Biomedical subjects

E Bianchi

Publications and source records attributed to E Bianchi.

At least 37 records · Page 2Linked to original sources

PubMed1999

Endogenous morphine levels increase in molluscan neural and immune tissues after physical trauma.

The aim of this study was to demonstrate by biochemical and immunocytochemical methods the presence of endogenous morphine in nervous and immune tissues of the freshwater snail, Planorbarius corneus. High performance liquid chromatography (HPLC) coupled to electrochemical detection performed on tissues from control snails, revealed that the CNS contains 6.20+/-2.0 pmol/g of the alkaloid, the foot tissue contains a much lower level, 0.30+/-0.03 pmol/g, whilst morphine is not detected in the hemolymph and hepatopancreas. In specimens that were traumatized, we detected a significant rise of the CNS morphine level 24 h later (43.7+/-5.2 pmol/g) and an initial decrease after 48 h (19.3+/-4.6 pmol/g). At the same times, we found the appearance of the opiate in the hemolymph (0.38+/-0.04 pmol/ml and 0.12+/-0.03 pmol/ml) but not in the hepatopancreas. Using indirect immunocytochemistry, a morphine-like molecule was localized to a number of neurons and a type of glial cell in the CNS, to some immunocytes in the hemolymph and to amoebocytes in the foot, as well as to fibers in the aorta wall. Simultaneously to the rise of morphine biochemical level following trauma, morphine-like immunoreactivity (MIR) increased in both intensity and the number of structures responding positively, i.e., neurons and fiber terminals. In another mollusc, the mussel Mytilus galloprovincialis, the same pattern of enhanced MIR was found after trauma. Taken together, the data suggest the presence of a morphinergic signaling in invertebrate neural and immune processes resembling those of classical messenger systems and an involvement in trauma response.

Animals↗

PubMed1999

Structural characterization of the interactions of optimized product inhibitors with the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein by NMR and modelling studies.

The interactions of peptide inhibitors, obtained by the optimization of N-terminal cleavage products of natural substrates, with the protease of human hepatitis C virus (HCV) are characterized by NMR and modelling studies. The S-binding region of the enzyme and the bound conformation of the ligands are experimentally determined. The NMR data are then used as the experimental basis for modelling studies of the structure of the complex. The S-binding region involves the loop connecting strands E2 and F2, and appears shallow and solvent-exposed. The ligand binds in an extended conformation, forming an antiparallel beta-sheet with strand E2 of the protein, with the P1 carboxylate group in the oxyanion hole.

Amino Acid Sequence↗

PubMed1999

A high-throughput radiometric assay for hepatitis C virus NS3 protease.

A novel radiometric in vitro assay for discovery of inhibitors of hepatitis C viral protease activity, suitable for high-throughput screening, was developed. The NS3 protein of hepatitis C virus (HCV) contains a serine protease, whose function is to process the majority of the nonstructural proteins of the viral polyprotein. The viral NS4A protein is a cofactor of NS3 protease activity in the cleavage of NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions. To establish an in vitro assay system we used NS3 proteases from different HCV strains, purified from Escherichia coli and a synthetic radiolabeled peptide substrate that mimics the NS4A-NS4B junction. Upon incubation with the enzyme the substrate was separated from the radiolabeled cleavage product by addition of an ion exchange resin. The assay was performed in a microtiter plate format and offered the potential for assaying numerous samples using a laboratory robot. Taking advantage of these features, we used the assay to optimize reaction conditions by simultaneously varying different buffer components. We showed that physicochemical conditions affect NS3 protease activity in a strain-specific way. Furthermore, the sensitivity of the assay makes it suitable for detection and detailed mechanistic characterization of inhibitors with low-nanomolar affinities for the HCV serine protease.

Buffers↗

PubMed1999

Regulation of lymphocyte traffic by adhesion molecules.

Lymphocytes are antigen specific cells whose effector function is acquired through complex differentiation pathways. This implies, firstly, antigen encounter and recognition at specific sites, and, subsequently, the transition from a naive to a memory/effector phenotype. Clonotypically expanded cells must then be capable of recirculating to the tissue where their effector function is needed. To this aim, defined receptor-counter receptor pairs are expressed on lymphocytes versus endothelial cells. Extravasation is therefore a key-process in this scenario. Indeed, different lymphocyte subsets display distinct recirculation patterns and capability to migrate into lymphoid and non-lymphoid tissues. As a general rule, naive lymphocytes preferentially migrate into secondary lymphoid organs, where all the requirements for effective antigen presentation and differentiation are available; in contrast, memory/effector lymphocytes preferentially migrate to peripheral tissues, such as skin and mucosa. We review here the molecular events that regulate leukocyte extravasation and the specific migration properties acquired by both naive and memory/effector lymphocytes under physiological and pathological conditions.

Animals↗

PubMed1999

Non specific aorto-arteritis. Clinical picture, diagnosis and therapy.

This study is aimed at analyzing clinical features, angiographic findings and evolution of Takayasu's arteritis and the criteria adopted to establish the indication for non-surgical versus operative treatment. Eighteen patients affected by non specific aortarteritis were observed and treated at our Department between 1973 and 1996. All patients met the American College of Rheumatology 1990 criteria of classification of Takayasu's arteritis. Sixteen patients were young females. Two patients were males. Nine patients underwent surgical procedures. One young female underwent a PTA. All eight asymptomatic patients were only medically treated. One patient died some days after an aorto-bicarotid bypass graft due to acute myocardial infarction. Two other patients died two and four years after intervention for renal and cardiac failure respectively. One patient after an aorto bicarotid bypass underwent a left hemiplegia due to thrombosis of the right graft branch. All the other 14 patients either surgically or medically treated are well and are under strict surveillance through rigorous follow-up. The 10 patients who underwent surgical or interventional radiological treatment were certainly the most seriously affected patients and were symptomatic (presented neurological disturbances or a severe hypertensive state). This fact explains, to some extent, the mortality and morbidity rate observed in this group. The seven medically treated patients were completely asymptomatic in spite of a major involvement of various vascular districts.

Adolescent↗

PubMed1998

Aggregates from mutant and wild-type alpha-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of beta-sheet and amyloid-like filaments.

Alpha-synuclein (alpha-syn) protein and a fragment of it, called NAC, have been found in association with the pathological lesions of a number of neurodegenerative diseases. Recently, mutations in the alpha-syn gene have been reported in families susceptible to an inherited form of Parkinson's disease. We have shown that human wild-type alpha-syn, mutant alpha-syn(Ala30Pro) and mutant alpha-syn(Ala53Thr) proteins can self-aggregate and form amyloid-like filaments. Here we report that aggregates of NAC and alpha-syn proteins induced apoptotic cell death in human neuroblastoma SH-SY5Y cells. These findings indicate that accumulation of alpha-syn and its degradation products may play a major role in the development of the pathogenesis of these neurodegenerative diseases.

Amyloid↗

PubMed1998

Potent peptide inhibitors of human hepatitis C virus NS3 protease are obtained by optimizing the cleavage products.

In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkühler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.

Amino Acid Sequence↗

PubMed1998

Interphysician variability in defining the planning target volume in the irradiation of prostate and seminal vesicles.

With the improvements in treatment accuracy the risk exists of over-reliance on the capability of the physician to estimate the extent of the tumour. We assessed the variability between six radiotherapists in defining the planning target volume (PTV) on CT slices for three prostate tumour cases. Percentage differences between measured volumes and mean values calculated for each case ranged from -53.64 to +60.48% (SD 36.00%). There is a considerable variation in delineating the PTV, both in the cranio-caudal direction and in the in-slice extension of the areas drawn on each slice (standard deviations ranged from 0.35 to 2.64 cm2). We also checked the uncertainty in the shape and position of the contours on each CT image. The analysis was performed on three slices of one test case. As we expected, the uncertainty seems largest for seminal vesicle slices and smallest for prostatic apex slices. These results endorse the need for uncertainty analysis of all departmental processes in order to define a detailed protocol and consequently to minimize the interphysician differences in PTV delineations.

Analysis of Variance↗

PubMed1998

Potassium-induced release of endogenous morphine from rat brain slices.

Endogenous morphine has been clearly demonstrated by gas chromatography/mass spectrometry in the brain, spinal fluid, adrenal glands, and liver of mammals. To clarify the role of endogenous morphine, its release from rat brain slices was studied in vitro in the presence of high potassium concentrations, with and without calcium in the medium. The perfusate was hydrolyzed, solid phase-extracted, and then analyzed by gas chromatography/mass spectrometry. Depolarization due to high potassium concentrations increased the release of the alkaloid manyfold with respect to the basal value, and the release was dependent on the presence of calcium in the medium. These results suggest that endogenous morphine might act as a neurotransmitter or neuromodulator in the rat CNS.

Animals↗

PubMed1998

[Malignant neoplasia of the small intestine. Apropos of 7 cases. Clinical discussion and review of the literature].

The authors report seven cases of primary malignancies of the small intestine observed and treated between 1989 and 1995 at the First Surgical Division of Treviglio Hospital. Three patients were affected by adenocarcinomas (two involving the duodenum and one the jejunum), two patients had non-endocrine carcinoid tumors of the ileum, one presented a jejunal liposarcoma and one a lymphoma of the ileum. Four patients died (respectively at three months, 15 months, 18 months and five years after surgical intervention). Three patients are still alive. The early clinical features of these malignancies are typically vague and non specific and for this reason the disease is often diagnosed at an advanced stage. The prognosis of these lesions is generally poor owing to the diffusion of the disease at the time of diagnosis. In fact these neoplasms are preoperatively identified only in 27-72%. The percentage of surgical removal ranges between 65 and 80% according to the recent literature. This study is aimed at analysing the clinical and pathologic characteristics of the observed cases and the difficulties of the diagnostic procedure. The adopted surgical techniques and the outcome of the operative treatment are also examined.

Adenocarcinoma↗

PubMed1997

Efficient signal transduction by a chimeric yeast-mammalian G protein alpha subunit Gpa1-Gsalpha covalently fused to the yeast receptor Ste2.

Saccharomyces cerevisiae uses G protein-coupled receptors for signal transduction. We show that a fusion protein between the alpha-factor receptor (Ste2) and the Galpha subunit (Gpa1) transduces the signal efficiently in yeast cells devoid of the endogeneous STE2 and GPA1 genes. To evaluate the function of different domains of Galpha, a chimera between the N-terminal region of yeast Gpa1 and the C-terminal region of rat Gsalpha has been constructed. This chimeric Gpa1-Gsalpha is capable of restoring viability to haploid gpa1Delta cells, but signal transduction is prevented. This is consistent with evidence showing that the C-terminus of the homologous Galpha is required for receptor-G protein recognition. Surprisingly, a fusion protein between Ste2 and Gpa1-Gsalpha is able to transduce the signal efficiently. It appears, therefore, that the C-terminus of Galpha is mainly responsible for bringing the G protein into the close proximity of the receptor's intracellular domains, thus ensuring efficient coupling, rather than having a particular role in transmitting the signal. To confirm this conclusion, we show that two proteins interacting with each other (such as Snf1 and Snf4, or Ras and Raf), each of them fused either to the receptor or to the chimeric Galpha, allow efficient signal transduction.

Animals↗

PubMed1997

Complex formation between the hepatitis C virus serine protease and a synthetic NS4A cofactor peptide.

The NS3 protein of the hepatitis C virus contains a serine protease that, upon binding to its cofactor, NS4A, is responsible for maturational cleavages that occur in the nonstructural region of the viral polyprotein. We have studied in vitro complex formation between the NS3 protease domain expressed in Escherichia coli and a synthetic peptide spanning the minimal domain of the NS4A cofactor. Complex dissociation constants in the low micromolar range were measured using different techniques such as activity titration, fluorescence titration, and pre-equilibrium analysis of complex formation. Cofactor binding was strictly dependent on the glycerol content of buffer solutions and was not significantly influenced by substrate saturation of the enzyme. NS4A peptide binding to NS3 was accompanied by changes in the circular dichroism spectrum in the region between 270 and 290 nm, as well as by an enhancement of tryptophan fluorescence. Conversely, no changes in the far UV region of the circular dichroism spectrum were detectable. These data are indicative of induced tertiary structure changes and suggest that the secondary structure content of the uncomplexed enzyme does not differ significantly from that of the NS3-cofactor complex. Pre-equilibrium measurements of complex formation showed very low values for k(on), suggesting conformational transitions to be rate limiting for the association reaction.

Amino Acid Sequence↗

PubMed1997

Substrate specificity of the hepatitis C virus serine protease NS3.

The substrate specificity of a purified protein encompassing the hepatitis C virus NS3 serine protease domain was investigated by introducing systematic modifications, including non-natural amino acids, into substrate peptides derived from the NS4A/NS4B cleavage site. Kinetic parameters were determined in the absence and presence of a peptide mimicking the protease co-factor NS4A (Pep4A). Based on this study we draw the following conclusions: (i) the NS3 protease domain has an absolute requirement for a small residue in the P1 position of substrates, thereby confirming previous modelling predictions. (ii) Optimization of the P1 binding site occupancy primarily influences transition state binding, whereas the occupancy of distal binding sites is a determinant for both ground state and transition state binding. (iii) Optimized contacts at distal binding sites may contribute synergistically to cleavage efficiency.

Alanine↗

PubMed1997

Metabolic effects of liver transplantation in cirrhotic patients.

To assess whether liver transplantation (LTx) can correct the metabolic alterations of chronic liver disease, 14 patients (LTx-5) were studied 5+/-1 mo after LTx, 9 patients (LTx-13) 13+/-1 mo after LTx, and 10 patients (LTx-26) 26+/-2 months after LTx. Subjects with chronic uveitis (CU) and healthy volunteers (CON) were also studied. Basal plasma leucine and branched-chain amino acids were reduced in LTx-5, LTx-13, and LTx-26 when compared with CU and CON (P < 0.01). The basal free fatty acids (FFA) were reduced in LTx-26 with respect to CON (P < 0.01). To assess protein metabolism, LTx-5, LTx-13, and LTx-26 were studied with the [1-14C]leucine turnover combined with a 40-mU/m2 per min insulin clamp. To relate changes in FFA metabolism to glucose metabolism, eight LTx-26 were studied with the [1-14C]palmitate and [3-3H]glucose turnovers combined with a two-step (8 and 40 mU/m2 per min) euglycemic insulin clamp. In the postabsorptive state, LTx-5 had lower endogenous leucine flux (ELF) (P < 0.005), lower leucine oxidation (LO) (P < 0.004), and lower non-oxidative leucine disposal (NOLD) (P < 0.03) with respect to CON (primary pool model). At 2 yr (LTx-26) both ELF (P < 0.001 vs. LTx-5) and NOLD (P < 0.01 vs. LTx-5) were normalized, but not LO (P < 0.001 vs. CON) (primary and reciprocal pool models). Suppression of ELF by insulin (delta-reduction) was impaired in LTx-5 and LTx-13 when compared with CU and CON (P < 0.01), but normalized in LTx-26 (P < 0.004 vs. LTx-5 and P = 0.3 vs. CON). The basal FFA turnover rate was decreased in LTx-26 (P < 0.01) and CU (P < 0.02) vs. CON. LTx-26 showed a lower FFA oxidation rate than CON (P < 0.02). Tissue glucose disposal was impaired in LTx-5 (P < 0.005) and LTx-13 (P < 0.03), but not in LTx-26 when compared to CON. LTx-26 had normal basal and insulin-modulated endogenous glucose production. In conclusion, LTx have impaired insulin-stimulated glucose, FFA, and protein metabolism 5 mo after surgery. Follow-up at 26 mo results in (a) normalization of insulin-dependent glucose metabolism, most likely related to the reduction of prednisone dose, and, (b) maintenance of some alterations in leucine and FFA metabolism, probably related to the functional denervation of the graft and to the immunosuppressive treatment.

Adult↗

PubMed1997

Surface topology of Minibody by selective chemical modifications and mass spectrometry.

The surface topology of the Minibody, a small de novo-designed beta-protein, has been probed by a strategy that combines selective chemical modification with a variety of reagents and mass spectrometric analysis of the modified fragments. Under appropriate conditions, the susceptibility of individual residues primarily depends on their surface accessibility so that their relative reactivities can be correlated with their position in the tertiary structure of the protein. Moreover, this approach provides information on interacting residues, since intramolecular interactions might greatly affect the reactivity of individual side chains by altering their pKa values. The results of this study indicate that, while overall the Minibody model is correct, the beta-sheet formed by the N- and C-terminal segments is most likely distorted. This is also in agreement with previous results that were obtained using a similar approach where mass spectrometry was used to identify Minibody fragments from limited proteolysis (Zappacosta F, Pessi A, Bianchi E, Venturini S, Sollazzo M, Tramontano A. Marino G, Pucci P. 1996. Probing the tertiary structure of proteins by limited proteolysis and mass spectrometry: The case of Minibody. Protein Sci 5:802-813). The chemical modification approach, in combination with limited proteolysis procedures, can provide useful, albeit partial, structural information to complement simulation techniques. This is especially valuable when, as in the Minibody case, an NMR and/or X-ray structure cannot be obtained due to insufficient solubility of the molecule.

Acetylation↗

PubMed1997

Through and beyond the wall: late steps in leukocyte transendothelial migration.

A combination of approaches has provided firm experimental support for a step-wise model of leukocyte adhesion to vascular endothelium under hemodynamic shear stress. However, leukocyte adhesion to vascular endothelium acquires functional significance only if coupled to transmigration and progression into extravascular tissues. As discussed here by Elisabetta Bianchi and colleagues, these latter processes involve the sequential activation of adhesion-dependent functional programs, carried out by structurally diverse multimolecular complexes.

Cell Adhesion↗

PubMed1997

Continuous infusion of carboplatin during conventional radiotherapy treatment in advanced squamous carcinoma of the cervix uteri IIB-IIIB (UICC). A phase I/II and pharmacokinetic study.

OBJECTIVE: A prospective, single-arm phase-I/II trial performed to assess the efficacy and toxicity of the concomitant use continuous infusion of low-dose carboplatin and pelvic conventional radiotherapy in patients with locally advanced squamous cell carcinoma of the cervix. MATERIALS AND METHODS: Between January and July 1994, a total of 12 patients consecutively diagnosed to have squamous cell carcinoma of the cervix uteri stages IIB-IIIB UICC-TNM (five patients, IIB; and seven patients, IIIB) entered the study. All patients were evaluated by a gynecologist and a radiation oncologist and were submitted to standard pretreatment staging procedures. Radiation was delivered with 10-MeV photon beams with the shrinking-field technique. The patients received 2 Gy radiotherapy daily, 5 fractions per week, up to a planned total of 60 Gy in 6 weeks to the primary tumor and 46 Gy in 4 weeks to the whole pelvis. Irradiation was performed using four fixed orthogonal fields. One intracavitary insertion, 8 Gy to point A (dose rate, 1.1 Gy/h), was performed immediately after external pelvic irradiation. Carboplatin (12 mg/m2/day) was also administered in a continuous infusion, starting 1 day before the first fraction of radiotherapy. The platinum in plasma and urine, as well as the platinum concentration in the cytosols of lymphocytes and tumor, was measured weekly. RESULTS: A complete response was seen in nine (75%) of the 12 patients. Of the nine patients who achieved a complete remission, only one had subsequent failure in the pelvis. The total pelvic failure rate was 33.3% (four of 12 patients). With a median follow-up time of 20 months, the actuarial survival rate at 24 months was 64.8%. All patients completed the treatment without major protocol violations. Grade-2 leukopenia (in nine patients) and grade-1 nausea and vomiting (in five) were the most common acute toxicities. There was one grade-3 hematologic toxicity. Grade-3 late complications were observed in 16.6% of cases (two of 12 patients). On days 28 and 42 of the treatment, the mean total platinum plasma concentrations were 491 micrograms/L (SD = 129) and 672 micrograms/L (SD = 160), and the ultrafilterable fraction was 8-10%. At the same time points, the concentration in lymphocytes was constant at 21 picograms (pg) platinum/lymphocyte. The levels of platinum concentration measured on days 14 and 28 in the cytosols of tumor cells were 0.3 microgram/g (SD = 0.1) and 0.93 microgram/g (SD = 0.2). CONCLUSION: The combination of continuous infusion of carboplatin and radiotherapy at the aforementioned doses in patients with locally advanced cervical carcinoma resulted in a relatively low frequency of significant acute and late complications. Platinum in normal tissue (picograms per lymphocyte) was stable from week 1 of treatment, whereas the platinum steady state in plasma and in tumor cells was not reached in 6 weeks and was below that required in vitro to produce radiopotentiation. Further studies to determine the optimal dose of carboplatin and irradiation are needed prior to the initiation of phase-III studies.

Adult↗

PubMed1997

Evidence for an inhibitory effect of physiological levels of insulin on the growth hormone (GH) response to GH-releasing hormone in healthy subjects.

It has been previously reported that in healthy subjects, the acute reduction of free fatty acids (FFA) levels by acipimox enhances the GH response to GHRH. In the present study, the GH response to GHRH was evaluated during acute blockade of lipolysis obtained either by acipimox or by insulin at different infusion rates. Six healthy subjects (four men and two women, 25.8 +/- 1.9 yrs old, mean +/- SE) underwent three GHRH tests (50 micrograms iv, at 1300 h) during: 1) iv 0.9% NaCl infusion (1200-1500 h) after oral acipimox administration (250 mg) at 0700 h and at 1100 h; 2) 0.1 mU.kg-1.min-1 euglycemic insulin clamp (1200-1500 h) after oral acipimox administration (250 mg at 0700 h and at 1100 h); 3) 0.4 mU.kg-1.min-1 euglycemic insulin clamp (1200-1500 h) after oral placebo administration (at 0700 and 1100 h). Serum insulin (immunoreactive insulin) levels were significantly different in the three tests (12 +/- 2, 100 +/- 10, 194 +/- 19 pmol/L, P < 0.06), plasma FFA were low and similar (0.04 +/- 0.003, 0.02 +/- 0.005, 0.02 +/- 0.003, not significant), and the GH response to GHRH was progressively lower (4871 +/- 1286, 2414 +/- 626, 1076 +/- 207 micrograms/L 120 min), although only test 3 was significantly different from test 1 (P < 0.05). Pooling the three tests together, a significant negative regression was observed between mean serum immunoreactive insulin levels and the GH response to GHRH (r = -0.629, P < 0.01). Our results indicate that in healthy subjects, acipimox and hyperinsulinemia produce a similar decrease in FFA levels and that at similar low FFA, the GH response to GHRH is lower during insulin infusion than after acipimox. These data suggest that insulin exerts a negative effect on GH release. Because the insulin levels able to reduce the GH response to GHRH are commonly observed during the day, for instance during the postprandial period, we conclude that the insulin negative effect on GH release may have physiological relevance.

Adult↗