Ingested soluble CD14 contributes to the functional pool of circulating sCD14 in mice.

Soluble CD14 (sCD14) is a pattern recognition receptor and Toll-like co-receptor observed in human milk (5-26µg/mL) and other bodily fluids such as blood (3µg/mL). The most well defined role of sCD14 is to recognize lipopolysaccharide of Gram-negative bacteria and signal an immune response through Toll-like receptor 4 (TLR4). Previous research has shown ingested sCD14 to transfer from the gastrointestinal tract and into the blood stream in neonatal rats. The contribution of human milk sCD14 to circulating levels in the infant and the functionality of the protein, however, remained unknown. Using CD14(-/-) mouse pups fostered to wild type (WT) mothers expressing sCD14 in their milk, we show herein that ingestion of sCD14 resulted in blood sCD14 levels up 0.16±0.09µg/mL. This represents almost one-third (26.7%) of the circulating sCD14 observed in WT pups fostered to WT mothers (0.60±0.14µg/mL). We also demonstrate that ingested-sCD14 transferred to the blood remains functional in its ability to recognize lipopolysaccharide as demonstrated by a significant increase in immune response (IL-6 and TNF-α) in CD14(-/-) pups fostered to WT mothers in comparison to control animals (P=0.002 and P=0.007, respectively). Using human intestinal cells (Caco-2), we also observed a significant decrease in sCD14 transcytosis when TLR4 was knocked down (P<0.001), suggesting sCD14 transfer involves TLR4. The bioavailability of human milk sCD14 established in this report confirms the importance of human milk proteins for the infant and demonstrates the need to improve infant formulas which are lacking in immune proteins such as sCD14.

Expression of the nos operon proteins from Pseudomonas stutzeri in transgenic plants to assemble nitrous oxide reductase.

Nitrous oxide (N(2)O) is a stable greenhouse gas that plays a significant role in the destruction of the ozone layer. Soils are a significant source of atmospheric N(2)O. It is important to explore some innovative and effective biology-based strategies for N(2)O mitigation. The enzyme nitrous oxide reductase (N(2)OR), naturally found in soil bacteria, is responsible for catalysing the final step of the denitrification pathway, conversion of N(2)O to dintrogen gas (N(2)). To transfer this catalytic pathway from soil into plants and amplify the abundance of this essential mechanism (to reduce global warming), a mega-cassette of five coding sequences was assembled to produce transgenic plants heterologously expressing the bacterial nos operon in plant leaves. Both the single-gene transformants (nosZ) and the multi-gene transformants (nosFLZDY) produced active recombinant N(2)OR. Enzymatic activity was detected using the methyl viologen-linked enzyme assay, showing that extracts from both types of transgenic plants exhibited N(2)O-reducing activity. Remarkably, the single-gene strategy produced higher reductase capability than the whole-operon approach. The data indicate that bacterial N(2)OR expressed in plants could convert N(2)O into inert N(2) without involvement of other Nos proteins. Silencing by homologous signal sequences, or cryptic intracellular targeting are possible explanations for the low activities obtained. Expressing N(2)OR from Pseudomonas stutzeri in single-gene transgenic plants indicated that such ag-biotech solutions to climate change have the potential to be easily incorporated into existing genetically modified organism seed germplasm.