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Comprehensive Study of the Influence of Altered Gravity on the Oxidative Burst of Mussel (Mytilus edulis) Hemocytes

by cfynanon 22 August 2016in Biology & Biotechnology No comment

Microgravity induces alterations in the function- ing of immune cell; however, the underlying mechanisms have not yet been identified. In this study, hemocytes (blood cells) of the blue mussel Mytilus edulis were investigated under altered gravity conditions. The study was conducted on the ground in preparation for the BIOLAB TripleLux- B experiment, which will be performed on the International Space Station (ISS). On-line kinetic measurements of reac- tive oxygen species (ROS) production during the oxidative burst and thus cellular activity of isolated hemocytes were performed in a photomultiplier (PMT)-clinostat (simulated microgravity) and in the 1g operation mode of the clino- stat in hypergravity on the Short-Arm Human Centrifuge (SAHC) as well as during parabolic flights. In addition to studies with isolated hemocytes, the effect of altered gravity conditions on whole animals was investigated. For this pur- pose, whole mussels were exposed to hypergravity (1.8 g) on a multi-sample incubator centrifuge (MuSIC) or to simu- lated microgravity in a submersed clinostat. After exposure for 48 h, hemocytes were taken from the mussels and ROS production was measured under 1 g conditions. The results from the parabolic flights and clinostat studies indicate that mussel hemocytes respond to altered gravity in a fast and reversible manner. Hemocytes (after cryo-conservation)exposed to simulated microgravity (μ g), as well as fresh hemocytes from clinorotated animals, showed a decrease in ROS production. Measurements during a permanent exposure of hemocytes to hypergravity (SAHC) show a decrease in ROS production. Hemocytes of mussels mea- sured after the centrifugation of whole mussels did not show an influence to the ROS response at all. Hypergravity dur- ing parabolic flights led to a decrease but also to an increase in ROS production in isolated hemocytes, whereas the cen- trifugation of whole mussels did not influence the ROS response at all. This study is a good example how ground- based facility experiments can be used to prepare for an upcoming ISS experiment, in this case the TRIPLE LUX B experiment.

Attachment of LcrV from Yersinia pestis at dual binding sites to human TLR-2 and human IFN-gamma receptor.

by cfynanon 22 August 2016in Biology & Biotechnology No comment

The virulence antigen (V-antigen, LcrV) of Yersinia pestis, the causative agent of bubonic plague, is an established protective antigen known to regulate, target, and mediate type III translocation of cytotoxic yersiniae outer proteins termed Yops; LcrV also prompts TLR2-dependent upregulation of anti-inflammatory IL-10. In this study, we determined the parameters of specific interaction of LcrV with TLR2 expressed on human transfected HEK293 cells (TLR2+/CD14-), VTEC2.HS cells (TLR2+/CD14-), primary monocytes (TLR2+/CD14+), and THP-1 cells (TLR2+/CD14+). The IRRL314-317 motif of the extracellular domain of human and mouse TLR2 accounted for high-affinity binding of LcrV. The CD14 co-receptor did not influence this interaction. LcrV did not bind to human U937 (TLR2-/CD14-) and alveolar macrophages (TLR2-/CD14+) in the absence of receptor-bound human IFN-gamma or a synthetic C-terminal fragment (hIFN-gamma132-143). The latter, but not mouse IFN-gamma (or synthetic control peptides), shared a GRRA138-141 site necessary for high-affinity specific binding. LcrV of Y. pestis shares the N-terminal LEEL32-35 binding site of Yersinia enterocolitica and also has an exposed internal DEEI203-206 binding site. Comparison of binding constants and consideration of steric restrictions indicate that binding is not cooperative and only the internal site binds LcrV to target cells. Both the LEEL32-35 and DEEI203-206 binding sites are removed by five amino acids from DKN residues associated with biological activity of bound LcrV. LcrV of Y. pestis promoted both TLR2/CD14-dependent and TLR2/CD14-independent amplification of IL-10 and concomitant downregulation of TNF-alpha in human target cells. The ability of LcrV to utilize human IFN-gamma (a major inflammatory effector of innate immunity) to minimize inflammation is insidious and may account in part for the severe symptoms of plague in man.

Interaction of Yersinia pestis Virulence Factors with IL-1R/TLR Recognition System

by cfynanon 22 August 2016in Biology & Biotechnology No comment

The virulence antigen (V-antigen, LcrV) of Yersinia pestis, the causative agent of bubonic plague, is an established protective antigen known to regulate, target, and mediate type III translocation of cytotoxic yersiniae outer proteins termed Yops; LcrV also prompts TLR2-dependent upregulation of anti-inflammatory IL-10. In this study, we determined the parameters of specific interaction of LcrV with TLR2 expressed on human transfected HEK293 cells (TLR2+/CD14-), VTEC2.HS cells (TLR2+/CD14-), primary monocytes (TLR2+/CD14+), and THP-1 cells (TLR2+/CD14+). The IRRL314-317 motif of the extracellular domain of human and mouse TLR2 accounted for high-affinity binding of LcrV. The CD14 co-receptor did not influence this interaction. LcrV did not bind to human U937 (TLR2-/CD14-) and alveolar macrophages (TLR2-/CD14+) in the absence of receptor-bound human IFN-γ or a synthetic C-terminal fragment (hIFN-γ132-143). The latter, but not mouse IFN-γ (or synthetic control peptides), shared a GRRA138-141 site necessary for high-affinity specific binding. LcrV of Y. pestis shares the N-terminal LEEL32-35 binding site of Yersinia enterocolitica and also has an exposed internal DEEI203-206 binding site. Comparison of binding constants and consideration of steric restrictions indicate that binding is not cooperative and only the internal site binds LcrV to target cells. Both the LEEL32-35 and DEEI203-206 binding sites are removed by five amino acids from DKN residues associated with biological activity of bound LcrV. LcrV of Y. pestis promoted both TLR2/CD14-dependent and TLR2/CD14-independent amplification of IL-10 and concomitant downregulation of TNF-α in human target cells. The ability of LcrV to utilize human IFN-γ (a major inflammatory effector of innate immunity) to minimize inflammation is insidious and may account in part for the severe symptoms of plague in man.

The V-Antigen of Yersinia Forms a Distinct Structure at the Tip of Injectisome Needles

by cfynanon 22 August 2016in Biology & Biotechnology No comment

Many pathogenic bacteria use injectisomes to deliver effector proteins into host cells through type III secretion. Injectisomes consist of a basal body embedded in the bacterial membranes and a needle. In Yersinia, translocation of effectors requires the YopB and YopD proteins, which form a pore in the target cell membrane, and the LcrV protein, which assists the assembly of the pore. Here we report that LcrV forms a distinct structure at the tip of the needle, the tip complex. This unique localization of LcrV may explain its crucial role in the translocation process and its efficacy as the main protective antigen against plague.

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Research Containing: innate immunity

Comprehensive Study of the Influence of Altered Gravity on the Oxidative Burst of Mussel (Mytilus edulis) Hemocytes

Attachment of LcrV from Yersinia pestis at dual binding sites to human TLR-2 and human IFN-gamma receptor.

Interaction of Yersinia pestis Virulence Factors with IL-1R/TLR Recognition System

The V-Antigen of Yersinia Forms a Distinct Structure at the Tip of Injectisome Needles