[A case of Xanthinuria in a patient with marked hypouricemia]. / Un caso di xantinuria in una paziente con marcata ipouricemia.

Xanthinuria is a rare autosomal recessive disorder associated with a deficiency of xanthine oxidoreductase (XOR), which normally catalyzes the conversion of hypoxanthine to uric acid. The effects of this deficit are an elevated concentration of hypoxanthine and xanthine in the blood and urine, hypouricemia, and hypouricuria. The deficit in XOR can be isolated (type I xanthinuria) or associated with a deficit in aldehyde oxidase (type II xanthinuria) and sulfite oxidase (type III xanthinuria). While the first two variants have a benign course, are often asymptomatic (20%), and clinically indistinguishable, type III xanthinuria is a harmful form that leads to infant death due to neurological damage. The clinical symptoms (kidney stones, CKD, muscle and joint pain, peptic ulcer) are the result of the accumulation of xanthine, which is highly insoluble, in the body fluids. We describe a case of type I xanthinuria in a 52-year-old woman who presented with hypouricemia, hypouricuria and kidney stones. The diagnosis was based on purine catabolite levels in urine and serum measured by 3 nonroutine methods: high-pressure liquid chromatography, mass spectrometry, and magnetic resonance imaging. To identify the type of xanthinuria the allopurinol test was used. We believe that these tests will facilitate the diagnosis of xantinuria especially in asymptomatic patients without the need for a biopsy of the liver or intestines, which is useful only for scientific purposes.

Mucuna pruriens Linn. seed extract pretreatment protects against cardiorespiratory and neuromuscular depressant effects of Naja sputatrix (Javan spitting cobra) venom in rats.

Mucuna pruriens has been used by native Nigerians as a prophylactic for snakebite. The protective effects of M. pruriens seed extract (MPE) were investigated against the pharmacological actions of N. sputatrix (Javan spitting cobra) venom in rats. The results showed that MPE-pretreatment protected against cardiorespiratory and, to a lesser extent, neuromuscular depressant effects of N. sputatrix venom. These may be explained at least in part by the neutralisation of the cobra venom toxins by anti-MPE antibodies elicited by the MPE pretreatment.

The protective effect of Mucuna pruriens seeds against snake venom poisoning.

ETHNOPHARMACOLOGICAL RELEVANCE: The seed, leaf and root of Mucuna pruriens have been used in traditional medicine for treatments of various diseases. In Nigeria, the seed is used as oral prophylactics for snakebite. AIM OF THE STUDY: To study the protective effects of Mucuna pruriens seed extract against the lethalities of various snake venoms. MATERIALS AND METHODS: Rats were pre-treated with Mucuna pruriens seed extract and challenged with various snake venoms. The effectiveness of anti-Mucuna pruriens (anti-MPE) antibody to neutralize the lethalities of snake venoms was investigated by in vitro neutralization. RESULTS: In rats, MPE pre-treatment conferred effective protection against lethality of Naja sputatrix venom and moderate protection against Calloselasma rhodostoma venom. Indirect ELISA and immunoblotting studies showed that there were extensive cross-reactions between anti-MPE IgG and venoms from many different genera of poisonous snakes, suggesting the involvement of immunological neutralization in the protective effect of MPE pre-treatment against snake venom poisoning. In vitro neutralization experiments showed that the anti-MPE antibodies effectively neutralized the lethalities of Asiatic cobra (Naja) venoms, but were not very effective against other venoms tested. CONCLUSIONS: The anti-MPE antibodies could be used in the antiserum therapy of Asiatic cobra (Naja) bites.

Purine nucleotide catabolism in rat liver. Certain preliminary aspects of uricase reaction.

We investigated the mechanism of action of uricase, which oxidizes uric acid to allantoin, in the rat. Allantoin may decompose chemically to urea and hydantoin, containing the carbons in positions 2 and 8 of the purine ring, respectively. These carbons are derived by formylation, catalyzed by formyltransferase, in two reactions of de novo synthesis. Since uric acid and allantoin are represented in equivalent amounts in the liver, we expected to find identical incorporation of radioactivity in C(2) and C(8) of both compounds after administration of (14)C-formate. In the case of (14)C-allantoin, this was true, but not for (14)C-uric acid extracted from rat liver. We interpret these results through a series of experiments and considerations.

Evidence of a new phosphoryl transfer system in nucleotide metabolism.

Crude rat liver extract showed AMP-AMP phosphotransferase activity which, on purification, was ascribed to a novel interaction between adenylate kinase, also known as myokinase (EC 2.7.4.3), and adenosine kinase (EC 2.7.1.20). The activity was duplicated using the same enzymes purified from recombinant sources. The reaction requires physical contact between myokinase and adenosine kinase, and the net reaction is aided by the presence of adenosine deaminase (EC 3.5.4.4), which fills the gap in the energy balance of the phosphoryl transfer and shifts the equilibrium towards ADP and inosine synthesis. The proposed mechanism involves the association of adenosine kinase and myokinase through non-covalent, transient interactions that induce slight conformational changes in the active site of myokinase, bringing two already bound molecules of AMP together for phosphoryl transfer to form ADP. The proposed mechanism suggests a physiological role for the enzymes and for the AMP-AMP phosphotransferase reaction under conditions of extreme energy drain (such as hypoxia or temporary anoxia, as in cancer tissues) when the enzymes cannot display their conventional activity because of substrate deficiency.

Proteomic analysis of the pathophysiological process involved in the antisnake venom effect of Mucuna pruriens extract.

Previously, we reported the antisnake venom properties of a Mucuna pruriens seed extract (MPE) and tested its in vivo efficacy against Echis carinatus venom (EV) in short- (1 injection) and long-term (three weekly injections) treatments. The aim of the present study was to investigate plasma proteome changes associated with MPE treatments and identify proteins responsible for survival of envenomated mice (CHALLENGED mice). Six treatment groups were studied. Three control groups: one saline, one short-term and one long-term MPE treatment. One group received EV alone. Two test groups received EV with either a short-term or long-term MPE treatment (CHALLENGED mice). The plasma from each group was analysed by 2-DE/MALDI-TOF MS. The most significant changes with treatment were: albumin, haptoglobin, fibrinogen, serum amyloid A and serum amyloid P. Most of these changes were explained by EV effects on coagulation, inflammation and haemolysis. However, MPE treatments prevented the EV-induced elevation in HPT. Consequently, HPT levels were similar to controls in the plasma of CHALLENGED mice. The plasma of CHALLENGED mice showed substantial proteomic modifications. This suggests the mechanism of MPE protection involves the activation of counterbalancing processes to compensate for the imbalances caused by EV.

Nitric oxide levels in patients with atheromatous carotid plaque.

Nitric oxide (NO), an unstable derived of nitrogen, is released by endothelium in response to physiological stimulus. Indeed, the endothelium is not only a barrier between the lumen and the inner side of the vessel wall but also a metabolically active organ with endocrine, paracrine and autocrine functions. Endothelial vascular cells play an important role in the regulating vasomotor tone, local homeostasis and vascular bed proliferation. NO mediates the vasodilation and inhibits platelet aggregation, expression of molecular adhesion of monocyte, neutrophils adhesion and smooth muscle growth. Atherosclerosis risk factors such as hypercholesteremia, high blood pressure, smoking and oxidative stress inhibit NO production, leading paradoxically to vasodilatation, which affects endothelial function and may lead to ischemic manifestations in patients with arterial pathology. Therapies that increase NO production may improve endothelial vasodilatation. To check whether a decrease or lower production of NO terminates with the initial formation of atheromatous plaque or whether it continues, we determined the content of NO in plasma and plaque of subjects undergoing carotid surgery and in plasma of control subjects.

Antidiabetic oligocyclitols in seeds of Mucuna pruriens.

Using a combination of chromatographic and NMR techniques, the presence of D-chiro-inositol and its two galacto-derivatives is demonstrated in Mucuna pruriens seeds. The quantities detected explain the well-established antiglycaemic effect of Mucuna pruriens seeds.

Protection of Mucuna pruriens seeds against Echis carinatus venom is exerted through a multiform glycoprotein whose oligosaccharide chains are functional in this role.

In a previous paper we demonstrated that extracts of Mucuna pruriens seeds (MPE) protect mice against Echis carinatus venom (EV) by an immunological mechanism. In this paper we demonstrate that the MPE immunogen generating the antibody that cross-reacts with the venom proteins is a multiform glycoprotein (gpMuc) whose immunogenic properties mainly reside in its glycan-chains. The glycoprotein was purified from the protein extract of M. pruriens seeds using Concanavalin A affinity chromatography. Using 2-D gel electrophoresis it separated into seven isoforms having MWs in the range from 20.3 to 28.7 kDa and pIs from 4.8 to 6.5. N-terminal sequencing of these spots revealed close similarity since all of them contained the consensus sequence DDREPV-DT found in soybean Kunitz-type trypsin inhibitor. We suggest that gpMuc contains both N- and O-glycans. Mild alkaline treatment but not PNGase F led to loss of reactivity, indicating that O-glycans are probably involved in the antigenicity of gpMuc.

Capillary electrophoresis in diagnosis and monitoring of adenosine deaminase deficiency.

BACKGROUND: The diagnosis and monitoring of severe combined immunodeficiency disease (SCID) attributable to adenosine deaminase (ADA) deficiency requires measurements of ADA, purine nucleoside phosphorylase (PNP), and S-adenosyl-L-homocysteine-hydrolase (SAHH) activity and of deoxyadenosine metabolites. We developed capillary electrophoresis (CE) methods for the detection of key diagnostic metabolites and evaluation of enzyme activities. METHODS: Deoxyadenosine metabolites were separated in 30 mmol/L sodium borate-10 mmol/L sodium dodecyl sulfate (pH 9.80) at 25 degrees C on a 60-cm uncoated capillary. For determination of enzyme activities, substrate-product separation and measurements were carried out in 20 mmol/L sodium borate (pH 10.00) at 25 degrees C on a 42-cm uncoated capillary. RESULTS: Deoxynucleotides and deoxyadenosine were readily detectable in erythrocytes and urine, respectively. Both methods were linear in the range 2-500 micro mol/L (r >0.99). Intra- and interassay CV were <4%. Enzyme activities were linear with respect to sample amounts in the incubation mixture and to incubation time (r >0.99 for both). In erythrocytes from healthy individuals, mean (SD) ADA activity was 5619 (2584) nmol/s per liter of packed cells. In erythrocytes of SCID patients at diagnosis, ADA activity was 56.9 (48.3) nmol/s per liter of packed cells; SAHH activity was also much reduced. PNP activity was similar in patients and controls. CONCLUSIONS: CE can be used to test ADA deficiency and enables rapid assessment of ADA expression in hematopoietic cells of SCID patients during therapy.

Lipid composition in atheromatous plaque: evaluation of the lipid three-phase percentage.

There is a renewed interest in the study of plaque lipid composition because it is recognized that it, rather than the luminal narrowing, influences the plaque stability and determines patient symptoms. At this purpose, we quantitatively evaluated in the carotid plaque of different categories of patients the expression of triglycerides, phospholipids, cholesterol, free cholesterol, esters of cholesterol, and the percentages of the three-phases (cholesterol, esters of cholesterol, phospholipids) by using the "Roozeboom triangle". Significant differences in the content of specific lipid and the percentage of the three-phases were detected among the different types of plaque evaluated in this study. The analysis of the three-phases by "Roozeboom triangle" may open a new approach in the study of atheromatous plaque and give new information on development of the disease.

Structure and function correlations between the rat liver threonine deaminase and aminotransferases.

The rat liver threonine deaminase is a cytoplasmic enzyme that catalyses the pyridoxal-phosphate-dependent dehydrative deamination of L-threonine and L-serine to ammonia and alpha-ketobutyrate and pyruvate, respectively, in vivo. During deamination, a molecule of the cofactor is converted to pyridoxamine phosphate. Recently, the ability of this enzyme to accomplish an inverse half-reaction, restoring pyridoxal-phosphate and L-alanine or L-aminobutyrate, respectively, from pyruvate or 2-oxobutyrate, was reported. In order to investigate the molecular mechanisms of this transaminating activity, a molecular model of rat liver threonine deaminase was constructed on the basis of sequence homology with the biosynthetic threonine deaminase of Escherichia coli, the crystal structure of which is known. The model has structural features shared by aminotransferases, suggesting that tertiary structural elements may be responsible for the transaminating activity observed for rat liver threonine deaminase.

Purine nucleotide catabolism in rat liver: labelling of uric acid and allantoin after administration of various labelled precursors.

Uric acid and allantoin are the key compounds of purine nucleotide catabolism formed in liver and many other organs of the rat. We observed that, after administration of 14C-formate, incorporation of radioactivity into uric acid and allantoin is not similar, as one would expect. The phenomenon was demonstrated to be specific to liver and perfused liver, and not to other organs such as heart, jejunal mucosa, lung, spleen, and kidney. To interpret these results, the specific radioactivity of uric acid and allantoin in rat liver were analysed comparatively, after administration of the following labelled precursors: 14C-glycine, 14C-formate, 14C-hypoxanthine, 14C-uric acid and 14C-adenine. After administration of 14C-formate the specific radioactivity of allantoin was higher than that of uric acid and the same behavior was observed after 14C-uric acid and 14C-hypoxanthine, but not after 14C-glycine and 14C-adenine administration. The results indicate that the rate of their incorporation into uric acid and allantoin, and the subsequent export of these compounds into serum, can only partially explain the observed phenomenon, while the presence of different pools of uric acid and allantoin may give a complete explanation.

Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning.

Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.

Proteins from Mucuna pruriens and enzymes from Echis carinatus venom: characterization and cross-reactions.

Mucuna pruriens seeds have been widely used against snakebite in traditional medicine. The antivenin property of a water extract of seeds was assessed in vivo in mice. The serum of mice treated with extract was tested for its immunological properties. Two proteins of Echis carinatus venom with apparent molecular masses of 25 and 16 kDa were detected by Western blot analysis carried out using IgG of mice immunized with extract or its partially purified protein fractions. By enzymatic in-gel digestion and electrospray ionization-mass spectrometry/mass spectrometry analysis of immunoreactive venom proteins, phospholipase A(2,) the most toxic enzyme of snake venom, was identified. These results demonstrate that the observed antivenin activity has an immune mechanism. Antibodies of mice treated with non-lethal doses of venom reacted against some proteins of M. pruriens extract. Proteins of E. carinatus venom and M. pruriens extract have at least one epitope in common as confirmed by immunodiffusion assay.