Biomedical subjects
G Zanetti
Publications and source records attributed to G Zanetti.
Structure of the mutant E92K of [2Fe-2S] ferredoxin I from Spinacia oleracea at 1.7 A resolution.
Ferredoxin I (Fd I) from Spinacia oleracea is composed of 97 amino-acid residues and a [2Fe-2S] cluster. The crystal structure of the E92K mutant of Fd I was solved by molecular replacement and refined to an R factor of 19.6% for 11755 reflections at 1.7 A resolution. The overall structure and the active centre of spinach Fd is highly conserved with respect to ferredoxins of known structure. The E92K mutation appears to disturb a hydrogen-bond network which stabilizes the loop bearing the [2Fe-2S] cluster. This observation provides a rationale for the reduced electron-transfer efficiency displayed by the E92K mutant. Inspection of the crystal packing reveals that the side chain of Lys92 is engaged in an intermolecular interaction with Asp26 of a symmetry-related molecule. This feature may explain why only the mutant E92K and not wild-type Fd I could be successfully crystallized.
Reaction of the NAD(P)H:flavin oxidoreductase from Escherichia coli with NADPH and riboflavin: identification of intermediates.
Flavin reductase catalyzes the reduction of free flavins by NAD(P)H. As isolated, Escherichia coli flavin reductase does not contain any flavin prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin substrate in a ternary complex prior to oxidoreduction. The reduction of riboflavin by NADPH catalyzed by flavin reductase has been studied by static and rapid kinetics absorption spectroscopies. Static absorption spectroscopy experiments revealed that, in the presence of riboflavin and reduced pyridine nucleotide, flavin reductase stabilizes, although to a small extent, a charge-transfer complex of NADP+ and reduced riboflavin. In addition, reduction of riboflavin was found to be essentially irreversible. Rapid kinetics absorption spectroscopy studies demonstrated the occurrence of two intermediates with long-wavelength absorption during the catalytic cycle. Such intermediate species exhibit spectroscopic properties similar to those of charge-transfer complexes of oxidized flavin and NAD(P)H, and reduced flavin and NAD(P)+, respectively, which have been identified as intermediates during the reaction of flavoenzymes of the ferredoxin-NADP+ reductase family. Thus, a minimal kinetic scheme for the reaction of flavin reductase with NADPH and riboflavin can be proposed. After formation of the Michaelis complex of flavin reductase with NADPH and riboflavin, a first intermediate, identified as a charge-transfer complex of NADPH and riboflavin, is formed. It is followed by a second charge-transfer intermediate of enzyme-bound NADP+ and reduced riboflavin. The latter decays, yielding the Michaelis complex of flavin reductase with NADP+ and reduced riboflavin, which then dissociates to complete the reaction. These results support the initial hypothesis of a structural similarity between flavin reductase and the enzymes of the ferredoxin-NADP+ reductase family and extend it at a functional level.
The recombinant alpha subunit of glutamate synthase: spectroscopic and catalytic properties.
As part of our studies of Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein, we have overproduced the two enzyme subunits separately in Escherichia coli. The beta subunit (53.2 kDa) was demonstrated to contain the site of NADPH oxidation of glutamate synthase and the FAD cofactor, which was identified as Flavin 1 of glutamate synthase, the flavin located at the site of NADPH oxidation. We now report the overproduction of the glutamate synthase alpha subunit (162 kDa), which is purified to homogeneity in a stable form. This subunit contains FMN as the flavin cofactor which exhibits the properties of Flavin 2 of glutamate synthase: reactivity with sulfite to yield a flavin-N(5)-sulfite addition product (Kd = 2.6 +/- 0.22 mM), lack of reactivity with NADPH, reduction by L-glutamate, and reoxidation by 2-oxoglutarate and glutamine. Thus, FMN is the flavin located at the site of reduction of the iminoglutarate formed on the addition of glutamine amide group to the C(2) carbon of 2-oxoglutarate. The glutamate synthase alpha subunit contains the [3Fe-4S] cluster of glutamate synthase, as shown by low-temperature EPR spectroscopy experiments. The glutamate synthase alpha subunit catalyzes the synthesis of glutamate from L-glutamine and 2-oxoglutarate, provided that a reducing system (dithionite and methyl viologen) is present. The FMN moiety but not the [3Fe-4S] cluster of the subunit appears to participate in this reaction. Furthermore, the isolated alpha subunit of glutamate synthase exhibits a glutaminase activity, which is absent in the glutamate synthase holoenzyme. These findings support a model for glutamate synthase according to which the enzymes prepared from various sources share a common glutamate synthase function (the alpha subunit of the bacterial enzyme, or its homologous polypeptide forming the ferredoxin-dependent plant enzyme) but differ for the chosen electron donor. The pyridine nucleotide-dependent forms of the enzyme have recruited a FAD-dependent oxidoreductase (the bacterial beta subunit) to mediate electron transfer from the NAD(P)H substrate to the glutamate synthase polypeptide. However, it appears that the presence of the enzyme beta subunit and/or of the additional iron-sulfur clusters (Centers II and III) of the bacterial glutamate synthase is required for communication between Center I (the [3Fe-4S] center) and the FMN moiety within the alpha subunit, and for ensuring coupling of glutamine hydrolysis to the transfer of the released ammonia molecule to 2-oxoglutarate in the holoenzyme.
Esophageal leiomyomatosis in children: report of a case and review of the literature.
The authors describe a case of a 5-years-old girl affected by diffuse oesophageal leiomyomatosis presenting with progressive dysphagia, cyanosis, recurrent pneumonia and retrosternal pain. She also suffered from occasional constipation due to perineal involvement by the disease and had a past history of recurrent microscopic hematuria, suggesting an association with an Alport-like syndrome. Only 24 cases of esophageal leiomyomatosis could be found in the medical literature in children aged less than 14 years, confirming the rarity of the disease. A clinical analysis of all cases reviewed from the literature is made, stressing the importance of an accurate preoperative diagnosis for the choice of proper surgical treatment to avoid recurrence.
ViVa: the virtual vascular project.
The aim of the virtual vascular project (ViVa) is to develop tools for the modern hemodynamicist and cardiovascular surgeon to study and interpret the constantly increasing amount of information being produced by noninvasive imaging equipment. In particular, we are developing a system able to process and visualize three-dimensional (3-D) medical data, reconstruct the geometry of arteries of specific patients, and simulate blood flow in them. The initial applications of the system will be for clinical research and training purposes. In a later stage, we will explore the application of the system to surgical planning. ViVa is based on an integrated set of tools, each dedicated to a specific aspect of the data processing and simulation pipeline: image processing and segmentation; real-time 3-D volume visualization; 3-D geometry reconstruction; 3-D mesh generation; and blood flow simulation and visualization.
Prospective randomized comparison of imipenem-cilastatin and piperacillin-tazobactam in nosocomial pneumonia or peritonitis.
Nosocomial pneumonia and acute peritonitis may be caused by a wide array of pathogens, and combination therapy is often recommended. We have previously shown that imipenem-cilastatin monotherapy was as efficacious as the combination of imipenem-cilastatin plus netilmicin in these two settings. The efficacy of imipenem-cilastatin is now compared to that of piperacillin-tazobactam as monotherapy in patients with nosocomial pneumonia or acute peritonitis. Three hundred seventy one patients with nosocomial pneumonia or peritonitis were randomly assigned to receive either imipenem-cilastatin (0.5 g four times a day) or piperacillin-tazobactam (4.5 g three times a day). Three hundred thirteen were assessable (154 with nosocomial pneumonia and 159 with peritonitis). For nosocomial pneumonia, clinical-failure rates in the piperacillin-tazobactam group (13 of 75 [17%]) and in the imipenem-cilastatin group (23 of 79 [29%]) were similar (P = 0.09), as were the numbers of deaths due to infection (6 in the imipenem-cilastatin group [8%], 7 in the piperacillin-tazobactam group [9%]) (P = 0.78). For acute peritonitis, clinical success rates were comparable (piperacillin-tazobactam, 72 of 76 [95%]; imipenem-cilastatin, 77 of 83 [93%]). For infections due to Pseudomonas aeruginosa, 45 patients had nosocomial pneumonia (21 in the piperacillin-tazobactam group and 24 in the imipenem-cilastatin group) and 10 had peritonitis (5 in each group). In the patients with nosocomial pneumonia, clinical failure was less frequent in the piperacillin-tazobactam group (2 of 21 [10%]) than in the imipenem-cilastatin [corrected] group (12 of 24 [50%]) (P = 0.004). Bacterial resistance to allocated regimen was the main cause of clinical failure (1 in the piperacillin-tazobactam group and 12 in the imipenem-cilastatin group). For the patients with peritonitis, no difference in clinical outcome was observed (five of five cured in each group). The overall frequencies of adverse events related to treatment in the two groups were similar (24 in the piperacillin-tazobactam group, 22 in the imipenem-cilastatin group). Diarrhea was significantly more frequent in the piperacillin-tazobactam group (10 of 24) than in the imipenem-cilastatin group (2 of 22). This study suggests that piperacillin-tazobactam monotherapy is at least as effective and safe as imipenem-cilastatin monotherapy in the treatment of nosocomial pneumonia or peritonitis. In P. aeruginosa pneumonia, piperacillin-tazobactam achieved a better clinical efficacy than imipenem-cilastatin, due to reduced development of microbiological resistance. Tolerance was comparable, with the exception of diarrhea, which was more frequent with piperacillin-tazobactam.
A study of dietary calcium and other nutrients in idiopathic renal calcium stone formers with low bone mineral content.
PURPOSE: Patients with calcium renal stone are reported to have lower bone mineral density. The state of bone density in patients with renal stones have different explanations but the role of nutritional factors seems to be crucial. A group of 48 consecutive male calcium renal stone formers was studied to investigate the relationship between bone density and dietary intake. MATERIALS AND METHODS: Patients completed a dietary diary for a 3-day period during normal diet. Nutrients and calories were calculated by food composition tables using a computerized procedure. Bone densitometry was assessed at the lumbar spine and femoral neck, and expressed as Z score. A blood sample was collected and was analyzed for serum biochemistry including alkaline phosphatase, parathyroid hormone and 1,25 vitamin D. A 24-hour urine sample was analyzed for calcium, phosphate, oxalate, citrate and other electrolytes. RESULTS: Dietary calcium intake was significantly lower (p < 0.01) in patients with low than in those with normal bone mineral density. There was no difference in serum parathyroid hormone levels, phosphate and alkaline phosphatase between the 2 groups. The results suggest that some renal stone formers seem to be unable to decrease renal excretion of calcium on a low calcium diet leading to a negative calcium balance. CONCLUSIONS: A primary abnormality of bone metabolism could be a reasonable explanation of reduced bone density observed in renal stone formers on a low calcium diet since serum parathyroid hormone levels are in the normal range. From a therapeutic point of view these data confirm that restriction of dairy products in renal stone formers should be avoided.
[Male and female urinary incontinence: treatment in day surgery].
Incontinence isn't itself a disease but the feature of possible urinary tract alterations or outside of it. Incontinence is frequent above all in the elderly but it can be on charge of both sexes at every age. In Italy, according to recent evaluations, people affected with this disease would be more than 4 millions. Incontinence is therefore an important failure for its health aspects but also for economic and social ones. The problem is to evaluate if incontinence can't be prevented and as consequence needs only an assistance management, or it can be considered a preventable disease able to be cured, as we deeply believe, suggested also by the positive results of new therapeutical procedures, in association with traditional surgery and rehabilitation such as injectables or mini-invasive quick operations such as colpocleisis or percutaneous vaginal colposuspension (PVC), matters of this presentation and always performed according to correct diagnosis and indication. Bovine dermal collagen highly purified, poorly viscous and easily injectable, despite traditional rehabilitation and surgery, is a further procedure, endoscopic and minimally invasive to treat stress incontinence. Collagen is employed to perform a bladder neck plasty, increasing urethrosphincterial competence, to obtain continence without the creation of an obstruction. Genital prolapse, that is hysterocolpocele or simple vaginal vault prolapse, has course in high proportion (37%) in elderly (after 80 years). Surgical management of severe failures of continence and often also of the voiding function, such as: hyscuria with vesicoureteral reflux, obstinate constipation related to severe genital prolapse with allied rectocele is often hardly performed in elderly owing to the age and general health conditions: colpoclesis is a vaginal surgical approach that can be easily performed by the urologist too, it is an effective alternative to permanent catheterization or maxipad to be offered to the patient to improve her quality of life. In between the above maintained procedures takes place the percutaneous vaginal colposuspension (PVC). It is an original technique made up in our Institute to treat incontinence by the bladder neck resuspension to Cooper ligament according to a complete miniinvasive retropubic tension free transvaginal colposuspension, in local anaesthesia and complementary light narcosis in Day Surgery. Urinary incontinence is today a disturbance easy to be cured thanks to injectables and to miniinvasive surgical procedures as reported in this presentation concerning the most advanced approaches to its management.
[Extracorporeal SWL in the treatment of reno-ureteral calculosis in day hospital].
Eighteen years after the first clinical shock wave lithotripsy (SWL), no doubt remains as to its therapeutic efficacy in ureterorenal lithiasis. The advent of lithotriptors with a large shock wave energy range and integration of both ultrasound and radiologic imaging equipment at the shock wave source has meant that outpatients treatment of urolithiasis is now feasible in a good proportion of cases. In our lithotripsy center, from January 1995 to August 1996, 208 out of 310 patients who underwent SWL treatment for renal and ureteral stones, were outpatients. Pretreatment manoeuvres were performed in 10.6% of the patients. No major complications occurred during the treatment. Only three patients (1.4%) were admitted to hospital because of fever, colics or perirenal haematoma in the first two days after SWL therapy. The stone free rate was 67 and 84% respectively one and three months after treatment. In our experience, the possibility of performing SWL treatments without anesthesia and even analgosedation, the absence of complications and the high success rate, make outpatient treatment of urolithiasis safe and suitable in a large number of patients.
[Urethral implantation of collagen in the treatment of urinary incontinence. Comparison of transurethral and periurethral approach].
BACKGROUND: In mini-invasive surgery for the treatment of urinary incontinence injectable substances such as politef, autologous fat or bovine collagen, which can increase urethral resistance or support the sphincteric one are particularly used. It's a simple procedure in which patients may undergo local anesthesia, in short time and in "day hospital" regimen. Collagen, used as an injectable bulking agent, is the gold standard for this surgical technique because of complete biocompatibility, no evidences of adverse immunogenic effects, foreign-body reaction, migration of injected material, and because of higher fluidity which allows the use of thin needles compared to Politef (polytetrafluoroethylene) where an injection device is requested. Bovine collagen is partially reabsorbed within 24-36 months and a reinjection is often necessary. Although autologous fat is cheaper than bovine collagen, its injection shows more difficulties due to the need of higher needle diameter and time to store and prepare the material to inject. METHODS: The results obtained by two different approaches: transurethral injection of collagen and periurethral injection of collagen have been studied. A total of 48 patients underwent collagen injection for treatment of urinary incontinence: 24 treated by transurethral approach and 24 by periurethral approach. Follow-up at 6, 12, 24 months after treatment includes: objective exam, PAD test, functional evaluation, complete urodynamic evaluation (uroflow, cystomanometry, LPP, pressure/flow study, UPP). RESULTS: No clinical differences between peri/trans-urethral approach were found although collagen injection via transurethral technique need fewer material, reducing cost of treatment: an efficacy of treatment in 80% of patients and restoration of a complete urinary continence in 50% of patients was obtained. Although no clinical differences were demonstrated between the two different sites of injection, transurethral approach can be used similarly in both sexes even if without a modified resector as "injection device" it's more difficult to inject collagen in the right position, at the level of bladder neck submucosa. Perineal approach, exclusive of female sex, realizes the procedure without bleeding of urethral mucosa and no waste of collagen, though a longer period of training by surgeon is requested. After four years of experience the authors agreed that with an adequate injection device (modified resector), transurethral approach should be preferred, thus depending on the possibility to inject collagen in a correct position, just under bladder neck mucosa, which is possible to manage with this technique, in order to reduce reabsorption process of collagen and to obtain a longer efficacy of treatment reducing the number of reinjection and of course the cost of treatment. Collagen injection should be considered within an integrated therapeutical picture together with topic and systemic pharmacotherapy, physiotherapy, not excluding surgery techniques such as implantation of an artificial sphincter device in man or a sling procedure in woman. CONCLUSIONS: The urethral collagen injection for the treatment of urinary incontinence is a safe, durable and valid technique to improve urinary continence through a non obstructive effect increasing urethral resistance. Performed under local anesthesia this procedure allows the treatment of patients who may not be candidates suitable for general anesthesia and conventional surgery.
A three-domain iron-sulfur flavoprotein obtained through gene fusion of ferredoxin and ferredoxin-NADP+ reductase from spinach leaves.
Ferredoxin and ferredoxin-NADP+ reductase are the two last partners of the photosynthetic electron-transfer chain, responsible for the final reduction of NADP+ to NADPH. Herein, we report the engineering and characterization of a novel protein molecule in which the electron-carrier protein (ferredoxin I) and the reductase (a flavoprotein) were covalently linked in a single polypeptide chain by gene fusion. The gene was obtained by joining the cDNAs encoding the respective proteins and subsequently by deleting the intervening sequence between them by site-directed mutagenesis. No extra amino acid residues were introduced between the C-terminus of ferredoxin I and the N-terminus of the flavoenzyme. The chimera was purified to homogeneity and characterized. The M(r) of the chimera apoprotein was 45,800 as determined by mass spectrometry, in agreement with the expected value of 45,846. Both flavin and iron-sulfur cluster were in 1:1 ratio with respect to the apoprotein. The chimera was found active as a diaphorase and, more interestingly, highly efficient as a cytochrome c reductase, without need for free ferredoxin addition in the assay medium. Several lines of evidence indicate that the ferredoxin and the reductase in the chimera assume a configuration quite similar to that in the dissociable physiological complex. Thus, the fusion protein could be a useful tool for studying the mechanism of protein-protein recognition and electron transfer in the ferredoxin-ferredoxin-NADP+ reductase system.
On the role of the acidic cluster Glu 92-94 of spinach ferredoxin I.
The role of the acidic cluster Glu 92-94 of spinach ferredoxin I in the interaction both with the photosystem I multisubunit complex and the ferredoxin-NADP+ reductase, either membrane-bound or purified, was studied by kinetic characterization of site-directed mutants. Three mutants of ferredoxin have been produced to evaluate the effects of elimination of one or two negative charges in the three specific positions of the acidic cluster. Kinetic characterization of the ferredoxin mutants E92A/E93A, E93A and E93A/E94A as electron carriers in the photosynthetic electron transport chain, allowed to establish that the two latter mutants were nearly indistinguishable from the wild-type protein in their ability to be photoreduced by photosystem I and as electron donor to the reductase in the NADP+ photoreduction with thylakoid membranes. The E92A/E93A ferredoxin mutant behaved very similarly to E92 mutants previously characterized. Thus, the elimination of the carboxyl groups adjacent to residue 92 did not further impaired ferredoxin I main function, i.e., as an electron carrier in NADP+ photoreduction. The two double mutants showed a reduced rate in the cross-linking of ferredoxin to the reductase promoted by a soluble carbodiimide, indicating an involvement of the acidic cluster in the formation of the active covalent complex between the two proteins.
[Community-acquired pneumonia: considerations on ambulatory management].
Community-acquired pneumonia is a frequent disease in outpatients; its incidence is highest among persons at the extremes of age. Hospitalization is rarely required. Since most clinical studies are performed with hospitalized patients, available data are not always representative of the general population. For instance, the importance of some organisms, such as Mycoplasma pneumoniae, is underestimated, and the severity of the prognosis is overestimated in the literature. Identification of the causative organism is usually not cost-effective in outpatients. Therefore, therapy is most often empirical. In Switzerland, the majority of patients can be successfully treated with macrolides. Antibiotics may also be chosen according to the most probable organisms in a given patient, based on his age and underlying diseases; this last strategy may be more logical from the point of view of public health.
Sepsis and septic shock.
Focal infectious processes may produce a systemic syndrome whose description has been recently standardized by the definitions of sepsis, severe sepsis and septic shock. This classification should only be used as an adjunct to the microbiological and clinical diagnosis of a given infection. The incidence of sepsis and septic shock has been increasing over recent decades, but the ratio of gram-negative to gram-positive causative organisms has remained largely similar (most often between 1:1 and 3:2). Recent advances in understanding of the pathophysiology of sepsis and septic shock have made it possible to delineate more clearly the role of bacterial products such as lipopolysaccharide, exotoxins or cell wall fragments. These products are able either to directly trigger inflammatory pathways, or to stimulate target cells (such as monocytic cells, PMN or endothelial cells) to produce pro-inflammatory cytokines. Management of the infectious process itself with antibiotics, and with surgery if needed, is the cornerstone of the therapy of sepsis and septic shock. More recent approaches aim at inhibiting the bioactivity of bacterial or pro-inflammatory mediators. Up to now, however, none of these approaches has led to therapeutic modalities that can be applied routinely to patients.
Influence of particle surface area on the toxicity of insoluble manganese dioxide dusts.
The objective of this study was to examine the influence of specific surface area on the biological activity of insoluble manganese dioxide (MnO2) particles. The biological responses to various MnO2 dusts with different specific surface area (0.16, 0.5, 17 and 62 m2/g) were compared in vitro and in vivo. A mouse peritoneal macrophage model was used to evaluate the in vitro cytotoxic potential of the particles via lactate dehydrogenase (LDH) release. In vivo, the lung inflammatory response was assessed by analysis of bronchoalveolar lavage after intratracheal instillation in mice (LDH activity, protein concentration and cellular recruitment). In both systems, the results show that the amplitude of the response is dependent on the total surface area which is in contact with the biological system, indicating that surface chemistry phenomena are involved in the biological reactivity. Freshly ground particles with a specific surface area of 5 m2/g were also examined in vitro. These particles exhibited an enhanced cytotoxic activity, which was almost equivalent to that of 62 m2/g particles, indicating that undefined reactive sites produced at the particle surface by mechanical cleavage may also contribute to the toxicity of insoluble particles. We conclude that, when conducting studies to elucidate the effect of particles on the lung, it is important for insoluble particles such as manganese dioxide to consider the administered dose in terms of surface area (e.g. m2/kg) rather than in gravimetric terms (e.g. mg/kg).
A series of 17 cases of congenital cystic adenomatoid malformation of the lung: management and outcome.
During a 5-year-period, 17 cases of congenital adenomatoid malformation of the lung were observed in the Department of Pediatric Surgery of Bologna University-S. Orsola-Malpighi Hospital; 16 cases were diagnosed in utero by ultra-sound. 13 patients had prenatal diagnosis between 21 and 23 weeks of gestation; 2 cases were diagnosed at 25 weeks and another one at 26 weeks. The other case came from another hospital, and the diagnosis was accidentally detected postnatally. In 3 cases the parents decided to terminate the pregnancy 3 cases had spontaneous improvement during the last weeks of gestation, and were perfectly normal at birth. One case was lost to follow-up; all remaining patients underwent resection. Among these patients only one was a nonsurvivor, while 9 have completely recovered. However, one patient developed persistent pulmonary hypertension, which could successfully be treated with drugs, another underwent laser treatment for laryngeal stenosis due to intubation. A special epidemiological information: 13 out 17 cases (76.5%) were female.
Chemical characterization and reactivity of iron chelator-treated amphibole asbestos.
Iron in amphibole asbestos is implicated in the pathogenicity of inhaled fibers. Evidence includes the observation that iron chelators can suppress fiber-induced tissue damage. This is believed to occur via the diminished production of fiber-associated reactive oxygen species. The purpose of this study was to explore possible mechanisms for the reduction of fiber toxicity by iron chelator treatments. We studied changes in the amount and the oxidation states of bulk and surface iron in crocidolite and amosite asbestos that were treated with iron-chelating desferrioxamine, ferrozine, sodium ascorbate, and phosphate buffer solutions. The results have been compared with the ability of the fibers to produce free radicals and decompose hydrogen peroxide in a cell-free system in vitro. We found that chelators can affect the amount of iron at the surface of the asbestos fibers and its valence, and that they can modify the chemical reactivity of these surfaces. However, we found no obvious or direct correlations between fiber reactivity and the amount of iron removed, the amount of iron at the fiber surface, or the oxidation state of surface iron. Our results suggest that surface Fe3+ ions may play a role in fiber-related carboxylate radical formation, and that desferrioxamine and phosphate groups detected at treated fiber surfaces may play a role in diminishing and enhancing, respectively, fiber redox activity. It is proposed that iron mobility in the silicate structure may play a larger role in the chemical reactivity of asbestos than previously assumed.