OXIDATIVE PHOSPHORYLATION AND ULTRASTRUCTURAL TRANSFORMATION IN MITOCHONDRIA IN THE INTACT ASCITES TUMOR CELL

We have examined the ultrastructure of mitochondria as it relates to energy metabolism in the intact cell. Oxidative phosphorylation was induced in ultrastructurally intact Ehrlich ascites tumor cells by rapidly generating intracellular adenosine diphosphate from endogenous adenosine triphosphate by the addition of 2-deoxyglucose. The occurrence of oxidative phosphorylation was ascertained indirectly by continuous and synchronous monitoring of respiratory rate, fluorescence of pyridine nucleotide, and 90° light-scattering. Oxidative phosphorylation was confirmed by direct enzymatic analysis of intracellular adenine nucleotides and by determination of intracellular inorganic orthophosphate. Microsamples of cells rapidly fixed for electron microscopy revealed that, in addition to oxidative phosphorylation, an orthodox → condensed ultrastructural transformation occurred in the mitochondria of all cells in less than 6 sec after the generation of adenosine diphosphate by 2-deoxyglucose. A 90° light-scattering increase, which also occurs at this time, showed a t ½ of only 25 sec which agreed temporally with a slower orthodox → maximally condensed mitochondrial transformation. Neither oxidative phosphorylation nor ultrastructural transformation could be initiated in mitochondria in intact cells by the intracellular generation of adenosine diphosphate in the presence of uncouplers of oxidative phosphorylation. Partial and complete inhibition of oxidative phosphorylation by oligomycin resulted in a positive relationship to partial and complete inhibition of 2-deoxyglucose-induced ultrastructural transformation in the mitochondria in these cells. The data presented reveal that an orthodox → condensed ultrastructural transformation is linked to induced oxidative phosphorylation in mitochondria in the intact ascites tumor cell.     

A single-step competitive binding assay for mapping of single DNA molecules

Optical mapping of genomic DNA is of relevance for a plethora of applications such as scaffolding for sequencing and detection of structural variations as well as identification of pathogens like bacteria and viruses. For future clinical applications it is desirable to have a fast and robust mapping method based on as few steps as possible. We here demonstrate a single-step method to obtain a DNA barcode that is directly visualized using nanofluidic devices and fluorescence microscopy. Using a mixture of YOYO-1, a bright DNA dye, and netropsin, a natural antibiotic with very high AT specificity, we obtain a DNA map with a fluorescence intensity profile along the DNA that reflects the underlying sequence. The netropsin binds to AT-tetrads and blocks these binding sites from YOYO-1 binding which results in lower fluorescence intensity from AT-rich regions of the DNA. We thus obtain a DNA barcode that is dark in AT-rich regions and bright in GC-rich regions with kilobasepair resolution. We demonstrate the versatility of the method by obtaining a barcode on DNA from the phage T4 that captures its circular permutation and agrees well with its known sequence.     

Zebrafish enhancer trap line recapitulates embryonic aquaporin 1a expression pattern in vascular endothelial cells

Aquaporin 1 (Aqp1) is a water channel protein, expressed widely in microvascular endothelial cells and implicated in mammalian tumor angiogenesis. However, its developmental expression has not yet been characterized in great detail. An enhancer trap screen was performed using a Tol2-derived GFP reporter in zebrafish embryos. An insertional Et(GBT-B1)tpl1 line was identified that has reporter insertion in the vicinity of the aqp1a gene. We further characterized the embryonic expression pattern of this GFP reporter line, as well as that of endogenous aqp1a. Both endogenous aqp1a and reporter GFP expression were restricted to the vascular endothelial cells within the dorsal aorta, cranial, intersegmental and other secondary vessels, but were absent in the axial venous vasculature. In addition, endogenous aqp1a expression was observed in both primitive and definitive hematopoietic erythroid progenitors, as well as in the otic vesicle, swim bladder, pneumatic duct, intestine and a subset of neurons within the retina and the midbrain-hindbrain region. We further show that gata1 and etsrp/etv2 function is required for hematopoietic and endothelial aqp1a expression, respectively. Aqp1a expression is restricted to endothelial and erythroid cells during early embryogenesis. The transgenic Et(GBT-B1)tpl1 line recapitulates endogenous endothelial aqp1a expression. Because currently very few reporter lines can differentiate between arterial and venous endothelial cells, the Et(GBT-B1)tpl1 transgenic line and characterization of the aqp1a expression pattern will be useful for future studies of endothelial and arterial-venous differentiation.     

Unique Substrates Secreted by the Type VI Secretion System of Francisella tularensis during Intramacrophage Infection

Gram-negative bacteria have evolved sophisticated secretion machineries specialized for the secretion of macromolecules important for their life cycles. The Type VI secretion system (T6SS) is the most widely spread bacterial secretion machinery and is encoded by large, variable gene clusters, often found to be essential for virulence. The latter is true for the atypical T6SS encoded by the Francisella pathogenicity island (FPI) of the highly pathogenic, intracellular bacterium Francisella tularensis. We here undertook a comprehensive analysis of the intramacrophage secretion of the 17 FPI proteins of the live vaccine strain, LVS, of F. tularensis. All were expressed as fusions to the TEM β-lactamase and cleavage of the fluorescent substrate CCF2-AM, a direct consequence of the delivery of the proteins into the macrophage cytosol, was followed over time. The FPI proteins IglE, IglC, VgrG, IglI, PdpE, PdpA, IglJ and IglF were all secreted, which was dependent on the core components DotU, VgrG, and IglC, as well as IglG. In contrast, the method was not directly applicable on F. novicida U112, since it showed very intense native β-lactamase secretion due to FTN_1072. Its role was proven by ectopic expression in trans in LVS. We did not observe secretion of any of the LVS substrates VgrG, IglJ, IglF or IglI, when tested in a FTN_1072 deficient strain of F. novicida, whereas IglE, IglC, PdpA and even more so PdpE were all secreted. This suggests that there may be fundamental differences in the T6S mechanism among the Francisella subspecies. The findings further corroborate the unusual nature of the T6SS of F. tularensis since almost all of the identified substrates are unique to the species.     

Hematopoietic stem cells are regulated by Cripto, as an intermediary of HIF-1α in the hypoxic bone marrow niche

Cripto has been known as an embryonic stem (ES)- or tumor-related soluble/cell membrane protein. In this study, we demonstrated that Cripto has a role as an important regulatory factor for hematopoietic stem cells (HSCs). Recombinant Cripto sustained the reconstitution ability of HSCs in vitro. Flow cytometry analysis uncovered that GRP78, one of the candidate receptors for Cripto, was expressed on a subset of HSCs and could distinguish dormant/myeloid-biased HSCs and active/lymphoid-biased HSCs. Cripto is expressed in hypoxic endosteal niche cells where GRP78(+) HSCs mainly reside. Proteomics analysis revealed that Cripto-GRP78 binding stimulates glycolytic metabolism-related proteins and results in lower mitochondrial potential in HSCs. Furthermore, conditional knockout mice for HIF-1α, a master regulator of hypoxic responses, showed reduced Cripto expression and decreased GRP78(+) HSCs in the endosteal niche area. Thus, Cripto-GRP78 is a novel HSC regulatory signal mainly working in the hypoxic niche.     

Automatisation de l’exploration plaquettaire: présentation des systèmes technologiques disponibles en biologie clinique

The global study of primary hemostasis process is regularly performed in case of bleeding syndrome. The study of the role of platelets in the pathogenesis of bleeding syndrome is frequently necessary but also in case of ischemic vascular disease. Classical platelet aggregation tests and cytometry analysis are not suited for routine used. Specialized assays will be planned in expert laboratories. Today several techniques are in use to measure platelet aggregation. The monitoring of antiplatelet treatments is up to date and requires reliable platelet function tests. The recent development of rapid whole blood point of care platelet function assays allows the monitoring of platelet inhibition in much larger populations than previously possible. The results are well correlated with turbidimetric aggregometry and receptor binding assays. Ready to use apparatus at any time without any previous preparation are useful in the investigation of haemostatic disorders and ischemic vascular diseases. But platelet aggregometry and cytometry assays remain gold standard methods in case of difficult diagnosis.     

Synthesis and structural study of pyrimidyl carbocyclic analogues of nucleosides based on cyclopentene rings

A series of 1,2-disubstituted carbonucleoside analogues of pyrimidine and 5-halopyrimidines with the unsaturated carbocycle cyclopentene was synthesized. AIM theory was applied to analyse the conformational and electronic effects of 5-halogenation.     

Induced repatterning of type XVIII collagen expression in ureter bud from kidney to lung type: association with sonic hedgehog and ectopic surfactant protein C

Epithelial-mesenchymal tissue interactions regulate the formation of signaling centers that play a role in the coordination of organogenesis, but it is not clear how their activity leads to differences in organogenesis. We report that type XVIII collagen, which contains both a frizzled and an endostatin domain, is expressed throughout the respective epithelial bud at the initiation of lung and kidney organogenesis. It becomes localized to the epithelial tips in the lung during the early stages of epithelial branching, while its expression in the kidney is confined to the epithelial stalk region and is lost from the nearly formed ureter tips, thus displaying the reverse pattern to that in the lung. In recombinants, between ureter bud and lung mesenchyme, type XVIII collagen expression pattern in the ureter bud shifts from the kidney to the lung type, accompanied by a shift in sonic hedgehog expression in the epithelium. The lung mesenchyme is also sufficient to induce ectopic lung surfactant protein C expression in the ureter bud. Moreover, the shift in type XVIII collagen expression is associated with changes in ureter development, thus resembling aspects of early lung type epigenesis in the recombinants. Respecification of collagen is necessary for the repatterning process, as type XVIII collagen antibody blocking had no effect on ureter development in the intact kidney, whereas it reduced the number of epithelial tips in the lung and completely blocked ureter development with lung mesenchyme. Type XVIII collagen antibody blocking also led to a notable reduction in the expression of Wnt2, which is expressed in the lung mesenchyme but not in that of the kidney, suggesting a regulatory interaction between this collagen and Wnt2. Respecification also occurred in a chimeric organ containing the ureter bud and both kidney and lung mesenchymes, indicating that the epithelial tips can integrate the morphogenetic signals independently. A glial cell line-derived neurotrophic factor signal induces loss of type XVIII collagen from the ureter tips and renders the ureter bud competent for repatterning by lung mesenchyme-derived signals. Our data suggest that differential organ morphogenesis is regulated by an intra-organ patterning process that involves coordination between inductive signals and matrix molecules, such as type XVIII collagen.