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G Finazzi

Publications and source records attributed to G Finazzi.

At least 37 records · Page 2Linked to original sources
PubMed1999

The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase.

We created a Qo pocket mutant by site-directed mutagenesis of the chloroplast petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY sequence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow in phototrophic conditions although it assembled wild-type levels of cytochrome b6f complexes. We demonstrated a complete block in electron transfer through the cytochrome b6f complex and a loss of plastoquinol binding at Qo. The accumulation of cytochrome b6f complexes lacking affinity for plastoquinol enabled us to investigate the role of plastoquinol binding at Qo in the activation of the light-harvesting complex II (LHCII) kinase during state transitions. We detected no fluorescence quenching at room temperature in state II conditions relative to that in state I. The quantum yield spectrum of photosystem I charge separation in the two state conditions displayed a trough in the absorption region of the major chlorophyll a/b proteins, demonstrating that the cells remained locked in state I. 33Pi labeling of the phosphoproteins in vivo demonstrated that the antenna proteins remained poorly phosphorylated in both state conditions. Thus, the absence of state transitions in the pwye mutant demonstrates directly that plastoquinol binding in the Qo pocket is required for LHCII kinase activation.

Amino Acid Sequence↗
PubMed1999

A new electrochemical gradient generator in thylakoid membranes of green algae.

Using a new method of delayed luminescence digital imaging, mutants of Chlorella sorokiniana lacking the chloroplast CF0CF1 ATP synthase were isolated for the first time. Biochemical characterization of these strains indicates a lack of detectable synthesis and accumulation of the ATP synthase subunits alpha-CF1 and beta-CF1. Functional characterization indicates the presence of a permanent electrochemical gradient (DeltaMu) across the thylakoid membrane in the dark-adapted state, which is not suppressed under anaerobic conditions. Contrary to what is observed in the presence of the CF0CF1 ATP synthase, this gradient is essentially due to an electric field component DeltaPsi with no detectable DeltapH component, under both aerobic and anaerobic conditions. Neither the CF0CF1 ATP synthase nor a respiratory process can thus be responsible for a permanent gradient detected under these conditions. The previous proposal of a new ATP-dependent electrogenic pump in thylakoid membranes is supported by these results that, in addition, indicate a specificity of this new pump for ions other than protons.

Aerobiosis↗
PubMed1999

Treatment of essential thrombocythemia with special emphasis on leukemogenic risk.

Acute leukemia and myelodysplastic syndromes are rare, but almost invariably fatal, evolutions of essential thrombocythemia (ET). Three major factors are associated with blastic transformation: cytogenetic abnormalities, myelofibrotic features, and the use of cytotoxic agents. Hematological malignancies have been reported in ET patients after treatment with alkylating agents, such as busulphan, as well as other cytoreductive drugs, such as hydroxyurea. Concerns about leukemogenicity have led some to suggest limiting the indications of these drugs to patients at higher risk of bleeding and thrombosis. Major risk factors for thrombosis are age above 60 years and a previous thrombotic event, whereas an increased bleeding tendency has been reported with platelet counts in excess of 1000-1500x10(9)/l. No myelosuppressive therapy is recommended for younger patients if they are asymptomatic or their platelet counts are below 1500x10(9)/l. The threshold of 1500x10(9)/l is controversial, however, and cytoreduction can be considered when platelets are above 1000x10(9)/l or in the presence of risk factors for cardiovascular disease. In the presence of thrombotic events or extreme thrombocytosis, young ET patients can be managed with cytoreductive agents theoretically devoid of leukemogenic risk, such as a-interferon or anagrelide. Nevertheless, the mutagenic risk of anagrelide has not been investigated in long-term follow-up studies, and the ultimate place of these 'new' drugs in the management of ET patients remains to be established in prospective and controlled clinical trials.

Antineoplastic Agents↗
PubMed1999

Diagnosis, pathogenesis and treatment of the myeloproliferative disorders essential thrombocythemia, polycythemia vera and essential megakaryocytic granulocytic metaplasia and myelofibrosis.

According to strict clinical, hematological and morphological criteria, the Philadelphia (Ph) chromosome negative chronic myeloproliferative disorders essential thrombocythemia (ET), polycythemia vera (PV), and agnogenic myeloid (megakaryocytic/granulocytic) metaplasia (AMM) or idiopathic myelofibrosis (IMF) are three distinct disease entities with regard to clinical manifestations, natural history and outcome in terms of life expectancy. As clonality studies have clearly demonstrated that fibroblast proliferation in AMM, as well as in many other conditions such as advanced stages of Ph(+)-essential thrombocythemia, Ph(+)-granulocytic leukemia, and Ph(-)-polycythemia vera, is polyclonal indicating that myelofibrosis is secondary to the megakaryocytic granulocytic metaplasia in these various conditions, AMM is illogically labeled as IMF. As abnormal megakaryocytic granulocytic metaplasia is the essential feature preceding the early prefibrotic stage of AMM, the term essential megakaryocytic granulocytic metaplasia (EMGM) can readily be used to characterize this condition more appropriately at the biological level. Clinical, hematological and morphological characteristics, in particular megakaryocytopoiesis and bone marrow cellularity, reveal diagnostic features, which enable a clear-cut distinction between ET, PV and EMGM or classical IMF. The characteristic increase and clustering of enlarged megakaryocytes with mature cytoplasm and multilobulated nuclei and their tendency to cluster in a normal or only slightly increased cellular bone marrow represent the hallmark of ET. The characteristic increase and clustering of enlarged mature and pleiomorphic megakaryocytes with multilobulated nuclei and proliferation of erythropoiesis in a moderate to marked hypercellular bone marrow with hyperplasia of dilated sinuses are the specific diagnostic features of untreated PV. EMGM, including the early prefibrotic stages as well as the various myelofibrotic stages of classical IMF appear to be a distinct neoplastic dual proliferation of abnormal megakaryopoiesis and granulopoiesis. The histopathology of the bone marrow in prefibrotic EMGM and in classical IMF is dominated by atypical, enlarged and immature megakaryocytes with cloud-like immature nuclei, which are not seen in ET and PV at diagnosis and during follow-up. Myelofibrosis in ET, PV and EMGM is graded into: no reticulin fibrosis (MF0), early reticulin fibrosis (MF1), advanced reticulin sclerosis with minor or moderate collagen fibrosis (MF2) and advanced collagen fibrosis with osteosclerosis (MF3). Myelofibrosis is not a feature of ET at diagnosis and during long-term follow-up. Myelofibrosis may be present in a minority of PV-patients at diagnosis and usually becomes apparent during long-term follow-up in the majority of PV-patients. Myelofibrosis secondary to the abnormal megakaryocytic and granulocytic myeloproliferation constitutes a prominent feature in the majority of EMGM/IMF at time of diagnosis and usually progresses more or less rapidly during the natural history of the disease. Life expectancy is normal in ET, normal during the 1st ten years and compromised during the 2nd ten years follow-up in PV, but significantly shortened in the prefibrotic stage of EMGM as well as in the various myelosclerotic stages of classical IMF. First line treatment options in prospective randomized clinical trials of newly diagnosed MPD-patients are control of platelet function with low-dose aspirin versus reduction of platelet count with anagrelide, interferon or hydroxyurea in ET; control of platelet and erythrocyte counts by interferon alone versus bloodletting plus hydroxyurea on indication in PV; interferon versus no treatment in the early stages of EMGM; a wait and see strategy in the fibrotic stages of EMGM or classical IMF with favorable prognostic factors, and bone marrow transplantation in classical IMF with poor prognostic factors at presentation or during short-term follow-up.

Aspirin↗
PubMed1999

Analysis of the nucleus-encoded and chloroplast-targeted rieske protein by classic and site-directed mutagenesis of Chlamydomonas.

Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron-sulfur protein 2Fe-2S subunit of the chloroplast cytochrome b(6)f complex have been characterized. One has a stable deletion that eliminates the protein; two others carry substitutions Y87D and W163R that result in low accumulation of the protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b(6)f complexes of the Y87D and W163R mutant Rieske proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe-2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske protein of photosynthesis.

Amino Acid Sequence↗
PubMed1999

Risk of venous thromboembolism and clinical manifestations in carriers of antithrombin, protein C, protein S deficiency, or activated protein C resistance: a multicenter collaborative family study.

Deficiencies of antithrombin (AT), protein C (PC) or protein S (PS), and activated protein C resistance (APCR) are very well-established coagulation defects predisposing to venous thromboembolism (VTE). We performed a retrospective cohort family study to assess the risk for VTE in individuals with AT, PC, or PS deficiency, or APCR. Five hundred thirteen relatives from 9 Italian centers were selected from 233 families in which the proband had had at least 1 episode of VTE. We calculated the incidence of VTE in the whole cohort and in the subgroups after stratification by age, sex, and defect. The overall incidence of VTE (per 100 patient-years) in the group of relatives was 0.52. It was 1.07 for AT, 0.54 for PC, 0.50 for PS, 0.30 for APCR, and 0.67 in the group with a double defect. The incidence was associated with age, but not with sex. The mean age at onset was between 30 and 40 years for all the coagulation defects. Women had the peak of incidence in the age range of 21 to 40 years, earlier than men. The lifetime risk for VTE was 4.4 for AT versus APCR, 2.6 for AT versus PS, 2.2 for AT versus PC, 1.9 for PC versus APCR, and 1.6 for PS versus APCR. AT deficiency seems to have a higher risk for VTE than the other genetic defects. There is a relation between age and occurrence of thrombosis for both men and women. The latter had the peak of incidence earlier than the former.

Activated Protein C Resistance↗
PubMed1999

The risk of thrombosis in patients with lupus anticoagulants is predicted by their specific coagulation profile.

Lupus anticoagulants belong to the family of antiphospholipid antibodies. They include two phospholipid-dependent inhibitors of coagulation that may be distinguished on the basis of specific coagulation profiles generated from the comparison of the ratios of the Kaolin Clotting Time (KCT) and the dilute Russell's Viper Venom Time (dRVVT): when the ratio of the KCT exceeds that of the dRVVT, the plasma is allocated to the "KCT" coagulation profile, when the opposite occurs, the plasma is defined to belong to the "dRVVT" coagulation profile group. We prospectively followed-up a historical cohort of 100 consecutive patients with lupus anticoagulants referred to our Institution between January 1988 and October 1997 to investigate the relationship between their coagulation profile at diagnosis and the development of thrombosis during a median follow-up time of 37.5 months (range 1-115 months). Fifty-six patients were allocated to the "dRVVT" coagulation profile, whereas the other 44 displayed the "KCT" profile. Lupus anticoagulants were transient in 17 patients, without differences between the two groups. None of these patients developed clinical events before disappearance of the phospholipid-dependent inhibitors of coagulation. The 83 cases with persistent lupus anticoagulants consistently displayed the same coagulation profile they had been allocated to at entry. Fourteen patients developed 18 thromboembolic events during the follow-up, with an overall rate of thrombosis of 4.2% patients-year. Twelve of them belonged to the "dRVVT" coagulation profile, whereas the other 2 to the "KCT" profile (p = 0.03). The "dRVVT" coagulation profile gave an odds ratio of thrombosis of 5.25 (95% confidence interval [C.I]: 1.17-23.50). Ten of the 14 patients who developed thrombosis during follow-up had already experienced thrombosis: a previous thrombotic event caused an odds ratio of recurrency of 2.72 (95% C.I.: 0.85-8.73) (p = 0.09). By multivariate analysis, the "dRVVT" coagulation profile was still associated with a trend to a higher risk of thrombosis, but the difference did not reach statistical significance. Increased levels of anticardiolipin antibodies (> 40 GPL and/or MPL units) were found in all the 14 patients (p = 0.0064). The "KCT" coagulation profile was significantly associated (p = 0.005) with moderate thrombocytopenia (platelets 50-150 X 10(9)/l). Neither profile was found to represent a risk factor for the development of recurrent miscarriages, neoplastic diseases and death. In conclusion, the "dRVVT" profile appears to have predictive value with respect to the thrombotic complications suffered by patients with antiphospholipid antibodies.

Adult↗
PubMed1999

Clinical parameters for determining when and when not to treat essential thrombocythemia.

Essential thrombocythemia (ET) is a myeloproliferative disorder that occurs primarily in middle-aged adults. ET is relatively benign compared with some of the other myeloproliferative disorders (MPDs), but there is still a high rate of morbidity and quality of life can be affected, especially by thrombotic and hemorrhagic complications. Two thirds of ET patients are symptomatic, but therapies are not benign. While alkylating agents, radioactive phosphorous, and hydroxyurea (HU) all have a leukemogenic risk, their use in high-risk and elderly patients may still be warranted in some circumstances. Use of nonmutagenic agents such as anagrelide, interferon-alpha (IFN), or low-dose aspirin has become an attractive alternative. However, the risks and benefits of treatment versus nontreatment should be based on clinical data and must be determined on an individual basis.

Humans↗
PubMed1998

Glu78, from the conserved PEWY sequence of subunit IV, has a key function in cytochrome b6f turnover.

We have investigated the structure to function relationship at the Qo site in cytochrome b6f complexes in vivo. To this end, we created site-directed mutants of Chlamydomonas reinhardtii, at position 78 in the sequence of subunit IV. The target glutamic acid, present in the highly conserved 77PEWY80 sequence, was changed to residues of different polarities which did not prevent the functional assembly of cytochrome b6f complexes. Spectroscopic analysis performed in anaerobic conditions in vivo revealed distinct alterations in cytochrome b6f function, depending on the nature of the substituted residue. The semiconservative E78D substitution, in which only the length of the side chain is reduced, retained the functional features of the wild-type configuration. The E78K and E78L substitutions caused a significant decrease, by factors of 3 and 5, respectively, in the rate of the concerted oxidation process at the Qo site without a change in the affinity of Qo for reduced plastoquinones. The E78Q and E78N substitutions modified the characteristics of cytochrome b6f turnover under repetitive flash illumination. They caused a large increase in the electrogenicity of the electron-transfer reactions through the mutated cytochrome b6f complex. This increase was specifically sensitive to the electrical component of the proton-motive force. Surprisingly, despite the larger number of charges translocated across the membrane per charge injected in the high potential chain, the reduction phase for cytochrome b6 became barely detectable in the mutants, unless inhibitors at the Qi site were present. We show that similar functional characteristics can be observed with the cytochrome b6f complex in the wild-type in anaerobic conditions, provided a single flash illumination regime is used. These observations suggest that cytochrome b6f turnover may involve a mechanism implying an extra proton pumping activity.

Amino Acid Sequence↗
PubMed1998

In vivo characterization of the electrochemical proton gradient generated in darkness in green algae and its kinetic effects on cytochrome b6f turnover.

When unicellular algal cells are placed under anaerobic conditions, a large electrochemical gradient is built in darkness across the thylakoid membranes. We have estimated, in vivo, the amplitude of the Delta pH component of this transmembrane potential and shown that the Delta pH is twice as large as the Delta Psi. The amplitude of the Delta mu tildeH+ (approximately 110-140 mV) fits well with estimations based on the ATP/ADP ratio measured in green algae under the same conditions, suggesting that an equilibrium state is established across the thylakoid membrane. Therefore, under anaerobic dark incubation of algae, the electrochemical transmembrane potential is determined only by the cellular ATP content. The existence of this Delta mu tildeH+ is expected to result in a constitutive amount of activated CFo-CF1 ATPase, thereby facilitating ATP synthesis under low light intensity illumination. We report also on the effects of this dark-existing electrochemical gradient on the cytochrome b6f complex turnover kinetics. We show that they are largely slowed by the presence of this electrochemical transmembrane potential. The pH component is mainly responsible for the kinetic slowing down of cytochrome b6f complex turnover, despite the fact that electrogenicity is associated with the reactions taking place within this complex. Therefore, in vivo, owing to the low lumenal pH, the oxidation of plastoquinol at the Qo site is limiting the turnover of the cytochrome b6f complex in the presence of the Delta pH, while in its absence the oxidation rate of the b6 hemes becomes rate-limiting.

Adenosine Triphosphate↗
PubMed1998

No treatment for low-risk thrombocythaemia: results from a prospective study.

Essential thrombocythaemia (ET) is a chronic myeloproliferative disorder characterized by the occurrence of thromboembolic episodes, particularly in patients aged > 60 years or with a previous history of thrombosis, and/or by haemorrhages in patients with an exceedingly high platelet count. In these subgroups of patients the use of cytoreductive therapy is beneficial in terms of risk/benefit ratio. Only limited anecdotal data are available on the thrombotic or haemorrhagic risk and survival in young asymptomatic ET patients with a platelet count < 1500 x 10(9)/l. Therefore the optimal management of these patients is unknown. To assess the incidence of thrombosis and haemorrhages in this group of patients we carried out a prospective observational study in a cohort of 65 patients with ET, aged < 60 years, with no history of thrombosis or haemorrhage and platelet count < 1500 x 10(9)/l, and in 65 age- and sex-matched controls. Patients were not treated with cytoreductive therapy until the occurrence of thrombosis or haemorrhage. Arterial or venous thrombotic events were objectively documented both in cases and in controls. The median follow-up was 4.1 years, with an incidence of thrombosis in patients and controls of 1.91 and 1.50 cases/100 patient-years, respectively. The age- and sex-adjusted risk rate ratio was 1.43 (95% CI 0.37-5.4). Only three minor haemorrhagic episodes occurred in patients, with an incidence of 1.12 cases/100 patient-years. Pregnancy and surgery were not associated with thrombosis in these patients. We conclude that the thrombotic risk in young ET patients, with no thrombotic history and a platelet count < 1500 x 10(9)/l, is not increased compared to the normal population and that a conservative therapeutic approach should therefore be considered in these patients.

Adolescent↗
PubMed1998

Treatment of polycythemia vera.

BACKGROUND AND OBJECTIVE: Current guidelines for the management of patients with polycythemia vera (PV) derive from a few clinical trials and several uncontrolled clinical studies. The purpose of this paper is to critically review the available evidence in literature for selecting the best treatment in the single patient. METHODS: The authors have been working in this field contributing original papers whose data have been used for this study. In addition, the material analyzed in this article includes papers published in the journals covered by the Science Citation index and Medline. RESULTS: Therapeutic strategies for patients with PV include both cytoreductive and antithrombotic drugs. Among cytoreductive treatments, phlebotomy is associated with poor compliance and an increased incidence of thrombosis in the first three-five years, whereas chemotherapy may induce an higher risk of secondary malignancies after seven-ten years of follow-up. New cytoreductive drugs virtually devoid of mutagenic risk include alpha-interferon and anagrelide, but their role in reducing thrombotic complications or mortality remains to be demonstrated. Antithrombotic drugs, such as aspirin, are frequently used in PV, despite doubts regarding safety and efficacy. CONCLUSIONS: The management of patients with PV is a difficult balance between the prevention of thrombotic complications and the risk of drug side effects and toxicity. Appropriate studies are needed and an European collaboration has been set up for launching a randomized, placebo-controlled clinical trial (European Collaboration on Low-Dose Aspirin-ECLAP study) aimed at testing the efficacy of low-dose aspirin in preventing thrombosis and prolonging survival in PV patients.

Humans↗
PubMed1998

Congenital resistance to activated protein C in patients with lupus anticoagulants: evaluation of two functional assays.

The R506Q mutation ("Factor V Leiden") is responsible for the resistance to activated Protein C (aPCR), that is evaluated by coagulation tests. Such tests cannot be used in patients with lupus anticoagulants (LAs), due to the interfering effect exerted by these antibodies on "in vitro" phospholipid-dependent coagulation tests. For this reason, assays have been developed to evaluate aPCR that are insensitive to the presence of LA antibodies. We evaluated two such coagulation tests in the plasma of 82 consecutive patients with LAs. By polymerase chain reaction 3 patients (3.6%) were found heterozygous for the R506Q mutation. aPCR was evaluated by two clotting assays, proposed to be "insensitive" to the presence of LAs: 1. aPCR-tissue factor-based assay, using Factor V deficient plasma and 1:40 diluted test plasma; 2. aPCR-dRVVT-based assay with highly concentrated phospholipids. Their interassay coefficient of variation was 28% and 6.2%, respectively. Compared to the polymerase chain reaction analysis, the 2 tests displayed the following characteristics: sensitivity 67% vs 100%, specificity 92% vs 96%, positive predictive value 25% vs 50%, negative predictive value 99% vs 100%. respectively. Among LA patients without the R506Q mutation, 5 scored positive in the aPCR-tissue factor-based assay, 2 in the aPCR-dRVVT-based assay and another one in both assays. Our findings suggest that the aPCR-dRVVT-based test is more reliable and sensitive than the aPCR-tissue factor-based one to the R506Q mutation in patients with LAs. Both assays, when negative, make unlikely the presence of the R506Q mutation. Polymerase chain reaction analysis remains, however, to be performed when either test is positive.

Activated Protein C Resistance↗
PubMed1997

The threshold for prophylactic platelet transfusions in adults with acute myeloid leukemia. Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto.

BACKGROUND: Prophylactic platelet transfusions are usually administered to patients receiving myelotoxic chemotherapy when their platelet count falls below 20,000 per cubic millimeter. Some observations suggest that lower platelet counts can be appropriate in patients in stable condition, but the safety of lower thresholds is uncertain. METHODS: We evaluated 255 adolescents and adults (age, 16 to 70 years) with newly diagnosed acute myeloid leukemia (but not acute promyelocytic leukemia), who were treated in 21 centers. One hundred thirty-five patients were randomly assigned to receive a transfusion when their platelet count fell below 10,000 per cubic millimeter (or 10,000 to 20,000 per cubic millimeter in those with a temperature above 38 degrees C, with active bleeding, or a need for invasive procedures), and 120 patients were assigned to receive a transfusion when their platelet count was less than 20,000 per cubic millimeter. RESULTS: Patients in the group with a threshold of 10,000 platelets per cubic millimeter received 21.5 percent fewer platelet transfusions than the patients in the group with a threshold of 20,000 platelets per cubic millimeter (P=0.001). The numbers of red-cell units transfused were not significantly different between groups. Major bleeding (defined as any bleeding more than petechiae or mucosal or retinal bleeding) occurred in 21.5 and 20 percent of patients, respectively (P=0.41), and on 3.1 and 2.0 percent of the days of hospitalization. One episode of fatal cerebral hemorrhage occurred in the group with a threshold of 10,000 platelets per cubic millimeter; none occurred in the other group (P= 0.95). Actuarial estimates of survival during induction chemotherapy, actuarial estimates of the absence of major bleeding, and the length of hospital stay were not significantly different in the two groups. CONCLUSIONS: The risk of major bleeding during induction chemotherapy in adolescents and adults with acute myeloid leukemia (except acute promyelocytic leukemia, which we did not study) was similar with platelet-transfusion thresholds of 20,000 per cubic millimeter and 10,000 per cubic millimeter (or 10,000 to 20,000 per cubic millimeter when body temperature exceeded 38 degrees C, there was active bleeding, or invasive procedures were needed). Use of the lower threshold reduced platelet use by 21.5 percent.

Adolescent↗