Loss of tumor suppressor IGFBP4 drives epigenetic reprogramming in hepatic carcinogenesis.

Genomic sequencing of hepatocellular carcinoma (HCC) uncovers a paucity of actionable mutations, underscoring the necessity to exploit epigenetic vulnerabilities for therapeutics. In HCC, EZH2-mediated H3K27me3 represents a major oncogenic chromatin modification, but how it modulates the therapeutic vulnerability of signaling pathways remains unknown. Here, we show EZH2 acts antagonistically to AKT signaling in maintaining H3K27 methylome through epigenetic silencing of IGFBP4. ChIP-seq revealed enrichment of Ezh2/H3K27me3 at silenced loci in HBx-transgenic mouse-derived HCCs, including Igfbp4 whose down-regulation significantly correlated with EZH2 overexpression and poor survivals of HCC patients. Functional characterizations demonstrated potent growth- and invasion-suppressive functions of IGFBP4, which was associated with transcriptomic alterations leading to deregulation of multiple signaling pathways. Mechanistically, IGFBP4 stimulated AKT/EZH2 phosphorylation to abrogate H3K27me3-mediated silencing, forming a reciprocal feedback loop that suppressed core transcription factor networks (FOXA1/HNF1A/HNF4A/KLF9/NR1H4) for normal liver homeostasis. Consequently, the in vivo tumorigenicity of IGFBP4-silenced HCC cells was vulnerable to pharmacological inhibition of EZH2, but not AKT. Our study unveils chromatin regulation of a novel liver tumor suppressor IGFBP4, which constitutes an AKT-EZH2 reciprocal loop in driving H3K27me3-mediated epigenetic reprogramming. Defining the aberrant chromatin landscape of HCC sheds light into the mechanistic basis of effective EZH2-targeted inhibition.

Yin Yang 1-mediated epigenetic silencing of tumour-suppressive microRNAs activates nuclear factor-κB in hepatocellular carcinoma.

Enhancer of zeste homolog 2 (EZH2) catalyses histone H3 lysine 27 trimethylation (H3K27me3) to silence tumour-suppressor genes in hepatocellular carcinoma (HCC) but the process of locus-specific recruitment remains elusive. Here we investigated the transcription factors involved and the molecular consequences in HCC development. The genome-wide distribution of H3K27me3 was determined by chromatin immunoprecipitation coupled with high-throughput sequencing or promoter array analyses in HCC cells from hepatitis B virus (HBV) X protein transgenic mouse and human cell models. Transcription factor binding site analysis was performed to identify EZH2-interacting transcription factors followed by functional characterization. Our cross-species integrative analysis revealed a crucial link between Yin Yang 1 (YY1) and EZH2-mediated H3K27me3 in HCC. Gene expression analysis of human HBV-associated HCC specimens demonstrated concordant overexpression of YY1 and EZH2, which correlated with poor survival of patients in advanced stages. The YY1 binding motif was significantly enriched in both in vivo and in vitro H3K27me3-occupied genes, including genes for 15 tumour-suppressive microRNAs. Knockdown of YY1 reduced not only global H3K27me3 levels, but also EZH2 and H3K27me3 promoter occupancy and DNA methylation, leading to the transcriptional up-regulation of microRNA-9 isoforms in HCC cells. Concurrent EZH2 knockdown and 5-aza-2'-deoxycytidine treatment synergistically increased the levels of microRNA-9, which reduced the expression and transcriptional activity of nuclear factor-κB (NF-κB). Functionally, YY1 promoted HCC tumourigenicity and inhibited apoptosis of HCC cells, at least partially through NF-κB activation. In conclusion, YY1 overexpression contributes to EZH2 recruitment for H3K27me3-mediated silencing of tumour-suppressive microRNAs, thereby activating NF-κB signalling in hepatocarcinogenesis.

Phosphorylation in the catalytic cleft stabilizes and attracts domains of a phosphohexomutase.

Phosphorylation can modulate the activities of enzymes. The phosphoryl donor in the catalytic cleft of α-D-phosphohexomutases is transiently dephosphorylated while the reaction intermediate completes a 180° reorientation within the cleft. The phosphorylated form of 52 kDa bacterial phosphomannomutase/phosphoglucomutase is less accessible to dye or protease, more stable to chemical denaturation, and widely stabilized against NMR-detected hydrogen exchange across the core of domain 3 to juxtaposed domain 4 (each by ≥ 1.3 kcal/mol) and parts of domains 1 and 2. However, phosphorylation accelerates hydrogen exchange in specific regions of domains 1 and 2, including a metal-binding residue in the active site. Electrostatic field lines reveal attraction across the catalytic cleft between phosphorylated Ser-108 and domain 4, but repulsion when Ser-108 is dephosphorylated. Molecular dynamics (MD) simulated the dephosphorylated form to be expanded due to enhanced rotational freedom of domain 4. The contacts and fluctuations of the MD trajectories enabled correct simulation of more than 80% of sites that undergo either protection or deprotection from hydrogen exchange due to phosphorylation. Electrostatic attraction in the phosphorylated enzyme accounts for 1) domain 4 drawing closer to domains 1 and 3; 2) decreased accessibility; and 3) increased stability within these domains. The electrostriction due to phosphorylation may help capture substrate, whereas the opening of the cleft upon transient dephosphorylation allows rotation of the intermediate. The long-range effects of phosphorylation on hydrogen exchange parallel reports on protein kinases, suggesting a conceptual link among these multidomain, phosphoryl transfer enzymes.

Promotion of enzyme flexibility by dephosphorylation and coupling to the catalytic mechanism of a phosphohexomutase.

The enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa catalyzes an intramolecular phosphoryl transfer across its phosphosugar substrates, which are precursors in the synthesis of exoproducts involved in bacterial virulence. Previous structural studies of PMM/PGM have established a key role for conformational change in its multistep reaction, which requires a dramatic 180° reorientation of the intermediate within the active site. Here hydrogen-deuterium exchange by mass spectrometry and small angle x-ray scattering were used to probe the conformational flexibility of different forms of PMM/PGM in solution, including its active, phosphorylated state and the unphosphorylated state that occurs transiently during the catalytic cycle. In addition, the effects of ligand binding were assessed through use of a substrate analog. We found that both phosphorylation and binding of ligand produce significant effects on deuterium incorporation. Phosphorylation of the conserved catalytic serine has broad effects on residues in multiple domains and is supported by small angle x-ray scattering data showing that the unphosphorylated enzyme is less compact in solution. The effects of ligand binding are generally manifested near the active site cleft and at a domain interface that is a site of conformational change. These results suggest that dephosphorylation of the enzyme may play two critical functional roles: a direct role in the chemical step of phosphoryl transfer and secondly through propagation of structural flexibility. We propose a model whereby increased enzyme flexibility facilitates the reorientation of the reaction intermediate, coupling changes in structural dynamics with the unique catalytic mechanism of this enzyme.

Compromised catalysis and potential folding defects in in vitro studies of missense mutants associated with hereditary phosphoglucomutase 1 deficiency.

Recent studies have identified phosphoglucomutase 1 (PGM1) deficiency as an inherited metabolic disorder in humans. Affected patients show multiple disease phenotypes, including dilated cardiomyopathy, exercise intolerance, and hepatopathy, reflecting the central role of the enzyme in glucose metabolism. We present here the first in vitro biochemical characterization of 13 missense mutations involved in PGM1 deficiency. The biochemical phenotypes of the PGM1 mutants cluster into two groups: those with compromised catalysis and those with possible folding defects. Relative to the recombinant wild-type enzyme, certain missense mutants show greatly decreased expression of soluble protein and/or increased aggregation. In contrast, other missense variants are well behaved in solution, but show dramatic reductions in enzyme activity, with kcat/Km often <1.5% of wild-type. Modest changes in protein conformation and flexibility are also apparent in some of the catalytically impaired variants. In the case of the G291R mutant, severely compromised activity is linked to the inability of a key active site serine to be phosphorylated, a prerequisite for catalysis. Our results complement previous in vivo studies, which suggest that both protein misfolding and catalytic impairment may play a role in PGM1 deficiency.

A CCRK-EZH2 epigenetic circuitry drives hepatocarcinogenesis and associates with tumor recurrence and poor survival of patients.

BACKGROUND & AIMS: Aberrant chromatin modification is a key feature of hepatocellular carcinoma (HCC), which is characterized by strong sexual dimorphism. Both enhancer of zeste homolog 2 (EZH2) and cell cycle-related kinase (CCRK) contribute to hepatocarcinogenesis, yet whether the two oncogenic factors have functional crosstalk is unknown. METHODS: Cellular proliferation and tumorigenicity upon transgenic expression and RNA interference were determined by colony formation and soft agar assays, xenograft, orthotopic and diethylnitrosamine-induced HCC models. Gene regulation was assessed by chromatin immunoprecipitation, site-directed mutagenesis, luciferase reporter, co-immunoprecipitation and expression analyses. Protein levels in clinical specimens were correlated with clinicopathological parameters and patient survival rates. RESULTS: Ectopic CCRK expression in immortalized human liver cells increased EZH2 and histone H3 lysine 27 trimethylation (H3K27me3) to stimulate proliferation and tumor formation. Conversely, knockdown of CCRK reduced EZH2/H3K27me3 levels and decreased HCC cell growth, which could be rescued by EZH2 over-expression. Mechanistically, GSK-3ß phosphorylation by CCRK activated a ß-catenin/TCF/E2F1/EZH2 transcriptional feedback loop to epigenetically enhance androgen receptor (AR) signaling. Simultaneously, the phosphorylation of AKT/EZH2 by CCRK facilitated the co-occupancy of CCRK promoter by EZH2-AR and its subsequent transcriptional activation, thus forming a self-reinforcing circuitry. Lentiviral-mediated knockdown of CCRK, which abrogated the phosphorylation-transcriptional network, prevented diethylnitrosamine-induced tumorigenicity. More importantly, the hyperactivation of the CCRK-EZH2 circuitry in human HCCs correlated with tumor recurrence and poor survival. CONCLUSIONS: These findings uncover an epigenetic vicious cycle in hepatocarcinogenesis that operates through reciprocal regulation of CCRK and EZH2, providing novel therapeutic strategy for HCC.

A survey of student opinions on ethical design standards in Taiwan.

Design ethics has been offered as a course in undergraduate design programs in Taiwan for over a decade, but research on teaching design ethics and the results of teaching these courses is scant. We conducted two tests to examine (1) the effect of an ethics course, and (2) the differences among the effects of design department, gender, and study year on student opinions regarding ethical design standards (EDSs) at the National Yunlin University of Science and Technology (YunTech) in Taiwan. The participants comprised 934 undergraduates (660 women and 274 men) from the five design departments at YunTech's College of Design from Years 1-4. The results confirmed the effect of an ethics course on student EDS opinions. In addition, we observed significant variations among students according to design departments, suggesting that the characteristics of the design departments also affected students' EDS opinions. The results indicated that gender did not significantly affect design students' EDS opinions; however, students in their early years of study produced higher scores than those in their advanced years of study did, based on the six EDS opinions. The implications of these results for teaching design ethics and future research are discussed in this paper.

Cell cycle-related kinase mediates viral-host signalling to promote hepatitis B virus-associated hepatocarcinogenesis.

BACKGROUND: Androgen receptor (AR) signalling contributes to male predominance in hepatocellular carcinoma (HCC), which is more pronounced in HBV-endemic areas. Cell cycle-related kinase (CCRK) is essential for AR-induced hepatocarcinogenesis but its molecular function in HBV-associated HCC remains obscure. OBJECTIVE: To determine the molecular function of CCRK in HBV-associated HCC. DESIGN: Transcriptional regulation was assessed by chromatin immunoprecipitation, promoter mutation and luciferase reporter assays. Hepatocellular proliferation and tumourigenesis were examined by colony formation, soft agar assays and using HBV X protein (HBx) transgenic mice with low-dose exposure to diethylnitrosamine. Protein expressions were examined in clinical samples and correlated with patient survival by log-rank Mantel-Cox test. RESULTS: Overexpression of CCRK, but not its kinase-defective mutant, activated ß-catenin/T cell factor signalling through phosphorylation of glycogen synthase kinase-3ß (GSK-3ß) at Ser9, led to upregulation of AR transcriptional activity and, subsequently, expression of HBx. The viral transactivator in turn induced CCRK expression through enhanced AR signalling, thus forming a positive regulatory loop. RNA interference silencing of CCRK, which suppressed the CCRK/GSK-3ß/ß-catenin/AR regulatory loop, significantly suppressed HBx-induced hepatocellular proliferation (p=0.001) and transformation (p<0.001) and remarkably reduced >80% diethylnitrosamine-mediated hepatocarcinogenesis in HBx transgenic mice. Finally, patients with HBV-associated HCC with concordant overexpression of CCRK, GSK-3ß phosphorylation at Ser9, active dephosphorylated ß-catenin and AR phosphorylation at Ser81 had poorer overall (HR=31.26, p<0.0001) and disease-free (HR=3.60, p<0.01) survival rates. CONCLUSIONS: Our findings highlight the critical role of CCRK in a self-reinforcing circuitry that regulates HBV-associated hepatocarcinogenesis. Further characterisation of this intricate viral-host signalling may provide new prognostic biomarkers and therapeutic targets for HCC treatment.

Solution NMR of a 463-residue phosphohexomutase: domain 4 mobility, substates, and phosphoryl transfer defect.

Phosphomannomutase/phosphoglucomutase contributes to the infectivity of Pseudomonas aeruginosa, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. (13)C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 α-helices, C-capping motifs, and 20 of the 22 ß-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These can be attributed to the phosphorylation state and possibly to conformational interconversions. The S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose 1-phosphate substrate by impairing k(cat). Addition of the glucose 1,6-bisphosphate intermediate accelerated this reaction by 2-3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft while preserving the secondary structure in solution. Diminished peak heights and faster (15)N relaxation suggest line broadening and millisecond fluctuations within four loops that can contact phosphosugars. (15)N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1-3, and with a different principal axis of diffusion. This adds to the crystallographic evidence of domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of the intermediate, substrate binding, and product release.

Craniocervical arterial dissection: a cause of childhood arterial ischemic stroke in Taiwan.

BACKGROUND/PURPOSE: To describe the clinical characteristics and imaging findings of craniocervical dissection in childhood ischemic stroke, in a tertiary medical center. METHODS: In this retrospective study, we investigated children (aged 1 month to 18 years) with symptoms and radiographic confirmation of ischemic stroke from January 1996 to January 2007. Stroke work-up included neuroimaging (magnetic resonance imaging, computed tomography, conventional angiography, and magnetic resonance angiography), cardiac assessment, prothrombotic assays, immunoassays, infection screening, and metabolic screening. RESULTS: Among 95 children with arterial ischemic stroke, arterial dissection was identified as the underlying risk factor in nine patients (7 boys and 2 girls; age range, 1.9 17.2 years). All the patients had focal neurological signs and two had warning symptoms. A history of trauma was noted in two patients and another two had stroke during physical exertion. The other five patients had spontaneous dissection. Six patients had anterior circulation arterial dissection. Three patients had posterior circulation arterial dissection, and the most common location was in the vertebral artery. Antiplatelet treatment was given to five patients and anticoagulants to one. Endovascular treatment was given to one patient with dissecting aneurysm. One patient died at the acute stage and another seven had neurological deficits after 9 months to 8 years follow-up. The ninth patient had no residual neurological impairment. No patients had recurrent stroke. CONCLUSION: Arterial dissection should be considered in childhood ischemic stroke. Spontaneous arterial dissection is an important factor in this group. Early investigation and treatment can improve the outcome.

[Traditional Chinese medicine dietary regimens for the elderly].

Food-based nutrition is a main source of physical and spiritual energy and the basis of maintaining healthy growth, development, and regular activity. Because the elderly experience organ degeneration and insufficient energy and nutrition, together with chronic diseases, they need a diet able to deliver proper nutrition and calories. Such diets can help strengthen the physical condition, prevent disease, and even control and treat disease. Therefore, dietary regimens have been viewed as the most basic form of geriatric care, and have also proven highly effective. Traditional Chinese medicine has accumulated abundant experience in the implementation of dietary regimens for the elderly for more than three thousand years. Main Chinese medicine principles addressed in this article include: eating at set times; eating food in appropriate quantities; eating plain food items; eating food that is cooked, soft and warm; chewing carefully and swallowing slowly; having a hearty breakfast, an adequate lunch, and a small dinner; maintaining a balanced diet; eating a mix of flavors; adjusting the diet as needed; choosing appropriate foods based on specific body needs / conditions / diseases; and taking regular walks, rinsing the mouth and massaging the abdomen gently after meals. These suggestions, if followed properly, can contribute significantly to elderly health.

Risk factors and outcomes of childhood ischemic stroke in Taiwan.

In this retrospective study, we reviewed the charts and collected clinical and radiographic data on children (age range, 1 month to 18 years) with symptoms and radiographic confirmation of ischemic stroke for the period of January 1996 to July 2006. Ninety-four children were enrolled. Eighty-eight had arterial ischemic stroke and six had sinovenous thrombosis. Twenty-nine percent of the children had seizures. Twenty-six percent had diffuse neurological signs and 76% had focal neurological signs. Risk factors included vascular disease (33%), infection (27%), metabolic disorders (18%), trauma (11%), prothrombotic states (13%), cardiac disease (10%), and mitochondrial disease (6%). Ten percent (n=9) had no identifiable cause. Twenty-two percent of the children had more than one risk factor. Anterior territory (70%) was more involved than posterior territory (18%) in arterial ischemic stroke. Unilateral infarctions were more common on the left side (51%) than on the right (24.5%). Neurological deficits were present in 45% (n=34/75) of the children; the most frequent deficit was motor impairment (24%). Seven children (9%) died in the acute stage. There were 12 children (16%) who had recurrent stroke and 8 children (8/12) who had underlying vascular disease. The vascular disease included moyamoya disease (5), CNS lupus (1) and ill-defined vasculopathy (2). The etiology pattern in Taiwan was different from that in Western countries. Vascular disease was a significant risk factor for recurrence in childhood ischemic stroke.

Agenesis of the corpus callosum in Turner's syndrome: report of a case and review of the literature.

Turner's syndrome (TS) is a genetic disorder caused by loss of entire or a substantial part of the X-chromosome, but association with central nervous system (CNS) abnormalities is rarely reported. A 32-year-old female with TS was found to have agenesis of the corpus callosum (ACC) and various clinical features including coarctation of aorta, hypertelorism, small jaw, short and webbed neck, cubitus valgus, and absence of the uterus. Karyotype analysis revealed X monosomy cell line (45, X). There have been only three other cases of TS associated with ACC. High prenatal lethality of TS fetuses with congenital CNS malformations may decrease the incidence of this association. Neuropsychological studies showed a normal intelligence neither prominent learning disability nor discrepancy between verbal and non-verbal items.

Antiviral therapy reduces risk of haemorrhagic stroke in patients with HCV infection: a nationwide cohort study.

BACKGROUND: The tendency for haemorrhagic stroke in patients with chronic HCV infection has emerged recently but the finding may be confounded by comorbidities. Proving the causality between HCV infection and haemorrhagic stroke is mandatory. Our study was designed to investigate the incidence of intracranial haemorrhage in HCV-infected patients with and without treatment. METHODS: In the 11-year and population-based retrospective study, we acquired data from the Taiwan National Health Insurance Research Database. The patients with major comorbidities were excluded and 97,198 HCV-infected patients were included for analysis. Treated and untreated cohorts were matched with propensity score to make the confounding factors in two groups comparable. Cox proportional hazard regression analysis was performed to evaluate the hazard ratio of haemorrhagic stroke in the cohorts. We applied survival analysis to compare the cumulative incidence of outcome events between the two cohorts. RESULTS: After matching, the incidence density (ID) of haemorrhagic stroke in the untreated cohort is significantly higher than in the treated cohort (ID: 1.0 versus 0.6 events per 1,000 person-years; P=0.0014). The adjusted hazard ratio (aHR) of haemorrhagic stroke is significantly reduced in the treated group (P<0.05). Cumulative incidence of haemorrhagic stroke is significantly lower in the treated group than in the untreated group (P=0.013). CONCLUSIONS: The study demonstrates that antiviral therapy significantly reduces the events of intracranial haemorrhage in HCV-infected patients and consolidates the novel concept that chronic HCV infection is a risk factor for haemorrhagic stroke.

Data on the phosphorylation state of the catalytic serine of enzymes in the α-D-phosphohexomutase superfamily.

Most enzymes in the α-D-phosphohexomutase superfamily catalyze the reversible conversion of 1- to 6-phosphosugars. They play important roles in carbohydrate and sugar nucleotide metabolism, and participate in the biosynthesis of polysaccharides, glycolipids, and other exoproducts. Mutations in genes encoding these enzymes are associated with inherited metabolic diseases in humans, including glycogen storage disease and congenital disorders of glycosylation. Enzymes in the superfamily share a highly conserved active site serine that participates in the multi-step phosphoryl transfer reaction. Here we provide data on the effects of various phosphosugar ligands on the phosphorylation of this serine, as monitored by electrospray ionization mass spectrometry (ESI-MS) data on the intact proteins. We also show data on the longevity of the phospho-enzyme under various solution conditions in one member of the superfamily from Pseudomonas aeruginosa, and present inhibition data for several ligands. These data should be useful for the production of homogeneous samples of phosphorylated or unphosphorylated proteins, which are essential for biophysical characterization of these enzymes.

Phosphorylation-Dependent Effects on the Structural Flexibility of Phosphoglucosamine Mutase from Bacillus anthracis.

Phosphoglucosamine mutase (PNGM) is an evolutionarily conserved bacterial enzyme in the peptidoglycan biosynthetic pathway, catalyzing the reversible conversion between glucosamine 1- and 6-phosphate. Previous structural studies of PNGM from the pathogen Bacillus anthracis revealed its dimeric assembly and highlighted the rotational mobility of its C-terminal domain. Recent studies of two other enzymes in the same superfamily have demonstrated the long-range effects on the conformational flexibility associated with phosphorylation of the conserved, active site phosphoserine involved in phosphoryl transfer. Building on this work, we use a combination of experimental and computational studies to show that the active, phosphorylated version of B. anthracis PNGM has decreased flexibility relative to its inactive, dephosphorylated state. Limited proteolysis reveals an enhanced and accelerated cleavage of the dephosphorylated enzyme. 15N transverse relaxation-optimized NMR spectra corroborate a conformational adjustment with broadening and shifts of peaks relative to the phospho-enzyme. Electrostatic calculations indicate that residues in the mobile, C-terminal domain are linked to the phosphoserine by lines of attraction that are absent in the dephosphorylated enzyme. Phosphorylation-dependent changes in protein flexibility appear linked with the conformational change and enzyme mechanism in PNGM, establishing this as a conserved theme in multiple subgroups of the diverse α-d-phosphohexomutase superfamily.

Dissecting the pleiotropic actions of HBx mutants against hypoxia in hepatocellular carcinoma.

Error-prone integration of the hepatitis B virus X protein (HBx) into the hepatocellular genome generates a multitude of mutants exerting diverse effects on the development and progression of hepatocellular carcinoma (HCC). A recent study by Lai and colleagues revealed the disparate regulatory activity of clinically-predominant HBx mutants towards hypoxia-inducible factor-1α (HIF-1α), a central regulator of tumor angiogenesis, proliferation, metastasis and differentiation. These findings have shed insight into specific viral contribution of hypoxic response during hepatocarcinogenesis.

Automatic detection and quantification of acute cerebral infarct by fuzzy clustering and histographic characterization on diffusion weighted MR imaging and apparent diffusion coefficient map.

Determination of the volumes of acute cerebral infarct in the magnetic resonance imaging harbors prognostic values. However, semiautomatic method of segmentation is time-consuming and with high interrater variability. Using diffusion weighted imaging and apparent diffusion coefficient map from patients with acute infarction in 10 days, we aimed to develop a fully automatic algorithm to measure infarct volume. It includes an unsupervised classification with fuzzy C-means clustering determination of the histographic distribution, defining self-adjusted intensity thresholds. The proposed method attained high agreement with the semiautomatic method, with similarity index 89.9 ± 6.5%, in detecting cerebral infarct lesions from 22 acute stroke patients. We demonstrated the accuracy of the proposed computer-assisted prompt segmentation method, which appeared promising to replace the laborious, time-consuming, and operator-dependent semiautomatic segmentation.

Identification of an essential active-site residue in the α-D-phosphohexomutase enzyme superfamily.

UNLABELLED: Enzymes in the α-d-phosphohexomutase superfamily catalyze the conversion of 1-phosphosugars to their 6-phospho counterparts. Their phosphoryl transfer reaction has long been proposed to require general acid-base catalysts, but candidate residues for these key roles have not been identified. In this study, we show through mutagenesis and kinetic studies that a histidine (His329) in the active site is critical for enzyme activity in a well-studied member of the superfamily, phosphomannomutase/phosphoglucomutase from Pseudomonas aeruginosa. Crystallographic characterization of an H329A mutant protein showed no significant changes from the wild-type enzyme, excluding structural disruption as the source of its compromised activity. Mutation of the structurally analogous lysine residue in a related protein, phosphoglucomutase from Salmonella typhimurium, also results in significant catalytic impairment. Analyses of protein-ligand complexes of the P. aeruginosa enzyme show that His329 is appropriately positioned to abstract a proton from the O1/O6 hydroxyl of the phosphosugar substrates, and thus may serve as the general base in the reaction. Histidine is strongly conserved at this position in many proteins in the superfamily, and lysine is also often conserved at a structurally corresponding position, particularly in the phosphoglucomutase enzyme sub-group. These studies shed light on the mechanism of this important enzyme superfamily, and may facilitate the design of mechanism-based inhibitors. DATABASE: Structural data have been deposited in the Protein Data Bank with accession number 4IL8.

A coevolutionary residue network at the site of a functionally important conformational change in a phosphohexomutase enzyme family.

Coevolution analyses identify residues that co-vary with each other during evolution, revealing sequence relationships unobservable from traditional multiple sequence alignments. Here we describe a coevolutionary analysis of phosphomannomutase/phosphoglucomutase (PMM/PGM), a widespread and diverse enzyme family involved in carbohydrate biosynthesis. Mutual information and graph theory were utilized to identify a network of highly connected residues with high significance. An examination of the most tightly connected regions of the coevolutionary network reveals that most of the involved residues are localized near an interdomain interface of this enzyme, known to be the site of a functionally important conformational change. The roles of four interface residues found in this network were examined via site-directed mutagenesis and kinetic characterization. For three of these residues, mutation to alanine reduces enzyme specificity to ~10% or less of wild-type, while the other has ~45% activity of wild-type enzyme. An additional mutant of an interface residue that is not densely connected in the coevolutionary network was also characterized, and shows no change in activity relative to wild-type enzyme. The results of these studies are interpreted in the context of structural and functional data on PMM/PGM. Together, they demonstrate that a network of coevolving residues links the highly conserved active site with the interdomain conformational change necessary for the multi-step catalytic reaction. This work adds to our understanding of the functional roles of coevolving residue networks, and has implications for the definition of catalytically important residues.