Duk-Soo Kim

Whole-genome sequencing and intensive analysis of the undomesticated soybean (Glycine soja Sieb. and Zucc.) genome

The genome of soybean (Glycine max), a commercially important crop, has recently been sequenced and is one of six crop species to have been sequenced. Here we report the genome sequence of G. soja, the undomesticated ancestor of G. max (in particular, G. soja var. IT182932). The 48.8-Gb Illumina Genome Analyzer (Illumina-GA) short DNA reads were aligned to the G. max reference genome and a consensus was determined for G. soja. This consensus sequence spanned 915.4 Mb, representing a coverage of 97.65% of the G. max published genome sequence and an average mapping depth of 43-fold. The nucleotide sequence of the G. soja genome, which contains 2.5 Mb of substituted bases and 406 kb of small insertions/deletions relative to G. max, is ∼0.31% different from that of G. max. In addition to the mapped 915.4-Mb consensus sequence, 32.4 Mb of large deletions and 8.3 Mb of novel sequence contigs in the G. soja genome were also detected. Nucleotide variants of G. soja versus G. max confirmed by Roche Genome Sequencer FLX sequencing showed a 99.99% concordance in single-nucleotide polymorphism and a 98.82% agreement in insertion/deletion calls on Illumina-GA reads. Data presented in this study suggest that the G. soja/G. max complex may be at least 0.27 million y old, appearing before the relatively recent event of domestication (6,000∼9,000 y ago). This suggests that soybean domestication is complicated and that more in-depth study of population genetics is needed. In any case, genome comparison of domesticated and undomesticated forms of soybean can facilitate its improvement.

Astroglial loss and edema formation in the rat piriform cortex and hippocampus following pilocarpine‐induced status epilepticus

In the present study we analyzed aquaporin‐4 (AQP4) immunoreactivity in the piriform cortex (PC) and the hippocampus of pilocarpine‐induced rat epilepsy model to elucidate the roles of AQP4 in brain edema following status epilepticus (SE). In non‐SE‐induced animals, AQP4 immunoreactivity was diffusely detected in the PC and the hippocampus. AQP4 immunoreactivity was mainly observed in the endfeet of astrocytes. Following SE the AQP4‐deleted area was clearly detected in the PC, not in the hippocampus. Decreases in dystrophin and α‐syntrophin immunoreactivities were followed by reduction in AQP4 immunoreactivity. These alterations were accompanied by the development of vasogenic edema and the astroglial loss in the PC. In addition, acetazolamide (an AQP4 inhibitor) treatment exacerbated vasogenic edema and astroglial loss both in the PC and in the hippocampus. These findings suggest that SE may induce impairments of astroglial AQP4 functions via disruption of the dystrophin/α‐syntrophin complex that worsen vasogenic edema. Subsequently, vasogenic edema results in extensive astroglial loss that may aggravate vasogenic edema. J. Comp. Neurol. 518:4612–4628, 2010.

Differential expressions of aquaporin subtypes in astroglia in the hippocampus of chronic epileptic rats.

In order to elucidate the roles of aquaporins (AQPs) in astroglial responses, we investigated AQP expressions in the experimental epileptic hippocampus. In control animals, AQP1 protein expression was restricted to the ventricular-facing surface of the choroid plexus. AQP4 was expressed in astrocyte foot processes near blood vessels and in ependymal and pial surfaces in contact with cerebrospinal fluid. AQP9 protein has been detected in cells lining the cerebral ventricles, and in astrocytes. Six to eight weeks after status epilepticus (SE), AQP1 expression was mainly, but not all, detected in vacuolized astrocytes, which were localized in the stratum radiatum of the CA1 region. AQP4 was negligible in vacuolized CA1 astrocytes, although AQP4 immunoreactivity in non-vacuolized astrocytes was increased as compared to control level. AQP9 expression was shown to be mainly induced in non-vacuolized CA1 astrocytes. Therefore, our findings suggest that AQP subunits may play differential roles in various astroglial responses (including astroglial swelling and astroglial loss) in the chronic epileptic hippocampus.

Levetiracetam inhibits interleukin-1β inflammatory responses in the hippocampus and piriform cortex of epileptic rats

Levetiracetam (LEV, 2S-(oxo-1-pyrrolidinyl)butanamide, Keppra, UCB Pharma) is a new anti-epileptic drug used to treat certain types of seizures in epilepsy patients. However, the pharmacodynamics of LEV is still controversial. Recently, interleukin-1 beta (IL-1 beta) has been reported to involve in epileptic phenomena. Therefore, we investigated the effects of LEV on IL-1 beta system in the hippocampus and piriform cortex of chronic epileptic rats. As compared to controls, typical reactive astrogliosis and microgliosis were observed in the hippocampus and piriform cortex of epileptic animals. In addition, both reactive astrocytes and reactive microglia showed strong IL-1 beta and interleukin-1 receptor subtype 1 (IL-1R1) immunoreactivities. LEV reduced reactive gliosis and expression levels of IL-1 beta system in the hippocampus and the piriform cortex, while valproic acid did not. These findings suggest that the LEV may have, at least in part, anti-inflammatory effect, particularly against IL-1 beta system in neuroglia within epileptic brains.

PEP-1–SIRT2 inhibits inflammatory response and oxidative stress-induced cell death via expression of antioxidant enzymes in murine macrophages

Sirtuin 2 (SIRT2), a member of the sirtuin family of proteins, plays an important role in cell survival. However, the biological function of SIRT2 protein is unclear with respect to inflammation and oxidative stress. In this study, we examined the protective effects of SIRT2 on inflammation and oxidative stress-induced cell damage using a cell permeative PEP-1-SIRT2 protein. Purified PEP-1-SIRT2 was transduced into RAW 264.7 cells in a time- and dose-dependent manner and protected against lipopolysaccharide- and hydrogen peroxide (H₂O₂)-induced cell death and cytotoxicity. Also, transduced PEP-1-SIRT2 significantly inhibited the expression of cytokines as well as the activation of NF-κB and mitogen-activated protein kinases (MAPKs). In addition, PEP-1-SIRT2 decreased cellular levels of reactive oxygen species (ROS) and of cleaved caspase-3, whereas it elevated the expression of antioxidant enzymes such as MnSOD, catalase, and glutathione peroxidase. Furthermore, topical application of PEP-1-SIRT2 to 12-O-tetradecanoylphorbol 13-acetate-treated mouse ears markedly inhibited expression levels of COX-2 and proinflammatory cytokines as well as the activation of NF-κB and MAPKs. These results demonstrate that PEP-1-SIRT2 inhibits inflammation and oxidative stress by reducing the levels of expression of cytokines and ROS, suggesting that PEP-1-SIRT2 may be a potential therapeutic agent for various disorders related to ROS, including skin inflammation.

Phospholipase C β4 in the Medial Septum Controls Cholinergic Theta Oscillations and Anxiety Behaviors

Anxiety is among the most prevalent and costly diseases of the CNS, but its underlying mechanisms are not fully understood. Although attenuated theta rhythms have been observed in human subjects with increased anxiety, no study has been done on the possible physiological link between these two manifestations. We found that the mutant mouse for phospholipase C β4 (PLC-β4−/−) showed attenuated theta rhythm and increased anxiety, presenting the first animal model for the human condition. PLC-β4 is abundantly expressed in the medial septum, a region implicated in anxiety behavior. RNA interference-mediated PLC-β4 knockdown in the medial septum produced a phenotype similar to that of PLC-β4−/− mice. Furthermore, increasing cholinergic signaling by administering an acetylcholinesterase inhibitor cured the anomalies in both cholinergic theta rhythm and anxiety behavior observed in PLC-β4−/− mice. These findings suggest that (1) PLC-β4 in the medial septum is involved in controlling cholinergic theta oscillation and (2) cholinergic theta rhythm plays a critical role in suppressing anxiety. We propose that defining the cholinergic theta rhythm profile may provide guidance in subtyping anxiety disorders in humans for more effective diagnosis and treatments.

Bidirectional modulation of fear extinction by mediodorsal thalamic firing in mice

The mediodorsal thalamic nucleus has been implicated in the control of memory processes. However, the underlying neural mechanism remains unclear. Here we provide evidence for bidirectional modulation of fear extinction by the mediodorsal thalamic nucleus. Mice with a knockout or mediodorsal thalamic nucleus–specific knockdown of phospholipase C β4 exhibited impaired fear extinction. Mutant mediodorsal thalamic nucleus neurons in slices showed enhanced burst firing accompanied by increased T-type Ca2+ currents; blocking of T channels in vivo rescued the fear extinction. Tetrode recordings in freely moving mice revealed that, during extinction, the single-spike (tonic) frequency of mediodorsal thalamic nucleus neurons increased in wild-type mice, but was static in mutant mice. Furthermore, tonic-evoking microstimulations of the mediodorsal thalamic nucleus, contemporaneous with the extinction tones, rescued fear extinction in mutant mice and facilitated it in wild-type mice. In contrast, burst-evoking microstimulation suppressed extinction in wild-type mice, mimicking the mutation. These results suggest that the firing mode of the mediodorsal thalamic nucleus is critical for the modulation of fear extinction.

Tat-glyoxalase protein inhibits against ischemic neuronal cell damage and ameliorates ischemic injury.

Methylglyoxal (MG), a metabolite of glucose, is the major precursor of protein glycation and induces apoptosis. MG is associated with neurodegeneration, including oxidative stress and impaired glucose metabolism, and is efficiently metabolized to S-D-lactoylglutathione by glyoxalase (GLO). Although GLO has been implicated as being crucial in various diseases including ischemia, its detailed functions remain unclear. Therefore, we investigated the protective effect of GLO (GLO1 and GLO2) in neuronal cells and an animal ischemia model using Tat-GLO proteins. Purified Tat-GLO protein efficiently transduced into HT-22 neuronal cells and protected cells against MG- and H2O2-induced cell death, DNA fragmentation, and activation of caspase-3 and mitogen-activated protein kinase. In addition, transduced Tat-GLO protein increased D-lactate in MG- and H2O2-treated cells whereas glycation end products (AGE) and MG levels were significantly reduced in the same cells. Gerbils treated with Tat-GLO proteins displayed delayed neuronal cell death in the CA1 region of the hippocampus compared with a control. Furthermore, the combined neuroprotective effects of Tat-GLO1 and Tat-GLO2 proteins against ischemic damage were significantly higher than those of each individual protein. Those results demonstrate that transduced Tat-GLO protein protects neuronal cells by inhibiting MG- and H2O2-mediated cytotoxicity in vitro and in vivo. Therefore, we suggest that Tat-GLO proteins could be useful as a therapeutic agent for various human diseases related to oxidative stress including brain diseases.

Protective effects of PEP-1-Catalase on stress-induced cellular toxicity and MPTP-induced Parkinson's disease.

Parkinson’s disease (PD) is a neurodegenerative disability caused by a decrease of dopaminergic neurons in the substantia nigra (SN). Although the etiology of PD is not clear, oxidative stress is believed to lead to PD. Catalase is antioxidant enzyme which plays an active role in cells as a reactive oxygen species (ROS) scavenger. Thus, we investigated whether PEP-1-Catalase protects against 1-methyl-4-phenylpyridinium (MPP+) induced SH-SY5Y neuronal cell death and in a 1-methyl-4-phenyl-1,2,3,6-trtrahydropyridine (MPTP) induced PD animal model. PEP-1-Catalase transduced into SH-SY5Y cells significantly protecting them against MPP+-induced death by decreasing ROS and regulating cellular survival signals including Akt, Bax, Bcl-2, and p38. Immunohistochemical analysis showed that transduced PEP-1-Catalase markedly protected against neuronal cell death in the SN in the PD animal model. Our results indicate that PEP-1-Catalase may have potential as a therapeutic agent for PD and other oxidative stress related diseases. [BMB Reports 2015; 48(7): 395-400]

The reverse roles of transient receptor potential canonical channel-3 and -6 in neuronal death following pilocarpine-induced status epilepticus.

Transient receptor potential canonical channel (TRPC) is a nonselective cation channel permeable to Ca2+, which is expressed in many cell types, including neurons. However, the alterations in TRPC receptor expressions in response to status epilepticus (SE) have not been explored. Therefore, the present study was designated to elucidate the roles of TRPC3 and TRPC6 in neuronal death following SE. In non-SE animals, TRPC3 and TRPC6 immunoreactivity was abundantly detected in the dendrites of pyramidal cells and the cell bodies of dentate granule cells. Following SE, TRPC3 expression was significantly elevated in CA1-, CA3 pyramidal cells, and dentate granule cells, while TRPC6 expression was reduced in these regions. Pyrazole-3 (a TRPC3 inhibitor) effectively prevented up-regulation of neuronal TRPC3 expression induced by SE. Hyperforin (a TRPC6 activator) effectively prevented down-regulation of neuronal TRPC6 expression induced by SE. In addition, both Pyr3 and hyperforin effectively protected neuronal damages from SE. Therefore, the present study yields novel information regarding the role of TRPC3 and 6 in epileptogenic insults and suggests that TRPC 3 and 6 may be involved in neurodegeneration following SE.