Bovine trophoblastic cell vesicle attachment to polarized endometrial epithelial cells in vitro

Summary Interactions between bovine trophoblastic cell vesicles and bovine endometrial epithelial cells were investigated by light and electron microscopy and lectin histochemistry in a cell culture model of early blastocyst attachment. Primary lines of bovine endometrial epithelial cells were polarized by subculturing on substrata and maintaining cultures at the air-medium interface. Trophoblastic cell vesicles were obtained from elongated Day 14 blastocysts. In co-cultures, trophoblastic cell vesicles adhered to endometrial epithelial cells through microvillus interdigitation and formation of primitive membrane junctional complexes. After 3 d in co-culture, a multilayered cellular plaque formed at the trophoblastic cell-endometrial epithelial cell interface. The type of cells contributing to this local proliferative response could not be identified specifically as trophoblastic or endometrial cells, and areas of membrane fusion between cells were noted. Ultrastructural features of vesicle adhesion in cultures were similar to features of conceptus attachment in vivo. Lectins bound to apical membranes of trophoblastic cells and endometrial epithelial cells in all locations except contact sites between vesicles and endometrial cells. These findings suggest that local cellular proliferation and membrane fusion between trophoblastic and endometrial epithelial cells may be early events in conceptus implantation in the cow and these events can be reproduced in culture. This work was supported by a grant from U.S. Department of Agriculture Animal Health and Disease Program, Washington, DC.     

Differences in metallothionein gene expression in primary cultures of rainbow trout hepatocytes and the RTH-149 cell line

Primary cultures of rainbow trout, Salmo gairdneri, hepatocytes were used to study the expression of metallothionein (MT) genes in response to steroid hormone treatment. The expression pattern was compared to that of an immortal cell line (RTH-149). MT mRNA accumulated in both cell cultures after exposure to zinc while 17 beta-oestradiol had no effect in either system. Treatment with cortisol and corticosterone resulted in a 2-fold increase of metallothionein mRNA levels in the primary cultures but had no effect in the RTH-149 cell culture. Primary cultures that were exposed to zinc or cortisol showed a high temporal correlation (r = 0.974) between MT mRNA and MT protein levels. The basal level expression was 3-4-fold higher in primary cultures than in RTH-149 cells. The present study demonstrates the inducibility of rainbow trout MT genes in response to glucocorticoids. It further indicates that primary cultures are to be preferred to immortal cell lines when investigating the inducibility of MT mRNA.     

Regulation of the xylan-degrading apparatus of Cellvibrio japonicus by a novel two-component system

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.     

Diel phytoplankton periodicity in Mikołajskie lake,Poland, as determined by different methods in parallel

Photosynthetic rates as measured by the oxygen light and dark bottle method were highly correlated with estimates using the ¹⁴C technique. The high O₂/¹⁴C ratios found are explained by algal respiration and extracellular release which are included in photosynthetic measurements by the oxygen technique, while the ¹⁴C method yields values close to net photosynthesis. Separation of net- and nannoplankton using a 50 μm plankton net for filtration was not comparable to distinctions made by microscopic examination. Separation of both by filtration caused a significant decrease in the photosynthetic activity of nannoplankton in 24-hour incubations, but had no detectable effect after 4 hours of exposure. “Bottle effects” in 24-hour measurements of photosynthesis were similar using both methods. Asymmetric photosynthetic time-curves as well as vertical phytoplankton migrations were the main reasons for errors in estimates of daily photosynthetic rates from part-day incubations which were extrapolated to the entire day.     

Evidence That Lipopolisaccharide May Contribute to the Cytokine Storm and Cellular Activation in Patients with Visceral Leishmaniasis

Background

Visceral leishmaniasis (VL) is characterized by parasite-specific immunosuppression besides an intense pro-inflammatory response. Lipopolisaccharide (LPS) has been implicated in the immune activation of T-cell deficient diseases such as HIV/AIDS and idiopathic lymphocytopenia. The source of LPS is gram-negative bacteria that enter the circulation because of immunological mucosal barrier breakdown. As gut parasitization also occurs in VL, it was hypothesized that LPS may be elevated in leishmaniasis, contributing to cell activation.

Methodology/Principal Findings

Flow cytometry analysis and immunoassays (ELISA and luminex micro-beads system) were used to quantify T-cells and soluble factors. Higher LPS and soluble CD14 levels were observed in active VL in comparison to healthy subjects, indicating that LPS was bioactive; there was a positive correlation between these molecules (r = 0.61;p<0.05). Interestingly, LPS was negatively correlated with CD4⁺ (r = −0.71;p<0.01) and CD8⁺ T-cells (r = −0.65;p<0.05). Moreover, higher levels of activation-associated molecules (HLA-DR, CD38, CD25) were seen on T lymphocytes, which were positively associated with LPS levels. Pro-inflammatory cytokines and macrophage migration inhibitory factor (MIF) were also augmented in VL patients. Consistent with the higher immune activation status, LPS levels were positively correlated with the inflammatory cytokines IL-6 (r = 0.63;p<0.05), IL-8 (r = 0.89;p<0.05), and MIF (r = 0.64;p<0.05). Also, higher plasma intestinal fatty acid binding protein (IFABP) levels were observed in VL patients, which correlated with LPS levels (r = 0.57;p<0.05).

Conclusions/Significance

Elevated levels of LPS in VL, in correlation with T-cell activation and elevated pro-inflammatory cytokines and MIF indicate that this bacterial product may contribute to the impairment in immune effector function. The cytokine storm and chronic immune hyperactivation status may contribute to the observed T-cell depletion. LPS probably originates from microbial translocation as suggested by IFABP levels and, along with Leishmania antigen-mediated immune suppression, may play a role in the immunopathogenesis of VL. These findings point to possible benefits of antimicrobial prophylaxis in conjunction with anti-Leishmania therapy.     

Mitochondrial DNA clonality in the dock: can surveillance swing the case?

Mitochondrial DNA (mtDNA) is a favoured tool of evolutionary biologists because its high mutation rate generates enough signal to make inferences about population history over short time frames. Furthermore, mtDNA inheritance is clonal, being transmitted only through the maternal line. This enables evolutionary histories to be assembled without the complexities introduced by biparental recombination. Recently, a single case of human biparental inheritance has been reported. Given this, and the role supposed clonal inheritance has had in shaping our knowledge of human population history, it is essential to establish a method for identifying any recombinant mtDNA molecules in our population. A reliable surveillance mechanism would either maintain our confidence in clonal inheritance or indicate the inaccuracy of our inferences.