Biodiversity in aerobic granule membrane bioreactor at high organic loading rates

Antibacterial peptide CM4 (ABP-CM4) is a small cationic peptide with broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. We report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antibacterial peptide ABP-CM4. The fusion protein expressed in a soluble form was purified to a purity of 90% by Ni-IDA chromatography and 112 mg protein of interest was obtained per liter of fermentation culture. After the SUMO–CM4 fusion protein was cleaved by the SUMO protease at 30 °C for 1 h, the cleaved sample was re-applied to a Ni-IDA. Finally, about 24 mg recombinant CM4 was obtained from 1 l fermentation culture with no less than 96% purity and the recombinant CM4 had similar antimicrobial properties to the synthetic CM4. Thus, the SUMO-mediated peptide expression and purification system potentially could be employed for the production of recombinant cytotoxic peptides.     

OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function

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Highlights? Identification of OTUB1 mutants with impaired ability to inhibit E2 enzymes ? Crystal structure of a ubiquitin-charged E2 bound to OTUB1 ? Molecular basis of Lys48-linked ubiquitin chain recognition by OTUB1 determined ? OTUB1 employs product inhibition mimicry to inhibit E2 enzymes     

The C-terminus of PARK2 is required for its self-interaction, solubility and role in the spindle assembly checkpoint

PARK2, an ubiquitin ligase closely correlated with Parkinson's disease and cancer, has been shown to accumulate at centrosomes to ubiquitinate misfolded proteins accumulated during interphase. In the present study, we demonstrated that PARK2 can also localize to centrosomes in mitosis and that the protein does not fluctuate through the S- to M-phase. A C-terminal truncation of PARK2 resulted in a spindle assembly checkpoint defect, characterized by HeLa cells able to bypass mitotic arrest induced by nocodazole and form multinucleated cells when overexpressing the C-terminal truncated PARK2 protein. The spindle assembly checkpoint defect may be due to a change in a biochemical or structural property of PARK2 caused by the C-terminal truncation, resulting in a loss of self-interaction between PARK2 proteins.     

The C-terminus of PARK2 is required for its self-interaction, solubility and role in the spindle assembly checkpoint

PARK2, an ubiquitin ligase closely correlated with Parkinson's disease and cancer, has been shown to accumulate at centrosomes to ubiquitinate misfolded proteins accumulated during interphase. In the present study, we demonstrated that PARK2 can also localize to centrosomes in mitosis and that the protein does not fluctuate through the S- to M-phase. A C-terminal truncation of PARK2 resulted in a spindle assembly checkpoint defect, characterized by HeLa cells able to bypass mitotic arrest induced by nocodazole and form multinucleated cells when overexpressing the C-terminal truncated PARK2 protein. The spindle assembly checkpoint defect may be due to a change in a biochemical or structural property of PARK2 caused by the C-terminal truncation, resulting in a loss of self-interaction between PARK2 proteins.     

Enhancing DNA vaccine potency by combining a strategy to prolong dendritic cell life with intracellular targeting strategies

We have recently shown that intradermal coadministration of DNA encoding Ag with DNA encoding inhibitors of apoptosis, including Bcl-x(L), prolongs dendritic cell (DC) life and thereby enhances the potency of DNA vaccines in vivo. We have also demonstrated that DNA vaccines targeting Ag to subcellular compartments, using proteins such as Mycobacterium tuberculosis heat shock protein 70, calreticulin, or the sorting signal of the lysosome-associated membrane protein type 1 (LAMP-1), enhanced DNA vaccine potency. In this study, we reasoned that the combination of a strategy to prolong DC life with intracellular targeting strategies might produce a more effective DNA vaccine against human papillomavirus E7. We showed that coadministration of DNA encoding Bcl-x(L) with DNA encoding E7/heat shock protein 70, calreticulin/E7, or Sig/E7/LAMP-1 resulted in further enhancement of the E7-specific CD8(+) T cell response for all three constructs. Of these strategies, mice vaccinated with Sig/E7/LAMP-1 DNA mixed with Bcl-x(L) DNA showed the greatest increase in E7-specific CD8(+) T cells ( approximately 13-fold increase). This combination of strategies resulted in increased CD8(+) T cell functional avidity, an increased E7-specific CD4(+) Th1 cell response, enhanced tumor treatment ability, and stronger long-term tumor protection when compared with mice vaccinated without Bcl-x(L) DNA. Therefore, DNA vaccines that combine strategies to enhance intracellular Ag processing and prolong DC life have potential clinical implications for control of viral infection and neoplasia.     

Process development for degradation of phenol by Pseudomonas putida in hollow-fiber membrane bioreactors

The degradation of phenol (100-2800 mg/L) by cells Pseudomonas putida CCRC14365 in an extractive hollow-fiber membrane bioreactor (HFMBR) was studied, in which the polypropylene fibers were prewetted with ethanol. The effects of flow velocity, the concentrations of phenol, and the added dispersive agent tetrasodium pyrophosphate on phenol degradation and cell growth were examined. It was shown that about 10% of phenol was sorbed on the fibers at the beginning of the degradation process. The cells P. putida fully degraded 2000 mg/L of phenol within 73 h when the cells were immobilized and separated by the fibers. Even at a level of 2800 mg/L, phenol could be degraded more than 90% after 95-h operation. At low phenol levels (< 400 mg/L) where substrate inhibition was not severe, it was more advantageous to treat the solution in a suspended system. At higher phenol levels (> 1000 mg/L), however, such HFMBR-immobilized cells could degrade phenol to a tolerable concentration with weak substrate-inhibition effect, and the degradation that followed could be completed by suspended cultures due to their larger degradation rate. The process development in an HFMBR system was also discussed.