Biomedical subjects

G Orsini

Publications and source records attributed to G Orsini.

At least 55 records · Page 3Linked to original sources

PubMed1994

Probing the structure of hirudin from Hirudinaria manillensis by limited proteolysis. Isolation, characterization and thrombin-inhibitory properties of N-terminal fragments.

Hirudin is the most potent and specific inhibitor of the blood-clotting enzyme thrombin so far known. Several hirudin variants were isolated mostly from Hirudo medicinalis and shown to be polypeptide chains of approximately 7 kDa with three internal disulfide bridges. In this study, limited proteolysis has been used to probe aspects of the structure and dynamics of a hirudin variant HM2 isolated from Hirudinaria manillensis. Proteolysis of the polypeptide chain of 64-amino-acid residues of hirudin HM2 by protease from Staphylococcus aureus V8, trypsin, thermolysin and subtilisin occurs at region 41-49 of the chain. The N-terminal fragments 1-41 and 1-47 were isolated to homogeneity and shown to maintain inhibitory action on thrombin, though much lower than the intact protein. The results were interpreted on the basis of a proposed three-dimensional structure of hirudin HM2 deduced by protein modelling the known structure of hirudin variant HV1 from Hirudo medicinalis (75% sequence similarity between HM2 and HV1). Both proteolysis experiments and protein modelling provide evidence for the existence in hirudin HM2 of a N-terminal well-structured domain (core) and a C-terminal flexible polypeptide segment. Determination of the accessible surface area of the three-dimensional model of hirudin HM2 showed that the sites of preferential cleavages are at the surface of the polypeptide molecule.

Amino Acid Sequence↗

PubMed1994

The asiA gene product of bacteriophage T4 is required for middle mode RNA synthesis.

The asiA gene of bacteriophage T4 encodes a 10-kDa peptide which binds strongly in vitro to the sigma 70 subunit of Escherichia coli RNA polymerase, thereby weakening sigma 70-core interactions and inhibiting sigma 70-dependent transcription. To assess the physiological role of this protein, we have introduced an amber mutation into the proximal portion of the asiA gene. On suppressor-deficient hosts, this mutant phage (amS22) produces minute plaques and exhibits a pronounced delay in phage production. During these mutant infections, T4 DNA synthesis is strongly delayed, suggesting that the AsiA protein plays an important role during the prereplicative period of phage T4 development. The kinetics of protein synthesis show clearly that while T4 early proteins are synthesized normally, those expressed primarily via the middle mode exhibit a marked inhibition. In fact, the pattern of protein synthesis after amS22 infection resembles greatly that seen after infection by amG1, an amber mutant in motA, a T4 gene whose product is known to control middle mode RNA synthesis. The amber mutations in the motA and asiA genes complement, both for phage growth and for normal kinetics of middle mode protein synthesis. Furthermore, primer extension analyses show that three different MotA-dependent T4 middle promoters are not recognized after infection by the asiA mutant phage. Thus, in conjunction with the MotA protein, the AsiA protein is required for transcription activation at T4 middle mode promoters.

Bacteriophage T4↗

PubMed1993

Biochemical characterization of the molecular interaction between recombinant basic fibroblast growth factor and a recombinant soluble fibroblast growth factor receptor.

The extracellular domain of human fibroblast growth factor receptor (XC-FGF-R) was expressed in Escherichia coli. The protein was purified to homogeneity and the interaction with basic fibroblast growth factor (bFGF), its physiological ligand, was examined. Using resins on which bFGF was reversibly bound, we analysed the characteristics of the binding between XC-FGF-R and immobilized bFGF. We also investigated the stoichiometry of the binding between XC-FGF-R and recombinant human bFGF (rhbFGF) applying non-denaturing gel electrophoresis, chemical cross-linking followed by SDS/PAGE, and gel-filtration chromatography. In cross-linking and gel-filtration chromatography experiments, a 1:1 complex between rhbFGF and XC-FGF-R was observed. The complex was separated from the non-complexed proteins using non-denaturing PAGE in the presence of 0.1% Triton X-100. The band corresponding to the complex was recognized by specific antibodies directed against bFGF and its receptor, blotted on poly(vinylidene difluoride) membranes and submitted to sequence and amino acid analysis. The data obtained from these determinations confirmed the formation of a 1:1 complex between rhbFGF and XC-FGF-R.

Fibroblast Growth Factor 2↗

PubMed1993

Implication of cysteine residues in the activity of single-chain urokinase-plasminogen activator.

Single-chain urokinase-plasminogen activator contains 24 cysteine residues involved in 12 disulfide bonds and distributed all along the three domains of the protein. In order to investigate the role of these disulfide bridges in the catalytic activities of scu-PA, we used site-specific mutagenesis to construct 10 mutants in which some cysteine residues were changed to serine residues. Each mutated DNA fragment was cloned into a procaryotic expression vector and the protein expressed in E. coli. Mutant proteins of the expected size were produced and analyzed for amidolytic and fibrinolytic activities. From this, it is shown that: i) the disulfide bonds in the epidermal growth factor (EGF)-like and in the kringle domains are not necessary. Moreover, disulfide bond deletion in the kringle domain improves those catalytic activities; ii) on the contrary, the disulfide bridges in the catalytic domain are essential for maintaining both activities.

Amino Acid Sequence↗

PubMed1993

Novel hirudin variants from the leech Hirudinaria manillensis. Amino acid sequence, cDNA cloning and genomic organization.

Novel hirudin variants isolated from the leech Hirudinaria manillensis, a leech more specialized for mammalian parasitism, are described. Isolation of antithrombin polypeptides was performed by ion-exchange chromatographies followed by an affinity chromatography step on immobilized thrombin. The major active component, antithrombin polypeptide peak 2 (HM2) and a second polypeptide, named HM1, were purified to homogeneity and their complete amino acid sequences were determined. The protein structure of the two hirudin variants include 64 amino acids with 6 cysteine residues at highly conserved positions. Comparison of the amino acid sequences of HM1 and HM2 with other known hirudins shows differences mainly in the central part and in the C-terminal region of the polypeptides. Particularly significant is the lack of a sulfated tyrosine residue in the C-terminal portion of the molecule which is replaced by aspartic acid. Polymerase chain reaction cloning techniques were used to isolate and characterize the cDNAs and determine the genomic structures of these hirudin-like polypeptides. The cDNA clones coding for the two variants indicate the expression of pre-hirudins of 84 amino acids where the first 20 residues constitute the signal peptide required for extracellular secretion. The leader sequence appears to be highly conserved for both isoforms and shares a complete similarity with the partial hirudin variant 2 (HV2) signal peptide sequence previously reported. The HM1 and HM2 gene fragments show the presence of four exons: the first one corresponding to a 20-amino-acid signal peptide while the other three exons share the full primary structure of the antithrombin polypeptides. HM2 was also efficiently produced in recombinant Escherichia coli by expressing a periplasmic construction containing the synthetic gene.

Amino Acid Sequence↗

PubMed1993

The asiA gene of bacteriophage T4 codes for the anti-sigma 70 protein.

The anti-sigma 70 factor of bacteriophage T4 is a 10-kDa (10K) protein which inhibits the sigma 70-directed initiation of transcription by Escherichia coli RNA polymerase holoenzyme. We have partially purified the anti-sigma 70 factor and obtained the sequence of a C-terminal peptide of this protein. Using reverse genetics, we have identified, at the end of the lysis gene t and downstream of an as yet unassigned phage T4 early promoter, an open reading frame encoding a 90-amino-acid protein with a predicted molecular weight of 10,590. This protein has been overproduced in a phage T7 expression system and partially purified. It shows a strong inhibitory activity towards sigma 70-directed transcription (by RNA polymerase holoenzyme), whereas it has no significant effect on sigma 70-independent transcription (by RNA polymerase core enzyme). At high ionic strength, this inhibition is fully antagonized by the neutral detergent Triton X-100. Our results corroborate the initial observations on the properties of the phage T4 10K anti-sigma 70 factor, and we therefore propose that the gene which we call asiA, identified in the present study, corresponds to the gene encoding this T4 transcriptional inhibitor.

Amino Acid Sequence↗

PubMed1992

Thrombomodulin is a cofactor for thrombin degradation of recombinant single-chain urokinase plasminogen activator "in vitro" and in a perfused rabbit heart model.

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) to a two-chain derivative (tcu-PA) fibrinolytically inactive. This reaction was accelerated in vitro by purified rabbit lung thrombomodulin in equimolar complex with thrombin. Polyclonal antibodies to rabbit thrombomodulin prevented this effect. We also observed that heparin and other sulfated polysaccharides had an accelerating effect on thrombin cleavage of recombinant scu-PA. Their effect was concentration-dependent and then reversed at high levels. The effect of heparin and heparan sulfate was independent and synergic with respect to thrombomodulin. All observations except the effect of heparin, could be confirmed in a Langendorff isolated rabbit heart model. From competition experiments carried out with scu-PA derivatives and mutants, we postulate that the amino-terminal sequence of rscu-PA, containing the epidermal growth factor (EGF)-like and the kringle domains is involved in the cofactor effect of thrombomodulin on scu-PA inactivation by thrombin. We conclude that a regulatory mechanism of scu-PA inactivation is present at the cell surface.

Animals↗

PubMed1991

Efficient renaturation and fibrinolytic properties of prourokinase and a deletion mutant expressed in Escherichia coli as inclusion bodies.

Prourokinase is a plasminogen activator of 411 amino acids which displays a clot-lysis activity through a fibrin-dependent mechanism, and which seems to be a promising agent for the treatment of acute myocardial infarction. The preparation of recombinant prourokinase in bacteria has been hampered by its insolubility and by difficulty in refolding the polypeptide chain. In this paper we describe the renaturation process of two recombinant proteins expressed in Escherichia coli as inclusion bodies: prourokinase and a deletion derivative (delta 125-prourokinase) in which 125 amino acids of the N-terminal region have been removed. Deletion of this sequence brings to higher refolding yields and faster kinetics (first-order rate constant of renaturation of 0.57 h-1 for delta 125-prourokinase and 0.25 h-1 for prourokinase). Our process involves sequential steps of denaturation, reduction and controlled refolding of the polypeptide chain. When applied to pure, non-glycosylated and active prourokinase, it gives a refolding yield of about 80%, demonstrating the efficiency of the renaturation procedure. Lower yields (15% and 30%, respectively, for prourokinase and delta 125-prourokinase) were obtained when the same refolding protocol was applied to inclusion bodies from bacteria. After purification to homogeneity (as shown by HPLC and SDS/PAGE) specific activities were 160,000 and 250,000 IU/mg protein, respectively, for prourokinase and delta 125-prourokinase. As with prourokinase, the deletion mutant delta 125-prourokinase displays a zymogenic nature, being activated by plasmin to the active two-chain form; however, this mutant is approximately fourfold more resistant than prourokinase to plasmin activation, and consequently shows a different fibrinolytic profile.

Amino Acid Sequence↗

PubMed1990

Arthrography of the prosthesetized painful hip: the importance of imaging and functional testing.

Thirty-four patients with painful prosthesetized hips were submitted to arthrography in order to evaluate the correlation between pain and the anatomical situation between the implants and the host bone. By injecting a local anaesthetic together with the contrast medium it was possible to perform functional tests comprising passive movements of the hip and full weightbearing for at least 5 minutes in order to ascertain whether or not the pain persisted. This yielded different results varying from total relief to persistence, depending on the type of mobilisation demonstrated by arthrography. In particular, pain was reduced in cases of mobilisation of the acetabular component, and persisted when mobilisation was in the femoral component. A correlation between functional testing, arthrography and bacteriological examination for possible low grade infection allowed for a closer study of the origin of pain, and more accurate planning of surgery when re-prosthesetization was being considered.

Aged↗

PubMed1989

Metal cementless prosthesis for vertebral body replacement of metastatic malignant disease of the cervical spine.

Sixteen patients affected with metastatic malignant disease of the cervical spine underwent cervical vertebral body replacement with an original metal cementless prosthesis. In all cases there was immediate spine stability postoperatively and pain relief. All patients were able to walk within a few days postoperatively without any rigid external support. Follow-up roentgenograms have shown nonprosthetic mobilization.

Adult↗

PubMed1989

Vertebral artery release in vertebro-basilar insufficiency due to cervical uncoarthrosis.

The authors report 31 cases submitted to transversectomy and uncosectomy according to the Jung and Kehr method for the treatment of vertebrobasilar insufficiency secondary to compression of the vertebral artery due to cervical uncoarthrosis. The results obtained were all classified as excellent or good. Emphasis is placed on several diagnostic aspects such as arteriographic investigation, and some details of the surgical technique used for the prevention of complications are discussed.

Adult↗

PubMed1988

Phage T4 DNA codes for two distinct 10-kDa proteins which strongly bind to RNA polymerase.

Radioactive proteins have been synthesized in vitro from a coupled transcription-translation system primed with bacteriophage T4 DNA. The labeled proteins were chromatographed on RNA polymerase-Sepharose affinity columns in order to identify a protein which comigrates with the 10-kDa anti-sigma subunit of T4-modified RNA polymerase. When we primed the in vitro system with specific restriction fragments of T4 DNA, we found that there seemed to be two widely separated genes which code for this protein. The products of these two genes were compared by two-dimensional electrophoresis; they were found to have different charges, even though they had the same molecular weight and strong affinity for RNA polymerase. One of these proteins exists in a form which has the same charge as the 10-kDa subunit isolated from purified T4-modified RNA polymerase. We report a preliminary mapping experiment within the 22.5-kb fragment harboring this gene.

Chromatography, Affinity↗

PubMed1988

Percutaneous lumbar nuclectomy according to the Onik method: indications, technique, first results.

The authors report their first experience with 20 cases of lumbar herniated disc treated by percutaneous nuclectomy according to the Onik method. The methods used for a precise preliminary assessment of the prolapse in order to establish correct indications are discussed. The technique of the operation is then described. The simplicity and relative absence of trauma in this type of surgery, associated with the patient's rapid recovery, drastically reduces hospitalization time, with consequent social and economic advantages. The clinical results at follow-up are sufficiently rewarding to suggest a wider use of this method, but it is essential to restrict it to the indications laid down, which are fully illustrated. The method is applicable only to contained hernias and is contra-indicated in extruded hernias.

Adult↗

PubMed1987

Contrast arthrography in symptomatic varus knees.

The authors carried out a study of contrast arthrography in 48 cases of primary genu varum, i.e. not secondary to trauma or to pathological conditions of the hip or foot. The patients all had symptoms and were classified according to the commonly used Trillat-Dejour method into four groups or stages depending on the degree of varus and the radiographic and arthrographic findings. Associated lesions secondary to the basic pathology were observed in all cases. These ranged from hypertrophic synovitis to torn medial or lateral menisci up to total compromise of the joint typical of stage 4 arthritic varus knee. In these patients, contrast arthrography is useful in planning surgery, particularly high tibial osteotomy. It also enables the surgeon to decide whether complementary intra-articular measures are indicated without having to resort to arthroscopy. This is always a more invasive method, necessitating general anaesthesia, as these knees are quite painful and the arthroscopic access is difficult.

Aged↗

PubMed1986

N-imidazolylchroman-4-ones, N-imidazolyl-1-tetralones, and their alcohols as hypolipemic agents raising high-density lipoproteins.

A series of 3-(1-imidazolyl)chroman-4-ones and 2-(1-imidazolyl)-1-tetralones II, some of their alcohols, and some related compounds were synthesized and tested for hypolipidemic activity. Compounds II, bearing appropriate lipophilic substituents on the phenyl ring, strongly reduced total serum cholesterol while raising high-density lipoprotein cholesterol in diet-induced hypercholesterolemic rats. 3-(1-Imidazolyl)chroman-4-ols and 2-(1-imidazolyl)-1-tetralols corresponding to II retained the hypolipidemic activity while removal of the carbonyl or hydroxy group adjacent to imidazole gave inactive compounds. Although many of the active compounds significantly increased liver weight, the one studied as a model, 6-chloro-3-(1-imidazolyl)-2,3-dihydro-4H-1-benzopyran-4-one (5), caused no peroxisome proliferation. Compound 5 and the corresponding alcohol 40, as representatives of the ketone and alcohol series, showed significant hypolipidemic activity in normolipemic rats. Some of the compounds assayed in cholesterol biosynthesis inhibited acetate incorporation but none inhibited HMG-CoA reductase. 5-Bromo-6-hydroxy-2-(1-imidazolyl)-3,4-dihydro-1(2H)-naphthalenone (38), which showed strong activity but caused little hepatomegaly in the rat, was chosen for further pharmacological evaluation.

Alcohols↗

PubMed1985

Effect of the antilipolytic compound acipimox on peroxisome marker enzymes, lipid pattern and biotransformation related functions in rat liver.

The antilipolytic drug acipimox (5-methylpyrazine-2-carboxylic acid 4-oxide) was given to male rats for 1 week at 500, 1000 and 2000 mg/kg/day and for 2, 6 and 7 months at 20, 100 and 500 mg/kg/day. The peroxisome proliferative effect was evaluated determining the activity of catalase and carnitine acetyltransferase, the rate of cyanide-insensitive palmitoyl CoA oxidation and the electrophoretic profile of liver polypeptides. Hepatic lipid content and distribution were evaluated after 2 and 6 months' treatment. The effect on liver detoxificating function was evaluated by assaying glutathione, cytochrome P-450, glutathione-S-transferase and glutathione-reductase activities after 7 months' treatment. Sub-acute and chronic treatment with a wide range of acipimox doses did not cause hepatomegaly, liver peroxisome proliferation or liver steatosis and did not change some important biochemical variables related to detoxification and biotransformation mechanisms. Acipimox given to rats does not have the negative side-effects of other compounds and seems a safe blood lipid lowering drug.

Animals↗

PubMed1985

The surgical treatment of metastatic tumours of the lumbar spine.

Thirty-three patients affected by metastases in the lumbar spine were treated surgically with the objective of stabilising the affected area while at the same time alleviating involvement of the neurological structures. The surgical technique was adapted to the type and site of the lesion. The lumbar spine differs from the dorsal and cervical spine in that there are two quite different levels of neurological risk above and below L2. Above L2 it is severe, of rapid onset, and usually irreversible. Below L2 it is more easily reversible, even if surgical intervention is not carried out immediately. In cases with neurological involvement, the results were always very much better in the cases operated on early. This led us to carry out the operation prophylactically with rewarding results because none of these patients subsequently developed neurological damage.

Adult↗

PubMed1984

Electron microscopy study of the aminoacyl-tRNA synthetase multienzymatic complex purified from rabbit reticulocytes.

The morphology of the high molecular weight complex of aminoacyl-tRNA synthetases purified from rabbit reticulocytes has been investigated by electron microscopy. To stabilize it against dissociation, the complex was also studied after chemical crosslinking. Freeze fracture, drying shadowing and negative staining were used. The reticulocyte complex appears as a moderately elongated object with no simple compact shape. Upon rapid drying, the native complex dissociates and shows the presence of approximately 8 globular components, the individual size of which is 80-100 A. The surface of the cross-linked complex shows several distinct globules which appear to extend out of a central core. The irregularly shaped crosslinked complex has a maximal dimension of 350 +/- 50 A. The morphology of the synthetase complex is discussed with respect to some of the properties of this type of multienzymatic system.

Amino Acyl-tRNA Synthetases↗