Crystal structures of apo-form and binary/ternary complexes of Podophyllum secoisolariciresinol dehydrogenase, an enzyme involved in formation of health-protecting and plant defense lignans.

(-)-Matairesinol is a central biosynthetic intermediate to numerous 8-8'-lignans, including the antiviral agent podophyllotoxin in Podophyllum species and its semi-synthetic anticancer derivatives teniposide, etoposide, and Etopophos. It is formed by action of an enantiospecific secoisolariciresinol dehydrogenase, an NAD(H)-dependent oxidoreductase that catalyzes the conversion of (-)-secoisolariciresinol. Matairesinol is also a plant-derived precursor of the cancer-preventative "mammalian" lignan or "phytoestrogen" enterolactone, formed in the gut following ingestion of high fiber dietary foodstuffs, for example.

Protocol optimization of multidetector computed tomography colonography using pig colonic phantoms.

Objective: The introduction of multidetector computed tomography (CT) increases the possibility of enhanced polyp detection using CT colonography. The effects of collimation and tube currents on polyp detection were investigated in pig colonic phantoms to determine the optimal scanning parameters.

Materials And Methods: Twenty pig colons were prepared, and polyps with diameters ranging from 3 mm to 15 mm were created and randomly distributed.

DNA hypomethylation is prevalent even in low-grade breast cancers.

Hypomethylation of some portions of the genome and hypermethylation of others are very frequent attributes of human cancer. We previously showed that cancer-associated DNA hypomethylation often involves satellite 2 (Sat2), the main DNA component of the large juxtacentromeric (centromere-adjacent) heterochromatin of chromosome 1. In this study, we compared methylation of Sat2 and centromeric satellite DNA (Satalpha) as well as overall genomic methylation in 41 breast adenocarcinomas of known tumor grade and stage, 16 non-neoplastic breast tissues (mostly fibroadenomas), and a variety of normal somatic tissue controls.

Purification, crystallization and preliminary crystallographic studies of the ligand-binding domain of a plant vacuolar sorting receptor.

Vacuolar sorting receptor (VSR) proteins bind soluble protein ligands in a sequence-specific manner and target them to the lytic vacuole in plant cells. A VSR from Arabidopsis thaliana, AtBP80b, has been successfully purified after heterologous expression in Drosophila S2 cells. The AtBP80b protein (560 amino acids) was crystallized by the hanging-drop method with a PEG 400-based precipitant.

Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: effects of side-chain size on reduction potential.

Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles. Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding. Our crystal structures of [V44L] (at 1.

DD1.5k, the gene preferentially expressed in bloodstream isolates of vancomycin-resistant Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium (VREFM) is becoming a threatening pathogen. We identified a gene called DD1.5K by differential display-PCR, which was preferentially expressed in the bloodstream isolates of VREFM.

The role of adipocytokines in adipocyte-related pathological processes.

It is becoming clear that adipose tissue is not merely a storage for excess energy but that it secretes a number of biologically active soluble factors collectively termed adipocytokines that control glucose and fatty acid metabolism. Of these adipocytokines, adiponectin and resistin have been the objects of intensive research, as they are implicated in obesity and diabetes-related diseases. In this review, we summarize recent advances in understanding the roles of adiponectin and resistin in the causation of metabolic diseases and consider the prospects for treating metabolic disorders by targeting these two adipocytokines.

Use of monoclonal antibodies that recognize p60 for identification of Listeria monocytogenes.

Listeria monocytogenes causes major food-borne outbreaks of disease worldwide. Specific identification of this microorganism is of utmost importance to public health and industry. Listeria species are known to secrete a 60-kDa protein collectively termed p60, which is encoded by the iap (invasion-associated protein) gene and secreted in large quantities into the growth media.

The unique hydrogen bonded water in the reduced form of Clostridium pasteurianum rubredoxin and its possible role in electron transfer.

Rubredoxin is a small iron-sulfur (FeS4) protein involved in oxidation-reduction reactions. The side chain of Leu41 near the iron-sulfur center has two conformations, which we suggested previously serve as a gate for a water molecule during the electron transfer process. To establish the role of residue 41 in electron transfer, an [L41A] mutant of Clostridium pasteurianum rubredoxin was constructed and crystallized in both oxidation states.

Adipocyte determination- and differentiation-dependent factor 1/sterol regulatory element-binding protein 1c regulates mouse adiponectin expression.

Adiponectin is exclusively expressed in differentiated adipocytes and plays an important role in regulating energy homeostasis, including the glucose and lipid metabolism associated with increased insulin sensitivity. However, the control of adiponectin gene expression in adipocytes is poorly understood. We show here that levels of adiponectin mRNA and protein are reduced in the white adipose tissue of ob/ob and db/db mice and that there is a concomitant reduction of the adipocyte determination- and differentiation-dependent factor 1 (ADD1)/sterol regulatory element-binding protein 1c (SREBP1c) transcription factor.

Comparing skeletal and cardiac calsequestrin structures and their calcium binding: a proposed mechanism for coupled calcium binding and protein polymerization.

Calsequestrin, the major calcium storage protein of both cardiac and skeletal muscle, binds and releases large numbers of Ca(2+) ions for each contraction and relaxation cycle. Here we show that two crystal structures for skeletal and cardiac calsequestrin are nearly superimposable not only for their subunits but also their front-to-front-type dimers. Ca(2+) binding curves were measured using atomic absorption spectroscopy.

Common genetic polymorphisms in the promoter of resistin gene are major determinants of plasma resistin concentrations in humans.

Aims/hypothesis: Resistin is thought to be an important link between obesity and insulin resistance. It has been suggested that genetic polymorphism in the promoter of resistin gene is a determinant of resistin mRNA expression and possibly associated with obesity and insulin resistance. In this study, we investigated the association between the genotype of resistin promoter and its plasma concentrations.

Dual-probe radiofrequency ablation: an in vitro experimental study in bovine liver.

Objective: To determine whether alternative radiofrequency (RF) application with dual probes has advantages over sequential or simultaneous RF application for creating larger coagulation necrosis.

Materials And Methods: Using 2 17-gauge internally cooled electrodes and a 200-watt generator (CC-3 model, Radionics), RF energy was applied sequentially (group A, n = 20), simultaneously (group B, n = 20), or alternately (group C, n = 20) to explanted bovine liver. After preliminary experiments with a probe distance of 2-5 cm, a distance of 2 cm and 3 cm were chosen for main experiments.

Plasma resistin concentrations measured by enzyme-linked immunosorbent assay using a newly developed monoclonal antibody are elevated in individuals with type 2 diabetes mellitus.

Resistin is an adipocyte-derived peptide that might play a role in obesity and insulin resistance. However, its role in humans is largely unclear. Although many studies have measured the expression of human resistin in tissues, the circulating concentrations of resistin and its relation to metabolic parameters in humans are unknown.

Cloning and characterization of GITR ligand.

The gene encoding the natural ligand of murine glucocorticoid-induced tumor necrosis factor receptor (GITR) was cloned and characterized. The putative GITR ligand (GITRL) is composed of 173 amino acids with features resembling those of type II membrane proteins and is 51% identical to the human activation-inducible TNF receptor (AITR) ligand, TL6. Expression of the GITRL is restricted to immature and mature splenic dendritic cells.

Identification of a ligand for glucocorticoid-induced tumor necrosis factor receptor constitutively expressed in dendritic cells.

Glucocorticoid-induced tumor necrosis receptor (GITR) has been implicated in regulation of T cell suppression by CD25(+)CD4(+) regulatory T cells (Tregs). We isolated a cDNA encoding GITR ligand (GITRL) from mouse endothelioma cells. When stably expressed in HEK293 cells, its specific interaction with GITR was confirmed by flow cytometry with the use of GITR-Fc.

Repression of phospho-JNK and infarct volume in ischemic brain of JIP1-deficient mice.

Mice lacking JIP1, a scaffold protein that organizes JNK pathway components, were constructed independently by two groups. The proposed in vivo function, however, remains contradictory; One study reported that targeted disruption of the jip1 caused embryonic death due to the requirement of JIP1 for fertilized eggs (Thompson et al. [2001] J.

Crystal structures of pinoresinol-lariciresinol and phenylcoumaran benzylic ether reductases and their relationship to isoflavone reductases.

Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.

Prolonged culture of normal chorionic villus cells yields ICF syndrome-like chromatin decondensation and rearrangements.

Untreated cultures from normal chorionic villus (CV) or amniotic fluid-derived (AF) samples displayed dramatic cell passage-dependent increases in aberrations in the juxtacentromeric heterochromatin of chromosomes 1 or 16 (1qh or 16qh). They showed negligible levels of chromosomal aberrations in primary culture and no other consistent chromosomal abnormality at any passage. By passage 8 or 9, 82 +/- 7% of the CV metaphases from all eight studied samples exhibited 1qh or 16qh decondensation and 25 +/- 16% had rearrangements in these regions.

Hypomethylation and hypermethylation of DNA in Wilms tumors.

We quantitatively analysed hypermethylation at CpG islands in the 5' ends of 12 genes and one non-CpG island 5' region (MTHFR) in 31 Wilms tumors. We also determined their global genomic 5-methylcytosine content. Compared with various normal postnatal tissues, approximately 40-90% of these pediatric kidney cancers were hypermethylated in four of the genes, MCJ, RASSF1A, TNFRSF12 and CALCA as determined by a quantitative bisulfite-based assay (MethyLight).

C-kit associated with the transmembrane 4 superfamily proteins constitutes a functionally distinct subunit in human hematopoietic progenitors.

The transmembrane 4 superfamily (TM4SF) has come into prominence for its association with a wide range of cell surface molecules, especially integrins. We report that TM4SF molecules CD9, CD63, and CD81 are physically associated with c-kit receptor tyrosine kinase in the human factor-dependent myeloid cell line, MO7e. We characterized this complex using coimmunoprecipitation and colocalization methods.

Role of the CC chemokine receptor 9/TECK interaction in apoptosis.

Chemokine receptors are members of the G protein coupled receptor (GPCR) supergene family whose expression is highly restricted to hematopoietic cells. Although the primary role of chemokine and chemokine receptor interaction is believed to be regulation of chemotaxis of leukocytes, subsequent information clearly suggests that multiple immune regulatory functions are attributed to chemokine receptor signaling. We recently showed that activation of the CC chemokine 9 receptor (CCR9), a thymus-specific chemokine receptor, led to potent cFLIP(L)-independent resistance to cycloheximide-induced apoptosis and modest resistance to Fas-mediated apoptosis possibly via activation of multiple signaling components involving Akt and glycogen synthase kinase 3beta.

An intronic silencer of the mouse perforin gene.

We have previously shown that the perforin gene locus is comprised of eight DNase I hypersensitive sites (DHS). Seven (DHS I-DHS VII) of them were CTL-specific whereas one (DHS VIII) in the second intron was expressed in a wide range of cell types. DHS VIII was highly AT-rich (75%) and was comprised of multiple sets of high mobility group (HMG)-I/Y binding site, two potential Special AT-rich Binding protein (SATB-1)-binding sites, and a long stretch of CTAT repeats, indicating that DHS VIII may relate to nuclear matrix-associated region (MAR).

Mechanisms of organoselenium compounds in chemoprevention: effects on transcription factor-DNA binding.

Data obtained on the effects of selenium compounds on regulatory transcription factor-DNA binding by other laboratories are briefly reviewed, and some of our own results in this area are also presented. We assessed the in vitro and in vivo effects of the organoselenium compound 1,4-phenylenebis(methylene)selenocyanate (p-XSC) on the binding activities of the transcription factors nuclear factor-kappa B (NF-kappa B), activator protein-1 (AP-1), Sp1, and Sp3 using the HCT-116 (human colorectal adenocarcinoma) cell line as a model system. Using nuclear extracts, electrophoretic mobility shift assays were carried out to determine the extent of binding of the transcription factors to their respective consensus recognition sites on radiolabeled oligonucleotides.