In vitro effects of undifferentiated callus extracts from Plantago major L, Rhodiola rosea L and Silybum marianum L in normal and malignant human skin cells.

Background and objectives: The occurrence of non-melanoma and melanoma skin cancers is currently increasing rapidly with one in every three cancers diagnosed as a skin cancer. A useful strategy to control the progression of skin cancer could be the use of plant flavonoids that suppress pro-inflammatory cytokines involved in tumor initiation and progression. In this study, the anti-inflammatory and antioxidant activity of undifferentiated callus extracts from Plantago major L, Silybum marianum L and Rhodiola rosea L was investigated both in normal and malignant skin cells. Methods: Antioxidant activity of the extracts was analyzed by using the Trolox Equivalent Antioxidant Capacity (TEAC) assay. High-Performance Thin-Layer Chromatography (HPTLC) was performed to demonstrate the phytochemical profile, and the total flavonoid content was analyzed with an aluminum chloride colorimetric method. The anti-inflammatory effect was investigated by cell treatments using the plant extracts. Thereafter, the possible suppression of induced IL-6 response was measured from the cultured skin cancer cell lines A2058 and A431, and normal primary keratinocytes with Enzyme-Linked Immunosorbent Assay (ELISA). Results: The HPTLC analysis assessed that the extracts contained a complex phytochemical profile that was rich in phenolic and flavonoid compounds. Dose response assays showed that concentrations between 15 and 125 µg/mL of all three plant extracts could be used to investigate an effect on the IL-6 production. The S. marianum extract had the most pronounced anti-inflammatory effect, which significantly inhibited induced IL-6 production in both normal keratinocytes and skin cells derived from epidermal carcinoma. The extract from S. marianum also had the highest flavonoid content and showed the highest antioxidant activity of the three extracts tested. Conclusion: All in all, we have confirmed that undifferentiated callus extracts of S. marianum possess properties such as antioxidant and anti-inflammatory activities in both normal and malignant keratinocytes, and thus could be a promising agent controlling the pro-inflammatory IL-6 production.

Gene expression changes during short day induced terminal bud formation in Norway spruce.

The molecular basis for terminal bud formation in autumn is not well understood in conifers. By combining suppression subtractive hybridization and monitoring of gene expression by qRT-PCR analysis, we aimed to identify genes involved in photoperiodic control of growth cessation and bud set in Norway spruce. Close to 1400 ESTs were generated and their functional distribution differed between short day (SD-12 h photoperiod) and long day (LD-24 h photoperiod) libraries. Many genes with putative roles in protection against stress appeared differentially regulated under SD and LD, and also differed in transcript levels between 6 and 20 SDs. Of these, PaTFL1(TERMINAL FLOWER LIKE 1) showed strongly increased transcript levels at 6 SDs. PaCCCH(CCCH-TYPE ZINC FINGER) and PaCBF2&3(C-REPEAT BINDING FACTOR 2&3) showed a later response at 20 SDs, with increased and decreased transcript levels, respectively. For rhythmically expressed genes such as CBFs, such differences might represent a phase shift in peak expression, but might also suggest a putative role in response to SD. Multivariate analyses revealed strong differences in gene expression between LD, 6 SD and 20 SD. The robustness of the gene expression patterns was verified in 6 families differing in bud-set timing under natural light with gradually decreasing photoperiod.

In vitro protective effects of Paeonia mascula subsp. hellenica callus extract on human keratinocytes.

Natural ingredients have been used to improve the state of health in humans. The genus Paeonia has been studied only limited yet it's reported to have many activities such as antioxidant and anti-inflammatory. To this context, here we focused on an endemic Paeonia species in Attica. This study aims to present the development of the Paeonia mascula subsp. hellenica callus extract and its pleiotropic bioactivity on human primary keratinocytes exploring its potential application as an active agent in skin-related products. This extract showed a high scavenging activity with high phenolic content and an interesting metabolic profile. At a molecular level, the study on the transcript accumulation of genes revealed that this extract exhibits in vitro skin-related protection properties by mediating mitochondrial energy, cell proliferation, immune and inflammatory response and positively regulates genes involved in epidermal and in stratum corneum function. Besides, the extract is proven not skin irritant on reconstructed human skin model. These findings indicate that the specific P. mascula subsp. hellenica extract possesses significant in vitro protection activity on human epidermis and provides new insights into its beneficial role in skin confirming that the advent of biotechnology contribution the past few decades.

Identification of PaCOL1 and PaCOL2, two CONSTANS-like genes showing decreased transcript levels preceding short day induced growth cessation in Norway spruce.

In woody plants of the temperate zone short photoperiod (SD) leads to growth cessation. In angiosperms CONSTANS (CO) or CO-like genes play an important role in the photoperiodic control of flowering, tuberisation and shoot growth. To investigate the role of CO-like genes in photoperiodic control of shoot elongation in gymnosperms, PaCOL1 and PaCOL2 were isolated from Norway spruce. PaCOL1 encodes a 3.9kb gene with a predicted protein of 444 amino acids. PaCOL2 encodes a 1.2kb gene with a predicted protein of 385 amino acids. Both genes consist of two exons and have conserved domains found in other CO-like genes; two zinc finger domains, a CCT and a COOH domain. PaCOL1 and PaCOL2 fall into the group 1c clade of the CO-like genes, and are thus distinct from Arabidopsis CO that belongs to group 1a. Transcript levels of both PaCOL-genes appear to be light regulated, an increasing trend was observed upon transition from darkness to light, and a decreasing trend during darkness. The increasing trend at dawn was observed both in needles and shoot tips, whereas the decreasing trend in darkness was most prominent in shoot tips, and limited to the late part of the dark period in needles. The transcript levels of both genes decreased significantly in both tissues under SD prior to growth cessation and bud formation. This might suggest an involvement in photoperiodic control of shoot elongation or might be a consequence of regulation by light.

Polyphenol-hydrogen peroxide reactions in skin: In vitro model relevant to study ROS reactions at inflammation.

Antioxidants are important to protect and maintain biological barriers, such as the skin. Antioxidant effects are often assessed using clinical trials, however these tests are costly and time consuming. In this work we introduce a skin membrane-covered oxygen electrode (SCOE) as an in vitro tool for monitoring H2O2 and antioxidant reactions in skin. The SCOE gives amperometric response to H2O2 concentrations down to 0.05 mM. More importantly, the electrode allows measurements of polyphenol penetration and reaction with H2O2 in skin. Measurements with SCOE show that lipophilic polyphenols such as quercetin, piceatannol, resveratrol, and plant extract from Plantago major impose their antioxidant effect in skin within 2-20 min. Rutin is however too hydrophilic to penetrate into stratum corneum and therefore cannot deliver its antioxidant effect during similar time interval. The measurements are interpreted considering polyphenol partition-penetration through stratum corneum and the reaction with the H2O2-catalase system in the skin. The contribution of other enzymes will be addressed in the future.

Genetics of avirulence genes in Blumeria graminis f.sp. hordei and physical mapping of AVR(a22) and AVR(a12).

Powdery mildew fungi are parasites that cause disease on a wide range of important crops. Plant resistance (R) genes, which induce host defences against powdery mildews, encode proteins that recognise avirulence (AVR) molecules from the parasite in a gene-for-gene manner. To gain insight into how virulence evolves in Blumeria graminis f.sp. hordei, associations between segregating AVR genes were established. As a prerequisite to the isolation of AVR genes, two loci were selected for further analysis. AVR(a22) is located in a tightly linked cluster comprising AVR(a10) and AVR(k1) as well as up to five other AVR genes. The ratio between physical and genetic distance in the cluster ranged between 0.7 and 35 kB/cM. The AVR(a22) locus was delimited by the previously isolated gene AVR(a10) and two cleaved amplified polymorphic sequence (CAPS) markers, 19H12R and 74E9L. By contrast, AVR(a12) was not linked to other AVR genes in two crosses. Bulk segregant analysis of over 100,000 AFLP fragments yielded two markers, ETAMTG-285 and PAAMACT-473, mapping 10 and 2cM from AVR(a12), respectively, thus delimiting AVR(a12) on one side. All markers obtained for AVR(a12) mapped proximal to it, indicating that the gene is located at the end of a chromosome. Three more AVR(a10) paralogues were identified at the locus interspersed among genes for metabolic enzymes and abundant repetitive elements, especially those homologous to the CgT1 class of retrotransposons. The flanking and close markers obtained will facilitate the isolation of AVR(a22) and AVR(a12) and provide useful tools for studies of the evolution of powdery mildew fungi in agriculture and nature.

Intron splicing in 5' untranslated region of the rolA transcript in transgenic apple.

The rolA gene encoded on the Ri plasmid of Agrobacterium rhizogenes causes developmental alterations, including dwarfing characteristics in the transgenic plants. In an attempt to introduce dwarfing characteristics into apple rootstocks for breeding purposes, the rolA gene was incorporated into the apple rootstock M26 and obtained four transgenic clones. All the clones exhibited reduced growth compared to untransformed control plants but different degree of dwarfing and wrinkled leaves. In the present study, expression of the rolA gene was further investigated by analysing the structure of the rolA transcript and the levels of the rolA mRNAs from these clones. The nucleotide (nt) sequence of the rolA transcript showed two forms of the transcript: one, the unspliced form, was co-linear with the rolA sequence in the genomic DNA; the other was spliced mRNA in which an 85-base pair (bp) intron sequence in the 5' untranslated region (5'UTR) was spliced out. The position of splicing is different from that in Arabidopsis thaliana but similar to the splicing site found in tobacco. The transcription start region of the rolA gene in apple was 206bp upstream of that in Arabidopsis and 277bp upstream to Nicotiana tabacum transcription start. A hairpin-like secondary structure and an upstream open reading frame (uORF) were revealed in the rolA 5'UTR. The levels of the rolA mRNA in the apple transgenic clones were analysed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed slight variation in the shoot tissues of the transgenic clones.