Association between polymorphisms rs2228001 and rs2228000 in XPC and genetic susceptibility to preeclampsia: a case control study.

BACKGROUND: Xeroderma pigmentosum complementation group C (XPC) is a DNA damage recognition protein that plays an important role in nucleotide excision repair and can reduce oxidative stress, which may be involved in the development of preeclampsia (PE). Therefore, the aim of this study was to explore whether XPC polymorphisms were relevant to the genetic susceptibility to PE in Chinese Han women. METHOD: A total of 1276 healthy pregnant women were included as the control group and 958 pregnant women with PE as the case group. DNA was extracted from peripheral blood samples to perform genotyping of loci rs2228001 and rs2228000 in XPC through real-time quantitative polymerase chain reaction (PCR). The relationship between XPC and susceptibility to PE was evaluated by comparing the genotypic and allelic frequencies between the two groups of pregnant women. RESULTS: Polymorphism of rs2228000 may be associated with PE risk and allele T may play a protective role (genotype, χ2 = 38.961, P < 0.001 and allele χ2 = 21.746 P < 0.001, odds ratio (OR) = 0.885, 95% confidence interval (CI) = 0.840-0.932). No significant difference was found between the two groups in rs2228001,(genotype χ2 = 3.148, P = 0.207 and allele χ2 = 0.59, P = 0.442, OR = 1.017, 95% CI = 0.974-1.062). When the frequencies of genotypes and alleles for early- and late-onset PE, mild PE and severe PE were compared with those of controls, the results were consistent with the large clinical sample. CONCLUSION: Our data suggest that the genetic variant rs2228000 in XPC may be associated with PE risk in Chinese Han women, and that pregnant women with the TT genotype have a reduced risk of PE. Further investigations are needed to confirm these findings in other regions or larger prospective populations.

Role of Toll-Like Receptor 3 Gene Polymorphisms in Preeclampsia.

BACKGROUND/AIMS: Accumulating evidence suggests that an excessive maternal systemic inflammatory response to pregnancy with exaggerated activation of the innate immune system plays a critical role in the development of preeclampsia (PE). In this study, we investigated whether polymorphisms in the Toll-like receptor 3 (TLR3) gene are associated with susceptibility to PE in the Chinese Han population. METHODS: We recruited 987 PE patients and 1227 healthy pregnant women. Two polymorphisms (rs3775291 and rs3775296) located in TLR3 were genotyped by TaqMan allelic discrimination real-time PCR. The association between the genotype or allele frequencies and PE was examined using chi-square tests. Clinical data were compared between cases and controls using Student's t test. RESULTS: No significant difference was determined in the genetic distribution of rs3775291 and rs3775296 between cases and controls. There were also no significant differences in the genotype and allele frequencies of either SNP between healthy pregnant women and patients with late or early onset PE, or with mild or severe PE. CONCLUSION: Although this is the first study of the association between TLR3 polymorphisms and preeclampsia, we found that TLR3 polymorphisms are unlikely to play a significant role in the development of preeclampsia in the Chinese Han population.

Analysis of ventricular-vascular properties during preeclampsia: an echocardiography study.

The aim of the study is to analyze ventricular-vascular properties with different ventricular-arterial coupling (VAC) ratio in the preeclamptic women. Seventy-seven pregnant women with preeclampsia and eighty-nine with normal pregnancy were performed echocardiography. VAC was defined as the ratio between aortic elastance (Ea) and left ventricular (LV) end-systolic elastance (Ees). Using the VAC value of 0.8 as the cut-off near uncoupling, the preeclampsia cases were divided into two subgroups: VAC ratio ≥ 0.8 and <0.8. Cardiac structure and function, VAC properties, as well as four components of the LV pressure-strain loop, including global myocardial work index (GWI), constructive work (GCW), wasted work (GWW), and work efficiency (GWE) were determined. The preeclampsia with VAC ≥ 0.8 had an enlarger indexed ventricular volume and a thicker relative ventricular wall than the VAC < 0.8. The Ees significantly increased in the subgroup with VAC < 0.8 and decreased in the VAC ≥ 0.8, while the Ea increased in both of them. The preeclampsia with VAC ≥ 0.8 showed an obvious augmentation in GWI, GCW and GWE, along with a similar GWW compared to those with VAC < 0.8. There were variable relationships between the LV pressure-strain components and VAC properties. Thus, the preeclampsia with VAC ≥ 0.8 undergoes a more adverse remodeling and a greater impact on cardiac contractility. The increased stiffness of the heart and arterial system, and increased resistance of peripheral vessels net lead to the deteriorative ventricular efficiency with elevated myocardial oxygen consumption during a preeclampsia pregnancy.

Development of Akt-activated GSK3ß inhibitory peptide.

Abnormal overexpression of GSK3ß has been implicated in insulin resistance. Although many potent GSK3ß inhibitors have been developed as drug candidates for anti-insulin resistance, the inhibitors are prone to show side effects because they interfere with normal GSK3ß function without regulation. Recently, it was reported that the PPPSPxS motifs in the Wnt coreceptor LRP6 were able to directly inhibit GSK3ß only when the motif was phosphorylated. Here, we generated a new GSK3ß inhibitory peptide that can be activated by Akt by combining the PPPSPxS motif and an Akt target sequence. The peptide exhibited an inhibitory effect on GSK3ß only when it was phosphorylated by Akt in a purified system and in cells when stimulated by insulin. Thus, our findings provide a novel concept for drugs against diseases that are involved in the abnormal GSK3ß activity, including type 2 diabetes mellitus.

Quantification of Myocardial Work by 2-D Echocardiography during a Normal Pregnancy: A Preliminary Study.

This study was aimed at investigating the adaptive changes in myocardial work (MyW) during normal pregnancy. Sequential 2D standard and speckle-tracking echocardiography was performed on 41 pregnant women in each gestational trimester and 4 to 11 mo after delivery. Thirty-eight age-matched, healthy, non-pregnant women served as controls. Four components of MyW-global myocardial work index (GWI), constructive work (GCW), wasted work (GWW) and work efficiency (GWE)-were calculated. GWI began to decline early in the first trimester and remained at a low level until delivery; GCW gradually decreased with gestational progression and reached its lowest level in the third trimester. When compared with the values for non-pregnant women and those postpartum, GWE and GWW remained unchanged in gestation. This study provides normal ranges of MyW during pregnancy. Despite the adapted decrease in GWI and GCW, the myocardium manages to work efficiently in the healthy pregnancy with drastic hemodynamic alternations.

Evaluating the characteristics of myocardial work by two-dimensional echocardiography during pre-eclampsia pregnancy.

This study aimed to analyze the changes in myocardial work (MyW) properties and the correlation of MyW with cardiovascular and clinical indices during the pre-eclampsia (PE) pregnancy. Standard two-dimensional and speckle-tracking echocardiography were sequentially performed on 77 women with PE and 89 with normal pregnancy. Four components of MyW: global myocardial work index (GWI), constructive work (GCW), wasted work (GWW), and work efficiency (GWE) were measured. The significant increased GWI, GCW and GWW were observed, while GWW elevated more than GCW with consequently resulting the decline in GWE among PE cases. Although there was a diverse relationship between MyW components and LV morphological as well as functional indices, MyW parameters were significantly correlated with the grades of arterial hypertension and the incidence of adverse outcome of PE. With the hypertension stages, GWI, GCW and GWW gradually increased but GWE decreased. Meanwhile, the higher GWI and GCW and the lower GWE, the more adverse events occurred in PE group. In conclusion, during the PE pregnancy, GWI, GCW and GWW increase, while GWW elevates more than GCW, which leads to the decrease in GWE. Moreover, the changes in MyW are associated with the hypertension grades and the poor prognosis in PE. The non-invasive manner for MyW assessment provides a new perspective on the myocardial biomechanics, cardio-metabolic conditions and pathophysiological changes in the condition of PE.

Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB-TolC.

Macrolide-specific efflux pump MacAB-TolC has been identified in diverse gram-negative bacteria including Escherichia coli. The inner membrane transporter MacB requires the outer membrane factor TolC and the periplasmic adaptor protein MacA to form a functional tripartite complex. In this study, we used a chimeric protein containing the tip region of the TolC α-barrel to investigate the role of the TolC α-barrel tip region with regard to its interaction with MacA. The chimeric protein formed a stable complex with MacA, and the complex formation was abolished by substitution at the functionally essential residues located at the MacA α-helical tip region. Electron microscopic study delineated that this complex was made by tip-to-tip interaction between the tip regions of the α-barrels of TolC and MacA, which correlated well with the TolC and MacA complex calculated by molecular dynamics. Taken together, our results demonstrate that the MacA hexamer interacts with TolC in a tip-to-tip manner, and implies the manner by which MacA induces opening of the TolC channel.

Structural and functional characterization of a highly specific serpin in the insect innate immunity.

The Toll signaling pathway, an essential innate immune response in invertebrates, is mediated via the serine protease cascade. Once activated, the serine proteases are irreversibly inactivated by serine protease inhibitors (serpins). Recently, we identified three serpin-serine protease pairs that are directly involved in the regulation of Toll signaling cascade in a large beetle, Tenebrio molitor. Of these, the serpin SPN48 was cleaved by its target serine protease, Spätzle-processing enzyme, at a noncanonical P1 residue of the serpin's reactive center loop. To address this unique cleavage, we report the crystal structure of SPN48, revealing that SPN48 exhibits a native conformation of human antithrombin, where the reactive center loop is partially inserted into the center of the largest ß-sheet of SPN48. The crystal structure also shows that SPN48 has a putative heparin-binding site that is distinct from those of the mammalian serpins. Ensuing biochemical studies demonstrate that heparin accelerates the inhibition of Spätzle-processing enzyme by a proximity effect in targeting the SPN48. Our finding provides the molecular mechanism of how serpins tightly regulate innate immune responses in invertebrates.

Morin attenuates tau hyperphosphorylation by inhibiting GSK3ß.

Alzheimer's disease (AD) is the major form of age-related dementia and is characterized by progressive cognitive impairment, the accumulation of extracellular amyloid ß-peptide (Aß), and intracellular hyperphosphorylated tau aggregates in affected brain regions. Tau hyperphosphorylation and accumulation in neurofibrillary tangles is strongly correlated with cognitive deficits, and is apparently a critical event in the dementia process because mutations in tau can cause a tangle-only form of dementia called frontotemporal lobe dementia. Among kinases that phosphorylate tau, glycogen synthase kinase 3ß (GSK3ß) is strongly implicated in AD pathogenesis. In the present study, we established an ELISA to screen for agents that inhibit GSK3ß activity and found that the flavonoid morin effectively inhibited GSK3ß activity and blocked GSK3ß-induced tau phosphorylation in vitro. In addition, morin attenuated Aß-induced tau phosphorylation and protected human neuroblastoma cells against Aß cytotoxicity. Furthermore, treatment of 3xTg-AD mice with morin resulted in reductions in tau hyperphosphorylation and paired helical filament-like immunoreactivity in hippocampal neurons. Morin is a novel inhibitor of GSK3ß that can reduce tau pathology in vivo and may have potential as a therapeutic agent in tauopathies.

Crystal structure and thermostability of a putative α-glucosidase from Thermotoga neapolitana.

Glycoside hydrolase family 4 (GH4) represents an unusual group of glucosidases with a requirement for NAD(+), Mn(2+), and reducing conditions. We found a putative α-glucosidase belonging to GH4 in hyperthermophilic Gram-negative bacterium Thermotoga neapolitana. In this study, we recombinantly expressed the putative α-glycosidase from T. neapolitana, and determined the crystal structure of the protein at a resolution of 2.0Å in the presence of Mn(2+) but in the absence of NAD(+). The structure showed the dimeric assembly and the Mn(2+) coordination that other GH4 enzymes share. In comparison, we observed structural changes in T. neapolitana α-glucosidase by the binding of NAD(+), which also increased the thermostability. Numerous arginine-mediated salt-bridges were observed in the structure, and we confirmed that the salt bridges correlated with the thermostability of the proteins. Disruption of the salt bridge that linked N-terminal and C-terminal parts at the surface dramatically decreased the thermostability. A mutation that changed the internal salt bridge to a hydrogen bond also decreased the thermostability of the protein. This study will help us to understand the function of the putative glucosidase and the structural features that affect the thermostability of the protein.

Crystallization and preliminary X-ray crystallographic analysis of Salmonella Typhimurium CueP.

Salmonella enterica serovar Typhimurium (S. Typhimurium) can survive in the phagosome of macrophages, causing serious medical and veterinary problems. CueP is uniquely found in S. Typhimurium and has been characterized as a major periplasmic copper-binding protein. Although cueP has been identified as being responsible for the copper resistance of the bacterium in vivo, the biochemical role and three-dimensional structure of CueP remain unknown. In this study, CueP from S. Typhimurium was overexpressed and the recombinant protein was purified using Ni-NTA affinity, anion-exchange and gel-filtration chromatographies. The purified CueP protein was crystallized using the vapour-diffusion method. A diffraction data set was collected to 2.5 Šresolution at 100 K. The crystal belonged to space group P2(1)2(1)2(1). To obtain initial phases, selenomethionyl-substituted protein was overproduced and purified. Optimization of crystallization conditions for the selenomethionyl-substituted protein is in progress.

Left Atrial Strain and Compliance Correlate with Diastolic Dysfunction Grades and Complications during Pre-eclampsia: A Speckle-Tracking Echocardiography Study.

This study aimed to investigate left atrium (LA) strain components in the assessment of cardiac function and its clinical correlates in pre-eclampsia (PE). With the use of speckle tracking echocardiography, phasic LA strain and (LASr)/(E/e'), the surrogate of LA compliance, were compared between healthy pregnant women (n = 70) and those with PE (n = 146) and among different diastolic dysfunction (DD) grades in PE. Receiver operating characteristic curves and logistic regression analysis were used to identify the role of strain components in distinguishing DD grades and predicting cardiac complications. LA reservoir strain, conduit strain and LA compliance reduced significantly in PE (p < 0.01). LASr/(E/e') gradually decreased with worsening DD and LASr/(E/e') <3.40 was the independent risk factor for cardiac events in PE (p < 0.01). This study observed significantly decreased LA strain and compliance in PE. Notably, LA compliance decreased progressively with the severity of DD, and LASr/(E/e') <3.40 is the independent risk factor for cardiac complications during PE pregnancy.

Functional relationships between the AcrA hairpin tip region and the TolC aperture tip region for the formation of the bacterial tripartite efflux pump AcrAB-TolC.

Tripartite efflux pumps found in Gram-negative bacteria are involved in antibiotic resistance and toxic-protein secretion. In this study, we show, using site-directed mutational analyses, that the conserved residues located in the tip region of the alpha-hairpin of the membrane fusion protein (MFP) AcrA play an essential role in the action of the tripartite efflux pump AcrAB-TolC. In addition, we provide in vivo functional data showing that both the length and the amino acid sequence of the alpha-hairpin of AcrA can be flexible for the formation of a functional AcrAB-TolC pump. Genetic-complementation experiments further indicated functional interrelationships between the AcrA hairpin tip region and the TolC aperture tip region. Our findings may offer a molecular basis for understanding the multidrug resistance of pathogenic bacteria.

Crystal structure and functional insight of HP0420-homolog from Helicobacter felis.

Helicobacter pylori infect more than half of the world's population and are considered a cause of peptic ulcer disease and gastric cancer. Recently, hypothetical gene HP0421 was identified in H. pylori as a cholesterol alpha-glucosyltransferase, which is required to synthesize cholesteryl glucosides, essential cell wall components of the bacteria. In the same gene-cluster, HP0420 was co-identified, whose function remains unknown. Here we report the crystal structure of HP0420-homolog of H. felis (HF0420) to gain insight into the function of HP0420. The crystal structure, combined with size-exclusion chromatography, reveals that HF0420 adopts a homodimeric hot-dog fold. The crystal structure suggests that HF0420 has enzymatic activity that involves a conserved histidine residue at the end of the central alpha-helix. Subsequent biochemical studies provide clues to the function of HP0420 and HF0420.

The tip region of the MacA alpha-hairpin is important for the binding to TolC to the Escherichia coli MacAB-TolC pump.

The tripartite efflux pump MacAB-TolC found in gram-negative bacteria is involved in resistance to antibiotics. We previously reported the funnel-like hexameric structure of the adaptor protein MacA to be physiologically relevant. In this study, we investigated the role of the tip region of its alpha-hairpin, which forms a cogwheel structure in the funnel-like shape of the MacA hexamer. Mutational and biochemical analyses revealed that the conserved residues located at the tip region of the alpha-hairpin of MacA play an essential role in the binding of TolC. Our findings offer a molecular basis for understanding the drug resistance of pathogenic bacteria.

Crystal structure of bacterioferritin from Rhodobacter sphaeroides.

Iron is essential for the survival of organisms, but either excess or deficient levels of iron induce oxidative stress, thereby causing cell damage. As a result, iron regulation is essential for proper cell growth and proliferation in most organisms. Bacterioferritin is a ferritin-like family protein that contains a heme molecule and a ferroxidase site at the di-iron center. This protein plays a primary role in intracellular iron storage for iron homeostasis, as well as in the maintenance of iron in a soluble and non-toxic form. Although several bacterioferritin structures have been determined, no structural studies have successfully elucidated the molecular function of the heme molecule and the ferroxidase center. Here, we report the crystal structure of bacterioferritin from Rhodobacter sphaeroides. This protein exists in a roughly spherical configuration via the assembly of 24 subunits. We describe the oligomeric arrangement, ferroxidase center and heme-binding site based on this structure. The protein contains a single iron-binding configuration in the ferroxidase center, which allows for the release of iron by His130 when the protein is in the intermediate state. The heme molecule in RsBfr is stabilized by shifting of the van der Waals interaction center between the porphyrin of the heme and Trp26. We anticipate that further structural analysis will provide a more complete understanding of the molecular mechanisms of members of the ferritin-like family.

Crystallization and preliminary X-ray crystallographic analysis of a highly specific serpin from the beetle Tenebrio molitor.

The Toll signalling pathway, which is crucial for innate immunity, is transduced in insect haemolymph via a proteolytic cascade consisting of three serine proteases. The proteolytic cascade is downregulated by a specific serine protease inhibitor (serpin). Recently, the serpin SPN48 was found to show an unusual specific reactivity towards the terminal serine protease, Spätzle-processing enzyme, in the beetle Tenebrio molitor. In this study, the mature form of SPN48 was overexpressed in Escherichia coli and purified. The purified SPN48 protein was crystallized using 14% polyethylene glycol 8000 and 0.1 M 2-(N-morpholino)ethanesulfonic acid pH 6.0 as the precipitant. The crystals diffracted X-rays to 2.1 A resolution and were suitable for structure determination. The crystals belonged to space group P2(1). The crystal structure will provide information regarding how SPN48 achieves its unusual specificity for its target protease.

The role of the Ser/Thr cluster in the phosphorylation of PPPSP motifs in Wnt coreceptors.

Wnt/beta-catenin signaling controls a variety of cellular processes, including cell growth, oncogenesis, and development. Upon Wnt stimulation, the intracellular region of the coreceptor, LRP6 or 5, is phosphorylated by the membrane-recruited GSK3beta and CK1. The cytoplasmic domain of LRP6/5 contains one Ser/Thr cluster and the PPPSP motifs, both of which are essential for propagation of the signal. While the phosphorylated PPPSP motifs are known to directly inhibit GSK3beta, the biochemical role of the phosphorylated Ser/Thr cluster remains to be elucidated. Herein, we reveal that the Ser/Thr cluster plays an important role in the phosphorylation of the PPPSP motif. Interestingly, we observe that GSK3beta activity on the PPPSP motif requires a high ATP concentration, close to that of the physiological condition. Taken together, these data suggest that the phosphorylated Ser/Thr cluster serves as a docking site for GSK3beta to promote the phosphorylation of the PPPSP motif. Our results provide insight into the molecular mechanism for the initial events of the Wnt/beta-catenin signaling.

Crystallization and preliminary X-ray crystallographic analysis of MacA from Actinobacillus actinomycetemcomitans.

Periplasmic membrane-fusion proteins (MFPs) are an essential component of the multidrug efflux pump in Gram-negative bacteria. They play a crucial role in bridging the outer membrane porin TolC and two distinct types of inner membrane transporters. The MFP MacA bridges the inner membrane ABC-type multidrug transporter MacB and the outer membrane porin TolC. MacA from the pathogenic bacterium Actinobacillus actinomycetemcomitans was expressed in Escherichia coli B834 (DE3) and the recombinant protein was purified using Ni-NTA affinity, Q anion-exchange and gel-filtration chromatography. The purified MacA protein was crystallized using the vapour-diffusion method. A MAD diffraction data set was collected to a resolution of 3.0 A at 100 K. The crystal belongs to space group P622, with unit-cell parameters a = b = 109.2, c = 255.4 A, alpha = beta = 90, gamma = 120 degrees , and contains one molecule in the asymmetric unit.

Overexpression and preliminary X-ray crystallographic analysis of prophenoloxidase activating factor II, a clip domain family of serine proteases.

A clip domain family of serine proteases has been identified in invertebrates as a crucial enzyme involved in diverse biological processes including immune responses and embryonic development. Although these proteins contain at least one clip domain at the N-terminal of the serine protease domain, the roles and three-dimensional structure of the clip domain are unknown. Prophenoloxidase activating factor-II (PPAF-II), a clip domain family of serine proteases, derived from the beetle Holotrichia diomphalia larvae, was overexpressed in the baculovirus system, and crystallized using the hanging-drop vapor-diffusion method. High-quality single crystals of PPAF-II were obtained in a precipitant solution containing 0.15 M ammonium sulfate, 1.25 M lithium sulfate monohydrate, and 0.1 M sodium citrate dehydrate (pH 5.5). These crystals belong to space group C2 with unit-cell parameters a=107.84, b=76.78, c=70.49 A and beta=113.93 degrees , and contain one or two molecules in the asymmetric unit. Determination of the three-dimensional structure of PPAF-II would clarify the functions of the clip domains.