There is a substantial list of pre-analytical variables that can alter the analysis of blood-derived samples. We have undertaken studies on some of these issues including choice of sample type, stability during storage, use of protease inhibitors, and clinical standardization. As there is a wide range of sample variables and a broad spectrum of analytical techniques in the HUPO PPP effort, it is not possible to define a single list of pre-analytical standards for samples or their processing. We present here a compendium of observations, drawing on actual results and sound clinical theories and practices. Based on our data, we find that (1) platelet-depleted plasma is preferable to serum for certain peptidomic studies; (2) samples should be aliquoted and stored preferably in liquid nitrogen; (3) the addition of protease inhibitors is recommended, but should be incorporated early and used judiciously, as some form non specific protein adducts and others interfere with peptide studies. Further, (4) the diligent tracking of pre-analytical variables and (5) the use of reference materials for quality control and quality assurance, are recommended. These findings help provide guidance on sample handling issues, with the overall suggestion being to be conscious of all possible pre-analytical variables as a prerequisite of any proteomic study.

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Two genes involved in aflatoxin B 1 (AFB1) biosynthesis in Aspergillus parasiticus, nor-1 and ver-1, were localized to a 35-kb region on one A. parasiticus chromosome and to the genomic DNA fragment carried on a single cosmid, NorA. A physical and transcriptional map of the 35-kb genomic DNA insert in cosmid NorA was prepared to help determine whether other genes located in the nor-1-ver-1 region were involved in aflatoxin synthesis. Northern (RNA) analysis performed on RNA isolated from A. parasiticus SU1 grown in aflatoxininducing medium localized 14 RNA transcripts encoded by this region. Eight of these transcripts, previously unidentified, showed a pattern of accumulation similar to that of nor-1 and ver-1, suggesting possible involvement in AFB1 synthesis. To directly test this hypothesis, gene-1, encoding one of the eight transcripts, was disrupted in A. parasiticus CS10, which accumulates the aflatoxin precursor versicolorin A, by insertion of plasmid pAPNVES4. Thin-layer chromatography revealed that gene-1 disruptant clones no longer accumulated versicolorin A. Southern hybridization analysis of these clones indicated that gene-1 had been disrupted by insertion of the disruption vector. These data confirmed that gene-1 is directly involved in AFB1 synthesis. The predicted amino acid sequence of two regions of gene-1 showed a high degree of identity and similarity with the ␤-ketoacyl-synthase and acyltransferase functional domains of polyketide synthases, consistent with a proposed role for gene-1 in polyketide backbone synthesis.

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It is established that the medically significant yersiniae require the presence of physiological levels of Ca2+ (ca. 2.5 mM) for sustained growth at 37°C and that this nutritional requirement is mediated by a shared ca. 70-kb Lcr plasmid. The latter also encodes virulence factors (Yersinia outer membrane proteins lYops] and V antigen) known to be selectively synthesized in vitro at 37°C in Ca2"-deficient medium. In this study, cells of Yersinia pestis KIM were first starved for Ca2+ at 37°C to prevent synthesis of bulk vegetative protein and then, after cell division had ceased, pulsed with [35S]methionine. After sufficient chase to ensure plasminogen activator-mediated degradation of Yops, the remaining major radioactive peptides were separated by conventional chromatographic methods and identified as Lcr plasmid-encoded V antigen and LcrH (and possibl LcrG), ca. 10-kb Pst plasmid-encoded pesticin and plasminogen activator, ca. 100-kb Tox plasmidencoded fraction 1 (capsular) antigen and murine exotoxin, and chromosomally encoded antigen 4 (pH 6 antigen) and antigen 5 (a novel hemin-rich peptide possessing modest catalase activity but not superoxide dismutase activity). Also produced at high concentration was a chromosome-encoded GroEL-like chaperone protein. Accordingly, the transcriptional block preventing synthesis of bulk vegetative protein at 37°C in Ca2+-deficient medium may not apply to genes encoding virulence factors or to highly conserved GroEL (known in other species to utilize a secondary stress-induced sigma factor).The medically significant yersiniae consist of Yersinia pestis, the causative agent of bubonic plague, and the closely related enteropathogenic species Yersinia pseudotuberculosis and Yersinia enterocolitica. Wild-type cells of these three species share an approximately 70-kb Lcr (low-calcium response) plasmid encoding a set of regulatory genes that mediate shutoff of cell division at 37°C in Ca2"-deficient media (Lcr+ phenotype) (4,8,14,46,59). Little is known about the mechanism responsible for this unique form of restriction other than that Ca2+-starved yersiniae remain viable while undergoing an ordered metabolic stepdown resulting in concomitant reduction of adenylate energy charge and shutoff of stable RNA synthesis (12, 66). As an indirect consequence of these events, the organisms become progressively blocked in synthesis of the bulk cellular protein needed for vegetative growth (12,34). It is therefore significant that this same Ca2+-deficient environment promotes selective expression of most Lcr plasmid-encoded virulence factors (4,8,14,46,59).These virulence factors consist of a series of released proteins termed Yersinia outer membrane proteins (Yops) (6) and a secreted ca. 38-kDa peptide designated LcrV (43) or V antigen (11). This peptide possesses the potential to express an internal secretion sequence (47) that may account for its observed exit from all three species of yersiniae without significant detectable accumulation at the cell surface (52, 59, 60). Evidence derived...

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Enteropathogenic yersiniae (Yersinia pseudotuberculosisand Yersinia enterocolitica) typically cause chronic disease as opposed to the closely related Yersinia pestis, the causative agent of bubonic plague. It is established that this difference reflects, in part, carriage by Y. pestis of a unique 9.6-kb pesticin or Pst plasmid (pPCP) encoding plasminogen activator (Pla) rather than distinctions between shared ∼70-kb low-calcium-response, or Lcr, plasmids (pCD in Y. pestisand pYV in enteropathogenic yersiniae) encoding cytotoxic Yops and anti-inflammatory V antigen. Pla is known to exist as a combination of 32.6-kDa (α-Pla) and slightly smaller (β-Pla) outer membrane proteins, of which at least one promotes bacterial dissemination in vivo and degradation of Yops in vitro. We show here that only α-Pla accumulates in Escherichia coli LE392/pPCP1 cultivated in enriched medium and that either autolysis or extraction of this isolate with 1.0 M NaCl results in release of soluble α and β forms possessing biological activity. This process also converted cell-bound α-Pla to β-Pla and smaller forms in Y. pestis KIM/pPCP1 and Y. pseudotuberculosis PB1/+/pPCP1 but did not promote solubilization. Pla-mediated posttranslational hydrolysis of pulse-labeled Yops in Y. pseudotuberculosis PB1/+/pPCP1 occurred more slowly than that in Y. pestis but was otherwise similar except for accumulation of stable degradation products of YadA, a pYV-mediated fibrillar adhesin not encoded in frame by pCD. Carriage of pPCP by Y. pseudotuberculosis did not significantly influence virulence in mice.