Application of Supercritical Fluid Extraction in Biotechnology

ABSTRACT

In the present paper recent investigations on the applications of supercritical fluid extraction (SCE) from post fermentation biomass or in situ extraction of inhibitory fermentation products as a promising method for increasing the yield of extraction have been reviewed. Although supercritical CO₂ (SC-CO₂) is unfriendly, or even toxic, for some living cells and precludes direct fermentation in dense CO₂, it does not rule out other useful applications for in situ extraction of inhibitory fermentation products and fractional extraction of biomass constituents. This technique is a highly desirable method for fractional extraction of biomass constituents, and intracellular metabolites due to the potential of system modification by physical parameters and addition of co-solvents to selectively extract compounds of different polarity, volatility and hydrophilicity without any contamination.     

Structure of the Blm10-20 S proteasome complex by cryo-electron microscopy. Insights into the mechanism of activation of mature yeast proteasomes

The 20 S proteasome is regulated at multiple levels including association with endogenous activators. Two activators have been described for the yeast 20 S proteasome: the 19 S regulatory particle and the Blm10 protein. The sequence of Blm10 is 20% identical to the mammalian PA200 protein. Recent studies have shown that the sequences of Blm10 and PA200 each contain multiple HEAT-repeats and that each binds to the ends of mature proteasomes, suggesting a common structural and biochemical function. In order to advance structural studies, we have developed an efficient purification method that produces high yields of stoichiometric Blm10-mature yeast 20 S proteasome complexes and we constructed a three-dimensional (3D) model of the Blm10-20 S complex from cryo-electron microscopy images. This reconstruction shows that Blm10 binds in a defined orientation to both ends of the 20 S particle and contacts all the proteasome alpha subunits. Blm10 displays the solenoid folding predicted by the presence of multiple HEAT-like repeats and the axial gates on the alpha rings of the proteasome appear to be open in the complex. We also performed a genetic analysis in an effort to identify the physiological role of Blm10. These experiments, however, did not reveal a robust phenotype upon gene deletion, overexpression, or in a screen for synthetic effects. This leaves the physiological role of Blm10 unresolved, but challenges earlier findings of a role in DNA repair.     

Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

Eukaryotic translation initiation factor 4E (eIF4E) is the mRNA 5' cap binding protein, which plays an important role in the control of translation. The activity of eIF4E is regulated by a family of repressor proteins, the 4E-binding proteins (4E-BPs), whose binding to eIF4E is determined by their phosphorylation state. When hyperphosphorylated, 4E-BPs do not bind to eIF4E. Phosphorylation of the 4E-BPs is effected by the phosphatidylinositol (PI) 3-kinase signal transduction pathway and is inhibited by rapamycin through its binding to FRAP/mTOR (FK506 binding protein-rapamycin-associated protein or mammalian target of rapamycin). Phosphorylation of 4E-BPs can also be induced by protein synthesis inhibitors. These observations led to the proposal that FRAP/mTOR functions as a "sensor" of the translational apparatus (E. J. Brown and S. L. Schreiber, Cell 86:517-520, 1996). To test this model, we have employed the tetracycline-inducible system to increase eIF4E expression. Removal of tetracycline induced eIF4E expression up to fivefold over endogenous levels. Strikingly, upon induction of eIF4E, 4E-BP1 became dephosphorylated and the extent of dephosphorylation was proportional to the expression level of eIF4E. Dephosphorylation of p70(S6k) also occurred upon eIF4E induction. In contrast, the phosphorylation of Akt, an upstream effector of both p70(S6k) and 4E-BP phosphorylation, was not affected by eIF4E induction. We conclude that eIF4E engenders a negative feedback loop that targets a component of the PI 3-kinase signalling pathway which lies downstream of PI 3-kinase.     

Dual Interactions of the Translational Repressor Paip2 with Poly(A) Binding Protein

The cap structure and the poly(A) tail of eukaryotic mRNAs act synergistically to enhance translation. This effect is mediated by a direct interaction of eukaryotic initiation factor 4G and poly(A) binding protein (PABP), which brings about circularization of the mRNA. Of the two recently identified PABP-interacting proteins, one, Paip1, stimulates translation, and the other, Paip2, which competes with Paip1 for binding to PABP, represses translation. Here we studied the Paip2-PABP interaction. Biacore data and far-Western analysis revealed that Paip2 contains two binding sites for PABP, one encompassing a 16-amino-acid stretch located in the C terminus and a second encompassing a larger central region. PABP also contains two binding regions for Paip2, one located in the RNA recognition motif (RRM) region and the other in the carboxy-terminal region. A two-to-one stoichiometry for binding of Paip2 to PABP with two independent K(d)s of 0.66 and 74 nM was determined. Thus, our data demonstrate that PABP and Paip2 could form a trimeric complex containing one PABP molecule and two Paip2 molecules. Significantly, only the central Paip2 fragment, which binds with high affinity to the PABP RRM region, inhibits PABP binding to poly(A) RNA and translation.     

Negamycin restores dystrophin expression in skeletal and cardiac muscles of mdx mice

The ability of aminoglycoside antibiotics to promote read-through of nonsense mutations has attracted interest in these drugs as potential therapeutic agents in genetic diseases. However, the toxicity of aminoglycoside antibiotics may result in severe side effects during long-term treatment. In this paper, we report that negamycin, a dipeptide antibiotic, also restores dystrophin expression in skeletal and cardiac muscles of the mdx mouse, an animal model of Duchenne muscular dystrophy (DMD) with a nonsense mutation in the dystrophin gene, and in cultured mdx myotubes. Dystrophin expression was confirmed by immunohistochemistry and immunoblotting. We also compared the toxicity of negamycin and gentamicin, and found negamycin to be less toxic. Furthermore, we demonstrate that negamycin binds to a partial sequence of the eukaryotic rRNA-decoding A-site. We conclude that negamycin is a promising new therapeutic candidate for DMD and other genetic diseases caused by nonsense mutations.