1,721,090 research outputs found
Epidermal inhibitory pentapeptide phosphorylated in vitro by calf thymus protein kinase NII is protected from serum enzyme hydrolysis.
No full textIn this paper, we demonstrate that synthetic epidermal mitosis inhibiting pentapeptide (pyroGlu-Glu-Asp-Ser-Gly) is phosphorylated in vitro at serine level by protein kinase NII isolated from calf thymus chromatin. A serum enzyme, which rapidly cleaves the synthetic epidermal mitosis inhibiting pentapeptide, also hydrolyses the synthetic transcription inhibiting pentapeptide (pyroGlu-Ala-Glu-Ser-Asn). The phosphorylated forms of both pentapeptides are protected from the serum enzyme activity
BIOMONITORAGGIO PER LA PRESENZA DI DISTRUTTORI ENDOCRINI NEL FIUME CHIENTI: DUE METODOLOGIE A CONFRONTO
No full textLe acque superficiali ricevono un continuo apporto di sostanze inquinanti mediante gli scarichi urbani, industriali e le acque di dilavamento dei suoli agricoli., Molti di questi inquinanti possono funzionare da distruttori endocrini e, come ormai è ben noto, tali composti rappresentano una classe di contaminanti a diffusione ubiquitaria. Nel presente lavoro è stata valutata la presenza di di distruttori endocrini ad azione estrogenica (xenoestrogeni) nel fiume Chienti (Macerata) utilizzando un approccio integrato basato sull’impiego di test in vivo ed in vitro. Sono stati analizzati tre differenti siti: uno prossimo alla sorgente, uno nel tratto intermedio ed uno prossimo alla foce. Per quanto riguarda gli studi in vivo è stata valutata la comparsa della vitellogenina in giovanili di Carassius auratus mantenuti, per un periodo di tre settimane, in acqua prelevata nei diversi siti monitorati. Per gli studi in vitro, invece, è stata utilizzata una coltura cellulare di carcinoma di mammella estrogeno-dipendente (MCF-7) trattata con la frazione organica (estratta mediate cromatografia con colonnine C-18) dei campioni di acqua prelevati nei tre siti.
I risultati ottenuti indicano chiaramente come tutti i campioni di acqua testati siano in grado di indurre la sintesi di VTG in C. auratus e stimolare la proliferazione nella linea cellulare MCF-7; si dimostra inoltre come la concentrazione degli xenoestrogeni aumenta passando dal tratto montano alla foce.
La presenza di xenoestrogeni nel tratto prossimo alla sorgente va valutata tenendo conto del fatto che una piccola parte del bacino idrografico del fiume Chienti si trova negli altopiani di Colfiorito (Perugia) nei quali sono presenti coltivazioni intensive.
Appare significativo il fatto che i due metodi di studio applicati mostrano un risultato comparabile; questo dato fornisce supporto all'utilizzo di sistemi in vitro come primo livello di indagine per il monitoraggio degli xenoestrogeni limitando il più possibile il sacrificio di animali.
Progetto finanziato dalla Provincia di Macerata con delibera n.277 del 04/06/2009 della Giunta Provinciale di Macerata
Synthetic seminal plasma peptide inhibits testosterone production in rat Leydig cells.
No full textNative peptides, extracted from human and bovine seminal plasma, were characterized at the endocrine and paracrine level as modulators of follicle stimulating hormone activity. Similarly to other peptides extracted from chromatin of various tissues, seminal plasma peptides show regulatory activity on RNA synthesis by exerting inhibitory effects on transcription in vitro and in neoplastic and fast-growing cells. Biochemical and mass spectrometry analysis of highly purified fraction from seminal plasma allowed to design and synthesize a peptide with the following amino acid sequence: pGlu-Val-Ala-Asp-Ser-Asp-Gln-Asn (Mancinelli et al. Biol. Chem. 380: 31-40, 1999). Preliminary data on the activity of synthetic peptide have shown an inhibition of testosterone production by frog testes incubated in presence of peptide. The aim of our research was to study the role of synthetic peptide in mammalian testicular steroidogenesis. Continuous, isoosmotic Percoll gradient was utilized to isolate highly purified Leydig cells from rat testicular interstitial cell suspensions. 5 x 104 cells were incubated with increasing doses of synthetic peptide in presence of 50 mU LH for 180 min at 37°C. Testosterone levels in the incubation medium were measured by radioimmunoassay. The results showed the inhibition of testosterone production by Leydig cells with a bell-shape curve. 10-8 M synthetic peptide exerted the highest testosterone inhibition (58%). The basal production of testosterone, without LH stimulation, remained unchanged after treatment of rat Leydig cells with synthetic seminal plasma peptide. Toxicological analysis of synthetic peptide was carried out on carcinoma cell line A431 with negative results. These data suggest a probable role of synthetic seminal plasma peptides in modulating testicular steroidogenesis
Antioxidant and prooxidant properties of Brassica oleracea L. var. sabauda sprout extracts.
No full textReactive oxygen species (ROS) play a critical role in cardiovascular diseases, inflammatory diseases, neurodegenerative disorders, cancer and aging. Diets rich in foods containing antioxidants, such as fruits and vegetables, could help to prevent these pathologies. Wheat and Brussels sprouts contain antioxidant compounds active in the protection of DNA against the oxidative stress induced by ROS. The aim of this work was to determine antioxidant activity of aqueous and ethanol extracts from savoy cabbage (Brassica oleracea L. var. sabauda) sprout powder. Significant values of antioxidant activity were found in both extracts using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging assays. 2’,7’-dichlorofluorescin-diacetate (DCFH-DA), a useful indicator of ROS, was used to determine the antioxidant and prooxidant activity of sprout extracts in a cell-based assay (human dermal cell line). Results show a dose-dependent prooxidant activity of aqueous sprout extract, higher than ethanol extract. The prooxidant activity was correlated with the inhibition of cell proliferation. Interestingly, the boiling of aqueous extract inhibits this prooxidant effect, restore cell proliferation without affecting the antioxidant effect in vitro
Comparison of ACE activity in amphibian tissues: Rana esculenta and Xenopus laevis
No full textAngiotensin converting enzyme (ACE) is the dipeptidyl-carboxypeptidase of the renin–angiotensin system involved in the control of blood pressure and hydromineral metabolism. It converts angiotensin I to angiotensin II, the biologically active octapeptide. Angiotensin converting enzyme-like activity has been demonstrated in a wide range of vertebrates. The presence of ACE was investigated in tissues of two amphibian species, the frog Rana esculenta and the toad Xenopus laevis. ACE activities were determined by specific substrate hydrolysis in gut, gonads, lung, kidney, heart, liver, skin, erythrocytes, and muscle homogenates and plasma by means of high performance liquid chromatography. Significant ACE activity was found in gut, gonads, lung and kidney, while that in heart, liver, skin, erythrocytes, muscle, and plasma was very low. Testis of toad contained the highest ACE activity, while that in erythrocytes of male and female frogs was notable
Antimitotic and cytotoxic activity of climacostol on tumoral mammalian cells.
No full textThe freshwater ciliate Climacostomum virens defends itself
against predators by discharging the toxin climacostol (1,3-dihydroxy-5-[(Z)-20-nonenyl]benzene), a resorcinolic lipid contained in its extrusomes (Miyake et al. 2003, Eur. J. Protistol. 39: 25–36). Like other natural chemically related compounds, exerts a dose-dependent cytotoxic effect on tumoral mammalian cell lines (Buonanno et al. 2005, J. Eukaryot. Microbiol. 52:38S). Based on this evidence, we have now examined the effects of chemically synthesized climacostol (Masaki et al. 2004, Tetrahedron 60: 7041–7048) on the proliferation of human promyelocytic leukaemia cells (HL60 line), and squamous carcinoma cells (A431 line) plus, as control, of non-tumoral cells derived from mice Leydig cells (TM3 line). It was observed that: (i) climacostol inhibits the
growth of all cell lines used in dose-dependent manner, but the HL60 and A431 cells are significantly more sensitive to the toxin than TM3 cells; (ii) the cytotoxicity of climacostol (used at a concentration of 10 mg/ml, 24 h of incubation, and detected by the LDH-cytotoxicity test) is appreciably stronger on the tumoral cell lines (60% and 41% cytotoxic activity for the A431 and HL60 cells, respectively) than on the TM3 cells (23% cytotoxic activity). It was in addition observed (by using a fluorescent dye technique) that climacostol induces apoptosis, rather than necrosis, in all the cell lines used. These results indicate that climacostol possesses interesting antitumoral biological activities and encourage further in vivo investigations to assess its potential use in cancer chemotherapy
Synthetic seminal plasma peptide inhibits testosterone production in frog testis in vitro.
No full textThe role of synthetic seminal plasma peptide, designed using biochemical and mass spectroscopy analyses of native peptides extracted from seminal plasma, was studied in amphibian (Rana esculenta) testicular steroidogenesis. Production of testosterone and prostaglandin F2α was determined by incubating frog testes with synthetic peptide in vitro. Analysis of the data showed a dose-dependent inhibition of testosterone production (43% at 10-5 M concentration) without prostaglandin F2α synthesis being affected. Determination of the peptide activity during the annual R. esculenta reproductive cycle showed inhibition of testosterone production in post-reproductive and recovery periods, suggesting a possible involvement of peptide in gonad steroidogenesis
Bioavailability of thymic humoral factor gamma2.
No full textDespite the considerable progress made in peptide drug research, many serious obstacles, the most important being that of obtaining sufficient bioavailability for maintaining pharmacological efficiency, still hinder the use of peptides as useful therapeutic agents. Thymic Humoral Factor gamma2 (THF-gamma2) is an immunoregulatory peptide present in thymic extract, identified as an octapeptide of the structure Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu. THF-gamma2 increases T-cell functions as the response to T-cell lectins and interleukin-2 production. THF-gamma2 has been used as an immunomodulator in clinical conditions associated with immune impairment and dysregulation. The degradation of THF-gamma2 by enzymes present in human plasma, has been investigated. The cleavage of THF-gamma2 was insensitive to classical proteinase inhibitors, but sensitive to metalloproteinase and aminopeptidase inhibitors. The degradation was completely blocked by specific inhibitors of angiotensin converting enzyme (ACE) and aminopeptidase. The sites of cleavage were identified by HPLC analysis and amino acid analysis. Leu1-Glu2 and Lys6-Phe7 bounds were cleaved by aminopeptidase and ACE, respectively. After human ACE purification, some kinetic parameters were determined. Km and Kcat values for THF-gamma2 were 0.273 mM and 107 s-1, respectively. The optimum of pH was 7.6 . I50 for captopril and lisinopril, two specific ACE inhibitors, were 1.052±0.140 nM and 25.770±1.079 nM. The data obtained are useful to modifying the peptide structure so as to reduce recognition by proteinases and to prolong the biological activity
Effects of climacostol on normal and tumoral mammalian cell lines
No full textClimacostol, 1,3-dihydroxy-5-[(Z)-20-nonenyl]benzene, is a
natural toxin contained in the extrusomal cortical granules of
the heterotrich ciliate Climacostomum virens. It is used for
chemical defence against predators such as the raptorial ciliate Dileptus margaritifer and its cytotoxic activity has been assessed on several species of ciliates such as Didinium nasutum, Paramecium caudatum, and Blepharisma japonicum (Miyake et al. 2003, Europ. J. Protistol., 39:25–36). On the basis of its chemical structure, climacostol may be classified into the large group of natural compounds known as resorcinolic lipids, that show antimicrobial, antiparasitic, and antitumoral activities (Kozubek et al. 2003, Cell Moll. Biol. Lett., 6:351–355). To explore the possibility to use climacostol in medical applications, we examined the
effects of chemically synthesized climacostol (Masaki et al.
2004, Tetrahedron, 60:7041–7048) on the growth and proliferation of tumoral and normal mammalian cell lines: (1) human promyelocytic leukaemia cells, HL60; (2) human squamous carcinoma cells, A431; and (3) non-tumoral cells derived from mice Leydig cells, TM3. It was observed that (1) a concentration of 10 mg/ml of climacostol exerts a strong cytotoxic activity on all cell lines used; (2) at lower concentrations of 10 ng/ml and 1 ng/ ml, the effect of climacostol is limited to the inhibition of the cell growth; and (3) the normal TM3 cells are more resistant to climacostol than the two tumoral HL60 and A431cell lines. The dose-dependent cytotoxic effects of climacostol encourage further
investigation on the potential use of this ciliate toxin as an anticancer chemical
Purification of swine serum angiotensin converting enzyme with high recovery of activity using lisinopril coupled to epoxy-activated sepharose 6B.
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