Engineering the primary substrate specificity of Streptomyces griseus trypsin.

Streptomyces griseus trypsin (SGT) was chosen as a model scaffold for the development of serine proteases with enhanced substrate specificity. Recombinant SGT has been produced in a Bacillus subtilis expression system in a soluble active form and purified to homogeneity. The recombinant and native proteases have nearly identical enzymatic properties and structures.

CD1d-expressing dendritic cells but not thymic epithelial cells can mediate negative selection of NKT cells.

Natural killer T (NKT) cells are a unique immunoregulatory T cell population that is positively selected by CD1d-expressing thymocytes. Previous studies have shown that NKT cells exhibit autoreactivity, which raises the question of whether they are subject to negative selection. Here, we report that the addition of agonist glycolipid alpha-galactosylceramide (alpha-GalCer) to a fetal thymic organ culture (FTOC) induces a dose-dependent disappearance of NKT cells, suggesting that NKT cells are susceptible to negative selection.

Factors affecting surgical risk in elderly patients with inflammatory bowel disease.

The operative treatment of elderly patients with inflammatory bowel disease (IBD) has often been avoided in favor of medical management because of a perceived increase in surgical risk. This study sought to define the following in the elderly IBD patient population: (1) the risk of surgical management and (2) those factors affecting risk. Thirty patients with IBD, aged 60 years or more, who were surgically managed by a single surgeon over a 10-year period, were retrospectively matched to 75 patients with IBD who were less than 60 years of age; patients were matched according to sex, date of surgery, and type of surgery performed.

Kottamides A-D: novel bioactive imidazolone-containing alkaloids from the New Zealand ascidian Pycnoclavella kottae.

Kottamides A-D (1-4), novel 2,2,5-trisubstituted imidazolone-containing alkaloids, were isolated from the New Zealand endemic ascidian Pycnoclavella kottae and structurally characterized using 15N natural abundance 2-D NMR in addition to standard spectroscopic methods. The kottamides exhibited anti-inflammatory and anti-metabolic activity as well as cytotoxicity toward tumor cell lines.

Cluster analysis of an extensive human breast cancer cell line protein expression map database.

In the current study, the protein expression maps (PEMs) of 26 breast cancer cell lines and three cell lines derived from normal breast or benign disease tissue were visualised by high resolution two-dimensional gel electrophoresis. Analysis of this data was performed with ChiClust and ChiMap, two analytical bioinformatics tools that are described here. These tools are designed to facilitate recognition of specific patterns shared by two or more (a series) PEMs.

Hyaluronidase gene profiling and role of hyal-1 overexpression in an orthotopic model of prostate cancer.

The mRNA levels of hyal-1, hyal-2, LUCA3 and PH20, the 4 hyaluronidases with demonstrated endoglucosaminidase activity, were extensively profiled in normal and tumor tissues and cell lines, using dot blot analysis and quantitative PCR. In normal tissues, hyal-1, hyal-2 and LUCA3 all showed unique patterns of mRNA expression, but were generally of widespread distribution, whereas PH20 mRNA was restricted to testes. In a small set of breast tumor samples, no elevations in hyal-1, hyal-2 or LUCA3 mRNA were seen.

Submillimeter evidence for the coeval growth of massive black holes and galaxy bulges.

The correlation, found in nearby galaxies, between black hole mass and stellar bulge mass implies that the formation of these two components must be related. Here we report submillimeter photometry of eight x-ray-absorbed active galactic nuclei that have luminosities and redshifts characteristic of the sources that produce the bulk of the accretion luminosity in the universe. The four sources with the highest redshifts are detected at 850 micrometers, with flux densities between 5.

Isolation and characterization of the new purine 1,3, 7-trimethylisoguanine from the New Zealand ascidian Pseudodistoma cereum.

The new purine 1,3,7-trimethylisoguanine (1) has been isolated from the ascidian Pseudodistoma cereum. The structure of 1 was elucidated by analysis of NMR spectroscopic and mass spectrometric data and by comparison with the regioisomeric purine 1,3,7-trimethylguanine (2).

Cd1d-restricted cellular lysis by peripheral blood lymphocytes: relevance to the inflammatory bowel diseases.

The CD1d molecule has been implicated to play a role in inflammatory bowel diseases (IBD), possibly through its presentation of an intestinal antigen trigger. To understand the role of the CD1d class I-like protein in IBD, we investigated the molecule's expression in diseased intestinal tissue and determined its potential to undergo specific recognition by intraepithelial and peripheral blood lymphocytes (PBLs) derived from IBD patients. We have observed an increase in precipitable CD1d in inflamed tissues, which suggests CD1d up-regulation in IBD; this was not accompanied by the occurrence of CD1d-specific cytotoxicity by lymphocytes isolated from the same tissue sites.

Mycalamide D, a new cytotoxic amide from the New Zealand marine sponge Mycale species.

A new mycalamide, mycalamide D (3), has been isolated from the New Zealand marine sponge Mycale sp. This new metabolite, in which the C13-O-methyl group of mycalamide A (1) is replaced by a hydrogen atom, was found to be cytotoxic to a range of mammalian cell lines, with a potency approximately 20-fold less than that of 1.

Absence of GNAI2 codon 179 oncogene mutations in inflammatory bowel disease.

The human GNAI2 gene coding for G protein, Galphai2, is located on chromosome 3p21 in proximity to the region where an inflammatory bowel disease (IBD) locus has been suggested. Galphai2-deficient mice develop a lethal diffuse colitis that resembles human ulcerative colitis (UC) and frequently progresses to colon adenocarcinoma. Furthermore, the human GNAI2 gene is subject to point mutations at certain positions, including three at codon 179, all of which have been reported in human endocrine tumors.

Increased gelsolin expression and retarded collagen lattice contraction with smooth muscle cells from Crohn's diseased intestine.

Intestinal smooth muscle cells (SMC) produce the fibrotic tissue, strictures, that characterize Crohn's disease. These SMC change their phenotype from a contractile muscle form to an inflammation-responsive form that migrates and synthesizes a collagen matrix. It is postulated that the inflammatory responsive SMC form associates differently with its surrounding collagen matrix compared to the normal SMC form.

Proteomic definition of normal human luminal and myoepithelial breast cells purified from reduction mammoplasties.

Normal human luminal and myoepithelial breast cells separately purified from a set of 10 reduction mammoplasties by using a double antibody magnetic affinity cell sorting and Dynabead immunomagnetic technique were used in two-dimensional gel proteome studies. A total of 43,302 proteins were detected across the 20 samples, and a master image for each cell type comprising a total of 1,738 unique proteins was derived. Differential analysis identified 170 proteins that were elevated 2-fold or more between the two breast cell types, and 51 of these were annotated by tandem mass spectrometry.

Gamma delta intraepithelial lymphocytes drive tumor necrosis factor-alpha responsiveness to intestinal iron challenge: relevance to hemochromatosis.

The dependence of intestinal epithelial cell (IEC) growth and differentiation on intraepithelial lymphocytes (IELs) expressing the gamma/delta (gamma delta) T-cell receptor (TCR), suggested a potential role for gamma delta + IELs in the regulation of iron absorption. We therefore examined the levels of hepatic iron and the IEL cytokine responses in C57BL/6J control and class I and TCR knockout lines (placed on a C57BL/6J genetic background) following the administration of supplemental dietary iron. The highest level of liver iron was found in the beta 2-microglobulin knockout (beta 2m-/-) mice followed by the TCR-delta knockout (TCR delta-/-) animals.

Proteomics: a major new technology for the drug discovery process.

Proteomics is a new enabling technology that is being integrated into the drug discovery process. This will facilitate the systematic analysis of proteins across any biological system or disease, forwarding new targets and information on mode of action, toxicology and surrogate markers. Proteomics is highly complementary to genomic approaches in the drug discovery process and, for the first time, offers scientists the ability to integrate information from the genome, expressed mRNAs, their respective proteins and subcellular localization.

A novel, orally administered nucleoside analogue, OGT 719, inhibits the liver invasive growth of a human colorectal tumor, C170HM2.

OGT 719 is a novel p.o. bioavailable nucleoside analogue in which galactose is incorporated onto the fluoropyrimidine moiety of the cytotoxic agent 5-fluorouracil (5-FU).

Factor V Leiden mutation: a nursing perspective.

The factor V Leiden mutation is a recently described autosomal dominant genetic risk factor for venous thromboembolism (VTE). Persons who are heterozygous or homozygous for this disorder are at 4 to 7 times and 50 to 100 times increased risk, respectively, for VTE. In particular, women have unique challenges because the presence of the Leiden mutation in combination with pregnancy or use of oral contraceptives results in an even greater increased risk for VTE.

New model of ErbB-2 over-expression in human mammary luminal epithelial cells.

The ErbB-2 receptor has been strongly implicated in the development of breast cancer. To establish a new model system to investigate the role of erbB-2 in tumorigenesis of the breast, the conditionally immortalised human mammary luminal epithelial cell line HB4a was transfected with erbB-2 cDNA. Biological and biochemical characterisation of the resulting cell lines demonstrated that high levels of ErbB-2 expression were sufficient to cause transformation in vitro but did not cause tumours in vivo.

Amelioration of graft versus host disease with anti-ICAM-1 therapy.

We have previously demonstrated an increase in the adhesion molecule lymphocyte function-associated antigen-1 (LFA-1) in GVHD after small bowel transplantation (SBTx) and a therapeutic effect for the monoclonal antibody (MoAb) to LFA-1 in the same model. The present study evaluated the role of MoAb to LFA-1's ligand, intercellular adhesion molecule-1 (ICAM-1) in GVHD. Methods.

Bowel permeability is improved in Crohn's disease after ileocolectomy.

Purpose: Numerous investigators have shown increased bowel permeability in patients with Crohn's disease. It is unclear whether this is a precondition affecting the entire intestine or a consequence of the inflammation and, therefore, only affecting the diseased bowel. The present study tested the hypothesis that resection of diseased bowel in patients with ileocolonic Crohn's disease would correct abnormalities in bowel permeability.

Cytomegalovirus enteritis: a highly lethal condition requiring early detection and intervention.

Unlabelled: Cytomegalovirus infection causing symptomatic enteritis is most usually associated with immunosuppressed transplant patients or patients positive for human immunodeficiency virus. Most reports studying this illness are small and do not clearly define the risk factors or mortality rates.

Methods: The present study retrospectively reviewed the charts of 67 patients with biopsy-proven cytomegalovirus enteritis (esophageal, gastric, small bowel, and colonic) to define and to investigate factors that influence survival.

Monoclonal antibody to lymphocyte function associated antigen-1 improves graft-versus-host disease.

Unlabelled: We previously showed that intestinal tissue expression of lymphocyte function associated antigen-1 is increased in animals with graft-versus-host disease after small-bowel transplantation.

Hypothesis: Treatment of rats with monoclonal antibody to lymphocyte function associated antigen-1 after small-bowel transplantation will lessen the severity of graft-versus-host disease.

Methods: Graft-versus-host disease was created in Lewis X Brown-Norway F1 rats by heterotopic vascularized small-bowel transplantation from Lewis donors.

A new retroviral vector, CA1, to identify and select for cells expressing an inserted gene in vitro and in vivo.

A new retroviral vector has been constructed that expresses genes encoding three different activities from a single transcript. This feature has been exploited to enable the efficient marking and selection of cells that express a gene of interest. The marker gene lacZ, encoding beta-galactosidase, and neo, encoding neomycin phosphotransferase, for selection by the antibiotic G418, are expressed as a fusion, beta Geo.