Hyoung Woo Park

Endotoxin-neutralizing antimicrobial proteins of the human placenta.

Microbial colonization and infection of placental tissues often lead to adverse pregnancy outcomes such as preterm birth, a leading cause of neonatal morbidity and mortality. The fetal membranes of the placenta, a physical and active barrier to microbial invasion, encapsulate the fetus and secure its intrauterine environment. To examine the innate defense system of the human placenta, antimicrobial peptides were isolated from the fetal membranes of human placenta and characterized biochemically. Two salt-resistant antimicrobial host proteins were purified to homogeneity using heparin-affinity and reversed-phase HPLC. Characterization of these proteins revealed that they are identical to histones H2A and H2B. Histones H2A and H2B showed dose-dependent inhibition of the endotoxin activity of LPS and inhibited this activity by binding to and therefore blocking both the core and lipid A moieties of LPS. Consistent with a role for histones in the establishment of placental innate defense, histones H2A and H2B were highly expressed in the cytoplasm of syncytiotrophoblasts and amnion cells, where the histone proteins were localized mainly to the epithelial surface. Furthermore, culturing of amnion-derived WISH cells led to the constitutive release of histone H2B, and histones H2A and H2B contribute to bactericidal activity of amniotic fluid. Our studies suggest that histones H2A and H2B may endow the epithelium of the placenta with an antimicrobial and endotoxin-neutralizing barrier against microorganisms that invade this immune-privileged site.

Timetable for Upper Eyelid Development in Staged Human Embryos and Fetuses

In this study, we examined the development of the upper eyelids to provide a basic understanding of gross anatomical structures and information relative to mechanisms of congenital anomalies in the upper eyelids. We studied the upper eyelids by external and histological observation in 48 human embryos and in fetuses from 5 to 36 weeks postfertilization. The upper eyelid fold began to develop at Stage 18. Upper and lower eyelids fused from the lateral cantus at Stage 22, and fusion was complete by 9 weeks of development. Mesenchymal condensations forming the orbital part of the orbicularis oculi (OO), tarsal plate, and the eyelashes and their appendages, were first seen at Week 9. Definite muscle structures of the upper eyelid, such as the orbital part of the OO and the levator palpebrae superioris and its aponeurosis, and the Müllers muscle were observed at 12 and 14 weeks, respectively. In addition, orbital septum, arterial arcade and orbital fat pad, and tarsal gland (TG) were apparent at 12, 14, and 18 weeks, respectively. Opening of the palpebral fissure was observed at Week 20. In addition, we defined the directional orientation between the levator aponeurosis and orbital septum and the growth pattern of the TG. Our results will be helpful in understanding the normal development of the upper eyelid and the origins of upper eyelid birth defects. Anat Rec, 2011.

The discomallear ligament and the anterior ligament of malleus: An anatomic study in human adults and fetuses

According to some reports, movement of the malleus, resulting from anterior hypertension on the discomallear ligament (DML), could produce aural symptoms related with damage to middle ear structures. The aim of this study was to examine the topographic relationship of the DML and the anterior ligament of malleus (ALM). Four fetuses and 16 adult hemi-sectioned heads were used to determine the anatomic-clinical relevance of DML and ALM in temporomandibular disorder. In fetal specimens, the DML was distinctly interposed between the malleus and the disc of the temporomandibular joint (TMJ), and the ALM had a structure apparently composed of the superior and inferior lamellae, running anteriorly in continuation with the sphenomandibular ligament (SML) through the future petrotympanic fissure (PTF). In all adult specimens, the DML was inserted into the malleus, and it expanded broadly toward the disc and capsular region of the TMJ in a triangular shape and inserted into the disc and capsule of the TMJ. The two-lamellae structure of the ALM was not distinguishable in adult specimens. The overstretched ALM resulted in movement of the malleus in five cases, but similar tension applied to the DML did not cause any movement of the malleus. This result provides an indication of the clinical significance of the ALM, a ligamentous structure continuous with the SML. It is apparent that the ALM has the potential to cause aural symptoms as a result of damage to the middle ear structure.

Early development of the nose in human embryos: a stereomicroscopic and histologic analysis.

Objectives/Hypothesis: To analyze the morphologic features of the nose in the human embryo from the 4th to 8th developmental week according to Carnegie stage.

The Third Helix of the Hoxc8 Homeodomain Peptide Enhances the Efficiency of Gene Transfer in Combination with Lipofectamine

Protein transduction domains (PTDs) have been shown to cross the biological cell membranes efficiently through a receptor and energy independent mechanism. Because of its ease in membrane transducing ability, PTDs could be used as a gene delivery vector. Since we already have shown that purified Hoxc8 homeoprotein has the ability to cross the cellular membrane, we analyzed the possibility of the third helix of the Hoxc8 homeodomain as a useful gene delivery vector. For that purpose, a 16-aa long synthetic oligopeptide Hoxc8 Protein Transduction Domain (HPTD) was chemically synthesized and then tested to see whether the HPTD could form a complex with DNA or not. Gel retardation analysis revealed that the HPTD interacts with plasmid DNA efficiently but failed to transfer the DNA into the cells. However, HPTD can enhance the efficiency of gene transfer in combination with Lipofectamine which doubled the gene transfer rate into COS-7 cells compared with the DNA/Lipofectamine control. An MTT assay indicated that the amount of HPTD used in the complex for the transfection did not show any cytotoxicty in COS-7 cells. The TEM studies showed compact particle formation in the presence of HPTD. These results indicate that the HPTD could be a good candidate adjuvant molecule to enhance the gene transfer efficiency of Lipofectamine in eukaryotic cells.

Hoxc8 represses BMP-induced expression of Smad6.

Proper regulation of bone morphogenetic protein (BMP) signaling is critical for correct patterning and morphogenesis of various tissues and organs. A well known feedback mechanism is a BMP-mediated induction of Smad6, an inhibitor of BMP signaling. Hoxc8, one of the Hox family transcription factors, has also been shown to negatively regulate BMP-mediated gene expression. Here we add another level of Hoxc8 regulation on BMP signaling. Our results show that Hoxc8, when over-expressed in C3H10T1/2 or C2C12 cells, suppressed basal Smad6 promoter activity and its mRNA expression. Activation of Smad6 transcription either by BMP2 treatment or Smad1 over-expression was also abolished by Hoxc8. When chromatin was precipitated from mouse embryos with anti-Smad1 or anti-Hoxc8 antibody, Smad6 promoter sequence was enriched, suggesting that Hoxc8 proteins make complexes with Smad1 in the Smad6 promoter region. Yet, a lack of Hox binding motifs in the Smad6 promoter sequence suggests that instead of directly binding to the DNA, Hoxc8 may regulate Smad6 expression via an indirect mechanism. Our results suggest that the Smad6-mediated negative feedback mechanism on BMP signaling may be balanced by the repression of Smad6 expression by Hoxc8.

The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells

Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 microM, and appeared to plateau above a concentration of 1 microM. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.

Membrane transducing activity of recombinant Hoxc8 protein and its possible application as a gene delivery vector.

In order to examine whether the Hoxc8 protein can deliver nucleic acid into mammalian cells, we designed several Hoxc8-derived recombinant proteins to be synthesized as glutathione S-transferase (GST) fused forms in E.coli (GST-Hoxc81-242, containing a full length of Hoxc8; GST-Hoxc8152-242, possessing a deletion of the acidic N-terminus of Hoxc8; GST-Hoxc8149-208, which contained the homeodomain only). After labeling these proteins with Oregon 488, we examined their membrane transduction ability under the fluorescence microscope and verified that all three proteins showed similar transduction efficiency. The ability of the proteins to form in vitro protein-DNA complexes was analyzed on agarose gel; both GST-Hoxc81-242 and GST-Hoxc8149-208 formed complexes. In contrast, the GST-Hoxc8152-242 protein did not form a complex. The GST-Hoxc8149-208 protein formed a complex with DNA at a mass ratio of 1 : 1 (DNA : protein), and GST-Hoxc81-242 formed a complex at a mass ratio of 1 : 5. When the DNA (pDsRed1-C1) and protein complexes were added to culture media containing mammalian cells, the cells uptook the complexes, which was indicated by red fluorescence expression under the fluorescent microscope. These results indicate that recombinant Hoxc8 derivatives that harbor a homeodomain are able to traverse the mammalian cellular membrane. DNA that is bound to the recombinant derivatives can be carried across the membrane as well. This process could be applied in the development of a useful delivery vector for gene therapy in the future.

A Great Teacher of Neurosurgery in Korea: Hun Jae Lee (1921–1983)

Hun Jae Lee was born on November 11, 1921 in Unsu-myeon, a village of Goryeong-gun, in the province of Gyeongbuk, Korea (Fig. 1). He was the eldest son of Bong Hwan Lee, a patriot who served 3 years in prison for his independence act, and Yeon Bun Ihm. He graduated from Severance Hospital Medical School in 1944 and completed his medical internship in Pyongyang Christian Hospital in 1945. Then, he was recruited to serve as professor in the Department of General Surgery at Daegu Medical University. In 1950, the Korean War began, and he joined the army as a surgeon, where he gained considerable experience in neurosurgery at the mobile army surgical hospital (Fig. 2). At that time, there was no neurosurgical institute in Korea. His experience in the Korean War helped lead him to become a neurosurgeon. In 1955, he went to the United States in order to learn neurosurgery. He underwent resident training at the University of Michigan Medical School from 1955 to 1957.1,2 He obtained the American Board of Neurological Surgery in 1958. Professor Edgar Kahn regarded him as excellent, and asked him to write a chapter on metastatic brain tumors in his book Correlative Neurosurgery.3 Although Kahn attempted to persuade him to stay in the United States, he returned home to develop neurosurgery in Korea. After his return in 1959, he practiced at Soo Do Medical College, which is now the Korea University College of Medicine. Then, he moved to Yonsei University in 1966 as Chairman of the Department of Neurosurgery. From that time, he dedicated 17 years to building a foundation for neurosurgery in Yonsei University, until he passed away in 1981. Over those 17 years, he performed stereotactic thalamotomy in a patient with Parkinsons disease using a Cooper stereotactic frame.4 He reported the follow-up results of chemothalamotomy for seven cases of Parkinsons disease and four cases of dystonia.4,5 He administered open cordotomy for cancer pain, retrogasserian rhizotomy, and percutaneous alcohol injection for trigeminal neuralgia.6,7,8,9 For the treatment of hydrocephalus, he performed an experiment on ventriculo-atrial shunt, and he applied it to a patient using a Holter valve.10 In addition, he presented on hypertensive intracerebral hemorrhage, microneurosurgery, and surgical treatment of cerebral aneurysms under hypotensive anesthesia. He had great interests and passion in the surgical treatment of intracranial aneurysms. As many essential surgical apparatuses were not available in Korea in the 1960s, he even developed his own surgical clips. To do so, he remodeled a remote cable releaser of a film camera into a remote temporary clip, and asked a jeweler to make metal spring clips from his own design.11 He began microneurosurgery in 1972.11 In 1973, the neurosurgical department got their own surgical microscope. By the early 1970s, the annual number of operating cases for cerebrovascular disease had already reached up to 100, and the first micro-neurosurgical training course was organized to foster competitive Korean neurosurgeons (Fig. 3).12,13 Not only did this accelerate progress in achieving better neurosurgical outcomes, but also it had a great effect on neurosurgical training. With access to a dedicated microscope in 1974, his surgical technique became much more sophisticated, especially in the surgery for schwannomas and pituitary adenomas. He operated on numerous cases of brain tumors, including glioma, pituitary adenoma, meningioma, schwannoma, craniopharyngioma, chordoma, etc. Fig. 1 Hun Jae Lee (Nov. 11, 1921–Mar. 13, 1983). Fig. 2 The Mobile Army Surgical Hospital under the United States Army medical unit served as a fully functional hospital in combat areas. The picture was taken at Chuncheon in 1952. Fig. 3 Dr. Lee in the classroom. Dr. Lee first established a system of subspecialties in the neurological department, which remains a tremendous legacy from the great pioneer. The strict sectioning enabled each section chief to concentrate on his major field and overtake advanced neurosurgery in a short time. In the early 1970s, he allocated professors to each section to help them to concentrate on their respective subjects. As a result, each section was operated by a specialized professor, including Kyu Chang Lee in cerebrovascular surgery, Sang Sup Chung in functional neurosurgery, Young Soo Kim in spine neurosurgery, and Joong Uhn Choi in pediatric neurosurgery (Fig. 4). Fig. 4 Department of Neurosurgery. International relationships were also important to Dr. Lee. He was selected as a member of the Harvey Cushing Society in 1961. In May 1974, he was selected as an Emeritus Invited Professor in West Germany Max-Plank Brain Institute. In 1975, he was appointed as a consultant neurosurgeon of the US 8th Army and a Korean representative to the International Neurosurgery Society. In September 1977, he was appointed as an Invited Professor of Michigan University. In October of the same year, he also won a medal of US private service from the United States government (Fig. 5). He was one of 16 founding members of the Korean Neurosurgical Society, which was established in 1961.14 In 1965, he was elected as President of the society.13 In July 1979, he was appointed as an academic director of the Korean Medical Association due to recognition of his academic achievements. Also, in November 1980, he was appointed as academic director of the Korean Microsurgery Society. Fig. 5 Dr. Hun Jae Lee was appointed as a consultant neurosurgeon for the US 8th Army. In October 1977, he also won a medal of US private service from the United States government. When Professor Hun Jae Lee selected residents, he always selected one resident from other provinces outside of Seoul. His intent was to change underdeveloped areas through his alumni. The policy was effective and many Yonsei Neurosurgery alumni have taken major roles in regional hospitals. The graduates have settled all over the country, leading many parts of the Korean Neurosurgical Society.5 The first alumni meeting was held in 1973 to promote mutual friendship and enhance scientific development between alumni members, and 16 alumni members started having regular meetings. In May 1979, an alumni meeting decided the official name of the Severance Neurosurgery Alumni Association, as Sei Baek Hoe (世栢會). Sei (世) was quoted from Yonsei (延世), and Baek (栢) was taken from the pen name of Professor Hun Jae Lee, Baek Je (栢齊). At present, Sei Baek Hoe has 313 regular members. The interests of Dr. Lee extended beyond neurosurgery. He was good at Chinese calligraphy and oriental paintings. He presented his calligraphy and paintings at various retirement, marriage, and clinic opening ceremonies. Collecting stones, however, was his most favorite hobby. The collected stones have been displayed on the lawn of Yonsei University until now (Fig. 6). He passed away at 60 years of age on March 13, 1983. His body was buried in his hometown of Unsu-myeon. Regretfully, our beloved chief was unable to spend more time sharing his passion for learning and developing neuroscience in South Korea. Fig. 6 Dr. Lee in front of his stone collection on the lawn of Yonsei University.

Establishment of stable melanoma cell line expressing a novel gene, jpk, using a tetracycline-controlled gene expression system.

Jpk, originally isolated as an associating factor with the position-specific regulatory element of Hoxa-7, was found to be toxic to Escherichia coli(1) and to F9 teratocarcinoma cells (2) when transiently transfected and expressed. To investigate the possibility of tumor gene therapy using Jpk, its effect was tested in B16F10 murine melanoma cells. Because Jpk reduces the viability of B16F10 cells when transiently expressed, the Jpk gene was cloned into a tetracycline-controlled gene expression vector, pRetro-On to circumvent the lethal effect in unwanted situations. The retroviral plasmid pRetroJpk purified from the packaging cell was infected into B16F10 melanoma cells and screened in the presence of puromycin. Out of a total of 53 stable clones selected with puromycin, two clones overexpressed Jpk at more than twice the level when induced by doxycycline, a tetracycline-derivative, which implies the amount of the Jpk exhibiting the toxicity is critical. Although these clones control only low levels of Jpk, overexpression of the established melanoma cell line may help us decipher the function of Jpk and apply it as a tumor therapeutic gene in the future.