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.

In vitro exposure of human osteoarthritic chondrocytes to ELF fields and new therapeutic application of musically modulated electromagnetic fields: biological evidence.

Osteoarthritis (OA) is the most frequently occurring rheumatic disease, caused by metabolic changes in chondrocytes, the cells that maintain cartilage. Treatment with electromagnetic fields (MF) produces benefits in patients affected by this pathology. Isolated human osteoarthritic (OA) chondrocytes were cultured in vitro under standard conditions or stimulated with IL-1beta or IGF-1, to mimic the imbalance between chondroformation and chondroresorption processes observed in OA cartilage in vivo. The cells were exposed for a specific time to extremely low frequency (ELF; 100-Hz) electromagnetic fields and to the Therapeutic Application of Musically Modulated Electromagnetic Fields (TAMMEF), which are characterized by variable frequencies, intensities, and waveforms. Using flow cytometry, we tested the effects of the different types of exposure on chondrocyte metabolism. The exposure of the cells to both systems enhances cell proliferation, does not generate reactive oxygen species, does not cause glutathione depletion or changes in mitochondrial transmembrane potential and does not induce apoptosis. This study presents scientific support to the fact that MF could influence OA chondrocytes from different points of view (viability, ROS production and apoptosis). We can conclude that both ELF and TAMMEF systems could be recommended for OA therapy and represent a valid non-pharmacological approach to the treatment of this pathology.

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

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

[Preliminary results of the effects of electromagnetic fields ELF and TAMMEF on human lymphocytes: evaluation of some biological parameters]. / Studio preliminare sugli effetti di campi elettromagnetici, ELF e TAMMEF, nei linfociti umani: valutazione di alcuni parametri biologici.

AIMS: The Authors have evaluated the effects of low frequency electromagnetic fields on human peripheral blood lymphocytes metabolism. MATERIALS AND METHODS: It has been assayed total proteins and the activities of some purine metabolism enzymes after exposure of the cells to sinusoidal ELF fields (100 Hz frequency) or to the new TAMMEF system fields, characterized by variable frequency, intensity and shape of wave. RESULTS: The protein lymphocytes content, increased after both treatments. Instead, the enzyme activities did not vary, with the exception of Myokinase activity, which, respect to the control, increased after ELF field treatment, and slightly decreased after TAMMEF treatment. This preliminary result was interpreted as a variation of the cellular electric charge stability, stimulated by ELF fields, which induce the Myokinase to increase its rate, to rearrange the correct equilibrium, while the TAMMEF field were useless to maintain the cellular electric charge at normal levels. CONCLUSIONS: We interpreted these preliminary results as follow: the ELF fields influence the cellular electric charge stability, so that the cells must increase MK activity to restore the correct equilibrium, while TAMMEF fields are useful to maintain and regulate cellular electric charge. The results obtained by this preliminary study opens interesting prospective to be further investigated.

Adenosine kinase from rat liver: new biochemical properties.

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

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.

Quantitative separation of uric acid and allantoin from rat liver tissue.

A simple procedure is described for the assay of liver uric acid and allantoin and their specific radioactivity after administration of a radioactive precursor. Uric acid was quantified by the uricase reaction in liver trichloroacetic acid (TCA) extracts. The 'true' allantoin content of the liver could be estimated only after precipitation with Hg-acetate, a step by which the standard allantoin was also quantitatively recovered. Crude extracts lead to the evaluation of 'apparent' allantoin. For the determination of specific radioactivity, the Hg-acetate precipitate was further purified by ion-exchange chromatography. The purity of the two metabolites was confirmed by ultraviolet absorbance spectra, HPLC, constancy of specific radioactivity and the absence of amino acids. The incorporation of [14C]formate into uric acid and allantoin in the liver was studied by this procedure. The radioactivity in allantoin was several-fold higher than that in uric acid up to 60 min after administration of the precursor. This quite unexpected result is not easily explained on the basis of current knowledge.

The influence of testosterone on purine nucleotide metabolism in rat liver.

Purine nucleotide metabolism was studied in rat liver by following the incorporation of 14C-formate into soluble nucleotides, uric acid and RNA ribonucleotides. After castration, GMP formation was less than that of AMP, and purine nucleotide catabolism and RNA synthesis decreased. Testosterone administration did not modify GMP or AMP synthesis, but restored purine nucleotide catabolism and RNA production to normal values. These results demonstrate the influence of testosterone on purine nucleotide metabolism in a non-reproductive organ.

The regulation of aminotransferase activity by carbamoyl-phosphate.

We studied the effect of carbamoylphosphate (CP) on L-aspartate aminotransferase (GOT) and L-alanine aminotransferase (GPT), compared to its effect on L-threonine deaminase (TD). GPT and GOT were slightly inhibited by CP, while TD was strongly inhibited. GPT and TD, but not GOT, were inactivated when preincubated with CP. Only GOT was enhanced by pyridoxal 5'-phosphate (PLP), but not when the coenzyme was preincubated with CP. When the enzymes were resolved by p-chloromercuribenzoate (PCMB) treatment to apoenzymes, only GOT retained 47% of the original activity. Reconstitution of the apoenzymes with PLP also followed different course; activities of GPT and TD were completely restored while GOT remained partially inactivated. Treatment of apoenzymes with CP resulted in impairment of their reconstitution except GPT, activity of which could be completely restored. When PLP was pre-treated with CP before reconstitution, however, even GPT was only partially restored. The data indicated that CP affect activities of these enzymes at different levels, holoenzymes, PLP and probably apoenzymes. Under a concentration of PLP, activity of GOT would be most enhanced, followed by TD then GPT. In the presence of CP, this effect would be eliminated.

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.

Influence of estrogen on cholesterol esterification and fatty acid composition in serum lipoproteins of castrated rats.

Total, free and esterified cholesterol and its fatty acid composition were measured in the serum lipoproteins of castrated rats after estradiol administration. In general, castration and treatment with estradiol led to a decrease in total esterified cholesterol content. However, estradiol induced an effect opposite to that of castration on the fatty acid composition in VLDL. The effects were variable in HDL and insignificant in LDL. Similarly, the ratios of essential fatty acid to non-essential fatty acid (EFA/NEFA) and that to monoenoic acid (EFA/ME) were affected differently in castration and estradiol treatment in VLDL, but not in HDL or LDL. The pattern of lipid metabolism in castrated and estradiol-treated rats thus appears opposite to that described in human pathology.

Fatty acid composition of plasma lipids in gout.

The changes occurring in the fatty acid composition of the plasma lipids were studied in gout. The major changes consisted in an increase in oleic acid and a decrease in linoleic and arachidonic acids in most lipid fractions of plasma; linoleic acid is unchanged in plasma cholesteryl esters and in FFA while arachidonic acid does not vary only in FFA. These changes are not related either to diet or to the age of patients and are similar to those reported in atherosclerotic and diabetic patients, as well as those with cardiac ischemia. The variations observed are directly influenced by the ratio essential fatty acids/monoenoic acids and all factors affecting this ratio.

Purine nucleotide metabolism in lymphocytes of B-cell chronic lymphocytic leukemia patients.

Purine nucleotides were studied in human peripheral blood lymphocytes from normal subjects and patients with chronic B-cell lymphocytic leukemia (B-CLL). Nucleotide content was determined by high performance liquid chromatography (HPLC). The overall rate of purine nucleotide synthesis was measured following the incorporation of 14C-formate into the nucleotides of a lymphocytic suspension. Results indicate a substantially reduced rate of purine nucleotide metabolism.

The excretion of oxypurines in normal subjects.

Reverse phase high performance liquid chromatography was used for the determination of urinary hypoxanthine and xanthine in normal subjects. The values observed in normal subjects agree with those reported by other authors. Excretion of hypoxanthine and xanthine was not related to sex. Urinary oxypurines were related to age, and, when corrected for body surface area (1.73 m2), they were significantly higher in normal children under 15 years of age than in adults.

Plasma oxypurines in gastric and colorectal cancer.

Reverse phase high performance liquid chromatography has been used for the determination of plasma hypoxanthine, xanthine, and uric acid in normal subjects and in patients with gastric and colorectal cancer. Plasma oxypurines are significantly elevated in either type of cancer, while uric acid concentration is only higher in gastric cancer. The variations are related to the stage of the tumors, and the physiopathology of their occurrence is discussed.

Enzyme activities controlling adenosine levels in normal and neoplastic tissues.

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

Synthesis and NMR properties of 2-(4-pyridyl) thiazolidine-4-carboxylic acids.

Various thiazolidine derivatives, obtained through condensation between pyridoxal or pyridoxal-5'-phosphate with several aminothiols, have been obtained and examined for their NMR spectra. For the compounds tested, we demonstrated the presence in solution of the two diasteroisomeric thiazolidines, A and C, which differ for the configuration at C2, and are interconverted via the open chain Schiff's base, with different rates, depending on the pH. The possibility that, when aminothiols react with PLP bound to the enzyme, only one of the two diasteroisomers may be formed, and the biological role of such derivatives is discussed.

The determination of urinary oxypurines as markers of gastrointestinal tumors.

Plasma levels and urinary excretion of oxypurines--hypoxanthine and xanthine--were evaluated by reverse-phase high-pressure liquid chromatography in 13 patients affected by gastric tumors and in 19 colorectal tumor-bearing patients. Preliminary results indicate higher values of urinary xanthine and an increase in the xanthine/hypoxanthine ratio in cancer patients. The increase was not generalized to all subjects, and did not appear related either to the stage of the disease or to CEA values. The limits within which the determination of urinary oxypurines can be employed as a tumor marker are discussed.

Some aspects of purine nucleotide metabolism in lymphocytes of B-CLL.

The authors studied the behavior of some enzymes involved in purine nucleotide metabolism in human peripheral blood lymphocytes from normal and B-cell chronic lymphocytic leukemia subjects. Determinations were made with radiochemical methods associated with high performance liquid chromatography. Results indicated a marked increase in de novo purine synthesis enzymes, particularly those of the "inosinic branch point". The latter were absent in normal lymphocytes, whereas they were well evident in leukemic lymphocytes, with the exception of AMP-S synthetase. Whereas the enzymes of the "salvage pathway" were spared in comparison to other proteins, those of the "catabolic pathway" significantly decreased. The authors discuss the possibility that such enzymes may be used as tumor markers.

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.