Gingipains inactivate a cell surface ligand on Porphyromonas gingivalis that induces TLR2-and TLR4-independent signaling

Arginine-specific gingipain and lysine-specific gingipain are two major cysteine proteinases produced by Porphyromonas gingivalis. To clarify the role of gingipains in the interaction between P. gingivalis and the innate immune system, CHO reporter cells expressing TLR2 or TLR4 were stimulated with wildtype or gingipain-deficient P. gingivalis cells and activation of nuclear factor-kappaB in these cells was examined. While CHO/CD14 cells and 7.19 cells, an MD-2-defective mutant derived from CHO/CD14 cells, failed to respond to wild-type P. gingivalis, they responded to gingipain-deficient P. gingivalis. On the other hand, CHO/CD14/TLR2 cells responded to both wild-type and gingipain-deficient P. gingivalis. These results suggested that gingipains have no effects on TLR2-dependent signaling from P. gingivalis but have inhibitory effects on TLR2-and TLR4-independent signaling in CHO cells. Indeed, the activity of gingipain-deficient P. gingivalis to induce the activation of 7.19 cells was diminished after treatment of the bacterial cells with gingipains. We next partially purified bacterial cell components activating 7.19 cells from gingipain-deficient P. gingivalis. The activity of the partially purified components was diminished by treatment with heat or gingipains. It is also noteworthy that anti-CD14 mAb inhibited the activation of 7.19 cells induced by the partially purified components. These results indicated that the components of P. gingivalis that were able to induce TLR2-and TLR4-independent signaling were inactivated by gingipains before being recognized by CD14. The inactivation of the components would be helpful for P. gingivalis to escape from the innate immune system.     

Effect of polyanions and polycations on adenosine 3',5'-monophosphate-binding and protein kinase activity.

Histone, protamine, poly-L-arginine, and poly-L-lysine enhance the binding of adenosine 3′,5′-monophosphate (cyclic AMP) to rat liver cyclic AMP-dependent protein kinase as determined by Millipore filtration assay. Poly-L-glutamic acid and poly-L-aspartic acid suppress cyclic AMP-binding stimulated by histone. Poly-L-glutamic acid and poly-L-aspartic acid are effective against protein kinase and result in decrease in initial reaction velocity when histone is used as a protein substrate. Incubation of cyclic AMP-dependent protein kinase with 6 μg poly-L-glutamic acid produces half-maximal inhibition of cyclic AMP-dependent protein kinase when 30 μg histone is used as substrate.     

Fruit Flies in Biomedical Research

Many scientists complain that the current funding situation is dire. Indeed, there has been an overall decline in support in funding for research from the National Institutes of Health and the National Science Foundation. Within the Drosophila field, some of us question how long this funding crunch will last as it demotivates principal investigators and perhaps more importantly affects the long-term career choice of many young scientists. Yet numerous very interesting biological processes and avenues remain to be investigated in Drosophila, and probing questions can be answered fast and efficiently in flies to reveal new biological phenomena. Moreover, Drosophila is an excellent model organism for studies that have translational impact for genetic disease and for other medical implications such as vector-borne illnesses. We would like to promote a better collaboration between Drosophila geneticists/biologists and human geneticists/bioinformaticians/clinicians, as it would benefit both fields and significantly impact the research on human diseases.     

Association of phosphorylated insulin-like growth factor-I receptor with the SH2 domains of phosphatidylinositol 3-kinase p85.

Insulin-like growth factor-I (IGF-I) stimulates the production of 3-inositides and markedly increases the phosphatidylinositol 3-kinase activity that is immunoprecipitated by anti-phosphotyrosine antibodies, a portion of which is also associated with the IGF-I receptor. In this study, recombinant p85, the regulatory subunit of phosphatidylinositol 3-kinase, and fusion proteins containing various subdomains were used to investigate the association of p85 with the IGF-I receptor and to demonstrate that p85 is a direct in vitro substrate of the IGF-I receptor kinase. Solubilized IGF-I receptor was immobilized on antireceptor antibody-agarose beads. Following in vitro receptor phosphorylation and incubation with cell lysate, immobilized receptor became associated with phosphatidylinositol 3-kinase activity and with protein bands with molecular masses of 85 and 110 kDa, which correspond to the known molecular masses of the subunits of phosphatidylinositol 3-kinase. These associations were inhibited by the addition of recombinant intact p85 or SH2-containing fusion proteins, but not by fusion proteins containing its SH3 domain or breakpoint cluster homology region. A fusion protein containing the SH2 domains of Ras GTPase-activating protein also inhibited the association of phosphatidylinositol 3-kinase activity with immobilized IGF-I receptor, although less effectively than p85, whereas a similar construct containing the SH2 domain of pp60src was without effect. When immobilized phosphorylated IGF-I receptor was incubated with intact p85 or the SH2-containing fusion proteins, it became associated with and phosphorylated these proteins. These results demonstrate that at least in vitro, a tight association occurs between phosphorylated IGF-I receptor and phosphatidylinositol 3-kinase, that the region of phosphatidylinositol 3-kinase that contains its SH2 domains is directly involved in this association, and that this region is a direct substrate for IGF-I receptor tyrosine kinase. Furthermore, these results suggest that Ras GTPase-activating protein can also interact with the IGF-I receptor and that different SH2 domain-containing proteins interact with the IGF-I receptor with widely differing affinities.     

Failure to Reject an Allografted Tumor after Elimination of Macrophages in Mice

After an i.p. transplantation of an allogeneic tumor (Meth A) to C57BL/6 mice, a macrophage (MΦ)-rich, non-T, non-NK cell population is induced as the major infiltrate and cytotoxic cells. We here evaluated the role of the MΦs in the rejection of allografted Meth A cells and characterized the MΦs in comparison with other well-known MΦs. At all time intervals after transplantation, the highest cytotoxic activities against Meth A tumor were obtained with the MΦ-rich population. In addition, the lymphocyte-rich population had a significant but low cytotoxic activity, whereas two other population types, granulocytes and large granular cells, were inactive. When the MΦ-rich or the T cell-depleted MΦ-rich population was i.p. transplanted simultaneously with Meth A cells into untreated C57BL/6 mice, the tumor cells were rejected without growth. After specific elimination of MΦs by in vivo application of dichloromethylene diphosphonate-containing liposomes, the cytotoxic activity against Meth A cells was hardly induced at the transplantation site of Meth A cells and the allografted Meth A tumor continued to grow, indicating that a type of MΦ is the effector cell essential for the rejection. In contrast to other well-known MΦs, the cytotoxic activity against Meth A cells was cell-to-cell contact dependent and soluble factor (e.g., NO and TNF-α) independent. Moreover, the cytotoxic activity of the MΦs (H-2^b) against ⁵¹Cr-labeled Meth A (H-2^d) cells was inhibited by the addition of unlabeled H-2^d, but not H-2^a, H-2^k or H-2^b, lymphoblasts as well as Meth A cells, implying the specific interaction of the MΦs with H-2^d cells.     

The ultrastructural basis of the permeability of arterial endothelium to horseradish peroxidase

The thoracic aorta and basilar artery, in which the incidence of atherosclerosis is known to be different, were examined to elucidate the correlation between the structure of the intercellular cleft junction between adjacent endothelial cells and its permeability to HRP. Tannic acid or HRP in the vessel lumen passed through the intercellular clefts of the thoracic aorta into the subendothelial space, whereas in the basilar artery they were unable to penetrate beyond the tight junction of the intercellular clefts. Freeze-fracture replicas revealed that the tight junctions of the thoracic aorta consisted of one to two junctional strands in most areas of the cleaved planes, with discontinuities in some places, whereas those of the basilar artery consisted of a continuous belt-like meshwork of six anastomosing junctional strands on average. These observations confirm that the structure of endothelial junctions in arteries has a close correlation with the permeability of the intercellular clefts to HRP.