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Biomedical subjects

M Magnani

Publications and source records attributed to M Magnani.

At least 145 records · Page 8Linked to original sources
PubMed1992

[The pediatric emergency department: assessment of clinical activity and interaction with local health authorities].

A perspective study was carried out at the Emergency Department of the Gaslini Institute on a sample of the children admitted from May to September 1990. The study aimed to describe types and modalities of admission. "G. Gaslini" Institute is a multidisciplinary children's hospital: 45% of patients admitted in the hospital come from the southern regions of Italy, while the admission at the Emergency Department were local in 82%. Among all children considered, 2080 (71.9% were admitted on request of their parents, who did not ask previously any physician; of those cases, 356 (17%) were hospitalized, 474 (25%) were either examined by specialist or treated and then discharged. In the remaining cases, no intervention was necessary. As a consequence, 58% of children were admitted without any clinically plausible reason. Parents' anxiety clearly played an important role in most cases. 840 children were addressed to the Emergency Department by a physician, 652 (22.3%) by the doctor in charge, 213 (32.7%) of which were sent back home by the doctor on duty, who did not think it necessary any treatment. Of the 120 (4.1%) children sent by different Emergency Departments, 95 (79.1%) were hospitalized. The reason why physicians addressed quite a large number of children to the Emergency Department, when they didn't need any intervention remain nucleo. Their diagnosis, in fact, agreed with those of the doctor on duty in 83% of cases. In conclusion, it is difficult to imagine a diagnostic or therapeutic problem which needs specific structures. Cooperative programs with physicians working in our region will be carried out in order to control, where possible, admission to Emergency Departments.

Adolescent↗
PubMed1992

Acetaldehyde, ethanol and acetone concentrations in blood of alcohol-treated mice receiving aldehyde dehydrogenase-loaded erythrocytes.

Blood levels of acetaldehyde (ACh), ethanol and acetone were investigated in mice treated with ethanol for 6 months and receiving compatible erythrocytes (RBCs) overloaded with aldehyde dehydrogenase (AlDH). Following an acute dose of ethanol, ACh levels were significantly lower in these animals than in alcohol-treated mice receiving AlDH-unloaded RBCs, and were similar to the ACh levels of normal mice. The peak ethanol concentration was higher in normal mice than in both groups of alcohol-treated animals, while acetone concentrations were not significantly different in the three groups of animals.

Acetaldehyde↗
PubMed1992

In vitro targeting of erythrocytes to cytotoxic T-cells by coupling of Thy-1.2 monoclonal antibody.

A method was explored to develop a general means to target erythrocytes to T-cells in vitro. Mouse erythrocytes were coupled with an anti-Thy-1.2 monoclonal antibody by two methods. Chromic chloride coupling of antibody was preferred to biotinylation. The morphology, osmotic fragility, and the hematological values of treated cells were normal compared with those of control erythrocytes. Antibody-coupled erythrocytes were incubated with cytotoxic T-lymphocytes (CTLL) in vitro at a 20:1 ratio. Approximately 60-70% of the CTLL formed rosettes. The cell mixture was subjected to gradient centrifugation and separated into four fractions. THe rosettes were clearly identified only in the treated group containing anti-Thy-1.2-coupled erythrocytes. No rosettes were found when aspecific monoclonal antibody was coupled to erythrocytes. Examination by scanning and transmission electron microscopy revealed CTLL with 4-5 erythrocytes attached to them but did not show any evidence of membrane fusion. Rosettes of CTLL incubated in vitro proliferated as well as CTLL alone and maintained their dependency on interleukin-2. Targeting of erythrocyte carriers to lymphocytes offers the potential for delivery of molecules directly to the target cell.

Animals↗
PubMed1992

Increased glucose metabolism by enzyme-loaded erythrocytes in vitro and in vivo normalization of hyperglycemia in diabetic mice.

The main metabolic properties of human red blood cells (RBC) overloaded with glucose catabolizing enzymes such as hexokinase and glucose oxidase were evaluated. Human erythrocytes loaded with human hexokinase metabolized 3.1 +/- 0.2 mumol/h/ml RBC of glucose, an amount double that consumed by normal and unloaded cells (1.46 +/- 0.16 mumol/h/ml RBC), while glucose oxidase-loaded erythrocytes consumed up to 5.5 +/- 0.5 mumol/h/ml RBC of glucose but with a time-dependent increase in methemoglobin formation due to the H2O2 produced in the glucose oxidase reaction. This methemoglobin production was greatly reduced while glucose consumption was increased (8.1 +/- 0.4 mumol/h/ml RBC) by coentrapment of hexokinase and glucose oxidase. Similar results were obtained in mouse red blood cells, although the role of hexokinase was less pronounced due to a higher basal level of this enzyme. When administered to diabetic mice the hexokinase/glucose oxidase-overloaded erythrocytes had a circulating half-life of 5 days and were able to regulate blood glucose at near physiological levels. A single intraperitoneal administration of 500 microliters of enzyme-loaded cells maintained a near-normal blood glucose concentration for 7 +/- 1 days, while repeated administrations at 10-day intervals were effective in the regulation of blood glucose levels for several weeks. These results suggest that enzyme-loaded erythrocytes can behave as circulating bioreactors and can provide a new way to reduce abnormally elevated blood glucose.

Animals↗
PubMed1992

[The activities of a pediatric emergency department. The emergency problems].

The authors reviewed records of admission at the Fist Ais-Emergency Service of "G. Gaslini" Children's Hospital, data referring both to in and outpatients. First of all we took into account epidemiological data analysing occurrence and types of diseases; at the same time a demographic study, which aimed to show a decrease in the child population in Genova, was performed. Secondly we compared these data with the real number of admitted patients: collected data showed that this service has been used excessively.

Age Factors↗
PubMed1991

Evidence for a particulate location of ubiquitin conjugates and ubiquitin-conjugating enzymes in rabbit brain.

Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this "mitochondrial fraction" shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.

Animals↗
PubMed1991

Cloning and expression of a new human polypeptide which regulates protein phosphorylation in Escherichia coli.

A 1,820 bp full-length clone encoding for a new human protein was isolated from a lambda gt11 placental cDNA library using anti-human hexokinase antibodies. The cDNA complete sequence includes a 12 bp 5' non-coding region, a single open reading frame encoding a protein of 55 KDa (HP-10) and a 177 bp non-coding with two putative polyadenylation signals upstream of 3' poly(A)tail. The deduced amino acid sequence reveals a sequence of 492 amino acids that contains a stretch of 7 glutamic acid from position 169 and one potential glycosylation site at position 274. Although antibodies against hexokinase recognize the fusion protein and antibodies against the fusion protein recognize hexokinase, HP-10 is not human hexokinase, by a number of criteria including the alignment of determined amino acid sequences. In searching for a possible functional role of HP-10 its cDNA was inserted into a procaryotic vector which allows the expression of the non-fused protein. Bacteria expressing the HP-10 encoded protein were isolated and found to have a dramatic increase in endogenous phosphorylated proteins. Since HP-10 does not have a protein kinase activity per se it should be considered a new regulatory phosphorylation protein which is active in E. coli.

Amino Acid Sequence↗
PubMed1991

Human hexokinase type I microheterogeneity is due to different amino-terminal sequences.

Human placenta hexokinase type I was previously shown to be present in two subtypes with similar isoelectric points but different molecular masses of 112 and 103 kDa, respectively. In order to exclude that these subtypes arise by artifact(s) occurring during the protein purification, we have developed a single-step immunoaffinity chromatography for the isolation of microgram quantities of hexokinase. The results obtained confirmed the presence of both hexokinase subtypes in human placenta. By Northern blot analysis a single mRNA species that hybridized with a hexokinase-I cDNA was found to be present in human placenta. Furthermore, in vitro translation of placenta mRNA in a rabbit reticulocyte lysate followed by hexokinase immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that only one hexokinase with apparent molecular mass of about 112 kDa is expressed in this tissue and suggests a post-translational modification as a probable cause of hexokinase I microheterogeneity. To further investigate this point we have purified the high and low Mr hexokinase and determined their NH2-terminal sequences. The results obtained show that when compared with the amino acid sequence deduced from a cDNA the high Mr hexokinase starts at amino acid 11 while the low Mr hexokinase starts at amino acid 103. Since the first 10 amino acids are involved in the binding of hexokinase to mitochondrial porin these data provide an explanation both for the inability of these hexokinases to bind to mitochondria and for their differences in Mr.

Amino Acid Sequence↗
PubMed1991

Free radicals promote "in vitro" a different intracellular decay of rabbit reticulocyte and erythrocyte glycolytic enzymes.

Rabbit red blood cells (RBC) were exposed in vitro to an oxygen-radical-generating system represented by iron and ascorbic acid. Under these experimental conditions we have investigated the effect of this system on some intracellular rabbit reticulocyte and erythrocyte enzymes. The results obtained have shown a pronounced decay of hexokinase activity both in the erythrocytes and reticulocytes when exposed to these radical species. We have found that the amount of hexokinase inactivated is at least three times higher in a blood sample with a percentage of reticulocytes of 50-60%. This different behaviour of the hexokinase decay in the erythrocytes and reticulocytes could be due to its different intracellular distribution related to the two distinct cells. In addition we have evaluated some important intracellular compounds involved in maintaining the redox and the energetic state of the cell such as the reduced glutathione and the adenine nucleotides and their degradation products, in order to understand if there is any correlation between the hexokinase decay and a change concerning the metabolic conditions of the rabbit reticulocytes and erythrocytes exposed to free radicals.

Animals↗
PubMed1991

In vitro toxicity and metabolism of 2',3'-dideoxycytidine, an inhibitor of human immunodeficiency virus infectivity.

U937 human monoblastoid cell growth was inhibited in a concentration-dependent manner by 2',3'-dideoxycytidine (ddCyd) (an antiretroviral drug) up to 500 microM. Cell growth inhibition was associated with a pronounced increase in cell volume, however this was not due to cell ATP or NAD+ depletion that could effect osmotic balance or DNA repair. This ddCyd toxicity paralleled the accumulation of ddCyd into acid soluble material where 2',3'-dideoxycytidine-5'-triphosphate (ddCTP) was the predominant labelled nucleotide up to an extracellular ddCyd concentration of 150 microM. At higher ddCyd concentrations, the amount of 2',3'-dideoxycytidine-5'-diphosphate (ddCDP) became predominant over ddCTP. This increase of phosphorylated dideoxycytidine in U937 cells was also associated with an increased incorporation of the drug into cell DNA suggesting a possible toxicity mechanism. That ddCyd does indeed become cytotoxic to human cell by incorporation into DNA was shown by incubating human resting and stimulated lymphocytes with ddCyd. While the drug does not affect cell viability in resting cells it strongly affects cell proliferation upon phytohemagglutinin (PHA) addition.

Adenosine Triphosphate↗
PubMed1991

Cell age dependent decay of human erythrocytes glutathione S-transferase.

Human red blood cells contain both glutathione S-transferase sigma (GST sigma) and glutathione S-transferase rho (GST rho). While the first isozyme does not change in red blood cell fractions of different mean density (age), GST rho, the main isozyme, shows a pronounced cell age dependent decay. Ion-exchange chromatographic experiments show that GST rho consists of only one isozymic form in young erythrocytes but is present in two components, with different electric charge, in mature and old cells. The "secondary" GST rho isozyme is more heat stable than the "primary" GST rho isozyme with the result that the total GST activity shows an apparent increase in heat stability during cell aging due to the formation of "secondary" isozymes. The kinetic properties and specificity of this enzyme do not show appreciable modifications during cell ageing. The data reported in this paper suggest that red blood cell aging is associated with a reduced detoxifying ability due to GST rho decay.

Erythrocyte Aging↗
PubMed1991

Glucose-6-phosphate dehydrogenase Lodi844C: a study on its expression in blood cells and muscle.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency was found in erythrocytes, lymphocytes and muscle of an Italian male, whose family has lived for at least three generations in Lodi (Lombardy, northern Italy). The subject was hospitalized for myalgia and dark urine after intense physical exercise, but no sign of anemia and chronic hemolysis were present at rest. Family studies revealed that the mother and the maternal aunt had the same enzymopathy. The enzyme-specific activity in red blood cells was 15% of control and the kinetic properties were the following: slower electrophoretic mobility; biphasic pH activity curve; slightly reduced thermal stability, and increased utilization of the substrate analogs. The analysis of our patient's DNA showed a G----C mutation at nucleotide 844 which causes an Asp----His amino acid change in position 282. This is the same mutation found by De Vita et al. in the G6PD Seattle-like variant. However, by following a new convention, we labelled our variant as G6PD Lodi844C. As far as the muscle is concerned, we found that the enzyme-specific activity in this tissue was 14% of control values, but cultured myotubes and myoblasts revealed a normal level of G6PD as well as skin fibroblasts. On the contrary in the same type of cultured cells obtained from G6PD Mediterranean subjects, the G6PD activity was about 20% of normal. Our results complete the characterization of this mutant enzyme, demonstrate the expression of the deficit in muscle and describe the enzyme behaviour in cultured cells.

Adult↗
PubMed1991

[Cerebral arteriovenous malformations (CAVM)].

Two infants with endocranic A-V malformations and irreversible picture of congestive cardiac failure are presented: the first is a newborn with a very large angioma, the second is a newborn with a large aneurysm of the vein of Galen. A review of the literature is presented: the salient age-related features required to make a diagnosis are discussed.

Cerebral Arteries↗
PubMed1991

Human red blood cells as bioreactors for the inactivation of harmful xenobiotics.

Human red blood cells are able to inactivate lipophilic electrophiles by conjugation with reduced glutathione. This metabolic ability was found to be limited by the rate of permeation of the xenobiotic into erythrocytes and by the amount of available reduced glutathione. By a procedure of hypotonic dialysis, isotonic resealing and reannealing human red blood cells were overloaded with increasing amounts of reduced glutathione up to three- to fourfold the normal level without modification of their metabolic functions or of their energetic state. These overloaded erythrocytes were able to conjugate increasing amounts of xenobiotics and to export the resulting conjugates from the cells. These properties of glutathione overloaded erythrocytes are significant for the use of carrier erythrocytes in cases of acute intoxication by lipophilic electrophiles.

Biotechnology↗
PubMed1990

Preparation and characterization of monoclonal antibodies to human hexokinase type I.

1. Three different immunization protocols and several screening procedures were used to prepare seven mouse monoclonal antibodies to human placenta hexokinase type I. None of these monoclonals were able to recognize the native enzyme but all detected hexokinase when adsorbed onto polystyrene plates or on immunoblots after SDS/polyacrylamide-gel electrophoresis. 2. All seven monoclonals recognize the two different subtypes of human hexokinase I equally well. Limited tryptic digestion of hexokinase followed by Western blotting and immunodetection show that these monoclonals recognize epitopes that lie in different tryptic peptides. 3. Comparative ELISA studies showed that human hexokinase types I and II have great immunological similarities while hexokinase I from different mammalian species and yeast hexokinase are recognized with different affinities.

Animals↗