Suhng Wook Kim

Crystal structures of the membrane-binding C2 domain of human coagulation factor V

Rapid and controlled clot formation is achieved through sequential activation of circulating serine proteinase precursors on phosphatidylserine-rich procoagulant membranes of activated platelets and endothelial cells. The homologous complexes Xase and prothrombinase, each consisting of an active proteinase and a non-enzymatic cofactor, perform critical steps within this coagulation cascade. The activated cofactors VIIIa and Va, highly specific for their cognate proteinases, are each derived from precursors with the same A1-A2-B-A3-C1-C2 architecture. Membrane binding is mediated by the C2 domains of both cofactors. Here we report two crystal structures of the C2 domain of human factor Va. The conserved β-barrel framework provides a scaffold for three protruding loops, one of which adopts markedly different conformations in the two crystal forms. We propose a mechanism of calcium-independent, stereospecific binding of factors Va and VIIIa to phospholipid membranes, on the basis of (1) immersion of hydrophobic residues at the apices of these loops in the apolar membrane core; (2) specific interactions with phosphatidylserine head groups in the groove enclosed by these loops; and (3) favourable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups.

Crystal Structure of Pfu, the High Fidelity DNA Polymerase from Pyrococcus Furiosus.

We have determined a 2.6A resolution crystal structure of Pfu DNA polymerase, the most commonly used high fidelity PCR enzyme, from Pyrococcus furiosus. Although the structures of Pfu and KOD1 are highly similar, the structure of Pfu elucidates the electron density of the interface between the exonuclease and thumb domains, which has not been previously observed in the KOD1 structure. The interaction of these two domains is known to coordinate the proofreading and polymerization activity of DNA polymerases, especially via H147 that is present within the loop (residues 144-158) of the exonuclease domain. In our structure of Pfu, however, E148 rather than H147 is located at better position to interact with the thumb domain. In addition, the structural analysis of Pfu and KOD1 shows that both the Y-GG/A and beta-hairpin motifs of Pfu are found to differ with that of KOD1, and may explain differences in processivity. This information enables us to better understand the mechanisms of polymerization and proofreading of DNA polymerases.

High-risk human papillomavirus infection is associated with premature rupture of membranes

BackgroundHuman papillomavirus (HPV) is known to be more prevalent in spontaneous abortions than in elective terminations of pregnancy. More recently, placental infection with HPV was shown to be associated with spontaneous preterm delivery. However, no study has evaluated the prevalence of HPV infection in pregnant Korean females and its association with adverse pregnancy outcomes.MethodsWe conducted a cross-sectional study of 311 females who gave birth at Korea University Medical Center. Our sample included 45 preterm deliveries, 50 cases of premature rupture of the membranes (PROM), 21 preeclampsia cases, and 8 gestational diabetes mellitus (GDM) patients. We used the Hybrid Capture II system to detect high-risk (HR)-HPV infection at six weeks postpartum.ResultsThe prevalence of HR-HPV infection was 14.1%. Women with HR-HPV infection had a higher incidence of PROM than those without HR-HPV. HR-HPV infection was associated with an increased risk of PROM (OR, 2.380; 95% CI, 1.103-5.134). The prevalence of preterm delivery, preeclampsia, or GDM was not different between the two groups.ConclusionsWe observed a high prevalence of HR-HPV infection in pregnant women. Moreover, HR-HPV infection was associated with a risk of PROM at term. Further studies are needed to evaluate mechanisms by which HR-HPV infection induces PROM.

Effects of environmental temperature change on mercury absorption in aquatic organisms with respect to climate warming.

Because of global warming, the quantity of naturally generated mercury (Hg) will increase, subsequently methylation of Hg existing in seawater may be enhanced, and the content of metal in marine products rise which consequently results in harm to human health. Studies of the effects of temperatures on Hg absorption have not been adequate. In this study, in order to observe the effects of temperature changes on Hg absorption, inorganic Hg or methylmercury (MeHg) was added to water tanks containing loaches. Loach survival rates decreased with rising temperatures, duration, and exposure concentrations in individuals exposed to inorganic Hg and MeHg. The MeHg-treated group died sooner than the inorganic Hg-exposed group. The total Hg and MeHg content significantly increased with temperature and time in both metal-exposed groups. The MeHg-treated group had higher metal absorption rates than inorganic Hg-treated loaches. The correlation coefficients for temperature elevation and absorption were significant in both groups. The results of this study may be used as basic data for assessing in vivo hazards from environmental changes such as climate warming.

Involvement of Oxidative Stress and Mitochondrial Apoptosis in the Pathogenesis of Pelvic Organ Prolapse

PURPOSE Biomechanical weakness of the pelvic supportive structures has been proposed to be a cause of pelvic organ prolapse. However, the molecular mechanism involved in these changes is not completely understood. In this investigation we evaluated oxidative stress biomarkers in the uterosacral ligaments of women with pelvic organ prolapse and compared them with those of women with normal support. In addition, mitochondrial apoptosis was examined. MATERIALS AND METHODS Samples were collected from 26 women with advanced stage pelvic organ prolapse and 29 age matched controls. The expression levels of 8-OHdG and 4-hydroxy-2-nonenal in the uterosacral ligaments were measured using immunohistochemistry. To assess mitochondrial apoptosis we performed TUNEL assay, immunohistochemistry for cleaved caspase-3 and cytochrome c, and Western blot analyses for cleaved caspase-3 and caspase-9. RESULTS The mean percentage of cells immunopositive for 8-OHdG, 4-hydroxy-2-nonenal, TUNEL, cleaved caspase-3 and cytochrome c in the uterosacral ligaments was significantly higher in patients with pelvic organ prolapse than in controls. Similarly, Western blot analysis revealed increased expression of cleaved caspase-3 and caspase-9 in patients with pelvic organ prolapse. Correlation analyses revealed significant positive correlations between the percentage of cells immunopositive for 8-OHdG or 4-hydroxy-2-nonenal and markers of mitochondrial apoptosis. Analyzing by pelvic organ prolapse quantification system stage according to C point, the mean percentage of cells immunopositive for 8-OHdG, 4-hydroxy-2-nonenal and cytochrome c was significantly higher in patients with pelvic organ prolapse compared to controls, regardless of stage. However, the mean percentage of TUNEL and cleaved caspase-3 positive cells was significantly higher only in patients with stage III or IV pelvic organ prolapse. CONCLUSIONS Oxidative stress and increased mitochondrial apoptosis may contribute to the pathological process of pelvic organ prolapse.

Rapid ABO Genotyping Using Whole Blood without DNA Purification

BACKGROUND ABO genotyping is commonly used in cases of an ABO discrepancy between cell typing and serum typing, as well as in forensic practice for personal identification and paternity testing. We evaluated ABO genotyping via multiplex allele-specific PCR (ASPCR) amplification using whole blood samples without DNA purification. METHODS A four-reaction multiplex ASPCR genotyping assay was designed to detect specific nucleotide sequence differences between the six ABO alleles A101, A102, B101, O01, O02, and cis-AB01. The ABO genotypes of 127 randomly chosen samples were determined using the new multiplex ASPCR method. RESULTS The genotypes of the 127 samples were found to be A101/A102 (n=1), A102/A102 (n=9), A101/O01 (n=3), A102/O01 (n=12), A102/O02 (n=14), B101/B101 (n=5), B101/O01 (n=18), B101/O02 (n=15), O01/O01 (n=14), O02/O02 (n=8), O01/O02 (n=14) and A102/B101 (n=14), from which phenotypes were calculated to be A (n=39), B (n=38), O (n=36) and AB (n=14). The multiplex ASPCR assay results were compared with the serologically determined blood group phenotypes and genotypes determined by DNA sequencing, and there were no discrepancies. CONCLUSIONS This convenient multiplex ASPCR assay, performed using whole blood samples, provides a supplement to routine serological ABO typing and might also be useful in other genotyping applications.

Efficient Reprogramming of Mouse Fibroblasts to Neuronal Cells including Dopaminergic Neurons

Somatic cells were directly converted to functional neurons through the use of a combination of transcription factors, including Ascl1, Brn2, and Myt1l. However, a major limitation is the lack of a reliable source of cell-replacement therapy for neurological diseases. Here, we show that a combination of the transcription factors Ascl1 and Nurr1 (AN) and neurotrophic factors including SHH and FGF8b directly reprogrammed embryonic mouse fibroblasts to induced neuronal (iN) cells: pan-neuronal cells and dopaminergic (DA) neurons under our systematic cell culture conditions. Reprogrammed cells showed the morphological properties of neuronal cells. Additionally, cells were analyzed using various markers, including Tuj1 and Map2 for neuronal cells and Lmx1a, Th, Aadc and Vmat2 for DA neurons in our immunostaining and reverse transcription (RT)-PCR experiments. We found that a combination of transcription factors and neurotrophic factors could directly reprogram fibroblasts to neuronal cells including DA neurons. Various types of reprogrammed cells are promising cell sources for cell-based therapy of neurological disorders like Parkinsons disease and spinal cord injury.

Sublingual administration of bacteria-expressed influenza virus hemagglutinin 1 (HA1) induces protection against infection with 2009 pandemic H1N1 influenza virus.

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.

Rapid and Direct Detection of Apolipoprotein E Genotypes Using Whole Blood from Humans

Polymerase chain reaction (PCR) is a powerful molecular biological tool in the field of toxicity testing and diagnostics. The use of PCR for large-scale genetic testing requires an effective method of sample processing. Unfortunately, isolation of PCR-quality DNA is time-consuming. PCR performed directly on whole blood is preferred because of time efficiency, cost of the procedure, and possible automation for large-scale toxicity evaluation and diagnosis. The apolipoprotein E (APOE) gene contains two single-nucleotide polymorphisms (SNP) located at codons 112 and 158, producing three APOE protein isoforms known to be associated with the risks of developing cardiovascular disease and susceptibility to Alzheimers disease. In the present study, an attempt was made to use the AnyDirect solution for APOE genotyping by PCR using whole blood directly without DNA purification. Results for two PCR methods, (1) conventional PCR using purified DNA and conventional buffer and (2) direct PCR using whole blood and AnyDirect solution, were compared in four different PCR-based APOE genotyping methods including PCR restriction-fragment-length polymorphism (PCR-RFLP), allele-specific PCR, SNaPshot mini-sequencing, and multiplex tetra-primer amplification refractory mutation system (T-ARMS) PCR. There was complete concordance in the APOE genotypes between conventional PCR and direct PCR, in all four different PCR-based APOE genotyping methods. Data demonstrated that the four different PCR-based APOE genotyping methods are able to determine the APOE genotypes successfully using whole blood directly with the use of AnyDirect solution. The direct multiplex T-ARMS PCR using whole blood may be the most rapid, simple, and inexpensive method for detecting APOE genotypes among four different APOE genotyping methods.

Immunological responses induced by asd and wzy/asd mutant strains of Salmonella enterica serovar Typhimurium in BALB/c mice

Attenuated bacteria have long been developed as vaccine candidates but can have some disadvantages, such as the potential for damage to immune organs due to insufficient clearance. To minimize these disadvantages, we generated Salmonella enterica serovar Typhimurium mutants SHJ2104 (asd::cm) and HTSaYA (wzy::km, asd::cm). The wzy gene codes for the O-antigen polymerase, which is involved in lipopolysaccharide (LPS) biosynthesis, and asd codes for aspartate β-semialdehyde dehydrogenase, which participates in cell wall formation. The strains synthesized LPS with a short-chain length, and showed lower cytotoxicity and reduced intracellular proliferation in animal cells compared to wild-type bacteria. After oral infection, the mutants were cleared in immune tissues, including the Peyer’s patch, mesenteric lymph node, and spleen, within 5 days. The LD50 of the mutants in Balb/c mice was estimated to be 106 higher than wild-type bacteria when administered either via an oral or i.p. route, indicating that the two strains are highly attenuated. To compare the immune response to and protective effects of the mutants against wild-type bacterial infection, we inoculated the mutants into mice via an oral (1×1010CFU) or i.p. (1×107 CFU) route once or twice at a two week interval. All immune responses, such as serum IgG and secretory IgA levels, cytokine production, and delayed hypersensitivity, were highly induced by two rounds of immunization. HTSaYA and SHJ2104 induced similar immune responses, and mice immunized with HTSaYA or SHJ2104 via an i.p. route were protected against wild-type Salmonella infection even at 100-fold of the LD50 (5×106 CFU). Taken together, these data indicate that HTSaYA and SHJ2104 could be developed as live attenuated Salmonella vaccine candidates.