Epidermal UV-screening in vascular plants from Svalbard (Norwegian Arctic)

Stratospheric ozone depletion is most pronounced at high latitudes, and the concurring increased UV-B radiation might adversely affect plants from polar areas. However, vascular plants may protect themselves against UV-B radiation by UV-absorbing compounds located in the epidermis. In this 3-year study, epidermal UV-B (_max 314 nm) and UV-A (_max 366 nm) screening was assessed using a fluorescence method in 12 vascular species growing in their natural environment at Svalbard. The potential for acclimation to increased radiation was studied with artificially increased UV-B, simulating 11% ozone depletion. Open-top chambers simulated an increase in temperature of 2–3°C in addition to the UV-B manipulation. Adaxial epidermal UV-B transmittance varied between 1.6 and 11.4%. Artificially increased UV-B radiation and temperature did not consistently influence the epidermal UV-B transmittance in any of the measured species, suggesting that they may not have the potential to increase their epidermal screening, or that the screening is already high enough at the applied UV-B level. We propose that environmental factors other than UV-B radiation may influence epidermal UV-B screening.     

Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.     

Tumor attenuation by 2(6-hydroxynaphthyl)-beta-D-xylopyranoside requires priming of heparan sulfate and nuclear targeting of the products

We have previously reported that the heparan sulfate-priming glycoside 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside selectively inhibits growth of transformed or tumor-derived cells. To investigate the specificity of this xyloside various analogs were synthesized and tested in vitro. Selective growth inhibition was dependent on the presence of a free 6-hydroxyl in the aglycon. Because cells deficient in heparan sulfate synthesis were insensitive to the xyloside, we conclude that priming of heparan sulfate synthesis was required for growth inhibition. In growth-inhibited cells, heparan sulfate chains primed by the active xyloside were degraded to products that contained anhydromannose and appeared in the nuclei. Hence the degradation products were generated by nitric oxide-dependent cleavage. Accordingly, nitric oxide depletion reduced nuclear localization of the degradation products and counteracted the growth-inhibitory effect of the xyloside. We propose that 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside entered cells and primed synthesis of heparan sulfate chains that were subsequently degraded by nitric oxide into products that accumulated in the nucleus. In vivo experiments demonstrated that the xyloside administered subcutaneously, perorally, or intraperitoneally was adsorbed and made available to tumor cells located subcutaneously. Treatment with the xyloside reduced the average tumor load by 70-97% in SCID mice. The present xyloside may serve as a lead compound for the development of novel antitumor strategies.     

Efficient gene transfer into murine embryonic stem cells by nucleofection

Genetic manipulation of embryonic stem (ES) cells is performed by non-viral as well as viral transfection methods. We tested the recently developed nucleofection method delivering plasmid DNA directly into the nucleus for the introduction of a plasmid encoding enhanced green fluorescent protein (EGFP) into murine ES cells. Cell viability decreased from 77% before to 40% 24 h after nucleofection. Transfection effciencies in viable stem cells were between 85% and 96% with high levels of EGFP expression [mean fluorescence intensity (MFI): 630 +/- 90] 24 h after nucleofection. After a two week culture in geneticin (G418) selection medium, nearly 50% of the stem cells were EGFP positive and continued transgene expression (MFIs: 120-240) for a two further weeks. We conclude that nucleofection is an efficient nonviral gene transfer method for the introduction of genes into murine ES cells.     

Tissue plasminogen activator is a potent activator of PDGF-CC

Tissue plasminogen activator (tPA) is a serine protease involved in the degradation of blood clots through the activation of plasminogen to plasmin. Here we report on the identification of tPA as a specific protease able to activate platelet-derived growth factor C (PDGF-C). The newly identified PDGF-C is secreted as a latent dimeric factor (PDGF-CC) that upon proteolytic removal of the N-terminal CUB domains becomes a PDGF receptor alpha agonist. The CUB domains in PDGF-CC directly interact with tPA, and fibroblasts from tPA-deficient mice fail to activate latent PDGF-CC. We further demonstrate that growth of primary fibroblasts in culture is dependent on a tPA-mediated cleavage of latent PDGF-CC, generating a growth stimulatory loop. Immunohistochemical analysis showed similar expression patterns of PDGF-C and tPA in developing mouse embryos and in tumors, indicating both autocrine and paracrine modes of activation of PDGF receptor-mediated signaling pathways. The identification of tPA as an activator of PDGF signaling establishes a novel role for the protease in normal and pathological tissue growth and maintenance, distinct from its well-known role in plasminogen activation and fibrinolysis.     

Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics

We describe the introduction of the yeasts Saccharomyces cerevisiae and Pichia pastoris as eukaryotic hosts for the routine production of recombinant proteins for a structural genomics initiative. We have previously shown that human cDNAs can be efficiently expressed in both hosts using high throughput procedures. Expression clones derived from these screening procedures were grown in bioreactors and the over-expressed human proteins were purified, resulting in obtaining significant amounts suitable for structural analysis. We have also developed and optimized protocols enabling a high throughput, low cost fermentation and purification strategy for recombinant proteins for both S. cerevisiae and P. pastoris on a scale of 5 to 10 mg. Both batch and fed batch fermentation methods were applied to S. cerevisiae. The fed batch fermentations yielded a higher biomass production in all the strains as well as a higher productivity for some of the proteins. We carried out only fed batch fermentations on P. pastoris strains. Biomass was produced by cultivation on glycerol, followed by feeding methanol as carbon source to induce protein expression. The recombinant proteins were expressed as fusion proteins that include a N-terminal His-tag and a C-terminal Strep-tag. They were then purified by a two-step chromatographic procedure using metal-affinity chromatography and StrepTactin-affinity chromatography. This was followed by gel filtration for further purification and for buffer exchange. This three-step purification procedure is necessary to obtain highly purified proteins from yeast. The purified proteins have successfully been subjected to crystallization and biophysical analysis.     

Seasonal locomotion and home-range characteristics of European hares (<Emphasis Type="Italic">Lepus europaeus</Emphasis>) in an arable region in central Germany

We studied seasonal use of space by 38 radio-tracked European hares in an arable region in central Germany over 5 years. Mean distance between successive daytime and nighttime fixes of a hare amounted to 226 m, and an average distance of 172 m was recorded for successive daytime fixes. The hares shifted the centres of their home ranges from one 2-month period to the next by an average distance of 131 m and over a time span of 11.2 months by 216 m. The size of their 2-month home ranges (MCP 95) averaged 21 ha; the variation between individuals was high. Night ranges were larger than day ranges. Mean size of seasonal home ranges—based on daytime and nighttime fixes—remained largely constant throughout the year. Home-range size increased as the day-to-night distance increased, as the number of used habitat elements increased and as frequency of use of inner field parts during the daytime increased. Home-range size was inversely related to population density. On average, 32% of the area of the home range of a hare overlapped with the home range of a neighbouring conspecific. When related to the population density of adult hares in spring, the home range of a single hare was estimated to overlap with 13–21 home ranges of adults as well as an indefinite number of juveniles. The large intraseasonal variation in locomotion and home-range characteristics is interpreted in relationship to the social structure of European hares.     

Bystander suppression of collagen-induced arthritis in mice fed ovalbumin

We wanted to assess whether B-cell and/or T-cell responses to collagen and thereby the course of collagen-induced arthritis could be suppressed by regulatory mechanisms associated with oral tolerance to an unrelated protein. DBA/1 mice were fed ovalbumin (OVA)-containing pellets ad libitum for 1 week and subsequently coimmunized twice, with a mixture of bovine collagen type II (BCII) and OVA in Freund's complete adjuvant. Mice fed OVA before coimmunization with BCII and OVA had significantly lower arthritic scores than mice immunized with BCII only. Their body weight increased during the study period and their anti-BCII antibody activity was significantly IgG_2a lower. The frequency of spleen cells producing IgG anti-BCII antibody was also reduced. Coimmunization per se slightly ameliorated the development of arthritis, resulting in an early, transient reduction. It resulted in significantly higher IgG₁ anti-BCII antibody activity and increased splenocyte secretion of IFN-γ and IL-10 in response to BCII. Our findings demonstrate that OVA-specific regulatory events induced by feeding OVA, i.e. bystander suppression, reduced the severity of arthritis in animals immunized with BCII and OVA. Anti-BCII specific antibody responses and cytokine secretion by types 1 and 2 T helper cells were also decreased.     

Effects of pretreatment with a xanthine oxidase inhibitor on free radical levels during carotid endarterectomy

Objective: Free radicals contribute to the tissue damage caused by ischaemia-reperfusion. The aim of the present study was to investigate whether preoperative antioxidant therapy (allopurinol) affects free radical levels in cerebral venous blood in connection with surgery for carotid artery stenosis.

Materials and methods: Twenty-five patients were randomised into the study. Thirteen were controls and 12 were pretreated with allopurinol the day before surgery. Before, during and after surgery, blood samples were drawn from the ipsilateral jugular vein. Radical levels were measured using the spin trap technique ex vivo using OXANOH as the spin trap. Multivariate statistics were used with Principal Component Analysis and Partial Least Square regression analysis.

Results: Radical levels increased with diabetes, high leukocyte count, high creatinine and a high degree of contralateral stenosis. Radical levels decreased with high age, blood pressure, collateral circulation as well as operation for left-side carotid artery stenosis. After pretreatment with allopurinol, several of the relationships noted in the control group were eliminated, i.e. leukocyte count, side of operation, Betapred pretreatment and collateral circulation.

Conclusions: Radical levels can be determined in connection with surgery for carotid artery stenosis using an ex vivo spin trap method. With preoperative antioxidant therapy the relationships between enhanced radical levels and clinical data, as seen in control subjects, disappeared. This might indicate a beneficial effect of preoperative pretreatment with antioxidants.