The Potential of Bovine Colostrum-Derived Exosomes to Repair Aged and Damaged Skin Cells.

In this study, we examined the potentially beneficial effects of bovine colostrum-derived exosomes on UV-induced aging and damage in three major resident skin cells including keratinocytes, melanocytes, and fibroblasts. The treatment with colostrum exosomes prevented the UV-induced generation of intracellular reactive oxygen species in epidermal keratinocytes. In UV-stimulated melanocytes, colostrum exosomes could also significantly reduce the production of the protective skin-darkening pigment melanin, which may help to reduce the risk of excessive melanin formation causing skin hyperpigmentation disorders. In the human dermal fibroblasts treated with colostrum exosomes, the expression of matrix metalloproteinases was suppressed, whereas increased cell proliferation was accompanied by enhanced production of collagen, a major extracellular matrix component of skin. Taken together, our findings indicate that bovine colostrum-derived exosomes having excellent structural and functional stability offer great potential as natural therapeutic agents to repair UV-irradiated skin aging and damage.

Cancer-Specific Production of N-Acetylaspartate via NAT8L Overexpression in Non-Small Cell Lung Cancer and Its Potential as a Circulating Biomarker.

In order to identify new cancer-associated metabolites that may be useful for early detection of lung cancer, we performed a global metabolite profiling of a non-small cell lung cancer (NSCLC) line and immortalized normal lung epithelial cells from the same patient. Among several metabolites with significant cancer/normal differences, we identified a unique metabolic compound, N-acetylaspartate (NAA), in cancer cells-undetectable in normal lung epithelium. NAA's cancer-specific detection was validated in additional cancer and control lung cells as well as selected NSCLC patient tumors and control tissues. NAA's cancer specificity was further supported in our analysis of NAA synthetase (gene symbol: NAT8L) gene expression levels in The Cancer Genome Atlas: elevated NAT8L expression in approximately 40% of adenocarcinoma and squamous cell carcinoma cases (N = 577), with minimal expression in all nonmalignant lung tissues (N = 74). We then showed that NAT8L is functionally involved in NAA production of NSCLC cells through siRNA-mediated suppression of NAT8L, which caused selective reduction of intracellular and secreted NAA. Our cell culture experiments also indicated that NAA biosynthesis in NSCLC cells depends on glutamine availability. For preliminary evaluation of NAA's clinical potential as a circulating biomarker, we developed a sensitive NAA blood assay and found that NAA blood levels were elevated in 46% of NSCLC patients (N = 13) in comparison with age-matched healthy controls (N = 21) among individuals aged 55 years or younger. Taken together, these results indicate that NAA is produced specifically in NSCLC tumors through NAT8L overexpression, and its extracellular secretion can be detected in blood. Cancer Prev Res; 9(1); 43-52. ©2015 AACR.

Neurochemical properties of dental primary afferent neurons.

The long belief that dental primary afferent (DPA) neurons are entirely composed of nociceptive neurons has been challenged by several anatomical and functional investigations. In order to characterize non-nociceptivepopulation among DPA neurons, retrograde transport fluorescent dye was placed in upper molars of rats and immunohistochemical detection of peripherin and neurofilament 200 in the labeled trigeminal ganglia was performed. As the results, majority ofDPA neurons were peripherin-expressing small-sized neurons, showing characteristic ofnociceptive C-fibers. However, 25.7% of DPA were stained with antibody against neurofilament 200, indicating significant portion of DPA neurons are related to large myelinated Aß fibers. There were a small number of neurons thatexpressed both peripherin and neurofilament 200, suggestive of Aδ fibers. The possible transition of neurochemical properties by neuronal injury induced by retrograde labeling technique was ruled out by detection of minimal expression of neuronal injury marker, ATF-3. These results suggest that in addition to the large population of C-fiber-related nociceptive neurons, a subset of DPA neurons is myelinated large neurons, which is related to low-threshold mechanosensitive Aß fibers. We suggest that these Aß fiber-related neurons might play a role as mechanotransducers of fluid movement within dentinal tubules.

Intracellular acidification is associated with changes in free cytosolic calcium and inhibition of action potentials in rat trigeminal ganglion.

The effect of intracellular acidification and subsequent pH recovery in sensory neurons has not been well characterized. We have studied the mechanisms underlying Ca(2+)-induced acidification and subsequent recovery of intracellular pH (pH(i)) in rat trigeminal ganglion neurons and report their effects on neuronal excitability. Glutamate (500 µM) and capsaicin (1 µM) increased intracellular Ca(2+) concentration ([Ca(2+)](i)) with a following decrease in pH(i). The recovery of [Ca(2+)](i) to the prestimulus level was inhibited by LaCl(3) (1 mM) and o-vanadate (10 mM), a plasma membrane Ca(2+)/ATPase (PMCA) inhibitor. Removal of extracellular Ca(2+) also completely inhibited the acidification induced by capsaicin. TRPV1 was expressed only in small and medium sized trigeminal ganglion neurons. mRNAs for Na(+)/H(+) exchanger type 1 (NHE1), pancreatic Na(+)-HCO(3)(-) cotransporter type 1 (pNBC1), NBC3, NBC4, and PMCA types 1-3 were detected by RT-PCR. pH(i) recovery was significantly inhibited by pretreatment with NHE1 or pNBC1 siRNA. We found that the frequency of action potentials (APs) was dependent on pH(i). Application of the NHE1 inhibitor 5'-(N-ethyl-N-isopropyl) amiloride (5 µM) or the pNBC1 inhibitor 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid (500 µM) delayed pH(i) recovery and decreased AP frequency. Simultaneous application of 5'-(N-ethyl-N-isopropyl) amiloride and 4',4'-di-isothiocyanostilbene-2',2'-sulfonic acid almost completely inhibited APs. In summary, our results demonstrate that the rise in [Ca(2+)](i) in sensory neurons by glutamate and capsaicin causes intracellular acidification by activation of PMCA type 3, that the pH(i) recovery from acidification is mediated by membrane transporters NHE1 and pNBC1 specifically, and that the activity of these transporters has direct consequences for neuronal excitability.

Effects of saccharin intake on hippocampal and cortical plasticity in juvenile and adolescent rats.

The sensory system is developed and optimized by experiences given in the early phase of life in association with other regions of the nervous system. To date, many studies have revealed that deprivation of specific sensory experiences can modify the structure and function of the central nervous system; however, the effects of sensory overload remains unclear. Here we studied the effect of overloading the taste sense in the early period of life on the synaptic plasticity of rat hippocampus and somatosensory cortex. We prepared male and female Sprague Dawley rats with ad libitum access to a 0.1% saccharin solution for 2 hrs per day for three weeks after weaning on postnatal day 22. Saccharin consumption was slightly increased in males compared with females; however, saccharin intake did not affect chow intake or weight gain either in male or in female rats. We examined the effect of saccharin-intake on long term potentiation (LTP) formation in hippocampal Schaffer collateral pathway and somatosensory cortex layer IV - II/III pathways in the 6-week old saccharin-fed rats. There was no significant difference in LTP formation in the hippocampus between the control group and saccharin-treated group in both male and female rats. Also in the somatosensory cortex, we did not see a significant difference in LTP among the groups. Therefore, we conclude that saccharin-intake during 3~6 weeks may not affect the development of physiological function of the cortical and hippocampal synapses in rats.

P2X7 Receptor-mediated Membrane Blebbing in Salivary Epithelial Cells.

High concentrations of ATP induce membrane blebbing. However, the underlying mechanism involved in epithelial cells remains unclear. In this study, we investigated the role of the P2X7 receptor (P2X7R) in membrane blebbing using Par C5 cells. We stimulated the cells with 5 mM of ATP for 1~2 hrs and found the characteristics of membrane blebbing, a hallmark of apoptotic cell death. In addition, 500 microM Bz-ATP, a specific P2X7R agonist, induced membrane blebbing. However, 300 microM of Ox-ATP, a P2X7R antagonist, inhibited ATP-induced membrane blebbing, suggesting that ATP-induced membrane blebbing is mediated by P2X7R. We found that ATP-induced membrane blebbing was mediated by ROCK I activation and MLC phosphorylation, but not by caspase-3. Five mM of ATP evoked a biphasic [Ca(2+)](i) response; a transient [Ca(2+)](i) peak and sustained [Ca(2+)](i) increase secondary to ATP-stimulated Ca(2+) influx. These results suggest that P2X7R plays a role in membrane blebbing of the salivary gland epithelial cells.

Effects of pilocarpine on the secretory acinar cells in human submandibular glands.

Pilocarpine has been used as a choice of drugs for treatment of impaired salivary flow. Although considerable data are available as to the stimulatory effect of pilocarpine on the salivary secretion in human, its underlying mechanism, at the cellular level, has not been rigorously studied. In this experiment, we studied the effect of pilocarpine on the ion channel activity, cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) and aquaporin (AQP)-5 expression, which play key roles in the secretary process and determine the capacity of fluid secretion. In human submandibular gland (SMG) acinar cells, 10(-5) M pilocarpine activated the outward rectifying-current, which was predominantly K(+) selective in the whole cell patch clamp study. The pilocarpine increased [Ca(2+)](i) in a concentration-dependent manner in the range of 10(-6) M to 10(-4) M. We found that both increases of [Ca(2+)](i) and outward rectifying- K(+) current were inhibited by 10(-5) M U-73122, a specific phospholipase C inhibitor. The magnitudes of pilocarpine-induced [Ca(2+)](i) transients were approximately 55% lower than those with the same concentration of carbachol (CCh). Pilocarpine also increased the amount of AQP-5 protein in the apical membrane (APM) in human SMG acinar cells. Our results suggest that pilocarpine induce salivary secretions in human by activating K(+) channels, increasing [Ca(2+)](i) via phospholipase C dependent pathway, and increasing AQP-5 protein expression in the APM of SMG acinar cells.

Expression of the Na+-HCO3- cotransporter and its role in pHi regulation in guinea pig salivary glands.

Patterns of salivary HCO(3)(-) secretion vary and depend on species and gland types. However, the identities of the transporters involved in HCO(3)(-) transport and the underlying mechanism of intracellular pH (pH(i)) regulation in salivary glands still remain unclear. In this study, we examined the expression of the Na(+)-HCO(3)(-) cotransporter (NBC) and its role in pH(i) regulation in guinea pig salivary glands, which can serve as an experimental model to study HCO(3)(-) transport in human salivary glands. RT-PCR, immunohistochemistry, and pH(i) measurements from BCECF-AM-loaded cells were performed. The amiloride-sensitive Na(+)/H(+) exchanger (NHE) played a putative role in pH(i) regulation in salivary acinar cells and also appeared to be involved in regulation in salivary ducts. In addition to NHE, NBC also played a role in pH(i) regulation in both acini and ducts. In the parotid gland, NBC1 was functionally expressed in the basolateral membrane (BLM) of acinar cells and the luminal membrane (LM) of ducts. In the submandibular gland, NBC1 was expressed only in the BLM of ducts. NBC1 expressed in these two types of salivary glands takes up HCO(3)(-) and is involved in pH(i) regulation. Although NBC3 immunoreactivity was also detected in submandibular gland acinar cells and in the ducts of both glands, it is unlikely that NBC3 plays any role in pH(i) regulation. We conclude that NBC1 is functionally expressed and plays a role in pH(i) regulation in guinea pig salivary glands but that its localization and role are different depending on the type of salivary glands.

Molecular cloning and functional expression of a sodium bicarbonate cotransporter from guinea-pig parotid glands.

We recently found that the concentration of HCO3- in guinea-pig saliva is very similar to that of human saliva; however, the entity that regulates HCO3- transport has not yet been fully characterized. In order to investigate the mechanism of HCO3- transport, we identified, cloned, and characterized a sodium bicarbonate (Na(+)/HCO3- cotransporter found in guinea-pig parotid glands (gpNBC1). The gpNBC1 gene encodes a 1079-amino acid protein that has 95% and 96% homology with human and mouse parotid NBC1, respectively. Oocytes expressing gpNBC1 were exposed to HCO3- or Na(+)-free solutions, which resulted in a marked change in membrane potentials (V(m)), suggesting that gpNBC1 is electrogenic. Likewise, a gpNBC1-mediated pH recovery was observed in gpNBC1 transfected human hepatoma cells; however, in the presence of 4, 4-diisothiocyanostilbene-2,2-disulfonic acid, a specific NBC1 inhibitor, such changes in V(m) and pH(i) were not observed. Together, the data show that the cloned guinea-pig gene is a functional, as well as sequence homologue of human NBC1.

Mechanosensitivity of voltage-gated K+ currents in rat trigeminal ganglion neurons.

We investigated the mechanosensitivity of voltage-gated K+ channel (VGPC) currents by using whole-cell patch clamp recording in rat trigeminal ganglion (TG) neurons. On the basis of biophysical and pharmacological properties, two types of VGPC currents were isolated. One was transient (I(K,A)), the other sustained (I(K,V)). Hypotonic stimulation (200 mOsm) markedly increased both I(K,A) and I(K,V) without affecting their activation and inactivation kinetics. Gadolinium, a well-known blocker of mechanosensitive channels, failed to block the enhancement of I(K,A) and I(K,V) induced by hypotonic stimulation. During hypotonic stimulation, cytochalasin D, an actin-based cytoskeletal disruptor, further increased I(K,A) and I(K,V), whereas phalloidin, an actin-based cytoskeletal stabilizer, reduced I(K,A) and I(K,V). Confocal imaging with Texas red-phalloidin showed that actin-based cytoskeleton was disrupted by hypotonic stimulation, which was similar to the effect of cytochalasin D. Our results suggest that both I(K,A) and I(K,V) are mechanosensitive and that actin-based cytoskeleton is likely to regulate the mechanosensitivity of VGPC currents in TG neurons.

Amide I vibrational circular dichroism of polypeptides: generalized fragmentation approximation method.

Fragment analyses of vibrational circular dichroic response of dipeptides were carried out recently [Choi and Cho, J. Chem. Phys. 120, 4383 (2004)]. In the present paper, by using a minimal size unit peptide containing two chiral carbons covalently bonded to the peptide group, a generalized fragmentation approximation method is discussed and applied to the calculations of infrared-absorption and vibrational circular dichroism (VCD) intensities of amide I vibrations in various secondary structure polypeptides. Unlike the dipole strength determining IR-absorption intensity, the rotational strength is largely determined by the cross terms that are given by the inner product between the transition electric dipole and the transition magnetic dipole of two different peptides. This explains why the signs and magnitudes of VCD peaks are far more sensitive to the relative orientation and distance between different peptide bonds in a given protein. In order to test the validity of fragmentation approximation, three different segments in a globular protein ubiquitin, i.e., right-handed alpha-helix, beta-sheet, and beta-turn regions, were chosen for density-functional theory (DFT) calculations of amide I vibrational properties and the numerically simulated IR-absorption and VCD spectra by using the fragmentation method are directly compared with DFT results. It is believed that the fragmentation approximation method will be of use in numerically simulating vibrational spectra of proteins in solutions.

Inhibitory effects of autoantibodies on the muscarinic receptors in Sjögren's syndrome.

Sjogren's syndrome (SS) is a systemic autoimmune disease that involves reduced salivary secretions. Recently, circulating autoantibodies from SS patients against the type 3 muscarinic cholinergic receptor (M3R) has been reported in the sera of SS patients. However, the role of these autoantibodies in the development of SS has not been elucidated. In this study, purified IgG was obtained from the sera of 11 SS patients, and its inhibitory effect on the M3R of the salivary glands was evaluated using RT-PCR, microspectrofluorimetry, immunohistochemistry, and Western blot analysis. Stimulation with carbachol (CCh) evoked a [Ca2+]i transient in the fura-2 loaded HSG cells. However, pretreatment of the cells with SS IgG (0.5 mg/ml) for 12 or 24 h significantly reduced the magnitude of the CCh-induced [Ca2+]i transient (CICT). We found that the magnitude of CICT was decreased by 62-45% when cells were pretreated with the SS IgG. However, the [Ca2+]i response to ATP was not altered by the pretreatment of SS IgG. The effect of SS IgG on CICT was abrogated by the inclusion of excessive competitive peptides that encode the amino-acid sequence of M3R, which was not recapitulated by nonspecific peptides. The inhibitory effect of SS IgG on the aquaporin (AQP)-5 expression was also examined. After confirming the apical localization of AQP-5 along with its increase by pilocarpine (10(-5) M), we examined whether SS IgG had an effect on pilocarpine-induced AQP-5 trafficking to the apical membrane (APM) using rat parotid acinar cells. After incubating the cells with SS IgG for 12 h, the amount of pilocarpine-induced AQP-5 significantly decreased compared to the control groups. In conclusion, autoantibodies from the SS patients inhibit the function of the human M3R that is mediated by Ca2+ mobilization and AQP-5 trafficking. Our results could partly explain the underlying mechanisms of glandular dysfunction and associated features of impaired autonomic function in SS patients.

Capsaicin modulates K+ currents from dissociated rat taste receptor cells.

Chili pepper is one of most widely used spices. The main active component of chili pepper is the capsaicin. The effects of capsaicin on sensory nerve endings are well known; however, little is known regarding the direct effect of capsaicin on taste receptor cells (TRCs). In this study, patch clamp methods were used to study the effects of capsaicin on the K(+) currents in TRCs isolated from the rat circumvallate papilla. Fura-2 microspectrofluorimetry was also used to determine the effects of capsaicin on the intracellular Ca(2+) concentration ([Ca(2+)](i)). In the resting state, whole-cell experiments identified outward-rectifying K(+) currents, which were inhibited by 5 mM tetraethylammonium (TEA(+)) chloride. Voltage-dependent K(+) channels with a conductance of 55+/-4 pS (mean+/-S.E.M.; n=3), were observed in cell-attached patches. Capsaicin (500 nM) completely inhibited the outward-rectifying K(+) current in the whole-cell recordings. In cell-attached patches 500 nM capsaicin significantly reduced the open probability (P(o)) of the K(+) channels from 0.401+/-0.052 (n=3) in the resting state, to 0.018+/-0.002 (n=3, P<0.05 by unpaired t-test). In the fura-2-loaded TRCs, micromolar concentrations of capsaicin increased [Ca(2+)](i) in a dose-dependent manner, e.g., 100 microM capsaicin consistently increased the 340:380 fluorescence ratio from 1.04+/-0.05 in the resting state to 1.40+/-0.05 (n=28). These results suggest that capsaicin can enhance or modify the gustatory sensation by inhibiting the K(+) currents of the TRCs directly.