Trehalose treatment accelerates the healing of UVB-irradiated corneas. Comparative immunohistochemical studies on corneal cryostat sections and corneal impression cytology

The UVB-irradiated cornea is damaged by oxidative stress. Toxic oxygen products induced by UVB radiation in the cornea are insufficiently removed by antioxidants, whose numbers decrease with increasing UVB irradiation. In addition, the UVB-irradiated cornea suffers from hypoxic conditions because damaged corneal cells cannot utilize oxygen normally, although the supply of oxygen to the cornea is unchanged (normal). This contributes to attenuated re-epithelialization, corneal neovascularization and apoptotic cell death. Our previous publications reported that trehalose applied on the corneal surface during irradiation significantly suppressed UVB-induced corneal oxidative damage. The results of this study provide for the first time important evidence that trehalose applied on the surface of corneas for two weeks following repeated UVB irradiation (312 nm, daily dose 0.5 J/cm2) accelerated corneal healing, restored corneal transparency and suppressed corneal neovascularization. Compared to buffered saline treatment, following which caspase-3, nitrotyrosine, malondialdehyde and urokinase-type plasminogen activator were still strongly expressed in the corneal epithelium two weeks after irradiation and corneal neovascularization was evident, apoptotic cell death was already significantly reduced after one week of trehalose application. The expression of other markers of injury returned to normal levels during two weeks of trehalose treatment. In conclusion, our results show that trehalose accelerated healing of the UVB irradiated cornea, very probably via suppression of hypoxia-response injury. In addition, immunohistochemical results on corneal cryostat sections corresponded with those obtained using corneal impression cytologies, thus confirming that corneal impression cytologies are useful for diagnostic purposes.     

Transient expression of <Emphasis Type="Italic">Human papillomavirus</Emphasis> type 16 L2 epitope fused to N- and C-terminus of coat protein of <Emphasis Type="Italic">Potato virus X</Emphasis> in plants

Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108–120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2_108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX CP- L2_108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue. Immunogenicity of L2_108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In animal sera the antibodies against the PVX CP and the L2_108-120 epitope were found after both methods of vaccine delivery.     

Kinetic and chemical analyses of the cytokinin dehydrogenase-catalysed reaction: correlations with the crystal structure

CKX (cytokinin dehydrogenase) is a flavoprotein that cleaves cytokinins to adenine and the corresponding side-chain aldehyde using a quinone-type electron acceptor. In the present study, reactions of maize (Zea mays) CKX with five different substrates (N6-isopentenyladenine, trans-zeatin, kinetin, p-topolin and N-methyl-isopentenyladenine) were studied. By using stopped-flow analysis of the reductive half-reaction, spectral intermediates were observed indicative of the transient formation of a binary enzyme-product complex between the cytokinin imine and the reduced enzyme. The reduction rate was high for isoprenoid cytokinins that showed formation of a charge-transfer complex of reduced enzyme with bound cytokinin imine. For the other cytokinins, flavin reduction was slow and no charge-transfer intermediates were observed. The binary complex of reduced enzyme and imine product intermediate decays relatively slowly to form an unbound product, cytokinin imine, which accumulates in the reaction mixture. The imine product only very slowly hydrolyses to adenine and an aldehyde derived from the cytokinin N6 side-chain. Mixing of the substrate-reduced enzyme with Cu2+/imidazole as an electron acceptor to monitor the oxidative half-reaction revealed a high rate of electron transfer for this type of electron acceptor when using N6-isopentenyladenine. The stability of the cytokinin imine products allowed their fragmentation analysis and structure assessment by Q-TOF (quadrupole-time-of-flight) MS/MS. Correlations of the kinetic data with the known crystal structure are discussed for reactions with different cytokinins.     

High Arctic vegetation after 70 years: a repeated analysis from Svalbard

We asked whether vegetation mapping repeated after 70 years revealed vegetation changes in the high Arctic. The study site is located at 78°38′N, 16°45′E, near Brucebyen at the Adolfbukta Bay (head of Billjefjorden) in central Spitsbergen (Svalbard), and encompasses an area of 2,042 × 521 m. The mapping carried out in 2008 did not reveal any changes in vegetation, since a previous study in 1936–1937, that could be attributed to climate change. We argue that our finding can be interpreted as evidence of a slow ecological response of constituent plants in such a harsh environment. Moreover, geographic isolation may limit establishment and expansion of new species. Some successional changes were only due to erosion–accumulation processes connected especially to stream activity.     

Restoration of spermatogenesis after transplantation of c-Kit positive testicular cells in the fowl

Transplantation of male germ line cells into sterilized recipients has been used in mammals for conventional breeding as well as for transgenesis. We have previously adapted this approach for the domestic chicken and we present now an improvement of the germ cell transplantation technique by using an enriched subpopulation of c-Kit-positive spermatogonia as donor cells. Dispersed c-Kit positive testicular cells from 16 to 17 week-old pubertal donors were transplanted by injection directly into the testes of recipient males sterilized by repeated gamma irradiation. We describe the repopulation of the recipient's testes with c-Kit positive donor testicular cells, which resulted in the production of functional heterologous spermatozoa.Using manual semen collection, the first sperm production in the recipient males was observed about nine weeks after the transplantation. The full reproduction cycle was accomplished by artificial insemination of hens and hatching of chickens.     

Broad Habitat Range of the Phylogenetically Narrow R-BT065 Cluster,Representing a Core Group of the Betaproteobacterial Genus Limnohabitans

The distribution of the phylogenetically narrow R-BT065 cluster (Betaproteobacteria) in 102 freshwater lakes, reservoirs, and various ponds located in central Europe (a total of 122 samples) was examined by using a cluster-specific fluorescence in situ hybridization probe. These habitats differ markedly in pH, conductivity, trophic status, surface area, altitude, bedrock type, and other limnological characteristics. Despite the broad ecological diversity of the habitats investigated, the cluster was detected in 96.7% of the systems, and its occurrence was not restricted to a certain habitat type. However, the relative proportions of the cluster in the total bacterioplankton were significantly lower in humic and acidified lakes than in pH-neutral or alkaline habitats. On average, the cluster accounted for 9.4% of the total bacterioplankton (range, 0 to 29%). The relative abundance and absolute abundance of these bacteria were significantly and positively related to higher pH, conductivity, and the proportion of low-molecular-weight compounds in dissolved organic carbon (DOC) and negatively related to the total DOC and dissolved aromatic carbon contents. Together, these parameters explained 55.3% of the variability in the occurrence of the cluster. Surprisingly, no clear relationship of the R-BT065 bacteria to factors indicating the trophic status of habitats (i.e., different forms of phosphorus and chlorophyll a content) was found. Based on our results and previously published data, we concluded that the R-BT065 cluster represents a ubiquitous, highly active segment of bacterioplankton in nonacidic lakes and ponds and that alga-derived substrates likely form the main pool of substrates responsible for its high growth potential and broad distribution in freshwater habitats.Heterotrophic bacterioplankton assemblages found in a broad variety of freshwater ecosystems are frequently dominated by representatives of a few phylogenetic clusters of Betaproteobacteria and Actinobacteria (1, 7, 9, 17, 21, 23, 34, 40). One of these clusters is the phylogenetically defined R-BT065 group (34), which is currently represented by >700 environmental ribosomal sequences deposited in the GenBank database. These sequences were obtained in several nonquantitative diversity studies of many European and North American freshwater habitats (5, 10, 18, 27, 30, 42). The R-BT065 cluster is characterized by a minimum level of intragroup 16S rRNA sequence similarity of 97.3%, and investigations of recently cultivated strains demonstrated that members of the group are heterotrophic bacteria (V. Kasalický, J. Jezbera, K. Šimek, and M. W. Hahn, submitted for publication). This cluster forms a phylogenetically distinct subgroup of the so-called “Rhodoferax sp. BAL47” cluster (42). The new genus Limnohabitans was recently established for some strains affiliated with the “Rhodoferax sp. BAL47” cluster (11), and it has to be assumed that bacteria belonging to the R-BT065 cluster are also affiliated with this genus (Kasalický and coworkers, submitted for publication).Bacteria belonging to the R-BT065 cluster in natural systems can be quantified using a homonymous fluorescence in situ hybridization (FISH) probe (34). This probe enabled intensive investigations of the ecology of R-BT065 bacteria; however, only a few freshwater systems (mainly representing larger pH-neutral lakes and reservoirs) have been quantitatively investigated to determine the presence of this bacterial group so far (2, 13, 24, 27, 30, 30). In the few habitats investigated, R-BT065 bacteria comprised 3 to 50% of the total bacteria. Importantly, these bacteria consistently had the highest net growth rates and showed high levels of substrate uptake in a freshwater reservoir (12, 13, 31, 32), as well as in an alpine lake (27). These investigations indicated that the ecology of the R-BT065 group is rather uniform and is characterized by (i) the potential for a rapid response to environmental changes, (ii) a high level of vulnerability to flagellate predation (14, 35), and (iii) a strong link with phytoplankton-derived organic material as the key growth substrate for the bacteria (24, 25, 33). However, these findings were based on quantitative investigations of only a few freshwater systems, and we can only speculate that the findings could be generalized for populations of this bacterial group in systems with different limnological characteristics. In particular, investigations of the habitat range, as well as investigations of environmental factors modulating the abundance of R-BT065 bacteria, have not been performed. Therefore, we set out to conduct a comprehensive survey of a broad variety of freshwater habitats that could support our hypothesis concerning the potential general importance of the cluster.The specific aims of this study were (i) to quantify the R-BT065 bacteria in the surface waters of a large variety of central European freshwater ecosystems (102 habitats) spanning broad ranges of system type and size (from large and deep alpine lakes to small and shallow ponds or various fishponds), type of bedrock, altitude, trophic status, pH, and humic substance content; (ii) to determine the proportion of R-BT065 bacteria in the total bacteria and in the total Betaproteobacteria in these habitats using FISH probes; and (iii) to suggest major physical, chemical, and biological factors that influence the occurrence of the R-BT065 phylotypes in the bacterioplankton.     

Switching p53-dependent growth arrest to apoptosis via the inhibition of DNA damage-activated kinases

Cisplatin and doxorubicin are widely used anticancer drugs that cause DNA damage, which activates the ATM-Chk2-p53 pathway in cancer cells. This activation leads to cell cycle block or apoptosis, depending on the nature of the DNA damage. In an attempt to enhance the effects of these agents, we inhibited ATM/ATR and Chk2, which are known upstream regulators of p53. The cancer cell lines A2780 and ARN8, bearing the wild-type p53 protein, were used to study changes in p53 activation and trans-activation. Our results suggest that the G₁-checkpoint, normally activated by DNA damage, is functionally overcome by the action of kinase inhibitors that sensitize cells to apoptosis. Both inhibitors show these effects, albeit with variable intensity in different cell lines, which is promising for other studies and theoretically for use in clinical practice.