Distinct Effects of Rotenone, 1-methyl-4-phenylpyridinium and 6-hydroxydopamine on Cellular Bioenergetics and Cell Death

Parkinson's disease is characterized by dopaminergic neurodegeneration and is associated with mitochondrial dysfunction. The bioenergetic susceptibility of dopaminergic neurons to toxins which induce Parkinson's like syndromes in animal models is then of particular interest. For example, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP(+)), and 6-hydroxydopamine (6-OHDA), have been shown to induce dopaminergic cell death in vivo and in vitro. Exposure of animals to these compounds induce a range of responses characteristics of Parkinson's disease, including dopaminergic cell death, and Reactive Oxygen Species (ROS) production. Here we test the hypothesis that cellular bioenergetic dysfunction caused by these compounds correlates with induction of cell death in differentiated dopaminergic neuroblastoma SH-SY5Y cells. At increasing doses, rotenone induced significant cell death accompanied with caspase 3 activation. At these concentrations, rotenone had an immediate inhibition of mitochondrial basal oxygen consumption rate (OCR) concomitant with a decrease of ATP-linked OCR and reserve capacity, as well as a stimulation of glycolysis. MPP(+) exhibited a different behavior with less pronounced cell death at doses that nearly eliminated basal and ATP-linked OCR. Interestingly, MPP(+), unlike rotenone, stimulated bioenergetic reserve capacity. The effects of 6-OHDA on bioenergetic function was markedly less than the effects of rotenone or MPP(+) at cytotoxic doses, suggesting a mechanism largely independent of bioenergetic dysfunction. These studies suggest that these dopaminergic neurotoxins induce cell death through distinct mechanisms and differential effects on cellular bioenergetics.     

Molecular cloning and characterization of a cDNA encoding a novel antibacterial peptide, defensin, from the mulberry longicorn beetle, Apriona germari.

A full-length cDNA clone with high homology (62% mature peptide sequence identity) to an Acalolepta luxuriosa antibacterial gene, possessing a conserved cysteine-stabilized alphabeta motif, was cloned by screening an Apriona germari cDNA library. This gene (AgCRP) had a total length of 360 bp with an open reading frame of 207 bp, and encoded a predicted peptide of 69 amino acid residues. The mature AgCRP peptide was 27 amino acid residues long and had a cysteine-stabilized alphabeta motif of C...CXXXC...C...CXC consensus sequence, similar to insect defensins. Northern blot analysis revealed that the AgCRP exhibited fat body-specific expression and was up-regulated by wounding, bacterial or fungal challenge.     

Synthesis of cholesteryl doxorubicin and its anti-cancer activity

Doxorubicin (dox) has been used as anti-cancer agent, but there are disadvantages such as rapid excretion, short retention time and cardiotoxicity. For giving lipophilic properties to dox, it was modified with cholesterol derivatives that were validated as a component of liposomal gene delivery. This article describes the synthesis of dox derivatives (lipo-dox A-D), their cytotoxicity and cellular uptake. In A549, HeLa, MCF7 and MDA MB 231 cell lines, lipo-dox A and lipo-dox B substituted at alcohol group showed similar anti-cancer effect as dox, but lipo-dox C and lipo-dox D substituted at amino group did not. As a result, the amino group of dox seems an important site for its cancer cell inhibition. Lipophilic property of lipo-dox A and lipo-dox B induced more accumulation in cells compared to parent drug. Therefore, the newly synthesized lipo-dox A and lipo-dox B would be a good candidate for anti-cancer agent.     

Identification and monitoring of Korean medicines derived from <Emphasis Type="Italic">Cinnamomum</Emphasis> spp. by using ITS and DNA marker

In this study, we identified and evaluated the genetic relationships among Cinnamomum plants, which are used in traditional medicine. We also attempted to monitor the distribution of traditional medicines derived from Cinnamomum cassia by using DNA barcoding and a species-specific DNA marker. Plants of the genus Cinnamomum, and in particular C. cassia, are commonly used as medicinal herbs in the form of Cinnamomi Ramulus, Cinnamomi Cortex, and Cassiae Cortex Interior. However, it is difficult to distinguish among different Cinnamomum species based on morphological features, and so to overcome this limitation, nucleotide sequences of the internal transcribed spacer (ITS) region of Cinnamomum DNA were determined and compared. On the basis of the discrepancy in determined ITS sequences, a 408-bp product, amplified by the primer pair CC F1/CC R3, was developed as a C. cassia-specific DNA marker. Using the developed DNA marker in combination with the ITS 2 nucleotide sequence, we monitored imported and commercially supplied medicinal products derived from Cinnamomum plants in markets in Korean, China, and Japan. The results revealed that most of the specimens monitored were derived from C. cassia.     

Development and validation of reverberation-chamber type whole-body exposure system for mobile-phone frequency

We developed whole-body exposure systems for in-vivo study at cellular (848.5 MHz) and Personal Communication System (PCS, 1,762.5 MHz) frequency, utilizing reverberation chamber. The field uniformities in the test area of the designed chambers were verified by simulation and measurement. In the whole-body exposure environment, Specific Absorption Rate (SAR) distributions inside of mice were calculated using Finite Difference Time Domain (FDTD) simulation. Key results are presented in this article.     

Enhanced glycogen synthase kinase-3β activity mediates podocyte apoptosis under diabetic conditions

Glycogen synthase kinase-3β (GSK-3β) is involved in the pathogenesis of various kidney diseases. This study was undertaken to examine the changes in GSK-3β activity in podocytes under diabetic conditions and to elucidate the functional role of GSK-3β in podocyte apoptosis. In vivo, 32 rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with 6-bromoindirubin-3′-oxime (BIO) for 3 months. In vitro, immortalized mouse podocytes were exposed to 5.6 mM glucose or 30 mM glucose (HG) with or without 10 μM BIO. Western blot analysis and TUNEL or Hoechst 33342 staining were performed to identify apoptosis. Urinary albumin excretion was significantly higher in DM rats, and this increase was significantly abrogated in DM rats by BIO treatment. The protein expression of Tyr216-phospho-GSK-3β was significantly increased in DM glomeruli and in cultured podocytes exposed to HG. Western blot analysis revealed that the protein expression of Bax and active fragments of caspase-3 were significantly increased, whereas phospho-Akt, β-catenin, and Bcl-2 protein expression were significantly decreased in DM glomeruli and HG-stimulated podocytes. Apoptosis, determined by TUNEL assay and Hoechst 33342 staining, was also significantly increased in podocytes under diabetic conditions. The changes in the expression of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG-stimulated podocytes were significantly ameliorated by BIO. These findings suggest that enhanced GSK-3β activity within podocytes under diabetic conditions is associated with podocyte loss in diabetic nephropathy.     

Differentiating Coptis chinensis from Coptis japonica and other Coptis species used in Coptidis Rhizoma based on partial trnL-F intergenic spacer sequences

Dried rhizomes of Coptis species are utilized as “Coptidis Rhizoma” (CR), an important herbal medicinal material in traditional Chinese medicine. Almost all CRs traded in the Korean herbal medicine market originate from Coptis chinensis (“Chun Hwang-Lyun” in Korean medical terminology). Other minor CRs originate from Coptis japonica (“Il Hwang-Lyun”). Although there is an obvious discrepancy in the price of traded CRs in the herbal market depending on the Coptis species, CRs originating from C. chinensis and C. japonica are often confused. Furthermore, the CR traded as “Chun Hwang-Lyun” is occasionally mixed with rhizomes of Coptis deltoidea and/or Coptis omeiensis. Therefore, we sought to discriminate C. chinensis from C. japonica, as well as C. deltoidea and C. omeiensis, by using nucleotide sequence differences in the partial trnL-F intergenic spacer. We developed an efficient real-time polymerase chain reaction (PCR)-based discrimination assay to separate samples of C. chinensis from those of C. japonica without the need to separate the DNA markers by using gel electrophoresis. In addition, we developed a multiplex PCR method with which we were able to discriminate samples of C. chinensis from those of C. deltoidea and C. omeiensis by amplifying the 153-bp DNA marker in C. chinensis in a single PCR process.     

Topographic and climatic controls on soil respiration in six temperate mixed-hardwood forest slopes, Korea

To better understand the effects of local topography and climate on soil respiration, we conducted field measurements and soil incubation experiments to investigate various factors influencing spatial and temporal variations in soil respiration for six mixed‐hardwood forest slopes in the midst of the Korean Peninsula. Soil respiration and soil water content (SWC) were significantly greater (P=0.09 and 0.003, respectively) on north‐facing slopes compared to south‐facing slopes, while soil temperature was not significantly different between slopes (P>0.5). At all sites, soil temperature was the primary factor driving temporal variations in soil respiration (r²=0.84–0.96) followed by SWC, which accounted for 30% of soil respiration spatial and temporal variability. Results from both field measurements and incubation experiments indicate that variations in soil respiration due to aspect can be explained by a convex‐shaped function relating SWC to normalized soil respiration rates. Annual soil respiration estimates (1070–1246 g C m^?2 yr^?1) were not closely related to mean annual air temperatures among sites from different climate regimes. When soils from each site were incubated at similar temperatures in a laboratory, respiration rates for mineral soils from wetter and cooler sites were significantly higher than those for the drier and warmer sites (n=4, P<0.01). Our results indicate that the application of standard temperature‐based Q₁₀ models to estimate soil respiration rates for larger geographic areas covering different aspects or climatic regimes are not adequate unless other factors, such as SWC and total soil nitrogen, are considered in addition to soil temperature.     

Identification and characterization of genes regulated by AqsR, a LuxR-type regulator in Acinetobacter oleivorans DR1

The complete genome of Acinetobacter oleivorans DR1 contains AqsR and AqsI genes, which are LuxR and LuxI homolog, respectively. In a previous study, we demonstrated that quorum sensing (QS) signals play an important role in biofilm formation and hexadecane biodegradation. However, the regulation of genes controlled by the QS system in DR1 remains unexplored. We constructed an aqsR mutant and performed RNA sequencing analysis to understand the QS system. A total of 353 genes were differentially expressed during the stationary phase of wild-type cells compared to that of the aqsR mutant. AqsR appears to be an exceptionally important regulator because knockout of aqsR affected global gene expression. Genes involved in posttranslational modification, chaperones, cell wall structure, secondary metabolites biosynthesis, and stress defense were highly upregulated only in the wild type. Among upregulated genes, both the AOLE_03905 (putative surface adhesion protein) and the AOLE_11355 (L-asparaginase) genes have putative LuxR binding sites at their promoter regions. Soluble AqsR proteins were successfully purified in Escherichia coli harboring both aqsR and aqsI. Comparison of QS signals in an AqsI–AqsR co-overexpression strain with N-acyl homoserine lactone standards showed that the cognate N-acyl homoserine lactone binding to AqsR might be 3OH C12HSL. Our electrophoretic mobility shift assays with purified AqsR revealed direct binding of AqsR to those promoter regions. Our data showed that AqsR functions as an important regulator and is associated with several phenotypes, such as hexadecane utilization, biofilm formation, and sensitivity to cumene hydroperoxide.