Carnitine Protects against MPP+-Induced Neurotoxicity and Inflammation by Promoting Primary Ciliogenesis in SH-SY5Y Cells.

Primary cilia help to maintain cellular homeostasis by sensing conditions in the extracellular environment, including growth factors, nutrients, and hormones that are involved in various signaling pathways. Recently, we have shown that enhanced primary ciliogenesis in dopamine neurons promotes neuronal survival in a Parkinson's disease model. Moreover, we performed fecal metabolite screening in order to identify several candidates for improving primary ciliogenesis, including L-carnitine and acetyl-L-carnitine. However, the role of carnitine in primary ciliogenesis has remained unclear. In addition, the relationship between primary cilia and neurodegenerative diseases has remained unclear. In this study, we have evaluated the effects of carnitine on primary ciliogenesis in 1-methyl-4-phenylpyridinium ion (MPP+)-treated cells. We found that both L-carnitine and acetyl-L-carnitine promoted primary ciliogenesis in SH-SY5Y cells. In addition, the enhancement of ciliogenesis by carnitine suppressed MPP+-induced mitochondrial reactive oxygen species overproduction and mitochondrial fragmentation in SH-SY5Y cells. Moreover, carnitine inhibited the production of pro-inflammatory cytokines in MPP+-treated SH-SY5Y cells. Taken together, our findings suggest that enhanced ciliogenesis regulates MPP+-induced neurotoxicity and inflammation.

Inhibition of BRD4 Promotes Pexophagy by Increasing ROS and ATM Activation.

Although autophagy regulates the quality and quantity of cellular compartments, the regulatory mechanisms underlying peroxisomal autophagy (pexophagy) remain largely unknown. In this study, we identified several BRD4 inhibitors, including molibresib, a novel pexophagy inducer, via chemical library screening. Treatment with molibresib promotes loss of peroxisomes selectively, but not mitochondria, ER, or Golgi apparatus in HeLa cells. Consistently, depletion of BRD4 expression also induced pexophagy in RPE cells. In addition, the inhibition of BRD4 by molibresib increased autophagic degradation of peroxisome ATG7-dependency. We further found that molibresib produced reactive oxygen species (ROS), which potentiates ATM activation. Inhibition of ROS or ATM suppressed the loss of peroxisomes in molibresib-treated cells. Taken together, our data suggest that inhibition of BRD4 promotes pexophagy by increasing ROS and ATM activation.

Effects of a new sustained-release microsphere formulation of exenatide, DA-3091, on obese and non-alcoholic fatty liver disease mice.

The aim of this study was to examine the effects of a new sustained-release (SR) microsphere formulation of exenatide, DA-3091, on body weight gain and hepatic injury in high fat diet (HFD)-induced obese mice and high sucrose diet (HSD)-induced non-alcoholic fatty liver disease (NAFLD) mice. Then, we determined whether DA-3091 has the potency as a drug for the treatment of metabolic disease. In obese mice, after 8-week treatment, the body weight gain was significantly more suppressed by both 1 mg/kg and 2 mg/kg of DA-3091, monthly subcutaneous administered, than by 10 mg/kg/day of sibutramin, a drug against obesity. In NAFLD mice, a significant reduction in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, representative markers of hepatic injury, was observed after biweekly subcutaneous administration of 1 mg/kg and 2 mg/kg of DA-3091 for 8 weeks. A significant reduction in hepatic lipid accumulation was observed in DA-3091 treated groups as well. Based on these results, it is demonstrated that DA-3091 has the potency as a drug for the treatment of metabolic disease.

Proteomic analysis on acetate metabolism in Citrobacter sp. BL-4.

Mass production of glucosamine (GlcN) using microbial cells is a worthy approach to increase added values and keep safety problems in GlcN production process. Prior to set up a microbial cellular platform, this study was to assess acetate metabolism in Citrobacter sp. BL-4 (BL-4) which has produced a polyglucosamine PGB-2. The LC-MS analysis was conducted after protein separation on the 1D-PAGE to accomplish the purpose of this study. 280 proteins were totally identified and 188 proteins were separated as acetate-related proteins in BL-4. Acetate was converted to acetyl-CoA by acetyl-CoA synthetase up-regulated in the acetate medium. The glyoxylate bypass in the acetate medium was up-regulated with over-expression of isocitrate lyases and 2D-PAGE confirmed this differential expression. Using (1)H-NMR analysis, the product of isocitrate lyases, succinate, increased about 15 times in the acetate medium. During acetate metabolism proteins involved in the lipid metabolism and hexosamine biosynthesis were over-expressed in the acetate medium, while proteins involved in TCA cycle, pentose phosphate cycle and purine metabolism were down-regulated. Taken together, the results from the proteomic analysis can be applied to improve GlcN production and to develop metabolic engineering in BL-4.

Efficacy of a new sustained-release microsphere formulation of exenatide, DA-3091, in Zucker diabetic fatty (ZDF) rats.

Exenatide must be administered serially by twice-daily subcutaneous (SC) injection due to its short half-life. The purpose of the present study is to develop an improved sustained-release exenatide formulation with a therapeutic efficacy comparable to serial, twice-daily injections of exenatide. A novel SR formulation of exenatide, DA-3091, was prepared by single-emulsion solvent evaporation using PLGA. It was administered by SC injection to ZDF rats in single exenatide doses of 0.1, 0.25, 0.5, 1 or 2mg/kg. On the 28th, 49th and 70th days, a 1 or 2mg/kg dose of DA-3091 was further administered to rats in dose groups of 1 or 2mg/kg. The efficacy of DA-3091 was then compared with that of serial, twice-daily SC injections of an exenatide solution for 13 weeks. NFBG and HbA1c concentrations were decreased both significantly and linearly as the exenatide dose in DA-3091 increased. In addition, food intake and body weight were suppressed both significantly and dose-dependently. In equivalent or half doses of exenatide, the efficacy of DA-3091 was comparable to that of twice-daily injections of exenatide solution for 13 weeks. In conclusion, DA-3091 has the potential to be clinically effective when administered every 3 weeks, or less frequently, which promises to significantly improve patient compliance.

Pharmacokinetics and efficacy of a biweekly dosage formulation of exenatide in Zucker diabetic fatty (ZDF) rats.

PURPOSE: To develop an improved sustained-release (SR) formulation of exenatide (a therapy for patients with type 2 diabetes mellitus) in a biweekly dosage form with therapeutic efficacy comparable to that achieved with twice-daily injections of the drug. METHODS: A SR formulation of exenatide, DA-3091, was prepared by single-emulsion solvent evaporation using poly(D,L-lactide-co-glycolide). Plasma exenatide, as well as plasma insulin, non-fasting blood glucose and HbA1c concentrations, and changes in food intake and body weight were evaluated in both Zucker diabetic fatty (ZDF) and ZDF lean control rats. RESULTS: After a single SC administration of DA-3091 (i.e., 2 mg/kg of exenatide), the plasma exenatide concentration increased and remained elevated in both groups. The concentrations of non-fasting blood glucose and HbA1c decreased significantly following a single SC injection of DA-3091 only in ZDF rats, indicating that the effects of exenatide are dependent on blood glucose concentration. On the other hand, both food intake and body weight gain were reduced in ZDF and ZDF lean control rats. A single injection of DA-3091 (i.e., 2 mg/kg of exenatide) lowered non-fasting blood glucose and HbA1c concentrations more effectively than 14 days of twice-daily administration of exenatide (i.e., 1.96 mg/kg of exenatide). CONCLUSION: DA-3091 has the potential to be used safely and efficaciously in a biweekly dosing regimen.

Morphological variation of Enterobacter sp. BL-2 in acetate-mediated pH environment for excretive production of cationic microbial polyglucosamine biopolymer.

Enterobacter sp. BL-2 excretively produced unique cationic polyglucosamine biopolymer PGB-1 comprised of more than 95% D-glucosamine in an acetate-mediated culture condition. The excretion of the biopolymer PGB- was closely associated with the cellular morphology Enterobacter sp. BL-2, a feature highly dependable on the pH of the medium. The initially formed uneven and irregular surface cells were aggregated into the cell-biopolymer network structure connected by the adhesion modules of the cell-bound biopolymer. The excretive production of the biopolymer PGB-1 coincided with the disruption of the cell-biopolymer network, most actively at the medium pH of 8.0.

Acetate-mediated pH-stat fed-batch cultivation of transconjugant Enterobacter sp. BL-2S over-expressing glmS gene for excretive production of microbial polyglucosamine PGB-1.

A unique cationic polyglucosamine biopolymer PGB-1 comprising more than 95% D-glucosamine was excretively produced from a new bacterial strain Enterobacter sp. BL-2 under acetate-mediated culture conditions. Since the biopolymer PGB-1 could be synthesized from the UDP-N-acetylglucosamine monomer derived from the hexosamine pathway, three glmS, glmM, and glmU genes in the hexosamine pathway were cloned from Enterobacter sp. BL-2, and their molecular structures were elucidated. The cloned glmS, glmM, and glmU genes were reintroduced into the parent strain Enterobacter sp. BL-2 through a conjugative transformation for the overproduction of the biopolymer PGB-1. The biopolymer production increased 1.5-fold in the transconjugant Enterobacter sp. BL-2S over-expressing the first-step glmS gene encoding glucosamine-6-phosphate synthase. The transconjugant Enterobacter sp. BL-2S was cultivated pH-stat fed-batch widely, while intermittently feeding an acetate solution to maintain a constant pH level of 8.0 for 72 h, resulting in 1.15 g/L of the extracellular polyglucosamine biopolymer PGB-1.

Polymeric and compositional properties of novel extracellular microbial polyglucosamine biopolymer from new strain of citrobacter sp. BL-4.

A novel polyglucosamine polymer, PGB-2, was produced extracellularly from a new strain Citrobacter sp. BL-4 using pH-stat fed batch cultivation. It was composed of 97.3% glucosamine and 2.7% rhamnose; its average molecular weight, solubility in 2% acetic acid and viscosity were 20 kDa, 5 g l(-1) and 2.9 cps, respectively. FT-IR and 1H NMR spectra of PGB-2 revealed a close identity with chitosan from crab shells.

Pharmacokinetics and biodistribution of a pGT2-VEGF plasmid DNA after administration in rats.

Intramyocardial administration of gene therapy vectors expressing angiogenic factors have been attempted as an alternative to conventional surgical methods for the management of myocardial ischemia. In this study, we have developed the pGT2-VEGF, a plasmid DNA vector expressing human VEGF165, for the management of ischemic cardiovascular disease and investigated in vivo pharmacokinetics and tissue distribution of pGT2-VEGF after intramyocardial and intravenous administration in rats. A high concentration of pGT2-VEGF was observed in the heart after intramyocardial injection of 300 microg, which is in line with the assumption that direct intramyocardial delivery enables extended localization at the administration site. Leakage of the pGT2-VEGF to the blood circulation was observed after intramyocardial injection, with an area under the curve (AUC) of 3.8 microg min/mL, as compared with 37.3 microg min/mL after intravenous injection of the same dose. The pGT2-VEGF concentration in blood peaked at 5 minutes after intramyocardial administration and declined rapidly to undetectable levels by 2 hours post-administration. In tissue distribution studies, pGT2-VEGF peaked at 5 minutes post-administration in various organs but was undetectable at 2 hours in all organs except heart, lung, and liver. Taken together, the results suggest that intramyocardial-delivered pGT2-VEGF was degraded rapidly in vivo and mainly persisted in target tissues, the heart. In addition, intramyocardial-administered pGT2-VEGF was expressed for longer periods than the persistence of the pGT2-VEGF plasmid DNA in a target tissue. Therefore, a direct myocardial injection of pGT2-VEGF might be useful for local therapeutic angiogenesis.