Biosynthesis, extraction, purification, and analysis of trisporoid sexual communication compounds from mated cultures of Blakeslea trispora.

The zygomycete Blakeslea trispora produces high amounts of the general zygomycete ß-carotene-derived sexual signal compounds, the trisporoids. These can be isolated from the culture medium and purified by extraction with organic solvents followed by thin layer chromatography. Concentration is determined spectrophotometrically using specific extinction coefficients established for some members of this compound family. The effect of the extraction and activity of the isolated compounds is best tested physiologically, exploiting the ability of trisporoids to induce the formation of sexually committed hyphae, the zygophores, in other zygomycete species. Methods for B. trispora culture, trisporoid extraction, and further analyses of trisporoids are described in this chapter.

Model of the synthesis of trisporic acid in Mucorales showing bistability.

An important substance in the signalling between individuals of Mucor-like fungi is trisporic acid (TA). This compound, together with some of its precursors, serves as a pheromone in mating between (+)- and (-)-mating types. Moreover, intermediates of the TA pathway are exchanged between the two mating partners. Based on differential equations, mathematical models of the synthesis pathways of TA in the two mating types of an idealised Mucor-fungus are here presented. These models include the positive feedback of TA on its own synthesis. The authors compare three sub-models in view of bistability, robustness and the reversibility of transitions. The proposed modelling study showed that, in a system where intermediates are exchanged, a reversible transition between the two stable steady states occurs, whereas an exchange of the end product leads to an irreversible transition. The reversible transition is physiologically favoured, because the high-production state of TA must come to an end eventually. Moreover, the exchange of intermediates and TA is compared with the 3-way handshake widely used by computers linked in a network.

Statoliths in Phycomyces: characterization of octahedral protein crystals.

To identify the molecular mechanisms of gravitropism in the fungus Phycomyces blakesleeanus we determined several biochemical and physical parameters of paracrystalline protein bodies, so-called octahedral crystals. The crystals, which are present throughout the central vacuoles of the sporangiophore, function as statoliths (Schimek et al., 1999a,b). They possess an average volume of 9.96 microm(3) and a specific mass of 1.26 g cm(-3). SDS-PAGE of purified crystals shows three major proteins with relative molecular masses of 16, 46.5, and 55 kDa. These proteins are absent in gravitropism mutants which lack the crystals. Phototropism mutants (genotype mad) which are graviresponsive (class 1) and those which are defective in gravitropism (class 2) contain the crystals and the three associated proteins. Absorption spectra of isolated crystals and in situ absorption spectra of growing zones indicate the presence of chromophores, probably oxidized and reduced flavins. The flavin nature of the chromophores is also indicated by their fluorescence properties. It appears likely that the chromophores represent an essential part of the statoliths and thus the gravitropic transduction chain.

Interaction between gravitropism and phototropism in sporangiophores of Phycomyces blakesleeanus.

The interaction between gravitropism and phototropism was analyzed for sporangiophores of Phycomyces blakesleeanus. Fluence rate-response curves for phototropism were generated under three different conditions: (a) for stationary sporangiophores, which reached photogravitropic equilibrium; (b) for sporangiophores, which were clinostated head-over during phototropic stimulation; and (c) for sporangiophores, which were subjected to centrifugal accelerations of 2.3g to 8.4g. For blue light (454 nm), clinostating caused an increase of the slope of the fluence rate-response curves and an increase of the maximal bending angles at saturating fluence rates. The absolute threshold remained, however, practically unaffected. In contrast to the results obtained with blue light, no increase of the slope of the fluence rate-response curves was obtained with near-ultraviolet light at 369 nm. Bilateral irradiation with near-ultraviolet or blue light enhanced gravitropism, whereas symmetric gravitropic stimulation caused a partial suppression of phototropism. Gravitropism and phototropism appear to be tightly linked by a tonic feedback loop that allows the respective transduction chains a mutual influence over each other. The use of tropism mutants allowed conclusions to be drawn about the tonic feedback loop with the gravitropic and phototropic transduction chains. The results from clinostating mutants that lack octahedral crystals (implicated as statoliths) showed that these crystals are not involved in the tonic feedback loop. At elevated centrifugal accelerations, the fluence-rate-response curves for photogravitropic equilibrium were displaced to higher fluence rates and the slope decreased. The results indicate that light transduction possesses a logarithmic transducer, whereas gravi-transduction uses a linear one.

Gravitropism in Phycomyces: a role for sedimenting protein crystals and floating lipid globules.

To elucidate the graviperception of the unicellular fungus, Phycomyces blakesleeanus, sporangiophores were inspected for intracellular structures which relocate with respect to gravity. Two structures, paracrystalline proteins (so-called octahedral crystals) and an aggregate of lipid globules, were identified which showed redistribution upon reorientation of the sporangiophore. Octahedral crystals occur throughout the sporangiophore, including the apical growing zone, and are localized inside vacuoles in which they reside singly or in clusters of up to 40 loosely associated individuals. Upon a 90 degrees reorientation of sporangiophores, crystal clusters sedimented in approximately 50-200 s from the upper to the lower side, corresponding to a speed of 0.5-2 micrometers s-1. Stage-4 sporangiophores (with sporangium) of three mutants which lack the crystals displayed anormal kinetics of gravitropism and substantially reduced bending angles in comparison to sporangiophores of the wild type. While horizontally placed wild-type sporangiophores reached the vertical position after 10-12 h, the crystal-lacking mutants bent maximally 40 degrees-50 degrees upward. In stage-1 sporangiophores a conspicuous aggregate of lipid globules is positioned about 50 micrometers below the apex. The globules floated upwards when the sporangiophore was placed horizontally forming in this way a cap-like aggregate. It is proposed that both the sedimenting protein crystals and the upward-floating globules are involved in gravisensing.

Protein crystals in Phycomyces sporangiophores are involved in graviperception.

The sporangiophores of the zygomycete fungus Phycomyces blakesleeanus contain octahedral crystals with diameters of up to 5 micrometers in their vacuole. The crystals are associated with the intracellular membrane system. In tilted or horizontally placed sporangiophores, the crystals sediment to the respective lower face of the vacuole with a velocity of up to 100 micrometers per minute. The sedimentation is completed within about 2 minutes, well within the latency period for the negative gravitropic response of Phycomyces. Crystal-lacking mutant strains display a smaller maximal bending angle and a reduced gravitropic bending rate in comparison to the wild type. We therefore conclude that the crystals serve as statoliths for gravitropism in Phycomyces.

Indeterminate results of the second-generation hepatitis C virus (HCV) recombinant immunoblot assay: significance of high-level c22-3 reactivity and influence of HCV genotypes.

A second-generation recombinant immunoblot assay (RIBA 2.0) is used in the United States to confirm infection with hepatitis C virus (HCV) in samples that are anti-HCV (enzyme immunoassay) positive. In some cases, indeterminate results of RIBA 2.0, which are defined as reactivity to a single antigen species or reactivity limited to two proteins derived from the same coding region of the HCV genome, are encountered. This study was performed to establish the significance of indeterminate RIBA 2.0 results in relation to HCV RNA detection, high positivity for the c22-3 band, and the HCV genotype as determined by direct DNA sequencing. Ninety-six samples with indeterminate RIBA 2.0 results were studied. HCV RNA was detected in 21 of 34 (62%) samples with high reactivity to c22-3 and in 8 of 62 (13%) samples with low reactivity to c22-3. The HCV genotype distribution in samples that were RIBA 2.0 indeterminate and HCV RNA positive was significantly different from that in samples of a control group with positive results for both the RIBA 2.0 and HCV PCR. These results suggest that highly positive c22-3 samples are likely to be associated with HCV viremia and that infection with less common HCV genotypes is more commonly associated with indeterminate RIBA 2.0 results.

Blood donors who are repeatedly reactive for hepatitis C virus on enzyme immunoassay and positive on recombinant immunoblot assay: evidence of failure to identify some risk factors.

BACKGROUND: Despite the introduction of surrogate testing and subsequent antibody testing of donor blood, transmission of hepatitis C virus (HCV) still occurs. The institution from which this report originates is a medical center, and many of the blood donors have also been seen as patients at the institution. This provided an opportunity for comparison of donor questionnaire responses and medical history information and for correlation of those findings with HCV test results. STUDY DESIGN AND METHODS: HCV polymerase chain reaction (PCR) testing was performed on nine stored frozen sera from donors or former donors with previous positive results on HCV enzyme immunoassay (EIA) (first- or second-generation) and recombinant immunoblot assay (RIBA) (first- or second-generation). The medical histories were also reviewed for 22 of 23 such HCV EIA-repeatedly reactive, RIBA-positive donors and 88 randomly chosen HCV-negative donors. The lifestyle information was compared with the donors' responses on the blood donation questionnaires. The data were then correlated with available clinical and laboratory evidence of HCV transmission to transfusion recipients. RESULTS: For eight donors, there were no recipients of their blood to be assessed. For 9 of the remaining 15 donors, there were recipients who were tested for HCV. Recipients of blood from 5 of these 9 donors were repeatedly reactive for HCV; while recipients of blood from 4 donors were not. Donor PCR positivity correlated with apparent transmission (p = 0.047). Twelve of 20 HCV EIA-positive donors for whom history and questionnaires were available for comparison had at least one suggestive lifestyle or established risk factor in their medical records, while none of 88 HCV-negative controls did (p < 0.0001). The data did not indicate that paid donation correlated with failure to disclose these factors. CONCLUSION: Despite more explicit and intrusive donor questioning, it is still not possible to identify all possible risk factors at donations, though many donors who do not disclose all their risk factors are eliminated from the pool by the increasingly sensitive donor tests. As long as tests are not completely foolproof, workers must be vigilant regarding the ability to elicit complete information on a donor's risk. Further study is required to determine the best way(s) to do so.

Frequency of antibody to hepatitis C virus in asymptomatic HBsAg-negative chronic active hepatitis.

To determine the frequency of antibodies to hepatitis C virus in asymptomatic patients with HBsAg-negative chronic active hepatitis, sera from 30 consecutive patients with few or no symptoms of liver disease were tested by an enzyme immunoassay. The reactivity of antibodies detected by enzyme immunoassay against hepatitis C virus encoded antigens was determined by recombinant immunoblot assay. Antibodies were detected in 11 of the 30 patients (37%) and eight of the seropositive sera (73%) were reactive by recombinant immunoblot assay. Nonreactive patients were weakly positive by enzyme immunoassay (sample/cutoff ratio, less than or equal to 1.9) in contrast to reactive patients (sample/cutoff ratio, greater than or equal to 6.3). The prevalence of immunoserologic markers was similar in patients with and without antibodies (78 vs. 87%) but high titers (greater than or equal to 1:160) were more common in seronegative patients (53 vs. 11%). Additionally, seronegative patients had smooth muscle antibodies (83 vs. 25%, p less than 0.05) and concurrent extrahepatic immunologic diseases (37 vs. 9%) more commonly than seropositive counterparts. We conclude that asymptomatic patients with HBsAg-negative chronic active hepatitis frequently have antibodies to hepatitis C virus. These antibodies commonly react to specific viral antigens, especially if the enzyme immunoassay is strongly positive. Seropositive patients infrequently have concurrent immunologic disorders or smooth muscle antibodies. Immunoserologic markers lack diagnostic specificity except in higher titer.

Frequency and significance of antibody to hepatitis C virus in severe corticosteroid-treated autoimmune chronic active hepatitis.

To determine the frequency and significance of antibody to hepatitis C virus (anti-HCV) in severe autoimmune chronic active hepatitis, we tested sera from 85 cortico-steroid-treated patients by an enzyme immunoassay. Seropositive patients were assessed for specific antibodies to hepatitis C virus-encoded antigens by recombinant immunoblot assay. The findings in patients with and without anti-HCV were contrasted, and the frequency of seropositivity was compared with that in patients who had other types of chronic liver disease and in normal adults. Only 5 of the 85 patients with autoimmune hepatitis (6%) were seropositive for anti-HCV, and only 2 of these patients were reactive by recombinant immunoblot assay. The frequency of seropositivity in autoimmune hepatitis was not significantly different from that in hepatitis B surface antigen-positive (9%) and cryptogenic (18%) disease, but it was significantly less than that in posttransfusion chronic active hepatitis (6% versus 75%; P less than 0.001). Two patients became seronegative after corticosteroid therapy; both had been nonreactive by recombinant immunoblot assay. Four of the seropositive patients entered remission during corticosteroid therapy, including three whose sera were nonreactive to virus-encoded antigens. We conclude that anti-HCV occurs infrequently in corticosteroid-treated severe autoimmune hepatitis and that antibodies detected by enzyme immunoassay may be nonreactive to hepatitis C virus-encoded antigens. Seropositive patients who are nonreactive by immunoblot assay may still respond to corticosteroid therapy and become seronegative during treatment.

Frequency and significance of antibody to hepatitis C virus in severe corticosteroid-treated cryptogenic chronic active hepatitis.

To determine the frequency and significance of antibody to hepatitis C virus (anti-HCV) in severe cryptogenic chronic active hepatitis (CAH), we tested sera from 17 corticosteroid-treated patients by an enzyme immunoassay. Specificity of the antibodies to HCV-encoded antigens was assessed by recombinant immunoblot assay. The findings in patients with and without anti-HCV were contrasted, and the frequency of seropositivity was compared with that in patients who had other types of chronic liver disease and in normal adults. Only three patients (18%) with severe cryptogenic CAH had anti-HCV. Sera from two of these patients were reactive by recombinant immunoblot assay; the other sample produced an indeterminate reaction. The frequency of seropositivity in patients with cryptogenic disease was not statistically different from that in patients with autoimmune CAH (6%), hepatitis B surface antigen-positive CAH (9%), or alcoholic liver disease (0%), but it was significantly less than in those with posttransfusion CAH (18% versus 75%; P less than 0.01). Seropositive patients tended to have lower serum aspartate aminotransferase, gamma-globulin, and bilirubin levels than seronegative counterparts, and they did not have histologic features of confluent necrosis at initial assessment. Two of the three seropositive patients, both of whom had been reactive by recombinant immunoblot assay, entered remission during therapy, and one, with an indeterminate reaction, died of liver failure. We conclude that anti-HCV occurs infrequently in severe corticosteroid-treated cryptogenic CAH. Seropositive patients may have less severe inflammatory activity than seronegative counterparts. Cryptogenic disease may improve during corticosteroid treatment, a result suggesting an underlying immunologic disorder in some patients.