GMDD: a database of GMO detection methods

Background  

Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed.     

Consequences of concurrent Ascaridia galli and Escherichia coli infections in chickens

   

Three experiments were carried out to examine the consequences of concurrent infections with Ascaridia galli and Escherichia coli in chickens raised for table egg production. Characteristic pathological lesions including airsacculitis, peritonitis and/or polyserositis were seen in all groups infected with E. coli. Furthermore, a trend for increased mortality rates was observed in groups infected with both organisms which, however, could not be confirmed statistically. The mean worm burden was significantly lower in combined infection groups compared to groups infected only with A. galli. It was also shown that combined infections of E. coli and A. galli had an added significant negative impact on weight gain.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

Dual structural requirements for multilineage hematopoietic-suppressive activity of chemokine-derived peptides

Many chemokines have direct suppressive activity in vitro and in vivo on primitive hematopoietic cells. However, few chemokine-derived peptides have shown a significant activity in inhibiting hematopoiesis. Interestingly, a peptide derived from the 34-58 sequence of the CXC chemokine platelet factor 4 (PF4) produced a 30-40% inhibition of proliferation of murine hematopoietic progenitors (CFU-MK, CFU-GM, and BFU-E) in vitro, at concentrations of 30-60-fold lower than PF4. The aim of the present work was to define the structural parameters and motifs involved in conferring biological activity to the peptide PF4(34-58). Both structural predictions and determinations revealed a new helical motif that was further localized between residues 38 and 46. This helix was necessary for binding of the peptide and for permitting the functional DLQ motif at position 54-56 to activate the putative receptor site. Peptides lacking either the helical or the DLQ motif were devoid of inhibitory activity on the hematopoietic progenitors in vitro. However, among inactive peptides, only those having the helical motif counteracted the inhibition induced by the active peptide PF4(34-58). This suggested that the helix might be required for peptide interactions with a putative receptor site, whereas the DLQ motif would be implicated in the activation of this receptor. These results identify for the first time the dual requirements for the design of chemokine-derived peptides with high suppressive activity on hematopoiesis, as well as for the design of molecules with antagonistic action.     

Probing conformational changes in the I-like domain and the cysteine-rich repeat of human beta 3 integrins following disulfide bond disruption by cysteine mutations: identification of cysteine 598 involved in alphaIIbbeta3 activation

We have investigated receptor function and epitope expression of recombinant alpha(IIb)beta(3) mutated at Cys(177) or Cys(273) in the I-like domain as well as Cys(598), located in the fourth repeat of the membrane-proximal cysteine-rich region and mutated in a Glanzmann's thrombasthenia type II patient. The beta(3) mutants beta(3)C177A, beta(3)C273A, and beta(3)C598Y exhibited a decreased electrophoretic mobility in SDS-polyacrylamide gel electrophoresis under nonreducing conditions, confirming the disruption of the respective disulfide loops. Despite reduced surface expression, the alpha(IIb)beta(3)C177A, alpha(IIb)beta(3)C273A, and alpha(IIb)beta(3)C598Y receptors mediated cell adhesion to immobilized fibrinogen and translocated into focal adhesion plaques. The beta(3)C598Y mutation, but not the beta(3)C177A or beta(3)C273A mutations, induced spontaneous binding of the ligand mimetic monoclonal antibody PAC-1, while the beta(3)C177A and beta(3)C273A mutants exhibited reduced complex stability in the absence of Ca(2+). Epitope mapping of function-blocking monoclonal antibodies (mAbs) allowed the identification of two distinct subgroups; mAbs A2A9, pl2-46, 10E5, and P256 did not interact with alpha(IIb)beta(3)C273A and bound only weakly to alpha(IIb)beta(3)C177A, while mAbs AP2, LM609 and 7E3 bound normally to mutant alpha(IIb)beta(3)C273A, but interacted only weakly with mutant alpha(IIb)beta(3)C177A. Furthermore, a cryptic epitope recognized by mAb 4D10G3 and not exposed on wild type alpha(IIb)beta(3) became accessible only on mutant alpha(IIb)beta(3)C177A and was mapped to the 60-kDa chymotrypsin fragment of beta(3). Finally, the ligand-induced binding site (LIBS) epitopes AP5, D3, LIBS1, and LIBS2 were spontaneously expressed on all three mutants independent of RGDS or dithiothreitol treatment. Our results provide evidence that disruption of a single cysteine disulfide bond in the cysteine-rich repeat domain, but not in the I-like domain, activates integrin alpha(IIb)beta(3). In contrast, disruption of each of the disulfide bonds in the two long insertions of the I-like domain predicted to be in close contact with the alpha subunit beta-propeller domain affect the stability of the alpha(IIb)beta(3) heterodimer and inhibit complex-specific mAb binding without affecting the RGD binding capacity of the metal ion-dependent adhesion site-like domain.     

Identification of an endothelial cell growth-inhibitory activity produced by human monocytes

The control of endothelial cell proliferation is important in a variety of processes including wound healing and tumor-induced angiogenesis. We have observed that normal unstimulated human monocytes isolated from the blood can inhibit human endothelial cell proliferation. Monocyte-conditioned medium was fractionated by gel filtration chromatography, yielding a 175-fold enrichment of a growth inhibitory activity, designated monocyte-derived endothelial cell inhibitory factor (MECIF). MECIF was found to be protease sensitive, resistant to acid treatment, and heat labile. When conditioned medium was subjected to HPLC gel filtration, the inhibitory activity was eluted as a single peak with a molecular weight of 50-70 kDa. Several characteristics distinguish MECIF from previously described monocyte/macrophage-derived inhibitory factors. Unlike TGF-beta, MECIF is heat labile and does not induce a mitogenic response in growth-arrested normal rat kidney cells. In addition, polyclonal antibodies specific for TGF-beta or INF-gamma do not inhibit MECIF activity. MECIF preparations show low levels of TNF-alpha, insufficient to promote the observed growth inhibitory effect. MECIF activity on human endothelial cells was found to be dose dependent and reversible. MECIF also appeared to be target cell selective in that it did not significantly alter the growth of human smooth muscle cells or skin fibroblasts. These data suggest that monocyte-derived factors may play a key role in inhibiting endothelial cell proliferation.     

Interaction of platelets with endothelial cells: Activation of a novel neutral protease

The activation of a new neutral protease of MW 85,000 was demonstrated on interaction between porcine aortic endothelial cells and human and porcine platelets in culture. The activity of this enzyme, PECAP (platelet endothelial cell activated protease), was detected by electrophoresis on polyacrylamide gels impregnated with the substrate casein. Our results showed that the platelet-endothelial cell interaction did not involve induction of synthesis of de novo enzyme, but rather an activation of a latent enzyme. PECAP cleaved casein and fibrinogen, but had no activity against gelatin or elastin. It was not inhibited by inhibitors of metalloproteases (EDTA, 1.10 phenanthroline), serine proteases (phenylmethylsulfonyl fluoride, elastatinal), or cysteine proteases (iodoacetate, N-ethylmaleimide) and it seems to be unrelated to the previously known proteases of mesenchymal and hematopoietic cells.