Identification of putative new tomato allergens and differential interaction with IgEs of tomato allergic subjects.

BACKGROUND: Tomato became one of the world-wide most consumed vegetables, unfortunately accompanied by an increasing risk of tomato allergy affecting certain people. As tomato allergic subjects show highly variable reactions in clinical allergy tests, it is difficult to identify cultivars or differentially treated tomato plants where a significant reduction in the allergenic potential over all subjects of a cohort can be detected. OBJECTIVE: This study was carried out to test the hypothesis that individual variability is based on differential reactions of single subjects to particular allergens in tomato fruits of plants with certain genetic background or cultivated under distinct conditions. METHODS: Proteins were extracted from tomato fruits of the previously investigated genotypes 76R, its mycorrhizal mutant RMC, and the cultivar Counter, fertilized with different forms of nitrogen in deficit or excess. 2-D immunoblots were carried out with sera of nine tomato allergic subjects, beforehand analysed in skin prick tests. RESULTS: In total, ten putative tomato allergens were identified in these immunoblots. No correlation was detected between individual skin prick test results and the quantity of positive reactions to putative allergens. IgEs of each subject showed reactions to nearly every identified putative allergen, but reactions were dependent on genotype and growth conditions. Among the ten putative tomato allergens, five new candidates were identified as follows: an endo-ß-mannanase, a pectinacetylesterase, a pectinesterase inhibitor, an aspartyl protease family protein and a protein of unknown function. CONCLUSION AND CLINICAL RELEVANCE: The hypothesis that high interindividual differences in allergic reactions are based on the interactions between the IgEs of allergic subjects with particular allergens has to be rejected. However, five proteins with putative clinical relevance as tomato allergens could be newly identified.

Application of metal-coded affinity tags (MeCAT): absolute protein quantification with top-down and bottom-up workflows by metal-coded tagging.

As the quantification of peptides and proteins extends from comparative analyses to the determination of actual amounts, methodologies for absolute protein quantification are desirable. Metal-coded affinity tags (MeCAT) are chemical labels for peptides and proteins with a lanthanide-bearing chelator as a core. This modification of analytes with non-naturally occurring heteroelements adds the analytical possibilities of inductively coupled plasma mass spectrometry (ICPMS) to quantitative proteomics. We here present the absolute quantification of recombinantly expressed aprotinin out of its host cell protein background using two independent MeCAT methodologies. A bottom-up strategy employs labeling of primary amino groups on peptide level. Synthetic peptides with a MeCAT label which are externally quantified by flow injection analysis (FIA)-ICPMS serve as internal standard in nanoHPLC-ESI-MS/MS. In the top-down approach, protein is labeled on cysteine residues and separated by two-dimensional gel electrophoresis. Flow injection analysis of dissolved gel spots by ICPMS yields the individual protein amount via its lanthanide label content. The enzymatic determination of the fusion protein via its ß-galactosidase activity found 8.3 and 9.8 ng/µg (nanogram fusion protein per microgram sample) for batches 1 and 2, respectively. Using MeCAT values of 4.0 and 5.4 ng/µg are obtained for top-down analysis, while 14.5 and 15.9 ng/µg were found in the bottom-up analysis.

Allergenic activity of different tomato cultivars in tomato allergic subjects.

BACKGROUND: Tomatoes (Solanum lycopersicum) are consumed worldwide and their amount of consumption is associated with the prevalence of tomato allergy. Therefore, identification of tomato cultivars with reduced allergenicity would potentially increase the quality of life of affected subjects. OBJECTIVE: In this study, we examined the allergenic and biological activity of two different tomato cultivars in tomato allergic subjects. METHODS: Twenty-five subjects with tomato allergy were identified using double-blind, placebo-controlled food challenges (DBPCFC). We applied skin prick test (SPT) and further DBPCFC to investigate the clinical differences between two tomato cultivars ('Reisetomate' and 'Matina'). To examine the molecular basis of allergenic activity, immunoblotting and basophil activation test (BAT) were performed. RESULTS: The cultivar 'Reisetomate' induced significantly less positive skin reactions (P = 0.045) and elicited fewer symptoms after oral challenge compared with 'Matina' (P = 0.047). Molecular assessment revealed that IgE-binding profiles were variable on an interindividual basis, but no major differences between 'Reisetomate' and 'Matina' were detectable. In contrast, BAT underpinned the clinical differences evoked by the different tomato cultivars and showed a left-shift of the dose-response curve obtained for 'Matina' extract (P = 0.046). CONCLUSIONS AND CLINICAL RELEVANCE: Tomato cultivars promote a distinct clinical reactivity in tomato allergic subjects, demonstrated using SPT, DBPCFC and BAT. The molecular background for these differences could not be clarified, as the IgE-binding profiles did not reveal significant alterations. This might be due to instabilities of physicochemical sensitive proteins and/or different isoform expression of allergens.

Microarray analysis of placental tissue in intrauterine growth restriction.

OBJECTIVE: Besides foetal or maternal disorders, placental dysfunction is a major cause of intrauterine growth restriction (IUGR). Although numerous macro- and histopathological changes have been described, little is known about the precise aetiology and the contribution of foetal/placental genes in this disorder. DESIGN: Placental tissues of 20 IUGR and control neonates were analysed by microarray technique. Four of the regulated genes with possible relevance in the pathogenesis of IUGR and its consequences were further studied in placentas of 27 IUGR and 35 control newborns. RESULTS: Elevated gene expression of leptin, corticotrophin-releasing hormone (CRH), and IGF-binding protein-1 (IGFBP-1) in IUGR placentas could be confirmed in the larger group by real-time PCR, whereas prolactin showed no significant difference. Accordingly, protein expression of leptin and IGFBP-1 depicted by Western blot was elevated in IUGR, prolactin was not different. Birthweight standard deviation score (SDS) correlated negatively to leptin, IGFBP-1, and CRH, whereas placental weight correlated only to IGFBP-1. Leptin correlated negatively to gestational age of IUGR patients and positively to placental score, a marker of severity of impaired foeto-placental circulation. CONCLUSIONS: As confirmed in a large group of IUGR and control samples, the up-regulated factors leptin, IGFBP-1, and CRH may serve as candidate genes for the prediction of subsequent metabolic consequences in IUGR newborns. These three factors may not only influence growth of the foetus, but might also interact with programming of its metabolic functions, which has to be determined in an ongoing study.

Carnitine status of pregnant women: effect of carnitine supplementation and correlation between iron status and plasma carnitine concentration.

BACKGROUND/OBJECTIVES: It has been shown that plasma carnitine concentrations markedly decline during gestation in women. The reason for this, however, is unknown. One objective of this study was to investigate the effect of carnitine supplementation on plasma carnitine concentrations in pregnant women. The second objective was to investigate the hypothesis that reduced plasma carnitine concentrations during gestation are caused by a reduced carnitine synthesis because of a diminished iron status. SUBJECTS/METHODS: Healthy pregnant women (n=26) were randomly assigned in two groups receiving either a L-carnitine supplement (500 mg L-carnitine per day as L-carnitine L-tartrate) (n=13) or placebo (n=13) from the 13th week of gestation to term. RESULTS: In the control group, there was a marked reduction of plasma carnitine concentration from the 12th week of gestation to term. This reduction was prevented by the supplementation of carnitine. In the control group, there was a positive relationship between the parameters of iron status (mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and ferritin) and plasma concentration of carnitine (P<0.05). Moreover, there were inverse correlations between the concentrations of ferritin and the carnitine precursor gamma-butyrobetaine in plasma, and between gamma-butyrobetaine and carnitine in plasma (P<0.05). CONCLUSIONS: This study confirms that plasma carnitine concentrations decline in the course of pregnancy, an effect that can be prevented by the supplementation of carnitine. Data of this study, moreover, suggest that the decline of plasma carnitine concentration during pregnancy could be caused by a reduced rate of carnitine biosynthesis, possibly because of an inadequate iron status.

[27-year old pregnant woman with syncope and dyspnea after aortic alloplastic heart valve replacement 15 years ago]. / 27-jährige Schwangere mit Synkope und Dyspnoe nach Aortenklappenersatz vor 15 Jahren.

A 27-year old pregnant woman presented in our emergency department with syncope, dyspnea and complaints about an overall impairment. She had received aortic alloplastic heart valve replacement due to a congenital, valvular stenosis in 1993. We diagnosed a prosthetic heart valve thrombosis associated with cardiac decompensation. Due to tachycardia and critical hypotension we decided to perform fibrinolytic therapy with tenecteplase. After fibrinolysis both prosthetic heart valve leaflets were separating in normal and regular function. The patient was initially anticoagulated with low molecular weight heparin, which was switched during the hospital stay to unfractionated heparin. Later oral anticoagulation with phenprocoumon was initiated. At 36 weeks of gestation an uneventful elective abdominal caesarean section was performed. The mother and the newborn were in healthy condition. Hypercoaguability in pregnancy is a serious problem, especially for patients who already have an existing indication for therapeutic anticoagulation. Fibrinolysis should definitely be considered an option during pregnancy in critical and life-threatening situations.

[Blood flow in the umbilical artery in maternal hypotension and after therapy with Pholedrine longo--a Doppler ultrasound study]. / Der Blutfluss in der A. umbilicalis bei mütterlicher Hypotonie und nach Therapie mit Pholedrin longo--Eine dopplersonographische Studie.

We performed a study in dopplersonography of the A. umbilicalis in pregnant women, who had either an untreated or a with Pholedrin longo (alpha receptor stimulating substance) treated hypotension. These study groups were compared with patients who had a normal blood pressure. In patients with hypotension we found higher values of the qualitative flow indices than women with normotension reflecting a low uterine perfusion. In the group with therapy of hypotension we could analyse a normalisation of the values without decrease of uteroplacental perfusion. These findings show, that hypotension is a high risk in pregnancy which we have to care for.

[Blood flow in the umbilical artery in maternal hypotonia]. / Der Blutfluss in der A. umbilicalis bei mütterlicher Hypotonie.

We performed a study in Doppler sonography in pregnant patients with hypotension compared with women in pregnancy with normal blood pressure. The flow was measured in the foetal aorta, the umbilical artery such as in the A. arcuate and A. cerebri media. Low blood pressure is followed by decreased uteroplacental perfusion. That means a haemodynamic danger for the foetus reflected in significantly increased flow parameters. The low central flow indices show a centralisation of the foetal blood circulating system. These findings describe the importance of hypotension as a risk of pregnancy we have to observe carefully.

[HELLP syndrome on the 6th puerperal day. Presentation of case reports]. / HELLP-Syndrom am 6. Wochenbettstag. Schilderung von Kasuistiken.

We show by two case reports, that the HELLP-syndrome is the most severe form of pre-eclampsia and associated with a relative high maternal and perinatal mortality because of the sudden onset of the complications. In the first case report we describe the development of this syndrome after two eclamptical convulsions on the 6th day of puerperal period. A similar case has not been published yet in all literature. In the second report a mother died by rupture of the capsule of liver because of the HELLP-syndrome 72 hours after Section caesarea. We want to show, that caesarean section cannot avoid this complication. It is not in all cases the most advantageous procedure of delivery in patients with HELLP-syndrome.

Identification of human myocardial proteins separated by two-dimensional electrophoresis using an effective sample preparation for mass spectrometry.

Peptide mass fingerprinting is a powerful tool for the identification of proteins separated by two-dimensional gel electrophoresis (2-DE). The identification of in-gel digested proteins by peptide mass fingerprinting was significantly improve in comparison to blot-digests by using a peptide-collecting device. This device allows the effective purification and concentration of enzymatic digests of low-intensity spots without expensive equipment and is described in detail. Sensitivity in the fmol range was demonstrated by unequivocal identification of bovine serum albumin after sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Furthermore the high performance liquid chromatography pattern of in-gel digests indicated a 2- to 3-fold higher yield of the separated peptides. Therefore, a higher amount of the peptides was available to perform N-terminal sequencing. The identification of 16 proteins from a high-resolution 2-DE gel map of human myocardium tissue has been achieved by means of this technique. Three of these proteins were associated with changes in spot intensity with dilated cardiomyopathy.

Identification and characterization of heat shock protein 27 protein species in human myocardial two-dimensional electrophoresis patterns.

Immunostaining of heat shock protein 27 (Hsp27) protein species on two-dimensional electrophoresis (2-DE) gels with enhanced sensitivity yields 59 spots reacting with anti-Hsp27 antibodies. Recombinant Hsp27 exists in 2-DE as two major protein species which comigrate in the human myocardial pattern with Hsp27 spots C754 and D899 as defined in the heart high-performance 2-DE database (http://www.mdc-berlin.de/emu/heart/). Preparative electrophoresis of human myocardial proteins and analysis of the enriched mass range 20-30 kDa by 2-DE revealed eight protein spots (C438, C582, C658, C697, C754, C595, C750) from the human myocardial database and a new spot not previously detected on silver-stained gels. These spots were identified as Hsp27 protein species by enzymatic in-gel-digestion and analysis by matrix assisted laser desorption-ionization (MALDI) peptide mass fingerprinting and, in part, MALDI-post source decay sequencing of single fragments. Possible post-translational modifications were investigated: immunostaining tests with anti-phospho-serine/-threonine/-tyrosine antibodies, although positive for other myocardial proteins, were negative for presumed Hsp27 protein species; likewise, periodate-glycostaining assays and biotinylation screening did not detect modifications in the investigated Hsp27 protein species.

Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS).

Identification of proteins separated by two-dimensional electrophoresis (2-DE) is a necessary task to overcome the purely descriptive character of 2-DE and a prerequisite to the construction of 2-DE databases in proteome projects. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has a sensitivity for peptide detection in the lower fmol range, which should be sufficient for an analysis of even weakly silver-stained protein spots by peptide mass fingerprinting. Unfortunately, proteins are modified by the silver staining procedure, leading to low sequence coverage. Omission of glutaraldehyde increased the sequence coverage, but this improved sequence coverage is still clearly below the sequence coverage starting with Coomassie Brilliant Blue (CBB) R-250-stained spots. Other factors additionally seem to modify proteins during silver staining. By decreasing the protein amount, the advantage of very sensitive detection on the gel is lost during identification, because the resulting low sequence coverage is not sufficient for secure identification. Low-quantity proteins can be identified better starting with CBB G-250 or Zn-imidazol-stained proteins. In contrast, for high-quantity CBB R-250-stained spots, a sequence coverage of up to 90% can be obtained by using only one cleaving enzyme, and up to 80% was reached for medium-quantity spots after combination of tryptic digest with Asp-N- and Glu-C digest.

Comparison of two-dimensional electrophoresis patterns of heat shock protein Hsp27 species in normal and cardiomyopathic hearts.

Heat shock protein Hsp27 occurs in a complex pattern in human myocardial tissue. Normal and failing explanted human heart from patients with dilated cardiomyopathy (DCM) or ischemic heart failure (IHF), respectively, were analyzed by high resolution two-dimensional electrophoresis (23x30 cm) and Hsp27 immunostaining. Twelve Hsp27 spots in DCM samples were significantly altered in intensity and ten of these were significantly changed in IHF. Four spots (h1, h2, h4, h5) in DCM samples and three spots (h2, h4, h5) in IHF at a molecular mass of 28 kDa were decreased in intensity. In this study, investigating left ventricles of human myocardium, spot h4 was only detected in normal heart samples. On the other hand, spots with a lower molecular mass of 27 kDa (h14, h15, h17, h20, h21) and 22-23 kDa (46, h47, h50) increased in intensity in failing hearts, suggesting that some form of Hsp27 degradation occurs during heart failure.

Altered expression of two light-dependent genes in a microcystin-lacking mutant of Microcystis aeruginosa PCC 7806.

Microcystin is a potent inhibitor of eukaryotic protein phosphatases and has been implicated in causing hepatotoxicity to humans and animals worldwide. It is produced primarily by the bloom-forming cyanobacterium Microcystis aeruginosa, although the function of the peptide in this micro-organism is unknown. In this study, a microcystin-related protein, MrpA, was identified using a microcystin-lacking mutant of M. aeruginosa, PCC 7806. Comparative two-dimensional protein electrophoresis showed that MrpA was strongly expressed in wild-type PCC 7806, but was not detectable in the mcyB mutant. MrpA showed similarity to the RhiA protein from Rhizobium leguminosarum, which is encoded by the rhiABC operon and controlled by quorum-sensing mediators. Sequencing of mrpA flanking regions in M. aeruginosa PCC 7806 revealed the presence of a rhiB homologue, mrpB, directly downstream of mrpA. Northern blot analyses of mrpA expression in cells exposed to different light conditions revealed a rapid decline of transcription under high light conditions. Most striking was a strong increase in transcript levels from cultures irradiated with blue light. The mrpA transcription level was strongly reduced in two independent microcystin-lacking mutants under all light conditions investigated.

A two-dimensional electrophoresis database of rat heart proteins.

More than 3000 myocardial protein species of Wistar Kyoto rat, an important animal model, were separated by high-resolution two-dimensional gel electrophoresis (2-DE) and characterized in terms of isoelectric point (pI) and molecular mass (Mr). Currently, the 2-DE database contains 64 identified proteins; forty-three were identified by peptide mass fingerprinting (PMF) using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), nine by exclusive comparison with other 2-DE heart protein databases, and in only 12 cases of 60 attempts N-terminal sequencing was successful. We used the Make2ddb software package downloaded from the ExPASy server for the construction of a rat myocardial 2-DE database. The Make2ddb package simplifies the creation of a new 2-DE database if the Melanie II software and a Sun workstation under Solaris are available. Our 2-DE database of rat heart proteins can be accessed at URL http://gelmatching.inf.fu-berlin.de/pleiss/2d.

High-performance human myocardial two-dimensional electrophoresis database: edition 1996.

The master gel of the human myocardial two-dimensional electrophoresis (2-DE) gel database contains about 3300 protein spots characterized in terms of isoelectric point (pI) and molecular mass. A high-performance technique was applied, using large gels (23 x 30 cm). Isoelectric focusing with anodic sample preparation and nonequilibrium running conditions (NEPHGE) was combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 15% acrylamide gels in the second dimension. The range of pI extends from pH 4.5 to 9.6. Seventy proteins were identified by combinations of amino acid analysis, N-terminal and internal sequencing, immunostaining, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) peptide mass fingerprinting, post-source decay MALDI-MS and ladder sequencing by carboxypeptidase P. The identification of additional proteins, not found in the master gel, was achieved by immunoblotting. Unequivocal identification with high sensitivity and good yield was obtained by combining internal sequencing and MALDI-MS. In-gel digestion, the concentration and purification of peptides in a peptide collecting device, and the improved FRAGMOD program for peptide mass fingerprinting have added to the security and sensitivity of identification. The high-performance human myocardial 2-DE database was built up with proteins detected by the TOPSPOT program. Spots within six sections of the whole pattern are clickable. Protein description includes detailed information about identification, characterization, and links to the related SWISS-PROT, other 2-DE databases and Medline entries. The database is constructed in accordance with four of the rules for a federated database.

Stability of some Cactaceae proteins based on fluorescence, circular dichroism, and differential scanning calorimetry measurements.

Characterization of three cactus proteins (native and denatured) from Machaerocereus gummosus (Pitahaya agria), Lophocereu schottii (Garambullo), and Cholla opuntia (Cholla), was based on electrophoretic, fluorescence, CD (circular dichroism), DSC (differential scanning calorimetry), and FT-IR (Fourier transform infrared) measurements. The obtained results of intrinsic fluorescence, DSC, and CD were dissimilar for the three species of cactus, providing evidence of differences in secondary and tertiary structures. Cactus proteins may be situated in the following order corresponding to their relative stability: Machaerocereus gummosus (Pitahaya agria) > Cholla opuntia (Cholla) > Lophocereu schottii (Garambullo). Thermodynamic properties of proteins and their changes upon denaturation (temperature of denaturation, enthalphy, and the number of ruptured hydrogen bonds) were correlated with the secondary structure of proteins and disappearance of alpha-helix.