Preferential expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato.

Root hairs as specialized epidermal cells represent part of the outermost interface between a plant and its soil environment. They make up to 70% of the root surface and, therefore, are likely to contribute significantly to nutrient uptake. To study uptake systems for mineral nitrogen, three genes homologous to Arabidopsis nitrate and ammonium transporters (AtNrt1 and AtAmt1) were isolated from a root hair-specific tomato cDNA library. Accumulation of LeNrt1-1, LeNrt1-2, and LeAmt1 transcripts was root-specific, with no detectable transcripts in stems or leaves. Expression was root cell type-specific and regulated by nitrogen availability. LeNrt1-2 mRNA accumulation was restricted to root hairs that had been exposed to nitrate. In contrast, LeNrt1-1 transcripts were detected in root hairs as well as other root tissues under all nitrogen treatments applied. Analogous to LeNrt1-1, the gene LeAmt1 was expressed under all nitrogen conditions tested, and root hair-specific mRNA accumulation was highest following exposure to ammonium. Expression of LeAMT1 in an ammonium uptake-deficient yeast strain restored growth on low ammonium medium, confirming its involvement in ammonium transport. Root hair specificity and characteristics of substrate regulation suggest an important role of the three genes in uptake of mineral nitrogen.

Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors.

Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 +/- 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with IL-12 cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional IL-12 gene transfections, could significantly inhibit systemic metastases, resulting in extended survival of test mice. These results suggest that gene gun-mediated in vivo delivery of IL-12 cDNA should be further developed for potential clinical testing as an approach for human cancer gene therapy.

An ultrastructural study of the relationship between the mite Floracarus perrepae Knihinicki & Boczek (Acariformes : Eriophyidae) and the fern Lygodium microphyllum (Lygodiaceae)

The ultrastructure of the mite Floracarus perrepae was investigated in relation to its host, Lygodium microphyllum, the Old World climbing fern. Floracarus perrepae has been suggested as a means of biological control for the fern, which is an aggressive weed in tropical areas. Feeding by the mite induces a change in the size of epidermal cells, and cell division is stimulated by mite feeding, causing the leaf margin to curl over into a roll with two to three windings. The enlarged epidermal layer greatly increases its cytoplasmic contents, which become a nutritive tissue for the mite and its progeny. Damage by the mite ultimately debilitates the fern. The structure and depth of stylet penetration by the mite, and the thickness of the epidermal cell wall of L. microphyllum, do not appear to account for the mite's differential ability to induce leaf rolling in its co-adapted host from south-east Queensland but not in the invasive genotype of the fern in Florida. F

Sunscreen penetration of human skin and related keratinocyte toxicity after topical application

Sunscreen skin penetration and safety assessment should be considered together in order to ensure that in vitro cytotoxicity studies examine relevant doses of these organic chemical UV filters to which viable epidermal cells are realistically exposed. In this study, we sought to determine whether sufficient topically applied sunscreens penetrated into human viable epidermis to put the local keratinocyte cell populations at risk of toxicity. The penetration and retention of five commonly used sunscreen agents ( avobenzone, octinoxate, octocrylene, oxybenzone and padimate O) in human skin was evaluated after application in mineral oil to isolated human epidermal membranes. Sunscreen concentration - human keratinocyte culture response curves were then defined using changes in cell morphology and proliferation ( DNA synthesis using radiolabelled thymidine uptake studies) as evidence of sunscreens causing toxicity. Following 24 h of human epidermal exposure to sunscreens, detectable amounts of all sunscreens were present in the stratum corneum and viable epidermis, with epidermal penetration most evident with oxybenzone. The concentrations of each sunscreen found in human viable epidermis after topical application, adjusting for skin partitioning and binding effects, were at least 5-fold lower, based on levels detected in viable epidermal cells, than those appearing to cause toxicity in cultured human keratinocytes. It is concluded that the human viable epidermal levels of sunscreens are too low to cause any significant toxicity to the underlying human keratinocytes. Copyright (C) 2005 S. Karger AG, Basel.

Postharvest infection of Freesia hybrida flowers by Botrytis cinerea

'Specking' on harvested freesia (Freesia hybrida) flowers is a problem worldwide. The disease is caused by the fungal pathogen Botrytis cinerea. This disease symptom detracts from appearance and reduces marketability of the flowers. Unlike other important cut flower crops (e.g. gerbera), the mode of infection and epidemiology of postharvest freesia flower specking caused by B. cinerea has not been reported. Epidemiological studies were carried out under simulated conditions typical of those occurring during postharvest handling of freesia flowers. Infection of freesia flowers by B. cinerea occurred when a conidium germinated, formed a germ tube(s) and penetrated epidermal cells. Fungal hyphae then colonised adjacent cells, resulting in visible lesions. Different host reactions were observed on freesia 'Cote d'Azur' petals at 20 degrees C compared to 5 degrees C. The infection process was relatively rapid at 20 degrees C, with visible lesions produced within 7 h of incubation. However, lesion expansion ceased after 24 h of incubation. Infection was slower at 5 degrees C, with visible lesions produced after 48 h of incubation. However, lesion development at 5 degrees C was continuous, with lesions expanding over 4 days. Light microscopy observations revealed increased host defence reactions during infection. These reactions involved production of phenolic compounds, probably lignin and/or callose, around infection sites. Such substances may play a role in restricting petal colonisation and lesion expansion. Disease severity and lesion numbers on freesia flowers incubated at 12 degrees C were higher, but not significantly higher (P > 0.05), than on those incubated at 20 degrees C. Disease severity and progression were differentially mediated by temperature and relative humidity (R. H.). Infection of freesia flowers was severe at 100% R. H. for all three incubation temperatures of 5, 12 and 20 degrees C. In contrast, no lesions were produced at 80 to 90% R. H. at either 5 or 20 degrees C.

Programmed cell death (PCD) processes begin extremely early in Alstroemeria petal senescence

-In the Liliaceous species Alstroemeria, petal senescence is characterized by wilting and inrolling, terminating in abscission 8-10 d after flower opening. -In many species, flower development and senescence involves programmed cell death (PCD). PCD in Alstroemeria petals was investigated by light (LM) and transmission electron microscopy (TEM) (to study nuclear degradation and cellular integrity), DNA laddering and the expression programme of the DAD-1 gene. -TEM showed nuclear and cellular degradation commenced before the flowers were fully open and that epidermal cells remained intact whilst the mesophyll cells degenerated completely. DNA laddering increased throughout petal development. Expression of the ALSDAD-1 partial cDNA was shown to be downregulated after flower opening. -We conclude that some PCD processes are started extremely early and proceed throughout flower opening and senescence, whereas others occur more rapidly between stages 4-6 (i.e. postanthesis). The spatial distribution of PCD across the petals is discussed. Several molecular and physiological markers of PCD are present during Alstroemeria petal senescence. © New Phytologist (2003).

Structural fruit coloration in Delarbrea michieana (Araliaceae)

The brilliant blue fruit color of Delarbrea michieana (F. Muell.) F. Muell. (Araliaceae), a Queensland understory rain forest tree, is caused by iridisomes (structures) in the epidermal cells that are produced beneath the cell wall and probably outside of the cytoplasm. Layers within these iridisomes are of such a thickness that they interfere constructively with light at 420–440 nm and produce the color. Such color production may aid in attracting mammals and large frugivorous birds (which may disperse the fruits) and may also allow ripe fruits to continue photosynthetic carbon assimilation.

Colour differentiation of high-lycopene tomato fruit through the addition of the colourless-epidermis (y) mutation

High-lycopene tomatoes (Solanum lycopersicum) are characterised by an intense red flesh-colour, due to an elevated concentration of the carotenoid, lycopene. However, this characteristic is only visible once fruit are cut open, making it impossible to differentiate intact high-lycopene fruit from standard tomato fruit, a clear market disadvantage. The reason that fruit colour of both high-lycopene and standard fruit looks almost identical from the outside is because tomato fruit normally contain the yellow flavonoid 'naringenin chalcone' in a thin layer of epidermal cells. It is this combination of naringenin chalcone and the underlying lycopene in the flesh that gives tomatoes their characteristic orange-red colour. By incorporation of the recessive colourless epidermis mutant allele 'y' (which prevents naringenin chalcone accumulation) into high-lycopene fruit, we have been able to create high-lycopene tomatoes (hp1.ogc.y) exhibiting a deep-pink colour visible from the outside. Hue angle of the skin of the high-lycopene 'y' mutant and a regular highlycopene tomato (hp1.ogc.Y) was 30 and 38°, respectively, while flesh values were similar at 31 and 32°, respectively. Removal of naringenin chalcone from the epidermis appeared to improve the visibility of underlying lycopene, such that fruit outer colour became a subsequent indicator of underlying flesh colour. The removal of epidermal pigmentation means that high-lycopene fruit can now be differentiated from standard tomato fruit in the market place without the need to cut fruit open.

Estudio molecular en dos hermanas afectadas por ictiosis congénita autosómica recesiva : descripción de una nueva mutación causal en TGM1

Se describe la variante homocigota c.320-2A>G de TGM1 en dos hermanas con ictiosis congénita autosómica recesiva. El clonaje de los transcritos generados por esta variante permitió identificar tres mecanismos moleculares de splicing alternativos.

Combining Xanthan And Chitosan Membranes To Multipotent Mesenchymal Stromal Cells As Bioactive Dressings For Dermo-epidermal Wounds.

The association between tridimensional scaffolds to cells of interest has provided excellent perspectives for obtaining viable complex tissues in vitro, such as skin, resulting in impressive advances in the field of tissue engineering applied to regenerative therapies. The use of multipotent mesenchymal stromal cells in the treatment of dermo-epidermal wounds is particularly promising due to several relevant properties of these cells, such as high capacity of proliferation in culture, potential of differentiation in multiple skin cell types, important paracrine and immunomodulatory effects, among others. Membranes of chitosan complexed with xanthan may be potentially useful as scaffolds for multipotent mesenchymal stromal cells, given that they present suitable physico-chemical characteristics and have adequate tridimensional structure for the adhesion, growth, and maintenance of cell function. Therefore, the purpose of this work was to assess the applicability of bioactive dressings associating dense and porous chitosan-xanthan membranes to multipotent mesenchymal stromal cells for the treatment of skin wounds. The membranes showed to be non-mutagenic and allowed efficient adhesion and proliferation of the mesenchymal stromal cells in vitro. In vivo assays performed with mesenchymal stromal cells grown on the surface of the dense membranes showed acceleration of wound healing in Wistar rats, thus indicating that the use of this cell-scaffold association for tissue engineering purposes is feasible and attractive.

Epidermal growth factor sensitizes cells to ionizing radiation by down-regulating protein mutated in ataxia-telangiectasia

Epidermal growth factor (EGF) has been reported to either sensitize or protect cells against ionizing radiation. We report here that EGF increases radiosensitivity in both human fibroblasts and lymphoblasts and downregulates both ATM (mutated in ataxia-telangiectasia (A-T)) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). No further radiosensitization was observed in A-T cells after pretreatment with EGF. The down-regulation of ATM occurs at the transcriptional level. Concomitant with the down-regulation of ATM, the DNA binding activity of the transcription factor Spl decreased. A causal relationship was established between these:observations by demonstrating that upregulation of Spl DNA binding activity by granulocyte/ macrophage colony-stimulating factor rapidly reversed the EGF-induced decrease in ATM protein and restored radiosensitivity to normal levels. Failure to radiosensitize EGF-treated cells to the same extent as observed for A-T cells ban be explained by induction of ATM protein and kinase activity with time post-irradiation, Although ionizing radiation damage to DNA rapidly activates ATM kinase and cell cycle checkpoints, we have provided evidence for the first time that alteration in the amount of ATM protein occurs in response to both EGF and radiation exposure. Taken together these data support complex control of ATM function that has important repercussions for targeting ATM to improve radiotherapeutic benefit.

T cell receptor specificity is critical for the development of epidermal gammadelta T cells.

A particular feature of gammadelta T cell biology is that cells expressing T cell receptor (TCR) using specific Vgamma/Vdelta segments are localized in distinct epithelial sites, e.g., in mouse epidermis nearly all gammadelta T cells express Vgamma3/Vdelta1. These cells, referred to as dendritic epidermal T cells (DETC) originate from fetal Vgamma3+ thymocytes. The role of gammadelta TCR specificity in DETC's migration/localization to the skin has remained controversial. To address this issue we have generated transgenic (Tg) mice expressing a TCR delta chain (Vdelta6.3-Ddelta1-Ddelta2-Jdelta1-Cdelta), which can pair with Vgamma3 in fetal thymocytes but is not normally expressed by DETC. In wild-type (wt) Vdelta6.3Tg mice DETC were present and virtually all of them express Vdelta6.3. However, DETC were absent in TCR-delta(-/-) Vdelta6.3Tg mice, despite the fact that Vdelta6.3Tg gammadelta T cells were present in normal numbers in other lymphoid and nonlymphoid tissues. In wt Vdelta6.3Tg mice, a high proportion of in-frame Vdelta1 transcripts were found in DETC, suggesting that the expression of an endogenous TCR-delta (most probably Vdelta1) was required for the development of Vdelta6.3+ epidermal gammadelta T cells. Collectively our data demonstrate that TCR specificity is essential for the development of gammadelta T cells in the epidermis. Moreover, they show that the TCR-delta locus is not allelically excluded.

Influence of epidermal growth factor on the maturation of fetal rat brain cells in aggregate culture. An immunocytochemical study.

Maturation of astrocytes, neurons, and oligodendrocytes was studied in serum-free aggregating cell cultures of fetal rat telencephalon by an immunocytochemical approach. Cell type-specific immunofluorescence staining was examined by using antibodies directed against glial fibrillary acidic protein (GFAP) and vimentin, two astroglial markers; neuron-specific enolase (NSE) and neurofilament (NF), two neuronal markers, and galactocerebroside (GC), an oligodendroglial marker. It was found that the cellular maturation in aggregates is characterized by distinct developmental increases in immunoreactivity for GFAP, vimentin, NSE, NF, and GC, and by a subsequent decrease of vimentin-positive structures in more differentiated cultures. These findings are in agreement with observations in vivo, and they corroborate previous biochemical studies of this histotypic culture system. Treatment of very immature cultures with a low dose of epidermal growth factor (EGF, 5 ng/ml) enhanced the developmental increase in GFAP, NSE, NF and GC immunoreactivity, suggesting an acceleration of neuronal and glial maturation. In addition, EGF was found to alter the cellular organization within the aggregates, presumably by influencing cell migration.

Epidermal growth factor (EGF) stimulation of cultured brain cells. I. Enhancement of the developmental increase in glial enzymatic activity.

Serum-free aggregating cell cultures of fetal rat telencephalon grown in the presence of 3 ng/ml (5 X 10(-10) M) epidermal growth factor (EGF) until day 12 showed 2- to 3-fold increased activities in the two glial enzymes, glutamine synthetase (GLU-S) and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). This effect was concentration-dependent, with maximal stimulation in cultures treated daily with 3 ng/ml EGF. Addition of EGF during the first 10 culture days was sufficient to produce a maximal stimulation of both GLU-S and CNPase on day 19, whereas treatments starting on day 12 were ineffective. The stimulation of GLU-S preceded that of CNPase. The EGF-induced increase in GLU-S activity was not directly dependent on the presence of insulin, triiodothyronine, or hydrocortisone in the medium, whereas insulin was required for the stimulation of CNPase. A single dose of 5 ng/ml EGF on day 2 caused a slight but significant decrease in DNA synthesis after day 6. The present results indicate that in serum-free aggregating cell cultures of fetal rat telencephalon EGF partially inhibits DNA synthesis, and stimulates an early step in glial differentiation.