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G Zanetti

Publications and source records attributed to G Zanetti.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of the flavins and the iron-sulfur centers of glutamate synthase from Azospirillum brasilense by absorption, circular dichroism, and electron paramagnetic resonance spectroscopies.

Azospirillum brasilense glutamate synthase has been studied by absorption, electron paramagnetic resonance, and circular dichroism spectroscopies in order to determine the type and number of iron-sulfur centers present in the enzyme alpha beta protomer and to gain information on the role of the flavin and iron-sulfur centers in the catalytic mechanism. The FMN and FAD prosthetic groups are demonstrated to be non-equivalent with respect to their reactivities with sulfite. Sulfite reacts with only one of the two flavins forming an N(5)-sulfite adduct with a Kd of approximately 1 mM. The enzyme-sulfite complex is reduced by NADPH, and the complexed sulfite is competitively displaced by 2-oxoglutarate, which suggests the reactive flavin to be at the imine-reducing site. These data are in agreement with the two-site model of the enzyme active center proposed on the basis of kinetic studies [Vanoni, M.A., Nuzzi, L., Rescigno, M., Zanetti, G., & Curti, B. (1991) Eur. J. Biochem. 202, 181-189]. Each enzyme protomer was found, by chemical analysis, to contain 12.1 +/- 0.5 mol of non-heme iron. Electron paramagnetic resonance spectroscopic studies on the oxidized and reduced forms of glutamate synthase demonstrated the presence of three distinct iron-sulfur centers per enzyme protomer. The oxidized enzyme exhibits an axial spectrum with g values at 2.03 and 1.97, which is highly temperature-dependent and integrates to 1.1 +/- 0.2 spin/protomer. This signal is assigned to a [3Fe-4S]1+ cluster (Fe-S)I. Reduction of the enzyme with an NADPH-regenerating system results in reduction of the [3Fe-4S]1+ center to a species with a g approximately 12 signal characteristic of the S = 2 spin state of a [3Fe-4S]0 cluster. The NADPH-reduced enzyme also exhibits an [Fe-S] signal at g values of 1.98, 1.95, and 1.88, which integrates to 0.9 spin/protomer and is due to a second cluster (Fe-S)II. Reduction of the enzyme with the light/deazaflavin method results in a signal characteristic of [Fe-S] clusters with g values of 2.03, 1.92, and 1.86 and an integrated intensity of 1.9 spin/protomer. This signal arises from reduction of the (Fe-S)II center and from that of the third, lower potential iron-sulfur center (Fe-S)III. Circular dichroism spectral data on the oxidized and reduced forms of the enzyme are more consistent with the assignment of (Fe-S)II and (Fe-S)III as [4Fe-4S] clusters rather than [2Fe-2S] centers.

Azospirillum brasilense↗
PubMed1992

Cytokine production after intravenous or peritoneal gram-negative bacterial challenge in mice. Comparative protective efficacy of antibodies to tumor necrosis factor-alpha and to lipopolysaccharide.

The production of TNF-alpha, IL-1, and IL-6 was measured in mice after bolus i.v. Escherichia coli O111 LPS injections and during bacteremia induced either by bolus i.v. or by i.p. challenges of live E. coli O111. High but transient TNF-alpha peaks were observed after bolus i.v. LPS or bacterial challenges. In contrast, the levels during lethal peritonitis increased progressively to values 50- to 100-fold lower than the peak values observed after i.v. injections, and remained sustained until death. Whereas after i.v. challenge with 1000 LD50 of LPS, anti-TNF-alpha antibody fully protected mice from death and reduced serum IL-1 and IL-6 levels, anti-TNF-alpha antibody did not improve the survival of mice nor reduced serum IL-1 and IL-6 levels after i.p. bacterial challenge. In contrast to anti-TNF-alpha antibodies, anti-LPS antibodies were protective in the peritonitis model. Protection was accompanied by a striking reduction of bacterial numbers and of TNF-alpha, IL-1, and IL-6 levels in the serum, but the levels of these cytokines were only marginally affected in the peritoneal lavage fluid. This latter observation demonstrates that the local peritoneal cytokines did not diffuse readily into the circulation, thus suggesting that at least part of the circulating cytokines are produced systemically. In conclusion, the striking differences between cytokine profiles as well as the divergent efficacy of anti-TNF-alpha antibody after i.v. bolus and after i.p. challenges suggest that TNF-alpha may not be as important in the pathogenesis of lethal peritonitis than after lethal acute bacteremia.

Animals↗
PubMed1992

Studies on the holoenzyme biogenesis of the spinach ferredoxin-NADP+ reductase.

An expression plasmid, pPreFNR, in which the DNA sequence coding for the spinach ferredoxin-NADP+ reductase precursor was under the control of prokaryotic transcription and translation initiation signals has been constructed. The plasmid directed the synthesis in Escherichia coli of a 43-kDa immunoreactive polypeptide which could be identified with the reductase preprotein. Analyses of bacterial extracts showed that the precursor was unstable and devoid of catalytic activities, suggesting that the presence of the transit peptide would not allow the assembly in E. coli of an active preholoenzyme. Furthermore, the reductase precursor was found to undergo a processing in E. coli. The proteolysed form, which retained 13 of the 55 residues of the transit peptide, was active, suggesting that removal of the first 42 residues of the presequence enabled the protein to properly fold and to bind the FAD prosthetic group in the bacterial host, as it was previously shown in the case of the mature form of the spinach reductase.

Amino Acid Sequence↗
PubMed1992

A case of Merkel-cell carcinoma metastatic to the tonsil.

Metastatic tumours are rare in the tonsil. We describe a 69-year-old male patient who had previously undergone a resection of a Merkel-cell tumour of the left forearm and subsequently presented with a left tonsillar tumour. Biopsy revealed a metastatic Merkel-cell carcinoma. Our patient is the first described case of Merkel-cell carcinoma metastasizing to the tonsil. The clinical and histopathological picture of this rare tumour is presented, along with a review of literature.

Aged↗
PubMed1992

Prognostic significance of prostate-specific antigen in endocrine treatment for prostatic carcinoma.

We have studied the prognostic significance of prostate-specific antigen (PSA), monitored monthly, in 24 patients with prostatic cancer (5 D1, 19 D2) on endocrine therapy. The pretreatment levels of PSA were high in all patients (mean value 41 ng/ml). It was found that PSA levels at the end of the first and sixth months of treatment were reliable prognostic indicators. At the first month evaluation PSA had decreased more than 50% from the initial values in the 16 patients with stable disease, while it had decreased less than 50% in those with progressing disease. At the end of 6 months, patients with stable disease had PSA levels within the normal range, while 8 of the patients who had progressing disease had levels higher than 10 ng/ml. Respectively 6 and 2 patients had also had increases in PSA levels at 3 and 6 months before scintigraphic demonstration of increased bone metastases.

Aged↗
PubMed1992

[Recurrence of lithiasis after extracorporeal lithotripsy, percutaneous surgery, and open surgery for calculi of the upper urinary tract].

The safety and short-term effectiveness of percutaneous nephrolithotomy (PCNL) and extracorporeal shock wave lithotripsy (ESWL) have already been well established. However, long-term follow-up studies are essential to prove that the recurrence rate after PCNL and/or ESWL is equal or even better than that of surgery. We reviewed 57 patients treated with ESWL monotherapy, 45 patients treated by PCNL (or by PCNL combined with ESWL for complete staghorn stones) and 59 patients treated by open surgery who all had at least three years of follow-up. The overall recurrence rate for ESWL was 37% for PCNL and for open surgery 39%. ESWL, PCNL and open surgery all showed a higher rate of recurrence and persistent bacteriuria when associated with residual stones. Therefore every effort should be made to remove residual fragments following stone treatment. We conclude that because of its efficacy and low morbidity ESWL should be the treatment of choice for most of renal calculi. Complete staghorn calculi are best treated with open surgery because the complete elimination of all calculous material and the eradication of infection are achieved at a higher rate than by PCNL and ESWL.

Adult↗
PubMed1992

[Metastasis and markers].

Tumor markers are antigens which can be associated with certain malignancies. A variety of markers have been demonstrated in genitourinary tumors. The best known examples are human chorionic gonadotropin (bHCG) and alpha-fetoprotein (AFP) for testicular tumors, prostatic acid phosphatase (PAP) and prostatic specific antigen (PSA) for prostatic cancer. The plasma levels of these substances are influenced by the tumor mass and therefore by the tumor stage. Markedly elevated plasma levels can be demonstrated when metastases are present, although a few patients without metastases may elaborate abnormal amount of markers. The removal of the primary tumor leads to a fall to normal levels: a still increased level indicates residual primary tumor or the presence of metastases. Measurements of markers are also of value in estimating the effects of medical treatment and in detecting local or distant recurrences.

Acid Phosphatase↗
PubMed1992

[Use of ultrasound-guided percutaneous nephrostomy before and after ESWL: 4 years of experience].

We evaluated the usefulness of percutaneous nephrostomy in 1700 patients treated for reno-ureteral stones by extracorporeal shock wave lithotripsy (SWL). Out of this group 81 patients (5.8%) underwent echo-guided percutaneous nephrostomy (EPCN): the procedure has been performed in local anesthesia in 38% of the cases (31 pts) before SWL and in 62% (50 pts) after. The majority of EPCN were carried out for the presence of acute or chronic ureteral obstruction with echographic evaluable dilation of pyelocaliceal system when retrograde ureteral manipulations failed or were considered unsuitable. EPCN before SWL was performed because of ureteral stone and uncomplicated pyelocalyceal dilation (19 pts); ureteral stone, pyelocaliceal dilation and fever > 38 degrees C (3 pts); ureteral stone, pyelocalyceal dilation and functional IVP exclusion (5 pts); pyelic stone in solitary kidney (1 pts); ureteral stones in pregnancy (2 pts). EPCN after SWL was performed because of uncomplicated, persistent pile-up (31 pts); ureteral pile-up complicated by fever and colics (9 pts); ureteral obstructing fragments (2 pts); double J obstruction by stone dust (3 pts); persistent ureteral pile-up around double J (3 pts); anuria in solitary kidney (1 pt). Out of the patients who underwent EPCN before SWL 13% were stone free and without drainage at discharge, 77% had passable stone fragments at discharge and drainage has been taken out at 15-30 days check up, 10% had unbroken stone and underwent with drainage to ureterolithotripsy.(ABSTRACT TRUNCATED AT 250 WORDS)

Humans↗
PubMed1992

[Postoperative impotence].

Increasing interest has developed in recent years about the preservation of sexual function after urological surgery. The pelvic plexus which is formed by parasympathetic visceral efferent preganglionic fibers that arise from the sacral center provides autonomic innervation to the corpora cavernosa. The cavernous nerves emerge from the pelvic plexus and then travel along the posterolateral portion of the seminal vesicle and prostate and then along the membranous urethra. The preservation of the nerve supply of the corpora during urological and pelvic procedures is of vital importance to preserve potency.

Erectile Dysfunction↗
PubMed1991

Mechanistic studies on Azospirillum brasilense glutamate synthase.

The reaction mechanism of Azospirillum brasilense glutamate synthase has been investigated by several approaches. 15N nuclear magnetic resonance studies demonstrate that the amide nitrogen of glutamine is reductively transferred to 2-oxoglutarate in an irreversible manner with no release of the transferred ammonia group into the medium. Identical results were obtained using thio-NADPH and acetylpyridine-NADPH, which are shown to be less efficient substrates of the enzyme than NADPH. Similarly, no exchange of the ammonia group being transferred with exogenous ammonium ion was observed during catalysis. The glutamate formed as the product of the iminoglutarate reduction was determined to be in the L configuration. The enzyme was also found to catalyze, under anaerobic conditions, the exchange of the 4proS H of NADPH with solvent both in the absence and in the presence of 2-oxoglutarate and glutamine. The reductive half-reaction is therefore a reversible segment of the overall irreversible amidotransferase reaction. 15N NMR studies also showed that the enzyme does not catalyze glutamate dehydrogenase/oxidase reactions or any observable glutaminase activity under neutral (pH 7.5) conditions. Glutaminase activity was also not observable with the reduced enzyme alone or in the presence of D-glutamate (a competitive inhibitor of glutamate synthase with respect to 2-oxoglutarate, with a Ki of about 11 microM) or with the oxidized enzyme in the presence of 2-oxoglutarate, D-glutamate, or NADP+. These data confirm species-dependent differences of A. brasilense glutamate synthase with respect to the enzyme from other sources.

Ammonia↗
PubMed1991

The kinetic mechanism of the reactions catalyzed by the glutamate synthase from Azospirillum brasilense.

The reactions catalyzed by glutamate synthase from Azospirillum brasilense have been investigated by a combination of absorption spectroscopy, steady-state kinetic measurements and experiments with stereospecifically labelled substrate. The data show that both L-glutamine-dependent and ammonia-dependent reactions of the glutamate synthase from A. brasilense follow an identical two-site uni-uni bi-bi kinetic mechanism, in which the enzyme is alternately reduced by NADPH and oxidized by the iminoglutarate formed on addition of ammonia to the C2 of 2-oxoglutarate. The spectroscopic experiments support the involvement of the enzyme chromophores (flavins and iron-sulfur centers) in both reactions. Finally, using stereospecifically labelled NADPH, we showed that the enzyme from Azospirillum is specific for the transfer of the 4S hydrogen of NADPH. During the catalysis of both L-glutamine-dependent and ammonia-dependent reactions, this hydrogen atom equilibrates with the solvent. The data obtained with glutamate synthase from A. brasilense, a diazotroph, differ significantly from those regarding the ammonia-dependent reaction of other glutamate synthases. The ammonia-dependent activity of glutamate synthase from Azospirillum is not physiologically significant, representing only a segment of the overall physiological L-glutamine-dependent activity and requiring the enzyme flavins and iron-sulfur centers. Finally, the data are not consistent with the hypothesis [Geary, L. E. & Meister, A. (1977) J. Biol. Chem. 252, 3501-3508] that the small subunit of glutamate synthase is endowed with a glutamate-dehydrogenase-like activity.

Ammonia↗
PubMed1991

Probing the role of lysine 116 and lysine 244 in the spinach ferredoxin-NADP+ reductase by site-directed mutagenesis.

Two mutants of the spinach ferredoxin-NADP+ reductase (FNR) were constructed, expressed by using a heterologous expression system previously described (Aliverti, A., Jansen, T., Zanetti, G., Ronchi, S., Herrmann, R. G., and Curti, B. (1990) Eur. J. Biochem. 191, 551-555), and purified to homogeneity. The mutant enzymes FNR-Lys116Gln and FNR-Lys244Gln were similar to the wild-type enzyme in the physicochemical properties measured; however, both enzymes showed a lower activity. Steady-state kinetic analyses and NADP+ binding measurements of the mutant proteins have revealed that the Lys116Gln enzyme had a catalytic efficiency (kcat/KmNADPH) 500-fold lower than that of the wild-type enzyme, and its interaction with NADP+ was greatly impaired. The Lys244Gln enzyme instead had almost the same kcat in the ferricyanide reductase reaction as the wild-type enzyme, while higher values of KmNADPH and Kd for NADP+ were observed. Thus, protein engineering has enabled us to define the role of Lys-116 and Lys-244 in ferredoxin-NADP+ reductase; whereas Lys-244 contributes to the stabilization of the Michaelis complex, the Lys-116 side chain plays an important role also in the further steps of the catalytic cycle, because both Km and kcat are highly affected by the mutation.

Binding Sites↗
PubMed1991

Identification of Lys116 as the target of N-ethylmaleimide inactivation of ferredoxin:NADP+ oxidoreductase.

Oxidized ferredoxin:NADP+ oxidoreductase (FNR) was slowly and irreversibly inactivated by N-ethylmaleimide. Complete protection against inactivation was afforded by saturating concentrations of NADP+. In the presence of NADPH, a rapid inhibition of the enzyme ensued; however, this inhibition was found to be reversible. In the tryptic map of the flavoprotein, modified with N-ethyl[2,3-14C]maleimide in oxidizing conditions, a unique radioactive peptide was found. Its sequence comprised residues 110-117 of the enzyme: Lys116 was shown to be the residue alkylated by N-ethylmaleimide. It is noteworthy that the same residue of FNR was found to be modified by 5-dimethylaminoaphthalene-1-sulfonyl(dansyl) chloride at the putative NADP(H)-binding site [Cidaria, D., Biondi, P. A., Zanetti, G. & Ronchi, S. (1985) Eur. J. Biochem. 146, 295-299]. Furthermore, the data reported here demonstrate that the sulfhydryl groups of FNR are not involved in enzyme inactivation by N-ethylmaleimide.

Amino Acids↗
PubMed1991

An unusual case of hepatic tumor.

A case is reported of a large hepatic tumor in a patient aged 71. Preoperative diagnostic techniques, including echography, CT and angiography, did not provide sufficient criteria for a precise diagnosis. The mass was removed with an extended right hepatectomy with no particular physiopathological consequences. Histological analysis revealed that this was a metastasis from a melonoma of the choroid, operated on 17 years previously.

Aged↗
PubMed1991

[Treatment of ureteral calculi with extracorporeal lithotripsy. Comparison between the original Dornier HM3 and the modified lithotriptor].

From January 1985 till June 1989, 222 patients with ureteral stones, underwent ESWL treatment. 109 patients were treated with the original Dornier HM3 under general or epidural anaesthesia and 113 patients were treated with the modified Dornier HM3 under anglosedation. 156 stones were in the upper ureter, 15 in the middle ureter, 51 in the lower ureter. We performed a total of 269 ESWL treatments. Out of 109 patients treated with the original HM3 and 113 patients treated by the modified HM3 12 (11%) and 28 (24.7%) needed, respectively, two or more ESWL sessions. In 89.9% and 88.5% of the patients, respectively, has been performed an ureteral manipulation before the treatment to push up or to localize the stone. The treatment was unsuccessful in 10 patients of whom 3 underwent anterograde lithotripsy by percutaneous access, 4 transureteral lithotripsy and 3 surgery. At three months the percentage of patients stone free was as follows: original HM3 patients 88% modified HM3 patients 92.9%. The extracorporeal lithotripsy is now-day the treatment of choice for the ureteral stone without any limitation, due to the stone localisation. The clinical use of modified HM3 on ureteral stones didn't low our success rate, but increased the shock wave out of session.

Humans↗
PubMed1990

Expression in Escherichia coli of ferredoxin:NADP+ reductase from spinach. Bacterial synthesis of the holoflavoprotein and of an active enzyme form lacking the first 28 amino acid residues of the sequence.

A cDNA clone for the preprotein of spinach ferredoxin:NADP+ reductase has been modified to allow the expression in Escherichia coli of the mature flavoprotein form the lacks the transit peptide. An expression vector, pFNR1, was constructed by subcloning the fragment into the plasmid pDS12/RBSII, SphI. In the crude extracts of transformed cells after induction, two active holoproteins of 35 kDa and 32 kDa, respectively, were found. The 32-kDa protein, purified by immunoaffinity chromatography, was found to lack the first 28 residues of the spinach protein sequence and to have a methionine as the N-terminal residue instead of Val29. A new expression plasmid, pFNR2, was obtained by in vitro mutagenesis of the codon GTG for Val29 to the synonymous GTT; in this case, only the 35-kDa protein was expressed by transformed cells. Both the 35-kDa and 32-kDa enzymes were purified and characterized. All the properties analyzed of the cloned 35-kDa enzyme were very similar to those of the spinach flavoprotein. The 32-kDa form showed the same catalytic efficiency of the spinach enzyme as a diaphorase but its interaction with oxidized ferredoxin was partially impaired.

Amino Acid Sequence↗
PubMed1990

Structure-function relationship in spinach ferredoxin-NADP+ reductase as studied by limited proteolysis.

Studies of limited proteolysis on purified ferredoxin-NADP+ reductase with various proteases were performed in the presence and absence of the flavoprotein ligands. Both the diaphorase and the ferredoxin-dependent activities of the enzyme were followed as well as the proteolytic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with further characterization of the polypeptides produced. These experiments revealed that only two regions of the flavoprotein are susceptible to the attack of the proteases used: (a) the N-terminal chain which can be cleaved only up to Lys35 and (b) the sequence segment 235-250. It can be inferred that these regions are on the surface of the protein molecule and presumably have a very flexible conformation adaptable to the protease active site. The deletion of the N-terminal region up to Thr36 of the native reductase (Mr 35,000) produced a truncated form (Mr about 31,000) which had full diaphorase activity but lost the capacity to catalyze the ferredoxin-dependent reaction. Proteolytic cleavage at the 235-250 segment of the sequence yielded a nicked protein (Mr about 30,000 by gel filtration; 23,000 plus 7,000 in denaturing electrophoresis) devoid of both activities. Protection by the flavoprotein ligands implies that the 23-35 region of the sequence is part of the binding site for ferredoxin and the 235-250 polypeptide segment is in the NADP(+)-binding site.

Amino Acid Sequence↗