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Biomedical subjects

G Ricci

Publications and source records attributed to G Ricci.

At least 19 recordsLinked to original sources
PubMed1996

Structural flexibility modulates the activity of human glutathione transferase P1-1. Role of helix 2 flexibility in the catalytic mechanism.

Presteady-state and steady-state kinetic studies performed on human glutathione transferase P1-1 (EC 2.5.1.18) with 1-chloro-2, 4-dinitrobenzene as co-substrate indicate that the rate-determining step is a physical event that occurs after binding of the two substrates and before the final sigma-complex formation. It may be a structural transition involving the ternary complex. This event can be related to diffusion-controlled motions of protein portions as kcat degrees /kcat linearly increases by raising the relative viscosity of the solution. Similar viscosity dependence has been observed for Km GSH, while Km CDNB is independent. No change of the enzyme structure by viscosogen has been found by circular dichroism analysis. Thus, kcat and Km GSH seem to be related to the frequency and extent of enzyme structural motions modulated by viscosity. Interestingly, the reactivity of Cys-47 which can act as a probe for the flexibility of helix 2 is also modulated by viscosity. Its viscosity dependence parallels that observed for kcat and Km GSH, thereby suggesting a possible correlation between kcat, Km GSH, and diffusion-controlled motion of helix 2. The viscosity effect on the kinetic parameters of C47S and C47S/C101S mutants confirms the involvement of helix 2 motions in the modulation of Km GSH, whereas a similar role on kcat cannot be ascertained unequivocally. The flexibility of helix 2 modulates also the homotropic behavior of GSH in these mutants. Furthermore, fluorescence experiments support a structural motion of about 4 A occurring between helix 2 and helix 4 when GSH binds to the G-site.

Binding Sites↗
PubMed1996

Structural flexibility modulates the activity of human glutathione transferase P1-1. Influence of a poor co-substrate on dynamics and kinetics of human glutathione transferase.

Presteady-state and steady-state kinetics of human glutathione transferase P1-1 (EC 2.5.1.18) have been studied at pH 5.0 by using 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, a poor co-substrate for this isoenzyme. Steady-state kinetics fits well with the simplest rapid equilibrium random sequential bi-bi mechanism and reveals a strong intrasubunit synergistic modulation between the GSH-binding site (G-site) and the hydrophobic binding site for the co-substrate (H-site); the affinity of the G-site for GSH increases about 30 times at saturating co-substrate and vice versa. Presteady-state experiments and thermodynamic data indicate that the rate-limiting step is a physical event and, probably, a structural transition of the ternary complex. Similar to that observed with 1-chloro-2, 4-dinitrobenzene (Ricci, G., Caccuri, A. M., Lo Bello, M., Rosato, N. , Mei, G., Nicotra, M., Chiessi, E., Mazzetti, A. P., and Federici, G.(1996) J. Biol. Chem. 271, 16187-16192), this event may be related to the frequency of enzyme motions. The observed low, viscosity-independent kcat value suggests that these motions are slow and diffusion-independent for an increased internal viscosity. In fact, molecular modeling suggests that the hydroxyl group of Tyr-108, which resides in helix 4, may be in hydrogen bonding distance of the oxygen atom of this new substrate, thus yielding a less flexible H-site. This effect might be transmitted to the G-site via helix 4. In addition, a new homotropic behavior exhibited by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole is found in Cys-47 mutants revealing a structural intersubunit communication between the two H-sites.

4-Chloro-7-nitrobenzofurazan↗
PubMed1996

HCV infection in a patient with hyper IgM syndrome.

The association between an acquired form of hyper-IgM syndrome and a chronic hepatitis C virus (HCV) infection in a 71-year-old female patient is described. Both diseases were diagnosed at the age of 58 years. She was started on intramuscular and then intravenous immunoglobulin replacement therapy. HCV RNA was detected in 1992. The patient remained in well-balanced clinical condition until 1994, when total and specific anti-HCV IgM levels increased and the patient developed an IgM kappa monoclonal gammopathy. Adherent cells and B cells were HCV RNA positive, while T cells were HCV RNA negative. Anti-IgM reactivity was specifically directed to the core antigen of the HCV. The patient we describe showed a picture of a late-onset form of hypogammaglobulinemia with a progressive increase in IgM antibodies, possibly due to the concomitant HCV infection. It is possible that the immunodeficiency might also result from the HCV infection, with formation of specific antibodies belonging to the IgM class, and that the worsening of the clinical condition may be directly related to the persistent viral infection.

Aged↗
PubMed1996

Pravastatin reduces carotid intima-media thickness progression in an asymptomatic hypercholesterolemic mediterranean population: the Carotid Atherosclerosis Italian Ultrasound Study.

PURPOSE: The Carotid Atherosclerosis Italian Ultrasound Study (CAIUS) was performed to test the effects of lipid lowering on the progression of carotid intima-media thickness (IMT) in 305 asymptomatic patients from a Mediterranean country. PATIENTS AND METHODS: Eligibility included hypercholesterolemia (baseline means: low-density lipoprotein [LDL] = 4.68 mmol/L, high-density lipoprotein [HDL] = 1.37 mmol/L), and at least one 1.3 < IMT < 3.5 mm in the carotid arteries. Patients (mean age 55 years, 53% male) were assigned to pravastatin (40 mg/day, n = 151) or placebo (n not equal to 154). Ultrasound imaging was used to quantify IMT at baseline, and semiannually thereafter for up to 3 years. The mean of the 12 maximum IMTs (MMaxIMT), was calculated for each patient visit, and used to determine each patient's longitudinal progression slope. The intention-to-treat group difference in the MMaxIMT progression was chosen a priori as the primary end point. RESULTS: Five serious cardiovascular events (1 fatal myocardial infarction), and 7 drop-outs for cancer were registered. In the pravastatin group, LDL decreased -0.22 after 3 months versus -0.01 in the placebo group, and remained substantially unchanged afterward (-0.23 versus +0.01 at 36 months, respectively). Progression of the MMaxIMT was 0.009 +/- 0.0027 versus -0.0043 +/- 0.0028 mm/year (mean +/- SE, P < 0.0007) in the placebo and pravastatin groups, respectively. IMT progression slopes diverged after 6 months of treatment. CONCLUSIONS: Pravastatin stops the progression of carotid IMT in asymptomatic, moderately hypercholesterolemic men and women. This finding extends the beneficial effects of cholesterol lowering to the primary prevention of atherosclerosis in a population with relatively low cardiovascular event rates, and suggests that this benefit is mediated by specific morphological effects on early stages of plaque development.

Anticholesteremic Agents↗
PubMed1996

The determination of plasma transferrin receptor as good index of erythropoietic activity in renal anemia and after renal transplantation.

Both the plasma determinations of erythropoietic (EPO) and transferrin receptor (TfR) would provide a good characterization of anemia especially when mixed erythron disorders underlie, such as in renal failure. Immunologic assays of EPO and TfR, as well as standard hematologic determinations (hematocrit, reticulocyte count, serum iron, transferrin, ferritin) were performed in patients with chronic renal failure (CRF), in regular dialysis treatment (RDT) and in transplanted (TX) patients. In nonanemic TX patients both EPO and TfR ranged normally, whereas in anemic TX ones (Hct < 40%) both values were increased suggesting the physiologic response both of the kidney and of the erythron to decreased red cell mass. In transitory posttransplant erythrocytosis the increased values of TfR, with normal EPO values, would hypothesize a defective feedback to EPO release. Both EPO and TfR values were found increased in TX patients with adult polycystic kidney disease with persistent erythrocytosis (Hct > 50%), thus confirming previous observations. In CRF and RDT patients, all anemic, both EPO and TfR were normal, even though significantly low with respect to the degree of anemia. In RDT seriously anemic patients, the administration of recombinant human EPO induced different patterns of bone marrow response. We conclude that the determination of TfR would provide further information on renal anemia since the receptor increase mostly preceded the rise of Hct, evidencing those patients who will not have an effective bone marrow response to the therapy.

Adult↗
PubMed1996

Gonadal function after multimodality treatment in men with testicular germ cell cancer.

We evaluated gonadal function in 63 patients with testicular cancer both within 1 month of unilateral orchiectomy before further treatment (pretreatment) and 3 years after treatment discontinuation (post-treatment). Sixteen patients underwent orchiectomy alone (group 1), nine patients underwent infradiaphragmatic radiotherapy (group 2) and 28 patients received four cycles (group 3) and 10 patients received six cycles (group 4) of cisplatin-based chemotherapy (cisplatin, vinblastine and bleomycin-PVB, or cisplatin, etoposide and bleomycin-PEB). Pretreatment semen analyses showed reduced sperm cell density, motility and impaired morphology of spermatozoa in all four groups (p > 0.05). At the same time elevated estradiol and decreased serum follicle-stimulating hormone (FSH) levels in 28.5% of subjects were correlated with high serum beta human chorionic gonadotropin concentrations. Semen analyses revealed the lowest values for all parameters after infradiaphragmatic radiotherapy. Sperm cell count, motility and morphology were significantly better in patients treated with orchiectomy alone or with a conventional dose of chemotherapy than in the groups that received radiotherapy or high doses of chemotherapy (p < 0.05). We also observed a correlation between serum FSH values and sperm cell density for both pretreatment and post-treatment in every group of patients (p < 0.05). Persistent subclinical Leydig cell dysfunction in groups treated with radiotherapy or high doses of chemotherapy was expressed by increased basal luteinizing hormone levels (78% of patients in group 2 vs 60% of patients in group 4) (p < 0.05) and by normal testosterone serum values (89% of patients in group 2 vs 80% of patients in group 4). Spermatogenesis and Leydig cell function are, therefore, persistently impaired in the majority of testicular cancer patients treated with radiotherapy or with more intensive chemotherapy.

Adolescent↗
PubMed1996

In vitro permeability evaluation and colonization of membranes for periodontal regeneration by Porphyromonas gingivalis.

The use of membranes for periodontal regeneration is well established. In clinical use, the exposure of membranes to the oral microflora may result in a pathway for periodontal infections. An important role in this process is played by Porphyromonas gingivalis. The purpose of the present study was to examine the colonization of 6 different bioresorbable and nonresorbable membranes for periodontal regeneration by the strain DSM 20709 of P. gingivalis and to determine the time needed by this microorganism to pass through the membranes. A device consisting of a tube sealed with the membranes and filled with a medium suitable for the growth of P. gingivalis was incubated in a bigger tube containing the same medium to study the process of colonization and the crossing of membranes. The outer tube was inoculated with 10(4) cells of P. gingivalis DSM 20709. The passage of bacteria through the membranes was monitored at 6, 24, and 48 hours by counting the number of cells in the inner tube. The colonized membranes were observed using a scanning electron microscope. Differences in the behavior of the 6 membranes analyzed were demonstrated.

Bacterial Adhesion↗
PubMed1996

Primary sclerosing cholangitis: clinical presentation, natural history and prognostic variables: an Italian multicentre study. The Italian PSC Study Group.

OBJECTIVE: Because large-scale reports of PSC in the Mediterranean area we are still lacking, in this study we evaluated by Kaplan-Meyer analysis the natural history of primary sclerosing cholangitis (PSC) in Italy and by means of other statistical methods we identified the variables most useful in predicting survival of such patients. DESIGN: Retrospective multicentre study of unselected patients with PSC. Several variables involving sex, age, associated diseases, clinical features, laboratory, cholangiographic and histological findings at presentation and clinical outcome at data recording were collected by means of a detailed questionnaire. SETTING: 16 Italian university and regional hospitals all over the country, thus giving a geographically representative population. PATIENTS: A total of 117 PSC patients (73 men and 44 women); median age 35 years. METHODS: Survival analysis was performed by the Kaplan-Meyer method; the prognostic influence on survival of collected data was evaluated by univariate chi(2) analysis with Wilcoxon and log-rank tests. The same prognostic variables were also evaluated by multivariate analysis (Cox model), using a stepwise regression procedure. All statistical analyses were performed using the SAS statistical software. RESULTS: At presentation 70% of patients were symptomatic; symptoms did not relate to liver histology. Both intra- and extrahepatic bile duct lesions were detected in 46% of patients at cholangiography. Inflammatory bowel disease was found in 54% of symptomatic patients, ulcerative colitis was 36% of total. Clinical outcome (91/117): 15 underwent liver transplantation or died from liver disease (cholangiocarcinoma). Survival at 10 years was 74%. Features of poor prognosis were cholesterol, aspartate aminotransferase (AST), haemoglobin and albumin. CONCLUSION: PSC in Italy mainly follows a benign course and among clinical features recorded at presentation, serum cholesterol, AST, haemoglobin and albumin may provide some objective criteria to assess disease severity.

Adult↗
PubMed1996

[Maternal-fetal transmission of human papillomavirus].

AIM: Human papilloma virus (HPV) infection is one of the most frequently observed sexually-transmitted diseases (10-60% of the general population). In pregnant women, as well as accelerating the evolution of dysplasia to cervical cancer, the infection may be transmitted to the fetus during gestation or at the time of birth. Children who have been infected at birth may develop laryngeal papillomatosis during the first 5 years of life that may, in some cases, spread to the point of causing aphonia or severe respiratory obstruction. There is also the risk, although it is very low, of a carcinomatous degeneration of the larynx in these subjects during adulthood. The hypothesis of the present study was to verify the prevalence of HPV infection in a population of pregnant women and the prevalence of maternal-fetal transmission. EXPERIMENTAL DESIGN: A prospective longitudinal design lasting 11 months was used for the study. It included the collection of an endocervical biopsy from population of pregnant women using a swab that was diluted in 3 cc of physiological solution, and the collection of oropharyngeal secretions from their respective neonates using a cottonwool bud. PARTICIPANTS AND METHODS: A total of 170 pregnant women attending the Obstetric Centre of the Obstetric and Gynecological Clinic of Trieste University were recruited in the study. An endocervical biopsy was taken during the 1st and/or 2nd trimester of gestation and/or at the start of labour. Of these subjects, 23 completed all the planned biopsies and a sample of oropharyngeal secretion was collected from their neonates. TESTS: From the material obtained the presence of HPV-DNA was analysed using a PCR (protein chain reaction) technique consisting of the following steps: 1) culture of human cells expressing sequences of HPV 16 and 18 used as positive controls; 2) preservation of tissue material washed in watery 4% formalin solution; 3) amplification and viral characterization in types 6-11-16-18-31-33-52. RESULTS: Positive HPV-DNA results in at least one of the three samples collected during the various periods of gestation was 31.2%, whilst in the population in which all the planned samples were performed the frequency of positive cases was 30.4%. Positive results for HPV-DNA in oropharyngeal secretions from neonates was 21.7%. The concordance of positivity for HPV-DNA in mothers at the time of labour and in their respective neonates was 57.14%. CONCLUSIONS: The trend of infection did not reveal substantial changes during the various gestational periods in which tests were performed. The possibility of HPV-DNA transmission from mother to fetus is high, above all when the maternal PCR test is positive at the time of birth, or in the presence of a high viral load. This justifies the need to monitor this infection in pregnancy in those affected by florid genital condylomatosis or with koilocytosis on cervical cytology. It is also appropriate to check all HPV-DNA positive neonates one year after birth.

Biopsy↗
PubMed1996

[Evoked otoacoustic emissions using clicks in detection of deafness in young children].

Click-evoked otoacoustic emissions (EOAEs) are sounds emitted by the cochlea due to active outer hair cell contraction following stimulation of the stereocilia. The purpose of this study was to evaluate the use of testing for EOAEs as a valid auditory screening method in newborns, and 1074 healthy fullterm babies (2148 ears) with an Apgar score higher than 8 at birth were examined. EOAEs were elicited 4 days after birth and a correlation index higher than 70% represented normal response criteria. If there was no response, EOAEs and brainstem response potentials were tested at the age of one month. On the fourth day after birth. EOAEs specificity and efficiency were found to be 92.3% and 92.4% respectively, while sensitivity was 100%. Specificity and efficiency at one month were rated at 100%. The use of EOAEs offers numerous advantages over traditional auditory screening methods, thus suggesting that this technique is suitable for the mass-screening of infants.

Acoustic Stimulation↗
PubMed1995

HLA-A, B, C, DR and DQ expression and hepatocellular carcinoma: study of 205 Italian subjects.

We have evaluated the frequency of HLA class I and II antigens in 205 Italian patients with hepatocellular carcinoma (HCC) and 749 blood donors (controls). Moreover, we have looked for correlations between HLA antigen frequencies and HBV and/or HCV infections in HCC patients. We found great differences in HLA antigen frequencies considering only two groups: HCC patients and controls. The polymorphism is smaller when we consider the different groups of HCC patients in regard to the previous viral infections (HBV and/or HCV). The most interesting finding is the higher frequency of Cw7, B8 and DR3 in almost all groups of HCC patients. It is well known, that the HLA A1, Cw7, B8, DR3 antigen haplotype is associated with a rapid decline of CD4 cells, and HLA B8, DR3 positive subjects may display some changes in immune parameters and are prone to develop several immunological diseases. Thus HCC might be the result of a lower sensitivity (genetically given) to mitogenic stimuli of HBV and HCV.

Aged↗
PubMed1995

Studies on the dissociation of cryptomonad biliproteins.

The spectroscopic properties of two biliproteins, phycocyanin 645 and phycoerythrin 566, have been studied by treating the proteins with two different agents, NaSCN at pH 6.0, or pH 4.0 without NaSCN. For phycoerythrin 566, treatment with NaSCN revealed that the visible CD spectrum of its chromophores was separated into a pair of different spectra, and each of these spectra was observed as a negative and one or more positive bands. For phycocyanin 645, two negative CD bands have been observed previously, together with two or more positive bands, in the dimer (alpha 2 beta 2) state, and NaSCN treatment caused elimination of one of these negative bands. The dimer was stable at pH 6.0, but at pH 4.0 the spectra of phycocyanin 645 had one less negative band than those at pH 6.0. Chromatography demonstrated that phycocyanin 645 was a monomer (alpha beta) at pH 4.0. Monomers of cryptomonad biliproteins have never been observed before. Excitation at 514 nm, in picosecond time-resolved fluorescence studies, produced lifetimes of 11.0 and 45.2 ps for dimers and monomers, respectively. Excitation at 566 nm yielded times of 1.38 and 1.24 ps, for dimers and monomers, respectively. CD in the far UV showed that monomers and dimers had very similar secondary structures. These results have been used to test an hypothesis that proposed two types of exciton splitting among the chromophores of phycocyanin 645, and perhaps phycoerythrin 566 could also have this chromophore organization.

Animals↗
PubMed1995

Site-directed mutagenesis of human glutathione transferase P1-1. Mutation of Cys-47 induces a positive cooperativity in glutathione transferase P1-1.

Glutathione transferase P1-1 (EC 2.5.1.18) is a dimeric enzyme composed of identical subunits each containing one binding site for GSH and a second for the co-substrate e.g. 1-chloro-2,4-dinitrobenzene. Steady-state kinetics are strictly hyperbolic toward both these substrates. Replacement of Cys-47 with alanine or serine decreases the affinity for GSH and triggers a positive kinetic cooperativity with respect to the substrate. Hill coefficients were 1.31 and 1.43 for the C47A and C47S mutants. C47A/C101S and C47S/C101S double mutants display lower affinity for GSH and higher Hill coefficients (1.57 and 1.56, respectively) when compared with C47A and C47S single mutants. Conversely, replacement of Cys-101 with alanine or serine does not yield any cooperativity and any marked change of kinetic parameters. Fluorometric experiments gave sigmoidal isothermic GSH binding curves for all the Cys-47 mutants, with Hill coefficients similar to that obtained by the kinetic approach. These data, together with the activation experiments performed in the presence of S-hexylglutathione, suggest that the substitution of Cys-47 yields a dimeric low-affinity enzyme which may be revealed by the lack of a peculiar electrostatic bond between the thiolate form of Cys-47 and the protonated amino group of Lys-54.

Cysteine↗
PubMed1995

Site-directed mutagenesis of human glutathione transferase P1-1. Spectral, kinetic, and structural properties of Cys-47 and Lys-54 mutants.

In the human placental glutathione transferase, Cys-47 possesses, at physiological pH values, a pK alpha value of 4.2 and may exist as an ion pair with the protonated epsilon-amino group of Lys-54. Using site-directed mutagenesis we investigate spectral, kinetic, and structural properties of Cys-47 and Lys-54 mutants. The results shown indicate that the thiolate ion detected at 229 nm should be assigned exclusively to Cys-47. The contribution of Lys-54 to the activation of Cys-47 is assessed by the spectral properties of the K54A mutant enzyme. The induced cooperativity toward glutathione, as a consequence of mutation of Lys-54 to alanine, clearly parallels that observed for the Cys-47 mutant enzymes (see the preceding paper (Ricci, G., Lo Bello, M., Caccuri, A. M., Pastore, A., Nuccetelli, M., Parker, M. W., and Federici, G. (1995) J. Biol. Chem. 270, 1243-1248) and points out the importance of this electrostatic interaction in shaping the correct spatial arrangement for the binding of glutathione and in anchoring the flexible helix alpha 2. When this ion pair is disrupted, by mutation of either residue, the flexibility of this region could be greatly increased, causing helix alpha 2 to come in contact with the other subunit and generating a structural communication, which is the basis of the observed cooperativity.

Base Sequence↗
PubMed1995

Epidemiology of gallstone disease in Italy: prevalence data of the Multicenter Italian Study on Cholelithiasis (M.I.COL.)

This study was designed to obtain a general overview of gallstone disease in Italy. A total of 18 cohorts in 10 Italian regions were enrolled in this survey. Four cohorts were excluded from analysis because of a participation rate of less than 50 percent. Field activities started in December 1984 and terminated in April 1987. A precoded questionnaire was administered to each subject by a trained member of the medical staff. Participants underwent an ultrasonographic examination of the upper abdomen and blood sampling. Each research group was provided with an identical ultrasonographic machine. Standardized criteria were established for both the examination and diagnosis. The inter- and intraobserver variation in the ultrasonographic procedure was assessed using a reliability study. The overall kappa score for interobserver agreement was good (0.649), while intraobserver agreement was good or excellent (kappa scores > 0.60) in 75 percent of the observers. A total of 29,739 of the 46,139 (64.4 percent) enrolled subjects were examined. Among those with a clear-cut definition of the gallbladder status, 6.5 percent of the males and 10.5 percent of the females had gallstones. Additional 3.0 and 8.4 percents, respectively, had already undergone cholecystectomy. Prevalence of gallstone disease (gallstones plus cholecystectomy) increased linearly with age in both sexes. Among subjects with gallstones, 76.7 percent of men and 72 percent of women were not aware of having gallstones; 87.0 percent of men and 84.9 percent of women were asymptomatic. This study confirms that gallstone disease is a highly prevalent condition. Gallstones rarely cause symptoms, and subjects are mostly unaware of their presence.

Adult↗
PubMed1995

Cytoplasmic and periplasmic production of human placental glutathione transferase in Escherichia coli.

An expression vector yielding large amounts of GST P1-1 in the cytoplasm of Escherichia coli was constructed. The recombinant enzyme, obtained after purification, was characterized in its physicochemical and kinetic properties and appeared to be indistinguishable from that purified from human placenta. However, N-terminal amino acid sequencing revealed that about 50% of the recombinant GST still contained methionine as the N-terminal amino acid. Such an incomplete processing was not simply due to overproduction of GST. In fact, under growth conditions that lead to a sharp decrease in the production of the protein the N-terminal methionine was not removed. GST was unable to translocate across the bacterial membrane when it was fused to the leader peptide of the pelB gene from Erwinia carotovora and accumulated in the cytoplasm in a soluble and active conformation. However, when this fusion protein was produced in a bacterial strain overexpressing the bacterial chaperonins GroEL and GroES, a fraction of GST was exported into the periplasmic space with the correct N-terminal sequence. The yield of correctly processed GST accounted for 12% of total GST present in the E. coli cells. Our results suggest that chaperonins are able to interact with nascent GST, thus maintaining the protein in an export-competent form and that E. coli strains with enhanced secretory characteristics may be obtained by genetic engineering technology.

Amino Acid Sequence↗