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1.  Prevention of Both Neutrophil and Monocyte Recruitment Promotes Recovery after Spinal Cord Injury 
Journal of Neurotrauma  2011;28(9):1893-1907.
Abstract
Strategies that block infiltration of leukocytes into the injured spinal cord improve sparing of white matter and neurological recovery. In this article, we examine the dependency of recovery on hematogenous depletion of neutrophils and monocytes. Mice were depleted of neutrophils or monocytes by systemic administration of anti-Ly6G or clodronate-liposomes. A third group was depleted of both subsets. Neurological improvement, based on a battery of tests of performance, and white matter sparing, occurred only in animals depleted of both neutrophils and monocytes. We also attempted to define the nature of the environment that was favorable to recovery. Hemeoxygenase-1 and malondialdehyde, markers of oxidative stress and lipid peroxidation, respectively, were reduced to similar levels in animals depleted of both neutrophils and monocytes, or only monocytes, but remained elevated in the group only depleted of neutrophils. Matrix metalloproteinase-9, a protease involved in early damage, was most strongly reduced in animals depleted of both leukocyte subsets. Finally, disruption of the blood–spinal cord barrier and abnormal nonheme iron accumulation were reduced only in animals depleted of both neutrophils and monocytes. Together, these findings indicate cooperation between neutrophils and monocytes in mediating early pathogenesis in the contused spinal cord and defining long-term neurological recovery.
doi:10.1089/neu.2011.1860
PMCID: PMC3172879  PMID: 21657851
functional recovery; monocytes; mouse; neutrophils; spinal cord injury
2.  Knock-in of Enhanced Green Fluorescent Protein or/and Human Fibroblast Growth Factor 2 Gene into β-Casein Gene Locus in the Porcine Fibroblasts to Produce Therapeutic Protein 
Transgenic animals have become important tools for the production of therapeutic proteins in the domestic animal. Production efficiencies of transgenic animals by conventional methods as microinjection and retrovirus vector methods are low, and the foreign gene expression levels are also low because of their random integration in the host genome. In this study, we investigated the homologous recombination on the porcine β-casein gene locus using a knock-in vector for the β-casein gene locus. We developed the knock-in vector on the porcine β-casein gene locus and isolated knock-in fibroblast for nuclear transfer. The knock-in vector consisted of the neomycin resistance gene (neo) as a positive selectable marker gene, diphtheria toxin-A gene as negative selection marker, and 5′ arm and 3′ arm from the porcine β-casein gene. The secretion of enhanced green fluorescent protein (EGFP) was more easily detected in the cell culture media than it was by western blot analysis of cell extract of the HC11 mouse mammary epithelial cells transfected with EGFP knock-in vector. These results indicated that a knock-in system using β-casein gene induced high expression of transgene by the gene regulatory sequence of endogenous β-casein gene. These fibroblasts may be used to produce transgenic pigs for the production of therapeutic proteins via the mammary glands.
doi:10.5713/ajas.2014.14222
PMCID: PMC4213711  PMID: 25358326
Knock-in; Homologous Recombination; Therapeutic Proteins; Porcine β-casein Gene
3.  Isolated Cerebellar Variant of Adrenoleukodystrophy with a de novo Adenosine Triphosphate-Binding Cassette D1 (ABCD1) Gene Mutation 
Yonsei Medical Journal  2014;55(4):1157-1160.
X-linked adrenoleukodystrophy (X-ALD) shows a wide range of phenotypic expression, but clinical presentation as an isolated lesion of the cerebellar white matter and dentate nuclei has not been reported. We report an unusual presentation of X-ALD only with an isolated lesion of the cerebellar white matter and dentate nuclei. The proband, a 37-year-old man presented with bladder incontinence, slurred speech, dysmetria in all limbs, difficulties in balancing, and gait ataxia. Brain magnetic resonance imaging showed an isolated signal change of white matter around the dentate nucleus in cerebellum. With high level of very long chain fatty acid, gene study showed a de novo mutation in exon 1 at nucleotide position c.277_296dup20 (p.Ala100Cysfs*10) of the adenosine triphosphate-binding cassette D1 gene. It is advised to consider X-ALD as a differential diagnosis in patients with isolated cerebellar degeneration symptoms.
doi:10.3349/ymj.2014.55.4.1157
PMCID: PMC4075381  PMID: 24954351
X-linked adrenoleukodystrophy; cerebellar; ABCD1; very long chain fatty acid
4.  miR-106b impairs cholesterol efflux and increases Aβ levels by repressing ABCA1 expression 
Experimental Neurology  2011;235(2):476-483.
ATP-binding cassette transporter A1 (ABCA1) is a cholesterol transporter that transfers excess cellular cholesterol onto lipid-poor apolipoproteins. Given its critical role in cholesterol homeostasis, ABCA1 has been studied as a therapeutic target for Alzheimer’s disease. Transcriptional regulation of ABCA1 by liver X receptor has been well characterized. However, whether ABCA1 expression is regulated at the posttranscriptional level is largely unknown. Identification of a novel pathway that regulates ABCA1 expression may provide new strategy for regulating cholesterol metabolism and amyloid β (Aβ) levels. Since ABCA1 has an unusually long 3′ untranslated region, we investigated whether microRNAs could regulate ABCA1 expression. We identified miR-106b as a novel regulator of ABCA1 expression and Aβ metabolism. miR-106b significantly decreased ABCA1 levels and impaired cellular cholesterol efflux in neuronal cells. Furthermore, miR-106b dramatically increased levels of secreted Aβ by increasing Aβ production and preventing Aβ clearance. Alterations in Aβ production and clearance were rescued by expression of miR-106b-resistant ABCA1. Taken together, our data suggest that miR-106b affects Aβ metabolism by suppressing ABCA1 expression.
doi:10.1016/j.expneurol.2011.11.010
PMCID: PMC3328628  PMID: 22119192
microRNA; miR-106b; ABCA1; Cholesterol; Amyloid β; Aβ; Alzheimer’s disease; Liver X receptor; LXR; Lipid
5.  Determination of Odor Release in Hydrocolloid Model Systems Containing Original or Carboxylated Cellulose at Different pH Values Using Static Headspace Gas Chromatographic (SHS-GC) Analysis 
Sensors (Basel, Switzerland)  2013;13(3):2818-2829.
Static headspace gas chromatographic (SHS-GC) analysis was performed to determine the release of 13 odorants in hydrocolloid model systems containing original or regio-selectively carboxylated cellulose at different pH values. The release of most odor compounds was decreased in the hydrocolloid solutions compared to control, with the amounts of 2-propanol, 3-methyl-1-butanol, and 2,3-butanedione released into the headspace being less than those of any other odor compound in the hydrocolloid model systems. However, there was no considerable difference between original cellulose-containing and carboxylated-cellulose containing systems in the release of most compounds, except for relatively long-chain esters such as ethyl caprylate and ethyl nonanoate. The release from the original and carboxylated cellulose solutions controlled to pH 10 was significantly higher than that from solutions adjusted to pH 4 and 7 in the case of some esters (ethyl acetate, methyl propionate, ethyl propionate, ethyl butyrate, butyl propionate, ethyl caproate) and alcohols (2-propanol, 3-methyl-1-butanol), in particular, ethyl butyrate and 3-methyl-1-butanol. In contrast, the release of 2,3-butanedione from both the original and carboxylated cellulose solutions was increased at pH 4 and 7 compared to that at pH 10 by about 70% and 130%, respectively. Our study demonstrated that the release of some odorants could be changed significantly by addition of both original and carboxylated cellulose in hydrocolloid model systems, but only minor effect was observed in pH of the solution.
doi:10.3390/s130302818
PMCID: PMC3658716  PMID: 23447013
cellulose; regio-selectively carboxylation; release; hydrocolloid; static headspace gas chromatographic (SHS-GC) analysis
6.  Porcine Knock-in Fibroblasts Expressing hDAF on α-1,3-Galactosyltransferase (GGTA1) Gene Locus 
The Galactose-α1,3-galactose (α1,3Gal) epitope is responsible for hyperacute rejection in pig-to-human xenotransplantation. Human decay-accelerating factor (hDAF) is a cell surface regulatory protein that serves as a complement inhibitor to protect self cells from complement attack. The generation of α1,3-galactosyltransferase (GGTA1) knock-out pigs expressing DAF is a necessary step for their use as organ donors for humans. In this study, we established GGTA1 knock-out cell lines expressing DAF from pig ear fibroblasts for somatic cell nuclear transfer. hDAF expression was detected in hDAF knock-in heterozygous cells, but not in normal pig cells. Expression of the GGTA1 gene was lower in the knock-in heterozygous cell line compared to the normal pig cell. Knock-in heterozygous cells afforded more effective protection against cytotoxicity with human serum than with GGTA1 knock-out heterozygous and control cells. These cell lines may be used in the production of GGTA1 knock-out and DAF expression pigs for xenotransplantation.
doi:10.5713/ajas.2012.12146
PMCID: PMC4093019  PMID: 25049505
Gene Targeting; Pig Fibroblasts; Xenotransplantation; Knock-out; Hyperacute Rejection)
7.  18F-FDG PET/CT Findings in a Breast Cancer Patient with Concomitant Tuberculous Axillary Lymphadenitis 
Although 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) is a sensitive modality for detecting a malignant lesion, increased 18F-FDG uptake is also seen in infected or inflammatory processes. Here, we report the case of a breast cancer patient with concomitant tuberculous axillary lymphadenitis that showed increased 18F-FDG uptake. A 39-year-old woman underwent preoperative 18F-FDG PET/computed tomography (CT) as a part of the work-up for right breast cancer. 18F-FDG PET/CT images showed a malignant lesion in the right breast with moderate 18F-FDG uptake, and multiple enlarged right axillary lymph nodes with intense 18F-FDG uptake. Subsequently, the patient underwent right mastectomy and right axillary lymph node dissection. Histopathological examination confirmed breast cancer and tuberculous lymphadenitis, and the patient was treated concomitantly with anti-tuberculous therapy.
doi:10.1007/s13139-011-0080-1
PMCID: PMC4043025  PMID: 24899996
Breast cancer; Tuberculosis; F-18 fluorodeoxyglucose; Positron emission tomography
8.  Matrix metalloproteinase-9 and stromal cell-derived factor-1 act synergistically to support migration of blood borne monocytes into the injured spinal cord 
The Journal of Neuroscience  2011;31(44):15894-15903.
The infiltration of monocytes into the lesioned site is a key event in the inflammatory response after spinal cord injury (SCI). We hypothesized that the molecular events governing the infiltration of monocytes into the injured cord involve cooperativity between the upregulation of the chemoattractant stromal cell-derived factor-1 (SDF-1)/CXCL12 in the injured cord and matrix metalloproteinase-9 (MMP-9/gelatinase B), expressed by infiltrating monocytes. SDF-1 and its receptor CXCR4 mRNAs were upregulated in the injured cord, while macrophages immmunoexpressed CXCR4. When mice, transplanted with bone marrow cells from green fluorescent protein (GFP) transgenic mice, were subjected to SCI, GFP+ monocytes infiltrated the cord and displayed gelatinolytic activity. In vitro studies confirmed that SDF-1α, acting through CXCR4, expressed on bone marrow-derived macrophages, upregulated MMP-9 and stimulated MMP-9 dependent transmigration across endothelial cell monolayers by 2.6 fold. There was a reduction in F4/80+ macrophages in spinal cord injured MMP-9 knockout mice (by 36%) or wild-type mice, treated with the broad-spectrum MMP inhibitor GM6001 (by 30%). Mice were adoptively transferred with myeloid cells and treated with the MMP-9/-2 inhibitor SB-3CT, the CXCR4 antagonist AMD3100, or a combination of both drugs. While either drug resulted in a 28–30% reduction of infiltrated myeloid cells, the combined treatment resulted in a 45% reduction, suggesting that SDF-1 and MMP-9 function independently to promote the trafficking of myeloid cells into the injured cord. Collectively, these observations suggest a synergistic partnership between MMP-9 and SDF-1 in facilitating transmigration of monocytes into the injured spinal cord.
doi:10.1523/JNEUROSCI.3943-11.2011
PMCID: PMC3265932  PMID: 22049432
9.  18F-FDG PET Demonstration of Cancer Recurrence Presenting as Dermatomyositis in a Rare Case of Primary Pleural Lymphoma 
Dermatomyositis (DM) or polymyositis (PM) are possibly considered to have an association with malignancies. We describe a case of dermatomyositis in which 18F-fluorodeoxy glucose (FDG)positron emission tomography (PET) was able to detect cancer recurrence earlier than any other modality in a patient with a history of primary pleural lymphoma, a very rare condition of malignancy. Further, a typical finding of dermatomyositis is diffuse hypermetabolism in the bilateral proximal shoulder and pelvic girdle areas was shown on 18F-FDG PET, which can implicate the inflammatory process in the skeletal muscle in dermatomyosistis. This case well illustrates the characteristic 18F-FDG findings of dermatomyositis as well as a capability of 18F-FDG PET in detection of recurrence of lymphoma, even in a rare condition.
doi:10.1007/s13139-010-0065-5
PMCID: PMC4042953  PMID: 24899983
Dermatomyositis; F-18-fluoro-deoxy glucose; Positiron emission tomography; Primary pleural lymphoma
10.  Cloning and Molecular Characterization of Porcine β-casein Gene (CNS2) 
The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Milk is presently the most mature system for production of therapeutic proteins from a transgenic animal. Specifically, β-casein is a major component of cow, goat and sheep milk, and its promoter has been used to regulate the expression of transgenic genes in the mammary gland of transgenic animals. Here, we cloned the porcine β-casein gene and analyzed the transcriptional activity of the promoter and intron 1 region of the porcine β-casein gene. Sequence inspection of the 5′-flanking region revealed potential DNA elements including SRY, CdxA, AML-a, GATA-3, GATA-1 and C/EBP β. In addition, the first intron of the porcine β-casein gene contained the transcriptional enhancers Oct-1, SRY, YY1, C/EBP β, and AP-1, as well as the retroviral TATA box. We estimated the transcriptional activity for the 5′-proximal region with or without intron 1 of the porcine β-casein gene in HC11 cells stimulated with lactogenic hormones. High transcriptional activity was obtained for the 5′-proximal region with intron 1 of the porcine β-casein gene. The β-casein gene containing the mutant TATA box (CATAAAA) was also cloned from another individual pig. Promoter activity of the luciferase vector containing the mutant TATA box was weaker than the same vector containing the normal TATA box. Taken together, these findings suggest that the transcription of porcine β-casein gene is regulated by lactogenic hormone via intron 1 and promoter containing a mutant TATA box (CATAAAA) has poor porcine β-casein gene activity.
doi:10.5713/ajas.2011.11240
PMCID: PMC4092958  PMID: 25049581
Lactogenic Hormones; HC11 Cells; TATA Box; Milk Protein; Porcine Casein Gene
11.  Ovarian Mass Mimicking Malignancy: A Case Report 
A 32-year-old female who suffered from abdominal pain underwent 18F-fluorodexoyglucose (FDG) positron emission tomography/computed tomography (PET/CT) for the diagnostic workup of pelvic mass lesions. Cystic mass lesions in the bilateral ovaries showed wall thickening and intense hypermetabolism along the rim. In addition, multifocal intense hypermetabolic lymphadenopathies were seen in the left paraaortic lymph node (LN), aortocaval LN, and both common iliac LNs. We interpreted these findings as bilateral ovarian cancer with retroperitoneal metastatic lymphadenopathies rather than endometriosis with reactive lymphadenopathies. However, histopathological examination confirmed the ovarian mass lesions as tubo-ovarian abscesses. We report a case that even if simultaneous hypermetabolic retroperitoneal LNs are seen, intense hypermetabolic lesions in both ovaries can be in consequence of inflammatory change.
doi:10.1007/s13139-010-0045-9
PMCID: PMC4042925  PMID: 24899966
PET/CT; Ovary; Retroperitoneal lymph node; Tubo-ovarian abscess
12.  The protective effect of early hypothermia on PTEN phosphorylation correlates with free radical inhibition in rat stroke 
We recently showed that intraischemic moderate hypothermia (30°C) reduces ischemic damage through the Akt pathway after permanent distal middle cerebral artery occlusion in rats. The only Akt pathway component preserved by hypothermia is phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (p-PTEN), which suggests that p-PTEN may have a central role in neuroprotection. Reactive oxygen species (ROS) are critically involved in mediating ischemic damage after stroke by interacting with signaling molecules, including Akt, PTEN, and δ-protein kinase C (PKC). We investigated the protective mechanisms of moderate hypothermia on these signaling proteins after transient focal ischemia in rats. Early moderate hypothermia (3 h) was administered 15 mins before reperfusion, and delayed moderate hypothermia (3 h) was applied 15 mins after reperfusion. Our results indicate that early hypothermia reduced infarction, whereas delayed hypothermia did not. However, both early and delayed hypothermia maintained levels of Mn-SOD (superoxide dismutase) and phosphorylated Akt and blocked δ-PKC cleavage, suggesting that these factors may not be critical to the protection of hypothermia. Nevertheless, early hypothermia preserved p-PTEN levels after reperfusion, whereas delayed hypothermia did not. Furthermore, ROS inhibition maintained levels of p-PTEN after stroke. Together, these findings suggest that phosphorylation levels of PTEN are closely associated with the protective effect of early hypothermia against stroke.
doi:10.1038/jcbfm.2009.81
PMCID: PMC3221613  PMID: 19553907
focal ischemia; hypothermia; neuroprotection; stroke
13.  Bone Scintigraphy Findings of A Case with Maffucci’s Syndrome 
Maffucci’s syndrome is a sporadic rare congenital disease that is characterized by enchondromatosis and soft tissue hemangiomas. A systemic evaluation should be considered because this syndrome is related to generalized mesodermal dysplasia, which has a high likelihood of a malignant transformation. Whole-body bone scintigraphy might be helpful for detecting skeletal involvement. We present a case of Maffucci’s syndrome using bone scintigraphy to evaluate the extent of the disease.
doi:10.1007/s13139-010-0022-3
PMCID: PMC4079783  PMID: 25013528
Maffucci’s syndrome; Bone scintigraphy; Enchondromatosis
14.  Monoclonal antibodies to recombinant Der p 2, a major house dust mite allergen: specificity, epitope analysis and development of two-site capture ELISA 
House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophagoides pteronyssinus, were produced. Four monoclonal antibodies produced were species-specific and did not cross-react to the D. farinae crude extract. Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides, A (aa residues 1-49), B (44-93), and C fragment (84-126). Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p 2 in house dust. The sensitivity limit was 4 ng/ml with rDer p 2 and 8 µg/ml with the D. pteronyssinus crude extract. The result suggested that the assay using monoclonal antibodies against rDer p 2 could be useful for the environmental studies and for the standardization of mite allergen extracts.
doi:10.3347/kjp.1999.37.3.163
PMCID: PMC2733146  PMID: 10507224
house dust mites; Dermatophagoides pteronyssinus; monoclonal antibodies; recombinant allergens; Der p 2; epitope
15.  Immunodiagnosis of clonorchiasis using a recombinant antigen 
A cDNA expression library of Clonorchis sinensis adult worm was constructed, and screened out immunologically. One clone, pBCs31, was selected in view of its predominant reactivity with an experimentally infected rabbit serum. Recombinant C. sinensis antigen with 28 kDa as a β-galactosidase fusion protein produced in Escherichia coli was identified by immunoblot analysis. The cloned gene was composed of 16 copies of a 30 base pair repeat and an additional 320 bases. The deduced amino acid sequence of the tandem repeat was AQPPKSGDGG. On RNA slot blot analysis. C. sinensis adult worm RNA showed a positive reaction with the cloned gene. Enzyme-linked immunosorbent assay using a purified recombinant antigen of pBCs31 showed high specificity for diagnosis of clonorchiasis.
doi:10.3347/kjp.1998.36.3.183
PMCID: PMC2732929  PMID: 9755589
Clonorchis sinensis; immunodiagnosis; recombinant antigen; repetitive sequence

Results 1-15 (15)