Fungicidal properties of two saponins from Capsicum frutescens and the relationship of structure and fungicidal activity.

Two steroidal saponins have been purified from cayenne pepper (Capsicum frutescens). Both have the same steroidal moiety but differ in the number of glucose moieties: the first saponin has four glucose moieties (molecular mass 1081 Da) and the second contains three glucose moieties (molecular mass 919 Da). Solubility in aqueous solution is less for the saponin containing three glucose moieties than for the one containing four glucose moieties. The larger saponin was slightly fungicidal against the nongerminated and germinating conidia of Aspergillus flavus, A. niger, A. parasiticus, A. fumigatus, Fusarium oxysporum, F. moniliforme, and F. graminearum, whereas, the second saponin (molecular mass 919 Da) was inactive against these fungi. Results indicate that the absence of one glucose molecule affects the fungicidal and aqueous solubility properties of these similar molecules.

Mechanisms of neutrophil damage to human alveolar extracellular matrix: the role of serine and metalloproteases.

Many syndromes of lung injury are associated with accumulation of neutrophils within the pulmonary parenchyma. These neutrophils have the capacity to produce lung injury by products including proteases and reactive oxygen species (ROS). We examined the ability of activated neutrophils to solubilize human alveolar extracellular matrix (ECM), and by use of scavengers and inhibitors, evaluated the role of ROS and proteases in this process. Supernatants of phorbol myristate acetate-activated neutrophils routinely solubilized 10.2% +/- 0.8% (n = 30) of collagen in human alveolar ECM, as measured by hydroxyproline release. Scavengers of ROS had no significant effect on ECM solubilization. Inhibitors of metalloproteases partially inhibited ECM solubilization (38.5% +/- 4.6% inhibition by ethylenediaminetetraacetic acid [n = 6], and 37.0% +/- 14.7% by 1,10-phenanthroline [n = 6]; p less than 0.05). Inhibitors of the neutrophil serine proteases, elastase and cathepsin G, markedly inhibited ECM solubilization (100.9% +/- 3.7% by alpha 1-protease inhibitor [alpha 1-PI] [n = 6] and 81.9% +/- 0.1% by soybean trypsin inhibitor [n = 6]; p less than 0.01). Since alpha 1-PI completely inhibited solubilization, metalloprotease activity appeared to be related to serine protease activity. This finding was confirmed by the observation that addition of a metalloenzyme activator, p-aminophenylmercuric acetate, in the presence of alpha 1-PI, restored solubilization to the same level as that inhibited by metal chelators. We conclude that human neutrophil metalloproteases and serine proteases directly solubilize human alveolar ECM. Furthermore, neutrophil serine proteases activate latent metalloproteases. However, ROS were not demonstrated to play a major role in ECM solubilization in our system.

Seasonal variation in aerobic bacterial populations and endotoxin concentrations in grain dusts.

Settled and respirable grain dust samples were collected at intervals over a 16-month period at two grain terminals on the lower Mississippi River. Total and gram-negative aerobic bacterial (GNB) populations, as well as endotoxin concentrations, were studied. Plate counts indicated that no viable bacteria were found to be present in the respirable samples. Endotoxin concentrations ranged from 0.0 to 6.4 ng (0.0 to 7.9 ng/m3 air) per respirable sample, indicating that bacteria were once viable in dust of respirable size or contamination occurred from larger, non-respirable particles. Numbers of bacteria from settled dusts, both total and GNB, remained relatively stable; however, the genera of the GNB populations varied. Enterobacter agglomerans was the predominant species in warm months, but not in winter. Pseudomonas and Klebsiella species increased in winter and decreased in summer. Other genera found were Citrobacter and Serratia. Endotoxin concentrations in settled dust samples ranged from 1.7 to 5.6 ng/mg dust.

Mesophilic microorganisms and endotoxin levels on developing cotton plants.

Healthy cotton leaf, bract, fiber and soil were collected twice each week during growing season. These samples were studied to determine the epiphytic total and gram-negative bacterial (GNB) populations and endotoxin levels from seedling development to harvest. Since bract is a significant trash component of raw cotton, a study of the epiphytic bract fungi was performed also. Bract and soil had the highest total bacterial count (TBC) until senescence, when the bract count increased significantly over that of the soil. Leaf TBC was usually third while fiber had the least TBC. Leaf senescence did not result in an increase in TBC as with bract. GNB counts paralleled, but were approximately 10-fold lower than that of the TBC. Enterobacter agglomerans was the most predominant bacterium on leaf and bract. Gram-positive rods were the most common bacteria on fiber with E. agglomerans second. Gram-positive rods were by far the predominant species in soil. Senescence affected the population levels of the various genera on leaf and bract. Endotoxin varied greatly for all sample types. Soil usually gave the highest amounts, followed by bract, fiber leaf surface. Statistical analysis showed some minor correlations between the presence of certain bacteria with other bacteria. No correlations were found between weather data and bacterial types present or endotoxin levels. The epiphytic fungal population on bract was studied through the growing season. Though different genera appeared, the predominant genus throughout the study was Cladosporium.

Separation of mycotoxin-containing sources in grain dust and determination of their mycotoxin potential.

Two distinct reservoirs of mycotoxins exist in fungal-infected cereal grains--the fungal spores and the spore-free mycelium-substrate matrix. Many fungal spores are of respirable size and the mycelium-substrate matrix can be pulverized to form particles of respirable size during routine handling of grain. In order to determine the contribution of each source to the level of mycotoxin contamination of dust, we developed techniques to harvest and separate mycelium-substrate matrices from spores of fungi. Conventional quantitative chromatographic analyses of separated materials indicated that aflatoxin from Aspergillus parasiticus, norsolorinic acid from a mutant of A. parasiticus, and secalonic acid D from Penicillium oxalicum were concentrated in the mycelium-substrate matrices and not in the spores. In contrast, spores of Aspergillus niger and Aspergillus fumigatus contained significant concentrations of aurasperone C and fumigaclavine C, respectively; only negligible amounts of the toxins were detected in the mycelium-substrate matrices of these two fungi.

Microscopical comparison of cotton, corn, and soybean dusts.

Specialized analytical methods are required for identification of components of agricultural dusts such as those generated in harvesting, transportation, storage, and processing of cotton, corn, and soybeans. The larger particles and trash components of the dusts can often be identified visually or with the aid of an optical microscope (OM). The respirable portion of the dust, that which causes lung dysfunction, retains few structural features for identification. Electron microscopy and X-ray microanalysis, together with special optical microscopical techniques, can be used to characterize these microdusts. Combination studies with scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) analysis of cotton dusts have shown the presence of mineralogical particles probably of a soil origin and materials that can be associated with plant parts. Even in screened and filtered cotton dusts, fibrillar fragments are usually present due to their ability to penetrate openings the size of their diameters. The corn and soybean dusts studied were different from the cotton dust in that the large fibrillar component of the cotton dust was absent in the screened grain dusts. However, these dusts consisted of structurally unrecognizable particles that appeared similar to those found in cotton dust. In addition they contained many spheroid particles identified as starch. Dusts from all three sources were found to agglomerate into larger particles, some of which were still less than 10 micron. This agglomeration could confuse the instrumental measurement of dust particle size.

Enhancement of influenza virus infections by secalonic acid D.

Secalonic acid D (SAD), a hepatotoxic, teratogenic, and slightly mutagenic metabolite of Penicillium oxalicum has been identified as a natural contaminant of grain dust. Secalonic acid D was administered intraperitoneally to male ICR mice that were exposed to influenza virus aerosols 5 days earlier. The mortality rate was significantly higher (p less than 0.001) in mice subjected to both influenza and SAD than those subjected to influenza alone. Virus titers in lung tissue samples at selected time intervals appeared similar for both influenza and influenza-SAD treated groups of mice for 9 days after exposure to the virus. After 9 days, influenza-SAD treated mice appeared to have higher virus titers. No difference in the pathological progression of pneumonia was discernible between these two groups of mice. The influenza-SAD group, in addition to pneumonia, exhibited severe hepatic necrosis characteristic of SAD administration. Mice infected with influenza virus followed by administration of SAD responded with significantly lower (p less than 0.05) antibody titers to influenza virus than mice exposed to influenza virus alone.

Gram-negative bacterial endotoxins in grain elevator dusts.

Twenty settled and 69 respirable grain dust samples were collected from 5 grain elevators along the lower Mississippi River. Studies were performed on 2-keto-3-deoxyoctonate (KDO) concentrations, total bacterial plate counts, gram-negative bacteria (GNB) count, identification of GNB, and endotoxin levels. KDO values were 52.1-510.0 ng/mg. A new method involving gas liquid chromatography was developed to analyze for KDO. No correlation could be made between KDO and endotoxin levels. Total bacterial plate counts for settled dusts ranged from 1.9 to 53.4 million/g, while the GNB count was 0.1-50.0 million/g. Two hundred and thirty GNB were isolated from all samples and identified. Sixty-one percent were Enterobacter agglomerans, followed by species of Pseudomonas (9.1%), Serratia (6.9%), Actinetobacter (6.9%) and other genera (16.2%). All but four of the airborne samples had no significant bacterial population. Twenty-three of sixty-nine airborne samples contained 0.6 ng of endotoxin while 3 filters had 6.0 ng. All settled dust samples were contaminated with endotoxin at levels ranging from 22.5 - 187.5 ng/mg. Based upon airborne endotoxin levels worker exposure range was extrapolated to be 0.0-0.2 ng/kg body weight per 8 hr period. This is much less than that necessary to elicit febrile and pulmonary responses. However, the heavily laden filters corresponded to an endotoxin dose of 1.6 ng/kg body weight.

Malathion and diazinon levels in grain dust from New Orleans area grain elevators.

Insecticides applied to grain may be present in dust generated during grain handling and could expose workers to high insecticide concentrations, or be fed to animals in the form of pelleted grain dust. Residual levels of two organophosphate insecticides, malathion and diazinon, were measured in 31 samples of grain dust collected from six terminal grain elevators along the Mississippi River in the New Orleans area. These grain elevator samples and additional spiked samples were extracted and analyzed by gas chromatography. The lower limit of detection for malathion was 0.1 micrograms/g and for diazinon, 0.01 micrograms/g. Recovery of malathion from samples spiked with less than 1 micrograms/g was erratic. Mean recoveries of both malathion and diazinon from spiked samples ranged from 83 to 92% at levels of 1 to 50 micrograms/g of dust. Samples of grain dust from the elevators contained 0.17 to 32 micrograms of malathion/g of dust, but diazinon was not detectable at the 0.01 micrograms/g limit of detection.

Preliminary study of mycoflora and mycotoxins in grain dust from New Orleans area grain elevators.

Dust particles arising from disintegration of grain during handling and shipping may be inhaled by exposed barge and elevator workers. Since the normal grain mycoflora usually contain mycotoxin-producing fungi, these toxins could be natural contaminants of grain dust. Known mycoflora of commercial grain include species of Penicillium, Aspergillus, and Fusarium-fungi that may produce ochratoxins, aflatoxins and zearalenone. A procedure was developed to extract simultaneously these toxins from grain dust. Initial extraction with methylene chloride and water was followed by specific cleanup procedures for each toxin and then by thin layer chromatographic quantitation. In 50-g samples the lowest level of detection for zearalenone was 50 ng of zearalenone/g of dust, for ochratoxin, 10 ng/g and for aflatoxin, 5 ng/g. Dust samples were collected at various locations in two grain elevators and their associated transfer facilities in the New Orleans area. Large amounts of dust that had settled upon floors, machinery and ledges were tested, as well as that which had been collected by dust control systems. Samples were analyzed for aflatoxins, ochratoxin A, and zearalenone. None of the 15 samples contained any detectable amount of aflatoxins or ochratoxin A, but 10 of the 15 samples contained zearalenone at levels from 25 to 100 ng/g.

Secalonic acid D: natural contaminant of corn dust.

The mycotoxin secalonic acid D was detected for the first time as a natural contaminant of corn dust obtained from grain storage elevators. Secalonic acid D amounts ranged from 0.3 to 4.5 ppm (0.3 to 4.5 micrograms/g), as determined by reversed-phase high-performance liquid chromatography.