Expression of RXFP1 in skin of scleroderma patients and control subjects.

OBJECTIVES: Relaxin (RLX) is involved in extracellular matrix and collagen remodelling. The therapeutic role of the circulating isoform RLX-2 as an anti-fibrotic factor in systemic sclerosis (SSc) has been investigated. Several RLX family peptide receptors (RXFPs) are recognized in humans: RLX-2 is a ligand for RXFP1/LGR7 and RXFP2/LGR8. The aim of this study was to define the pattern of expression of LGR7 in different types of human skin cells and to compare normal skin with lesional and unaffected skin from patients with limited SSc (lSSc). METHOD: We analysed RXFP1 immunolocalization on skin biopsies and cultured fibroblasts from lSSc patients and control subjects. Western blot analysis was carried out on fibroblast lysates. RESULTS: RXFP1 showed cytoplasmic localization on skin cells from control subjects and non-lesional skin from lSSc patients: keratinocytes, gland epithelial cells, endothelium, smooth muscle cells, and fibroblasts. Immunogold electron microscopy confirmed a diffuse epithelial cytoplasmic localization of RXFP1. A substantially lower RXFP1 expression was observed in scleroderma skin, with a lack of staining in most cells. Occasional weak reactivity was observed in cultured scleroderma fibroblasts, while control fibroblasts showed a diffuse cytoplasmic immunoreactivity of RXFP1, confirmed by Western blot analysis. CONCLUSIONS: The decreased cellular expression of RLX-2 receptor RXFP1 in scleroderma skin might represent a pro-fibrotic factor and contribute to the substantial inefficacy of RLX treatment in SSc, as reported in the literature. The pathophysiology of the decrease in RXFP1 may be linked to high RLX-2 serum levels previously detected in SSc, but it has yet to be elucidated.

Serum proteome of patients with systemic sclerosis: molecular analysis of expression and prevalence of haptoglobin alpha chain isoforms.

Haptoglobin (Hpt) is an acute phase protein characterized by three major phenotypes (Hpt 1-1, Hpt 2-1 and Hpt 2-2). The Hpt 2-2 phenotype is associated with increased prevalence of various systemic diseases, including autoimmune disorders. Moreover, the Hpt 2-2 phenotype induces a shift from Th1 to Th2 response and increases fibrotic processes. On this basis, we performed serum proteomic analysis of patients with Systemic Sclerosis (SSc), a connective tissue disorder associated with Th2-type immune response and characterized by interstitial and perivascular fibrosis due to different factors (including genetic, environmental, immunological and microchimeric factors). Serum of 23 SSc outpatients patients (4 males, 19 females, mean age 54+-5.3 years) diagnosed according to the American Rheumatism Association (ARA) criteria, were considered for the proteomic analysis and compared to serum of 21 control subjects. Serum depleted of HAP was analyzed by 2-DE, and Hpt chain spots were identified by WB. The expression frequency of each Hpt α chain in SSc patients and controls was compared and quantitative analysis of spot expression (percent Vol) was performed. Above all,, our study amplifies the limited data in the literature on proteomic analysis in SSc, also confirming previous data that revealed a significant increase of haptoglobin type 2-2 and a concomitant decrease of the 1-1 phenotype in SSc patients. Moreover, our results demonstrate that c spots are more prevalent in SSc patients than in controls (91.3% vs 55.5%, p<0.05), while the expression frequency of a and b spots does not change. In patients Hpt 2-1 or Hpt 1-1 e spot is less abundant. According to our results, the c and e spots can be considered markers for SSc and thus be of use for the early diagnosis of connective tissue disorders and in establishing appropriate treatment.

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.

Identification of a mitochondrial inhibitor of rat liver L-threonine dehydrogenase.

In a previous paper, we showed inhibition of rat liver L-threonine dehydrogenase by a preparation obtained by dialysis and concentration from rat liver mitochondria stored at -20 degrees C for 7-10 days (Pagani, R., Leoncini, R., Guerranti, R. and Marinello, E. (1990) It. J. Biochem. 39, 106-114). The chemical composition of the fraction containing the unknown 'inhibitor' has now been studied and identified as D-3-hydroxybutyrate (D3HB).

Inhibition and regulation of rat liver L-threonine dehydrogenase by different fatty acids and their derivatives.

Rat liver L-threonine dehydrogenase is a mitochondrial enzyme which transforms L-threonine either into aminoacetone or into acetyl-CoA. We show that it is inhibited by several fatty acids and their derivatives: short chain fatty acids, L-2-hydroxybutyrate and D-3-hydroxybutyrate, long chain fatty acids, such as lauric acid, myristic acid, palmitic and stearic acids, bicarboxylic acids such as malonic acid and its derivatives methyl- and hydroxymalonic acids. The inhibition occurs at low and physiological concentrations of such compounds, which are normally present and metabolized in mitochondria. It presumably plays a role in the physiology of acetyl-CoA-dependent formation of fatty acids and ketobodies, in L-threonine-dependent gluconeogenesis, and in the regulation of L-threonine metabolism by L-threonine dehydrogenase and L-threonine deaminase.

Restoration of rat liver L-threonine dehydratase activity by pyridoxamine 5'-phosphate: the half-transaminating activity of L-threonine dehydratase and its regulatory role.

When a highly purified preparation of rat liver l-threonine deaminase (l-TDH, EC 4.2.1.16) was 99% inactivated by dialysis, removing bound pyridoxal 5'-phosphate (PLP), the apoenzyme was reactivated not only by PLP but also by pyridoxamine 5'-phosphate (PMP). When purified by HPLC, the commercial PMP used in the incubation mixture was found to contain only extremely small amounts of PLP, which could not account for restoration of l-threonine dehydratase activity. HPLC analysis of the assay mixtures showed that during incubation, sufficient PLP had been formed for reactivation of the apoenzyme. The apoenzyme evidently bound PMP and triggered transamination between PMP and the keto acids, which either contaminated, or were formed by the minimal amount of PLP-holoenzyme always present even in the dialyzed preparation. When sufficient PLP was formed, the PLP-holoenzyme and the original 'true' l-threonine dehydratase activity were restored. When PMP was incubated with the apoenzyme in the presence of small quantities of keto acids (pyruvate or 2-oxobutyrate) small amounts of l-alanine or l-aminobutyrate were formed. The reaction was not reversible; l-alanine and l-aminobutyrate did not react with the PLP-holoenzyme. No transaminating activity occurred with other amino acids. These results show that l-threonine dehydratase exists in two forms: the well known stable apoenzyme-PLP (hydrolase deaminating) and the transient apoenzyme-PMP (non-reversible half-transaminating). Half-transamination has the biological role of keeping the activity of the 'true' l-TDH constant and of regulating intracellular levels of pyruvate, alanine, oxobutyric acid, l-aminobutyric acid, l-threonine and l-serine.

MtDNA mutagenesis impairs elimination of mitochondria during erythroid maturation leading to enhanced erythrocyte destruction.

Haematopoietic progenitor cells show special sensitivity to mitochondrial DNA (mtDNA) mutagenesis, which suggests that increased mtDNA mutagenesis could underlie anemias. Here we show that elevated mtDNA mutagenesis in mice with a proof-reading deficient mtDNA polymerase (PolG) leads to incomplete mitochondrial clearance, with asynchronized iron loading in erythroid precursors, and increased total and free cellular iron content. The resulting Fenton chemistry leads to oxidative damage and premature destruction of erythrocytes by splenic macrophages. Our data indicate that mitochondria actively contribute to their own elimination in reticulocytes and modulate iron loading. Asynchrony of this sequence of events causes severe mitochondrial anaemia by depleting the organism of red blood cells and the bone marrow of iron. Our findings account for the anaemia development in a progeroid mouse model and may have direct relevance to the anemias associated with human mitochondrial disease and ageing.

5'-nucleotidase activity in lymphocytes from patients affected by B-cell chronic lymphocytic leukemia.

OBJECTIVES: The activity of membrane-bound ecto-5'-nucleotidase and soluble e-Ns and c-N-II 5'-nucleotidases was evaluated on lymphocytes from patients affected by B-cell chronic lymphocytic leukemia (B-CLL). A statistically significative decrease in ecto-5'-nucleotidase, e-Ns, and c-N-II activities was observed in peripheral blood lymphocytes and in B and T populations from affected individuals. DESIGN AND METHODS: For the assay of ecto-5'-nucleotidase, e-Ns, and c-N-II activity we used a radioactive procedure coupled to HPLC. Since the ecto-5'-nucleotidase is identified as CD73 antigen, we performed immunofluorescence analysis using a specific monoclonal antibody. We analyzed ecto-5'-nucleotidase mRNA by RT-PCR to ascertain the possibility of an alteration in the transcription of its gene. RESULTS: A decrease in ecto-5'-nucleotidase activity was correlated to reduction in ecto-5'-nucleotidase positive cells (CD73+) in leukemia patients. RT-PCR produced a fragment of the expected size and the specific mRNA was found expressed in both healthy subjects and leukemia patients. CONCLUSIONS: The decrease in ecto-5'-nucleotidase activity in patients with B-CLL is not due to loss of transcription of the specific mRNA. The presence of point mutations, splicing alteration, or posttranslational modifications must be investigated. If a defect at DNA or RNA level will be detected, the molecular analysis will be considered for diagnosis of B-cell chronic lymphocytic leukemia.

Purine nucleotide catabolism in rat liver: labelling of uric acid and allantoin after treatment with oxonic acid and allopurinol.

In our previous experiments on rat liver we found that 15' after intraperitoneal administration of 14C-formate the specific radioactivity of allantoin was always higher than that of uric acid. The present experiments have been carried out to interpret this unexpected result, which was only observed in liver and we studied: a) the incorporation of 14C-glycine into uric acid and allantoin; b) the effects of two competitive inhibitors of xanthine oxidase and uricase, oxonic acid and allopurinol respectively, on levels of uric acid and allantoin in liver and on their specific radioactivity after administration of labelled precursor. The results suggested: a) that under normal conditions, the formation of allantoin is so fast that it exceedes export from liver to serum, and thus the radioactivity of labelled precursors accumulates in allantoin; b) that when allopurinol or oxonic acid are administered, the rate of export exceeds that of allantoin formation and the incorporation of radioactivity into allantoin is lower; c) that not all the data, however, could be interpreted on this basis, but seems to require the existence of different pools of uric acid, which are transformed separately into allantoin.

Fatty acid composition of phospholipids, triglycerides and cholesterol in serum of castrated and estradiol treated rats.

We have studied the levels of phospholipids, triglycerides, cholesterol esters, and their fatty acid composition in serum for normal, castrated and estradiol treated rats. The sex hormones did not greatly affect the levels of the various lipid fractions which did not undergo great significant variations, under the different treatments. More evident variations occurred in the percent composition of fatty acid and in the content of the various saturated (SAT), unsaturated (UNSAT), essential (EFA) and non essential fatty acids (NEFA). We studied the most important ratios: EFA/NEFA; UNS/SAT; 16:0/16:1; 18:0/18:1, 18:2/18:3; 18:2/20:4. 16:0/16:1; 18:0/18:1 represent the delta9 desaturase, one specific for palmitic, the other for stearic acid. 18:2/18:3 ratio is an index of the delta6 desaturase activity: 18:2/20:4 ratio of delta5 desaturase-elongase. Most changes were evident in triglycerides. We observed a different behaviour of the UNS/SAT and EFA/NEFA ratios in phospholipids and cholesterol esters, which may reflect either an effect of the sex hormones on the exchange of fatty acids between the same lipid fractions, or a redistribution of lipids among different tissues. Great variations were observed of the ratios 16:0/16:1; 18:0/18:1; 18:2/18:3; 18:2/20:4, which are ascribed a different effect of the sex hormones of delta9, delta6, delta5 desaturases.

Some chemical properties and biological role of thiazolidine compounds.

In this study we have investigated some chemical properties and the biological role of thiazolidine compounds, obtained by condensation of aminothiols (L- or D-cysteine, cysteamine) with pyridoxal-5'-phosphate. These products have been tested in presence of rat liver extracts (supernatant and mitochondria); bacterial suspensions and enzymes (L- or D-aminoacid oxidase, xanthine oxidase) with interesting results which gives evidence to a biological role. Their formation in vivo may represent the regulation of intracellular levels of pyridoxal-5'-phosphate and aminothiols. Moreover, we have analysed the two diastereoisomers of the thiazolidine compounds derived from L-cysteine and D-cysteine: we have succeeded to distinguish by NMR analysis the cis and the trans forms, concluding that the interconversion of the free forms is extremely rapid at pH 7: thus, it may be relevant for the protein bound forms.

Determination, activity and biological role of adenylosuccinate lyase in blood cells.

Adenylosuccinate lyase deficiency, which is associated with severe mental retardation and autistic features, was discovered in 1984. Since then this enzyme has been analyzed in many human tissues and it is now generally agreed that screening for this enzyme defect should be performed in all unexplained neurological diseases. The aim of the present study was to analyze adenylosuccinate lyase activity in blood cells by a fast simple method adaptable to screening purposes. The activity was also analyzed in B-lymphocytes from patients with B-cell chronic lymphocytic leukemia. The biological role of adenylosuccinate lyase and its importance in regulating cellular levels of AMP is discussed.

An improved method for purification of L-threonine deaminase from rat liver.

L-threonine deaminase was obtained at a high degree of purity from rat liver. Two main steps were added to the previously reported procedure: cryoprecipitation and chromatofocusing (in the presence of a specific KCl concentration). The purification factor was 3,090 and the specific activity 989. The method is very reproducible and convenient. It gives the highest specific activity and the highest degree of purity of the enzyme recorded to date.

Effects of Mucuna pruriens extract on activation of prothrombin by Echis carinatus venom.

Mucuna pruriens (L.) DC has long been used as a medicinal plant by traditional healers. The validity of the claims made for this plant has also been tested scientifically. Some of its properties are probably linked to high concentrations of dopa since it is useful in the treatment of Parkinson's disease. The antisnake properties of an extract of Mucuna pruriens' seeds (MP101UJ) in vivo were recently demonstrated and one is now investigating its biochemical mechanism. Echis carinatus venom (EV) contains a mixture of proteins that affect the coagulative cascade, causing severe bleeding and haemorrhage. Here the effect of an extract of MP101UJ in prothrombin activation by EV in vitro by clotting and chromogenic assay is studied. An increase in procoagulant activity was found. This could explain the protective effect in vivo.

Activation and inhibition of rat liver L-threonine dehydrogenase.

We have studied some characteristics of mitochondrial rat liver L-threonine dehydrogenase during freezing: the enzyme shows a net increase in activity and a "shift" in the pH optimum after storage at -20 degrees C of mitochondrial preparations. An unknown effector, which inhibited significantly the dehydrogenase activity, was isolated from the same preparations kept for 7-10 days at -20 degrees C. Some properties of the inhibitor have been determined.

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.