Stromal Cells in the Pathogenesis of Inflammatory Bowel Disease.

Up till now, research on inflammatory bowel disease [IBD] has mainly been focused on the immune cells present in the gastrointestinal tract. However, recent insights indicate that stromal cells also play an important and significant role in IBD pathogenesis. Stromal cells in the intestines regulate both intestinal epithelial and immune cell homeostasis. Different subsets of stromal cells have been found to play a role in other inflammatory diseases [e.g. rheumatoid arthritis], and these various stromal subsets now appear to carry out also specific functions in the inflamed gut in IBD. Novel potential therapies for IBD utilize, as well as target, these pathogenic stromal cells. Injection of mesenchymal stromal cells [MSCs] into fistula tracts of Crohn's disease patients is already approved and used in clinical settings. In this review we discuss the current knowledge of the role of stromal cells in IBD pathogenesis. We further outline recent attempts to modify the stromal compartment in IBD with agents that target or replace the pathogenic stroma.

Xenotransplantation of human intestine into mouse abdomen or subcutaneous tissue: Novel platforms for the study of the human enteric nervous system.

BACKGROUND: Current efforts to develop stem cell therapy as a novel treatment for neurointestinal diseases are limited by the unavailability of a model system to study cell transplantation in the human intestine. We propose that xenograft models support enteric nervous system (ENS) development in the fetal human intestine when transplanted into mice subcutaneously or intra-abdominally. METHODS: Fetal human small and large intestine were grafted onto the small intestinal mesentery and into the subcutaneous tissue of immunodeficient mice for up to 4 months. Intestinal cytoarchitecture and ENS development were studied using immunohistochemistry. KEY RESULTS: In both abdominal and subcutaneous grafts, the intestine developed normally with formation of mature epithelial and mesenchymal layers. The ENS was patterned in two ganglionated plexuses containing enteric neurons and glia, including cholinergic and nitrergic neuronal subtypes. c-Kit-immunoreactive interstitial cells of Cajal were present in the gut wall. CONCLUSIONS & INFERENCES: Abdominal xenografts represent a novel model that supports the growth and development of fetal human intestine. This in vivo approach will be a useful method to study maturation of the ENS, the pathophysiology of neurointestinal diseases, and the long-term survival and functional differentiation of neuronal stem cells for the treatment of enteric neuropathies.

Enhanced immunostimulatory activity of in silico discovered agonists of Toll-like receptor 2 (TLR2).

BACKGROUND: Emergent therapies in anticancer vaccination use Toll-like receptors (TLRs) agonists as dendritic cell (DC) vaccine adjuvants. DCs from the patient are isolated, stimulated with TLR agonists and tumor antigens ex vivo and then infused back into the patient. Although some TLR ligands have been tested in clinical trials, novel TLR agonists with improved immunomodulatory properties are essential to optimize treatment success. We report on the discovery of small-molecule TLR2 agonists, with favorable properties as synthetic adjuvants. METHODS: We performed a shape- and featured-based similarity virtual screening against a commercially available compound library. The selected virtual hits were experimentally tested in TLR2-reporter cells and their activity in phagocytes and DCs was characterized. A binding model of the compounds to TLR2 (docking studies) was proposed. RESULTS: Through a virtual screening approach against a library of three million compounds four virtual hits (AG1, AG2, AG3, AG4) were found to synergistically augment the NF-kB activation induced by the lipopeptide ligand Pam3CSK4 in luciferase reporter assays using HEK293-TLR2 cells. Biacore experiments indicated that AG1-AG4 are ago-allosteric modulators of TLR2 and AG2 bound TLR2 with high affinity (KD 0.8µM). The compounds induced TNF-α production in human peripheral blood mononuclear cells (PBMCs) and they activated DCs as indicated by IL-12 production and upregulation of CD83/CD86. CONCLUSIONS: Following a combined in silico/in vitro approach we have discovered TLR2-agonists (AG1-AG4) that activate human and mouse immune cells. GENERAL SIGNIFICANCE: We introduce four novel TLR2 ago-allosteric modulators that stimulate myeloid cell activity and constitute promising candidates as synthetic adjuvants.

Lasing of Tamm states in highly efficient organic devices based on small-molecule organic semiconductors.

We discuss approaches to increase the light outcoupling efficiency in organic microcavity (MC) lasers and organic light-emitting diodes (OLEDs). We find that the introduction of metals into the cavities leads to additional Tamm-plasmon polariton modes, while the corrugation of metal contacts, such as perforated µ-size holes or a periodic array of metal stripes, leads to 2D confinement of the cavity modes, which in turn reduces the lasing threshold in MCs. Furthermore, we elucidate light loss mechanisms in OLEDs and reveal how external dielectric layers and periodic gratings can be used to enhance outcoupling from the OLED cavity.

Usage of international standards for integrating extramural monitoring and personal health device data into medical information infrastructure.

Integrating extramural measured devices data into medical information systems is becoming more and more attractive for integrated medical care. A lot of devices already have the ability to transfer measured data to mobile devices or computers and a few systems offer submitting data to a centralized information database or information system. Unfortunately, all of these devices use proprietary protocols and processes which makes integration into other systems a major problem. To address this problem the Healthy Interoperability project has been created with the objective of creating a framework for transferring health data based on international standards. The paper outlines how the framework architecture takes full advantage from the definitions of the international standards ISO 11073, HL7, IHE and CEN 13606. Even the definition of the user profiles and the security framework is based on standards from ETSI, ISO and CEN. By using these standards the framework can also perfectly be used for intramural communication.

Flip-flop-induced relaxation of bending energy: implications for membrane remodeling.

Cellular and organellar membranes are dynamic materials that underlie many aspects of cell biology. Biological membranes have long been thought of as elastic materials with respect to bending deformations. A wealth of theory and experimentation on pure phospholipid membranes provides abundant support for this idea. However, biological membranes are not composed solely of phospholipids--they also incorporate a variety of amphiphilic molecules that undergo rapid transbilayer flip-flop. Here we describe several experimental systems that demonstrate deformation-induced molecular flip-flop. First we use a fluorescence assay to track osmotically controlled membrane deformation in single component fatty acid vesicles, and show that the relaxation of the induced bending stress is mediated by fatty acid flip-flop. We then look at two-component phospholipid/cholesterol composite vesicles. We use NMR to show that the steady-state rate of interleaflet diffusion of cholesterol is fast relative to biological membrane remodeling. We then use a Förster resonance energy transfer assay to detect the transbilayer movement of cholesterol upon deformation. We suggest that our results can be interpreted by modifying the area difference elasticity model to account for the time-dependent relaxation of bending energy. Our findings suggest that rapid interleaflet diffusion of cholesterol may play a role in membrane remodeling in vivo. We suggest that the molecular characteristics of sterols make them evolutionarily preferred mediators of stress relaxation, and that the universal presence of sterols in the membranes of eukaryotes, even at low concentrations, reflects the importance of membrane remodeling in eukaryotic cells.

The recent decline of Montastraea annularis (complex) coral populations in western Curaçao: a cause for concern? / La reciente disminución de las poblaciones de coral Montastraea annularis (complejo) en el oeste de Curazao: ¿un motivo de preocupación?

Shallow leeward reefs off the western end of Curaçao are dominated by extensive populations of M. annularis (complex). These species are larger in size (mean= 66 cm diameter) than all other species, with few small colonies (<30 cm) and notable absence of recruits. In 1998, colonies of M. annularis (complex) accounted for more then 45% of all species >10 cm observed within transects, and most exhibited low levels of partial mortality (mean= 22.5%). These species were less abundant (38% of all colonies) in 2005. Partial mortality among live colonies of M. annularis and M. faveolata increased by 85% (mean = 42% partial mortality) and numerous dead colonies of M. faveolata and M. annularis were observed; M. franksi colonies were generally in excellent condition (14% partial tissue mortality). A high prevalence of coral diseases (3-30%) was documented among M. annularis and M. faveolata, while all other species were less frequently affected. Yellow band disease (YBD) emerged shortly after the 1995 bleaching event, and rapidly spread throughout all depths, with the highest prevalence between 1997-1999. YBD caused slow rates of mortality (=1 cm/month), but multiple focal lesions appeared on individual colonies, and these progressively radiated outward as they killed the colonies. By 2005, 44% of the tagged corals were dead; the remainder exhibited active YBD infections (21%) or were in remission (31.6%) but were missing on average >90% of their tissue. Although the incidence of YBD has declined since 2000, white plague (WP) prevalence was unusually high (4-12%) in 2001 and 2005, with affected colonies exhibiting recent mortality of up to 70%. Dead Montastraea spp. surfaces are being colonized by other corals, including poritids, agaricids, and other faviids, while recruits of M. annularis (complex) are absent. If diseases and other biotic stressors persist on these reefs, M. annularis and M. faveolata populations may undergo a decline similar to that observed in the 1980s among Caribbean acroporids. Rev. Biol. Trop. 54 (Suppl. 3): 45- 58. Epub 2007 Jan. 15.

Los arrecifes someros a sotavento del oeste de Curaçao están dominados por extensas poblaciones de Montastraea annularis (complejo). Estas especies son mayores en tamaño (promedio= 66 cm de diámetro) que las otras especies, con algunas colonias pequeñas (<30 cm) y una notable ausencia de reclutas. En 1998, las colonias de Montastraea annularis (complejo), representaban más del 45% de todas las especies de >10 cm observadas en transectos y la mayoría exhibió bajos niveles de mortalidad parcial (prom= 22.5%). En el 2005, fueron menos abundantes (38% de las colonias). La mortalidad parcial en colonias vivas de M. annularis y M. faveolata se incrementó en un 85% (promedio= 42% de mortalidad parcial) y se observaron numerosas colonias de M. annularis y M. faveolata muertas; las colonias de M. franski generalmente se encontraron en excelentes condiciones (14% de mortalidad parcial). Se documentó una alta prevalencia de enfermedades de coral (3-30%) en M. annularis y M. faveolata, mientras que las otras especies se vieron afectadas con menor frecuencia. La enfermedad de banda amarilla (BA) emergió poco después del blanqueamiento de 1995 y se dispersó rápidamente en todas las profundidades, alcanzando su mayor prevalencia entre 1997-1999. La BA causó mortalidades lentas (= 1cm/mes), con aparición de múltiples lesiones focales en colonias individuales; estas lesiones fueron creciendo progresivamente al tiempo que mataban las colonias. Para el año 2005, el 44% de los corales que se etiquetaron habían muerto; los restantes exhibían infecciones activas de BA (21%), o estaban en remisión (31.6%) pero habían perdido un promedio de >90% de su tejido. Aunque la incidencia de BA disminuyó a partir del 2000, la prevalencia de la plaga blanca (PB) fue inusualmente alta (4-12%) en el 2001 y 2005, provocando mortalidades recientes de hasta un 70% en las colonias afectadas. Las superficies muertas de Montastraea spp. han sido colonizadas por otros corales, incluyendo porítidos, agarícidos y otros fávidos, mientras que continúa la ausencia de reclutas de M. annularis (complejo). Si las enfermedades y otros estresores bióticos persisten en estos arrecifes, las poblaciones de M. annularis y M. faveolata podrían decaer de forma similar a como lo hicieron los acropóridos del Caribe durante la década de 1980.

Cytotoxicity of chloral-derived beta-carbolines is not specific towards neuronal nor dopaminergic cells.

beta-Carbolines structurally related to the selective dopaminergic neurotoxin 1-methyl-4- phenylpyridinium (MPP(+)) may contribute to dopaminergic neurodegeneration in Parkinson's disease. The chloral-derived mammalian alkaloid derivative 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) is formed endogenously by a Pictet-Spengler condensation from the biogenic amine tryptamine (Ta) and the hypnotic aldehyde chloral (Clo). Here we examine the dopaminergic toxicity of TaClo and related compounds by testing their differential cytotoxicities in dopaminergic SH-SY5Y and non-dopaminergic murine Neuro2A neuroblastoma cell lines as well as in heterologous expression systems of the dopamine transporter (DAT) using both HEK-293 and Neuro2A cells. All TaClo derivatives showed significant cytotoxicity in all cell lines after 72 hours with the following rank order of toxic potency: 1-Tribromomethyl-1,2,3,4-tetrahydro-beta-carboline (TaBro) > TaClo > MPP(+) > 1,2,3,4-tetrahydro-beta-carboline (THbetaC) > 2[N]-methyl-TaClo > 2[N]-methyl-THbetaC. In contrast to MPP(+), there was no selectivity towards dopaminergic cells or cells ectopically expressing the DAT in vitro. Our results suggest that TaClo and related analogs are strong cytotoxins without selectivity towards dopaminergic cells.

Characterization of the single superoxide dismutase of Staphylococcus xylosus.

Staphylococcus xylosus is a facultative anaerobic bacterium used as a starter culture for fermented meat products. In an attempt to analyze the antioxidant capacities of this organism, the superoxide dismutase (SOD) was characterized. S. xylosus contains a single cytoplasmic SOD, which was not inhibited by H2O2. The SOD activity in crude extracts was completely lost upon metal depletion, but it could be recovered by manganese and very weakly by iron. It is therefore suggested that the S. xylosus SOD is a manganese-preferring enzyme. The corresponding gene, sod, was isolated from a genomic library of S. xylosus DNA and complemented the growth defect of an Escherichia coli SOD-deficient mutant. As deduced from the nucleotide sequence, sod encodes a protein of 199 amino acids with a molecular mass of 22.5 kDa. Two transcriptional start sites 25 and 120 bp upstream of the sod start codon were identified. A terminator-like structure downstream of the gene suggested a monocistronic sod mRNA. Regulation of sod expression was studied using fusions of the sod promoters to a genomic promoterless beta-galactosidase gene. The sod expression was not affected by manganese and increased slightly with paraquat. It was induced during stationary phase in a complex medium but not in a chemically defined medium. To investigate the physiological role of SOD, a mutant devoid of SOD activity was constructed. Growth experiments showed that sod is not essential for aerobic growth in complex medium. However, in chemically defined medium without leucine, isoleucine, and valine, the sod mutant hardly grew, in contrast to the wild-type strain. In addition, the mutant was sensitive to hyperbaric oxygen and to paraquat. Therefore, sod plays an important role in the protection of S. xylosus from oxidative stress.

Myeloid/natural killer cell precursor blast crisis of chronic myelogenous leukemia with two Philadelphia (Ph-1) chromosomes.

We report on a 30-year-old patient with blast crisis of a chronic myelogenous leukemia (CML) that shows immunophenotypic features similar to those of the myeloid/natural killer (NK) cell precursor leukemia previously described. Expression of CD13/CD33/CD65 as well as MPO+/LF- blasts was classified as a myelogenous blast crisis of a CML. In addition, the blasts were positive for CD7/CD56. Other lymphoid markers were not expressed. Cytogenetic and molecular cytogenetic examinations showed two Philadelphia (Ph-1) chromosomes and a trisomy 8. Similar to expression of the myeloid/NK cell precursor phenotype in acute myelogenous leukemia (AML), it is possible to exhibit this phenotype in Ph-1-positive CML. Only one case report of myeloid/NK precursor phenotype blast crisis of CML was found in the literature. Therefore, it is not clear whether this phenotype is a distinct biologic and clinical disease entity of CML, as is the case in the respective AML phenotype.

Analysis of catabolite control protein A-dependent repression in Staphylococcus xylosus by a genomic reporter gene system.

A single-copy reporter system for Staphylococcus xylosus has been developed, that uses a promoterless version of the endogenous beta-galactosidase gene lacH as a reporter gene and that allows integration of promoters cloned in front of lacH into the lactose utilization gene cluster by homologous recombination. The system was applied to analyze carbon catabolite repression of S. xylosus promoters by the catabolite control protein CcpA. To test if lacH is a suitable reporter gene, beta-galactosidase activities directed by two promoters known to be subject to CcpA regulation were measured. In these experiments, repression of the malRA maltose utilization operon promoter and autoregulation of the ccpA promoters were confirmed, proving the applicability of the system. Subsequently, putative CcpA operators, termed catabolite-responsive elements (cres), from promoter regions of several S. xylosus genes were tested for their ability to confer CcpA regulation upon a constitutive promoter, P(vegII). For that purpose, cre sequences were placed at position +3 or +4 within the transcribed region of P(vegII). Measurements of beta-galactosidase activities in the presence or absence of glucose yielded repression ratios between two- and eightfold. Inactivation of ccpA completely abolished glucose-dependent regulation. Therefore, the tested cres functioned as operator sites for CcpA. With promoters exclusively regulated by CcpA, signal transduction leading to CcpA activation in S. xylosus was examined. Glucose-dependent regulation was measured in a set of isogenic mutants showing defects in genes encoding glucose kinase GlkA, glucose uptake protein GlcU, and HPr kinase HPrK. GlkA and GlcU deficiency diminished glucose-dependent CcpA-mediated repression, but loss of HPr kinase activity abolished regulation. These results clearly show that HPr kinase provides the essential signal to activate CcpA in S. xylosus. Glucose uptake protein GlcU and glucose kinase GlkA participate in activation, but they are not able to trigger CcpA-mediated regulation independently from HPr kinase.

Effect of 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline (TaClo) on human serotonergic cells.

The tryptamine-derived dopaminergic neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline ('TaClo'), which was found to occur in humans after intake of the hypnotic chloral hydrate, was also shown to strongly disturb serotonergic cells. Incubation experiments using the human serotonergic cell line JAR clearly revealed TaClo to significantly reduce serotonin (5-HT) uptake (IC(50) = 59 microM) and to induce a distinct loss of cellular viability at increasing TaClo concentrations. In contrast to well-known serotonergic neurotoxins such as amphetamines, however, TaClo toxicity is not mediated by the 5-HT transporter (5-HTT). In the presence of the specific 5-HTT inhibitor imipramine, the uptake of TaClo into JAR cells was not reduced, hinting at an exclusively passive penetration of this highly lipophilic beta-carboline through cell membranes. Similar toxic effects towards JAR cells were also observed for the 5-HT-related TaClo analog 6-hydroxy-1-trichloromethyl-1,2,3,4-tetrahydro-beta-carboline ('6-OH-TaClo') (IC50 = 26 gM). The dopamine-derived alkaloid-type heterocycle 6,7-dihydroxy-1-trichloromethyl-1,2,3,4-tetrahydroisoquinoline ('DaClo'), by contrast, was found to be less toxic, showing only a weak inhibitory activity (IC50 = 260 microM) on 5-HT uptake. The pronounced toxicitiy of TaClo and 6-OH-TaClo against serotonergic cells became also evident from morphological findings: Dose-dependently, the survival of JAR cells was significantly impaired, while human dopaminergic IMR-32 cells were only moderately affected at similar toxin concentrations.

Bromal-derived tetrahydro-beta-carbolines as neurotoxic agents: chemistry, impairment of the dopamine metabolism, and inhibitory effects on mitochondrial respiration.

The mammalian alkaloids tryptoline (1) and eleagnine (2) as well as the highly halogenated (X = F, Cl, Br) tetrahydro-beta-carbolines (THbetaCs) 3-5, structurally similar to the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 6), were found to have a common feature of inducing a severe impairment of the nigrostriatal dopamine metabolism and inhibiting complex I of the mitochondrial respiratory chain highly selectively. Within the series of compounds tested, 1-tribromomethyl-1,2,3,4-tetrahydro-beta-carboline ('TaBro', 5), which was prepared in high yields from the biogenic amine tryptamine ('Ta', 7) and the unnatural aldehyde bromal ('Bro', 8) by a Pictet-Spengler cyclization reaction, turned out to be the most potent toxin in vitro and in vivo. As demonstrated by voltammetric measurements on rats, for all the THbetaCs 1-5 investigated, intranigral application of a single dose of 10 microg resulted in a significant reduction of the dopaminergic activity in the striatum, with the strongest effect being observed for TaBro (5). Using rat brain homogenates, again 5 (IC50 = 200 microM) as well as its dehydrohalogenation product 11 (IC50 = 150 microM) exhibited the most pronounced inhibitory potential on mitochondrial respiration. The halogen-free THbetaCs 1 and 2 as well as the MPTP metabolite 1-methyl-4-phenylpyridinium ion (MPP+), by contrast, showed only a moderate inhibition at concentrations in the millimolar range (e.g. for MPP+: IC50 = 3.5 mM). For an elucidation of the role of hydrophobic portion in the inhibitory action against complex I activity, several N-acyl derivatives (15-21) of 5 were synthesized and tested. An X-ray diffraction study on the 3-dimensional structure of trifluoroacetylated highly halogenated THbetaCs (12-14) revealed the tetrahydropyrido part to adopt a nearly planarized half-chair conformation. Because of the steric demand of the trihalogenmethyl moiety (CF3 < CCl3 < CBr3), the N-substituent is dramatically pushed out of that ring 'plane'.

Kinetic and mechanistic studies of signal peptidase I from Escherichia coli.

Signal peptidases of prokaryotic organisms reside in the outer leaflet of the cytoplasmic membrane and catalyze the hydrolytic cleavage of a specific peptide bond of membrane-imbedded preproteins to liberate mature proteins for secretion. In this manuscript, we report new and efficient peptide substrates for SPase and their use to explore features of this enzyme's reaction mechanism. The enzyme used in this study was recombinant SPase I of Escherichia coli that had been solubilized with Triton X-100 and purified to near homogeneity. Our new substrates are based on the fluorogenic peptide reported by Zhong and Benkovic [(1998) Anal. Biochem. 255, 66], Y(NO2)FSASALA approximately KIK(Abz)-NH(2) (Y(NO2), 3-nitro-L-tyrosine; K(Abz), epsilon-(2-aminobenzoyl)-L-Lys; hydrolysis at A approximately K). We found that when a signal peptide-like sequence is appended onto the N-terminus of this peptide to produce K(5)-L(10)-Y(NO2)FSASALA approximately KIK(Abz)-NH(2), k(c)/K(m) increases from 85 to 2.5 x 10(6) M(-)(1) s(-)(1). k(c)/K(m) decreases with increasing concentration of Triton X-100 micelles under the condition [Triton X-100](micelle) > [S](0) > [E](0). We explain this apparent inhibition with a model of surface dilution kinetics in which "empty" micelles compete with substrate-containing micelles for micelle-bound enzyme. Fusion of micelle-bound enzyme with a substrate-containing micelle leads to formation of productive E:S substrate complexes while fusion of micelle-bound enzyme with an "empty" micelle is nonproductive and inhibitory. The dependence of steady-state kinetic parameters for the SPase-catalyzed hydrolysis of K(5)-L(10)-Y(NO2)FSASALA approximately KIK(Abz)-NH(2) on [Triton X-100](micelle) supports this model. Product inhibition and solvent isotope effects were also investigated and could be interpreted in the context of this model.

A novel translocation (17;19)(p13;p13) in a patient with acute myelomonocytic leukemia.

We report on a patient with acute myeloid leukemia (AML M4) and a so far unrecorded translocation (17;19). The leukemia transformed from a myeloproliferative disorder (MPD) and showed a progressive fatal course. Following transformation, all leukemic cells showed an apparently balanced translocation (17;19)(p13;p13). The breakpoint regions harbor genes such as TP53 (17p13) and E2A, ENL, or LYL1 (19p13), which could be relevant in leukemogenesis. We suspect that the translocation (17;19)(p13;p13) may be a prognostic factor for transformation from chronic MPD to acute leukemia.

Characterization of an HPr kinase mutant of Staphylococcus xylosus.

The Staphylococcus xylosus gene hprK, encoding HPr kinase (HPrK), has been isolated from a genomic library. The HPrK enzyme, purified as a His(6) fusion protein, phosphorylated HPr, the phosphocarrier protein of the bacterial phosphotransferase system, at a serine residue in an ATP-dependent manner, and it also catalyzed the reverse reaction. Therefore, the enzyme constitutes a bifunctional HPr kinase/phosphatase. Insertional inactivation of the gene in the genome of S. xylosus resulted in the concomitant loss of both HPr kinase and His serine-phosphorylated-HPr phosphatase activities in cell extracts, strongly indicating that the HPrK enzyme is also responsible for both reactions in vivo. HPrK deficiency had a profound pleiotropic effect on the physiology of S. xylosus. The hprK mutant strain showed a severe growth defect in complex medium upon addition of glucose. Glucose uptake in glucose-grown cells was strongly enhanced compared with the wild type. Carbon catabolite repression of three tested enzyme activities by glucose, sucrose, and fructose was abolished. These results clearly demonstrate the prominent role of HPr kinase in global control to adjust catabolic capacities of S. xylosus according to the availability of preferred carbon sources.

Identification of a new repetitive element in Staphylococcus aureus.

The Staphylococcus aureus repeat (STAR) element is a sequence identified in two intergenic regions in S. aureus. The element is found in 13 to 21 copies in individual S. aureus strains, and elements in the homologous intergenic location are variable in length. The element sequence consists of several small and unusually GC-rich direct repeats with recurring intervening sequences. In addition, STAR-like elements may be present in related staphylococcal species.

Identification of a gene in Staphylococcus xylosus encoding a novel glucose uptake protein.

By transposon Tn917 mutagenesis, two mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild type. Both transposons integrated in a gene, designated glcU, encoding a protein involved in glucose uptake in S. xylosus, which is followed by a glucose dehydrogenase gene (gdh). Glucose-mediated repression of beta-galactosidase, alpha-glucosidase, and beta-glucuronidase activities was partially relieved in the mutant strains, while repression by sucrose or fructose remained as strong as in the wild type. In addition to the pleiotropic regulatory effect, integration of the transposons into glcU reduced glucose dehydrogenase activity, suggesting cotranscription of glcU and gdh. Insertional inactivation of the gdh gene and deletion of the glcU gene without affecting gdh expression showed that loss of GlcU function is exclusively responsible for the regulatory defect. Reduced glucose repression is most likely the consequence of impaired glucose uptake in the glcU mutant strains. With cloned glcU, an Escherichia coli mutant deficient in glucose transport could grow with glucose as sole carbon source, provided a functional glucose kinase was present. Therefore, glucose is internalized by glcU in nonphosphorylated form. A gene from Bacillus subtilis, ycxE, that is homologous to glcU, could substitute for glcU in the E. coli glucose growth experiments and restored glucose repression in the S. xylosus glcU mutants. Three more proteins with high levels of similarity to GlcU and YcxE are currently in the databases. It appears that these proteins constitute a novel family whose members are involved in bacterial transport processes. GlcU and YcxE are the first examples whose specificity, glucose, has been determined.

Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family.

Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.

Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.

We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.