Molecular Characterization of Recombinant Mus a 5 Allergen from Banana Fruit

Allergy to banana fruit appears to have become an important cause of fruit allergy in Europe. Among five allergens that have been found, beta-1,3-glucanase denoted as Mus a 5 was identified as a candidate allergen for the component-resolved allergy diagnosis of banana allergy. Because of the variations in protein levels in banana fruit, in this study Mus a 5 was produced as a fusion protein with glutathione-S-transferase in Escherichia coli. The recombinant Mus a 5 was purified under native conditions by a combination of affinity, ion-exchange, and reversed phase chromatography. N-terminal sequence was confirmed by Edman degradation and 55 % of the primary structure was identified by mass fingerprint, while the secondary structure was assessed by circular dichroism spectroscopy. IgG reactivity of recombinant protein was shown in 2-D immunoblot with anti-Mus a 5 antibodies, while IgG and IgE binding to natural Mus a 5 was inhibited with the recombinant Mus a 5 in immunoblot inhibition test. IgE reactivity of recombinant Mus a 5 was shown in ELISA within a group of ten persons sensitized to banana fruit. Recombinant Mus a 5 is a novel reagent suitable for the component-resolved allergy diagnosis of banana allergy.     

Direct analysis of α- and β-chains of hemoglobins from mammalian blood samples by nanoESI mass spectrometry during in-capillary proteolytic digestion

α- and β-chains of hemoglobins derived from several species were analyzed directly from diluted blood samples by simultaneous in-capillary proteolytic digestion and nanoESI MS and MS/MS analysis. Starting from fresh or frozen and thawed blood samples, sequence coverages of >80% were usually obtained. Only 2 h after resuspension of a dried blood spot, human origin could be demonstrated from data obtained by in-capillary tryptic digestion, nanoESI mass spectrometric analysis, and data base search. A fast and facile differentiation of closely related species by hemoglobin-derived proteolytic “marker peptides” was demonstrated for Asian (Elephas maximus) and African elephants (Loxodonta africana). Finally, amino acid sequences deduced from collision-induced dissociation experiments during in-capillary proteolytic digestion of the corresponding blood samples allowed de novo sequencing of previously unknown sequences of hemoglobin chains of the Patagonian cavy (Dolichotum patagona) and the Persian gazelle (Gazella subgutturosa subgutturosa). 100% of the α-chain sequences and more than 85% of the β-chain sequences were covered for both the species. Additionally, sequence data derived from tandem MS experiments obtained with the Q-Tof analyzer were confirmed by high resolution Fourier-transform ion cyclotron resonance mass spectrometric experiments. Accurate protein mass determination of the intact hemoglobin chains directly from the corresponding blood samples by use of a Fourier-transform ion cyclotron resonance mass spectrometer corroborated the deduced sequences of the respective α-chains. The present study demonstrates that in-capillary digestion allows fast characterization and/or sequencing of hemoglobin chains directly from blood samples.     

Tracking Molecular Recognition at the Atomic Level with a New Protein Scaffold Based on the OB-Fold

The OB-fold is a small, versatile single-domain protein binding module that occurs in all forms of life, where it binds protein, carbohydrate, nucleic acid and small-molecule ligands. We have exploited this natural plasticity to engineer a new class of non-immunoglobulin alternatives to antibodies with unique structural and biophysical characteristics. We present here the engineering of the OB-fold anticodon recognition domain from aspartyl tRNA synthetase taken from the thermophile Pyrobaculum aerophilum. For this single-domain scaffold we have coined the term OBody. Starting from a naïve combinatorial library, we engineered an OBody with 3 nM affinity for hen egg-white lysozyme, by optimising the affinity of a naïve OBody 11,700-fold over several affinity maturation steps, using phage display. At each maturation step a crystal structure of the engineered OBody in complex with hen egg-white lysozyme was determined, showing binding elements in atomic detail. These structures have given us an unprecedented insight into the directed evolution of affinity for a single antigen on the molecular scale. The engineered OBodies retain the high thermal stability of the parental OB-fold despite mutation of up to 22% of their residues. They can be expressed in soluble form and also purified from bacteria at high yields. They also lack disulfide bonds. These data demonstrate the potential of OBodies as a new scaffold for the engineering of specific binding reagents and provide a platform for further development of future OBody-based applications.     

Sprouted and Freeze‐Dried Wheat and Oat Seeds – Phytochemical Profile and in Vitro Biological Activities

This research was carried out to study phytochemical profile, in vitro antioxidant capacity, reducing power, anti‐hyperglycemic, anti‐inflammatory activities and simulated gastrointestinal digestion of 7‐day old cereal sprouts: spelt wheat ‘Nirvana’ (WSSpe), wheat ‘Simonida’ (WSSim), oat ‘Golozrni’ (OSG) and oat ‘Jadar’ (OSJ). OSG expressed significantly higher (P ≤ 0.05) total phenols (TPC) and flavonoids content (TFC), antioxidant capacities (DPPH and ABTS assays) and reducing power (EC₅₀^DPPH = 2.12 mg/ml; EC₅₀^ABTS = 0.87 mg/ml; EC_0.5^RP = 12.24 mg/ml) as well as anti‐hyperglycemic activity (EC₅₀^AHgA = 0.96 mg/ml). WSSpe had the highest content of chlorophyll (131.23 mg/100 g) and carotenoids (22.84 mg/100 g). WSSim possessed the most potent anti‐inflammatory activity (2.71 mg/ml), though not significantly different from OSG (2.77 mg/ml). The in vitro simulation of gastro‐intestinal digestion showed higher release of phenolic compounds in intestinal than in gastric fluid.     

Development of calcium bodies in Hylonsicus riparius (Crustacea: Isopoda)

Calcium bodies are internal epithelial sacs found in terrestrial isopods of the family Trichoniscidae that contain a mineralized extracellular matrix that is deposited and resorbed in relation to the molt cycle. Calcium bodies in several trichoniscids are filled with bacteria, the function of which is currently unknown. The woodlouse Hyloniscus riparius differs from other trichoniscids in that it possesses two different pairs of calcium bodies, the posterior pair being filled with bacteria and the anterior pair being devoid of bacteria. We explored the development of these organs and bacterial colonization of their lumen during the postmarsupial development with the use of optical clearing and whole-body confocal imaging of larval and juvenile stages. Our results show that calcium bodies are formed as invaginations of the epidermis in the region of intersegmental membranes during the postmarsupial development. The anterior pair of calcium bodies is generated during the first postmarsupial manca stage, whereas the posterior calcium bodies first appear in juveniles and are immediately colonized by bacteria, likely through a connection between the calcium body lumen and the body surface. Mineral is deposited in calcium bodies as soon as they are present.     

Mapping of Mhc class I and class II regions to different linkage groups in the zebrafish, Danio rerio

 The mammalian major histocompatibility complex (Mhc) consists of three closely linked regions, I, II, and III, occupying a single chromosomal segment. The class I loci in region I and the class II loci in region II are related in their structure, function, and evolution. Region III, which is intercalated between regions I and II, contains loci unrelated to the class I and II loci, and to one another. There are indications that a similar Mhc organization exists in birds and amphibians. Here, we demonstrate that in the zebrafish (Danio rerio), a representative of the teleost fishes, the class II loci are divided between two linkage groups which are distinct from the linkage group containing the class I loci. The β₂-microglobulin-encoding gene is loosely linked to one of the class II loci. The gene coding for complement factor B, which is one of the region III genes in mammals, is linked neither to the class I nor to the class II loci in the zebrafish. These results, combined with preliminary data suggesting that the class I and class II regions in another order of teleost fish are also in different linkage groups, indicate that close linkage of the two regions is not necessary either for regulation of expression or for co-evolution of the class I and class II loci. They also raise the question of whether linkage of the class I and class II loci in tetrapods is a primitive or derived character. Received: 16 December 1996 / Revised: 6 February 1997     

Structural studies of gangliosides from the YAC-1 mouse lymphoma cell line by immunological detection and fast atom bombardment mass spectrometry

YAC-1 cells were propagated in bioreactors in 11 and 7.51 volumes. The cells were metabolically labelled withd-[1-¹⁴C]galactose andd-[1-¹⁴C]glucosamine. The ganglioside fraction, purified by DEAE-Sepharose and silica gel column chromatography, showed on thin layer chromatography four major bands with mobilities between G_M1 and G_D1a. Gangliosides, obtained by further purification steps including high performance liquid chromatography on silica gel 60 columns with a gradient system of isopropanol:hexane:water, and preparative high performance TLC were characterized by (1) immunostaining of corresponding asialogangliosides obtained by mild acid hydrolysis and neuraminidase treatment and (2) fast atom bombardment mass spectrometry of native and permethylated samples and methylation analysis of G_M1b ganglioside. As well as small amounts of G_M2 and G_M1, the major gangliosides found in the complex mixture were G_M1b and GalNAc-G_M1b. The structural heterogeneity of these gangliosides was cased by (a) substitution of the ceramide moiety by fatty acids of different chain length and degree of unsaturation (C_16:0, C_24:0, C_24:1) and (b) N-substitution of the sialic acid moieties with either acetyl or glycolyl groups. Disialogangliosides were detected only in low amounts and will be the subject of further investigation. A polyclonal chicken antiserum was raised against IVNeuAc-GgOse₅Cer. The antiserum was highly specific for gangliosides (IVNeuAc and IVNeuGc) and asialogangliosides with a GgOse₅Cer backbone. No cross-reaction with G_M1b or GgOse₄Cer was observed. Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography, HPTLC, high performance thin layer chromatography; NK, natural killer; SIM, selective ion monitoring; TIC, total ion current. NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid. The designation of the following glycosphingolipids follows the IUB-IUPAC recommendations. GgOse₃Cer or gangliotriaosylceramide or asialo G_M2, GalNAc1-4Gal1-4GlcCer; GgOse₄Cer or gangliotetraosylceramide or asialo G_M1, Gal1-3GalNAc1-4Gal1-4GlcCer; GgOse₅Cer organgliopentaosylceramide, GalNAc1-4Gal1-3GalNAc1-4Gal1-4GlcCer; II³NeuAc-GgOse₄Cer or G_M1; IV³NeuAcGgOse₄Cer or G_M1b; IV³NeuAc-GgOse₅Cer or GalNAc-G_M1b; IV³NeuAc, II³NeuAc-GgOse₄Cer or G_D1a; II³(NeuAc)₂-GgOse₄Cer or G_D1b; IV³(NeuAc)₂-GgOse₄Cer or G_D1c; IV³NeuAc,III⁶NeuAc-GgOse₄Cer or G_D1a; IV³NeuAc,II³(NeuAc)₂-GgOse₄Cer or G_T1b;Vibrio cholerae and Arthrobacter ureafaciens neuraminidase (EC 3.2.1.18).     

Profile and multidrug resistance determinants of Chryseobacterium indologenes from seawater and marine fauna

The aim of this study was to investigate the prevalence and genetic basis of multidrug resistance in Chryseobacterium indologenes from seawater and marine invertebrates used for human consumption, in Ka?tela Bay, Adriatic Sea, Croatia. Out of 16 samples of seawater, Mediterranean mussel (Mytilus galloprovincialis Lam.), Rayed Mediterranean limpets (Patella caerulea L.) and Purple sea urchins (Paracentrotus lividus Lam.) collected, 15 were positive for C. indologenes. In total, 41 isolates were randomly selected and tested for antibiotic susceptibility by disc-diffusion and broth microdilution methods. PCR was used to detect alleles encoding extended-spectrum (ESBLs) and metallo-β-lactamases (MBLs). The clonality of β-lactamase-producing strains was evaluated by random amplified polymorphic DNA (RAPD) analysis. All C. indologenes isolates showed multiple resistance to at least 9 out of 16 antibiotics tested. Lowest resistance rates were found for piperacillin (9.7 %) and ciprofloxacin (24.4 %), whereas only piperacillin/tazobactam and trimethoprim/sulfamethoxazole showed 100 % activity. More than half of isolates carried bla _IND-type gene, including 2 isolates carrying bla _IND-2 and 21 carrying bla _IND-7, that was identified as a major MBL genotype in isolates from Adriatic Sea. RAPD typing of IND-producing isolates revealed 6 major groups with no predominant clone in population. The presence of multidrug resistant and IND-producing C. indologenes in marine environment, including marine fauna, pose a risk for transmitting this opportunistic pathogen to humans through recreation or consummation of seafood. In addition, the antibiotic susceptibility test results have practical relevance for empirical treatment of C. indologenes infections.     

Task and Spatial Frequency Modulations of Object Processing: An EEG Study

Visual object processing may follow a coarse-to-fine sequence imposed by fast processing of low spatial frequencies (LSF) and slow processing of high spatial frequencies (HSF). Objects can be categorized at varying levels of specificity: the superordinate (e.g. animal), the basic (e.g. dog), or the subordinate (e.g. Border Collie). We tested whether superordinate and more specific categorization depend on different spatial frequency ranges, and whether any such dependencies might be revealed by or influence signals recorded using EEG. We used event-related potentials (ERPs) and time-frequency (TF) analysis to examine the time course of object processing while participants performed either a grammatical gender-classification task (which generally forces basic-level categorization) or a living/non-living judgement (superordinate categorization) on everyday, real-life objects. Objects were filtered to contain only HSF or LSF. We found a greater positivity and greater negativity for HSF than for LSF pictures in the P1 and N1 respectively, but no effects of task on either component. A later, fronto-central negativity (N350) was more negative in the gender-classification task than the superordinate categorization task, which may indicate that this component relates to semantic or syntactic processing. We found no significant effects of task or spatial frequency on evoked or total gamma band responses. Our results demonstrate early differences in processing of HSF and LSF content that were not modulated by categorization task, with later responses reflecting such higher-level cognitive factors.