Study of Malformin C,a Fungal Source Cyclic Pentapeptide,as an Anti-Cancer Drug

Malformin C, a fungal cyclic pentapeptide, has been claimed to have anti-cancer potential, but no in vivo study was available to substantiate this property. Therefore, we conducted in vitro and in vivo experiments to investigate its anti-cancer effects and toxicity. Our studies showed Malformin C inhibited Colon 38 and HCT 116 cell growth dose-dependently with an IC₅₀ of 0.27±0.07μM and 0.18±0.023μM respectively. This inhibition was explicated by Malformin C’s effect on G2/M arrest. Moreover, we observed up-regulated expression of phospho-histone H2A.X, p53, cleaved CASPASE 3 and LC3 after Malformin C treatment, while the apoptosis assay indicated an increased population of necrotic and late apoptotic cells. In vivo, the pathological study exhibited the acute toxicity of Malformin C at lethal dosage in BDF1 mice might be caused by an acute yet subtle inflammatory response, consistent with elevated IL-6 in the plasma cytokine assay. Further anti-tumor and toxicity experiments proved that 0.3mg/kg injected weekly was the best therapeutic dosage of Malformin C in Colon 38 xenografted BDF1 mice, whereas 0.1mg/kg every other day showed no effect with higher resistance, and 0.9mg/kg per week either led to fatal toxicity in seven-week old mice or displayed no advantage over 0.3mg/kg group in nine-week old mice. Overall, we conclude that Malformin C arrests Colon 38 cells in G2/M phase and induces multiple forms of cell death through necrosis, apoptosis and autophagy. Malformin C has potent cell growth inhibition activity, but the therapeutic index is too low to be an anti-cancer drug.     

Wild Narcissus species as a source of resistance to Fusarium oxysporum f.sp. narcissi

Thirty three species and varieties of Narcissus plus seventeen cultivars with recent species parentage were screened for resistance to Fusarium oxysporum f.sp. narcissi. Accessions were either wholly resistant (bulbs remaining uninfected), partially resistant (a variable portion of the bulbs infected) or susceptible (all of the bulbs infected). Although a pattern of resistance was found among the species tested the genetic basis of the resistance is not known.     

The elimination of viruses from Narcissus tazetta cv. Grand Soleil d''Or, and rapid multiplication of virus-free clones

Arabis mosaic virus and narcissus degeneration virus were eliminated from Narcissus tazetta L. cv. Grand Soleil d'Or by meristem-tip culture. Cultures grew equally well in two liquid nutrient media, but tubes 25times75 mm size and temperature c. 12 ^oC were optimal for growth and survival. Virus-free bulbs grew more vigorously than infected bulbs, and gave more and larger flowers with a better colour. To increase the normal multiplication rate, either sector cuts were made in the basal plate, or bulbs were divided into chips (c. 1 g), before planting. Multiplication was greater by chip-cutting. Five virus-free bulbs were propagated to over 2000 in 8 years; theoretically, 20000000 bulbs should be produced in a further 5–6 years, sufficient to replant the Isles of Scilly with virus-free stock.     

Effect of malformin on phytochrome- and Ethrel-mediated responses

On etiolated cuttings of Phaseolus vulgaris malformin inhibitedseveral phytochromemediated responses. This included hypocotylhook-opening, leaf expansion, and inhibition of stem elongation.Malformin also inhibited anthocyanin synthesis by sorghum, buthad no effect upon lettuce seed germination in the light ordark. Malformin alleviated Ethrel-induced hook-retention, inhibitionof stem elongation, and root curvatures, but not Ethrel-inducedstimulation of lettuce seed germination in the dark. (Received February 19, 1975; )     

The effect of age, season and growth conditions on anti-inflammatory activity in Eucomis autumnalis (Mill.) Chitt. Plant extracts

Eucomis (Family Hyacinthaceae) bulbs are greatly valued in traditionalmedicine for the treatment of a variety of ailments, predominantly thoseinvolving pain, fever and inflammation. The COX-1 assay was used to screenethanolic extracts prepared from the dried leaves, bulbs and roots of E. autumnalis (subspecies autumnalis) to determine the variation ofanti-inflammatory activity with age and season of harvest. Young plantswere found to have large amounts of COX-1 inhibitory activity, particularlyin the leaves. As the plant matured, greater activity was associated with thebulb and root extracts. The anti-inflammatory activity of the leaf, bulb androot extracts varied slightly throughout the year, with the greatest levelsdetected towards the end of the growing season, shortly before the onsetof dormancy. A seaweed application (Kelpak) decreased the anti-inflammatory activity of the leaf, bulb and root extracts, while increasedtemperature/increased light intensity had no significant effect on theCOX-1 inhibitory activity of the leaf extracts. The bulb extracts fromtreated plants harvested towards the end of the growing season showed asignificant decrease in anti-inflammatory activity, while the anti-inflammatory activity of the corresponding not root extracts increased.     

Augusta disease in tulip - a reassessment

In an experiment in which the roots of field-grown tulip were commonly infected with tobacco necrosis virus (TNV), Augusta disease did not develop in the year of infection or when progeny bulbs were grown in the field or glass-house. When tulip bulbs of other stocks, including grades of 11 and 12 cm circumference, were forced, the disease developed sporadically, in some instances as the result of infection with TNV from the soil in which they were planted and in others as a result of infection by bulb-borne virus. The incidence of disease produced by current year infection was increased by warming the plunge bed. Different strains of TNV were obtained from field-grown plants with Augusta disease and different strains of the virus produced the disease when inoculated to tulip. Some, but not all, naturally diseased plants contained satellite virus, which therefore does not cause or prevent disease development. The disease was produced in some plants by TNV transmitted by Olpidium brassicae, but neither a vector nor a non-vector isolate of O. brassicae completed its life cycle in tulip. However, Olpidium-like zoospores were observed in some washings of tulip roots from TNV-infested soils. TNV was not obtained from all tulip plants with necrotic leaf symptoms resembling Augusta disease. Some were infected with tomato bushy stunt virus or cucumber mosaic virus, or with another agent that was transmitted by inoculation of sap to Nicotiana clevelandii and Chenopodium quinoa, and carried by bulbs of up to 11 cm circumference.     

Seasonal variation in antimicrobial and phytochemical properties of frequently used medicinal bulbous plants from South Africa

The growing popularity of traditional medicine and the unrestricted collection of medicinal plants from the wild have put many of the slow growing bulbous plant species at the risk of over-exploitation and extinction in South Africa. This study was aimed at comparing the phytochemical composition and biological (antibacterial and anticandidal) activities of bulb and leaf extracts of Tulbaghia violacea, Hypoxis hemerocallidea, Drimia robusta and Merwilla plumbea between spring, summer, autumn and winter seasons, with the view of promoting the use of leaves, as a conservation strategy. Antibacterial and anticandidal activities of petroleum ether (PE), dichloromethane (DCM), 80% ethanol and water extracts of bulbs and leaves were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae bacteria and the fungus Candida albicans using the microdilution bioassay. Spectrophotometric methods were used to evaluate saponin and phenolic compositions for the four seasons. Leaf and bulb extracts exhibited comparable anticandidal activity (MIC < 1 mg/ml) in all the plant species in all seasons. Only ethanol and water extracts of H. hemerocallidea corms (autumn and winter) showed correspondingly good fungicidal activity amongst the bulbs tested. Antibacterial activity was fairly comparable between bulbs and leaves with at least one extract of each plant species showing some good MIC values in most of the seasons. The best antimicrobial activities were recorded in winter and autumn seasons, with MIC values as low as 0.2 mg/ml in the DCM bulb extracts of T. violacea (winter) against K. pneumoniae and S. aureus. The amounts of total phenolic compounds in all plant samples were generally higher in spring compared to the other seasons. Condensed tannin, gallotannin and flavonoid levels, depending on the sample, were either higher in spring or winter except for H. hemerocallidea (corm) which had higher gallotannin levels in autumn. Total saponin levels were higher in winter in all plant samples. Although variation was observed in the phytochemical concentrations between the bulbs and leaves of each plant species, their antimicrobial activities were fairly comparable. Leaves may be used as substitutes for bulbs in the treatment of bacterial and fungal ailments.     

A comparison of the pharmacological properties of garden cultivated and muthi market-sold Bowiea volubilis

Biological activities of petroleum ether (PE), dichloromethane (DCM), 70% ethanol (EtOH) and water extracts of Botanical Garden-grown (BG) and muthi market-sourced (MM) Bowiea volubilis bulbs were compared. Bulb extracts were subjected to the microdilution technique using five test organisms for antimicrobial activity and cyclooxygenase (COX-1 and -2) inhibition as well as the Ames test for potential mutagenicity. Overall, both the MM and BG bulb extracts demonstrated a comparatively weak antimicrobial potency. The best minimum inhibitory concentration (MIC: 1.56 mg/ml) was detected in the MM bulb water extract against Candida albicans. In both MM and BG bulbs, 63% of the extracts, particularly the non-polar solvent extracts, exhibited a high (> 70% inhibition) COX-1 and -2 inhibitory activity. Both MM and BG bulb extracts were not mutagenic against the Salmonella typhimurium TA98 tester strain. Current findings indicate the potential substitution of cultivated B. volubilis bulbs (BG) for the wild population (MM) which is often utilized and preferred in traditional medicine. Inevitably, this will contribute to the conservation of the species as the strain on the wild population due to overharvesting will be alleviated.     

Antihemolytic and antioxidant activities of <Emphasis Type="Italic">Allium paradoxum</Emphasis>

Antioxidant activity of the aerial part and bulbs of Allium paradoxum was investigated by eight in vitro assay systems. Extracts showed good antioxidant activity. IC₅₀ for 1,1-diphenyl-2-picryl hydrazyl radical-scavenging activity was 890.9±43.2 and 984.9±33.5 μg/ml for the aerial part and bulbs, respectively. The aerial parts have better reducing power than bulb extracts but not comparable with Vitamin C (P>0.001). Extracts showed weak Fe²⁺ chelating ability, the IC₅₀ being 959±47 and 530±24 μg/ml for bulbs and aerial parts, respectively. Both tested extracts exhibited good hydrogen peroxide scavenging in a concentration dependent manner. They exhibited good antioxidant activity against the hemoglobin-induced linoleic acid system that was comparable with vitamin C (P>0.01). They showed good activity against cumene hydro peroxide induced hemolysis in RBCs. In addition, they possessed antihemolytic activity. The extract from aerial parts had significantly higher total phenol and flavonoid content than did bulbs. Amounts of eight elements (Cu, Mn, Zn, Fe, Ni, Pb, Cd and Cr) were also determined in the bulb and aerial part using atomic absorption spectroscopy. They contained higher Fe and Mn contents than other elements.     

Transmission characteristics and some other properties of bean yellow vein-banding virus, and its association with pea enation

Bean yellow vein-banding virus (BYVBV) has been found occasionally in mixed infection with pea enation mosaic virus (PEMV) in spring-sown field beans (Vicia faba minor) in southern England. Glasshouse tests confirmed that, like PEMV, BYVBV is transmissible by manual inoculation and by aphids in the persistent manner. However, BYVBV can be transmitted by aphids only from plants that are also infected with a helper virus, usually PEMV. Thus after separation from PEMV by passage through Phaseolus vulgaris it was no longer aphid-transmissible. It became aphid-transmissible again only after re-mixing in plants with PEMV or with a substitute helper, bean leaf roll virus (BLRV). It was not transmitted by aphids that fed sequentially on plants singly infected with PEMV and BYVBV. Thus the interaction between BYVBV and PEMV (or BLRV) that enables BYVBV to be transmitted by aphids seems to occur only in doubly infected plants. However, it was not transmitted by aphids from plants doubly infected with BYVBV and broad bean wilt virus (BBWV). BYVBV and PEMV were transmitted more readily by Acyrthosiphon pisum than by Myzus persicae; neither virus was transmitted by Aphis fabae. Phenol extracts of BYVBV-infected leaves were more infective than phosphate buffer or bentonite-clarified extracts and were sometimes infective when diluted to 1/1000. The infectivity of BYVBV in phosphate buffer extracts of leaves singly infected with BYVBV, unlike that in extracts of leaves doubly infected with BYVBV and PEMV (or BLRV), was destroyed by treatment with organic solvents. BYVBV infected 11 of 28 plant species that were inoculated with phenol extracts; seven of the infected species were legumes. No transmission of BYVBV was detected through seed harvested from infected field bean plants. Isometric particles c. 30 nm in diameter were seen in extracts of plants doubly infected with BYVBV and PEMV but not in extracts of plants infected with BYVBV alone. Leaves of plants infected with BYVBV, alone or with PEMV, contained membrane-bound structures c. 50–90 nm in diameter associated with the tonoplast in cell vacuoles. These structures were not found in healthy leaves. BYVBV has several properties in common with other known aphid-borne viruses that are helper-dependent and transmitted in a persistent manner. Possibly, as suggested for some of them, aphid transmission of BYVBV depends on the coating of its nucleic acid with helper virus coat protein.     

Effect of malformin on adventitious root formation and metabolism of indoleacetic acid-2-14C by Phaseolus vulgaris

Malformin inhibited rooting on cuttings of Phaseolus vulgaris.IAA antagonized malformin-induced inhibition of rooting, butmalformin inhibited IAA-induced swelling on the base of thecuttings. It was suggested that IAA-induced swelling was mediatedby ethylene. Malformin did not inhibit transport of root-promotingsubstances from upper portions of the cuttings or polar transportof IAA-2-¹⁴C, nor did it alter the melting point of DNA or thebinding of DNA to histone. Although malformin appeared to alterthe metabolism of IAA-2-¹⁴C, the effect may have been the resultof a marked and selective stimulation of efflux of IAA-2-¹⁴Cmetabolites by malformin. Efflux of IAA or its metabolites maycontribute toward inhibition of rooting by malformin. ¹ Journal Paper No. 4688 of the Purdue Agricultural ExperimentStation. Supported in part by grant GB-7158 from the NationalScience Foundation. ² Present address: Botanisches Institut der Technischen UniversitätBraunschweig, 3300 Braunschweig, Humboldtstraße 1. (Received March 9, 1972; )     

Multiplication and distribution of iris severe mosaic virus in secondarily-infected iris bulbs after stress induced by heat and wounding

In freshly-lifted iris bulbs infected with iris severe mosaic virus (ISMV), virus was not always detected in the basal plate and rarely in bulb scale tissue. After exposing the bulbs to stress (wounding or high-temperature treatment) the sensitivity of virus detection was enhanced. The improved detection of viral antigen after local stress (wounding) coincided with an increase of viral RNA synthesis. When general stress (high-temperature treatment) was applied, the virus could be reliably detected in the basal plate, and usually in vascular bundles and surrounding tissue. Virus was detected in the upper part of the bulb scale when such tissues were detached from the basal plate. Thus, virus must have been present in the scales in localised spots, albeit at a very low concentration, and multiplication is likely to be the main factor involved in the improved sensitivity of viral detection. The distribution of ISMV in the bulb after local or general stress treatment is discussed.     

The seasonal carry-over of Botrytis tulipae (Lib.) Lind., the cause of tulip fire

Damage done by Botrytis tulipae developed in a variety of ways after planting naturally and artificially inoculated bulbs, the fungus sometimes rapidly invading developing shoots, preventing their emergence. In other instances (a) leaves emerged above ground, but not before being severely infected (the traditional ‘primaries’), (b) leaves emerged uninfected but the flower stalks were attacked at a later stage causing mechanical weakness (and hence frequent snapping), or (c) leaves and flower stalks remained uninfected, with the development of B. tulipae being restricted to its spread from mother to daughter bulbs, which also occurred in (a) and (b). More emerging shoots were infected when inoculated bulbs were grown at 10° and 15.5° than at 4 °C—an effect suggesting that greater numbers of foliar primaries would develop in mild winters. The underground transmission of B. tulipae indicates the need for eradicating infections in the bulb, the primary sources of this pathogen.     

In vitro mRNA degradation system to study the virion host shutoff function of herpes simplex virus.

The virion host shutoff (vhs) gene of herpes simplex virus encodes a virion polypeptide that induces degradation of host mRNAs at early times and rapid turnover of viral mRNAs throughout infection. To better investigate the vhs function, an in vitro mRNA degradation system was developed, consisting of cytoplasmic extracts from HeLa cells infected with wild-type herpes simplex virus type 1 or a mutant encoding a defective vhs polypeptide. Host and viral mRNAs were degraded rapidly in extracts from cells productively infected with wild-type herpes simplex virus type 1 but not in extracts from mock-infected cells or cells infected with the mutant vhs1. In contrast, 28S rRNA was stable in all three kinds of extract. Accelerated turnover of host mRNAs was also observed in extracts from cells infected with wild-type virus in the presence of dactinomycin, indicating that the activity was induced by a structural component of the infecting virions. The in vitro vhs activity was inactivated by heat or proteinase K digestion but was insensitive to brief treatment of the extracts with micrococcal nuclease. It was not inhibited by placental RNase inhibitor, it exhibited a strong dependence upon added Mg2+, it was active at concentrations of K+ up to 200 mM, and it did not require the components of an energy-generating system. In summary, the in vitro mRNA degradation system appears to accurately reproduce the vhs-mediated decay of host and viral mRNAs and should be useful for studies of the mechanism of vhs action.     

Effect of divalent cations on malformin-induced efflux and biological activity

The effect of malformin on permeability was determined by efflux.Malformin increased membrane permeability to various organicand inorganic compounds or ions and was inhibited by severaldivalent cations. From the time required to increase permeabilityand the amount of material effluxed malformin primarily increasedtonoplast permeability. Calcium inhibited malformin-inducedstem collapse, abscission, and to a slight extent, inhibitionof primary leaf expansion. Although calcium inhibited malformin-inducedroot curvatures, malformin did not enhance permeability of rootslices at concentrations optimal for the curvature response. (Received May 6, 1975; )     

First Report of Fusarium proliferatum Causing Rot of Stored Garlic Bulbs (Allium sativum L.) in Italy

During 2011, Fusarium rot of stored garlic was detected on bulbs of ‘Aglio Bianco’ (white garlic) in Piacenza, Ferrara and Rovigo districts. Bulbs, harvested in July, were asymptomatic. During conservation in the drying sheds, approximately thirty percent of bulbs appeared emptied and softened. Fusarium proliferatum was consistently recovered from infected bulbs. The morphological identification was confirmed by Translation Elongation Factor 1‐alpha gene sequencing. Koch postulates were checked through pathogenicity tests. The disease has already been reported in Serbia, Germany, Spain, United States, China and India, but to our knowledge, this is the first report of F. proliferatum garlic bulb rot in Italy.     

Potentiation and inhibition of the effects of 2-chloroethylphosphonic Acid by malformin

Malformin completely inhibited Ethrel-induced swelling and fresh weight increase on the basal stem portion of Phaseolus vulgaris L. cuttings, but markedly potentiated Ethrel- or ethylene-induced abscission. With regard to abscission, malformin reacted synergistically with ethylene and dark aging, and in a manner which appeared to differ from that of ethylene and dark aging. The numerous effects of malformin on plant growth and development cannot be explained in simple terms of enhanced ethylene production.     

A rapid serological test for detecting Fusarium oxysporum f.sp. narcissi in Narcissus

Polyclonal antiserum was elicited against a strain of Fusarium oxysporum f.sp. narcissi (GCRI80/26) and a specific and sensitive enzyme-linked immunosorbent assay developed. Antiserum raised to cell wall fractions gave better recognition than that to cytoplasmic fractions. Recognition was equally good in artificially and naturally infected bulbs. Little cross-reactivity in bulb tissue was shown by three other bulb-rotting fungi. Nine isolates of F. oxysporum f.sp. narcissi from a wide geographic area gave similar results in an indirect ELISA of mycelial extracts, although some cross-reactivity was observed with two other Fusarium spp. Four Fusarium spp. and four other fungi showed little cross-reactivity. Ten days after inoculation the pathogen was readily detected in the base plate area of three Narcissus cultivars and points remote from the inoculation site in the most susceptible cultivar. A direct correlation was observed between positive results in the enzyme-linked immunosorbent assay and recovery of the pathogen on selective medium.     

Alterations in peroxidase activity and peroxidase isozymes in virus-infected plants

Peroxidase activity and isozyme patterns were investigated in two leguminous species infected with viruses which produced either local necrotic or systemic chlorotic symptoms. Highest peroxidase activity was recorded when the hosts reacted to infection with necrotic local lesions. No new virus-specific isozymes were found as a result of infection, but some isozymes, apparently associated with senescence, appeared earlier in extracts from leaves showing necrosis than in extracts from healthy leaves, or from infected leaves showing only very mild chlorosis. Increase in peroxidase activity was accompanied by alteration in isozyme pattern.     

The production by species of Allium of alkyl sulphides and their effect on germination of sclerotia of Sclerotium cepivorum Berk

A number of n -propyl and allyl sulphides were shown to stimulate germination of sclerotia of Sclerotium cepivorum in soil. Several of these sulphides were evolved by chopped garlic cloves and onion bulbs, and by their extracts, as well as by distillates and condensates prepared from extracts. Sulphides produced by garlic were methyl and allyl compounds, whereas those of onion were methyl and n -propyl derivatives. Few or no volatile sulphides were detected by gas chromatography of head spaces of intact Allium plants although it is considered that small quantities of sulphides may be produced from these plants in soil. The stimulatory activity of distillates and condensates appears to be entirely due to their alkyl sulphide content and that of extracts at least partially so.