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The use of lasers has been of great importance in the field of endoscopy and surgery for their applications in coagulation and the ability to vaporize tissue. In the 1990s, new machines were introduced based on a different technology, the argon-plasma-coagulation (APC) system. This technology causes different biological effects without direct contact. An example is the hemostasis of bleeding. In the literature, several cases of complications have been reported during endoscopic treatment with APC. In this study, we report our experience of a case with colon explosion during an APC procedure for bleeding due to radiotherapy and also review the literature on the complications of APC treatment.

The thermodynamics of Cu(II) to Cu(I) reduction and the kinetics of the electron transfer (ET) process for Rhus vernicifera stellacyanin (STC) immobilized on a decane-1-thiol coated gold electrode have been measured through cyclic voltammetry at varying pH and temperature, in the presence of urea and in D(2)O. Immobilized STC undergoes a limited conformational change that mainly results in an enhanced exposure of one or both copper binding histidines to solvent which slightly stabilizes the cupric state and increases histidine basicity. The large immobilization-induced increase in the pK(a) for the acid transition (from 4.5 to 6.3) makes this electrode-SAM-protein construct an attractive candidate as a biomolecular ET switch operating near neutral pH in molecular electronics. Such a potential interest is increased by the robustness of this interface against chemical unfolding as it undergoes only moderate changes in the reduction thermodynamics and in the ET rate in the presence of up to 8 M urea. The sensitivity of these parameters to solvent H/D isotope effects testifies to the role of protein solvation as effector of the thermodynamics and kinetics of ET.

The low-pH conformational equilibria of ferric yeast iso-1 cytochrome c (ycc) and its M80A, M80A/Y67H, and M80A/Y67A variants were studied from pH 7 to 2 at low ionic strength through electronic absorption, magnetic circular dichroism, and resonance Raman spectroscopies. For wild-type ycc, the protein structure, axial heme ligands, and spin state of the iron atom convert from the native folded His/Met low-spin (LS) form to a molten globule His/H(2)O high-spin (HS) form and a totally unfolded bis-aquo HS state, in a single cooperative transition with an apparent pK(a) of ~3.0. An analogous cooperative transition occurs for the M80A and M80A/Y67H variants. This is preceded by protonation of heme propionate-7, with a pK(a) of ~4.2, and by an equilibrium between a His/OH(-)-ligated LS and a His/H(2)O-ligated HS conformer, with a pK(a) of ~5.9. In the M80A/Y67A variant, the cooperative low-pH transition is split into two distinct processes because of an increased stability of the molten globule state that is formed at higher pH values than the other species. These data show that removal of the axial methionine ligand does not significantly alter the mechanism of acidic unfolding and the ranges of stability of low-pH conformers. Instead, removal of a hydrogen bonding partner at position 67 increases the stability of the molten globule and renders cytochrome c more susceptible to acid unfolding. This underlines the key role played by Tyr67 in stabilizing the three-dimensional structure of cytochrome c by means of the hydrogen bonding network connecting the Ω loops formed by residues 71-85 and 40-57.

Background: Microparticles (MP) are vesicles released from activated or apoptotic cells. Endothelial MP (EMP) are derived from injured endothelium, platelet MP (PMP) from activated platelets, and Annexin V positive MP (AMP) from apoptotic endothelial cells. The aim was to assess the release of MP and its association with inflammation and atherosclerotic burden., Methods and Results: AMP, EMP and PMP were measured on admission (Day 0) in 33 patients with stable angina (SA) and 43 patients with acute coronary syndrome (ACS) undergoing percutaneous coronary interventions (PCI). In SA, peripheral artery disease (PAD) was assessed by ultrasound examination. In 30 of the 76 patients (20 ACS and 10 SA), MP, high-sensitivity-C-reactive protein (hs-CRP), and troponin T (TnT) levels were also assessed 24h (Day 1) and 48 h (Day 2) after PCI. AMP, EMP, and PMP were higher in ACS than in SA (all P<0.01). In the SA group, AMP, PMP, and EMP were similar in patients with or without PAD. In the ACS group, AMP increased until Day 2 (P=0.001), while EMP and PMP peaked on Day 1 (P<0.01) then decreased to baseline values. Day 2 AMP correlated with Day 2 TnT levels (r=0.43, P=0.01) while Day 1 EMP and PMP correlated with Day 1 hs-CRP (r=0.37, P=0.04 and r=0.33, P=0.05; respectively)., Conclusions: Higher MP levels were observed in ACS than in SA. Atherosclerotic burden did not affect MP levels in stable patients.

Presently, orthotopic liver transplant is the major therapeutic option for patients affected by primary liver diseases. This procedure is characterized by major invasive surgery, scarcity of donor organs, high costs, and lifelong immunosuppressive treatment. Transplant of hepatic precursor cells represents an attractive alternative. These cells could be used either for allogeneic transplantation or for autologous transplant after ex vivo genetic modification. We used stromal cells isolated from adipose tissue (AT-SCs) as platforms for autologous cell-mediated gene therapy. AT-SCs were transduced with lentiviral vectors expressing firefly luciferase, allowing for transplanted cell tracking by bioluminescent imaging (BLI). As a complementary approach, we followed circulating human α1-antitrypsin (hAAT) levels after infusion of AT-SCs overexpressing hAAT. Cells were transplanted into syngeneic mice after CCl(4)-induced hepatic injury. Luciferase bioluminescence signals and serum hAAT levels were measured at different time points after transplantation and demonstrate persistence of transplanted cells for up to 2 months after administration. These data, along with immunohistochemical analysis, suggest engraftment and repopulation of injured livers by transplanted AT-SCs. Moreover, by transcriptional targeting using cellular tissue-specific regulatory sequences, we confirmed that AT-SCs differentiate towards a hepatogenic-like phenotype in vitro and in vivo. Additionally, in transplanted cells reisolated from recipient animals' livers, we detected activation of the α-fetoprotein (AFP) promoter. This promoter is normally transcriptionally silenced in adult tissues but can be reactivated during liver regeneration, suggesting commitment towards hepatogenic-like differentiation of engrafted cells in vivo. Our data support AT-SC-mediated gene therapy as an innovative therapeutic option for disorders of liver metabolism.

In anterior resection of rectum, the section level of inferior mesenteric artery is still subject of controversy between the advocates of high and low tie. The low tie is the division and ligation to the branching of the left colic artery and the high tie is the division and ligation at its origin at the aorta. We intend to assess current scientific evidence in literature and to establish the differences comparing technique, anatomy and physiology. The aim of this protocol is to achieve a meta-analysis that tests safety and feasibility of the two procedures with several types of outcome measures.

Introduction: The literature suggests several techniques to ensure the optimal anesthetic procedure for carotid surgery. The aim of our study is to combine, in the carotid endarterectomy, a model of general anesthesia with the loco-regional anesthesia in order to test the safety of the method, the eventual brain damage and hemodynamic stability. We also wanted to test whether the immediate postoperative analgesia was adequate., Material and Methods: Seventy eight (78) patients were enrolled in the study (from 60 to 75 years), ASA II-III, candidates for unilateral carotid endarterectomy in a period from June 2008 to January 2009. During the procedure data about perioperative neurologic and hemodynamic complications were collected. At the end of the surgical treatment the pain at the awakening moment through verbal numerical scale VNS was estimated. Data are presented as mean values +/- sd and validated using the chi2 test, when required. Significance was accepted at a value of P < or = 0.05., Results: 85% of patients reported a VNS between 1 and 2 and 10% equal to 3-5. In the study 95% of patients (P<0.05) appreciated the technique and only 3 patients reported their dissatisfaction. There was no mortality and in 5 patients a postoperative morbidity was registered., Discussion: The study demonstrated the advantages in combining loco-regional anesthesia (valid monitoring of neurological and hemodynamic stability) with general anesthesia (complete airways control and patient comfort) during carotid surgery.

The 16-kDa diheme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in Escherichia coli and investigated through UV-vis, magnetic circular dichroism, and (1)H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides diheme cytochrome c (Rs-DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three α-helices and a compact overall folding. The C-terminal domain is less helical than the corresponding domain of Rs-DHC. The two heme groups are bridged by Tyr26 in correspondence with the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid-base equilibria with pK (a) values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and a pH-linked conformational change of the protein, respectively. Reduction potentials of -0.144 and -0.257 V (vs. the standard hydrogen electrode), were determined for the C- and N-terminal heme groups, respectively. An approach based on the extended Debye-Hückel equation was applied for the first time to a two-centered metalloprotein and was found to reproduce successfully the ionic strength dependence of E°'.

Recent studies have underscored a role for the epicardium as a source of multipotent cells. Here, we investigate the myogenic potential of adult human epicardium-derived cells (EPDCs) and analyze their ability to undergo skeletal myogenesis when cultured with differentiating primary myoblasts. Results are compared to those obtained with mesenchymal stromal cells (MSCs) and with endothelial cells, another mesodermal derivative. We demonstrate that EPDCs spontaneously fuse with pre-existing myotubes with an efficiency that is significantly higher than that of other cells. Although at a low frequency, endothelial cells may also contribute to myotube formation. In all cases analyzed, after entering the myotube, nonmuscle nuclei are reprogrammed to express muscle-specific genes. The fusion competence of nonmyogenic cells in vitro parallels their ability to reconstitute dystrophin expression in mdx mice. We additionally show that vascular cell adhesion molecule 1 (VCAM1) expression levels of nonmuscle cells are modulated by soluble factors secreted by skeletal myoblasts and that VCAM1 function is required for fusion to occur. Finally, treatment with interleukin (IL)-4 or IL-13, two cytokines released by differentiating myotubes, increases VCAM1 expression and enhances the rate of fusion of EPDCs and MSCs, but not that of endothelial cells.

Chronic ulcers represent a major health problem in diabetic patients resulting in pain and discomfort. Conventional therapy does not guarantee adequate wound repair. In diabetes, impaired healing is partly due to poor endothelial progenitor cells mobilisation and homing, with altered levels of the chemokine stromal-derived factor-1 (SDF-1) at the wound site. Adipose tissue-associated stromal cells (AT-SCs) can provide an accessible source of progenitor cells secreting proangiogenic factors and differentiating into endothelial-like cells. We demonstrated that topical administration of AT-SCs genetically modified ex vivo to overexpress SDF-1, promotes wound healing into diabetic mice. In particular, by in vivo bioluminescent imaging analysis, we monitored biodistribution and survival after transplantation of luciferase-expressing cells. In conclusion, this study indicates the therapeutic potential of AT-SCs administration in wound healing, through cell differentiation, enhanced cellular recruitment at the wound site, and paracrine effects associated with local growth-factors production.

Background: We conducted a systematic review to evaluate the role of Ultrasonic dissector (UAS) versus conventional clamp and tie in thyroidectomy., Materials and Methods: We searched for all published RCT in into electronic databases. To be included in the analysis, the studies had to compare thyroidectomy with UAS versus conventional vessel ligation and tight (conventional technique = CT). The following outcomes were used to compare the total thyroidectomy group with UAS versus CT group: operative duration, operative blood loss, overall drainage volume during the first 24 hours, transiet laryngeal nerve palsy, permanent laryngeal nerve palsy, transiet hypocalcaemia and permanent hypocalcaemia., Results: There are currently 7 RCT on this issue to compare thyroidectomy with UAS versus CT. From the analysis of these studies it was possible to confront 608 cases: 303 undergoing to thyroidectomy with UAS versus 305 that were treated with CT. Actually, it was shown a relevant advantage of cost-effectiveness in patients treated with UAS; there is a statistically significant reduction of the operative duration (weighted mean difference [WMD], -18.74 minutes; 95% confidence interval [CI], (-26.97 to -10.52 minutes) (P = 0.00001), intraoperative blood loss (WMD, -60.10 mL; 95% CI, -117.04 to 3.16 mL) (P = 0.04) and overall drainage volume (WMD, -35.30 mL; 95% CI, -49.24 to 21.36 mL) (P = 0.00001) in the patients underwent thyroidectomy with UAS. Although the analysis showed that the patients who were treated with USA presented more favourable results in incidence of post-operative complications (transient laryngeal nerve palsy: P = 0.11; permanent laryngeal nerve palsy: not estimable; transient hypocalcaemia: P = 0.24; permanent hypocalcaemia: P = 0.45), these data didn't present statistical relevance., Conclusion: This meta-analysis shown a relevant advantage only in terms of cost-effectiveness in patients treated with UAS; it is subsequent to statistically significant reduction of operation duration, intraoperative blood loss and of overall drainage volume during the first 24 hours. Although the analysis showed that the patients who were treated with UAS presented more favourable results in incidence of post-operative complications (transiet laryngeal nerve palsy; transiet hypocalcaemia and permanent hypocalcaemia), these data didn't present statistical relevance.

Introduction: The prognosis of patients with melanoma varies according to the staging of disease at the moment of diagnosis. Melanoma can metastasize to every organ or tissue, but the most common site involved is locoregional. In selected patients surgery plays a central role with the possibility of changing the prognosis at distance., Case Report: A 65-year-old man with a diagnosis of neoplasm of the left thyroid lobe and metastatic melanoma with unknown primitive localization. Since 2003 the patient has undergone many operations to remove metastatic melanoma. Currently he is in good conditions and performs neoadjuvant palliative treatments., Discussion: The indication for surgery in cases of patients with distant disease (IV stage) or metastases in transit (IIIc stage) is linked to the possibility of surgical removal of individual lesions. In particular are candidates for surgery patients who have a visceral localization less than or equal to 2 sites, a number less than or equal to 8 metastases, in good health and having a melanoma-specific survival estimated more than 3 months; surgical approach requires an accurate and early identification by imaging study. The case report shows that in selected cases, some patients may benefit from aggressive surgery, especially in terms of survival at distance.

We have studied the effect of urea-induced unfolding on the electron transfer process of yeast iso-1-cytochrome c and its mutant K72AK73AK79A adsorbed on electrodes coated by mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol self-assembled monolayers. Electrochemical measurements, complemented by surface enhanced resonance Raman studies, indicate two distinct states of the adsorbed proteins that mainly differ with respect to the ligation pattern of the haem. The native state, in which the haem is axially coordinated by Met80 and His18, displays a reduction potential that slightly shifts to negative values with increasing urea concentration. At urea concentrations higher than 6 M, a second state prevails in which the Met80 ligand is replaced by an additional histidine residue. This structural change in the haem pocket is associated with an approximately 0.4 V shift of the reduction potential to negative values. These two states were found for both the wild-type protein and the mutant in which lysine residues 72, 73 and 79 had been substituted by alanines. The analysis of the reduction potentials, the reaction enthalpies and entropies as well as the rate constants indicates that these three lysine residues have an important effect on stabilising the protein structure in the adsorbed state and facilitating the electron transfer dynamics.

Aim: To evaluate the convenience in terms of recurrence and fecal incontinence rates of fibrin glue versus surgical treatment in the management of fistula-in-ano., Materials and Methods: Randomized controlled trials (RCTs) and non-randomized studies (CCTs) comparing conventional surgical treatment versus fibrin glue treatment in patients with perianal fistulae were identified using a predefined search strategy. The post treatment anal incontinence rate and the fistula recurrence rates between the two operations were compared by using the methods provided by the Cochrane Handbook for Systematic Reviews of Interventions. The lack of homogeneity of results between the different studies did not allow to analyze other secondary outcomes. Patients with cryptoglandular and Crohn's anal fistula were enrolled in the analysis. The employed fibrin glue came from commercial kits: Beriplast (Aventis Behring, Sussex, United Kingdom) and Tisseal or Tissucol (Baxter, Inc, Mississauga, Ontario). Surgical conventional treatment consisted of fistulotomy, placement of a cutting or loose latex seton and advancement mucosal flap closure. All patients were followed up at 6 and 12 weeks, the longest follow up was 6 months., Results: Two RCTs (106 patients) and 1 non randomized studies (232 patients) were identified. The recurrence rate is higher, although still not statistically significant, in those patients who underwent fibrin glue injection (44/81) versus conventional surgical treatment (108/230), (OR: 0.44; 95 %CI: 0.12-1.68; P = 0.23). Furthermore in the analysis of the subgroup of RCTs alone there were not significant differences with the previous results of RCTs with CCT analysis (OR: 0.33; 95 %CI: 0.03-3.66; P = 0.37). In the same way the analysis of the subgroup of RCTs with complex anal fistulae were not statistically significant and similar to the previous results regarding all type of fistulas (OR: 0.86; 95 %CI: 0.01-72.36; P = 0.95). The analysis of post-operative anal incontinence showed no difference between the group who underwent fibrin glue injection (9/230) and the conventional surgical treatment group (10/81), (OR: 1.00; 95 %CI: 0.43-2.34; P = 1.00). A very low heterogeneity in the analysis was detected (Chi-square = 0.04 - P = 0%)., Conclusion: Our statistical analysis does not show any significant statistical difference between fibrin glue treatment versus conventional surgical treatment for all perianal fistulae in terms of recurrence (P = 0.23) and anal incontinence (P = 1.00).

We analyzed a homogeneous sample of 671 patients underwent total thyroidectomy for various pathologies evaluating the final histological diagnosis and seeking the association between thyroiditis and cancer. As is known to the literature the incidence of association between autoimmune disease and cancer is not exceptional. In our experience, we have shown it in 39 cases (18.6%). We also considered the genetic background RET/PTC which is more prevalent in cases of papillary carcinoma in an inflammatory environment; could be stimulant the study of specific molecular markers to identify targets to inhibit the inflammatory status in thyroid cancer prevention.

Replacement of the axial Met80 heme ligand in electrode-immobilized cytochrome c with a noncoordinating Ala residue and alteration of the hydrogen bonding network in the region nearby following substitution of Tyr67 were investigated as effectors of the thermodynamics and kinetics of the protein-electrode electron transfer (ET) and the heme-mediated electrocatalytic reduction of H(2)O(2). To this end, the voltammetry of the Met80Ala, Met80Ala/Tyr67His, and Met80Ala/Tyr67Ala variants of yeast iso-1-cytochrome c chemisorbed on carboxyalkanethiol self-assembled monolayers was measured at varying temperature and hydrogen peroxide concentration. The thermodynamic study shows that insertion of His and Ala residues in place of Tyr67 results mainly in differences in protein-solvent interactions at the heme crevice with no relevant effects on the E degrees' values at pH 7, which for single and double variants range from approximately -0.200 to -0.220 V (vs SHE). On the contrary, both double variants show much lower ET rates compared to Met80Ala, most likely as a consequence of a change in the ET pathways. In the present nondenaturing immobilizing conditions, and with hydrogen peroxide concentrations in the micromolar range, the variants catalyze H(2)O(2) reduction at the electrode, whereas wild-type cytochrome c does not. H(2)O(2) electrocatalysis occurs with an efficient mechanism likely involving a fast catalase-like process followed by electrocatalytic reduction of the resulting dioxygen at the electrode. Comparison of Met80Ala/Tyr67His with Met80Ala/Tyr67Ala shows that the presence of a general acid-base residue for H(2)O(2) recognition and binding through H-bonding in the distal heme site is a key requisite for the reductive turnover of this substrate.

The recombinant diheme cytochrome c(4) from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 and its Met64Ala and Met164Ala variants, which feature a hydroxide ion axially bound to the heme iron at the N- and C-terminal domains, respectively, were found to exchange electrons efficiently with a gold electrode coated with a SAM of 11-mercapto-1-undecanoic acid. The mutation-induced removal of the redox equivalence of the two heme groups and changes in the net charge of the protein lobes yield two-centered protein systems with unprecedented properties in the electrode-immobilized state. The heterogeneous and intraheme electron transfer processes were characterized for these species in which the high- and low-potential heme groups are swapped over in the bilobal protein framework and experience a constrained (M64A) and unconstrained (M164A) orientation toward the electrode. The reduction thermodynamics for the native and mutated hemes were measured for the first time for a diheme cytochrome c. In the diffusing regime, they reproduce closely those for the corresponding centers in single-heme class-I cytochromes c, despite the low sequence identity. Larger differences are observed in the thermodynamics of the immobilized species and in the heterogeneous electron transfer rate constants. T-dependent kinetic measurements show that the proteins are positioned approximately 7 A from the HOOC-terminated SAM-coated electrode. Protein-electrode orientation and efficient intraheme ET enable the His,OH(-)-ligated heme A of the immobilized Met64Ala variant to carry out the reductive electrocatalysis of molecular oxygen. This system therefore constitutes a novel two-centered heme-based biocatalytic interface to be exploited for "third-generation" amperometric biosensing.

The adsorption of native, wild-type, and engineered cytochrome c on sodium-exchanged kaolinite was investigated by spectroscopic means. The variants of yeast cytochrome c were obtained replacing surface lysines in positions 72, 73, and 79 with alanine residues. All proteins are strongly adsorbed onto kaolinite. In particular, the presence of the lysine residue in position 73 remarkably favors adsorption. A detailed characterization of the thermodynamic aspects of the adsorption process has been performed. Most notably, adsorbed cytochrome c maintains its moderate peroxidase activity against guaiacol. This investigation is prodromal to the exploitation of the catalytic activity of engineered cytochrome c immobilized on a polydisperse system.

The 20-kDa di-heme cytochrome c (4) from the psycrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 was cloned and expressed in Escherichia coli and investigated through UV-vis and (1)H NMR spectroscopies and protein voltammetry. The model structure was computed using the X-ray structure of Pseudomonas stutzeri cytochrome c (4) as a template. The protein shows unprecedented properties within the cytochrome c (4) family, including (1) an almost nonpolar surface charge distribution, (2) the absence of high-spin heme Fe(III) states, indicative of a thermodynamically stable and kinetically inert axial heme His,Met coordination, and (3) identical E degrees ' values for the two heme centers (+0.322 V vs the standard hydrogen elecrode). At pH extremes, both heme groups undergo the "acid" and "alkaline" conformational transitions typical of class I cytochromes c, involving ligand-exchange equilibria, whereas at intermediate pH values their electronic properties are sensitive to several residue ionizations.

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the "LID" domain. The sequence 129Cys-X5-His-X15-Cys-X2-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain.

At the embryonic or fetal stages, autonomously myogenic cells (AMCs), i.e., cells able to spontaneously differentiate into skeletal myotubes, have been identified from several different sites other than skeletal muscle, including the vascular compartment. However, in the adult animal, AMCs from skeletal muscle-devoid tissues have been described in only two cases. One is represented by thymic myoid cells, a restricted population of committed myogenic progenitors of unknown derivation present in the thymic medulla; the other is represented by a small subset of adipose tissue-associated cells, which we recently identified. In the present study we report, for the first time, the presence of spontaneously differentiating myogenic precursors in the pancreas and in other skeletal muscle-devoid organs such as spleen and stomach, as well as in the periaortic tissue of adult mice. Immunomagnetic selection procedures indicate that AMCs derive from Flk-1(+) progenitors. Individual clones of myogenic cells from nonmuscle organs are morphologically and functionally indistinguishable from skeletal muscle-derived primary myoblasts. Moreover, they can be induced to proliferate in vitro and are able to participate in muscle regeneration in vivo. Thus, we provide evidence that fully competent myogenic progenitors can be derived from the Flk-1(+) compartment of several adult tissues that are embryologically unrelated to skeletal muscle.

Objectives: The aim of the study was to analyze the molecular mechanisms activated during postinfarction remodeling in human hearts., Background: The molecular mechanisms of initial response to ischemic insult in the heart and the pathways involved in compensation and remodeling are still largely unknown., Methods: Up-regulation or down-regulation of gene expression in the human viable peri-infarct (vs. remote) myocardial region was investigated by complementary deoxyribonucleic acid array technology and confirmed at a single-gene/protein level with reverse transcriptase polymerase chain reaction and immunohistochemistry. An in vitro model of cardiomyocyte hypoxia in HL1 cells was used to validate anti-apoptotic effects of the candidate gene/protein and to assess the associated downstream cascade. Finally, a mouse model of myocardial infarction was used to test the in vivo effects of exogenous transfection with the candidate gene/protein., Results: Protein disulfide isomerase (PDI), a member of the unfolded protein response, is 3-fold up-regulated in the viable peri-infarct myocardial region, and in a postmortem model, its expression is significantly inversely correlated with apoptotic rate and with presence of heart failure (HF) and biventricular dilatation. Induced PDI expression in HL1 cells conferred protection from hypoxia-induced apoptosis. Adenoviral-mediated PDI gene transfer to the mouse heart resulted in 2.5-fold smaller infarct size, significantly reduced cardiomyocyte apoptosis in the peri-infarct region, and smaller left ventricular end-diastolic diameter versus mice treated with a transgene-null adenoviral vector., Conclusions: These results suggest that PDI promotes survival after ischemic damage and that zinc-superoxide dismutase is one of the PDI molecular targets. Pharmacological modulation of this pathway might prove useful for future prevention and treatment of HF.

Untrimethylated yeast iso-1-cytochrome c (cytc) and its single and multiple Lys to Ala variants at the surface lysines 72, 73, and 79 were adsorbed on carboxyalkanethiol self-assembled monolayers (SAMs) on gold, and the thermodynamics and kinetics of the heterogeneous protein-electrode electron-transfer (ET) reaction were determined by voltammetry. The reaction thermodynamics were also measured for the same species freely diffusing in solution. The selected lysine residues surround the heme group and contribute to the positively charged domain of cytc involved in the binding to redox partners and to carboxyl-terminated SAM-coated surfaces. The E degrees' (standard reduction potential) values for the proteins immobilized on SAMs made of 11-mercapto-1-undecanoic acid and 11-mercapto-1-undecanol on gold were found to be lower than those for the corresponding diffusing species owing to the stabilization of the ferric state by the negatively charged SAM. For the immobilized proteins, Lys to Ala substitution(s) do not affect the surface coverage, but induce significant changes in the E degrees' values, which do not simply follow the Coulomb law. The results suggest that the species-dependent orientation of the protein (and thereby of the heme group) toward the negatively charged SAM influences the electrostatic interaction and the resulting E degree' change. Moreover, these charge suppressions moderately affect the kinetics of the heterogeneous ET acting on the reorganization energy and the donor-acceptor distance. The kinetic data suggest that none of the studied lysines belong to the interfacial ET pathway.

The thermodynamics of the alkaline transition which influences the spectral and redox properties of the type 1 copper center in phytocyanins has been determined spectroscopically. The proteins investigated include Rhus vernicifera stellacyanin, cucumber basic protein and its Met89Gln variant, and umecyanin, the stellacyanin from horseradish roots, along with its Gln95Met variant. The changes in reaction enthalpy and entropy within the protein series show partial compensatory behavior. Thus, the reaction free energy change (hence the pK (a) value) is rather variable. This indicates that species-dependent differences in reaction thermodynamics, although containing an important contribution from changes in the hydrogen-bonding network of water molecules in the hydration sphere of the protein (which feature enthalpy-entropy compensation), are to a large extent protein-based. The data for axial ligand variants are consistent with the hypothesis of a copper-binding His as the deprotonating residue responsible for this transition.

Differently from some lower vertebrates, which can completely regenerate their heart, in higher vertebrates cardiac injury generally leads to progressive failure. Induction of cycle re-entry in terminally differentiated cardiomyocytes and stem-cell transplantation are strategies to increase the regenerative potential of the heart. As experimental and clinical studies progress, demonstrating that adult stem-cell administration has a favorable impact on myocardial function, the identification of cardiac stem cells suggests that some endogenous repair mechanisms actually exist in the mammalian heart. However, a deeper understanding of the mechanism that drives cardiomyocyte proliferation and stem-cell-mediated cardiac repair is required to translate such strategies into effective therapies.

Direct protein electrochemistry was used to obtain the thermodynamic parameters of transition from the native (state III) to the alkaline (state IV) conformer for untrimethylated Saccharomyces cerevisiae iso-1-cytochrome c expressed in E. coli and its single and multiple lysine-depleted variants. In these variants, one or more of the lysine residues involved in axial Met substitution (Lys72, Lys73, and Lys79) was mutated to alanine. The aim of this work is to determine the thermodynamic affinity of each of the substituting lysines for the heme iron and evaluate the interplay of enthalpic and entropic factors. The equilibrium constants for the deprotonation reaction of Lys72, 73, and 79 were computed for the minimized MD average structures of the wild-type and mutated proteins, applying a modified Tanford-Kirkwood calculation. Solvent accessibility calculations for the substituting lysines in all variants were also performed. The transition enthalpy and entropy values within the protein series show a compensatory behavior, typical of a process involving extensive solvent reorganization effects. The experimental and theoretical data indicate that Lys72 most readily deprotonates and replaces M80 as the axial heme iron ligand, whereas Lys73 and Lys79 show comparably higher pKa values and larger transition free energies. A good correlation is found within the series between the lowest calculated Lys pKa value and the corresponding experimental pKa value, which can be interpreted as indicative of the deprotonating lysine itself acting as the triggering group for the conformational transition. The triple Lys to Ala mutant, in which no lysine residues are available for heme iron binding, features transition thermodynamics consistent with a hydroxide ion replacing the axial methionine ligand.

For many streptomycetes, a distinct dependence on the "bioavailability" of copper ions for their morphological development has been reported. Analysis of the Streptomyces coelicolor genome reveals a number of gene products encoding for putative copper-binding proteins. One of these appears as an unusual copper-binding protein with a lipoprotein signal sequence and a cupredoxin-like domain harboring a putative Type-1 copper-binding motif. Cloning of this gene from S. coelicolor and subsequent heterologous expression in Escherichia coli has allowed for a thorough spectroscopic interrogation of this putative copper-binding protein. Optical and electron paramagnetic resonance spectroscopies have confirmed the presence of a "classic" Type-1 copper site with the axial ligand to the copper a methionine. Paramagnetic NMR spectroscopy on both the native Cu(II) form and Co(II)-substituted protein has yielded active-site structural information, which on comparison with that of other cupredoxin active sites reveals metal-ligand interactions most similar to the "classic" Type-1 copper site found in the amicyanin family of cupredoxins. Despite this high structural similarity, the Cu(II)/(I) midpoint potential of the S. coelicolor protein is an unprecedented +605 mV vs normal hydrogen electrode at neutral pH (amicyanin approximately +250 mV), with no active-site protonation of the N-terminal His ligand observed. Suggestions for the physiological role/function of this high-potential cupredoxin are discussed.

Adipose-tissue-derived mesenchymal stem cells can be directed towards a myogenic phenotype in vitro by the addition of specific inductive media. However, the ability of these or other adipose-tissue-associated cells to respond to ;natural' myogenic cues such as a myogenic environment has never been investigated in detail. Here, we provide evidence that a restricted subpopulation of freshly harvested adipose-tissue-derived cells possesses an intrinsic myogenic potential and can spontaneously differentiate into skeletal muscle. Conversion of adipose-tissue-derived cells to a myogenic phenotype is enhanced by co-culture with primary myoblasts in the absence of cell contact and is maximal when the two cell types are co-cultured in the same plate. Conversely, in vitro expanded adipose-tissue-derived mesenchymal stem cells require direct contact with muscle cells to generate skeletal myotubes. Finally, we show that uncultured adipose-tissue-associated cells have a high regenerative capacity in vivo since they can be incorporated into muscle fibers following ischemia and can restore significantly dystrophin expression in mdx mice.

In this work, we have determined the thermodynamic parameters of the reduction of four different variants of Thiobacillus versutus amicyanin by electrochemical techniques. In addition, the thermodynamic parameters were determined of the low-pH conformational change involving protonation of the C-terminal histidine ligand and the concomitant dissociation of this histidine from the Cu(I) ion. In these variants, the native C-terminal loop containing the Cys, His, and Met copper ligands has been replaced with the corresponding polypeptide segments of Pseudomonas aeruginosa azurin, Populus nigra plastocyanin, Alcaligenes faecalis S-6 pseudoazurin, and Thiobacillus ferrooxidans rusticyanin. For the reduction reaction, each loop invariably holds an entropic "memory" of the mother protein. The thermodynamics of the low-pH transition vary in a fashion that is species-dependent. When present, the memory effect again shows a large entropic component. In particular, loop elongation tends to favor the formation of the Cu(I)-His bond (hence disfavors His protonation, yielding lower pK(a) values) probably due to an increased flexibility of the loop in the reduced state. Overall, it appears that both reduction and low-pH transition are loop-responsive processes. The spacing between the ligands mostly affects the change in the conformational freedom that accompanies the reaction.

The L12E, L12K, Q88E, and Q88K variants of spinach plastocyanin have been electrochemically investigated. The effects of insertion of net charges near the metal site on the thermodynamics of protonation and detachment from the copper(I) ion of the His87 ligand have been evaluated. The mutation-induced changes in transition enthalpy cannot be explained by electrostatic considerations. The existence of enthalpy/entropy (H/S) compensation within the protein series indicates that solvent-reorganization effects control the differences in transition thermodynamics. Once these compensating contributions are factorized out, the resulting modest differences in transition enthalpies turn out to be those that can be expected on purely electrostatic grounds. Therefore, this work shows that the acid transition in cupredoxins involves a reorganization of the H-bonding network within the hydration sphere of the molecule in the proximity of the metal center that dominates the observed transition thermodynamics and masks the differences that are due to protein-based effects.

The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.

Compensation phenomena between the enthalpy and entropy changes of the reduction reaction for all classes of electron transport metalloproteins, namely cytochromes, iron-sulfur, and blue copper proteins, are brought to light. This is the first comprehensive report on such effects for biological redox reactions. Following Grunwald's approach for the interpretation of H/ S compensation for solution reactions, it is concluded that reduction-induced solvent reorganization effects involving the hydration shell of the molecule dominate the reduction thermodynamics in these species, although they have no net effect on the E degrees values, owing to exact compensation. Thus the reduction potentials of these species are primarily determined by the selective enthalpic stabilization of one of the two oxidation states due to ligand binding interactions and electrostatics at the metal site and by the entropic effects of reduction-induced changes in protein flexibility.

The 1H NMR spectra of the fully oxidized Rhus vernicifera laccase and of its 1:1 and 2:1 azide adducts are reported for the first time. These spectra, which are the first so far reported for a multi copper oxidase, contain a number of broad hyperfine-shifted resonances in the high frequency region of the spectrum, which are attributed to the metal binding residues of the mononuclear T1 center. The differences between the patterns of the hyperfine resonances of the free enzyme and its azide derivatives suggest that the alterations in the structural properties of the T3 site induced by the binding of the first azide molecule induce a limited alteration of the spin density distribution over the T1 copper ligands. Overall, these data demonstrate that 1H NMR can be fruitfully applied to characterize the electronic properties of the metal sites of blue oxidases at room temperature.

Well-differentiated thyroid neoplasms may be included among the most frequently occurring thyroid carcinomas. Papillary ca. is without doubt the best behaved type. The aim of the present work is to perform a retrospective case history study to assess patients with con papillary ca. who have been treated surgically over the last 17 years and have been subjected to periodic checks. A sample of patients was therefore extrapolated who had all undergone total thyroidectomy for papillary ca. of the thyroid. The incidence of local recurrence of the disease was verified, together with the results at distance. Furthermore, the assessments performed were evaluated and compared. From the sample of patients observed we inferred that papillary carcinoma of the thyroid can have a good prognosis over time provided periodic random checks are carried out.

Introduction: Breast cancer is the most frequent cancer among female sex, above all in rich countries where it occurs ten times more than the others. The most incidence of breast cancer is in female over 45 yrs and over 70 yrs with a plateau during menopause. So it is really very uncommon before 30 yrs. It is well known that breast cancer is related to several risk factors: these have always to be well evaluated before any treatment, especially in surgery. In the last years we can see an improvement of techniques to make diagnosis of breast cancer. Also surgery has improved in that way, so it can ensure better results to patients than before, also from the esthetical point of view. There are lot of discussions on which is the best surgical treatment to do, if lymphoadenectomy of the armpit is always required and if it has to be considered only as a prognostic element or also a therapeutical one. It is also important to plan radio, chemio and hormonal therapy, well chosen on the base of histological exams, and of the grading made either before either during surgery. Last studies moreover evaluate the specificity, the importance and the prognostic value of searching the so called "sentinel lymph node" who is always the first one to be interested in a metastatic process., Study and Discussion: Through a random and retrospective study among all our female patients (Department of Surgical Science in Rome University "La Sapienza") who had surgery for breast cancer in the last ten years (approving all the hystological exams) we have related the kind of surgical treatments to overall survival, to the therapies, and to any possible local recurrence of disease. We analyzed 270 female patients (age between 19 and 83). We considered all surgical treatments used in breast cancer: from the "simple" quadrantectomy up to radical mastectomy with the association of lymphoadenectomy of the armpit (both armpits in case of recurrence to disease). All these patients had then their own therapy (radio, chemo or hormonal one, it depended on each case) and follow up of the length of at least five years. The overall survival was of 82.2%. From these study we made important considerations about all the factors involved in breast cancer. The first step is to consider always all of these to have a well surgical approach, and to get the best compliance and performance from patients in order to have at distance the best results.

Regionally restricted expression patterns of Hox genes in developing embryos rely on auto-, cross-, and para-regulatory transcriptional elements. One example is the Hoxb1 auto-regulatory element (b1-ARE), which drives expression of Hoxb1 in the fourth rhombomere of the hindbrain. We previously showed that HOXB1 and PBX1 activate transcription from the b1-ARE by binding to sequences required for the expression of a reporter gene in rhombomere 4 in vivo. We now report that in embryonal carcinoma cells, which retain characteristics of primitive neuroectodermal cells, the b1-ARE displays higher basal and HOX/PBX-induced activities than in other cell backgrounds. We have identified a bipartite-binding site for SOX/OCT heterodimers within the b1-ARE that accounts for its cell context-specific activity and is required for maximal transcriptional activity of HOX/PBX complexes in embryonal carcinoma cells. Furthermore, we found that in an embryonal carcinoma cell background, HOXB1 has a significantly higher transcriptional activity than its paralog HOXA1. We map the determinants for this differential activity within the HOXB1 N-terminal transcriptional activation domain. By using analysis in transgenic and HOXA1 mutant mice, we extended these findings on the differential activities of HOXA1 and HOXB1 in vivo, and we demonstrated that they are important for regulating aspects of HOXB1 expression in the hindbrain. We found that mutation of the SOX/OCT site and targeted inactivation of Hoxa1 both impair the response of the b1-ARE to retinoic acid in transgenic mice. Our results show that Hoxa1 is the primary mediator of the response of b1-ARE to retinoic acid in vivo and that this function is dependent on the binding of SOX/OCT heterodimers to the b1-ARE. These results uncover novel functional differences between Hox paralogs and their modulators.

Cell type-specific expression of the human alpha2(V) collagen (COL5A2) gene depends on a cis-acting element that consists of two contiguous protein binding sites (FPA and FPB) located between nucleotides -149 and -95, relative to the transcription start site. The present study focused on the characterization of the FPB-bound complex. DNA binding assays and cell transfection experiments revealed that the bipartite core sequence of FPB (5'-ATCAATCA-3') binds the PBX1/2, PREP1, and HOXB1 proteins, and this in turn leads to promoter transactivation. In the presence of all three nuclear factors, cooperative interactions between recombinant PBX1 and PREP1 or PBX1 and HOXB1 result in binding of the heterodimers to FPB in vitro. Similarly, overexpression of different combinations of PBX1, PREP1, and HOXB1 transactivates FPB-driven transcription. In contrast to the composition of the FPB complex purified from COL5A2-positive cells, the FPB complex from COL5A2-negative cells contains PBX2 and PREP1 but lacks PBX1. However, PBX1 exogenously introduced into COL5A2-negative cells cannot stimulate FPB-driven transcription unless co-expressed with PREP1. Within the intrinsic limitations of the experimental model, our results indicate that combinatorial interactions among PBX and PREP or HOX proteins are involved in regulating tissue-specific production of collagen V.

Hox proteins control developmental patterns and cell differentiation in vertebrates by acting as positive or negative regulators of still unidentified downstream target genes. The homeodomain and other small accessory sequences encode the DNA-protein and protein-protein interaction functions which ultimately dictate target recognition and functional specificity in vivo. The effector domains responsible for either positive or negative interactions with the cell transcriptional machinery are unknown for most Hox proteins, largely due to a lack of physiological targets on which to carry out functional analysis. We report the identification of the transcriptional activation domains of three human Hox proteins, HOXB1, HOXB3, and HOXD9, which interact in vivo with the autoregulatory and cross-regulatory enhancers of the murine Hoxb-1 and human HOXD9 genes. Activation domains have been defined both in a homologous context, i.e., within a HOX protein binding as a monomer or as a HOX-PBX heterodimer to the specific target, and in a heterologous context, after translocation to the yeast Gal4 DNA-binding domain. Transfection analysis indicates that activation domains can be identified in different regions of the three HOX proteins depending on the context in which they interact with the DNA target. These results suggest that Hox proteins may be multifunctional transcriptional regulators, interacting with different cofactors and/or components of the transcriptional machinery depending on the structure of their target regulatory elements.

The p21ras small GTP binding proteins participate in signal transduction from cell surface receptors and affect neoplastic transformation and development in many different cell types. In the present study, we examined the relationship between ras transformation and differentiation of human B lymphocytes. We show that the constitutive expression of the T24 Ha-ras oncogene in EBV-immortalized B lymphoblasts was associated with the induction of the interleukin 2 receptor alpha subunit, with an impaired immunoglobulin gene expression, altered adhesion properties and increased survival in serum-free medium. Since induction of the IL-2 receptor alpha subunit is a hallmark of lymphocyte activation, we suggest that p21ras naturally triggers B cell activation. The ras-transformed lymphocytes displayed a fully functional IL-2r, as assessed by c-fos induction following treatment with IL-2; nevertheless, they were not growth stimulated by this lymphokine. The decreased expression of immunoglobulin genes indicates that the ras oncogene blocks terminal differentiation to plasma cells, possibly by inhibiting the activity of lymphocyte-specific transcription factors. Somewhat unexpectedly, the constitutive p21ras activity did not cause an increased DNA binding of transcription factors PEA1 (AP1), PEA3, Oct-2 or NF-kB.