Regulation of CO2 by the sea in areas around Latin America in a context of climate change.

Anthropogenic activities are increasing the atmospheric carbon dioxide (CO2); around a third of the CO2 emitted by these activities has been taken up by the ocean. Nevertheless, this marine ecosystem service of regulation remains largely invisible to society, and not enough is known about regional differences and trends in sea-air CO2 fluxes (FCO2), especially in the Southern Hemisphere. The objectives of this work were as follows: first to put values of FCO2 integrated over the exclusive economic zones (EEZ) of five Latin-American countries (Argentina, Brazil, Mexico, Peru, and Venezuela) into perspective regarding total country-level greenhouse gases (GHG) emissions. Second, to assess the variability of two main biological factors affecting FCO2 at marine ecological time series (METS) in these areas. FCO2 over the EEZs were estimated using the NEMO model, and GHG emissions were taken from reports to the UN Framework Convention on Climate Change. For each METS, the variability in phytoplankton biomass (indexed by chlorophyll-a concentration, Chla) and abundance of different cell sizes (phy-size) were analyzed at two time periods (2000-2015 and 2007-2015). Estimates of FCO2 at the analyzed EEZs showed high variability among each other and non-negligible values in the context of greenhouse gas emissions. The trends observed at the METS indicated, in some cases, an increase in Chla (e.g., EPEA-Argentina) and a decrease in others (e.g., IMARPE-Peru). Evidence of increasing populations of small size-phytoplankton was observed (e.g., EPEA-Argentina, Ensenada-Mexico), which would affect the carbon export to the deep ocean. These results highlight the relevance of ocean health and its ecosystem service of regulation when discussing carbon net emissions and budgets.

A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis phages.

Here, we present pRH030, a new CRISPR-Cas9 tool for the genetic engineering of Bacillus phages and beyond. It is based on the Streptococcus pyogenes cas9 with its native constitutive promoter, tracrRNA, and a gRNA precursor. The constitutive expression of Cas9 was conducive to the inactivation of viral attackers and enhanced phage mutagenesis efficiency up to 100%. The gRNA precursor can be built up to an artificial CRISPR array with up to 5 spacers (target sequences) assembled from ordinary oligonucleotides and directly cloned into pRH030. Required time and resources remain comparable to a single gRNA cloning. These properties make pRH030 an attractive new system for the modification of Bacillus phages and qualify it for research beyond genetic construction.

Characterization of fibrillar collagen types using multi-dimensional multiphoton laser scanning microscopy.

In dermal photodamage the ratio of the collagen types III to I changes. This makes the investigation of the fibrillar collagen type characteristics interesting for skin research. In this study collagen types were characterized using 5-dimensional multiphoton laser scanning microscopy (5D-IVT) that can be applied in vivo. Second harmonic generation (SHG) signals and fluorescence lifetimes of the collagen autofluorescence were analysed. Collagen type I generates a higher SHG intensity and a longer fluorescence lifetime compared to collagen type III. Thus, the SHG intensity decrease found in photodamaged skin might be explained by the increase in collagen type III. Calculating the in vivo relevant increase of collagen type III gives a negligible difference in fluorescence lifetime not qualifying this method for the determination of collagen type changes in dermal photodamage in vivo in human skin. However, for pathologies that exhibit higher differences in collagen types 5D-IVT analysis might be a suitable method.

The presence of extreme feather peckers in groups of laying hens.

Feather pecking is a serious economic and welfare problem in laying hens. Feather damage occurs mainly through severe feather pecking (SFP). Selection experiments have proved that this behavior is heritable and lines have been divergently selected for high (HFP) and low feather pecking (LFP). The number of bouts of SFP per hen follows a Poisson distribution with a maximum nearby 0. A few studies indicate that the distribution within flocks is not homogenous but contains sub-groups of birds showing extremely high levels of feather pecking (EFP). It was the aim of the current study to re-analyze data on SFP of lines selected for HFP/LFP and their F2 cross so as to uncover hidden sub-populations of EFP birds. Data of seven selection generations of HFP and LFP selection lines as well as their F2 cross have been used. We fitted a two-component mixture of Poisson distributions in order to separate the sub-group of EFP from the remaining birds. HFP and LFP lines differed mainly in mean bouts per bird. The proportion of EFP was only marginal in the LFP as compared with the HFP and the F2 population. Selection for LFP did not result in total elimination of EFP. The presence of even small proportions of EFP may play an important role in initiating outbreaks of feather pecking in large flocks. Further studies on feather pecking should pay special attention to the occurrence of EFP sub-groups.

Quantitative genetic analysis of causal relationships among feather pecking, feather eating, and general locomotor activity in laying hens using structural equation models.

The objective of this research was to analyze the relationship between feather pecking (FP) and feather eating (FE) as well as general locomotor activity (GLA) using structural equation models, which allow that one trait can be treated as an explanatory variable of another trait. This provides an opportunity to infer putative causal links among the traits. For the analysis, 897 F2-hens set up from 2 lines divergently selected for high and low FP were available. The FP observations were Box-Cox transformed, and FE and GLA observations were log and square root transformed, respectively. The estimated heritabilities of FE, GLA, and FP were 0.36, 0.29, and 0.20, respectively. The genetic correlation between FP and FE (GLA) was 0.17 (0.04). A high genetic correlation of 0.47 was estimated between FE and GLA. The recursive effect from FE to FP was [Formula: see text], and from GLA to FP [Formula: see text] These results imply that an increase of FE leads to an increased FP behavior and that an increase in GLA results in a higher FP value. Furthermore, the study showed that the genetic correlation among the traits is mainly caused by indirect effects.

Urokinase induces proliferation of human ovarian cancer cells: characterization of structural elements required for growth factor function.

Ovarian cancer metastasis is associated with an increase in the urokinase-type plasminogen activator (uPA) and its receptor uPAR. We present evidence that binding of uPA to uPAR provokes a mitogenic response in the human ovarian cancer cell line OV-MZ-6 in which endogenous uPA production had been significantly reduced by stable uPA 'antisense' transfection. High molecular weight (HMW) uPA, independent of its enzymatic activity, produced an up to 95% increase in cell number concomitant with 2-fold elevated [3H]thymidine incorporation as did the catalytically inactive but uPAR binding amino-terminal fragment of uPA, ATF. uPA-induced cell proliferation was significantly decreased by blocking uPA/uPAR interaction by the monoclonal antibody IIIF10 and by soluble uPAR. The efficiency of the uPAR binding synthetic peptide cyclo19,31 uPA19-31 to enhance OV-MZ-6 cell growth proved this molecular domain to be the minimal structural determinant for uPA mitogenic activity. Dependence of uPA-provoked cell proliferation on uPAR was further demonstrated in Raji cells which do not express uPAR and were thus not induced by uPA. However, upon transfection with full-length uPAR, Raji cells acquired a significant growth response to HMW uPA and ATF.

Reduction of breast carcinoma tumor growth and lung colonization by overexpression of the soluble urokinase-type plasminogen activator receptor (CD87).

The serine protease urokinase-type plasminogen activator, uPA, when bound to its specific receptor, uPAR (CD87), plays a significant role in tumor cell invasion and metastasis. In breast cancer, enhanced uPA antigen in the primary tumor is correlated with poor prognosis of the patient. In an in vivo nude mouse model, we tested tumor growth and metastasis of human breast carcinoma cells that had been transfected with an expression plasmid encoding a soluble form of uPAR (suPAR). We explored, whether suPAR/uPA interaction reduces the binding of uPA to cell surface-associated uPAR, and, as a consequence, could suppress tumor growth and metastasis of the human breast cancer cell line MDA-MB-231 BAG. Overexpressed, secreted suPAR was shown to bind and thus scavenge the uPA secreted by the transfected lines suPAR3 and suPAR10. In vitro, an overexpression of suPAR did not alter the proliferation rate of the transfected tumor cells, nor did it affect the expression of uPA. Overexpression of suPAR led to a reduction in the plasminogen activation-related proteolytic activity of breast carcinoma cells. Primary tumor growth in the mammary fat pad of nude mice was followed up for 52 days. Overexpression of suPAR correlated with a reduction in tumor growth (from day 21, reaching 30% by day 34) as well as lung colonization (lung metastasis-positive mice in suPAR3: 4 of 17; suPAR10: 3 of 10; parental MDA-MB-231 BAG: 13 of 18). We conclude that suPAR overexpression leading to effective scavenge of uPA impairs proteolysis as well as the tumor growth and metastatic potential of breast carcinoma cells in vivo.

Multifunctional potential of the plasminogen activation system in tumor invasion and metastasis (review).

Tumor cell migration and invasion into the surrounding tissue depend on the invasive capacity of cells leading to the loosening of cell-cell and cell-substratum contacts via cell surface associated proteolytic enzyme systems. Plasmin is one of the enzymes involved in these complex events. It is generated by the cleavage of the proenzyme plasminogen upon the action of the urokinase-type plasminogen activator (uPA). uPA is synthesized and secreted by tumor cells and normal cells and interacts with a specific cell surface receptor (uPAR) thereby focalizing enzymatic activity to the cell surface. The activity of uPA is controlled by plasminogen activator inhibitors type-1 and type-2. A strong statistically independent prognostic impact has been attributed to uPA and its inhibitor PAI-1 in a variety of malignancies. Besides its proteolytic activity, uPA in concert with uPAR exert biological effects characteristic for molecules with signal transducing properties including chemotaxis, migration/invasion, adhesion, and mitogenesis.

New ELISA for quantitation of human urokinase receptor (CD87) in cancer.

The serine protease urokinase-type plasminogen activator (uPA), its inhibitor (PAI-1), and its receptor (uPAR; CD87) facilitate cancer cell invasion and metastasis. Whereas uPA and PAI-1 antigen levels determined in tumor tissue extracts of breast cancer patients correlate with disease recurrence and overall survival, the prognostic relevance of uPAR is still a matter of debate. We established two new sandwich-type enzyme-linked immunosorbent assay (ELISA) formats (HU/IIIF10-ELISA and HU/HD13-ELISA) using the epitope-defined monoclonal antibody (mAb) IIIF10 or the conformation-dependent mAb HD13.1, a polyclonal chicken antibody (HU277), and recombinant soluble uPAR (CHO-suPAR) as the standard. The lower detection limit of the assays was at 0.16 ng/ml, with a linear dose-response up to 5 ng/ml of uPAR antigen. Both ELISA formats showed good reproducibility and recovery. The intra-assay and the inter-assay variation coefficients were respectively 4.3% and 11.7% (HU/IIIF10-ELISA) and 4.0% and 10.7% (HU/HD13-ELISA). The recovery rate of uPAR in cell lysates spiked with CHO-suPAR was above 82% and 88%, respectively. With these new ELISA formats, uPAR antigen content in breast cancer tissue extracts and tumor cell lysates was determined and compared to a commercially available ELISA (ADI-ELISA). By all of the three uPAR ELISA formats CHO-suPAR and uPAR present in lysates of non-malignant epithelial cells and stimulated monocytes were quantified with similar sensitivity. Interestingly, in breast cancer cell lines of epitheloid origin a higher uPAR antigen content was determined by the HU/IIIF10-ELISA than the HU/HD13- or ADI-ELISA formats. In lysates of fibroblastic breast cancer cell lines similar uPAR values were obtained with the HU/IIIF10- and ADI-ELISA formats, whereas with the HU/HD13-ELISA significantly lower uPAR concentrations were determined. The prognostic relevance of tumor uPAR antigen was evaluated in 199 primary breast cancer patients with a median follow-up of 24 months. uPAR antigen values above the cut-off of 3.33 ng/mg protein as determined by the HU/IIIF10-ELISA were significantly correlated with short disease-free survival (p=0.025). Results obtained by the other two ELISA formats (HU/HD13-ELISA and ADI-ELISA) were not associated with prognosis. Our findings stress the need of well-characterized antibodies, which detect both uPAR of non-malignant and tumor cells, in setting up a uPAR-ELISA useful for assessing breast cancer patient prognosis.

Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression, enzyme properties and cell-type specific expression in plants.

We investigated the P450 dependent flavonoid hydroxylase from the ornamental plant Catharanthus roseus. cDNAs were obtained by heterologous screening with the CYP75 Hf1 cDNA from Petunia hybrida. The C. roseus protein shared 68-78% identity with other CYP75s, and genomic blots suggested one or two genes. The protein was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus. Enzyme assays showed that it was a flavonoid 3', 5'-hydroxylase, but 3'-hydroxylated products were also detected. The substrate specificity was investigated with the C. roseus enzyme and a fusion protein of the Petunia hybrida CYP75 with the C. roseus P450 reductase. Both enzymes accepted flavanones as well as flavones, dihydroflavonols and flavonols, and both performed 3'- as well as 3'5'-hydroxylation. Kinetics with C. roseus cultures on the level of enzyme activity, protein and RNA showed that the F3'5'H was present in dark-grown cells and was induced by irradiation. The same results were obtained for cinnamic acid 4-hydroxylase and flavanone 3beta-hydroxylase. In contrast, CHS expression was strictly dependent on light, although CHS is necessary in the synthesis of the F3'5'H substrates. Immunohistochemical localization of F3'5'H had not been performed before. A comparison of CHS and F3'5'H in cotyledons and flower buds from C. roseus identified CHS expression preferentially in the epidermis, while F3'5'H was only detected in the phloem. The cell-type specific expression suggests that intercellular transport may play an important role in the compartmentation of the pathways to the different flavonoids.

[Comparative study of the bioavailability and pharmacokinetics of isosorbide dinitrate formulations in retard form and in standard preparations by determination of isosorbide-5-mononitrate]. / Vergleichende Prüfung der Bioverfügbarkeit und Pharmakokinetik von Isosorbiddinitrat-Formulierungen in Retardform gegen Standardpräparate mit Bestimmung des Isosorbid-5-Mononitrats.

The aim of the study was to determine the relative bioavailability and the pharmacokinetic parameters following administration of a slow-release formulation of isosorbide dinitrate (ISDN, Isdin) capsules (20, 40 and 60 mg). A gas chromatographic method was used to determine the plasma concentrations of ISDN and isosorbide-5-mononitrate (IS-5-MN). All of the required pharmacokinetic parameters were ascertained. The study was performed on 12 healthy volunteers in a steady-state crossover design. A comparison of the areas under the plasma concentration/time curves of the test preparations and the commercial preparations showed higher values for the test preparations in all of the investigations. However, these values were only statistically significant for the 40 and 60 mg formulations ISDN and the 60 mg formulation IS-5-MN.

High level synthesis of recombinant soluble urokinase receptor (CD87) by ovarian cancer cells reduces intraperitoneal tumor growth and spread in nude mice.

Focussing of the serine protease urokinase (uPA) to the tumor cell surface via interaction with its receptor (uPAR) is an important step in tumor invasion and metastasis. The human ovarian cancer cell line OV-MZ-6#8 was stably transfected with expression plasmids either encoding cell-associated uPAR (GPI-uPAR) or a soluble form of uPAR (suPAR) lacking its glycan lipid anchor. In vitro, high level synthesis of functionally active recombinant suPAR inhibited cell proliferation and led to reduced cell-associated fibrin matrix degradation, whereas fibrinolytic activity was increased in OV-MZ-6#8 cells overexpressing GPI-uPAR. Both OV-MZ-6#8-derived clones were inoculated into the peritoneum of nude mice and tested for tumor growth and spread. High level synthesis of recombinant suPAR (without altering the physiological expression levels of GPI-uPAR and uPA in these cells) resulted in a significant reduction of tumor burden (up to 86%) in the xenogeneic mouse model. In contrast, overexpression of GPI-uPAR in tumor cells did not affect tumor growth. Our results demonstrate that high levels of suPAR in the ovarian cancer cell vicinity can act as a potent scavenger for uPA, thereby significantly reducing tumor cell growth and cancer progression in vivo.

Cellular glycosylphosphatidylinositol-specific phospholipase D regulates urokinase receptor shedding and cell surface expression.

The glycosylphosphatidylinositol (GPI)-anchored, multifunctional receptor for the serine proteinase, urokinase plasminogen activator (uPAR, CD87), regulates plasminogen activation and cell migration, adhesion, and proliferation. uPAR occurs in functionally distinct, membrane-anchored and soluble isoforms (s-uPAR) in vitro and in vivo. Recent evidence indicates that s-uPAR present in the circulation of cancer patients correlates with tumor malignancy and represents a valuable prognostic marker in certain types of cancer. We have therefore analyzed the mechanism of uPAR shedding in vitro. We present evidence that uPAR is actively released from ovarian cancer cells since the rate of receptor shedding did not correlate with uPAR expression. While s-uPAR was derived from the cell surface, it lacked the hydrophobic portion of the GPI moiety indicating anchor cleavage. We show that uPAR release is catalyzed by cellular GPI-specific phospholipase D (GPI-PLD), an enzyme cleaving the GPI anchor of the receptor. Thus, recombinant GPI-PLD expression increased receptor release up to fourfold. Conversely, a 40% reduction in GPI-PLD activity by GPI-PLD antisense mRNA expression inhibited uPAR release by more than 60%. We found that GPI-PLD also regulated uPAR expression, possibly by releasing a GPI-anchored growth factor. Our data suggest that cellular GPI-PLD might be involved in the generation of circulating prognostic markers in cancer and possibly regulate the function of GPI-anchored proteins by generating functionally distinct, soluble counterparts.