Innervation of the arteriovenous anastomoses in the dog tongue

Summary Profiles of nerve plexuses in the arteriovenous anastomoses of the dog tongue were investigated by both transmission and scanning electron microscopy. Three-dimensional morphology of the vascular nerves was examined after removal of the connective tissue components by the HCl-hydrolysis method. Tight bending and a rich nerve supply were the most characteristic features of the anastomosing channels. The tunica media consisted of an outer circular layer of typical smooth-muscle cells and an inner region containing longitudinal plicae of ramified smoothmuscle cells. The tunica adventitia was exclusively occupied by nerve bundles; fibroblasts were poorly developed. Numerous nerve bundles of variable size were coiled around the anastomosing channels, and occasional bundles ran crosswise over the U-shaped bent vessels.     

Identification of the agg1 mutation responsible for negative phototaxis in a “wild-type” strain of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a model organism for various studies in biology. CC-124 is a laboratory strain widely used as a wild type. However, this strain is known to carry agg1 mutation, which causes cells to swim away from the light source (negative phototaxis), in contrast to the cells of other wild-type strains, which swim toward the light source (positive phototaxis). Here we identified the causative gene of agg1 (AGG1) using AFLP-based gene mapping and whole genome next-generation sequencing. This gene encodes a 36-kDa protein containing a Fibronectin type III domain and a CHORD-Sgt1 (CS) domain. The gene product is localized to the cell body and not to flagella or basal body.     

Metabolic engineering to improve 1,5-diaminopentane production from cellobiose using β-glucosidase-secreting Corynebacterium glutamicum

Microbial production of 1,5-diaminopentane (DAP) from renewable feedstock is a promising and sustainable approach for the production of polyamides. In this study, we constructed a β-glucosidase (BGL)-secreting Corynebacterium glutamicum and successfully used this strain to produce DAP from cellobiose and glucose. First, C. glutamicum was metabolically engineered to produce l -lysine (a direct precursor of DAP), followed by the coexpression of l -lysine decarboxylase and BGL derived from Escherichia coli and Thermobifida fusca YX (Tfu0937), respectively. This new engineered C. glutamicum strain produced 27 g/L of DAP from cellobiose in CGXII minimal medium using fed-batch cultivation. The yield of DAP was 0.43 g/g glucose (1 g of cellobiose corresponds to 1.1 g of glucose), which is the highest yield reported to date. These results demonstrate the feasibility of DAP production from cellobiose or cellooligosaccharides using an engineered C. glutamicum strain.     

Comparison of autophagy inducibility in various tyrosine kinase inhibitors and their enhanced cytotoxicity via inhibition of autophagy in cancer cells in combined treatment with azithromycin

Tyrosine kinase inhibitors (TKIs) induce autophagy in many types of cancer cells. We previously reported that gefitinib (GEF) and imatinib (IMA) induce autophagy in epidermal growth factor receptor (EGFR) knock-out A549 and non-BCR-ABL-expressing leukemia cell lines, respectively. This evidence suggests that TKI-induced autophagy is independent of the original target molecules. The present study compared the autophagy-inducing abilities of various TKIs, regardless of their targets, by quantitative autophagy flux assay. We established stable clones expressing the GFP-LC3-mCherry-LC3ΔG plasmid in A549, PC-9, and CAL 27 cell lines and assessed autophagy inducibility by monitoring the fluorescent ratios of GFP-LC3 to mCherry-LC3ΔG using an IncuCyte live cell imaging system during exposure to TKIs viz; GEF, osimertinib (OSI), lapatinib (LAP), lenvatinib (LEN), sorafenib (SOR), IMA, dasatinib (DAS), and tivantinib (TIV). Among these TKIs, DAS, GEF, and SOR exhibited prominent autophagy induction in A549 and PC-9 cells. In CAL 27 cells, IMA, SOR, and LEN, but not GEF, TIV, or OSI, exhibited autophagy induction. In the presence of azithromycin (AZM), which showed an inhibitory effect on autophagy flux, TKIs with prominent autophagy inducibility exhibited enhanced cytotoxicity via non-apoptotic cell death relative to effects of TKI alone. Therefore, autophagy inducibility of TKIs differed in the context of cancer cells. However, once induced, they appeared to have cytoprotective functions. Thus, blocking TKI-induced autophagy with AZM may improve the therapeutic effect of TKIs in cancer cells.     

Flow cytometric and biochemical analysis of dose-dependent effects of sodium butyrate on human endometrial adenocarcinoma cells.

Sodium butyrate (SB) treatment was previously shown to produce seven-fold increases in estrogen hormone receptor binding sites of human endometrial adenocarcinoma (IK) cells. Flow cytometric analysis and histone gel electrophoresis were used to examine cell cycle, cell metabolism, and nuclear histone fractions in IK cells treated with different concentrations of SB. SB-treated cells stained with fluorochromes specific for DNA, RNA, or general protein were analyzed by flow cytometry (FCM). Changes in accessibility to three DNA stains and gel electrophoresis were used to analyze rearrangements in chromatin structure. SB caused an accumulation of cells in the G1 phase and inhibited DNA synthesis, but not cellular levels of RNA and protein. Hoechst accessibility to A-T rich regions on DNA was dramatically increased after removal of SB. H1 histones were dephosphorylated and core histones were acetylated during SB-treatment. Information obtained in these studies may be useful for correlating cellular and biochemical events with SB-induced increases in nuclear steroid hormone binding sites.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in glucose transport

Cultured Chinese hamster ovary (CHO) cells possess an insulin-sensitive facilitated diffusion system for glucose transport. Mutant clones of CHO cells defective in glucose transport were obtained by repeating the selection procedure, which involved mutagenesis with ethyl methanesulfonate, radiation suicide with tritiated 2-deoxy-D-glucose, the polyester replica technique and in situ autoradiographic assaying for glucose accumulation. On the first selection, we obtained mutants exhibiting about half the glucose uptake activity of parental CHO-K1 cells and half the amount of a glucose transporter, the amount of which was determined by immunoblotting with an antibody to the human erythrocyte glucose transporter. The second selection, starting from one of the mutants obtained in the first-step selection, yielded a strain, GTS-31, in which both glucose uptake activity and the quantity of the glucose transporter were 10-20% of the levels in CHO-K1 cells, whereas the responsiveness of glucose transport to insulin, and the activities of leucine uptake and several glycolytic enzymes remained unchanged. GTS-31 cells grew slower than CHO-K1 cells at both 33 and 40 degrees C, and in a medium containing a low concentration of glucose (0.1 mM), the mutant cells lost the ability to form colonies. All the three spontaneous GTS-31 cell revertants, which were isolated by growing the mutant cells in medium containing 0.1 mM glucose, exhibited about half the glucose uptake activity and about half the amount of glucose transporter, as compared to in CHO-K1 cells, these characteristics being similar to those of the first-step mutant. These results indicate that the decrease in glucose uptake activity in strain GTS-31 is due to a mutation which induces a reduction in the amount of the glucose transporter, providing genetic evidence that the glucose transporter functions as a major route for glucose entry into CHO-K1 cells.