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M Ruoppolo

Publications and source records attributed to M Ruoppolo.

At least 37 records · Page 2Linked to original sources
PubMed1995

Refolding by disulfide isomerization: the mixed disulfide between ribonuclease T1 and glutathione as a model refolding substrate.

Protein folding, associated with isomerization of disulfide bonds, was studied using the mixed disulfide between glutathione and reduced ribonuclease T1 (GS-RNase T1) as a stable soluble and homogeneous starting material; conditions were selected to model those within the lumen of the endoplasmic reticulum where native disulfide bonds are formed in protein biosynthesis. Folding was initiated by addition of free glutathione (GSH +/- GSSG) to promote thiol-disulfide interchange and was monitored by intrinsic protein fluorescence, appearance of native ribonuclease activity, HPLC, and nonreducing SDS-PAGE. All the analyses indicated that native RNase T1 was recovered in high yield in a variety of redox conditions. Appearance of native activity followed first-order kinetics; kinetic analysis of the intrinsic fluorescence changes indicated an additional rapid process in some conditions, interpreted as the formation of a nonnative intermediate state. Analysis by HPLC and SDS-PAGE also indicated the formation of transient intermediates. In 1.5 M NaCl, GS-RNase T1 adopts a compact native-like conformation; refolding by thiol-disulfide interchange in these conditions was accelerated approximately 2-fold. Refolding of GS-RNase T1 was catalyzed by protein disulfide isomerase (PDI); substoichiometric quantities of PDI accelerated refolding several-fold. GS-RNase T1 refolding was inhibited by BiP; refolding was completely blocked in presence of a 5-fold molar excess of BiP, and the yield of refolding was substantially reduced by equimolar concentrations of BiP; the refolding was then restored by the addition of ATP. GS-RNase T1 is a convenient model substrate for studying protein folding linked to native disulfide formation in conditions comparable to those within the lumen of the endoplasmic reticulum.

Animals↗
PubMed1995

[Benign prostatic hypertrophy. Optimized diagnosis].

The optimal diagnostics of B.P.H. must be efficient, cheap and of spare invasiveness either physical than psychological. The optimization of diagnosis is the obtainment of high quality in the services provided, matched to a correct and shrewd utilisation of resources. To optimise a diagnosis efficiency is necessary, which means the largest degree of standardization of diagnostical procedures, with responsible management of economical factors. A course of standardization, cause his excessive simplification, cannot shared to a lot of Urologists, because in their opinion there is a risk to lose informations in every single patient, but is essential to use a universal language that make easy the comparison with patients and with results whether in order to clinics or trails. The standardisation of diagnostics consists to obtain the maximum of informations from present methodology and that is possible to realize in two way: improving the technique of execution and including the diagnostic test in the appropriate point of algorithm.

Age Factors↗
PubMed1995

[TURP: what and when. Indications, results, complications: our experience].

Transurethral resection of the prostate (T.U.R.P.) has indeed replaced open surgery in the great majority of cases, particularly when the gland is estimated to weight 50-60 g or less. After prostatectomy the patient resumes a normal voiding pattern and obstructive symptoms quickly disappear, although irritative symptoms tend to persist for some time.

Humans↗
PubMed1995

[Radical prostatectomy: our experience].

Between July 1989 and June 1994 32 men (48-73 years old) underwent radical retropubic prostatectomy for prostate cancer. 26 patients (70%) presented with symptoms of bladder outflow obstruction. The primary tumour was understaged preoperatively in 5 patients (15.5%). At follow-up (6-66 months, average 24) significant urinary incontinence not occurred in our patients; sixty-two per cent reported a substantial problem with reduced or absent erection. A total of 23 patients was free of clinical or biochemical progression, observed in 28% of cases as distant or local progression. Radical prostatectomy is being performed with increasing frequency: trends in morbidity have been identified.

Aged↗
PubMed1995

[External devices: for which patients?].

Vacuum therapy is a reversible, non-invasive form of treatment for partial impotence, with great success in USA. The story of vacuum therapy begins about 1960, when Osbon developed a vacuum tumescence device which he personally used for more than 20 years. The device was made commercially available many years ago and has been marketed under several names. The newer systems have incorporated a negative pressure pump to achieve vacuum. Osbon's system was patented in 1983, sale is permitted by the U.S. Food and Drug Administration, and it is available by prescription only. More than 10,000 units have been sold. There are four different types of vacuum therapy: 1) loading cone + constriction band; 2) external splint + negative pressure; 3) Negative pressure + constriction band; 4) Negative pressure + intracavernous injections, without the use of constriction band. Each basic system will be described. The authors make a comparison between the use of negative pressure devices plus tension band and the use of negative pression devices without tension band (plus C.I.D. with vaso-active agents). In the second case there's a sort of synergistic action between vaso-active agents and vacuum therapy, representing a sort of "vaso-active exercise" of the erectile tissue. There are no absolute contraindications to use of external penile devices and potential contraindications are few. The external penile devices described represent a reversible therapeutic modality that can augment an inadequate erection and they should prove useful in any man who needs erectile enhancement. These devices appear to be particularly effective in men with partial impotence in whom only erectile enhancement is needed.

Constriction↗
PubMed1994

Analysis of RNase A refolding intermediates by electrospray/mass spectrometry.

Electrospray/mass spectrometry (ES/MS) was extensively used to obtain information on disulphide-containing intermediates formed during refolding of bovine pancreatic ribonuclease A. The analysis showed the existence of an equilibrated population of disulphide bonded intermediates, and indicates that intermediates containing two intramolecular S-S are predominant until late stages of the refolding process. Mixed disulphides with exogenous glutathione were also detected, supporting previous evidence of conformational restrictions on the ability of RNase A to form intramolecular disulphides. The results indicate that ES/MS is a suitable technique to detect and characterize refolding intermediates.

Animals↗
PubMed1994

Protein-S-S-glutathione mixed disulfides as models of unfolded proteins.

Mixed disulfides between glutathione and the reduced forms of disulfide-bonded proteins were generated and characterized to explore their suitability as models of the unfolded state of newly-synthesized secretory proteins. RNase T1 and alpha-lactalbumin were reduced and converted to mixed disulfide derivatives, named GS-RNase T1 and GS-alpha-lactalbumin, in good yield; the molecular masses of the derivatives were confirmed by electrospray mass spectrometry. The intrinsic fluorescence of the derivatives and the binding of the hydrophobic fluorescent dye ANS were characteristic of fully unfolded proteins. Fluorescence studies and enzyme activity data indicated that GS-RNase T1 could be refolded to a nativelike state at NaCl concentrations greater than 1.5 M, as was previously demonstrated for the reduced, carboxymethylated derivative of this protein. The [NaCl]-dependent folding/unfolding equilibrium for GS-RNase T1 was reversible and could be influenced by urea. Fluorescence studies indicated that GS-alpha-lactalbumin showed a [NaCl]-dependent partial shift toward a more nativelike state, which was enhanced by the presence of Ca2+ ions. Both of the GS derivatives stimulated the ATPase activity of BiP, with apparent affinities in the range 0.1-1.0 mM. The results indicate that these GS-S-protein mixed disulfide derivatives are ideal model unfolded proteins that can be used as substrates for detailed studies on secretory protein folding in vitro and on the interactions between unfolded proteins and facilitators of protein folding.

Adenosine Triphosphatases↗
PubMed1994

[Penile nuclear magnetic resonance (NMR)].

Nuclear Magnetic Resonance (NMR) is a new diagnostic technique with great opportunities of application in the field of the penile pathologies. A new interest for this diagnostic technique was born when the use of vasoactive agents, like papaverine or PGE1, and the use of para-magnetic contrast agents, like gadolinium, were introduced. The introduction of dynamic NMR in andrology allowed a better definition of anatomical details and a better knowledge of penile micro-circulation. N.M.R. is showing a great diffusion, because of a little invasiveness (X-rays are not used in this technique). The Authors show a wide spread of possible applications of NMR in penile pathologies, helping in the interpretation of the images. In conclusion the authors describe NMR as a diagnostic technique with great possibilities of improvement, even if the high costs don't allow a better diffusion until now.

Condylomata Acuminata↗
PubMed1993

Disulfide isoform intermediates in the reoxidation of recombinant human basic fibroblast growth factor.

The reoxidation of human recombinant basic fibroblast growth factor was investigated following treatment of the protein with a mixture of reduced and oxidized glutathione, both in the absence and in the presence of protein disulfide isomerase. The oxidative process took place throughout the formation of two transient intermediates and yielded a stable bFGF derivative, GS2-bFGF. All of these components were separated by HPLC and accurately characterized at the molecular level by advanced mass spectrometric procedures. When the reoxidation was carried out in the presence of PDI, a 4-fold increase in the reaction rate was estimated. A mixed disulfide with a single glutathione molecule was shown to occur in the two transient intermediates, each of which has different cysteine residues involved in the linkage. The final product GS2-bFGF was structurally different from other bFGF derivatives previously described [Thompson, S. A. (1992) J. Biol. Chem. 267, 2269-2273; Caccia et al. (1992) Eur. J. Biochem. 204, 649-655]. The four cysteine residues are all involved in disulfide bridges; Cys 34 and Cys 78 are linked to exogenous glutathione, whereas Cys 91 and Cys 101 form an intramolecular S-S bridge.

Chromatography, High Pressure Liquid↗
PubMed1993

Structural characterization of a biologically active human lipocortin 1 expressed in Escherichia coli.

Lipocortin or annexin 1 is a calcium-dependent phospholipid-binding protein which probably acts as a glucocorticoid- regulated anti-inflammatory factor. cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl thio-beta-D-galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 30 mg/l bacterial culture. Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N-terminal alanine; tryptic peptide mapping by fast-atom-bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge. Recombinant human lipocortin 1 reduces the carrageenin-induced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase A2 activity in vitro; in both cases, a dose-related response is observed.

Amino Acid Sequence↗
PubMed1993

[Urologic laparoscopy: preliminary experience].

Laparoscopy is a safe and reliable procedure for treatment of cryptorchid testicle, varix ligation, staging of lymph nodes in prostatic and in bladder cancer. Recently this procedure is adopted in management of renal cysts. From 2/92 to 7/92, we performed laparoscopic assessment in 31 patients: in 15 cases for varicocelectomy, in 9 for renal cysts, in 2 for lymph nodes dissection, in 3 for preoperatory evaluation, in 1 for unpalpable testicle and in 1 case for ileal perforation in orthotopic neobladder. In 1 case the procedure was unsuccessful because of damage of the spleen. All the patients were mobilized within 1 day postoperative treatment and discharged within 3 days. There are many present and potential application of laparoscopic surgery in urologic pathology, but we think that this technique must be carefully applied in order to avoid complications.

Female↗
PubMed1992

Characterization by mass spectrometry of a recombinant hepatitis delta virus antigen and its proteolytic products.

The recombinant hepatitis delta virus antigen was obtained as a chimaeric protein fused to the C-terminus of the phage MS2 RNA polymerase. Following induction of the temperature-sensitive promoter, two major polypeptides of about 34 kDa and 29 kDa, and two minor peptides about 21 kDa and 18 kDa, were obtained on PAGE. The 34-kDa protein was identified as the expected recombinant protein by confirming 82% of the primary structure using fast-atom-bombardment mass spectrometry. The most represented degradation product, i.e. the 29-kDa polypeptide, was also characterized by means of mass spectrometry and found to be produced by cleavage between amino acids 261 and 265. The presence of two main protein bands, with a similar difference in size, is also a typical feature of delta antigens, both extracted and recombinant, and it is considered to be derived either from heterogeneity of viral sequences, which can encode hepatitis delta antigen proteins of 195 and 214 amino acids, or from proteolysis of a single precursor. Since the data were obtained with a single viral sequence coding for 195 amino acids fused to 106 residues from MS2 polymerase, there is direct evidence that intrinsic structural properties of the protein sequence are able to cause a specific proteolysis resulting in the presence of two major forms, of which the smaller is 35-40 amino acids at the C-terminus. The recombinant protein can be used as an antigenic substitute of viral antigens both for immunoassays and for the preparation of anti-(hepatitis delta virus) antisera.

Amino Acid Sequence↗
PubMed1992

Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues.

The production of recombinant human basic fibroblast growth factor (rhbFGF) in Escherichia coli cells yielded active forms of this polypeptide which, however, displayed a high degree of instability towards oxidative processes. Biochemical studies in our laboratory and those of others indicated that the reactivity of the four cysteine residues was the main cause of the observed instability. Several attempts to obtain more stable derivatives of rhbFGF were carried out by modification of the sulfhydryl groups. Among these, treatment of rhbFGF with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm-FGF). Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group. The remaining cysteines (34 and 101) were not modified under the conditions used and were found to be in the reduced form. Cm-FGF and unmodified rhbFGF showed similar affinity both for heparin and for high-affinity receptors. Cm-FGF was more stable than the unmodified molecule as measured by HPLC and SDS/PAGE analysis. Interestingly, Cm-FGF was more active than unmodified rhbFGF in stimulating proliferation of endothelial cells and DNA synthesis in 3T3 fibroblasts. This new derivative could represent a desirable complementation to rhbFGF for the development of more stable pharmaceutical formulations in wound healing applications.

Animals↗
PubMed1992

Study of interaction of styrene oxide with angiotensin by mass spectrometry.

The present study describes how mass spectrometry was extensively applied to the characterization and quantification of modified amino acids within the polypeptide chain of Angiotensin I, chosen as model substrate, combining the use of fast atom bombardment mass spectrometry with gas chromatography--mass spectrometry. The reaction products after in vitro incubation of Angiotensin I with styrene oxide, a well known carcinogen, under different conditions, have been characterized: a prominent reactivity of several potential nucleophilic sites of Angiotensin I was shown, including two histidine residues and a tyrosine residue; it is worth noting that it has never been stated that tyrosine is highly reactive with styrene oxide. The results obtained demonstrate the usefulness of mass spectrometry for the structural determination of chemically modified amino acids in peptides and proteins, and the presence of a reliable relationship between reaction conditions and the production of alkylated amino acids. This characterization procedure offers the possibility of identifying reactive sites following exposure to unknown alkylating agents.

Angiotensin I↗
PubMed1992

[Intracavernous infusion of drugs].

In 1982 Virag presented the initial experience of use in diagnosis and treatment of impotence. PGE1 has been showed a safe and effectiveness drug. From september 1989 to october 1991 210 patients with erectile disorders were managed by I.C.I. with PGE1 with minimal side effects.

Alprostadil↗
PubMed1991

[Endocavernous drug infusions revisited].

From the beginning of the eighties papaverine was well established as the drug of choice for diagnosis and management of vasculogenic impotence. In the second half of the eighties decade many authors referred on experimental use of PGE1 in erection disease. PGE1 has been showed a safe and effectiveness drug. During the period September 1989-October 1990 at our Institution 98 pts. with erectile disorders were managed by I.C.I. with PGE1. Out of 98 cases 88 had success. Prolonged erection occurred in 4 pts. (4.2%). 10 pts. showed only tumescence. So there are been treated by I.C.I. with combined papaverine and PGE1 with better results. No side effects were formed in 10 pts. managed by self-injection of PGE1.

Alprostadil↗
PubMed1990

Urinary NAG excretion after anesthesia-free extracorporeal lithotripsy of renal stones: a marker of early tubular damage.

Second generation lithotripters require a higher number of shocks per session as well as an increased rate of secondary treatments for complete stone disintegration compared to the original spark gap lithotripter. The clinical relevance of biological side effects caused by such treatment are less known. We evaluated urinary excretion of N-acetyl-glucosaminidase (NAG) before and after lithotripsy in 50 patients treated with a low pressure spark gap lithotripter (Dornier HM3) and in 36 patients treated with a piezoelectric lithotripter (Wolf Piezolith 2200) in an attempt to evaluate their side effects on renal tissue. The urinary excretion of NAG increased after both spark gap lithotripsy using the modified HM3 and piezoelectric lithotripsy. These changes may be associated with slight tubular damage that would occur after anesthesia-free lithotripsy in patients subjected both to a high number of shocks and to secondary treatments.

Acetylglucosaminidase↗