Molecular recognition of pseudodistamine isomeric precursors trans-3(4)-aminopiperidin-4(3)-ols by EI mass spectrometry.

Synthesis of drugs, biologically active compounds or their derivatives always requires precise and reliable method of their identification, including differentiation of the possible isomers. Pseudodistamines and their precursors became a matter of elevated attention due to their different enzymatic inhibition. This paper deals with one of the groups of the pseudodistamine precursors - trans-3(4)-aminopiperidin-4(3)-ols. Their synthesis brings to a mixture of 2 regioisomers, resulting in the necessity of their reliable recognition. NMR spectroscopy commonly used by organic chemists requires advance knowledge and experience to analyse the spectra of these regioisomers. Therefore, we herein proposed a simpler way to recognize trans-3(4)-aminopiperidin-4(3)-ols using mass spectrometry with electron ionization. Fragmentation of 4 pairs of aminopiperidinol regioisomers with variation of amine moiety was studied. The obtained results allowed defining a group of 3 ions ([M-18]+., [M-19]+, [M-43]+) related only to the structure of trans-4-aminopiperidin-3-ols and 1 ion (m/z 100) related to the structure of trans-3-aminopiperidin-4-ols. Besides, interrogation of intensity of ions common for spectra of both regioisomers allows making differentiation as well.

Performance of a polymer coated silicon microarray for simultaneous detection of food allergen-specific IgE and IgG4.

BACKGROUND: Microarray-based component-resolved diagnostics (CRD) has become an accepted tool to detect allergen-specific IgE sensitization towards hundreds of allergens in parallel from one drop of serum. Nevertheless, specificity and sensitivity as well as a simultaneous detection of allergen-specific IgG4 , as a potential parameter for tolerance development, remain to be optimized. OBJECTIVE: We applied the recently introduced silicon chip coated with a functional polymer named copoly(DMA-NAS-MAPS) to the simultaneous detection of food allergen-specific IgE and IgG4 , and compared it with ImmunoCAP and ImmunoCAP ISAC. Inter- and intraslide variation, linearity of signal and working range, sensitivity and application of internal calibrations for IgE and IgG4 were assessed. METHODS: Native and recombinant allergenic proteins from hen's egg and cow's milk were spotted on silicon chips coated with copoly(DMA-NAS-MAPS) along with known concentrations for human IgE and IgG4 . A serum pool and 105 patient samples were assessed quantitatively and semi-quantitatively with the ImmunoCAP and ImmunoCAP ISAC and correlated with IgE- and IgG4 -specific fluorescence on silicon microarrays. RESULTS: Allergen-specific IgE and IgG4 were detected in parallel using two fluorescent dyes with no crosstalk. Results from the ImmunoCAP correlated better with microarray fluorescence than with ImmunoCAP ISAC except for the allergen ovomucoid. The working range of the silicon microarray for total hen's egg-specific IgE was comparable to the range of 0.1 to >100 kUA /L of the ImmunoCAP system, whereas for total cow's milk, the silicon microarray was less sensitive. Detectable allergen-specific IgG4 could be determined only for low concentrations, but still correlated positively with ImmunoCAP results. CONCLUSIONS: We confirmed the ability of the polymer coated silicon microarray to be comparably sensitive to the ImmunoCAP ISAC for various food allergens. This suggests that the copoly(DMA-NAS-MAPS) microarray is a low-cost, self-producible alternative to the commercial ImmunoCAP ISAC in allergy research.

[The role and place of high-dose immunosuppressive therapy and autologous transplantation of hematopoietic stem cells for autoimmune diseases].

AIM: To determine the possible boundaries of high-dose immunosuppressive therapy and autologous hematopoietic stem cell transplantation (HDIT-autoHSCT) for autoimmune diseases (AUDs), such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS). SUBJECTS AND METHODS: A long-term trial was conducted at one center to evaluate the efficiency and safety of HDIT-autoHSCT in patients with AUDs. The previous standard therapy was noted to be resistant or lowly effective. The age of 10 patients with systemic connective tissue diseases was 27.6±2.8 years; the pre-HDIT-autoHSCT disease duration was 5.9±1.3 years; the median posttransplantation follow-up was 39.3 months. The age of 49 patients with MS reached 34.9±1.33 years; the pretransplantation disease duration was 8.4±0.69 years; the median post-HDIT-autoHSCT follow-up was 42 months. The efficiency of transplantation was evaluated on the basis of clinical findings, by using scales, laboratory tests, and magnetic resonance imaging. Pretransplantation conditioning was carried out according to the protocols: a) BEAM + antilymphocyte globulin (ALG); b) fludarabine + melphalan + ALG. No fatal outcomes due to a transplant procedure were observed. RESULTS: Overall 5-year survival after transplantation was 80% for systemic connective tissue diseases and 95% for MS; 5-year progression-free survival rates were 30% in the RA and SLE groups and 45% in the MS group. HDIT-autoHSCT turned out safe and reduced the activity of the process and further disease progression for a long period of time, as confirmed by regression of clinical symptoms and/or status stabilization in 9 patients with SLE or RA and in all patients with MS. CONCLUSION: The favorable factors associated with the results of transplantation are age younger than 35 years in collagenoses with their short-term duration and moderate signs; age younger than 40 years in MS with a disease duration of less than 10 years and expanded disability status scale scores of not more than 6.5. Of importance are functional system scores, duration of first remission, and an index of disease progression in different types of MS.

Identification and characterization of DC-SIGN-binding glycoproteins in allergenic foods.

BACKGROUND: DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin) is a C-type lectin receptor expressed on macrophages and dendritic cells. DC-SIGN has high affinity for fucosylated glycans in several plant glycoproteins and pathogens. DC-SIGN is thought to be crucial for the development of allergic sensitization. However, the precise role of DC-SIGN in food allergy pathogenesis is not yet understood. OBJECTIVE: We sought to characterize DC-SIGN-binding glycoproteins in a panel of allergenic and non-allergenic foods. METHODS: Fluorescent-labeled peanut and soy extracts were used to test protein binding to human monocyte-derived dendritic cells (DCs) by flow cytometry. DC-SIGN-blocking assays were performed by incubating DCs with food extracts followed by staining with anti-DC-SIGN antibody. Using a DC-SIGN-Fc chimera, food extracts were tested for binding by ELISA and autoradiography. IgE immunoblotting was performed with pooled sera from food-allergic subjects. DC activation and maturation were assessed by flow cytometry. RESULTS AND CONCLUSIONS: We demonstrate that peanut agglutinin, a minor peanut allergen, is a novel ligand for DC-SIGN. Peanut agglutinin activates DCs to induce the expression of costimulatory molecules in vitro. We present a comprehensive report on the characterization of DC-SIGN-binding proteins in common allergenic foods such as peanut, soy, tree nuts, egg, and milk. Foods that rarely induce allergy, such as pine nuts, chickpea, and corn, showed no binding to DC-SIGN. Several DC-SIGN-binding proteins show reactivity in serum IgE immunoblots. We have also identified novel non-IgE-binding proteins that interact with DC-SIGN; these proteins may be important for regulating immune responses to these foods.

[Influence of the nitric oxide donors on the microcirculation in infusion therapy of the experimental hemorrhagic shock].

Infusion of the nitric oxide donors L-Arginine (150 mg/kg bolus) and Oxacom (3,2 µM/ kg bolus) with saline solution has been shown improves cardiovascular and metabolic changes in animal model of hemorrhagic shock. As a result improves survival rats. These data made this effect clinically attractive.

Use of nitric oxide producer L-arginine during infusion therapy of experimental hemorrhagic shock.

Experiments on rats showed that infusion of NO precursor L-arginine before bleeding enhanced their tolerance to hemorrhagic shock. When infused after blood loss as a component of saline solution, L-arginine improved efficiency of infusion therapy for hemorrhagic shock and increased survival rate of the animals.

Is epitope recognition of shrimp allergens useful to predict clinical reactivity?

BACKGROUND: Shrimp is a frequent cause of severe allergic reactions world-wide. Due to issues such as cross-reactivity, diagnosis of shrimp allergy is still inaccurate, requiring the need for double-blind, placebo-controlled food challenges (DBPCFC). A better understanding of the relationship between laboratory findings and clinical reactivity is needed. OBJECTIVE: To determine whether sensitization to certain shrimp allergens or recognition of particular IgE epitopes of those allergens are good biomarkers of clinical reactivity to shrimp. METHODS: Thirty-seven consecutive patients were selected with clinical histories of shrimp allergy. Skin prick test, specific IgE determinations, DBPCFC and immunoblot assays to shrimp extract were performed. IgE binding to synthetic overlapping peptides representing the sequence of the four allergens from the Pacific white shrimp (Litopenaeus vannamei) identified to date (Lit v1, Lit v2, Lit v3 and Lit v4) was analysed. RESULTS: Of 37 (46%) patients, 17 had a positive challenge to shrimp (11 children and 6 adults). By microarray, patients with positive challenges showed more intense binding to shrimp peptides than those with negative challenges. Statistically significant differences in terms of the frequency and intensity of IgE binding to some epitopes were observed between the two groups. Diagnostic efficiency was higher for individual epitopes than for proteins. Particularly, efficiency was highest for certain Lit v 1 and Lit v 2 epitopes, followed by Lit v 3 and Lit v 4 epitopes. CONCLUSION AND CLINICAL RELEVANCE: Patients with positive shrimp challenges present in general more intense and diverse epitope recognition to all four shrimp allergens. IgE antibodies to these shrimp epitopes could be used as biomarkers for prediction of clinical reactivity in subjects with sensitization to shrimp. Patients with positive shrimp challenges show more intense sensitization and more diverse epitope recognition. Several IgE-binding shrimp epitopes could be used as biomarkers for predicting clinical reactivity in subjects with sensitization to shrimp.

[Use of regulators of the nitric oxide synthesis in experimental hemorrhagic shock and its infusion therapy].

In a model of volume-controlled hemorrhagic shock in rats bolus injection prior to hemorrhagic of non-selective inhibitor of the nitric oxide synthases--N-Nitro-L-Arginine at the dose 250 mg/kg promotes considerable blood flow redistribution and rapid death of animals. However the donor of nitric oxide--L-Arginine (300 mg/kg) enchances stability of animals in hemorrhagic shock. Infusion of L-Arginine (300 mg/kg) with physiological salt solution after bleeding restored cardiac function and microcirculation in the serous membrane of the small intestine of rats. These data suggest that generation of nitric oxide in early stage of hemorrhagic shock may be protective reaction for supporting of normal perfusion of tissues.

Identification of two pistachio allergens, Pis v 1 and Pis v 2, belonging to the 2S albumin and 11S globulin family.

BACKGROUND: IgE-mediated allergic reactions to pistachio appear to be occurring more frequently; however, little is known about its allergenic proteins. OBJECTIVE: We attempted to identify pistachio allergens and to clone the encoding genes. METHODS: Pistachio proteins were extracted and separated by SDS-PAGE. Immunolabelling was performed with sera from 28 pistachio-allergic individuals. Proteins of interest were further analysed by Edman sequencing and mass spectrometry/mass spectrometry (MS/MS). In parallel, a cDNA library was generated from immature pistachios and screened with primers designed on the basis of internal sequences and peptide spectra. Full-length cDNA clones were isolated from the library and sequenced. Recombinant proteins were expressed and tested with sera from pistachio-allergic patients. RESULTS: Nineteen out of 28 patients (68%) showed IgE binding to a 7 kDa protein fraction, while 14 (50%) showed specific IgE to a 32 kDa protein fraction. Analysis by Edman sequencing and MS/MS revealed that these proteins were homologue to the cashew nut allergens Ana o 3 and Ana o 2, respectively. Screening of the pistachio cDNA library resulted in isolation of novel protein cDNAs. Open-reading frame translation provided the complete amino acid sequences of two new allergenic pistachio proteins. Recombinant proteins were recognized by six out of six selected patients. Therefore, these new allergens were named Pis v 1 and Pis v 2 by the Allergen Nomenclature Subcommittee. CONCLUSION: Novel allergens in pistachio, Pis v 1 and Pis v 2, which belong to 2S albumin and 11S globulin family, respectively, were isolated and the genes encoding these allergens were identified.

A surface-exposed region of G(salpha) in which substitutions decrease receptor-mediated activation and increase receptor affinity.

The mechanism by which receptors activate G proteins is unclear because a connection between the receptor and the nucleotide binding site has not been established. To investigate this mechanism, we evaluated the roles in receptor interaction of three potential receptor contact sites in alpha(s): the alpha2/beta4, alpha3/beta5, and alpha4/beta6 loops. Substitutions of alpha(i2) homologs for alpha(s) residues in the alpha2/beta4 loop and alanine substitutions of residues in the alpha4/beta6 loop do not affect activation by the beta(2)-adrenergic receptor. However, replacement of five alpha(s) residues in the alpha3/beta5 loop region with the homologous alpha(i2) residues decreases receptor-mediated activation of alpha(s) and increases the affinity of G(s) for this receptor. The substitutions do not alter guanine nucleotide binding or hydrolysis, or activation by aluminum fluoride, indicating that the effects on receptor interaction are not due to a destabilization of the guanine-nucleotide bound state. In a model of the receptor-G protein complex, the alpha3/beta5 loop maps near the second and third intracellular loops of the receptor. The effects of the alpha3/beta5 substitutions suggest that the wild-type residues may be receptor contact sites that are optimized to ensure the reversibility of receptor-G protein interactions. Furthermore, the alpha3/beta5 region corresponds to an exchange factor contact site in both EF-Tu and Ras, suggesting that the mechanisms by which seven-transmembrane receptors and exchange factors catalyze nucleotide exchange may share common elements.

Peptides from bovine brain: structure and biological role.

Fractionation of bovine brain extracts followed by automatic Edman sequencing of individual components resulted in identification of 107 endogenous peptides formed from functional proteins (haemoglobin, myelin basic protein, cytochrome c oxidase, etc) or unknown precursors. Several of the newly identified brain peptides demonstrate different types of biological activity; some of the substances show considerable overlap with the known biologically active peptides. It is suggested that these peptides should participate in regulation of extracellular and intracellular biochemical processes. A concept of 'tissue-specific peptide pool' is formulated describing a novel system of peptidergic regulation, complementary to the conventional hormonal and neuromodulatory systems. According to that description functional proteins provide their proteolytically derived fragments for maintaining the tissue homeostasis by modulating the availability of peptide receptors to respective 'true' ligands.

Mutations at the domain interface of GSalpha impair receptor-mediated activation by altering receptor and guanine nucleotide binding.

G protein alpha subunits consist of two domains, a GTPase domain and a helical domain. Receptors activate G proteins by catalyzing replacement of GDP, which is buried between these two domains, with GTP. Substitution of the homologous alphai2 residues for four alphas residues in switch III, a region that changes conformation upon GTP binding, or of one nearby helical domain residue decreases the ability of alphas to be activated by the beta-adrenergic receptor and by aluminum fluoride. Both sets of mutations increase the affinity of alphas for the beta-adrenergic receptor, based on an increased amount of high affinity binding of the beta-adrenergic agonist, isoproterenol. The mutations also decrease the rate of receptor-mediated activation and disrupt the ability of the beta-adrenergic receptor to increase the apparent affinity of alphas for the GTP analog, guanosine 5'-O-(3-thiotriphosphate). Simultaneous replacement of the helical domain residue and one of the four switch III residues with the homologous alphai2 residues restores normal receptor-mediated activation, suggesting that the defects caused by mutations at the domain interface are due to altered interdomain interactions. These results suggest that interactions between residues across the domain interface are involved in two key steps of receptor-mediated activation, promotion of GTP binding and subsequent receptor-G protein dissociation.

Receptor-mediated activation of Gsalpha: evidence for intramolecular signal transduction.

To investigate the mechanism by which cell surface receptors activate heterotrimeric G proteins, we applied a scanning mutagenesis approach to the carboxyl-terminal 40% of alphas (residues 236-394) to identify residues that play a role in receptor-mediated activation. We identified four regions of sequence in which mutations significantly impaired receptor-dependent stimulation of cAMP synthesis in transiently transfected cyc- S49 lymphoma cells, which lack endogenous alphas. Residues at the carboxyl terminus are likely to be receptor contact sites. Buried residues near the bound GDP are connected to the carboxyl terminus by an alpha helix and may regulate GDP affinity. Residues in two adjacent loops of the GTPase domain at the interface with the helical domain, one of which includes a region, switch III, that changes conformation on GTP binding, are positioned to relay the receptor-initiated signal across the domain interface to facilitate GDP release. Consistent with this hypothesis, replacing the helical domain of alphas with that of alphai2 in an alphas/alphai2/alphas chimera corrects the defect in receptor-mediated activation caused by alphai2 substitutions on the GTPase side of the interface. Thus, complementary interactions between residues across the domain interface seem to play a role in receptor-catalyzed activation.

Identification of common and distinct residues involved in the interaction of alphai2 and alphas with adenylyl cyclase.

The G protein alpha subunits, alphas and alphai2, have stimulatory and inhibitory effects, respectively, on a common effector protein, adenylyl cyclase. These effects require a GTP-dependent conformational change that involves three alpha subunit regions (Switches I-III). alphas residues in three adjacent loops, including Switch II, specify activation of adenylyl cyclase. The adenylyl cyclase-specifying region of alphai2 is located within a 78-residue segment that includes two of these loops but none of the conformational switch regions. We have used an alanine-scanning mutagenesis approach within Switches I-III and the 78-residue segment of alphai2 to identify residues required for inhibition of adenylyl cyclase. We found a cluster of conserved residues in Switch II in which substitutions cause major losses in the abilities of both alphai2 and alphas to modulate adenylyl cyclase activity but do not affect alpha subunit expression or the GTP-induced conformational change. We also found two regions within the 78-residue segment of alphai2 in which substitutions reduce the ability of alphai2 to inhibit adenylyl cyclase, one of which corresponds to an effector-activating region of alphas. Thus, both alphai2 and alphas interact with adenylyl cyclase using: 1) conserved Switch II residues that communicate the conformational state of the alpha subunit and 2) divergent residues that specify particular effectors and the nature of their modulation.

Localization of the effector-specifying regions of Gi2alpha and Gqalpha.

Heterotrimeric G proteins transmit hormonal and sensory signals received by cell surface receptors to effector proteins that regulate cellular processes. Members of the highly conserved family of alpha subunits specifically modulate the activities of a diverse array of effector proteins. To investigate the determinants of alpha subunit-effector specificity, we localized the effector-specifying regions of alphai2, which inhibits adenylyl cyclase, and alphaq, which stimulates phosphoinositide phospholipase C using chimeric alpha subunits. The chimeras were generated using an in vivo recombination method in Escherichia coli. The effector-specifying regions of both alphai2 and alphaq were localized within the GTPase domain. An alphaq/alphai2/alphaq chimera containing only 78 alphai2 residues within the GTPase domain robustly inhibited adenylyl cyclase. This alphai2 segment includes regions corresponding to two of the three regions of alphas that activate adenylyl cyclase, but does not include any of the alpha subunit regions that switch conformation upon binding GTP. Replacement of the alphaq residues that comprise the helical domain with the homologous alphai2 residues resulted in a chimeric alpha subunit that activated phospholipase C. Combined with previous studies of the effector-specifying residues of alphas and alphat, our results suggest that the effector specificity of alpha subunits is generally determined by the GTPase and not the helical domain.

Duodenase, a new serine protease of unusual specificity from bovine duodenal mucosa. Primary structure of the enzyme.

The complete amino acid sequence of duodenase, a new serine endopeptidase from bovine duodenal mucosa, has been determined. The sequence was reconstructed by the automated sequence analysis of the peptides obtained after cleavage with trypsin, Staphylococcus aureus V8 protease, cyanogen bromide and duodenase. The enzyme is composed of 226 amino acid residues yielding a molecular mass of 29.06 kDa. The presence of six cysteine residues and one potential sugar-chain-binding site at Asn50 was revealed. A predicted catalytic triade characteristic of the serine proteases was traced in the duodenase primary structure at the corresponding positions (His44, Asp87 and Ser181 in the sequence). Comparison of the sequence of duodenase with the other known primary structures of mammalian serine proteinases reveales the duodenase identity to granzymes from human and mice, human cathepsin G and mast cell chymases from rat, and gives an overall sequence identity of 47-55% with the mentioned enzymes. Alignment of the known serine protease and duodenase primary structures showed unique amino acid residues within the duodenase substrate-binding pocket at positions 189 (Asn) and 226 (Asp) (the bovine chymotrypsinogen A numbering). These results are discussed with respect to the relation between the duodenase unique residues within the primary specificity pocket S1 and the unusual dual specificity of the enzyme.

Serological survey on hepatitis E virus infection in an endemic area: diagnosis potential of enzyme immunoassay for detection of IgG antibody.

BACKGROUND: Hepatitis E is a major cause of acute icteric disease widespread in tropical and sub-tropical regions but rarely occurring in industrialized countries. Recently solid-phase enzyme immunoassays with recombinant antigens have been introduced for diagnosis of this infection. OBJECTIVES: The aim of this study was to evaluate the diagnostic potential of a newly developed Abbott test for the detection of IgG class antibodies to hepatitis E virus (anti-HEV IgG) in hepatitis patients and 'normal' individuals. STUDY DESIGN: Sera taken from hepatitis patients and individuals without liver disorders in endemic (Kirghizstan and Uzkbekistan) versus non-endemic (Moscow) areas were investigated. In parallel IgG class antibodies to hepatitis A virus (anti-HAV IgG) were determined by an enzyme immunoassay with native HAV antigen. RESULTS: In five groups comprising altogether 86 suspected hepatitis E patients from endemic area the rate of anti-HEV IgG seropositivity varied from 85% to 17%. In Moscow anti-HEV IgG was found in one patient (who also had acute hepatitis B) out of 19. Anti-HEV IgG persisted in an experimentally infected volunteer for at least 12 years after the acute disease. Among the individuals without liver disorders eight out of 173 (4.6%) showed anti-HEV IgG seropositivity in Kirghizstan while there was only one seropositive out of 165 (0.6%) in Moscow. In contrast, anti-HAV IgG were frequently present in the residents of both areas: in Kirghizstan over 90% of individuals from young age groups already had these antibodies; in Moscow the rate of anti-HAV IgG seropositivity constantly increased from 31% in the youngest age group to almost 85% in the oldest one. CONCLUSION: Prevalence of anti-HEV antibodies was unexpectedly low in endemic area; in Moscow anti-HEV IgG was found only in single cases. Anti-HEV IgG seropositivity in a single serum sample could be of certain diagnostic value in non-endemic areas.

Recombinant human insulin. III. High-performance liquid chromatography and high-performance capillary electrophoresis control in the analysis of step-by-step production of recombinant human insulin.

The production of recombinant human insulin consists of five main stages, accompanied by considerable transformation of molecules, concerning size, secondary structure and the presence of charged groups. The application of different methods, i.e., size-exclusion, ion-exchange and reversed-phase high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE) (capillary zone electrophoresis and micellar electrokinetic capillary chromatography), to the analysis of insulin, insulin-related and non-insulin-related substances was studied. A combined HPLC-HPCE system for the step-by-step control of recombinant human insulin production technology is suggested. The advantages and shortcomings of these methods are discussed.