Biomedical subjects
Publications and source records attributed to R T Riley.
All of us realize that these are times of high change in virtually every health care system, regardless of size or location. As part of this change process, many health care systems are taking a serious look at the area that has evolved as knowledge management. While change may be mandatory, progress is definitely optional.
The first discovered naturally occurring inhibitor of de novo sphingolipid biosynthesis was fumonisin B1. There are now 11 identified fungal inhibitors of ceramide synthase or 'fumonisin B1-like' compounds. With the exception of the australifungins, all other fungal ceramide synthase inhibitors are structurally sphingoid-like. There are several recently discovered fungal inhibitors of another enzyme in the de novo sphingolipid biosynthesis pathway: serine palmitoyltransferase (SPT). One of the SPT inhibitors is named ISP-I. While ceramide synthase inhibitors are toxic to animals, plants and fungi, the SPT inhibitors are not known to cause animal or plant disease, but are potent inhibitors of fungal growth. Very little is known about their toxicity in animals. There are at least 24 fungal SPT inhibitors produced by a variety of fungi. Given that the fungal inhibitors of sphingolipid biosynthesis are chemically and biologically diverse, two bioassays have been developed to screen for fumonisin-like or ISP-I-like activity in naturally contaminated products or fungal culture materials. These bioassays are based on the changes in free sphingoid base concentration that occur when the ceramide synthase or SPT are inhibited. The bioassays have the advantage that they are functionally rather than chemically specific and thus will detect ceramide synthase and SPT inhibitors regardless of their chemical structure.
Fumonisins are mycotoxins produced by several species of Fusaria. They are found on corn and in corn-based products, can cause fatal illnesses in some animals and are suspected human esophageal carcinogens. Fumonisins are believed to cause toxicity by blocking ceramide synthase, a key enzyme in sphingolipid biochemistry which converts sphinganine (or sphingosine) and fatty acyl CoA to ceramide. Relatively few fungal species have been evaluated for their ability to produce fumonisins. Fewer have been studied to determine if they produce ceramide synthase inhibitors, whether fumonisin-like structures or not, therefore potentially having toxicity similar to fumonisins. We analyzed corn cultures of 49 isolates representing 32 diverse species of fungi for their ability to produce fumonisins. We also evaluated the culture extracts for ceramide synthase activity. Only cultures prepared with species reported previously to produce fumonisins--Fusarium moniliforme and F. proliferatum--tested positive for fumonisins. Extracts of these cultures inhibited ceramide synthase, as expected. None of the other fungal isolates we examined produced fumonisins or other compounds capable of inhibiting ceramide synthase. Although the fungi we selected for these studies represent only a few of the thousands of species that exist, they share the commonality that they are frequently associated with cereal grasses, including corn, either as pathogens or as asymptomatic endophytes. Thus, these results should be encouraging to those attempting to find ways to genetically manipulate fumonisin-producing fungi, to make corn more resistant, or to develop biocontrol measures because it appears that only a relatively few fungal contaminants of corn can produce fumonisins.
Adenovirus has been used in vivo and in vitro as a vector to carry a foreign gene for gene transfer. Two kinds of replication defective human recombinant adenovirus vectors were used in this study, the first containing beta-galactosidase reporter gene (AdCMVLac-Z) and the second carrying a gene for porcine leptin gene (AdCMVpLeptin). AdCMVLac-Z was tested for its ability to transfer DNA into pig kidney and pituitary cells. These cells expressed Lac-Z transiently 48 hours after the infection. In addition, when the pig kidney cells expressing the Lac-Z were replated with low density for the formation of colonies from each cell, colonies of blue cells expressing Lac-Z were observed. These results demonstrate that human recombinant adenovirus can be used as a transducing viral vector for inducing long-term expression in pig kidney cells. We also constructed a recombinant adenovirus (AdCMVpLeptin) which contained a pig leptin gene for the expression of pig leptin in vitro in the 293 human kidney cell line. 293 cells transfected with AdCMVpLeptin produced both a 15 KDa of a secretory form of porcine leptin and an 18 KDa long form containing signal peptide. Our study demonstrated that the recombinant adenovirus system offers a method for gene transfer and expression in pig cells.
Fumonisin B1 (FB1) is a frequently encountered mycotoxin that inhibits ceramide synthase, the enzyme that acylates sphinganine, sphingosine and other "sphingoid" bases. Exposure of rats, rabbits, pigs and nonhuman primates to fumonisin-contaminated feed elevates sphingoid base amounts in urine; therefore, this study examined the time course and reversibility of these changes. When an AIN-76 diet supplemented with >/=5 microg FB1/g was fed to male Sprague-Dawley rats, there was a significant increase in sphinganine (ca. 50-fold in urine from rats fed 50 microg FB1/g diet) and smaller changes in sphingosine within 5 to 7 d, compared to rats fed the same diet without FB1. No change occurred in sphingoid bases upon feeding 1 microg FB1/g for up to 60 d. When rats were fed FB1 (10 microg FB1/g diet for 10 d), then changed to the same diet minus FB1, urinary sphingoid bases returned to normal within 10 d. However, if the rats were fed 10 microg FB1/g for 10 d, then changed to 1 microg FB1/g, the amounts of sphingoid bases in urine were the same as for rats that were continuously fed 10 microg FB1/g. These results establish that consumption of FB1 causes dose-dependent and reversible elevations in the amounts of urinary sphingoid bases. The finding that 1 microg FB1/g (which does not, alone, alter urinary sphingoid bases) will sustain the elevation caused by previous exposure to 10 microg FB1/g raises the possibility that even low levels of fumonisins could be deleterious when an animal is occasionally exposed to higher amounts.
Fumonisin B1 (FB1) is a common contaminant of corn worldwide and is responsible for several diseases of animals. In the preparation of tortillas, corn is treated with lime (producing nixtamal) that when heated hydrolyzes at least a portion of the FB1 to the aminopentol backbone (AP1), another known toxin. This study analyzed the amounts of FB1 and AP1 in tortillas and nixtamal from two communities in the central highlands of Guatemala where corn is a major dietary staple (Santa Maria de Jesus, Sacatepequez, and Patzicia, Chimaltenango). The amounts of FB1 and AP1 in tortillas from Santa Maria de Jesus were, respectively, 0.85 +/- 2.0 and 26.1 +/- 38.5 microg/g dry weight (mean +/- SD), and from Patzicia were 2.2 +/- 3.6 and 5.7 +/- 9.4 microg/g dry weight. Less than 6% of the tortillas from both locations contained > or = 10 microg FB1/g dry weight; whereas, 66% of the samples from Santa Maria de Jesus and 29% from Patzicia contained > or = 10 microg AP1/g dry weight. The highest amount of AP1 (185 microg/g dry weight) was found in tortillas from Santa Maria de Jesus. The highest amounts of FB1 were 6.5 and 11.6 microg/g dry weight in tortillas from Santa Maria de Jesus and Patzicia, respectively. The mean concentration of FB1 in nixtamal was significantly higher in Santa Maria de Jesus compared to Patzicia. Surprisingly, AP1 was not detected in any of the nixtamal samples. The human impact of exposure to these amounts of fumonisins is not known. However, based on findings with other animals, where corn is a dietary staple, long-term consumption of FB1 and AP1 (especially at > or = 10 microg/g of the diet) may pose a risk to human health.
Fumonisin B1 is a mycotoxin produced by Fusarium moniliforme, a common fungus in corn. It is known to cause a variety of diseases, including hepatic and renal degeneration in many species of laboratory and domestic animals. The known biochemical events in fumonisin B1 toxicity involve inhibition of ceramide synthase leading to disruption of sphingolipid metabolism. The effect of fumonisin B1 on ceramide and more complex sphingolipids in mice is not known. Groups of five male BALB/c mice each were injected with fumonisin B1 subcutaneously at doses of 0, 0.25, 0.75, 2.25, and 6.75 mg/kg body weight daily for 5 days. This protocol has been shown to produce a dose-dependent increase in apoptosis in liver and kidney of these animals. In the present study, liver, kidney, and brain were sampled and analyzed for free sphingoid bases and complex sphingolipids one day after the last treatment. A dose-related accumulation of free sphinganine and sphingosine was observed in liver and kidney, but not brain. The maximal increase in free sphinganine in kidney was 10-fold greater than in liver. Total phospholipids increased only in liver, whereas ceramide levels were not consistently altered in liver, kidney, or brain. In liver and kidney, fumonisin B1 treatment increased the sphinganine-containing complex sphingolipids, but no effect was observed on sphingosine-containing complex sphingolipids. No changes in complex sphingolipids were observed in brain. In liver, there was a close correlation between the extent of free sphinganine accumulation, and apoptosis and hepatopathy. This correlation was also evident in kidney but to a lessor extent. Nonetheless, the apoptosis and nephropathy occurred with little or no change in the levels of ceramide or more complex sphingolipids.
Ultrastructural effects of AAL-toxin TA from Alternaria alternata on black nightshade (Solanum, nigrum L.) leaf discs and correlation with biochemical measures of toxicity. In black nightshade (Solanum nigrum L.) leaf discs floating in solutions of AAL-toxin TA (0.01-200 microM) under continuous light at 25 degrees C, electrolyte leakage, chlorophyll loss, autolysis, and photobleaching were observed within 24 h. Electrolyte leakage, measured by the conductivity increase in the culture medium, began after 12 h with 200 microM AAL-toxin T(A), but was observed after 24 h with 0.01 to 50 microM AAL-toxin T(A), when it ranged from 25%) to 63% of total releasable electrolytes, respectively. After 48 h incubation, leakage ranged from 39% to 79% of total for 0.01 to 200 microM AAL-toxin T(A), respectively, while chlorophyll loss ranged from 5% to 32% of total, respectively. Ultrastructural examination of black night-shade leaf discs floating in 10 microM AAL-toxin TA under continuous light at 25 degrees C revealed cytological damage beginning at 30 h, consistent with the time electrolyte leakage and chlorophyll reduction were observed. After 30 h incubation chloroplast starch grains were enlarged in control leaf discs, but not in AAL-toxin T(A)-treated discs, and the thylakoids of treated tissue contained structural abnormalities. After 36-48 h incubation with 10 microM AAL-toxin T(A), all tissues were destroyed with only cell walls, starch grains, and thylakoid fragments remaining. Toxicity was light-dependent, because leaf discs incubated with AAL-toxin T(A) in darkness for up to 72 h showed little phytotoxic damage. Within 6 h of exposure to > or =0.5 microM toxin, phytosphingosine and sphinganine in black nightshade leaf discs increased markedly, and continued to increase up to 24 h exposure. Thus, phy siological and ultrastructural changes occurred in parallel with disruption of sphingolipid synthesis, consistent with the hypothesis that AAL-toxin T(A) causes phytotoxicity by interrupting sphingolipid biosynthesis, thereby damaging cellular membranes.
Fumonisins are mycotoxins produced by Fusarium moniliforme, F. proliferatum, and related Fusarium species found on corn. They occur naturally in corn-based feeds and foods and are suspected human esophageal carcinogens. Fumonisin B1 (FB1), the most common homologue, causes the animal diseases associated with F. moniliforme. Hepato- and nephrotoxicities, disrupted sphingolipid metabolism, and liver cancer have been found in rats fed FB1. To determine the in vivo effects of diets containing fumonisins B2 (FB2) or B3 or (FB3), male rats were fed culture materials (CM) of FB1 non-producing F. moniliforme isolates to provide low (4.6-6.7 ppm), mid (32-49 ppm) or high (219-295 ppm) dietary levels of either FB2 (FB2CM) or FB3 (FB3CM). Other groups were fed culture material of an FB1 producing isolate (FB1CM) providing 6.9, 53 or 303 ppm total fumonisins (FB1: FB2: FB3 = 1.0: 0.38: 0.15) and a tenth group was fed a control diet having no detectable fumonisins. One-half (n = 5/group) the animals were killed after three weeks, at which time the toxicological and histopathological effects of the three culture materials were similar, mimicked the effects of FB1, and included decreased body weight gains, serum chemical indicators of hepatotoxicity, decreased kidney weights, and apoptosis of hepatocytes and kidney tubular epithelium. FB1CM, FB2CM, and FB3CM affected sphingolipids, causing increased sphinganine to sphingosine ratios (Sa/So) in both liver and kidneys. The remaining animals (n = 5/group0 were fed a control diet for three additional weeks. All body weight and tissue specific effects, including increased Sa/So, induced by the FB2Cm, FB3CM and low level FB1CM diets were absent following the recovery period. Except for mild biliary lesions found in the high dose of FB1CM group and a few apoptotic hepatocytes present in one mid- and two high-dose FB1CM rats, no evidence of toxicity remained in these groups, following the recovery period.
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The purpose of this study was (i) to determine whether pure fumonisin B1 could be incorporated into, recovered, and detected by high-pressure liquid chromatographic analysis from the semipurified Oregon test diet (OTD) used in rainbow trout feeding studies, and (ii) to determine if the incorporated fumonisin B1 was biologically available using the change in free sphingoid bases in liver, kidney, and serum as a mechanism-based biomarker. The results indicate that fumonisin is not easily quantified in the OTD. Recoveries ranged from 12 to 81% of the calculated concentrations based on the fumonisin B1 added to the OTD. However, the fumonisin B1 in the OTD was readily absorbed and biologically active as evidenced by marked increases in free sphinganine in liver, kidney, and serum. The magnitude of the increase in free sphinganine at 100 ppm in the OTD was comparable to that known to be associated with liver toxicity in rats, pigs, and ponies.
Positive outcomes come to health care organizations that are doing the right things well, that is, their organizational strategies are aligned with their environments and they are executing those strategies well. Likewise, the informatics strategies must also be aligned with the organization's strategies. Without this congruence, informatics does not have the potential to have a substantial positive impact on the overall organizational outcomes. The execution of the informatics strategies must also be exemplary; however, the change processes required for achieving the desired informatics and organizational outcome goals are demanding and complicated. Implementing them in extremely complex organizations that operate on a 7-day by 24 hour basis is not easy. However, we are constantly learning more about complex change processes and the ways we can better manage them to improve our needed informatics outcomes. The challenge is to build upon the existing research base to more us even further ahead.
Fumonisin B1 is a toxic product of Fusarium moniliforme, which inhibits ceramide synthase, leading to accumulation of free sphingoid bases. Despite its known biochemical action, the mechanism of toxicity is not fully understood. Male BALB/c mice were injected subcutaneously with 0 to 6.75 mg/kg/day of fumonisin B1 for 5 days. One day after the last treatment, spleens were collected, and peritoneal macrophages were obtained from separate groups after an intraperitoneal injection of thioglycolate broth. Peripheral leukocyte counts were increased and kidney weights were decreased by fumonisin B1 treatment. Presence of apoptotic cells in the liver and kidney of treated mice was confirmed by enzymatic immunoassay. Macrophages cultured with lipopolysaccharide indicated an increased secretion of tumor necrosis factor-alpha (TNF-alpha) but not of interleukin-1alpha. No effect was seen on interferon-gamma production when splenocytes were incubated with concanavalin A. Elevation of leukocyte and reticulocyte counts was abrogated by pretreatment with anti-TNF-alpha antibody before a single dose of fumonisin B1 (25 mg/kg), supporting the hypothesis that the fumonisin B1 toxicity involves TNF-alpha. Cultures of J774A.1 cells, when treated with fumonisin B1, produced TNF-alpha in vitro. Results indicate that fumonisin B1 toxicity may involve secretion of TNF-alpha by TNF-alpha-producing cells without altering interleukin-1alpha or interferon-gamma. The influence on TNF-alpha-production may be a contributing factor to fumonisin B1-induced apoptosis and other observed toxic effects in animals.
The "sphingosin" backbone of sphingolipids was so named by J. L. W. Thudichum in 1884 for its enigmatic ("Sphinx-like") properties. Although still an elusive class of lipids, research on the involvement of sphingolipids in the signal transduction pathways that mediate cell growth, differentiation, multiple cell functions, and cell death has been rapidly expanding our understanding of these compounds. In addition to the newly discovered role of ceramide as an intracellular second messenger for tumor necrosis factor-alpha, IL-1beta, and other cytokines, sphingosine, sphingosine-1-phosphate, and other sphingolipid metabolites have recently been demonstrated to modulate cellular calcium homeostasis and cell proliferation. Perturbation of sphingolipid metabolism using synthetic and naturally occurring inhibitors of key enzymes of the biosynthetic pathways is aiding the characterization of these processes; for examples, inhibition of cerebroside synthase has indicated a role for ceramide in cellular stress responses including heat shock, and inhibition of ceramide synthase (by fumonisins) has revealed the role of disruption of sphingolipid metabolism in several animal diseases. Fumonisins are currently the focus of a FDA long-term tumor study. This review summarizes recent research on (i) the role of sphingolipids as important components of the diet, (ii) the role of sphingoid base metabolites and the ceramide cycle in expression of genes regulating cell growth, differentiation, and apoptosis, (iii) the use of cerebroside synthase inhibitors as tools for understanding the role of sphingolipids as mediators of cell cycle progression, renal disease, and stress responses, and (iv) the involvement of disrupted sphingolipid metabolism in animal disease and cellular deregulation associated with exposure to inhibitors of ceramide synthase and serine palmitoyltransferase, key enzymes in de novo sphingolipid biosynthesis. These findings illustrate how an understanding of the function of sphingolipids can help solve questions in toxicology and this is undoubtedly only the beginning of this story.
Fumonisin B1 (FB1) is the predominant member of a family of toxic metabolites produced by several species of Fusarium and is commonly found on corn. FB1 is a potent competitive inhibitor of ceramide synthase, which catalyzes the conversion of sphinganine and sphingosine to ceramide. The resultant accumulation of free sphingoid bases and the disruption of sphingolipid metabolism is believed to be the mechanism of toxicity of the fumonisins. The objectives of this study were to determine the relative potency of analogs of FB1 to inhibit ceramide synthase and to determine whether the inhibition is specific to mycotoxins with fumonisin-like structures. Fumonisins B1, B2, B3, B4, C4, and TA toxin (a structurally similar mycotoxin produced by the tomato pathogen, Alternaria alternata f. sp. lycopersici) were approximately equipotent inhibitors. Hydrolyzed fumonisins B1, B2, and B3, which lack the tricarballylic side chains, were only 30-40% as potent as the parent toxins. N-acetylated FB1 (FA1) did not block ceramide synthase, suggesting that FA1 is nontoxic. Inhibition of ceramide synthase by fumonisin analogs did not appear to be related to the lipophilicity of the compounds, as determined by computer estimation of log P values. The ability of relatively high (10 and 100 microm) doses of other mycotoxins that bear no structural similarity to fumonisins, including aflatoxin B1, cyclopiazonic acid, beauvericin, T-2 toxin, sterigmatocystin, luteoskyrin, verrucarin A, scirpentriol, and zearalenone, to block ceramide synthase was also determined. All of the toxins tested were negative in the bioassay with the exception of fumonisins, indicating that disruption of sphingolipid metabolism is a specific cytotoxic response.
Sphingolipids are highly bioactive compounds that participate in the regulation of cell growth, differentiation, diverse cell functions, and apoptosis. They are present in both plant and animal foods in appreciable amounts, but little is known about their nutritional significance. Recent studies have shown that feeding sphingomyelin to female CF1 mice treated with a colon carcinogen (1,2-dimethylhydrazine) reduced the number of aberrant colonic crypt foci; longer-term feeding also affected the appearance of colonic adenocarcinomas. Therefore, dietary sphingolipids should be considered in studies of the relationships between diet and cancer. Sphingolipids have also surfaced as important factors in understanding the mechanism of action of a recently discovered family of mycotoxins, termed fumonisins. Fumonisins are produced by fungi commonly found on maize and a few related foods, and their consumption can result in equine leukoencephalomalacia, porcine pulmonary edema and a number of other diseases of veterinary animals and, perhaps, humans. A cellular target of fumonisins is the enzyme ceramide synthase, and disruption of sphingolipid metabolism by fumonisins has been established by studies with both cells in culture and animals that have consumed these toxic mycotoxins. These findings underscore the ways in which sphingolipids and agents that affect sphingolipid utilization should be given consideration in selecting animal diets for nutritional and toxicological studies.
Fumonisins are inhibitors of the biosynthesis of sphingosine and more complex sphingolipids. In eucaryotic cells, fumonisin inhibition of sphingolipid biosynthesis is a result of inhibition of the enzyme ceramide synthase. Large increase in free sphinganine concentration in plant and animal cells are observed within a few hours after exposure to fumonisins and/or Alternaria toxins (AAL-toxins). Some of the sphinganine is metabolized to other bioactive intermediates, and some is released from cells. In animals, free sphinganine accumulates in tissues and quickly appears in blood and urine. Free sphingoid bases are toxic to most cells, and complex sphingolipids are essential for normal cell growth. Fumonisin B1 stimulates sphinganine-dependent DNA synthesis in Swiss 3T3 cells, but is mitoinhibitory in other cell types. In cultured cells the accumulation of bioactive long-chain sphingoid bases and depletion of complex sphingolipids are clearly contributing factors in growth inhibition, increased cell death, and (in Swiss 3T3 cells) mitogenicity of fumonisins. While disruption of sphingolipid metabolism directly affects cells, it may indirectly affect some tissues. For example, fumonisin B1 impairs the barrier function of endothelial cells in vitro. Adverse effects on endothelial cells could indirectly contribute to the neurotoxicity and pulmonary edema caused by fumonisins. It is hypothesized that fumonisin-induced changes in the sphingolipid composition of target tissues could directly or indirectly contribute to all Fusarium moniliforme-associated diseases.
Fumonisins are mycotoxins produced by the fungi Fusarium moniliforme, F. proliferatum, and other Fusarium species. Fumonisin B1, the most commonly found fumonisin, causes the fatal diseases equine leukoencephalomalacia and porcine pulmonary edema. Fumonisins are suspected human carcinogens because of the extraordinarily high incidences of esophageal cancer coincidentally found in areas of southern Africa and China where F. moniliforme-contaminated corn is consumed as a dietary staple. The subchronic (up to 90 days) effects of F. moniliforme-contaminated corn, corn cultures of this fungus, and purified fumonisin B1 (FB1) in rats and mice were systematically studied to determine target organs, characterize organ-specified lesions, and obtain dose-response data. The liver is a target organ in both species. Serum chemical findings indicative of hepatocellular injury and morphological findings, including apoptosis, appeared qualitatively similar in both species. The kidney is also a target organ in rats, but not mice. Lesions which include apoptosis and cellular degeneration are predominately found in the outer medella. Results of several investigations showed that the kidney was consistently affected at lower doses than the liver. The "no-observed-effect" level for nephropathy in rats was also consistently lower in males than females, suggesting a sex-related difference in nephrotoxic response to fumonisins. Other findings suggest that toxigenesis may be mediated by disruption of de novo sphingolipid biosynthesis. Hepatic and renal sphingolipid profiles, specifically sphinganine concentration and sphinganine-to-sphingosine ratio, were altered in rats fed FB1 at levels that did not cause serum chemical, organ weight, or histopathological evidence of toxicity.