PubMed · 5964531
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K Motegi. 1966. [Alkaline phosphatase].. https://pubmed.ncbi.nlm.nih.gov/5964531/
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Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus, the two most abundant phototrophs on Earth, thrive in oligotrophic oceanic regions. While it is well known that specific lineages are exquisitely adapted to prevailing in situ light and temperature regimes, much less is known of the molecular machinery required to facilitate occupancy of these low-nutrient environments. Here, we describe a hitherto unknown alkaline phosphatase, Psip1, that has a substantially higher affinity for phosphomonoesters than other well-known phosphatases like PhoA, PhoX, or PhoD and is restricted to clade III Synechococcus and a subset of high light I-adapted Prochlorococcus strains, suggesting niche specificity. We demonstrate that Psip1 has undergone convergent evolution with PhoX, requiring both iron and calcium for activity and likely possessing identical key residues around the active site, despite generally very low sequence homology. Interrogation of metagenomes and transcriptomes from TARA oceans and an Atlantic Meridional transect shows that psip1 is abundant and highly expressed in picocyanobacterial populations from the Mediterranean Sea and north Atlantic gyre, regions well recognized to be phosphorus (P)-deplete. Together, this identifies psip1 as an important oligotrophy-specific gene for P recycling in these organisms. Furthermore, psip1 is not restricted to picocyanobacteria and is abundant and highly transcribed in some α-proteobacteria and eukaryotic algae, suggesting that such a high-affinity phosphatase is important across the microbial taxonomic world to occupy low-P environments.
The development of insect cells expressing recombinant proteins in a stable continuous manner is an attractive alternative to the BEV system for recombinant protein production. High cell density fed batch and continuous perfusion processes can be designed to maximize the productivity of stably transformed cells. A cell line (Sf-9SEAP) expressing high levels of the reporter protein SEAP stably was obtained by lipid-mediated transfection of Sf-9 insect cells and further selection and screening. The expression of the Sf-9SEAP cells was compared with the BEVS system. It was observed that, the yield obtained in BEVS was similar to the batch Sf-9SEAP at 8 and 7 IU/mL, respectively. The productivity of this foreign gene product with the stable cells was enhanced by bioprocess intensification employing the fed-batch and perfusion modes of culture to increase the cell density in culture. The fed batch process yielded a maximum cell density of 28 x 10(6) cells/mL and 12 IU/mL of SEAP. Further improvements in the productivity could be made using the perfusion process, which demonstrated a stable production rate for extended periods of time. The process was maintained for 43 days, with a steady-state cell density of 17-20 x 10(6) cells/mL and 7 IU/mL SEAP. The total yield obtained in the perfusion process (394 IU) was approximately 22 and 8 times higher than that obtained in a batch (17.6 IU) and fed batch (46.1 IU) process, respectively.
Uniform distribution of cells and their extracellular matrix is essential for the in vivo success of bone tissue engineering constructs produced in vitro. In this study, the effects of biphasic calcium phosphate (BCP) granules embedded into chitosan scaffolds on the distribution, morphology, and phenotypic expression of osteoblastic cells were investigated. Mesenchymal stem cells (MSCs) and preosteoblasts were cultured on chitosan scaffolds with and without BCP under osteoblastic differentiation/maturation conditions for periods up to 4 weeks. The addition of 25 wt % BCP to chitosan created a uniform layer of calcium phosphate (CaP) precipitation similar to bone mineral on the scaffold surfaces as determined by scanning electron microscopy and X-ray spectroscopy. Scaffolds with this CaP layer yielded more uniform and complete cell and ECM distribution than chitosan scaffolds without BCP. The suggestion of chemotaxis in the appearance of this response was confirmed by successive experiments in a Boyden chamber. The CaP layer also altered morphology of cells initially attached to the scaffold surfaces, leading to higher expression of marker proteins of osteoblastic phenotype including alkaline phosphatase and osteocalcin. The use of chitosan/BCP scaffolds for culture of MSCs and preosteoblasts enhances bone tissue development in vitro.