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G Zanetti

Publications and source records attributed to G Zanetti.

At least 73 records · Page 4Linked to original sources
PubMed1997

Alterations in protein glycosylation in PMA-differentiated U-937 cells exposed to mineral particles.

Carbohydrate moieties of cell glycoconjugates play a pivotal role in molecular recognition phenomena involved in the regulation of most biological systems and the changes observed in cell surface carbohydrates during cell activation or differentiation frequently modulate certain cell functions. Consequently, some aspects of macrophage response to particle exposure might conceivably result from alterations in glycosylation. Therefore, the effect of mineral particles on protein glycosylation was investigated in phorbol myristate acetate (PMA)-differentiated U-937. Jacalin, a lectin specific for O-glycosylated structures, showed a global increase in O-glycosylation in particle-treated cells. In contrast, no significant modifications were observed with concanavalin A, a lectin that recognizes certain N-glycosylated structures. The sialic acid-specific lectins Sambucus nigra agglutinin and Maackia amurensis agglutinin and the galactose-specific lectin Ricinus communis agglutinin revealed a complex pattern of alterations in glycoprotein glycosylation after crystalline silica or manganese dioxide treatments. Expression of sialyl Lewis(x), a glycosylated structure implicated in leukocyte trafficking, could not be detected in control or treated cells. This finding was consistent with the decrease in sialyl Lewis(x) expression observed during PMA-induced differentiation. In conclusion, various treatments used in this study induced quantitative as well as qualitative changes in protein glycosylation. Whether these changes are due to glycosidase release or to an alteration in glycosyltransferase expression remains to be determined. The potential functional implications of these changes are currently under investigation.

Blotting, Western↗
PubMed1997

Recombinant wild-type and mutant complexes of ferredoxin and ferredoxin:NADP+ reductase studied by isothermal titration calorimetry.

The interaction of spinach ferredoxin:NADP+ reductase (FNR) with ferredoxin (Fd) is driven by a favorable change of entropy and shows almost no change in enthalpy. The change in heat capacity between the free proteins and the complex is -0.47 +/- 0.1 kJ mol(-1) K(-1), a value indicating a relatively small surface area buried in the complex. A single proton is taken up from the environment when the ferredoxin:FNR complex forms. In the complex, the protonated residue(s) is (are) probably located in the vicinity of E92 of Fd because charge reversal in Fd(E92K) quenches protonation. Substitution of K88 by Q in FNR(K88Q) destabilizes the complex by a 7 kJ mol(-1) reduction in binding entropy, which indicates that dehydration of the complex interface contributes to stability.

Calorimetry↗
PubMed1997

Renal stone fragments following shock wave lithotripsy.

PURPOSE: We describe a select group of asymptomatic patients with fragments and dust 3 months after extracorporeal treatment, who were followed to evaluate the long-term outcome and therapeutic implications. MATERIALS AND METHODS: A total of 129 patients with dust and residual fragments (less than 4 mm.) at 3 months was re-examined at 12 months, and 95 were also evaluated at 24 months. Followup examinations consisted of radiographic studies, renal ultrasonography and urine culture. Dust and residual fragments were sought, and patients were defined as free or as having persistent lithiasis or stone regrowth. At 24 months recurrences in the patients stone-free at 12 months also were considered. RESULTS: At the 12-month followup 60 patients (46.5%) were stone-free and 56 (43.5%) still had dust or residual fragments. The localization of the stones or fragments at 3 months and their sizes did not have a significant influence on the stone-free rate but regrowth was greater in patients with stones larger than 10 mm. (11 of 40 patients, 27.5% versus 2 of 89, 2.2%, p = 0.001). The probability of eliminating residual lithiasis at 12 months was significantly greater in patients with dust than in those with residual fragments (42 of 79 patients, 58% versus 18 of 50, 36%, p = 0.026). Regrowth of residual lithiasis was observed in 13 patients (10%). CONCLUSIONS: Based on our results, we do not believe that patients with fragments require systematic re-treatment in the short term but they may be followed long term and re-treated if symptoms persist or stones recur.

Adult↗
PubMed1996

Analysis of the oxidation-reduction potentials of recombinant ferredoxin-NADP+ reductase from spinach chloroplasts.

Midpoint oxidation-reduction potentials for the two-electron reduction of the bound FAD in spinach ferredoxin-NADP+ reductase were measured by potentiometry (Em = -342 +/- 1 mV at pH 7 and 10 degrees C). They were used with the semiquinone formation constant, obtained by spectroscopic measurement of the semiquinone concentration, to calculate values for the redox potentials of the two one-electron steps in the reduction. The redox potential for the oxidized enzyme/enzyme semiquinone couple (EOX/SQ) at pH 7 is -350 +/- 2 mV (10 degrees C) while the value for the enzyme semiquinone/enzyme hydroquinone couple (ESQ/HQ) under the same conditions is -335 +/- 1 mV. These values correspond to a semiquinone formation constant of 0.55. Measurement of the effects of pH on the potentials showed that EOX/SQ varies linearly with pH (slope -46 +/- 4 mV), while ESQ/HQ is independent of pH at high pH values, but below about pH 7.5 the potential becomes less negative with decreasing pH. indicating that there is a redox-linked protonation of the fully reduced enzyme (pKa = 7.2, 10 degrees C). The absorption spectrum of the fully reduced enzyme was found to depend on pH with the changes giving a calculated pKa of 7.5 (at 15 degrees C). The spectrum at high pH is similar to that of the anionic form of free flavin hydroquinone. The observations suggest that at physiological pH, the enzyme FAD cycles between the three redox states: oxidized, neutral semiquinone and hydroquinone anion.

Anions↗
PubMed1996

Properties of the recombinant beta subunit of glutamate synthase.

Glutamate synthase is a complex iron-sulfur flavoprotein containing one molecule each of FAD and FMN and three distinct iron-sulfur centers/alpha beta protomer. Production of the beta subunit was observed in total extracts of Escherichia coli BL21 (DE) cells harbouring a pT7-7 derivative carrying gltD, the gene encoding the Azospirillum brasilense glutamate synthase beta subunit. The protein was soluble, and the identity of the purified protein with the Azospirillum glutamate synthase beta subunit was confirmed by N-terminal sequence analysis. The kinetic and spectroscopic characterization of the glutamate synthase beta subunit confirmed that it contains the NADPH binding site, but, in contrast with earlier proposals that assigned both FAD and FMN binding sites to the alpha subunit of glutamate synthase, the beta subunit was shown to contain stoichiometric amounts of FAD. No iron-sulfur centers were detected by EPR spectroscopy measurements of the recombinant beta subunit. Under steady-state conditions, the glutamate synthase beta subunit can catalyze the NADPH-dependent reduction of several synthetic electron acceptors but no glutamate synthase or glutamate dehydrogenase reactions in either direction. The results are in agreement with previous data from our laboratory and, together with the absence of amino acid sequence similarity between glutamate synthase beta subunit and glutamate dehydrogenases, are against the hypothesis that glutamate synthase is evolutionarily derived from the association of an ancestral glutamate dehydrogenase (the beta subunit) and an amidotransferase (the alpha subunit). The protein-bound FAD is reduced by NADPH at a rate much faster than turnover with synthetic electron acceptors, leading to formation of a stable reduced flavin-NADP+ charge-transfer complex. The rate of reduction of the bound FAD by NADPH is also similar to the rate at which one of the flavins is reduced in the native glutamate synthase, as measured in a stopped-flow spectrophotometer under pre-steady-state conditions. The ability of FAD bound to the beta subunit of glutamate synthase to react with NADPH and the lack of reactivity with sulfite lead us to conclude that FAD is Flavin 1 of glutamate synthase [Vanoni, M.A., Edmondson, D.E., Zanetti, G. & Curti, B. (1992) Biochemistry 31, 4613-4623].

Anaerobiosis↗
PubMed1996

Mutations of Glu92 in ferredoxin I from spinach leaves produce proteins fully functional in electron transfer but less efficient in supporting NADP+ photoreduction.

Ferredoxin I in spinach chloroplasts fulfils the role of distributing electrons of low redox potential produced by photosystem I to several metabolic routes, NADP+ reduction being the major output. To investigate the role of Glu92, which is conserved in the chloroplast-type ferredoxins, mutations of this residue to either Gln, Ala or Lys were obtained through site-directed mutagenesis. A Glu93Ala mutant was also designed. The four mutants of ferredoxin I were overproduced in Escherichia coli, purified and characterised. The different migration in nondenaturing gel electrophoresis of wild-type and mutant proteins confirmed that the desired mutation was present in the expressed proteins. Spectral and physical properties of the mutants were similar to those of wild-type ferredoxin; electron-transfer properties were, however, quite different in the case of the mutants at position 92. Unexpectedly, these mutant ferredoxins were found to be twice as active as the wild-type protein in supporting the NADPH--cytochrome c reductase reaction catalysed by ferredoxin--NADP+ reductase. However, interactions of the mutant ferredoxins with the isolated thylakoid membranes deprived of endogenous ferredoxin showed that the mutants were less capable of supporting NADP+ photoreduction than the wild-type protein: both V and the apparent Km for reduced ferredoxin were influenced. On the other hand, the Kd values for the complex between oxidised ferredoxin and the reductase, measured at low ionic strength, were substantially changed only in the case of the Glu-->Lys mutation. With this mutant the rate of cross-linking between the two proteins induced by a carbodiimide was also decreased. It was found that the redox potentials of the iron-sulfur cluster of the mutants were more positive by 73-93 mV than that of ferredoxin I. Thus, the behavior of the ferredoxin mutants can be rationalised in terms of the effect of the side-chain replacement on the electrochemical properties of the [2Fe-2S] cluster and of an impairment in the interaction with the reductase under physiological conditions.

Base Sequence↗
PubMed1996

Ectopic cervical thymus: case report.

Ectopic cervical thymus is rarely considered in the differential diagnosis of cervical masses: this lesion is essentially asymptomatic and generally occupies a position in the neck along the carotid sheath, underneath the sterno-cleido-mastoid muscle. It is supposed that most of these masses arise as a consequence of migration defects during glandular embryogenesis. Ectopic thymus rarely invades contiguous structures but in the literature some cases of malignant transformation of aberrant cervical thymus have been reported. Some non-invasive investigations (MRI, ultrasonography) are useful but accurate diagnosis depends eventually on surgical excision and histologic examination.

Choristoma↗
PubMed1996

Extracorporeal shock wave lithotripsy.

The first clinical application of extracorporeal SWL dates back to 1980. Since then the use of this method has spread widely and its indications have been extended progressively so that it way now be considered the treatment of choice in 80-90% of cases of ureterorenal lithiasis. Treatments without anesthesia or analgesics have been associated with an increase of retreatments from 5-14% (original HM3) to 45-60% (lithotripters not requiring anesthesia or analgesia). However, almost all lithotripter succeed in fragmenting stones sufficiently. The stone free rate varies with different lithotripters in the different series: 90-56% for stones of maximum diameter < 1 cm, 78-30% for stones of maximum diameter of 1-2 cm. and 52.5-10% for stones of maximum diameter of 2-3 cm. (the last figure was obtained with a piezoelectric lithotripter). Extracorporeal lithotripsy as monotherapy of staghorn stones has yelded a stone free rate varying between 31% and 55% with high percentages of residual fragments in about 50% of case of the various series. The stone free rate after treatment varies according to stone site: it is between 75% and 84% of caliceal stones for upper caliceal calculi and falls to under 60% for lower caliceal ones. The frequency of recurrences, that is, of new stones in patients stone free after SWL, is between 4% and 10% annually. Adding the percentage of true recurrences reported by the various authors at 19 to 42 months of follow-up (6.2-13.8%) to the fragment regrowth rate (17.2-22.3%) gives a total new stone rate of 23.4% and 36%. These figures are not greatly different from those reported in a population of untreated stone formers (10-15% per year). Extracorporeal lithotripsy seems thus not to influence lithiasis recurrence significantly.

Equipment Design↗
PubMed1996

Extracorporeal shock wave lithotripsy in the treatment of ureteral lithiasis: methodological controversies and therapeutic efficacy.

The treatment of ureteral stones has undergone a radical change in the last 15 years. First, the increased use of endoscopic procedures and then the introduction of extracorporeal lithotripsy relegated traditional surgery to a marginal role for this type of disorder. The best available treatment modality for ureteral lithiasis, particularly distal ureteral stones, is still a matter of great controversy among urologist. With the introduction in clinical use of second- and third generation lithotripters, which are even less invasive and require no anesthesia, interest has increased in treating patients by extracorporeal lithotripsy, reducing endoscopic monoeuvres to a minimum. The absolute contraindications to extracorporeal lithotripsy for ureteral stones are the same as those for renal stones: intractable hemostatic alterations, pregnancy, physical structure that limits positioning and altered patency of the urinary tract. From June 1990 to December 1994, 270 patients with ureteral stones were treated by extracorporeal lithotripsy at our center. The Dornier MPL 9000 lithotripter was used in 68 cases (25%) and the modified HM3 Dornier in 202 (75%). Pretreatment manoeuvres were performed in 130 patients (48%). Endoscopic manoeuvres were not performed in 140 patients treated in situ. 18 patients (13%) treated initially in situ subsequently underwent post-treatment manoeuvres which were required only in 3 patients who had undergone pretreatment. All patients were examined as outpatients 3 months after the treatment. A total of 241 patients (89%) were stone free, 121 who had undergone pretreatment manoeuvres and 119 who had been treated in situ. 29 patients (11%) were not stone free: 23 patients subsequently underwent endoscopic lithotripsy, 2 surgery and 4 stone removal by Dormia probe. The possibility of performing treatment without anesthesia, the absence of complications and the high proportion of successes make extracorporeal lithotripsy, particularly the in situ procedure, the treatment of choice for ureteral stones. Ureterorenoscopy has been proposed by some authors as the first treatment for mid and pelvic ureteral stones which are difficult to localize with the lithotripter. However, although this method is very efficacious and less expensive, the percentage of complications is greater and patient compliance is less.

Evaluation Studies as Topic↗
PubMed1996

[Ileal neobladder in women: Milano's technique].

In the female patient with transitional cell carcinoma the risk of urethral recurrence is very low, when the bladder neck is histologically free of tumour. On the other hand urinary continence can be maintained if the lower half of the urethra together with the nerve supply is preserved. On this basis orthotopic bladder reconstruction was applied also in a female patient. In order to preserve urinary continence minimal dissection was performed anterior to the proximal urethra; in addition pubourethral ligaments were left intact. On the other hand to prevent chronic urinary retention arising from downward displacement of the reservoir a colposacropexy was performed. At 3 weeks the filling cystogram demonstrated a well shaped and compliant reservoir. No downward displacement of the reservoir was observed in upright position. Neither reflux nor residual urine could be demonstrated by voiding cystourethrogram. The woman achieved diurnal and nocturnal continence at 3 month.

Carcinoma, Transitional Cell↗
PubMed1995

Direct electrochemistry and EPR spectroscopy of spinach ferredoxin mutants with modified electron transfer properties.

Mutations of the conserved residue Glu-92 to lysine, glutamine, and alanine have been performed in the recombinant ferredoxin I of spinach leaves. The purified ferredoxin mutants were found twice as active with respect to wild-type protein in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase in the presence of ferredoxin. Cyclic voltammetry and EPR measurements showed that the mutations cause a change in the [2Fe-2S] cluster geometry, whose redox potential becomes approximately 80 mV less negative. These data point to a role of the Glu-92 side-chain in determining the low redox potential typical of the [2Fe-2S] cluster of chloroplast and cyanobacterial ferredoxins. Also a ferredoxin/ferredoxin-NADP+ reductase chimeric protein obtained by gene fusion was overproduced in Escherichia coli and purified. Fusion of the ferredoxin with its reductase causes only minor effects to the iron-sulfur cluster, as judged by cyclic voltammetry and EPR measurements.

Electric Conductivity↗
PubMed1995

Involvement of serine 96 in the catalytic mechanism of ferredoxin-NADP+ reductase: structure--function relationship as studied by site-directed mutagenesis and X-ray crystallography.

The crystal structure of ferredoxin-NADP+ reductase (FNR) suggests that Ser96 is directly involved in hydride transfer between the isoalloxazine moiety of FAD and the nicotinamide ring of NADP(H). To probe its role, Ser96 has been mutated to valine (S96V) and glycine (S96G). These mutations primarily affected the interaction of the nicotinamide ring with the flavin. Absorbance, fluorescence, and circular dichroism spectra and the crystal structure of FNR-S96V indicate that this mutant folds properly. FNR-S96V shows only 0.05% of wild-type activity, while the affinities for both ferredoxin and NADP+ are virtually unchanged. However, spectral perturbations induced by NADP+ binding to FNR-S96V strongly resemble those elicited by the binding of 2'-monophosphoadenosine-5'-diphosphoribose, a substrate analog lacking the nicotinamide ring, both to the mutant and wild-type enzymes. Rapid reaction studies on the valine mutant failed to detect charge-transfer intermediates during flavin reduction by NADPH. In addition, no semiquinone formation was seen during photoreduction of FNR-S96V. The three-dimensional structure of the valine mutant shows small, albeit definite, changes only in the isoalloxazine microenvironment. The glycine mutant of FNR displays behavior intermediate between that of wild-type enzyme and that of the valine mutant. It maintains ca. 2% of the wild-type activity as well as the ability to form the charge-transfer species between reduced FNR and NADP+. In photoreduction experiments, the same degree of flavin semiquinone stabilization was observed with FNR-S96G and with the wild-type enzyme. NADP+ binding to the glycine mutant was very similar to that observed in the case of the valine mutant.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Spinach ferredoxin i: overproduction in Escherichia coli and purification.

Ferredoxin I is the most abundant form of photosynthetic-type ferredoxin present in spinach chloroplasts. A cDNA clone encoding the precursor of spinach ferredoxin I has been engineered to synthesize the mature form of the plant protein in Escherichia coli. Among several different plasmid constructions, the expression system based on phage T7 promoter (vector pET-11d) was found to be the most efficient for spinach ferredoxin overproduction. Upon induction, ferredoxin I accounted for about 2.5% of soluble E. coli protein. A rapid procedure for the purification of the recombinant protein, which yielded at least 1 mg of homogeneous ferredoxin I per gram of cells (fresh wt), was developed. The recombinant protein was found to be identical to ferredoxin I isolated from spinach, both by mass spectrometry analysis and by N-terminal protein sequencing, indicating in vivo removal of the N-terminal methionine. Ferredoxin I was synthesized as the holoprotein, correctly assembled with the [2Fe-2S] cluster as judged by its absorption spectrum, and was fully active in the assay with its physiological partner (ferredoxin-NADP+ reductase). The expression system described here is amenable to the structure-function relationship study of spinach ferredoxin I through site-directed mutagenesis and NMR spectroscopy.

Amino Acid Sequence↗
PubMed1995

Automatic reading of hybridization filter images.

We describe a new technique for the automatic detection and characterization, in terms of location, size and intensity, of positive hybridization spots on hybridization filter images. We will also discuss how this information can be used to reconstruct the spot deposition pattern on the filter and thus identify the spots. The part of the algorithm dedicated to the detection of spots is derived from a technique used in astronomy for the automatic recognition of galaxies on photographic plates. This technique has been modified so that it becomes able to cope with the strongly varying background typical of hybridization filter images. The technique does not require any human intervention.

Algorithms↗
PubMed1995

Andrological laparoscopy.

The laparoscopic technique has well defined indications for some andrological procedures such as the diagnosis and the treatment of cryptorchidism, but its role remains controversial in varix ligation for which laparoscopy is however the newest development. At the Institute of Urology of the University of Milan from January 1992 to June 1994 five adults patients with undescended and unpalpable testis underwent laparoscopy. In 4 cases laparoscopic orchiopexy (2 direct and 2 staged procedures) and in one case laparoscopic orchiectomy have been performed. In the same period 20 cases of varicocele (6 bilateral) have been observed and treated by laparoscopic varix ligation. For cryptorchidism after the identification of the testis we decide on the basis of parenchimal trophism wheter to perform orchidopexy or orchiectomy. A single step laparoscopic orchiopexy can be performed if the undescended testis is located proximal to the internal inguinal ring and if the mobilization of the spermatic vessels allows it. A Fowler-Stephens staged orchiopexy is indicated for intra abdominal testicle with short spermatic vessels. In the first stage the spermatic vessels are isolated and divided relying on the compensation offered by the deferential and extrafunicular vessels. After six months, once the testis trophism has been ascertained, the testis can be placed in the scrotum. For varicocelectomy the peritoneum is incised at the projection of the spermatic cord from the internal inguinal ring. A blunt and gentle dissection prepares the spermatic vascular bundle, the spermatic artery is identified and isolated and the vein are clipped and divided.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗