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PubMed2006

Use of laccase as a novel, versatile reporter system in filamentous fungi.

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.

Amino Acid Sequence↗
PubMed1983

Isolation of satratoxins from the bedding straw of a sheep flock with fatal stachybotryotoxicosis.

During a period of several weeks, more than 100 sheep died at a Hungarian farm. The animals exhibited fleece loosing, and hemorrhaging was the most important autopsy finding. Pasteurella haemolytica was cultured from various organs. The bedding straw was abundantly covered with Stachybotrys atra, and removal of the straw stopped the disease. Methanol extraction of the bedding straw followed by solvent partitioning, column chromatography, preparative thin-layer chromatography, and high-pressure liquid chromatography led to the isolation of satratoxins G and H, which were characterized by thin-layer chromatography, high-pressure liquid chromatography, and mass spectroscopy. This is the first isolation and characterization of toxins from a field sample of material responsible for an outbreak of stachybotryotoxicosis.

Animals↗
PubMed1999

Prediction of toxigenic fungal growth in buildings by using a novel modelling system.

There is growing concern about the adverse effects of fungal bioaerosols on the occupants of damp dwellings. Based on an extensive analysis of previously published data and on experiments carried out within this study, critical limits for the growth of the indoor fungi Eurotium herbariorum, Aspergillus versicolor, and Stachybotrys chartarum were mathematically described in terms of growth limit curves (isopleths) which define the minimum combination of temperature (T) and relative humidity (RH) at which growth will occur. Each growth limit curve was generated from a series of data points on a T-RH plot and mathematically fitted by using a third-order polynomial equation of the form RH = a(3)T(3) + a(2)T(2) + a(1)T + a(0). This fungal growth prediction model was incorporated within the ESP-r (Environmental Systems Performance [r stands for "research"]) computer-based program for transient simulation of the energy and environmental performance of buildings. For any specified location, the ESP-r system is able to predict the time series evolution of local surface temperature and relative humidity, taking explicit account of constructional moisture flow, moisture generation sources, and air movement. This allows the predicted local conditions to be superimposed directly onto fungal growth curves. The concentration of plotted points relative to the curves allows an assessment of the risk of fungal growth. The system's predictive capability was tested via laboratory experiments and by comparison with monitored data from a fungus-contaminated house.

Air Microbiology↗
PubMed2000

Mycotoxins in crude building materials from water-damaged buildings.

We analyzed 79 bulk samples of moldy interior finishes from Finnish buildings with moisture problems for 17 mycotoxins, as well as for fungi that could be isolated using one medium and one set of growth conditions. We found the aflatoxin precursor, sterigmatocystin, in 24% of the samples and trichothecenes in 19% of the samples. Trichothecenes found included satratoxin G or H in five samples; diacetoxyscirpenol in five samples; and 3-acetyl-deoxynivalenol, deoxynivalenol, verrucarol, or T-2-tetraol in an additional five samples. Citrinine was found in three samples. Aspergillus versicolor was present in most sterigmatocystin-containing samples, and Stachybotrys spp. were present in the samples where satratoxins were found. In many cases, however, the presence of fungi thought to produce the mycotoxins was not correlated with the presence of the expected compounds. However, when mycotoxins were found, some toxigenic fungi usually were present, even if the species originally responsible for producing the mycotoxin was not isolated. We conclude that the identification and enumeration of fungal species present in bulk materials are important to verify the severity of mold damage but that chemical analyses are necessary if the goal is to establish the presence of mycotoxins in moldy materials.

Aspergillus↗
PubMed2003

Quantifying mold biomass on gypsum board: comparison of ergosterol and beta-N-acetylhexosaminidase as mold biomass parameters.

Two mold species, Stachybotrys chartarum and Aspergillus versicolor, were inoculated onto agar overlaid with cellophane, allowing determination of a direct measurement of biomass density by weighing. Biomass density, ergosterol content, and beta-N-acetylhexosaminidase (3.2.1.52) activity were monitored from inoculation to stationary phase. Regression analysis showed a good linear correlation to biomass density for both ergosterol content and beta-N-acetylhexosaminidase activity. The same two mold species were inoculated onto wallpapered gypsum board, from which a direct biomass measurement was not possible. Growth was measured as an increase in ergosterol content and beta-N-acetylhexosaminidase activity. A good linear correlation was seen between ergosterol content and beta-N-acetylhexosaminidase activity. From the experiments performed on agar medium, conversion factors (CFs) for estimating biomass density from ergosterol content and beta-N-acetylhexosaminidase activity were determined. The CFs were used to estimate the biomass density of the molds grown on gypsum board. The biomass densities estimated from ergosterol content and beta-N-acetylhexosaminidase activity data gave similar results, showing significantly slower growth and lower stationary-phase biomass density on gypsum board than on agar.

Agar↗
PubMed1974

Mycotoxins: toxicity, carcinogenicity, and the influence of various nutritional conditions.

Toxicologic diseases of man and animals, associated with molds growing on foods, have been recognized for centuries. Only in recent years, however, have these mycotoxicoses received the attention of many laboratories and skilled scientists around the world in a broad inter-disciplinary effort. This review covers the literature on mycotoxicoses but centers on those about which most is known, particularly the diseases associated with metabolites elaborated by some strains of Aspergilli, Penicillia, Fusaria, Stachybotrys, and Claviceps. The ubiquitous nature of the aflatoxins, toxic metabolites produced by Aspergillus flavus, make them important to public health, especially since it is now known that certain areas of endemic liver disease coincide with consumption of aflatoxins and, often, malnutrition. The older disease of ergotism, the scourge of Europe for centuries, is considered in detail. Alimentary toxic aleukia, which has caused enormous suffering in Russian human and animal populations, is better understood as a result of relatively recent experimental investigations. Stachybotryotoxicosis, a disease previously considered to be of significance only to man has now been identified in domestic animals.Finally, Japanese studies have clearly revealed the hepatotoxicity of certain metabolites of Penicillium molds. Factors that influence susceptibility to mycotoxins and the hazards they present to man are also reviewed.

Aflatoxins↗
PubMed2006

Airborne mold and endotoxin concentrations in New Orleans, Louisiana, after flooding, October through November 2005.

BACKGROUND: The hurricanes and flooding in New Orleans, Louisiana, in October and November 2005 resulted in damp conditions favorable to the dispersion of bioaerosols such as mold spores and endotoxin. OBJECTIVE: Our objective in this study was to assess potential human exposure to bioaerosols in New Orleans after the flooding of the city. METHODS: A team of investigators performed continuous airborne sampling for mold spores and endotoxin outdoors in flooded and nonflooded areas, and inside homes that had undergone various levels of remediation, for periods of 5-24 hr during the 2 months after the flooding. RESULTS: The estimated 24-hr mold concentrations ranged from 21,000 to 102,000 spores/m3 in outdoor air and from 11,000 to 645,000 spores/m3 in indoor air. The mean outdoor spore concentration in flooded areas was roughly double the concentration in nonflooded areas (66,167 vs. 33,179 spores/m3 ; p < 0.05) . The highest concentrations were inside homes. The most common mold species were from the genera of Cladosporium and Aspergillus/Penicillium ; Stachybotrys was detected in some indoor samples. The airborne endotoxin concentrations ranged from 0.6 to 8.3 EU (endotoxin units) /m3 but did not vary with flooded status or between indoor and outdoor environments. CONCLUSIONS: The high concentration of mold measured indoors and outdoors in the New Orleans area is likely to be a significant respiratory hazard that should be monitored over time. Workers and returning residents should use appropriate personal protective equipment and exposure mitigation techniques to prevent respiratory morbidity and long-term health effects.

Air Pollution, Indoor↗
PubMed2003

Mold: "tsara'at," Leviticus, and the history of a confusion.

The noun tsara'at appears about two dozen times in the Hebrew Bible, almost exclusively in Leviticus, where it is used to describe a state of ritual defilement manifested as a scaly condition of the skin, a condition of cloth, leather, and the walls of houses. In the Septuagint, the Greek translation of the Hebrew Bible, negac tsara'at was translated as aphe lepras; in the Latin Vulgate, this became plega leprae. These words in Greek and Latin implied a condition that spread over the body, not a term of ritual impurity. Tsara'at has continued to be translated as "leprosy," even though this term is not appropriate, as there was no leprosy as we know it in the Middle East during the time period the Hebrew Bible was written. Others have suggested that the proper translation of tsara'at is "mold." The recent identification of a specific mold (Stachybotrys sp.) that contaminates buildings and causes respiratory distress, memory loss, and rash, and the fact that mold has been present for millennia, lend support to the translation of tsara'at as "mold."

Ancient Lands↗
PubMed2005

A simple polymerase chain reaction/restriction fragment length polymorphism assay capable of identifying medically relevant filamentous fungi.

Because of the accumulating evidence that suggests that numerous unhealthy conditions in the indoor environment are the result of abnormal growth of the filamentous fungi (mold) in and on building surfaces, it is necessary to accurately reflect the organisms responsible for these maladies and to identify them in precise and timely manner. To this end, we have developed a method that is cost effective, easy to perform, and accurate. We performed a simple polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) analysis on multiple members of species known to negatively influence the indoor environment. The genera analyzed were Stachybotrys, Penicillium, Aspergillus, and Cladosporium. Each organism underwent PCR with universal primers that amplified ribosomal sequences generating products from 550 to 600 bp followed by enzymatic digestion with EcoRI, HaeIII, MspI, and HinfI. Our results show that using this combination of restriction enzymes enables the identification of these fungal organisms at the species level.

Aspergillus↗
PubMedDate not supplied

Brainstem auditory evoked response in adolescents with acoustic mycotic neuroma due to environmental exposure to toxic molds.

Indoor air contamination with toxic opportunistic molds is an emerging health risk worldwide. Some of the opportunistic molds include: Stachybotrys chartarum, Aspergillus species (A. fumigatus, A. flavus, A. niger, A. versicolor etc.), Cadosporium, Alternaria, Penicillium, Trichoderma, Fusarium graminearum etc. These molds flourish in homes that are moist and damp. Reports of floods are now evident in many parts of the world. With these global changes in climatic conditions that favor the opportunistic mode of living among these molds, some health authorities are beginning to feel concerned about the diversity and the extent to which opportunistic molds can cause adverse health effects in humans. Mycotoxicosis is the collective name for all the diseases caused by toxic molds. Frequently, we have cases of acoustic neuroma due to mycotoxicity in our Center. Mycotic neuroma probably has not been reported before and the application of brainstem auditory evoked response (BAER) techniques in acoustic mycotic neuroma have not been reported either. The aim of this study, therefore, was to report cases and measurements of acoustic mycotic neuroma in adolescents using the brainstem auditory evoked response. The patients' case history, clinical neurological and neurobehavioral questionnaires were assessed. Then, the BAERs were recorded between Cz and Ai, with a second channel, Cz-Ac. The case histories and the questionnaires were analyzed in conjunction with the outcome of the objective brainstem auditory evoked response measurements. The prevalent subjective findings in the patients were headaches, memory loss, hearing loss, lack of concentration, fatigue, sleep disturbance, facial swelling, rashes, nosebleeds, diarrhea, abdominal pains and respiratory difficulties. Objective BAER showed overall abnormalities in all the patients. Although the waveform abnormalities varied, 1-3 interpeak latencies were abnormal in all the patients. Overall results showed the presence of acoustic mycotic neuroma and confirmed the sensitivity and usefulness of BAER in screening acoustic mycotic neuroma and sensorineural auditory dysfunction.

Adolescent↗
PubMed2002

The presence of fungi associated with sick building syndrome in North American zoological institutions.

A total of 110 sites from five zoological institutions were examined to determine whether fungi associated with sick building syndrome (SBS) were prevalent in the exhibits or night-time holding facilities and to investigate whether the presence of these organisms was associated with declining breeding rates or increases in morbidity and mortality (or both). Each site was sampled with an Andersen two-stage air sampler using Sabourauds dextrose agar media and a Burkard personal volumetric air sampler. Suspect surfaces were also sampled. High levels of airborne Penicillium chrysogenum, a fungal species associated with poor indoor air quality, were recovered from 16 sites out of all five institutions. Five culturable growth sites of Stachybotrys chartarum, a species strongly associated with SBS and commonly known as "black mold," were recovered from surfaces at two institutions. A wide range of other fungal species was recovered in low numbers from all institutions. A Fisher exact test analysis showed a significant nonrandom association between high levels of P. chrysogenum and sites with records of poor animal health. This study indicated that significant numbers of airborne fungi associated with SBS and poor indoor air quality are present in zoological institutions and that they could affect animal health and reproduction rates and zoo staff.

Air Microbiology↗
PubMed1996

Staplabin, a novel fungal triprenyl phenol which stimulates the binding of plasminogen to fibrin and U937 cells.

A novel triprenyl phenol, designated staplabin, has been isolated from a culture of Stachybotrys microspora IFO 30018 by solvent extraction and successive chromatographic fractionation using silica gel, Sephadex LH-20 and silica ODS columns. By a combination of spectroscopic analyses, the structure of staplabin is proposed to be 5-(2-(5,7-dihydroxy-8-methyl-8-(4,8-dimethyl-3,7-nonadienyl)-3-oxo -7, 8-dihydro-6H-pyrano[2,3-e][1,3]dihydroisoindolyl)pentanoic acid. Staplabin stimulated the binding of plasminogen, the zymogen of the fibrinolytic serine protease plasmin, to both fibrin and U937 cells. Binding was elevated 2-fold at a concentration of 0.3 approximately 0.5 mM.

Benzopyrans↗
PubMed1998

Isolation of SMTP-3, 4, 5 and -6, novel analogs of staplabin, and their effects on plasminogen activation and fibrinolysis.

Four novel triprenyl phenol metabolites, designated SMTP-3, -4, -5, and -6, have been isolated from cultures of Stachybotrys microspora IFO 30018 by solvent extraction and successive chromatographic fractionation using silica gel and silica ODS columns. A combination of spectroscopic analyses showed that SMTP-3, -4, -5, and -6 are staplabin analogs, containing a serine, a phenylalanine, a leucine or a tryptophan moiety in respective molecules in place of the N-carboxybutyl portion of the staplabin molecule. SMTP-4, -5, and -6 were active at 0.15 to 0.3 mM in enhancing urokinase-catalyzed plasminogen activation and plasminogen binding to fibrin, as well as plasminogen- and urokinase-mediated fibrinolysis. On the other hand, the concentration of staplabin required to exert such effects was 0.4 to 0.6 mM, and SMTP-3 was inactive at concentrations up to 0.45 mM.

Benzopyrans↗
PubMed2000

Activation of fibrinolysis by SMTP-7 and -8, novel staplabin analogs with a pseudosymmetric structure.

Two novel staplabin analogs, SMTP-7 and -8, have been isolated from cultures of Stachybotrys microspora IFO 30018. Spectroscopic analyses showed that the SMTP-7 molecule consisted of two identical staplabin core structures and ornithine which bridges the two partial structures. In the SMTP-8 molecule, the bridging unit was lysine. At concentrations of 80 approximately 150 microM, the two compounds caused 2- to 12-fold increase in urokinase-catalyzed plasminogen activation, fibrin binding of plasminogen, and urokinase- and plasminogen-mediated fibrinolysis. These activities of SMTP-7 and -8 were two to ten times higher than those of staplabin and previously isolated SMTPs, which exerted such effects at concentrations ranging from 150 to 800 microM.

Benzopyrans↗
PubMedDate not supplied

[Copper ions sorption by soil micromycetes].

This study gives a comparison of copper ions sorption ability of micromycetes (72 strains) isolated from both modern and archaeological soils polluted with heavy metals. Copper sorption ability was inter- and intra-specific with Cladosporium cladosporioides 77, C. cladosporioides 716 and Stachybotrys chartarum 357, S. chartarum 424 strains being the most efficient sorbents. In general, copper sorption was significantly higher for strains of fungi isolated from modern soils compared to those from archaeological soils. Melanised strains of fungi sorbed copper significantly better than non-melanised fungi.

Archaeology↗
PubMed1979

An anticomplementary agent, K-76 monocarboxylic acid: its site and mechanism of inhibition of the complement activation cascade.

A monocarboxylic acid derivative (K-76 COOH) of K-76, purified from the culture filtrate of Stachybotrys complement I nov. sp. K-76, inhibits complement (C) activity. Its inhibitory action is mainly on C5 step. It strongly inhibits the generation of EAC1,4b,2a,3b,5b from C5 and EAC1,4b,2a,3b, and accelerates the decay of EAC1,4b,2a,3b,5b. It also causes some inhibition of the reactions of the reactions of C2,C3,C6,C7 and C9 with their respective preceding intermediate cells. It has no effect on the generation of EAC1,4b from C4 and EAC1, or of EAC-8 from C8 and EAC-7, and apparently increases the generation of EAC1,4b from C1 and EAC4b probably by inhibiting transfer or turnover of C1. It does not affect the rate of decay of EAC1,4b,2a or the T max of generation of EAC1,4b,2a, and it inhibits immune adherence only at high concentration. K-76 COOH also strongly inhibits hemolysis through the alternative pathway of C activation by cobra venom factor, but it does not seem to inhibit the early steps of the alternative pathway, because it has little affect on the consumption of C3 or the conversion of beta 1C to beta 1A on treatment of C serum with zymosan. K-76 COOH probably combines with C5 molecules, forming the inactive complexes, or it causes the structural alteration of C5.

Animals↗
PubMed1979

A field outbreak of suspected stachybotryotoxicosis in sheep.

An outbreak of mortality in a flock of mutton merino sheep in which 109 out of 568 sheep died in the south-western Cape Province, is described. It was characterized by haemorrhagic septicaemia, anaemia, leucocytopaenia and haemorrhagic tendencies. Mortalities followed unseasonal and heavy summer rain, extended over a period of 6 months and were associated with the uninterrupted consumption of sheep cubes processed on the farm severly fungus-infested wheat, barley and rye straw for a period of at least one month. The main clinical signs occurred in two phases: an elevated body temperature, listlessness, epistaxis and intermittent haemorrhagic diarrhoea during the first phase of the outbreak, and a progressively worsening anaemia, leucocytopaenia and less severe haemorrhagic tendencies and a terminally elevated body temperature during the second phase. The predominant autopsy findings were purpuric haemorrhage on serosal and mucosal surfaces and in most of the organs, enterorrhagia and severe pulmonary congestion and oedema during the first stage; anaemia was the predominant sign during the second stage - widespread haemorrhage still occurred but was less extensive. Pasteurella haemolytica was isolated from most of the animals autopsied during the first stage. Histologically the most salient features were atrophy and necrosis of the lymphoid tissue, aplastic anaemia and a markedly impaired inflammatory response. Extensive post-natal lamb mortalities, probably due to an Escherichia coli infection precipitated by the toxicosis, occurred during the outbreak. Toxigenic strains of Stachybotrys chartarum were isolated from the wheat and barley straw. Diethyl ether extracts of the wheat straw, sheep cubes and S. chartarum culture material elicited positive skin tests in rats following intradermal injection and the presence of 12,13-epoxytrichothecenes in these extracts were confirmed by thin layer chromatography. In feeding trials sheep cubes and wheat straw caused the death of 4/4 one-day-old Pekin ducklings and weanling Wistar rats in six and nine days, respectively. This is the first description of an outbreak of disease in sheep associated with the ingestion of S. chartarum-infested food components in the Republic of South Africa.

Animal Feed↗