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PubMed2005

Cloning, bacterial expression, purification and structural characterization of N-terminal-repetitive domain of gamma-Gliadin.

The gene encoding the repetitive domain located in the N-terminal half of gamma-Gliadin from wheat endosperm has been subcloned into a thioredoxin expression system (pET102/D-Topo). It was over-expressed as fusion protein with thioredoxin in Escherichia coli. Thioredoxin was removed by enterokinase cleavage or by acid cleavage at the respective engineered recognition sites. The soluble N-terminal half of gamma-Gliadin was purified by affinity and reverse-phase chromatography. While, the enterokinase cleavage leaded to only one species detectable by mass spectroscopy, the acid cleavage resulted in a three different length polypeptides, due to the presence of the same number of acid cleavage sites. The secondary structure of the purified protein domain was analysed by circular dichroism, showing an spectral shape common to a Poly(Pro) II conformation. The spectrum is dominated by a large negative peak centred around 201 nm and a broad shoulder centred around 225 nm. Also, the temperature denaturation process was studied. The differences observed in the spectra show two main tendencies, the increment of the shoulder intensity, and the drop of the intensity of the peak around 201. When the sample was cooled down, the change on intensity of the shoulder around 225 was completely reversible and that around the 201 nm peak reached a reversibility of 90%. Such structure and thermal behaviour are characteristic of the repetitive domains of the wheat prolamins.

Chromatography, High Pressure Liquid↗
PubMed1993

Structural alterations of the chloroplast genome found in grasses are not common in monocots.

The distribution of structural rearrangements of the chloroplast genome found in grass cpDNA in comparison to that of tobacco was systematically checked in the cpDNAs of representative monocots. The physical map of lily cpDNA, which shares a key position in the diversity of monocotyledonous plants, was constructed to assess whether three inversions found in grass cpDNA are common in monocots. Specific probes for the detection of (1) intron loss in the rpoC1 gene, (2) insertional sequence gain in rpoC2, (3) deletion of ORF2280 in the inverted repeats, (4) non-reciprocal translocation of rpl23, and (5) rearrangements of ORF512, were hybridized to cpDNAs of lily, onion, spiderwort, two turf grasses, and wheat. The existence of intervening sequences in the rpoC1 and rpoC2 genes was also confirmed by PCR analysis. All markers used in the study revealed that structural rearrangements of the chloroplast genome were restricted to grasses, indicating that drastic structural alterations of the chloroplast genome had occurred in the ancestor(s) of grasses. These results also suggest that structural analysis of the chloroplast genome is applicable to the phylogenetic reconstruction of related plants.

Base Sequence↗
PubMed1987

Vascular and leptomeningeal projections of the subcommissural organ in reptiles. Lectin-histochemical, immunocytochemical, and ultrastructural studies.

In the snake, Natrix maura, and the turtle, Mauremys caspica, the basal processes of the ependymal cells of the subcommissural organ project toward the local blood vessels and the leptomeninges. These processes and their endings were studied using aldehyde-fuchsin (AF), periodic-acid Schiff (PAS), periodic-acid silver-methenamine (PA-SM), concanavalin A (ConA), wheat germ agglutinin (WGA), immunoperoxidase staining (employing an antiserum against bovine Reissner's fiber; AFRU), and conventional transmission electron microscopy. For the purposes of comparison, the ventricular cell pole was also analyzed. The secretory material located in the ventricular cell pole and that present in ependymal endings had only a few staining properties in common, i.e., affinity for AF, ConA, and AFRU at a dilution of 1:1000. On the other hand, PAS, PA-SM, WGA, and AFRU at a dilution of 1:200,000 stained the apical (ventricular) secretory material but not the secretory material of the ependymal processes. The histochemical features of the secretory material located in the terminals of ependymal processes, as well as the presence at these sites of numerous rough-endoplasmic-reticulum cisternae and secretory granules, suggest that secretory material may be synthesized in these terminals. The probable fate of this material, i.e., release to the perivascular and leptomeningeal spaces or transport to the ventricular cell pole, is discussed.

Animals↗
PubMed1987

A monoclonal antibody against a family of nuclear pore proteins (nucleoporins): O-linked N-acetylglucosamine is part of the immunodeterminant.

Using nuclear envelopes from Chinese hamster ovary (CHO) cells as an antigen, a mouse monoclonal antibody (IgM; designated mAb CHON211) that specifically binds to components of the nuclear pore complex (nucleoporins) was isolated. Immunofluorescence localization of the antigen recognized by mAb CHON211 revealed a punctate pattern restricted to the nuclear envelope; this pattern changed dramatically during the cell cycle. When examined by electron microscopy, the antigen was largely restricted to the nucleoplasmic and cytoplasmic faces of the nuclear pore complex. Immunoblots showed that mAb CHON211 bound to a series of polypeptides enriched in the nuclear envelope fraction with apparent molecular masses ranging from 110 to 35 kDa. Using galactosyltransferase and the lectin wheat germ agglutinin as probes, we have previously shown that proteins bearing O-linked N-acetylglucosamine (GlcNAc) are restricted to the cytoplasmic and nucleoplasmic faces of the nuclear pore complex. The nuclear membrane proteins recognized by mAb CHON211 had properties very similar to those identified by galactosyltransferase and wheat germ agglutinin labeling. Removal of O-linked GlcNAc residues with beta-hexosaminidase greatly reduced the binding of mAb CHON211, strongly suggesting that O-linked GlcNAc moieties are part of the immunodeterminant. This monoclonal antibody defines a new family of antigens that are restricted to the nuclear pore and bear a common modification: O-linked GlcNAc. mAb CHON211 will be useful for defining the role of the nucleoporins in nucleo-cytoplasmic exchange.

Acetylglucosamine↗
PubMed2002

Pet food and feed applications of inulin, oligofructose and other oligosaccharides.

Prebiotics may be considered as functional food ingredients. They are attracting considerable interest from pet owners, pet food manufacturers, livestock producers and feed manufacturers. The most common forms of prebiotics are nondigestible oligosaccharides (NDO), including inulin, oligofructose mannanoligosaccharides, gluco-oligosaccharides, and galacto-oligosaccharides. These NDO are nondigestible by enzymes present in the mammalian small intestine, but are fermented by bacteria present in the hindgut of nonruminants. Inulin and oligofructose are present in measurable quantities in feed ingredients like wheat, wheat by-products, barley, and peanut hulls. Consumption of prebiotic oligosaccharides elicits several purported health benefits. In companion animals, prebiotics have been shown to improve gut microbial ecology and enhance stool quality. In production livestock and poultry, prebiotics are employed to control pathogenic bacteria, reduce faecal odour, and enhance growth performance. Research to date indicates positive effects of prebiotics on health status and performance of companion animals, livestock, and poultry.

Animal Feed↗
PubMed2002

Differences in histoplasmosis in patients with acquired immunodeficiency syndrome in the United States and Brazil.

Demographic and clinical parameters among patients with acquired immunodeficiency syndrome and histoplasmosis in Brazil and United States were compared. The Brazilian isolates were typed by restriction-fragment length polymorphism analysis and were DNA fingerprinted by random amplification of polymorphic DNA (RAPD)-polymerase chain reaction (PCR). Skin lesions occurred in 66% of Brazilian case patients, compared with 1%-7% of US case patients. Of 21 treated case patients, 4 (19%) died, a rate similar to that of the US case patients (5%-13%). By nuclear gene typing, the Brazilian isolates were equally divided between South American classes 5 and 6, and RAPD-PCR showed 18 distinct genetic fingerprints in 20 isolates. Skin lesions are more common in infection with class 5 or 6 organisms than with class 2 Histoplasma capsulatum. The role of genetic differences in the organism as a cause for the clinical differences requires investigation.

AIDS-Related Opportunistic Infections↗
PubMed1988

[Is protective therapy possible in occupational asthma? Role of immunotherapy].

Specific immunotherapy (ITS) can be a preventative measure for occupational allergy when allergens cannot be fully eliminated. Many allergens provoke IgE-mediated reactions, but when they are haptens and the physiopathology is complex, then the value of ITS is restricted. However, with common allergens whose density increases with occupation, such as house dust and house dust mites for domestic employees, pollens for florists and animal epithelia for breeders, then ITS is effective. On the other hand, when the allergen is specific for the job, of which there are two well documented examples in bakers asthma from wheat flour and laboratory worker's asthma to small laboratory animals there are disparate results. Overall, there is only a modest place for ITS in the treatment of professional asthma, because of the lack of purified, well-standardised extracts.

Asthma↗
PubMed1996

Molecular characterization of Hor v 9. Conservation of a T-cell epitope among group IX pollen allergens and human VCAM and CD2.

We have cloned, sequenced and expressed a recombinant group IX pollen allergen from barley (Hordeum vulgare). Hor v 9 is a polypeptide of 313 amino acids. The Hor v 9 cDNA clone was engineered into the E. coli protein expression vector pMAL and expressed as a fusion of maltose binding protein and truncated Hor v 9. Polyclonal antibodies to the fusion protein were raised in mice. Cross-reactive proteins, RNA and DNA homologues were found in many agricultural species including wheat, rye, triticale, oats, maize, sunflower and flax. The presence of group IX-like proteins in a variety of agricultural crops may represent a previously uncharacterized aeroallergenic occupational hazard. Sequence comparisons of the barley allergen, Hor v 9, with Poa p 9 and other cloned group IX pollen allergens revealed putative structural domains common to all. These include a signal peptide, two conserved immunoglobulin-like motifs, a 150 amino acid highly conserved carboxyterminal domain and a carboxyterminal transmembrane helix. This structural arrangement is also found in cell adhesion molecules. The highly conserved T-cell epitope previously characterized and mapped in group IX allergens (and present in Hor v 9) was found in several human cell adhesion molecule sequences (VCAM, NCAM and CD2). This T-cell epitope corresponded to the most highly conserved amino acid residues common to all group IX homologues sequenced to date. CD2 and VCAM are known to play a role in allergic inflammation: VCAM is involved in the recruitment of lymphocytes to sites of inflammation, and cross-linking CD2 leads to T-cell activation. We anticipate that the similar structural arrangement of group IX allergens and human cell adhesion molecules, as well as the presence of a T-cell epitope common to group IX pollen allergens and cell adhesion molecules, will have important consequences in the natural history of the atopic immune response.

Allergens↗
PubMed1993

Analysis of mannoproteins from blastoconidia and hyphae of Candida albicans with a common epitope recognized by anti-complement receptor type 2 antibodies.

Mannoproteins of approximately 50 kDa from blastoconidia and 60 kDa from hyphae of Candida albicans reacted in Western blots (immunoblots) with either a polyclonal rabbit antiserum (CA-7) or a monoclonal antibody (CA-A) to the C. albicans C3d-binding protein (complement receptor type 2). The glycosylated nature of these proteins was demonstrated by their reactivity with concanavalin A and by selective labeling with the biotin-hydrazide reagent following periodate oxidation. Differences in the oligosaccharides of these proteins were observed in regard to their reactivity with lectin-peroxidase reagents and sensitivity to glycosidases such as N-glycanase or endoglycosidase F (but not endoglycosidase H). The 60-kDa mannoprotein reacted with wheat germ agglutinin, while the 50-kDa mannoprotein did not. Treatment of the 60-kDa mannoprotein with the glycosidases mentioned above resulted in its conversion into a species of 40 to 45 kDa. Enzyme treatment had no obvious effect on the electrophoretic mobility of the 50-kDa species from blastoconidia. Both the 50- and 60-kDa glycoproteins remained immunoreactive after treatment with the glycosidases. Reactivities of the two mannoproteins to neuraminidase also differed. Finally, the 50-kDa (blastoconidia) and the 60-kDa (hyphae) mannoproteins were purified by using ion-exchange chromatography and electroelution. The purified proteins differed in net charge, the 60-kDa species having a more acidic pI. Functional activity of the purified mannoproteins was demonstrated, as each inhibited the rosetting of antibody-sensitized sheep erythrocytes conjugated with iC3b or C3d by hyphae. Thus, an epitope(s) common to both a mycelial and blastoconidial mannoprotein is associated with a structurally different oligosaccharide for each growth form.

Animals↗
PubMed1996

Natural human antibodies to dietary lectins.

Natural antibodies to self and non-self proteins, including dietary proteins, are a significant part of the immune repertoire of humans. Antibodies to three structurally related legume lectins (Erythrina corallodendron lectin (ECorL), peanut agglutinin (PNA), and soybean agglutinin (SBA)) and to one cereal lectin (wheat germ agglutinin (WGA)) were purified by affinity chromatography from human sera and their binding specificity examined. The anti-SBA, anti-ECorL and anti-WGA antibodies exhibited high specificity, whereas the anti-PNA antibodies were polyreactive. Although the anti-WGA antibodies were highly specific for WGA, they also crossreacted slightly toward some other proteins. The anti-ECorL antibodies bound to native SBA, but the anti-SBA antibodies failed to bind to the native ECorL. Although the anti-SBA and anti-ECorL antibodies both exhibited specificity when interacting with native lectins, they bound to a wider range of denatured lectins, indicating a common or universal epitope which is recognized by many natural antibodies. Interestingly, the natural antibodies did not interfere with the agglutination properties of the lectins. These findings may provide a basis for studying the in vivo biological effects of anti-dietary protein antibodies, including those against carbohydrate-binding proteins.

Antibodies↗
PubMed2005

Development of time-resolved chemiluminescence for the determination of antu in river water, wheat, barley, and oat grain samples.

A novel chemiluminescence method for the determination of antu has been developed based on the reaction between potassium permanganate in acid medium with this rat-poison in the presence of formaldehyde as an emission enhancer. The main feature of the system used is that the recording of the whole chemiluminescence intensity-vs-time profiles can be obtained, using the stopped-flow technique in a continuous-flow system. This enables the use of three quantitative parameters adjustable via software settings, one of them a typically kinetic parameter, such as rate of the light-decay reaction, and the others conventional parameters, such as maximum emission intensity and total emission area, which are proportional to the analyte concentration. The optimum chemical conditions for the chemiluminescence emission were investigated. The effect of common emission enhancers, such as formic acid, formaldehyde, glutaraldehyde, acetaldehyde, quinine, fluorescein, rhodamine B, and rhodamine 6G, was studied. The parameters selected were sulfuric acid 4.0 mol L(-)(1), permanganate 0.1 mmol L(-)(1), and formaldehyde 1.0 mol L(-)(1). The calibration graphs obtained with each one of the measurement parameters were linear for the concentration range from 0.05 to 3.00 microg mL(-)(1). The detection limits ranged from 0.005 to 0.010 microg mL(-)(1), and RSD values (n = 10) of 0.99-1.79% at a 0.30 microg mL(-)(1) concentration level and 1.71-2.22% at a 1.0 microg mL(-)(1) concentration level were obtained. The present chemiluminescence procedures were applied to the determination of antu in different kinds of samples, such as river water, wheat, barley, and oat grain samples. Recovery values not significantly different from the spiked amount were found for these determinations.

Avena↗
PubMed1988

Comparative topography of projections from the mesodiencephalic junction to the inferior olive, vestibular nuclei, and upper cervical cord in the cat.

Distributions of neurons located in the central rostral mesencephalon and caudal diencephalon that project to the upper cervical spinal cord, vestibular nuclei, or inferior olive were studied in the cat by using retrograde axonal transport of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP). Afferent sources to all of these targets were observed in the interstitial nucleus of Cajal (INC), the region surrounding the fasciculus retroflexus (PF), and the nucleus of the fields of Forel (NFF). Three-dimensional reconstruction revealed differences in densities of cells projecting from these common areas. Spinal projecting cells were present in slightly greater numbers in the caudal two-thirds of the INC, whereas those projecting to the vestibular complex were more numerous in the rostral two-thirds of this nucleus. A relatively smaller number of olivary projecting cells were dispersed throughout the INC. Olivary afferent sources outnumber those with spinally directed or vestibularly directed axons in the PF region. In the fields of Forel, cells projecting to the vestibular nuclei or inferior olive were concentrated medially, whereas cells projecting to the spinal cord appeared both medially and laterally. Each type of afferent source was also seen in the nucleus of the posterior commissure and the posterior ventral lateral hypothalamic area. Unique sources of afferents to the inferior olive were observed in the parvicellular red nucleus (ipsilateral to the injections) and the anterior and posterior pretectal nuclei. A large number of labeled neurons was seen in the nucleus of Darkschewitsch after injections of tracer into the inferior olive, but this projection did not appear to be unique, as small numbers of labeled cells were also seen after injections into the cervical spinal cord. The Edinger-Westphal nucleus and the adjacent somatic oculomotor nucleus contained cells which projected separately to the spinal cord or the vestibular complex, and the superior colliculus contained cells which projected separately to the contralateral spinal cord or the contralateral inferior olive. In this study, it was also noted that neurons in the medial terminal nucleus of the accessory optic tract projected to the ipsilateral inferior olive or to the contralateral vestibular complex. These differences in locations and densities of cells projecting to the cervical spinal cord, vestibular complex, and inferior olive may underlie functional specializations in these areas in relation to vertical eye and head movement control and to neural systems controlling postural adjustments accompanying limb movements.

Afferent Pathways↗
PubMed1976

Isolation of the major intrinsic transmembrane protein of the human erythrocyte membrane.

The major intrinsic protein of the human erythrocyte membrane commonly referred to as "Band 3", was isolated by a multi-step procedure. Extraction of ghost membranes in dilute solutions of lithium diiodosalicylate removed most of the proteins considered to be extrinsic to the membrane. The resulting membrane fragments were solubilized in sodium dodecyl sulfate, and the major sialoglycoprotein (glycophorin A) was removed by wheat germ agglutinin-Sepharose affinity chromatography. Gel filtration in sodium dodecyl sulfate was used as the final step to yield the band 3 polypeptide in electrophoretically homogeneous form.

Biological Transport, Active↗
PubMed2003

Antifungal activity of volatile compounds generated by essential oils against fungi commonly causing deterioration of bakery products.

AIMS: To investigate the volatile fractions of 16 essential oils for activity against the more common fungi causing spoilage of bakery products, Eurotium amstelodami, E. herbariorum, E. repens, E. rubrum, Aspergillus flavus, A. niger and Penicillium corylophilum. METHODS AND RESULTS: The study applied 50 microl of pure essential oils in a sterilized filter paper, were carried out at pH 6 and at different water activity levels (0.80-0.90). First, a wheat flour based agar medium was used, where cinnamon leaf, clove, bay, lemongrass and thyme essential oils where found to totally inhibit all microorganisms tested. These five essential oils were then tested in sponge cake analogues, but the antifungal activity detected was much more limited. CONCLUSION: Five essential oils showed potential antifungal capacity against all species tested, over a wide range of water availability. Their activity, however, seems to be substrate-dependent. More research is needed to make them work in real bakery products, as in the preliminary study limited effectiveness was found. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the cinnamon leaf, clove, bay, lemongrass and thyme essential oils against species belonging to Eurotium, Aspergillus and Penicillium genus has been demonstrated.

Agar↗
PubMed2001

Airway epithelial cell wound repair mediated by alpha-dystroglycan.

Dystroglycans (DGs) bind laminin matrix proteins in skeletal and cardiac muscle and are expressed in other nonmuscle tissues. However, their expression in airway epithelial cells has not been demonstrated. We examined expression of DGs in the human airway epithelial cell line 1HAEo(-), and in human primary airway epithelial cells. Expression of the common gene for alpha- and beta-DG was demonstrated by reverse transcriptase/ polymerase chain reaction in 1HAEo(-) cells. Protein expression of beta-DG was demonstrated by both Western blot and flow cytometry in cultured cells. Localization of alpha-DG, using both a monoclonal antibody and the alpha-DG binding lectin wheat-germ agglutinin (WGA), was to the cell membrane and nucleus. We then examined the function of DGs in modulating wound repair over laminin matrix. Blocking alpha-DG binding to laminin in 1HAEo(-) monolayers using either glycosyaminoglycans or WGA attenuated cell migration and spreading after mechanical injury. alpha-DG was not expressed in epithelial cells at the wound edge immediately after wound creation, but localized to the cell membrane in these cells within 12 h of injury. These data demonstrate the presence of DGs in airway epithelium. alpha-DG is dynamically expressed and serves as a lectin to bind laminin during airway epithelial cell repair.

Binding, Competitive↗
PubMed2005

Elimination of Listeria monocytogenes from ready-to-eat turkey and cheese tortilla wraps using ionizing radiation.

Listeria monocytogenes is a common postprocess contaminant on ready-to-eat foods including premade ready-to-eat sandwiches. One popular type of sandwich product is the tortilla wrap, which contains sliced luncheon meats and cheeses rolled within a flour tortilla. This study determined the radiation resistance of L. monocytogenes surface inoculated onto two types of commercially available wheat flour tortillas, processed cheese slices, and deli turkey meat. The D10-values for L. monocytogenes (the radiation dose required to inactivate 1 log of the pathogen) were 0.27 kGy when inoculated onto two flour tortilla types, 0.28 and 0.30 kGy when inoculated onto two types of sliced processed cheeses, and 0.58 and 0.65 kGy when inoculated onto two types of sliced deli turkey meat. When two types of tortilla wraps were assembled from the individual components and L. monocytogenes was inoculated into the interfaces between the individual components, the D10-values were 0.27 to 0.37 kGy in the tortilla and cheese interfaces, 0.33 to 0.41 kGy in the cheese and turkey interfaces, and 0.25 to 0.33 kGy in the turkey and tortilla interfaces. The ability of ionizing radiation to reduce pathogen levels on the complex tortilla, cheese, and luncheon meat product was limited by the higher radiation resistance of L. monocytogenes when inoculated onto the ready-to-eat turkey-meat component.

Bread↗
PubMedDate not supplied

Infection and diabetes mellitus.

Most physicians believe that diabetic individuals are predisposed to infections and that infection complicates the control of the diabetes. However, only bacteriuria can be documented to occur with increased frequency in diabetic compared with nondiabetic patients. Although most bacteriuric diabetic patients are asymptomatic, severe infections such as emphysematous pyelonephritis, papillary necrosis, perinephric abscess, and candida pyelonephritis may occur. Tuberculosis, once a proven threat to diabetic individuals, is a less serious problem now that effective screening and chemoprophylaxis programs have been initiated. Several unusual infections such as malignant external otitis, rhinocerebral mucormycosis, emphysematous pyelonephritis, and emphysematous cholecystitis occur also exclusively in diabetics. Foot infections are very important in diabetic patients; successful treatment requires accurate assessment of the extent and etiology of the infections and often involves surgery as well as broad antibiotic coverage. The important problem of infection in diabetic patients deserves careful evaluation. Questions such as do diabetic individuals have a higher incidence of infection, why are diabetic patients predisposed to infection, why is necrosis common in several of the infections, what is the course of asymptomatic bacteriuria, who do diabetic patients develop foot infections, and how should foot infections be prevented and treated should be topics of clinical investigation.

Cellulitis↗
PubMed2005

Leaf tip necrosis, molecular markers and beta1-proteasome subunits associated with the slow rusting resistance genes Lr46/Yr29.

Resistance based on slow-rusting genes has proven to be a useful strategy to develop wheat cultivars with durable resistance to rust diseases in wheat. However this type of resistance is often difficult to incorporate into a single genetic background due to the polygenic and additive nature of the genes involved. Therefore, markers, both molecular and phenotypic, are useful tools to facilitate the use of this type of resistance in wheat breeding programs. We have used field assays to score for both leaf and yellow rust in an Avocet-YrA x Attila population that segregates for several slow-rusting leaf and yellow rust resistance genes. This population was analyzed with the AFLP technique and the slow-rusting resistance locus Lr46/Yr29 was identified. A common set of AFLP and SSR markers linked to the Lr46/Yr29 locus was identified and validated in other recombinant inbred families developed from single chromosome recombinant populations that segregated for Lr46. These populations segregated for leaf tip necrosis (LTN) in the field, a trait that had previously been associated with Lr34/Yr18. We show that LTN is also pleiotropic or closely linked to the Lr46/Yr29 locus and suggest that a new Ltn gene designation should be given to this locus, in addition to the one that already exists for Lr34/Yr18. Coincidentally, members of a small gene family encoding beta-1 proteasome subunits located on group 1L and 7S chromosomes implicated in plant defense were linked to the Lr34/Yr18 and Lr46/Yr29 loci.

Basidiomycota↗