Biomedical subjects
Publications and source records attributed to G Giardina.
All denitrifiers can keep the steady-state concentrations of nitrite and nitric oxide (NO) below cytotoxic levels by controlling the expression of denitrification gene clusters by redox signalling through transcriptional regulators belonging to the CRP (cAMP receptor protein)/FNR (fumarate and nitrate reductase regulator) superfamily.
In denitrifying bacteria, the concentration of NO is maintained low by a tight control of the expression and activity of nitrite and NO reductases. Regulation involves redox-linked transcription factors, such as those belonging to the CRP-FNR (cAMP receptor protein-fumarate and nitrate reductase regulator) superfamily, which act as oxygen and N-oxide sensors. Given that few members of this superfamily have been characterized in detail, we have cloned, expressed and purified the dissimilative nitrate respiration regulator from Pseudomonas aeruginosa. To gain insights on the structural properties of the dissimilative nitrate respiration regulator, we have also determined the aggregation state of the purified protein and its ability to bind hydrophobic compounds such as 8-anilino-1-naphthalenesulphonic acid.
Human kallikrein 6 protein is a newly discovered human kallikrein. We determined the amount of human kallikrein 6 in extracts of 182 ovarian tumours and correlated specific activity (ng hK6 mg(-1) total protein) with clinicopathological variables documented at the time of surgical excision and with outcome (progression free survival, overall survival) monitored over a median interval of 62 months. Thirty per cent of the tumours were positive for human kallikrein 6 (>35 ng hK6 mg(-1) total protein). Human kallikrein 6-specific immunohistochemical staining of four ovarian tissues that included benign, borderline and malignant lesions indicated a cytoplasmic location of human kallikrein 6 in tumour cells of epithelial origin, although the intensity of staining was variable. Tumour human kallikrein 6 (ng hK6 mg(-1) total protein) was higher in late stage disease, serous histotype, residual tumour >1 cm and suboptimal debulking (>1 cm) (P<0.05). Univariate analysis revealed that patients with tumour human kallikrein 6 positive specific activity were more likely to suffer progressive disease and to die (hazard ratio 1.71 (P=0.015) and 1.88 (P=0.022), respectively). Survival curves demonstrated the same (P=0.013 and 0.019, respectively). Multivariate analysis revealed that human kallikrein 6 positivity was retained as an independent prognostic variable in several subgroups of patients, namely those with (low) grade I and II tumours (hazard ratio progression free survival 4.3 (P=0.027) and overall survival 4.1 (P=0.023)) and those with optimal debulking (hazard ratio progression free survival 3.8 (P=0.019) and overall survival 5.6 (P=0.011)). We conclude that tumour kallikrein 6 protein levels have utility as an independent adverse prognostic marker in a subgroup of ovarian cancer patients with otherwise apparently good prognosis.
Many members of the human kallikrein gene family were found to be differentially expressed in various malignancies and some are useful cancer diagnostic/prognostic markers. KLK9 is a newly discovered human kallikrein gene that is expressed in several tissues including thymus, testis, spinal cord, salivary gland, ovary, and skin. Like other kallikreins, the KLK9 gene was found to be regulated by steroid hormones in cancer cell lines. Our purpose is to examine whether quantitative analysis of KLK9 expression has prognostic value in ovarian cancer. We studied the expression of KLK9 by quantitative reverse transcription-PCR in 168 consecutive ovarian tumors of different stages, grades, and histological types, and correlated the expression with clinicopathological parameters, response to chemotherapy, and patients' survival. We found that KLK9 expression was significantly higher in patients with early disease stages (I or II; P = 0.044) and in patients with optimal debulking (P = 0.019). Kaplan-Meier survival curves demonstrated that patients with KLK9-positive tumors have substantially longer progression-free and overall survival (P < 0.001 and P = 0.016, respectively). When the Cox proportional hazard regression analysis was applied to subgroups of patients, KLK9 expression was found to be a significant predictor of progression-free survival in the subgroup of patients with low-grade tumors [hazard ratio (HR), 0.13; P = 0.0015], early stage (HR, 0.099; P = 0.031); and those with optimal debulking (HR, 0.26; P = 0.012). After adjusting for other known prognostic variables, KLK9 retained its independent prognostic value in all of these subgroups of patients. A negative correlation was found between the expression levels of CA125 and KLK9 (rs, 0.350; P = 0.002). Our results indicate that KLK9 is under steroid hormone regulation in ovarian and breast cancer cell lines. Immmunohistochemically, human kallikrein protein (hK9) was localized in the cytoplasm, but not in the nuclei, of the epithelial cells of ovarian cancer tissues. We conclude that KLK9 is a potential new independent favorable prognostic marker for early stage, low-grade, optimally debulked ovarian cancer patients.
Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.
Sequential administration of the association of 5-fluorouracil, epirubicin and cyclophosphamide (FEC) and paclitaxel could be better tolerated than the association of an anthracycline and paclitaxel while having a similar antitumour effect. 69 patients with advanced breast cancer previously untreated with anthracyclines or paclitaxel entered a phase II multicentre study in which FEC was followed by paclitaxel. Both regimens were administered 4 times every 21 days. The median follow-up is 20 months and 38/69 patients have died. Grade III-IV toxicity was acceptable. Leukopenia occurred in 26% of patients, thrombocytopenia in 2% and anaemia in 4%. One patient had reversible heart failure during FEC therapy. Peripheral neuropathy and arthralgia-myalgia occurred in 9% and 4% of patients, respectively and one patient had respiratory hypersensitivity during paclitaxel treatment. 9 patients did not complete therapy because of: treatment refusal (n = 1), cardiac toxicity (n = 1), early death during FEC chemotherapy (n = 1), major protocol violations (n = 4), hypersensitivity reaction (n = 1) and early death during paclitaxel chemotherapy (n = 1). The overall response rate was 65% (95% CI = 53-76), and 7% of patients had stable disease. Therapy was defined as having failed in 28% of patients because they were not evaluable (13%) or had progressive disease (15%). The median time to progression and survival are 13.2 and 23.5 months, respectively. Sequential FEC-paclitaxel is a suitable strategy for patients with metastatic breast cancer who have not been previously treated with anthracyclines and/or taxanes. In fact, it avoids major haematologic toxicity and has a good antitumour effect.
The two-neutron halo nucleus (14)Be has been investigated in a kinematically complete measurement of the fragments ((12)Be and neutrons) produced in dissociation at 35 MeV/nucleon on C and Pb targets. Two-neutron removal cross sections, neutron angular distributions, and invariant mass spectra were measured, and the contributions from electromagnetic dissociation (EMD) were deduced. Comparison with three-body model calculations suggests that the halo wave function contains a large nu(2s(1/2))(2) admixture. The EMD invariant mass spectrum exhibited enhanced strength near threshold consistent with a nonresonant soft-dipole excitation.
OBJECTIVE: The aim of the study was to analyze the benefit/toxicity profile of a second-line treatment with carboplatin alone or carboplatin plus another non-cross-resistant drug (epidoxorubicin) in ovarian cancer patients sensitive to cisplatin-based chemotherapy at first-line treatment. METHODS: We conducted a randomized clinical trial. Women with epithelial ovarian cancer FIGO Stage II--IV who had a complete or partial response to first-line treatment with cisplatin or carboplatin-based regiments and subsequently progressed or relapsed more than 6 months after discontinuation of first-line treatment were eligible for the study. A total of 190 subjects entered the study. They were randomly allocated to either 300 mg/m(2) of carboplatin every 28 days for five cycles (95 patients) or 120 mg/m(2) of epidoxorubicin and 300 mg/m(2) of carboplatin every 28 days for five cycles (95 patients). RESULTS: A complete response was reported, respectively, in 32 (36%) women allocated to carboplatin alone and in 28 (31.8%) of those allocated to carboplatin plus epidoxorubicin. The corresponding figures for partial response were 18 (20.2%) and 26 (29.9%). Comparing the frequency of complete response, partial response, no change, and progression, the differences between the two groups were not significant (chi(2)(3) 5.10, P = 0.16). The median duration of response was 16 months in the carboplatin alone and 20 months in the carboplatin plus epidoxorubicin group (P = not significant). The 3-year percentage of survival was 29% in the carboplatin alone and 42% in the carboplatin plus epidoxorubicin group; this difference was not statistically significant. The frequency of leukopenia, anemia, and thrombocytopenia grade 3-4 was higher in the epidoxorubicin plus carboplatin than in the carboplatin alone group. Alopecia G3 was present in 88% of women treated with epidoxorubicin plus carboplatin. CONCLUSIONS: The general results of this study do not show any marked differences in response to second-line treatment among women treated with single-agent (carboplatin) or multiagent (carboplatin plus epidoxorubicin) schedules. Toxicity, particularly hematological, was more relevant in women treated with the multiagent schedule.
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Members of a family of intracellular molecular switches, the small GTPases, sense modifications of the extracellular environment and transduce them into a variety of homeostatic signals. Among small GTPases, Ras and the Rho family of proteins hierarchically and/or coordinately regulate signaling pathways leading to phenotypes as important as proliferation, differentiation and apoptosis. Ras and Rho-GTPases are organized in a complex network of functional interactions, whose molecular mechanisms are being elucidated. Starting from the simple concept of linear cascades of events (GTPase-->activator--> GTPase), the work of several laboratories is uncovering an increasingly complex scenario in which upstream regulators of GTPases also function as downstream effectors and influence the precise biological outcome. Furthermore, small GTPases assemble into macromolecular machineries that include upstream activators, downstream effectors, regulators and perhaps even final biochemical targets. We are starting to understand how these macromolecular complexes work and how they are regulated and targeted to their proper subcellular localization. Ultimately, the acquisition of a cogent picture of the various levels of integration and regulation in small GTPase-mediated signaling should define the physiology of early signal transduction events and the pathological implication of its subversion.
In this study, the activity of the delta-opioid receptor subtype-selective agonist, SB 227122, was investigated in a guinea pig model of citric acid-induced cough. Parenteral administration of selective agonists of the delta-opioid receptor (SB 227122), mu-opioid receptor (codeine and hydrocodone), and kappa-opioid receptor (BRL 52974) produced dose-related inhibition of citric acid-induced cough with ED(50) values of 7.3, 5.2, 5.1, and 5.3 mg/kg, respectively. The nonselective opioid receptor antagonist, naloxone (3 mg/kg, i.m.), attenuated the antitussive effects of codeine or SB 227122, indicating that the antitussive activity of both compounds is opioid receptor-mediated. The delta-receptor antagonist, SB 244525 (10 mg/kg, i.p.), inhibited the antitussive effect of SB 227122 (20 mg/kg, i.p.). In contrast, combined pretreatment with beta-funaltrexamine (mu-receptor antagonist; 20 mg/kg, s.c.) and norbinaltorphimine (kappa-receptor antagonist; 20 mg/kg, s.c.), at doses that inhibited the antitussive activity of mu- and kappa-receptor agonists, respectively, was without effect on the antitussive response of SB 227122 (20 mg/kg, i.p.). The sigma-receptor antagonist rimcazole (3 mg/kg, i.p.) inhibited the antitussive effect of dextromethorphan (30 mg/kg, i.p.), a sigma-receptor agonist, but not that of SB 227122. These studies provide compelling evidence that the antitussive effects of SB 227122 in this guinea pig cough model are mediated by agonist activity at the delta-opioid receptor.
The small guanine nucleotide (GTP)-binding protein Rac regulates mitogen-induced cytoskeletal changes and c-Jun amino-terminal kinase (JNK), and its activity is required for Ras-mediated cell transformation. Epistatic analysis placed Rac as a key downstream target in Ras signalling; however, the biochemical mechanism regulating the cross-talk among these small GTP-binding proteins remains to be elucidated. Eps8 (relative molecular mass 97,000) is a substrate of receptors with tyrosine kinase activity which binds, through its SH3 domain, to a protein designated E3b1/Abi-1. Here we show that Eps8 and E3b1/Abi-1 participate in the transduction of signals from Ras to Rac, by regulating Rac-specific guanine nucleotide exchange factor (GEF) activities. We also show that Eps8, E3b1 and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro. We propose a model in which Eps8 mediates the transfer of signals between Ras and Rac, by forming a complex with E3b1 and Sos-1.
We investigated whether recombinant human (rHu-IFN-beta) (IFN-beta) could counteract the inhibition of natural killer (NK) activity caused by antitumor agents. Peripheral blood lymphocytes (PBL) were incubated with different antitumor agents alone or in combination with IFN-beta for 3 days and then tested in a cytotoxicity assay against the K562 cell line. The following drugs were used, all of which caused a dose-dependent inhibition of NK activity: etoposide, camptothecin, doxorubicin, cis-DDP, tallimustine, and L-PAM. Concomitant treatment with (1000 U/ml) IFN-beta counteracted the inhibitory effect of etoposide and camptothecin but had no consistent effect on the inhibition mediated by the other drugs. Mean values of inhibition of NK activity at 1 microM camptothecin was 48%+/-3.4% and with IFN-beta was 10%+/-4.9%. With 100 microM etoposide, mean value of inhibition was 78%+/-3.3%, and with IFN-beta, it was 18%+/-1.5%. Cell viability, assessed by vital dye exclusion, and drug uptake, assessed with radiolabeled etoposide, were similar in cells treated with or without IFN-beta. The protective effect of IFN-beta on NK function was rather selective, as IFN-beta did not counteract the drug-mediated inhibition of PBL proliferation when stimulated by phytohemagglutinin (PHA). Other cytokines, IFN-alpha, IFN-gamma, and interleukin-2 (IL-2), had similar protective effect, although IFN-beta, was slightly more potent. On the other hand, IL-6, a cytokine sharing some properties with IFNs was ineffective. Camptothecin inhibited the expression of mRNA for granzyme B, a lytic protein involved in lymphoid-mediated cytotoxicity. Combined treatment with IFN-beta restored-at least in part-the transcription of granzyme B mRNA. These results show that the immunosuppressive effect of some antitumor agents could be partly counteracted by treatment with IFN-beta.
NK cells migrate in response to C-C chemokines, including monocyte chemotactic protein-1 (MCP-1) and MCP-3. Increased migration was observed in IL-2-activated NK cells. It was therefore of interest to define the expression in resting and activated NK cells of the MCP-1 receptor (CCR2) for which two cDNAs (A and B) have been described. Specific oligonucleotides and reverse-transcriptase PCR revealed the presence in activated NK cells and mononuclear phagocytes of the fragments expected on the basis of the reported cDNAs. In addition, amplification with a common A/B- and an A-specific oligonucleotide yielded an unexpected, abundant, 1649-bp fragment. Sequence analysis as well as Northern blotting and RNase protection with different probes revealed that the CCR2 gene is expressed in activated NK cells and mononuclear phagocytes as a predominant long transcript (3.4 kb) consisting of CCR2B, followed by a novel sequence (X), corresponding to an intron in the genome, and by a CCR2A-specific portion. The predominant long transcript is polyadenylated and present in the cytoplasm. The augmented migratory capacity of IL-2 activated vs resting NK cells was associated with increased CCR2 transcript levels.
Interleukin-15 (IL-15) is a recently described cytokine with IL-2-like stimulating activities on T lymphocytes and natural killer (NK) cells. IL-15 mediates its function through the beta- and gamma-chains of the IL-2 receptor. In this work, we have investigated the effect of IL-15 on the directional migration of NK cells in chemotaxis assays and on the ability of NK cells to bind to vascular endothelium. IL-15 (10-20 ng/mL) had chemotactic effects on freshly isolated resting NK cells as well as on long-termed IL-2-cultured NK cells. A checkerboard experiment demonstrated that migration in response to IL-15 was observed only in the presence of a positive gradient (chemotaxis). Overnight treatment of freshly isolated NK cells with IL-15 (10-20 ng/mL) augmented their binding to cultured endothelial cells (EC) in vitro, especially to resting EC. IL-15-activated NK cells bound to resting and tumor necrosis factor-activated EC by use of LFA-1/ICAM-1 and VLA-4/VCAM-1 adhesion pathways, essentially as untreated NK cells do. The fact that IL-15 increased NK cell binding to ICAM-1-transfected NIH-3T3 fibroblasts, together with the finding that IL-15 did not increase binding to extracellular matrix proteins, where the major molecules involved are VLA proteins, indicated that IL-15 primarily stimulates LFA-1-dependent adhesion. By increasing NK cell adhesion to vascular endothelium and migratory response, IL-15 is an important determinant of NK cell recruitment in tissues.
The efficacy and tolerability of granisetron in the management of acute and delayed emesis was compared with that of a multiple antiemetic drug combination regimen, including metoclopramide, dexamethasone, lorazepam and orphenadrine. The trial was a randomized, cross-over study involving 111 patients with gynecological cancers undergoing chemotherapy with cisplatin. Granisetron was significantly more effective than the combination regimen during the first 24 h after chemotherapy; complete response, rates were 67 and 48%, respectively (p = 0.002). There was a significant reduction in the effectiveness of the combination during the second treatment cycle, compared with the first. In contrast, the efficacy of granisetron did not differ between the two cycles. The response rate during the 6 days after chemotherapy was 40.8% in both groups. At the end of the study, 55% of patients preferred granisetron and 23% preferred the combination (p < 0.001). Granisetron was well tolerated. The principal adverse event was headache, which was reported in 7% of patients. The results of this study confirm that granisetron is effective in the treatment of cisplatin-induced nausea and vomiting during the 24 h after chemotherapy.
To assess the efficacy and the compliance of weekly cisplatin as neoadjuvant chemotherapy in locally advanced cervical cancer, 23 patients, FIGO stage IB-IIA > 4 cm-IIB, were recruited. Treatment consisted of four weekly courses of cisplatin (1.5 mg/kg): 91% of the patients received the planned therapy without dose reduction or delay. Toxicity was mild: delayed emesis was the most common side effect. Overall clinical response rate was 85% with a complete response in 28%. Nineteen patients underwent surgery without any undue increase in morbidity. Histologic analysis of surgical specimens revealed a complete response in 25% of stage IB-IIA patients; in only one case of stage IIB, was residual parametrial involvement present. In conclusion, weekly cisplatin is highly effective, with lower cost and less toxicity compared to other neoadjuvant chemotherapy regimens.
BACKGROUND: The purpose of the study was to analyze the determinants of long term survival in women with advanced ovarian cancer and negative second-look laparotomy. METHODS: A series of 140 advanced (Stage III-IV) ovarian cancer patients (median age, 54 years; range, 22-74 years) with negative second-look laparotomy after primary surgery and chemotherapy is included in the analysis. At first diagnosis, all patients were treated with radical or debulking surgery. After primary surgery, the patients were treated with a chemotherapy regimen based on cisplatin or carboplatin alone or in combination with other drugs. All second-look laparotomies were performed 6-8 months after first surgery. RESULTS: The overall survival rates were 76% at 3 years, 66% at 5 years, and 51% at 8 years. The corresponding rates for disease free survival were 57, 50, and 43%, respectively. Survival rates were better for women with a residual tumor 1 cm or less after primary surgery. The 5-year probability of survival was 78% in this group, compared with 55% in women with a residual tumor more than 1 cm (log rank test, P < 0.05). Survival rates for women with tumor Grade 3 tended to be worse than Grades 1-2, but the difference was only of borderline statistical significance. No relationship emerged between survival and age, histotype, and presence of ascites at diagnosis. Women with a residual tumor 1 cm or less and positive lymph nodes had a 66% 5-year probability of survival, compared with 85% for women with a residual tumor 1 cm or less and negative lymph nodes. This difference was significant (log rank test, P = 0.05). The 5-year survival probabilities were 47 and 58%, respectively, in women with a residual tumor more than 1 cm and positive or negative lymph nodes. CONCLUSIONS: This analysis shows a favorable long term survival rate for women with advanced ovarian cancer and complete pathologic response after debulking surgery and postoperative chemotherapy. It further suggests that lymph nodal status is a prognostic factor for women with minimal residual tumor after surgery.