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Biomedical subjects

A Lombardi

Publications and source records attributed to A Lombardi.

At least 91 records · Page 5Linked to original sources
PubMed1999

De novo design and structural characterization of proteins and metalloproteins.

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.

Metalloproteins↗
PubMed1998

From synthetic coiled coils to functional proteins: automated design of a receptor for the calmodulin-binding domain of calcineurin.

A series of synthetic receptors capable of binding to the calmodulin-binding domain of calcineurin (CN393-414) was designed, synthesized and characterized. The design was accomplished by docking CN393-414 against a two-helix receptor, using an idealized three-stranded coiled coil as a starting geometry. The sequence of the receptor was chosen using a side-chain re-packing program, which employed a genetic algorithm to select potential binders from a total of 7.5x10(6) possible sequences. A total of 25 receptors were prepared, representing 13 sequences predicted by the algorithm as well as 12 related sequences that were not predicted. The receptors were characterized by CD spectroscopy, analytical ultracentrifugation, and binding assays. The receptors predicted by the algorithm bound CN393-414 with apparent dissociation constants ranging from 0.2 microM to >50 microM. Many of the receptors that were not predicted by the algorithm also bound to CN393-414. Methods to circumvent this problem and to improve the automated design of functional proteins are discussed.

Algorithms↗
PubMed1998

Neuronorm is a potent and water soluble neurokinin A receptor antagonist.

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.

Animals↗
PubMed1998

A novel super-potent neurokinin A receptor antagonist containing dehydroalanine.

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.

Alanine↗
PubMed1998

Effect of 3,5-di-iodo-L-thyronine on the mitochondrial energy-transduction apparatus.

We examined the effect of a single injection of 3,5-di-iodo-L-thyronine (3,5-T2) (150 microg/100 g body weight) on the rat liver mitochondrial energy-transduction apparatus. We applied 'top-down' elasticity analysis, which allows identification of the site of action of an effector within a metabolic pathway. This kinetic approach considers oxidative phosphorylation as two blocks of reactions: those generating the mitochondrial inner-membrane potential (DeltaPsi; 'substrate oxidation') and those 'consuming' it ('proton leak' and 'phosphorylating system'). The results show that 1 h after the injection of 3,5-T2, state 4 (respiratory state in which there is no ATP synthesis and the exogenous ADP added has been exhausted) and state 3 (respiratory state in which ATP synthesis is at maximal rate) of mitochondrial respiration were significantly increased (by approx. 30%). 'Top-down' elasticity analysis revealed that these increases were due to the stimulation of reactions involved in substrate oxidation; neither 'proton leak' nor the 'phosphorylating system' was influenced by 3,5-T2. Using the same approach we divided the respiratory chain into two blocks of reactions: cytochrome c reducers and cytochrome c oxidizers. We found that both cytochrome c reducers and cytochrome c oxidizers are targets for 3,5-T2. The rapidity with which 3,5-T2 acts in stimulating the mitochondrial respiration rate suggests to us that di-iodo-L-thyronine may play an important role in the physiological conditions in which rapid energy utilization is required, such as cold exposure or overfeeding.

Animals↗
PubMed1998

Miniaturized hemoproteins.

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.

Amino Acid Sequence↗
PubMed1998

Conformational behaviour of C(alpha,alpha)-diphenylglycine: folded vs. extended structures in DphiG-containing tripeptides.

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.

Glycine↗
PubMed1998

Hirunorms are true hirudin mimetics. The crystal structure of human alpha-thrombin-hirunorm V complex.

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.

Antithrombins↗
PubMed1998

3,5-Diiodo-L-thyronine and 3,5,3'-triiodo-L-thyronine both improve the cold tolerance of hypothyroid rats, but possibly via different mechanisms.

The effects of 3,5-diiodo-L-thyronine (3,5-T2, 2.5-10 microg/100 g BW) on cold tolerance, energy expenditure and oxidative capacity of four metabolically very active tissues (brown adipose tissue, skeletal muscle, liver and heart) were determined in hypothyroid, cold-exposed rats. Hypothyroid rats survived cold for only 3-4 days. 3,5-T2 improved survival dose dependently; with 10 microg/100 g BW the rats survived 3 weeks (limit of observation). This effect was paralleled by an increased energy expenditure of the whole animal for the entire 3 weeks. Similar effects were observed in hypothyroid rats treated with 3,3',5-triiodo-L-thyronine (T3). 3,5-T2 stimulated the specific oxidative capacity (expressed as cytochrome oxidase activity per milligram protein) of all four tissues dose dependently. When the oxidative capacity was expressed as total activity (cytochrome oxidase activity times organ weight), the percentage increases were of the same order. T3 exerted similar effects, but the changes in total activity were much greater than in specific activity, indicating an effect on the tissue trophism. The effect of 3,5-T2 on cold tolerance thus mimics the effect of T3, but via different cellular mechanisms. T3 seems to act primarily on the trophism of the tissues, while 3,5-T2 may act directly on mitochondria without an effect on tissue trophism.

Adaptation, Physiological↗
PubMed1998

Effect of 3,5-diiodo-L-thyronine on thyroid stimulating hormone and growth hormone serum levels in hypothyroid rats.

We have investigated the biological effects of physiological doses of 3,5-diiodo-L-thyronine (3,5-T2) and 3,3'-diiodo-L-thyronine (3,3'-T2) (at doses from 2.5 to 10 microg/100 g BW) on serum TSH and GH levels in rats made hypothyroid by propylthiouracil and iopanoic acid administration. In such animals deiodinase activities were inhibited and thyroid hormones serum levels strongly reduced. The effects of T2s were compared with those elicited by 3,5,3'-triiodo-L-thyronine (T3) (2.5 microg/100 g BW).The serum TSH level was much greater in hypothyroid rats than in euthyroid ones. T3 administration suppressed TSH by 88% compared to control (i.e, the level in hypothyroid rats); it thus reached a value not significantly different from that seen in the euthyroid rats. 3,5-T2 produced a similar effect, suppressing the TSH level by about 75% compared to control; it thus reached values not significantly different from those of the euthyroid and T3-treated rats. By contrast, 3,3'-T2 had no effect on TSH, whatever the dose. The serum GH level was much lower in hypothyroid rats than in euthyroid ones. T3 administration increased the GH level by about 5-fold, restoring it to the value seen in euthyroid rats. 3,5-T2-treated hypothyroid rats, at all the doses used (from 2.5 to 10 microg/100 g BW), showed increased serum GH levels: at a dose of 10 microg/100 g BW the level reached a value about 5-fold higher than that in hypothyroid rats. This value was not significantly different from those of euthyroid and T3-treated rats. 3,3'-T2 did not affect GH levels whatever the dose. Thus, 3,5-T2 (but not 3,3'-T2) seems to mimic the effects of T3 on serum TSH and GH levels in rats.

Animals↗
PubMed1998

Molecular identification and cluster analysis of homofermentative thermophilic lactobacilli isolated from dairy products.

Twenty-five strains of thermophilic lactobacilli isolated from yoghurt and from semi-hard and hard cheeses (in parallel with nine type or reference strains) were identified and grouped according to their genetic relatedness. Strains were identified by sugar fermentation patterns using the "API 50 CHL" galleries, by species-specific DNA probes in dot-blot hybridization experiments, by amplification and restriction analysis of the 16S rRNA gene (ARDRA) and by polymerase chain reaction (PCR) using species-specific oligonucleotide primers. Strains were classified as Lactobacillus delbrueckii subsp. lactis and subsp. bulgaricus, L. helveticus, and L. acidophilus. Strains which were atypical by sugar fermentation patterns were also identified. Most of the strains could not be grouped using carbohydrate fermentation profiles. PCR fingerprinting was used to identify DNA profiles for the 25 lactobacilli. Experimentally obtained PCR profiles enabled discrimination of all strains, which were grouped according to the similarities in their combined patterns. In general, the clustering of the strains corresponded well with species delineation obtained by molecular identification. The dendrogram of genetic relatedness enabled the unambiguous identification of most of the strains which were shown to be atypical by the sugar fermentation profile, except for a discrepancy in one L. delbrueckii subsp. lactis strain and one atypical Lactobacillus sp. strain.

Bacterial Typing Techniques↗
PubMed1998

Modulating effect of lonidamine on response to doxorubicin in metastatic breast cancer patients: results from a multicenter prospective randomized trial.

Previous results from our preclinical studies have shown that lonidamine (LND) can positively modulate the antiproliferative activity of doxorubicin (DOX) on breast cancer cell lines. To evaluate the effect of LND in a clinical setting, a multicenter randomized trial was carried out on patients with advanced breast cancer. From September 1991 to July 1993, 181 patients were enrolled in the trial and received an initial treatment of DOX at 75 mg/m2 for 3 cycles. The 137 patients who reached complete remission, partial remission, or stable disease were randomized to receive either DOX alone (75 mg/m2 day 1) (arm A) or DOX plus LND (600 mg orally/day) (arm B). The patients enrolled in the two arms were fairly homogeneous in terms of major clinical characteristics. Toxicity was similar in both arms except for myalgia: WHO grade > or=2 was observed in 57% of arm B patients. Overall response rate to DOX + LND was 50% and to DOX alone 38% in evaluable patients, and 48% vs 37% in all registered patients, as determined by an intention-to-treat analysis. The differences did not reach statistical significance. Conversely, in agreement with previous findings, we observed a significant difference in response rate in the subgroup of patients with liver metastases, regardless of the extent of hepatic involvement (DOX + LND 68% vs DOX 33%, p=0.03). This observation makes LND an important tool in association with anthracyclines in the treatment of this subgroup of patients.

Adult↗
PubMed1998

Pelvic ultrasonography in normal girls and in girls with pubertal precocity.

UNLABELLED: This prospective study sought to evaluate the role of pelvic ultrasonography in differentiating between various types of pubertal precocity. A control group of 117 normal girls (aged 1.1-15.6) was studied and compared with 87 girls with premature sexual maturation (aged 1.1-9.2 y). Of these patients 19 had central precocious puberty (CPP), 48 had isolated premature thelarche (IPT) and 20 had premature adrenarche (IPA). Pelvic ultrasound variables evaluated were: (i) uterus: longitudinal diameter (uterine length), cross-sectional area (CSA) and fundo-cervical ratio; and (ii) ovaries: volume and morphology. Ovarian morphology was subdivided in 6 different appearances: solid, microcystic, paucicystic, multicystic, macrocystic, and major isolated cyst. In normal control girls, uterine length and CSA increased with age, although no cut-off values could be defined between different age ranges, and they were correlated with breast stage; fundo-cervical ratio was stable through childhood and increased after age 9. Ovarian volume was significantly greater in pubertal girls with breast stage 2 than in those with only pubic and/or axillary hair. There was a clear predominance of solid ovarian appearances in the age range 2-7, with the multicystic appearance being seen only after age 7, a minority being macrocystic. After age 10 all the different patterns were observed, and after age 13 the frequency of a macrocystic pattern increased. Significantly more mature ovarian appearances were observed in subjects with breast development compared with those without, independently of the presence of pubic hair. Patients with IPT had no significant differences in pelvic ultrasound measurements when compared with age-matched controls. All the different morphological ovarian appearances were observed in IPT, in contrast to age-matched controls, where only the less mature patterns (solid, micro- and paucicystic) were seen. Patients with CPP had significantly more mature patterns of ovarian morphology compared with age-matched controls, but did not differ from pubertal pre-menarcheal controls. Those patients with IPA differed from age-matched controls only in having significantly greater uterine length and CSA. Comparison of the pelvic ultrasound parameters between patient groups (IPT, CPP, IPA) and age-matched controls revealed significantly higher values in CPP for uterine length, uterine CSA and ovarian volume. Ovarian volume was also greater in IPT than in IPA. Ovarian morphology was significantly different in patients (IPT, CPP, IPA) compared with age-matched controls, but none of the ovarian morphological appearances was exclusive to a single condition. IN CONCLUSION: (i) pelvic ultrasound parameters increase progressively from birth to maturity, but no clear cut-off values can be established between age ranges; (ii) pelvic ultrasound variables reach adult values during puberty, with differences in the timing that may reflect geographical variations; (iii) the multicystic ovarian appearance occurs just before the onset of puberty; (iv) pelvic ultrasonography cannot always differentiate clearly between different disturbances of puberty and therefore cannot supersede other observations and investigations in the evaluation of pubertal disorders; and (v) in this study we propose a more detailed pelvic ultrasound terminology that can avoid apparent confusion in defining ovarian ultrasound appearance.

Analysis of Variance↗
PubMed1998

Delayed acquisition of high-avidity anti-cytomegalovirus antibody is correlated with prolonged antigenemia in solid organ transplant recipients.

Previous studies have demonstrated that maturation of cytomegalovirus (CMV)-specific antibodies in solid organ transplant recipients is delayed after primary CMV infection. To investigate the clinical significance of this finding, the avidity indices of anti-CMV antibody were determined in parallel with other serologic and virologic parameters in serial serum samples from 24 solid organ transplant recipients who had primary CMV infection that began during the first 3 months after transplantation. The data obtained show that a delay in antibody maturation is significantly correlated with a long persistence of positive antigenemia.

Adult↗
PubMed1998

Effects of alendronate on gastric and duodenal mucosa.

OBJECTIVES: This single-center, double-blind, randomized study assessed the effect of alendronate 5 and 10 mg on the gastroduodenal mucosa. METHODS: Overall, 95 postmenopausal women without a recent history of major upper gastrointestinal (GI) disease and not taking gastric-irritant drugs, were screened with an upper GI endoscopy. Fourteen women (15% of the total) were found to have baseline endoscopic gastric and/or duodenal abnormalities, including mucosal hemorrhages (n = 4), erosions (n = 11), and ulcers (n = 3). Two additional women had baseline esophageal abnormalities. Thus, 79 postmenopausal women (mean age 51 yr, range 41-64 yr), free of esophageal, gastric and/or duodenal erosions or ulcer, were enrolled. Subjects received placebo, alendronate 5 mg/day or 10 mg/day, or aspirin 650 mg q.i.d. for 14 days. Endoscopy was repeated on Day 8 and on Day 15. Gastric and duodenal mucosae were graded separately using a 5-point scale for erosive mucosal injury. RESULTS: The proportions of subjects with a gastric or duodenal erosion score > or = 2 (presence of at least one mucosal erosion) on either Day 8 or 15 were four of 22 (18.2%) in the placebo group; four of 22 (18.2%) in the alendronate 5 mg group; five of 21 (23.8%) in the alendronate 10 mg group; and 14 of 14 (100.0%) in the aspirin group. Thirty-five of 76 (46%) subjects were H. pylori-positive (Pyloritek test), and were equally distributed across treatment groups. CONCLUSIONS: Alendronate 5 and 10 mg/day for 2 wk was associated with a lower incidence of gastric erosions than aspirin. The incidence of gastric erosions in the alendronate groups did not differ significantly from the placebo group. In this study, unlike aspirin, alendronate did not induce gastric erosions.

Adult↗