A tryptophan-based assay method to search regulatory compounds for transcriptionally controlled tumor protein.

Transcriptionally controlled tumor protein (TCTP) is a highly conserved protein performing a large number of cellular functions by binding with various partner proteins. The importance of its roles in many diseases requires an assay method to find regulatory compounds. However, the molecular characteristics of TCTP made it difficult to search for chemicals interacting with it. In this study, a tryptophan-based assay method was designed and Y151W mutant TCTP was constructed to search binding chemicals. Since there is no tryptophan in the native sequence of TCTP, the incorporation of tryptophan in the Y151W mutant was very effective to establish the method. A flavonoid library was employed to the assay with the method. With the native and Y151W mutant TCTPs, three flavonoids such as morin, myricetin and isobavachalcone have been found to interact with TCTP. Combined with native gel electrophoresis, the binding region of isobavachalcone was suggested to be the flexible loop of TCTP. This approach can be easily applicable to find binding compounds of proteins with similar molecular characteristics of TCTP.

Development of disease-specific growth charts for Korean children with Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is an epigenetic overgrowth syndrome. Despite its distinctive growth pattern, the detailed growth trajectories of children with BWS remain largely unknown. We retrospectively analyzed 413 anthropometric measurements over an average of 4.4 years of follow-up in 51 children with BWS. We constructed sex-specific percentile curves for height, weight, and head circumference using a generalized additive model for location, scale, and shape. Males with BWS exhibited greater height at all ages evaluated, weight before the age of 10, and head circumference before the age of 9 than those of the general population. Females with BWS showed greater height before the age of 7, weight before the age of 4.5, and head circumference before the age of 7 than those of the general population. At the latest follow-up visit at a mean 8.4 years of age, bone age was significantly higher than chronological age. Compared to paternal uniparental disomy (pUPD), males with imprinting center region 2-loss of methylation (IC2-LOM) had higher standard deviation score (SDS) for height and weight, while females with IC2-LOM showed larger SDS for head circumference. These disease-specific growth charts can serve as valuable tools for clinical monitoring of children with BWS.

Ochratoxin A induces hepatic and renal toxicity in mice through increased oxidative stress, mitochondrial damage, and multiple cell death mechanisms.

Ochratoxin A (OTA) is a widespread food toxin produced by Aspergillus ochraceus and other molds. In this study, we developed and established acute OTA toxicity conditions in mice, which received daily oral doses of OTA between 0.5 up to 8 mg/kg body weight up to 7 days and were subjected to histological and biochemical analysis to characterize renal and hepatic damage. Oral administration of OTA for 7 days resulted in loss of body weight in a dose-dependent manner and increased the levels of serum biomarkers of hepatic and renal damage. The kidney was more sensitive to OTA-induced damage than the liver. In addition to necrosis, OTA induced hepatic and renal apoptosis in dose- and time-dependent manners. Especially, a high dose of OTA (8 mg/kg body weight) administered for 7 days led to necroptosis in both liver and kidney tissues. OTA dose-dependently increased the oxidative stress levels, including lipid peroxidation, in the liver and kidneys. OTA disrupted mitochondrial dynamics and structure in hepatic and renal cells, leading to the dysregulation of mitochondrial homeostasis. OTA increased transferrin receptor 1 and decreased glutathione peroxidase 4 levels in a dose- and time-dependent manner. These results suggest the induction of ferroptosis. Collectively, this study highlighted the characteristics of acute OTA-induced hepatic and renal toxicity in mice in terms of oxidative stress, mitochondrial damage, and multiple cell death mechanisms, including necroptosis and ferroptosis.

Whey Peptide Alleviates Muscle Atrophy by Strongly Regulating Myocyte Differentiation in Mice.

Background and Objectives: Muscle atrophy occurs when protein degradation exceeds protein synthesis, resulting in imbalanced protein homeostasis, compromised muscle contraction, and a reduction in muscle mass. The incidence of muscle atrophy is increasingly recognized as a significant worldwide public health problem. The aim of the current study was to evaluate the effect of whey peptide (WP) on muscle atrophy induced by dexamethasone (DEX) in mice. Materials and Methods: C57BL/6 mice were divided into six groups, each consisting of nine individuals. WPs were orally administered to C57BL/6 mice for 6 weeks. DEX was administered for 5-6 weeks to induce muscle atrophy (intraperitoneal injection, i.p.). Results: Microcomputer tomography (CT) analysis confirmed that WP significantly increased calf muscle volume and surface area in mice with DEX-induced muscle atrophy, as evidenced by tissue staining. Furthermore, it increased the area of muscle fibers and facilitated greater collagen deposition. Moreover, WP significantly decreased the levels of serum biomarkers associated with muscle damage, kidney function, and inflammatory cytokines. WP increased p-mTOR and p-p70S6K levels through the IGF-1/PI3K/Akt pathway, while concurrently decreasing protein catabolism via the FOXO pathway. Furthermore, the expression of proteins associated with myocyte differentiation increased noticeably. Conclusions: These results confirm that WP reduces muscle atrophy by regulating muscle protein homeostasis. Additionally, it is believed that it helps to relieve muscle atrophy by regulating the expression of myocyte differentiation factors. Therefore, we propose that WP plays a significant role in preventing and treating muscle wasting by functioning as a supplement to counteract muscle atrophy.

Dimerization Tendency of 3CLpros of Human Coronaviruses Based on the X-ray Crystal Structure of the Catalytic Domain of SARS-CoV-2 3CLpro.

3CLpro of SARS-CoV-2 is a promising target for developing anti-COVID19 agents. In order to evaluate the catalytic activity of 3CLpros according to the presence or absence of the dimerization domain, two forms had been purified and tested. Enzyme kinetic studies with a FRET method revealed that the catalytic domain alone presents enzymatic activity, despite it being approximately 8.6 times less than that in the full domain. The catalytic domain was crystallized and its X-ray crystal structure has been determined to 2.3 Å resolution. There are four protomers in the asymmetric unit. Intriguingly, they were packed as a dimer though the dimerization domain was absent. The RMSD of superimposed two catalytic domains was 0.190 for 182 Cα atoms. A part of the long hinge loop (LH-loop) from Gln189 to Asp197 was not built in the model due to its flexibility. The crystal structure indicates that the decreased proteolytic activity of the catalytic domain was due to the incomplete construction of the substrate binding part built by the LH-loop. A structural survey with other 3CLpros showed that SARS-CoV families do not have interactions between DM-loop due to the conformational difference at the last turn of helix α7 compared with others. Therefore, we can conclude that the monomeric form contains nascent enzyme activity and that its efficiency increases by dimerization. This new insight may contribute to understanding the behavior of SARS-CoV-2 3CLpro and thus be useful in developing anti-COVID-19 agents.

Importance of extracutaneous organ involvement in determining the clinical severity and prognosis of incontinentia pigmenti caused by mutations in the IKBKG gene.

Incontinentia pigmenti (IP) is a rare X-linked skin disease caused by mutations in the IKBKG gene, which is required for activation of the nuclear factor-kappa B signalling pathway. Multiple systems can be affected with highly variable phenotypic expressivity. We aimed to clarify the clinical characteristics observed in molecularly confirmed Korean IP patients. The medical records of 25 females confirmed as IP by molecular genetic analysis were retrospectively reviewed. The phenotypic score of extracutaneous manifestations was calculated to assess the disease severity. The IKBKG gene partial deletion or intragenic mutations were investigated using long-range PCR, multiplex ligation-dependent probe amplification and direct sequencing methods. Among the 25 individuals, 18 (72%) were sporadic cases. All patients showed typical skin manifestations at birth or during the neonatal period. Extracutaneous findings were noted in 17 (68%) patients; ocular manifestations (28%), neurological abnormalities (28%), hair abnormalities (20%), dental anomalies (12%), nail dystrophy (8%). The common exon 4-10 IKBKG deletion was observed in 20 (80%) patients. In addition, five intragenic sequence variants were identified, including three novel variants. The phenotype scores were highly variable, ranging from abnormal skin pigmentation only to one or more extracutaneous features, although no significant difference was observed for each clinical characteristic between the group with sequence variants and that with common large deletion. Our cohort with IP showed heterogeneity of extracutaneous manifestations and high incidence of sporadic cases. Long-term monitoring with multidisciplinary management is essential for evaluating the clinical status, providing adequate genetic counselling and understanding the genotype-phenotype correlation in IP.

Autonomic Dysfunction is Associated with Increased Cardiometabolic Risk in Patients with Childhood-Onset Craniopharyngioma.

Patients with craniopharyngioma are susceptible to autonomic dysfunction as a result of hypothalamic damage. We evaluated indices of heart rate variability (HRV) in patients with childhood-onset craniopharyngioma to investigate autonomic function and its relationship with components of the metabolic syndrome (MetS). This cross-sectional, case-only study included 53 patients (10-30 years of age). We measured the standard deviation of all normal R-R intervals (SDNN) and total power indicating overall HRV, the root-mean square of the difference of successive R-R intervals (RMSSD) and high frequency indicating parasympathetic modulation, and low frequency. These indices were compared according to the presence of the MetS. During the mean 10.8 years of follow-up, 25% of patients were diagnosed with the MetS. Patients with the MetS showed significantly lower levels of SDNN (29.0 vs. 40.6 ms), total power (416.1 vs. 1129.6 ms2), RMSSD (20.1 vs. 34.5 ms), high frequency (94.7 vs. 338.5 ms2), and low frequency (94.5 vs. 289.4 ms2) than those without (p <0.05, for all). Individual components of the MetS including insulin resistance, serum triglycerides levels, and systolic blood pressure were inversely associated with SDNN, total power, RMSSD and high frequency. Higher overall variability and parasympathetic modulation were related to decreased odds ratios for having the MetS (OR 0.91, p=0.029 for SDNN; OR 0.91, p=0.032 for total power). In conclusion, autonomic dysfunction, as evidenced by reduced HRV indices, is associated with increased cardiometabolic risk in patients with childhood-onset craniopharyngioma.

Guanosine tetra- and pentaphosphate increase antibiotic tolerance by reducing reactive oxygen species production in Vibrio cholerae.

The pathogen Vibrio cholerae is the causative agent of cholera. Emergence of antibiotic-resistant V. cholerae strains is increasing, but the underlying mechanisms remain unclear. Herein, we report that the stringent response regulator and stress alarmone guanosine tetra- and pentaphosphate ((p)ppGpp) significantly contributes to antibiotic tolerance in V. cholerae We found that N16961, a pandemic V. cholerae strain, and its isogenic (p)ppGpp-overexpressing mutant ΔrelAΔspoT are both more antibiotic-resistant than (p)ppGpp0 (ΔrelAΔrelVΔspoT) and ΔdksA mutants, which cannot produce or utilize (p)ppGpp, respectively. We also found that additional disruption of the aconitase B-encoding and tricarboxylic acid (TCA) cycle gene acnB in the (p)ppGpp0 mutant increases its antibiotic tolerance. Moreover, expression of TCA cycle genes, including acnB, was increased in (p)ppGpp0, but not in the antibiotic-resistant ΔrelAΔspoT mutant, suggesting that (p)ppGpp suppresses TCA cycle activity, thereby entailing antibiotic resistance. Importantly, when grown anaerobically or incubated with an iron chelator, the (p)ppGpp0 mutant became antibiotic-tolerant, suggesting that reactive oxygen species (ROS) are involved in antibiotic-mediated bacterial killing. Consistent with that hypothesis, tetracycline treatment markedly increased ROS production in the antibiotic-susceptible mutants. Interestingly, expression of the Fe(III) ABC transporter substrate-binding protein FbpA was increased 10-fold in (p)ppGpp0, and fbpA gene deletion restored viability of tetracycline-exposed (p)ppGpp0 cells. Of note, FbpA expression was repressed in the (p)ppGpp-accumulating mutant, resulting in a reduction of intracellular free iron, required for the ROS-generating Fenton reaction. Our results indicate that (p)ppGpp-mediated suppression of central metabolism and iron uptake reduces antibiotic-induced oxidative stress in V. cholerae.

Identification of integrin heterodimers functioning on the surface of undifferentiated porcine primed embryonic stem cells.

In vitro expansion of undifferentiated porcine primed embryonic stem (ES) cells is facilitated by use of non-cellular niches that mimic three-dimensional (3D) microenvironments enclosing an inner cell mass of porcine blastocysts. Therefore, we investigated the integrin heterodimers on the surface of undifferentiated porcine primed ES cells for the purpose of developing a non-cellular niche to support in vitro maintenance of the self-renewal ability of porcine primed ES cells. Immunocytochemistry and a fluorescence immunoassay were performed to assess integrin α and ß subunit levels, and attachment and antibody inhibition assays were used to evaluate the function of integrin heterodimers. The integrin α3 , α5 , α6 , α9 , αV , and ß1 subunits, but not the α1 , α2 , α4 , α7 , and α8 subunits, were identified on the surface of undifferentiated porcine primed ES cells. Subsequently, significant increase of their adhesion to fibronectin, tenascin C, and vitronectin were observed and functional blocking of integrin heterodimer α5 ß1 , α9 ß1 , or αV ß1 showed significantly inhibited adhesion to fibronectin, tenascin C, or vitronectin. No integrin α6 ß1 heterodimer-mediated adhesion to laminin was detected. These results demonstrate that active α5 ß1 , α9 ß1 , and αV ß1 integrin heterodimers are present on the surface of undifferentiated porcine primed ES cells, together with inactive integrin α3 (presumed) and α6 subunits.

Selenoprotein Gene Nomenclature.

The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Down-regulation of MsrB3 induces cancer cell apoptosis through reactive oxygen species production and intrinsic mitochondrial pathway activation.

Methionine sulfoxide reductase B3 (MsrB3) is a protein repair enzyme that specifically catalyzes the reduction of methionine-R-sulfoxide residues and has an antioxidant function. We have previously shown that depletion of MsrB3 suppresses the proliferation of normal mammalian cells by arresting cell cycle. In this study, we report the crucial role of MsrB3 in cancer cell death. Deficiency of MsrB3 induced cancer cell death, while MsrB3 overexpression stimulated cancer cell proliferation. MsrB3 depletion resulted in apoptotic cancer cell death through the activation of the intrinsic mitochondrial pathway. MsrB3 deficiency increased the levels of cellular reactive oxygen species (ROS) and led to redox imbalance, and also increased the Bax to Bcl-2 ratio and cytochrome c release, leading to caspase activation. Treatment of MsrB3-depleted cells with N-acetylcysteine, an ROS scavenger, prevented cell death, suggesting that MsrB3 deficiency-induced cell death is associated with increased ROS production. In addition, MsrB3 depletion activated poly(ADP ribose) polymerase-1 (PARP-1) and led to the translocation of apoptosis-inducing factor (AIF) to the nucleus. Taken together, our results suggest that MsrB3 plays an important role in cancer cell survival through the modulation of the intrinsic apoptosis pathway.

Methionine sulfoxide reductase A deficiency exacerbates acute liver injury induced by acetaminophen.

Acetaminophen (APAP) overdose induces acute liver injury via enhanced oxidative stress and glutathione (GSH) depletion. Methionine sulfoxide reductase A (MsrA) acts as a reactive oxygen species scavenger by catalyzing the cyclic reduction of methionine-S-sulfoxide. Herein, we investigated the protective role of MsrA against APAP-induced liver damage using MsrA gene-deleted mice (MsrA-/-). We found that MsrA-/- mice were more susceptible to APAP-induced acute liver injury than wild-type mice (MsrA+/+). The central lobule area of the MsrA-/- liver was more impaired with necrotic lesions. Serum alanine transaminase, aspartate transaminase, and lactate dehydrogenase levels were significantly higher in MsrA-/- than in MsrA+/+ mice after APAP challenge. Deletion of MsrA enhanced APAP-induced hepatic GSH depletion and oxidative stress, leading to increased susceptibility to APAP-induced liver injury in MsrA-deficient mice. APAP challenge increased Nrf2 activation more profoundly in MsrA-/- than in MsrA+/+ livers. Expression and nuclear accumulation of Nrf2 and its target gene expression were significantly elevated in MsrA-/- than in MsrA+/+ livers after APAP challenge. Taken together, our results demonstrate that MsrA protects the liver from APAP-induced toxicity. The data provided herein constitute the first in vivo evidence of the involvement of MsrA in hepatic function under APAP challenge.

Methionine sulfoxide reductase A protects hepatocytes against acetaminophen-induced toxicity via regulation of thioredoxin reductase 1 expression.

Thioredoxin reductase 1 (TXNRD1) is associated with susceptibility to acetaminophen (APAP)-induced liver damage. Methionine sulfoxide reductase A (MsrA) is an antioxidant and protein repair enzyme that specifically catalyzes the reduction of methionine S-sulfoxide residues. We have previously shown that MsrA deficiency exacerbates acute liver injury induced by APAP. In this study, we used primary hepatocytes to investigate the underlying mechanism of the protective effect of MsrA against APAP-induced hepatotoxicity. MsrA gene-deleted (MsrA-/-) hepatocytes showed higher susceptibility to APAP-induced cytotoxicity than wild-type (MsrA+/+) cells, consistent with our previous in vivo results. MsrA deficiency increased APAP-induced glutathione depletion and reactive oxygen species production. APAP treatment increased Nrf2 activation more profoundly in MsrA-/- than in MsrA+/+ hepatocytes. Basal TXNRD1 levels were significantly higher in MsrA-/- than in MsrA+/+ hepatocytes, while TXNRD1 depletion in both MsrA-/- and MsrA+/+ cells resulted in increased resistance to APAP-induced cytotoxicity. In addition, APAP treatment significantly increased TXNRD1 expression in MsrA-/- hepatocytes, while no significant change was observed in MsrA+/+ cells. Overexpression of MsrA reduced APAP-induced cytotoxicity and TXNRD1 expression levels in APAP-treated MsrA-/- hepatocytes. Collectively, our results suggest that MsrA protects hepatocytes from APAP-induced cytotoxicity through the modulation of TXNRD1 expression.

MsrB3 deficiency induces cancer cell apoptosis through p53-independent and ER stress-dependent pathways.

We have previously shown that down-regulation of methionine sulfoxide reductase B3 (MsrB3) induces cancer cell apoptosis through the activation of the intrinsic mitochondrial pathway. However, the mechanism through which MsrB3 deficiency results in cancer cell death is poorly understood. In this study, we investigated whether p53 and endoplasmic reticulum (ER) stress are involved in MsrB3 deficiency-induced cancer cell apoptosis using breast and colon cancer cells. MsrB3 depletion resulted in p53 down-regulation at the post-transcriptional level. MsrB3 deficiency induced cell death to a similar extent in both p53 wild-type (p53+/+) and null (p53-/-) cancer cells, suggesting that MsrB3 deficiency-induced apoptosis occurs independently of p53. MsrB3 deficiency significantly increased ER stress, which resulted in apoptosis. In addition, MsrB3 depletion activated the pro-apoptotic Bim molecule, which is essential for ER stress-induced apoptosis. MsrB3 deficiency increased cytosolic calcium levels, suggesting that MsrB3 down-regulation leads to a disturbance of calcium homeostasis in the ER, which consequently triggers ER stress. MsrB3 overexpression in MsrB3-depleted cells reduced ER stress, and was accompanied by at least partial recovery of cell viability. Taken together, our results suggest that MsrB3 plays a critical role in cancer cell apoptosis through the modulation of ER stress status.

Methionine sulfoxide reductase A protects against lipopolysaccharide-induced septic shock via negative regulation of the proinflammatory responses.

Methionine sulfoxide reductase A (MsrA) is a major antioxidant enzyme that specifically catalyzes the reduction of methionine S-sulfoxide. In this study, we used MsrA gene-knockout (MsrA-/-) mice and bone marrow-derived macrophages (BMDMs) to investigate the role of MsrA in the regulation of inflammatory responses induced by lipopolysaccharide (LPS). MsrA-/- mice were more susceptible to LPS-induced lethal shock than wild-type (MsrA+/+) mice. Serum levels of the proinflammatory cytokines IL-6 and TNF-α induced by LPS were higher in MsrA-/- than in MsrA+/+ mice. MsrA deficiency in the BMDMs also increased the LPS-induced cytotoxicity as well as TNF-α level. Basal and LPS-induced reactive oxygen species (ROS) levels were higher in MsrA-/- than in MsrA+/+ BMDMs. Phosphorylation levels of p38, JNK, and ERK were higher in MsrA-/- than in MsrA+/+ BMDMs in response to LPS, suggesting that MsrA deficiency increases MAPK activation. Furthermore, MsrA deficiency increased the expression and nuclear translocation of NF-κB and the expression of inducible nitric oxide synthase, a target gene of NF-κB, in response to LPS. Taken together, our results suggest that MsrA protects against LPS-induced septic shock, and negatively regulates proinflammatory responses via inhibition of the ROS-MAPK-NF-κB signaling pathways.

Selenoprotein MsrB1 deficiency exacerbates acetaminophen-induced hepatotoxicity via increased oxidative damage.

Acetaminophen (APAP) overdose induces acute liver damage and failure via reactive oxygen species production and glutathione (GSH) depletion. Methionine sulfoxide reductase B1 (MsrB1) is an antioxidant selenoenzyme that specifically catalyzes the reduction of methionine R-sulfoxide residues. In this study, we used MsrB1 gene-knockout mice and primary hepatocytes to investigate the effect of MsrB1 on APAP-induced hepatotoxicity. Analyses of histological alterations and serum indicators of liver damage showed that MsrB1-/- mice were more susceptible to APAP-induced acute liver injury than wild-type (MsrB1+/+) mice. Consistent with the in vivo results, primary MsrB1-/- hepatocytes displayed higher susceptibility to APAP-induced cytotoxicity than MsrB1+/+ cells. MsrB1 deficiency increased hepatic oxidative stress after APAP challenge such as hydrogen peroxide production, lipid peroxidation, and protein oxidation levels. Additionally, basal and APAP-induced ratios of reduced-to-oxidized GSH (GSH/GSSG) were significantly lower in MsrB1-/- than in MsrB1+/+ livers. Nrf2 nuclear accumulation and heme oxygenase-1 expression levels after APAP challenge were lower in MsrB1-/- than in MsrB1+/+ livers, suggesting that MsrB1 deficiency attenuates the APAP-induced activation of Nrf2. Collectively, the results of this study suggest that selenoprotein MsrB1 plays a protective role against APAP-induced hepatotoxicity via its antioxidative function.

Contributions of CAG repeat length in the androgen receptor gene and androgen profiles to premature pubarche in Korean girls.

The CAG repeat length of the androgen receptor (AR) gene, which exhibits an inverse relationship to AR sensitivity, might influence the development of the pubarche along with hyperandrogenemia. There are ethnic differences in the AR CAG repeat length, however, no Asian studies on premature pubarche (PP) have been reported, including Korea. Our objectives were to examine the hormone levels and AR CAG repeat length, and to assess their contributions to PP in Korean girls. Subjects with PP (n=16) and normal pubarche (NP, n=16), and normal controls (NC, n=16) were enrolled. The levels of dehydroepiandrosterone (DHEA), dehydroepiandrosterone-sulfate (DHEAS), 17-hydroxyprogesterone (17-OHP), and free testosterone (FT) were checked. The methylation-weighted (MW) average CAG repeat lengths were analyzed. The median ages at pubarche were 7.4 and 8.9 years in the PP and NP groups, respectively, and the levels of 17-OHP, DHEAS, and FT were similar in both groups. The PP group exhibited a higher DHEAS:DHEA ratio than the NP group (P=0.014). The medians of the MW average CAG repeat length of the AR gene were 22.4 for all subjects and did not differ among the PP (22.3), NP (22.4), and NC (22.2) groups. The AR CAG repeat lengths in the PP and NP groups did not correlate with DHEAS or FT levels. These results suggest that the AR CAG repeat length was not involved in the development of PP in Korean girls. However, excessive adrenal androgen levels, particularly those caused by increased sulfotransferase activity, might be important in the pathogenesis of PP.

The Role of Overweight and Obesity on Bone Health in Korean Adolescents with a Focus on Lean and Fat Mass.

As the associations between pediatric overweight/obesity and bone health remain controversial, we investigated the effects of overweight/obesity as well as lean mass (LM) and fat mass (FM) on bone parameters in adolescents. Bone parameters were evaluated using dual-energy X-ray absorptiometry (DXA) data of 982 adolescents (aged 12-19 years) from the Korea National Health and Nutrition Examination Survey (2009-2010). Z-scores for LM, FM, bone mass, bone mineral density (BMD), and bone mineral apparent density (BMAD) using Korean pediatric reference values were used for analysis. Adolescents with overweight/obesity had significantly higher bone mass and density of the total-body-less-head (TBLH), lumbar spine, and femur neck than underweight or normal-weight adolescents (P < 0.001) after adjusting for vitamin D deficiency, calcium intake, and insulin resistance in both sexes. LM was positively associated with bone parameters at all skeletal sites in both sexes (P < 0.001). FM was negatively related to TBLH BMD in boys (P = 0.018) but was positively associated to BMD and BMAD of the lumbar spine and femur neck in girls. In conclusion, overweight/obesity and LM play a positive role in bone health in adolescents. The effect of FM on bone parameters is sex- and site-specific.

Essential Role of the Linker Region in the Higher Catalytic Efficiency of a Bifunctional MsrA-MsrB Fusion Protein.

Many bacteria, particularly pathogens, possess methionine sulfoxide reductase A (MsrA) and B (MsrB) as a fusion form (MsrAB). However, it is not clear why they possess a fusion MsrAB form rather than the separate enzymes that exist in most organisms. In this study, we performed biochemical and kinetic analyses of MsrAB from Treponema denticola (TdMsrAB), single-domain forms (TdMsrA and TdMsrB), and catalytic Cys mutants (TdMsrAB(C11S) and TdMsrAB(C285S)). We found that the catalytic efficiency of both MsrA and MsrB increased after fusion of the domains and that the linker region (iloop) that connects TdMsrA and TdMsrB is required for the higher catalytic efficiency of TdMsrAB. We also determined the crystal structure of TdMsrAB at 2.3 Å, showing that the iloop mainly interacts with TdMsrB via hydrogen bonds. Further kinetic analysis using the iloop mutants revealed that the iloop-TdMsrB interactions are critical to MsrB and MsrA activities. We also report the structure in which an oxidized form of dithiothreitol, an in vitro reductant for MsrA and MsrB, is present in the active site of TdMsrA. Collectively, the results of this study reveal an essential role of the iloop in maintaining the higher catalytic efficiency of the MsrAB fusion enzyme and provide a better understanding of why the MsrAB enzyme exists as a fused form.