Calcitonin gene-related peptide regulates mitogen-activated protein kinase pathway to decrease transforming growth factor β1-induced hepatic plasminogen activator inhibitor-1 mRNA expression in HepG2 cells

Transforming growth factor (TGF) β1-induced plasminogen activator inhibitor (PAI)-1 is one of factors associated with the development of hepatic fibrosis. Calcitonin gene-related peptide (CGRP) shows hepatoprotective effect during hepatic injuries, including fibrosis. However, the effects of CGRP on PAI-1 expression induced by TGFβ1 are unknown. In this study, we investigated the effect of CGRP on TGFβ1-induced PAI-1 expression and its regulatory mechanisms in HepG2 cells. CGRP inhibited TGFβ1-induced PAI-1 expression. H89, a protein kinase A inhibitor, abolished the inhibition of TGFβ1-induced PAI-1 expression by CGRP. TGFβ1 activated mitogen-activated protein kinase (MAPK), including extracellular signal-regulated kinase, c-jun NH₂-terminal kinase, and p38, and this activation was abolished by CGRP. These results show that the CGRP-induced cAMP/PKA activation suppresses activation of MAPK induced by TGFβ1, leading to decreased PAI-1 expression in HepG2 cells.     

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.     

Validity of ultrasound muscle thickness measurements for predicting leg skeletal muscle mass in healthy Japanese middle-aged and older individuals

Background

The skeletal muscle mass of the lower limb plays a role in its mobility during daily life. From the perspective of physical resources, leg muscle mass dominantly decreases after the end of the fifth decade. Therefore, an accurate estimate of the muscle mass is important for the middle-aged and older population. The present study aimed to clarify the validity of ultrasound muscle thickness (MT) measurements for predicting leg skeletal muscle mass (SM) in the healthy Japanese middle-aged and older population.

Findings

MTs at four sites of the lower limb and the bone-free lean tissue mass (LTM) of the right leg were determined using brightness-mode ultrasonography and dual-energy X-ray absorptiometry (DXA), respectively, in 44 women and 33 men, 52- to 78-years old. LTM was used as a representative variable of leg skeletal muscle mass. In the model-development group (30 women and 22 men), regression analysis produced an equation with R² and standard error of the estimate (SEE) of 0.958 and 0.3 kg, respectively: LTM (kg) = 0.01464 × (MT_SUM×L) (cm²) - 2.767, where MT_SUM is the sum of the product of MTs at four sites, and L is length of segment where MT is determined. The estimated LTM (7.0 ± 1.7 kg) did not significantly differ from the measured LTM (7.0 ± 1.7 kg), without a significant systematic error on a Bland-Altman plot. The application of this equation for the cross-validation group (14 women and 11 men) did not yield a significant difference between the measured (7.2 ± 1.6 kg) or estimated (7.2 ± 1.6 kg) LTM and systematic error.

Conclusion

The developed prediction equation may be useful for estimating the lean tissue mass of the lower extremity for the healthy Japanese middle-aged and older population.     

The role of cullin 5-containing ubiquitin ligases

The suppressor of cytokine signaling (SOCS) box consists of the BC box and the cullin 5 (Cul5) box, which interact with Elongin BC and Cul5, respectively. SOCS box-containing proteins have ubiquitin ligase activity mediated by the formation of a complex with the scaffold protein Cul5 and the RING domain protein Rbx2, and are thereby members of the cullin RING ligase superfamily. Cul5-type ubiquitin ligases have a variety of substrates that are targeted for polyubiquitination and proteasomal degradation. Here, we review the current knowledge on the identification of Cul5 and the regulation of its expression, as well as the signaling pathways regulated by Cul5 and how viruses highjack the Cul5 system to overcome antiviral responses.     

NOTCH3 Is Induced in Cancer-Associated Fibroblasts and Promotes Angiogenesis in Oral Squamous Cell Carcinoma

Recent studies have shown that Notch signaling is involved in many types of cancers, including oral squamous cell carcinomas (OSCCs). However, the role of Notch signaling in the tumor microenvironment is not yet fully understood. In this study, we investigated the roles of NOTCH3 signaling in cancer associated fibroblasts (CAFs) in OSCCs. Immunohistochemical study of 93 human tongue OSCC cases indicated that about one third of OSCCs showed NOTCH3 expression in CAFs, and that this expression significantly correlated with tumor-size. In vitro study showed that OSCC cell lines, especially HO1-N-1 cells stimulated NOTCH3 expression in normal human dermal fibroblasts (NHDFs) through direct cell-to-cell contact. Immunohistochemical and morphometric analysis using human OSCC samples demonstrated that NOTCH3 expression in CAFs significantly correlated with micro-vessel density in cancer stroma. In vitro angiogenesis assays involving co-culture of NHDFs with HO1-N-1 and human umbilical endothelial cells (HUVECs), and NOTCH3 knockdown in NHDFs using siRNA, demonstrated that HO1-N-1 cells significantly promoted tube formation dependent on NOTCH3-expression in NHDFs. Moreover, NOTCH3 expression in CAFs was related to poor prognosis of the OSCC patients. This work provides a new insight into the role of Notch signaling in CAFs associated with tumor angiogenesis and the possibility of NOTCH3-targeted molecular therapy in OSCCs.     

The existence of cyanobactericidal bacteria and growth-inhibiting bacteria on water plants in Lake Ohnuma,Japan

Limnology - Microorganisms such as bacteria are considered to be control agents against toxic cyanobacterial blooms such as Microcystis aeruginosa. We investigated cyanobactericidal bacteria and...     

Gliadain, a gibberellin-inducible cysteine proteinase occurring in germinating seeds of wheat, Triticum aestivum L., specifically digests gliadin and is regulated by intrinsic cystatins

We cloned a new cysteine proteinase of wheat seed origin, which hydrolyzed the storage protein gliadin almost specifically, and was named gliadain. Gliadain mRNA was expressed 1 day after the start of seed imbibition, and showed a gradual increase thereafter. Gliadain expression was suppressed when uniconazol, a gibberellin synthesis inhibitor, was added to germinating seeds. Histochemical detection with anti-gliadain serum indicated that gliadain was present in the aleurone layer and also that its expression intensity increased in sites nearer the embryo. The enzymological characteristics of gliadain were investigated using recombinant glutathione S-transferase (GST)-progliadain fusion protein produced in Escherichia coli. The GST-progliadain almost specifically digested gliadin into low molecular mass peptides. These results indicate that gliadain is produced via gibberellin-mediated gene activation in aleurone cells and secreted into the endosperm to digest its storage proteins. Enzymologically, the GST-progliadain hydrolyzed benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH(2)-Mec) at K(m) = 9.5 microm, which is equivalent to the K(m) value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC(50) values of 1.7 x 10(-8) and 5.0 x 10(-8) m, respectively. These values are comparable with those found for GST-progliadain inhibition by E-64 and egg-white cystatin, and are consistent with the possibility that, in germinating wheat seeds, gliadain is under the control of intrinsic cystatins.     

Interfacial friction and substrate deformation mediate long-range signal propagation in tissues

Biomechanics and Modeling in Mechanobiology - Tissue layers can generally slide at the interface, accompanied by the dissipation due to friction. Nevertheless, it remains elusive how force could...