Copper complex derived from <Emphasis Type="Italic">S</Emphasis>-benzyldithiocarbazate and 3-acetylcoumarin induced apoptosis in breast cancer cell

Copper complexes have been widely studied for the anti-tumour application as cancer cells are reported to take up greater amounts of copper than normal cells. Preliminary study revealed that the newly synthesised copper complex [Cu(SBCM)₂] displayed marked anti-proliferative towards triple-negative MDA-MB-231 breast cancer cells. Therefore, Cu(SBCM)₂ has great potential to be developed as an agent for the management of breast cancer. The present study was carried out to investigate the mode of cell death induced by Cu(SBCM)₂ towards MDA-MB-231 breast cancer cells. The inhibitory and morphological changes of MDA-MB-231 cells treated with Cu(SBCM)₂ was determined by using MTT assay and inverted light microscope, respectively. The safety profile of Cu(SBCM)₂ was also evaluated towards human dermal fibroblast (HDF) normal cells. Confirmation of apoptosis and cell cycle arrest were determined by flow cytometry analysis. The expression of p53, Bax, Bcl-2 and MMP2 protein were detected with western blot analysis. Cu(SBCM)₂ significantly inhibited the growth of MDA-MB-231 cells in a dose-dependent manner with GI₅₀ 18.7?±?3.06 µM. Indeed, Cu(SBCM)₂ was less toxic towards HDF normal cells with GI₅₀ 31.8?±?4.0 µM. Morphological study revealed that Cu(SBCM)₂-treated MDA-MB-231 cells experienced cellular shrinkage, membrane blebbing, chromatin condensation and formation of apoptotic bodies, suggesting that Cu(SBCM)₂ induced apoptosis in the cells, which was confirmed by Annexin-V/PI flow cytometry analysis. It was also found that Cu(SBCM)₂ induced G₂/M phase cell cycle arrest towards MDA-MB-231 cells. The induction of apoptosis and cell cycle arrest in the present study is possibly due to the down-regulation of the mutant p53 and MMP2 protein. In conclusion, Cu(SBCM)₂ can be developed as a targeted therapy for the treatment of triple-negative breast cancer.     

Comparative analysis of core collection sampling methods for mandarin germplasm based on molecular and phenotypic data

Gene banks have been established to conserve the genetic diversity of crop species. Large germplasm collections lead to management problems (space, maintenance costs, etc.), especially in collections involving species with recalcitrant seeds that must be maintained as growing plants. Core collections (CCs) are thus developed to reduce the size of large germplasm collections while keeping the maximum variability. This also facilitates fine phenotypic evaluation. In this study, several software packages (DARwin, PowerMarker and MSTRAT) and methods (Max length subtree, M strategy, simulated annealing and MinSD) were compared to define a mandarin (Citrus reticulata) CC. One hundred and sixty‐seven accessions were sampled from two germplasm collections, which were genotyped with 50 SSR, 24 InDel and 68 single nucleotide polymorphism markers. All the CC obtained were tested for the maintenance of the genetic variability parameters (Ho and He) of the initial collection, the level of linkage disequilibrium (LD) and the phenotypic diversity retention. The Max length subtree function from DARWin seemed to be the most appropriate method for establishing a CC in C. reticulata. It maintained 96.82% of the allelic richness and 17.96% of the size of the initial collection with only 30 accessions. Besides it did not increase the LD (r² value) of the initial collection and retained the vast majority of the phenotypic variability. However, a CC with 70 accessions would be more helpful for genetic association studies.     

Synthesis of Highly Functionalized Fluorinated Cispentacin Derivatives

Fluorinated highly functionalized cispentacin derivatives were synthetised starting from an unsaturated bicyclic β‐lactam through C?C bond functionalization via the dipolar cycloaddition of a nitrile oxide, isoxazoline opening, and fluorination by OH/F exchange.     

Crystal structure and magnetic properties of a tetramer with a step-like Cu4N4O2 core containing asymmetric bridged dimeric subunits related to the hemocyanin copper active site

The Cu(II)-derivate of the macrocycle L derived from 2-hydroxy-5-methylisophthalaldehyde and 3- dimethylamino-1-propylamine forms tetranuclear {[LCu₂(N₃)₃](ClO₄)₂}₂ units. The crystal structure determined by X-ray crystallography is based on a step-like Cu₄N₄O₂-core with interdimeric μ(1)-azido bridging. The tetramer is formed from two dimeric subunits which are connected by a center of symmetry. The subunits are asymmetric bridged dkneric complexes with a phenolate and μ(1)-azido bridge. The coordination polyhedra around the two copper atoms are 4 + 2 and 4 + 1 including one coordinating perchlorate ion.The magnetic susceptibility of the complex has been measured in the temperature range 4.2 K to 315.6 K. The theoretical analysis using models for tetrameric and dimeric interactions revealed only a small interdimeric interaction. The magnetic exchange coupling of the asymmetric bridged dimeric unit (2J = ?528 cm^?1) is comparable to that of known di-oxygen bridged dimers. The results are related to the magnetic behaviour of the asymmetric bridges found in the binuclear copper sites in hemocyanin.Infrared measurements have been taken and there is a discussion of the asymmetric vibrations of the three different azido-groups.     

Glucose-6-phosphate dehydrogenase alloenzymes and their relationship to pigmentation in Serratia marcescens

A comparative study of environmental and clinical isolates of Serratia marcescens was undertaken with regard to glucose-6-phosphate dehydrogenase (G6PD) electrophoretic mobility and the production of prodigiosin. Two electromorphs of G6PD with electrophoretic mobilities of 0.22 and 0.30 were detected. G6PD electrophoretic type showed a good correlation with the ability to produce prodigiosin.     

Photophysical study of photoinduced electron transfer in a bis-thiophene substituted peryleneimide.

Based on femtosecond time-resolved spectroscopy and single photon timing experiments, intramolecular photoinduced charge transfer has been investigated in two systems containing a peryleneimide chromophore (P) and thiophene (T) groups. The first compound bearing a single thiophene ring (PT1) is used as model and shows a behavior similar to P, studied previously, while in the compound with two thiophene rings attached (PT2) electron transfer from the thiophene donor to the peryleneimide acceptor is observed in benzonitrile. Femtosecond fluorescence upconversion and femtosecond transient absorption experiments in benzonitrile indicate that this ion-pair state formation occurs in 19 ps. This ion-pair state then decays with two time constants of 1400 and 820 ps, probably corresponding to different conformations of the thiophene rings.     

A new bioassay method for quantitative analysis of tetracyclines

A new method is described for quantitative analysis of tetracycline, based on the decrease in external pH of bacterial suspensions after the addition of a glucose pulse. The decrease in external pH of these suspensions was inversely proportional to the concentration of tetracycline. The correlation coefficient of standard response lines derived from the bioassay was 0.99. Tetracycline potency was determined in six tetracycline HCl samples by the sugar pulse bioassay and a turbidimetric method. The turbidimetric assay result varied from the glucose-pulse data by no more than 7 and 3% at 3 and 7 min, respectively. The procedure is rapid, precise and quantitative, and requires minimal preparation and use of media, with savings in laboratory resources and time.     

High-throughput automated refolding screening of inclusion bodies

One of the main stumbling blocks encountered when attempting to express foreign proteins in Escherichia coli is the occurrence of amorphous aggregates of misfolded proteins, called inclusion bodies (IB). Developing efficient protein native structure recovery procedures based on IB refolding is therefore an important challenge. Unfortunately, there is no "universal" refolding buffer: Experience shows that refolding buffer composition varies from one protein to another. In addition, the methods developed so far for finding a suitable refolding buffer suffer from a number of weaknesses. These include the small number of refolding formulations, which often leads to negative results, solubility assays incompatible with high-throughput, and experiment formatting not suitable for automation. To overcome these problems, it was proposed in the present study to address some of these limitations. This resulted in the first completely automated IB refolding screening procedure to be developed using a 96-well format. The 96 refolding buffers were obtained using a fractional factorial approach. The screening procedure is potentially applicable to any nonmembrane protein, and was validated with 24 proteins in the framework of two Structural Genomics projects. The tests used for this purpose included the use of quality control methods such as circular dichroism, dynamic light scattering, and crystallogenesis. Out of the 24 proteins, 17 remained soluble in at least one of the 96 refolding buffers, 15 passed large-scale purification tests, and five gave crystals.     

Characterization of ''basparin A,'' a prothrombin-activating metalloproteinase,from the venom of the snake Bothrops asper that inhibits platelet aggregation and induces defibrination and thrombosis

A prothrombin activator, named 'basparin A,' was isolated from the venom of the crotaline snake Bothrops asper, the species responsible for the majority of snakebite cases in Central America. It is an acidic (pI 5.4), 70kDa, single chain P-III metalloproteinase comprising, in addition to the metalloproteinase domain, disintegrin-like, and high-cysteine domains. Basparin A is a glycoprotein displaying immunological cross-reactivity with BaH1, a P-III hemorrhagic metalloproteinase isolated from the same venom. It activates prothrombin through the formation of meizothrombin, without requiring additional cofactors; it is, therefore, a class A snake venom prothrombin activator. In contrast with most venom metalloproteinases, it does not degrade components of the extracellular matrix. Apart from its clotting activity, basparin A inhibits collagen-dependent platelet aggregation in vitro, an effect that does not depend on proteolytic activity. Clotting activity on human plasma is not abrogated by the plasma proteinase inhibitors alpha(2) macroglobulin and murinoglobulin, whereas activity is completely inhibited by Costa Rican polyvalent (Crotalinae) anti-venom. Basparin A does not induce local tissue alterations, such as hemorrhage, myonecrosis, and edema, in mice. Moreover, it does not induce systemic hemorrhage, thrombocytopenia nor prolongation of the bleeding time following intravenous administration. At low doses, the only observed effect induced by basparin A, when injected intravenously or intramuscularly into mice, is defibrin(ogen)ation. At higher doses, intravenous administration resulted in sudden death due to numerous occluding thrombi in pulmonary vessels. Basparin A is likely to play an important role in the coagulopathy associated with B. asper envenoming.