The major antigenic determinant of influenza A and B virus is haemagglutinin (HA). The HA content is an important specification of influenza vaccines. HA in vaccines has typically been quantified by single-radial-immunodiffusion (SRID). However, SRID is a laborious and low throughput assay. Moreover, sensitivity, accuracy, and precision, especially for non-purified (in-process) influenza virus is relatively low. We present a novel method for quantification of HA in influenza viral cultures as well as for the identification of HA from individual influenza strains in trivalent vaccines. The method is based on the separation of HA(1), the hydrophilic subunit of HA, from the more hydrophobic viral and matrix components by reversed-phase high performance liquid chromatography (RP-HPLC). The HA(1) peak area is demonstrated to be proportional to the level of HA in non-purified, semi-purified and purified vaccine products of various epidemic and pandemic influenza A and B strains propagated in PER.C6((R)) cell cultures. The RP-HPLC assay selectivity allows for the simultaneous identification and quantification of HA(1) from influenza A and B strains in the yearly revised trivalent vaccines for epidemic outbreaks.

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Our knowledge of the yeast cell wall has increased rapidly in the past few years, allowing for the first time a description of its structure in molecular terms. Two types of cell wall proteins (CWPs) have been identified that are covalently linked to beta-glucan, namely GPI-CWPs and Pir-CWPs. Both define a characteristic supramolecular complex or structural unit. The GPI building block has the core structure GPI-CWP-->beta1,6-glucan-->beta1,3-glucan, which may become extended with one or more chitin chains. The Pir building block is less well characterized, but preliminary evidence points to the structure, Pir-CWP-->beta1,3-glucan, which probably also may become extended with one or more chitin chains. The molecular architecture of the cell wall is not fixed. The cell can make considerable adjustments to the composition and structure of its wall, for example, during the cell cycle or in response to environmental conditions such as nutrient and oxygen availability, temperature, and pH. When the cell wall is defective, dramatic changes can occur in its molecular architecture, pointing to the existence of cell wall repair mechanisms that compensate for cell damage. Finally, evidence is emerging that at least to a considerable extent the cell wall of Saccharomyces cerevisiae is representative for the cell wall of the Ascomycetes.

Our knowledge of the yeast cell wall has increased rapidly in the past few years, allowing for the first time a description of its structure in molecular terms. Two types of cell wall proteins (CWPs) have been identified that are covalently linked to β-glucan, namely GPI-CWPs and Pir-CWPs. Both define a characteristic supramolecular complex or structural unit. The GPI building block has the core structure GPI-CWP→β1,6-glucan→β1,3-glucan, which may become extended with one or more chitin chains. The Pir building block is less well characterized, but preliminary evidence points to the structure, Pir-CWP→β1,3-glucan, which probably also may become extended with one or more chitin chains. The molecular architecture of the cell wall is not fixed. The cell can make considerable adjustments to the composition and structure of its wall, for example, during the cell cycle or in response to environmental conditions such as nutrient and oxygen availability, temperature, and pH. When the cell wall is defective, dramatic changes can occur in its molecular architecture, pointing to the existence of cell wall repair mechanisms that compensate for cell damage. Finally, evidence is emerging that at least to a considerable extent the cell wall of Saccharomyces cerevisiae is representative for the cell wall of the Ascomycetes.

The yeast Saccharomyces cerevisiae is the first fungus for which the structure of the cell wall is known at the molecular level. It is a dynamic and highly regulated structure. This is vividly illustrated when the cell wall is damaged and a salvage pathway becomes active, resulting in compensatory changes in the wall.

To isolate the putative linkage region between chitin and beta(1-->3)-glucan, Saccharomyces cerevisiae cell walls were digested with beta(1-->3)-endoglucanase and the reducing ends of the enzyme-resistant glucose chain stubs were labeled by reduction with borotritide. The radioactive material was further digested with exochitinase to remove the bulk of the chitin, and the liberated oligosaccharides were fractionated on a sizing column. A single peak (compound I) was found to consist of N-acetylglucosamine, glucose, and glucitol residues in the ratio 1:2:1. By digestion with beta-N-acetylglucosaminidase and by NMR spectroscopy, N-acetylglucosamine was identified as the nonreducing terminus, linked to laminaritriitol by a beta(1-->4) bond. Five additional oligosaccharides were recovered, two being analogs of compound I, with 1 or 3 glucose units, respectively; the remaining three were shown to be the reduced analogs of laminaribiose, laminaritriose, and laminaritetraose. The presence of N-acetylglucosamine-containing oligosaccharides arises from the activity of chitinase in cleaving 2 sugar units sequentially in those chains containing an odd number of N-acetylglucosamine residues; correspondingly, oligosaccharides containing only glucose and sorbitol derived from even-numbered chitin chains, a result implying that chitinase can hydrolyze the linkage between N-acetylglucosamine and glucose. It is concluded that the terminal reducing residue of a chitin chain is attached to the nonreducing end of a beta(1-->3)-glucan chain by a beta(1-->4) linkage. Experiments with appropriate mutants showed that synthesis of the chitin combined with glucan is catalyzed by chitin synthetase 3. The timing and possible mechanism of formation of the chitin-glucan linkage is discussed.

The sensitivity of cytochrome-P450-dependent sterol 14alpha-demethylase (P450(14DM)) to prochloraz and several prochloraz analogues was studied in a cell-free assay of Botrytis cinerea Pers. ex Fr. The EC50 values (concentrations which inhibited radial growth of B. cinerea by 50 %) of the compounds tested ranged from 3-3 x 10(-8) to 1.7 x 10(-5) m. The IV50 values (concentrations which inhibited cell-free C4-demethyl sterol synthesis by 50 %) in cell-free assays of B. cinerea ranged from 2.6 x 10(-9) to 4.4 x 10(-7) M. Ranking compounds in terms of their relative inhibitory potencies showed quite similar trends to the order of fungitoxicity, but the IC50 values did not quantitatively reflect the differences in toxicity. Therefore, the differential inhibition of cell-free P450(14DM) activity by these compounds cannot fully account for their differences in activity towards B. cinerea. Additional mechanisms must be involved.The compounds tested were generally more potent in the B. cinerea assay than in similar assays developed for Penicillium italicum Wehmer and, in particular, Saccharomyces cerevisiae Meyen. This correlated with the relatively higher activity of most test compounds to B. cinerea. Results suggest that the cell-free assay of B. cinerea is more useful to evaluate candidate fungicides as inhibitors of sterol 14alpha-demethylase activity than similar assays from model organisms.The present study confirms that the affinity of prochloraz analogues for P450(14DM) depends on the nature of the N-1 substituent of the imidazole and the azole ring. It was also found that addition of an amino group at C-2 of the imidazole moiety of prochloraz gave a compound (6) which inhibited 4,4-demethyl sterol biosynthesis in B. cinerea at a different site from the P450(14DM). This was confirmed by the observation that laboratory-generated triadimenol-resistant isolates of B. cinerea showed reduced sensitivity to triadimenol and prochloraz, but not to compound 6.

An assay for measuring ergosterol synthesis in cell-free extracts of the filamentous plant pathogen Botrytis cinerea is described. The extracts capable of synthesizing C4-desmethyl sterols from [2-C-14]mevalonate were derived by mechanical disruption of young conidial germlings in a Bead-Beater apparatus. The C4-desmethyl sterol fraction consisted of three distinct compounds and totalled 39% of the non-saponifiable lipids formed. Ergosterol accounted for 63% of the C4-desmethyl sterols. Only small amounts of C4-monomethyl sterols were synthesized, while C4,4-dimethyl sterols made up 29% of the non-saponifiable lipids. The latter fraction mainly consisted of lanosterol (54%) and eburicol (28%). The cell-free system had a narrow pH optimum for synthesis of C4-desmethyl sterols of pH 7.3-7.4. Cell-free synthesis of C4-desmethyl sterols was inhibited by the imidazole fungicide imazalil, concomitant with an accumulation of eburicol. The IC50 value (concentration of fungicide which inhibited cell-free synthesis of C4-desmethyl sterols by 50%) was 9.1 x 10(-9) m. These results are consistent with the hypothesis that imazalil is a potent inhibitor of the cytochrome P450-dependent sterol 14alpha-demethylase of B. cinerea. The method described may be used to screen compounds biochemically for inhibition of sterol synthesis in an agriculturally important plant pathogen.

The interaction of various sterol demethylation inhibitors (DMIs) and experimental compounds with cytochrome-P450-dependent sterol 14alpha-demethylase (P450(14DM)) from Penicillium italicum was studied by difference spectroscopy using a preparation of microsomal P450 isozymes and assays with cell-free extracts capable of synthesizing ergosterol from [C-14]mevalonate. The EC50 values (concentrations of compounds which inhibited radial growth of P. italicum by 50%) of the compounds ranged from 3 x 10(-8) M to levels higher than 10 (-3) M. All the compounds investigated gave type II difference spectra and interfered with the binding of carbon monoxide (CO) to microsomal P450 isozymes. However, the differences in the IC50 values (concentrations of compounds which caused 50% of maximal type II spectral change) between compounds and their inhibition of CO binding did not correlate with the fungicidal activity of the compounds. Hence, neither type II difference spectra nor CO-displacement tests with microsomal preparations from this fungus can be used for studying the selective fungitoxicity of azoles. Data obtained using the cell-free sterol 14alpha-demethylase assay revealed larger differences between the compounds in their inhibition of sterol 14alpha-demethylation. The I50 values (concentrations of compounds which inhibited cell-free sterol biosynthesis by 50%) varied from 4-3 x 10(-9) to 4.4 x 10(-5) M. This assay was able to rank the compounds in order of fungitoxicity, but the I50 and EC50 values did not correlate quantitatively. Consequently, the observed differential inhibition of P450(14DM)) activity between the compounds cannot fully explain their selective fungitoxicity. Additional mechanisms must be involved. The present study supports the general opinion that the affinity of azoles for P450(14DM) depends on the nature of their N1 substituent. However, it was demonstrated that the nature of the azole moiety was also of importance in determining the affinity of DMIs for P450(14DM).

The selective fungitoxic actions of prochloraz (an imidazole) and a triazole fungicide, quinconazole (3-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-4(3H)-quinazolinone: II), were studied with selected phytopathogenic fungi. With the exception of Ustilago maydis, all the fungi tested were more sensitive to prochloraz than to II. A number of DMI-resistant mutants of Penicillium digitatum and P. italicum showed positive cross-resistance to both DMIs, but except for P. italicum isolate H17, the levels of resistance to II were much higher than to prochloraz.The generally higher toxicity of prochloraz to the fungi investigated, as compared to II, could not be ascribed to the slightly higher accumulation of prochloraz. With regard to prochloraz, there was no general correlation between the sensitivity of the fungi tested and the amount of fungicide accumulated. A similar situation was evident for II. However, the DMI-resistant mutants of P. italicum did show a reduced accumulation of both azoles, which may account for a low level of acquired DMI-resistance in this fungus. Since accumulation levels of the test compounds in the isolates with different degrees of resistance were the same, additional mechanisms of resistance may be involved in isolates with relatively high degrees of DMI-resistance.No detectable amounts of fungicide metabolites were found in most fungi tested over a 16-hour incubation period. Therefore, fungal metabolism is not generally responsible for the differences in sensitivity between fungi to each azole tested. It also does not generally explain the differential toxicities of prochloraz and II to each individual species. The exception to this was Rhizoctonia solani which metabolized prochloraz to a non-fungitoxic compound. This correlated with its low prochloraz sensitivity.

A procedure for the isolation of microsomes containing cytochrome-P450 isozymes from Ustilago maydis is described. Yields of P450 amount to approximately 19(+/- 6) pmol mg-1 of microsomal protein. The wavelength of maximum absorbance of the reduced carbon monoxide difference spectrum is 448-449 nm. The azole fungicides prochloraz, etaconazole, imazalil, triadimefon and 3-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-4(3H)-quinazoline, which differ markedly in toxicity to U. maydis, all induce type II binding difference spectra at extremely low concentrations (10(-9)-10(-8) M). The DMI concentrations which cause half saturation of type II binding difference spectra (IC50) do not correlate with the fungicidal activities of the azoles. Binding of carbon monoxide to ferrous cytochrome-P450 was only slightly inhibited to different degrees by the DMIs tested. However, the inhibition of carbon monoxide binding also does not correlate with fungitoxicity of the DMIs. The results in this paper suggest that the spectrophotometric studies with this preparation are not useful for evaluating selective toxicity of DMIs to intact sporida of U. maydis.