Biosynthesis of poly(3-hydroxyalkanoate) from amino acids

It was found that an optically active copolyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), denoted as P(3HB-co-3HV), is synthesized by Alcaligenes eutrophus H16 from several amino acids under various fermentation conditions. The optimum condition for the biosynthesis from one amino acid, threonine, was investigated and its biosynthetic pathway was discussed on the basis of the relation between the fermentation condition and the co-monomer composition of the produced polyesters.     

Structure and biosynthesis of the xylose-containing carbohydrate moiety of rice alpha-amylase

Suspension-cultured cells of rice secrete alpha-amylase into the culture medium. It has been shown that the mature form of the alpha-amylase contains xylose-bearing N-linked oligosaccharide: (formula; see text) We demonstrate that suspension-cultured cells of rice secrete alpha-amylase containing oligomannose-type oligosaccharides in the presence of 1-deoxymannojirimycin or tris(hydroxymethyl)aminomethane. On the other hand, alpha-amylase purified from germinated rice seedlings contains several kinds of oligomannose-type and N-acetyllactosamine-type oligosaccharides. The processing pathway of oligosaccharide moieties in rice cells is discussed on the basis of a comparison of these oligosaccharides structures.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.     

Protocadherin-1 is expressed in the notochord of mouse embryo but is dispensable for its formation

Notochord is an embryonic midline structure that serves as mechanical support for axis elongation and the signaling center for the surrounding tissues. Precursors of notochord are initially induced in the dorsal most mesoderm region in gastrulating embryo and separate from the surrounding mesoderm/endoderm tissue to form an elongated rod-like structure, suggesting that cell adhesion molecules may play an important role in this step. In Xenopus embryo, axial protocadherin (AXPC), an orthologue of mammalian Protocadherin-1 (PCDH1), is indispensable for the assembly and separation from the surrounding tissue of the notochord cells. However, the role of PCDH1 in mammalian notochord remains unknown. We herein report that PCDH1 is expressed in the notochord of mouse embryo and that PCDH1-deficient mice form notochord normally. First, we examined the temporal expression pattern of pcdh1 and found that pcdh1 mRNA was expressed from embryonic day (E) 7.5, prior to the stage when notochord cells detach from the surrounding endoderm tissue. Second, we found that PCDH1 protein is expressed in the notochord of mouse embryos in addition to the previously reported expression in endothelial cells. To further investigate the role of PCDH1 in embryonic development, we generated PCDH1-deficient mice using the CRISPR-Cas9 system. In PCDH1-deficient embryos, notochord formation and separation from the surrounding tissue were normal. Structure and marker gene expression of notochord were also unaffected by loss of PCDH1. Major vascular patterns in PCDH1-deficient embryo were essentially normal. These results suggest that PCDH1 is dispensable for notochord formation, including the tissue separation process, in mammalian embryos. We successfully identified the evolutionary conserved expression of PCDH1 in notochord, but its function may differ among species.     

Cellular Auxin Homeostasis under High Temperature Is Regulated through a SORTING NEXIN1–Dependent Endosomal Trafficking Pathway

High-temperature-mediated adaptation in plant architecture is linked to the increased synthesis of the phytohormone auxin, which alters cellular auxin homeostasis. The auxin gradient, modulated by cellular auxin homeostasis, plays an important role in regulating the developmental fate of plant organs. Although the signaling mechanism that integrates auxin and high temperature is relatively well understood, the cellular auxin homeostasis mechanism under high temperature is largely unknown. Using the Arabidopsis thaliana root as a model, we demonstrate that under high temperature, roots counterbalance the elevated level of intracellular auxin by promoting shootward auxin efflux in a PIN-FORMED2 (PIN2)-dependent manner. Further analyses revealed that high temperature selectively promotes the retrieval of PIN2 from late endosomes and sorts them to the plasma membrane through an endosomal trafficking pathway dependent on SORTING NEXIN1. Our results demonstrate that recycling endosomal pathway plays an important role in facilitating plants adaptation to increased temperature.     

In Vitro Evaluation of the Effect of Stimulation with Magnetic Fields on Chondrocytes

Magnetic fields (MFs) have been used as an external stimulus to increase cell proliferation in chondrocytes and extracellular matrix (ECM) synthesis of articular cartilage. However, previously published studies have not shown that MFs are homogeneous through cell culture systems. In addition, variables such as stimulation times and MF intensities have not been standardized to obtain the best cellular proliferative rate or an increase in molecular synthesis of ECM. In this work, a stimulation device, which produces homogeneous MFs to stimulate cell culture surfaces was designed and manufactured using a computational model. Furthermore, an in vitro culture of primary rat chondrocytes was established and stimulated with two MF schemes to measure both proliferation and ECM synthesis. The best proliferation rate was obtained with an MF of 2 mT applied for 3 h, every 6 h for 8 days. In addition, the increase in the synthesis of glycosaminoglycans was statistically significant when cells were stimulated with an MF of 2 mT applied for 5 h, every 6 h for 8 days. These findings suggest that a stimulation with MFs is a promising tool that could be used to improve in vitro treatments such as autologous chondrocyte implantation, either to increase cell proliferation or stimulate molecular synthesis. Bioelectromagnetics. 2020;41:41–51 © 2019 Bioelectromagnetics Society     

Liposome Vector Containing Biosurfactant-Complexed DNA as Herpes Simplex Virus Thymidine Kinase Gene Delivery System

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N′,N′–dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as β-sitosterol β-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.     

Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (K_m = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (K_m = 3.6 μM; k_cat = 283 s⁻¹) in preference to flavin adenine dinucleotide (FAD) (K_m = 19 μM; k_cat = 128 s⁻¹). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE_25–1 for 2,5-DKCMO-1 and camE_25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE₃₆). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.