JNK1-Dependent Phosphorylation of GAP-43 Serine 142 is a Novel Molecular Marker for Axonal Growth

Mammalian axon growth has mechanistic similarities with axon regeneration. The growth cone is an important structure that is involved in both processes, and GAP-43 (growth associated protein-43 kDa) is believed to be the classical molecular marker. Previously, we used growth cone phosphoproteomics to demonstrate that S96 and T172 of GAP-43 in rodents are highly phosphorylated sites that are phosphorylated by c-jun N-terminal protein kinase (JNK). We also revealed that phosphorylated (p)S96 and pT172 antibodies recognize growing axons in the developing brain and regenerating axons in adult peripheral nerves. In rodents, S142 is another putative JNK-dependent phosphorylation site that is modified at a lower frequency than S96 and T172. Here, we characterized this site using a pS142-specific antibody. We confirmed that pS142 was detected by co-expressing mouse GAP-43 and JNK1. pS142 antibody labeled growth cones and growing axons in developing mouse neurons. pS142 was sustained until at least nine weeks after birth in mouse brains. The pS142 antibody could detect regenerating axons following sciatic nerve injury in adult mice. Comparison of amino acid sequences indicated that rodent S142 corresponds to human S151, which is predicted to be a substrate of the MAPK family, which includes JNK. Thus, we confirmed that the pS142 antibody recognized human phospho-GAP-43 using activated JNK1, and also that its immunostaining pattern in neurons differentiated from human induced pluripotent cells was similar to those observed in mice. These results indicate that the S142 residue is phosphorylated by JNK1 and that the pS142 antibody is a new candidate molecular marker for axonal growth in both rodents and human.

     

Small‐molecule auxin inhibitors that target YUCCA are powerful tools for studying auxin function

Auxin is essential for plant growth and development, this makes it difficult to study the biological function of auxin using auxin‐deficient mutants. Chemical genetics have the potential to overcome this difficulty by temporally reducing the auxin function using inhibitors. Recently, the indole‐3‐pyruvate (IPyA) pathway was suggested to be a major biosynthesis pathway in Arabidopsis thaliana L. for indole‐3‐acetic acid (IAA), the most common member of the auxin family. In this pathway, YUCCA, a flavin‐containing monooxygenase (YUC), catalyzes the last step of conversion from IPyA to IAA. In this study, we screened effective inhibitors, 4‐biphenylboronic acid (BBo) and 4‐phenoxyphenylboronic acid (PPBo), which target YUC. These compounds inhibited the activity of recombinant YUC in vitro, reduced endogenous IAA content, and inhibited primary root elongation and lateral root formation in wild‐type Arabidopsis seedlings. Co‐treatment with IAA reduced the inhibitory effects. Kinetic studies of BBo and PPBo showed that they are competitive inhibitors of the substrate IPyA. Inhibition constants (K_i) of BBo and PPBo were 67 and 56 nm , respectively. In addition, PPBo did not interfere with the auxin response of auxin‐marker genes when it was co‐treated with IAA, suggesting that PPBo is not an inhibitor of auxin sensing or signaling. We propose that these compounds are a class of auxin biosynthesis inhibitors that target YUC. These small molecules are powerful tools for the chemical genetic analysis of auxin function.     

Possible Involvement of Palmitate in Pathogenesis of Periodontitis

Type 2 diabetes (T2D) is characterized by decreased insulin sensitivity and higher concentrations of free fatty acids (FFAs) in plasma. Among FFAs, saturated fatty acids (SFAs), such as palmitate, have been suggested to promote inflammatory responses. Although many epidemiological studies have shown a link between periodontitis and T2D, little is known about the clinical significance of SFAs in periodontitis. In this study, we showed that gingival fibroblasts have cell‐surface expression of CD36, which is also known as FAT/fatty acid translocase. Moreover, CD36 expression was increased in gingival fibroblasts of high‐fat diet‐induced T2D model mice, compared with gingival fibroblasts of mice fed a normal diet. DNA microarray analysis revealed that palmitate increased mRNA expression of pro‐inflammatory cytokines and chemokines in human gingival fibroblasts (HGF). Consistent with these results, we confirmed that palmitate‐induced interleukin (IL)‐6, IL‐8, and CXCL1 secretion in HGF, using a cytokine array and ELISA. SFAs, but not an unsaturated fatty acid, oleate, induced IL‐8 production. Docosahexaenoic acid (DHA), which is one of the omega‐3 polyunsaturated fatty acids, significantly suppressed palmitate‐induced IL‐6 and IL‐8 production. Treatment of HGF with a CD36 inhibitor also inhibited palmitate‐induced pro‐inflammatory responses. Finally, we demonstrated that Porphyromonas gingivalis (P.g.) lipopolysaccharide and heat‐killed P.g. augmented palmitate‐induced chemokine secretion in HGF. These results suggest a potential link between SFAs in plasma and the pathogenesis of periodontitis. J. Cell. Physiol. 230: 2981–2989, 2015. © 2015 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

     

Hair follicle stem cell progeny heal blisters while pausing skin development

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that HF development can be sacrificed for the sake of epidermal wound regeneration. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.     

Effects of magnesium sulfate on the luminescence of Vibrio fischeri under nutrient-starved conditions

In this study, we investigated the relationship between MgSO(4) and luminescence in Vibrio fischeri under nutrient-starved conditions. When V. fischeri was cultured in an artificial seawater medium, the luminescence intensity was low relative to that observed under normal growth conditions. It decreased during the initial 14 h, and then increased slightly at 24 h. This regulation of luminescence was not dependent on the quorum-sensing mechanism, because the cell densities had not reached a critical threshold concentration. Under MgSO(4)-starved conditions, luminescence was not fully induced at 14 h, and decreased at 24 h. In contrast, induction of luminescence occurred under MgSO(4)-supplemented conditions, but MgSO(4) alone was insufficient to induce luminescence, and required NaHCO(3) or KCl. These results suggest that the luminescence of V. fischeri is controlled by an exogenous sulfur source under nutrient-starved conditions. In addition, they indicate that the induction of sulfur-dependent luminescence is regulated by the NaHCO(3) or KCl concentration.     

Recovery of time on limits of stability from functional fatigue in Division II collegiate athletes

Health and fitness professionals working with athletes could establish effective and safe practice and training programs if recovery time on dynamic balance from exertion was available. Research investigating the time needed to recover dynamic limits of stability (LOS) from exertion has not been reported. The purpose of this study was to determine the recovery timeline on LOS from functional fatigue in collegiate athletes. Eighteen athletes (11 men, 7 women) from Division II collegiate soccer team who passed prescreening tests to identify their fitness levels were randomly tested on 2 different days by condition (fatigue or nonfatigue). Functional fatigue was determined by using the Borg 15-point rating of perceived exertion (RPE) scale. Subjects were tested on LOS on the Biodex Balance System pre, post, 10, 15, and 20 minutes for each condition. The main effect for condition was not significant (F() = 0.004, p = 0.948), whereas the main effect for time was significant (F(4,64) = 6.167, p < 0.001). The RPE scoring revealed the significant main effect in FATIGUE (F(2.69, 45.73) = 234.8, p < 0.001). In conclusion, 20 minutes of functional activity will likely have a negative influence on dynamic balance, with balance recovery occurring within 10 minutes after the cessation of exercise in Division II collegiate soccer athletes. Moreover, the level of exertion measured by RPE would correspond to athletes' ability to control their center of mass.