Cloning, expression, and antigenic characterization of recombinant protein of Mycoplasma gallisepticum expressed in Escherichia coli.

Mycoplasma gallisepticum (MG) is a member of the most important avian mycoplasmas, causing chronic respiratory disease in chickens and leading to important economic losses in the poultry industry. Recombinant technology represents a strategic approach used to achieve highly reliable and specific diagnostic tests in veterinary diseases control: in particular this aspect is crucial for confirming mycoplasma infection and for maintaining mycoplasma-free breeder flocks. In this study, we identified a component of the pyruvate dehydrogenase dihydrolipoamide acetyltransferase (i.e., E2) protein by 2-dimensional electrophoresis (2-DE), characterized it in immunoblotting assays, and analyzed its recombinant (r-E2) in a rec-ELISA test. For full-length protein expression in Escherichia coli (EC) a point mutation was introduced. A rabbit antiserum produced against r-E2 was tested in a Western Blot using different samples of Mycoplasma species. The results showed the applicability of site-directed mutagenesis, with a good yield of the r-E2 after purification. Also, anti-E2 serum reacted with all the tested MG strains showing no cross reaction with other mycoplasmas. The developed E2 ELISA test was capable of detecting MG antibodies in the sera examined. Those results demonstrate the antigenic stability of the E2 protein which could represent a recombinant antigen with potential diagnostic applications.

Shrimp allergy beyond Tropomyosin in Italy: clinical relevance of Arginine Kinase, Sarcoplasmic calcium binding protein and Hemocyanin.

BACKGROUND: Little is known about the prevalence and clinical relevance of sensitization to shrimp allergens other than tropomyosin. OBJECTIVE: We detected the prevalence of arginine kinase and sarcoplasmic calcium binding protein sensitization, and identified a high molecular weight allergen that is frequently recognized by Italian shrimp-allergic patients. METHODS: Sera from 40 shrimp-allergic patients underwent the detection of IgE specific for arginine kinase (rPen m 2) and sarcoplasmic calcium-binding protein (rPen m 4) by ISAC 112 Microarray platform and immunoblot analysis. A high molecular weight shrimp allergen was identified by N-terminal amino acid sequencing. RESULTS: IgE to rPen m 2 and rPen m 4 were found in 4/40 (10%) and 6/40 (15%) sera, respectively; two sera reacted to both allergens. Clinically, 6/8 Pen m 2 and/or Pen m 4 reactors experienced severe allergies to shrimp. On immunoblot, 4/6 rPen m 4-positive sera showed IgE reactivity at about 20 kDa, whereas no rPen m 2-positive serum reacted at about 40 kDa. Nineteen (47%) sera showed IgE reactivity at molecular weights > 60 kDa. Such profile was not associated with IgE reactivity to rPen m 2 or rPen m 4. N-terminal amino acid sequencing of the high molecular weight allergen led to the identification of hemocyanin. CONCLUSION: Shrimp arginine kinase and sarcoplasmic calcium-binding protein are minor allergens sensitizing only 10%-15% of Italian shrimp-allergic patients, but are clinically relevant. Hemocyanin is a clinically relevant high molecular weight shrimp allergen possibly cross-reacting to house dust mite.

Mugwort-fennel-allergy-syndrome associated with sensitization to an allergen homologous to Api g 5.

BACKGROUND: The cross-reactive allergen responsible for the so called "mugwort-celery-spice-syndrome", a pollen-food allergy that occurs in a minority of mugwort pollen-allergic patients, is still undefined. OBJECTIVE: To identify the allergen responsible for the cross-reactivity between mugwort pollen and plant-derived foods. METHODS: The serum from one index patient with both fennel and mugwort pollen allergy was used to identify IgE-reactive allergens by direct ELISA and Immunoblot analysis. Cross-reactivity between mugwort pollen and fennel was checked by cross-inhibition experiments. Fennel and mugwort allergens selected on the basis of IgE reactivity and inhibition tests were excised from SDS-PAGE gels and microsequenced. The amino acid sequences obtained were used to screen the NCBI database using the protein BLAST software. RESULTS: On ELISA inhibition experiments, serum absorption with fennel extract completely inhibited the IgE response to mugwort. On immmunoblot analysis periodate treatment caused the disappearance of all bands of IgE reactivity except one at about 60 kDa. The 60 kDa bands from both mugwort and fennel PAGE-SDS gels revealed the presence of distinct proteins. The N-terminal amino acid sequencing gave the same major amino acid sequence corresponding to an Api g 5-like allergen. The MS/MS spectra were analyzed and a provided evidence of a fennel-specific protein with sequence similarity to phosphoglyceromutase from Apium graveolens. CONCLUSION: A 60 kDa allergen, highly homologous to Api g 5, was recognized in fennel by patient's IgE. Inhibition experiments showed a high degree of cross-reactivity between this fennel allergen and the homologous mugwort pollen allergen. This allergen might be responsible for the mugwort-celery-spice syndrome.

Prenatal diagnosis and molecular characterization of an interstitial 1q24.3-31.3 deletion: case report and review.

We describe a foetus with an interstitial deletion of 1q detected in amniotic fluid cells and we review the literature of similar pre- and postnatal cases, in order to identify prognostic factors useful for prenatal counselling. Foetal/parents karyotyping and FISH with whole chromosome 1 paint and BAC clone specific for 1q23-32 region were performed. Further 100 Kb resolution array-CGH analysis was executed after pregnancy termination on DNA extracted from foetal skin fibroblasts. Cytogenetic analyses revealed a de novo interstitial deletion involving the long arm of chromosome 1. FISH analysis confirmed that the deletion involves the intermediate 1q31.2 region. Foetal ultrasound (US), performed at 21 weeks of gestation, showed intrauterine growth restriction, shortening of the long bones, echogenic intracardiac focus and mild cerebral ventriculomegaly. Array-CGH localized the deletion in a DNA sequence of about 21 Mb in the 1q24.3-q31.3 region. Our findings, together with available data on patients with 1q deletion, suggest that the most severe phenotypes are not simply associated with larger deletion, and that the results of prenatal US assessment, rather than a fine molecular characterization of the deletion, should be taken into account for prognostic evaluation.

Authentication of food allergen quality by physicochemical and immunological methods.

Purified allergens are required to detect cross-contamination with other allergenic foods and to understand allergen interaction with other components of the food matrix. Pure allergens are also used for the diagnosis and treatment of food allergies. For example, serological methods are being developed to improve the quality of diagnosis, and to reduce the need for food challenge tests. In addition, recombinant allergens are being evaluated as candidate vaccines for safe and efficacious specific immunotherapy. Pure allergens are indispensable as reference materials for the calibration and standardization of methods between different laboratories and operators for risk assessment in the food industry. Therefore, there is a need for well-defined purified food allergens. In this context, a panel of 46 food allergens from plant and animal sources has been purified, from either the food sources or as recombinant forms, within the EU-funded EuroPrevall project. These allergens have been characterized by a battery of diagnostic tests demonstrating that they constitute an authentic, well-defined library of comparable quality. The review summarizes the applications, potentials and limitations of key techniques used for the characterization and authentication of these allergen preparations, with a special emphasis on protein purity and identity, folding, post-translational modifications and immunochemical properties. One key area identified is the development of powerful analytical techniques, such as mass spectrometry and nuclear magnetic resonance, to improve the authentication of allergens for routine applications in allergy management.

Identification of a caleosin associated with hazelnut (Corylus avellana L.) oil bodies.

Caleosins are involved in several cellular and biological processes that are closely associated with the synthesis, degradation and stability of oil bodies (OB). Because of the importance and the multiple roles of these OB-associated proteins, in silico identification of sequences corresponding to putative caleosins in the hazelnut genome has been performed, and the association with seed OB was verified using a proteomic approach. Five full-length sequences (CavCLO-H1, CavCLO-H2, CavCLO-H3, CavCLO-L1, CavCLO-L2), belonging to the two groups of caleosins (H and L), have been identified in the hazelnut genome. The number of identified caleosins is in agreement with that previously observed in other plant species, confirming that caleosins comprise small gene families in plants. A proteomic approach allowed us to verify only the presence of CavCLO-H1 in hazelnut OB, suggesting that several members inside this family could have different roles during plant growth and development. In silico analysis also suggests that CavCLO-H1 may act as a peroxygenase.

Influence of ethanol, malate and arginine on histamine production of Lactobacillus hilgardii isolated from an Italian red wine.

Wine, like other fermented foods, may contain biogenic amines produced by lactic acid bacteria via amino acids decarboxylation. The most relevant amines from the toxicological standpoint are histamine and tyramine. The complexity of fermented substrates makes it difficult to suggest a priori how variables can modulate amine production. Lactobacillus hilgardii ISE 5211 was isolated from an Italian red wine. Besides producing lactate from malate, this strain is also able to convert arginine to ornithine and histidine to histamine. In the present investigation we studied the influence of malate, arginine and ethanol on histamine accumulation by L. hilgardii ISE 5211. Ethanol concentrations above 13% inhibit both histamine accumulation and bacterial growth; concentrations below 9% affect neither growth nor histamine production. However, an ethanol concentration of 11% allows a low but continuous accumulation of histamine to occur. Arginine also delays histamine accumulation, while malate appears to have no effect on histidine-histamine conversion.

Proteolysis of milk fat globule membrane proteins in preterm milk: a transient phenomenon with a possible biological role?

Milk fat globule membrane (MFGM) proteins constitute a milk fraction currently of great interest, as they appear to significantly contribute to milk protective role. We investigated these proteins in human preterm colostrum and milk. For the former we found a peculiar 2-DE pattern, with a spot concentration at low molecular weight, which mass spectrometry analysis showed to be fragments belonging to some MFGM proteins with a well-known biological and especially immunological role: lactadherin, membrane-associated lactoferrin, butyrophilin, clusterin and heavy-chain immunoglobulin. Since we were able to rule out protease activity after specimen collection, we hypothesize the localization of the proteolytic enzymes in the alveolar cell membranes of the mammary gland. This mechanism is probably under hormonal control and the unexpected advent of preterm delivery would not allow hormonal conditions typical of lactation to occur immediately, causing a delay in enzymatic inhibition. This hypothesis is supported by some of our results, picturing a peculiar transient phenomenon of adaptation of the mammary-gland-membrane proteins after preterm delivery. Further studies will be required to verify whether the presence of protein fragments exerts a specific biological and immuno-defensive role in preterm infants, thus adding evidence to the outstanding biological role and benefits of mother's own milk in feeding preterm infants.

Identification of latex UDP glucose pyrophosphorylase (Hev b UDPGP) as a novel cause of latex fruit allergy syndrome.

Based on a recently published unusual ase of food allergy in a latex-allergic patients, the present study identifies Hev b UDPGP as a novel allergen in natural rubber latex able to cause latex-fruit allergy syndrome and as a novel, potential pan-allergen in vegetable foods.

Isolation of a new ligand-carrying casein fragment from bovine mammary gland microsomes.

Whilst looking for components involved in retinol metabolism in secreting mammary gland cells, a 12 kDa protein was isolated. This protein had bound a ligand with characteristics of retinol. N-Terminal sequencing and amino acid analysis showed that this protein is highly homologous with an alpha-s1-casein fragment. No ligand was found for beta-lactoglobulin, previously thought to be involved in retinol metabolism.

Amino acid sequence and crystal structure of buffalo alpha-lactalbumin.

Isolation, purification, amino acid sequence determination and X-ray crystal structure of buffalo alpha-lactalbumin were performed in order to gain further knowledge of the molecular basis of alpha-lactalbumin in the lactose synthase complex. The deduced amino acid sequence differs at one position from the bovine alpha-lactalbumin sequence (at position 17). The refined crystal structure at 2.3 A is very similar to those previously reported for human and baboon alpha-lactalbumins. However, a portion of the molecule (residues 105-109) exhibits different conformation. It forms a 'flexible loop', and appears to be a functionally important region in forming the lactose synthase complex.

Detection of specific IgE to human milk proteins in sera of atopic infants.

Specific IgE (sIgE) for cow's milk proteins (CMP) have been reported to be present in blood sera of exclusively breast-fed infants. The aim of this study was to find whether the presence of sIgE to human milk proteins in the sera of exclusively breast-fed infants could explain the apparent detection of sIgE to CMP in infants that were never previously in contact with cow's milk. sIgE for human milk whey proteins were found in the blood sera of atopic infants, and these sIgE strongly cross-reacted with the corresponding CMP. In none of the sera examined were sIgE to bovine beta-lactoglobulin detected.

Identification of the human beta-casein C-terminal fragments that specifically bind to purified antibodies to bovine beta-lactoglobulin.

The presence of foreign proteins in human milk after the ingestion of bovine dairy products is thought to be one of the possible causes of allergic sensitization in exclusively breast-fed predisposed infants. The immunologic determination of bovine beta-lactoglobulin (LG) concentration in human milk has been reported by several researchers, but the results are conflicting. Moreover, a strong cross-reactivity between antibodies to bovine beta-LG and human milk proteins and peptides was reported, throwing doubt on the reliability of radioimmunoassay and enzyme-linked immunosorbent assay detection and quantification assays for bovine beta-LG in human milk. Thus, the goal of this study was to isolate human milk peptides with a molecular mass >or= 1,000 Da cross-reactive with antibodies to bovine beta-LG in order to identify possible common epitopes between human and bovine milk proteins. The proteins were first isolated by affinity chromatography with purified polyclonal antibodies to bovine beta-LG, followed by gel filtration fast phase liquid chromatography and reverse phase-high performance liquid chromatography purification of the components specifically bound in the affinity separation step. Affinity-bound peptides were identified by determining their amino acid sequence. All the sequenced peptides belonged to the C-terminal part of human beta-casein, which confirms the cross-reactivity of human milk proteins and peptides with antibodies to bovine beta-LG and allows the identification of possible common epitopes between the two proteins. No bovine beta-LG peptides with a molecular mass >or= 1,000 Da were found in our milk samples from healthy mothers on a diet rich in bovine milk and dairy products.

Direct identification and characterization of llama (Lama glama L.) whey proteins by microsequencing after western blotting.

Amino acid sequence determination is the most reliable and powerful tool to identify a protein or to classify a new one by comparison of its primary structure with already known sequences. A rapid and simple purification procedure is an essential pre-requisite for routine sequence determination. Structural characterization of llama whey proteins was undertaken for evolutionary as well as economic purposes. N-terminal sequence analyses directly on an immobilon polyvinylidene difluoride (PVDF) membrane, following Western blotting of both native and SDS-denatured llama whey proteins after polyacrylamide gel electrophoresis, revealed three different forms of glycosylated alpha-lactalbumin, and a protein with a high degree of homology with a camel whey protein of unknown function. Furthermore, by immunoblotting techniques, the electrophoretic band corresponding to serum albumin was identified.

Olive oil adulterated with hazelnut oils: simulation to identify possible risks to allergic consumers.

According to European Union Regulation EC 1531/2001, olive oil labelled as "extra-virgin" should be cold-pressed and contain no refined oil or oil from other oleaginous seeds or nuts. Adulteration of extra virgin olive oil (EVOO) with hazelnut oil (HAO) is a serious concern both for oil suppliers and consumers. The high degree of similarity between the two fats complicates the detection of low percentages of HAO in EVOO. Many analytical approaches have been developed in recent years to trace HAO in EVOO, principally based on chromatographic analyses, differential scanning calorimetry or nuclear magnetic resonance. In addition adulteration of EVOO with HAO may introduce hazelnut-derived allergens. The aim of this work was to analyse the protein and allergen content of EVOO intentionally spiked with raw cold-pressed HAO or solvent-extracted HAO. SDS-PAGE analysis confirmed the presence of hazelnut proteins in solvent-extracted HAO with molecular masses ranging 10-60 kDa. In contrast, cold-pressed HAO showed no traces of protein. In spiked EVOO, solvent-extracted HAO was still detectable at a 1% contamination level. Several bands on SDS-PAGE migrated at apparent molecular masses coinciding with known allergens, such as Cor a 1 (approximately 17 kDa), Cor a 2 (approximately 14 kDa), Cor a 8 (approximately 12 kDa), oleosin (approximately 17 kDa) and Cor a 9 (approximately 60 kDa). MALDI-TOF MS analysis confirmed the presence of two oleosin isoforms and of Cor a 9. Immunoblotting demonstrated that an allergic patient with known reactivity to Cor a 1 and Cor a 2 recognized a 17-kDa band in solvent-extracted HAO. In conclusion, we have shown that adulteration of extra virgin olive oil with solvent-extracted hazelnut oil can be traced by simple SDS-PAGE analysis, and that adulteration introduces a potential risk for hazelnut allergic patients.

Mare's colostrum globules stimulate fibroblast growth in vitro: a biochemical study.

The wound repair function of mare's milk and colostrum was investigated. Mare's colostrum improved wound healing in vivo; thus fibroblast growth activation by mare's milk and colostrum was examined. As expected, colostrum was more effective than milk. To establish the biochemical nature of the bioactive molecules involved, colostrum was fractionated into whey, casein, and fat globules, and the efficacy of these fractions on fibroblast proliferation was studied. The fat globule fraction provided the strongest stimulation; its composition was studied and compared with the less-active milk fat globule fraction. The lipid pattern highlighted several differences between mare's colostrum and milk; in particular, total lipid, linoleic acid, linolenic acid, ganglioside, and glycolipid contents were higher in colostrum. A proteomic investigation revealed some differences between the protein composition of colostrum and milk fat globules. Adipophylin and lactadherin were significantly overexpressed in colostrum fat globules. The role of specific lipids on skin wound repair and that of the epidermal growth factor-like domain, embedded within the lactadherin molecule and probably released in conditions stimulating proteolysis, are discussed.

Development of recombinant chimeric antigen expressing immunodominant B epitopes of Leishmania infantum for serodiagnosis of visceral leishmaniasis.

Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3'-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n = 180) and canine (n = 343) control groups, while sensitivity was higher in canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k = 0.95, 95% confidence interval = 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts.

Isolation and characterization of full and truncated forms of human breast carcinoma protein BA46 from human milk fat globule membranes.

We have isolated and characterized two proteins of 50 and 30 kDa from human milk fat globule membranes of healthy donors. N-terminal and internal sequencing revealed that the 50-kDa protein is the full-length human breast carcinoma protein BA46 that is highly expressed in human breast tumors. The 30-kDa protein is a truncated form of protein BA46 which consists of the C-terminal factor V/VIII-like domain of BA46 and which appears to anchor BA46 to the milk fat globule membrane. Defective release of the epidermal growth factor domain containing a surface RGD motif may be related to involvement of BA46 in breast cancer.

Human milk components cross-reacting with antibodies against bovine beta-lactoglobulin.

The human whey components cross-reacting with antibodies raised against bovine and/or equine beta-lactoglobulin were screened systematically. The milk of six women on a normal diet was collected within 72 h of confinement and whey components were fractionated by high-speed size exclusion chromatography and reversed-phase techniques. The fractions which were immunoreactive in double diffusion experiments with antisera anti-bovine and/or equine beta-lactoglobulin were subsequently purified by native PAGE and then electroblotted on Pro-blott membrane (Western blotting). Pro-blot membranes were stained in parallel with Coomassie and by immunostaining using antibodies against bovine and/or equine beta-lactoglobulin as first antibody solution. The immunoreactive bands were cut out from the membrane and N-terminally sequenced; all the immunoreactive components were clearly identified as human beta-casein or its (mainly tryptic) fragments. The strong antigenic similarity between human beta-casein and beta-lactoglobulin (bovine and equine) might be of immunological importance; it could mean that breast-fed neonates risk being sensitized to beta-lactoglobulin irrespective of the presence of cow's milk in the mother's diet.