Biomedical subjects
Publications and source records attributed to V Pavone.
We report on a patient with thalassaemia which was refractory to blood transfusions. The clinical picture was striking, and highlights the potential severity of intrathecal bone reactions after chronic intractable haemolytic anaemia.
Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme N-acetylgalactosamine-4-sulfatase (arylsulfatase B, ASB). Mutation analysis in Maroteaux-Lamy syndrome resulted in the identification of approximately 40 molecular defects underlying a great genetic heterogeneity. Here we report five novel mutations in Italian subjects: S65F, P116H, R315Q, Q503X, P531R; each defect was confirmed by restriction enzyme or amplification refractory mutation system (ARMS) analysis. We also performed a three-dimensional (3-D) structure analysis of the alterations identified by us, and of an additional 22 point mutations reported by other groups, in an attempt to draw helpful information about their possible effects on protein conformation.
A large number of potent and selective therapeutic agents, useful for the treatment of several diseases, have been isolated from natural sources. For example, the most active thrombin inhibitors are those secreted by the salivary glands of leeches. One peculiar feature of these agents is the lack of any significant inhibitory cross-reaction with other serine proteinases. Hence, the knowledge of the exact mechanism of action of these molecules provides the basis for the development of new and efficient synthetic drugs. For this reason, many studies have been undertaken on the structure-activity relationships of natural thrombin inhibitors, and a large amount of detailed information has been obtained by the crystal structures of these inhibitors when complexed with thrombin. In this paper, we review natural and synthetic multisite thrombin inhibitors, whose structural aspects have been determined in detail. We also report here the approach used by us to develop a new class of synthetic, multisite directed thrombin inhibitors, named hirunorms, designed to mimic the distinctive binding mode of hirudin.
OBJECTIVE: To investigate the involvement of opioid tone, obesity, and hyperinsulinemia in GH secretion in women with polycystic ovary syndrome (PCOS). DESIGN: Controlled clinical study. SETTING: Catholic University of Sacred Heart School of Medicine in Rome, Italy. PATIENT(S): Twenty-two patients with PCOS and 14 healthy, normally ovulating volunteers, matched for age and body mass index. INTERVENTION(S): Patients underwent a GH-releasing hormone (GHRH) test and an oral glucose tolerance test before and after 4-5 weeks of treatment with 50 mg/d of naltrexone. MAIN OUTCOME MEASURE(S): Serum concentrations of GH, insulin, glucose, steroids, and gonadotropins, as well as the GH area under the curve (AUC-GH) and the insulin area under the curve (AUC-I), were measured before and after naltrexone treatment. RESULT(S): In patients with PCOS, the administration of naltrexone increased the GH response to the GHRH test without interfering with the insulin response to the oral glucose tolerance test. However, the GH response to the GHRH test was improved significantly only in lean patients with PCOS, whereas obese patients with PCOS did not show any improvement in GH secretion. In obese control subjects, the treatment reduced plasma basal insulin concentrations and increased the AUC-GH, whereas in lean control subjects, the treatment reduced the GHRH-induced response. In normoinsulinemic patients with PCOS, the GH response to the GHRH test increased significantly after treatment, whereas the AUC-I was not affected. In hyperinsulinemic patients with PCOS, treatment with naltrexone significantly reduced the AUC-I, whereas the AUC-GH increased only in lean hyperinsulinemic patients with PCOS. CONCLUSION(S): Naltrexone treatment improves GHRH-induced GH secretion in patients with PCOS. However, this GH response is heterogeneously represented in relation to both obesity and hyperinsulinism.
OBJECTIVE: To evaluate the influence of body mass on the hypothalamic-pituitary-adrenal (HPA)-axis response to naloxone in polycystic ovary syndrome (PCOS). DESIGN: Controlled clinical study. SETTING: Academic research environment. PATIENT(S): Ten lean and 10 obese women with PCOS compared with 7 lean and 8 obese control subjects matched for body mass index. INTERVENTION(S): Each patient received an IV bolus of naloxone at a dosage of 125 microg/kg. MAIN OUTCOME MEASURE(S): Samples were collected 30 minutes before and 0, 15, 30, 60, 90, and 120 minutes after injection: ACTH and cortisol levels were measured in all plasma samples. RESULT(S): No significant differences were found in the ACTH and cortisol responses to opioid blockade between lean women with PCOS and lean as well as obese control subjects; conversely, obese patients with PCOS showed a higher ACTH and cortisol responses to naloxone compared with all other groups. CONCLUSION(S): Hypothalamic-pituitary-adrenal-axis abnormalities of PCOS may be central in origin and abdominal obesity seems to play a key role in the HPA-axis hyperactivity of women with PCOS when naloxone is administered.
To assess the differential impact of the insulin secretory pattern and obesity on the endocrinometabolic features of the polycystic ovary syndrome (PCOS), we studied 110 PCOS women. Patients underwent a gonadotropin-releasing hormone (GnRH) test, an oral glucose tolerance test (OGTT), and basal evaluation of hormonal and biochemical parameters. Basal androgens and lipids, basal and stimulated gonadotropins, insulin, and glucose levels were measured. Patients were classified into four groups according to the body mass index (BMI) and insulin secretion: normoinsulinemic-lean ([NL] n = 24), normoinsulinemic obese ([NO] n = 24), hyperinsulinemic lean ([HL] n = 17), hyperinsulinemic obese ([HO] n = 45). HL patients showed a higher luteinizing hormone (LH) area under curve (AUC) after GnRH stimulus compared with NL patients (HL v NL, 4,285 +/- 348 v 3,377 +/- 314 IU/L x 120 min, P < .05), whereas we failed to find a statistically significant difference in a similar comparison among obese subjects (HO v NO, 3,606 +/- 302 v 3,129 +/- 602 IU/L x 120 min). A trend toward increased plasma testosterone and decreased sex hormone-binding globulin (SHBG) was found in relation to hyperinsulinemia and obesity, thus resulting in a higher free androgen index (FAI) in groups HL and NO versus NL (HL, 5.54 +/- 0.51; NO, 5.64 +/- 0.49; NL, 4.13 +/- 0.33; P < .05 and P < .01, respectively). The presence of both exaggerated insulin secretion and obesity resulted in a synergistic additive effect on the FAI in the HO group (6.81 +/- 0.34). Concerning the lipoprotein lipid profile, the NL group showed lower plasma triglyceride levels compared with the other three groups, whereas no significant differences were found for nonesterified fatty acid (NEFA) concentrations. Higher low-density lipoprotein cholesterol (LDL-C) and very-low-density lipoprotein cholesterol (VLDL-C) and lower high-density lipoprotein cholesterol (HDL-C) levels were found in the obese groups compared with the lean counterparts, whereas the same parameters did not significantly differ in a comparison between normoinsulinemic and hyperinsulinemic groups. In conclusion, our data suggest an important role of hyperinsulinemia in the LH response to a GnRH stimulus and an independent and synergistic additive effect of obesity and hyperinsulinemia on the FAI in PCOS.
We report on a girl with familial neurofibromatosis type 1 (Nf1) who at age 11 had multiple osteolytic lesions of the right mandible and the distal femoral and proximal tibial metaphyses bilaterally. No other skeletal component was affected, and no abnormalities were present on skeletal radiographs of her parents. Histologic examination of the mandibular and right femoral lesions revealed a "multinucleated giant cell process." Results of repeated routine laboratory and bone metabolic studies were within normal limits. The lack of reliable histologic criteria and the diagnostic problems in distinguishing among central giant cell granulomata, giant cell tumors of the jaw, cherubism, brown tumors resulting from (occult) hyperparathyroidism, Jaffe-Campanacci syndrome, McCune-Albright syndrome, Noonan-like/multiple giant cell lesion syndrome, and multiple nonossifying fibromas of bone are discussed.
The genital tract as a primary site of malignant lymphoma in women is extremely rare. This report concerns a 64 year old patient with a primary vaginal non-Hodgkin lymphoma (large cell B lineage according to the REAL classification--centroblastic type according to the Kiel classification--"G" according Working Formulation) with an unusual clinical presentation--pelvic discomfort accompanied by frequent ureteral-like colic. Due to gynecological onset symptoms and the rarity of this extranodal primary site misinterpretation of a primary vaginal lymphoma as a benign inflammatory disease or endometriosis may occur. We emphasize the importance of their recognition and also the differential diagnosis of cervical lymphoma from other neoplastic and non-neoplastic lesions.
Uncertainties regarding the pathogenetic changes underlying the polycystic ovarian syndrome (PCOS) have been reported. The aim of this study was to investigate the endocrine and metabolic features of PCOS patients in relation to luteinizing hormone (LH) secretion. Androgen assays, oral glucose tolerance tests, hyperinsulinaemic euglycaemic clamps and gonadotrophin releasing hormone (GnRH) tests were performed in 100 patients. Sixty-six patients scheduled as hyperinsulinaemic and 34 as normoinsulinaemic showed similar concentrations of LH, follicle stimulating hormone (FSH), LH/FSH ratio, and LH response to GnRH testing. Hyperinsulinaemic subjects showed higher body mass index (BMI), insulin resistance, testosterone and free androgen index levels compared with those of normoinsulinaemic subjects; when clustered in relation to their LH basal concentrations, the two groups obtained differed only in androstenedione concentrations. Considering both insulin and LH plasma concentrations, four groups were obtained. Hyperinsulinaemia and hyper-LH secretion were not related in 54% and coexisted in the same subjects in 26% of cases. Hyperinsulinaemia as well as hyper-LH secretion affected the expression of the syndrome; the insulinaemia was directly correlated with testosterone concentrations and all metabolic parameters that affected the free androgen index. The LH concentrations were related to androgen production and were independent of BMI and insulin concentrations. It is concluded that the degree of hormonal alteration is the final sum of such pathogenetic factors.
EST2, a thermophilic carboxylesterase from Alicyclobacillus acidocaldarius, belonging to the HSL group of the esterase/lipase superfamily, has been crystallized for the first time. Ammonium sulfate was used as a precipitant and the crystallization proceeded at pH 7.8. The crystals belong to space group P41212 or its enantiomorph P43212, with unit-cell parameters a = b = 78.8, c = 106. 4 A. A complete data set has been collected at the synchrotron source Elettra in Trieste to 2.4 A resolution, using a single frozen crystal.
The X-ray crystal structure of the human alpha-thrombin-hirunorm IV complex has been determined at 2.5 A resolution, and refined to an R-factor of 0.173. The structure reveals an inhibitor binding mode distinctive of a true hirudin mimetic, which justifies the high inhibitory potency and the selectivity of hirunorm IV. This novel inhibitor, composed of 26 amino acids, interacts through the N-terminal end with the alpha-thrombin active site in a nonsubstrate mode, and binds specifically to the fibrinogen recognition exosite through the C-terminal end. The backbone of the N-terminal tripeptide Chg1"-Arg2"-2Na13" (Chg, cyclohexyl-glycine; 2Na1, beta-(2-naphthyl)-alanine) forms a parallel beta-strand to the thrombin main-chain segment Ser214-Gly216. The Chg1" side chain occupies the S2 site, Arg2" penetrates into the S1 specificity site, while the 2Na13" side chain occupies the aryl binding site. The Arg2" side chain enters the S1 specificity pocket from a position quite apart from the canonical P1 site. This notwithstanding, the Arg2" side chain establishes the typical ion pair with the carboxylate group of Asp189.
De novo protein design has recently emerged as an attractive approach for studying the structure and function of proteins. This approach critically tests our understanding of the principles of protein folding; only in de novo design must one truly confront the issue of how to specify a protein's fold and function. If we truly understand proteins, it should be possible to design receptors, enzymes, and ion channels from scratch. Further, as this understanding evolves and is further refined, it should be possible to design proteins and biomimetic polymers with properties unprecedented in nature.
BACKGROUND AND OBJECTIVE: Trisomy 12 is the most common numerical chromosomal aberration in patients with B-cell chronic lymphocytic leukemia (B-CLL). Fluorescence in situ hybridization (FISH) has improved the detection of this cytogenetic abnormality and has made detection possible in all phases of the cell cycle. The presence of the trisomy 12 positive (+12) cell population has generally been investigated in leukemic cells obtained from the peripheral blood of CLL patients. To ascertain whether trisomy 12 is expressed homogeneously in cells of different hemopoietic tissues, we applied FISH to lymph node, peripheral blood and bone marrow samples obtained simultaneously from 23 untreated B-CLL patients. DESIGN AND METHODS: Twenty-three newly diagnosed patients with B-CLL, 15 in stage B and 8 in stage C, were included in the present study. Peripheral blood smears, bone marrow aspirate smears and lymph node touch imprints were collected from each patient at diagnosis. Cytologic preparations were examined by light microscopy in order to assess the lymphocyte morphology. Immunophenotyping was performed by cytofluorimetric analysis of the peripheral blood, bone marrow and lymph node mononuclear cell suspensions. The diagnosis was supported in all cases by histologic findings in bone marrow biopsy and lymph node biopsy specimens. Fluorescence in situ hybridization was performed on smears of blood and aspirated bone-marrow and lymph node touch imprints obtained by fresh tissue apposition. RESULTS: In 6 of the 23 cases (26%) trisomy 12 was clearly present in all tissues examined. A comparative analysis of the three different hemopoietic tissues was performed. A higher percentage of leukemic CD5+CD23+ cells was detected in lymph nodes than in peripheral blood and bone marrow. A significantly higher proportion of trisomic cells was observed in lymph nodes samples than in peripheral blood or bone marrow smears of trisomy 12 positive CLL patients. INTERPRETATION AND CONCLUSIONS: Several previous reports show that only a proportion of malignant B-CLL cells carry trisomy 12 when analyzed by interphase FISH. The higher proportion of +12 cells in lymph nodes than in peripheral blood or bone marrow of CLL patients with trisomy 12 could reflect different cell distributions in different tissues, or lymph node specific tropism, or proliferative advantage in selected tissue. At present, the role of trisomy 12 in the pathogenesis of lymphoproliferative disorders is unclear.
We report here the synthesis and preliminary pharmacological characterization of a novel water soluble Neurokinin A receptor antagonist named Neuronorm. The synthesis was achieved in high yield by a combination of classical peptide synthesis methodologies, in both solution and solid phase. The pharmacological properties as neurokinin A receptor antagonist were assessed in in vitro experiments on rat vas deferens and guinea pig trachea, and were compared to those of MEN10627.
We report here the synthesis and preliminary pharmacological characterization of a novel Neurokinin A receptor antagonist. This molecule contains a dehydroalanine residue. It displays a high conformational rigidity and possesses very high activity. Its pharmacological properties as a neurokinin A receptor antagonist were assessed in in vitro experiments on rat vas deferens and were compared to those of Neuronorm and MEN10627.
The present paper highlights and reviews current research in the field of hemoprotein models. Hemoproteins have been extensively studied in order to understand structure-function relationships, and to design new molecules with desired functions. A wide number of synthetic analogues have been developed, using quite different approaches. They differ in molecular structures, ranging from simple meso-substituted tetraaryl-metalloporphyrins and peptide-porphyrin conjugates. In this paper we summarize the state of the art on peptide based hemoprotein models. We also report here the approach used by us to develop a new class of molecules, named mimochromes. They can be regarded as miniaturized hemoproteins, because mimochromes are low molecular weight compounds with some structural and functional properties common to those of the parent high molecular weight protein. The basic structure of mimochromes is a deuteroporphyrin ring covalently linked to two helical peptide chains. Two molecules of this series have been fully characterized. All the information derived from their structural analysis has been applied to the design of new analogues with additional functions.
The crystal structures of three fully protected tripeptides containing the Dphi g residue (C[alpha,alpha]-diphenylglycine) in the central position are reported, namely Z-Gly-Dphi g-Gly-OMe (a), Z-Gly-Dphi g-Aib-OMe (b) and Z-Aib-Dphi g-Aib-OMe (c). The molecular conformations are quite unusual because the Dphi g residue adopts a folded conformation in the 3(10)-helical region when the following residue adopts a folded conformation of opposite handedness (peptides b and c). In contrast, the Dphi g residue adopts the more frequently observed fully extended conformation when the following residue adopts a semi-extended conformation (peptide a). These findings are in agreement with the theoretical calculations on Ac-Dphi g-Aib-NHCH3 and Ac-Aib-Dphi g-NHCH3 also reported in this work.
A novel class of synthetic, multisite-directed thrombin inhibitors, known as hirunorms, has been described recently. These compounds were designed to mimic the binding mode of hirudin, and they have been proven to be very strong and selective thrombin inhibitors. Here we report the crystal structure of the complex formed by human alpha-thrombin and hirunorm V, a 26-residue polypeptide containing non-natural amino acids, determined at 2.1 A resolution and refined to an R-factor of 0.176. The structure reveals that the inhibitor binding mode is distinctive of a true hirudin mimetic, and it highlights the molecular basis of the high inhibitory potency (Ki is in the picomolar range) and the strong selectivity of hirunorm V. Hirunorm V interacts through the N-terminal tetrapeptide with the thrombin active site in a nonsubstrate mode; at the same time, this inhibitor specifically binds through the C-terminal segment to the fibrinogen recognition exosite. The backbone of the N-terminal tetrapeptide Chg1"-Val2"-2-Nal3"-Thr4" (Chg, cyclohexyl-glycine; 2-Nal, beta-(2-naphthyl)-alanine) forms a short beta-strand parallel to thrombin main-chain residues Ser214-Gly219. The Chg1" side chain fills the S2 subsite, Val2" is located at the entrance of S1, whereas 2-Nal3" side chain occupies the aryl-binding site. Such backbone orientation is very close to that observed for the N-terminal residues of hirudin, and it is similar to that of the synthetic retro-binding peptide BMS-183507, but it is opposite to the proposed binding mode of fibrinogen and of small synthetic substrates. Hirunorm V C-terminal segment binds to the fibrinogen recognition exosite, similarly to what observed for hirudin C-termninal tail and related compounds. The linker polypeptide segment connecting hirunorm V N-and C-terminal regions is not observable in the electron density maps. The crystallographic analysis proves the correctness of the design and it provides a compelling proof on the interaction mechanism for this novel class of high potency multisite-directed synthetic thrombin inhibitors.