Cloning,characterization and heterelogous expression of the INU1 gene from Cryptococcus aureus HYA

The INU1 gene (Accession number: JX073660) encoding exo-inulinase from Cryptococcus aureus HYA was cloned and characterized. The gene had an open reading frame (ORF) of 1653 bp long encoding an inulinase. The coding region of the gene was not interrupted by any intron. It encoded 551 amino acid residues of a protein with a putative signal peptide of 23 amino acids and the calculated molecular mass of 59.5 kDa. The protein sequence deduced from the inulinase structural gene contained the inulinase consensus sequences (WMNDPNGL), (RDP), ECP, FS and Q. It also had two conserved putative N-glycosylation sites. The inulinase from C. aureus HYA was found to be closely related to that from Kluyveromyces marxianus and Pichia guilliermondii. The inulinase gene without the signal sequence was subcloned into pPICZaA expression vector and expressed in Pichia pastoris X-33. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 60 kDa was found. Enzyme activity assay verified the recombinant protein as an inulinase. A maximum inulinase activity of 16.3 ± 0.24 U/ml was obtained from the culture supernatant of P. pastoris X-33 harboring the inulinase gene. The optimal temperature and pH for action of the enzyme were 50 °C and 5.0, respectively. A large amount of monosaccharides were detected after the hydrolysis of inulin with the purified recombinant inulinase.     

Design and discovery of novel quinazolinedione-based redox modulators as therapies for pancreatic cancer

Background

Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species (ROS) in tumors make them more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger ROS-mediated cell death. Recently, we have discovered a novel class of quinazolinediones that exert their cytotoxic effects by modulating ROS-mediated signaling.

Methods

Cytotoxic potential was determined by colorimetric and colony formation assays. An XF24 Extracellular Flux Analyzer, and colorimetric and fluorescent techniques were used to assess the bioenergetics and oxidative stress effects, respectively. Mechanism was determined by Western blots.

Results

Compound 3a (6-[(2-acetylphenyl)amino]quinazoline-5,8-dione) was identified through a medium throughput screen of ~ 1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics. Further structural optimizations led to the discovery of a more potent analog, 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) that displayed anti-proliferative activities in low micromolar range in both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt activation resulting in increased cellular oxygen consumption and oxidative stress in pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity.

Conclusion

In conclusion, our novel quinazolinediones are promising lead compounds that selectively induce ROS-mediated cell death in cancer cells and warrant further preclinical studies.

General significance

Since 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) exerts Akt-dependent ROS-mediated cell death, it might provide potential therapeutic options for chemoresistant and Akt-overexpressing cancers.     

Recruitment facilitation can promote coexistence and buffer population growth in metacommunities

Although positive species interactions are ubiquitous in nature, theory has generally focused on the role of negative interactions to explain patterns of species diversity. Here, we incorporate recruitment facilitation, a positive interaction prevalent in marine and terrestrial systems, into a metacommunity framework to assess how the interplay between colonisation, competition and facilitation mediates coexistence. We show that when subordinate species facilitate the recruitment of dominant species, multi-species metacommunities can persist stably even if the colonisation rate of the dominant species is greater than that of the subordinate species. In addition, recruitment facilitation can buffer population growth from changes in colonisation rates, and thus explain the paradoxical mismatch between patterns of abundance and recruitment in marine systems. Overall, our results demonstrate that recruitment facilitation can have profound effects on the assembly, dissolution and regulation of metacommunities by mediating the relative influence of local and regional processes on population abundance and species diversity.     

Somatic alterations in mitochondrial DNA produce changes in cell growth and metabolism supporting a tumorigenic phenotype

There have been many reports of mitochondrial DNA (mtDNA) mutations associated with human malignancies. We have observed allelic instability in UV-induced cutaneous tumors at the mt-Tr locus encoding the mitochondrial tRNA for arginine. We examined the effects of somatic alterations at this locus by modeling the change in a uniform nuclear background by generating cybrids harboring allelic variation at mt-Tr. We utilized the naturally occurring mtDNA variation at mt-Tr within the BALB/cJ (BALB) and C57BL/6J (B6) strains of Mus musculus to transfer their mitochondria into a mouse ρ⁰ cell line that lacked its own mtDNA. The BALB haplotype containing the mt-Tr 9821insA allele produced significant changes in cellular respiration (resulting in lowered ATP production), but increased rates of cellular proliferation in cybrid cells. Furthermore, the mtDNA genotype associated with UV-induced tumors endowed the cybrid cells with a phenotype of resistance to UV-induced apoptosis and enhanced migration and invasion capabilities. These studies support a role for mtDNA changes in cancer.     

Comparative study of mature and zymogen mite cysteine protease stability and pH unfolding

Background

Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes.

Methods

To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1.

Results

We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen.

General significance

Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis.     

The aspartic acid of Fyn at 390 is critical for neuronal migration during corticogenesis

The mammalian cerebral cortex develops through the coordinated migration of postmitotic neurons. Fyn, a member of the Src tyrosine kinase family (SFKs), is involved in the neuronal migration and the absence of Fyn leads to abnormal migration. However, the molecular mechanism whereby Fyn acts on migrating neurons has remained unclear. Here, we employed two Fyn mutants (Fyn259T and FynD390A) to investigate the function of Fyn kinase domain in neuronal migration. Using in utero electroporation, we co-transfected the migrating neurons in embryonic cortex with these mutants combined with plasmid expressing GFP. Interestingly, although both of them impaired neuronal migration, FynD390A, rather than Fyn259T, induced remarkable morphology change. Our work provides in vivo and in vitro evidence that the aspartic acid of Fyn at 390 is indispensable for the radial migration, and it is required for precise cooperation with focal adhesion kinase.     

Generating novel recombinant prokaryotic lectins with altered carbohydrate binding properties through mutagenesis of the PA-IL protein from Pseudomonas aeruginosa

Background

Prokaryotic lectins offer significant advantages over eukaryotic lectins for the development of enhanced glycoselective tools. Amenability to recombinant expression in Escherichia coli simplifies their production and presents opportunities for further genetic manipulation to create novel recombinant prokaryotic lectins (RPLs) with altered or enhanced carbohydrate binding properties. This study explored the potential of the α-galactophilic PA-IL lectin from Pseudomonas aeruginosa for use as a scaffold structure for the generation of novel RPLs.

Method

Specific amino acid residues in the carbohydrate binding site of a recombinant PA-IL protein were randomly substituted by site-directed mutagenesis. The resulting expression clones were then functionally screened to identify clones expressing rPA-IL proteins with altered carbohydrate binding properties.

Results

This study generated RPLs exhibiting diverse carbohydrate binding activities including specificity and high affinity for β-linked galactose and N-acetyl-lactosamine (LacNAc) displayed by N-linked glycans on glycoprotein targets. Key amino acid substitutions were identified and linked with specific carbohydrate binding activities. Ultimately, the utility of these novel RPLs for glycoprotein analysis and for selective fractionation and isolation of glycoproteins and their glycoforms was demonstrated.

Conclusions

The carbohydrate binding properties of the PA-IL protein can be significantly altered using site-directed mutagenesis strategies to generate novel RPLs with diverse carbohydrate binding properties.

General significance

The novel RPLs reported would find a broad range of applications in glycobiology, diagnostics and in the analysis of biotherapeutics. The ability to readily produce these RPLs in gram quantities could enable them to find larger scale applications for glycoprotein or biotherapeutic purification.     

Characterization of chicken endoglin, a member of the zona pellucida family of proteins, and its tissue expression

Endoglin is a TGF-β co-receptor expressed in endothelial cells, where it plays a crucial role in angiogenesis, cardiovascular development and vascular remodeling. In humans, mutations in the endoglin gene give rise to Hereditary Hemorrhagic Telangiectasia type 1 (HHT1), an autosomal dominant disorder associated with vascular lesions in skin, mucosa and internal organs. So far, endoglin cDNA has been sequenced in several species from mammals, amphibians and birds. While in mammals the characterization of endoglin protein expression and function is well documented, little is known about the protein homologue in birds. In silico analysis by multiple sequences alignment showed a low homology score of 30-33 between the full length chicken endoglin protein and several mammalian homologues. However, a high homology score (80-85) was observed with the cytoplasmic and transmembrane regions and the overall structure of the zona pellucida (ZP) and orphan domains of the extracellular region appear to be conserved. Transient expression of chicken endoglin allowed the identification of a 180-kDa disulfide linked homodimer similar to the mammalian homologues. To further characterize its tissue expression, the novel specific monoclonal antibody (mAb) 7H5A8 was generated against chicken endoglin transfectant cells. The mAb 7H5A8 specifically recognized chicken endoglin by western blot, immunoprecipitation, immunofluorescence flow cytometry as well as immunofluorescence microscopy assays and displayed a positive staining of the endothelium in veins and arteries from frozen tissue sections of lung and bursa of Fabricius. These results may help to further understand the endoglin expression in vertebrates.