Surface molecules regulating rolling and adhesion to endothelium of neutrophil granulocytes and MDA-MB-468 breast carcinoma cells and their interaction

The extravasation of leukocytes and tumor cells is a multi-step process with the involvement of various adhesion molecules depending on the three steps rolling, adhesion, and diapedesis. We have developed an in vitro model, by which we investigated the rolling and adhesion of neutrophil granulocytes and MDA-MB-468 human breast carcinoma cells to lung endothelial cells under physiological flow-conditions. We found that norepinephrine had an inhibitory function on the fMLP-promoted adhesion of neutrophil granulocytes due to a down-regulation of β2-integrin. Furthermore, neutrophil granulocytes serve as linking cells for the interaction of the MDA-MB-468 cells with the endothelium, which are both β2-integrin negative, but express the β2-integrin ligand ICAM-1. In addition, we show here that N-cadherin is up-regulated on the endothelial cells and on neutrophil granulocytes in response to fMLP. This up-regulation resulted in a significant increase of adherent MDA-MB-468 cells, which are also N-cadherin positive. Received 3 September 2007; received after revision 17 October 2007; accepted 22 October 2007     

The regulation of ion channels and transporters by glycolytically derived ATP

Glycolysis is an evolutionary conserved metabolic pathway that provides small amounts of energy in the form of ATP when compared to other pathways such as oxidative phosphorylation or fatty acid oxidation. The ATP levels inside metabolically active cells are not constant and the local ATP level will depend on the site of production as well as the respective rates of ATP production, diffusion and consumption. Membrane ion transporters (pumps, exchangers and channels) are located at sites distal to the major sources of ATP formation (the mitochondria). We review evidence that the glycolytic complex is associated with membranes; both at the plasmalemma and with membranes of the endo/sarcoplasmic reticular network. We examine the evidence for the concept that many of the ion transporters are regulated preferentially by the glycolytic process. These include the Na⁺/K⁺-ATPase, the H⁺-ATPase, various types of Ca²⁺-ATPases, the Na⁺/H⁺ exchanger, the ATP-sensitive K⁺ channel, cation channels, Na⁺ channels, Ca²⁺ channels and other channels involved in intracellular Ca²⁺ homeostasis. Regulation of these pumps, exchangers and ion channels by the glycolytic process has important consequences in a variety of physiological and pathophysiological processes, and a better understanding of this mode of regulation may have important consequences for developing future strategies in combating disease and developing novel therapeutic approaches. Received 20 July 2007; received after revision 30 July 2007; accepted 17 August 2007     

Alcohol dehydrogenase 2 is a major hepatic enzyme for human retinol metabolism

The metabolism of all-trans- and 9-cis-retinol/ retinaldehyde has been investigated with focus on the activities of human, mouse and rat alcohol dehydrogenase 2 (ADH2), an intriguing enzyme with apparently different functions in human and rodents. Kinetic constants were determined with an HPLC method and a structural approach was implemented by in silico substrate dockings. For human ADH2, the determined K_m values ranged from 0.05 to 0.3 μM and k_cat values from 2.3 to 17.6 min⁻¹, while the catalytic efficiency for 9-cis-retinol showed the highest value for any substrate. In contrast, poor activities were detected for the rodent enzymes. A mouse ADH2 mutant (ADH2Pro47His) was studied that resembles the human ADH2 setup. This mutation increased the retinoid activity up to 100-fold. The K_m values of human ADH2 are the lowest among all known human retinol dehydrogenases, which clearly support a role in hepatic retinol oxidation at physiological concentrations. Received 12 October 2006; received after revision 6 December 2006; accepted 8 January 2007     

tRNase Z: the end is not in sight

Although the enzyme tRNase Z has only recently been isolated, a plethora of data has already been acquired concerning the enzyme. tRNase Z is the endonuclease that catalyzes the removal of the tRNA 3′ trailer, yielding the mature tRNA 3′ end ready for CCA addition and aminoacylation. Another substrate cleaved by tRNase Z is the small chromogenic phosphodiester bis(p-nitrophenyl)phosphate (bpNPP), which is the smallest tRNase Z substrate known so far. Hitherto the biological function as tRNA 3′-end processing enzyme has been shown only in one prokaryotic and one eukaryotic organism, respectively. This review summarizes the present information concerning the two tRNase Z substrates pre-tRNA and bpNPP, as well as the metal requirements of tRNase Z enzymes. Received 29 March 2007; received after revision 15 May 2007; accepted 21 May 2007     

Cellular internalization of proinsulin C-peptide

Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37°C for up to 1 h, and was reduced at 4°C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action. Received 31 October 2006; received after revision 27 December 2006; accepted 30 December 2006     

Variation in FTO contributes to childhood obesity and severe adult obesity

We identified a set of SNPs in the first intron of the FTO (fat mass and obesity associated) gene on chromosome 16q12.2 that is consistently strongly associated with early-onset and severe obesity in both adults and children of European ancestry with an experiment-wise P value of 1.67 x 10(-26) in 2,900 affected individuals and 5,100 controls. The at-risk haplotype yields a proportion of attributable risk of 22% for common obesity. We conclude that FTO contributes to human obesity and hence may be a target for subsequent functional analyses.     

The Shwachman-Bodian-Diamond syndrome protein mediates translational activation of ribosomes in yeast

The autosomal recessive disorder Shwachman-Diamond syndrome, characterized by bone marrow failure and leukemia predisposition, is caused by deficiency of the highly conserved Shwachman-Bodian-Diamond syndrome (SBDS) protein. Here, we identify the function of the yeast SBDS ortholog Sdo1, showing that it is critical for the release and recycling of the nucleolar shuttling factor Tif6 from pre-60S ribosomes, a key step in 60S maturation and translational activation of ribosomes. Using genome-wide synthetic genetic array mapping, we identified multiple TIF6 gain-of-function alleles that suppressed the pre-60S nuclear export defects and cytoplasmic mislocalization of Tif6 observed in sdo1Delta cells. Sdo1 appears to function within a pathway containing elongation factor-like 1, and together they control translational activation of ribosomes. Thus, our data link defective late 60S ribosomal subunit maturation to an inherited bone marrow failure syndrome associated with leukemia predisposition.     

Association scan of 14,500 nonsynonymous SNPs in four diseases identifies autoimmunity variants

We have genotyped 14,436 nonsynonymous SNPs (nsSNPs) and 897 major histocompatibility complex (MHC) tag SNPs from 1,000 independent cases of ankylosing spondylitis (AS), autoimmune thyroid disease (AITD), multiple sclerosis (MS) and breast cancer (BC). Comparing these data against a common control dataset derived from 1,500 randomly selected healthy British individuals, we report initial association and independent replication in a North American sample of two new loci related to ankylosing spondylitis, ARTS1 and IL23R, and confirmation of the previously reported association of AITD with TSHR and FCRL3. These findings, enabled in part by increased statistical power resulting from the expansion of the control reference group to include individuals from the other disease groups, highlight notable new possibilities for autoimmune regulation and suggest that IL23R may be a common susceptibility factor for the major 'seronegative' diseases.