ROS-Mediated ABA Signaling

In plants, reactive oxygen species (ROS) are short-lived molecules produced through various cellular mechanisms in response to biotic and abiotic stimuli. ROS function as second messengers for hormone signaling, development, oxygen deprivation, programmed cell death, and plant–pathogen interactions. Recent research on ROS-mediated responses has produced stimulating findings such as the specific sources of ROS production, molecular elements that work in ROS-mediated signaling and homeostasis, and a ROS-regulated gene network (Neill et al., Curr Opin Plant Biol 5:388–395, 2002a; Apel and Hirt, Annu Rev Plant Biol 55:373–399, 2004; Mittler et al., Trends Plant Sci 9:490–498, 2004; Mori and Schroeder, Plant Physiol 135:702–708, 2004; Kwak et al., Plant Physiol 141:323–329, 2006; Torres et al., Plant Physiol 141:373–378, 2006; Miller et al., Physiol Plant 133:481–489, 2008). In this review, we highlight new discoveries in ROS-mediated abscisic acid (ABA) signaling. Drs. Daeshik Cho and June M. Kwak are the corresponding authors for this paper.     

Hydrogen sulfide removal by immobilized Thiobacillus novellas on SiO2 in a fluidized bed reactor

The removal of hydrogen sulfide (H2S) from aqueous media was investigated using Thiobacillus novellas cells immobilized on a SiO2 carrier (biosand). The optimal growth conditions for the bacterial strain were 30 degrees C and initial pH of 7.0. The main product of hydrogen sulfide oxidation by T. novellus was identified as the sulfate ion. A removal efficiency of 98% was maintained in the three-phase fluidized-bed reactor, whereas the efficiency was reduced to 90% for the two-phase fluidized-bed reactor and 68% for the two-phase reactor without cells. The maximum gas removal capacity for the system was 254 g H2S/m3/h when the inlet H2S loading was 300 g/m3/h (1,500 ppm). Stable operation of the immobilized reactor was possible for 20 days with the inlet H2S concentration held to 1,100 ppm. The fluidized bed bioreactor appeared to be an effective means for controlling hydrogen sulfide emissions.     

Lung hyperinflation in isolated dog lungs during high-frequency oscillation

To study the phenomenon of lung hyperinflation (LHI), i.e., an increase in lung volume without a concomitant rise in airway pressure, we measured lung volume changes in isolated dog lungs during high-frequency oscillation (HFO) with air, He, and SF6 and with mean tracheal pressure controlled at 2.5, 5.0, and 7.5 cmH2O. The tidal volume and frequency used were 1.5 ml/kg body wt and 20 Hz, respectively. LHI was observed during HFO in all cases except for a few trials with He. The degree of LHI was inversely related to mean tracheal pressure and varied directly with gas density. Maximum expiratory flow rate (Vmax) was measured during forced expiration induced by a vacuum source (-150 cmH2O) at the trachea. Vmax was consistently higher than the peak oscillatory flow rate (Vosc) during HFO, demonstrating that overall expiratory flow limitation did not cause LHI in isolated dog lungs. Asymmetry of inspiratory and expiratory impedances seems to be one cause of LHI, although other factors are involved.     

Detection of an intermediate during the unfolding process of the dimeric ketosteroid isomerase

Failure to detect the intermediate in spite of its existence often leads to the conclusion that two-state transition in the unfolding process of the protein can be justified. In contrast to the previous equilibrium unfolding experiment fitted to a two-state model by circular dichroism and fluorescence spectroscopies, an equilibrium unfolding intermediate of a dimeric ketosteroid isomerase (KSI) could be detected by small angle X-ray scattering (SAXS) and analytical ultracentrifugation. The sizes of KSI were determined to be 18.7A in 0M urea, 17.3A in 5.2M urea, and 25.1A in 7M urea by SAXS. The size of KSI in 5.2M urea was significantly decreased compared with those in 0M and 7M urea, suggesting the existence of a compact intermediate. Sedimentation velocity as obtained by ultracentrifugation confirmed that KSI in 5.2M urea is distinctly different from native and fully-unfolded forms. The sizes measured by pulse field gradient nuclear magnetic resonance (NMR) spectroscopy were consistent with those obtained by SAXS. Discrepancy of equilibrium unfolding studies between size measurement methods and optical spectroscopies might be due to the failure in detecting the intermediate by optical spectroscopic methods. Further characterization of the intermediate using (1)H NMR spectroscopy and Kratky plot supported the existence of a partially-folded form of KSI which is distinct from those of native and fully-unfolded KSIs. Taken together, our results suggest that the formation of a compact intermediate should precede the association of monomers prior to the dimerization process during the folding of KSI.     

Isolation and Characterization of Antifungal Substances from Burkholderia sp. Culture Broth

A new antagonistic Burkholderia strain, designated MP-1 and producing antifungal activities against various filamentous plant pathogenic fungi, was isolated from the rhizoshere in the Naju area. Cultural characteristic studies strongly suggested that this strain belongs to the genus Burkholderia. The nucleotide sequence of the 16S rRNA gene (1491 pb) of strain MP-1 exhibited close similarity (99% to 100%) with other Burkholderia 16S rRNA genes. Extraction of fermentation broth of Burkholderia sp. MP-1 and various separations and purification steps led to isolation of four pure active molecules. The chemical structure of these four compounds—named phenylacetic acid, hydrocinnamic acid, 4-hydroxyphenylacetic acid, and 4-hydroxyphenylacetate methyl ester—was established on the basis on their gas chromatography–electron impact–mass spectrometry (GC-EI-MS) and trimethylsilation GC-EI-MS data. The four isolated compounds inhibited filamentous fungal growth on potato dextrose agar medium supplemented with 100 mg/L of phenylacetic acid, hydrocinnamic acid, 4-hydroxyphenylacetic acid and 4-hydroxyphenylacetate methyl ester individually.     

Facilitation of Expression and Purification of an Antimicrobial Peptide by Fusion with Baculoviral Polyhedrin in Escherichia coli

Several fusion strategies have been developed for the expression and purification of small antimicrobial peptides (AMPs) in recombinant bacterial expression systems. However, some of these efforts have been limited by product toxicity to host cells, product proteolysis, low expression levels, poor recovery yields, and sometimes an absence of posttranslational modifications required for biological activity. For the present work, we investigated the use of the baculoviral polyhedrin (Polh) protein as a novel fusion partner for the production of a model AMP (halocidin 18-amino-acid subunit; Hal18) in Escherichia coli. The useful solubility properties of Polh as a fusion partner facilitated the expression of the Polh-Hal18 fusion protein (~33.6 kDa) by forming insoluble inclusion bodies in E. coli which could easily be purified by inclusion body isolation and affinity purification using the fused hexahistidine tag. The recombinant Hal18 AMP (~2 kDa) could then be cleaved with hydroxylamine from the fusion protein and easily recovered by simple dialysis and centrifugation. This was facilitated by the fact that Polh was soluble during the alkaline cleavage reaction but became insoluble during dialysis at a neutral pH. Reverse-phase high-performance liquid chromatography was used to further purify the separated recombinant Hal18, giving a final yield of 30% with >90% purity. Importantly, recombinant and synthetic Hal18 peptides showed nearly identical antimicrobial activities against E. coli and Staphylococcus aureus, which were used as representative gram-negative and gram-positive bacteria, respectively. These results demonstrate that baculoviral Polh can provide an efficient and facile platform for the production or functional study of target AMPs.     

Inhibition of Endoplasmic Reticulum Stress-induced Apoptosis by Silkworm Storage Protein 1

The endoplasmic reticulum (ER) plays essential roles indispensable for cellular activity and survival, including functions such as protein synthesis, secretory and membrane protein folding, and Ca²⁺ release in cells. The ER is sensitive to stresses that can lead to the aggregation and accumulation of misfolded proteins, which eventually triggers cellular dysfunction; severe or prolonged ER stress eventually induces apoptosis. ER stress-induced apoptosis causes several devastating diseases such as atherosclerosis, neurodegenerative diseases, and diabetes. In addition, the production of biopharmaceuticals such as monoclonal antibodies requires the maintenance of normal ER functions to achieve and maintain the production of high-quality products in good quantities. Therefore, it is necessary to develop methods to efficiently relieve ER stress and protect cells from ER stress-induced apoptosis. The silkworm storage protein 1 (SP1) has anti-apoptotic activities that inhibit the intrinsic mitochondrial apoptotic pathway. However, the role of SP1 in controlling ER stress and ER stress-induced apoptosis has not been investigated. In this paper, we demonstrate that SP1 can inhibit apoptosis induced by a well-known ER stress inducer, thapsigargin, by alleviating the decrease in cell viability and mitochondrial membrane potential. Interestingly, SP1 significantly blocked increases in CHOP and GRP78 expression as well as ER Ca2+ leakage into the cytosol following ER stress induction. This indicates that SP1 protects cells from ER stressinduced apoptosis by functioning as an upstream inhibitor of apoptosis. Therefore, studying SP1 function can offer new insights into protecting cells against ER stress-induced apoptosis for future applications in the biopharmaceutical and medicine industries.