Identification and characterisation of mutations associated with von Willebrand disease in a Turkish patient cohort.

Several cohort studies have investigated the molecular basis of von Willebrand disease (VWD); however, these have mostly focused on European and North American populations. This study aimed to investigate mutation spectrum in 26 index cases (IC) from Turkey diagnosed with all three VWD types, the majority (73%) with parents who were knowingly related. IC were screened for mutations using multiplex ligation-dependent probe amplification and analysis of all von Willebrand factor gene (VWF) exons and exon/intron boundaries. Selected missense mutations were expressed in vitro. Candidate VWF mutations were identified in 25 of 26 IC and included propeptide missense mutations in four IC (two resulting in type 1 and two in recessive 2A), all influencing VWF expression in vitro. Four missense mutations, a nonsense mutation and a small in-frame insertion resulting in type 2A were also identified. Of 15 type 3 VWD IC, 13 were homozygous and two compound heterozygous for 14 candidate mutations predicted to result in lack of expression and two propeptide missense changes. Identification of intronic breakpoints of an exon 17-18 deletion suggested that the mutation resulted from non-homologous end joining. This study provides further insight into the pathogenesis of VWD in a population with a high degree of consanguineous partnerships.

Bacterial nitric oxide detoxification prevents host cell S-nitrosothiol formation: a novel mechanism of bacterial pathogenesis.

S-nitrosylation is an important mediator of multiple nitric oxide-dependent biological processes, including eukaryotic cellular events such as macrophage apoptosis and proinflammatory signaling. Many pathogenic bacteria possess NO detoxification mechanisms, such as the nitric oxide reductase (NorB) of Neisseria meningitidis and the flavohemoglobins (Hmp) of Salmonella enterica and Escherichia coli, which serve to protect the microorganism from nitrosative stress within the intracellular environment. In this study, we demonstrate that expression of meningococcal NorB increases the rate at which low-molecular-weight S-nitrosothiol (SNO) decomposes in vitro. To determine whether this effect occurs in cells during infection by bacteria, we induced SNO formation in murine macrophages by activation with lipopolysaccharide and gamma-interferon and observed a reduced abundance of SNO during coincubation with N. meningitidis, S. enterica, or E. coli. In each case, this effect was shown to be dependent on bacterial NO detoxification genes, which act to prevent SNO formation through the removal of NO. This may represent a novel mechanism of host cell injury by bacteria.

FNR-mediated regulation of hyp expression in Escherichia coli.

The hypA-E operon is involved in the maturation of all three NiFe hydrogenases in Escherichia coli. Two hyp promoters have been described; a sigma54-dependent promoter upstream of hypA, and a sigma70-dependent promoter (PhypA) within the hypA coding region. Here it is shown that the oxygen-responsive transcription factor FNR regulates PhypA under anaerobic conditions only. PhypA does not possess a canonical FNR recognition sequence, but two FNR half-sites are present. Studies using PHYPA::lacZ fusions carrying lesions in one or both FNR half-sites indicated that although some residual anaerobic activity was retained by the promoter containing only the downstream FNR half-site, both half-sites are required for maximal PhypA activity in vivo. In vitro gel retardation analysis suggested that the primary interaction occurs at the downstream FNR half-site. Possible explanations for these observations and the implications for other FNR-regulated promoters are discussed.

Analysis of intracellular storage and regulated secretion of 3 von Willebrand disease-causing variants of von Willebrand factor.

The rapid exocytosis of von Willebrand factor (VWF) in response to vascular injury can be attributed to the fact that VWF is stored in the Weibel-Palade bodies (WPBs) of endothelial cells. We describe a system for examining the ability of VWF to drive both the formation of a storage compartment and the function of that compartment with respect to regulated secretion. Transient transfection of HEK293 cells with wild-type human VWF cDNA leads to the formation of numerous elongated organelles that resemble WPBs. These "pseudo-WPBs" exhibit the internal structure, as well as the ability to recruit membrane proteins including P-selectin, of bona fide WPBs. Finally, VWF was efficiently secreted upon stimulation by phorbol ester. We used this system to examine 3 VWF mutations leading to von Willebrand disease that affect VWF multimerization and constitutive secretion. Surprisingly we find that all 3 mutants can, to some extent, make pseudo-WPBs that recruit appropriate membrane proteins and that are responsive to secretagogues. The most striking defects are a delay in formation and a reduction in the length and number of pseudo-WPBs in proportion to the clinical severity of the mutation. Studies of pseudo-WPB formation in this system thus yield insights into the structure-function relationships underpinning the ability of VWF to form functional WPBs.

Expression of the Escherichia coli yfiD gene responds to intracellular pH and reduces the accumulation of acidic metabolic end products.

The YfiD protein of Escherichia coli has been reported to be an acid-inducible protein. Here it is shown that expression of a yfiD::lac reporter fusion is enhanced up to 3 small middle dot5-fold during acidic growth. The anaerobic transcription factor FNR was confirmed as the major regulator of yfiD expression, and ArcA was found to enhance anaerobic yfiD expression, probably by displacing a repressing FNR dimer in the -93 small middle dot5 region of the promoter. Moreover, the pyruvate sensor PdhR was shown to act as a minor anaerobic repressor of yfiD expression. On the basis of its strong homology to the C-terminal region of pyruvate formate-lyase (PFL) it was predicted that YfiD would be a radical-containing enzyme. The YfiD radical was found to be introduced by the PFL-activase enzyme, but unlike PFL, AdhE did not deactivate radicalized YfiD. The extent of radical activation of YfiD was enhanced by low intracellular pH, and thus it was concluded that both yfiD expression and YfiD activity are affected by growth at low pH. The yfiD mutant strain JRG4033 excreted increased levels of organic acids compared to the parental strain when grown in chemostat culture under oxygen-starved conditions, consistent with the acid-inducibility of yfiD expression and the recently reported ability of YfiD to rescue the activity of oxygenolytically cleaved PFL.