Mary C. Hurley

Identification of blood-protein carriers of the CA 19-9 antigen and characterization of prevalence in pancreatic diseases.

The current best serum marker for pancreatic cancer, CA 19‐9, detects a carbohydrate antigen on multiple protein carriers. Better knowledge of the protein carriers of the CA 19‐9 antigen in various disease states may lead to improved diagnostic tests. To identify proteins that carry the CA 19‐9 antigen, we immunoprecipitated the CA 19‐9 antigen from pooled sera and identified the associated proteins using MS. Among the high‐confidence identifications, we confirmed the presence of the CA 19‐9 antigen on Apolipoprotein B‐100 by antibody arrays and Western blot and on kininogen, ARVCF, and Apolipoprotein E by antibody arrays. We characterized the frequency and levels of the CA 19‐9 antigen on the four proteins across various patient groups (pancreatic cancer, pancreatitis, and healthy controls) using antibody arrays. Nearly, 10–25% of the subjects showed elevations of the antigen on each protein, but the elevations were not associated with disease state or total CA 19‐9 levels. These results contribute to our knowledge of the carrier proteins of an important functional glycan and the rate at which the glycan is displayed. This work also demonstrates a strategy for using the complementary methods of MS and antibody microarrays to identify protein carriers of glycans and assess the diagnostic value of measuring glycans on individual proteins.

Insulin and glucagon secretion in essential fatty acid deficient rats

SummaryInsulin and glucagon secretion in response to common secretagogues were ascertained in the perfused pancreas isolated from essential fatty acid deficient rats. The pattern of insulin secretory response to glucose (16.7 mmol/l) by isolated rat pancreas perfused for 30 min was biphasic in EFA-deficient and control rat pancreas. The amplitude of glucose-stimulated acute secretion (phase I) was significantly greater (p<0.01) in magnitude and amplitude in EFA-deficient rats than in the control rats. There was no significant difference in the second phase of glucosestimulated insulin secretion in the two groups. Glucagon secretion in EFA-deficient and control rats was inhibited by glucose (16.7 mmol/l). Glucagon secretion induced by L-arginine (10 mmol/l) was not significantly different in EFA-deficient and in control rat pancreata (p>0.05). However, arginine (10 mmol/l)-stimulated insulin release was significantly higher in EFA-deficient than in control rats. Growth hormone (100 nmol/l)-induced glucagon and insulin secretion was variable in the two groups but significantly higher than basal secretion. The level of L-leucine (10 mmol/l)-stimulated glucagon and insulin secretion in EFA-deficient rats was minimal but significant. Our results show that isolated pancreata of rats devoid of precursors for endogenous prostaglandin synthesis secreted insulin and glucagon in response to common secretagogues. On the basis of our data, it is concluded that endogenous prostaglandins are probably not obligatory for normal secretory functions of islets of Langerhans.

Comparative analysis of the extracellular proteomes of two Clostridium sordellii strains exhibiting contrasting virulence

Clostridium sordellii is a toxin-producing anaerobic bacillus that causes severe infections in humans and livestock. C. sordellii infections can be accompanied by a highly lethal toxic shock syndrome (TSS). Lethal toxin (TcsL) is an important mediator of TSS. We recently obtained a clinical strain of C. sordellii (DA-108) lacking the TcsL-encoding tcsL gene, which was not fatal in rodent models of infection, in contrast to a tcsL(+) reference strain (ATCC9714). Protein preparations derived from cell-free, stationary phase cultures obtained from ATCC9714 were lethal when injected into mice, while those obtained from DA-108 were not, a difference that was attributed to the unique presence of TcsL in the ATCC9714-derived proteins. We questioned whether there were other major differences between the extracellular proteomes of these two strains, apart from TcsL. Two-dimensional gel electrophoresis was conducted using crude cell-free supernatants from these strains and 14 differentially expressed proteins were subjected to mass spectrometric analysis. Nine of these 14 proteins were more highly expressed by DA-108 and 5 by ATCC9714. Twelve of the 14 proteins isolated from the 2-D gels were putatively identified by mass spectrometry. Several of these proteins were identical, possibly reflecting enzymatic cleavage, degradation, and/or post-translational modifications. Excluding identical sequences, only 5 unique proteins were identified. Four proteins (ferredoxin-nitrite reductase; formate acetyltransferase; Translation Elongation Factor G; and purine nucleoside phosphorylase) were over-expressed by DA-108 and 1 (N-acetylmuramoyl-l-alanine amidase) by ATCC9714. These results support the concept that TcsL is the major determinant of C. sordellii TSS during infection.

Measurement of nucleoside kinases in crude tissue extracts

The measurements of deoxyadenosine kinase, adenosine kinase, and deoxycytidine kinase were examined in human placental cytosol to achieve a valid and reliable assay linear with time and protein. Our studies confirm the need to inhibit deaminase enzymes, since deoxyadenosine and deoxycytidine undergo extensive deamination and phosphorolysis. The use of a uniformly labeled nucleoside substrate introduced an artifact because the chromatographic behavior of the deoxyribose-1-phosphate, formed during the assay, was difficult to distinguish from the deoxynucleoside phosphate product. Accurate product identification was also essential. Finally, the substitution of GTP in place of ATP as the phosphate donor, the addition of a sulfhydryl reducing agent and a monovalent cation need to be considered when an assay is optimized. The use of these methods have lead to valid assays in placental cytosol that are linear with time and protein. Consideration of these important principles are necessary when establishing a valid and reliable nucleoside kinase assay in a crude tissue preparation.

Human Placental Nucleoside Kinase Activitiesa

Our studies have indicated that normal human placental cytosol contains a complex mixture of nucleoside kinase enzymes, some of which conform to previously characterized activities. Deoxyadenosine is phosphorylated by deoxycytidine kinase, adenosine kinase, and two as yet uncharacterized activities. Deoxyguanosine phosphorylation is associated with deoxycytidine kinase. More complete and detailed studies will be necessary to characterize these enzymes fully.

Dissociation of vascular resistance with endocrine pancreas secretion: The effects of epoxymethano analogs of PGH2

The epoxymethano analogs of PGH2 caused rapid and persistent increase in perfusion pressure in isolated rat pancreata without significant effect on glucagon and insulin secretory responses to PGH2 and PGE2. The changes in perfusion pressure are interpreted as alterations in vascular resistance since the flow rate was kept constant at 2.5 mL per min. PGH2 alone caused significant elevation in pressure. However, PGH2 administration superimposed upon an infused epoxymethano analog of PGH2, decreased perfusion pressure significantly, whereas PGH2 induced hormone release was not decreased. The analogs neither stimulated nor inhibited the endocrine pancreas secretion. These studies provide evidence for complete dissociation of vascular constriction from pancreatic hormone release and further suggest that the effects of PGH2 on islet hormone secretion may result from the conversion of PGH2 to other prostanoids.

Abstract 5567: Pancreatic disease-specific carriers of the CA 19-9 antigen identified by antibody microarrays and mass spectrometry

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Distinguishing patients with malignant pancreatic tumors from those with benign conditions is critical for doctors to make proper treatment decisions. However, this process is usually costly and invasive due to the lack of a simple detection method such as a blood test. The current standard serological biomarker of pancreatic cancer, the CA 19-9 antigen, is not accurate enough for early diagnosis due to its occasional elevation in benign pancreatic diseases. The CA 19-9 antigen is a carbohydrate epitope found on multiple protein carriers. We sought to determine whether the protein carriers of the CA 19-9 antigen are different between benign and malignant pancreatic disease, and whether these differences could be harnessed to develop improved biomarkers. Two approaches were used to investigate this question. In the first, we used antibody microarrays to capture multiple different mucins from the sera of early-stage pancreatic cancer patients (n = 33), late-stage pancreatic cancer patients (n = 17), pancreatitis patients (n = 38), and healthy control subjects (n = 20), and we probed the level of the CA 19-9 antigen on the captured proteins using a monoclonal antibody. Pancreatic cancer progression was associated with a shift in CA 19-9 carriers from MUC16 to MUC1 and MUC5AC. Certain monoclonal antibodies captured glycoforms of MUC1 that predominantly carried CA 19-9 in the cancer patients. Because of that relationship, the detection of CA 19-9 on specific protein glycoforms improved the discrimination of cancer from control subjects over total CA 19-9 (AUC= 0.93 vs. AUC=0.86. In the second approach, we immunoprecipitated CA19-9 carrier proteins from pooled sera of late-stage cancer, early-stage cancer, pancreatitis, and control subjects, followed by deglycosylation and mass spectrometry (MALDI/TOF/TOF). Several proteins were identified in the sera of the cancer patients but not in the sera of pancreatitis or control subjects, indicating the possible identification of state-specific carriers of CA 19-9. Ongoing studies are aimed at verifying these identifications and testing performance as biomarkers of pancreatic cancer. These findings may lead to more accurate methods of discriminating benign from malignant pancreatic disease, which would improve the ability to render proper care to patients at the earliest possible stages. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5567.

34 PURIFICATION AND PROPERTIES OF HUMAN T-LYMPHOBLAST DEOXYCYTIDINE KINASE

Nucleoside kinases phosphorylate endogenous nucleosides and nucleoside analogs which are anticancer and antiviral drugs. dCytidine kinase was purified from cultured human T-lymphoblasts to a specific activity of 8 umol/min/mg protein and 92% purity. The molecular weight was 60 kD and the stokes radius was 32 A. The subunit molecular weight was 30.5 kD. dGuanosine, dadenosine and cytidine phosphorylating activities copurified with dcytidine kinase to final specific activities of 7.2, 13.5 and 4 umol/min/mg protein, respectively. This enzyme had apparent Km values of 1.5, 430, 500, 450 and 40 uM for dcytidine, dguanosine, dadenosine, cytidine and cytosine arabinoside, respectively. The pH optimum ranged from 6.5-9.0. Mg2+ and Mn2+ were the preferred divalent cations. ATP, GTP, dGTP, ITP, dITP, TTP and XTP were substrates for the enzyme. This highly purified enzyme will facilitate our delineation of the role for this enzyme in nucleoside or nucleoside analog phosphorylation and the structural basis for the regulation of this enzyme.