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Inflammatory cardiomyopathy diagnosed by endomyocardial biopsy (EMB) is common in non-ischemic heart failure (HF) and might be associated with adverse outcome. We aimed to identify markers predicting myocardial inflammation in HF. We screened 517 patients with symptomatic non-ischemic HF who underwent EMB; 397 patients (median age 54 [IQR 43/64], 28.7% females) were included in this study. 230 patients were diagnosed with myocardial inflammation, defined as ≥ 7.0 CD3 + lymphocytes/mm 2 and/or ≥ 35.0 Mac1 macrophages/mm 2 and were compared to 167 inflammation negative patients. Patients with myocardial inflammation were more often smokers (52.4% vs. 39.8%, p = 0.013) and had higher C-reactive protein (CRP) levels (5.4 mg/dl vs. 3.7 mg/dl, p = 0.003). In logistic regression models CRP ≥ 8.15 mg/dl (OR 1.985 [95%CI 1.160-3.397]; p = 0.012) and Troponin I (TnI) ≥ 136.5 pg/ml (OR 3.011 [1.215-7.464]; p = 0.017) were independently associated with myocardial inflammation, whereas no association was found for elevated brain natriuretic peptide (OR 1.811 [0.873-3.757]; p = 0.111). In prognostic performance calculation the highest positive predictive value (90%) was detected for the combination of Global longitudinal strain (GLS) ≥ -13.95% and TnI ≥ 136.5 pg/ml (0.90 (0.74-0.96)). Elevated CRP, TnI and GLS in combination with TnI can be useful to detect myocardial inflammation. Smoking seems to predispose for myocardial inflammation.

Objective: Glucose-dependent insulinotropic polypeptide (GIP), unlike glucagon-like peptide (GLP)-1, lacks glucose-lowering properties in patients with type 2 diabetes. We designed this study to elucidate the underlying pathophysiology., Research Design and Methods: Twenty-two insulin-naïve subjects with type 2 diabetes were given either synthetic human GIP (20 ng x kg(-1) x min(-1)) or placebo (normal saline) over 180 min, starting with the first bite of a mixed meal (plus 1 g of acetaminophen) on two separate occasions. Frequent blood samples were obtained over 6 h to determine plasma GIP, GLP-1, glucose, insulin, glucagon, resistin, and acetaminophen levels., Results: Compared with placebo, GIP induced an early postprandial increase in insulin levels. Intriguingly, GIP also induced an early postprandial augmentation in glucagon, a significant elevation in late postprandial glucose, and a decrease in late postprandial GLP-1 levels. Resistin and acetaminophen levels were comparable in both interventions. By immunocytochemistry, GIP receptors were present on human and mouse alpha-cells. In alphaTC1 cell line, GIP induced an increase in intracellular cAMP and glucagon secretion. CONCLUSIONS; GIP, given to achieve supraphysiological plasma levels, still had an early, short-lived insulinotropic effect in type 2 diabetes. However, with a concomitant increase in glucagon, the glucose-lowering effect was lost. GIP infusion further worsened hyperglycemia postprandially, most likely through its suppressive effect on GLP-1. These findings make it unlikely that GIP or GIP receptor agonists will be useful in treating the hyperglycemia of patients with type 2 diabetes.

The pattern of expression of glutaminase isoenzymes in tumour cells has been investigated to clarify its role in the malignant transformation and the prospect of its use as a clinically relevant factor. Using leukaemia cells from medullar blood of human patients and several established human cancer cell lines, we have developed a competitive RT (reverse transcriptase)-PCR assay to quantify simultaneously K-type (kidney-type) and L-type (liver-type) glutaminase mRNAs. Co-expression of both transcripts and higher amounts of L-type mRNA were always found in all cancer cell types analysed. However, mature lymphocytes from the medullar blood of a patient suffering aplasia did not express the K-type transcript and showed a 15-fold increase of L-type transcript. Co-expression was also confirmed at the protein level using isoform-specific antibodies; nevertheless, it did not correlate with the relative abundance of glutaminase transcripts and strong K-type protein signals were detected. On the other hand, marked differences were found with regard to glutamate inhibition and phosphate activation of tumour glutaminase activity. Taken together, the protein data suggest that K isoform would account for the majority of glutaminase activity in these human tumour cells. The results confirm that simultaneous expression of both isoenzymes in human cancer cells is a more frequent event than previously thought. Furthermore, the present work and other previous data suggest that K isoform is up-regulated with increased rates of proliferation, whereas prevalence of the L isoform seems to be related with resting or quiescent cell states.

Sporadic Amyotrophic Lateral Sclerosis (SALS) is a fatal neurologic disease characterized by degeneration of motor neurons in the spinal cord, brainstem and cortex. While familial cases of ALS exist, the sporadic form accounts for the majority of adult-onset cases. It has been hypothesized that the neurodegenerative mechanisms underlying SALS might arise from glutamate-mediated excitotoxicity and mitochondrial dysfunction. Studies on autopsied SALS spinal cord and brain have reported decreased cytochrome oxidase activity, decreased astrocytic glutamate-transporter protein, and alterations of glutamate levels and glutamate metabolizing enzyme activities. We conjectured that if alterations in glutamate metabolism and cytochrome oxidase activity occur in the SALS central nervous system these alterations may also be manifested in peripheral tissues such as platelets in living SALS patients. In this study we compared the activities of cytochrome oxidase, citrate synthase, glutamate dehydrogenase and glutaminase in platelets from SALS and control subjects. We found that there were no differences in any of the enzyme activities measured between the two groups. Our data argue against generalized ubiquitous biochemical alterations of these enzymes in SALS patients.

The capacity of rat neutrophils to utilize glutamine was investigated by 1) determination of oxygen consumption in the presence of glucose or glutamine, 2) measurement of maximal activity of phosphate-dependent glutaminase, 3) Northern blot, Western blot, and immunocytochemical detection of glutaminase, and 4) measurement of glutamine utilization and also production of ammonia, glutamate, aspartate, alanine, and lactate and decarboxylation of [U-14C]glutamine in cells incubated for 1 h. The rate of respiration by isolated neutrophils in the absence of added substrate was 5.0 nmol x min(-1) x 10(7) cells(-1). Maximal activity of phosphate-dependent glutaminase was 56 nmol x min(-1) x mg protein(-1) in freshly obtained neutrophils; the Michaelis-Menten constant was 3.5 mM for glutamine. This enzyme activity was inhibited by 2 mM glutamate, 2 mM oxoglutarate, and 2 mM NH4Cl. The presence of glutaminase protein (65 kDa) was confirmed by Western blot and immunocytochemical detection and the presence of the mRNA (6.0 kb) by Northern blot analysis. Glutamine was utilized by neutrophils incubated for 1 h at a rate of 12.8 nmol x min(-1) x mg protein(-1) when the amino acid was added to the medium at 2 mM, which is three to four times higher than the physiological concentration. In the presence of 0.5 mM glutamine, the amino acid was utilized at a rate of 2.9 nmol x min(-1) x mg protein(-1). The addition of 0.5 mM glutamate to the incubation medium caused a marked reduction (by 70%) in glutamine utilization by neutrophils. Glucose was utilized at 7.7 nmol x min(-1) x mg protein(-1) when cells were incubated in 5 mM glucose. The conversion of [U-14C]glutamine to 14CO2 was very low: <1% was totally oxidized. The formation of ammonia was approximately 27% of glutamine utilization, and the conversion of glutamine to glutamate, aspartate, alanine, and lactate accounted for approximately 84.6% of the total amino acid utilized by neutrophils. In this study, evidence is presented that, in addition to lymphocytes and macrophages, neutrophils also utilize glutamine.

Among all the glutamine deaminating enzymes the highest activity was exhibited by phosphate-dependent glutaminase (72% of the total activity) in kidney cortex and blood serum of healthy dogs and by phosphate-independent glutaminase (47% of the total activity)--in liver tissue. A distinct decrease in the activity of both isoforms of glutaminase I (phosphate-dependent and phosphate-independent enzymes) and of glutaminase II (pyruvate-dependent enzyme) occurred in kidney under conditions of acute and chronic kidney insufficiency. The phosphate-independent glutaminase activity was decreased and, by contrast, activities of phosphate- and pyruvate-dependent glutaminases were increased in liver tissue under the conditions of acute kidney insufficiency. In blood serum under conditions of acute and chronic insufficiency the glutaminase I (both forms) activity was distinctly dcreased, but the glutaminase II activity had a marked trend to elevation. Possible mechanisms responsible for an alteration of glutamine deaminating enzymes in kidney, liver tissue and blood serum of dogs under acute and chronic kidney insufficiency are discussed.

A simple and sensitive method is developed to determine the glutaminase activity. Conditions are selected for detecting enzymatically formed ammonia in the presence of such a high-labile substrate as glutamine. The method is simple in performance, readily reproducible and high-sensitive.

Acinetobacter glutaminase-asparaginase was chemically modified by succinylation and glycosylation with glycopeptides from human fibrin and gamma-globulin. These modifications markedly prolonged the half-lives of the enzyme in mice, rats, and rabbits. The plasma half-life in mice increased with decreasing isoelectric point. Glycosylation caused greater prolongation in rodents than succinylation. The kinetic properties of the modified enzymes were unchanged. Succinylation protected the enzyme from trypsin digestion. Glycosylated preparations had less heat inactivation than native and succinylated enzyme. Sedimentation equilibrium studies on a succinylated preparation showed reversible dissociation to a dimer (71, 400 g/mol) with an association constant of 1.3 times 10-6 liters/mol. This dissociation was identical with native enzyme, except for a 3% increase in molecular weight due to succinate groups. Sedimentation equilibrium studies on glycosylated preparations showed mixtures of molecular weight from 60, 000 to over 180, 000. Gel filtration and active enzyme sedimentation showed active polymers, but no active species smaller than tetramer.

Crystalline glutaminase-asparaginase which is effective against solid as well as ascites tumors was prepared from soil isolate organism Pseudomonas 7A. This enzyme has a ration of Vmax for L-glutamine and L-asparagine of 2.0. The presence of glutamic acid in the growth medium is essential for optimal enzyme production and glucose inhibits the production of glutaminase-asparaginase. The purification procedure provides an overall yield of 40 to 45% from crude cell extract to homogeneous glutaminase-asparaginase and is adaptable to large scale production of the enzyme. The specific activity of homogeneous enzyme is 160 +/- 15 i.u./mg of protein and the E1% 280 is 9.8. No disulfide or sulfhydryl groups appear to be present on the enzyme. The isoelectric point of glutaminase-asparaginase by isoelectric focusing on ampholine polyacrylamide gel plates is 5.8. The Km values for L-glutamine and L-asparagine are 4.6 and 4.4 X 10(-6) M, respectively. The enzyme catalyzes the hydrolysis of the D isomers of glutamine and asparagine at 87 and 69% the rate of the respective L isomers. L-Glutamic acid gamma-monohydroxamate is hydrolyzed at approximately the same rate as L-glutamine. The enzyme is not inhibited by ethylenediaminetetraacetate (0.1 mM), L-glutamate (30 mM), or L-aspartate (30 mM). Ammonium sulfate (10 mM) inhibits the enzymatic activity. The plasma half-life of Pseudomonas 7A glutaminase-asparaginase if 13 hours in normal mice and 43 hours in mice infected with the lactate dehydrogenase-elevating virus.

Three enzymes used in cancer chemotherapy (asparaginases from Escherichia coli and Erwinia carotovora and glutaminase from Achromobacter) were each reacted with four amino specific reagents (ethyl acetimidate, O-methylisourea, succinic anhydride, and formaldehyde/sodium borohydride). The half-lives of the modified enzymes measured in the blood of rats showed that guanidation, acetimidation and reductive alkylation were more likely to increase the persistence of the native enzymes than succinylation. However, the improvement in the persistence of any one enzyme after any one modification could not be predicted from the results with the others. It was concluded that changes in persistence caused by each modification were due to the different effects on the tertiary structure of each native enzyme. The advantages of chemical modification for increasing the persistence of enzymes over other methods such as encapsulation or aggregation are discussed.

A glutaminase-asparaginase enzyme from Achromobacter sp has antitumour activity in vitro and in animals. Glutaminase was administered in doses of 3500-20 000 IU/m2 body surface area/day to six patients with acute lymphoblastic leukaemia (ALL) and three patients with acute myeloid leukaemia (AML). The enzyme had a blood half life of 80 minutes but depletion of blood glutamine persisted for 12 hours after single doses. Seven patients, including four (two with AML and two with ALL) resistant to asparaginase, received repeated doses of glutaminase. Antileukaemic effects were observed in all seven; one elderly patient developed metabolic acidosis. Study of this new antileukaemic agent in patients with acute leukaemia at an earlier stage of their disease is now justified.

Treatment with a chemically modified glutaminase was lethal to rabbits and Rhesus monkeys at all but the lowest doses. Changes in the serum levels of triglycerides, glucose, creatinine, urea, cholesterol and protein and in the activities of some serum enzymes were the probable result of the development of lesions in liver, kidney and intestine observed at necropsy. Treatment with unmodified glutaminase induced similar changes in rabbits but not in Rhesus monkeys.

The erythrocyte enzymes of glutamic acid metabolism (glutaminase I, glutaminase II, glutamic acid decarboxylase, glutamine synthetase, and transaminases) and related amino acids (glutamine, glutamic acid, aspartic acid, alanine, and gamma-aminobutyric acid) were estimated in 69 children with protein-energy malnutrition, 13 with nephrosis, and 10 with Indian childhood cirrhosis. Twenty-one apparently healthy children served as controls. There was a significant increase in the activities of erythrocytic glutaminase I, glutaminase II, glutamic acid decarboxylase, and glutamine synthetase in all the three hypoproteinemic states, while the activities of the transaminases showed a decrease in all the conditions. The concentrations of all the amino acids were significantly increased in both the varieties of protein-energy malnutrition (edematous and nonedematous). In nephrosis and Indian childhood cirrhosis, aspartic acid, alanine, and gamma-aminobutyric acid showed a significant rise. The concentration of glutamic acid was also significantly increased in nephrosis. The observations of the present study suggest an increase in intracellular production of glutamic acid in hypoproteinemia.

The extent of genetic determination of platelet glutaminase was evaluated by sampling 13 monozygotic and 11 dizygotic adult male twin pairs. Intraclass correlation coefficients of 0.96 for monozygotic and 0.53 for dizygotic twins together with high heritability estimates indicate a strong genetic component.

The activity of adenosine desaminase AMP-desaminase and glutaminase was studied in blood serum and the liver tissue at the normal state and with acute, subacute experimental affection of the liver as well as with the CCl4-induced liver cirrhosis and obturation jaundice in different periods. It is shown that the degree and trend of changes in the enzyme activity in the affected liver depend on the character and duration of the affecting agents action. The acute and subacute affection of the liver and one-day obturation jaundice are accompanied by a decrease in the activity of all the studied enzymes in the liver tissue. With cirrhosis induced by a multiple administration of CCl4 for three months or with a month obturation jaundice the activity of glutaminase in the liver tissue lowers and that of adenosine desaminase and AMP-desaminase increased sharply. In blood serum the activity of adenosine desaminase and AMP-desaminase increases more considerably with acute affection and cirrhosis, especially with billar one.