Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism

The inhibitory effect of the triterpeneglycosides aescin and avenacin on growth capability, leakage of organic and inorganic substances, and uptake of potassium ions in Ophiobolus graminis and Neurospora crassa was antagonized by the cations Ca²⁺ and Mg²⁺, the former having the greatest effect. The effect was different in the two fungi and was influenced by the buffer system.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism

Aescin in phosphate buffer reduced, to some extent, the production of ¹⁴CO₂ from uniformly labelled glucose in mycelia of Ophiobolus graminis and Neurospora crassa, whereas aescin in succinate buffer had no effect. The enzymatic hydrolysis of sucrose was, however, severely affected, no production of ¹⁴CO₂ from labelled sucrose being found after treatment of mycelia of O. graminis with 100 mg/l of aescin and N. crassa with 300 mg/l for 90 and 300 min, respectively. In Aspergillus niger the production of ¹⁴CO₂ from glucose or sucrose was not affected. The ATPase in whole cells and isolated plasma membranes was not inhibited by the aescin treatment, on the contrary, the ATPase in whole cells of N. crassa was somewhat stimulated.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism

A venacin, the resistance factor in oat roots against Ophio-bolus graminis var. graminis, and a related triterpeneglycoside, aescin, induced a rapid release of K⁺ from mycelia of Opbio-bolus graminis and Neurospora crassa, suspended in phosphate buffer. N. crassa also released Mg²⁺ whereas no outflux of Mg²⁺ was found from O. graminis. The inhibitors induced a release of inorganic phosphate into acetate buffer from Neurospora crassa. The amount of inorganic phosphate in the mycelia decreased when O. graminis and N. crassa were treated with the inhibitors in phosphate buffer. In other media the inhibitors had weak or no effects on the ion contents of the mycelia. The effect of aescin was low in Aspergillus niger and nil in Pythium irregulare. However, high amounts of K⁺, Mg²⁺, and phosphate ions were lost to the medium when the mycelium of P. irregulare, washed with distilled water, was suspended in different buffers. The ions lost were reabsorbed during the experimental period. The leakage of ions indicates that the plasma membrane of the growth sensitive fungi is severely affected by the inhibitors, while a corresponding effect on the growth insensitive fungi does not take place.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism

Avenacin, the resistance factor ioat roots against Ophiobolus graminisi ν graminis, and a related triterpeneglycoside, aescin, inhibited growth of various fungi, while the growth of a few fungi was unaffected. The triterpenoidal saponins tested seemed to be fungicidal, as the mycelia did not grow after exposure to the inhibitors. The fungicidal effect, however, depended on the medium used. The inhibitors did not seem to act primarily on the endogenous respiration, since the oxygen uptake, although much reduced, continued after the time required to destroy the growth capacity. The leakage of UV-absorbing substances from fungi, sensitive to the inhibitors, and the decrease of UV-absorbing substances in the transient pool of the cells indicate a change in the cell membrane.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism 8. Induced Leakage of Nucleotide Materials

Addition of aescin to mycelia of Ophiobolus graminis and Neurospora crassa induced leakage of ribonucleotide material together with pentose phosphate or pentose. In O. graminis a rapid release of mono- and dinucleotides together with nucleosides and pentose occurred. In addition some oligonucleotides were released during the first 30 min of the treatment. A slow leakage of polynucleotides was also observed. In N. crassa aescin induced only slight leakage during the first hour, and the mono- and dinucleotide leakage was actually decreasing. From that time on, however, a rapid release of mono- and dinucleotides together with nucleosides and pentose phosphate or pentose was observed. Only small amounts of poly- and oligonucleotides leaked and the cell content of oligonucleotides seemed to be released only during the last hour of the treatment period. In Aspergillus niger, which was insensitive to the inhibitor, aescin only induced leakage of pentose or pentose phosphate and small amounts of mono- and dinucleotides. Loss of viability in mycelia of O. graminis and N. crassa seemed to be correlated with the loss of oligonucleotides from the cells.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism 3. Effect on Uptake of Potassium, Magnesium and Inorganic Phosphate

A venacin, the resistance factor in oat roots against Ophiobolus graminis var. graminis, and a related triterpeneglycoside, aescin, inhibited the uptake of K⁺ and Mg²⁺ in the fungal mycelium both in phosphate and succinate buffers. The uptake of the cations in Neurospora crassa was similarly inhibited when the inhibitors were dissolved in phosphate or acetatebuffer, while no decrease in the uptake of K⁺ and Mg²⁺ was observed when the inhibitors were dissolved in succinate buffer. The uptake of cations in Aspergillus niger and Pythium irregulare was more or less unaffected by aescin. The uptake of inorganic phosphate was in no case inhibited, but some decrease of the accumulation of inorganic phosphate in Ophiobolus graminis and Ncurospora crassa due to inhibitor treatment in phosphate buffer was observed. No accumulation of Ca²⁺ was observed in any of the tested fungi.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism 6. The Effect of Aescin on Fungi with Reduced Sterol Contents

The amount of sterols in the mycelia of Ophiobolus gra-minis and Neurospora crassa was reduced by cultivating the fungi in the presence of inhibitors of the sterol synthesis. The hypocholesteraemic compounds β-diethylaminoethyl-(2.2-diphenylpentanoate) hydrochloride (SK & F 525-A) and 2.2-diphenyl -1 -(β- dimethylaminoethoxy)pentane hydrocchloride (SK & F 3301-A) were particularly effective in reducing the sterol contents. At the same time, the growth yield was reduced. Aescin had a reduced inhibitory effect on the radial growth of the mycelia with decreased sterol contents, the leakage of UV-absorbing substances and K⁺ ions was reduced to small amounts, and the inhibition of the K⁺ uptake was nullified.     

Fungal Metabolism of n-Alkylbenzenes

Isolates of Paecilomyces, Verticillium, Beauveria, and Penicillium species were tested for ability to metabolize a variety of n-alkylbenzenes. Minimum side chain lengths were required for metabolism of these substrates. These were C₄ for the Paecilomyces sp., C₈ for the Verticillium sp., and C₉ for the other two isolates. Growth on dodecylbenzene yielded benzoic and phenylacetic acids as transient intermediates, and these acids supported growth of the isolates.     

Fungal Metabolism of Sorbic Acid

The metabolism of sorbic acid (S A) by 11 species of Mucor and 14 other fungi in addition to Mucor sp. A–73 were studied. All species of Mucor metabolized SA to trans-4-hexenol (4–HEL) without exception. The metabolic pathway was investigated precisely by using the resting cells of Mucor sp. A–73. The reduction of SA to 4–HEL by the fungus demanded glucose. The reduction of SA to sorbic alcohol (SAL) was catalyzed by the inducible enzymic system and that of SAL to 4–HEL by the constitutive enzyme. In the phosphate buffer, a part of 4–HEL produced from SA was further transformed into mono-trans-4-hexenyl phosphate by Mucor sp. A–73.

The reductive action of the fungus upon several trans-2-alkenoic acids was also examined.     

Tumor Necrosis Factor Inhibitors and Fungal Infections

The number of commercially available tumor necrosis factor (TNF)-α inhibitors has been increasing, including two new agents licensed since 2008. In addition to an expanding number of agents, there are also increasing licensed and “off label” clinical applications for the TNF inhibitors for the treatment of a variety of inflammatory or granulomatous disorders. Unfortunately, use of the TNF inhibitors has been associated with a wide variety of opportunistic infections, including fungal infections. Higher rates of morbidity and mortality from fungal infection in TNF inhibitor-treated patients have been observed, likely due to a delay in the diagnosis of invasive fungal infections and a tendency for these patients to develop severe and disseminated disease. Therefore, the US Food and Drug Administration issued a “black box” warning for clinicians in September 2008 to alert providers to the risks of fungal infections in patients treated with TNF-α inhibitors.     

Vital Fluorochromes as Tracers for Fungal Growth Studies

Eight fluorescent dyes were tested for staining the spores or mycelia of six fungi and for their translocation into new growth when the preloaded spores or mycelia were incubated on agar coated coverslips. The dyes studied were Cellufluor, Nile red, fluorescein diacetate (FDA), carboxyfluorescein diacetate (CFDA), chloromethylfluorescein diacetate (CMFDA), aminochloromethyl coumarin (CMAC), and the carbocyanines DiIC₁₈(3) and DiOC₁₈(3). The fungi on which the dyes were tested included Botrytis cinerea, Fusarium oxysporum f.sp. lycopersici, Idriella bolleyi, Pythium oligandrum, Sclerotium cepivorum and Trichoderma. harzianum Most of the fluorochromes gave good initial staining of mycelia or spores; however, FDA fluorescence faded rapidly during excitation, making it impractical for use. Also, the spores and mycelia of B. cinerea and T. harzianum sometimes gave weak fluorescence with Nile red, and the spores and mycelia of I. bolleyi gave unusually weak fluorescence with Cellufluor. There were other variations of staining among the different dye/fungus combinations, but each fungus showed strong fluorescence with at least one dye. Cellufluor, CMFDA, CMAC and, to a lesser extent, CFDA and Nile red, were efficiently translocated into new growth from preloaded spores or mycelia, whereas FDA, DiIC₁₈(3) and DiOC₁₈(3) were not. The extent of translocation ranged from 0.1 to 1.2 mm in germ tubes arising from spores, and from 0.9 to 9.2 mm in mycelia extending from dye-loaded agar blocks. The findings suggest that fluorescent dyes could be used as markers or tracers in studies of fungal growth and differentiation.     

Carbonic Anhydrase Inhibitors: Aromatic Sulfonamides and Disulfonamides Act as Efficient Tumor Growth Inhibitors

Abstract

Aromatic/heterocyclic sulfonamides generally act as strong inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). Here we report the unexpected finding that potent aromatic sulfonamide inhibitors of CA, possessing inhibition constants in the range of 10^?-⁸-^?10^?-⁹ M (against all the isozymes), also act as efficient in vitro tumor cell growth inhibitors, with GI₅₀ (molarity of inhibitor producing a 50% inhibition of tumor cell growth) values of 10 nM-35 μM against several leukemia, non-small cell lung cancer, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines. The investigated compounds were sulfanilyl-sulfanilamide-, 4-thioureido-benzenesulfonamide- and benzene-1.3-disulfonamide-derivatives. The mechanism of antitumor action with these sulfonamides is unknown, but it might involve either inhibition of several CA isozymes (such as CA IX, CA XII, CA XIV) predominantly present in tumor cells, a reduced provision of bicarbonate for the nucleotide synthesis (mediated by carbamoyl phosphate synthetase II), the acidification of the intracellular milieu as a consequence of CA inhibition or uncoupling of mitochondria and potent CA V inhibition among others. A combination of several such mechanisms is also plausible. Optimization of such derivatives from the SAR point of view, might lead to the development of effective novel types of anticancer agents/therapies.     

Evaluation of Chalcone Derivatives as Photosynthesis and Plant Growth Inhibitors

We report the evaluation of chalcone derivatives as photosystem II (PSII) and plant growth inhibitors. Chalcone derivatives were evaluated as PSII inhibitors through Chl a fluorescence measurement. (E)-Chalcone ( 6a ) and (E)-3-(4-bromophenyl)-1-(4-fluorophenyl)prop-2-en-1-one ( 6j ) showed the best results, reducing the performance index on absorption basis parameter (PI_abs) by 70 %. Additionally, the decrease of TR₀/RC and ET₀/RC parameters indicates that the chalcone derivatives limited the number of active PSII reaction centers and the amount of trapped energy within them. Compounds 6a and 6j both act as post-emergent herbicides at 50 μM, reducing the root biomass of the Ipomoea grandifolia weed by 72 % and 83 %, respectively, corroborating the fluorescence results. The selectivity against weeds as compared to valuable crops by compounds 6a and 6j were evaluated employing Zea mays and Phaseolus vulgaris plants. In these, our newly synthesized compounds showed no effects on biomass accumulation of roots and aerial parts when compared to the control, providing valuable evidence for the role of these compounds as selective inhibitors of the growth of undesired weeds.     

Growth and Metabolism of Soybean as Affected by Paclobutrazol

Paclobutrazol, an experimental growth retardant, was soil-appliedat the rate of 125 or 250 µg active ingredient per 10cm pot to 19 day-old soybean plants. This compound considerablyreduced plant height, leaf area, and stem dry weight. In addition,paclobutrazol-treated plants had numerous thickened lateralroots at the soil surface and had increased chlorophyll andsoluble protein contents compared to controls. During the first14 days after treatment, paclobutrazol increased the activitiesof NAD- and NADP-glyceraldehyde-3-phosphate dehydrogenase andaminotransferases but decreased the activity of nitrate reductase.Net photosynthesis (P_n) of the first and second trifoliatesof treated plants remained fairly constant throughout the studywhile control P_n declined during the latter portion of the experimentalperiod presumably due to leaf senescence. This decline of P_nin controls was accompanied by a decrease in the activitiesof NAD- and NADP-glyceraldehyde-3-phosphate dehydrogenase, nitratereductase, aminotransferases, and NAD malate dehydrogenase.Activities of these enzymes also tended to decline in paclobutrazol-treatedplants, but were still considerably higher than in controlsat the end of the experiment. The activities of RNase, protease,and glutamic dehydrogenase were higher in controls than in treatedplants. Our results suggest that paclobutrazol not only modifiesthe activity of a number of soybean enzymes but also delaysthe onset of senescence, thereby prolonging the period of normalmetabolic activity in a given leaf. ¹ Permanent address: Department of Botany, University of Jodhpur,India.     

Metabolism of Monoamine Oxidase Inhibitors

1. The principal routes of metabolism of the following monoamine oxidase inhibitors (MAOIs) are described: phenelzine, tranylcypromine, pargyline, deprenyl, moclobemide, and brofaromine.2. Acetylation of phenelzine appears to be a minor metabolic pathway. Phenelzine is a substrate as well as an inhibitor of MAO, and major identified metabolites of phenelzine include phenylacetic acid and p-hydroxyphenylacetic acid. Phenelzine also elevates brain GABA levels, and as yet unidentified metabolites of phenelzine may be responsible for this effect. -Phenylethylamine is a metabolite of phenelzine, and there is indirect evidence that phenelzine may also be ring-hydroxylated and N-methylated.3. Tranylcypromine is ring-hydroxylated and N-acetylated. There is considerable debate about whether or not it is metabolized to amphetamine, with most of studies in the literature indicating that this does not occur.4. Pargyline and R(–)-deprenyl, both propargylamines, are N-demethylated and N-depropargylated to yield arylalkylamines (benzylamine, N-methylbenzylamine, and N-propargylbenzylamine in the case of pargyline and amphetamine, N-methylamphetamine and N-propargylamphetamine in the case of deprenyl). These metabolites may then undergo further metabolism, e.g., hydroxylation.5. Moclobemide is biotransformed by C- and N-oxidation on the morpholine ring and by aromatic hydroxylation. An active metabolite of brofaromine is formed by O-demethylation. It has been proposed that another as yet unidentified active metabolite may also be formed in vivo. 6. Preliminary results indicate that several of the MAOIs mentioned above are substrates and/or inhibitors of various cytochrome P450 (CYP) enzymes, which may result in pharmacokinetic interactions with some coadministered drugs.     

Relationship between Secondary Metabolism and Fungal Development

Filamentous fungi are unique organisms—rivaled only by actinomycetes and plants—in producing a wide range of natural products called secondary metabolites. These compounds are very diverse in structure and perform functions that are not always known. However, most secondary metabolites are produced after the fungus has completed its initial growth phase and is beginning a stage of development represented by the formation of spores. In this review, we describe secondary metabolites produced by fungi that act as sporogenic factors to influence fungal development, are required for spore viability, or are produced at a time in the life cycle that coincides with development. We describe environmental and genetic factors that can influence the production of secondary metabolites. In the case of the filamentous fungus Aspergillus nidulans, we review the only described work that genetically links the sporulation of this fungus to the production of the mycotoxin sterigmatocystin through a shared G-protein signaling pathway.     

Investigations on Growth and Metabolism of Plant Cells: III. Evidence for Growth Inhibitors in Certain Mature Tissues

Using the carrot explant technique, with added coconut-milkfactor, evidence has been obtained of inhibitors of growth inpotato tubers, onion bulbs, and maple buds. In discussion itis suggested that the regulation of growth in such tissues asmature parenchyma may thus be due to absence of necessary growth-factorsor the presence of growth inhibitors.     

Growth and Metabolism of Senna as Affected by Salt Stress

Pot culture experiments were conducted using different NaCl concentrations to assess their impact on the growth and metabolic changes in senna (Cassia angustifolia Vahl.). Five treatments (0, 40, 80, 120, and 160 mM NaCl) were given to the plants at three phenological stages, i.e. at pre-flowering, (45 days after sowing, DAS); flowering (75 DAS) and post-flowering (90 DAS) stages. A significant reduction in the biomass and length of the roots and shoots, photosynthetic rate, stomatal conductance, the total chlorophyll content, protein content, nitrate reductase activity, and reduced nitrogen content of the leaves was observed at each phenological stage with each salt concentration applied. Contrary to this, proline and nitrate contents of the leaves increased markedly. The post-flowering stage was most sensitive to NaCl treatment.     

Serpins Identified as Cell Growth Inhibitors in Human Plasma

We have identified a new factor, CFX, in human serum and plasma that inhibits the growth of cultured human and mouse cell lines. CFX was determined to be a negatively charged, hydrophobic glycoprotein, with a native molecular weight of 110–120 kDa and a minimal active subunit of 55 kDa. It is precipitated by 60% ammonium sulfate and is resistant to heat treatment at 100°C for 30 min. CFX was purified from human plasma to a single band on a gel which retained the cell growth inhibitory activity. Amino acid sequence analysis of the CFX band revealed sequences from four human glycoproteins, α1-antichymotrypsin, C1-esterase inhibitor, α1-antitrypsin, and α2-antiplasmin, all members of the superfamily of serpins. Of the four, C1-esterase inhibitor was shown to be the most potent cell growth inhibitor. These results suggest that serpins may play a cell growth inhibitory role in vivo, in addition to their role as protease inhibitors.