<Emphasis Type="Italic">In vitro</Emphasis> shoot induction and plant regeneration from flower buds in <Emphasis Type="Italic">Paphiopedilum</Emphasis> orchids

Paphiopedilum species are recalcitrant in tissue culture, and no explant from mature plants has been successfully mass propagated in vitro. This study was aimed at inducing shoots and regenerating plants from the flowering plants of a sequentially flowering Paphiopedilum Deperle and a single floral Paphiopedilum Armeni White. By using cross-sectioned flower buds (FBs), we found that in both species, only sections that contained the base tissue of FBs were able to produce shoots and plants. We have also found that sections of FBs between 1.5 and 3.0 cm from Paphiopedilum Deperle were able to produce shoots, but only sections of FBs >2.5 cm from Paphiopedilum Armeni White were regenerable. Our microscopic observations revealed that the small bract at the FB base harbored a new miniature FB, which further harbored a primitive FB with dome-shaped meristem-like tissues that presumably led to the plant induction. The reiteration of this pattern resulted in a scorpioid cyme inflorescence architecture in the multifloral Paphiopedilum species, and its failure to reiterate resulted in a single flower. The induction rates were 57–75%, and all plants survived in a greenhouse. This method is potentially applicable for the micropropagation and conservation of slipper orchids.     

Evaluation of Pathological Effects in Broilers During Fumonisins and Clays Exposure

This study was conducted to evaluate the possible protector effect of bentonite and zeolite in Bovans chicks fed a diet containing 59 mg kg(-1) of fumonisin B1 (FB1) during 3 weeks. A total of 200 one-day-old male chicks were treated varying the amount of bentonite and zeolite. Chick weight was registered weekly. At the end of the experiment, all the chicks were killed, and the livers were analyzed for gross examination and histopathological changes. Plasmatic activity of alanine amino transferase and aspartate amino transferase (AST) were also determined. Sphinganine and the sphinganine-to-sphingosine ratio in serum were evaluated. Both, bentonite and zeolite showed a protector effect against FB1 adsorption in the digestive tract of chicks. Chicks fed with FB1-contaminated feed, amended either with zeolite or bentonite, were heavier, and no macroscopic lesions were observed in the livers. AST activity might be considered as an indicator for FB1 exposition because AST levels were affected when only FB1 was present in the basal diet. These results indicate that both, zeolite and bentonite can be added into feed to diminish the effects of FB1.     

Exclusive rewards in mutualisms: ant proteases and plant protease inhibitors create a lock–key system to protect Acacia food bodies from exploitation

Myrmecophytic Acacia species produce food bodies (FBs) to nourish ants of the Pseudomyrmex ferrugineus group, with which they live in an obligate mutualism. We investigated how the FBs are protected from exploiting nonmutualists. Two‐dimensional gel electrophoresis of the FB proteomes and consecutive protein sequencing indicated the presence of several Kunitz‐type protease inhibitors (PIs). PIs extracted from Acacia FBs were biologically active, as they effectively reduced the trypsin‐like and elastase‐like proteolytic activity in the guts of seed‐feeding beetles (Prostephanus truncatus and Zabrotes subfasciatus), which were used as nonadapted herbivores representing potential exploiters. By contrast, the legitimate mutualistic consumers maintained high proteolytic activity dominated by chymotrypsin 1, which was insensitive to the FB PIs. Larvae of an exploiter ant (Pseudomyrmex gracilis) taken from Acacia hosts exhibited lower overall proteolytic activity than the mutualists. The proteases of this exploiter exhibited mainly elastase‐like and to a lower degree chymotrypsin 1‐like activity. We conclude that the mutualist ants possess specifically those proteases that are least sensitive to the PIs in their specific food source, whereas the congeneric exploiter ant appears partly, but not completely, adapted to consume Acacia FBs. By contrast, any consumption of the FBs by nonadapted exploiters would effectively inhibit their digestive capacities. We suggest that the term ‘exclusive rewards’ can be used to describe situations similar to the one that has evolved in myrmecophytic Acacia species, which reward mutualists with FBs but safeguard the reward from exploitation by generalists by making the FBs difficult for the nonadapted consumer to use.     

Accumulation of fumonisins B1 and B2 in freshly harvested Brazilian commercial maize at three locations during two nonconsecutive seasons

Fifty-six Brazilian commercial maize cultivars were examined for FB1 and FB2 accumulation after two non-consecutive growing seasons. During the 94/95 growing season 35 cultivars were planted at three locations in the state of S?o Paulo, Brazil. All samples (total of 105) were contaminated (0.10 micro/g-6.58 microg/g FB1 and 0.04 microg/g-2.15 microg/g FB2). During the 97/98 growing season, 8 of the cultivars used during 94/95 and 21 others were replanted at the same locations. All 87 samples were contaminated (1.15 microg/g-43.80 microg/g FB1 and 0.08 microg/g-11.65 microg/g FB2). One cultivar accumulated significantly less fumonisins in all locations during both growing seasons, indicating that some degree of selection may be possible even in climates that favor F. moniliforme (verticillioides) infection of maize. The presence of water surplus in soil from kernel maturity to harvest correlated with concentrations of FB1 in the grain for the 8 cultivars planted during both seasons at three locations. Observed trends indicated that water excesses and deficits from silking to harvest increased fumonisin levels. The difference in the incidence of FB1, FB2, and FB1 + FB2 was significant between growing seasons, planting locations and between cultivars. Neither the level of hybridization, nor the type of endosperm, nor the length of the vegetative cycle showed any effect on the FB1 contamination.     

Anatomy, ultrastructure and chemical composition of food bodies of Hovenia dulcis (Rhamnaceae)

Background and Aims

Food bodies (FBs) are structures that promote mutualism between plants and ants, which help protect them against herbivores. The present study aims to describe the anatomical organization, ultrastructure and chemical composition of the FBs in Hovenia dulcis, which represent the first structures of this type described in Rhamnaceae.

Methods

Leaves in various stages of development were collected and fixed for examination under light, transmission and scanning electron microscopy. Samples of FBs were subjected to chemical analysis using thin-layer chromatography and nuclear magnetic resonance of ¹H and ¹³C.

Key Results

The FBs vary from globose to conical and are restricted to the abaxial leaf surface, having a mixed origin, including epidermis and parenchyma. The FB epidermis is uniseriate, slightly pilose and has a thin cuticle. The epidermal cells are vacuolated and pigments or food reserves are absent. The parenchyma cells of immature FBs have dense cytoplasm showing mitochondria, endoplasmic reticulum and plastids. Mature FB cells store oils, which are free in the cytosol and occupy a large portion of the cell lumen. In these cells the plastids accumulate starch.

Conclusions

The lipids present in FBs are glycerin esters characteristic of plant energy reserves. Ants were observed collecting these FBs, which allows us to infer that these structures mediate plant–ant interactions and can help protect the young plants against herbivores, as these structures are prevalent at this developmental stage.Key words: Ant–plant interactions, cell ultrastructure, food body, Hovenia dulcis, lipid, myrmecophily, Rhamnaceae     

Fumonisins production by Fusarium proliferatum strains isolated from asparagus crown

The present work deals with the capability for producing fumonisin by Fusarium proliferatum strains isolated from asparagus in China. Fifty of F. proliferatum strains were randomly selected and incubated on cultures of maize grain and asparagus spear, respectively. Fumonisin levels (FB₁ and FB₂) were determined by high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The results showed that all 50 strains produced fumonisins in maize culture within a wide range of concentrations, 10–11,499 μg/g and 2–6,598 μg/g for FB₁ and FB₂, respectively. On culture of asparagus spear,48 strains (96%) produced fumonisins in the range 0.2–781.6 μg/g and no detected to 40.3 μg/g for FB₁ and FB₂, respectively. All of F. proliferatum strains produced much higher levels of FB₁, FB₂ and total fumonisins (FB₁ + FB₂) in maize grain culture than in asparagus spear culture. Meanwhile, fumonisin B₃ (FB₃) was identified in all maize culture extracts and most of asparagus spear culture extracts. This is the first study carried out the fumonisin-producing ability of F. proliferatum strains isolated from asparagus in China. The information obtained is useful for assessing the risk of fumonisins contamination in asparagus spear. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Fiber-optic immunosensor for mycotoxins

Evanescent wave-based fiber-optic immunosensors were studied for the detection of fumonisins and aflatoxins in maize. Two formats, competitive and non-competitive, were used. A competitive format was used to measure fumonisin B1 (FB1) in both spiked and naturally contaminated maize samples. Fumonisin monoclonal antibodies were covalently coupled to an optical fiber and the competition between FB1 and FB1 labeled with fluorescein (FB1-FITC) for the limited number of binding sites on the fiber was assessed. The signal generated in the assay was inversely proportional to the FB1 concentration. For samples, the concentration causing an inhibition of binding by 50% (IC50) was dependent upon the clean-up procedure used. Simple dilution of methanolic maize extracts yielded an assay with an IC50 equivalent to 25 microg FB1 g(-1) maize with a limit of detection of 3.2 microg g(-1) maize. Affinity column clean-up yielded an assay with an IC50 equivalent to 5 microg FB1 g(-1) maize (limit of detection 0.4 microg FB1 g(-1)). An HPLC method and the immunosensor method agreed well for naturally contaminated maize samples except when large amounts of other fumonisins that cross-react with the immunosensor were present. The second sensor format, for the mycotoxin aflatoxin B1 (AFB1), was a non-competitive assay using the native fluorescence of this mycotoxin. Because the fluorescence of AFB1 itself was detected, the response of the sensor was directly proportional to the toxin concentration. The sensor, while capable of detecting as little as 2 ng ml(-1) of AFB1 in solution was technically not an immunosensor, since the attachment of aflatoxin specific antibodies was not required. Sensors of the formats described have the potential to rapidly screen individual maize samples but require coupling with a clean-up technique to be truly effective.     

Maternal fumonisin exposure and risk for neural tube defects: mechanisms in an in vivo mouse model

BACKGROUND: Fumonisin B1 (FB1) is a mycotoxin produced by the fungus Fusarium verticillioides, a common contaminant of corn worldwide. FB1 disrupts sphingolipid biosynthesis by inhibiting the enzyme ceramide synthase, resulting in an elevation of free sphingoid bases and depletion of downstream glycosphingolipids. A relationship between maternal ingestion of FB1-contaminated corn during early pregnancy and increased risk for neural tube defects (NTDs) has recently been proposed in human populations around the world where corn is a dietary staple. The current studies provide an in vivo mouse model of FB1 teratogenicity. METHODS: Pregnant LM/Bc mice were injected with increasing doses of FB1 on GD 7.5 and 8.5, and exposed fetuses were examined for malformations. Sphingolipid profiles and (3)H-folate concentrations were measured in maternal and fetal tissues. Immunohistochemical expression of the GPI-anchored folate receptor (Folbp1) and its association with the lipid raft component, ganglioside GM1, were characterized. Rescue experiments were performed with maternal folate supplementation or administration of gangliosides. RESULTS: Maternal FB1 administration (20 mg/kg of body weight) during early gestation resulted in 79% NTDs in exposed fetuses. Sphingolipid profiles were significantly altered in maternal and embryonic tissues following exposure, and (3)H-folate levels and immunohistochemical expression of Folbp1 were reduced. Maternal folate supplementation partially rescued the NTD phenotype, whereas GM1 significantly restored folate concentrations and afforded almost complete protection against FB1-induced NTDs. CONCLUSIONS: Maternal FB1 exposure altered sphingolipid metabolism and folate concentrations in LM/Bc mice, resulting in a dose-dependent increase in NTDs that could be prevented when adequate folate levels were maintained.     

Simple method for the simultaneous isolation and determination of fumonisin B1 and its metabolite aminopentol-1 in swine liver by liquid chromatography--fluorescence detection

An analytical method based on high-performance liquid chromatography (HPLC) combined with fluorescence detection (FL) has been developed for the simultaneous determination of fumonisin B1 (FB1) and its totally hydrolized metabolite aminopentol-1 (AP1) in pig liver. The sample preparation is based on a single solid phase extraction (SPE). o-Phthalaldehyde (OPA) was used for pre-column derivatization before the programmed reversed-phase analysis on phenylhexyl column. The developed method shows good repeatibility for inter- and intra-day precision as well as adequate linearity of calibration curves (r2 was 0.9855 for FB1 and 0.9831 for AP1). Average recoveries from the matrix were 93.6% for FB1 and 95.3% for AP1. The limit of quantification (LOQ) in swine liver was 75 microg/kg for FB1 and 42 microg/kg for AP1.     

Plants feed ants: food bodies of myrmecophytic Piper and their significance for the interaction with Pheidole bicornis ants

Several species of Piper (Piperaceae) live in symbiosis with Pheidole bicornis (Formicidae-Myrmicinae) on the southern Pacific slope of Costa Rica. These plants produce small single-celled food bodies (FBs) in leaf domatia, formed by the petiole bases and roofing leaf sheaths. In the present study the dependency of ants on FBs of Piper fimbriulatum as a food source was analysed by comparing the natural abundance of ¹³C and ¹⁵N in ants and FBs. Both '¹³C and '¹⁵N values were very similar between FBs and Pheidole bicornis ants but differed substantially between the plant and other ant species. Therefore we suggest that FBs are a main food source for Pheidole bicornis ants. To strengthen this suggestion, the chemical composition of FBs of four myrmecophytic Piper species was analysed, with special emphasis on the nutritional requirements of inhabiting Pheidole bicornis ants. Standard chemical methods were modified and combined to a novel analysis scheme by which all major FB constituents could be quantified from minute [3-10 mg dry mass (DM)] quantities. Piper FBs mainly consisted of lipids (41-48% of DM) and proteins (17-24% of DM). Soluble carbohydrates and amino acids proved to be quantitatively unimportant. N was predominantly stored as soluble protein and, thus, was easily available to the ants. FBs proved to be a high-energy food source (up to 23 kJ g^-1 DM), with a chemical composition that meets well the nutritional needs of the inhabiting ants.     

Role of functional blocks in the evolution of exotrophy in vertebrates (as exemplified by fish and mammals)

The review reports the data on the presence in fish and mammals of the identical elementary or universal functional blocks (FBs)—molecules and supramolecular complexes providing exotrophic processes, with a special focus on those involved in the regulation of feeding behavior, digestion and transport of nutrients. When describing FBs implementing symbiotic digestion and induced autolysis, the data on the bacterial and mammalian enzymes have been analyzed. It is assumed that the structural and functional similarity of FBs in different organisms, which maintains the existence of trophic networks, is based on the need to perform similar functions and hence to develop similar mechanisms of FB adaptation in organisms phylogenetically distant from each other (bacteria, invertebrates, fish and mammals).     

FUM cluster divergence in fumonisins-producing Fusarium species

Fumonisins are polyketide-derived mycotoxins, produced by several Fusarium species, and its biosynthetic pathway is controlled by the FUM cluster--a group of genes exhibiting a common expression pattern during fumonisin biosynthesis. The most common are the B analogues with fumonisin B(1) (FB(1)) being the most prevalent. At least a part of the inter- and intraspecific variation in FBs synthesis level can be explained by the sequence differences inside FUM cluster. The aim of our study was to evaluate the toxin production and sequence variability in FUM genes and intergenic regions among thirty isolates of seven species reported as potential fumonisins producers: Fusarium anthophilum, Fusarium fujikuroi, Fusarium nygamai, Fusarium oxysporum, Fusarium proliferatum, Fusarium subglutinans and Fusarium verticillioides, particularly with respect to FBs synthesis. Fumonisins were produced in high amounts (over 1mg g(-1)) by one isolate of F. subglutinans, three of F. verticillioides and all F. proliferatum isolates except one, regardless of the host organism. The remaining isolates produced low amounts of FBs and two F. verticillioides isolates didn't produce it at all. The lowest variation in amount of toxin produced was found among F. proliferatum isolates. Based on the translation elongation factor 1α (tef-1α) sequence of F. fujikuroi, a species-specific marker was developed. The intergenic region presents similar opportunity for F. nygamai identification. The phylogenetic reconstruction based on FUM1 gene generally reflects the scenario presented by tef-1α sequences. Although the sequence similarities for intergenic regions were lower than in coding regions, there are clearly conserved patterns enabling separation of different subsets of species, including the non-producer species.     

Effects of fluid shear stress on adventitial fibroblast migration: implications for flow-mediated mechanisms of arterialization and intimal hyperplasia

The involvement of vascular fibroblasts (FBs) and smooth muscle (SM)-like cells in physiological and pathological processes in large vessels (intimal hyperplasia) and microvessels (capillary arterialization), and the realization that these cells are exposed to interstitial flow shear stress (SS), motivate this study of SS on FB migratory activity. Rat adventitial FBs were grown to either 30-50% confluence (subconfluent FBs; SFBs) or full confluence (confluent FBs; CFBs) in culture. Immunofluorescence and Western blotting assays were conducted to evaluate the expression of two phenotype markers: SM alpha-actin and SM myosin heavy chain (MHC). Both assays indicated a significant increase in SM alpha-actin expression in CFBs compared with SFBs, suggesting a phenotype difference between the two cell populations. SFBs and CFBs both expressed minimal SM MHC. Both cell populations were seeded on Matrigel-coated cell culture inserts and exposed to 4 h of either 1 or 20 dyn/cm(2) SS via a rotating disk apparatus in the presence of the chemoattractant platelet-derived growth factor-BB to quantify the effect of SS on SFB and CFB migration. Four hours of 20 dyn/cm(2) SS significantly enhanced SFB migration while it suppressed CFB migratory activity. Four hours of 1 dyn/cm(2) SS did not significantly alter either SFB or CFB migration levels. Because of the distinct migratory responses of SFBs and CFBs in response to SS, phenotype modulation appears to be one way to regulate their involvement in both physiological and pathological remodeling processes.     

Histopathological changes in avian kidney caused by Bothrops insularis (jararaca ilh a) venom and a phospholipase A2-containing fraction

The histopathological changes induced in avian kidney by the intramuscular injection of Bothrops insularis (jararaca ilh a) venom and its phospholipase A2 (PLA2)-containing fraction were examined. Acute experiments (3 h and 24 h) with B. insularis crude venom (20 microg and 80 microg) or its PLA2-contaning fraction (10 microg and 40 microg) resulted in significant structural damage to the kidneys of 5-12-day-old chicks. Histopathological analysis indicated that the venom and its fraction acted on the renal tubules and glomeruli. The morphological changes, although widespread, varied in intensity from cell to cell, and from tubule to tubule in venom-injected chicks. The tubular and glomerular changes produced by the venom and its PLA2-containing fraction may be the result of a direct cytotoxic effect potentiated by ischemia-related disturbances in the regional hemodynamics. The venom and its fraction affected more segments along reptilian-type nephrons than along mammalian ones. This divergent sensitivity to the venom and its fraction may reflect the species-specific characteristics of B. insularis snake, an example of geographical isolation influencing its diet which is almost exclusively avian.     

The effect of heavy metal accumulation on metallothionein content in selected tissues of bank voles and yellow-necked mice caught near a steelworks and zinc smelter

The effect of cadmium, zinc, and copper accumulation on metallothionein content in the selected tissues of bank voles and yellow-necked mice trapped near the Sendzimir Steelworks in Krakow and the zinc smelter in Bukowno were analysed. The Borecka Forest was chosen as a control area. The highest cadmium concentration, 32.98 microg g(-1) dry weight, was detected in the kidneys of the bank voles caught in the Bukowno area. Zinc and copper concentrations in the tissues did not exceed the critical values. Metallothionein content in the liver and kidneys was associated with heavy metal accumulation in the tissues. The highest content of sulphydryl groups was detected in the livers of the bank voles trapped within the neighbourhood of the zinc smelter in Bukowno. The highest level of disulphide bonds was found in the kidneys of the bank voles from the same area.     

A fumonisin biosynthetic gene cluster in Fusarium oxysporum strain O-1890 and the genetic basis for B versus C fumonisin production

Most species of Fusarium that produce fumonisin mycotoxins produce predominantly B fumonisins (FBs). However, Fusarium oxysporum strain O-1890 produces predominantly C fumonisins (FCs). In this study, the nucleotide sequence of the fumonisin biosynthetic gene (FUM) cluster in strain O-1890 was determined. The order and orientation of FUM genes were the same as in the previously described clusters in Fusarium verticillioides and Fusarium proliferatum. Coding regions of F. oxysporum and F. verticillioides FUM genes were 88-92% identical, but regions flanking the clusters did not share significant identity. The FUM cluster gene FUM8 encodes an alpha-oxoamine synthase, and fum8 mutants of F. verticillioides do not produce fumonisins. Complementation of a fum8 mutant with the F. verticillioidesFUM8 restored FB production. Complementation with F. oxysporumFUM8 also restored production, but the fumonisins produced were predominantly FCs. These data indicate that different orthologues of FUM8 determine whether Fusarium produces predominantly FBs or FCs.     

Rapid intensification of an established CHO cell fed-batch process

Currently, the mammalian biomanufacturing industry explores process intensification (PI) to meet upcoming demands of biotherapeutics while keeping production flexible but, more importantly, as economic as possible. However, intensified processes often require more development time compared with conventional fed-batches (FBs) preventing their implementation. Hence, rapid and efficient, yet straightforward strategies for PI are needed. In this study we demonstrate such a strategy for the intensification of an N-stage FB by implementing N-1 perfusion cell culture and high inoculum cell densities resulting in a robust intensified FB (iFB). Furthermore, we show successful combination of such an iFB with the addition of productivity enhancers, which has not been reported so far. The conventional CHO cell FB process was step-wise improved and intensified rapidly in multi-parallel small-scale bioreactors using N-1 perfusion. The iFBs were performed in 15 and 250 ml bioreactors and allowed to evaluate the impact on key process indicators (KPI): the space–time yield (STY) was successfully doubled from 0.28 to 0.55 g/L d, while product quality was maintained. This gain was generated by initially increasing the inoculation density, thus shrinking process time, and second supplementation with butyric acid (BA), which reduced cell growth and enhanced cell-specific productivity from ~25 to 37 pg/(cell d). Potential impacts of PI on cell metabolism were evaluated using flux balance analysis. Initial metabolic differences between the standard and intensified process were observed but disappeared quickly. This shows that PI can be achieved rapidly for new as well as existing processes without introducing sustained changes in cellular and metabolic behavior.     

Determination of scutellarin in mouse plasma and different tissues by high-performance liquid chromatography

A simple HPLC-UV method was developed for the determination of scutellarin in plasma and different tissues of mice (heart, liver, spleen, lungs and kidneys). The separation was achieved by HPLC on a Hypersil C(18) column with a mobile phase composed of methanol-water-glacial acetic acid (40:60:1). UV detection was used at 335 nm. The calibration curves were linear in all matrices (r(2)>0.997) in the concentration range of 0.1-10 microg/ml for plasma and 0.1-20 microg/g for tissue homogenates, respectively. The method described is suitable for studies on the distribution of scutellarin in different tissues of mice.