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.

     

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.     

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.     

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.     

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.     

RFLP mapping of the dwarfing (Ddw1) and hairy peduncle (Hp) genes on chromosome 5 of rye (Secale cereale L.)

An F₂ population was established for mapping the two dominant genes for dwarfness (Ddw1) and hairy peduncle (Hp) on chromosome 5R. The location of both genes was shown to be on the segment of chromosome 5RL which was ancestrally translocated and is homoeologous to Triticeae 4L. Hp cosegregated with the wheat gDNA probe WG199, localised in wheat on chromosomes 5AL, 4BL and 4DL. No segregation was observed between the traits hairy peduncle and hairy leaf sheath. The locus for Ddw1 was found to map distally to Hp/Xwg199 but proximal to the isozyme marker -amy-R1. The genetical distances were 5.6 cM between Hp/Xwg199 and Ddw1 and ll.ScM between Ddw1 and -amy-R1, respectively. The map position of Ddw1 suggests that it is homoeologous to the wheat dominant dwarfing gene Rht12, present on chromosome 5AL and linked to -amy-A1.     

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.     

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.     

Serum protein groups (Hp, GC, C3) in patients with gastric carcinoma.

The phenotypes and gene frequencies of 3 serum protein systems (Hp, GC and C3) were studied in 114 consecutive patients from all over Greece with gastric carcinoma. Healthy Greeks studied previously in our Department served as controls. No significant differences were found between patients and controls concerning Hp. Significant differences were found in the GC and C3 systems; GC 2-1 and C3F phenotype as well as C3*F gene frequencies were significantly higher in patients than in controls.     

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.     

Studies of association between some genetic systems (ABO, MN, EsD, GLO, C3 and Hp)

Using random population data on the ABO, MN, esterase D (EsD), glyoxalase I, complement (C3) and haptoglobin markers in the population of Madras City associations were studied between these genetic systems. Out of a total of fifteen comparisons one significant association (chi 2 = 11.0; d.f. 4; 0.05 greater than p greater than 0.02) was found between the EsD and C3 phenotypes.     

Cytotoxicity of PP(Arg)(2)- and Hp(Arg)(2)-mediated photodynamic therapy and early stage of apoptosis induction in prostate carcinoma in vitro

Porphyrin photosensitizers tend to localize in mitochondria. The depolarization of mitochondrial membrane is one of the early stages of apoptosis and Laser Scanning Fluorescence Microscopy allows to determine changes in transmembrane mitochondrial potential under influence of PDT depending on the kind of photosensitizer (PP(Arg)(2), Hp(Arg)(2)), the energy dose (5, 10, 30 and 50 J/cm(2)) and time periods (24 and 48 hours after irradiation) in the LNCaP (lymphonodal metastasis of prostate carcinoma, the androgen dependent cell line). Cyototoxicity induced by PP(Arg)(2)- and Hp(Arg)(2)-based PDT depending on energy dose and time after irradiation in prostate carcinoma is determined with MTT. Generally, it was shown that lower energy doses induce greater changes in transmembrane mitochondrial potential. Hp(Arg)(2)-based PDT was more effective causing greater mitochondrial membrane depolarization and cell viability decrease in comparison to PP(Arg)(2)-mediated PDT (in the case of maximal nontoxic photosensitizer doses used).     

Serum protein polymorphisms in a village community from the Gambia,West Africa (Hp,Tf, and Gc)

Summary Serum samples from 857 inhabitants of the village of Keneba, The Gambia, West Africa, were examined by means of polyacrylamide gel electrophoresis. In 203 cases no haptoglobin could be detected, whilst in the remaining 654 samples the three common haptoglobin phenotypes were found with gene frequencies of 0.651 (Hp¹) and 0.349 (Hp²). The D₁ transferrin variant gene was found with a frequency of 0.025. In the serum Gc system the fast variant Gc-Ab was detected, the gene frequencies being: Gc¹, 0.943; Gc², 0.044; and Gc^Ab, 0.013.     

Die Serumgruppen-Systeme Lp(a), Gm(a), Gc und Hp bei Cerebralsklerotikern

Zusammenfassung Im genetisch determinierten Lp(a)-System war die Häufigkeit von Lp(a+) bei 307 Cerebralsklerosefällen statistisch signifikant erhöht im Vergleich zur Lp(a+)-Häufigkeit der gleichen Population. In den Systemen Gm(a), Gc und Hp wurde dagegen keine Veränderung der Genotypenhäufigkeit infolge der arteriosklerotischen Erkrankung festgestellt.

---

In the genetically determined Lp(a) system the frequency of Lp(a+) showed with 45.6 per cent a statistically significant raise in 307 patients with cerebral sclerosis compared with 38.7 per cent in normal population. The increase of Lp(a+) is almost exclusively confined to weakly positive precipitation and is discussed in connection with the often observed increase of -lipoprotein concentration in the serum of arteriosclerotic persons. As expected no changes of genotype frequency due to arteriosclerotic diseases has been stated in the Gm(a), Gc and Hp system.

---

---

Herrn Medizinalrat Dr. Wieder, Ärztlicher Direktor des Bezirkskrankenhauses, sind wir für die Überlassung der Blutproben zu besonderem Dank verpflichtet.

---

(Direktor: Obermedizinalrat Dr. med. K. Thomas)     

The association of Hp 1 and sickle cell disease

The distribution of gene frequencies and types for haptoglobin (Hp) were studied in a normal black population and one with sickle cell disease. The statistical analysis of our findings indicated a significant association between sickle cell disease and the Hp 1 type.