Identification and characterization of a novel bifunctional Δ<Superscript>12</Superscript>/Δ<Superscript>15</Superscript>-fatty acid desaturase gene from <Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis>

Objectives

To elucidate the biosynthesis pathway of linoleic acid and α-linolenic acid in Rhodosporidium kratochvilovae YM25235 and investigate the correlation of polyunsaturated fatty acids with its cold adaptation.

Results

A 1341 bp cDNA sequence, designated as RKD12, putatively encoding a Δ¹²-desaturase was isolated from YM25235. Sequence analysis indicated that this sequence comprised a complete ORF encoding 446 amino acids of 50.6 kDa. The encoded amino acid sequence shared higher similarity to known fungal Δ¹²-desaturases that are characteristic of three conserved histidine-rich motifs. RKD12 was further transformed into Saccharomyces cerevisiae INVScl for functional characterization. Fatty acid analysis showed the yeast transformants accumulated two new fatty acids: linoleic acid and α-linolenic acid. Furthermore, mRNA expression level of RKD12 and the content of linoleic acid and α-linolenic acid were increased significantly with the culture temperature downshift from 30 to 15 °C, which might be helpful for the cold adaptation of YM25235.

Conclusion

RKD12 is a novel bifunctional ?¹²/?¹⁵-desaturase gene, and the increased RKD12 mRNA expression level and PUFAs content at low temperature might be helpful for the cold adaptation of YM25235.

     

Parthenolide induces apoptosis and autophagy through the suppression of PI3K/Akt signaling pathway in cervical cancer

Objective

To investigate the effect of parthenolide on apoptosis and autophagy and to study the role of the PI3K/Akt signaling pathway in cervical cancer.

Results

Parthenolide inhibits HeLa cell viability in a dose dependent-manner and was confirmed by MTT assay. Parthenolide (6 µM) induces mitochondrial-mediated apoptosis and autophagy by activation of caspase-3, upregulation of Bax, Beclin-1, ATG5, ATG3 and down-regulation of Bcl-2 and mTOR. Parthenolide also inhibits PI3K and Akt expression through activation of PTEN expression. Moreover, parthenolide induces generation of reactive oxygen species that leads to the loss of mitochondrial membrane potential.

Conclusion

Parthenolide induces apoptosis and autophagy-mediated growth inhibition in HeLa cells by suppressing the PI3K/Akt signaling pathway and mitochondrial membrane depolarization and ROS generation. Parthenolide may be a potential therapeutic agent for the treatment of cervical cancer.

     

Molecular understandings on ‘the never thirsty’ and apomictic <Emphasis Type="Italic">Cenchrus</Emphasis> grass

The genus Cenchrus comprises around 25 species of ‘bristle clade’ grasses. Cenchrus ciliaris (buffel grass) is a hardy, perennial range grass that survives in poor sandy soils and limiting soil moisture conditions and, due to the very same reasons, this grass is one of the most prevalent fodder grasses of the arid and semi-arid regions. Most of the germplasms of Cenchrus produce seeds asexually through the process of apomeiosis. Therefore, the lack of sufficient sexual lines has hindered the crop improvement efforts in Cenchrus being confined to simple selection methods. Many attempts have been initiated in buffel grass to investigate the various molecular aspects such as genomic signatures of different species and genotypes, molecular basis of abiotic stress tolerance and reproductive performance. Even though it is an important fodder crop, molecular investigations in Cenchrus lack focus and the molecular information available on this grass is scanty. Cenchrus is a very good gene source for abiotic stress tolerance and apomixis studies. Biotechnological interventions in Cenchrus can help in crop improvement in Cenchrus as well as other crops through transgenic technology or marker assisted selection. To date no consolidated review on biotechnological interventions in Cenchrus grass has been published. Therefore we provide a thorough and in depth review on molecular research in Cenchrus focusing on molecular signatures of evolution, tolerance to abiotic stress and apomictic reproductive mechanism.     

Identification and characterization of a novel antioxidant peptide from feather keratin hydrolysate

Objectives

To improve the potential value of feather, which is a valuable protein resource, we have separated and identified antioxidant peptide(s) from feather hydrolysate.

Results

Feather hydrolysate was prepared by fermentation with Bacillus subtilis S1–4. Antioxidative peptides were separated by sequential acid precipitation, cation exchange, and reversed-phase fast performance liquid chromatography. Finally, a peptide with antioxidative activity was identified as Ser-Asn-Leu-Cys-Arg-Pro-Cys-Gly by MALDI time-of-flight (TOF)/TOF analysis, and determined to represent a portion of feather keratin near its N-terminal. A synthesized peptide with the same sequence was used to characterize its antioxidative properties, including scavenging free radicals, reducing power, and Fe²⁺ chelation. In terms of the peptide’s amino acid composition, the antioxidative activity might be mainly attributed to Cys and other amino acid residues.

Conclusion

Feather keratin is a good source for the quantitative preparation of antioxidative peptides.

     

Morphology engineering of basidiomycetes for improved laccase biosynthesis

Objective

This work is the first application of a morphological engineering technique called microparticle-enhanced cultivation (MPEC) aimed at the facilitation of laccase production in the submerged cultures by two basidiomycetes species Cerrena unicolor and Pleurotus sapidus.

Results

The positive effect of the applied 10 μm Al₂O₃ microparticles at concentrations from 5 to 30 g Al₂O₃ l^?1 was shown. Laccase activity increased 3.5-fold for C. unicolor and 2-fold for P. sapidus at 15 g Al₂O₃ l^?1 on 9 and 14 day of the cultivation, respectively, compared to the control culture without microparticles. The increase of laccase activity in the cultivation broths was caused by the action of Al₂O₃ microparticles on the agglomeration of hyphae. It led to the decrease of the size of the pellets, (on average by 2 mm for C. unicolor), the change of their shape (star-shaped pellets for C. unicolor) and the change of their structure (more compact pellets for P. sapidus).

Conclusions

Application of MPEC for the submerged cultures of two laccase-producing basidiomycetes proved successful in increasing of enzyme production.

     

Calcium regulates glutamate dehydrogenase and poly-γ-glutamic acid synthesis in <Emphasis Type="Italic">Bacillus natto</Emphasis>

Objective

To study the effect of Ca²⁺ on glutamate dehydrogenase (GDH) and its role in poly-γ-glutamic acid (γ-PGA) synthesis in Bacillus natto HSF 1410.

Results

When the concentration of Ca²⁺ varied from 0 to 0.1 g/l in the growth medium of B. natto HSF 1410, γ-PGA production increased from 6.8 to 9.7 g/l, while GDH specific activity and NH₄Cl consumption improved from 183 to 295 U/mg and from 0.65 to 0.77 g/l, respectively. GDH with α-ketoglutarate as substrate primarily used NADPH as coenzyme with a K _m of 0.08 mM. GDH was responsible for the synthesis of endogenous glutamate. The specific activity of GDH remained essentially unchanged in the presence of CaCl₂ (0.05–0.2 g/l) in vitro. However, the specific activity of GDH and its expression was significantly increased by CaCl₂ in vivo. Therefore, the regulation of GDH and PGA synthesis by Ca²⁺ is an intracellular process.

Conclusion

Calcium regulation may be an effective approach for producing γ-PGA on an industrial scale.

     

High level soluble expression and one-step purification of IBDV VP2 protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To improve the expression of soluble IBDV VP2 protein by using different tagged vectors in Escherichia coli.

Results

Fusion tags, Grifin, MBP, SUMO, thioredoxin, γ-crystallin, ArsC and PpiB, enhanced the expression and solubility of VP2 protein. The fusion proteins were purified by Ni–NTA chromatography, MBP-VP2 showed the highest purity about 90 %. After removing the MBP tag, VP2 self-assembled into virus-like particles, ~25 nm diam. Results from AGP suggested the recombinant IBDV VP2 protein identified by reference serum like IBDV.

Conclusion

All the seven tags enhanced the expression and solubility of IBDV VP2 protein. The recombinant protein self-assembly into virus like particles and possess antigenicity as reference IBDV.