Won Hee Jang

Mitochondrial localization of DJ-1 leads to enhanced neuroprotection

Mutations in DJ‐1 (PARK7) cause recessively inherited Parkinsons disease. DJ‐1 is a multifunctional protein with antioxidant and transcription modulatory activity. Its localization in cytoplasm, mitochondria, and nucleus is recognized, but the relevance of this subcellular compartmentalization to its cytoprotective activity is not fully understood. Here we report that under basal conditions DJ‐1 is present mostly in the cytoplasm and to a lesser extent in mitochondria and nucleus of dopaminergic neuroblastoma SK‐N‐BE(2)C cells. Upon oxidant challenge, more DJ‐1 translocates to mitochondria within 3 hr and subsequently to the nucleus by 12 hr. The predominant DJ‐1 species in both mitochondria and nucleus is a dimer believed to be the functional form. Mutating cysteine 106, 53, or 46 had no impact on the translocation of DJ‐1 to mitochondria. To study the relative neuroprotective activity of DJ‐1 in mitochondria and nucleus, DJ‐1 cDNA constructs fused to the appropriate localization signal were transfected into cells. Compared with 30% protection against oxidant‐induced cell death in wild‐type DJ‐1‐transfected cells, mitochondrial targeting of DJ‐1 provided a significantly stronger (55%) cytoprotection based on lactate dehydrogenase release. Nuclear targeting of DJ‐1 preserved cells equally as well as the wild‐type protein. These observations suggest that the time frame for the translocation of DJ‐1 from the cytoplasm to mitochondria and to the nucleus following oxidative stress is quite different and that dimerized DJ‐1 in mitochondria is functional as an antioxidant not related to cysteine modification. These findings further highlight the multifaceted functions of DJ‐1 as a cytoprotector in different cellular compartments.

The -238 tumor necrosis factor-α promoter polymorphism is associated with decreased susceptibility to cancers

We investigated the potential association of tumor necrosis factor-alpha (TNF-alpha) promoter polymorphisms with cancers. The study included 169 patients with gastric cancer, uterine cervical cancer, colorectal cancer, or renal cell carcinoma and 92 healthy controls. The -308 and -238 polymorphisms in the TNF-alpha promoter were analyzed by PCR-restriction fragment length polymorphism (RFLP). The proportion of individuals carrying the TNF-238A allele was significantly lower in the cancer group than in the control group. The odds ratio for cancer in subjects with the TNF-238A allele was 0.25 (95% CI, 0.10-0.64). No association was found between the -308 polymorphism and cancers. These results suggest that the -238A allele has a protective function against cancers.

Apoptosis signal-regulating kinase 1 mediates MPTP toxicity and regulates glial activation.

Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase 3 family, is activated by oxidative stress. The death-signaling pathway mediated by ASK1 is inhibited by DJ-1, which is linked to recessively inherited Parkinsons disease (PD). Considering that DJ-1 deficiency exacerbates the toxicity of the mitochondrial complex I inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we sought to investigate the direct role and mechanism of ASK1 in MPTP-induced dopamine neuron toxicity. In the present study, we found that MPTP administration to wild-type mice activates ASK1 in the midbrain. In ASK1 null mice, MPTP-induced motor impairment was less profound, and striatal dopamine content and nigral dopamine neuron counts were relatively preserved compared to wild-type littermates. Further, microglia and astrocyte activation seen in wild-type mice challenged with MPTP was markedly attenuated in ASK1−/− mice. These data suggest that ASK1 is a key player in MPTP-induced glial activation linking oxidative stress with neuroinflammation, two well recognized pathogenetic factors in PD. These findings demonstrate that ASK1 is an important effector of MPTP-induced toxicity and suggest that inhibiting this kinase is a plausible therapeutic strategy for protecting dopamine neurons in PD.

Lipase and its modulator from Pseudomonas sp strain KFCC 10818: Proline-to-glutamine substitution at position 112 induces formation of enzymatically active lipase in the absence of the modulator

A lipase gene, lipK, and a lipase modulator gene, limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the known Pseudomonas Lifs, suggesting that Pseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonas species. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro(112) residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.

Ex vivo organ culture of adipose tissue for in situ mobilization of adipose-derived stem cells and defining the stem cell niche

In spite of the advances in the knowledge of adipose‐derived stem cells (ASCs), in situ location of ASCs and the niche component of adipose tissue (AT) remain controversial due to the lack of an appropriate culture system. Here we describe a fibrin matrix‐supported three‐dimensional (3D) organ culture system for AT which sustains the ASC niche and allows for in situ mobilization and expansion of ASCs in vitro. AT fragments were completely encapsulated within the fibrin matrix and cultured under dynamic condition. The use of organ culture of AT resulted in a robust outgrowth and proliferation in the fibrin matrix. The outgrown cells were successfully recovered from fibrin by urokinase treatment. These outgrown cells fulfilled the criteria of mesenchymal stem cells, adherence to plastic, multilineage differentiation, and cell surface molecule expression. In vitro label retaining assay revealed that newly divided cells during the culture resided in interstitium between adipocytes and capillary endothelial cells. These interstitial stromal cells proliferated and outgrew into the fibrin matrix. Both in situ mobilized and outgrown cells expressed CD146 and α‐smooth muscle actin (SMA), but no endothelial cell markers (CD31 and CD34). The structural integrity and spatial approximation of CD31−/CD34−/CD146+/SMA+ interstitial stromal cells, adipocytes, and capillary endothelial cells were well preserved during in vitro culture. Our results suggest that ASCs are natively associated with the capillary wall and more specifically, belong to a subset of pericytes. Furthermore, organ culture of AT within a fibrin matrix‐supported 3D environment can recapitulate the ASC niche in vitro. J. Cell. Physiol. 224: 807–816, 2010.

Enhanced thermal stability of an alkaline protease, AprP, isolated from a Pseudomonas sp. by mutation at an autoproteolysis site, Ser-331.

The thermal stability of the alkaline protease extracellular subtilisin‐type serine protease (AprP) from Pseudomonas sp. KFCC 10818 was improved by altering an amino acid residue at an autoproteolytic cleavage site. N‐terminal sequence analysis of the autoproteolytic products of the protein revealed the presence of two cleavage sites, Ser‐307 and Ser‐331. To increase the thermal stability of the enzyme, serine residues of these sites were replaced with aspartate. The S331D mutant enzyme was successfully purified and characterized whereas the S307D mutant was not. The half‐lives of the S331D mutant at 55 °C and 60 °C were 1.5 and 2.4 times longer than that of the wild‐type enzyme, respectively. In addition, the catalytic efficiency was also enhanced.

Interleukin-4 C-590T polymorphism is associated with protection against nasal polyps in a Korean population.

Background Although many studies have implicated interleukin (IL)-4 promoter polymorphisms as potential determinants of disease susceptibility, there are no reports on the association between IL-4 promoter polymorphisms and nasal polyps. The aim of this study was to investigate the relationship between an IL-4 promoter polymorphism and nasal polyps. Methods The C-590T promoter polymorphism of the IL-4 gene was genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis in 106 Korean chronic rhinosinusitis patients with or without nasal polyps and 70 healthy Korean subjects. Results The frequency of the T allele at position -590 of the IL-4 gene in a Korean population was 0.85, which was significantly higher than those of other ethnic groups, and the T/T allele at position -590 of IL-4 was associated with protection against nasal polyps if compared with the C/C allele (relative risk, 0.529; 95% confidence interval, 0.307–0.912; p = 0.028). Conclusion T-590, which is dominant at position -590 of the IL-4 promoter, appears to be associated with a protective mechanism against nasal polyps in Korean populations.

The isolation and in situ identification of MSCs residing in loose connective tissues using a niche-preserving organ culture system.

Mesenchymal stem cells (MSCs) have been discovered in a multitude of organs, but their distribution and identity are still uncertain. Furthermore, loose connective tissue (LCT) is dispersed throughout virtually all organs, but its biological role in tissue homeostasis is unclear. Here, we describe a unique organ culture system to explore the omnipresence and in situ identity of MSCs among the LCTs. This culture system included the use of the fibrin hydrogel coupled with dynamic culture conditions, using native LCTs obtained from various organs as starting materials. This culture allowed MSC outgrowth into the hydrogel to be robustly supported, while maintaining the structural integrity of LCTs during in vitro culture. Subcultured outgrown cells fulfilled the minimal requirements for defining MSCs on the basis of clonogenicity, multipotency, and immunophenotypic characteristics. In vitro label-retaining assay demonstrated that the numbers of mobilized and proliferated cells in situ increased in the pericapillary region and expressed both MSCs and pericytes markers, indicating that the in situ identity of MSCs represents a certain population of pericapillary pericytes. Our results indicate that this culture system affords a unique strategy for both isolating MSCs and recapitulating their niche in LCTs.

Fibrin matrix-supported three-dimensional organ culture of adipose tissue for selective outgrowth, expansion, and isolation of adipose-derived stem cells

Conventional systems for isolating adipose-derived stem cells (ASC) require enzymatic digestion of adipose tissue (AT), followed by monolayer culture to the enrich the stem cell population. However, these systems are hindered by low cell yields and a lack of reproducibility. The present study was aimed at developing a unique strategy for isolating ASC based on fibrin matrix-supported three-dimensional (3-D) organ culture of native AT. Furthermore, we tried to optimize the fibrin composition by adjusting the fibrinogen and thrombin concentrations to allow rapid outgrowth and proliferation of ASC in the 3-D fibrin matrix. Human cutaneous AT fragments were encapsulated within the fibrin matrix to construct a 3-D environment and cultured under dynamic conditions. During in vitro culture the fibrin matrix provided physical support for the AT and also allowed selective outgrowth of ASC from embedded AT fragments. In situ expanded outgrown cells were recovered from the fibrin matrix by selective fibrinolysis and propagated under monolayer culture conditions. The cultured cells fulfilled the following criteria for ASC: adhesion to culture plastic, multipotent differentiation, correct immunophenotypic profile. Fibrin matrix-supported 3-D organ culture produced ASC that with high competency in terms of growth and differentiation capabilities, and resulted in a larger and more consistent cell yield than obtained with conventional culture systems. The fibrinogen and thrombin concentrations inversely affected spreading, migration, and ASC outgrowth from native AT. Our results indicate that this 3-D organ culture system for AT can be used as an efficient and reproducible method for ASC isolation.

Expression of pro-angiogenic cytokines and their inhibition by dexamethasone in an ex vivo model of nasal polyps.

We used an organ culture of nasal polyps (NP) to provide ex vivo model to study the expression of pro-angiogenic cytokines and the effect of glucocorticoids in the pathogenesis of NP. Glucocorticoids are the drugs of choice for clinical treatment of NP; however, their mechanism of action is not fully understood. The cell-cell and cell-matrix integrity is well maintained in cultured NP. Expression of IL-6, IL-8, bFGF, GRO, and MCP-1 was increased in cultured NP compared to pre-cultured NP. Expression levels of IL-6, bFGF, and GRO in cultured NP were downregulated by dexamethasone (DEX) treatment, while MCP-1 expression was not suppressed. Further, RT-PCR and immunohistochemical analysis showed that HIF-1alpha and VEGF were suppressed in DEX-treated NP compared to untreated NP. Taken together, these results suggest that ex vivo organ culture can be considered a useful model to study the pathogenesis and regulation of pro-angiogenic cytokines in nasal polypogenesis.