Tissue distribution and biosynthesis of 1,2-saturated pyrrolizidine alkaloids in Phalaenopsis hybrids (Orchidaceae)

Phalaenopsis hybrids contain two 1,2-saturated pyrrolizidine monoesters, T-phalaenopsine (necine base trachelanthamidine) and its stereoisomer Is-phalaenopsine (necine base isoretronecanol). T-Phalaenopsine is the major alkaloid accounting for more than 90% of total alkaloid. About equal amounts of alkaloid were genuinely present as free base and its N-oxide. The structures were confirmed by GC-MS. The quantitative distribution of phalaenopsine in various organs and tissues of vegetative rosette plants and flowering plants revealed alkaloid in all tissues. The highest concentrations were found in young and developing tissues (e.g., root tips and young leaves), peripheral tissues (e.g., of flower stalks) and reproductive organs (flower buds and flowers). Within flowers, parts that usually attract insect visitors (e.g., labellum with colorful crests as well as column and pollinia) show the highest alkaloid levels. Tracer feeding experiments with (14)C-labeled putrecine revealed that in rosette plants the aerial roots were the sites of phalaenopsine biosynthesis. However active biosynthesis was only observed in roots still attached to the plant but not in excised roots. There is a slow but substantial translocation of newly synthesized alkaloid from the roots to other plant organs. A long-term tracer experiment revealed that phalaenopsine shows neither turnover nor degradation. The results are discussed in the context of a polyphyletic molecular origin of the biosynthetic pathways of pyrrolizidine alkaloids in various scattered angiosperm taxa. The ecological role of the so called non-toxic 1,2-saturated pyrrolizidine alkaloids is discussed in comparison to the pro-toxic 1,2-unsaturated pyrrolizidine alkaloids. Evidence from the plant-insect interphase is presented indicating a substantial role of the 1,2-saturated alkaloids in plant and insect defense.     

GFP-labelling of 26S proteasomes in living yeast: insight into proteasomal functions at the nuclear envelope/rough ER

26S proteasomes are multisubunit protease complexes that play the central role in the ubiquitin-dependent protein degradation pathway. The proteolytically active core is formed by the 20S proteasome. Regulatory subunits, principally the 19S cap complex, confer the specificity towards ubiquitinated substrates and an ATP-dependence on proteolysis. Green fluorescence protein (GFP)-tagged versions of either an -subunit of the 20S core or an ATPase subunit of the 19S cap complex were functionally incorporated into the protease complex, thus allowing to monitor the subcellular distribution of 26S proteasomes in living yeast. Our localization studies suggest that proteasomal proteolysis mainly occurs at the nuclear envelope (NE)/rough ER. Implications of proteasomal functions at the NE/rough ER are discussed in the context of published work on ER degradation and with regard to possible targeting mechanisms.     

Gene expression analysis of ischaemia and reperfusion in human microsurgical free muscle tissue transfer

The aim of this study was to analyse various gene expression profiles of muscle tissue during normoxia, ischaemia and after reperfusion in human muscle free flaps, to gain an understanding of the occurring regulatory, inflammatory and apoptotic processes on a cellular and molecular basis. Eleven Caucasian patients with soft tissue defects needing coverage with microsurgical free muscle flaps were included in this study. In all patients, the muscle samples were taken from free myocutaneous flaps. The first sample was taken before induction of ischaemia in normoxia (I), another one after ischaemia (II), and the last one was taken after reperfusion (III). The samples were analysed using DNA‐microarray, real‐time‐quantitative‐PCR and immunohistochemistry. DNA‐microarray analysis detected multiple, differentially regulated genes when comparing the different groups (I–III) with statistical significance. Comparing ischaemia (II) versus normoxia (I) educed 13 genes and comparing reperfusion (III) versus ischaemia (II) educed 19 genes. The comparison of reperfusion (III) versus normoxia (I) yielded 100 differentially regulated genes. Real‐time‐quantitative‐PCR confirmed the results of the DNA‐microarrays for a subset of four genes (CASP8, IL8, PLAUR and S100A8). This study shows that ischaemia and reperfusion induces alterations on the gene expression level in human muscle free flaps. Data may suggest that the four genes CASP8, IL8, PLAUR and S100A8 are of great importance in this context. We could not confirm the DNA‐microarry and real‐time‐quantitative‐PCR results on the protein level. Finally, these findings correspond with the surgeon’s clinical experience that the accepted times of ischaemia, generally up to 90 min., are not sufficient to induce pathophysiological processes, which can ultimately lead to flap loss. When inflammatory and apoptotic proteins are expressed at high levels, flap damage might occur and flap loss is likely. The sole expression on mRNA level might explain why flap loss is unlikely.     

Functional and structural insight into properdin control of complement alternative pathway amplification

Properdin (FP) is an essential positive regulator of the complement alternative pathway (AP) providing stabilization of the C3 and C5 convertases, but its oligomeric nature challenges structural analysis. We describe here a novel FP deficiency (E244K) caused by a single point mutation which results in a very low level of AP activity. Recombinant FP E244K is monomeric, fails to support bacteriolysis, and binds weakly to C3 products. We compare this to a monomeric unit excised from oligomeric FP, which is also dysfunctional in bacteriolysis but binds the AP proconvertase, C3 convertase, C3 products and partially stabilizes the convertase. The crystal structure of such a FP-convertase complex suggests that the major contact between FP and the AP convertase is mediated by a single FP thrombospondin repeat and a small region in C3b. Small angle X-ray scattering indicates that FP E244K is trapped in a compact conformation preventing its oligomerization. Our studies demonstrate an essential role of FP oligomerization in vivo while our monomers enable detailed structural insight paving the way for novel modulators of complement.     

The Relationship between Social Capital and Quality Management Systems in European Hospitals: A Quantitative Study

Background

Strategic leadership is an important organizational capability and is essential for quality improvement in hospital settings. Furthermore, the quality of leadership depends crucially on a common set of shared values and mutual trust between hospital management board members. According to the concept of social capital, these are essential requirements for successful cooperation and coordination within groups.

Objectives

We assume that social capital within hospital management boards is an important factor in the development of effective organizational systems for overseeing health care quality. We hypothesized that the degree of social capital within the hospital management board is associated with the effectiveness and maturity of the quality management system in European hospitals.

Methods

We used a mixed-method approach to data collection and measurement in 188 hospitals in 7 European countries. For this analysis, we used responses from hospital managers. To test our hypothesis, we conducted a multilevel linear regression analysis of the association between social capital and the quality management system score at the hospital level, controlling for hospital ownership, teaching status, number of beds, number of board members, organizational culture, and country clustering.

Results

The average social capital score within a hospital management board was 3.3 (standard deviation: 0.5; range: 1-4) and the average hospital score for the quality management index was 19.2 (standard deviation: 4.5; range: 0-27). Higher social capital was associated with higher quality management system scores (regression coefficient: 1.41; standard error: 0.64, p=0.029).

Conclusion

The results suggest that a higher degree of social capital exists in hospitals that exhibit higher maturity in their quality management systems. Although uncontrolled confounding and reverse causation cannot be completely ruled out, our new findings, along with the results of previous research, could have important implications for the work of hospital managers and the design and evaluation of hospital quality management systems.     

Pulmonary surfactant protein A enhances endolysosomal trafficking in alveolar macrophages through regulation of Rab7

Surfactant protein A (SP-A), the most abundant pulmonary soluble collectin, modulates innate and adaptive immunity of the lung, partially via its direct effects on alveolar macrophages (AM), the most predominant intra-alveolar cells under physiological conditions. Enhanced phagocytosis and endocytosis are key functional consequences of AM/SP-A interaction, suggesting a SP-A-mediated modulation of small Rab (Ras related in brain) GTPases that are pivotal membrane organizers in both processes. In this article, we show that SP-A specifically and transiently enhances the protein expression of endogenous Rab7 and Rab7b, but not Rab5 and Rab11, in primary AM from rats and mice. SP-A-enhanced GTPases are functionally active as determined by increased interaction of Rab7 with its downstream effector Rab7 interacting lysosomal protein (RILP) and enhanced maturation of cathepsin-D, a function of Rab7b. In AM and RAW264.7 macrophages, the SP-A-enhanced lysosomal delivery of GFP-Escherichia coli is abolished by the inhibition of Rab7 and Rab7 small interfering RNA transfection, respectively. The constitutive expression of Rab7 in AM from SP-A(-/-) mice is significantly reduced compared with SP-A(+/+) mice and is restored by SP-A. Rab7 blocking peptides antagonize SP-A-rescued lysosomal delivery of GFP-E. coli in AM from SP-A(-/-) mice. Activation of Rab7, but not Rab7b, by SP-A depends on the PI3K/Akt/protein kinase Cζ (PKCζ) signal transduction pathway in AM and RAW264.7 macrophages. SP-A induces a Rab7/PKCζ interaction in these cells, and the disruption of PKCζ by small interfering RNA knockdown abolishes the effect of SP-A on Rab7. The data demonstrate a novel role for SP-A in modulating endolysosomal trafficking via Rab7 in primary AM and define biochemical pathways involved.     

Inhibition of multidrug resistance transporters in the diatom Thalassiosira rotula facilitates dye staining.

Cells are protected by multidrug resistance transporters, which remove potentially harmful chemicals entering the cells from the environment or originating endogenously from the cellular metabolism. Multidrug resistance transporters have not been investigated so far in marine eukaryotic algae like diatoms. We investigated the uptake of a calcium-sensitive dye, Fura 2 acetoxymethylester (AM), by the marine diatom Thalassiosira rotula in the presence and absence of substances known to inhibit multidrug resistance transporters (ATP-binding cassette transporters, ABC). Three inhibitors known to block transporters in living organisms were tested in the marine diatom T. rotula. We applied verapamil, which blocks multidrug resistance P-glycoprotein (MDR1), probenecid as an inhibitor of organic anion transport and the specific inhibitor of multidrug resistance-associated protein (MRP), MK571, obtaining positive results with the highly specific MK571. This leads to the assumption that the cells of T. rotula possess MRP transporters. Marine diatom cells can now be loaded by incubation with a calcium-sensitive dye, which facilitates measurements of cellular calcium signals without using methods risking injury of the cell membrane. This opens an avenue for investigation on diatom calcium signalling and perhaps how they process environmental signals.     

Effects of progesterone on iNOS, COX-2, and collagen expression in the cervix.

This study examines the relationship between inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the control of cervical ripening and parturition under normal (normal term pregnancy) and abnormal (preterm labor and prolongation of pregnancy) conditions by (a) measuring changes in the collagen both visually and quantitatively, (b) localizing and characterizing iNOS and COX-2 under normal conditions, and (c) characterizing the changes in iNOS and COX-2 under abnormal conditions. Cervices are obtained from estrus and timed pregnant Sprague-Dawley rats (n=4-10 per group). Preterm labor is induced with Onapristone (3 mg/rat; progesterone antagonist) and the prolongation of pregnancy with progesterone (2.5 mg, twice daily). Collagen changes are measured and visualized with the picrosirius polarization method. RT-PCR is used to characterize the mRNA expression (p<0.05), and immunohistochemistry is used to localize the protein expression for iNOS and COX-2. The organization and birefringence of the collagen during pregnancy decreased and is supported by changes in the luminosity (p<0.001). The iNOS and COX-2 enzymes were localized in cervical smooth muscle, vascular smooth muscle, and epithelium. Under normal conditions, iNOS mRNA levels decreased as COX-2 mRNA levels increased demonstrating an inverse correlation (Spearman r = -0.497; p=0.00295). Onapristone stimulated preterm labor, increasing the iNOS and COX-2 mRNA (p<0.05). The increase demonstrated a positive correlation (Spearman r = 0.456; p=0.03). Progesterone prolonged pregnancy, decreasing the iNOS and COX-2 mRNA (p=0.036). In conclusion, there may be an interaction between the nitric oxide and prostaglandin pathways in cervical ripening and parturition.     

In silico protein interaction analysis using the global proteome machine database

Experiments to probe for protein-protein interactions are the focus of functional proteomic studies, thus proteomic data repositories are increasingly likely to contain a large cross-section of such information. Here, we use the Global Proteome Machine database (GPMDB), which is the largest curated and publicly available proteomic data repository derived from tandem mass spectrometry, to develop an in silico protein interaction analysis tool. Using a human histone protein for method development, we positively identified an interaction partner from each histone protein family that forms the histone octameric complex. Moreover, this method, applied to the α subunits of the human proteasome, identified all of the subunits in the 20S core particle. Furthermore, we applied this approach to human integrin αIIb and integrin β3, a major receptor involved in the activation of platelets. We identified 28 proteins, including a protein network for integrin and platelet activation. In addition, proteins interacting with integrin β1 obtained using this method were validated by comparing them to those identified in a formaldehyde-supported coimmunoprecipitation experiment, protein-protein interaction databases and the literature. Our results demonstrate that in silico protein interaction analysis is a novel tool for identifying known/candidate protein-protein interactions and proteins with shared functions in a protein network.