New TRAP1 and Hsp90 chaperone inhibitors with cationic components: Preliminary studies on mitochondrial targeting.

TRAP1 (Hsp75) is the mitochondrial paralog of the Hsp90 molecular chaperone family. Due to structural similarity among Hsp90 chaperones, a potential strategy to induce apoptosis through mitochondrial TRAP1 ATPase inhibition has been envisaged and a series of compounds has been developed by binding the simple pharmacophoric core of known Hsp90 inhibitors with various appendages bearing a permanent cationic head, or a basic group highly ionizable at physiologic pH. Cationic appendages were selected as vehicles to deliver drugs to mitochondria. Indeed, masses of new derivatives were evidenced to accumulate in the mitochondrial fraction from colon carcinoma cells and a compound in the series, with a guanidine appendage, demonstrated good activity in inhibiting recombinant TRAP1 ATPase and cell growth and in inducing apoptotic cell death in colon carcinoma cells.

In vitro antiproliferative study of novel adamantyl pyridin-4-ones.

The preparation of several N-aryl-substituted (phenyl, p-methylphenyl, p-methoxyphenyl, p-nitrophenyl, p-aminophenyl, p-hydroxyphenyl) 3-hydroxy-2-methylpyridin-4-ones as well as their adamantyl derivatives is described, and their in vitro antitumor properties were investigated. The compounds were synthesized in good yields using efficient synthetic routes and methods. Prepared derivatives were evaluated in an antiproliferative in vitro study on 4 cancer cell lines, namely HCT 116 (colon carcinoma), H 460 (lung carcinoma), MCF-7 (breast carcinoma) and K562 (chronic myelogenous leukemia). All tested compounds showed antiproliferative activity ranging from moderate to strong on all inspected cell lines with 4 adamantane containing derivatives being active and selective at low micromolar IC[Formula: see text] concentrations on HCT 116, H 460 and MCF-7. LDH cytotoxicity assay revealed that cytotoxic effects occur after 48 h of exposure. It was shown that there was no change in caspase activity in the treated cells, but there were changes in the cell cycle. All treated samples showed reduced number of cells in the S phase with increased G0/G1 (4b, 5a, 5b) and G2/M (4a) phase.

Effect of proteases against biofilms of Staphylococcus aureus and Staphylococcus epidermidis.

UNLABELLED: Biofilms play a key role in bacterial resistance against antibacterial agents-an issue that causes multiple problems in medical fields, particularly with Staphylococcus biofilms that colonize medical indwelling devices. The literature reports several anti-biofilm strategies that have been applied in medicine. Disrupting the biofilm formation process creates new sites open to colonization by treatment-generated planktonic bacteria, so efforts have turned to focus on strategies to prevent and control the initial Staphylococci adhesion. Here, we investigated the preventive activities of three commercial proteases (Flavourzyme, Neutrase and Alcalase) against biofilm formation by two Staphylococcus strains. Some proteolytic extracts revealed interesting results with Staphylococcus epidermidis and Staphylococcus aureus aureus biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Three proteases were tested against Staphylococcus aureus and Staphylococcus epidermidis biofilms in standard conditions. The Flavourzyme containing a mix of Aspergillus orizae endo- and exoproteases demonstrated significant efficacy against Staph. epidermidis biofilm formation. These results could prove valuable in the effort to develop simple anti-biofilm methods.

A new tool to detect high viscous exopolymers from microalgae.

Microalgae are microorganisms often surrounded by a slime layer made of secreted polymeric substances sometimes including polysaccharides. These polysaccharides, weakly described in the literature, can constitute value-added molecules in several industrial areas. The aim of this article is to show that a new tool, the BioFilm Ring Test®, can be used to detect viscous microalgal exopolymers. Two red microalgal strains (Rhodella violacea and Porphyridium purpureum), one cyanobacterium (Arthrospira platensis) and their excreted polymeric fractions were studied. R. violacea and P. purpureum induced a positive response with the BioFilm Ring Test® contrary to A. platensis. Finally, the understanding of the fractions viscosity involvement in the BRT response was performed by a rheological study.

Antioxidant Activities of Peptoid-Grafted Chitosan Films.

The aim of this study was to investigate the possibility of immobilizing peptoid on chitosan film in order to generate new active material. Chitosan films have been grafted for the first time with short-length peptoid oligomers displaying antioxidant activities. The antioxidant activity of the selected peptoids was initially investigated with the DPPH assay and hydroxyl radical procedure. The metal chelating capacity of peptoids was also evaluated prior to their covalent attachment to chitosan. The benefit of chitosan functionalization with respect to its intrinsic antioxidant properties was finally evaluated in the present study. Interestingly, an increase of up to 90 % of the antioxidant activity of chitosan was observed.

Analysis of the individual contributions of immunoglobulin heavy and light chains to the binding of antigen using cell transfection and plasmon resonance analysis.

We have cloned the Tg10 murine monoclonal antibody, which is specific for a human thyroglobulin (hTg) epitope targeted by autoantibodies in several thyroid pathologies. Transfection of COS-7 cells with plasmids expressing Tg10H and -kappa chains combined with surface plasmon resonance analysis (BIAcore) of culture supernatants showed that the entire cloned Tg10 antibody displays an affinity comparable to that of the parental antibody. This approach also permitted determination of the probable role of each chain to the recognition of the cognate epitope due to the ability of COS-7 cells to secrete independently each of the two constituting immunoglobulin chains. Tg10 heavy chain recognizes hTg in the absence of the light chain, but with a ten-fold lower affinity mainly due to an increase in kappaoff. In contrast, the light chain is unable to bind hTg on its own. This suggests that the latter is probably involved in stabilization rather than in initiating the formation of the antibody/antigen complex and that the specificity of Tg10 is mostly, if not exclusively, carried by the heavy chain. The potential applications of combined cell transfection and surface plasmon resonance to our understanding of antigen/antibody interactions are discussed.

[Clinical and capillaroscopic evaluation of chronic uncomplicated venous insufficiency with procyanidins extracted from vitis vinifera]. / Valutazione clinica e capillaroscopica del trattamento dell'insufficienza venosa cronica non complicata con oligomeri procianidolici, estratti da semi di vitis vinifera.

BACKGROUND: The pharmacological treatment of non-complicated chronic venous insufficiency is a current and well-debated topic. The introduction of new products with action on the venous system, improved knowledge on the physiopathology of venous insufficiency and the possibility provided by new analytical instruments, have given new impulse to the consolidation of the clinical value of phlebotonics in this indication. METHODS: In light of this, 24 patients with non-complicated chronic venous insufficiency were treated with oral administration of Oligomeric Proanthocyanidins (Pycnogenols-OPC) 100 mg/day. To evaluate the therapeutic efficacy of the treatment, an instrumental evaluation by optical probe capillaroscope was employed in addition to the traditional subjective clinical parameters: swelling, itching, heaviness and pain. The videocapillaroscope examination was performed at the lower third of the leg and the first toe. Edema in the capillaroscopic field, the number of observable capillaries and the capillary dilatation were the parameter chosen to evaluate the efficacy of treatment. All patients completed the study with no reports of adverse events during the period of observation. RESULTS: The results obtained show a positive clinical response (improved or absent symptoms) in over 80% of patients, with significant improvement of symptoms already evident after the first 10 days of treatment. The mechanism of action of the OPCs explains the rapid reduction of the swelling of the lower limbs and correlated with this are the other evaluable symptoms: heaviness and itching. Particularly striking results were observed for itching and pain which completely disappeared during the course of therapy in 80% and 53% of the patients respectively. Noteworthy is the good correlation between the clinical and instrumental data, with improvement in a total of 70% of patients. CONCLUSIONS: The results obtained in the course of this clinical experience, with evident improvement already during the first weeks of treatment, the absence of adverse events added to the benefit of a once-a-day administration, justify the use of OPC in the treatment of non-complicated chronic venous insufficiency.

A proposed multi-detector method for prompt 90Sr detection in post-accident environmental matrices.

Strontium-90 is of relevant biological importance among fission products released during a fission reactor accident. Rapid information on its concentration in environmental matrices is extremely valuable, yet this implies chemical separations and handling with a shift in daughter equilibrium and therefore the need to wait a sufficient time for the equilibrium to be reestablished. The work concerns a feasibility study of a multi-detector system for a prompt evaluation of the 90Sr activity or, at least, a prompt determination of its order of magnitude in the presence of other pure beta and beta-gamma emitters; their interferences are examined on the basis of the Chernobyl releases and their decay properties. The technique is based on a plastic scintillator beta detector and a guard ring of BGO counters which can be logically connected in coincidence and anticoincidence. The evaluations show that a few hours are sufficient to determine a specific activity comparable with the Maximum Permissible Concentration in air by sampling 10 m3.

The state of the art of non-invasive diagnostics for bone densitometry. Methods of photonic absorption.

The authors examine densitometric diagnostics by photonic absorption for the in-vivo determination of bone mass. Based on an analysis of the literature a comparison is made for values for accuracy, repeatability, and dosimetry offered by techniques such as single-energy photon absorptiometry (SPA), dual photon absorptiometry (DPA), dual-energy X-ray absorptiometry (DXA), or quantitative computer tomography (QCT). Also discussed are economic aspects and daily management, recent developments and future possibilities. None of the densitometric diagnostics examined is capable of entirely satisfying the different needs of measurement, and nearly all of the methods of photonic absorption reported here have specific indications. The coexistence of different systems will increase the priority of the definition of standardized protocols of calibration, so as to obtain densitometric data that may be compared.

Long-term risk of fibroid recurrence after laparoscopic myomectomy.

OBJECTIVE: The use of laparoscopic myomectomy as a surgical treatment for uterine leiomyoma is associated with low intraoperative morbidity and short hospitalization. Limited data about the long-term outcome of this surgical approach are available. The aims of this study were to estimate the risk of uterine fibroid recurrence after laparoscopic myomectomy and to identify factors contributing to the rate of fibroid relapse. STUDY DESIGN: Between 1996 and 2003, 331 patients underwent laparoscopic myomectomy to treat uterine leiomyoma in our hospital; 224 of these patients consented to participate in our 2009 follow-up survey. Clinical symptomatic uterine leiomyoma recurrence was defined as relapse. Recurrence rates at 24 and 60 months post-operatively were calculated for the study population. Fisher's exact tests were used to examine the impacts of factors previously linked to an increased risk of fibroid recurrence, including (1) patient age at the time of initial surgery, (2) pre-operative body mass index, (3) number and localization of uterine leiomyoma removed, and (4) pregnancy and (5) delivery after laparoscopic myomectomy on fibroid recurrence in our study cohort. RESULTS: We observed 75 recurrences in 224 patients. The cumulative risk of recurrence was 4.9% at 24 months and 21.4% at 60 months post-operatively. An age of 30-40 years and the presence of more than one fibroid at the time of initial laparoscopic myomectomy were identified as factors significantly increasing the risk of symptomatic recurrence after laparoscopic myomectomy (31.25% and 38.71%, respectively; both p<0.01). CONCLUSION: Patients with multiple uterine leiomyoma and those in the third decade of life should be counselled thoroughly about the risk of recurrence prior to laparoscopic myomectomy. The low observed recurrence rate in peri- and postmenopausal patients in our study may support the use of laparoscopic myomectomy as a uterus-preserving surgical alternative beyond the reproductive period.

Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options.

The incidence of fungal infections has increased significantly, so contributing to morbidity and mortality. This is caused by an increase in antimicrobial resistance and the restricted number of antifungal drugs, which retain many side effects. Candida species are major human fungal pathogens that cause both mucosal and deep tissue infections. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth. Biofilms are biological communities with a high degree of organization, in which micro-organisms form structured, coordinated and functional communities. These biological communities are embedded in a self-created extracellular matrix. Biofilm production is also associated with a high level of antimicrobial resistance of the associated organisms. The ability of Candida species to form drug-resistant biofilms is an important factor in their contribution to human disease. The study of plants as an alternative to other forms of drug discovery has attracted great attention because, according to the World Health Organization, these would be the best sources for obtaining a wide variety of drugs and could benefit a large population. Furthermore, silver nanoparticles, antibodies and photodynamic inactivation have also been used with good results. This article presents a brief review of the literature regarding the epidemiology of Candida species, as well as their pathogenicity and ability to form biofilms, the antifungal activity of natural products and other therapeutic options.

New perspectives for Lactobacilli exopolysaccharides.

Lactobacilli have the ability to produce different kinds of exopolysaccharides (EPS) exhibiting a wide diversity of structures. EPS are classified, according to their composition into homopolysaccharides and heteropolysaccharides. One of their most described applications is their utilization as texturing agents naturally synthesized in the fermented food products. Nowadays, in regard to demand of modern consumers focusing towards safe and healthy food without additives, new perspectives of development appear for these biopolymers. The GRAS (Generally Recognized As Safe) and probiotic status of some lactobacilli give to them more preference for consumable EPS production. The main drawbacks limiting their industrial expansion are their low yields of production and the validation of their healthy allegations. Moreover, the texturing role of these exopolysaccharides, notably in dairy products, is actually a controversial issue. This review focuses on the novel ways of EPS production employing Lactobacillus spp. and their potential as nutraceuticals.

A new method to screen polysaccharide cleavage enzymes.

The activity of polysaccharide cleavage enzymes has usually been evaluated by qualitative plate screening methods and quantitative colorimetric or chromatographic assays. The recent development of protein engineering has shown the limits of these techniques when applied to high throughput screening. Here we propose a microplate method to measure the activity of polysaccharide cleavage enzymes through small variations in viscosity. Polysaccharide solutions are co-incubated with magnetic particles in enzyme buffers. The cleavage action of polymer-degrading enzymes increases the mobility of the particles in a magnetic field, even at low levels of enzyme activities. This reproducible, sensitive technique was used to evaluate enzymatic specificity towards substrates. BioFilm indices (BFI) determined by associated software were used to follow enzyme kinetics and measure the usual variables.

New method showing the influence of matrix components in Leuconostoc mesenteroides biofilm formation.

Studying biofilm formation and influence of the matrix composition was heavy because only old and long methods were employed up to now: confocal microscopy, fluorescent chemical markers, and/or dying techniques. In this context, an innovative tool, the BioFilm Ring Test, was here employed to explore the role of exopolysaccharides, proteins, and nucleic acids in the formation of biofilm by Leuconostoc mesenteroides. The principle is to add magnetic particles in the culture medium. When a biofilm is formed, particles are unable to migrate in the media to form a ring when a magnet is brought nearer to the well. Therefore, culture media supplemented with proteases, glycanases, and/or nucleases allowed us to identify the involvement of these substances in L. mesenteroides biofilm formation. The results permitted to demonstrate that dextran, proteins, and nucleic acids are implied in biofilm formation.

Expression in Escherichia coli of soluble and M13 phage-displayed forms of a single-chain antibody fragment specific for digoxin: assessment in a novel drug immunoassay.

A high affinity anti-digoxin single-chain Fv antibody fragment (scFv) was cloned from the mouse 2C2 hybridoma cell line and was functionally expressed both in the Escherichia coli periplasm as a soluble molecule and at the surface of the filamentous M13 bacteriophage as a fusion protein with the gene III minor coat protein. The 2C2 scFv sequence significantly differs from that of all the other anti-digoxin antibodies previously described. The 2C2 scFv shares with its parental monoclonal antibody a high specificity for digoxin, a cross-reactivity with active digoxin metabolites, but none with inactive metabolites. M13 phages displaying the 2C2 scFv at their surface have a high apparent affinity constant for digoxin (6.6 x 10(8) M-1) and were directly used to set up a novel type of immunoenzymatic assay for monitoring digoxin in sera of patients treated for either congestive heart failure or cardiac arrythmias. We thus report for the first time that phages displaying scFv may constitute a large source of important new reagents in the field of immunodiagnosis.

Systematic exploration of the antigen binding activity of synthetic peptides isolated from the variable regions of immunoglobulins.

Sets of short (12 residues) cellulose-bound synthetic overlapping peptides derived from the sequences of the variable regions of the heavy and light chains of three different antibodies (an anti-thyroglobulin antibody, the HyHEL-5 anti-lysozyme antibody, and an anti-angiotensin II antibody) were used to systematically assess the antigen binding capacity of peptides from the antibody paratope outside their natural molecular context. Peptides enclosing one or several of the complementarity determining region (CDR) residues had antigen binding activity, although the most active peptides were not necessarily those bearing the greatest number of CDR residues. Several residues from the framework region, preceding or following the CDR, were found to play a role in binding. Affinity constants from 4.1 x 10(-7) to 6.7 x 10(-8) M-1 for the soluble form of 9 lysozyme-binding dodecapeptides were measured by BIAcore analysis. Alanine scanning of lysozyme-binding hexapeptides from the HyHEL-5 sequence identified 38 residues important for binding, of which 22 corresponded to residues that had been shown by x-ray crystallography to be at the interface between HyHEL-5 and lysozyme. Our results could be of interest for the rational identification of biologically active peptides derived from antibody sequences and in providing an experimental basis for mutagenesis of the antibody paratope.