Biomedical subjects

Giovana Seno Di Marco

Publications and source records attributed to Giovana Seno Di Marco.

9 recordsLinked to original sources

PubMed2005

Expression and localization of N-domain ANG I-converting enzymes in mesangial cells in culture from spontaneously hypertensive rats.

The angiotensin-converting enzyme (ACE) profile in urine of hypertensive patients and spontaneously hypertensive rats (SHR; 90- and 65-kDa N-domain ACEs) is different from that of healthy subjects and Wistar rats (190 and 65 kDa). In addition, four ACE isoforms were purified from mesangial cells (MC) of Wistar rats in the intracellular compartment (130 and 68 kDa) and as secreted forms (130 and 60 kDa). We decided to characterize ACE forms from SHR MC in culture. Analysis of the ACE gene showed that SHR MC are able to express ACE mRNA. The concentrated medium and cell homogenate were separately purified by gel filtration and then subjected to lisinopril-Sepharose chromatography. The molecular masses of purified enzymes, 90 kDa for ACEm1A and 65 kDa for ACEm2A (secreted enzymes) and 90 kDa for ACEInth1A and 65 kDa for ACEInth2A (intracellular), were different from those of Wistar MC. The purified enzymes are Cl- dependent, inhibited by enalaprilat and captopril, and able to hydrolyze AcSDKP. Immunofluorescence and cell fractionation followed by Western blotting showed predominant immunoreaction of the 9B9 antiserum for N-domain ACE in the nuclei. The N-domain ACE was localized in the glomerulus from Wistar rats and SHR. ANG II and ANG-(1-7) were localized in the cell cytoplasm and nuclei. The 90-kDa N-domain ACE, described recently as a possible genetic marker of hypertension, was found inside the cell nuclei of SHR MC colocalized with ANG II and ANG-(1-7). The presence of ANG II in the cell nuclei could suggest an important role for this peptide in the transcription of new genes.

Angiotensin II↗

PubMed2005

Sirolimus quantification by high-performance liquid chromatography with ultraviolet detection.

The need to adapt optimal conditions of sirolimus blood level monitoring in laboratories led us to optimize an high-performance liquid chromatography-ultraviolet method and compare the elution performances using the mobile phase A, 68% MeOH/2% acetonitrile (ACN)/30% H(2)O and mobile phase B, 30% MeOH/42% ACN/28% H(2)O. Samples were assayed with 1-chlorobutane, redissolved in MeOH/water and injected onto a C-18 column at 50 degrees C. The assay achieved sensitivity of 2.5-150 ng/ml (CV = 10.6%) and recovery of 92-103.6%. The intra- and interassay precisions ranged from 3.3% to 13% and from 5.9% to 15% for quality controls of 7.5, 60 and 120 ng/ml. The mobile phase A was unable to elute and recover sirolimus and internal standard in the expected retention time and concentration. Under our working conditions, the assay was precise, accurate and sensible, stressing the importance of establishing for the best working conditions according to the staff and demands of the laboratory.

Chromatography, High Pressure Liquid↗

PubMed2005

Cyclosporine A and NAC on the inducible nitric oxide synthase expression and nitric oxide synthesis in rat renal artery cultured cells.

BACKGROUND: The immunosuppressor cyclosporine A (CsA) presents the nephrotoxicity as its major side effect that is mostly attributed to a renal vasoconstriction. This may be due to an excessive generation of vasoconstrictors like reactive oxygen species (ROS), or due to a reduction of vasodilators such as the nitric oxide, which in turn, can be caused by increased amounts of ROS. We evaluated the effect of CsA and the antioxidant N-acetylcysteine (NAC) on inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide synthesis, in rat renal artery vascular smooth muscle cells (rVSMCs) primary culture. METHODS: In cells treated during 72 hours with CsA (10 microg/mL), its vehicle (control) (10 microL/mL), Escherichia coli lipopolysaccharide (LPS) (100 microg/mL), CsA + LPS, NAC (6.13 mmol/L), or CsA + NAC, we determined the nitric oxide synthesis (Griess and chemiluminescence methods), iNOS expression [reverse transcription-polymerase chain reaction (RT-PCR)] and cell viability (acridine orange method). RESULTS: In rVSMCs, LPS increased nitric oxide and iNOS expression; CsA decreased basal and LPS-induced nitric oxide and iNOS expression; NAC increased nitric oxide and blunted the nitric oxide reduction caused by CsA, with no effect on iNOS. CsA reduced cell viability. CONCLUSION: In this study, CsA reduced nitric oxide synthesis in rVSMCs, both through iNOS down-regulation and reduction of cell viability, which could be responsible for the vasoconstrictive effect of the CsA. In the effect of CsA on nitric oxide, probably a role is also played by free radical production, as this effect was blunted by NAC.

Acetylcysteine↗

PubMed2004

Urinary neopterin quantification by reverse-phase high-performance liquid chromatography with ultraviolet detection.

Neopterin plays an important role in the malignant disease diagnostics. However, the methods employed in neopterin determination are generally difficult and/or time consuming. The aim of this work was to standardize a practical method to quantify neopterin using high-performance liquid chromatography-ultraviolet (HPLC-UV) and quantify it in patients with systemic lupus erythematosus (SLE). Urine was collected from healthy subjects (n= 49), patients with inactive (n= 15), active (n= 28), and highly active SLE (n= 6). The HPLC was performed using two coupled reverse-phase columns eluted with 150 mM sodium phosphate, pH 4.0, under a flow rate of 0.8 ml/min, with UV detector set at 353 nm and 100-fold diluted urines. The inter- and intra-assay studies presented an imprecision of 12.5% and 12.9% for quality controls of 3.94 and 1.1 micromol/ml, respectively. Recovery from 79.5% to 82% was observed throughout the assay's linear range. Subjects with active (874.2 +/- 165.38 micromol/mol creatinin) and highly active SLE (1753.8 +/- 453.9 micromol/mol creatinin) showed three- and sixfold increased neopterin levels, respectively, compared to subjects with inactive SLE (314.3 +/- 121.3 micromol/mol creatinin) and healthy subjects (294.6 +/- 178.6 micromol/mol creatinin) (P< 0.05). Briefly, the proposed method was precise, specific, and reproducible, not invasive and allows the urinary neopterin quantification only with UV detection.

Biomarkers↗

PubMed2003

NADPH oxidase and enhanced superoxide generation in intrauterine undernourished rats: involvement of the renin-angiotensin system.

OBJECTIVE: We previously reported that intrauterine undernutrition increased the oxidative stress by decreasing superoxide dismutase activity. In the present study, we tested whether NADPH oxidase, xanthine oxidase, cyclooxygenase or nitric oxide synthase are responsible for the increased O(2)(-) generation observed in rats submitted to intrauterine undernutrition. In addition, we investigated the effect of angiotensin II (ANG II) on O(2)(-) production via activation of NADPH oxidase. METHODS: Female pregnant Wistar rats were fed either normal or 50% of the normal intake diets, during the whole gestational period. At 16 weeks of age, the rats were used for the study of intravital fluorescence microscopy; microvascular reactivity, local ANG II concentration and AT(1), p22(phox) and gp91(phox) gene expression. In this study only the male offspring was used. RESULTS: Treatment of mesenteric arterioles with the xanthine oxidase inhibitor oxypurinol, the nitric oxide synthase inhibitor L-NAME or the cyclooxygenase inhibitor diclofenac did not significantly change superoxide production. Thus, these vascular sources of superoxide were not responsible for the increased superoxide concentration. In contrast, treatment with the NADPH oxidase inhibitor apocynin significantly decreased superoxide generation and improved vascular function. On the other hand, intrauterine undernutrition did not alter the gene expression for p22(phox) and gp91(phox). The fact that the local ANG II concentration was increased and the attenuation of oxidative stress by blocking AT(1) receptor with losartan, led us to suggest that ANG II induces O(2)(-) generation in intrauterine undernourished rats. CONCLUSION: Our study shows that NADPH oxidase inhibition attenuated superoxide anion generation and ameliorated vascular function in rats submitted to intrauterine undernutrition. Although it is not clear which mechanisms are responsible for the increase in NADPH oxidase activity, a role for ANG II-mediated superoxide production via activation of NADPH oxidase is suggested.

Acetophenones↗

PubMed2003

Mesangial cells are able to produce catecholamines in vitro.

Mesangial cells (MC) participate in the control of the glomerular function due to their ability to synthesize hormones and induce cell contraction. Since MC can produce various kinds of hormones, the purpose of the present study was to determine if they are able to synthesize catecholamines. For this evaluation, the levels of norepinephrine, epinephrine, dopamine, and biopterin, the enzymatic cofactor of tyrosine hydroxylase (TH), were analyzed by HPLC in the intracellular compartment and in the medium of primary cultured MC. To identify and locate the enzymes responsible for monoamine synthesis, TH, dopa decarboxylase, and dopamine beta-hydroxylase, Western blotting and immunocytochemistry were employed using monoclonal and polyclonal antibodies. Concentrations of NE = 57 +/- 8, EPI = 82 +/- 10, and DA = 52 +/- 9 pg/mg protein (X +/- SEM) were found in the cell homogenate. The culture medium showed concentrations of NE = 25 +/- 3, EPI = 33 +/- 3, and DA = 62 +/- 15 pg/mg protein. Western blotting analysis and immunocytochemistry evidenced the presence of all enzymes. Moreover, biopterin was also detected in the intracellular compartment and in the medium (0.28 +/- 0.03 and 5.70 +/- 2 nmol/mg cell protein, respectively). Overall, the data indicate that MC have the biosynthetic machinery necessary to produce catecholamines, suggesting that they can act as a paracrine/autocrine hormone system, contributing to the regulation of glomerular hemodynamic and renal microcirculation.

Animals↗

PubMed2003

Determination of sirolimus blood concentration using high-performance liquid chromatography with ultraviolet detection.

BACKGROUND: Different HPLC methods have been developed and used to determined sirolimus blood concentrations. These methods show different performance characteristics, mostly related to peak interference, recovery, assay sensitivity, and turnaround times. OBJECTIVE: We adapted, improved, and validated an HPLC method with UV detection for measurement of sirolimus in whole blood clinical samples. METHODS: The standards, quality controls, or patient samples (0.25 or 0.5 mL) and internal standard (desmethoxysirolimus) were extracted with 1-chlorobutane. After evaporation, the extract was reconstituted in a 70% acetonitrile/water mixture and analyzed onto a reverse-phase C18 column at 50 degrees C under a flow rate of 1.0 mL/min in the HPLC system. Ultraviolet detection was performed at 278 nm, with sensitivity setting of 0.010 AUFS. Identification of peaks of interest was by retention time; quantification of sirolimus was based on a peak area ratio. RESULTS: Analytic recovery ranging from 96 to 120% (CV = 3.7 to 16.8%; bias = -4.2 to 16.7%) was observed throughout the assay's linear range (2.5-150.0 ng/mL). The lower limit of quantification for both sample volumes (0.25 or 0.5 mL) was 2.5 ng/mL (CV = 12 and 15%, bias = -1.2 and 4%, respectively). The intra- and interassay imprecision ranged from 6.2 to 14.4% and from 9.1 to 18.6%, with bias ranging from 1.3 to 12.9% and -1.8% to 7.1, for quality control levels of 3, 10, and 20 ng/mL. Whole blood and extracted samples are stable at room temperature and at 4 and -20 degrees C for 1 week and 3 days, respectively. Chromatograms showed good separation free of interfering peaks. A set of 45 samples can be extracted in 2 h, allowing results within 24 h. CONCLUSION: This HPLC-UV method shows good and reproducible performance, satisfying all requirements of an assay designated to be applied in therapeutic drug monitoring strategies after organ transplantation.

Chromatography, High Pressure Liquid↗

PubMed2003

Neutral endopeptidase expression in mesangial cells.

In the kidney, neutral endopeptidase (NEP) is implicated in the metabolism of several peptides involved in blood pressure and sodium homeostasis control, such as the atrial natriuretic peptide, bradykinin and angiotensin I. Due to its physiological importance in the modulation of pressor responses, the presence of NEP in mouse mesangial cells has been investigated, since these cells control glomerular function and are able to synthesise components of the renin-angiotensin system. A NEP-like activity (NEP-like) that cleaves the fluorogenic substrates Abz-BKQ-EDDnp and Abz-DRRL-EDDnp was purified from mesangial cell lysate by ion-exchange, followed by gel filtration chromatography. The enzyme was able to hydrolyse bradykinin at the G4-F5 peptide bond and was inhibited by thiorphan. A pH study established that enzyme activity was maximal at pH 7.5 and the determined K(m) was 4.86 M using Abz-DRRL-EDDnp as substrate. NEP-like was recognised by monoclonal anti-NEP and had a molecular mass of 95 kDa. The purified enzyme was sequenced and showed similarity with human, rat, mouse and rabbit NEPs. We isolated, for the first time, NEP-like from mesangial cells. This enzyme could have an important role in the renal physiology by its action upon different peptides that are able to alter renal haemodynamics.

Amino Acid Sequence↗

PubMed2002

Purification and characterization of the active form of tyrosine hydroxylase from mesangial cells in culture.

The capacity of mesangial cells (MC) to produce catecholamines (CAs) has been investigated in our laboratory. To study the CA cascade, it is necessary to examine some steps in their metabolic pathway. Tyrosine hydroxylase (TH) catalyzes the rate-limiting step in the biosynthesis of these biogenic amines (dopamine (DA), norepinephrine (NE), and epinephrine (EPI)). Since the glomerular mesangium is their target in the regulation of renal sodium transport and renin secretion, the aim of the study was to determine the presence of TH in these cells in culture. The CA levels were detected in immortalized MC by high-performance liquid chromatography with electrochemical detection. The following concentrations were found in the intracellular region and in the medium, respectively: NE = 284 +/- 31 and 134 +/- 22, EPI = 75 +/- 14 and 22 +/- 5, and DA = 42 +/- 14, 40 +/- 20 pg/mg cell protein. The enzymatic activity of the cell lysate and medium was measured based on L-dopa formation. In the presence of o-phenanthroline, both samples presented 39% inhibition. The biopterin was detected in the intracellular and in the medium (64.87 and 631.99 pmol/mg protein, respectively) using high-performance liquid chromatography with ultraviolet detection. The cell lysate was submitted to a DEAE-Sephacel column, followed by gel filtration, and Heparin-Sepharose. TH was purified 613.16-fold with a specific activity of 466.0 pg/mg cell protein. Immunoblotting using monoclonal antibody revealed the presence of TH in the different purification steps. Purified TH was sequenced, presenting an alignment with amino-terminal sequence of mouse enzyme. Our results demonstrated the presence of active TH in MC, suggesting that these cells are able to produce CA "in vivo", and establishing a convenient purification method for TH that can be applied to the study of the molecular properties of the enzyme modified "in vivo" by different physiological and pathophysiological stimuli.

Animals↗