host''s stomach. Although Hp''s glycans are linked to pathogenesis, Hp''s glycome remains poorly understood; only the two flagellin glycoproteins have been firmly characterized in Hp. Evidence from our laboratory suggests that Hp synthesizes a large number of as-yet unidentified glycoproteins. Here we set out to discover Hp''s glycoproteins by coupling glycan metabolic labeling with mass spectrometry analysis. An assessment of the subcellular distribution of azide-labeled proteins by Western blot analysis indicated that glycoproteins are present throughout Hp and may therefore serve diverse functions. To identify these species, Hp''s azide-labeled glycoproteins were tagged via Staudinger ligation, enriched by tandem affinity chromatography, and analyzed by multidimensional protein identification technology. Direct comparison of enriched azide-labeled glycoproteins with a mock-enriched control by both SDS-PAGE and mass spectrometry-based analyses confirmed the selective enrichment of azide-labeled glycoproteins. We identified 125 candidate glycoproteins with diverse biological functions, including those linked with pathogenesis. Mass spectrometry analyses of enriched azide-labeled glycoproteins before and after cleavage of O-linked glycans revealed the presence of Staudinger ligation-glycan adducts in samples only after beta-elimination, confirming the synthesis of O-linked glycoproteins in Hp. Finally, the secreted colonization factors urease alpha and urease beta were biochemically validated as glycosylated proteins via Western blot analysis as well as by mass spectrometry analysis of cleaved glycan products. These data set the stage for the development of glycosylation-based therapeutic strategies, such as new vaccines based on natively glycosylated Hp proteins, to eradicate Hp infection. Broadly, this report validates metabolic labeling as an effective and efficient approach for the identification of bacterial glycoproteins.Helicobacter pylori (Hp)¹ infection poses a significant health risk to humans worldwide. The Gram-negative, pathogenic bacterium Hp colonizes the gastric tract of more than 50% of humans (1). Approximately 15% of infected individuals develop duodenal ulcers and 1% of infected individuals develop gastric cancer (2). Current treatment to clear infection requires “triple therapy” (3), a combination of multiple antibiotics that is often associated with negative side effects (4). Because of poor patient compliance and the evolution of antibiotic resistance, existing antibiotics are no longer effective at eradicating Hp infection (4). New treatment methods are needed to eliminate Hp from the human gastric tract.Recent work has focused on gaining insights into the pathogenesis of Hp to aid the development of new treatments. The most recent findings in this area have conclusively revealed that glycosylation of proteins in Hp is required for pathogenesis. Hp use complex flagella, comprised of flagellin proteins, to navigate the host''s gastric mucosa (5, 6). The flagellin proteins are heavily glycosylated with the unusual nine-carbon sugar pseudaminic acid, found exclusively in mucosal-associated pathogens (Hp (7), Campylobacter jejuni (8) and Pseudomonas aeruginosa (9)). This modification is absolutely essential for the formation of functional flagella on Hp (7, 10). Deletion of any one of the enzymes in the pseudaminic acid biosynthetic pathway results in Hp that lack flagella, are nonmotile, and are unable to colonize the host''s stomach (7). Although pseudaminic acid is critical for Hp virulence, it is absent from humans (11, 12). Therefore, insights into Hp''s pathogenesis have revealed that Hp''s glycan pseudaminic acid is a bona fide target of therapeutic intervention. This is one of a number of examples linking protein glycosylation to virulence in medically significant bacterial pathogens (13, 14).Despite these findings, Hp''s glycome remains poorly understood overall. Only the two flagellin glycoproteins have been firmly characterized in Hp (7) to date. Nine other candidate glycoproteins have been identified in Hp, but their glycosylation status has not been biochemically confirmed (15). The relative paucity of information regarding Hp''s glycoproteins is due in part to the previously held belief that protein glycosylation could not occur in bacteria (13, 16, 17). However, even after Szymanski (18, 19), Koomey (20), Guerry (21), Logan (7), Comstock and others (13, 16, 17) disproved this belief by firmly establishing the synthesis of glycoproteins in bacteria, the study of bacterial glycoproteins has presented unique challenges for analytical study (14, 22). For example, the unusual structures of bacterial glycans, which often contain amino- and deoxy-carbohydrates exclusively found in bacteria (12, 23–25), hampers their identification using existing tools. Though methods such as the use of glycan-binding reagents (20, 24, 26, 27) and periodic acid/hydrazide glycan labeling (15) have successfully detected glycoproteins in a range of bacteria, they present limitations. Glycan binding-based methods are often limited because of the unavailability of lectins or antibodies with binding specificity for glycosylated proteins in the bacteria of interest (14, 22). Periodic acid/hydrazide-based labeling is plagued by a lack of specificity for glycosylated proteins (15). Thus, an efficient and robust approach to discover Hp''s glycoproteins is needed.In previous work, we established that the chemical technique known as metabolic oligosaccharide engineering (MOE), which was developed by Bertozzi (28, 29), Reutter (30), and others for the study of mammalian glycoproteins, is a powerful approach to label and detect Hp''s glycoproteins (31). Briefly, Hp metabolically processes the unnatural, azide-containing sugar peracetylated N-azidoacetylglucosamine (Ac₄GlcNAz) (32), an analog of the common metabolic precursor N-acetylglucosamine (GlcNAc), into cellular glycoproteins (Fig. 1). Elaboration of azide-labeled glycoproteins via Staudinger ligation (33) with a phosphine probe conjugated to a FLAG peptide (Phos-FLAG) (34) followed by visualization with an anti-FLAG antibody (Fig. 1) revealed a glycoprotein fingerprint containing a large number of as-yet unidentified Hp glycoproteins that merit further investigation (31).Open in a separate windowFig. 1.Metabolic oligosaccharide engineering facilitates labeling and detection of Hp''s glycoproteins. Supplementation of Hp with Ac₄GlcNAz leads to metabolic labeling of Hp''s N-linked and O-linked glycoproteins with azides. Azide-modified glycoproteins covalently labeled with Phos-FLAG can be detected via Western blot analysis with anti-FLAG antibody to yield Hp''s glycoprotein fingerprint, which contains a large number of as-yet unidentified glycoproteins.Here we describe a glycoproteomic identification strategy for the selective detection, isolation, and discovery of Hp''s glycoproteins. In particular, we demonstrate that glycan metabolic labeling coupled with mass spectrometry analysis is an efficient and robust chemical approach to identify novel glycoproteins in Hp. This work characterizes glycosylated virulence factors in Hp, thus opening the door to new vaccination and antibiotic therapies to eradicate Hp infection. Broadly, this work validates metabolic oligosaccharide engineering as a complementary method to discover bacterial glycoproteins.     

Evidence of tandem repeat and extra thiol-groups resulted in the polymeric formation of bovine haptoglobin: a unique structure of Hp 2-2 phenotype

Human plasma Hp is classified as 1-1, 2-1, and 2-2. They are inherited from two alleles Hp 1 and Hp 2, but there is only Hp 1 in almost all the animal species. Hp 2-2 molecule is extremely large and heterogeneous associated with the development of inflammatory-related diseases. In this study, we expressed entire bovine Hp in E. coli as a alphabeta linear form. Interestingly, the antibodies prepared against this form could recognize the subunit of native Hp. In stead of a complicated column method, the antibody was able to isolate bovine Hp via immunoaffinity and gel-filtration columns. The isolated Hp is polymeric containing two major molecular forms (660 and 730 kDa). Their size and hemoglobin binding complex are significantly larger than that of human Hp 2-2. The amino-acid sequence deducted from the nucleotide sequence is similar to human Hp 2 containing a tandem repeat over the alpha chain. Thus, the Hp 2 allele is not unique in human. We also found that there is one additional -SH group (Cys-97) in bovine alpha chain with a total of 8 -SH groups, which may be responsible for the overall polymeric structure that is markedly different from human Hp 2-2. The significance of the finding and its relationship to structural evolution are also discussed.     

Serum haptoglobin concentrations in ringed seals (Pusa hispida) from Svalbard, Norway

Haptoglobin (Hp) levels were measured in blood serum from 185 apparently healthy ringed seals (Pusa hispida) from Svalbard (age range, 1-30 yr) collected during the spring seasons of 2002 through 2004. The Hp concentration was 0.26 +/- 0.02 g/l (mean +/- SE; range, 0.0-1.5 g/l). Maturity status, body condition index (CI), interactions between CI and maturity status, and sex and maturity status all had significant influences on Hp levels. Mature males had significantly higher Hp concentrations compared with mature females (0.30 +/- 0.03 vs. 0.17 +/- 0.01 g/l, F(1,16) = 14.9, P<0.01). No differences were found between Hp levels in immature male and female seals (0.33 +/- 0.06 g/l). The Hp levels increased significantly in immature seals when the CI decreased. Despite the fact that mature males had a significantly lower CI than mature females, no direct correlation was found between Hp concentration and CI among adults. The elevated Hp values found in mature males might be related to injuries inflicted among males while fighting during the mating season or to high stress levels related to mating competition. The Hp concentrations of ringed seals measured in this study are useful baseline data for development of a diagnostic tool for future monitoring of the general health of this and other ringed seal populations.     

Canine haptoglobin: a unique haptoglobin subunit arrangement.

1. Isolated canine haptoglobin behaved identically to the alpha 2 beta 2 structure typical of human haptoglobin type 1-1 on alkaline polyacrylamide gel electrophoresis and on gel filtration. 2. In the presence of urea or sodium dodecyl sulphate canine haptoglobin dissociated into alpha beta subunits that separated into alpha and beta chains after reduction with 2-mercaptoethanol. 3. Compositional analysis identified one less half-cystine in canine alpha chain when compared to human alpha 1 chain. 4. These results provide evidence that there is no inter alpha chain disulphide in canine haptoglobin comparable to the alpha 1 20-alpha 1 20 disulphide in human haptoglobin that links the two alpha beta subunits.     

Relationship between Hp1S and Hp2 gene frequencies among human populations

In this work, we present new data on the Hp1alpha- and Hp2alpha-chains polymorphism in different populations. We confirm the singularity of the geographical distribution of the Hp2 alleles in our samples. The analysis of the results shows that a significant correlation exists in the population between the Hp1S and Hp2 gene frequencies. An additional Hp1alpha-chain variant is described in a Pyrenean sample.     

The obesity and inflammatory marker haptoglobin attracts monocytes via interaction with chemokine (C-C motif) receptor 2 (CCR2)

Background

Phylogeographic reconstruction of some bacterial populations is hindered by low diversity coupled with high levels of lateral gene transfer. A comparison of recombination levels and diversity at seven housekeeping genes for eleven bacterial species, most of which are commonly cited as having high levels of lateral gene transfer shows that the relative contributions of homologous recombination versus mutation for Burkholderia pseudomallei is over two times higher than for Streptococcus pneumoniae and is thus the highest value yet reported in bacteria. Despite the potential for homologous recombination to increase diversity, B. pseudomallei exhibits a relative lack of diversity at these loci. In these situations, whole genome genotyping of orthologous shared single nucleotide polymorphism loci, discovered using next generation sequencing technologies, can provide very large data sets capable of estimating core phylogenetic relationships. We compared and searched 43 whole genome sequences of B. pseudomallei and its closest relatives for single nucleotide polymorphisms in orthologous shared regions to use in phylogenetic reconstruction.

Results

Bayesian phylogenetic analyses of >14,000 single nucleotide polymorphisms yielded completely resolved trees for these 43 strains with high levels of statistical support. These results enable a better understanding of a separate analysis of population differentiation among >1,700 B. pseudomallei isolates as defined by sequence data from seven housekeeping genes. We analyzed this larger data set for population structure and allele sharing that can be attributed to lateral gene transfer. Our results suggest that despite an almost panmictic population, we can detect two distinct populations of B. pseudomallei that conform to biogeographic patterns found in many plant and animal species. That is, separation along Wallace's Line, a biogeographic boundary between Southeast Asia and Australia.

Conclusion

We describe an Australian origin for B. pseudomallei, characterized by a single introduction event into Southeast Asia during a recent glacial period, and variable levels of lateral gene transfer within populations. These patterns provide insights into mechanisms of genetic diversification in B. pseudomallei and its closest relatives, and provide a framework for integrating the traditionally separate fields of population genetics and phylogenetics for other bacterial species with high levels of lateral gene transfer.