Activities of 2,4-dihydroxy-6-n-pentylbenzoic acid derivatives

Esters of 2-hydroxy-4-methoxy-6-n-pentylbenzoic acid (2-8) (methyl, ethyl, butyl, pentyl, isopropyl, sec-butyl and benzyl), olivetol (9), methyl, ethyl, butyl perlatolates (10-12), 2,4-dihydroxy-6-n-pentylbenzoic acid (15), and methyl and ethyl esters of (15) were prepared through structural modifications of perlatolic acid (1) with the aim to detect new antifungal and antibacterial substances and also to evaluate the toxicity by the brine shrimp lethality assay against Artemia salina. The antifungal assays were carried out against the fungus Cladosporium sphaerospermum through the bioautography method, and methyl 2,4-dihydroxy-6-n-pentylbenzoate (13) showed the highest antifungal activity (2.5 yg). Olivetol (9) and 2,4-dihydroxy-6-n-pentylbenzoic acid (15) are also potent inhibitors of the growth of the fungus (5.0 microg). Except for methyl (10), the ethyl (11) and butyl (12) perlatolates were less active than perlatolic acid (1). The activities presented by methyl (2) and ethyl (3) 2-hydroxy-4-methoxy-6-n-pentylbenzoates and methyl (13) and ethyl (14) 2,4-dihydroxy-6-n-pentylbenzo-ates suggest that compounds with a free hydroxy group in the aromatic ring (C-4) have a more pronounced effect against C. sphaerospermum. Antibacterial activities were tested by the disc diffusion method using pathogenic strains of S. aureus and E. coli. The compounds were weakly active with inhibition zones between 9-15 mm. The 2-hydroxy-4-methoxy-6-n-pentylbenzoic esters 2-8 and alkyl perlatolates 10-12 were selective against E. coli. Perlatolic acid (1) and methyl 2-hydroxy-4-methoxy-6-n-pentylbenzoate (2) were the most active with LD50 values of 24.1 microM and 27.2 microM, respectively. The other compounds were not toxic to Artemia salina larvae.     

Decreased permeability as a mechanism of resistance to methyl benzimadazol-2-yl carbamate (MBC) in Sporobolomyces roseus.

Mutants of Sporobolomyces roseus resistant to benzimidazole fungicides varied in their responses to 2-(thiazol-4-yl) benzimidazole (thiabendazole, TBZ), methyl 1-(butylcarbamoyl)-benzimidazol-2-yl carbamate (benomyl) and methyl benzimidazol-2-yl carbamate (carbendazim, MCB). Incorporation of [14C] MBC into trichloroacetic acid extracts of the sensitive strain S4 increased during a 2 h incubation period, whereas incorporation into the resistant mutant M55 was unchanged. [14C] MBC uptake by S4 cells was five times higher than that by M55. MBC was identified as the main radioactive compound inside the S4 cells and reached a level of 2.4 mug/100 mg dry wt. The compound MBC enters the cells of Sp. roseus by a temperature-, energy-, pH- and concentration-dependent transport system which may be specific for compounds containing a benzimidazole nucleus. It is suggested that tolerance of M55 to MBC is due to decreased permeability of the cell to this compound.     

青阳参组织培养及愈伤组织的成分分析

用青阳参(Cynanchum otophyllum)的嫩枝和芽在Ms 2．0mg／L2,4-D 0．1mg／L KIN的培养基上诱导愈伤组织。通过不同的培养基和激素配比实验,发现6,7-V 2．0mg／L2,4．D 0．3mg／LKIN最适合愈伤组织的生长。但在6,7-V 1．0mg／L2,4．D 0．1mg／L KIN培养基中的愈伤组织次生代谢物含量最高。愈伤组织的生长周期为27d,但在33d时次生代谢产物的含量最高。从愈伤组织中分离到7个化合物：(1)9,10,11-三羟基-十八碳-12(Z)-烯酸甲酯(methyl9,10,11-trihydroxy-12-octadecencate),(2)胡萝卜甙(daucosterol),(3)β-谷甾醇(β-sitoster01),(4)华木酸(betuliniic acid),(5)齐端果酸(oleamlic acid),(6)棕榈酸(hexadecanoic acid),(7)十八碳-9-烯酸(9-octadecenoic acid)。首次报道从植物愈伤组织中分离到多羟基十八碳烯酸,并讨论了化合物（1）对植物细胞生长的可能影响。     

青阳参组织培养及愈伤组织的成分分析

用青阳参（Cynanchum otophyllum）的嫩枝和芽在MS + 2.0 mg/L 2,4-D + 0.1 mg/L KIN的培养基上诱导愈伤组织。通过不同的培养基和激素配比实验,发现 6,7-V + 2.0 mg/L 2,4-D + 0.3 mg/L KIN 最适合愈伤组织的生长。但在6,7-V + 1.0 mg/L 2,4-D + 0.1 mg/L KIN 培养基中的愈伤组织次生代谢物含量最高。愈伤组织的生长周期为27 d,但在33 d时次生代谢产物的含量最高。从愈伤组织中分离到7个化合物：⑴9,10,11-三羟基-十八碳-12(Z)-烯酸甲酯 (methyl 9,10,11-trihydroxy-12-octadecenoate),(2) 胡萝卜甙 (daucosterol),(3)β 谷甾醇 (β sitosterol),（4） 华木酸 (betulinic acid),（5）齐端果酸 (oleanolic acid),（6）棕榈酸 (hexadecanoic acid),（7）十八碳-9-烯酸 (9-octadecenoic acid)。首次报道从植物愈伤组织中分离到多羟基十八碳烯酸,并讨论了化合物（1）对植物细胞生长的可能影响。     

The thiazolobenzimidazole TBZE-029 inhibits enterovirus replication by targeting a short region immediately downstream from motif C in the nonstructural protein 2C

TBZE-029 {1-(2,6-difluorophenyl)-6-trifluoromethyl-1H,3H-thiazolo[3,4-a]benzimidazole} is a novel selective inhibitor of the replication of several enteroviruses. We show that TBZE-029 exerts its antiviral activity through inhibition of viral RNA replication, without affecting polyprotein processing. To identify the viral target of TBZE-029, drug-resistant coxsackievirus B3 (CVB3) was selected. Genotyping of resistant clones led to the identification of three amino acid mutations in nonstructural protein 2C, clustered at amino acid positions 224, 227, and 229, immediately downstream of NTPase/helicase motif C. The mutations were reintroduced, either alone or combined, into an infectious full-length CVB3 clone. In particular the mutations at positions 227 and 229 proved essential for the altered sensitivity of CVB3 to TBZE-029. Resistant virus exhibited cross-resistance to the earlier-reported antienterovirus agents targeting 2C, namely, guanidine hydrochloride, HBB [2-(alpha-hydroxybenzyl)-benzimidazole], and MRL-1237 {1-(4-fluorophenyl)-2-[(4-imino-1,4-dihydropyridin-1-yl)methyl]benzimidazole hydrochloride}. The ATPase activity of 2C, however, remained unaltered in the presence of TBZE-029.     

Effect of jasmonates and exogenous polysaccharides on production of alkannin pigments in suspension cultures of Alkanna tinctoria

Summary The conditions for the efficient production of alkannin pigments by a suspension culture of Alkanna tinctoria were established. Pectin, polygalacturonic acid sodium salt and galactan increased the pigment production but not as much as agar did. A marked increase in the pigment content in cells and medium of suspension cultures after treatment with methyl jasmonate was observed. It was shown, applying a two-layer culture method, that mineral and olive oils intensified the pigment secretion from cells to the medium but did not enhance significantly their synthesis. Thin layer chromatography and high performance liquid chromatography methods showed that two main esters of alkannin are responsible for the characteristic colour of A. tinctoria suspension cultures.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole 3-acetic acid - NAA 1-naphthaleneacetic acid - MeJA methyl jasmonate - TLC thin layer chromatography - HPLC high performance liquid chromatography     

Somatic embryogenesis and subsequent plant regeneration from inflorescence callus of Bambusa beecheyana Munro var. beecheyana

Somatic embryos of bamboo, Bambusa beecheyana Munro var. beecheyana were developed in callus derived from young florets and adventive roots obtained from floret callus. The medium was a modified Murashige and Skoog medium (1962) supplemented with 3 mg/l 2,4-dichlorophenoxyacetic acid, 2 mg/l kinetin, a high content of sucrose (6%) and 0.7% agar. The embryoids germinated spontaneously to yield whole plantlets on this medium with or without the hormonal adjuvants.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - MS Murashige and Skoog's (1962) medium     

A rapid method to screen degradation ability in chlorophenoxyalkanoic acid herbicide-degrading bacteria

AIMS: An agar medium containing a range of related chlorophenoxyalkanoic acid herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), racemic mecoprop, (R)-mecoprop and racemic 2,4-DP (2-(2,4-dichlorophenoxy) propionic acid) was developed to assess the catabolic activity of a range of degradative strains. METHODS AND RESULTS: The medium was previously developed containing 2,4-D as a carbon source to visualise degradation by the production of dark violet bacterial colonies. Strains isolated on mecoprop were able to degrade 2,4-D, MCPA, racemic mecoprop, (R)-mecoprop and racemic 2,4-DP, whereas the 2,4-D-enriched strains were limited to 2,4-D and MCPA as carbon sources. Sphingomonas sp. TFD44 solely degraded the dichlorinated compounds, 2,4-D, racemic 2,4-DP and 2,4-DB (2,4-dichlorophenoxybutyric acid). However, Sphingomonas sp. AW5, originally isolated on 2,4,5-T, was the only strain to degrade the phenoxybutyric compound MCPB (4-chloro-2-methylphenoxybutyric acid). CONCLUSION: This medium has proved to be a very effective and rapid method for screening herbicide degradation by bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This method reduces the problem of assessing the biodegradability of this family of compounds to an achievable level.     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

Facilitated diffusion of glucosamine-6-phosphate synthase inhibitors enhances their antifungal activity

N3-(4-Methoxyfumaroyl)-L-2,3-diaminopropanoic acid (FMDP) and 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP) are strong inhibitors of the essential fungal enzyme, glucosamine-6-phosphate synthase, but their antifungal activity is poor, due to slow penetration of these agents through the cytoplasmic membrane. In the present studies we have exploited the possibility of enhancement of ADGP and FMDP antifungal activity by improving their transport properties. It has been found that membrane-permeabilising polyene macrolides amphotericin B (AMB) and its N-methyl-N-fructosyl methyl ester derivative (MF-AME), at subinhibitory concentrations, facilitate diffusion of ADGP through the fungal cell membrane, thus allowing a decrease of its minimal inhibitory concentration (MIC). Synergistic effects have been observed for combinations of ADGP with AMB or MF-AME. Fractional inhibitory concentration (FIC) indexes, determined against a number of Candida spp., have been in the 0.18-0.81 range. Weak antifungal synergistic effects have been found for combinations of FMDP with AMB or MF-AME. ADGP can be easily encapsulated into unilamellar lipid vesicles. Liposomal preparations of ADGP demonstrated stronger antifungal activity against some fungal strains than free ADGP.     

Pigment Production of Cryptococcus neoformans Grown with Extracts of Guizotia abyssinica

Brown pigment(s) formed in Cryptococcus neoformans when grown on media containing extracts of the seeds of Guizotia abyssinica cannot be extracted by common organic solvents or by 6 n HCl or 2 n NaOH. A similar pigmentation was observed in C. neoformans when grown on a medium containing caffeic acid isolated from the hydrolyzed methanol extract of G. abyssinica seeds. Its methyl ester and the diacetate thereof, as well as the following structurally related compounds, 3-hydroxytyramine, 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylethanolamine, and 4-hydroxy-3,5-dimethoxycinnamic acid, brought about similar pigmentation. However, 2,4-, 2,5-, 2,6-, and 3,5-dihydroxybenzoic acids, tyrosine, phenylalanine, cinnamic acid, 4-hydroxycinnamic acid, and 4-hydroxy-3-methoxycinnamic acid did not cause coloration in C. neoformans.     

Cell cycle-dependent disruption of microtubules by methyl jasmonate in tobacco BY-2 cells

Summary Methyl jasmonate, a growth-regulating substance that is ubiquitous in the plant kingdom, was found to disrupt cortical microtubules in tobacco cultured cells. It exerted a microtubule-disrupting effect only in cells at the S phase of the cell cycle. Neither microtubules in preprophase bands, spindles and phragmoplasts nor cortical microtubules at stages of the cell cycle other than the S phase were disrupted by methyl jasmonate. Jasmonic acid was as effective as methyl jasmonate in disrupting cortical microtubules.Abbreviations BUdR 5-bromo-2-deoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - EGTA ethylene glycol bis(2-aminoethyl ether)-tetraacetic acid - FITC fluorescein isothiocyanate - FUdR 5-fluoro-2-deoxyuridine - JA jasmonic acid - JA-Me methyl jasmonate - PBS phosphate-buffered saline - PMSF phenylmethylsulfonyl fluoride     

Plant regeneration of grapevine (Vitis sp.) protoplasts isolated from embryogenic tissue

Summary Protoplasts with high embryogenic competence could be isolated from leaf-disk-derived embryos and embryoids of Vitis sp. cv. Seyval blanc. After a 4-week induction treatment in NN-69 medium supplemented with 4.0mg/l naphthoxyacetic acid (NOA) and 0.9mg/l thidiazuron (TDZ) and subsequent subcultivation in hormone-free medium, 38.5% of the developed microcalluses showed somatic embryogenesis. In contrast, only few formed somatic embryos after induction in CPW-13 medium with either 1.0mg/l 2,4-dichlorophenoxyacetic acid and 0.5mg/l benzylaminopurine treatment (13.8%) or NOA/TDZ treatment (1.4%). Up to 30% of these embryos germinated and about half of them regenerated into typical in vitro grapevines when transferred onto LS-medium in culture tubes.Abbreviations BAP benzylaminopurine - BSA bovine serum albumin - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-[N-morpholino]-ethanesulfonic acid - NOA naphthoxyacetic acid - PCR polymerase chain reaction - PVP polyvinylpyrrolidone - TDZ thidiazuron     

Indole alkaloids from a culture of the fungus Aporpium caryae

In a screening for antifungal metabolites, two indole compounds of mixed biogenesis, 1H-indole-3-carboxylic acid, 1-(1,1-dimethyl-2-propenyl) methyl ester and 1H-indole-3-carboxylic acid, 1-(2,3-dihydroxy-1,1-dimethylpropyl) methyl ester were isolated from a culture of the basidiomycete Aporpiums caryae. The structural elucidation of these compounds was accomplished by spectroscopic methods.     

Antifungal diterpenoids of Pseudolarix kaempferi, and their structure-activity relationship study

The in vitro antifungal activities of 19 structurally diversified analogues of pseudolaric acids tested against the major pathogenic fungus Candida albicans has led to the establishment of a very clear structure-activity relationship of pseudolaric acids derivatives. Pseudolaric acid A was first found to be a potent antifungal component comparable with pseudolaric acid B. Among the tested 19 diterpenoids, pseudolaric acids A2 (1), B2 (3), B3 (4) and methyl pseudolarate A2 (2) are new isolates of the root bark of Pseudolarix kaempferi, and their structures were elucidated mainly by 2D-NMR techniques and chemical methods. Compounds 12-19 were first semi-synthesized by efficient routines from pseudolaric acid B.     

Phomenoic acid and phomenolactone, antifungal substances from Phoma lingam (Tode) Desm.: kinetics of their biosynthesis, with an optimization of the isolation procedures.

Phomenoic acid and phomenolactone, previously isolated from the fungus Phoma lingam (Tode) Desm., have shown moderate antifungal and antibacterial properties in vitro. To rationalize the production of phomenoic acid, a kinetic study of its biosynthesis in the mycelium was performed. Phomenoic acid and phomenolactone appear in the mycelium after a prolonged incubation, a phenomenon which may be of particular interest for the production of these substances or in the study of the mechanism of their biosynthesis. The isolation procedure was optimized for phomenoic acid. Through a series of SiO2 column chromatographies, high-pressure liquid chromatography, and transformation of the mixture of phomenoic acid and phomenolactone into methyl phomenoate (BF3), the final yield of phomenoic acid reached 160 mg/liter of culture medium. An alternative method for the isolation of both phomenoic acid and phomenolactone is also reported in detail.     

Plantlets from somatic callus tissue of the East Indian Rosewood (Dalbergia latifolia Roxb.)

Callus-mediated shoot bud formation was demonstrated in Dalbergia latifolia Roxb. (East Indian Rosewood). Cultures were raised from shoot explants of six year-old plants on Murashige and Skoog (MS) medium supplemented with naphthaleneacetic acid (NAA) and benzyladenine (BA). A sequential treatment of callus with increasing BA levels and decreasing NAA ensured shoot bud induction. Rooting of shoots was achieved by a three-step culture procedure involving 1) White's(W) liquid medium containing indoleacetic acid (IAA), naphthaleneacetic acid and indolebutyric acid (IBA), 2) half-strength MS agar-solidified medium with charcoal (0.25%) and 3) half-strength MS liquid medium.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - MS Murashige and Skoog - NAA a-naphthaleneacetic acid - PVP Polyvinylpyrrolidone - W White's medium - 2,4-D 2,4-dichlorophenoxyacetic acid     

Rapid and repetitive plant regeneration in sweetpotato via somatic embryogenesis

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and petiole expiants has been developed in sweetpotato [Ipomoea batatas L. (Lam.)]. The optimal somatic embryogenic response was obtained in the genotype PI 318846-3 with a two-step protocol: (1) stage I-incubation of expiants in the dark for 2 weeks on Murashige Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg/l) and 6-benzylaminopurine (0.25 mg/l) and, (2) stage II-culture in the light on MS medium with abscisic acid (ABA) (2.5 mg/l). The addition of ABA was critical for enhanced production of somatic embryos. Secondary somatic embryos were produced from the primary embryos cultured on MS medium with 2,4-D at 0.2 mg/l. The somatic embryos were converted into normal plantlets when cultured on basal MS medium. Upon transfer to soil, plants grew well and appeared normal with no mortality. The system of somatic embryogenesis described here will facilitate tissue culture, germplasm conservation and gene transfer research of sweetpotato due to its rapidity (6 to 10 weeks), prolific plant production by direct embryogenesis, ease of secondary somatic embryo production and reproducibility.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine, 2,4-D-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - KIN kinetin - MS medium of Murashige and Skoog (1962) - NAA 1-naph-thaleneacetic acid - PIC picolinic acid - TDZ thidiazuron     

Phomenoic acid and phomenolactone, antifungal substances from Phoma lingam (Tode) Desm.: kinetics of their biosynthesis, with an optimization of the isolation procedures

Phomenoic acid and phomenolactone, previously isolated from the fungus Phoma lingam (Tode) Desm., have shown moderate antifungal and antibacterial properties in vitro. To rationalize the production of phomenoic acid, a kinetic study of its biosynthesis in the mycelium was performed. Phomenoic acid and phomenolactone appear in the mycelium after a prolonged incubation, a phenomenon which may be of particular interest for the production of these substances or in the study of the mechanism of their biosynthesis. The isolation procedure was optimized for phomenoic acid. Through a series of SiO2 column chromatographies, high-pressure liquid chromatography, and transformation of the mixture of phomenoic acid and phomenolactone into methyl phomenoate (BF3), the final yield of phomenoic acid reached 160 mg/liter of culture medium. An alternative method for the isolation of both phomenoic acid and phomenolactone is also reported in detail.     

Effect of antifungal agents on biological fitness of Lygus hesperus (Heteroptera: Miridae)

Artificial diets have become important components of rearing systems for insects that are used for research purposes and in commercial production. Because the rearing conditions for insects also provide ideal settings for mold growth, antifungal additives are often used to reduce diet contamination. However, the antifungal agents must not only be effective in mold suppression, they must also be safe to the target insects of the rearing programs. The toxicity of five commonly used antifungal agents (benzoic acid, formalin, methyl paraben, propionic acid, and sorbic acid) was tested using diet bioassays on Lygus hesperus Knight, and the effect on biological fitness was measured. Biological fitness was defined as total number of survivors, mean biomass (dry weight) accumulated per cage over the total treatment period, egg production, time to adult emergence, and time to start of egg laying. Methyl paraben and formalin were found to have significant negative effects on these measurements of biological fitness. Challenge tests to determine the ability of the antifungal agents to suppress mold growth when inoculated into the diet medium are currently in progress.