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Biomedical subjects

F Pagani

Publications and source records attributed to F Pagani.

At least 55 records · Page 3Linked to original sources
PubMed1997

Cysteine residues in human lysosomal acid lipase are involved in selective cholesteryl esterase activity.

Human lysosomal acid lipase (LAL) catalyses the deacylation of triacylglycerol and cholesteryl esters in the acidic lysosomal compartment. Treatment of LAL with the reducing agent dithiothreitol affected the triacylglycerol and cholesteryl esterase activities differentially, suggesting the involvement of cysteine residues in determining substrate specificity. To identify the residues involved, human LAL cDNA, under the control of the T7 promoter and tagged with a herpes simplex virus coding epitope, was specifically mutated in order to introduce single amino acid substitutions of each of the six cysteine residues of mature LAL. All Cys-227 mutants showed selectively decreased activity towards cholesteryl oleate, while preserving that towards trioleylglycerol. Substitutions of Cys-236, Cys-240 and Cys-244 affected catalysis towards the two substrates to a variable degree, depending on the side chain of the amino acid introduced. The replacement of Cys-41 or Cys-188 did not result in the preferential cleavage of either one of the two substrates. These data indicate that Cys-227, Cys-236, Cys-240 and Cys-244 play a crucial role in determining LAL substrate specificity. We propose that these cysteine residues are involved in the hydrolysis of cholesteryl ester by affecting selectively the access of this substrate to the catalytic active site. In addition, the fact that the catalytic activity is never completely abolished in cysteine mutants demonstrates that LAL is not a thiol enzyme.

Cysteine↗
PubMed1997

Rapid and specific immunoassay for cardiac troponin I in the diagnosis of myocardial damage.

We evaluated a new immunometric assay for the quantitation of cardiac troponin I with the Behring Opus analyzer. All assay steps are performed automatically by the analyzer, the complete procedure requiring 20 min for the first test result. Total precision (coefficient of variation) varied between 5.6% and 13.0%. Comparison with the Pasteur immunoenzymometric assay showed good correlation (r = 0.959), but the Opus assay gave approximately tenfold higher values. Cardiac troponin I was undetectable (< 0.5 microgram/l) in sera from healthy subjects (n = 46), patients with severe skeletal muscle damage (n = 8), and all but 1 patient with chronic renal failure (n = 39). In patients with myocardial infarction (n = 21), investigated for 4 days after onset by frequent sampling, cardiac troponin I peaked at 20.8 +/- 8.1 h after onset with a mean concentration of 164.8 +/- 361.3 micrograms/l, remaining elevated in 89% of patients on the 4th day. In patients with unstable angina (n = 15), cardiac troponin I measurement was valuable in predicting the type of lesion morphology on coronary angiography and the short-term outcome.

Adult↗
PubMed1997

Protection by amylin of gastric erosions induced by indomethacin or ethanol in rats.

1. The effect of amylin on gastric ulcers induced by oral administration of indomethacin (Indo, 20 mg kg-1 at a dosing volume of 5 ml) or ethanol 50% (EtOH, 1 ml/rat) was investigated in conscious rats. 2. Amylin given intracerebroventricularly (0.22, 0.66 and 2.2 micrograms/rat, i.c.v.) demonstrated a dose-dependent cytoprotective effect against both Indo and EtOH-induced ulcers. In contrast, amylin, given subcutaneously at doses effective in inhibiting acid gastric secretion (2.5, 10 and 40 micrograms kg-1, s.c.), did not show any cytoprotective effect. 3. The interaction between amylin and endogenous nitric oxide (NO) in the maintenance of gastric mucosal integrity was investigated by pretreating the rats with a selective inhibitor of NO-synthesis, NG-nitro-L-arginine methyl ester (L-NAME, 25 and 70 mg kg-1, s.c.). Administration of L-NAME to rats did not significantly increase the degree of the Indo-induced ulcer index and was not able to remove the protective effect of amylin on Indo-induced ulcers, thus excluding a role for endogenous NO in mediating the protective effect of this peptide. 4. To determine whether the cytoprotective effect of amylin was mediated by endogenous prostaglandins, we studied the effect of amylin (2.2 micrograms/rat, i.c.v.) on EtOH- induced ulcers in rats pretreated with Indo (10 mg kg-1, s.c.) to inhibit prostanoid biosynthesis; Indo was injected 30 min before amylin and EtOH after a further 30 min. Pretreatment with Indo did not significantly increase the ulcer index induced by EtOH but counteracted the ability of amylin to prevent the ulcer formation. 5. These findings suggest that amylin exerts a gastroprotective activity that is not strictly related to inhibition of acid gastric secretion and can be partly explained through a prostaglandin-dependent mechanism mediated by receptors for the peptide in the brain. Amylin might be considered as a new brain-gut peptide.

Amyloid↗
PubMed1996

L273S missense substitution in human lysosomal acid lipase creates a new N-glycosylation site.

Human lysosomal acid lipase (LAL), when expressed in HeLa cells using the Vaccinia T7 expression system, showed four major molecular forms ranging from 42 to 54 kDa. Treatment with endoglycosidase H resulted in a 42 kDa protein, indicating that the molecular weight variations were due to N-glycosylation. A missense substitution, L273S, previously detected in a patient with cholesteryl ester storage disease (CESD), produced catalytically inactive LAL showing a largest molecular mass form of 56 kDa instead of 54 kDa. Analysis of the amino acid sequence in the close proximity of the mutation (NMS- NML) indicated that the L273S mutation creates an additional N-glycosylation consensus (N-X-S/T) in this region. Two site directed mutants disrupting this consensus, QMS and QML, when expressed in HeLa cells, did not show the 56 kDa form but the normal 54 kDa band whereas deglycosylation always resulted in the major 42 kDa form, as observed with normal LAL and the L273S mutant. These data confirmed that an additional N-glycosylation at N271 was responsible for the 56 kDa form of the protein produced from the L273S allele. Furthermore, deglycosylation of normal LAL reduced the acid hydrolase activity towards both tri-oleyl glycerol and cholesteryl oleate by 50%, strongly suggesting that N-linked carbohydrate residues are important for optimal catalytic activity.

Amino Acid Sequence↗
PubMed1996

Modified method for Novacor left ventricular assist device implantation.

We have developed and applied in 7 cases an alternative "orthodromic" technique for Novacor left ventricular assist device implantation. After sternotomy, the pocket is tailored, dividing the insertion of the diaphragm to the lower ribs, for easier bleeding control. On cardiopulmonary bypass, the aorta is cross-clamped and cardioplegia administered. Apical cannulation is performed first on a dry, still field. The device is then easily deaired, with blood flowing in the physiologic direction. The aorta is declamped and the outflow conduit is anastomosed. Before the suture is tied, the final deairing is obtained. This technique allows extreme precision in apical cannulation, easier control of bleeding, and accurate deairing of the pump. The ischemic time is short and damage to the right ventricle negligible.

Heart-Assist Devices↗
PubMed1996

Induction of human endothelial cell growth by mildly oxidized low density lipoprotein.

The aim of the present study was to examine whether endothelial growth could be modulated by mildly oxidized low density lipoprotein. When human endothelial cells were cultured in the presence of mildly oxidized low density lipoprotein (1 microgram/ml), a significant induction of endothelial cell growth was observed, whereas native low or high density lipoprotein were ineffective. Further, treatment of endothelial cells with mildly oxidized low density lipoprotein modulated the expression of cytokines and growth factors which may be relevant in atherogenesis. Endothelial cells chronically exposed to mildly oxidized low density lipoprotein underwent a more rapid onset of cellular senescence. Since senescence is associated with endothelial dysfunction, the novel finding showing that mildly oxidized low density lipoprotein induces endothelial cell growth may be relevant in the development and evolution of the atherosclerotic lesions.

Arteriosclerosis↗
PubMed1996

Surgery for atrial fibrillation.

OBJECTIVE: The mechanisms of atrial fibrillation arc multiple reentry circuits spinning around the atrial surface, and these baffle any attempt to direct surgical interruption. The purpose of this article is to report the surgical experience in the treatment of isolated and concomitant atrial fibrillation at the Cardiac Surgical Institute of the University of Pavia. METHODS: In cases of atrial fibrillation secondary to mitral/valve disease, surgical isolation of the left atrium at the time of mitral valve surgery can prevent atrial fibrillation from involving the right atrium, which can exert its diastolic pump function on the right ventricle. Left atrial isolation was performed on 205 patients at the time of mitral valve surgery. Atrial partitioning ("maze operation") creates straight and blind atrial alleys so that non-recentry circuits can take place. Five patients underwent this procedure. In eight-cases of atrial fibrillation secondary to atrial septal defect, the adult patients with atrial septal defect and chronic or paroxysmal atrial fibrillation underwent surgical isolation of the right atrium associated which surgical correction of the defect, in order to let sinus rhythm govern the left atrium and the ventricles. "Lone" atrial fibrillation occurs in hearts with no detectable organic disease. Bi-atrial isolation with creation of an atrial septal internodal "corridor" was performed on 14 patients. RESULTS: In cases of atrial fibrillation secondary to mitral valve disease, left atrial isolation was performed on 205 patients at the time of mitral valve surgery with an overall sinus rhythm recovery of 44%. In the same period, sinus rhythm was recovered and persisted in only 19% of 252 patients who underwent mitral valve replacement along (P < 0.001). Sinus rhythm was less likely to recover in patients with right atriomegaly requiring tricuspid valve annuloplasty: 59% vs 84% (P < 0.001). Restoration of the right atrial function raised the cardiac index from 2.25 +/- 0.55 1/min per m2 during atrial fibrillation to 2.54 +/- 0.58 1/min per m2, with a mean percentage increase in cardiac index of 13.5% (P < 0.00018). Atrial partitioning ("maze operation") was performed on five patients with an immediate sinus rhythm recovery of 100%, but with two patients requiring pacemaker implant. Seven out of eight patients (87.5%), with atrial fibrillation secondary to atrial septal defect, who underwent surgical isolation of the right atrium at the time of surgery were free from atrial fibrillation and without medications. 2-52 months after operation. Thirteen of 14 patients with "lone" atrial fibrillation who underwent corridor procedure remained in sinus rhythm with a sinus rhythm recovery rate of 92%. CONCLUSIONS: Different surgical options can be chosen for different cases of atrial fibrillation, according to the underlying cardiac disease.

Adult↗
PubMed1996

Expression of lysosomal acid lipase mutants detected in three patients with cholesteryl ester storage disease.

Lysosomal acid lipase (LAL) gene mutations were identified in three patients with cholesteryl ester storage disease (CESD). Direct sequencing of genomic DNA revealed that: patient 1 was a compound heterozygote for a P181L mutation and an A to G3' splice site substitution that causes skipping of exon 7, with a loss of 49 amino acids from LAL (delta 205-253); patient 2 was a compound heterozygote for a G66V mutation and a 5' splice site mutation (G to A) that leads to skipping of exon 8 (delta 254-277); and patient 3 was a compound heterozygote for a L273S mutation and an unidentified null allele. Furthermore, patients 2 and 3 showed a novel G-2A polymorphism that could be detected by an Xbal restriction fragment length polymorphism. All these mutants and a previously reported H274Y allele were expressed in vitro in HeLa cells using the vaccinia T7 expression system. The resulting recombinant proteins were inactive towards cholesteryl oleate and trioleylglycerol, demonstrating the direct involvement of these mutations in the pathogenesis of CESD. Immunoblotting of normal LAL expressed in HeLa cells revealed four major molecular forms, at least two of high molecular mass (54 and 50-51 kDa) and two of low molecular mass (42 and 43 kDa). L273S and P181L substitutions and delta 254-277 were shown to result in altered LAL molecular forms, some of which suggest that post-translational processing may interfere with the catalytic activity of LAL.

Child↗
PubMed1996

Biological variation in urinary excretion of pyridinium crosslinks: recommendations for the optimum specimen.

We assessed the analytical and biological variation of pyridinium crosslinks in early morning, 2 h fasting, and 24 h urine specimens from 14 healthy adults over a 1 month period. The results were expressed both in terms of pyridinoline concentration and pyridinoline/creatinine ratio. The data obtained were used to select the optimum specimen for clinical purposes. We found that: (a) early morning specimens are preferred; (b) results should be expressed as pyridinoline/creatinine ratio; (c) reference intervals should be stratified according to gender; (d) the necessary analytical imprecision (CV < or = 9%), derived from biological variation, is not easily achieved by current methods; (e) the difference between serial results from an individual must be > 50% to be statistically significant; and (f) assessment of risk for osteoporotic fracture by means of the pyridinium crosslink assay would, in a significant number of patients, require analysis of multiple urine specimens.

Adult↗
PubMed1996

Role of the neuronal histaminergic system in the regulation of somatotropic function: comparison between the neonatal and the adult rat.

To study possible age-related differences in the role of neuronal histaminergic pathways in the control of GH secretion, the effects of alpha-fluoromethylhistidine (alpha-FMH), an irreversible inhibitor of histamine (HA) synthesis, were examined on basal and opioid-induced GH release in neonatal and adult rats. The mechanisms involved in such effects were evaluated by measuring pituitary GH mRNA levels and hypothalamic levels of GH-releasing hormone (GHRH) and somatostatin (SRIF) mRNAs. Daily injection of alpha-FMH (20 mg/kg, s.c.) in pups of either sex, from birth until 10 days of age, caused a significant increase in baseline plasma GH and potentiated the GH response to the [Met5]-enkephalin analog FK 33-824 (1 mg/kg, s.c.) administered 3 h after the last alpha-FMH injection. GH and SRIF mRNA levels were significantly higher in alpha-FMH-treated pups than in controls, whereas no difference was observed in GHRH mRNA levels. In young adult male rats, acute administration of alpha-FMH (100 mg/kg, s.c., 3 h before) did not change significantly basal GH levels but potentiated FK 33-824 (0.3 mg/kg, intracarotid)-induced stimulation of GH secretion. Repeated administration of alpha-FMH (200 micrograms/rat, i.c.v., for 3 days) failed to modify basal and FK 33-824-induced GH secretion, caused a significant reduction in hypothalamic GHRH mRNA levels and left SRIF and GH mRNAs unchanged. These findings indicate that HA exerts an inhibitory effect on GH secretion in both neonatal and adult rats. The different effects of short-term HA depletion on hypothalamic and pituitary indices of somatotropic function observed at the two age periods may be ascribed to the immaturity of the HA system in early postnatal life and to a different functional role of GH-regulatory factors during ontogeny.

Aging↗
PubMed1996

[Anesthesiology problems for implant and explant of left ventricular Novacor systems].

OBJECTIVES: To review experience with anaesthetic management in patients undergoing a left ventricular assist system (LVAS) Novacor implant as a bridge to cardiac transplantation. DESIGN: Retrospective clinical study. SETTING: Cardiothoracic operating room in a university hospital. PATIENTS: Nine, aged 37-62 years, suffering from 8 dilated and 1 postischemic cardiomyopathy at end stage heart failure. INTERVENTIONS OR METHODS: Under general anaesthesia and cardio-pulmonary bypass the LVAS pump is placed in the abdomen and connected to the left ventricle through an inflow cannula and to the aorta through an outflow cannula. The pump has an output of 5-7 l/min and works in synchronous counterpulsation with the left ventricle. RESULTS: The mean Novacor support has been 75.56 days (DS +/- 85.95). Five out of 9 patients have been transplanted, while 3 are still waiting at home. Pretransplant CI was on average 1.66 l/min/m2 (DS +/- 0.27) and REF 11.44% (DS +/- 5.29); after the Novacor implant CI was 3.44 (DS +/- 0.49) and REF 22.22% (DS +/- 4.49). CONCLUSIONS: A LVAS can bridge patients to heart transplantation while improving or preserving their health conditions. Some factors are to be taken into account for the anaesthesiological management of the implant/explant of a LVAS: the patients' poor clinical conditions; the knowledge of the components and functioning of the LVAS that conditions the choice of the anaesthesiological drugs and volemia; the unassisted right ventricle function that has to be pharmacologically supported; the bleeding problem because of the adherences during the explant surgery.

Adult↗