The Functional Interrelationship between Gap Junctions and Fenestrae in Endothelial Cells of the Liver Organoid

Functional intact liver organoid can be reconstructed in a radial-flow bioreactor when human hepatocellular carcinoma (FLC-5), mouse immortalized sinusoidal endothelial M1 (SEC) and A7 (HSC) hepatic stellate cell lines are cocultured. The structural and functional characteristics of the reconstructed organoid closely resemble the in vivo liver situation. Previous liver organoid studies indicated that cell-to-cell communications might be an important factor for the functional and structural integrity of the reconstructed organoid, including the expression of fenestrae. Therefore, we examined the possible relationship between functional intact gap junctional intercellular communication (GJIC) and fenestrae dynamics in M1-SEC cells. The fine morphology of liver organoid was studied in the presence of (1) irsogladine maleate (IM), (2) oleamide and (3) oleamide followed by IM treatment. Fine ultrastructural changes were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and compared with control liver organoid data. TEM revealed that oleamide affected the integrity of cell-to-cell contacts predominantly in FLC-5 hepatocytes. SEM observation showed the presence of fenestrae on M1-SEC cells; however, oleamide inhibited fenestrae expression on the surface of endothelial cells. Interestingly, fenestrae reappeared when IM was added after initial oleamide exposure. GJIC mediates the number of fenestrae in endothelial cells of the liver organoid.     

Foraging interactions between native and exotic bumblebees: enclosure experiments using native flowering plants

To assess the impact of Bombus terrestris invasion on the foraging efficiency of native Japanese bumblebees, consumption and acquisition of floral resources during foraging on flowers of native Japanese plant species were investigated using enclosures with three treatments: one with only B. terrestris (exotic), one with both B. terrestris and native Japanese bumblebee species (mixed), and one with only Japanese species (native), but with the bumblebee density held constant. Changes in the body mass of queens and the nest mass of colonies for two days did not significantly differ among four combinations of the species and treatment, B. terrestris in the exotic and mixed treatments and Japanese species in the mixed and native treatments. Thus, it is not clear that B. terrestris has higher foraging efficiency than native species and that B. terrestris individuals more negatively affect the foraging efficiency of native species than individuals of the native species themselves. The nectar standing crop of Cirsium kamtschaticum was smaller in the exotic treatment than in the mixed and native treatments. However, this may have been an artifact of differences in the numbers of flowers in the various treatments. T. Nagamitsu and T. Kenta contributed equally to this work     

Quantitative evaluation of adhesion of lactobacilli isolated from human intestinal tissues to human colonic mucin using surface plasmon resonance (BIACORE assay)

AIMS: To isolate lactobacilli from the mucus layer of the human intestine and evaluate their adhesion abilities using a BIACORE assay. METHODS AND RESULTS: Thirty strains of lactobacilli were isolated from the mucus layer of normal human intestinal tissues using conventional plate culture. The strains were identified using homology comparisons of the 16S rDNA sequence to databases as Lactobacillus salivarius (26%), Lactobacillus fermentum (13%), Lactobacillus gasseri (10%), Lactobacillus paracasei (7%), Lactobacillus casei (3%), Lactobacillus mucosae (3%) and Lactobacillus plantarum (3%). Lactobacillus plantarum LA 318 shows the highest adhesion to human colonic mucin (HCM) using the BIACORE assay at 115.30 +/- 12.37 resonance unit (RU). The adhesion of cell wall surface proteins from strain LA 318 was significantly higher to HCM than to bovine serum albumin (BSA; P < 0.05). CONCLUSIONS: We isolated 30 strains of lactobacilli. Lactobacillus salivarius was the predominant species of lactobacilli isolated in this study. The adhesion of strain LA 318 isolated from human transverse colon to its mucin was shown. The adhesion could be mediated by lectin-like components on the bacterial cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study where lactobacilli were isolated from human intestinal tissues and shown to adhere to HCM.     

Mutations in the helix termination motif of mouse type I IRS keratin genes impair the assembly of keratin intermediate filament

Two classical mouse hair coat mutations, Rex (Re) and Rex wavy coat (Re(wc)), are linked to the type I inner root sheath (IRS) keratin genes of chromosome 11. An N-ethyl-N-nitrosourea-induced mutation, M100573, also maps close to the type I IRS keratin genes. In this study, we demonstrate that Re and M100573 mice bear mutations in the type I IRS gene Krt25; Re(wc) mice bear an additional mutation in the type I IRS gene Krt27. These three mutations are located in the helix termination motif of the 2B alpha-helical rod domain of a type I IRS keratin protein. Immunohistological analysis revealed abnormal foam-like immunoreactivity with an antibody raised to type II IRS keratin K71 in the IRS of Re/+ mice. These results suggest that the helix termination motif is essential for the proper assembly of types I and II IRS keratin protein complexes and the formation of keratin intermediate filaments.     

A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud

Mammal-fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog (Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements.     

Synthesis, structure-activity relationships, and mechanism of action of anti-HIV-1 lamellarin α 20-sulfate analogues

Lamellarin α and six different types of lamellarin α 20-sulfate analogues were synthesized and their structure–activity relationships were investigated using a single round HIV-1 vector infection assay. All lamellarin sulfates having pentacyclic lamellarin core exhibited anti-HIV-1 activity at a 10 μM concentration range regardless of the number and position of the sulfate group. On the other hand, non-sulfated lamellarin α and ring-opened lamellarin sulfate analogues did not affect HIV-1 vector infection in similar concentrations. The lamellarin sulfates utilized in this study did not exhibit unfavorable cytotoxic effect under the concentrations tested (IC₅₀ > 100 μM). Confocal laser scanning microscopic analysis indicated that hydrophilic lamellarin sulfates were hardly incorporated in the cell. HIV-1 Env-mediated cell–cell fusion was suppressed by lamellarin sulfates. These results suggested that lamellarin sulfates have a novel anti-HIV-1 activity besides the previously reported integrase activity inhibition, possibly at a viral entry step of HIV-1 replication.     

Vasorelaxant effects of macrocyclic bis(bibenzyls) from liverworts

Vasorelaxant effects of a series of bis(bibenzyls) from liverworts such as Marchantia polymorpha and Marchantia paleacea on rat aorta demonstrated that they relaxed phenylephrine (PE)-induced contractions, which may be mediated through the increased release of NO from endothelial cells as well as opening of K(+) channels, and inhibition of Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCs) and/or receptor-operated Ca(2+) channels (ROCs). Structure-activity relationship based on their structures was discussed. The presence of two aromatic rings which can be connected through two atoms bridge spacer may play an important role for vasorelaxant effect.     

Plant regeneration via direct and indirect adventitious shoot formation and chromosome-doubled somaclonal variation in Titanotrichum oldhamii (Hemsl.) Solereder

The gesneriaceous perennial plant Titanotrichum oldhamii has beautiful foliage and attractive bright yellow flowers. However, breeding of T. oldhamii by conventional sexual hybridization may be difficult because sexual reproduction of this species is very rare. In the present study, plant regeneration systems via both direct and indirect formation of adventitious shoots from leaf explants were established as the first step toward breeding T. oldhamii by using biotechnological techniques. Adventitious shoots were formed efficiently on medium containing 0.1 mg l⁻¹ benzyladenine. Histological observation showed that shoot formation on this medium occurred directly from leaf epidermal cells without callus formation. On the other hand, leaf explants formed calluses on medium containing 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid. The calluses could be maintained by monthly subculturing to fresh medium of the same composition. When the calluses were transferred to plant growth regulator-free medium, they formed adventitious shoots. Directly and indirectly formed shoots rooted well on medium containing 0.1 mg l⁻¹ indole-3-butyric acid. Plantlets thus obtained were successfully acclimatized and grew vigorously in the greenhouse. Flow cytometry analysis indicated that no variation in the ploidy level was observed in plants regenerated via direct shoot formation, whereas chromosome doubling occurred in several plants regenerated via indirect shoot formation. Regenerated plants with the same ploidy level as the mother plants showed almost the same phenotype as the mother plants, whereas chromosome-doubled plants showed apparent morphological alterations: they had small and crispate flowers, and round and deep green leaves.     

Engagement of overexpressed Her2 with GEP100 induces autonomous invasive activities and provides a biomarker for metastases of lung adenocarcinoma

Overexpression of Her2/ErbB2/Neu in cancer is often correlated with recurrent distant metastasis, although the mechanism still remains largely elusive. We have previously shown that EGFR, when tyrosine-phosphorylated, binds to GEP100/BRAG2 to activate Arf6, which induces cancer invasion and metastasis. We now show that overexpressed Her2 in lung adenocarcinoma cells also employs GEP100. Like EGFR-GEP100 binding, this association is primarily mediated by the pleckstrin homology (PH) domain of GEP100 and Tyr1139/Tyr1196 of Her2. Tyr1139/Tyr1196 are autonomously phosphorylated, when Her2 is overexpressed. Accordingly, invasive activities mediated by the Her2-GEP100 pathway are not dependent on external factors. Blocking Her2-GEP100 binding, as well as its signaling pathway all inhibit cancer invasive activities. Moreover, our clinical study indicates that co-overexpression of Her2 with GEP100 in primary lung adenocarcinomas of patients is correlated with the presence of their node-metastasis with a statistical significance. Since the GEP100 PH domain interacts with both Her2 and EGFR, targeting this domain may provide novel cancer therapeutics.