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Recent findings suggest that inhibition of AMP-deaminase (AMPD) could be effective therapeutic strategy in heart disease associated with cardiac ischemia. To establish experimental model to study protective mechanisms of AMPD inhibition we developed conditional, cardiac specific knock-outs in Cre recombinase system. AMPD3 floxed mice were crossed with Mer-Cre-Mer mice. Tamoxifen was injected to induce Cre recombinase. After two weeks, hearts, skeletal muscle, liver, kidney, and blood were collected and activities of AMPD and related enzymes were analyzed using HPLC-based procedure. We demonstrate loss of more than 90% of cardiac AMPD activity in the heart of AMPD3 -/- mice while other enzymes of nucleotide metabolism such as adenosine deaminase, purine nucleoside phosphorylase were not affected. Surprisingly, activity of AMPD was also reduced in the erythrocytes and in the kidney by 20%–30%. No change of AMPD activity was observed in the skeletal muscle and the liver.

Introduction Human placenta mitochondria produces huge amounts of progesterone necessary for maintaining the pregnancy. Lipid peroxidation in human placental mitochondria inhibits progesterone synthesis and that inhibition can be reversed by superoxide dismutase and other antioxidants. Paraquat (PQ) a highly toxic herbicide generates superoxide radical inside cells and induces lipid peroxidation. Hence, it is supposed to stimulate lipid peroxidation in human placental mitochondria and in consequence to inhibit a placental mitochondrial steroidogenesis. Methods Placentas were obtained from normal pregnancies. All experiments were done using isolated human placental mitochondria. Mitochondrial lipid peroxidation was determined as tiobarbituric acid reactive substances (TBARS). A conversion of cholesterol to pregnenolone or pregnenolone to progesterone was measured using radiolabeled steroids and thin layer chromatography. Results PQ enhanced the iron-dependent lipid peroxidation as also PQ heightened the inhibitory action of this process on progesterone synthesis in isolated human placental mitochondria. Paradoxically, a superoxide dismutase (SOD) reversed the inhibition of progesterone synthesis only minimally although it strongly inhibited PQ stimulated iron-dependent lipid peroxidation. When iron was absent, PQ stimulated only negligible lipid peroxidation but strongly inhibited progesterone synthesis. SOD had no effect on inhibition of progesterone synthesis by PQ. PQ strongly inhibited of the conversion of cholesterol to pregnenolone but had not got any influence on the enzymatic activity of mitochondrial 3β-hydroxysteroid dehydrogenase. PQ strongly decreased the efficiency of NADPH-dependent cytochrome P450 reduction as well as it promoted the rapid oxidation of the pre-reduced mitochondrial cytochrome P450. However PQ has not inhibited combined activity of adrenodoxin reductase and adrenodoxin. Discussion We conclude that the most important reason of the inhibition of progesterone synthesis by PQ is the escape of electrons from cytochrome P450scc to that compound what leads to cytochrome oxidation and, in consequence the inhibition of the reaction catalyzed by it. The action of PQ described here should be considered as potentially harmful for pregnancy and fetal development.

During pregnancy the isoform composition of human placental AMP-deaminase changes. This may reflect the adaptation of enzyme to changing metabolic requirements of the growing fetus. In this paper kinetic and regulatory properties of AMP-deaminase purified from human preterm (∼25 week of gestation) placenta were described and compared with these of the enzyme purified from term placenta. AMP-deaminase from preterm placenta was less sensitive to pH changes and in contrast to the enzyme from the term organ, at low range of substrate concentrations was not inhibited but activated by physiological concentrations of orthophosphate. This may significantly improve the catalytic efficiency of enzyme at early phase of the pregnancy.

Myasthenia gravis (MG) is characterized clinically by skeletal muscle fatigue following the excessive exercise. Interestingly most of MG patients manifest parallely also some abnormalities of the thymus.AMP-deaminase (AMPD) from human thymus was not a subject of studies up to now. In this paper, mRNA expression and some physico-chemical and immunological properties of AMPD purified from the thymus of MG patients were described. Experiments performed identified the liver isozyme (AMPD2) as the main isoform of AMPD expressed in this organ. The activity of AMPD found in this organ was higher than in other human non-(skeletal) muscle tissues indicating on role the enzyme may play in supplying of guanylates required for the intensive multiplication of thymocytes.

Objectives: Possession of the C34T (Glu12Stop) nonsense mutation in the AMP-deaminase 1 ( AMPD1 ) gene has been shown to be associated with improved prognosis in heart failure and ischemic heart disease. The most likely event leading to these clinical effects is a reduced capacity of the AMP deamination pathway and increased production of cardio-protective adenosine. However, since AMPD1 is predominantly expressed in skeletal muscle, the protective effects could be related not only to local cardiac changes, but also to a systemic mechanism. In the present study we evaluated the effect of the C34T mutation on cardiac AMP-deaminase activity and on the systemic changes in adenosine production. Methods: The presence of the C34T mutation was assayed by single-stranded conformational polymorphism (SSCP). Analysis of the AMPD1 genotype and measurement of enzyme activities was performed on 27 patients with heart failure (HF). In addition, blood adenosine concentration was measured by liquid chromatography/mass spectrometry (LC/MS) in 21 healthy subjects with established AMPD1 genotype at rest and following exhaustive exercise. Results: Cardiac AMP-deaminase activity in heterozygotes (C/T) was 0.59±0.02 nmol/min/g wet wt—about half of the activity found in normal wild-type (C/C) individuals (1.06±0.09 nmol/min/g wet wt, P =0.003). There were no significant differences in the activities of any other enzymes between subjects with the C/T or C/C genotype. Resting venous blood adenosine concentration was similar in subjects with C/C, C/T and homozygous for the mutated allele (T/T) genotype. Following exercise, a significant increase in adenosine was observed in T/T subjects (by 0.013±0.009 μmol/l, P =0.035) but not in C/C (0.003±0.009 μmol/l) or C/T (−0.002±0.011 μmol/l). Conclusions: Our findings indicate that the C34T mutation of AMPD1 leads to a decrease in cardiac enzyme activity of AMP-deaminase without changes in any other adenosine-regulating enzymes, highlighting the importance of local cardiac metabolic changes. Systemic (blood) changes in adenosine concentration were apparent only in homozygous subjects and therefore may play a relatively small part in cardio-protection.

AMP-deaminase from human term placenta was chromatographed on a phosphocellulose column and physico-chemical and immunological properties of the purified enzyme were investigated. At physiological pH7.0, in the absence of regulatory ligands (control conditions) studied AMP-deaminase manifested sigmoid-shaped substrate saturation kinetics, with half-saturation parameter (S0.5) value of about 7 mM. Addition of important allosteric effectors (ATP, ADP or orthophosphate) modified kinetic properties of studied AMP-deaminase, influencing mainly the value of S0.5 parameter. Micromolar concentrations of stearylo-CoA inhibited potently the enzyme making it no longer sensitive towards 1 mM ATP-induced activation. SDS-PAGE electrophoresis of the purified enzyme revealed presence of 68 kDa protein fragment, reacting with anti-(human) liver AMP-deaminase antibodies. Experimental results presented indicate that ‘liver type’ of AMP-deaminase is an enzyme form present in human term placenta.

AMP-deaminase (EC 3.5.4.6) is a key enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in the liver. Mechanisms regulating activity of the enzyme are not completely elucidated, till now. In this paper experimental data indicating on the potential regulatory significance of changes in oligomeric structure of the enzyme are presented. SDS-PAG electrophoresis of human liver AMP-deaminase revealed the presence of three enzyme fragments. Only largest of them (the protein fragments weighing 68 kDa) reacted immunologically with monoclonal anti- (human liver) AMP-deaminase antibodies. At physiological pH 7.0, in the absence of regulatory ligands, reaction catalysed by human liver AMP-deaminase was strongly dependent on enzyme concentration used, with half-saturation constant (S0.5) values increasing significantly with the degree of enzyme dilution. Preincubation with activated long-chain fatty acids – substances promoting dissociation of oligomeric enzymes, inhibited the activity of AMP-deaminase studied nearly completely. Gel filtration on Sepharose CL-6B column demonstrated existence of at least three active oligomeric forms of human liver AMP-deaminase. We postulate that oligomeric structure of the enzyme is a factor determining regulatory profile of AMP-deaminase studied.

The paper presents a case of acute, accidental sertindole poisoning. Intoxication had a stormy clinical course with symptoms of cardiovascular, respiratory and nervous system. A relatively small dose of ingested preparation and severe overdose course may indicate a low therapeutic drug index.

We describe deployment of electronic toxicological information database in poison control center of Pomeranian Center of Toxicology. System was based on Google Apps technology, by Google Inc., using electronic, web-based forms and data tables. During first 6 months from system deployment, we used it to archive 1471 poisoning cases, prepare monthly poisoning reports and facilitate statistical analysis of data. Electronic database usage made Poison Center work much easier.

Nucleotide metabolism and signalling is directly linked to myocardial function. Therefore analysis how diversity of genes coding nucleotide metabolism related proteins affects clinical progress of heart disease could provide valuable information for development of new treatments. Several studies identified that polymorphism of AMP deaminase 1 gene (AMPD1), in particular the common C34T variant of this gene was found to benefit patients with heart failure and ischemic heart disease. However, these findings were inconsistent in subsequent studies. This prompted our detailed analysis of heart transplant recipients that revealed diverse effect: improved early postoperative cardiac function associated with C34T mutation in donors, but worse 1-year survival. Our other studies on the metabolic impact of AMPD1 C34T mutation revealed decrease in AMPD activity, increased production of adenosine and de-inhibition of AMP regulated protein kinase. Thus, genetic, clinical and biochemical studies revealed that while long term attenuation of AMPD activity could be deleterious, transient inhibition of AMPD activity before acute cardiac injury is protective. We suggest therefore that pharmacological inhibition of AMP deaminase before transient ischemic event such as during ischemic heart disease or cardiac surgery could provide therapeutic benefit.

Metabolic acidosis (lactic and ketoacidosis) are frequent cause of sudden, unexplained deaths of alcohol abusers with negative results of pathomorphological and toxicological determinations. We described the pathomechanism leading to the acidosis.

Manganism is a neurodegenerative disease of brain caused by intoxication by manganese and its excessive accumulation in this tissue. Some of the clinical symptoms observed in this disease resemble these observed in Parkinson disease.

Methoxetamine (MXE) is an analogue of ketamine.We present a 25-year-old male who, after getting an information from the Internet, started to use MXE to avoid the excitement connected with recreational codeine abuse. For about 8 - 10 months he injected about 100 mg of MXE intramuscularly. On the day of admission the patient decided to take much higher dose of 750 mg of MXE. For the first 3-4 hours of hospitalization the profound agitation, which demanded the usage of high doses of benzodiazepines, was observed every several minutes. After 6-7 hours of supportive treatment the patient returned to his baseline mental status.MXE presents the new healthcare threat because of easy accessibility via Internet, and lack of legal restrictions in many countries. The low dose of MXE can cause "peace and serenity", however, higher dose may act opposite.

We present a case of a 22-year-old male who, in a suicide attempt, ingested approximately 200 g of potassium chlorate. Upon admission to the hospital, he presented in full respiratory failure with cyanosis. Methylene blue antidote was given but found to be ineffective. The patient was intubated and mechanical ventilation was initiated. Because of renal failure with anuria, intermittent haemodialysis (iHD) followed by continuous venovenous hemodiafiltration (CVVHDF) was performed. His hospital stay was also complicated by hemolysis, disseminated intravascular coagulation, and atrial fibrillation. Transfusions of packed red blood cells, platelets, and fresh frozen plasma were necessary to correct the deficits. He also developed liver failure and required two sessions of molecular adsorbent recirculating system (MARS) therapy. On day 14 of his hospitalization, he regained consciousness, as well as full respiratory and circulatory function. There are no controlled studies addressing management of potassium chlorate poisoning. We suggest that early renal replacement therapy should be strongly considered.

Reactive oxygen species, which plays a role in pathogenesis of many neurodegenerative diseases, seems to be important also in pathogenesis of the Parkinson's disease. Experiments performed recently, revealed in the cerebrum of patients suffering from this disease (induced by the oxidative stress) elevated levels of 3,4-dihydroxyphenylacetaldehyde (DOPAL)--a strong endogenous neurotoxin to dopamine neurons.

Pathomechanism of vitamin D intoxication is still unclear. Experiments on animals showed that intoxication is accompanied by distinctly elevated serum level of vitamin D precursor [25(OH)D3] but not by that of hormonally, metabolic active form of it [1alpha,25(OH)2D3]. The most probable explanation of this phenomenon is that vitamin D precursor releases hormonal form of vitamin from its binding with serum transporting protein (DBP) stimulating in this way transcription of genes in cells of target tissues.

A C34T mutation in the AMPD1 gene is proposed to cause local or systemic augmentations in blood adenosine level and improvement of prognoses in heart diseases like congestive heart failure or heart ischemic disease. This study examines some physico-chemical properties of AMP-deaminase isolated from cardiac muscle of a 10-year-old boy heterozygote for this mutation.

Human placental mitochondria might be a significant source of NADPH- and iron-dependent production of reactive oxygen species (ROS). Preeclampsia is believed to be a consequence of overproduction of ROS in human placenta. The experimental results presented here show that melatonin inhibits NADPH- and iron-dependent lipid peroxidation of human placental mitochondria in a concentration-dependent manner. At 1.5 mm concentration, melatonin suppressed this process nearly completely. Melatonin does not influence significantly the iron oxidation at this conditions, indicating that free radical scavenging rather than metal-chelating phenomenon is the basis of its antioxidant action. The fact of inhibition of lipid peroxidation by melatonin at conditions excluding iron participation also supports this hypothesis. Elucidation of the nature of common interaction among melatonin, ascorbate, and alpha-tocopherol in human placental mitochondria was the main aim of this study. In presence of 90 mum ascorbate, the inhibition of lipid peroxidation by melatonin was strong and had a feature of synergistic interaction. At presence of 30 mum ascorbate, which stimulated lipid peroxidation, melatonin caused a loss of pro-oxidant effect of ascorbate. While the interaction of melatonin with ascorbate indicated synergism, the joint action of melatonin and alpha-tocopherol was additive. When all three antioxidants were applied together, the strongest inhibition of lipid peroxidation was observed. The experimental results presented here indicated that melatonin could be considered as an effective component of antioxidant treatment of preeclampsia, allowing the use of reduced doses of vitamin C and E owing to elevated efficiency of their antioxidant activity in placenta when used in combination.

Human placental mitochondria might be a significant source of NADPH- and iron-dependent production of reactive oxygen species (ROS). Preeclampsia is believed to be a consequence of overproduction of ROS in human placenta. The experimental results presented here show that melatonin inhibits NADPH- and iron-dependent lipid peroxidation of human placental mitochondria in a concentration-dependent manner. At 1.5 mm concentration, melatonin suppressed this process nearly completely. Melatonin does not influence significantly the iron oxidation at this conditions, indicating that free radical scavenging rather than metal-chelating phenomenon is the basis of its antioxidant action. The fact of inhibition of lipid peroxidation by melatonin at conditions excluding iron participation also supports this hypothesis. Elucidation of the nature of common interaction among melatonin, ascorbate, and alpha-tocopherol in human placental mitochondria was the main aim of this study. In presence of 90 mum ascorbate, the inhibition of lipid peroxidation by melatonin was strong and had a feature of synergistic interaction. At presence of 30 mum ascorbate, which stimulated lipid peroxidation, melatonin caused a loss of pro-oxidant effect of ascorbate. While the interaction of melatonin with ascorbate indicated synergism, the joint action of melatonin and alpha-tocopherol was additive. When all three antioxidants were applied together, the strongest inhibition of lipid peroxidation was observed. The experimental results presented here indicated that melatonin could be considered as an effective component of antioxidant treatment of preeclampsia, allowing the use of reduced doses of vitamin C and E owing to elevated efficiency of their antioxidant activity in placenta when used in combination.

During pregnancy the activity of AMP-deaminase in developing human placenta gradually decreases, being in homogenates of mature, term placenta (approximately 40 week of gestation) one fourth to one third of that in homogenates of immature (approximately 25 week of gestation) organ. The gradual decrease of activity correlates inversely with the increasing presence of the form of enzyme predominating in homogenates of the mature placenta. The discrepancy observed indicate that isozymic pattern of AMP-deaminase in developing human placenta changes.

AMP-deaminase from human kidney (cortex and medulla) was purified and the physicochemical properties were characterized. The enzyme from both portions of the kidney exhibited identical kinetics and regulatory properties. At optimal pH (6.6), the AMP-deaminase studied exhibited a distinctly sigmoidal substrate saturation kinetics, with the half-saturation parameter (S0.5) as high as 10 m m . ATP at 1 m m strongly activated the enzyme, decreasing S0.5 nearly 10-fold. The activating effect of ADP was less strong. Orthophosphate inhibited the enzyme, but the inhibition observed was weak (Ki ≈ 16 m m ) and had a pure competitive character. At pH 7.2, physiological for the kidney cortex, orthophosphate inhibition became even weaker and became partially competitive. Variations in the adenylate energy charge had potent effects on the activity of AMP-deaminase, depending on the size of the total adenine nucleotide pool examined. The results of gel filtration and SDS-PAGE indicated that human kidney AMP-deaminase is an oligomeric enzyme composed of four, probably identical, subunits weighing about 37 kDa each.

The strong positive relationship between cholesterolaemia and ischaemic heart disease is unquestioned and comes from several lines of evidence. In this paper results of questionnaire studies concerning cholesterolemia performed among patients of the Cardiology Ward of a Regional Hospital in Wejherowo are presented.

The main target of acetaminophen application is bifunctional enzyme--prostaglandin endoperoxide H2 synthase (PGHS)--which has cyclo-oxygenase and peroxidase activities and synthesizes initial intermediates in prostanoid synthesis. The reaction catalyzed by PGHS is radical-based and it is initiated and then maintained by the constant presence of peroxides especially peroxynitrate, which generate so-called "peroxide tone" in the enzyme surrounding. Currently it is known that inhibitory effect of acetaminophen on PGHS activity is directly connected with the elimination of "peroxide tone". High concentrations of reactive compounds (e.g. peroxynitrate and lipid peroxides)--produced by cellular defending mechanism at inflammatory sites--significantly decrease inhibitory impact of acetaminophen on PGHS activity. Such observation allows explanation of weak antiinflammatory effect of acetaminophen together with its strong analgesic and antipyretic properties.

Daily heart consumption of energy (about 6 kg of ATP) exceeds significantly energy consumption of other organs. Deprivation of energy is considered as one of the main factors in development of heart failure. Currently available research methods permit not only to evaluate more precisely the role of impaired energy metabolism in the heart failure, but also give a hope for the future metabolic therapy.

We have described three physicians aged 65-70 years, who had noticed during their insulintherapy significant, periodically recurrent variations of insulin requirement (max. 50 i.u./24h vs 25 i.u./24h) in the periods of 4-16 weeks. They had been characterized by similar diet and physical activity, stable normal body mass (av. BMI 24.6) and normal glycosylated hemoglobin (av. HbA1c 5.9%). The hypothesis was put forward that the cause of this phenomenon may be periodical changes in the incretin system activity or increased proliferation of pancreatic beta cells leading to decrease of insulin requirement.

AMP-deaminase from human uterine smooth muscle has been isolated, and properties of the enzyme were characterized. At pH 7.0, and in the presence of 100 mM potassium chloride the enzyme manifests a distinctly sigmoidal type of kinetics, with S0.5 parameter value about 12 mM. 1 mM ATP strongly activates the enzyme, and diminishes the value of S0.5 to 1.2 mM. In contrast to that 2.5 mM orthophosphate slightly inhibits the activity of AMP-deaminase studied and increases the S0.5 to about 14 mM. Similarly to ATP, orthophosphate does not influence the maximum velocity of the reaction. Electrophoresis in the presence of sodium dodecyl sulphate revealed that the molecular weight of human smooth muscle AMP-deaminase subunit is close to 37 kDa.

1. 1. AMP-deaminases from fish heart and skeletal muscle have been isolated, and their kinetic and regulatory properties compared. 2. 2. The results obtained indicate that the enzyme variants present in fish heart and skeletal muscle, in contrast to their mammalian counterparts, show very similar chromatographic, kinetic and regulatory characteristics. 3. 3. The above may reflect evolutionary programmed differences in AMP-deaminase gene(s) organization.

Because mutation of AMP deaminase 1 gene leading to reduced AMP deaminase activity may result in protection of cardiac function in patients with heart disease, inhibitors of AMP deaminase (AMPD) may have therapeutic applications. This study evaluated the effect of a specific inhibitor of AMP deaminase 3-[2-(3-carboxy-4-bromo-5,6,7,8-tetrahydronaphthyl)ethyl]-3,6,7,8-tetrahydroimidazo [4,5-d][1,3]diazepin-8-ol (AMPDI) on the isolated human enzyme and on nucleotide catabolism in rat cardiomyocytes. AMPDI effectively inhibited isolated human AMPD with an IC(50) = 0.5 micro M. AMPDI was much less effective with isolated cardiomyocytes (IC(50) = 0.5 mM). AMPDI is a very effective inhibitor of AMPD that despite lower efficiency in the cell system examined could be useful for in vivo studies.

Background AMP-deaminase (EC 3.5.4.6) and 5'-nucleotidase (EC 3.1.3.5) are enzymes responsible for the maintenance of cellular adenine nucleotides pool. Both exist in several isoforms that differ in kinetic properties and tissue distribution. Profile of isoforms of these enzymes in human placenta has not been analyzed so far while this could be important for understanding of pathology of placental ischemia such as in preeclampsia. Our aim was therefore to analyze expression of AMPD and CN-I genes in human term placenta. Methods RT-PCR analysis was used for determine expression of AMPD1, AMPD2, AMPD3 and CN-I. Results and conclusion The experimental results presented here indicate that genes coding "AMP-preferring", cytosolic isozyme of 5'-nucleotidase (cN-I) as well as "muscle-type" isozyme of AMP-deaminase (AMPD1) are not expressed in human term placenta. Among other AMPD family genes, only these coding "liver-type" isozyme (AMPD2) and, in lesser degree, "erythrocyte-type" isozyme (AMPD3) of AMP-deaminase are expressed in this organ. The expression level of AMPD3 was a half of that presented by AMPD2. We conclude that high abundance of AMP-deaminase 2 transcript suggest that this particular isoform is a predominant pathway of adenine nucleotides degradation in human term placenta that follows liver-type regulation of this process.

In the result of liver detoxification, xenobiotics change into more water soluble and thus easier for excretion from the body. It is convenient to consider this process as occurring in two phases. In phase I, the major reactions involved are hydroxylation, catalyzed by monoxygenases. In phase II, the preliminary modified xenobiotics after conjugation with some specific metabolites are transformed into less toxic and more soluble end-products. Recently, antiporter activity of MDR1 (MultiDrug Resistence) gene products in enterocytes was recognized as important stage in detoxification of xenobiotics, and definied as phase III of this process.

Heme oxygenase (HO) is the rate limiting enzyme in the breakdown of heme into carbon monoxide (CO), iron and biliverdin. To date three mammalian HO isozymes have been identified, and only one of them (HO-1) is inducible. The products of HO-catalyzed reaction, particularly CO and biliverdin/bilirubin have been shown to exert protective effects in the liver against different noxious stimuli. The results of experimental data show that induction of HO-1 expression contributes to protection against liver damage induced by several chemical compounds such as acetaminophen.

After the transplantation of hearts from female donors into male recipients, cells migrating from the host to the transplanted heart demonstrate the presence of the Y chromosome (up to 18 percent of all cardiomyocytes). Most probably, this kind of migration occurs in all transplantations. However, postnatal origins of pancreatic beta-cells remain still controversial. The extensive experiments on mice question the existence of adult pancreatic endocrine stem cells. In our opinion one of the methods to elucidate this still open problem is carrying out the similar investigation in sex-mismatched transplanted pancreas. The finding of the chromosome Y in Langerhans islets taken from a male recipient's pancreas which has been transplanted from a female donor can confirm the existence of pancreatic endocrine stem cells.

Carbon monoxide is a side product of enzymatic degradation of heme--a reaction catalyzed by heme oxygenase (HO). There are three independent molecular forms (isozymes) of heme oxygenase in man. Isoforms HO-2 and HO-3 are constitutive. More detailed studied isoform HO-2 is especially abundant in the human brain. In contrast to isoforms: HO-2 and HO-3, isoform HO-1 is an inducible species of heme oxygenase. Seen in other tissues, expression of HO-1 is also manifested in the endothelium and smooth muscle of blood vessels. Accumulated experimental data indicate more and more clearly on the possible role of endogenous carbon monoxide in cell cytoprotection, and shows it as a probable factor participating in modulation of the vascular tonus in hypoxia.

AMP-deaminase from human liver was purified by two-step phosphocellulose chromatography, and SDS-PAG electrophoresis of the most active enzyme fraction eluted has been performed. The largest of the protein fragments revealed had a size (92 kDa) of an apparent full-size enzyme subunit, and reacted positively with antibodies produced against specific human ampd2 gene product. Three-day storage at cold room temperature modified significantly the electrophoretical pattern of the enzyme, evidencing continuous and progressive degradation of its structure. This is a first report evidencing the presence of apparent full-size form of human liver AMP-deaminase in preparation obtained from endogenous source.

In the present paper physico-chemical properties of AMP-deaminase purified from human liver neoplasm-hepatocellular carcinoma (HCC) were investigated and compared with these obtained for the enzyme from normal, unaffected tissue.

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.

Myasthenia Gravis (MG) is a primary disorder of neuromuscular transmission, and patients suffering from this disease manifest specific, progressive weakness of several groups of skeletal muscles. Myoadenylate deaminase deficiency (MADD) is an inborn disease of skeletal muscles, the clinical symptoms of which may mimic symptoms observed in MG. Preliminary results of studies presented in this paper suggest that simultaneous occurrence of MG and MADD may modify energetic metabolism of skeletal muscle.

The importance of dyslipidemia in the development of cardiovascular disease is now recognized as a central factor of equal, if not greater significance than any other risk factor. Although correction of high level of low-density lipoproteins (LDL) has been regarded now as the main goal of therapy, it has now been reaffirmed that the contribution of low level of high-density lipoproteins (HDL) to the risk of ischaemic heart disease should also be considered. In the therapy of dislipidemias with hipertriglyceridemia and decreased level of HDL lipoprotein fibrates play an especially important role. In the article the molecular mechanism of fibrates action is presented.

Clinical studies indicate that treatment with statins significantly reduces the incidence of myocardial infarction and death from cardiovascular diseases. Quite recently statins were found to exert a direct cardiovascular effect, which is independent on their well-known cholesterol lowering action. In this article current views on the role of statins in improvement of function of vascular endothelium are presented.

Leiomyomas are common, benign tumors which may occur at any location containing smooth muscle cells (1). Most of these tumors originate in the female genital tracts, and in particular, in the uterus. Leiomyomas are relatively avascular and tightly compacted tumors of smooth muscle cells and fibroblasts. Usually they are surrounded by a pseudocapsule of alveolar tissue and have two relatively large nourishing vessels. The majority of Leiomyomas are of normal karyotype, but their cytogenic profile is strinkingly similar to lipomas (2).

AMP-deaminase (EC 3.5.4.6) - the enzyme catalyzing hydrolytic deamination of adenylic acid is widely distributed in vertebrate tissues.

1. 1. The values of the substrate constant ( K s ) for the reaction catalysed by rabbit skeletal muscle AMP-deaminase at 5 different temperatures were calculated and compared with the estimated values of the Michaelis constant. The validity of the relation: K m = K s for this enzyme has been verified. 2. 2. pH optimum for the reaction catalysed by rabbit skeletal muscle AMP-deaminase has been found to be dependent both on potassium and substrate concentrations. 3. 3. In contrast to the maximum velocity, the Michaelis constant for this enzyme was independent of pH for the pH range of 5.9–7.3. 4. 4. At pH 6.1 the rate of the reaction was not sensitive to the temperature, independent of KC1 concentration in the incubation medium. The enzyme reaction became much more sensitive to temperature as the pH increased.

1. 1. Kinetic properties of the inhibitory effect of inorganic phosphate and fluoride and of the activatory effect of potassium ion on human skeletal muscle AMP-deaminase (E.C. 3.5.4.6) have been investigated 2. 2. It has been shown that phosphate is a competitive inhibitor ( K 1 , ≈0.8 × 10 −3 M) and fluoride a noncompetitive inhibitor ( K 1 ≈3.2 × 10 −3 M) of human muscle AMP-deaminase. 3. 3. The changes of potassium ion concentration between 20 and 200 mM did not influence the Michaelis constant which was about 0.9 x 10 −3 M at 30°C. Also the change of substrate concentration in the range 40–300 μM did not influence the activation constant for potassium ( K a ≈0.4 × 10 −1 M). 4. 4. Higher concentraion of potassium (200mM) was found to diminish the “temperature sensitivity” of the enzyme activity. 5. 5. The energy of activation (E) in the presence of 150 mM KC1 calculated from Arrhenius plot was about 4600 cal/mole of substrate. The heat of the enzyme-substrate complex formation obtained from the plot of log K m vs T −1 was shown to have positive value (+2200 cal/mole) at the temperatures lower than 23°C and negative value (—4100 cal/mole) at the temperatures higher than 23°C.

1. 1. AMP-deaminase (EC 3.5.4.6. AMP-aminohydrolase) isolated from beef heart was found to show sigmoid-shaped kinetics at a high (100mM) and low (10mM) potassium ions concentration. Addition of ATP or ADP to the incubation medium changed the kinetics of the reaction to hyperbolic. 2. 2. In the presence of high potassium ions concentration ATP and ADP caused a significant increase of the enzyme-substrate affinity. The calculated values of maximum velocity of the reaction were the same both in the presence and in the absence of activating nucleotides. 3. 3. When 10 mM potassium was present in the incubation mixture, the activating effect of ATP and ADP was more complexed. Both these nucleotides increased not only the enzyme-substrate affinity. but also the maximum velocity and thus catalytic efficiency of the enzyme in these conditions. 4. 4. ATP was shown to be a much more efficient activator of the enzyme than ADP both at low and high potassium concentrations. 5. 5. It is concluded that at a high potassium ions concentration AMP-deaminase from beef heart is regulated by ATP and ADP in the way corresponding to the “ K m -type” of enzyme, whereas a mixed “ K m V-type” of the regulation is observed at low potassium ions concentration.

1. 1.|The influence of temperature on the rate of the reaction catalyzed by purified rat muscle AMP-deaminase in the presence ADP, ATP, GTP and two different KCl concentrations was investigated. 2. 2.|It has been found that the same effector may exhibit a different effect on the enzyme depending on temperature at which the reaction is being carried out. 3. 3.|In the presence of low KCl concentration (7 mM) the curve of Michaelis-Menten depedence is sigmoidal and the Hill plot line is diphasic in these conditions. 4. 4.|The presence of high KCl concentration (150 mM) influenced the extent and direction of the effects exhibited by the nucleotides. 5. 5.|Apparent activation energies μ for the enzyme-catalysed reaction in the presence of the effectors were calculated. The Arrhenius plots were diphasic under all the conditions employed; lowest positive values of the apparent activation energy were found in the presence of high concentration of potassium.

1. 1. Temperature dependence of the rate of reaction catalysed by AMP-deaminase from skeletal muscle of rat at four stages of postnatal development was investigated. 2. 2. In newborn rat the reaction rate increased steadily with the increase of temperature from 10 to 50°C, whereas in older rat a distinct temperature optimum at 40 or 35°C was visible. 3. 3. These results are discussed and compared with experiments on AMP-deaminase from developing chick muscle, which have been described previously in this journal.

1. 1. Lactate dehydrogenase has been purified from skeletal muscle of 14-day chick embryo, of chick i day after hatching and of adult hen. 2. 2. The reaction rate measured at low substrate concentrations (IO−4 M pyruvate) did not depend on temperature between 10° and 50° C when catalyzed by the enzyme from adult muscle. The rate showed a sharp temperature optimum at 35° and 40° C when the reaction was catalyzed by the enzyme from embryonic and I-day chick muscle enzyme respectively. 3. 3. The dependence of Km on temperature for embryonic and adult enzyme was also different; the value of Km for adult enzyme increased steadily with temperature whereas the value of Km for embryonic enzyme increased sharply with the rise of temperature from 35° to 50° C, showing little change in the temperature range 10°–35°C. 4. 4. These results are discussed and compared with experiments on AMP-aminohydrolase, another enzyme the kinetics of which have been investigated previously in developing chick muscle.

1. 1. AMP-aminohydrolase has been purified from skeletal muscle of chick embryo, I-day-old chick, and adult hen. 2. 2. The reaction rates measured at low substrate concentrations (5.5 × 10 −5 M ) at temperatures between 10 and 50° C. showed that ‘ adult ’ enzyme has a temperature optimum of 35° C., whereas ‘ embryonic ’ enzyme has no temperature optimum in this temperature range, the rate increasing steadily with the rise of temperature. 3. 3. The dependence of K m on temperature was different for ‘ adult’ and for ‘ embryonic ’ enzyme, indicating that the differences mentioned above consist of different enzymesubstrate affinity rather than of different molecular activity of the enzyme.