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Biomedical subjects

E Bianchi

Publications and source records attributed to E Bianchi.

At least 73 records · Page 4Linked to original sources
PubMed1994

High level expression and rational mutagenesis of a designed protein, the minibody. From an insoluble to a soluble molecule.

We recently described the design and chemical synthesis of the minibody, a 61-residue metal binding beta-protein with a novel fold. Characterization of the polypeptide by circular dichroism spectroscopy, size exclusion chromatography, and metal binding studies showed the molecule to be folded, monomeric, globular and able to bind metals. The main obstacle which prevented a more detailed characterization was the very low solubility of the protein in water (about 10 microM). To address this problem, we used two independent approaches: (1) mutagenesis of the beta-sheet framework residues and (2) addition of a solubilizing motif, made of three lysine residues, at the N or C termini. Engineering and production of mutants was facilitated by the achievement of high level expression of the protein in Escherichia coli. Both approaches led to minibody variants with a solubility ranging from tenfold higher up to millimolar levels. For the best-characterized variant obtained so far, the thermodynamic stability calculated from denaturant-induced transition is identical to that of the parent, poorly soluble, molecule.

Amino Acid Sequence↗
PubMed1994

The urokinase receptor is expressed in invasive breast cancer but not in normal breast tissue.

We have studied expression of the urokinase receptor (u-PAR) in paraffin-embedded breast tissues at various stages of malignant progression. Forty-nine of 59 invasive cancers studied showed varying degrees of reactivity with our polyclonal antibody. The staining pattern was variable from case to case, although strong surface staining of tumor-associated macrophages was evident in most of these sections. In several cases, blood vessels in selected tumor areas were stained, as confirmed by treatment of adjacent sections with an anti-factor VIII antibody. These could represent regions of recent angiogenesis. Staining of tumor cells was observed in 21 of 59 cases and was extensive in 5 cases but confined to a small percentage of cells in the remaining 16 samples. In contrast with the cancer sections, all normal breast tissue (12 cases) was negative, as well as all fibroadenomas (4 cases), papillomas (5 cases), and hyperplasia with atypia (2 cases) studied. Seven carcinomas in situ examined were also negative for u-PAR, with the exception of few macrophages in two cases, suggesting that u-PAR expression may be associated with invasive tumor. The presence of u-PAR in human breast cancer and its absence from nonmalignant breast tissue supports the idea that plasminogen activation plays an important role in the process of cancer invasion. Expression of u-PAR on macrophages, endothelial cells, and cancer cells suggests the existence of complex paracrine interactions between tumor cells and stroma.

Base Sequence↗
PubMed1994

The making of the minibody: an engineered beta-protein for the display of conformationally constrained peptides.

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost the drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61-residue beta-protein aimed at retaining some desirable features of immunoglobulin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of the main chain conformation of the same regions, based on the 'canonical structures' model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical synthesis and expression in E. coli and characterized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low solubility. We were able to overcome this problem both by further mutagenesis of the framework and by addition of a solubilizing motif. The minibody is being used to select constrained human IL-6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.

Amino Acid Sequence↗
PubMed1994

The search for a gene involved in the determination of limited duplicative capacity in human cells.

On the assumption that EBV integration into the genome of human B lymphocyte might lead to the inactivation of a hypothetical gene determining the limited duplicative capacity and consequently participate to the cell immortalization, a search for the human-virus junction was done. This led to the identification of a site of integration in the central part of the heavy chain of the immunoglobulin region. The coincidence of the involvement of the site in lymphomatogenesis with the first complete characterization of an integration site led to the speculation that the heavy chain gene itself might be an important controller of cell duplication in the B lymphocyte.

B-Lymphocytes↗
PubMed1994

Immunocytochemical localization of endogenous codeine and morphine.

Experiments carried out by indirect immunofluorescence and unlabelled antibody enzyme procedures revealed the presence of morphine-like immunoreactive material in the perikarya, fibers, and terminals of neurons in different, discrete areas of rat and human brain. The monoclonal and polyclonal anti-morphine antibodies used do not distinguish between morphine and codeine. Endogenous morphine seems to be stored in neurons as the 3-ethereal sulphate conjugate. This possibility is supported by the finding that, although active uptake of [3H]morphine has not been detected in brain synaptosomes, long-term i.c.v. injection of the tritiated opiate results in the accumulation of radioactivity inside the same neurons in which the endogenous alkaloids have been detected. Finally, striatal slices exposed to high K+ concentrations showed a rapid disappearance of the morphine-like immunoreactive material from neurons, indicating that endogenous alkaloids are released from neurons by depolarization.

Animals↗
PubMed1994

Persistence of counter-regulatory abnormalities in insulin-dependent diabetes mellitus after pancreas transplantation.

Conventional insulin therapy does not correct the counter-regulatory abnormalities of insulin-dependent diabetes mellitus. Pancreas transplantation is an alternative therapy that restores the endogenous insulin secretion in diabetes. In this study, the effects of segmental pancreas transplantation on counter-regulation to mild hypoglycaemia were evaluated. Glucose kinetics and the counter-regulatory hormonal responses were assessed in eight insulin-dependent diabetics with end-stage renal failure who had received pancreas and kidney transplantation 1 year previously, seven diabetic uraemic subjects (candidates for combined transplantation), five patients with chronic uveitis on immunosuppressive therapy comparable to pancreas recipients and 10 normal subjects. Insulin (0.3 mU kg-1 min-1) was infused for 2 h to induce mild hypoglycaemia (plasma glucose 3.2-3.5 mmol l-1) and exogenous glucose was infused as required to prevent any glucose decrease below 3.1 mmol l-1. After transplantation, two of eight recipients had hypoglycaemic episodes reported in their medical records. During the study, hepatic glucose production was rapidly suppressed in the controls and in the patients on immunosuppression (-80 +/- 7 and -54 +/- 7%, P < 0.001 vs. basal), and rebounded to the baseline values within 1 h (-3 +/- 1 and -6 +/- 2%, P = NS vs. basal). The transplant recipients had similar suppression in the first hour (-88 +/- 8%, P < 0.001 vs. basal), but the suppression persisted in the second hour (-69 +/- 11%, P < 0.001 vs. basal) indicating a lack of glucose counter-regulatory response.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Combined pancreas and kidney transplantation normalizes protein metabolism in insulin-dependent diabetic-uremic patients.

In order to assess the combined and separate effects of pancreas and kidney transplant on whole-body protein metabolism, 9 insulin-dependent diabetic-uremic patients (IDDUP), 14 patients after combined kidney-pancreas transplantation (KP-Tx), and 6 insulin-dependent diabetic patients with isolated kidney transplant (K-Tx), were studied in the basal postabsorptive state and during euglycemic hyperinsulinemia (study 1). [1-14C]Leucine infusion and indirect calorimetry were utilized to assess leucine metabolism. The subjects were studied again with a combined infusion of insulin and amino acids, given to mimic postprandial amino acid levels (study 2). In the basal state, IDDUP demonstrated with respect to normal subjects (CON): (a) higher free-insulin concentration (17.8 +/- 2.8 vs. 6.8 +/- 1.1 microU/ml, P < 0.01) (107 +/- 17 vs. 41 +/- 7 pM); (b) reduced plasma leucine (92 +/- 9 vs. 124 +/- 2 microM, P < 0.05), branched chain amino acids (BCAA) (297 +/- 34 vs. 416 +/- 10 microM, P < 0.05), endogenous leucine flux (ELF) (28.7 +/- 0.8 vs. 39.5 +/- 0.7 mumol.m-2.min-1, P < 0.01) and nonoxidative leucine disposal (NOLD) (20.7 +/- 0.2 vs. 32.0 +/- 0.7 mumol.m-2. min-1, P < 0.01); (c) similar leucine oxidation (LO) (8.0 +/- 0.1 vs. 7.5 +/- 0.1 mumol.m-2.min-1; P = NS). Both KP-Tx and K-Tx patients showed a complete normalization of plasma leucine (116 +/- 5 and 107 +/- 9 microM), ELF (38.1 +/- 0.1 and 38.5 +/- 0.9 mumol.m-2.min-1), and NOLD (28.3 +/- 0.6 and 31.0 +/- 1.3 mumol.m-2.min-1) (P = NS vs, CON). During hyperinsulinemia (study 1), IDDUP showed a defective decrease of leucine (42% vs. 53%; P < 0.05), BCAA (38% vs. 47%, P < 0.05), ELF (28% vs. 33%, P < 0.05), and LO (0% vs. 32%, P < 0.05) with respect to CON. Isolated kidney transplant reverted the defective inhibition of ELF (34%, P = NS vs. CON) of IDDUP, but not the inhibition of LO (18%, P < 0.05 vs. CON) by insulin. Combined kidney and pancreas transplanation normalized all kinetic parameters of insulin-mediated protein turnover. During combined hyperinsulinemia and hyperaminoacidemia (study 2), IDDUP showed a defective stimulation of NOLD (27.9 +/- 0.7 vs. 36.1 +/- 0.8 mumol.m-2.min-1, P < 0.01 compared to CON), which was normalized by transplantation (44.3 +/- 0.8 mumol.m-2.min-1).

Adult↗
PubMed1994

[Clinical considerations in Raynaud's phenomenon. How many and which patients are treated?].

Among the multiple aspects about which little is still known about Raynaud's phenomenon (RP), is that, in the author's opinion, regarding the question: how many and which patients are sufficiently troubled by this pathology to seek medical treatment from their own GP or specialist, and what are the results of any treatment commenced. The following questions were put to a group of 180 patients affected by RP attending the out-patient clinic for the first time: 1) how many had already received treatment; 2) which patients were receiving pharmacological treatment and/or had undergone surgery. In this context, the authors assessed the influence of the following parameters on the fact that they had or had not been treated: a) the age of patients; b) the duration of disease; c) the presence of stenosing or obliterating arterial lesions (RP on an organic basis); d) the co-presence of pathologies considered to be the possible etiological cause of RP in literature; e) the severity of symptoms; 3) what results were obtained by medical and/or surgical treatment; 4) what was the course of RP which failed to respond to therapeutic measures. The study examined a series of 180 subjects including 139 (77.22%) females (mean age 47.6% years) and 41 (22.78) males (mean age 42.4 years). Symptoms had been present for a period ranging between 2 and 44 years (mean 10.01). In order to ascertain the possible presence of associated diseases considered by many authors to be causal factors, all patients underwent hemotochemical and instrumental test.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Endogenous codeine and morphine are stored in specific brain neurons.

Codeine and morphine have been detected in mammalian brain by radioimmunoassay (RIA), and in brain and other tissues by gas-chromatography/mass-spectrometry (GCMS) in different laboratories. It has been also shown that rat liver can synthesize the skeleton of the morphine molecule, thus suggesting that this alkaloid, which is the prototype of mu-receptor agonists, plays a physiological role in brain. We report the presence of morphine-like immunoreactive compounds inside the cell body, fibers and terminals of neurons in different brain areas. Moreover, neurons localized in the same brain areas were capable of accumulating and storing [3H]morphine slowly infused intracerebroventricularly (i.c.v.) through an osmotic minipump.

Animals↗
PubMed1993

A designed metal-binding protein with a novel fold.

A major challenge in protein design is to create stable scaffolds into which tailored functions can be introduced. Here we present the design, synthesis and characterization of a 61-residue all-beta protein: the minibody. We used a portion of the heavy chain variable domain of an immunoglobulin as a template, obtaining a molecule with a novel beta-sheet scaffold and two regions corresponding to the hypervariable loops H1 and H2. To exploit the potential for creating functional centres in the minibody, we engineered a metal-binding site into it. This site is formed by one histidine in H1 and two in H2. The protein is folded, compact and able to bind metal, thus representing the first designed beta-protein with a novel fold and a tailored function. By randomizing the sequence of the hypervariable loops, we are using the minibody scaffold to construct a conformationally constrained peptide library displayed on phage.

Amino Acid Sequence↗
PubMed1993

Chemical synthesis of a designed beta-protein through the flow-polyamide method.

A designed, 61-residue long, metal-binding protein was synthesized through the flow-polyamide method. This protein, named Minibody (Mini-antibody), contains a beta-sheet scaffold and two regions corresponding to immunoglobulin hypervariable loops (H1 and H2), onto which a metal binding site was engineered. The protein is extremely hydrophobic, with a 70% beta structure. Accordingly, it was anticipated, and actually found, to be a "difficult sequence" for all its length. Comparison of the standard, "minimum redundancy" protocol (single coupling, low excess of activated species) with a different protocol (double and triple coupling plus capping) showed that the two produced approximately the same amount of full-length product (3.7% after extensive purification). Capping was effective in blocking the unreacted amino groups, converting most failure sequences into truncated ones, a possible aid to implement affinity-type chromatographic protocols. Purification was complicated by the very low solubility of the molecule, coupled to a high tendency to aggregate even in concentrated chaotropic media (8 M urea). Nevertheless, a multi-dimensional purification scheme produced a highly homogeneous product, with the expected Ion Spray mass spectrum. The Minibody produced by recDNA methods showed identical chromatographic, spectroscopic and biochemical properties to those of the synthetic product.

Amino Acid Sequence↗
PubMed1993

Infantile mitochondria encephalomyopathies: report on 4 cases.

Four muscle biopsies from the quadriceps femoris muscle of children with symptoms suggesting mitochondrial encephalomyopathy were examined for morphological and biochemical differences. From a biochemical point of view, our patients showed a derangement of mitochondrial metabolism and in particular a marked deficit in the activity of cytochrome c oxidase. In all patients there was an abnormally high rise of lactate and pyruvic acid levels in blood before and after glucose loading. Results of morphological and enzyme histochemical studies showed smaller muscle fibre diameters than in normal children. Typical red ragged fibres and ultrastructural abnormalities in muscle mitochondria were present in 1 case only. Three patients showed focal changes in the distribution of some sarcoplasmic oxidative enzyme activities. The present findings suggest that the main metabolic disorder, pointed out by biochemical studies, is scarcely reflected by morphological and ultrastructural studies in the early stages of mitochondriopathies.

Biopsy↗
PubMed1993

[The irradiated volume in stage III non-small-cell carcinoma of the lung. Comparison of outdated and new techniques].

March 1988 through April 1992, three hundred and ninety-six patients affected with bronchogenic carcinoma were treated at the Department of Radiation Oncology of the S. Anna Hospital, Como, Italy. A hundred and ten patients presenting stage-III non-small-cell lung carcinoma were evaluable. All evaluable patients underwent radiation therapy alone, with either palliative or curative purposes. Two main periods can be distinguished: in period A, before June 30th, 1990, treatment planning included conventional techniques, with no simulators; the patients were treated with opposing anteroposterior fields only. In period B, after July 1st, 1990, either the simulator alone was used or a simulator, a CT unit and a treatment planning computer system were combined; anteroposterior opposing fields or multiportal technique were used. Median overall survival was 10 months. Independent of treatment goals, the irradiated volume was markedly different in the patients treated in period A than in those treated in period B. A marked and statistically significant increase in survival was observed in group B. Survival also increased in patients treated with doses > 40 Gy, but only if treatment planning had used adequate technology and accuracy. To conclude, better survival can be achieved only by improving treatment accuracy and quality.

Adult↗
PubMed1993

Affinity purification of a difficult-sequence protein. Implications for the inclusion of capping in synthetic protocols.

A biotinylated derivative of a designed, difficult-sequence protein (the Minibody, Bianchi, E., Tramontano, A., Sollazzo, M. & Pessi, A. (1993) Int. J. Peptide Protein Res. 41, 385-393) which represented only 3.7% of the crude, cleaved material was quantitatively recovered with about 70% purity, in a single step, by affinity chromatography on immobilised avidin. Purification to homogeneity was then easily achieved by preparative HPLC. This highly effective purification scheme must be contrasted with the previously shown multidimensional, low-yield chromatographic protocol. Since no facilitation of the purification had been obtained by capping alone, this result suggests that capping is useful only in conjunction with affinity chromatography.

Amino Acid Sequence↗