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D G Ward

Publications and source records attributed to D G Ward.

At least 19 recordsLinked to original sources
PubMed2006

Preclinical and post-treatment changes in the HCC-associated serum proteome.

SELDI-based proteomic profiling of body fluids is currently in widespread use for cancer biomarker discovery. We have successfully used this technology for the diagnosis of hepatocellular carcinoma (HCC) in hepatitis C patients and now report its application to serial serum samples from 37 hepatitis C patients before development of HCC, with HCC and following radiofrequency ablation of the tumour. As with alpha-fetoprotein, an accepted biomarker for HCC, we hypothesised that HCC-associated proteomic features would 'return to normal' following successful treatment and the primary aim of our study was to test this hypothesis. Several SELDI peaks that changed significantly during HCC development were detected but they did not reverse following treatment. These data may be interpreted to suggest that the characteristic SELDI profile is not linearly related to tumour burden but may result from the progression of underlying liver disease or from the emergence of precancerous lesions. beta2-Microglobulin, a protein previously reported to be markedly elevated in patients with HCV related HCC, was also the most significantly HCC associated proteomic feature (m/z 11720) in this study.

Aged↗
PubMed2006

Identification of serum biomarkers for colon cancer by proteomic analysis.

Colorectal cancer (CRC) is often diagnosed at a late stage with concomitant poor prognosis. Early detection greatly improves prognosis; however, the invasive, unpleasant and inconvenient nature of current diagnostic procedures limits their applicability. No serum-based test is currently of sufficient sensitivity or specificity for widespread use. In the best currently available blood test, carcinoembryonic antigen exhibits low sensitivity and specificity particularly in the setting of early disease. Hence, there is great need for new biomarkers for early detection of CRC. We have used surface-enhanced laser desorbtion/ionisation (SELDI) to investigate the serum proteome of 62 CRC patients and 31 noncancer subjects. We have identified proteins (complement C3a des-arg, alpha1-antitrypsin and transferrin) with diagnostic potential. Artificial neural networks trained using only the intensities of the SELDI peaks corresponding to identified proteins were able to classify the patients used in this study with 95% sensitivity and 91% specificity.

Adenocarcinoma↗
PubMed2006

Changes in the serum proteome associated with the development of hepatocellular carcinoma in hepatitis C-related cirrhosis.

Early diagnosis of hepatocellular carcinoma (HCC) is the key to the delivery of effective therapies. The conventional serological diagnostic test, estimation of serum alpha-fetoprotein (AFP) lacks both sensitivity and specificity as a screening tool and improved tests are needed to complement ultrasound scanning, the major modality for surveillance of groups at high risk of HCC. We have analysed the serum proteome of 182 patients with hepatitis C-induced liver cirrhosis (77 with HCC) by surface-enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI). The patients were split into a training set (84 non-HCC, 60 HCC) and a 'blind' test set (21 non-HCC, 17 HCC). Neural networks developed on the training set were able to classify the blind test set with 94% sensitivity (95% CI 73-99%) and 86% specificity (95% CI 65-95%). Two of the SELDI peaks (23/23.5 kDa) were elevated by an average of 50% in the serum of HCC patients (P<0.001) and were identified as kappa and lambda immunoglobulin light chains. This approach may permit identification of several individual proteins, which, in combination, may offer a novel way to diagnose HCC.

Amino Acid Sequence↗
PubMed2005

Liver tumors in wild flatfish: a histopathological, proteomic, and metabolomic study.

Fish play host to viral, bacterial, and parasitic diseases in addition to non-infectious conditions such as cancer. The National Marine Monitoring Programme (NMMP) provides information to the U.K. Government on the health status of marine fish stocks. An aspect of this work relates to the presence of tumors and other pathologies in the liver of the offshore sentinel flatfish species, dab (Limanda limanda). Using internationally agreed quality assurance criteria, tumors and pre-tumors are diagnosed using histopathology. The current study has expanded upon this work by integrating these traditional diagnostic approaches with ones utilizing modern technologies for analysis of proteomic and metabolomic profiles of selected lesions. We have applied SELDI and FT-ICR technologies (for proteomic and metabolomic analyses, respectively) to tumor and non-tumor samples resected from the liver of dab. This combined approach has demonstrated how these technologies are able to identify protein and metabolite profiles that are specific to liver tumors. Using histopathology to classify "analysis groups" is key to the success of such an approach since it allows for elimination of spurious samples (e.g., those containing parasite infections) that may confuse interpretation of "omic" data. As such, the pathology laboratory plays a central role in collating information relating to particular specimens and in establishing sampling groups relative to specific diagnostic questions. In this study, we present pilot data, which illustrates that proteomics and metabolomics can be used to discriminate fish liver tumors and suggest future directions for work of this type.

Adenoma, Liver Cell↗
PubMed2001

Additional PKA phosphorylation sites in human cardiac troponin I.

We used mass spectrometry to monitor cAMP-dependent protein kinase catalysed phosphorylation of human cardiac troponin I in vitro. Phosphorylation of isolated troponin I by cAMP-dependent protein kinase resulted in the covalent incorporation of phosphate on at least five different sites on troponin I, and a S22/23A troponin I mutant incorporated phosphates on at least three sites. In addition to the established phosphorylation sites (S22 and S23) we found that S38 and S165 were the other two main sites of phosphorylation. These 'overphosphorylation' sites were not phosphorylated sufficiently slower than S22 and S23 that we could isolate pure S22/23 bisphosphorylated troponin I. Overphosphorylation of troponin I reduced its affinity for troponin C, as measured by isothermal titration microcalorimetry. Phosphorylation of S22/23A also decreased its affinity for troponin C indicating that phosphorylation of S38 and/or S165 impedes binding of troponin I to troponin C. Formation of a troponin I/troponin C complex prior to cAMP-dependent protein kinase treatment did not prevent overphosphorylation. When whole troponin was phosphorylated by cAMP-dependent protein kinase, however, [(32)P]phosphate was incorporated only into troponin I and only at S22 and S23. Mass spectrometry confirmed that overphosphorylation is abolished in the ternary complex. Troponin I bisphosphorylated exclusively at S22 and S23 troponin I showed reduced affinity for troponin C but the effect was diminished with respect to overphosphorylated troponin I. These results show that care should be exercised when interpreting data obtained with troponin I phosphorylated in vitro.

Binding Sites↗
PubMed2000

The importance of the carboxyl-terminal domain of cardiac troponin C in Ca2+-sensitive muscle regulation.

The interactions between troponin I and troponin C are central to the Ca(2+)-regulated control of striated muscle. Using isothermal titration microcalorimetry we have studied the binding of human cardiac troponin C (cTnC) and its isolated domains to human cardiac troponin I (cTnI). We provide the first binding data for these proteins while they are free in solution and unmodified by reporter groups. Our data reveal that the C-terminal domain of cTnC is responsible for most of the free energy change upon cTnC.cTnI binding. Importantly, the interaction between cTnI and the C-terminal domain of cTnC is 8-fold stronger in the presence of Ca(2+) than in the presence of Mg(2+), suggesting that the C-terminal domain of cTnC may play a modulatory role in cardiac muscle regulation. Changes in the affinity of cTnI for cTnC and its isolated C-terminal domain in response to ionic strength support this finding, with both following similar trends. At physiological ionic strength the affinity of cTnC for cTnI changed very little in response to Ca(2+), although the thermodynamic data show a clear distinction between binding in the presence of Ca(2+) and in the presence of Mg(2+).

Binding Sites↗
PubMed1998

Affinity labeling of two nucleotide sites on Na,K-ATPase using 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-[alpha-32P]diphosphate (TNP-8N3-[alpha-32P]ADP) as a photoactivatable probe. Label incorporation before and after blocking the high affinity ATP site with fluorescein isothiocyanate.

ATP and its analogues act on the minimal functional unit of Na, K-ATPase, the alpha beta protomer, with high and low affinity effects. Fluorescein isothiocyanate (FITC) irreversibly blocks the high affinity, or catalytic, ATP site, and yet the surviving K+-phosphatase activity of soluble FITC-modified alphabeta protomers can be photoinactivated by 2'(3')-O-trinitrophenyl (TNP)-8N3-ADP (Ward, D. G., and Cavieres, J. D. (1998) J. Biol. Chem. 273, 14277-14284). We have now used TNP-8N3-[alpha-32P]ADP as a photoaffinity label for Na,K-ATPase. The native enzyme can be photolabeled at 5 microM TNP-8N3-[alpha-32P]ADP, and ATP or FITC treatment prevents labeling of the alpha chain. At 25 microM, however, TNP-8N3-[alpha-32P]ADP can be incorporated in the FITC-modified alpha chain, concurrently with the inactivation of the K+-phosphatase activity, to an extrapolated level of 0.5-1.2 mol of 32P-probe per mol of alpha chain. Photoinactivation and labeling are prevented by TNP-ADP, vanadate, or strophanthidin and are promoted by Na+ or Mg2+, but not K+. The cation effects suggest that the fluorescein-modified enzyme incorporates the TNP-8N3-[alpha-32P]ADP. Mg complex preferentially, and the free probe when in the E1 enzyme form and after occupation of a low-affinity Na+ site. Partial trypsinolysis reveals that the point of TNP-8N3-[alpha-32P]ADP attachment is on the C-terminal 58-kDa fragment of the FITC-modified alpha chain. The affinity labeling of the fluorescein enzyme by TNP-8N3-[alpha-32P]ADP endorses the view that two nucleotide sites can be occupied simultaneously in each alpha subunit of Na,K-ATPase.

Adenosine Triphosphate↗
PubMed1998

Photoinactivation of fluorescein isothiocyanate-modified Na,K-ATPase by 2'(3')-O-(2,4,6-trinitrophenyl)8-azidoadenosine 5'-diphosphate. Abolition of E1 and E2 partial reactions by sequential block of high and low affinity nucleotide sites.

The Na,K-ATPase activity of the sodium pump exhibits apparent multisite kinetics toward ATP, a feature that is inherent to the minimal enzyme unit, the alpha beta protomer. We have argued that this should arise from separate catalytic and noncatalytic sites on the alpha beta protomer as fluorescein isothiocyanate (FITC) blocks a high affinity ATP site on all alpha subunits and yet the modified Na, K-ATPase retains a low affinity response to nucleotides (Ward, D. G., and Cavieres, J. D. (1996) J. Biol. Chem. 271, 12317-12321). We now find that 2'(3')-O-(2,4,6-trinitrophenyl)8-azido-adenosine 5'-diphosphate (TNP-8N3-ADP), a high affinity photoactivatable analogue of ATP, can inhibit the K+-phosphatase activity of the FITC-modified enzyme during assays in dimmed light. The inhibition occurs with a Ki of 140 microM at 20 mM K+; it requires the adenine ring as 2'(3')-O-(2,4 6-trinitrophenyl) (TNP)-UDP or TNP-uridine are less potent and 2,4,6-trinitrobenzene-sulfonate is ineffective. Under irradiation with UV light, TNP-8N3-ADP inactivates the K+-phosphatase activity of the fluorescein-enzyme and also its phosphorylation by [32P]Pi. The photoinactivation process is stimulated by Na+ or Mg2+, and is inhibited by K+ or excess TNP-ADP. In the presence of 50 mM Na+ and 1 mM Mg2+, TNP-8N3-ADP photoinactivates with a K0.5 of 15 microM. Furthermore, TNP-8N3-ADP photoinactivates the FITC-modified, solubilized alpha beta protomers, even more effectively than the membrane-bound fluorescein-enzyme. These results strongly suggest that catalytic and allosteric ATP sites coexist on the alpha beta protomer of Na,K-ATPase.

Adenosine Diphosphate↗
PubMed1996

Binding of 2'(3')-O-(2,4-6-trinitrophenyl) ADP to soluble alpha beta protomers of Na, K-ATPase modified with fluorescein isothiocyanate. Evidence for two distinct nucleotide sites.

The overall reaction of well-defined solubilized protomers of Na,K-ATPase (one alpha plus one beta subunit) retains the dual ATP dependence observed with the membrane-bound enzyme, with distinctive ATP effects in the submicromolar and submillimolar ranges (Ward, D. G., and Cavieres, J. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5332-5336). We have now found that the K+/-phosphatase activity of the alpha beta protomers is still inhibited by 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP). What is most significant is that the TNP-ADP effect can be observed clearly with protomeric enzyme whose high affinity ATP site has been blocked covalently with fluorescein isothiocyanate. We conclude that nucleotides can bind at two discrete sites in each protomeric unit of Na,K-ATPase.

Adenosine Diphosphate↗
PubMed1996

A deep-tissue hemangioma presenting as a rapidly progressive expanding mass and thrombocytopenia in an infant.

OBJECTIVE: We present a case of Kasabach-Merritt syndrome with discussion of hemangiomas of childhood relevant to the emergency physician. DESIGN: This is a case report. SETTING: The case is described in the setting of a tertiary care hospital level I pediatric emergency department. PATIENTS: A single case is discussed. INTERVENTIONS: The patient's condition was diagnosed and referred to the appropriate hematology service. Methylprednisolone (2 mg/kg/day) therapy was instituted. CONCLUSIONS: Children may present to the emergency department with a mass requiring diagnosis. The emergency physician must efficiently and economically identify the cause and complications from a variety of etiologies. Hemangiomas with complicating thrombocytopenia and microangiopathic hemolysis are rare and must be correctly identified. The emergency physician should occasionally review such unusual diagnoses to maintain their differential diagnosis skills.

Anti-Inflammatory Agents↗
PubMed1993

Solubilized alpha beta Na,K-ATPase remains protomeric during turnover yet shows apparent negative cooperativity toward ATP.

A prominent feature of the Na,K-ATPase reaction is an ATP dependence that suggests high- and low-affinity ATP requirements during the enzymic cycle. As only one ATP-binding domain has been identified in the alpha subunit and none has been identified in the beta subunit, it has seemed likely that the apparent negative cooperativity results from subunit interactions in an (alpha beta)2 diprotomer. To test this possibility, we have examined the behavior of solubilized alpha beta protomers of Na,K-ATPase down to 50 nM [gamma-32P]ATP. Active-enzyme analytical ultracentrifugation shows that the protomer is the active species and that no oligomerization occurs during turnover. However, we find that dual ATP effects can be clearly demonstrated and that nonhydrolyzable ATP analogs can stimulate the Na,K-ATPase activity of the soluble protomer. We conclude that the apparent negative cooperativity is inherent to the alpha beta protomer and that this should explain some of the complexities found with membrane-bound Na,K-ATPase and, perhaps, other P-type cation pumps.

Adenosine Triphosphate↗
PubMed1993

Irreversible effects of calcium ions on the plasma membrane calcium pump.

The calcium pump of human red cells can be irreversibly activated by preincubation of the membranes in the presence of calcium ions, with a pattern reminiscent of that produced by controlled trypsin attack. With 1 mM Ca2+, the activity of the basal enzyme increases three to fourfold over 30 to 60 min, to levels about half those obtained in the presence of calmodulin. On the whole, the effect occurs slowly, with a very low Ca2+ affinity at 37 degrees C and is unaffected by serine-protease inhibitors. The activation caused by 1 mM Ca2+ is little affected by leupeptin (a thiol-protease inhibitor) and that obtained at 10 microM Ca2+ is not inhibited. Preincubations at 0 degrees C also lead to activation, to a level up to half that seen at 37 degrees C, and the effect is not affected by leupeptin or antipain. No activation is observed by preincubating soluble purified Ca,Mg-ATPase in Ca(2+)-containing solutions at 37 degrees C. Instead, calcium ions protect the detergent-solubilized enzyme from thermal inactivation, the effect being half-maximal between 10 and 20 microM Ca2+. We conclude that the activation of the membrane-bound Ca,Mg-ATPase by Ca2+ should result from an irreversible conformational change in the enzyme and not from attack by a membrane-bound protease, and that this change presumably arises from the release of inhibitory particles existing in the original membrane preparations.

Binding Sites↗
PubMed1992

Relaxin increases rat heart rate by a direct action on the cardiac atrium.

Relaxin (Rlx) is best understood as a protein hormone of pregnancy that can influence pelvic and cervical connective tissue as well as uterine smooth muscle activity. Thus, it was unexpected that dense Rlx binding sites would be found in the rat cardiac atrium. To functionally characterize this finding, isolated rat atria were challenged with Rlx (0.3 to 30 ng/ml), and they responded with an increased rate (+36%) and force (+38%) of contraction Further studies in conscious normotensive and spontaneously hypertensive rats established by minipump circulating Rlx levels of about 0.5 and 5 ng/ml over 1 to 2 wks. There were significant increases in heart rate of 10-15%, with no consistent changes in blood or urine volume, water or food intake, and mean arterial pressure. We conclude that Rlx can directly stimulate rat cardiac atrial activity in vitro and cause chronotropy in vivo.

Animals↗
PubMed1991

Lack of cardiovascular and vasopressin responses to human relaxin in conscious, late-pregnant rats.

Measurements of arterial pressure, heart rate, and plasma vasopressin were obtained in unanesthetized late-pregnant rats after administration of human relaxin (hRlx) alone or in conjunction with hemorrhage. Forty-two timed-pregnant rats were prepared with chronic femoral cannulas on the 17th day of pregnancy for measurements on the 19th day. In three separate sets of experiments, mean arterial pressure and heart rate were measured for 10 min before administration of 2 mg/kg hRlx, 100 micrograms/kg hRlx, or vehicle and for 20 h thereafter; plasma vasopressin was determined 20 min before and 3 min after administration of hRlx or vehicle and 20 min after performing a 15-ml/kg 3-min hemorrhage. Neither mean arterial pressure nor heart rate was significantly different among rats administered 2 mg/kg hRlx, 100 micrograms/kg hRlx, or vehicle. Plasma vasopressin was not significantly different among rats administered 2 mg/kg hRlx, 100 micrograms/kg hRlx, or vehicle. The decreases and subsequent compensatory changes in mean arterial pressure and heart rate after hemorrhage and the increases in plasma vasopressin were not significantly different among rats administered vehicle or hRlx.

Animals↗
PubMed1989

Neurons in the parabrachial nuclei respond to hemorrhage.

To examine the organization of pathways in the dorsal rostral pons that process information from cardiovascular receptors, 62 neurons in the parabrachial nuclei were tested, in rats anesthetized with urethane, for their response to hemorrhage (10 ml/kg.min) and reinfusion. Of these neurons, 21 exhibited patterns of activity in response to hemorrhage that were significantly different from those seen prior to hemorrhage. The patterns of activity appeared to depend, at least in part, on the location of the neurons within the parabrachial nuclei. The activity of 8 neurons in the external lateral and central lateral parabrachial nuclei (LT) and adjacent Kolliker-Fuse nucleus (KF) increased during hemorrhage and remained elevated during reinfusion. Multivariate regression analysis indicated that the activity of these neurons was best predicted by mean arterial pressure. In contrast, the activity of 8 neurons in the dorsal cap of the central lateral parabrachial nucleus (DR) and in the caudal medial parabrachial nucleus (MD) increased during hemorrhage and decreased during reinfusion, and appeared to be best predicted by blood volume. The activity of 5 neurons in the region between the caudal medial parabrachial nucleus and the locus coeruleus (BT) responded inversely to those in the caudal medial parabrachial nucleus, and was predicted well both by blood volume and by mean arterial pressure. Together, these data reveal a complex processing of hemodynamic signals within the parabrachial nuclei which may play a critical role in the control of neuroendocrine and sympathetic responses in relation to regulation of arterial pressure, blood volume and fluid balance.

Action Potentials↗
PubMed1988

Stimulation of the parabrachial nuclei with monosodium glutamate increases arterial pressure.

In the urethane-anesthetized rat both electrical stimulation (20 microA, 0.2 ms, 30 s) through micropipettes and glutamate injections (0.1 M, 100 nl) within an area including the dorsal lateral parabrachial nucleus, the adjacent central lateral parabrachial nucleus, the external lateral parabrachial nucleus, the Kölliker-Fuse nucleus, and the adjacent medial parabrachial nucleus led to increases in mean arterial pressure (electrical, 34.2 +/- 18.6 mm Hg; glutamate, 14.0 +/- 8.3 mm Hg). The magnitude of the glutamate responses appeared to be inversely related to the distance between the ventrolateral tip of the brachium conjunctivum and the site of injection. In contrast, electrical stimulation within an area between the caudal medial parabrachial nucleus and the locus coeruleus led to increases in mean arterial pressure (24.4 +/- 12.5 mm Hg), whereas glutamate injections within this area led to decreases in mean arterial pressure (-14.8 +/- 5.3 mm Hg).

Animals↗