• Korean Journal of Microbiology
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• v.45 no.4
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• pp.291-299
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• 2009

To elucidate HtrA3's functional roles in the HtrA3 mediated cellular processes, it is necessary to investigate its biochemical characteristics. In the present study, we constructed the plasmids encoding putative mature HtrA3 proteins (M1-HtrA3 and M2-HtrA3) based on the putative maturation sites of highly homologous HtrA1 and mouse HtrA3. We used the pGEX bacterial expression system to develop a simple and rapid purification for the recombinant HtrA3 protein. Although yields of the mature HtrA3 proteins were slightly low as 10~50 ${\mu}g$/L, the amounts and purity of M1- and M2-HtrA3 were enough to investigate their proteolytic activities. The putative mature HtrA3 proteins have proteolytic activity which could cleave $\beta$-casein as an exogenous substrate. We compared the proteolytic activity between the HtrA family, HtrA1, HtrA2, and HtrA3. The cleavage activity of HtrA3 and HtrA2 were 2 folds higher than that of HtrA1, respectively. Our study provides a method for generating useful reagents to identify natural substrates of HtrA3 in the further studies.

• Journal of Life Science
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• v.16 no.7 s.80
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• pp.1133-1140
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• 2006

Human HtrA1 (High temperature requirement protein A1) is a homologue of the E. coli periplasmic serine protease HtrA. A recent study has demonstrated that HtrA1 is a serine protease involved in processing of insulin like growth factor binding protein (ICFBP), indicating that it serves as an important regulator of IGF activity. Additionally, several lines of evidence suggest a striking correlation between proteolytic activity of HtrA1 serine protease and the pathogenesis of several diseases; however, physiological roles of HtrA1 remain to be elucidated. We used the pGEX bacterial expression system to develop a simple and rapid method for purifying HtrA1, and the recombinant HtrA1 protein was utilized to investigate the optimal conditions in executing its proteolytic activity. The proteolytically active HtrA1 was purified to approximately 85% purity, although the yield of the recombinant HtrA1 protein was slightly low $460{\mu}g$ for 1 liter E. coli culture). Using in vitro endoproteolytic cleavage assay, we identified that the HtrA1 serine protease activity was dependent on the enzyme concentration and the incubation time and that the best reaction temperature was $42^{\circ}C$ instead of $37^{\circ}C$. We arbitrary defined one unit of proteolytic activity of the HtrA1 serine protease as 200nM of HtrA1 that cleaves half of $5{\mu}M\;of\;{\beta}-casein$ during 3 hr incubation at $37^{\circ}C$. Our study provides a method for generating useful reagents to investigate the molecular mechanisms by which HtrA1 serine protease activity contributes in regulating its physiological function and to identify natural substrates of HtrA1.

• Journal of Life Science
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• v.18 no.4
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• pp.522-529
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• 2008

High-temperature requirement A2(HtrA2) has been known as a human homologue of bacterial HtrA that has a molecular chaperone function. HtrA2 is mitochondrial serine protease that plays a significant role in regulating the apoptosis; however, the physiological function of HtrA2 still remains elusive. To establish experimental system for the investigation of new insights into the function of HtrA2 in mammalian cells, we first obtained $HtrA2^{+/+}$ and $HtrA2^{-/-}$ MEF cells lines and identified those cells based on the expression pattern and subcellular localization of HtrA2, using immunoblot and biochemical assays. Additionally, we observed that the morphological characteristics of $HtrA2^{-/-}$ MEF cells are different form those of $HtrA2^{+/+}$ MEF cells, showing a rounded shape instead of a typical fibroblast-like shape. Growth rate of $HtrA2^{-/-}$ MEF cells was also 1.4-fold higher than that of $HtrA2^{+/+}$ MEF cells at 36 hours. Furthermore, we verified both MEF cell lines induced caspsase-dependent cell death in response to apoptotic stimuli such as heat shock, staurosporine, and rotenone. The relationship between HtrA2 and heat shock-induced cell death is the first demonstration of the research field of HtrA2. Our study suggests that those MEF cell lines are suitable reagents to further investigate the molecular mechanism by which HtrA2 regulates the balance between cell death and survival.

• Journal of Life Science
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• v.16 no.5
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• pp.716-722
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• 2006

Superoxide dismutase (SOD) is physiologically important in regulating cellular homeostasis and apoptotic cell death, and its mutations are the cause of familial amyotrophic lateral sclerosis (FALS). Mitochondrial serine protease HtrA2 has a pro-apoptotic function and has known to be associated with neurodegenerative disorders. To investigate the relationship between genes associated with apoptotic cell death, such as HtrA2 and SOD1, we utilized the pGEX expression system to develop a simple and rapid method for purifying wild-type and ALS-associated mutant SOD1 proteins in a suitable form for biochemical studies. We purified SOD1 and SOD1 (G93A) proteins to approximately 90% purity with relatively high yields (3 mg per liter of culture). Consistent with the result in mammalian cells, SOD1 (G93A) was more insoluble than wild-type SOD1 in E. coli, indicating that research on the aggregate formation of SOD1 may be possible using this pGEX expression system in E. coli. We investigated the HtrA2 serine protease activity on SOD1 to assess the relationship between two proteins. Not only wild-type SOD1 but also ALS-associated mutant SOD1 (G93A) were cleaved by HtrA2, resulting in the production of the 19 kDa and 21 kDa fragments that were specific for anti-SOD1 antibody. Using protein gel electrophoresis and immunoblot assay, we compared the relative molecular masses of thrombin-cleaved GST-SOD1 and HtrA2-cleaved SOD1 fragments and can predict that the HtrA2-cleavage sites within SOD1 are the peptide bonds between leucine 9-lysine 10 (L9-K10) and glutamine 23-lysine 24 (Q23-K24). Our study indicates that SOD1 is one of the substrate for HtrA2, suggesting that both HtrA2 and SOD1 may be important for modulating the HtrA2-SOD1-mediated apopotic cell death that is associated with the pathogenesis of neurodegenerative disorder.

• Molecules and Cells
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• v.20 no.1
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• pp.83-89
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• 2005

HtrA2/Omi is a mammalian mitochondrial serine protease homologous to the E. coli HtrA/DegP gene products. Recently, HtrA2/Omi was found to have a dual role in mammalian cells, acting as an apoptosis-inducing protein and being involved in maintenance of mitochondrial homeostasis. By screening a human brain cDNA library with $A{\beta}$ peptide as bait in a yeast two-hybrid system, we identified HtrA2/Omi as a binding partner of $A{\beta}$ peptide. The interaction between $A{\beta}$ peptide and HtrA2/Omi was confirmed by an immunoblot binding assay. The possible involvement of HtrA2/Omi in $A{\beta}$ peptide metabolism was investigated. In vitro peptide cleavage assays showed that HtrA2/Omi did not directly promote the production of $A{\beta}$ peptide at the ${\beta}/{\gamma}$-secretase level, or the degradation of $A{\beta}$ peptide. However, overexpression of HtrA2/Omi in K269 cells decreased the production of $A{\beta}40$ and $A{\beta}42$ by up to 30%. These results rule out the involvement of HtrA2/Omi in the etiology of Alzheimer's disease. However, the fact that overexpression of HtrA2/Omi reduces the generation of $A{\beta}40$ and $A{\beta}42$ suggests that it may play some positive role in mammalian cells.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.22 no.1
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• pp.83-85
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• 2000

The purpose of this study is to evaluate the effect of $HTR^{TM}$ (hard tissue replacement, Bioplant Inc, U.S.A) polymer on short-term healing as a grafting material for alveolar ridge augmentation. A 48-year-old female presented insufficient bone height and width for the placement of implants. $HTR^{TM}$ polymer was used for ridge augmentation. Bone biopsy was harvested 8 months after the ridge augmentation procedure. $HTR^{TM}$ polymer displayed rapid bone regeneration and mature lamellar and trabecular bone redevelopment. Clinical and histologic observation from the treatment of the patient presented suggest that $HTR^{TM}$ polymer seems to be a appropriate material for alveolar ridge augmentation.

• Journal of the Institute of Electronics Engineers of Korea TC
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• v.41 no.9
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• pp.55-65
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• 2004

ATM was recommended by the ITU and ATM Forum as a means of transportation for B-ISDN. At this time, due to the comprehensive mature of ATM protocol, ATM has been adapted as the backbone system for carrying Internet traffi $c^{[1,2,3,4]}$. But major conceptsregarding the ATN protocol will be used on future technology. This paper presents preventive congestion control mechanisms for detecting HTR(Hard-To Reach) code in ATM systems, in particular for an improved HTR call registration method using network signaling information will discussed. In high speed circuit switching system environments, a fast HTR control mechanism is necessary. We present research results for improving HTR call registration and control methods using network signaling information and fuzzy control mechanisms. We concluded that it showed fast congestion avoidance mechanisms with a fewer system load maximized the efficiency of network resources by restricting ineffective machine attempts.

• Biomolecules & Therapeutics
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• v.28 no.1
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• pp.83-91
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• 2020

Tryptamines are monoamine alkaloids with hallucinogenic properties and are widely abused worldwide. To hasten the regulations of novel substances and predict their abuse potential, we designed and synthesized four novel synthetic tryptamine analogs: Pyrrolidino tryptamine hydrochloride (PYT HCl), Piperidino tryptamine hydrochloride (PIT HCl), N,N-dibutyl tryptamine hydrochloride (DBT HCl), and 2-Methyl tryptamine hydrochloride (2-MT HCl). Then, we evaluated their rewarding and reinforcing effects using the conditioned place preference (CPP) and self-administration (SA) paradigms. We conducted an open field test (OFT) to determine the effects of the novel compounds on locomotor activity. A head-twitch response (HTR) was also performed to characterize their hallucinogenic properties. Lastly, we examined the effects of the compounds on 5-HTR1a and 5-HTR2a in the prefrontal cortex using a quantitative real-time polymerase chain reaction (qRT-PCR) assay. None of the compounds induced CPP in mice or initiated SA in rats. PYT HCl and PIT HCl reduced the locomotor activity and elevated the 5-HTR1a mRNA levels in mice. Acute and repeated treatment with the novel tryptamines elicited HTR in mice. Furthermore, a drug challenge involving a 7-day abstinence from drug use produced higher HTR than acute and repeated treatments. Both the acute treatment and drug challenge increased the 5-HTR2a mRNA levels. Ketanserin blocked the induced HTR. Taken together, the findings suggest that PYT HCl, PIT HCl, DBT HCl, and 2-MT HCl produce hallucinogenic effects via 5-HTR2a stimulation, but may have low abuse potential.

• Journal of Photoscience
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• v.9 no.2
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• pp.314-316
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• 2002

In halobacterial membrane, pharaonis phoborhodopsin (or pharaonis sensory rhdopsin II, psRII) forms a complex with its transducer pHtrII. Flash-photolyis of D75N mutant did not yield M-intermediate but an O-like intermediate is observed. We examined the interaction between D75N of ppR and t-Htr (truncated pHtrII). These formed a complex in the presence of n-dodecyl-$\beta$-D-maltoside, and the association accelerated the decay of the 0 of D75N from 15 to 56 s$\^$-1/. From the decay time constants under varying ratios of D75N and t-Htr, n, the molar ratio of D75N/t-Htr in the complex, and K$\_$D/, the dissociation constant, were estimated. The value of n was unity and K$\_$D/ was estimated to 146 nM. This K$\_$D/ value can be considered as the association between the photo-intermediate and t-Htr, which is deduced by the method of estimation. Previously we (Photochem. Photobiol. 74, 489-494 (2001)) reported K$\_$D/ of 15 $\mu$M for the interaction between the wild-type and t-Htr by means of the change of M-decay rates. Therefore, this value should be the K$\_$D/ value for the interaction between M of the wild-type and t-Htr.

• Interdisciplinary Bio Central
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• v.4 no.3
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• pp.8.1-8.7
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• 2012

The motor neuron degeneration 2 (mnd2) mice carry a point mutation of A to T nucleotide transversion at the serine 276 residue of high temperature requirement A2 (HtrA2), resulting in losses of an AluI restriction enzyme site (5'AGCT3') and the HtrA2 serine protease activity. Moreover, dysfunctions of HtrA2 are known to be intimately associated with the pathogenesis of neurodegenerative diseases, including Parkinson's disease. Thus, this mnd2 mouse is an invaluable model for understanding the physiological role of HtrA2 and its pathological role in neurodegenerative diseases. Nevertheless, many molecular and cellular biologists in this field have limited experience in working with mutant mouse models due to the necessity of acquired years of the special techniques and knowledges. Herein, using the mnd2 mouse model as an example, we describe easy-to-use standard protocols for web-based analyses of target genes, such as HtrA2, and a novel approach for simple and accurate PCR-AluI-RFLP-based genotype analysis of mnd2 mice. In addition, band resolution of AluI-RFLP fragments was improved in 12% polyacrylamide gel running in 1X Tris-Glycine SDS buffer. Our study indicates that this PCR-AluI-RFLP genotype analysis method can be easily applied by the molecular and cellular biologist to conduct biomedical science studies using the other mutant mouse models.