Purification, Characterization and Comparison between Two New L-asparaginases from Bacillus PG03 and Bacillus PG04.

BACKGROUND: L-asparaginase has been used as a chemotherapeutic agent in treatment of lymphoblastic leukemia. In the present investigation, Bacillus sp. PG03 and Bacillus sp. PG04 were studied. METHODS: L- asparaginases were produced using different culture media and were purified using ion exchange chromatography. RESULTS: Maximum productivity was obtained when asparagine was used as the nitrogen source at pH 7 and 48 h after cultivation. New intracellular L-asparaginases showed an apparent molecular weight of 25 kDa and 30 kDa by SDS-PAGE respectively. These enzymes were active in a wide pH range (3-9) with maximum activity at pH 6 for Bacillus PG03 and pH 7 for Bacillus PG04 L-asparaginase. Bacillus PG03 enzyme was optimally active at 37 ˚C and Bacillus PG04 maximum activity was observed at 40˚C. Kinetic parameters km and Vmax of both enzymes were studied using L-asparagine as the substrate. Thermal inactivation studies of Bacillus PG03 and Bacillus PG04 L-asparaginase exhibited t1/2 of 69.3 min and 34.6 min in 37 ˚C respectively. Also T50 and ∆G of inactivation were measured for both enzymes. CONCLUSION: The results revealed that both enzymes had appropriate characteristics and thus could be a potential candidate for medical applications.

Synthesis and investigation of new Hesperadin analogues antitumor effects on HeLa cells.

Hesperadin is one of the indolinones that was designed against the ATP-binding site of Aurora kinase. This molecule inhibits Aurora B kinase by phosphorylation of histone H3. In this study, new derivatives of Hesperadin containing an amide group in their structures were synthesized through sequential Ugi/palladium-catalyzed approach and in vitro antitumor activity of new compounds were evaluated by cell proliferation assay. The results show that compounds 6f, 6i, 6l, and 6o were dose-dependently inhibited in different concentrations, and IC50 values were between 35 and 43 nM. It seems that lipophilic substitution on the indolinone core with the ability to form additional hydrogen bond might lead to increased stability of structure and activity of new Hesperadin analogues.

Helicobacter pylori FliD protein is a highly sensitive and specific marker for serologic diagnosis of H. pylori infection.

Screening for H. pylori in large populations continues to be a challenging task, since available tests have limited sensitivity and specificity, which, in population-based approaches, leads to significant numbers of false positive and false negative results. Various H. pylori proteins associated with virulence are highly immunogenic and therefore candidates to detect the infection. There are currently no defined markers that are recognized in all H. pylori infected patients and that do not show cross-reactivity with other bacterial proteins. We identified the H. pylori "hook-associated protein 2 homologue", FliD (UniProtKB/Swiss-Prot: P96786.4) as a novel marker of infection for serological analysis. The H. pylori FliD protein is an essential element in the assembly of the functional flagella. However, this virulence factor has not yet been tested as a diagnostic marker in serology. For this purpose FliD was recombinantly expressed in E. coli, purified by affinity chromatography and gel filtration and used to coat ELISA plates or immobilized on nitrocellulose stripes. To evaluate its antigenicity we screened a defined panel of patient sera. The recombinant H. pylori FliD protein reacted with a high percentage of human sera. Among 318 samples reported positive by histology, 310 (97.4%) were tested positive by FliD Line assay, and 165 out of 170 samples were tested positive by ELISA (97%). We could also reconfirm 297 out of 300 (99%) negative sera by Line assay and 73 from 76 (96%) by ELISA. Taken together, application of FliD in serological diagnosis of H. pylori infection presents a high specificity of up to 99% and a sensitivity of up to 97%. This makes especially the FliD ELISA a simple, cost effective and highly efficient tool to detect H. pylori infection in developing countries where prevalence is high and other screening methods are either not available or are unaffordable.

Comparison of five diagnostic methods for Helicobacter pylori.

BACKGROUND AND OBJECTIVES: Invasive and non-invasive techniques are used to diagnose H. pylori infection. Some factors influence the choice of a diagnostic test, such as the sensitivity and specificity of the tests, the clinical circumstances and the cost-effectiveness of the testing strategy. The aim of this study was to reveal the relationship between different H. pylori infection diagnosis methods, and clarify the application scope of each diagnosis method. MATERIALS AND METHODS: patients were included in the study, and specimens including biopsies, blood and stool were taken. Biopsies were evaluated by hematoxylin and eosin, and Giemsa staining. A sequence of 294 bp in the ureC (glmM) gene was amplified. The rapid urease test (RUT) was performed using a non-commercial validated test. Stool samples were analyzed using a polyclonal ELISA stool antigen test. A serological assay for IgG antibodies was performed by a commercial Helicobacter pylori IgG ELISA kit. RESULTS: According to the predefined criteria, a total of 46 (50.5%) patients tested were positive by at least 2 of the 3 biopsy-based methods. The best sensitivity (95.6%) belonged to histology and RUT. The sensitivities of other tests including PCR, serology and stool antigen test were 93.5%, 91.3% and 73.9%, respectively. RUT showed the best specificity (100%), and the specificities of the other tests, including PCR, stool antigen test, histology and serology, were 95.6%, 86.7%, 77.8% and 55.6%, respectively. CONCLUSION: In view of the better results obtained for invasive vs non-invasive tests, for a more accurate diagnosis, it is advisable not to solely rely on non-invasive methods of H. Pylori diagnosis.

Active targeting of HER2-positive breast cancer cells by Herceptin-functionalized organically modified silica nanoparticles.

Normal micelle microemulsion method was utilized for fabrication of organically modified silica (ORMOSIL) nanoparticles. The void and dye-doped nanoparticles were synthesized in nonpolar core of two different surfactants including Aerosol OT and Tween 80. The nanoparticles were characterized using transmission electron microscopy, dynamic light scattering, and zeta potential analysis. Our results revealed that the type of surfactant molecules has a dramatic impact on the size and size distribution range, surface charge, and surface functionalization of the nanoparticles. The particles fabricated using Tween 80 had very smaller size with narrow size distribution and very lower amount of zeta potential. For specific delivery of functionalized nanoparticles to breast cancer cell line SKBR3, overexpressing human epidermal growth factor receptor 2 (HER2), both dye-doped nanoparticles fabricated with Aerosol OT or Tween 80, was conjugated to Herceptin. In vitro studies using fluorescent microscopy demonstrated that the surfactant used for preparation of the nanoparticles can affect the uptake of the particles by cells. The dye-doped functionalized ORMOSIL nanoparticles prepared with Aerosol OT showed better efficiency in the process of active targeting of HER2 receptor. Herceptin-functionalized ORMOSIL nanoparticles can be used for differentiation of HER2-positive from HER2-negative breast cancer cells or specific delivery of therapeutic and diagnostic agents and also other nanoparticles such as magnetic nanoparticles and quantum dots to breast cancer cells. FigureOrmosil bioconjugation.

Anticancer activity of a new gonadotropin releasing hormone analogue.

Gonadotropin releasing hormone (GnRH) has a pivotal role in the biology of reproduction processes. In extrapituitary compartments GnRH and its receptor act as a part of the autocrin regulatory system of cell proliferation, resulting in its anticancer activity. Here the anticancer activity of a new analogue of GnRH has been investigated. Results indicate that proliferation of human breast and ovarian cancer cell lines is dose-dependently inhibited. The inhibitory efficiency of this new analogue is proved to be higher than the original triptorelin. In addition to its antimitogenic activity, evidence was found for the involvement of the apoptotic mechanism in the action of the new analogue. Furthermore the presence of chemical groups in the peptide sequence is thought to increase the protease stability of the new analogue in comparison with triptorelin. Consequently our new analogue can be considered as a good pharmaceutical candidate.

Conjugation of Monoclonal Antibodies to Super Paramagnetic Iron Oxide Nanoparticles for Detection of her2/neu Antigen on Breast Cancer Cell Lines.

Conjugation of monoclonal antibodies to super paramagnetic nanoparticles is an effective method for cancer diagnosis and treatment. In this study the humanized anti her2/neu monoclonal antibody- Herceptin- was conjugated to super paramagnetic iron oxide (SPIO) nanoparticles using EDC method. The concentration of the conjugated antibodies was measured by Bradford assay. The antibody-nanoparticle conjugates were incubated with SKBR-3 and T47D human breast carcinoma cell lines and the presence of the conjugates on cell surface was confirmed by Prussian blue iron staining method. Conjugation of Herceptin to SPIO resulted in a precipitate-free conjugate containing 20µg antibody/mg SPIO. Prussian blue iron-staining of cells showed successful binding of the conjugates to the cell surfaces. Conjugation of monoclonal antibodies to SPIO may be a useful method for detection of tumor cells, especially by MRI techniques.

The anticancer activity compared between triptorelin and a new gonadotropin releasing hormone analogue.

Gonadotropin releasing hormone (GnRH) plays a key role in reproduction. This decapeptide is synthesized and released by hypothalamus and induces the pituitary gonadotrop cells to release pituitary gonadotropin hormones. In some extrapituitary compartments GnRH and its receptor act as part of the autocrine regulatory system of cell proliferation. The anticancer activity of GnRH and its analogues has been observed by many researchers. In this study the anticancer activity of a new analogue of GnRH and triptorelin was investigated by cell proliferation assay. Results indicate that proliferation of human breast and ovarian cancer cell lines are dose-dependently inhibited. The inhibitory efficiency of the new analogue is proved to be higher than the original triptorelin. In addition to its antimitogenic activity, evidence was found for the involvement of the apoptotic mechanism in the action of the new analogue and triptorelin. In conclusion, the new analogue can be considered as a good pharmaceutical candidate.