Synergistic effect of tumor necrosis factor-alpha and interferon-gamma on enterocyte shedding of syndecan-1 and associated decreases in internalization of Listeria monocytogenes and Staphylococcus aureus

Syndecan-1 is a heparan sulfate proteoglycan expressed on epithelia, and its ectodomain can be shed into the extracellular milieu, affecting a variety of cellular functions. Using two bacteria known to react with heparan sulfate, Listeria monocytogenes and Staphylococcus aureus, experiments were designed to clarify the effect of syndecan-1 shedding on bacterial internalization by human HT-29 enterocytes. Mature enterocytes were incubated with tumor necrosis factor (TNF)-alpha and/or interferon (IFN)-gamma for 16h prior to addition of bacteria. These cytokines acted synergistically to decrease syndecan-1 expression, assessed by visual observations of syndecan-1 expression on enterocytes using immunohistochemistry and a monoclonal antibody to the syndecan-1 core protein, by quantifying this fluorescent intensity, and by quantifying the concentration of shed syndecan-1 using an enzyme-linked immunoabsorbent assay. Neither IFN-gamma nor TNF-alpha alone had a noticeable effect on L. monocytogenes internalization, but a mixture of both cytokines resulted in decreased (P<0.01) internalization. Enterocyte preincubation with TNF-alpha alone, and with both cytokines, was associated with decreased S. aureus internalization, at P<0.05 and P<0.01, respectively. Thus, TNF-alpha and IFN-gamma acted synergistically to shed syndecan-1 ectodomains from HT-29 enterocytes, and shedding was associated with decreased internalization of two pathogenic bacteria, suggesting that syndecan-1 shedding may modulate the pathogenesis of specific microbes.     

HEPN: a common domain in bacterial drug resistance and human neurodegenerative proteins

A novel domain - HEPN (higher eukarytoes and prokaryotes nucleotide-binding domain) - found in several bacterial species is also present in the human protein, sacsin, a chaperonin implicated in an early-onset neurodegenerative disease. The distant structural similarity suggests that this domain might be involved in nucleotide binding.     

Pleiotrophin expression during odontogenesis

Pleiotrophin (PTN) is an extracellular matrix-associated growth factor and chemokine expressed in mesodermal and ectodermal cells. It plays an important role in osteoblast recruitment and differentiation. There is limited information currently available about PTN expression during odontoblast differentiation and tooth formation, and thus the authors aimed to establish the spatiotemporal expression pattern of PTN during mouse odontogenesis. Immortalized mouse dental pulp (MD10-D3, MD10-A11) and odontoblast-like (M06-G3) and ameloblast-like (EOE-3M) cell lines were grown and samples prepared for immunocytochemistry, Western blot, and conventional and quantitative PCR analysis. Effects of BMP2, BMP4, and BMP7 treatment on PTN expression in odontoblast-like M06-G3 cells were tested by quantitative PCR. Finally, immunohistochemistry of sectioned mice mandibles and maxillaries at developmental stages E16, E18, P1, P6, P10, and P28 was performed. The experiments showed that PTN, at both the mRNA and protein level, was expressed in all tested epithelial and mesenchymal dental cell lines and that the level of PTN mRNA was influenced differentially by the bone morphogenetic proteins. The authors observed initial expression of PTN in the inner enamel epithelium with prolonged expression in the ameloblasts and odontoblasts throughout their stages of maturation and strong expression in the terminally differentiated and enamel matrix-secreting ameloblasts and odontoblasts of the adult mouse incisors and molars.     

Regulated endocrine-specific protein 18 (RESP18) is localized to and regulated in A-like cells and G-cells in rat stomach

The regulated endocrine-specific protein 18 (RESP18) has previously been localized to different endocrine cells and neurons, in particular the pituitary gland and hypothalamus. It is found in the lumen of the endoplasmic reticulum and is degraded at the post-ER pre-Golgi compartment, and a role in processing of secreted peptides has been hypothesized. The present study examines localization of RESP18 in the gastrointestinal mucosa of rats by immunohistochemistry, and expression and regulation in response to hypergastrinemia induced by acid inhibition (pantoprazole), gastrin antagonism (YF476), fasting-refeeding and octreotide by mRNA measurements. RESP18 was mainly found in the gastric mucosa, but could also be detected in a few, scattered cells in the lower small intestine and in colon. In the antral mucosa, all RESP18 immunoreactivity was localized to ghrelin-producing A-like cells and gastrin-producing G-cells. In the corpus mucosa, a significant fraction, but not all of the RESP18 immunoreactive cells, were A-like cells. In both antrum and corpus, Resp18 mRNA seemed to vary similarly with the activation of the A-like cells, and in the antrum also with stimulation of the G-cells. This study demonstrates, for the first time, the localization of RESP18 to specific neuroendocrine cells of the gastrointestinal mucosa and that it seems to be regulated synchronously with the peptides secreted from these cells. This suggests that Resp18 may indeed have a functional role in the synthesis or storage of these gastrointestinal peptides.     

Structural comparison of bacterial and human iron-dependent phenylalanine hydroxylases: similar fold,different stability and reaction rates

Structure determination of bacterial homologues of human disease-related proteins provides an efficient path to understanding the three-dimensional fold of proteins that are associated with human diseases. However, the precise locations of active-site residues are often quite different between bacterial and human versions of an enzyme, creating significant differences in the biological understanding of enzyme homologs. To study this hypothesis, phenylalanine hydroxylase from a bacterial source has been structurally characterized at high resolution and comparison is made to the human analog. The enzyme phenylalanine hydroxylase (PheOH) catalyzes the hydroxylation of l-phenylalanine into l-tyrosine utilizing the cofactors (6R)-l-erythro-5,6,7,8 tetrahydrobiopterin (BH(4)) and molecular oxygen. Previously determined X-ray structures of human and rat PheOH, with a sequence identity of more than 93%, show that these two structures are practically identical. It is thus of interest to compare the structure of the divergent Chromobacterium violaceum phenylalanine hydroxylase (CvPheOH) ( approximately 24% sequence identity overall) to the related human and rat PheOH structures. We have determined crystal structures of CvPheOH to high resolution in the apo-form (no Fe-added), Fe(III)-bound form, and 7,8-dihydro-l-biopterin (7,8-BH(2)) plus Fe(III)-bound form. The bacterial enzyme displays higher activity and thermal melting temperature, and structurally, differences are observed in the N and C termini, and in a loop close to the active-site iron atom.     

Inhibition of in vitro attachment of Giardia trophozoites by mucin

An enzyme-linked immunoabsorbent assay was developed to quantify the number of Giardia sp. trophozoites attached to a substratum. Trophozoites were allowed to attach for 5 or 10 min to untreated or modified substratum, or allowed to attach in the presence of cytoskeletal inhibitors. Microtubule inhibitors, cochicine and nocodazole, were ineffective in blocking attachment, although the actin-disrupting agent cytochalasin B produced significant inhibition of attachment. Three different mucins were associated with significant inhibition of Giardia trophozoite attachment, which was reversed by treating the mucin-coated wells with 0.1% poly-L-arginine. Examination of mucin-treated substrata by high-resolution field emission scanning electron microscopy showed the presence of thin linear fibrils, whereas treatment of mucin-coated wells with poly-L-arginine revealed similar fibrils but of thicker dimensions. These data support the concept that mucin may inhibit Giardia sp. trophozoite attachment in vitro by electrostatic repulsion and that this can be eliminated by treatment of mucin-coated surfaces with polycationic agents, such as poly-L-arginine or poly-L-lysine.     

Tolerability and efficacy of single dose albendazole,diethylcarbamazine citrate (DEC) or co-administration of albendazole with DEC in the clearance of <Emphasis Type="Italic">Wuchereria bancrofti</Emphasis>in asymptomatic microfilaraemic volunteers in Pondicherry,South India: a hospital-based study

Background  

The tolerability and efficacy of single dose albendazole (400 mg), diethylcarbamazine citrate (DEC) (6 mg/kg bodyweight) or co-administration of albendazole (400 mg) + DEC (6 mg/kg bodyweight) was studied in 54 asymptomatic Wuchereria bancrofti microfilaraemic volunteers in a double blind hospital-based clinical study.