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Studies on Alternaria allergens. I. Isolation of allergens from Alternaria tenuis and Alternaria solani.

Extracts of Alternaria tenuis and Alternaria solani were separated into dialyzable (molecular weight less than 10,000) and non-dialyzable forms. The latter was further fractionated by gel filtration through Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The dialyzable material was fractionated by gel filtration through Sephadex G-50. The allergenic activities of the fractions obtained from the A. tenuis extract was measured in vitro by the radioallergosorbent test assay and the allergenic potency was measured by radioallergosorbent test inhibition assay. Allergenic activity was detected in most of the non-dialyzable fractions, the majority of the activity being in the last G-100 fraction (MW approximately 20,000) which was predominantly protein in nature. The same component may be responsible for the activity found in the dialyzate and its first G-50 fraction since the immunodiffusion studies indicated that the last G-100 fraction has antigenic components in common with those of the first G-50 fraction. In addition, cross-reactions between A. tenuis and A. solani extracts show that the two species share common antigenic determinants.

Allergens

Studies on Alternaria allergens. I. Establishment of the radioallergosorbent test for measurement of Alternaria allergens.

The ability to covalently couple Alternaria allergens to microcrystalline cellulose particles has permitted not only the measurement of IgE antibodies to Alternaria in patient serums but also the identification of allergenic fractions from crude Alternaria extracts. Crude aqueous Alternaria extracts from 3 commerical suppliers were coupled to cellulose but failed to bind more than 5% of total radioactive counts (TRC) when reacted with serums from highly sensitive patients. Fractionation of a commercial extract through Sephadex G-25 showed that almost all allergenic activity was located in a protein- and carbohydrate-containing peak eluting at the column void volume. These fractions were pooled and coupled to cellulose to yield a RAST polymer which produced up to 20% TRC binding when tested with serums from over 100 Alternaria-sensitive patients, and only up to 1% TRC binding with 17 nonallergic serums. The study of commercial Alternaria extracts by chromatographic and Rast inhibition techniques showed that present extracts are neither qualitatively or quantitatively comparable.

Allergens

Tenuazonic acid production by Alternaria alternata and Alternaria tenuissima isolated from cotton.

Cultures of Alternaria alternata (three isolates) and Alternaria tenuissima (three isolates) obtained from cottonseeds and bolls were toxigenic when cultured on various laboratory media. A mycotoxin was isolated and identified as tenuazonic acid by using solvent partition, thin-layer chromatography, and instrument analyses. Toxicity was monitored with brine shrimp and chicken embryo bioassays. All cultures except A. alternata 938 produced tenuazonic acid when grown on cottonseed and on yeast extract-sucrose broth. The most toxin (266 mg/kg) was produced by A. tenuissima 843 on cottonseed.

Alternaria

Studies on Alternaria allergens. II. Measurement of the relative potency of commercial Alternaria extracts by the direct RAST and by RAST inhibition.

The relative potency of 12 commercial Alternaria extracts was analyzed by end-point skin test titrations and compared to in vitro measurements of potency, including (1) the radioallergosorbent test (RAST) inhibition procedure; (2) the direct RAST procedure; and (3) the protein nitrogen unit (PNU) content. Potencies determined by skin testing 10 sensitive patients were strongly correlated among the various patients. Measurements of potency by both RAST inhibition and direct RAST assay were strongly correlated to potency as measured by skin testing. In contrast, neither the weight:volume nor the PNU content bore any relationship to allergenic potency as measured by skin testing or by either of the RAST procedures. Extracts differed by as much as 3,000-fold in allergen content by skin testing. Moreover, the extracts appeared to contain different allergenic determinants when tested by RAST inhibition. RAST inhibition offered several technical advantages over the direct RAST procedure, in that only one solid-phase RAST reagent was required, slopes of dose-response curves could be more easily compared, and a greater discrimination in allergenic potencies among extracts could be made. The RAST appears to offer an excellent method for measuring the potency of allergy extracts, pending the isolation and characterization of actual allergens.

Allergens

Alternaria IgG precipitins and adverse reactions.

Late reactions consisting of fever, malaise, and swelling at the site, 4 to 6 hr after injections of Alternaria extract occurred in several patients receiving immunotherapy with Alternaria. These patients had in common serum IgG precipitins and exquisite leukocyte histamine release sensitivity to Alternaria. Such precipitins were 3 times more frequent in patients receiving Alternaria immunotherapy than a control group of patients receiving immunotherapy with other antigens. A prospective study revealed that 5 of 23 Alternaria-sensitive persons had precipitins before immunotherapy and another 6 developed precipitins during therapy. Only one of the 23 experienced a late Alternaria reaction. Thus, precipitins to Alternaria are common and do not seem to be the basis for the late reactions we observed. The finding of precipitins does not contraindicate immunotherapy.

Alternaria

Systematic isolation of the genus Alternaria in the atmosphere of the "Grande Sao Paulo" area (Brazil).

The genus Alternaria has been isolated from the atmosphere of several countries and its spores are considered as having a strong allergenic power. The description of the genus isolation with a relative frequency was noted in the following countries: Mexico, the United States of America, Canada, Great Britain, Spain, South Africa, Denmark and Argentina. Many Brazilian authors have pointed out the rare incidence of this genus excluding Faria (5) who recorded in the city of Belo Horizonte, State of Minas Gerais, a frequency of 21.1%. Several authors have failed to detect Alternaria sp. spores in the atmosphere of the city of Sao Paulo. As we were studying in our laboratory the airborne fungi of a region called Greater Sao Paulo, where the collections were made in some boroughs of the main city and its neighborhood, we began to make findings of the Alternaria sp. This gave us the opportunity of studying its incidence in that area during a period of one year. We chose 11 areas of this region where we made weekly plate expositions performing 429 trials in all. The technique of collection was the usual one with the exposition of the Petri dishes containing Sabouraud Agar distributed 72 hours before. The collection was made from September 1973 to August 1974. The exposition period was of 5 minutes duration in the open air or in a protected place when it rained. The exposition height was 1 meter from the ground. The identification was made through the colony macroscopic morphology and through the Riddell technique. The Alternaria sp. was isolated in the mentioned area through all the months of the year with its highest frequency in August (35.8%) and its lowest in December 3.7%). The average relative frequency obtained was of 17%. The highest isolation frequency was attained in Winter, the lowest in Spring; in Summer and Autumn, it remained at a medium level. In relation to the studied areas, the Alternaria sp. was isolated in all of them with a minimum frequency of 8.3% in Campos Eliseos and with a maximum frequency of 28.9% in Osasco. The authors would like to emphasize this unusual finding because in the region studied the Alternaria sp. was previously considered absent.

Air Microbiology

Influence of IgG antibody and glycopeptide allergens on the correlation between the radioallergosorbent test (RAST) and skin testing or bronchial challenge with alternaria.

The radioallergosorbent test (RAST) for alternaria was compared to skin tests and bronchial challenges in children suffering from chronic intractable asthma. In contrast to when such children were tested with a timothy grass pollen extract, the bronchial challenge and skin test results against alternaria did not correlate significantly. When alternaria allergens were coupled to cyanogen bromide-activated microcrystalline cellulose, the RAST correlated with the results of skin testing but not bronchial challenge. It was demonstrated by column immunabsorption that some allergic sera contained sufficient IgG antibody against alternaria to competitively inhibit the RAST. When Sepharose 2B was substituted for cellulose as the insoluble support, the inhibition by IgG antibody was largely overcome and then the RAST correlated with both skin test and bronchial challenge results. Glycopeptides contribute significantly to the allergenicity of alternaria, and when these materials were coupled to a Sepharose 2B conjugate by mild oxidation, the RAST correlated with bronchial challenge, but not skin test, results. It was concluded that in this group of steroid-dependent asthmatic children, the correlation of the RAST with the in vivo challenges was strongly influenced by the presence of IgG antibody in the allergic sera and the chemical nature of the mould allergens investigated.

Absorption

Air-borne fungi in the air of Barcelona (Spain). II. The genus alternaria.

The genus Alternaria Nees formed one of the more important components of the fungus population of the air at Barcelone (Spain), during a two-year study, from February 1976 through January 1978. Results were based only on studied of colonies obtained by gravity-exposed plates. The occurrence of this genus was greatly affected by climatic conditions. In general. however, Alternaria appeared to prefer warmer weather. A total of six species of Alternaria was identified, of which, Alternaria alternata (Fries) Keissler was by far the commonest, representing 71.3% of all colonies.

Air Microbiology

[Spore concentration in cities of the European Economic Community. II. Spores of Cladosporium and Alternaria].

A comparative account of Alternaria and Cladosporium spore concentrations in four cities in the European Economic Community (EEC), is given for the years 1973 and 1974. Sijilar volumetric air sampling methods were employed in each of the cities, and concentrations of 3000 Cladosporium spores and 100 Alternaria spores/m3 were taken as the threshold concentrations for clinical significance. With Cladosporium, although highest concentrations were observed in Brussels, the differences were less marked at the other stations. Highest Alternaria concentrations were reocrded in Brussels, followed by London, Leiden and Munich. During the 2 years studied, the average number of days with spore concentrations greater than the threshold value for Cladosporium were 59 in Brussels, 36 in Leiden, 38 in London and 46 in Munich and for Alternaria, 48 in Brussels, 13 in Leiden, 33 in London and 5 in Munich.

Air Pollutants

Primary interaction between antibody and components of Alternaria. II. Antibodies in sera from normal, allergic, and immunoglobulin-deficient children.

Antibodies in sera from normal, allergic, and immunoglobulin-deficient children were studied for binding to radiolabeled components of Alternaria tenuis. Significant binding levels were found in 103 of 105 sera from normal children. The levels were age-dependent, rising from a low point in the 7- to 12-month age group to adult levels by the age of 8 years. Levels of binding to two antigens, a culture filtrate derivative (125I-CLF) and a mycelial derivative (125-I-HS), were similar. Sera from asthmatic children with strong immediate skin test reactions to Alternaria extracts bound significantly higher levels of 125-I-CLF than did sera from allergic children with negative skin tests or from control children. Binding levels in sera from children with hypogammaglobulinemia were significantly less than binding levels in sera from normal children in any age group. Sera from children with selective IgA deficiency bound 125-I-CLF at normal levels. The almost universal occurrence of anti-Alternaria antibodies in children was partly explained by the finding of partial cross-reactivity and/or shared antigens among several fungal species, including A. tenuis, Stemphylium sp., Curvularia sp., and Aspergillus fumigatus. The biological significance of these antibodies is not clear, but the procedures described lend themselves to further investigations.

Adolescent

Alternaria spore and mycelium sensitivity in allergic patients: in vivo and in vitro studies.

Extracts from Alternaria spores and mycelia were prepared to evaluate their allergenic potencies in allergic patients. Twelve Alternaria-sensitive patients, with histories of rhinitis or asthma, were submitted to skin prick tests and five of 12 patients received nasal challenges with spores and mycelia. In vitro, the allergenic activity of each extract was determined by RAST, basophil histamine-release and RAST-inhibition. All patients demonstrated skin reactivity to both extracts while skin reactivity to mycelia was higher than that induced by the spore extract (P < .005). Of the 12 patients, 11 had positive mycelia-RAST and 9/12 had positive spore-RAST. It was found that mycelium-IgE antibody levels were higher than spore-IgE antibody levels (P < .005). Nine RAST-positive patients had positive histamine release tests (> 50%) and basophils challenged with mycelia appeared 10-fold more sensitive compared to the spore challenge. In four of five patients subjected to nasal provocation tests, immediate-type rhinitis was elicited either after spore or mycelium challenge. The patients exhibited a higher nasal reactivity with the mycelium challenge than with spores. RAST-inhibition studies demonstrated that mycelial extracts shared common allergens with spore. These results indicated that Alternaria spore and mycelium were potent allergens in allergic patients and there was a variability in the pattern of in vivo and in vitro reactions between the patients for each allergen.

Adolescent

Characterization and genomic analysis of Bacillus halotolerans G3-2: a potential biocontrol agent against apple Alternaria leaf blotch disease.

BACKGROUND: Apple Alternaria leaf blotch (ALB) is a devastating disease threatening the apple industry worldwide. Biocontrol offers an effective and environmentally friendly alternative for disease management. RESULTS: Bacillus strain G3-2 exhibits strong antagonistic activity against Alternaria alternata (a major causal pathogen of ALB). In dual-culture assays, G3-2 inhibited A.&#x2009;alternata by 88.39%; in detached-leaf inoculation assays, it reduced the lesion area by >88%. 16S rRNA sequencing and phylogenetic analysis identified this strain as Bacillus halotolerans. Oxford Nanopore Technology (ONT) sequencing generated a 4.18-Mb complete genome (43.8% G&#x2009;+&#x2009;C) containing 4149 protein-coding genes, 30 rRNAs and 86 tRNAs. CAZy annotation identified 182 genes encoding carbohydrate-active enzymes (CAZymes), including glycoside hydrolases, glycosyltransferase, and carbohydrate esterases, suggesting potential for glycosylated secondary metabolite production. AntiSMASH analysis detected nine biosynthetic gene clusters, including those for surfactin, fengycin, bacillaene and laterocidine. Plate assays confirmed that G3-2 has the ability to produce protease, cellulase and siderophore. Moreover, it exhibits ~70% inhibition against several other phytopathogenic fungi. CONCLUSIONS: These findings demonstrate that G3-2 suppresses A.&#x2009;alternata through antibiosis (lipopeptides and polyketides), nutrient competition (siderophores) and cell-wall degradation (proteases and cellulases). Moreover, our study revealed that it has great potential to be used as a broad-spectrum, environmentally friendly biocontrol agent. &#xa9; 2026 Society of Chemical Industry.

Alternaria

Pulmonary hypersensitivity to Alternaria and Aspergillus in baker's asthma.

In two cases of baker's asthma pulmonary hypersensitivity was found to the fungi Alternaria and Aspergillus. Provocative bronchial challenge revealed a dual response; an immediate and an Arthus type hypersensitivity to Aspergillus in the first case. A primary binding assay revealed high titres of anti-Aspergillus antibody in the serum. In the second case intradermal and bronchial challenge suggested an immediate type I hypersensitivity response to Alternaria. The suspected organisms were present in the room air of the bakeries. It is suggested that an immunological response to these airborne fungi may have contributed to the pathogenesis of baker's asthma.

Alternaria

[Factors influencing the biosynthesis of beta-galactosidase in Alternaria tenuis].

The effect of pH, carbon sources, and some organic substances on the biosynthesis of beta-galactosidase was studied in Alternaria tenuis on a defined medium that had been optimized by the method of mathematical planning of experiments. Optimal conditions for the production of the enzyme and its liberation into the cultural broth were maintained by adding 2 per cent soya flour to the medium and 0.1 M phosphate-citrate buffer pH 3.0. The production of the enzyme was increased by 3 to 4 times. The biosynthesis of beta-galactosidase by Alternaria tenuis is of an induced nature.

Alternaria

Whole-genome sequencing and analysis of the endophytic fungus Alternaria alternata Y-2 from Leymus chinensis.

To explore the genetic basis and functional potential of beneficial symbiosis between the endophytic fungus Alternaria alternata Y-2 and its host Leymus chinensis, we performed Illumina-based draft whole-genome sequencing and systematic bioinformatic analysis. Although this assembly does not reach telomere-to-telomere completeness, it provides high-quality gene-level information for gene prediction, functional annotation, carbohydrate-active enzyme (CAZyme) identification, and secondary metabolite biosynthetic gene cluster analysis. The final genome size of A. alternata Y-2 was 34,383,676&#xa0;bp with a GC content of 51.0%, containing 12,724 predicted protein-coding genes, 90 tRNAs, and 12 rRNAs. BUSCO assessment showed 98.9% completeness, supporting the high quality of this draft genome. A total of 12,627 genes were successfully annotated in the NCBI NR database, and 17,183 genes were functionally categorized using GO terms. In total, 448 CAZyme genes and 21 secondary metabolite biosynthetic gene clusters were identified, which are potentially involved in lignocellulose degradation, cellular redox homeostasis and biosynthesis of bioactive metabolites. Based on ITS sequence alignment, NR annotation, and phylogenetic analysis of single-copy orthologous genes, the strain was confidently identified as A. alternata. This study firstly reports the draft genome of an endophytic A. alternata strain derived from L. chinensis and provides valuable genetic resources for exploring the endophytic lifestyle, stress tolerance, and bioactive metabolite potential of this fungus.

Alternaria

Mass spectrometry of Alternaria mali toxins and related cyclodepsipeptides.

The structures of AM-toxins I, II and III, host specific phytotoxic metabolites of Alternaria mali, can be readily deduced from low and high resolution mass spectral data, since the amino acids and their sequences are demonstrated by this technique. Additionally, the general fragmentation of these compounds by electron impact is discussed by comparing the spectra of analogous synthetic compounds.

Alanine

Linear, non-mitochondrial plasmids of Alternaria alternata.

Three plasmids, with sizes of 7.0 kbp, 6.8 kbp, and 5.0 kbp and designated pAal-1, pAal-2 and pAal-3 respectively, have been found in a tentoxin-producing isolate of Alternaria alternata. Exonuclease digestions show these plasmids to be linear with blocked 5' ends. Plasmid pAal-1 does not hybridize to nuclear DNA, mitochondrial DNA, or double-stranded RNA from a mycovirus found in the isolate, but does hybridize weakly to a series of linear DNAs which are not visible on gels and may include pAal-2 and pAal-3. Cellular fractionation shows that, unlike other linear fungal plasmids, these plasmids are not localized in the mitochondria. Plasmids have not been found in other tentoxin-producing isolates and there is no evidence that these plasmids have any effect on the production of tentoxin.

Alternaria

Nuclear distribution in Alternaria tenuis.

Nuclear distribution and behaviour during vegetative growth and spore formation in Alternaria tenuis was studied utilising the HC1-Giemsa staining technique. The vegetative mycelium and conidia are predominantly monokaryotic. Anastomoses, followed by nuclear migrations, have been recorded. Intercellular nuclear migrations have only been observed in germinating conidia. Nuclear behaviour during conidial formation indicates that the conidia are homokaryotic. Cytological differences have been found in different monocoidal isolates of A. tenuis.

Alternaria