Diversity of expression of non-muscle actin in amphibia

The number and relative ratio of different actins present in various amphibian non-muscle cells and tissues were determined by the characterization of the S-sulphone-¹⁴C]carboxymethylated actin amino-terminal tryptic peptides. The arrangement of aspartic acid and glutamic acid residues in positions two to four was inferred from electrophoretic properties of secondary cleavage products of the isolated amino-terminal peptides (Vandekerckhove & Weber, 1981).Cultured kidney epithelial cells, brain and liver of the toad Xenopus laevis reveal at least three forms of actin characterized by the following amino-terminal sequences: X-Glu-Asp-Asp- (type 4), X-Asp-Glu-Glu (type 5) and X-Glu-Glu-Glu-(type 8), with X being a blocking group recognized as an acetyl group in other actins. The same three actins are also expressed in X. laevis oocytes, where they are found in the same molar ratio in manually isolated cortices and nuclei. Three different non-muscle actins with the following amino-terminal sequences: X-Asp-Asp-Asp- (type 1), X-Asp-Glu-Asp- (type 3) and X-Glu-Glu-Glu- (type 8) are found in cultured cells of the newt, Triturus cristatus. Brain and liver tissues of the frog, Rana pipiens, only show two actin forms, type 4 and type 5, while liver tissue of the newt, Pleurodeles waltlii, shows only a single major actin form.The results indicate that non-muscle actins are more complex and variable in amphibia than in the warm-blooded vertebrates, where only two non-muscle actins (β and λ; type 1 and type 8) have been found in a number of different species.     

Study of a new group of bioregulators isolated from the greater plantain (<Emphasis Type="Italic">Plantago major</Emphasis> L.)

Proteins with physicochemical properties and biological activity similar to those of membranotropic homeostatic tissue-specific bioregulators that had been found earlier in various animal tissues were discovered in leaves of the common plantain (Plantago major L.). To study the specific activity of these plant proteins, we developed an experimental model for organotypic roller cultivation of newt (Pleurodeles waltl) skin tissue in vitro. We showed that the plant proteins of interest exert the wound-healing effect, which is characteristic of this plant, on the skin of vertebrates both in vitro and in vivo.     

Neural cell differentiation from retinal pigment epithelial cells of the newt: An organ culture model for the urodele retinal regeneration

Transdifferentiation from retinal pigment epithelium (RPE) to neural retina (NR) was studied under a new culture system as an experimental model for newt retinal regeneration. Adult newt RPEs were organ cultured with surrounding connective tissues, such as the choroid and sclera, on a filter membrane. Around day 7 in vitro, lightly pigmented “neuron‐like cells” with neuritic processes were found migrating out from the explant onto the filter membrane. Their number gradually increased day by day. BrdU‐labeling study showed that RPE cells initiated to proliferate under the culture condition on day 4 in vitro, temporally correlating to the time course of retinal regeneration in vivo. Histological observations of cultured explants showed that proliferating RPE cells did not form the stratified structure typically observed in the NR but they rather migrated out from the explants. Neuronal differentiation was examined by immunohistochemical detection of various neuron‐specific proteins; HPC‐1 (syntaxin), GABA, serotonin, rhodopsin, and acetylated tubulin. Immunoreactive cells for these proteins always possessed fine and long neurite‐like processes. Numerous lightly pigmented cells with neuron‐like morphology showed HPC‐1 immunoreactivity. Fibroblast growth factor‐2 (FGF‐2), known as a potent factor for the transdifferentiation of ocular tissues in various vertebrates, substantially increased the numbers of both neuron‐like cells and HPC‐1‐like immunoreactive cells in a dose‐dependent manner. These results indicate that our culture method ensures neural differentiation of newt RPE cells in vitro and provides, for the first time, a suitable in vitro experimental model system for studying tissue‐intrinsic factors responsible for newt retinal regeneration. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 209–220, 2002; DOI 10.1002/neu.10031     

Direct evidence for transformation of differentiated iris epithelial cells into lens cells

Although it is generally assumed that the lens regenerated in the newt eye after complete lentectomy is formed by cells derived from the dorsal iris epithelium, experimental evidence so far obtained for this transformation does not rule out participation of cells from the dorsal iris stroma. When the normal dorsal iris epithelium of adult Notophthalmus (Triturus) viridescens was isolated and cultured in the presence of frog retinal complex, newt lens tissue was produced in 88% of cultures. These lens tissues were positive for immunofluorescence for lens-fiber-specific gamma crystallins as well as for total lens protein. On the basis of a study of stromal cells contaminating the samples of dorsal iris epithelium and a test for the lens-forming capacity in vitro of the dorsal iris stroma in the presence of frog retinal complex, it is concluded that lens formation observed in the above experiment is not dependent on the contaminating stromal cells. This implies that, in Wolffian lens regeneration, fully differentiated adult cells completely withdrawn from the cell cycle are transformed into another cell type. An additional culture experiment demonstrated that lens-forming capacity is not restricted to the dorsal half of the iris epithelium, but extends into its ventral half.     

Haradamyces foliicola anam. gen. et sp. nov., a cause of zonate leaf blight disease in Cornus florida in Japan

A fungus causing zonate leaf blight diseases in various evergreen and deciduous woody plant species in Japan was characterized by a discoid multicellular propagule arising from a hyaline sclerotium-like structure in the leaf tissue and dark-coloured microconidia produced enteroblastically from the terminal cells on the surface of the discoid propagules. Myrioconium-like microconidiophores also producing microconidia were occasionally produced in culture. No teleomorphic characteristics were observed on the fungus. Molecular analysis based on the partial nu-rDNA sequence data revealed that the fungus was phylogenetically related to the Sclerotiniaceae, Leotiomycetes, and Ascomycota. Because the morphology and sequence data of this fungus does not coincide with those of any known anamorphic fungi, Haradamyces foliicola is proposed here as a new anamorphic genus and species for this fungus.     

Production of zearalenone,nivalenol, moniliformin,and wortmannin from toxigenic cultures ofFusarium obtained from pasture soil samples collected in New Zealand

One culture ofF avenaceum, 4 cultures ofF oxysporum, and 11 cultures of Fsambucinum were isolated from soil samples of pasture in New Zealand in 1987. All cultures, when grown on rice media and fed to rats caused a weight loss in rats as well as toxic signs including hemorrhaging and congestion, uterine enlargement, and hematuria. 6 out of 16 cultures caused death in rat feeding tests.F oxysporum #1 killed rats (feeding test) within 5-12hrs. 10 cultures produced zearalenone (19 to 8,849 ppm), 8 cultures produced nivalenol (32 to 117 ppm), 1 culture,F sambucinum #8, produced wortmannin (40 ppm), and 5 cultures produced moniliformin (19 to 9,000ppm). We report for the first time the co-occurrence of zearalenone, nivalenol, and moniliformin produced byF sambucinum #3 in culture.F avenaceum #1 andF oxysporum cultures (nos 1, 2, and 3) produced moniliformin alone.F oxysporum #4 produced zearalenone alone as well.F sambucinum #5 caused erythema in the small intestine of rats and 100% mortality and did not produce any known toxin(s). Nivalenol when administered to the stomach of rats orally at levels 10, 20, and 40mg/kg body weight caused inflammation in the intestines, coma, and death. The mycotoxins T-2 toxin, HT-2 toxin, T-2 tetraol, diacetoxyscirpenol (DAS), monoacetoxyscirpenol (MAS), deoxynivalenol (DON), 3-acetyl-and 15-acetyldeoxynivalenol, depoxynivalenol, fusarenon-X, alpha-and beta-zearalenone, and fusarochromanone (TDP-1) were not detected in the extracts of these cultures.     

A new non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells

Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, “tissue incubation”, for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7–10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3–4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α6 and laminin α5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.     

Influence of the pituitary on Wolffian lens regeneration

Removal of the pituitary 3 days before lentectomy retards Wolffian lens regeneration in the adult newt, Notophthalmus viridescens, by two stages over a 21-day period. Hypophysectomy 5 or 10 days after lentectomy does not alter the progress of regeneration during the subsequent 10-day period. Hypophysectomy 3 days before lentectomy also significantly decreases the incorporation of [³H]thymidine by iris epithelial nuclei 5 days after lentectomy but has no statistically significant effect on the incorporation 7 days after lentectomy.Pituitary tissue from newts or frogs enhances the regenerative activity of newt iris epithelial cells in vitro and in many cases promotes lens fiber formation. To a lesser extent, other tissues, such as nerve ganglion, also enhance the production of lens fiber cells from iris epithelium in vitro, whereas muscle tissue does not; and under certain conditions iris epithelial cells were found to depigment and redifferentiate into lens cells in the absence of other tissues in vitro.     

Extracellular biosynthesis of silver nanoparticles using a novel and non-pathogenic fungus,Neurospora intermedia: controlled synthesis and antibacterial activity

In the present study, the biosynthesis of silver nanoparticles (AgNPs) using Neurospora intermedia, as a new non-pathogenic fungus was investigated. For determination of biomass harvesting time, the effect of fungal incubation period on nanoparticle formation was investigated using UV–visible spectroscopy. Then, AgNPs were synthesized using both culture supernatant and cell-free filtrate of the fungus. Two different volume ratios (1:100 and 1:1) of the culture supernatant to the silver nitrate were employed for AgNP synthesis. In addition, cell-free filtrate and silver nitrate were mixed in presence and absence of light. Smallest average size and highest productivity were obtained when using equal volumes of the culture supernatant and silver nitrate solution as confirmed by UV–visible spectra of colloidal AgNPs. Comparing the UV–visible spectra revealed that using cell-free filtrate for AgNP synthesis resulted in the formation of particles with higher stability and monodispersity than using culture supernatant. The absence of light in cell-free filtrate mediated synthesis led to the formation of nanoparticles with the lowest rate and the highest monodispersity. The presence of elemental silver in all prepared samples was confirmed using EDX, while the crystalline nature of synthesized particles was verified by XRD. FTIR results showed the presence of functional groups which reduce Ag⁺ and stabilize AgNPs. The presence of nitrate reductase was confirmed in the cell-free filtrate of the fungus suggesting the potential role of this enzyme in AgNP synthesis. Synthesized particles showed significant antibacterial activity against E. coli as confirmed by examining the growth curve of bacterial cells exposed to AgNPs.     

MALDI-TOF mass spectrometric identification of novel intercellular space peptides

We performed the matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF) analysis of the peptides entering into the composition of not yet explored bioregulators derived from the extracellular matrix of the tissues of the various organs of the mammals, and also plants and fungi. The study included 15 different mammalian tissues, 13 species of plants, and 2 species of fungi. Exploring the bioregulators derived from eye tissues, we demonstrated that their composition includes peptide components with the same values of the molecular weight. The composition of the bioregulators derived from the tissues of various organs of mammals or different species of plants and fungi includes the peptides with different values of molecular weight. Obtained data indicate the growing evidence of the assumptions about the major function of the bioregulators of this group—their involvement in the regulation of tissue-organ homeostasis in the biological systems.     

FGF2 signaling pathway components in tissues of the posterior eye sector in the adult newt Pleurodeles waltl

The FGF2 signaling pathway components in tissues of the posterior wall in the normal and regenerating eye of the adult Pleurodeles waltl newt were detected for the first time. The fgf2 gene expression was found in the retina, retinal pigment epithelium, and choroid using polymerase chain reaction (PCR). A high homology of the mRNA nucleotide sequence of the most conservative fgf2 gene region in the P. waltl with the fgf2 orthologs in other vertebrates was proved. The Fgf2 protein amino acid sequence of the P. waltl newt demonstrates even more homology with this growth factor in other vertebrates. The Fgf2 protein with a molecular weight 35 kDa was found in the studied eye tissues using Western blot hybridization. Localization of the Fgf2 protein and its Fgfr receptors was immunohistochemically studied in the pigment epithelium, choroid, central and growth retina regions of the newt native eye, and in the connective cilium of photoreceptors. Using real-time PCR and immunohistochemistry methods, it was found that the fgf2 gene down-regulation and a decrease in the intensity of the immunochemical reaction of its protein product (Fgf2) occur in the early period after the retina removal (in 4–8 days) (as compared with those in the same department of the unoperated eye).     

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw ¹. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month ^2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) ^4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.     

Combining microtomy and confocal laser scanning microscopy for structural analyses of plant–fungus associations

The serious problem of extended tissue thickness in the analysis of plant–fungus associations was overcome using a new method that combines physical and optical sectioning of the resin-embedded sample by microtomy and confocal microscopy. Improved tissue infiltration of the fungal-specific, high molecular weight fluorescent probe wheat germ agglutinin conjugated to Alexa Fluor® 633 resulted in high fungus-specific fluorescence even in deeper tissue sections. If autofluorescence was insufficient, additional counterstaining with Calcofluor White M2R or propidium iodide was applied in order to visualise the host plant tissues. Alternatively, the non-specific fluorochrome acid fuchsine was used for rapid staining of both, the plant and the fungal cells. The intricate spatial arrangements of the plant and fungal cells were preserved by immobilization in the hydrophilic resin Unicryl?. Microtomy was used to section the resin-embedded roots or leaves until the desired plane was reached. The data sets generated by confocal laser scanning microscopy of the remaining resin stubs allowed the precise spatial reconstruction of complex structures in the plant–fungus associations of interest. This approach was successfully tested on tissues from ectomycorrhiza (Betula pendula), arbuscular mycorrhiza (Galium aparine; Polygala paniculata, Polygala rupestris), ericoid mycorrhiza (Calluna vulgaris), orchid mycorrhiza (Limodorum abortivum, Serapias parviflora) and on one leaf–fungus association (Zymoseptoria tritici on Triticum aestivum). The method provides an efficient visualisation protocol applicable with a wide range of plant–fungus symbioses.     

Cystomyces antheropori,a new rust fungus on Antheroporum glaucum from Thailand

A rust fungus was found on leaves of Antheroporum glaucum (Fabaceae, Faboideae) in Provinces of Petchaburi and Ratchaburi, western Thailand. No rust fungus was previously reported on this legume tree species. The new rust fungus was apparently microcyclic, producing telia surrounding dense aggregates of spermogonia, on the abaxial leaf surface. Teliospores were produced by sympodial proliferation from a basal sporogenous cell and mostly composed of three fertile cells subtended by two hygroscopic cysts. A germ slit was present near the septum in two of three fertile cells. A simple persistent pedicel was attached to the teliospore cysts. Because of resemblance in the teliospore morphology, this fungus was compared to Bibulocystis, Caetea, Cystomyces, and Spumula. Radially symmetric teliospores in the horizontal plane with three more or less globose fertile cells subtended by two hygroscopic cysts and the presence of a germ slit near the septum in fertile cells are the characteristics of the genus Cystomyces. Therefore, it was concluded that the fungus should be classified in Cystomyces, and a new name, C. antheropori was proposed.     

Study of a new group of bioregulators isolated from the greater plantain (Plantago major L.)

Proteins with physicochemical properties and biological activity similar to those of membrano-tropic homeostatic tissue-specific bioregulators that had been found earlier in various animal tissues were discovered in leaves of the common plantain (Plantago major L.). To study the specific activity of these plant proteins, we developed an experimental model for organotypic roller cultivation of newt (Pleurodeles waltl) skin tissue in vitro. We showed that the plant proteins of interest exert the wound-healing effect, which is characteristic of this plant, on the skin of vertebrates both in vitro and in vivo.     

Culture of newt cells from different tissues and their expression of a regeneration-associated antigen

We have established culture conditions for cells from normal limb, early limb regenerate (blastema), heart, and liver of the newt Notophthalmus viridescens. Whereas heart and liver cells had a relatively short life in culture, limb cells have shown no sign of senescence over more than 1 year in culture. Cultured cells from all these tissues express to differing extents the regeneration-associated antigen 22/18. The antigen is intracellular and filamentous, and its expression appears to be regulated by culture density. Furthermore, 22/18 antigen is turned off in limb and blastemal cultures following differentiation into muscle, as also occurs in vivo.     

Production of 4,15-diacetylnivalenol by <Emphasis Type="Italic">Fusarium Sambucinum</Emphasis> Fuckel var. <Emphasis Type="Italic">minus</Emphasis>

Fusarium sambucinum Fuckel var. minus isolate produced unusual for F. sambucinum Fuckel trichothecene metabolite 4,15-diacetylnivalenol (9 mg/l) in conditions of deep cultivation on Myro medium. This compound was identified by TLC, GLC, HPLC, and ¹H NMR spectroscopy. Other trichothecenes, 4-acetylnivalenol (3 mg/l) and nivalenol (1 mg/l), were also found in the culture. The observed feature of the studied isolate is assumed to be due to the presence of an additional gene, which encodes cytochrome P450 oxygenase responsible for the introduction of keto group at C-8 and hydroxyl group at C-7 of the trichothecene structure.     

Experimental histoplasmosis capsulati in athymic nude mice

Granuloma formation in nude (nu/nu) mice and their heterozygous littermates (nu/+ mice) against Histoplasma capsulatum var. capsulatum infection was studied.A culture of H. capsulatum var. capsulatum, isolated from a granuloma in the nasal cavity of a Japanese patient, was used in this experiment. Sixteen specific-pathogen-free male nu/nu and 32 nu/+ mice were used in this study.The nu/+ mice were divided into two groups. Sixteen nu/+ mice in one group and 16 nu/nu mice were inoculated intraperitoneally with 10⁶ yeast cells of the fungus, those in the other group of nu/+ mice were inoculated intravenously with the same number of the yeast cells. Two mice out of each group were sacrificed 2, 3, 7, 11, 14, 18, 25 and 30 days after inoculation, and each of their organs was examined histopathologically. In addition, pieces of these tissues were cultured on Sabouraud's dextrose agar slants.In the nu/+ mice inoculated intraperitoneally, although the fungus was recovered from the spleen, kidney and lymph nodes during the initial course of the infection, lesions were not detected in their histopathological sections. In the nu/+ mice inoculated intravenously, colonies were recovered from all of the organs examined, other than the brain and thymus, 7 days after inoculation.Histopathologically, a few microfoci consisting chiefly of mononuclear cells with or without yeast cells were found in the liver sections 4 days after inoculation. Seven and 11 days after inoculation the number of lesions had increased. They had large accumulations of mononuclear cells. From day 14 on, almost all of the yeast cells had lost most of their staining affinity or were destroyed in the granuloma. From day 25 on, the granulomatous lesions changed gradually to fibrous tissue.In the nu/nu mice the fungus was readily recovered from the spleen, liver, kidney and lymph nodes. Histopathologically, a few microfoci consisting of mononuclear cells were present in the liver sections 4 days after inoculation. That is to say, during the initial course of infection granulomas were formed. In the liver, from day 7 on, the lesions were large and their number increased. However, there was a definite difference between the nu/nu and nu/+ mice. In the former, the yeast cells were not killed, and they continued to multiply within the granulomas. These granulomas were never transformed into fibrous tissue.     

The fungus Phoma multirostrata is a host-specific pathogen and a potential biocontrol agent for a broadleaf weed

Since the increasing prevalence of herbicide-resistant weeds and herbicide bans, the use of biological controls with mycoherbicides become an innovative approach of weed control. In this study, we verified the pathogenicity of Phoma multirostrata TBRC 12769 against the common weed in Thailand, tridax daisy (Tridax procumbens), with its mechanism of infection unveiled by fluorescence microscopy. P. multirostrata directly penetrated through epidermal cells, stomata, and trichomes at 48 h post-inoculation. The hyphae also propagated in the lumen of the trichome, enabling the fungus to grow subcuticular to neighboring weed tissues at the bases of leaf trichomes. The necrotic pattern emerged around the trichome. During necrosis, unicellular chlamydospores were also detected inside the leaf trichomes, suggesting an overwintering stage under stress and nutrient-depleting conditions. Trichomes of weed leaves were found to be key infection sites for pathogenesis. Topical application of conidial suspension on T. procumbens potted plants led to 60–98% and 65 and 87% disease incidence under laboratory and greenhouse conditions, respectively, on days 15–20 post-inoculation. The 16-h dew period incubation results in a sharp increase by 37% in the pathogenicity rate. The greenhouse trials verified that the fungus is non-pathogenic to eight crops. Our LC-MS analysis indicated that norharman, a known bioherbicidal compound, and other compounds were detected in the supernatant fraction of fungal culture, of which resulted in a blight symptom on T. procumbens leaves. This study demonstrated that the P. multirostrata isolate is an effective mycoherbicide for this broadleaf weed.     

Synthetic bioregulators of poly-cis carotenoid biosynthesis

Seventeen new bioregulators were synthesized and tested for their ability to induce the biosynthesis of poly-cis carotenes in the flavedo of Marsh white seedless grapefruit. The effects of these new bioregulators are the same as that of the previously reported dibenzylamines, but several of the new compounds are more effective and cause the accumulation of up to 162 μg/g dry wt of poly-cis carotenes in the flavedo as compared to the maximum of 74 μg/g dry wt observed previously. The compounds tested were substituted N-benzyl furfurylamines, N-benzyl, N-methyl furfurylamines and N-alkyl, N-methyl benzylamines. They demonstrate the ability of tertiary as well as secondary amines to stimulate the formation of poly-cis carotenes. The interaction of N-(4-bromobenzyl) furfurylamine, one of the more effective of the new compounds, with lycopene and β-carotene inducers is also reported.