Genetic mosaics in the massive persisting rhizosphere colony “shiro” of the ectomycorrhizal basidiomycete Tricholoma matsutake

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies matsutake on a massive persisting rhizosphere aggregate of mycelia and mycorrhizas called shiro. Using inter-retrotransposon amplified polymorphism analysis, we attempted to explore the potential diversity within the population of T. matsutake isolated from small Pinus densiflora woodlands located in various parts of Japan. In general, random phylogenetic relationship was noted among T. matsutake tested. The population from each limited sampling area was highly heterogeneous. Even some isolates from fruit bodies produced in the same shiro and those from spores in the same fruit bodies were found to be genetically diverse, indicating the occurrence of genetic mosaics in shiro. In a mosaic shiro, heterologous genets produced their fruit bodies concurrently. Data suggested that the dispersal of spores through sexual reproduction may have been more prevalent than generally accepted in T. matsutake to bring mosaicism and coordination of heterologous genets within the shiro. Implementation of management taking such diversity into consideration is urgently needed for the restoration of devastated matsutake fields in Japan. Exploration of individual clones in mosaic fungal resources that promote colonization and fruit body production is necessary for it.     

Phylogenetic position of the ectomycorrhizal basidiomycete Tricholoma dulciolens in relation to species of Tricholoma that produce “matsutake” mushrooms

Tricholoma dulciolens is an ectomycorrhizal basidiomycete that produces “matsutake”-like mushrooms in association with Picea abies in Fennoscandia. The phylogenetic position of T. dulciolens relative to matsutake species is, however, unknown. In the present study, we demonstrated that T. dulciolens is phylogenetically independent from T. matsutake and its allied species. In addition, herbarium specimens identified as T. caligatum from Abies alba forests in Spain were phylogenetically related to but distinct from T. dulciolens. Based on ribosomal internal transcribed spacer DNA sequence data, T. dulciolens appears to exist in North America, as well. This is the first phylogeny of T. dulciolens in Fennoscandia and its related species in southern Europe.     

Ectomycorrhizal symbiosis in vitro between Tricholoma matsutake and Pinus densiflora seedlings that resembles naturally occurring ‘shiro’

We established an in vitro ectomycorrhizal symbiosis between Tricholoma matsutake and Pinus densiflora. Mycorrhiza formed in a substrate of Modified Norkrans' C medium and granite-based soil had features similar to those observed previously only in naturally occurring mycorrhizal system called ‘shiro,’ and promoted the growth of plants with smaller root/shoot ratios. The in vitro formation of ‘shiro’ is essential for the development of T. matsutake system to produce mushrooms and is useful for the propagation and plantation of the mycorrhizal seedlings.     

Tricholoma matsutake– an assessment of in situ and in vitro infection by observing cleared and stained whole roots

 Structures present within field-collected Tricholoma matsutake/Pinus densiflora ectomycorrhizas and in vitro infections of P. densiflora roots by T. matsutake were observed by clearing, bleaching and staining whole lateral roots and mycorrhizas. Field mycorrhizas were characterized by a lack of root hairs, by the presence of a sparse discontinuous mantle composed of irregularly darkly staining hyphae over the root surface, primarily behind the root cap, and by the presence of Hartig net mycelium within the root cortex. Hartig net 'palmettis' were classified into three basic structures, each with distinctive morphologies. Aerial hyphae, bearing terminal swellings, were observed emanating from the mantle. Cleared, bleached and stained in vitro-infected roots possessed multibranched hyphal structures within the host root cortex and aerial hyphae bearing terminal swellings were observed arising from the mycelium colonizing the root surface. T. matsutake on P. densiflora conforms to the accepted morphology of an ectomycorrhiza. This staining protocol is particularly suited to the study of Matsutake mycorrhizal roots and gives rapid, clear, high-contrast images using standard light microscopy while conserving spatial relationships between hyphal elements and host tissues. Accepted: 26 August 1999     

Highly polymorphic DNA markers to specify strains of the ectomycorrhizal basidiomycete Tricholoma matsutake based on σ marY1 , the long terminal repeat of gypsy-type retroelement marY1

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies—matsutake—in Pinus sp. forest. Here we report that PCR with outward facing primers designed based on sequences comprising _marY1, the long terminal repeat of the gypsy-type retroelement marY1, specifies strains of T. matsutake. PCR with a primer based on the 22-bp sequence conserved at the 5-end of _marY1 conferred 73 reliable bands overall whose profiles depend upon strains of T. matsutake and T. magnivelare, the latter known as American matsutake. This PCR system gave no detectable band in any other species of Tricholoma tested, including T. bakamatsutake and T. fulvocastaneum, symbionts closely related to T. matsutake, as well as a host plant, Pinus densiflora. Similarly, PCR with a set of primers based on 26-bp and 28-bp sequences at bp 48–73 and bp 281–308 of _marY1, internal regions that are mutated in a variant of marY1, conferred 90 reliable bands only in strains of T. matsutake. Theoretically, PCR with the 22-bp primer would allow generation of 2⁷³, or 9.4×10²¹, types of polymorphism, and PCR with a combination of 26- and 28-bp primers, 2⁹⁰, or 1.2×10²⁷ types. The probability of falsely specifying two different isolates as the same strain is <1/10²¹. While polymorphisms conferred by the primer based on the 5 end of _marY1 rather exhibit genetic conservation of a group of T. matsutake, those resulting from primers based on the internal sequences more clearly demonstrate intra-specific diversification. Both systems revealed that T. matsutake is divergent within the species. Ectomycorrhizas formed between P. densiflora and T. matsutake were identified by the PCR systems developed in the present study. This method, using _marY1 as a genetic marker, is useful in analyzing the diversity of T. matsutake, monitoring the behavior of individual mycorrhizas, and specifying the ecological background of fruit bodies traded in markets.     

The (oxalato)aluminate complex as an antimicrobial substance protecting the “shiro” of Tricholoma matsutake from soil micro-organisms

Tricholoma matsutake, a basidiomycete, forms ectomycorrhizas with Pinus densiflora as the host tree. Its fruiting body, “matsutake” in Japanese, is an edible and highly prized mushroom, and it grows in a circle called a fairy ring. Beneath the fairy ring of T. matsutake, a whitish mycelium-soil aggregated zone, called “shiro” in Japanese, develops. The front of the shiro, an active mycorrhizal zone, functions to gather nutrients from the soil and roots to nourish the fairy ring. Bacteria and sporulating fungi decrease from the shiro front, whereas they increase inside and outside the shiro front. Ohara demonstrated that the shiro front exhibited antimicrobial activity, but the antimicrobial substance has remained unidentified for 50 years. We have identified the antimicrobial substance as the (oxalato)aluminate complex, known as a reaction product of oxalic acid and aluminum phosphate to release soluble phosphorus. The complex protects the shiro from micro-organisms, and contributes to its development.     

Mobile DNA distributions refine the phylogeny of “matsutake” mushrooms, Tricholoma sect. Caligata

“Matsutake” mushrooms are formed by several species of Tricholoma sect. Caligata distributed across the northern hemisphere. A phylogenetic analysis of matsutake based on virtually neutral mutations in DNA sequences resolved robust relationships among Tricholoma anatolicum, Tricholoma bakamatsutake, Tricholoma magnivelare, Tricholoma matsutake, and Tricholoma sp. from Mexico (=Tricholoma sp. Mex). However, relationships among these matsutake and other species, such as Tricholoma caligatum and Tricholoma fulvocastaneum, were ambiguous. We, therefore, analyzed genomic copy numbers of σ _marY1 , marY1, and marY2N retrotransposons by comparing them with the single-copy mobile DNA megB1 using real-time polymerase chain reaction (PCR) to clarify matsutake phylogeny. We also examined types of megB1-associated domains, composed of a number of poly (A) and poly (T) reminiscent of RNA-derived DNA elements among these species. Both datasets resolved two distinct groups, one composed of T. bakamatsutake, T. fulvocastaneum, and T. caligatum that could have diverged earlier and the other comprising T. magnivelare, Tricholoma sp. Mex, T. anatolicum, and T. matsutake that could have evolved later. In the first group, T. caligatum was the closest to the second group, followed by T. fulvocastaneum and T. bakamatsutake. Within the second group, T. magnivelare was clearly differentiated from the other species. The data suggest that matsutake underwent substantial evolution between the first group, mostly composed of Fagaceae symbionts, and the second group, comprised only of Pinaceae symbionts, but diverged little within each groups. Mobile DNA markers could be useful in resolving difficult phylogenies due to, for example, closely spaced speciation events.     

Response of “criollo” maize to single and mixed species inocula of arbuscular mycorrhizal fungi

We tested the effect of two single species inocula and a mixed inoculum of the native population of arbuscular mycorrhizal (AM) fungi on the growth response of criollo maize (Zea mays L.). To determine the inocula that produced the highest response on maize growth, we conducted a greenhouse experiment at 3 levels of P fertilization (0, 40 and 80 kg ha^–1). Inoculation with Glomus mosseae (Nicolson and Gerdemann) Gerd. and Trappe (LMSS) produced the greatest shoot growth rates at the two lowest P fertilization levels. Inoculation with Acaulospora bireticulata Rothwell and Trappe (ABRT) and the native population (NP) resulted in similar shoot growth rates at all P levels. These rates were higher than the non-mycorrhizal control rate at the lowest P level but lower than the control at the highest P level. Also, ABRT and NP had significantly lower shoot growth rates than the inoculation treatment with G. mosseae at all P levels. The non-mycorrhizal control had the lowest growth rate at the lowest P level but its growth rate increased linearly with increased P fertilization. Inoculation with G. mosseae and A. bireticulata produced similar colonization rates which were lower than the native population colonization rate. There was no correlation between colonization and shoot growth rates.     

Spectral diagnostics of the interaction between pyridoxine hydrochloride and bovine serum albumin in vitro

The interaction between pyridoxine hydrochloride (VB6) and bovine serum albumin (BSA) were studied by spectroscopic methods including fluorescence spectroscopy and UV-visible absorption spectra. The quenching mechanism of fluorescence of BSA by VB6 was discussed. The number of binding sites n and observed binding constant K(b) was measured by fluorescence quenching method. The thermodynamic parameters DeltaH(theta), DeltaG(theta), DeltaS(theta) at different temperatures were calculated and the results indicate the binding reaction is mainly entropy-driven and hydrophobic interaction played major role in the reaction. The distance r between donor (BSA) and acceptor (VB6) was obtained according to FOrster theory of non-radiation energy transfer. Synchronous fluorescence and three-dimensional fluorescence spectra were used to investigate the structural change of BSA molecules with addition of VB6, the result indicates that the secondary structure of BSA molecules is changed in the presence of VB6.     

Design,synthesis and in vitro cytotoxicity studies of novel β-carbolinium bromides

A series of novel β-carbolinium bromides has been synthesized from easily accessible β-carbolines and 1-aryl-2-bromoethanones. The newly synthesized compounds were evaluated for their in vitro anticancer activity. Among the synthesized derivatives, compounds 16l, 16o and 16s exhibited potent anticancer activity with IC₅₀ values of <10 μM against tested cancer cell lines. The most potent analogue 16l was broadly active against all the tested cancer cell lines (IC₅₀ = 3.16–7.93 μM). In order to test the mechanism of cell death, we exposed castration resistant prostate cancer cell line (C4-2) to compounds 16l and 16s, which resulted in increased levels of cleaved PARP1 and AO/EB staining, indicating that β-carbolinium salts induce apoptosis in these cells. Additionally, the most potent β-carbolines 16l and 16s were found to inhibit tubulin polymerization.     

The role of arbuscular mycorrhizal fungi in plant community establishment at Samphire Hoe, Kent, UK – the reclamation platform created during the building of the Channel tunnel between France and UK

Samphire Hoe is a newly-created land platform comprising the sub-seabed material excavated during the construction of the Channel tunnel. It represents a unique resource where the arrival and establishment of arbuscular mycorrhizal fungi (AMF) within a sown plant community on a low nutrient substrate can be monitored. Arbuscular mycorrhizal fungi invasion was monitored in a number of ways: by assessing the degree of root colonisation within the roots of plants on the site, by using a successive trap culture technique to determine AMF species richness, and by using sterile substrate bins to determine the extent of wind-borne and rain-dispersed immigration of AMF propagules into the site. Levels of colonisation of indigenous plants by AMF were high in May–June (the pre-flowering phase of growth for many plants) reflecting the important role of the mycorrhizal symbiosis in dry, low nutrient soils. Twelve species of AMF were identified, representing a relatively high diversity for a recently deposited subsoil. An on-site experiment indicated that inoculum of AMF could enter the site within 8 months and that wind dispersal and/or rain were possible vectors. A field experiment compared the outplanting performance of commercially-produced Elymus pycnanthus seedlings (in a commercial compost with added nutrients) with seedlings produced in a low nutrient substrate and inoculated with AMF isolated from the site (a mixture of 5 species of Glomus) or left uninoculated. After 14 months in the field seedlings, inoculated with the indigenous AMF, had the same tiller production as commercially-produced plants, despite slower initial growth. In contrast, non-mycorrhizal controls grew very poorly with a greater frequency of plant mortality compared with the other treatments. Elymus seedlings inoculated with the indigenous AMF ultimately produced approximately seven times the mean number of seed spikes per surviving plant as commercially-produced seedlings and five times greater weight of seed spike. A phyto-microbial approach to the revegetation of nutrient-poor soils is proposed to stimulate plant successional processes as a economically-viable sustainable input for landscaping anthropogenic sites.     

In vitro synthesis of RNA with “aggregate” enzyme,chromatin, and DNA from liver of methylazoxymethanol acetate-treated rats

“Aggregate” enzyme, chromatin and DNA preparations were isolated from livers of rats treated with the carcinogen, methylazoxymethanol (MAM) acetate. DNA template activity for RNA synthesis in vitro was unimpaired while the template activity of chromatin was slightly reduced. There was a marked inhibition of UTP incorporation into RNA, however, when the “aggregate” enzyme preparation was the source of both template and RNA polymerase. Circular dichroism analysis of the “aggregate” enzyme preparation indicated a change in conformation of the protein component. The results suggest that MAM acetate interacts with nuclear proteins and produces conformational changes which result in a decreased RNA synthesis.     

Design,synthesis, in vitro,and in silico studies of novel diarylimidazole-1,2,3-triazole hybrids as potent α-glucosidase inhibitors

In this work, new derivatives of diarylimidazole-1,2,3-triazole 7a-p were designed, synthesized, and evaluated for their in vitro α-glucosidase inhibitory activity. All compounds showed potent inhibitory activity in the range of IC₅₀ = 90.4–246.7 µM comparing with acarbose as the standard drug (IC₅₀ = 750.0 µM). Among the synthesized compounds, compounds 7b, 7c, and 7e were approximately 8 times more potent than acarbose. The kinetic study of those compounds indicated that they acted as the competitive inhibitors of α-glucosidase. Molecular docking studies were also carried out for compounds 7b, 7c, and 7e using modeled α-glucosidase to find the interaction modes responsible for the desired inhibitory activity.     

Design,synthesis, in vitro inhibition and toxicological evaluation of human carbonic anhydrases I,II and IX inhibitors in 5-nitroimidazole series

Abstract

With the aim to obtain novel compounds possessing both strong affinity against human carbonic anhydrases and low toxicity, we synthesised novel thiourea and sulphonamide derivatives 3, 4 and 10, and studied their in vitro inhibitory properties against human CA I, CA II and CA IX. We also evaluated the toxicity of these compounds using zebrafish larvae. Among the three compounds, derivative 4 showed efficient inhibition against hCA II (KI = 58.6?nM). Compound 10 showed moderate inhibition against hCA II (KI = 199.2?nM) and hCA IX (KI = 147.3?nM), whereas it inhibited hCA I less weakly at micromolar concentrations (KI = 6428.4?nM). All other inhibition constants for these compounds were in the submicromolar range. The toxicity evaluation studies showed no adverse effects on the zebrafish larvae. Our study suggests that these compounds are suitable for further preclinical characterisation as potential inhibitors of hCA I, II and IX.     

Rational design,synthesis and in vitro evaluation of novel exo-methylene butyrolactone salicyloylamide as NF-κB inhibitor

(?)-Dehydroxymethylepoxyquinomicin ((?)-DHMEQ, 1) is a specific inhibitor of NF-κB. It binds to SH group in the specific cysteine residue of NF-κB components with its epoxide moiety to inhibit DNA binding. In the present research, we have designed and synthesized an epoxide-free analog called (S)-β-salicyloylamino-α-exo-methylene-?-butyrolactone (SEMBL, 3). SEMBL inhibited DNA binding of NF-κB component p65 in vitro. It inhibited LPS-induced NF-κB activation, iNOS expression, and inflammatory cytokine secretions. It also inhibited NF-κB and cellular invasion in ovarian carcinoma ES-2 cells. Moreover, its stability in aqueous solution was greatly enhanced compared with (?)-DHMEQ. Thus, SEMBL has a potential to be a candidate for a new anti-inflammatory and anticancer agent.     

Design,synthesis and in vitro biological evaluation of quinazolinone derivatives as EGFR inhibitors for antitumor treatment

Abstract

In this paper, a series of novel 3-methyl-quinazolinone derivatives was designed, synthesised and evaluated for antitumor activity in vitro on wild type epidermal growth factor receptor tyrosine kinase (EGFR^wt-TK) and three human cancer cell lines including A549, PC-3, and SMMC-7721. The results displayed that some of the compounds had good activities, especially 2-{4-[(3-Fluoro-phenylimino)-methyl]-phenoxymethyl}-3-methyl-3H-quinazolin-4-one (5?g), 2-{4-[(3,4-Difluoro-phenylimino)-methyl]-phenoxymethyl}-3-methyl-3H-quinazolin-4-one (5k) and 2-{4-[(3,5-Difluoro-phenylimino)-methyl]-phenoxymethyl}-3-methyl-3H-quinazolin-4-one (5?l) showed high antitumor activities against three cancer cell lines. Moreover, compound 5k could induce late apoptosis of A549 cells at high concentrations and arrest cell cycle of A549 cells in the G2/M phase at tested concentrations. Also, compound 5k could inhibit the EGFR^wt-TK with IC₅₀ value of 10?nM. Molecular docking data indicates that the compound 5k may exert inhibitory activity by forming stable hydrogen bonds with the R817, T830 amino acid residues and cation-Π interaction with the K72 residue of EGFR^wt-TK.     

Design,synthesis, and evaluation of Trolox-conjugated amyloid-β C-terminal peptides for therapeutic intervention in an in vitro model of Alzheimer’s disease

Two hallmarks of Alzheimer’s disease (AD) observed in the brains of patients with the disease include oxidative injury and deposition of protein aggregates comprised of amyloid-β (Aβ) variants. To inhibit these toxic processes, we synthesized antioxidant-conjugated peptides comprised of Trolox and various C-terminal motifs of Aβ variants, TxAβ_x–n (x = 34, 36, 38, 40; n = 40, 42, 43). Most of these compounds were found to exhibit anti-aggregation activities. Among them, TxAβ_36–42 significantly inhibited Aβ_1–42 aggregation, showed potent antioxidant activity, and protected SH-SY5Y cells from Aβ_1–42-induced cytotoxicity. Thus, this method represents a promising strategy for developing multifunctional AD therapeutic agents.     

Ethers and esters derived from apocynin avoid the interaction between p47phox and p22phox subunits of NADPH oxidase: evaluation in?vitro and in silico

NOX (NADPH oxidase) plays an important role during several pathologies because it produces the superoxide anion (O₂^•−), which reacts with NO (nitric oxide), diminishing its vasodilator effect. Although different isoforms of NOX are expressed in ECs (endothelial cells) of blood vessels, the NOX2 isoform has been considered the principal therapeutic target for vascular diseases because it can be up-regulated by inhibiting the interaction between its p47phox (cytosolic protein) and p22phox (transmembrane protein) subunits. In this research, two ethers, 4-(4-acetyl-2-methoxy-phenoxy)-acetic acid (1) and 4-(4-acetyl-2-methoxy-phenoxy)-butyric acid (2) and two esters, pentanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester (3) and heptanedioic acid mono-(4-acetyl-2-methoxy-phenyl) ester (4), which are apocynin derivatives were designed, synthesized and evaluated as NOX inhibitors by quantifying O₂^•− production using EPR (electron paramagnetic resonance) measurements. In addition, the antioxidant activity of apocynin and its derivatives were determined. A docking study was used to identify the interactions between the NOX2′s p47phox subunit and apocynin or its derivatives. The results showed that all of the compounds exhibit inhibitory activity on NOX, being 4 the best derivative. However, neither apocynin nor its derivatives were free radical scavengers. On the other hand, the in silico studies demonstrated that the apocynin and its derivatives were recognized by the polybasic SH3A and SH3B domains, which are regions of p47phox that interact with p22phox. Therefore this experimental and theoretical study suggests that compound 4 could prevent the formation of the complex between p47phox and p22phox without needing to be activated by MPO (myeloperoxidase), this being an advantage over apocynin.     

Phenomenon of “Siamese embryos” in cereals in vivo and in vitro: Cleavage polyembryony and fasciations

The genesis of wheat microsporial polyembryoids in vitro was analyzed in detail. The nature of different phenotypes of cereal polymeric embryos was identified. They represent the class “multiple shoot meristems,” which results from a cleavage polyembryony and is accompanied by organ fasciations of all known types (radial, flat, or ring). The morphological nature of cereal embryonic organs has been clarified: shoot meristem—axial organ; scutellum—lateral outgrowth of this axis; coleoptile—derivative of shoot meristem but fused with scutellum; terminality of scutellum—the result of linear fasciation that occurred historically. An explanation is given on how the structural model of an auxin polar transport works during the establishment of bilateral symmetry in a cereal embryo that is associated with the inverted polarization of the carrier protein PIN1 on cell membranes and, correspondingly, with the inverted auxin transport performed by this carrier (Fischer-Iglesias et al., 2001; Forestan et al., 2010).     

The development of in vitro and in vivo anti-tumor cytotoxicity in noncytotoxic,MOPC-315-tumor-bearer,spleen cells “educated” in vitro with MOPC-315 tumor cells

Summary Spleen cells from mice bearing various sizes of MOPC-315 plasmacytoma tumors were not cytotoxic in the ⁵¹Cr release assay or the local adoptive transfer assay. These noncytotoxic spleen cells became cytotoxic, however, upon in vitro cocultivation with MOPC-315 tumor cells. The maximal level of in vitro anti-tumor cytotoxicity (⁵¹Cr release) with in vitro educated tumor-bearer spleen cells was obtained on the fifth day of the cocultivation and was equal to or lower than the level of cytotoxicity seen with in vitro educated normal spleen cells. On the other hand, the level of in vivo anti-tumor cytotoxicity (Winn assay) achieved with tumor-bearer spleen cells educated in vitro was at least equal to, but usually greater than the level of anti-tumor cytotoxicity obtained with normal spleen cells educated in vitro. Thus, in vitro education can generate anti-tumor cytotoxicity in autochthonous lymphoid cells from tumor-bearing hosts. Such educated histocompatible cells should be useful for immunotherapy regimens that might be applicable to man.