Cloning, tissue expression, and inducibility of CYP 3A79 from sea bass (Dicentrarchus labrax).

Multiple members of the CYP3A subfamily have been identified and intensively studied in mammals as they represent prominent CYP enzymes involved in drug metabolism. Also in fish, some CYP3A genes have been identified by cDNA cloning and immunological techniques, but relatively little is known about their function, distribution, and inducibility. In this study, a novel CYP3A, designated as CYP3A79 was isolated from adult male sea bass, an economically valuable species in fisheries. The sea bass CYP3A79 that was cloned contained an open-reading frame of 1512 bp that encoded a 504 amino acid protein and shared a high-sequence identity with medaka, killifish, and trout CYP3As. Interestingly, CYP3A79 also shares five of six substrate recognition sites (SRS) with the SRS of other fish CYP3As, suggesting an evolutionary conservation of the function of these enzymes. In this fish, we also investigated the expression of CYP3A79 and its susceptibility to induction by various compounds including clotrimazole and dehydroepiandrosterone, two strong ligands of zebrafish PXR. The expression of CYP3A79 mRNA was detected by RT-PCR only in the intestine and liver. The immunoblot analysis by antitrout CYP3A27 confirmed the presence of a CYP3A-like protein in the microsomes of these tissues, but, in addition, a immunoreactive protein with this antibody was also observed in the heart microsomes, suggesting the presence of other CYP3A isoforms in this fish. Accordingly, the southern blot analysis of genomic DNA indicated that multiple CYP 3As may be present in sea bass. All attempts to induce 6beta-testosterone hydroxylase, as a marker of CYP3A79, by dexametasone, 17beta-estradiol, pregnenolone 16alpha-carbonitrile, corticosterone, clotrimazole, and dehydroepiandrosterone failed. On the contrary, the administration of 17beta-estradiol, pregnenolone 16alpha-carbonitrile, and corticosterone strongly inhibited this activity and, in parallel, reduced the expression of CYP3A79 transcript. Thus, the sea bass CYP3A79 appears to be resistant to induction, suggesting that this enzyme and likely other CYP3As are regulated differently compared to those of mammals.

[Farmacoeconomic impact of anti-TNF-alpha]. / Impatto farmacoeconomico degli anti-TNF-alpha.

The anti-TNF-alpha are undoubtedly an efficacious cure in the treatment of rheumatoid arthritis, but their costs are so high that a thorough pharmacoeconomical evaluation is needed in order to identify the specific conditions in which their use is to be considered convenient. For this reason there are related the most important experiences that have studied the cost-efficacy and cost-utility relationships of anti-TNF-alpha drugs, which have been made marketable in Italy. The data available, unfortunately, are too various to allow a final settlement of the chart of convenience between the different therapeutic alternatives. Moreover the socio-medical reality in Italy is so much different from the ones in other countries that it is impossible to try and use the foreign experiences. In a country of high social commitment like Italy, a fair judgment can thereafter be made only when the issue is considered related to our society, taking in account the summation of the medical costs endured by the National Health System, the patient's expenses and the ones that are a consequence of the loss of productivity.

Effects of liquid and freeze-dried grapefruit juice on the pharmacokinetics of praziquantel and its metabolite 4'-hydroxy praziquantel in beagle dogs.

Grapefruit juice changes the pharmacokinetic parameters of a variety of drugs metabolized primarily by cytochrome P450 3A. In a three-phase crossover study, six male beagle dogs were administered 100 ml of water (control), 100 ml of commercial liquid grapefruit juice, or 10 g of freeze-dried grapefruit juice (equivalent to 100 ml of liquid grapefruit juice) with 100 ml of water, followed after 2 h by single oral dose of praziquantel (30 mg kg(-1)). After treatment, the dogs were sampled at different times. Determination of praziquantel and its metabolite 4'-hydroxy praziquantel (identified by GC/MS) was performed by HPLC. Liquid and freeze-dried grapefruit juice preadministration increased the C(max) of praziquantel about three-fold and the AUC 2.5- and 2.3-fold, respectively. The T(max) (0.75 h) was unaffected by liquid or freeze-dried grapefruit juice, while T(1/2) was 2.3- and 1.7-fold higher compared with controls. The amount of 4'-hydroxy praziquantel was also affected by both liquid and freeze-dried grapefruit juice administration: the AUC and C(max) increased four- and three-fold, respectively and the T(max) was significantly enhanced. These findings demonstrate that both freeze-dried grapefruit juice and commercial liquid grapefruit juice significantly increase plasma concentrations and T(1/2) of praziquantel in dogs.

Inhibition of cytochrome p450 enzymes by enrofloxacin in the sea bass (Dicentrarchus labrax).

Currently, there are no reports on the effects of enrofloxacin (EF), a fluoroquinolone antibiotic, on the cytochrome p450 enzymes in fish, although its use as antimicrobial agent in aquaculture has been put forward. Therefore, the in vivo and in vitro effects of EF on hepatic p450 enzymes of sea bass, a widespread food-producing fish, have been evaluated. Sea bass pretreated with a single dose of EF (3 mg/kg i.p.) or with three daily doses of EF (1 mg/kg i.p.) markedly depressed the microsomal N-demethylation of aminopyrine, erythromycin, the O-deethylation of 7-ethoxycoumarin, ethoxyresorufin and the 6beta-testosterone hydroxylase. In vitro experiments showed that EF at 10 microM inhibited the above-mentioned activities and, in particular, the erythromycin N-demethylase (ERND) and 6beta-testosterone-hydroxylase, likely dependant on a p450 3A isoform. When the nature of ERND inhibition by EF was specifically studied with sea bass liver microsomes, it was found that EF is a potent mechanism-based inhibitor, with K(i) of 3.7 microM and a K(inact) of 0.045 min(-1). An immunoblot analysis with anti p450 3A27 of trout showed that the p450 3A isoform, constitutively expressed in sea bass, is particularly susceptible to inactivation by EF. In vitro experiments with sea bass microsomes have also demonstrated that EF is oxidative deethylated by the p450 system to ciprofloxacin (CF) and that this compound maintains the ability to inactivate the p450 enzymes. The mechanism by which EF or CF inactivate the p450 enzymes has not been studied but an attack of p450 on the cyclopropan ring, present, both in EF and CF structure, with the formation of electrophilic intermediates (i.e. radicals) has been postulated. In conclusion, the EF seems to be a powerful inhibitor of p450s in the sea bass. Therefore, the clinical use of this antibiotic in aquaculture has to be considered with caution.

Yield and post-yield behavior of mussel byssal thread: a self-healing biomolecular material.

Byssal threads provide marine mussels with the tenacity to remain sessile in habitats of high flow. Under uniaxial tension, byssal threads are typical of other biological and synthetic fibers in exhibiting an initial linear region followed by yield. They differ, however, in their capacity to recover or "self-heal" following yield. We have examined the effect of urea, dithiothreitol (DTT), and metal-chelating agents such as ethylenediamine tetraacetic acid (EDTA) in perturbing the modulus, yield point, and energy dissipated in distal byssal threads stretched cyclically in seawater to a strain of 0.7. Threads stretched in the presence of 8 M urea or DTT show a complete abolition of yield point, while those washed clean of urea and DTT prior to stretching approach native controls. Threads stretched in the presence of EDTA show no effect; however, preincubation of distal threads in EDTA for 24 h results in a loss of yield point if stretched in metal-deficient seawater and normal behavior in natural seawater. The results indicate that while protein unfolding and disruption of disulfide linkages or chelate complexes compromises the yield strength of distal byssal fibers, there is typically a rapid recovery in natural sea water.

Oxidative stress and the mechanical properties of naturally occurring chimeric collagen-containing fibers.

The byssal threads of marine mussels are a fiber-reinforced composite material. Fibers are continuous, separated by matrix, and consist of chimeric collagens that encompass within the same primary protein structure domains corresponding to collagen, polyhistidine, and either elastin or dragline spider silk. The elastic modulus (stiffness) of the proximal portion of byssal threads was measured by cyclic stress-strain analysis at 50% extension. Before measurement, the threads were conditioned by various treatments, particularly agitation in aerated or nitrogen-sparged seawater. Stiffness can be permanently increased by more than two times, e.g., from 25 MPa to a maximum of 65 MPa, by simple agitation in aerated seawater. Much but not all of this stiffening can be prevented by agitation under nitrogen. Reversible strain stiffening would seem to be a useful adaptation to lower residual stresses arising from the deformation of two joined materials, i.e., distal and proximal portions with rather different elastic moduli. The permanent strain stiffening that characterizes proximal byssal threads subjected to oxidative stress is probably due to protein cross-linking. In the short term, this results in a stronger thread but at the expense of dynamic interactions between the molecules in the structure.

Blotting patterns of IgG anti-(U1)RNP antibodies in mixed connective tissue disease.

Serum reactivities towards individual U1 snRNP proteins were determined by immunoblotting in 32 patients with mixed connective tissue disease (MCTD). Time persistence of immunoblot profiles and clinical significance of anti-(U1)RNP antibody specificities were also investigated. IgG anti-(U1)RNP antibodies were found in the sera of 29 out of 32 patients (90.6%): 21 (65.6%) reacted with the 70-kD protein, 25 (78.1%) with A, 23 (71.9%) with C and 20 (62.5%) with B/B' proteins. None were reactive with the Sm-D peptide. Seventy kilodalton antibody specificity was strongly associated with a higher antinuclear antibody titre (> 160) and slightly associated with disease activity; anti-B/B' specificity was associated with lymphadenopathy. Anti-A, -C and -B/B' antibodies were negatively associated with systemic lupus erythematosus (SLE) skin rashes. Two types of anti-(U1)RNP blotting patterns were selected: "full spectrum" (53.1% of cases) and a "partially/no reactive" one (46.9%). Such patterns were unchanged over time in 14 out of 16 cases prospectively examined (87.5%), while the pattern shifted from "full spectrum" to "partially/no reactive" in 2 cases (12.5%): in 1 after a prolonged clinical remission (> or = 4 years) and in the other following immunosuppressive therapy. The anti-(U1)RNP antibody immunoblot profile in MCTD patients consisted of various reactivities and remained unchanged over time in most cases. Antibody reactivity against the 70-kD protein represented the major U1 snRNP specificity. The various anti-(U1)RNP specific reactivities demonstrated poor clinical significance within MCTD. Thus, MCTD seems to be characterized by a longstanding serological heterogeneity whose reactivities do not apparently correspond to distinct features within the broad clinical spectrum of MCTD.

Fibrinolysis and coagulation abnormalities in systemic lupus erythematosus. Relationship with Raynaud's phenomenon, disease activity, inflammatory indices, anticardiolipin antibodies and corticosteroid therapy.

Endothelial cell damage in systemic lupus erythematosus (SLE) was evaluated by measuring fibrinolytic activity and von Willebrand factor levels. Tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor (PAI) activity, and von Willebrand factor antigen (vWF:Ag) and activity (vWF:RCof) were measured in 21 SLE patients (12 of whom were therapy free) and 22 controls. In addition, the relationship between such parameters and Raynaud's phenomenon, disease activity [according to personal criteria, Systemic Lupus Activity Measure (SLAM) and European Consensus Lupus Activity Measurement (ECLAM) scores] inflammatory indices [ESR, C-reactive protein (CRP), alpha 2-globulin], anticardiolipin antibodies and corticosteroid therapy was investigated. Lower levels of t-PA antigen (P = 0.003) and higher levels of vWF:Ag (P = 0.001) were found in SLE patients in comparison with controls. Moreover, t-PA antigen was lower (P = 0.02) in steroid-free patients in comparison with those taking steroids. No relationship was found between fibrinolysis and coagulation abnormalities and Raynaud's phenomenon, disease activity, inflammatory indices and anticardiolipin antibodies. Endothelial cell damage is probably a common feature in SLE patients; nevertheless, we were unable to clarify the nature of such abnormality. It is worth noting that low doses of steroids seem to be effective in improving endothelial cell function in SLE patients.

Therapy of lupus nephritis. A two-year prospective study.

The aim of our study was to compare the efficacy of 3 different therapeutic protocols in the treatment of patients with WHO class IV lupus nephritis and normal renal function. We carried out a randomized prospective trial. The treatment programs consisted of a standard therapy regimen alone (protocol A), plus plasmapheresis (protocol B) or pulse methylprednisolone (protocol C), followed by a slow (protocols A and B) or fast (protocol C) prednisone tapering schedule. Statistical analysis was performed, using univariate survival analysis according to Kaplan Meier and Breslow's test to compare survival curves. Eighteen patients entered the study: 6 protocol A, 5 protocol B and 7 protocol C. No patients developed renal insufficiency. Moreover, no statistical differences in the probability of inducing partial or complete disease remission and in reducing 24-hour urinary protein excretion to < or = 2 g per day were observed among the groups. Protocols A and B were more effective in comparison with protocol C in decreasing 24-hour urinary protein excretion to < or = 0.5 g and < or = 0.2 g per day. In conclusion, a slow prednisone tapering schedule is more effective in reducing 24-hour urinary protein excretion to < or = 0.5 and < or = 0.2 g per day as compared with a fast prednisone tapering schedule, even if it is preceded by methylprednisolone pulse therapy.

[Evaluation of cardiac involvement in systemic lupus erythematosus. Clinical and echographic study]. / Evaluation de l'atteinte cardiaque au cours du lupus érythémateux disséminé: étude clinique et échocardiographique.

Thirty-five consecutive patients with systemic lupus erythematosus were enrolled in a prospective study. Investigations included a physical evaluation, tests for antinuclear antibodies and antiphospholipid antibodies, an electrocardiogram, a plain chest film, a 2D echocardiogram and a Doppler study. Clinical cardiac manifestations and alterations of the electrocardiogram were infrequent (17% and 11% of patients, respectively) and no patients had abnormal chest film findings. In contrast, echocardiographic abnormalities were common (82% of patients), although moderate in most instances. Pericardial involvement was found in 15 patients (42.8%); a pericardial effusion was seen in 9 of the 14 patients with inactive disease (p < 0.003), whereas thickening of the pericardium was visible in 4 patients with active disease and 2 of the 21 patients with inactive disease. Valve abnormalities were found in 17 patients (48.5%), but were not related to the presence of antiphospholipid antibodies; valve alterations included verrucous endocarditis in one case, valve thickening in one case, mitral prolapse in five cases, and mild or moderate regurgitation in 15 cases (aortic in 2 cases, mitral in 7 cases, pulmonary in 3 cases and tricuspid in 7 cases). Alterations in ventricular chamber size and kinetics were also fairly common, albeit of uncertain pathogenetic significance. These data confirm the value of 2D echocardiography for identifying and monitoring cardiac involvement in systemic lupus erythematosus, even in patients with no overt clinical manifestations.

Cyclosporin A in a pregnant patient affected with systemic lupus erythematosus.

Few data regarding the use of cyclosporin A (CyA) in pregnancy are available and those available refer mainly to transplant recipients and not to patients with connective tissue diseases. We report the case of a patient with systemic lupus erythematosus (SLE), taking CyA before, during and after her pregnancy at a dose of 4 mg/kg per day. CyA was effective in controlling SLE activity and no side effects were observed in mother or baby. The lack of teratogenicity in this case was in keeping with previous reports in experimental systems, animals and human transplant recipients. If our observation is confirmed by further studies, CyA might become useful in the treatment of pregnant patients with SLE.

[Changes of plasma renin activity during perfusion with dextran 40. Functional study; statistical evaluations and cl-nical considerations]. / Variazioni della attività plasmatica reninica in corso di perfusione con dextran "40". Studio funzionale; valutazioni statistiche e considerazioni cliniche.

Changes in RAP, AP (max and min), Ht and R.R. induced by daily perfusion with 250 and 500 ml Dextran 40 in a 10% isotonic solution were examined in two groups of patients with peripheral obstructive arteriopathy. Systemic AP showed no appreciable changes, whereas RAP and Ht fell in direct proportion of the dose given. This effect tended to disappear after 96 hr. Administration of this plasma volume extender 4 days per week is recommended.

[Relations between athero-arteriosclerosis and chronic liver diseases. Anatomopathological and statistical studies]. / Sui rapporti tra atero-arteriosclerosi ed epatopatie croniche. Indagine anatomopatologica e statistica.

Starting from well known anatomopathological premises and on the basis of relative physiopathological concepts, particularly lipid metabolism and the lipidaemic fractions in patients suffering from chronic hepatopathy and athero-arteriosclerosis, a statistical investigation was carried out into the relations between the two disease conditions on the basis of the anatomopathological material collected over a period of six years in the S. Giovanni and S. Giacomo Hospitals in Rome. The conclusion is that there is a smaller incidence of athero-arteriosclerotic pathology in patients suffering from chronic hepatopathy up to the sixth decade of life.

[Changes in some plasma parameters in the course of dextran 40]. / Sulle modificazioni di talune varianti plasmatiche in corso di terapia perfusionale con Dextran 40

Changes in certainplasma parameters (Hc, arterial and venous VP, creatinine clearance, potassium, PA and FC) induced by Dextran 40 perfusions (500 ml at 40 drops/min and 250 ml at 20 drops/min) were examined in 50 patients. The expander effect was more intense, though less protracted when the larger quantity was used. A rebound effect 24 hr after the test was also more frequent percentage-wise in this group. Significant changes in Hc were not observed for 24 hr and 96 hr respectively with the higher and the lower dose. The venous district was primarily concerned. There were also increases in blood potassium, FC and max PA, while min PA and blood proteins fell. No relation could be demonstrated between creatinine clearance and diuresis at the end of the test with respect to the amount of Dextran employed. None of these latter modifications was significant. There were no signs of intolerance.