Young-Ho Kim

Expression and Purification of Enzymatically Active Forms of the Human Lysyl Oxidase-like Protein 4

The lysyl oxidase-like protein 4 (LOXL4) is the latest member of the emerging family of lysyl oxidases, several of which were shown to function as copper-dependent amine oxidases catalyzing lysine-derived cross-links in extracellular matrix proteins. LOXL4 contains four scavenger receptor cysteine-rich domains in addition to the characteristic domains of the LOX family, including the copper-binding domain, the cytokine receptor-like domain, and the residues of the lysyl-tyrosyl quinone cofactor. In an effort to assess its amine oxidase activity, we expressed LOXL4 as recombinant forms attached with hexa-histidine residues at the carboxyl terminus by using an Escherichia coli expression system. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis. The purified LOXL4 proteins showed β-aminopropionitrile-inhibitable activity of 0.022-0.032 units/mg toward a nonpeptidyl substrate, benzylamine. These results indicate that LOXL4, with the four scavenger receptor cysteine rich domains, may also function as an active amine oxidase. Availability of the pure and active forms of LOXL4 will be significantly helpful in functional studies related to substrate specificity and crystal structure of this amine oxidase, which should provide significant insights into functional differences within the LOX family members.

Neutrophil lactoferrin upregulates the human p53 gene through induction of NF-κB activation cascade

Neutrophil lactoferrin (Lf) was previously shown to act as a transcriptional activator in various mammalian cells. Here, we describe that Lf specifically transactivates the p53 tumor suppressor gene through the activation of nuclear factor-κB (NF-κB) and consequently regulates p53-responsive oncogenes. In HeLa cervical carcinoma cells stably expressing Lf (HeLa-Lf), expression of mdm2 and p21waf1/cip1 as well as p53 was greatly enhanced. Transient expression of Lf also markedly transactivates transcription of a p53 promoter-driven reporter and NF-κB-driven reporters in various mammalian cells. However, mutation of the NF-κB site or treatment with an NF-κB inhibitor abrogated the transactivation, suggesting that NF-κB should play an essential role in the Lf-induced transactivation. Increased binding activity and nuclear translocation of p65 in response to Lf strongly support these findings. Furthermore, Lf-mediated NF-κB activation is diminished in IKKα- or IKKβ-deficient mouse embryonic fibroblast cells. The activation of both IKKs and NF-κB by Lf is over-ridden by the expression of dominant-negative mutants of NIK, MEKK1, IKKα and IKKβ. Collectively, we conclude that overexpressed Lf directly relays signals to upstream components responsible for NF-κB activation, thereby leading to the activation of NF-κB target genes.

Purification of enzymatically active human lysyl oxidase and lysyl oxidase-like protein from Escherichia coli inclusion bodies

Lysyl oxidase (LOX) is an extracellular copper dependent enzyme catalyzing lysine-derived cross-links in extracellular matrix proteins. Recent molecular cloning has revealed the existence of a LOX family consisting of LOX and four lysyl oxidase-like proteins (LOXLs; LOXL, LOXL2, LOXL3, and LOXL4). Each member of the LOX family contains a copper-binding domain, residues for lysyl-tyrosyl quinone, and a cytokine receptor-like domain. Very recently, novel functions, such as tumor suppression, cellular senescence, and chemotaxis, have been attributed to this family of amine oxidases, but functional differences among the family members have yet to be determined. For efficient expression and purification, we cloned the cDNAs corresponding to proteolytically processed forms of LOX (LOX-p) and LOXL (LOXL-p1 and LOXL-p2) into a bacterial expression vector pET21a with six continuous histidine codons attached to the 3 of the gene. The recombinant proteins were purified with nickel-chelating affinity chromatography and converted into enzymatically active forms by stepwise dialysis in the presence of N-lauroylsarcosinate and Cu2+. The purified LOX-p, LOXL-p1, and LOXL-p2 proteins showed specific amine oxidase activity of 0.097, 0.054, and 0.150 U/mg, respectively, which was inhibited by beta-aminopropionitrile (BAPN), a specific inhibitor of LOX. Availability of these pure and active forms of LOX and LOXLs will be significantly helpful in functional studies related to substrate specificity and crystal structures of this family of amine oxidases.

Clinical Characteristics and Molecular Analysis of PIT1, PROP1,LHX3, and HESX1 in Combined Pituitary Hormone Deficiency Patients with Abnormal Pituitary MR Imaging

Background/Aims: Many genes encoding pituitary transcription factors involved in the formation of the pituitary gland are identified. Different mutations in these genes have been reported in patients with familial combined pituitary hormone deficiency (CPHD). This study was undertaken to analyze PIT1, PROP1, LHX3, and HESX1 in 12 CPHD patients with abnormal pituitary magnetic resonance imaging (MRI). Since embryonic development of the pituitary requires the coordinated expression of specific transcription factors, we postulated the presence of mutations in PIT1, PROP1, LHX3, and HESX1 genes. Methods: Anterior pituitary function was evaluated. Each gene was PCR amplified exon by exon, and subsequently sequenced. Results: In all cases, MRI examination showed abnormal pituitary gland development featuring ectopic neurohypophysis, hypoplastic anterior lobe, empty sella, and septo-optic dysplasia. Endocrinologically, all patients revealed multiple pituitary hormone deficiency including growth hormone, thyroid stimulating hormone, luteinizing hormone, follicular stimulating hormone and adrenocorticotropin. They were all sporadic cases without a positive family history. None of disease-causing specific mutations were identified in PIT1, PROP1, LHX3, and HESX1 genes of 12 sporadic CPHD patients with abnormal pituitary imaging. However, 2 novel polymorphisms were found in PROP1 gene: IVS1+3 A→G and 27 T→C (Ala9Ala) in exon 1. Their allele frequencies in patients and normal controls were not statistically different. Overall, allele frequencies of these polymorphisms were as follows: for the IVS1+3 A→G polymorphism, the allele frequency of A was 54%, and 46% for G, with 58% of an A/G heterozygosity. For the 27 T→C (Ala9Ala) polymorphism, the allele frequency of T was 46%, and 54% for G, with 42% of a T/C heterozygosity. Conclusions: Mutations of PIT1, PROP1, LHX3, and HESX1 genes are very rare in sporadic CPHD patients with abnormal pituitary MRI.

Myosin heavy chain composition of rat middle ear muscles.

Objective To quantitatively analyze myosin heavy chain (MHC) mRNA composition in two rat middle ear muscles (the tensor tympani and stapedius) using competitive polymerase chain reaction (PCR). Material and Methods An exogenous template, including oligonucleotide sequences specific for the seven rat MHCs (2A, 2B, 2X, 2L/EOM, embryonic, neonatal and β-cardiac) as well as β-actin, was constructed and used as the competitor. Results The tensor tympani and stapedius contained all MHC isoforms except 2L. The tensor tympani contained approximately equal proportions of 2X (40.4%±6.5%) and 2A (34.0%±1.3%) MHCs, with a smaller percentage of 2B (16.6%±1.5%) and neonatal (7.5%±0.6%) MHCs, while β-cardiac and embryonic MHCs were minimally expressed. The stapedius contained predominantly 2X (58.0%±4.2%) and 2A (32.3%±6.7%) MHCs, with a smaller percentage of 2B (7.4%±0.2%) and β-cardiac (1.9%±0.1%) MHCs. Neonatal and embryonic MHCs were detected at very low levels. Conclusion These results suggest that two middle ear muscles, which are mainly composed of two fast-twitching myosins (2X and 2A MHCs), contract fast and are fatigue-resistant.

Clinical phenotypes of bronchial hyperresponsiveness in school-aged children

BACKGROUNDnBronchial hyperresponsiveness (BHR), one of the key features of asthma, has a diverse natural course in school-aged children, but studies on BHR phenotypes are lacking.nnnOBJECTIVEnTo classify BHR phenotypes according to onset age and persistence in children and investigate the characteristics and factors associated with each phenotype in a longitudinal study.nnnMETHODSnThis study analyzed 1,305 elementary school children from the Childrens Health and Environmental Research (CHEER) study, a 4-year, prospective, follow-up study with 2-year intervals starting at a mean age of 7years. Total serum IgE levels and blood eosinophil counts were measured, and allergy workup, including methacholine challenge tests with the International Study of Asthma and Allergies in Childhood questionnaire, was performed at each survey.nnnRESULTSnThe 4 BHR phenotypes were classified as non-BHR (nu202f=u202f942 [72.2%]), early-onset transient BHR (nu202f=u202f201 [15.4%]), late-onset BHR (nu202f=u202f87 [6.7%]), and early-onset persistent BHR (nu202f=u202f75 [5.7%]). Early-onset persistent BHR is characterized by an increased eosinophil count, total serum IgE level, sensitization rate, decreased lung function, and increased risk of newly diagnosed asthma during follow-up (adjusted odds ratio, 3.89; 95% confidence interval, 1.70-8.88). The 2 early-onset phenotypes were associated with peripheral airway dysfunction. The late-onset BHR phenotype was related to increased risks of allergic rhinitis symptoms at baseline and later sensitization against inhalant allergens.nnnCONCLUSIONnThe early-onset persistent BHR phenotype in school-aged children is associated with high atopic burden and increased risk of newly diagnosed asthma, whereas the late-onset BHR phenotype related with later sensitization and allergic rhinitis symptoms. Diverse BHR phenotypes in children have specific characteristics that require targeted follow-ups.

Prenatal 25-hydroxyvitamin D deficiency affects development of atopic dermatitis via DNA methylation

Due to the hypomethylation and consequently increased expression of oxidative stress-promoting genes in severely vitamin D-deficient fetus, the resulting offspring is predisposed to subsequent AD development and its severity.

Prevalence, Risk Factors and Cutoff Values for Bronchial Hyperresponsiveness to Provocholine in 7-Year-Old Children

Background A US Food and Drug Administration (FDA)-approved drug methacholine chloride (Provocholine®) was recently introduced to Korea where it is now widely used in clinical practice. We aimed to evaluate the prevalence, risk factors and cutoff value of bronchial hyperresponsiveness (BHR) to Provocholine in 7-year-old children. Methods Six hundred and thirty-three children from the Panel Study on Korean Children who visited 16 regional hospitals were evaluated. Skin prick tests, spirometry and bronchial provocation tests for Provocholine as well as a detailed history and physical examinations were performed. The bronchial provocation test was reliably performed on 559 of these children. Results The prevalence of ever-diagnosed asthma via medical records was 7.7%, and that of current asthma (wheezy episode in the last 12 months + diagnosed asthma by physicians) was 3.2%. The prevalence of BHR to Provocholine was 17.2% and 25.8%, respectively, for a PC20 < 8 and < 16 mg/mL. The risk factors for BHR (PC20 < 16 mg/mL) were atopic dermatitis diagnosis and current dog ownership, whereas those for current asthma were allergy rhinitis diagnosis, a history of bronchiolitis before the age of 3, recent use of analgesics/antipyretics and maternal history of asthma. The BHR prevalence trend showed an increase along with the increased immunoglobulin E (IgE) quartile. The cutoff value of PC20 for the diagnosis of current asthma in children at age 7 was 5.8 mg/mL (sensitivity: 47.1%, specificity: 87.4%). Conclusions BHR to Provocholine (PC20 < 8 mg/mL) was observed in 17.2% of 7-year-olds children from the general population and the cutoff value of PC20 for the diagnosis of current asthma was 5.8 mg/mL in this age group. The risk factors for BHR and current asthma showed discrepancies suggesting different underlying mechanisms. Bronchial provocation testing with Provocholine will be a useful clinical tool in the future.

A Study of Cosmetic Sustainability Evaluation of Powder Base Make-up Products

파우더 베이스 메이크업 제품은 피부에 도포되어 8 ~ 12시간 동안 부착되며 피부에서 분비되는 땀이나 피지 등의 분비물에 의해 화장막이 변화된다. 따라서 메이크업 화장의 지속성은 중요한 품질 요소 중 하나이며 평가도 중요하다. 본 연구는 파우더 제품에서 지속성 평가를 위해 여러 가지 방법으로 파우더 반제품의 흡...

Corticotropin-Releasing Factor Down-Regulates Hair Growth-Related Cytokines in Cultured Human Dermal Papilla Cells

Corticotropin-releasing factor (CRF) is involved in the stress response and there is increasing evidence that stress influences skin disease such as hair loss. In cultured human hair follicles, CRF inhibits hair shaft elongation, induces premature regression and promotes the apoptosis of hair matrix keratinocytes. We investigated whether CRF influences the dermal papilla cells (DPC) that play pivotal roles in hair growth and cycling. Human DPCs were treated with CRF, adrenocorticotropic hormone (ACTH) and cortisol, key stress hormones along the hypothalamic-pituitary -adrenal (HPA) axis for 1-24 h. Interestingly, CRF modulated the expression of cytokines related to hair growth (KGF, Wnt5a, TGFβ-2, Nexin) and increased cAMP production in cultured DPCs. CRF receptors were down-regulated by negative feedback systems. Pretreatment of CRF receptor antagonists or protein kinase A (PKA) inhibitor prevented the CRF-induced modulation. Since the CRF induces proopiomelanocortin (POMC) expression through the cAMP/PKA pathway, we analyzed POMC mRNA. CRF stimulated POMC expression in cultured human DPCs, yet we were unable to detect ACTH levels by western blot. These results indicate that CRF operates within DPCs through CRF receptors along the classical CRF signaling pathway and CRF receptor antagonists could serve as potential therapeutic and cosmetic agents for stress-induced hair loss.