Jae-Hoon Shim

Alleviating Neuropathic Pain Hypersensitivity by Inhibiting PKMζ in the Anterior Cingulate Cortex

Pain in the Brain One of the major challenges in pain research is finding ways to reverse chronic pain. Synaptic long-term potentiation (LTP) at spinal or cortical levels is a cellular model of chronic pain. X.-Y. Li. et al. (p. 1400) studied the role of the enzyme protein kinase M zeta (PKMζ) in neurons of the anterior cingulate cortex (ACC) in the maintenance of LTP and for enhanced pain sensitivity after peripheral nerve injury in mice. Nerve injury appeared to lead to the up-regulation and phosphorylation of PKMζ. This triggered LTP at some synapses in the ACC by increasing the number of AMPA receptors. LTP was restricted to ACC neurons that were activated by nerve injury. Blocking PKMζ in the ACC days after nerve injury normalized pain behavior. Thus, PKMζ may represent a promising target for the treatment of chronic pain. Nerve injury increases the activity of an enzyme in the brain and contributes to chronic pain–related cortical sensitization. Synaptic plasticity is a key mechanism for chronic pain. It occurs at different levels of the central nervous system, including spinal cord and cortex. Studies have mainly focused on signaling proteins that trigger these plastic changes, whereas few have addressed the maintenance of plastic changes related to chronic pain. We found that protein kinase M zeta (PKMζ) maintains pain-induced persistent changes in the mouse anterior cingulate cortex (ACC). Peripheral nerve injury caused activation of PKMζ in the ACC, and inhibiting PKMζ by a selective inhibitor, ζ-pseudosubstrate inhibitory peptide (ZIP), erased synaptic potentiation. Microinjection of ZIP into the ACC blocked behavioral sensitization. These results suggest that PKMζ in the ACC acts to maintain neuropathic pain. PKMζ could thus be a new therapeutic target for treating chronic pain.

Horizontal distribution of butyltins in surface sediments from an enclosed bay system, Korea.

Tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT) compounds were quantitatively determined from surface sediment samples (total 59 stations) covering a whole basin where harbors, shipyards, and aquaculture farms were located. Butyltin compounds were detected from all the stations covering 640 km(2) of an enclosed bay system. TBT concentrations ranged from 4 to 382 ng/g as tin on a dry weight basis, and total butyltin concentrations, from 27 to 1763 ng/g. Horizontal distribution of TBT concentration showed apparent negative gradients from harbors and shipyards, indicating that its contamination was closely related to boating and dry-docking activities. However, TBT concentrations were decreased steeply from source areas. Elevated DBT and MBT levels in creeks imply the possible input of DBT from industrial wastewater. Total butyltin concentrations in sediments are significantly correlated with particulate organic carbon concentration for the subset of stations that are distant from source areas.

A cellular model of memory reconsolidation involves reactivation-induced destabilization and restabilization at the sensorimotor synapse in Aplysia

The memory reconsolidation hypothesis suggests that a memory trace becomes labile after retrieval and needs to be reconsolidated before it can be stabilized. However, it is unclear from earlier studies whether the same synapses involved in encoding the memory trace are those that are destabilized and restabilized after the synaptic reactivation that accompanies memory retrieval, or whether new and different synapses are recruited. To address this issue, we studied a simple nonassociative form of memory, long-term sensitization of the gill- and siphon-withdrawal reflex in Aplysia, and its cellular analog, long-term facilitation at the sensory-to-motor neuron synapse. We found that after memory retrieval, behavioral long-term sensitization in Aplysia becomes labile via ubiquitin/proteasome-dependent protein degradation and is reconsolidated by means of de novo protein synthesis. In parallel, we found that on the cellular level, long-term facilitation at the sensory-to-motor neuron synapse that mediates long-term sensitization is also destabilized by protein degradation and is restabilized by protein synthesis after synaptic reactivation, a procedure that parallels memory retrieval or retraining evident on the behavioral level. These results provide direct evidence that the same synapses that store the long-term memory trace encoded by changes in the strength of synaptic connections critical for sensitization are disrupted and reconstructed after signal retrieval.

Role of Maltogenic Amylase and Pullulanase in Maltodextrin and Glycogen Metabolism of Bacillus subtilis 168

The physiological functions of two amylolytic enzymes, a maltogenic amylase (MAase) encoded by yvdF and a debranching enzyme (pullulanase) encoded by amyX, in the carbohydrate metabolism of Bacillus subtilis 168 were investigated using yvdF, amyX, and yvdF amyX mutant strains. An immunolocalization study revealed that YvdF was distributed on both sides of the cytoplasmic membrane and in the periplasm during vegetative growth but in the cytoplasm of prespores. Small carbohydrates such as maltoheptaose and beta-cyclodextrin (beta-CD) taken up by wild-type B. subtilis cells via two distinct transporters, the Mdx and Cyc ABC transporters, respectively, were hydrolyzed immediately to form smaller or linear maltodextrins. On the other hand, the yvdF mutant exhibited limited degradation of the substrates, indicating that, in the wild type, maltodextrins and beta-CD were hydrolyzed by MAase while being taken up by the bacterium. With glycogen and branched beta-CDs as substrates, pullulanase showed high-level specificity for the hydrolysis of the outer side chains of glycogen with three to five glucosyl residues. To investigate the roles of MAase and pullulanase in glycogen utilization, the following glycogen-overproducing strains were constructed: a glg mutant with a wild-type background, yvdF glg and amyX glg mutants, and a glg mutant with a double mutant (DM) background. The amyX glg and glg DM strains accumulated significantly larger amounts of glycogen than the glg mutant, while the yvdF glg strain accumulated an intermediate amount. Glycogen samples from the amyX glg and glg DM strains exhibited average molecular masses two and three times larger, respectively, than that of glycogen from the glg mutant. The results suggested that glycogen breakdown may be a sequential process that involves pullulanase and MAase, whereby pullulanase hydrolyzes the alpha-1,6-glycosidic linkage at the branch point to release a linear maltooligosaccharide that is then hydrolyzed into maltose and maltotriose by MAase.

Bidirectional modulation of hyperalgesia via the specific control of excitatory and inhibitory neuronal activity in the ACC

Neurons in the anterior cingulate cortex (ACC) are assumed to play important roles in the perception of nociceptive signals and the associated emotional responses. However, the neuronal types within the ACC that mediate these functions are poorly understood. In the present study, we used optogenetic techniques to selectively modulate excitatory pyramidal neurons and inhibitory interneurons in the ACC and to assess their ability to modulate peripheral mechanical hypersensitivity in freely moving mice. We found that selective activation of pyramidal neurons rapidly and acutely reduced nociceptive thresholds and that this effect was occluded in animals made hypersensitive using Freunds Complete Adjuvant (CFA). Conversely, inhibition of ACC pyramidal neurons rapidly and acutely reduced hypersensitivity induced by CFA treatment. A similar analgesic effect was induced by activation of parvalbumin (PV) expressing interneurons, whereas activation of somatostatin (SOM) expressing interneurons had no effect on pain thresholds. Our results provide direct evidence of the pivotal role of ACC excitatory neurons, and their regulation by PV expressing interneurons, in nociception.

β-Propeller Phytase Hydrolyzes Insoluble Ca2+-Phytate Salts and Completely Abrogates the Ability of Phytate To Chelate Metal Ions

Phytate is an antinutritional factor that influences the bioavailability of essential minerals by forming complexes with them and converting them into insoluble salts. To further our understanding of the chemistry of phytates binding interactions with biologically important metal cations, we determined the stoichiometry, affinity, and thermodynamics of these interactions by isothermal titration calorimetry. The results suggest that phytate has multiple Ca(2+)-binding sites and forms insoluble tricalcium- or tetracalcium-phytate salts over a wide pH range (pH 3.0-9.0). We overexpressed the β-propeller phytase from Hahella chejuensis (HcBPP) that hydrolyzes insoluble Ca(2+)-phytate salts. Structure-based sequence alignments indicated that the active site of HcBPP may contain multiple calcium-binding sites that provide a favorable electrostatic environment for the binding of Ca(2+)-phytate salts. Biochemical and kinetic studies further confirmed that HcBPP preferentially recognizes its substrate and selectively hydrolyzes insoluble Ca(2+)-phytate salts at three phosphate group sites, yielding the final product, myo-inositol 2,4,6-trisphosphate. More importantly, ITC analysis of this final product with several cations revealed that HcBPP efficiently eliminates the ability of phytate to chelate several divalent cations strongly and thereby provides free minerals and phosphate ions as nutrients for the growth of bacteria. Collectively, our results provide significant new insights into the potential application of HcBPP in enhancing the bioavailability and absorption of divalent cations.

Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling

DNA shuffling was used to improve the thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2. Two highly thermostable mutants, III‐1 and III‐2, were generated after three rounds of shuffling and recombination of mutations. Their optimal reaction temperatures were all 80 °C, which was 10 °C higher than that of the wild‐type. The mutant enzyme III‐1 carried seven mutations: N147D, F195L, N263S, D311G, A344V, F397S, and N508D. The half‐life of III‐1 was about 20 times greater than that of the wild‐type at 78 °C. The mutant enzyme III‐2 carried M375T in addition to the mutations in III‐1, which was responsible for the decrease in specific activity. The half‐life of III‐2 was 568 min while that of the wild‐type was <1 min at 80 °C. The melting temperatures of III‐1 and III‐2, as determined by differential scanning calorimetry, increased by 6.1 °C and 11.4 °C, respectively. Hydrogen bonding, hydrophobic interaction, electrostatic interaction, proper packing, and deamidation were predicted as the mechanisms for the enhancement of thermostability in the enzymes with the mutations.

Overexpression and characterization of an extremely thermostable maltogenic amylase, with an optimal temperature of 100 °C, from the hyperthermophilic archaeon Staphylothermus marinus

A gene encoding a hyperthermostable maltogenic amylase of Staphylothermus marinus (SMMA) was cloned and overexpressed in Escherichia coli. SMMA consisted of 696 amino acids with a predicted molecular mass of 82.5 kDa. The enzyme was active in acidic conditions (pH 3.5-5.0), with an optimal pH of 5.0, and was extremely thermostable, with a temperature optimum of 100 degrees C and a melting temperature of 109 degrees C, both of which extremely favored the starch conversion process. SMMA hydrolyzed linear malto-oligosaccharides, starch, cyclodextrins, and cycloamylose, primarily to maltose and glucose, and showed highest activity toward acarbose and pullulan, hydrolyzed to acarviosine-glucose and panose, respectively. Investigation of the cleavage mode using (14)C-maltoheptaose revealed that SMMA preferentially hydrolyzed the first and second glycosidic bonds from the reducing end. To our knowledge, this enzyme is the most thermostable maltogenic amylase yet reported, and might be of potential value in the food and starch industries.

Microbial production of palatinose through extracellular expression of a sucrose isomerase from Enterobacter sp. FMB-1 in Lactococcus lactis MG1363

Sucrose isomerase (SIase) has been used to produce palatinose, a structural isomer of sucrose, which has many beneficial health properties, such as low-glycemic and low-insulinemic indices. A gene corresponding to SIase from Enterobacter sp. FMB-1 was expressed in Lactococcus lactis MG1363 using the P170 expression system. The autoinducible promoter (P170) and an optimized signal peptide (SP310mut2) were used to induce and secrete SIase in L. lactis. One-step Ni-NTA affinity chromatography and Western blot analysis demonstrated that SIase was successfully secreted to the culture supernatant, although 60% of the recombinant enzymes were retained inside the cells. The production of the recombinant SIase was highly correlated with pH (pH 6) and glucose concentration (30g/L) of the medium. The extracellularly produced recombinant SIase was functionally active, effectively transforming 50g/L sucrose to 36g/L palatinose, with a conversion rate of 72% in the culture supernatant.

Modulation of Substrate Preference of Thermus Maltogenic Amylase by Mutation of the Residues at the Interface of a Dimer

To elucidate the relationship between the substrate size and geometric shape of the catalytic site of Thermus maltogenic amylase, Gly50, Asp109, and Val431, located at the interface of the dimer, were replaced with bulky amino acids. The k cat/K m value of the mutant for amylose increased significantly, whereas that for amylopectin decreased as compared to that of the wild-type enzyme. Thus, the substituted bulky amino acid residues modified the shape of the catalytic site, such that the ability of the enzyme to distinguish between small and large molecules like amylose and amylopectin was enhanced.