Antifungal unsaturated cyclic Mannich ketones and amino alcohols: study of mechanism of action.

The antifungal activity of some known unsaturated Mannich ketones and their amino alcohols has been reported and the mechanism of antifungal action has been studied. The inhibition of the fungal ergosterol, chitin, protein synthesis and pseudohypha formation was investigated. According to our results, Mannich ketones can influence the development of pseudohyphae of Candida albicans strains. In addition, they are able to induce significant changes in the protein composition of this fungal strain. Some of our Mannich ketones have shown inhibitory effect on the fungal chitin synthase enzyme. QSAR studies were also carried out.

Field evaluation of novaluron, a chitin synthesis inhibitor larvicide, against mosquito larvae in polluted water in urban areas of Bangkok, Thailand.

Novaluron, an insect growth regulator, a benzoylphenyl urea insecticide, was evaluated in the field against the larvae of polluted-water mosquitoes. The study was carried out in highly polluted sites infested with populations of mosquito larvae, mostly Culex quinquefasciatus Say, in low-income communities in urban areas of Bangkok, Thailand. An EC10 formulation was premixed in water and applied by pressurized spray tank to plots ranging from 180 to 1,000 m2 at the rate of 0.1 ml EC 10/m2 (equal to 10 mg a.i./m2) of the breeding sites. Assessments were made by sampling mosquito larvae and pupae to determine the trends of immature populations before treatment and weekly after treatment. Reduction of the populations in percents were then computed by comparing counts of immature mosquitoes (larvae and pupae) to the pretreatment counts at each particular site. It was found that the immature populations of mosquitoes in the treated areas were dramatically suppressed and remained at extremely low levels for 3-7 weeks after the treatment depending on the prevailing conditions of each experimental site. No negative impact on fishes or aquatic plants in the treated areas were detected during the study period and three months after the experiment was discontinued. Novaluron is an effective agent to control immature populations of polluted-water mosquitoes, especially Cx. quinquefasciatus in habitats in urban areas. This IGR larvicide may play an important role in vector control programs in terms of effectiveness, environmental friendliness and strategies for insecticide-resistance management in vector mosquitoes.

Differential inhibitory effects of protoberberines on sterol and chitin biosyntheses in Candida albicans.

The anti-Candida potentials of 12 Korean medicinal plants were explored: methanol extracts from Coptis rhizoma and Phellodendron amurense caused significant inhibition of growth of Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis. The predominant active components of the extracts were the protoberberines berberine and palmatine; the most potent inhibition of growth was exhibited by berberine on C. krusei (MIC <4 mg/L) and palmatine on C. parapsilosis (MIC 16 mg/L). Both berberine and palmatine inhibited the in-vivo rate of incorporation of L-[methyl-14C]methionine into C-24 of ergosterol in C. albicans (50% inhibition concentration (IC50 values), 25 microM and 300 microM, respectively); this result suggests that sterol 24-methyl transferase (24-SMT) is one of the cellular targets for the antifungal activity of the protoberberines. In-vitro 24-SMT activity in microsomes from the yeast growth form of C. albicans was inhibited by both berberine (inhibition constant (Ki) 232 microM) and palmatine (Ki 257 microM) in a non-competitive manner; inhibition of 24-SMT was more marked for the mycelial form than for the yeast growth form of this organism. Palmatine inhibited chitin synthase from both the yeast and mycelial growth phases of C. albicans in a non-competitive manner (Ki 780 microM). The effects of protoberberines, extracted from established medicinal plants, on both sterol and cell wall biosyntheses in pathogenic fungi indicate that the potential of these compounds, or their semi-synthetic derivatives, as a novel class of antifungal agents should be investigated more fully.

Lufenuron, a chitin-synthesis inhibitor, interrupts development of Drosophila melanogaster.

The chitin-synthesis inhibitor lufenuron was administered to Drosophila melanogaster to better understand the effects of chitin-synthesis interruption during the development and reproduction of this insect. When larvae were fed a diet containing a low concentration (< 1 ppm) of lufenuron, mortality was observed during either larval or pupal development, depending on the dose. Survivor adults were unable to fly, probably due to abnormal cuticular development in the wing hinge regions of the thorax. Larvae fed a higher concentration (10 ppm) completed development within that instar, but died during ecdysis to the next instar, presumably due to inadequate cuticle synthesis. Third instar larvae pupariated, but the puparium was abnormal, and pupation did not occur. Adults fed 10 ppm showed normal viability but slightly depressed oogenesis; additionally, although their eggs were fertilized, they failed to hatch. Examination of the embryos showed that they completed development but were unable to perforate the surrounding vitelline membrane, probably due to a weakened chitinous mouth hook assembly that was insufficiently rigid to effect hatching. These results identify stages during D. melanogaster development when chitin synthesis and deposition are critical. This information will be useful for identifying chitin-synthesis mutants of this insect.

Entamoeba invadens: characterization of cysteine proteinases.

Cysteine proteinases have a number of important functions in the life cycle of protozoan parasites. Based on our previous studies demonstrating the role of cysteine proteinases in invasion by Entamoeba histolytica, we evaluated the cysteine proteinases of E. invadens, a related protozoan which causes invasive disease of reptiles. E. invadens readily encysts in axenic culture and provides a model to investigate the role of cysteine proteinases in encystation. Broad bands of approximately 130-230, 55, and 35 kDa were detected on gelatin substrate gels and were inhibited with specific cysteine proteinase inhibitors. Maximal enzymatic activity was detected with peptide substrates containing arginine in the P2 position. A 567-bp fragment containing the active site of an E. invadens cysteine proteinase gene was amplified by PCR and had 37.7, 79.1, and 67.9% identity to the derived amino acid sequences of the acp 1, 2, and 3 genes, respectively, of E. histolytica. The PCR product hybridized with a single band of 1.1 kb on a Southern blot of EcoRI-restricted E. invadens genomic DNA. Long-term inhibition of cysteine proteinase activity during encystation resulted in significantly fewer cysts (P < 0.02); however, this effect appeared to be secondary to decreased trophozoite cell division. No difference in chitin synthase activity was detected between controls and encysting cells with inhibited cysteine proteinases, suggesting that these proteinases are not critical for activation of a zymogen form of chitin synthase. These studies demonstrate that cysteine proteinases may be critical for the survival of E. invadens, and specific inhibition may ultimately interrupt transmission.

An electron microscope and electron diffraction study of the effect of calcofluor and congo red on the biosynthesis of chitin in vitro.

The structure of chitin made in vitro by chitin synthetase was studied by electron microscopy and electron diffraction. Two different forms of chitin synthetase from the fungus Mucor rouxii were tested: chitosomes and 16 S particles. The long chitin fibrils produced by chitosomes had a high degree of crystallinity as revealed by electron diffraction. Specimen regions made of largely parallel microfibril bundles produced distinct fiber diagrams, an indication that the chitin chains were aligned along the fibril axis. Microdiffractometry of the shorter chitin crystals synthesized by 16 S particles also showed a similar alignment of chitin chains. Calcofluor and Congo red were powerful inhibitors of chitin synthetase and had profound effects on the color, macroscopic texture, electron microscopic morphology, and crystal structure of the biosynthesized chitin. Chitin made in the presence of Congo red had a bright red color; the one made in the presence of Calcofluor was strongly fluorescent and had a distinctly blue hue when illuminated by daylight. The dyes were tightly bound to the chitin and could not be removed by washing with water or ethanol. At low dye concentration, a mixture of two kinds of crystals was produced by 16 S particles: some were of the same dimensions as those made in the absence of dyes, but others were much thinner. At high dye concentration, there were only thin crystals. With increasing concentrations of Calcofluor or Congo red, the typical electron diffraction reflections of alpha-chitin, particularly the strong equatorial band at 0.466 nm became fainter and a new additional reflection centered at 0.40 nm arose as the dominant feature of the patterns. We regard the gel-like material, synthesized at highly inhibitory dye concentrations, as an apposition complex where the dye does not form part of the crystal structure of chitin and the bulk of the complex consists of molecular stacks of dye associated with nascent chitin chains or narrow chitin microfibrils.

Chitin synthase in encysting Entamoeba invadens.

Although the cyst wall of Entamoeba invadens contains chitin, synthesis of this structural polymer during encystation has not been described before. Here we report that conditions which stimulate encystation of the parasite lead to increased chitin synthase (ChS) activity, measured by incorporation of [3H]GlcNAc ([3H]N-acetylglucosamine) from UDP-GlcNAc. The radiolabelled product was precipitable by trichloroacetic acid or ethanol and identified as chitin because it was digested by purified chitinase to radioactive chitobiose and GlcNAc. Cell fractionation indicated that approx. 60% of the enzyme is in the high-speed supernatant. pH-activity profiles showed that soluble ChS has an optimum at 6.0, whereas particulate ChS has a peak at pH 7.0-7.5. Both the activities were dependent on bivalent metal ions, especially Mn2+ and Mn2+ plus Co2+. In contrast with the ChS of other organisms, neither the particulate nor the soluble ChS of E. invadens was activated by trypsin treatment. Soluble and particulate ChS were also stimulated by digitonin and phosphatidylserine, whereas phosphatidylethanolamine stimulated only the soluble ChS. The enzyme activities were inhibited by UDP, UDP-glucose and UDP-GalNAc, but not by the analogues Polyoxin-D or Nikkomycin. This is the first report of an enzyme which is developmentally regulated during encystation of the primitive eukaryotic genus Entamoeba.