Non-destructive insights into photosynthetic and photoprotective mechanisms in Arabidopsis thaliana grown under two light regimes.

Probing insights into understanding photosynthetic processes via non-invasive means has an added advantage when used in phenotyping or precision agriculture. We employed Raman spectroscopy and fluorescence-based methods to investigate both the changes in the photosynthetic processes and the underlying protective mechanisms on Arabidopsis thaliana wild-type (WT), and ros1, which is a mutant of a repressor of transcriptional gene silencing, both grown under low light (LL: 100 µmol m-2s-1) and high light (HL: 400 µmol m-2s-1) regimes. Raman imaging detected a lower carotenoid intensity after two weeks in those plants grown under HL, compared to those grown under the LL regime; we interpret this as the result of oxidative damage of ß-carotene molecules. Further, the data revealed a significant depletion in carotenoids with enhanced phenolics around the midrib and tip of the WT leaves, but not in the ros1. On the contrary, small necrotic zones appeared after two weeks of HL in the ros1 mutant, pointing to the starting oxidative damage. The lower maximum quantum yield of the photochemistry (Fv/Fm) in the WT as well as in the ros1 mutant grown in HL (compared to those in the LL two weeks post-exposure), indicates the HL partially inactivated photosystems. Chlorophyll a fluorescence imaging further showed high non-photochemical quenching (NPQ) in the plants grown under the HL regime for both the WT and the ros1 mutant, but the spatial heterogeneity of NPQ images was much higher in the HL-grown ros1 mutant. Fluorescence screening methods revealed significantly high values of chlorophyll proxies in the WT as well as in the ros1 mutant two weeks after in the HL compared to those under LL. The data generally revealed an increased accumulation of phenolics under HL in both the WT and ros1 mutant plants, but the proxies of anthocyanin and flavonols were significantly lower in the ros1 mutant than in the WT. The comparatively low accumulation of anthocyanin in the ros1 mutant compared to the WT supports the Raman data. We conclude that integrated use of these techniques can be efficiently applied for a better understanding of insights into photosynthetic mechanisms.

Interactive effects of nitrogen, UV and PAR on barley morphology and biochemistry are associated with the leaf C:N balance.

Environmental conditions to which plants acclimate prior exposure to abiotic or biotic stressors can greatly affect their subsequent resilience. This may have a significant impact on the response to ongoing climate change and can be useful for increasing the food security under adverse weather conditions associated with climate change.Within this study, we tested the hypothesis that plant morphological and biochemical acclimation to radiation conditions and nitrogen (N) availability is closely linked with carbon (C) and N balance. Four barley (Hordeum vulgare) varieties, differing in their morphological characteristics and sensitivity to photooxidative stress, were grown at two levels of N supply and four radiation regimes combining distinct levels of ultraviolet (UV) and photosynthetically active radiation (PAR). Changes in root and shoot morphology, accumulation of phenolic compounds, amino acids, and sugars were studied together with the analysis of C and N content in leaves. Both UV and PAR reduced leaf length and increased root-to-shoot ratio (R:S). Such effect was more pronounced under high N availability. High N supply reduced R:S, but this effect showed significant interactions with UV and PAR, and also with barley variety. Changes in R:S were positively related to C:N ratio in leaves that varied in response to both N availability and radiation treatments. UV radiation, particularly in combination with high PAR intensity, led to increases in most phenolic compounds (particularly flavones such as saponarin, homoorientin and isovitexin) which was also closely associated with changes in C:N ratio, while specifically phenolic acids (vanillic and syringic acids) decreased under high levels of UV and PAR, and hydroxycinnamic acids responded positively mainly to PAR. Although high N availability generally reduced the accumulation of phenolic compounds, this effect was genotype-specific and modulated by the radiation regime. A similar antagonistic effect of radiation treatment and N availability was also found for the accumulation of sugars (pentoses), resulting in a close relationship between the accumulation of pentoses and C:N ratio. The accumulation of most amino acids, in contrary to phenolic compounds, increases at high N and is also stimulated by high PAR and UV intensities. We conclude that radiation conditions and N availability have opposite effects on plant morphology and accumulation of most phenolic compounds and modulate the amino acid and sugar metabolism. Strong associations of these responses with changes in C:N ratio indicates that plant stoichiometry integrates acclimation processes and induction of relevant defence mechanisms.

Caspase-9 inhibition decreases expression of Mmp9 during chondrogenesis.

Besides cell death, caspase-9 participates in non-apoptotic events, including cell differentiation. To evaluate a possible impact on the expression of chondrogenic/osteogenic factors, a caspase-9 inhibitor was tested in vitro. For this purpose, mouse forelimb-derived micromass cultures, the most common chondrogenic in vitro model, were used. The following analyses were performed based on polymerase chain reaction (PCR) arrays and real-time PCR. The expression of several chondrogenesis-related genes was shown to be altered, some of which may impact chondrogenic differentiation (Bmp4, Bmp7, Sp7, Gli1), mineral deposition (Alp, Itgam) or the remodelling of the extracellular matrix (Col1a2, Mmp9) related to endochondral ossification. From the cluster of genes with altered expression, Mmp9 showed the most significant decrease in expression, of more than 50-fold. Additionally, we determined the possible impact of caspase-9 downregulation on the expression of other Mmp genes. A mild increase in Mmp14 was observed, but there was no change in the expression of other studied Mmp genes (-2, -3, -8, -10, -12, -13). Interestingly, inhibition of Mmp9 in micromasses led to decreased expression of some chondrogenic markers related to caspase-9. These samples also showed a decreased expression of caspase-9 itself, suggesting a bidirectional regulation of these two enzymes. These results indicate a specific impact of caspase-9 inhibition on the expression of Mmp9. The localisation of these two enzymes overlaps in resting, proliferative and pre-hypertrophic chondrocytes during in vivo development, which supports their multiple functions, either apoptotic or non-apoptotic. Notably, a coincidental expression pattern was identified in Pik3cg, a possible candidate for Mmp9 regulation.

Interactive effects of ultraviolet radiation and elevated CO2 concentration on photosynthetic characteristics of European beech saplings during the vegetation season.

To test the hypothesis that ultraviolet radiation (UV) modulates photosynthetic responses to elevated CO2 concentration ([CO2]) in plants, saplings of European beech were grown for two vegetation seasons under ambient (400 ppm) and elevated (700 ppm) atmospheric [CO2]. From April to November the saplings were exposed to (i) ambient UV radiation, (ii) excluded and (iii) enhanced UV (150% of ambient). Gas-exchange and chlorophyll fluorescence techniques were used throughout the second vegetation season together with biochemical analyses of the amount and activity of the Rubisco enzyme. We found support for the hypothesis that an impact of elevated [CO2] on photosynthesis is substantially modulated by UV radiation. Moreover, we found that the [CO2] × UV interaction is changing along the vegetation season: an enhanced UV radiation stimulated a positive effect of elevated [CO2] on plant photosynthesis at the beginning of the vegetation season (short-term effect), whilst long-term cultivation reduced the stimulatory effect of elevated [CO2] (a clear down-regulation of photosynthesis). Down-regulation was, however, not found in plants grown under the conditions of excluded UV radiation. We found evidence that the down-regulation of photosynthesis is associated with a complex acclimation at different hierarchical and functional levels, including an acclimation of primary photochemical reactions, carboxylation activity of Rubisco enzyme, and stomatal conductance.

Coupling activation of pro-apoptotic caspases with autophagy in the Meckel´s cartilage.

Mammalian Meckel´s cartilage is a temporary structure associated with mandible development. Notably, its elimination is not executed by apoptosis, and autophagy was suggested as the major mechanism. Simultaneous reports point to pro-apoptotic caspases as novel participants in autophagic pathways in general. The aim of this research was to find out whether activation of pro-apoptotic caspases (-2, -3, -6, -7, -8 and -9) was associated with autophagy of the Meckel´s cartilage chondrocytes. Active caspases were examined in serial histological sections of mouse mandible using immunodetection and were correlated with incidence of autophagy based on Beclin-1 expression. Caspase-2 and caspase-8 were found in Beclin-1 positive regions, whereas caspase-3, -6, -7 and -9 were not present. Caspase-8 was further correlated with Fas/FasL and HIF-1alpha, potential triggers for its activation. Some Fas and FasL positivity was observed in the chondrocytes but caspase-8 activation was found also in FasL deficient cartilage. HIF-1alpha was abundantly present in the hypertrophic chondrocytes. Taken together, caspase-8 activation in the Meckel´s cartilage was demonstrated for the first time. Caspase-8 and caspase-2 were the only pro-apoptotic caspases detected in the Beclin-1 positive segment of the cartilage. Activation of caspase-8 appears FasL/Fas independent but may be switched on by HIF-1alpha.

Expression of Fas, FasL, caspase-8 and other factors of the extrinsic apoptotic pathway during the onset of interdigital tissue elimination.

Elimination of the interdigital web is considered to be the classical model for assessing apoptosis. So far, most of the molecules described in the process have been connected to the intrinsic (mitochondrial) pathway. The extrinsic (receptor mediated) apoptotic pathway has been rather neglected, although it is important in development, immunomodulation and cancer therapy. This work aimed to investigate factors of the extrinsic apoptotic machinery during interdigital regression with a focus on three crucial initiators: Fas, Fas ligand and caspase-8. Immunofluorescent analysis of mouse forelimb histological sections revealed abundant expression of these molecules prior to digit separation. Subsequent PCR Array analyses indicated the expression of several markers engaged in the extrinsic pathway. Between embryonic days 11 and 13, statistically significant increases in the expression of Fas and caspase-8 were observed, along with other molecules involved in the extrinsic apoptotic pathway such as Dapk1, Traf3, Tnsf12, Tnfrsf1A and Ripk1. These results demonstrate for the first time the presence of extrinsic apoptotic components in mouse limb development and indicate novel candidates in the molecular network accompanying the regression of interdigital tissue during digitalisation.

Detection of Bim and Puma in mouse hair follicles using immunofluorescence and TUNEL assay double staining.

Apoptosis in hair follicles often is studied under pathological conditions; little is known about apoptotic mechanisms during normal hair follicle formation and maintenance. We investigated proteins of intrinsic apoptotic pathway, Bim and Puma, during hair follicle development and the first catagen stage using immunofluorescence to describe their expression patterns and to correlate them with apoptosis as determined by TUNEL assay. Both proteins were found in developing follicles. Bim and Puma overlapped apoptosis only partially during physiological apoptotic stage and they were present in non-apoptotic parts of the follicles. Our findings suggest that these primary apoptotic molecules participate in postnatal development and maintenance of hair follicles.

Mouse Incisor Stem Cell Niche and Myb Transcription Factors.

Dental hard tissues are formed particularly by odontoblasts (dentin) and ameloblasts (enamel). Whereas the reparation of dentin is often observed, enamel does not regenerate in most species. However, in mouse incisor, a population of somatic stem cells in the cervical loop is responsible for the incisor regeneration. Understanding of the specificities of these cells is therefore of an interest in basic research as well as regenerative therapies. The Myb transcription factors are involved in essential cellular processes. B-Myb is often linked to the stem cell phenotype, and c-Myb expression marks undifferentiated and proliferating cells such as the stem cells. In the presented study, temporo-spatial expression of B-Myb and c-Myb proteins was correlated with localisation of putative somatic stem cells in the mouse incisor cervical loop by immunohistochemistry. B-Myb expression was localised mostly in the zone of transit-amplifying cells, and c-Myb was found in the inner enamel epithelium, the surrounding mesenchyme and in differentiated cells. Taken together, neither B-Myb nor c-Myb was exclusively present or abundant in the area of the incisor stem cell niche. Their distribution, however, supports recently reported novel functions of c-Myb in differentiation of hard tissue cells.

Recent approaches in tooth engineering research.

Tooth absence and defects caused by various reasons are frequent events in humans. They are not life threatening but may bring about social consequences. Recent dentistry provides solutions in the form of prosthetics or dental implants; however, several complications and distinct limitations favour bioengineering of dental and periodontal structures. At least two types of cells (epithelial and mesenchymal) have to be recombined to produce a new functional tooth. Moreover, the tooth must be vascularized, innervated and properly anchored in the bone. To study these issues, different approaches have been established in both basic and applied research. In this review, recent strategies and techniques of tooth engineering are comprehensively summarized and discussed, particularly regarding manipulation using stem cells.

Mybs in mouse hair follicle development.

The Myb transcription factors are involved in essential cellular processes, such as cell proliferation, differentiation and cell death. Biological functions carried out by specific Myb proteins are distinct. Hair follicles are ectodermal-derived organs with cycling character of the growth resulting from the presence of somatic stem cells. In this study, we followed the expression of the Myb proteins in developing hair follicles and in the hair follicle stem cell niche by immunofluorescence staining. During hair follicle development, B-Myb was present in a few cells located in the area of cell division; c-Myb was abundant postanally in dividing cells but also in keratinizing zone. In addition, c-Myb was also detected in cells under the hair follicle bulge. These findings indicate possible involvement of c-Myb in regulation of activated stem cells leaving the niche.

Complete proteinogram of cerebrospinal fluid and its contribution to the diagnostics of inflammatory and autoimmune diseases of the central nervous system.

Complete evaluation of cerebrospinal fluid proteinogram represents a routine request of the clinician in the analysis of CSF in the Czech Republic. It comprises the measurement of concentrations of acute phase proteins (CRP, orosomucoid, haptoglobin, transferrin, prealbumin), immunoglobulins (IgG, IgA, IgM), compressive markers (albumin, fibrinogen), markers of CNS tissue destruction (apolipoproteins A-I, A-II, Apo B), complement components (C3, C4), alpha-1-microglobulin, beta-2-microglobulin, and proteinase inhibitors (alpha-1-antitrypsin, antithrombin III). Therefore, 19 CSF proteins of precisely verified clinical relevance are routine parameters for the assessment of the functional state of the blood-CSF barrier, presence of the intrathecal synthesis of immunoglobulins, inflammatory changes and verification of CNS tissue destruction. Evidence of these clinically relevant and independent parameters enabled the detection of the presence of autoimmune and neuroinfective diseases of CNS, even in clinical cases where the basic CSF parameters do not express relevant changes, or they are of a bordering or non-specific character. Clinically typical and the most significant abnormalities in the CSF proteinogram represent themselves a new access to a contemporary CSF analysis. Despite the fact that assessment of CSF proteins and their analysis is quite a difficult field in laboratory medicine, it is now routinely requested and routinely performed in the Czech Republic.