Prolonged interferon-gamma exposure decreases ion transport,NKCC1, and Na+-K+-ATPase expression in human intestinal xenografts in vivo

IFN-gamma is elevated in intestinal inflammation and alters barrier and transport functions in human colonic epithelial cell lines, but its effects on normal human small intestinal epithelium in vivo are poorly defined. We investigated effects of prolonged IFN-gamma exposure on ion transport and expression of transporters by using human fetal small intestinal xenografts. Xenograft-bearing mice were injected with IFN-gamma, and 24 h later xenografts were harvested and mounted in Ussing chambers. Baseline potential difference (PD) was not affected by IFN-gamma treatment. However, conductance was enhanced and agonist-stimulated ion transport was decreased. IFN-gamma also decreased expression of the Na+-K+-2Cl- cotransporter and the alpha-subunit of Na+-K+-ATPase compared with controls, whereas levels of the calcium-activated Cl- channel and CFTR were unaltered. Thus prolonged exposure to IFN-gamma leads to decreased ion secretion due, in part, to decreased ion transporter levels. These findings demonstrate the implications of elevated IFN-gamma levels in human small intestine and validate the human intestinal xenograft as a model to study chronic effects of physiologically relevant stimuli.     

Interaction of Sclerotinia sclerotiorum with Brassica napus: cloning and characterization of endo- and exo-polygalacturonases expressed during saprophytic and parasitic modes

Five major and several minor PG isoenzymes were identified in a Sclerotinia sclerotiorum isolate from Brassica napus by isoelectric focusing and pectin gel overlays. Using a combination of degenerate PCR and expressed sequence tags (ESTs) four endo-polygalacturonase (PG) genes, designated as sspg1d, sspg3, sspg5, and sspg6, and two exo-PG genes, ssxpg1 and ssxpg2, were identified. SSPG1d is a member of the PG gene family previously described by Fraissinet-Tachet et al. [Curr. Genet. 29 (1995) 96]. The mature SSPG1d is a neutral PG, whereas fully processed SSPG3, SSPG5, and SSPG6 are acidic enzymes. Under saprophytic growth conditions, sspg1d, sspg3, sspg5, and ssxpg1 expression was induced by pectin and galacturonic acid and subject to catabolite repression by glucose. Conditions could not be identified under which sspg6 or ssxpg2 were expressed well. Transfer of mycelia from liquid media to solid substrates induced expression of sspg1d suggesting that it may also be regulated by thigmotrophic interactions. Under pathogenic conditions, sspg1d was highly expressed during infection. sspg3 was also expressed during infection, albeit at lower levels than sspg1d, whereas sspg5, sspg6, and ssxpg1 were expressed only weakly.     

Processing of seminal plasma hCAP-18 to ALL-38 by gastricsin: a novel mechanism of generating antimicrobial peptides in vagina

The human cathelicidin, hCAP-18, is expressed both in neutrophils and in epithelial cells. hCAP-18 is processed to the antimicrobial peptide LL-37 by proteinase 3 in neutrophils. hCAP-18 is highly expressed in the epididymis with a subsequent high concentration in seminal plasma where the protein is present in its unprocessed and antimicrobially inactive form. We report here that hCAP-18 in seminal plasma is processed to generate a 38-amino acid antimicrobial peptide ALL-38 by the prostate-derived protease gastricsin when incubated at a pH corresponding to the vaginal pH. In accordance with this, seminal plasma derived hCAP-18 was found in its processed form in the vagina following sexual intercourse. The antimicrobial activity of ALL-38 against a variety of microorganisms tested is equal to that of LL-37. This enzymatic activation of a proantimicrobial substance in seminal plasma following exposure to the vaginal milieu represents a novel mechanism to prevent infection following sexual intercourse.     

A genomic timescale for the origin of eukaryotes

Background  

Genomic sequence analyses have shown that horizontal gene transfer occurred during the origin of eukaryotes as a consequence of symbiosis. However, details of the timing and number of symbiotic events are unclear. A timescale for the early evolution of eukaryotes would help to better understand the relationship between these biological events and changes in Earth's environment, such as the rise in oxygen. We used refined methods of sequence alignment, site selection, and time estimation to address these questions with protein sequences from complete genomes of prokaryotes and eukaryotes.     

NS-417, a Novel Compound with Neurotrophic-Like Effects

NS-417 (5-(4-Chlorophenyl)-8-methyl-6-7-8-9-tetrahydro-1-H-pyrrolo[3.2-h]isoquinoline-2,3-dione-3-oxim hydrochloric acid salt) belongs to a new chemical series of compounds. NS-417 rescued differentiated PC12 cells from death induced by withdrawal of serum and nerve growth factor. Furthermore, NS-417 stimulated neurotrophic factor-induced neurite outgrowth in undifferentiated PC12 cells. In accordance with this observation, NS-417 potentiated NGF-induced signaling, such as activation of the extracellular signal-regulated kinases ERK1 and ERK2 and the Akt kinase. NS-417 also enhanced ERK activation induced by 10 minutes stimulation with NGF, bFGF or EGF in PC12 cells. In addition to the effect in PC12 cells, NS-417 increased the number of tyrosine hydroxylase (TH) positive cells in cultures established from dissociated E14 rat ventral mesencephali.     

Intermediary glucan structures formed during starch granule biosynthesis are enriched in short side chains, a dynamic pulse labeling approach

The formation of intermediary glucans, mature starch, and phytoglycogen was studied using leaves of Arabidopsis thaliana wild type and dbe mutant, which lacks plastidic isoamylase (Zeeman, S. C., Umemoto, T., Lue, W. L., Au-Yeung, P., Martin, C., Smith, A. M., and Chen, J. (1998) Plant Cell 10, 1699-1711). A new approach to the study of starch biosynthesis was developed based on "very short pulse" labeling of leaf starch through photosynthetic fixation of (14)CO(2). This allowed selective analysis of the structure of starch formed within a 30-s period. This time frame is shorter than the period required for the formation of a single crystalline amylopectin lamella and consequently permits a direct analysis of intermediary structures during granule formation. Analysis of chain length distribution showed that the most recently formed outer layer of the granules has a structure different from the mature starch. The outer layer is enriched in short chains that are 6-11 glucose residues long. Side chains with 6 glucose residues are the shortest abundant chains formed, and they are formed exclusively by transfer from donor chains of 12 glucose residues or longer. The labeling pattern shows that chain transfer resulting in branching is a rapid and efficient process, and the preferential labeling of shorter chains in the intermediary granule bound glucan is suggested to be a direct consequence of efficient branching. Although similar, the short chain intermediary structure is not identical to phytoglycogen, which is an even more highly branched molecule with very few longer chains (more than 40 glucose residues). Pulse and chase labeling profiles for the dbe mutant showed that the final structure is more highly branched than the intermediary structures, which implies that branching of phytoglycogen occurs over a longer time period than branching of starch.     

Comparative vaccine studies in HLA-A2.1-transgenic mice reveal a clustered organization of epitopes presented in hepatitis C virus natural infection

A polyepitopic CD8(+)-T-cell response is thought to be critical for control of hepatitis C virus (HCV) infection. Using transgenic mice, we analyzed the immunogenicity and dominance of most known HLA-A2.1 epitopes presented during infection by using vaccines that carry the potential to enter clinical trials: peptides, DNA, and recombinant adenoviruses. The vaccines capacity to induce specific cytotoxic T lymphocytes and interferon gamma-producing cells revealed that immunogenic epitopes are clustered in specific antigens. For two key antigens, flanking regions were shown to greatly enhance the scope of epitope recognition, whereas a DNA-adenovirus prime-boost vaccination strategy augmented epitope immunogenicity, even that of subdominant ones. The present study reveals a clustered organization of HCV immunogenic HLA.A2.1 epitopes and strategies to modulate their dominance.     

Expressed sequence tags from roots and nodule primordia of Lotus japonicus infected with Mesorhizobium loti

Messenger RNA from young Lotus japonicus roots carrying root nodule primordia appearing after inoculation with Mesorhizobium loti bacteria were used to construct a cDNA expression library. Single-pass sequencing employing colony-polymerase chain reaction (PCR) and analysis of PCR products established a total of 2,397 new expressed sequence tags (ESTs). We have putatively identified 1,236 known and 484 hypothetical proteins coded by the corresponding mRNAs. The remaining cDNAs are unknown (316) or redundant overlapping cDNAs (361). We hope that this batch of ESTs will assist in the recognition of plant genes involved during development of nitrogen-fixing root nodules.     

An epidemiological study of respiratory syncytial virus associated hospitalizations in Denmark

Respiratory syncytial virus (RSV) is the most common viral pathogen that causes lower respiratory tract infections in infants. Studies have implicated severe RSV infections early in life as a risk factor for subsequent development of reactive airway disease. We are conducting a study to validate RSV-associated diagnoses in the Danish National Patient Registry, to assess whether the incidence of severe RSV infection is increasing in Denmark, to identify predisposing and protective factors for RSV-associated hospitalization in Denmark, and to examine the association of severe RSV infection with reactive airway disease. The influence of various biological, social and environmental factors on hospitalization for RSV infection will be studied through several population-based registers, including the Danish National Birth Cohort: 'Better health for mothers and children'. The RSV hospitalization cases will be compared with control individuals selected within the same population groups on a case-control or a cohort basis in order to produce estimates of age-adjusted and sex-adjusted relative risks (odds ratio and relative risk) for hospitalization associated with various risk factors. Using register linkage and unique registration of exposures collected through interviews and blood samples from the Danish National Birth Cohort, we will be able to resolve the issues referred to above in a very large sample of Danish children.