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McCune–Albright syndrome (MAS) is a rare sporadic condition defined by the classic triad of fibrous dysplasia of bone, café au lait skin macules, and hyperfunctioning endocrinopathies. The molecular basis of MAS has been ascribed to the post-zygotic somatic gain-of-function mutations in the GNAS gene, which encodes the alpha subunit of G proteins, leading to constitutive activation of several G Protein-Coupled Receptors (GPCRs). The co-occurrence of two of the above-mentioned cardinal clinical manifestations sets the diagnosis at the clinical level. In this case report, we describe a 27-month-old girl who presented with gonadotropin-independent precocious puberty secondary to an estrogen-secreting ovarian cyst, a café au lait skin macule and growth hormone, and prolactin excess, and we provide an updated review of the scientific literature on the clinical features, diagnostic work-up, and therapeutic management of MAS.

Complex ecosystems characterize the Arctic Region, which experiences rapid environmental changes due to climate change. These changes are happening spatially as much as temporally. Spatiotemporal interactive three-dimensional visualization of environmental phenomena is eye-catching, while creating the foundations for the rapid understanding of Arctic complexity. The Arctic is a circumpolar region; cylinders seem suitable visual media for related data. In this case study, the visualization of anomalies of the sea ice illustrates abnormal changes, helping the viewer/user to focus on that filtered information rather than becoming lost in a multitude of information. A holistic view of dynamic environmental changes in a stand-alone visual could impact the quick understanding, response and policymaking regarding the visualized phenomena.

Background Nintedanib represents an antifibrotic compound able to slow down disease progression of patients with idiopathic pulmonary fibrosis (IPF). Objective To investigate the safety and efficacy of nintedanib in patients with IPF in a real-life setting. Methods This was a multicentre, retrospective, observational, real-life study for patients with IPF receiving nintedanib between October 2014 and October 2016. Results We identified 94 patients with IPF receiving nintedanib (72 males, mean age±SD: 73.8 ± 7.5, mean%FVC±SD = 68.1 ± 18.3, mean%DLCo±SD = 44.4 ± 14.5). Diarrhea (n = 52, 55.3%) was the most commonly reported adverse event. Twenty patients (21.2%) had to permanently discontinue nintedanib due to severe adverse events. In the 6-months follow-up, median decline in %FVC predicted and %DLCO predicted were 1.36 (95%Cl: 0 to 2.97) and 4.00 (95%Cl: 2.01 to 6.20), respectively, when deaths were censored and excluded from the analysis. At 12 months, mean%FVC±SD and mean%DLCo±SD were 64.5 ± 19.1 and 43.7 ± 15.4, respectively. With regards to mortality, 17 patients (18.1%) died over a study period of 730 days. Conclusion Nintedanib demonstrated an acceptable safety and efficacy profile in our real-world observational study. Prospective observational studies in the context of registries that collect well-defined supporting data over time are sorely needed to answer residual questions on drug's performance.

The aim of the present study was to determine the level of underlying DNA damage in females with PCOS. Twenty-two women with PCOS and twenty-two healthy controls were included in this study. Patients were further categorized into three phenotypic subgroups: Subgroup A, oligo/anovulation (ANOV) and polycystic ovaries (PCO); Subgroup B, hyperandrogenism (HA) as a main characteristic (HA + ANOV or HA + PCO); Subgroup C, all three conditions present (HA + ANOV + PCO). The frequency of sister chromatid exchanges (SCEs) was used as an index of cytogenetic damage. Proliferation rate index (PRI), mitotic index (MI), average generation time (AGT) and population doubling time (PDT) were also evaluated. A significant (p < 0.01) increase in SCE levels along with a significant (p < 0.01) reduction in PRIs and MIs were observed in women with PCOS compared with healthy subjects. Additionally, subgroup C demonstrated statistically significant differences compared with others, while subgroup A had similar results with healthy females. Our results suggest that females with PCOS show increased chromosomal instability in peripheral lymphocytes and a consequent inability of the cells to promote their own mitotic cycle. A positive correlation between DNA damage and PCOS phenotypes is also reported.

Human Angiogenin (hAng) is a member of the ribonuclease A superfamily and a potent inducer of neovascularization. Protein interactions of hAng in the nucleus and cytoplasm of the human umbilical vein cell line EA.hy926 have been investigated by mass spectroscopy. Data are available via ProteomeXchange with identifiers PXD006583 and PXD006584. The first gel-free analysis of hAng immunoprecipitates revealed many statistically significant potential hAng-interacting proteins involved in crucial biological pathways. Surprisingly, proliferating cell nuclear antigen (PCNA), was found to be immunoprecipitated with hAng only in the cytoplasm. The hAng-PCNA interaction and colocalization in the specific cellular compartment was validated with immunoprecipitation, immunoblotting, and immunocytochemistry. The results revealed that PCNA is predominantly localized in the cytoplasm, while hAng is distributed both in the nucleus and in the cytoplasm. hAng and PCNA colocalize in the cytoplasm, suggesting that they may interact in this compartment.

The present study investigated the nomological validity of the Amotivation Inventory–Physical Education (Shen, Wingert, Li, Sun, & Rukavina, 2010b) scores by examining the associations of ability, effort, value, and task characteristics amotivation beliefs with self-determination theory variables. Data were collected from 401 fifth- and sixth-grade students, 416 middle-school students, and 401 high-school students. After providing support for the correlated four-factor structure, the hierarchical structure, internal consistency, and composite reliability of the Amotivation Inventory–Physical Education scores, large negative correlations emerged with perceived autonomy support by the PE teacher; needs for autonomy, competence, and relatedness; identified regulation; intrinsic motivation; and students' subjective vitality. Strong positive correlations were obtained with unidimensional amotivation and external regulation, and weak correlations were obtained with introjected regulation. Also, multidimensional...

alpha-D-glucose is a weak inhibitor (Ki = 1.7 mM) of glycogen phosphorylase (GP) and acts as physiological regulator of hepatic glycogen metabolism; it binds to GP at the catalytic site and stabilizes the inactive T state of the enzyme promoting the action of protein phosphatase 1 and stimulating glycogen synthase. The three-dimensional structures of T state rabbit muscle GPb and the GPb-alpha-D-glucose complex have been exploited in the design of better regulators of GP that could shift the balance between glycogen synthesis and glycogen degradation in favour of the former. Close examination of the catalytic site with alpha-D-glucose bound shows that there is an empty pocket adjacent to the beta-1-C position. beta-D-glucose is a poorer inhibitor (Ki = 7.4 mM) than alpha-D-glucose, but mutarotation has prevented the binding of beta-D-glucose in T state GP crystals. A series of beta-D-glucose analogues has been designed and tested in kinetic and crystallographic experiments. Several compounds have been discovered that have an increased affinity for GP than the parent compound.

Glycogen phosphorylase is a molecular target for the design of potential hypoglycemic agents. Structure-based design pinpointed that the 3′-position of glucopyranose equipped with a suitable group has the potential to form interactions with enzyme's cofactor, pyridoxal 5′-phosphate (PLP), thus enhancing the inhibitory potency. Hence, we have investigated the binding of two ligands, 1-(β-d-glucopyranosyl)5-fluorouracil (GlcFU) and its 3′-CH 2OH glucopyranose derivative. Both ligands were found to be low micromolar inhibitors with K i values of 7.9 and 27.1μm, respectively. X-ray crystallography revealed that the 3′-CH 2OH glucopyranose substituent is indeed involved in additional molecular interactions with the PLP γ-phosphate compared with GlcFU. However, it is 3.4 times less potent. To elucidate this discovery, docking followed by postdocking Quantum Mechanics/Molecular Mechanics - Poisson-Boltzmann Surface Area (QM/MM-PBSA) binding affinity calculations were performed. While the docking predictions failed to reflect the kinetic results, the QM/MM-PBSA revealed that the desolvation energy cost for binding of the 3′-CH 2OH-substituted glucopyranose derivative out-weigh the enthalpy gains from the extra contacts formed. The benefits of performing postdocking calculations employing a more accurate solvation model and the QM/MM-PBSA methodology in lead optimization are therefore highlighted, specifically when the role of a highly polar/charged binding interface is significant.

C5 halogen substituted glucopyranosyl nucleosides (1-(β-D-glucopyranosyl)-5-X-uracil; X=Cl, Br, I) have been discovered as some of the most potent active site inhibitors of glycogen phosphorylase (GP), with respective K(i) values of 1.02, 3.27, and 1.94 μM. The ability of the halogen atom to form intermolecular electrostatic interactions through the σ-hole phenomenon rather than through steric effects alone forms the structural basis of their improved inhibitory potential relative to the unsubstituted 1-(β-D-glucopyranosyl)uracil (K(i) =12.39 μM), as revealed by X-ray crystallography and modeling calculations exploiting quantum mechanics methods. Good agreement was obtained between kinetics results and relative binding affinities calculated by QM/MM-PBSA methodology for various substitutions at C5. Ex vivo experiments demonstrated that the most potent derivative (X=Cl) toward purified GP has no cytotoxicity and moderate inhibitory potency at the cellular level. In accordance, ADMET property predictions were performed, and suggest decreased polar surface areas as a potential means of improving activity in the cell.

Objectives Given instances of less than optimal internal consistency levels of the Perceived Locus of Causality (PLOC) scale when applied to school-based physical education (PE), the aim of the study was to examine the psychometrics of a revised PLOC instrument for use in PE. Design A cross-sectional study in which self-report data on behavioral regulations, perceived autonomy support by the PE teacher, and subjective vitality were collected from 1729 students in relation to their PE participation at the elementary school (5th and 6th grade), middle school, and high school levels, including boys and girls. Results The revised instrument (PLOC-R) demonstrated satisfactory levels of internal consistency; a sound factor structure; evidence in support of a simplex-like structure; configural, metric, strong, and strict measurement invariance across boys and girls, students who participate or not in out-of-school sport activities, and across the three school grade levels; and nomological validity. Conclusions Initial evidence has emerged in favor of the PLOC-R as a scale appropriate for use with 5th and 6th grade elementary, middle school, and high school students to measure the degree of self-determined motivation for participation in compulsory school-based physical education classes.

The present study reported on the modification of the Basic Psychological Needs in Exercise Scale (Vlachopoulos & Michailidou, 2006) to assess students’ psychological need fulfillment in elementary school, middle school, and high school compulsory physical education classes. Data were collected from 817 5th and 6th grade students, 862 middle school students and 844 high school students, boys and girls. The findings supported an a priori correlated 3-factor structure of the Basic Psychological Needs in Physical Education scale (BPN-PE) with strong internal reliability for all three school grade levels. Support was also obtained for the nomological validity of the scale responses. Further, measurement invariance emerged for BPN-PE scores across boys and girls and across students who participated or not in out-of-school sports within each school grade level as well as across all three school grade levels.

The patterns of MHC diversity were studied at UAA and DAB1 loci and the two domains involved in the recognition of antigenic peptides (α2 and β1, respectively) in eight Ladigesocypris ghigii populations inhabiting streams and a concrete reservoir, in order to understand the significance of these genes in bottlenecked populations of an endemic species and develop conservation rationale. In agreement with previous study employing RAPD and mtDNA markers (Mamuris et al., Freshw Biol 50:1441–1453, 2005), both loci exhibited a very low level of polymorphism with only two and four alleles detected for UAA and DAB1, respectively. The functional MHC diversity was even lower since UAA alleles were distinguished by a single synonymous substitution. The type of habitat did not affect the level of polymorphism. Our data suggest that DAB1 polymorphism might be the outcome of the positive selection, imposed by the temporal and spatial variation of pathogen load, and the genetic drift as a result of successive habitat shrinkage and deterioration by water abstraction year after year. The populations studied were significantly less diverged at MHC loci than expected based on nuclear and mtDNA markers, suggesting that common parasites might act as causative factors to homogenize selection. Sufficient epidemiological data are required for the interpretation of the results and decision-making on suitable conservation actions.

The function of the central nervous system largely depends on growth and differentiation (neurite outgrowth) of neural cells and it is well established that growth factors, especially nerve growth factor NGF stimulate neurite outgrowth. However, additional factors are implicated in this process notably the redox state of the cells. For the first time we could demonstrate that the application of recombinant thioredoxin stimulates neurite outgrowth of PC12 cells to the same extend as NGF. Thioredoxin, a small redox protein is a major player in the cellular protein reduction system. An increased expression and secretion of thioredoxin is achieved by the application of the novel sialic acid precursor N-propionylmannosamine (ManNProp). From earlier studies it is known that this N-acylmannosamine analog stimulates significantly the neurite outgrowth in cell cultures. This finding would give new insights into the mechanism of the nerve-stimulatory action of ManNProp and demonstrates the novel role of thioredoxin during the regulation of nerve growth, encouraging further studies.

Sialic acids usually represent the terminal monosaccharide of glycoconjugates and are directly involved in many biological processes. The cellular concentration of their nucleotide-activated form is one pacemaker for the highly variable sialylation of glycoconjugates. Hence, the determination of CMP-sialic acid levels is an important factor to understand the complex glycosylation machinery of cells and to standardize the production of glycotherapeutics. We have established a highly sensitive strategy to quantify the concentration of nucleotide-activated sialic acid by a combination of reduction and fluorescent labeling using the fluorophore 1,2-diamino-4,5-methylenedioxybenzene (DMB). The labeling with DMB requires free keto as well as carboxyl groups of the sialic acid molecule. Reduction of the keto group prior to the labeling process precludes the labeling of nonactivated sialic acids. Since the keto group is protected against reduction by the CMP-substitution, labeling of nucleotide-activated sialic acids is still feasible after reduction. Subsequent combination of the DMB-high-performance liquid chromatography (HPLC) application with mass spectrometric approaches, such as matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF-MS) and electrospray-ionization (ESI)-MS, allows the unambiguous identification of both natural and modified CMP-sialic acids and localization of potential substituents. Thus, the described strategy offers a sensitive detection, identification, and quantification of nucleotide-activated sialic acid derivatives in the femtomole range without the need for nucleotide-activated standards.

In the quest for the rational design of selective and potent inhibitors for members of the pancreatic ribonuclease A (RNase A) family of biomedical interest, the binding of uridine 5'-phosphate (U5P) and uridine 5'-diphosphate (UDP) to RNase A have been investigated using kinetic studies and X-ray crystallography. Both nucleotides are competitive inhibitors of the enzyme, with K(i) values of 4.0 and 0.65 mM, respectively. They bind to the active site of the enzyme by anchoring two molecules connected to each other by hydrogen bonds and van der Waals interactions. While the first of the inhibitor molecules binds with its nucleobase in the pyrimidinyl-binding subsite, the second is bound at the purine-preferring subsite. The unexpected binding of a pyrimidine at the purine-binding subsite has added new important elements to the rational design approach for the discovery of new potent inhibitors of the RNase A superfamily.

Time- and dose-dependent measurements of metabolites of galactose (with glucose as control) in various organs of rats are discussed. Not only the liver but especially the brain and to a lesser extent the muscles also have the capacity to take up and metabolize galactose. Primarily, the concentrations of UDP-galactose, a pivotal compound in the metabolism of galactose, and UDP-glucose are measured. An important feature lies in the demonstration that galactose and glucose are metabolized to amino acids and that the only increases observed in the brain appear in the concentrations of glutamate, glutamine, GABA measured after acute galactose loads. In addition the increase in the amino acid concentrations after galactose has been administered persists for longer periods of time than after glucose administration. This conversion of hexoses, especially galactose, to amino acids requires the consumption of ammonia equivalents in the brain ; this finding might stimulate the use of galactose as a new means of removal of this neurotoxic compound from the brain in patients suffering from hepatic encephalopathy or Alzheimer's disease.

Fanconi anemia is a fatal, hereditary chromosome instability syndrome of early childhood with progressive pancytopenia and cancer-proneness. Hypersensitivity to alkylating agents points to DNA repair inefficiency. Excess reactive oxygen intermediates and hypersensitivity to oxygen, all features of Fanconi anemia cells, give evidence for a disturbed oxidative metabolism. Here, we report that expression of the inducible heme oxygenase-1, an essential antioxidative defense protein, is impaired in Fanconi anemia cells and can be reinstated with the transfection of Fanconi A wild-type cDNA. A causative interaction of Fanconi anemia proteins with transcription of selected proteins is indicated. The results enlighten the oxygen sensitivity in Fanconi anemia.

Sialic acid precursors are mediators of the sialic acid pathway. In this manuscript we present evidence that the application of sialic acid a precursor modulates gene expression and cell differentiation. The concept that sugars are involved in cellular transcription was first proposed by Jacob and Monod nearly 40 years ago studying the regulation of the lac-operon in prokaryotes. Surprisingly, these findings have never been transferred to eukaryotic systems. For our studies we have chosen PC12 cells. PC12-cells differentiate after application of NGF into a neuron-like phenotype. It is shown that treatment of PC12 cells with two different sialic acid precursors N-acetyl- or N-propanoylmannosamine, without application of NGF also induces neurite outgrowth. Moreover, the PC12 cells show the same morphology as the NGF-treated cells. Surprisingly, after application of both sialic acid precursors the phosphorylation and translocation of erk1/2 into the nucleus are activated, thus influencing the expression of genes involved in the differentiation of cells, such as the transcription factor c-Jun or TOAD-64/Ulip/CRMP (Turned ON After Division, 64 kd/ unc-33-like phosphoprotein/Collapsin Response Mediator Protein). These are the first experimental data showing that the sialic acid metabolism is closely associated with signal transduction and regulation of neuronal differentiation.

Sialic acid (Sia) is expressed as terminal sugar in many glycoconjugates and plays an important role during development and regeneration. Addition of homopolymers of Sia (polysialic acid; polySia/PSA) is a unique and highly regulated post-translational modification of the neural cell adhesion molecule (NCAM). The presence of polySia affects NCAM-dependent cell adhesion and plays an important role during brain development, neural regeneration, and plastic processes including learning and memory. PolySia-NCAM is expressed on several neuroendocrine tumors of high malignancy and correlates with poor prognosis. Two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV, catalyze the biosynthesis of polySia. This review summarizes recent knowledge on Sia biosynthesis and the correlation between Sia biosynthesis and polysialylation of NCAM and report on approaches to modify the degree of polySia on NCAM in vitro and in vivo. First, we describe the inhibition of polysialylation of NCAM in ST8SiaII-expressing cells using synthetic Sia precursors. Second, we demonstrate that the key enzyme of the Sia biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) regulates and limits the synthesis of polySia by controlling the cellular Sia concentration.

Received 11 March 2005/Accepted 25 May 2005 Two strains of the parvovirus minute virus of mice (MVM), the immunosuppressive (MVMi) and the prototype (MVMp) strains, display disparate in vitro tropism and in vivo pathogenicity. We report the crystal structures of MVMp virus-like particles (MVMpb) and native wild-type (wt) empty capsids (MVMpe), determined and refined to 3.25 and 3.75 A resolution, respectively, and their comparison to the structure of MVMi, also refined to 3.5 A resolution in this study. A comparison of the MVMpb and MVMpe capsids showed their structures to be the same, providing structural verification that some heterologously expressed parvovirus capsids are indistinguishable from wt capsids produced in host cells. The structures of MVMi and MVMp capsids were almost identical, but local surface conformational differences clustered from symmetry-related capsid proteins at three specific domains: (i) the icosahedral fivefold axis, (ii) the “shoulder” of the protrusion at the icosahedral threefold axis, and (iii) the area surrounding the depression at the icosahedral twofold axis. The latter two domains contain important determinants of MVM in vitro tropism (residues 317 and 321) and forward mutation residues (residues 399, 460, 553, and 558) conferring fibrotropism on MVMi. Furthermore, these structural differences between the MVM strains colocalize with tropism and pathogenicity determinants mapped for other autonomous parvovirus capsids, highlighting the importance of common parvovirus capsid regions in the control of virus-host interactions. Viral tissue tropism and pathogenesis are highly dependent on well-orchestrated and defined interactions that occur between the viral pathogen and its host. The single-stranded Parvoviridae infect a broad range of natural hosts that include invertebrates and mammals, and pathogenic members cause serious disease in the young and immunocompromised adults. However, disparities in tissue tropism and in vivo pathogenicity can be observed between highly homologous strains for several members of the Parvoviridae. The combination of molecular analysis with disease outcomes for a number of strains of the Aleutian mink disease parvovirus (AMDV) (9, 10), canine parvovirus (CPV), and feline panleukopenia virus (FPV) (46, 61, 62), and for porcine parvovirus (PPV) (8, 41), has provided

Cancer cells have high levels of thioredoxin (Trx) and of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cells from patients with the cancer-prone disease Fanconi anemia (FA) exhibit reduced Trx levels. We found the activity of GAPDH to correlate directly with the endogenous Trx content and mRNA transcripts for GAPDH and TRx reduced in FA cells. The treatment of cells with reduced human Trx stimulated the synthesis of GAPDH mRNA. Similarly, the transfection of cells with an expression plasmid for Trx increased GAPDH mRNA synthesis. Trx treatment of cells and subsequent analysis of the differential gene expression by human cDNA arrays containing about 50 000 different PCR products resulted in more than 300 up- or downregulated genes. Two representative genes, GAPDH and IkappaBalpha/MAD-3, were further investigated to confirm their stimulation by Trx. Trx besides being the major carrier of redox potential of cells is also a regulator of gene expression on the transcriptional level. By regulation via Trx, cells are able to adapt to the prevailing redox conditions. These findings also enlighten the pathophysiology of FA in the respect that the characteristic diminution of Trx that results in the dysregulation of gene expression is a basis for the major symptoms of this disease.

Fanconi anemia (FA) is a hereditary disease of unknown pathogenic mechanisms, although mutations in seven different genes can be causative. Six of these genes have been cloned and sequenced. Only slight homology to the DNA of any other known gene has been found with the exception of FANCG which is identical to XRCC9. The function of these genes, including XRCC9, is presently unknown. Since pADP ribosyl transferase (pADPRT) plays a role in apoptosis, and apoptosis is affected in FA cells, we studied the correlation between pADPRT and FA cells. We reinvestigated the previously reported lack of pADPRT activity in fibroblasts from patients with Fanconi anemia. Here we describe the role of the lower redox potential of FA cells and demonstrate that this is an efficient strategy in the prevention of cell death due to the lack of energy under oxidative stress. This strategy is advantageous for the cells under the nonreplicative condition of confluency in which the risk of mutation is low and the prevention of apoptosis permits cell survival. pADPRT is not diminished to the same extent in all complementation groups of FA. It is prominent in FANCA, FANCG and FANCF cells, indicating that these genes control pADPRT diminution. Our experiments suggest that the pADPRT level is linked with the oxidoreduction reactions seen in FA.

The cause of the molecular defect of Fanconi anemia (FA) remains unknown. Cells from patients with FA exert an elevated spontaneous chromosomal instability which is further triggered by mitomycin C. The induced lability is reduced by overexpression of thioredoxin which is not the case for spontaneous instability. However, both are eliminated by overexpression of thioredoxin cDNA with an added nuclear localization signal. This implies that thioredoxin is lacking in the nuclei of FA cells. The total thioredoxin content in all FA cells tested is reduced. The resultant lack of nuclear thioredoxin can be the explanation for the major symptomatology in FA. Since thioredoxin is known to be the reactive cofactor of ribonucleotid reductase its shortcoming reduces the supply of deoxyribonucleotides thus hindering the DNA and replication repair with resultant chromosomal breaks. Furthermore, depression of tyrosine hydroxylase, the key enzyme of melanine synthesis, could be the basis for the pathognomotic 'café au lait' spots of FA. The observation of thioredoxin reduction in FA cells permits insight into the molecular phathophysiology of FA.

Entire Helicobacter Pylori Neutrophil Activated Protein (HPNAP) and its truncated forms NH2-terminal region HPNAP1–57 and C-terminal region HPNAP58–144 after cloning into pET29c vector, purification and removal of LPS traces were subjected to human neutrophil activation. Our results revealed that the C-terminal region of HPNAP is indispensable for human neutrophil stimulation and their further adhesion to endothelial cells – a step necessary to H. pylori inflammation – in a ratio equal to that exhibited by the entire protein. In addition, experiments concerning the implication of Arabino-Galactan-Proteins (AGPs) derived from Chios Mastic Gum (CMG), the natural resin of the plant Pistacia lentiscus var. Chia revealed the inhibition of neutrophil activation and therefore their adhesion to endothelial cells, in vitro. Both, the involvement of HPNAP C-terminal region in stimulation-adhesion of neutrophils to endothelial cells as well as the inhibition of this process by AGPs have to be further investigated and may be exploited in a future anti-inflammatory therapy for H. pylori patients.

Sialic acids represent a family of 9-carbon acidic amino sugars expressed mainly as terminal monosaccharides on most mammalian glycoconjugates. Sialic acids play an outstanding role during cellular processes, such as development and regeneration, as they are involved in a variety of molecular interactions. Sialic acids are synthesized in the cytosol starting from UDP-N-acetylglucosamine by the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE), which is the key enzyme in the biosynthesis of sialic acid that catalyzes the generation of N-acetylmannosamine, which in turn is an intermediate of the sialic acid pathway that represents the natural molecular precursor of all sialic acids. Of increasing interest are the influence of the sialic acid precursor N-acetylmannosamine (or related N-acylmannosamines), GNE, and sialic acids themselves on cellular processes such as proliferation, gene expression, or cell differentiation. Here, we present recent data and review indications that N-acylmannosamines (the direct precursors of all sialic acids) may act as signaling molecules, and that the key enzyme of the sialic acid metabolism is directly involved in the regulation of cell proliferation and cell differentiation.

Objective.To examine the effect of heterogeneous case mix for a benchmarking analysis and interhospital comparison of the prevalence rates of nosocomial infection.Design.Cross-sectional survey.Setting.Eleven hospitals located in Cyprus and in the region of Crete in Greece.Methods.The survey included all inpatients in the medical, surgical, pediatric, and gynecology-obstetrics wards, as well as those in intensive care units. Centers for Disease Control and Prevention criteria were used to define nosocomial infection. The information collected for all patients included demographic characteristics, primary admission diagnosis, Karnofsky functional status index, Charlson comorbidity index, McCabe- Jackson severity of illness classification, use of antibiotics, and prior exposures to medical and surgical risk factors. Outcome data were also recorded for all patients. Case mix-adjusted rates were calculated by using a multivariate logistic regression model for nosocomial infection risk and an indirect standardization method.Results.The overall prevalence rate of nosocomial infection was 7.0% (95% confidence interval, 5.9%–8.3%) among 1,832 screened patients. Significant variation in nosocomial infection rates was observed across hospitals (range, 2.2%–9.6%). Logistic regression analysis indicated that the mean predicted risk of nosocomial infection across hospitals ranged from 3.7% to 10.3%, suggesting considerable variation in patient risk. Case mix-adjusted rates ranged from 2.6% to 12.4%, and the relative ranking of hospitals was affected by case-mix adjustment in 8 cases (72.8%). Nosocomial infection was significantly and independently associated with mortality (adjusted odds ratio, 3.6 [95% confidence interval, 2.1–6.1]).Conclusion.The first attempt to rank the risk of nosocomial infection in these regions demonstrated the importance of accounting for heterogeneous case mix before attempting interhospital comparisons.

Axonal outgrowth is a prerequisite for the development of the most complex organ, the brain. It depends partially on the attachment of sialic acid on glycans of (sialo)-glycoproteins expressed on the plasma membrane. In our study, we showed that nerve growth factor-induced neurite outgrowth of PC12-cells enhances the expression of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE), the key enzyme for the biosynthesis of sialic acid. Furthermore, we could show that overexpression of GNE induces neurite outgrowth in PC12 cells. The neurite-outgrowth promoting activity of overexpressed GNE, however, does not lead to an increased biosynthesis of sialic acid. These data suggest a novel role of GNE during neurite outgrowth, which is independent to its specific enzymatic activity.

Objective To evaluate the impact of fetal diagnosis of life-threatening congenital heart disease (CHD) on mode of delivery and early neonatal outcomes. Study Design 1-year retrospective cohort study of life-threatening CHD cases at a regional fetal cardiology unit (born June 2013–June 2014). Delivery outcomes were compared with 7630 deliveries over the same period in the tertiary obstetric unit. Results Forty-eight cases were diagnosed prenatally. There were ten terminations of pregnancy (21%) and three intrauterine deaths (6%). One of IUDs was unexpected following CVS biopsy. There were 35 livebirths; with 33 born at the intended location and 2 born elsewhere. Of the 33 babies, the mean gestation 37.6 weeks, mean birth-weight 3180g and male-to-female ratio 3:1. In CHD group (N=33), only six (18%) had spontaneous onset of labour (3 were not documented). Four (12%) had elective C-section (CS). Twenty inductions of labour (IOL) were carried out. IOL was significantly commoner in the CHD group than controls (77% vs 31% p < 0.05). Out of nine non-elective C-sections, two were semi-elective due to failed inductions; six were emergencies with one case not clearly documented. Overall CS rate was not significantly higher in the CHD group than controls (39% vs 32% p = 0.46). 20% (4/20) of vaginal deliveries were instrumental. At birth, 3 babies (9%) required some airway support (IPPV) with no significance difference in spontaneous delivery or CS groups (p = 0.8). The majority (26/33, 78.8%) were transferred to cardiac surgical unit within the first 72 h of life. Of these 26 babies, two (8%) required surgery and five (19%) required emergency balloon atrial septostomy within the first 72 h. Survival of the CHD cohort was 97% at 30-days and survival to discharge was 94%. There was no difference (p = 0.75) in survival-at-hospital-discharge comparing vaginal delivery (12/13) with CS (19/20). Conclusion The rate of IOL in CHD is higher than those found in another study.1 Despite higher induction rate, the rate of CS was no different compared to controls. Mode of delivery does not seem to impact on early outcomes. In comparison to other studies,2,3 survival for this group of antenatally-diagnosed CHD patients appears acceptable. REFERENCES 1. Walsh CA, MacTiernan A, Farrell S, Mulcahy C, McMahon CJ, Franklin O, Coleman D, Mahony R, Higgins S, Carroll S, McParland P, McAuliffe FM. Mode of delivery in pregnancies complicated by major fetal congenital heart disease: a retrospective cohort study. J Perinatol. 2014 29. doi: 10.1038/jp.2014.104 2. Trento L, Pruetz JD, Ruey K, et al. Prenatal diagnosis of congenital heart disease: impact of mode of delivery on neonatal outcome. Prenatal Diagnosis 2012;32: 1250–1255 3. Levey A, Glickstein JS, Kleinman CS, et al. The impact of prenatal diagnosis of complex congenital heart disease on neonatal outcomes. Pediatr Cardiol 2010;31:587–97

Metallo-beta-lactamases (MBLs) are considered an emerging family of Zn2+-dependent enzymes that significantly contribute to the resistance of many nosocomial pathogens against beta-lactam antimicrobials. Since these plasmid-encoded enzymes constitute specific molecular targets for beta-lactams, their exact mode of action is greatly important in deploying efficient anti-infective treatments and for the control of severe multi-resistant nosocomial infections, which becomes a global problem. A novel hybrid VIM-1/VIM-2-type beta-lactamase (named VIM-12) has recently been identified in a clinical isolate of Klebsiella pneumoniae in Greece. The sequence of this enzyme is highly similar with that of VIM-1 at its N-terminal region and with that of VIM-2 at its C-terminal region, raising the question of whether this sequence similarity reflects also a similar functional role. Moreover, the possible contribution of this novel beta-lactamase to the overall antibiotic resistance of this specific clinical isolate was investigated. The gene encoding VIM-12 was cloned and expressed, and the recombinant enzyme was used for detailed kinetic analysis, using a variety of beta-lactam antibiotics. VIM-12 was found to exhibit narrow substrate specificity, compared to other known beta-lactamases, limited mainly to penicillin and to a much lesser extent to imipenen. Interestingly, meropenem was found to act as a noncompetitive inhibitor of the enzyme, although the active site of VIM-12 exhibited complete conservation of residues among VIM enzymes. We conclude that VIM-12 represents a novel and unique member of the family of known metallo-beta-lactamases, exhibiting atypical substrate specificity.

To explore the potential role of computed tomographic cholangiography (CTC) in relation to magnetic resonance cholangiography (MRC) in cases in which knowledge of biliary kinetics and functional information are important for therapeutic decisions, 31 patients (14 men and 17 women) underwent MRC followed by CTC. We examined nine post-cholecystectomy cases with right upper quadrant abdominal pain, six cases with a previous biliary-enteric anastomosis and clinical evidence of cholangitis, eight biliary strictures with pain or symptoms of cholangitis, four cases with strong clinical evidence of sclerosing cholangitis, three cases with suspected post-laparoscopic cholecystectomy bile leakage, and one case with chronic pancreatitis and a common bile duct stent associated with cholangitis. In relation to MRC, CTC provided additional biliary functional information as follows: abnormal biliary drainage through the ampulla in 7/9 cholecystectomy cases, impaired drainage in 3/6 biliary-enteric anastomoses, and complete obstruction in 2/8 biliary strictures. CTC diagnosed early sclerosing cholangitis in 4/4 cases and confirmed suspected bile leakage in 1/3 post-laparoscopic cholecystectomy patients, and the patency of the biliary stent in the patient with chronic pancreatitis. Thus, CTC provides clinically important information about the function and kinetics of bile and complements findings obtained by MRC.

Electrophoretic mobility shift assays (EMSA) revealed that under standard cell culture conditions NF-kappaB was induced in Fanconi anemia fibroblasts in contrast to control cells. Dithiothreitol, a potent synthetic redox potential-delivering compound, when added to growing cells, prevented this induction of NF-kappaB and, simultaneously, chromosomal instability was reduced. Fanconi anemia cells possess low endogenous levels of the naturally occurring antioxidant thioredoxin. Transfection of Fanconi anemia cells with thioredoxin cDNA containing a nuclear localization signal prevented both spontaneous as well as mitomycin C-induced chromosomal instability. A promotor construct with two NF-kappaB binding sites in front of the CAT gene induced little CAT expression in cells with low thioredoxin content in spite of induced NF-kappaB. In cells with higher thioredoxin content CAT expression was increased. Cotransfection of the NF-kappaB-dependent CAT plasmid with the Trx/nuc-plasmid into FA fibroblasts increased the CAT expression to almost that of control cells, indicating that in this model system with diminished thioredoxin content NF-kappaB requires thioredoxin for binding to its specific promotor. Since Fanconi anemia cells have low thioredoxin contents, NF-kappaB-dependent genes are expressed insufficiently. This explains part of the pathophysiological processes observed in Fanconi anemia.

Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to alpha-D-glucose (Ki = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of beta-D-glucopyranosylamine, N-acetyl-beta-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (Ki = 32 microM) and the a (Ki = 35 microM) forms of the enzyme with respect to glucose 1-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase a and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-1-GlcNAc-IMP complex were grown in space group P4(3)2(1)2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 A resolution. 1-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of alpha-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-alpha-D-glucose complex structure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.

Several inhibitors of the large regulatory enzyme glycogen phosphorylase (GP) have been studied in crystallographic and kinetic experiments. GP catalyses the first step in the phosphorylysis of glycogen to glucose-l-phosphate, which is utilized via glycolysis to provide energy to sustain muscle contraction and in the liver is converted to glucose. alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase form b (GPb, K(i) = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several N-acetyl glucopyranosylamine derivatives have been synthesized and tested in a series of crystallographic and kinetic binding studies with GPb. The structural results of the bound enzyme-ligand complexes have been analysed together with the resulting affinities in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. Comparison of the N-methylacetyl glucopyranosylamine (N-methylamide, K(i) = 0.032 mM) with the analogous beta-methylamide derivative (C-methylamide, K(i) = 0.16 mM) illustrate the importance of forming good hydrogen bonds and obtaining complementarity of van der Waals interactions. These studies also have shown that the binding modes can be unpredictable but may be rationalized with the benefit of structural data and that a buried and mixed polar/non-polar catalytic site poses problems for the systematic addition of functional groups. Together with previous studies of glucose analogue inhibitors of GPb, this work forms the basis of a training set suitable for three-dimensional quantitative structure-activity relationship studies. The molecules in the training set are void of problems and potential errors arising from the alignment and bound conformations of each of the ligands since the coordinates were those determined experimentally from the X-ray crystallographic refined ligand-enzyme complexes. The computational procedure described in this work involves the use of the program GRID to describe the molecular structures and the progam GOLPE to obtain the partial least squares regression model with the highest prediction ability. The GRID/GOLPE procedure performed using 51 glucose analogue inhibitors of GPb has good overall predictivity [standard deviation of error predictions (SDEP) = 0.98 and Q(2) = 0.76] and has shown good agreement with the crystallographic and kinetic results by reliably selecting regions that are known to affect the binding affinity.

alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase b (Ki = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that, in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several alpha- and beta-anhydroglucoheptonic acid derivatives and 1-deoxy-1-thio-beta-D-glucose analogues have been synthesized and tested in a series of crystallographic and kinetic binding studies with glycogen phosphorylase. The structural results of the bound enzyme-ligand complexes have been analyzed, together with the resulting affinities, in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. This work has shown the following: (i) Similar affinities may be obtained through different sets of interactions. Specifically, in the case of the alpha- and beta-glucose-C-amides, similar Ki's (0.37 and 0.44 mM, respectively) are obtained with the alpha-anomer through interactions from the ligand via water molecules to the protein and with the beta-anomer through direct interaction from the ligand to the protein. Thus, hydrogen bonds through water can contribute binding energy similar to that of hydrogen bonds directly to the protein. (ii) Attempts to improve the inhibition by additional groups did not always lead to the expected result. The addition of nonpolar groups to the alpha-carboxamide resulted in a change in conformation of the pyranose ring from a chair to a skew boat and the consequent loss of favorable hydrogen bonds and increase in the Ki. (iii) The addition of polar groups to the alpha-carboxamide led to compounds with the chair conformation, and in the examples studied, it appears that hydration by a water molecule may provide sufficient stabilization to retain the chair conformation. (iv) The best inhibitor was N-methyl-beta-glucose-C-carboxamide (Ki = 0.16 mM), which showed a 46-fold improvement in Ki from the parent beta-D-glucose. The decrease in Ki may be accounted for by a single hydrogen bond from the amide nitrogen to a main-chain carbonyl oxygen, an increase in entropy through displacement of a water molecule, and favorable van der Waals contacts between the methyl substituent and nonpolar protein residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Werner’s syndrome is a rare disease of premature ageing. The WRN gene product defective in this disorder belongs to the RecQ helicase family and is thought to be involved in DNA metabolism. Another protein, which plays an important role in both DNA replication and repair, is the poly-ADP ribosyl transferase. Here we demonstrate an interaction of these two proteins resulting in ADP-ribosylation of the WRN protein. These results imply that WRN is involved in DNA replication and in DNA repair.

The Fab fragment of a rat monoclonal antibody (no. 192) with very high affinity for the main immunogenic region of the human muscle nicotinic acetylcholine receptor (AChR) has been purified, characterised and crystallised using vapour diffusion techniques. Its Kd for human AChR was determined to be 5×10−11 M. Its cross-reactivity pattern suggests that residue α23 of the AChR strongly affects its epitope. Crystals suitable for X-ray analysis, obtained by micro- and macroseeding techniques, belong to the orthorhombic space group C2221 and they diffract to 2.8 A resolution using synchrotron radiation. The unit cell dimensions are α = 83.4 Å, b =110.0 Å and c = 212.2 Å and there are two Fab molecules per asymmetric unit.

Haemostasis depends on the balanced participation of von Willebrand factor (vWF), a large multimeric and multidomain glycoprotein with essential role during the initial steps of blood clotting. Mature vWF circulates in plasma with the form of multimers comprised of several domains with diverse functions. More specifically, the A1 domain of vWF plays crucial role in haemostasis, regulating the mechanism of platelet adhesion in sites of vascular injury while A2 domain regulates the normal turnover of vWF. Recent studies have implied that an intramolecular interaction between A1 and A2 domains exists, which prevents platelets adhesion and subsequently inhibits the initial step of the blood coagulation mechanism. In an effort to elucidate the essential nature of the interaction between these two domains, we produced and purified the corresponding recombinant unmodified polypeptides. The secondary structure of the two domains was studied individually and as a mixture using circular dichroism spectroscopy. The observed interaction was verified by ELISA competition assays using antibodies and their ability to form productive interactions was further characterized kinetically. In silico analysis (docking and molecular dynamics simulations) of the A1-A2 binding indicated three possible structural models highlighting the crucial, for this interaction, region.