The cellular and molecular toxicity of sporidesmin.

The fungal metabolite sporidesmin is responsible for the hepatogenous photosensitising disease facial eczema in livestock. Toxicity is due to a sulfur-bridged epidithiodioxopiperazine ring that has wide biological reactivity. The ways in which the toxin causes hepatobiliary and other tissue damage have not been established. Hypotheses include direct interaction with cellular thiols including protein cysteine residues or production of reactive oxygen species resulting in oxidative stress. Comparison with the cellular effects of the structurally related compound gliotoxin suggests additional mechanisms including interaction with cell adhesion complexes and possible downstream consequences for regulated necrosis as a response to tissue injury. Revision of hypotheses of how sporidesmin affects cells has the potential to generate new strategies for control of facial eczema including through identification of proteins and genes that are associated with resistance to the disease.

Effects of sporidesmin on cultured biliary tract cells from Romney lambs that differed in their sensitivity to sporidesmin.

AIMS To investigate the effects of sporidesmin on cells cultured from the epithelial surface of sheep gallbladder walls, and to examine cellular responses to sporidesmin using cultures prepared from the gallbladders of sheep from selection lines that differed in sensitivity to sporidesmin-induced liver damage. METHODS Gallbladders were obtained following slaughter of lambs that were selected for resistance or susceptibility to sporidesmin-induced liver damage, or were not selected (controls). Monolayer cell cultures were established after incubation of excised gallbladders with protease to detach the lining epithelial cells from the muscular and connective tissue of the gallbladder wall. Released cells were harvested and transferred to culture flasks or dishes, then incubated with 1 µg/mL sporidesmin and were examined at 5 minute intervals, up to 3 hours, using light microscopy to monitor loss of attachment of cultured cells. Immunofluorescence staining of cell cultures was used to identify cytokeratin 19 as a marker for biliary epithelial cells, and to characterise sporidesmin-induced change in the cellular distribution of actin microfilaments. Gallbladder size was also measured. RESULTS In cultures incubated with sporidesmin, cells at the margins of sheets of cells showed the first signs of change, becoming unanchored from the culture vessels while remaining attached to the cell mass. This was followed by progressive detachment of sheets of cells and clumps of rounded cells. Disruption of cytoplasmic actin microfilaments with accumulation of actin in the cytoplasm adjacent to the plasma membrane preceded major detachment of cells. Cells from susceptible line lambs were extensively rounded up within 1 hour with complete or almost complete detachment within 2 hours, whereas cultures from resistant line lambs generally only contained detaching rounded-up cells at the periphery of monolayers within 2 hours; detachment observed in cells from the control line lambs was intermediate. There was a trend for gallbladders to be smaller in male lambs from the resistant line compared to the control or susceptible lines. CONCLUSIONS Altered cell adhesion and disruption of microfilament actin in biliary cell cultures incubated with sporidesmin suggest that biliary tract pathology may be due to the effects of the toxin on cytoplasmic and cell surface protein networks that affect the integrity of the epithelial lining of the biliary tract. These effects can be interpreted in terms of the hepatobiliary pathology of facial eczema, including potential differences in sensitivity of biliary tract cells that may contribute to inherited resistance and susceptibility to sporidesmin and hence facial eczema.

Survival of Neisseria meningitidis outside of the host: environmental effects and differences among strains.

Neisseria meningitidis is a gram-negative bacterium that lives as a commensal in the human nasopharynx. Meningococci are generally non-invasive, but can invade the nasopharyngeal epithelia and enter the bloodstream causing life-threatening illnesses. It is generally thought that meningococci do not survive for long outside the host, and that transmission requires relatively close contact between hosts. There are some reports, however, that meningococci can survive drying on surfaces, including glass, plastic and cloth. Our examination of N. meningitidis strains dried on glass showed differences in survival of isolates belonging to serogroups B, C and W135, including persistence of Cuban, New Zealand, and Norwegian epidemic strains up to 8 days, depending on temperature and humidity. Survival of a New Zealand epidemic strain isolate NZ98/254 under ambient conditions in the laboratory was greatest in winter suggesting that environmental factors impacted survival. For most isolates, including NZ98/254, survival under controlled conditions at 30 °C was greater at 22% than 30% relative humidity. There were also some differences in survival between carriage and invasive strains. The results suggest that N. meningitidis could be transmitted through contact with surfaces outside the host, potentially including contact through shared drinking vessels.

Differential patterns of liver proteins in experimental murine hepatosplenic schistosomiasis.

Schistosoma mansoni eggs produced by adult worms in the mesenteric vasculature become trapped in the liver, where they induce granulomatous lesions and strong immune responses. Infected individuals suffer from intestinal schistosomiasis (INT) in 90% of cases, whereas the remaining 10% present with severe hepatosplenic schistosomiasis (HS). The CBA/J mouse model mimics human disease, with 20% of infected mice developing hypersplenomegaly syndrome (HSS) that resembles HS and 80% developing moderate splenomegaly syndrome (MSS) similar to INT. We studied differential patterns of protein expression in livers of 20-week-infected CBA/J mice with MSS or HSS to understand the molecular changes that underlie these two disease forms. Using differential in-gel electrophoresis to identify differentially expressed protein spots, we found 80 protein spots significantly changed with infection and 35 changes specific to severe disease. In particular, the abundances of prohibitin 2, transferrin isoforms, and major urinary protein isoforms were significantly altered in HSS mice. Furthermore, annexin 5, glutathione S-transferase pi class, and S. mansoni phosphoenolpyruvate carboxykinase expression levels changed significantly with schistosome infection. Additionally, levels of major urinary protein decreased and levels of transferrin increased significantly in the sera of HSS mice compared to levels in sera of MSS or control mice, and these differences correlated to the degree of splenomegaly. These findings indicate that the liver protein abundances differ between MSS and HSS mice and may be used for the development of diagnostic markers for the early detection of hepatosplenic schistosomiasis.

Segmental axonopathy of Merino sheep in New Zealand.

AIM: To investigate an axonopathy of Merino sheep that caused progressive hindlimb ataxia and slight to moderate paresis, with the purpose of understanding its pathogenesis. METHODS: Tissues were fixed in buffered paraformaldehyde or paraformaldehyde and glutaraldehyde, processed into wax and epoxy resin, respectively, and examined by light and electron microscopy. Fresh frozen spinal cord and trigeminal nerve roots were subjected to homogenisation, centrifugation and two-dimensional electrophoresis. Selected protein spots were identified using matrix-assisted laser desorption ionisation (MALDI) mass spectrometry. RESULTS. By light microscopy, there were large pale foamy spheroidal axonal swellings affecting peripheral as well as central axons. By electron microscopy, these were shown to contain many membrane-bound vesicles. The main abnormalities in expressed proteins involved cytoskeletal elements and myosin heavy chain, the latter interpreted as associated with the molecular motor myosin Va. CONCLUSIONS: The disorder is the same as that described in Merinos in Australia as segmental axonopathy, and believed to have an inherited aetiology. The lesions and protein changes indicate abnormalities of the cytoskeleton, its relationship with the myelin sheath, and myosin Va molecular motor. The consequence appears to be abnormal axonal transport and inability to maintain the integrity of axons and their myelin sheaths.

Submitochondrial distribution and delayed proteolysis of subunit c of the H+-transporting ATP-synthase in ovine ceroid-lipofuscinosis.

The neuronal ceroid-lipofuscinose (NCL) are recessively inherited lysosomal storage diseases in children and animals. The major stored protein in many of these diseases is subunit c of the mitochondrial inner membrane H+-transporting ATP-synthase. Previous studies of naturally occurring ovine ceroid-lipofuscinosis (OCL) in South Hampshire sheep showed that the genes and transcripts for subunit c were normal and inferred that this protein was expressed normally in mitochondria prior to storage in lysosomes. Accumulation in mitochondria has not been conclusively established and we have therefore used the South Hampshire model to demonstrate approximately 1.8-fold normal levels of subunit c in mitochondrial inner membranes prepared from liver. Other mitochondrial inner membrane and ATP-synthase proteins that could be detected by mass spectrometry (MS) or two-dimensional electrophoresis (2-DE) were present in normal amounts. The accumulating subunit c showed normal post-translational modification but was abnormally resistant to proteolysis. These results are consistent with the hypothesis that OCL may result from a mitochondrial disorder that affects turnover of correctly expressed subunit c, although we cannot exclude the possibility that a postmitochondrial defect delays processing of subunit c out of mitochondria.

Application of proteomics for determining protein markers for wool quality traits.

The technique of two-dimensional electrophoresis (2-DE) has been under investigation for its usefulness in identifying protein markers for wool quality traits in sheep. However, before this could be achieved, unique problems relating to the detection and quantitation of wool proteins needed to be overcome so that 2-DE protein maps could be examined using computational programs like Melanie II. Four protein staining regimes were examined. Colloidal Coomassie Blue G-250 was found to be superior to Coomassie Blue R-250 and gave satisfactory staining of all protein classes. Silver staining detects minor strings of keratinous proteins, but unfortunately it negatively stains intermediate filament proteins, the major high sulphur proteins (HSPs) and the high glycine tyrosine proteins and the latter two classes can only be seen by overstaining the background of the gel. In contrast, labeling reduced keratins with [14C]iodoacetamide, followed by autoradiography detection, results in a protein map with low background and all protein spots stained positively. 2-DE has been used to obtain wool protein maps of Lincoln/Merino chimeric sheep to examine wool originating from two genotypes grown with different crimp frequencies within the same fleece. Between fleece, variations have also been examined. Work to date suggests that several major HSPs may be associated with the fibre curvature trait known as crimp frequency. From matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectral mapping, one of these proteins has been identified as being from the B2A family from the HSP class.

Disease-specific pathology in neurons cultured from sheep affected with ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCL, Batten disease) are a group of inherited neurodegenerative storage diseases in children. Mutations in different genes underlie different forms. Subunit c of mitochondrial ATP synthase is specifically stored in autofluorescent bodies in most of them, including a form in sheep. Mature bodies are lysosomal but the initial site of storage is not known, nor is it known how this leads to the characteristic neurodegeneration. Neurons were cultured in serum-free medium from control and affected sheep fetuses at 90 days gestation. They showed positive microtubule-associated protein staining, developed neurites, and had typical neuron morphology. Time-dependent accumulation of subunit c and of fluorescent storage bodies was observed in affected cells by immunocytochemistry and confocal microscopy. A small number of autofluorescent bodies were apparent after 4 days in culture. After 10 days these bodies were more numerous, more intensely autofluorescent, and often larger in size. By 14 and 21 days many neurons were packed with autofluorescent material. These bodies were not seen in control cultures. Immunocytochemistry revealed subunit c-positive storage material only in affected neurons and not in affected glial cells. Confocal microscope analysis, using organelle-specific dyes, demonstrated colocalization of autofluorescent bodies with lysosomes, not with mitochondria. Survival rates of the affected cells were unaffected by the storage body accumulation over a 3-month period. These cultures can now be used to study the mechanism of subunit c accumulation and of neurodegeneration and to test therapeutic possibilities.

Immunolocalization of basic fibroblast growth factor (bFGF) in human periodontal ligament (PDL) tissue.

The distribution of basic fibroblast growth factor (bFGF) in periodontal ligament (PDL) tissue was investigated in samples which were obtained from freshly extracted human teeth. The PDL tissue was collected by scraping, and bFGF was identified and localized by immunohistochemistry. Fibroblasts, endothelial cells, some fibrocytes and extracellular matrix (ECM) stained positively for bFGF. It was observed that cells from healthy PDL stained more intensely than those from PDL of teeth associated with chronic periodontitis; histological cell counts revealed that the numbers of fibroblasts was greater (p < or = 0.0005) in healthy PDL than in diseased PDL tissue. The results of this study show that bFGF is produced primarily by PDL fibroblasts and endothelial cells in the PDL and that bFGF levels may be decreased in tissue associated with chronic periodontal lesions.

Characteristics of the chemiluminescence from the blood plasma of a normal human population.

Oxygen-dependent chemiluminescence was detected from human blood plasma. The intensity of the chemiluminescence increased about three-fold under oxygenation and decreased almost to the background (zero) level under a nitrogen atmosphere. The blood plasma from a sample (n = 100) of donors was tested to determine the variability of several properties of the chemiluminescence in a normal population. No statistically significant difference in blood plasma chemiluminescence between genders was found, but there was a slight increase in luminescence intensity with age. However, the results were found to be dependent on a number of other factors, such as diet, smoking and the length of time between the donor's last meal and the sampling of the blood. Some of the trends in the results coincide with similar trends in the plasma lipoprotein levels and thus support the suggestion that the chemiluminescence arises from the decomposition of lipid hydroperoxides. These factors must all, therefore, be taken into account when using chemiluminescence as an indicator of illness.

Variant proteins in ovine ceroid-lipofuscinosis.

Two-dimensional polyacrylamide gel electrophoresis has been used to search for disease-related protein variation in South Hampshire sheep with ovine ceroid-lipofuscinosis. Several hundred proteins in homogenates and subcellular fractions from livers have been examined, using isoelectric focusing as the first dimension separation, and SDS PAGE in the second dimension. Under these circumstances it was not possible to detect subunit c of the Fo region of ATP synthase, as this protein did not enter the isoelectric focusing gels. However, our studies emphasize the selective nature of misprocessing of subunit c, as we have not been able to detect any other consistent variation between affected and control animals for over 200 mitochondrial fraction proteins. Comparison of the presence or absence, and abundance, of proteins from isolated storage bodies with their counterparts in subcellular fractions from normal liver indicated that storage bodies contained a small subset of mitochondrial proteins, in addition to subunit c, with possible minor contributions from lysosomal, microsomal, and soluble proteins. Analysis of extramitochondrial proteins showed greater than 10-20-fold accumulation of ferritin light chains in microsomes, and partial loss of a putatively lysosomal protein, in ovine ceroid-lipofuscinosis. In addition, senescence marker protein was more abundant in the cytosolic fraction of controls, compared with affected individuals. We are currently investigating the basis and significance of these differences.

Assessment of DNA fingerprinting for determining genetic diversity in sheep populations.

Genomic DNA, prepared from 12 animals from four sheep flocks, was digested with either HaeIII or HinfI and probed with three DNA fingerprinting probes. Mean DNA fingerprint band sharing and band frequency calculated for each flock were used to estimate genetic diversity. Each of the DNA fingerprinting systems showed the same trend in diversity within the sampled flocks, and greater diversity between the flocks than within the flocks. DNA fingerprinting therefore provides a useful measure of genetic diversity in sheep.

Mouse strain differences in susceptibility to sporidesmin-induced biliary tract injury.

Biliary tract injury was examined in four inbred strains of mice orally dosed with 500 micrograms of the fungal toxin sporidesmin. Semiquantitative histological analysis was used to assess the grade of necroinflammatory changes in the gall bladder, intra- and extrahepatic biliary tree and lobular parenchyma. Injury was greatest in the C57BL/6 and C3H strain mice and was least in SJL/J mice. In these strains injury was greatest at 4 days and had regressed by 10 days. In BALB/c mice the damage, although similar to that in SJL/J mice at 4 days, persisted at the same severity at day 10 and was accompanied by periductal fibrosis and occasionally by obliteration of ducts typical of sclerosing cholangitis. Analysis of the time-course of development of the lesions in C57BL/6 mice showed that the primary target for the toxin is the biliary epithelium. The severity of the lesions within the liver increased centripetally and the worst affected ducts were found at the confluence of the lobar ducts with the common bile duct. The variation in the degree of damage and rate of healing between strains may be due to differences in sporidesmin excretion in bile or interactions with biliary epithelial cells and/or efficacy of protective cellular repair mechanisms.

A double-label two-dimensional procedure for the analysis of membrane proteins.

A previously described double-label two-dimensional electrophoresis procedure (Wheeler et al., Anal. Biochem. 1986 159, 1-7) for the analysis of differences between two complex mixtures of soluble proteins has been modified to allow analysis of proteins requiring detergent for aqueous solubility. The samples are first disrupted by sonication and the insoluble proteins concentrated by high-speed centrifugation. The proteins are then solubilized with sodium dodecyl sulfate and further concentrated in a centrifugal concentrator to achieve protein mixtures suitable for labeling with 14C and 3H by reductive methylation and subsequent two-dimensional electrophoresis. The sample concentration step is quick, minimizes the concentration of sodium dodecyl sulfate in the final sample, and avoids the potential difficulties associated with lyophilization or precipitation. The modified procedure was applied to the analysis of erythrocyte membranes, platelets and isolated placental microvilli. The high resolving power of two-dimensional gel electrophoresis is retained and the procedure is sensitive because the conditions of labeling allow substantial incorporation of radioactivity into protein despite the presence of detergent.

Double-label two-dimensional electrophoresis of serum and plasma proteins from patients with multiple sclerosis.

A double-label two-dimensional electrophoretic procedure has been used to search for abnormal proteins in the serum and plasma of patients with definite multiple sclerosis. The procedure possesses high resolving power and is particularly valuable in comparative studies of complex mixtures of proteins since differences due to gel-to-gel variations in protein mobilities are eliminated. Proteins present in serum and plasma at a concentration of 5-10 micrograms/ml are detected routinely. No consistent differences were observed between patients with multiple sclerosis and normal subjects in comparisons using pooled or individual specimens of serum or plasma. The absence of consistent differences in serum and plasma proteins between normal subjects and MS patients applied even when the sensitivity of the procedure was increased several-fold and when the possibly obscuring effect of albumin in the electrophoretic gels was eliminated. To our knowledge, this study combines the most extensive and sensitive search for consistent abnormalities in serum and plasma proteins in multiple sclerosis sera thus far reported.

Antibody to liver membrane antigens in chronic active hepatitis. IV. Exclusion of specific reactivity to polypeptides and glycolipids by immunoblotting.

The reactivity of sera was examined in patients with autoimmune chronic active hepatitis and other liver diseases by immunoblotting. Polypeptides and glycolipids of liver plasma membrane, liver-specific lipoprotein and kidney membrane were separated and probed with sera from patients and from a rabbit immunized with mouse liver plasma membrane. Chronic active hepatitis sera reacted with a number of polypeptides in the liver plasma membrane preparations; similar but weaker reactivity was observed with sera from patients with other diseases and in some healthy subjects. Chronic active hepatitis sera did not react with glycolipids from liver plasma membrane. The immune rabbit serum reacted with two polypeptides of 180 kd present in liver plasma membrane but absent from kidney membrane, with two polypeptides of 50 kd which were nonliver-specific but species-specific, and with three major glycolipid components of liver plasma membrane: this reactivity thus differed markedly from that of the chronic active hepatitis sera. In studies using dot-blotting, it was found that solubilization of liver plasma membrane in detergents resulted in a marked reduction of the reactivity to liver plasma membrane of chronic active hepatitis sera, but little change in the reactivity of the chronic active hepatitis and other sera with liver-specific lipoprotein by immunoblotting indicated that liver-specific lipoprotein consisted of constituents of liver plasma membrane together with intracellular proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and culture of biliary epithelial cells from the biliary tract fraction of normal rats.

A method has been developed for the isolation of a population of cells enriched in epithelial lining cells from the bile ducts of normal rats. The procedure utilized digestion by pronase of the white strands of biliary and connective tissue which remained after hepatocytes had been mechanically removed from collagenase-perfused liver. The resulting cell population was enriched in cells whose ultrastructure resembled that of the epithelial cells of intrahepatic bile ducts. Contamination with hepatocytes, hepatocyte nuclei and erythrocytes was less than 2%. The cells have been maintained in short-term culture. The major morphological change during the first 2 days of culture was proliferation of microvilli, but cell protein composition was unchanged when analysed by polyacrylamide gel electrophoresis. A rabbit antiserum against bovine hoof prekeratin was used to immunohistochemically stain the intermediate filaments of biliary epithelium and was shown to stain more than 90% of the cells in the isolated cell population.

A double-label two-dimensional gel electrophoresis procedure specifically designed for serum or plasma protein analysis.

A double-label two-dimensional electrophoresis procedure has been developed which is specifically designed for the comparison of serum or plasma proteins in two different samples. Proteins are labeled by reductive methylation with [14C]- or [3H] formaldehyde. The procedure is economical because small quantities of relatively inexpensive isotopes are used and it is at least as sensitive as silver staining in detecting proteins. A fourfold increase in the sensitivity of autoradiography over existing methods was obtained by performing autoradiography before processing the gel for fluorography. A spot in the electrophoretic gel that contains 17-28 ng of labeled protein is detectable. This corresponds to proteins present in serum at a concentration of 5-10 micrograms/ml. Even greater sensitivity can be achieved, at greater expense, by increasing the quantities of the radioisotopes in the labeling reaction. The particular value of the double label approach is that complex mixtures from two different sources are resolved together thus eliminating the possibility of differences arising from the resolving procedure itself. The procedure was applied to a mixture of serum and plasma from a single subject and a number of qualitative and quantitative differences were observed.

Sporidesmin and gliotoxin induce cell detachment and perturb microfilament structure in cultured liver cells.

Changes in cell morphology and cell adhesion occurred when cultured cells from the rat liver cell strain C3 were exposed to the fungal toxins, sporidesmin or gliotoxin. Both toxins caused loss of attachment of the cells to the plastic of tissue culture plates and this effect was preceded by loss of actin cables. Other changes included cytoplasmic vacuolation and blocked entry into S-phase of the cell cycle. Under these conditions [3H]thymidine incorporation into the cells was also diminished but changes were not detected in the amount of cellular actin, or in the accessibility of cell surface proteins to iodination carried out by the Bolton and Hunter method. The observations suggest that disruption of microfilaments is one of the earliest effects of these toxins on eukaryotic cells.