The membrane bound bacterial lipocalin Blc is a functional dimer with binding preference for lysophospholipids

Lipocalins, a widespread multifunctional family of small proteins (15-25kDa) have been first described in eukaryotes and more recently in Gram-negative bacteria. Bacterial lipocalins belonging to class I are outer membrane lipoproteins, among which Blc from E. coli is the better studied. Blc is expressed under conditions of starvation and high osmolarity, conditions known to exert stress on the cell envelope. The structure of Blc that we have previously solved (V. Campanacci, D. Nurizzo, S. Spinelli, C. Valencia, M. Tegoni, C. Cambillau, FEBS Lett. 562 (2004) 183-188.) suggested its possible role in binding fatty acids or phospholipids. Both physiological and structural data on Blc, therefore, point to a role in storage or transport of lipids necessary for membrane maintenance. In order to further document this hypothesis for Blc function, we have performed binding studies using fluorescence quenching experiments. Our results indicate that dimeric Blc binds fatty acids and phospholipids in a micromolar K(d) range. The crystal structure of Blc with vaccenic acid, an unsaturated C18 fatty acid, reveals that the binding site spans across the Blc dimer, opposite to its membrane anchored face. An exposed unfilled pocket seemingly suited to bind a polar group attached to the fatty acid prompted us to investigate lyso-phospholipids, which were found to bind in a nanomolar K(d) range. We discuss these findings in terms of a potential role for Blc in the metabolism of lysophospholipids generated in the bacterial outer membrane.     

Surface pretreatment for prolonged survival of cemented tibial prosthesis components: full- vs. surface-cementation technique]

Aseptic loosening of tibial components due to degradation of the interface between bone cement and metallic tibial shaft component is still a persistent problem, particularly for surface-cemented tibial components. The surface cementation technique has important clinical meaning in case of revision and for avoidance of stress shielding. This study was done to prove crack formation in the bone cement near the metallic surface when this is not coated. We propose a newly developed coating process by SiOx-PVD layering to avoid crack formation. A biomechanical model for a vibration fatigue test was done to prove that crack formation can be significantly reduced in the case of coated surfaces. It was found that coated tibial components showed a highly significant reduction of cement cracking near the metal/bone cement interface (p < 0.01) and a significant reduction of gap formation in the metal-to-bone cement interface (p < 0.05). Coating dramatically reduces hydrolytic- and stress-related crack formation at the prosthesis metal/bone cement interface. This leads to a more homogenous load transfer into the cement mantle which should reduce the frequency of loosening in the metal/bone cement/bone interfaces. With surface coating of the tibial component it should become possible that surface-cemented TKAs reveal similar loosening rates as TKAs both surface- and stem-cemented. This would be an important clinical advantage since it is believed that surface cementing reduces metaphyseal bone loss in the case of revision and stress shielding for a better bone health.     

Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling

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Paracrine effect of CXCR4‐overexpressing mesenchymal stem cells on ischemic heart injury

It has been reported that CXCR4‐overexpressing mesenchymal stem cells (MSC^CX4) can repair heart tissue post myocardial infarction. This study aims to investigate the MSCCX4‐derived paracrine cardio‐protective signaling in the presence of myocardial infarction. Mesenchymal stem cells (MSCs) were divided into 3 groups: MSC only, MSC^CX4, and CXCR4 gene‐specific siRNA‐transduced MSC. Mesenchymal stem cells were exposed to hypoxia, and then MSCs‐conditioned culture medium was incubated with neonatal and adult cardiomyocytes, respectively. Cell proliferation–regulating genes were assessed by real‐time polymerase chain reaction (RT‐PCR). In vitro: The number of cardiomyocytes undergoing DNA synthesis, cytokinesis, and mitosis was increased to a greater extent in MSC^CX4 medium‐treated group than control group, while this proproliferative effect was reduced in CXCR4 gene‐specific siRNA‐transduced MSC–treated cells. Accordingly, the maximal enhancement of vascular endothelial growth factor, cyclin 2, and transforming growth factor‐β2 was observed in hypoxia‐exposed MSC^CX4. In vivo: MSCs were labeled with enhanced green fluorescent protein (EGFP) and engrafted into injured myocardium in rats. The number of EGFP and CD31 positive cells in the MSC^CX4 group was significantly increased than other 2 groups, associated with the reduced left ventricular (LV) fibrosis, the increased LV free wall thickness, the enhanced angiogenesis, and the improved contractile function. CXCR4 overexpression can mobilize MSCs into ischemic area, whereby these cells can promoted angiogenesis and alleviate LV remodeling via paracrine signaling mechanism.     

Differential expression and genetic variation of hepatic messenger RNAs from genetically lean and fat chickens

Although excessive adiposity has become a major drawback in meat type chicken production, few of the genes involved in this process have been characterized so far. In order to identify putative genes involved in adiposity, we performed differential display analysis of RNAs extracted from the liver of divergently selected lean and fat chickens. Twenty-six differential products were selected and purified by single strand conformation polymorphism gel electrophoresis before sequencing and Northern blot analyses. An orthologous sequence of a mammalian cytochrome P450 2C subfamily member was proven to be differentially expressed in the liver of lean and fat chickens and could play an important role in the regulation of adiposity. In mammals, these genes are involved in detoxification of xenobiotics and metabolism of some important biological compounds. Four other genes were found differentially expressed to a lower extent. Some unidentified products were shown to be lean or fat specific, with sequence polymorphism and liver specific expression, strongly suggesting that the related gene could be directly involved in adiposity. Our data indicate that differential display can evidence genes with differential expression and with sequence polymorphism, making this strategy more accurate for differential analysis of messenger RNAs.     

Proteomics of light-harvesting proteins in different plant species. Analysis and comparison by liquid chromatography-electrospray ionization mass spectrometry. Photosystem I

The light-harvesting proteins (Lhca) of photosystem I (PSI) from four monocot and five dicot species were extracted from plant material, separated by reversed-phase high-performance liquid chromatography (HPLC) and subsequently identified on the basis of their intact molecular masses upon on-line hyphenation with electrospray ionization mass spectrometry. Although their migration behavior in gel electrophoresis was very similar, the elution times among the four antenna types in reversed-phase-HPLC differed significantly, even more than those observed for the light-harvesting proteins of photosystem II. Identification of proteins is based on the good agreement between the measured intact molecular masses and the values calculated on the basis of their nucleotide-derived amino acid sequences, which makes the intact molecular masses applicable as intact mass tags. These values match excellently for Arabidopsis, most probably because of the availability of high-quality DNA sequence data. In all species examined, the four antennae eluted in the same order, namely Lhca1 > Lhca3 > Lhca4 > Lhca2. These characteristic patterns enabled an unequivocal assignment of the proteins in preparations from different species. Interestingly, in all species examined, Lhca1 and Lhca2 were present in two or three isoforms. A fifth antenna protein, corresponding to the Lhca6 gene, was found in tomato (Lycopersicon esculentum). However PSI showed a lower heterogeneity than photosystem II. In most plant species, Lhca2 and Lhca4 proteins are the most abundant PSI antenna proteins. The HPLC method used in this study was found to be highly reproducible, and the chromatograms may serve as a highly confident fingerprint for comparison within a single and among different species for future studies of the PSI antenna.     

An iterative calibration method with prediction of post-translational modifications for the construction of a two-dimensional electrophoresis database of mouse mammary gland proteins

Protein databases serve as general reference resources providing an orientation on two-dimensional electrophoresis (2-DE) patterns of interest. The intention behind constructing a 2-DE database of the water soluble proteins from wild-type mouse mammary gland tissue was to create a reference before going on to investigate cancer-associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (M(r)) and isoelectric point (pI) by high resolution 2-DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix-assisted laser desorption/ionization-mass spectrometry. Another method was N-terminal sequencing by Edman degradation. By N-terminal sequencing M(r) and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post-translational modifications of some proteins. The mouse mammary gland 2-DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib-berlin.mpg.de/2D-PAGE.