Chanil Moon

Plunc, a Member of the Secretory Gland Protein Family, Is Up-regulated in Nasal Respiratory Epithelium after Olfactory Bulbectomy

Subtraction suppression hybridization was used with high throughput screening to identify transcripts of genes that are differentially expressed in nasal epithelium following lesioning of the olfactory bulb, termed bulbectomy. We isolated the rat homologue ofplunc, a murine gene highly expressed in lung and nasopharyngeal regions, by this method. Rat plunc encodes a 270-amino acid protein containing a putative signal peptide.plunc up-regulation in respiratory epithelium was confirmed by Northern blot and in situ hybridization.plunc mRNA was expressed in nasal epithelium, heart, lung, thymus, and salivary gland in adult rodent. plunc was expressed in nasal epithelium, thymus, and salivary gland during embryogenesis. Antibodies against Plunc detected a 31-kDa protein in lung, heart, and spleen. Rat nasal epithelium displayed robust immunoreactivity that was highly localized to the microvilli layer of respiratory epithelium. The expression of plunc was up-regulated after bulbectomy in respiratory epithelium. We also detected secreted plunc in rat and human mucus. Sequence and homology analyses suggest that Plunc is a member of the secretory gland protein family with putative bactericidal/bacteriostatic function. This is the first protein found in respiratory epithelium whose expression is regulated by olfactory neuronal injury and may provide protection against infection subsequent to injury.

Exendin-4 protects against sulfonylurea-induced β-cell apoptosis.

Sulfonylurea is one of the commonly used anti-diabetic drugs that stimulate insulin secretion from β-cells. Despite their glucose lowering effects in type 2 diabetes mellitus, long-term treatment brought on secondary failure characterized by β-cell exhaustion and apoptosis. ER stress induced by Ca2+ depletion in endoplasmic reticulum (ER) is speculated be one of the causes of secondary failure, but it remains unclear. Glucagon like peptide-1 (GLP-1) has anti-apoptotic effects in β-cells after the induction of oxidative and ER stress. In this study, we examined the antiapoptotic action of a GLP-1 analogue in β-cell lines and islets against ER stress induced by chronic treatment of sulfonylurea. HIT-T15 and dispersed islet cells were exposed to glibenclamide for 48 h, and apoptosis was evaluated using Annexin/PI flow cytometry. Expression of the ER stress-related molecules and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 2/3 was determined by real-time PCR and western blot analysis. Chronic exposure to glibenclamide increased apoptosis by depletion of ER Ca2+ concentration through reduced expression of SERCA 2/3. Pretreatment with Exendin-4 had an anti-apoptotic role through ER stress modulation and ER Ca2+ replenishing by SERCA restoration. These findings will further the understanding of one cause of glibenclamide-induced β-cell loss and therapeutic availability of GLP-1-based drugs in secondary failure by sulfonylurea during treatment of diabetes.

Erythropoietin promotes hair shaft growth in cultured human hair follicles and modulates hair growth in mice

BACKGROUND Recent studies have shown that erythropoietin (EPO)/erythropoietin receptor (EPOR) signaling exist in both human and mouse hair follicles (HFs). OBJECTIVE To investigate whether dermal papilla cells (DPCs) express functional EPOR and, if so, to investigate effects of EPO on hair shaft growth in cultured human scalp hair follicles and hair growth in mice. METHODS EPOR expression in DPCs and follicular keratinocytes was examined by RT-PCR and immunoblot. Phosphorylation of EPOR signaling pathway mediators by EPO treatment was examined by immunoblot. MTT assay was employed to check cell viability after EPO treatment. Hair shaft growth was measured in the absence or presence of EPO and matrix keratinocyte proliferation was examined by Ki-67 immunostaining in cultured hair follicles. Agarose beads containing EPO were implanted into dorsal skin of C57BL/6 mice to examine effects of EPO on hair growth in vivo. RESULTS EPOR mRNA and protein are expressed in cultured human DPCs. EPOR signaling pathway mediators such as EPOR and Akt are phosphorylated by EPO in DPCs. EPO significantly promoted the growth of DPCs and elongated hair shafts with increased proliferation of matrix keratinocytes in cultured human hair follicles. In addition, EPO not only promoted anagen induction from telogen but also prolonged anagen phase. CONCLUSIONS EPO may modulate hair growth by stimulating DPCs that express functional EPOR.

Identification of peptides that selectively bind to myoglobin by biopanning of phage displayed-peptide library

Biopanning of phage displayed-peptide library was performed against myoglobin, a marker for the early assessment of acute myocardial infarction (AMI), to identify peptides that selectively bind to myoglobin. Using myoglobin-conjugated magnetic beads, phages that bound to myoglobin were collected and amplified for the next round of screening. A 148-fold enrichment of phage titer was observed after five rounds of screening relative to the first round. After phage binding ELISA, three phage clones were selected (3R1, 3R7 and 3R10) and the inserted peptides were chemically synthesized. The analysis of binding affinity showed that the 3R7 (CPSTLGASC) peptide had higher binding affinity (Kd=57 nM) than did the 3R1 (CNLSSSWIC) and 3R10 (CVPRLSAPC) peptide (Kd=125 nM and 293 nM, respectively). Cross binding activity to other proteins, such as bovine serum albumin, troponin I, and creatine kinase-MB, was minimal. In a peptide-antibody sandwich ELISA, the selected peptides efficiently captured myoglobin. Moreover, the concentrations of myoglobin in serum samples measured by a peptide-peptide sandwich assay were comparable to those measured by a commercial antibody-based kit. These results indicate that the identified peptides can be used for the detection of myoglobin and may be a cost effective alternative to antibodies.

In vivo imaging of myocardial cell death using a peptide probe and assessment of long-term heart function

During acute myocardial infarction (AMI), both apoptosis and necrosis of myocardial cells could occur and lead to left ventricular (LV) functional decline. Here we determined whether in vivo imaging signals of myocardial cell death by ApoPep-1 (CQRPPR), a peptide probe that binds to apoptotic and necrotic cells through histone H1, at an early stage after AMI showed correlation with the long-term heart function. AMI was induced using a rat model of ischemia and reperfusion (I/R) injury. Fluorescence-labeled ApoPep-1 was administered by intravenous injection into rats 2h after reperfusion. Ex vivo imaging of hearts isolated 2h after peptide injection showed higher levels of near-infrared fluorescence (NIRF) signals at hearts of I/R rats than those of sham-operated rats. The fluorescent peptide was rapidly cleared from the blood and did not bind to red and white blood cells. Localization of fluorescent ApoPep-1 at the area of cell death was demonstrated by co-staining of myocardial tissue with TUNEL. The intensity of in vivo NIRF imaging signals by homing of ApoPep-1 to injured myocardium of I/R rats obtained 2h after peptide injection (equivalent to 4h after injury) showed strong and moderate correlation with the change in the LV ejection fractions (r(2)=0.82) and the size of the fibrotic area (r(2)=0.64), respectively, observed at four weeks after injury. These results suggest that ApoPep-1-mediated in vivo imaging signals of myocardial cell death, including both apoptosis and necrosis, at an early stage of AMI could be a potential biomarker for assessment of long-term outcome of heart function.

The plasma membrane redox enzyme NQO1 sustains cellular energetics and protects human neuroblastoma cells against metabolic and proteotoxic stress

The plasma membrane redox system (PMRS) of nicotinamide adenine dinucleotide (NADH)-related enzymes plays a key role in the maintenance of cellular energetics. During the aging process, neural cells are particularly sensitive to impaired energy metabolism and oxidative damage, but the involvement of the PMRS in these processes is unknown. Here, we used human neuroblastoma cells with either elevated or reduced levels of the PMRS enzyme NADH-quinone oxidoreductase 1 (NQO1) to investigate how the PMRS regulates neuronal stress responses. Cells with elevated NQO1 levels were more resistant to death induced by 2-deoxyglucose, potassium cyanide (energetic stress), and lactacystin (proteotoxic stress), but were not protected from being killed by H2O2 and serum withdrawal. The NAD+(an oxidized form of NADH)/NADH ratio was maintained at a significantly higher level in cells overexpressing NQO1, consistent with enhanced levels of NQO1 activity. Levels of the neuroprotective transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells and nuclear factor (erythroid-derived 2)-like 2, and the protein chaperone HSP70 were elevated in cells overexpressing NQO1. Cells in which NQO1 levels were decreased by RNA interference exhibited increased vulnerability to death induced by 2-deoxyglucose and lactacystin. Thus, a higher NAD+/NADH ratio and activation of adaptive stress response pathways are enhanced by the PMRS in neuroblastoma cells, enabling them to maintain redox homeostasis under conditions of energetic and proteotoxic stress. These findings have implications for the development of therapeutic interventions for neural tumors and neurodegenerative conditions.

Protective Effect of Aminophylline against Cigarette Smoke Extract–Induced Apoptosis in Human Lung Fibroblasts (MRC-5 Cells)

Cigarette smoking is the principal cause of chronic obstructive pulmonary disease (COPD), especially emphysema, which is characterized by alveolar wall destruction and airspace enlargement. Apoptosis of lung structural cells is involved in the pathogenesis of COPD. Xanthine derivatives (aminophylline or theophylline) have been used for the treatment of COPD as a bronchodilator. But the effects of xanthine derivatives on apoptosis of the lung structural cells remain poorly understood, even though it is known that theophylline protects against ultraviolet irradiation-induced cell death in corneal epithelial cells. This study was designed to determine whether aminophylline would protect against cigarette smoke extract (CSE)-induced apoptosis in lung fibroblasts. We demonstrated that aminophylline protected against apoptosis of MRC-5 cells at a relatively lower therapeutic range (10 μg/ml), resulting in a significant increase in cell viability occurring at 20% concentration after 8-hr exposure. Annexin staining decreased from 68 ± 4% of the control to 12 ± 2% of aminophylline (10 μg/ml) pre-treatment after 20% CSE exposure for 12 hr (p < 0.05). Aminophylline decreased caspase 3 and 8 activities and nuclear condensation or fragmentation in MRC-5 cells after exposure to 20% CSE for 12 hr compared with control and high levels of aminophylline (>50 μg/ml) pre-treatment. These findings suggest that aminophylline protected apoptosis of MRC-5 cells through the inactivation of caspases 3 and 8 and could be an effective agent to reduce cigarette smoking-induced lung structural cell apoptosis.

Recombinant Human Erythropoietin in Amyotrophic Lateral Sclerosis: A Pilot Study of Safety and Feasibility

Background and Purpose It has been shown that erythropoietin is neuroprotective in animal models of neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). The aim of this study was to determine the safety and feasibility of repetitive high-dose recombinant human erythropoietin (rhEPO) therapy in ALS patients. Methods Two consecutive studies were conducted. We first recruited 26 subjects for an initial single-arm safety study. After a lead-in period of 3 months to assess the disease progression, rhEPO was infused intravenously (35,000 IU) once per month for 3 months, and the subjects were followed for an additional 3 months. The ALS Functional Rating Scale-Revised (ALSFRS-R) was used for clinical assessment. After confirming the safety of rhEPO, 60 subjects were recruited for the second controlled study (rhEPO and control groups), which involved a total of 6 infusions at a rate of 1/month. Results There were no serious adverse events in the first study. The mean rate of decline in the ALSFRS-R score was lower during the treatment period than during the lead-in period (mean±SD: 2.6±1.8 and 3.7±2.6, respectively; p=0.02). However, the rate of decline during the subsequent 3 months returned to that observed in the lead-in period. In the second study, the mean rate of decline in ALSFRS-R score was significantly lower in the rhEPO group than in the control group (during months 0-3, 1.8±1.7 vs. 3.1±2.3, p=0.03; during months 4-6, 2.1±2.2 vs. 3.5±2.3, p=0.02). Conclusions Intravenous high-dose rhEPO is both safe and feasible for the treatment of ALS. Further investigation using different intervals and doses should be considered.

The erythropoietin-derived peptide MK-X and erythropoietin have neuroprotective effects against ischemic brain damage

Erythropoietin (EPO) has been well known as a hematopoietic cytokine over the past decades. However, recent reports have demonstrated that EPO plays a neuroprotective role in the central nervous system, and EPO has been considered as a therapeutic target in neurodegenerative diseases such as ischemic stroke. Despite the neuroprotective effect of EPO, clinical trials have shown its unexpected side effects, including undesirable proliferative effects such as erythropoiesis and tumor growth. Therefore, the development of EPO analogs that would confer neuroprotection without adverse effects has been attempted. In this study, we examined the potential of a novel EPO-based short peptide, MK-X, as a novel drug for stroke treatment in comparison with EPO. We found that MK-X administration with reperfusion dramatically reduced brain injury in an in vivo mouse model of ischemic stroke induced by middle cerebral artery occlusion, whereas EPO had little effect. Similar to EPO, MK-X efficiently ameliorated mitochondrial dysfunction followed by neuronal death caused by glutamate-induced oxidative stress in cultured neurons. Consistent with this effect, MK-X significantly decreased caspase-3 cleavage and nuclear translocation of apoptosis-inducing factor induced by glutamate. MK-X completely mimicked the effect of EPO on multiple activation of JAK2 and its downstream PI3K/AKT and ERK1/2 signaling pathways, and this signaling process was involved in the neuroprotective effect of MK-X. Furthermore, MK-X and EPO induced similar changes in the gene expression patterns under glutamate-induced excitotoxicity. Interestingly, the most significant difference between MK-X and EPO was that MK-X better penetrated into the brain across the brain–blood barrier than did EPO. In conclusion, we suggest that MK-X might be used as a novel drug for protection from brain injury caused by ischemic stroke, which penetrates into the brain faster in comparison with EPO, even though MK-X and EPO have similar protective effects against excitotoxicity.

Differential cell death and Bcl-2 expression in the mouse retina after glutathione decrease by systemic D,L-buthionine sulphoximine administration.

Glutathione (GSH) plays a critical role in cellular defense against unregulated oxidative stress in mammalian cells including neurons. We previously demonstrated that GSH decrease using [D, L]-buthionine sulphoximine (BSO) induces retinal cell death, but the underlying mechanisms of this are still unclear. Here, we demonstrated that retinal GSH level is closely related to retinal cell death as well as expression of an anti-apoptotic molecule, Bcl-2, in the retina. We induced differential expression of retinal GSH by single and multiple administrations of BSO, and examined retinal GSH levels and retinal cell death in vivo. Single BSO administration showed a transient decrease in the retinal GSH level, whereas multiple BSO administration showed a persistent decrease in the retinal GSH level. Retinal cell death also showed similar patterns: transient increases of retinal cell death were observed after single BSO administration, whereas persistent increases of retinal cell death were observed after multiple BSO administration. Changes in the retinal GSH level affected Bcl-2 expression in the retina. Immunoblot and immunohistochemical analyses showed that single and multiple administration of BSO induced differential expressions of Bcl-2 in the retina. Taken together, the results of our study suggest that the retinal GSH is important for the survival of retinal cells, and retinal GSH appears to be deeply related to Bcl-2 expression in the retina. Thus, alteration of Bcl-2 expression may provide a therapeutic tool for retinal degenerative diseases caused by retinal oxidative stress such as glaucoma or retinopathy.