A 57-gene expression signature in B-cell chronic lymphocytic leukemia.

B-CLL is the most frequent type of leukemia in the Western countries. The disease, common among the elderly, follows a variable course in terms of survival time and symptoms. There is evidence that the accumulation of lymphocytes in peripheral blood and bone marrow is due to a cell resistance to apoptosis rather than to highly proliferative cells. Genetic mechanisms that lead to the development and progression of disease are mainly unknown, although a number of prognostically and diagnostically important genetic markers have been identified. The aim of this study is to investigate the gene expression profile, by a specific chip for microarray analysis, in B-CLL lymphocytes with regard to factors involved in apoptosis cascade, signal transduction, purine metabolism enzymes, interleukin expression, enzymes involved in the responses to oxidative stress. We found relevant results in a set of 19 of the 57 genes considered. IMP dehydrogenase, adenine phosphoribosyltransferase, adenylosuccinate lyase, adenylate kinase, ADORA1, G-protein-coupled receptor kinase 6, Bcl-2-like 1 isoform 2, caspase 6, and 8 were found underexpressed; while ADORA3, Gars-Airs-Gart, adenylate kinase 3, adenylate deaminase, NMN adenylyltransferase, CD26, CD38, interleukins 18 and 4 were found overexpressed. The microarray technique is a powerful method for identification of potential important diagnostic and prognostic markers, besides giving prominence to genes candidate for further studies.

Antioxidant status and purine bases in carotid artery plaque.

Free radical excess and oxidative stress are implicated in the formation and progression of atherosclerotic plaque through actions on susceptible vascular cells, such as by activating xanthine oxidase. Purine bases and other antioxidant compounds could play important protective roles in atherogenesis, as could nonenzymatic low molecular weight thiol defenses, not previously evaluated in carotid artery plaque. Therefore, we measured purine catabolites (hypoxanthine, xanthine, uric acid, allantoin) and antioxidant compounds (total sulphydryl groups, homocysteine, cysteine, and glutathione) in advanced carotid artery plaque and found a high ratio of allantoin to uric acid, suggesting a ongoing local oxidative stress.

A kinetic study of the rat liver adenosine kinase reverse reaction.

Adenosine kinase is an enzyme catalyzing the reaction: adenosine + ATP --> AMP + ADP. We studied some biochemical properties not hitherto investigated and demonstrated that the reaction can be easily reversed when coupled with adenosine deaminase, which transforms adenosine into inosine and ammonia. The overall reaction is: AMP + ADP --> ATP + inosine + NH(3). The exoergonic ADA reaction shifts the equilibrium and fills the energy gap necessary for synthesis of ATP. This reaction could be used by cells under particular conditions of energy deficiency and, together with myokinase activity, may help to restore physiological ATP levels.

Synthetic gel of carotid artery plaque.

Atherosclerosis is a complex disease that affects medium and large arteries, leading to the formation and progression of plaque. In this process the proteins play an essential role and as a consequence, proteomic-based strategies examining the protein content of cells or tissues could offer a useful approach for the study of plaque proteins. Due to the heterogeneous cell composition of plaque, proteome analysis of whole lesions is difficult, besides being also complicated by the presence of plasma proteins that cannot be completely eliminated. A good way to study variations in protein expression among series of gels is to construct a synthetic gel. This type of gel is obtained by averaging the positions, shapes and optical densities of spots in a given set of gels. To be included in the synthetic gel, spots must be found in at least three gels. To obtain a profile representative of the proteome of atherosclerotic plaque, cancelling its high variability, we constructed a synthetic gel using an average of ten carotid plaque samples. We then compared it with an equivalent synthetic gel constructed using ten plasma samples from the same carotid surgery patients. For the comparison of two synthetic gels (plasma/plaque) we could discriminate plasma proteins from plaque proteins. Besides analysis of spots common to plasma, the synthetic gel is useful to detect spots exclusive to plaque, thus simplifying a very complex mixture.

Nitric oxide generation is associated with an unbalance of protein tyrosine phosphatases during liver transplantation.

Organ dysfunction secondary to ischemia-reperfusion (I/R) injury still represents a major problem in liver transplantation. Apoptosis has been observed in hepatocytes and sinusoidal endothelial cell, following I/R injury and it has been postulated as a contributing factor in ischemia-reperfusion graft dysfunction, involving a complex series of events, as changes of protein tyrosine-kinase phosphorylation. We evaluated hepatic purine metabolites, protein tyrosine phosphatases (PTPs), nitrate plus nitrite levels (NOx), caspase-3 (C-3) activity and DNA fragmentation in the time course of twelve pig orthotopic liver transplantation. Biopsies were taken before explantation (t0), after cold ischemic storage (t1) and 30 min from reperfusion (t2). During the ischemic period we observed a reduction of high energy phosphates and an increase of purine bases; PTP activity was largely increased. At t2 high energy phosphates showed a tendency to increase with respect to t1, with a partial restoration of phosphorylation potential, measured as ATP/ADT ratio. PTP activity was significantly reduced, with a concomitant increase of NOx production and C-3 activity; in a considerable number of cases we observed a sustained DNA fragmentation. We speculate that NOx production could be related to nitrosative stress, which in turn leads to dynamic alteration in PTP balance and cell signalling, regulating the activity of a number of proteins implicated in apoptotic cell death. These findings could be of interest in new potential strategy to prevent and treat I/R injury.

Circulating gastrin and ghrelin levels in patients with colorectal cancer: correlation with tumour stage, Helicobacter pylori infection and BMI.

Many studies have pointed out a possible role of gut peptides, including gastrin and ghrelin, in the pathogenesis and natural history of gastrointestinal malignancies, one of the most common death cause in the Western world. The objective of this work is to check gastrin and ghrelin serum levels in patients with colorectal cancer according to tumour's location, stage, Helicobacter pylori infection and BMI, in order to understand the two peptides' behaviour through the tumour's natural history and evaluate their assay's use in research and clinical practice. Twenty-nine subjects affected by colorectal cancer and 50 healthy controls were studied. Circulating gastrin and ghrelin levels and H. pylori serum antibodies were assessed by radioimmunologic assay and ELISA method. Gastrin and ghrelin serum levels were respectively slightly higher and significantly lower in colon cancer patients than in controls. Gastrin levels were higher in patients carrying left colon cancer and H. pylori infection while ghrelin levels were lower in both these groups. Both hormones' serum levels decreased from tumour earlier to later stages. Significant differences persisted in the correlation between BMI and ghrelin levels in controls but not in patients. Additional studies are necessary to ascertain the significance of gastrin and ghrelin opposite behaviour in colon cancer probably linked with interferences in endocrine pathways involving other gut peptides in this compromised condition.

Adenosine kinase from rat liver: new biochemical properties.

Adenosine kinase is a well-known enzyme which catalyzes the phosphorylation of adenosine to AMP: Its metabolic and kinetic properties are well studied. Here, we report new properties of rat liver enzyme, demonstrating a new reaction: ADP can be a phosphate donor instead ATP, according to the reaction: adenosine + ADP --> 2AMP) demonstrating the efficiency of AdK to phosphorylate adenosine, also starting from ADP. Cells could exploited this property in situations in which ATP levels are strongly decreased and ADP decreases slowly.

Purine metabolism in B-cell lymphocytic leukemia: a microarray approach.

B-cell chronic lymphocytic leukemia (B-CLL) is an adult-onset highly heterogeneous malignancy characterized by a cells resistance to apoptosis rather than to highly proliferative cells. In previous research, we evidenced an imbalance of purine metabolism in B-CLL cells. Since the extracellular adenosine has been proved to induce apoptosis via A2b receptor, enzymes involved in adenosine metabolism could play an important role in apoptosis resistance of B-CLL cells. We prepared a microarray chip for the analysis of 50 selected genes that could be of interest in B-CLL: enzymes of purine de-novo, salvage and catabolic pathway, oxidative stress enzymes, and apoptotis-related proteins. Preliminary results identify many genes of purine metabolism that exhibit low or high expression, while genes involved in signal transduction and apoptosis exhibit lower alterations even if of remarkable interest. This application of microarray technique seems promising and at least a subset of these genes will be valid candidates for further studies.

Purine catabolism in advanced carotid artery plaque.

This study was carried out on carotid artery plaque and plasma of 50 patients. We analyzed uric acid, hypoxanthine, xanthine, and allantoin levels to verify if enzymatic purine degradation occurs in advanced carotid plaque; we also determined free radicals and sulphydryl groups to check if there is a correlation between oxidant status and purine catabolism. Comparing plaque and plasma we found higher levels of free radicals, hypoxanthine, xanthine, and a decrease of some oxidant protectors, such as sulphydryl groups and uric acid, in plaque. We also observed a very important phenomenon in plaque, the presence of allantoin due to chemical oxidation of uric acid, since humans do not have the enzyme uricase. The hypothetical elevated activity of xanthine oxidase in atherosclerosis could be reduced by specific therapies using its inhibitors, such as oxypurinol or allopurinol.

[Evaluation of ambulatory medical care in Milan]. / Analisi del consumo di prestazioni ambulatoriali nella città di Milano.

Ambulatory procedures are an essential component of health care both for their number and for their cost. We aim therefore to identify and to quantify the volume of ambulatory procedures performed in Milan during 2003. The data come from the Outpatient Care Information Report that collect much information by characteristics of patient, health care institution and procedure categories. The analyses in this report are based on absolute measures, utilization rates and concentration index. During the year 2003 nearly 20 millions of ambulatory procedures were performed in the public and private outpatient facilities of Milan for the resident population. The average annual rate was 15 procedures per person. Utilization varied according to patient age and sex, higher in women and in the elderly group, statistically significant variability is observed (p<0.001). Outpatient care accounts for high density supply (124 specialty points for 100,000 inhabitants). Utilization rate was not homogenous both for the medical specialties and for the geographical district distribution, all that, it seems depending on the activity concentration differences and complexity supply of services than on a different distribution of health needs. This analysis may be useful as methodological starting point for further investigating current outpatient care data and for identifying those sectors in need of corrective actions.

EPR spin trapping of a radical intermediate in the urate oxidase reaction.

Urate oxidase, or uricase (EC 1.7.3.3), is a peroxisomal enzyme that catalyses the oxidation of uric acid to allantoin. The chemical mechanism of the urate oxidase reaction has not been clearly established, but the involvement of radical intermediates was hypothesised. In this study EPR spectroscopy by spin trapping of radical intermediates has been used in order to demonstrate the eventual presence of radical transient urate species. The oxidation reaction of uric acid by several uricases (Porcine Liver, Bacillus Fastidiosus, Candida Utilitis) was performed in the presence of 5-diethoxyphosphoryl-5-methyl-pyrroline-N-oxide (DEPMPO) as spin trap. DEPMPO was added to reaction mixture and a radical adduct was observed in all cases. Therefore, for the first time, the presence of a radical intermediate in the uricase reaction was experimentally proved.

Evaluation of ADA gene expression and transduction efficiency in ADA/SCID patients undergoing gene therapy.

A capillary electrophoresis (CE) method was developed for ADA/SCID diagnosis and monitoring of enzyme replacement therapy, as well as for exploring the transfection efficiency for different retroviral vectors in gene therapy.

Liver transplant: adenosine metabolism and apoptosis.

Apoptosis and necrosis coexist in ischemia-reperfusion (I/R) injury following organ transplant. During experimental liver transplant we evidenced a deep alteration in energy and antioxidant status. The activity of purine catabolic enzymes was also altered. Caspase-3 (C-3), protein tyrosine phosphatase (PTP) showed significative alterations that lead to DNA fragmentation. These findings could be of interest in new potential strategy to prevent and treat I/R injury.

Serum folate and vitamin B12 levels in children from Mozambique.

In order to investigate the behaviour of biochemical parameters in children from Mozambique, we have determined the serum levels of folic acid and vitamin B12, two well known markers of nutritional anemia. We have correlated their values with other blood parameters and have evidenced potential interesting relationship between folate content and platelets count.

Metabolism of adenosine in human colorectal tumour.

The aim of this work is to analyse the activities of the enzymes metabolising adenosine in fragments of neoplastic and normal-appearing mucosa, surrounding the tumour in 20 patients affected by colorectal cancer. The results show that the activities of the enzymes are markedly higher in tumour in comparison to normal mucosa to coope with the accelerated purine metabolism in cancerous tissues.

Effect of testosterone on purine nucleotide metabolism in rat liver.

In previous studies, we found that castration induced interesting morphological and biochemical changes in rat liver. For the present study, we have examined the effects of testosterone on the kinetics of purine nucleotide metabolism with the aim of determining the steps affected by testosterone deficiency. A biomathematical model of purine nucleotide metabolism was used to analyze the many reactions involved. The model simplifies purine nucleotide metabolism to four main steps: 1) de novo synthesis from PRPP to IMP; 2) the inosinic branch point from IMP to GMP or AMP; 3) catabolism of IMP, AMP and GMP to uric acid; 4) RNA and DNA formation from AMP and GMP. We evaluated rate constants from each step from variations in specific radioactivity of metabolites labelled with (14)C-formate, a precursor of de novo synthesis. The model was applied to the liver of normal and castrated rats before and after testosterone treatment. All four steps were slowed after castration, and were not completely restored by androgen administration. The model can give a clear representation of the kinetics of the reactions involved in the liver nucleotide metabolism investigated here, and we propose that a similar approach could be useful whenever a quantitative evaluation of the results obtained in vivo after administration of labelled precursors is required.

Enzyme activities controlling adenosine levels in normal and neoplastic tissues.

Adenosine is known to be associated with effects such as inhibition of immune response, coronary vasodilation, stimulation of angiogenesis, and inhibition of inflammatory reactions. Some authors suggest that adenosine may also have similar functions in tumor tissues. Tissue levels of adenosine are under close regulation by different enzymes acting at different levels. Adenosine is produced from AMP by the action of 5'-nucleotidase (5'-NT) and is converted back into AMP by adenosine kinase (AK) or into inosine by adenosine deaminase (ADA). Inosine is converted into purine catabolites by purine nucleoside phosphorylase (PNP), whereas AMP is converted into ADP and ATP by adenylate kinase (MK). The aim of this study was to analyze the activities of the above enzymes in fragments of neoplastic and apparently normal mucosa, obtained less than 5 cm and at least 10 cm from tumors, in 40 patients with colorectal cancer. The results showed much higher activities of ADA, AK, 5'-NT, and PNP in tumor tissue than in neighboring mucosa (p > 0.01 for ADA, AK, and PNP; p > 0.05 for 5'-NT), suggesting that the activities of purine metabolizing enzymes increase to cope with accelerated purine metabolism in cancerous tissue. The simultaneous increase in ADA and 5'-NT activities might be a physiological attempt by cancer cells to provide more substrate to accelerate salvage pathway activity.

Purine bases and atheromatous plaque.

In this work we determined hypoxanthine (HX), xanthine (X), uric acid (UA), allantoin (ALL) and free radicals in atheromatous plaques to improve the comprehension of oxidative stress, a phenomenon which characterizes the evolution of atherosclerotic lesions. Carotid artery plaque were obtained from subjects undergoing endoarterectomy. Pulverized plaque, extracted by water, was used for analysis of oxidative stress factors (allantoin, uric acid, xanthine, hypoxanthine, free radicals). The peroxidation UA-->ALL was very high in the plaque, as was the level of free radicals. The results show that oxidative degradation of nucleotides, such as LDL oxidation, plays a specific role not only in the progression of atherosclerotic lesions but also in the advanced plaque.

Cardiac surgery: myocardial energy balance, antioxidant status and endothelial function after ischemia-reperfusion.

Myocardial and endothelial damage is still a widely debated problem during the ischemia-reperfusion sequence in heart surgery. We evaluated myocardial purine metabolites, antioxidant defense mechanisms, oxidative status and endothelial dysfunction markers in 14 patients undergoing coronary artery by-pass graft (CABG). Heart biopsies were taken before aortic cross-clamping (t1), before clamp removal (t2) and 30 min after reperfusion (t3); perchloric extracts of the tissue were analyzed for glutathione, NAD, nucleotide nucleoside and base content by capillary electrophoresis (CE). In plasma samples from the coronary sinus we evaluated: nitrate and nitrite concentrations by CE, plasma glutathione peroxidase (plGPx) by ELISA, endothelin-1 (ET-1) by RIA and reactive oxygen metabolites (ROM) by colorimetric assay. During the ischemic period (t2) we observed a reduction in cellular NAD and GSH levels, as well as nitrate, nitrite and plGPx. ATP and GTP levels decreased and their catabolic products AMP, GMP, IMP, adenosine, inosine and hypoxanthine accumulated. The energy charge, ATP/ADP ratio, and nucleotide/(nucleoside + base) ratios decreased. At t3, levels of plasma ET-1 increased and monophosphate nucleotides tended to return to basal values. The energy charge did not increase but the nucleotide/(nucleoside + nucleobase) ratio recovered to some extent. Levels of nitrates plus nitrites continued to decrease. No significant variation in ROM levels was observed. Our data indicate that oxidative stress and endothelial damage are major events during CABG, overwhelming the scavenging capacity of the myocyte and preventing restoration of the normal energy balance for 30 min after reperfusion. The AMP deaminase pathway leading to IMP production is active during ischemia and adenosine is not the main compound derived from ATP break-down in the human heart. The possible role of extracorporeal circulation is also discussed.