In this study, a standard method by gas chromatography coupled with mass spectrometry (GC/MS) for the analysis of classical organic explosives was developed. This method was validated in the EI mode, based on the XPT 90-210 standard method. Detection limits (LOD) and quantitation limits (LOQ) were both determined using electronic impact (EI) and negative ion chemical ionization (NICI) modes. These were compared and results showed that in the NICI mode, detection limits were lower than in the EI mode, thus NICI mode appeared to be the best way to analyze nitrate esters. Results of ion trap MS detection were then compared with those obtained in a previous study with single quadrupolar technology. Major ions that were obtained using ion trap MS detection in these two modes were reported.
Most purification schemes of calpain (CANP) involve a number of chromatographic steps. The final preparations often contain impurities, including degradation fragments. Two peptide‐affinity columns were developed, using peptides of 27 amino acids and 30 amino acids, corresponding to the products of exons 1B and 1C, respectively, of the natural inhibitor (calpastatin) gene, coupled to CNBr‐activated Sepharose® 4B. Crude preparations of calpain, isolated by anion‐exchange chromatography on a DEAE‐Sepharose® column, were incubated with a reversible or an irreversible synthetic inhibitor which blocks the catalytic subunit of the enzyme in the inactive 80‐kDa form. The crude preparation was then loaded onto the peptide column in the presence of calcium. Calpain was eluted with an EGTA‐containing buffer. Using the two peptide‐affinity columns connected in tandem, calpain was isolated with a high degree of purity, suitable for structural and mechanistic studies, i.e. as an 80/30‐kDa heterodimer or in the form of dissociated monomers.
The membrane-bound form of Fas ligand (FasL) signals apoptosis in target cells through engagement of the death receptor Fas, whereas the proteolytically processed, soluble form of FasL does not induce cell death.However, soluble FasL can be rendered active upon cross-linking.Since the minimal extent of oligomerization of FasL that exerts cytotoxicity is unknown, we engineered hexameric proteins containing two trimers of FasL within the same molecule.This was achieved by fusing FasL to the Fc portion of immunoglobulin G1 or to the collagen domain of ACRP30/adiponectin.Trimeric FasL and hexameric FasL both bound to Fas, but only the hexameric forms were highly cytotoxic and competent to signal apoptosis via formation of a death-inducing signaling complex.Three sequential early events in Fas-mediated apoptosis could be dissected, namely, receptor binding, receptor activation, and recruitment of intracellular signaling molecules, each of which occurred independently of the subsequent one.These results demonstrate that the limited oligomerization of FasL, and most likely of some other tumor necrosis factor family ligands such as CD40L, is required for triggering of the signaling pathways.
We have used a subtractive hybridization procedure to isolate cDNA clones for proteins that are produced by human fibroblasts, but not by their SV40-transformed counterparts.With this technique we found, in addition to fibronectin and collagen VI, a novel GTP-binding protein.Sequencing of overlapping cDNA clones demonstrated that this protein is composed of 364 amino acids with a molecular mass of 41 kDa and a calculated isoelectric point of 9.4.It contains the five sequence motifs Gl-G5 that are conserved in all GTP-binding proteins.Apart from these characteristic motifs the amino acid sequence differs substantially from those of the well characterized G-proteins, but it is similar to those of some recently identified proteins from Caenorhabditis elegans, from Schizosaccharomyces pombe, and from an archaebacterium, suggesting the existence of a new subfamily within the superfamily of the GTP-binding proteins.The striking conservation of the primary structure between distantly related species indicates a fundamental function of the new protein.Since it is produced in normal, but not in virally transformed fibroblasts, it may play a role in the expression of the transformed phenotype or in growth control.
The catalytic subunit (L-microCANP) of human calpain I (muCANP, the high Ca2+ affinity form) and two of its mutants were expressed in Escherichia coli or using the baculovirus Sf9 system. The mutants lacked domain III (L-mu CANPDelta3) and the calmodulin-like domain IV (L-mu CANPDelta4), respectively. The bacterially expressed proteins were solubilized from the inclusion bodies and refolded with polyethylene glycol. In Sf9 cells, co-expression of the inhibitor calpastatin was necessary to prevent autolysis of L-muCANP, whereas co-expression of the regulatory subunit enhanced it. Only very low levels of mRNA of the truncated form L-mu CANPDelta4 were found in bacmid-transfected Sf9 cells, and it proved impossible to isolate this mutant using the baculovirus expression system. While the apparent Km(Ca2+) of freshly isolated human erythrocyte muCANP was about 60 microM, the recombinant monomeric forms L-mu CANP and L-mu CANPDelta3 required 65-215 and 400-530 microM Ca2+, respectively. Bacterially expressed L-mu CANPDelta4 was Ca2+-independent; the presence of inhibitors during its renaturation was necessary to prevent its autolysis. A chimeric form (L-mu mCANP) composed by domains I-III of muCANP and domain IV of calpain II (mCANP, the low Ca2+ affinity form) was also expressed in Sf9 cells. This mutant required less Ca2+ (about 50 microM) than native erythrocyte calpain for half-maximal activity and had the highest specific activity of all calpains tested. Domain III proved unnecessary for the activity of the recombinant catalytic subunit, but its absence raised the Km(Ca2+) and removed its inactivation at high Ca2+ concentrations. All recombinant proteins were active as monomers in polyethylene glycol-containing buffers; the in vitro association with the regulatory subunit enhanced only slightly the Vmax and the Ca2+ dependence of the expressed proteins. Activation by Ca2+ promoted the separation of the two subunits of the expressed recombinant proteins.
