Receptor-mediated endocytosis of Neisseria gonorrhoeae into primary human urethral epithelial cells: the role of the asialoglycoprotein receptor

Urethral epithelial cells are invaded by Neisseria gonorrhoeae during gonococcal infection in men. To understand further the mechanisms of gonococcal entry into host cells, we used the primary human urethral epithelial cells (PHUECs) tissue culture system recently developed by our laboratory. These studies showed that human asialoglycoprotein receptor (ASGP-R) and the terminal lactosamine of lacto-N-neotetraose-expressing gonococcal lipooligosaccharide (LOS) play an important role in invasion of PHUECs. Microscopy studies showed that ASGP-R traffics to the cell surface after gonococcal challenge. Co-localization of ASGP-R with gonococci was observed. As ASGP-R-mediated endocytosis is clathrin dependent, clathrin localization in PHUECs was examined after infection. Infected PHUECs showed increased clathrin recruitment and co-localization of clathrin and gonococci. Preincubating PHUECs in 0.3 M sucrose or monodansylcadaverine (MDC), which both inhibit clathrin-coated pit formation, resulted in decreased invasion. N. gonorrhoeae strain 1291 produces a single LOS glycoform that terminates with Gal(beta1-4)Glc-Nac(beta1-3)Gal(beta1-4)Glc (lacto-N-neotetraose). Invasion assays showed that strain 1291 invades significantly more than four isogenic mutants expressing truncated LOS. Sialylation of strain 1291 LOS inhibited invasion significantly. Preincubation of PHUECs in asialofetuin (ASF), an ASGP-R ligand, significantly reduced invasion. A dose-response reduction in invasion was observed in PHUECs preincubated with increasing concentrations of NaOH-deacylated 1291 LOS. These studies indicated that an interaction between lacto-N-neotetraose-terminal LOS and ASGP-R allows gonococcal entry into PHUECs.

Genotype-by-management interactions for grain yield and grain protein concentration of wheat

The magnitude of genotype-by-management (G x M) interactions for grain yield and grain protein concentration was examined in a multi-environment trial (MET) involving a diverse set of 272 advanced breeding lines from the Queensland wheat breeding program. The MET was structured as a series of management-regimes imposed at 3 sites for 2 years. The management-regimes were generated at each site-year as separate trials in which planting time, N fertiliser application rate, cropping history, and irrigation were manipulated. irrigation was used to simulate different rainfall regimes. From the combined analysis of variance, the G x M interaction variance components were found to be the largest source of G x E interaction variation for both grain yield (0.117 +/- 0.005 t(2) ha(-2); 49% of total G x E 0.238 +/- 0.028 t(2) ha(-2)) and grain protein concentration (0.445 +/- 0.020%(2); 82% of total G x E 0.546 +/- 0.057%(2)), and in both cases this source of variation was larger than the genotypic variance component (grain yield 0.068 +/- 0.014 t(2) ha(-2) and grain protein 0.203 +/- 0.026%(2)). The genotypic correlation between the traits varied considerably with management-regime, ranging from -0.98 to -0.31, with an estimate of 0.0 for one trial. Pattern analysis identified advanced breeding lines with improved grain yield and grain protein concentration relative to the cultivars Hartog, Sunco and Meteor. It is likely that a large component of the previously documented G x E interactions for grain yield of wheat in the northern grains region are in part a result of G x M interactions. The implications of the strong influence of G x M interactions for the conduct of wheat breeding METs in the northern region are discussed. (C) 2001 Elsevier Science B.V. All rights reserved.

Microbial interactions with tannins: Nutritional consequences for ruminants

Polyphenolics are widely distributed in the plant kingdom and are often present in the diet of herbivores. The two major groups of plant polyphenolic compounds other than lignin are condensed and hydrolysable tannins. These compounds can have toxic and/or antinutritional effects on the animal. It is well established that tannins complex with dietary proteins can reduce nitrogen supply to the animal, but the ability of gastrointestinal microorganisms to metabolise these compounds and their effects on microbial populations have received little attention. In this paper, we review recent literature on the topic as well as present research from our laboratories on the effect of condensed tannins on rumen microbial ecology and rumen metabolism. Interactions of tannins with dietary components and endogenous protein in the rumen and post-ruminally, and their impact on the nutrition of the animal are considered. (C) 2001 Elsevier Science B.V. All rights reserved.

Conservative management of mesenchymal hamartomas of the chest wall

Mesenchymal hamartomas of the chest wall are rare benign lesions usually discovered in infancy. The authors present their experience with 3 cases. All of these cases were managed initially conservatively, although 1 child required a thoracotomy and partial tumour resection at 5 months of age because of respiratory compromise. The other 2 children have now reached 5 and 6 years of age with the tumors becoming less prominent. The authors believe many cases can be managed conservatively because malignant change has not been reported, and the lesions often become relatively smaller as the child grows. J Pediatr Surg 36:1346-1349, Copyright (C) 2001 by W.B. Saunders Company.

A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface, We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodo- main of the interleukin-2 alpha (IL-2 alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK1 epithelial cells, Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin, Truncation mutants unable to bind beta -catenin were correctly targeted, showing, contrary to current understanding, that beta -catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine mediated targeting is maintained in UC-PK, cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line, These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.

Initiation of cell locomotility is a morphogenetic checkpoint in thyroid epithelial cells regulated by ERK and PI3-kinase signals

Epithelial locomotility is a fundamental determinant of tissue patterning that is subject to strict physiological regulation. The current, study sought to identify cellular signals that initiate cell migration in cultured thyroid epithelial cells. Porcine thyroid cells cultured as 3-dimensional follicles convert to 2-dimensional monolayers when deprived of agents that stimulate cAMP/PKA signaling. This morphogenetic event is driven by the activation of cell-on-substrate locomotility, providing a convenient assay for events that regulate the initiation of locomotion. In this system, the extracellular signal regulated kinase (ERK) pathway became activated as follicles converted to monolayer, as demonstrated by immunoblotting for activation-specific phosphorylation and nuclear accumulation of ERK. Inhibition of ERK activation using the drug PD98059 effectively prevented cells from beginning to migrate. PD98059 inhibited cell spreading, actin filament reorganization and the assembly of focal adhesions, cellular events that mediate the initiation of thyroid cell locomotility. Akt (PKB) signaling was also activated during follicle-to-monolayer conversion and the phosphoinositide 3-kinase (PI3-kinase) inhibitor, wortmannin, also blocked the initiation of cell movement. Wortmannin did not, however, block activation of ERK signaling. These findings, therefore, identify the ERK and PI3-kinase signaling pathways as important stimulators of thyroid cell locomotility. These findings are incorporated into a model where the initiation of thyroid cell motility constitutes a morphogenetic checkpoint regulated by coordinated changes in stimulatory (ERK, PI3-kinase) and tonic inhibitory (cAMP/PKA) signaling pathways. Cell Motil. Cytoskeleton 49:93-103, 2001. (C) 2001 Wiley-Liss, Inc.

Evaluation of synergistic interactions between the Colorado potato beetle (Coleoptera : Chrysomelidae) pathogen Beauveria bassiana and the insecticides, imidacloprid, and cyromazine

Laboratory studies investigated the interaction between the fungal entomopathogen Beauveria bassiana (Balsamo) Vuillemin and sublethal doses of the insecticides imidacloprid and cyromazine when applied to larvae of the Colorado potato beetle, Leptinotarsa decemlinenta (Say). When second instars were fed potato leaf discs treated with sublethal doses of imidacloprid and a range of doses of B. bassiana, a synergistic action was demonstrated. Similar results were observed when larvae were sprayed directly with B. bassiana conidia and immediately fed leaf discs treated with imidacloprid. No synergistic interaction was detected when larvae were fed leaf discs treated with sublethal doses of imidacloprid 24 h after application of R. bassiana conidia to larvae. However, a synergistic interaction was detected when larvae were fed leaf discs treated with imidacloprid and sprayed with B, bassiana conidia 24 h later. Although sublethal doses of both imidacloprid and the triazine insect growth regulator (IGR) cyromazine prolonged the duration of the second instar, only imidacloprid interacted with B. bassiana to produce a synergistic response in larval mortality. In leaf consumption studies, the highest dose of B, bassiana tested promoted feeding in inoculated second instars. Feeding was inhibited when larvae were fed foliage treated with sublethal doses of imidacloprid and significantly reduced when fed foliage treated with a sublethal dose of cyromazine. Starvation of larvae for 24 h immediately after B. bassiana treatment produced a similar result to the combined treatment of B. bassiana and imidacloprid and increased the level of mycosis when compared with B. bassiana controls. Imidacloprid treatment affected neither the rate of germination of B. bassiana conidia on the insect cuticle nor the rate at which conidia were removed from the integument after application. The statistical analysis used to detect synergism and the possible role of starvation-induced stress factors underlying the observed synergistic interactions are discussed.

MUC13, a novel human cell surface mucin expressed by epithelial and hemopoietic cells

Transmembrane mucins are glycoproteins involved in barrier function in epithelial tissues. To identify novel transmembrane mucin genes, we performed a tblastn search of the GenBank(TM) EST data bases with a serine/ threonine-rich search string, and a rodent gene expressed in bone marrow was identified. We determined the cDNA sequence of the human orthologue of this gene, MUC13, which localizes to chromosome band 3q13.3 and generates 3.2-kilobase pair transcripts encoding a 512-amino acid protein comprised of an N-terminal mucin repeat domain, three epidermal growth factor-like sequences, a SEA module, a transmembrane domain, and a cytoplasmic tail (GenBank(TM) accession no. AF286113), MUC13 mRNA is expressed most highly in the large intestine and trachea, and at moderate levels in the kidney, small intestine, appendix, and stomach, In situ hybridization in murine tissues revealed expression in intestinal epithelial and lymphoid cells. Immunohistochemistry demonstrated the human MUC13 protein on the apical membrane of both columnar and goblet cells in the gastrointestinal tract, as well as within goblet cell thecae, indicative of secretion in addition to presence on the cell surface. MUC13 is cleaved, and the beta -subunit containing the cytoplasmic tail undergoes homodimerization, Including MUC13, there are at least five cell surface mucins expressed in the gastrointestinal tract.

Genetic and physical interactions between Microphthalmia transcription factor and PU.1 are necessary for osteoclast gene expression and differentiation

The microphthalmia transcription factor (MITF), a basic-helix-loop-helix zipper factor, regulates distinct target genes in several cell types. We hypothesized that interaction with the Ets family factor PU.1, whose expression is limited to hematopoietic cells, might be necessary for activation of target genes like tartrate-resistant acid phosphatase (TRAP) in osteoclasts. Several lines of evidence were consistent with this model. The combination of MITF and PU.1 synergistically activated the TRAP promoter in transient assays. This activation was dependent on intact binding sites for both factors in the TRAP promoter. MITF and PU.1 physically interacted when coexpressed in COS cells or in vitro when purified recombinant proteins were studied. The minimal regions of MITF and PU.1 required for the interaction were the basic-helix-loop-helix zipper domain and the Ets DNA binding domain, respectively. Significantly, mice heterozygous for both the mutant mi allele and a PU.1 null allele developed osteopetrosis early in life which resolved with age. The size and number of osteoclasts were not altered in the double heterozygous mutant mice, indicating that the defect lies in mature osteoclast function. Taken in total, the results afford an example of how lineage-specific gene regulation can be achieved by the combinatorial action of two broadly expressed transcription factors.

Characterization of the EphA1 receptor tyrosine kinase: Expression in epithelial tissues

The Eph family (of receptor tyrosine kinases plays a crucial role during development and is implicated in oncogenesis. Using a partial cDNA clone of an Eph-related kinase (Esk) we isolated the complete coding region of a gene which we show to be murine EphA1 by both structural and functional criteria. The chromosomal localization is shown to be syntenic to hEphA1 and the genomic organization also shows distinct features found in the hEphA1 gene. Functionally, in keeping with findings for the human homologue, both soluble recombinant and native mEphA1 show preferential binding to ephrin A1. However, we also observed significant binding to other A-type ligands as has been observed for other Eph receptors. We analysed the expression of mEphA1 mRNA by in situ hybridization on tissue sections. mEphA1 was expressed in epithelial elements of skin, adult thymus, kidney and adrenal cortex. Taken together with previous Northern blotting data these results suggest that mEphA1 is expressed widely in differentiated epithelial cells.

The linker domain of the Ha-Ras hypervariable region regulates interactions with exchange factors, Raf-1 and phosphoinositide 3-kinase

Ha-Ras and Ki-Ras have different distributions across plasma membrane microdomains. The Ras C-terminal anchors are primarily responsible for membrane microlocalization, but recent work has shown that the interaction of Ha-Ras with lipid rafts is modulated by GTP loading via a mechanism that requires the hypervariable region (HVR). We have now identified two regions in the HVR linker domain that regulate Ha-Ras raft association. Release of activated Ha-Ras from lipid rafts is blocked by deleting amino acids 173-179 or 166-172. Alanine replacement of amino acids 173-179 but not 166-172 restores wild type micro-localization, indicating that specific N-terminal sequences of the linker domain operate in concert with a more C-terminal spacer domain to regulate Ha-Ras raft association. Mutations in the linker domain that confine activated Ha-RasG12V to lipid rafts abrogate Raf-1, phosphoinositide 3-kinase, and Akt activation and inhibit PC 12 cell differentiation. N-Myristoylation also prevents the release of activated Ha-Ras from lipid rafts and inhibits Raf-1 activation. These results demonstrate that the correct modulation of Ha-Ras lateral segregation is critical for downstream signaling. Mutations in the linker domain also suppress the dominant negative phenotype of Ha-RasS17N, indicating that HVR sequences are essential for efficient interaction of Ha-Ras with exchange factors in intact cells.

Mast cell/T cell interactions in oral lichen planus

Lichen planus is a disorder characterized by lesions of the skin and oral mucous membranes. Although many patients have involvement of both skin and oral mucosa at some stage during the progress of the disease, a larger group has oral involvement alone. It has been reported that oral lichen planus (OLP) affects one to two percent of the general population and has the potential for malignant transformation in some cases (1, 2). Like many chronic inflammatory skin diseases, it often persists for many years. Numerous disorders may be associated with OLP such as graft-vs.-host disease and Hepatitis C virus infection (3), however, it is unclear how such diverse influences elicit the disease and indeed whether they are identical to idiopathic OLP Available evidence supports the view that OLP is a cell-mediated immunological response to an induced antigenic change in the mucosa (4-6). Studies of the immunopathogenesis of OLP aim to provide specific novel treatments as well as contributing to our understanding of other cell-mediated inflammatory diseases. In this paper, the interactions between mast cells and T cells are explored from the standpoint of immune regulation. From these data, a unifying hypothesis for the immunopathogenesis of OLP is then developed and presented.

Intra-epithelial CD8+ T cells and basement membrane disruption in oral lichen planus

Background: We investigated basement membrane (BM) disruption and the distribution of mast cells (MCs) and T cell subsets, in oral lichen planus (OLP) and normal buccal mucosa (NBM) using immunohistochemistry. In OLP, there were increased numbers of tryptase(+) MCs in areas of BM disruption (P