Cloning, expression, purification and three-dimensional structure prediction of haloalkane dehalogenase from a recently isolated Ancylobacter aquaticus strain UV5.

Haloalkane dehalogenase (DhlA) converts 1,2-dichloroethane (1,2-DCA) to 2-chloroethane in the genus Ancylobacter and Xanthobacter autotrophicus GJ10 (XaDhlA) and allows these organisms to utilise 1,2-DCA and some other halogenated alkanes for growth. The DhlA encoding gene (dhlA) was PCR-amplified from the genomic DNA of a recently isolated Ancylobacter aquaticus UV5 strain, cloned and overexpressed in Escherichiacoli BL21 (DE3). The recombinant enzyme was purified by using Amicon ultra-15 centrifugal filter units, an anion-exchange QFF column followed by a gel-filtration column (Sephacryl HR100). Enzyme activity was determined by using 1,2-DCA as a substrate. Three-dimensional structure of the enzyme was predicted using SWISS-MODEL workspace and the biophysical properties were predicted by submitting the amino acid sequence of DhlA on ExPASy server. DhlA (Mr 35kDa) exhibited optimum activity at temperature 37°C and pH 9.0. The enzyme retained approximately 50% of its activity after 1h of incubation at 50°C, and showed moderate stability against denaturing agent urea. The DhlA displayed a Km value of 842µM and kcat/Km ratio of 168mM(-)(1)min(-)(1) for its substrate 1,2-DCA. This DhlA was found to belong to the α/ß hydrolase family with a catalytic triad composed of Asp-His-Asp in its active site. This is the first study reporting on the characterisation and reaction kinetics of purified DhlA from A.aquaticus UV5 indigenous to contaminated site in Africa.

Less interference fluorescence analytical strategy: Bridging substance-triggered ratiometric sensor with convenient preparation and application.

High interference and narrow application range are key of bottleneck of recent fluorescence analysis methods, which limit their wide application in the sensing field. Therefore, to overcome these disadvantages, a ratiometric fluorescence sensing system utilizing berberine (BER) and silver nanoclusters protected by dihydrolipoic acid (DHLA-AgNCs) was constructed for the first time in this work, to achieve determination of BER and daunorubicin (Dau). BER aqueous solution (non-planar conformation) has no fluorescence emission. When it was mixed with DHLA-AgNCs, the conformation of BER became planar, producing fluorescence emission at 515 nm besides the fluorescence emission peak of DHLA-AgNCs at 653 nm. With the increase of BER concentration added in system, the fluorescence intensity of BER (planar conformation) at 515 nm increased obviously and the fluorescence intensity of DHLA-AgNCs decreased slightly. Therefore, the dual emission fluorescence sensing system was constructed based on a fluorescence substance and non fluorescence substance, to achieve determination of BER. Meanwhile, based on the bridging effect of BER and fluorescence resonance energy transfer effect from Dau, the altering of two peaks intensity was utilized to achieve determination of Dau. Thus, this dual emission sensing system can not only be used for fluorescence analysis of BER and its analogues, but also based on the bridging effect of BER, allowing the determination of Dau and its analogues that could not be directly measured with silver nanoclusters, expanding the application range of traditional dual emission detection systems. Meanwhile, this system has strong anti-interference ability and low toxicity to the human body and less pollution to the sample and environment. This provides a new direction and universal research strategy for the construction of new fluorescence sensing systems in the future for the analysis of target substances that cannot be directly detected with conventional fluorescence analysis methods.

Lipoic Acid Decreases the Viability of Breast Cancer Cells and Activity of PTP1B and SHP2.

BACKGROUND: Protein tyrosine phosphatases PTP1B and SHP2 are potential targets for anticancer therapy, because of the essential role they play in the development of tumors. PTP1B and SHP2 are overexpressed in breast cancer cells, thus inhibition of their activity can be potentially effective in breast cancer therapy. Lipoic acid has been previously reported to inhibit the proliferation of colon, breast and thyroid cancer cells. MATERIALS AND METHODS: We investigated the effect of alpha-lipoic acid (ALA) and its reduced form of dihydrolipoic acid (DHLA) on the viability of MCF-7 cancer cells and on the enzymatic activity of PTP1B and SHP2 phosphatases. RESULTS: ALA and DHLA decrease the activity of PTP1B and SHP2, and have inhibitory effects on the viability and proliferation of breast cancer cells. CONCLUSION: ALA and DHLA can be considered as potential agents for the adjunctive treatment of breast cancer.

Targeting tumor micro-environment for design and development of novel anti-angiogenic agents arresting tumor growth.

Angiogenesis: a process of generation of new blood vessels has been proved to be necessary for sustained tumor growth and cancer progression. Inhibiting angiogenesis pathway has long been remained a significant hope for the development of novel, effective and target orientated antitumor agents arresting the tumor proliferation and metastasis. The process of neoangiogenesis as a biological process is regulated by several pro- and anti-angiogenic factors, especially vascular endothelial growth factor, fibroblast growth factor, epidermal growth factor, hypoxia inducible factor 1 and transforming growth factor. Every endothelial cell destined for vessel formation is equipped with receptors for these angiogenic peptides. Moreover, numerous other angiogenic cytokines such as platelet derived growth factor (PGDF), placenta growth factor (PGF), nerve growth factor (NGF), stem-cell factor (SCF), and interleukins-2, 4, 6 etc. These molecular players performs critical role in regulating the angiogenic switch. Couple of decade's research in molecular aspects of tumor biology has unraveled numerous structural and functional mysteries of these angiogenic peptides. In present article, a detailed update on the functional and structural peculiarities of the various angiogenic peptides is described focusing on structural opportunities made available that has potential to be used to modulate function of these angiogenic peptides in developing therapeutic agents targeting neoplastic angiogenesis. The data may be useful in the mainstream of developing novel anticancer agents targeting tumor angiogenesis. We also discuss major therapeutic agents that are currently used in angiogenesis associated therapies as well as those are subject of active research or are in clinical trials.

Cytoprotective effects of dihydrolipoic acid and lipoic acid on the oxidative stress in cultured rat cortical neurons

In brain hypoxic-ischemia, an excess release of glutamate and a marked production of reactive oxygen species (ROS) occur in neuronal and non-neuronal cells. The present study investigated the effect of the biological antioxidants dihydrolipoic acid (DHLA) and lipoic acid (LA) on N-methyl-D-aspartate (NMDA)and ROS-induced neurotoxicity in cultured rat cortical neurons. DHLA enhanced NMDA-evoked rises in intracellular calcium concentration ((Ca2+)i). In contrast, LA did not alter the NMDA-evoked calcium responses but decreased after a brief treatment of dithiothreitol (DTT), which possesses a strong reducing potential. Despite the modulation of NMDA receptor-mediated rises in (Ca2+)i, neither DHLA nor LA altered the NMDA receptor-mediated neurotoxicity, as assessed by measuring the amount of lactate dehydrogenase released from dead or injured cells. DHLA, but not LA, prevented the neurotoxicity induced by xanthine/xanthine oxidase-generated superoxide radicals. Both DHLA and LA decreased the glutathione depletion-induced neurotoxicity. The present data may indicate that biological antioxidants DHLA and LA protect neurons from ischemic injuries via scavenging oxygen free radicals rather than modulating the redox modulatory site(s) of NMDA receptor.