Identification and genetic characterisation of orthopaedic Staphylococcus isolates collected in Italy by automated EcoRI ribotyping

The aim of this study was to evaluate the possibility to use automated EcoRI ribotyping to address, during the same analysis, both identification and genetic characterisation of 38 Staphylococcus aureus and 64 coagulase-negative staphylococci collected from surgical injuries. The ribotyping identification results confirmed those obtained using the API Staph system for 96% of the isolates. All strains were successfully genotyped and the ribotyping discriminatory power, calculated using the Simpson's index of discrimination, was very high for both groups of staphylococci tested. The same, as well as different biotypes, were identified among isolates with the identical ribotyping profile.     

Chemotaxonomic features of lipophilic components inAdesmia species (Leguminosae)

Studies were performed on GC-MS to assess the lipophilic composition of sixAdesmia species representing two subgenera and three series. Normal fatty acids and hydrocarbons were mainly found, as well as acetylenic compounds, dibasic acids, cyclic hydrocarbons, high molecular weight alcohols and one sterol.     

Changes in polyamines, c-myc and c-fos gene expression in osteoblast-like cells exposed to pulsed electromagnetic fields

Pulsed electromagnetic field (PEMF) stimulation promotes the healing of fractures in humans, though its effect is little known. The processes of tissue repair include protein synthesis and cell differentiation. The polyamines (PA) are compounds playing a relevant role in both protein synthesis processes and cell differentiation through c-myc and c-fos gene activation. Since several studies have demonstrated that PEMF acts on embryonic bone cells, human osteoblast-like cells and osteosarcoma TE-85 cell line, in this study we analyzed the effect on cell PAs, proliferation, and c-myc and c-fos gene expression of MG-63 human osteoblast-like cell cultures exposed to a clinically useful PEMF. The cells were grown in medium with 0.5 or 10% fetal calf serum (FCS). c-myc and c-fos gene expressions were determined by RT-PCR. Putrescine (PUT), spermidine (SPD), or spermine (SPM) levels were evaluated by HPLC. [(3)H]-thymidine was added to cultures for DNA analysis. The PEMF increased [(3)H]-thymidine incorporation (P < or = .01), while PUT decreased after treatment (P < or = .01); SPM and SPD were not significantly affected. c-myc was activated after 1 h and downregulated thereafter, while c-fos mRNA levels increased after 0.5 h and then decreased. PUT, SPD, SPM trends, and [(3)H]-thymidine incorporation were significantly related to PEMF treatment. These results indicate that exposure to PEMF exerts biological effects on the intracellular PUT of MG-63 cells and DNA synthesis, influencing the genes encoding c-myc and c-fos gene expression. These observations provide evidence that in vitro PEMF affects the mechanisms involved in cell proliferation and differentiation.     

Laser pressure catapulting (LPC): optimization LPC-system and genotyping of colorectal carcinomas

Genotype analysis is becoming more and more useful in clinical practice, since specific mutations in tumors often correlate with prognosis and/or therapeutic response. Unfortunately, current molecular analytical techniques often require time-consuming and costly steps of analysis, thus making their routine clinical use difficult. Moreover, one of the most difficult problems arising during tumor research is that of their cell heterogeneity, which depends on their clear molecular heterogeneity. SSCP analysis discriminates by means of aberrant electrophoresis migration bands, mutated alleles which may represent as little as 15-20% of their total number. Nevertheless, in order to identify by sequencing the type of alteration revealed by this technique, only the mutated allele must be isolated. The advent of laser microdissection is a procedure which easily solves these problems of accuracy, costs, and time. The aims of this study were to perfect the system of laser pressure catapulting (LPC) laser microdissection for the assessment of the mutational status of p53 and k-ras genes in a consecutive series of 67 patients with colorectal carcinomas (CRC), in order to compare this technique with that involving hand-dissection and to demonstrate that since the LPC system guarantees more accurate biomolecular analyses, it should become part of clinical routine in this field. The LPC-system was perfected with the use of mineral oil and the LPC-membrane. To compare the techniques of hand- and LPC-microdissection, alcohol-fixed, paraffin-embedded tissue from 67 cases of CRC were both hand- and laser-microdissected. In either case, dissected samples were analyzed by SSCP/sequencing and direct sequencing for k-ras and p53 gene mutations. LPC-microdissection made it possible to pick up mutations by direct sequencing or SSCP/sequencing, whereas hand-microdissection mutations were identified only by means of SSCP followed by sequencing; direct sequencing did not reveal any mutation. In the 67 patients examined by either method, 36% (24/67) showed p53 mutations, 32 of which identified. Seventy-eight percent (25/32) were found in the conserved areas of the gene, while 12% (4/32) were in the L2 loop, 50% (16/32) were in the L3 loop, and 12% (4/32) in the LSH motif of the protein. Moreover, of the 67 cases examined, 40% (27/67) showed mutations in k-ras, with a total of 29 mutations identified. Of these, 14 (48%) were found in codon 12 and 15 (52%) in codon 13. The modifications which we brought to the LPC system led to a vast improvement of the technique, making it an ideal substitution for hand-microdissection and guaranteeing a considerable number of advantages regarding facility, accuracy, time, and cost. Furthermore, the data obtained from the mutational analyses performed confirm that the LPC system is more efficient and rapid than hand-microdissection for acquiring useful information regarding molecular profile and can therefore be used with success in clinical routine.     

Highly efficient production of anti-HER2 scFv antibody variant for targeting breast cancer cells

In the present study, cultivation conditions and medium components were optimized using statistical design and analysis to enhance the production of Chi21702, a cold-active extracellular chitinase from the Antarctic bacterium Sanguibacter antarcticus KOPRI 21702. Identification of significant carbon sources and other key elements was performed using a statistical design technique. Chitin and glycerol were selected as main carbon sources, and the ratio of complex nitrogen sources to carbon sources was determined to be 0.5. Among 15 mineral components included in basal medium, NaCl, Fe(C₆H₅O₇), and MgCl₂ were found to have the most influence on Chi21702 production. The optimal parameters of temperature, initial pH, and dissolved oxygen level were found to be 25°C, 6.5, and above 30% of air saturation, respectively. The maximum Chi21702 activity obtained under the optimized conditions was 90 U/L. Through statistical optimization methods, a 7.5-fold increase in Chi21702 production was achieved over unoptimized conditions. Chi21702 showed relatively high activity, even at low temperatures close to 0°C. The information obtained in the present study could be applied to the production of cold-active endochitinase on a large scale, suitable for a process at low temperature in industry.     

Chirality driven self-assembly in a fluorescent organogel

In this work, we present the characterization of an enantiomeric pair of fluorescent dye organogelators and the properties of their stable gel at low concentration in organic solvents. The gels of both enantiomers and of their mixtures were analyzed by differential scanning calorimetry, circular dichroism (CD), atomic force microscopy, UV-vis absorption, and fluorescence. The acquired data were supported by molecular modeling of the helical assembly of the gelators and by the simulation of their CD spectra by means of DeVoe method, and suggested the occurrence of an enantiomeric discrimination process during the formation of the gels.     

Crystal structure of Mycobacterium tuberculosis zinc-dependent metalloprotease-1 (Zmp1), a metalloprotease involved in pathogenicity

Mycobacterium tuberculosis, the causative agent of tuberculosis, parasitizes host macrophages. The resistance of the tubercle bacilli to the macrophage hostile environment relates to their ability to impair phagosome maturation and its fusion with the lysosome, thus preventing the formation of the phago-lysosome and eventually arresting the process of phagocytosis. The M. tuberculosis zinc-dependent metalloprotease Zmp1 has been proposed to play a key role in the process of phagosome maturation inhibition and emerged as an important player in pathogenesis. Here, we report the crystal structure of wild-type Zmp1 at 2.6 Å resolution in complex with the generic zinc metalloprotease inhibitor phosphoramidon, which we demonstrated to inhibit the enzyme potently. Our data represent the first structural characterization of a bacterial member of the zinc-dependent M13 endopeptidase family and revealed a significant degree of conservation with eukaryotic enzymes. However, structural comparison of the Zmp1-phosphoramidon complex with homologous human proteins neprilysin and endothelin-converting enzyme-1 revealed unique features of the Zmp1 active site to be exploited for the rational design of specific inhibitors that may prove useful as a pharmacological tool for better understanding Zmp1 biological function.     

Evidence of calcium- and SNARE-dependent release of CuZn superoxide dismutase from rat pituitary GH3 cells and synaptosomes in response to depolarization

The antioxidant enzyme CuZn superoxide dismutase (SOD1) is secreted by many cell lines. However, it is not clear whether SOD1 secretion is only constitutive or can be regulated in an activity-dependent fashion. Using rat pituitary GH(3) cells that express voltage-dependent calcium channels and are subjected to Ca(2+) oscillations, we found that treatment with high K(+)-induced SOD1 release that was significantly higher than the constitutive secretion. Evoked SOD1 release was correlated with depolarization-dependent calcium influx and was virtually abolished by removal of extracellular calcium with EGTA or by pre-incubation of GH(3) cells with Botulinum toxin A that cleaves the SNARE protein SNAP-25. Immunofluorescence experiments performed in GH(3) cells and rat brain synaptosomes showed that K(+)-depolarization induced a marked depletion of intracellular SOD1 immunoreactivity, an effect that was again abolished in the absence of extracellular calcium or after treatment with Botulinum toxin A. Subcellular fractionation analysis showed that SOD1 was present in large dense core vesicles. These data clearly show that, in addition to the constitutive SOD1 secretion, depolarization induces an additional rapid calcium-dependent SOD1 release in GH(3) cells and in rat brain synaptosomes. This likely occurs through exocytosis from SOD1-containing vesicles operated by the SNARE complex.