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We synthesized ridaifen-B boron dipyrromethene (RID-B-BODIPY) using 2methyl-6-nitro benzoic anhydride (MNBA)-mediated dehydration condensation reaction between amino alkyl-tethered RID and BODIPY FL. Comparative experiments between dicyclohexylcarbodiimide (DCC) and MNBA for their coupling reactions demonstrated that MNBA is an effective condensation reagent for amines and BODIPY FL. A cell staining study with RID-B-BODIPY showed intracellular localization of BODIPY FL fluorescence, attributed to the RID-B structure, indicating the successful development of a tool for analyzing intracellular molecular behavior efficiently. [ABSTRACT FROM AUTHOR]

Background: CpG oligodeoxynucleotides (ODNs), resembling bacterial DNA, are currently tested in clinical trials as vaccine adjuvants. They have the nuclease-resistant phosphorothioate bond; the immune responses elicited differ according to the CpG ODN sequence and vaccination method. To develop a CpG ODN that can induce plasmacytoid dendritic cell (pDC)-mediated TH1 immunity through the mucosa, we constructed phosphodiester G9.1 comprising one palindromic CpG motif with unique polyguanosine-runs that allows degradation similar to naturally occurring bacterial DNA. Methods: TH1 and TH2 immunity activation was evaluated by cytokine production pattern and T-bet/GATA-3 ratio in human peripheral blood mononuclear cells and mouse bone marrow cells. Adjuvanticity was evaluated in mice administered G9.1 with diphtheria toxoid (DT) through nasal vaccination. Results: G9.1 exhibited stronger IFN-α-inducing activity than A-class CpG ODN2216 and increased T-bet/GATA-3 ratio by enhancing T-bet expression. Nasally administered G9.1 plus DT induced DT-specific mucosal IgA and serum IgG, but not IgE, responses with antitoxin activity in C57BL/6 and BALB/c mice, possibly due to IFN/BAFF production. Induction of TH1, but not TH2, -type Abs depended completely on pDCs, the first in vivo demonstration by CpG ODNs. Conclusions: G9.1 is a promising mucosal adjuvant for induction of pDC-mediated TH1 immunity. [ABSTRACT FROM AUTHOR]

While some intracellular bacterial and viral proteins secreted into host cell possess ubiquitin ligase (E3) activity for their profit, it has not been reported whether intracellular parasites secrete such molecules. We identified a gene that encodes a protein containing a secretory signal peptide and a RING finger domain in the intracellular protozoan parasite, Trypanosoma cruzi. This gene was specific to T. cruzi and was designated spring ( rotein with finger domain). An in vitro ubiquitination assay showed that SPRING possessed E3 activity in a RING finger domain-dependent manner. SPRING could utilize human ubiquitin-activating enzymes (E2), UbcH5 and UbcH13. Although SPRING was found to be a secretory protein, the signal peptide-cleaved mature form of SPRING was localized in the nucleus of host cells, indicating that SPRING may function in the host cell nuclei. Yeast two-hybrid screening identified 52 putative SPRING interactors in HeLa cells, suggesting that SPRING affects the stability or function of a number of host proteins. Furthermore, a co-immunoprecipitation assay showed that breast cancer-associated protein 3 interacted with SPRING, as well as being ubiquitinated by SPRING in vitro. These findings are the first to show that this protozoan parasite secretes an ubiquitin ligase-related protein into host cells. [ABSTRACT FROM AUTHOR]

Death receptor-mediated host cell apoptosis, a defense strategy for elimination by the immune system of parasite-infected cells, is inhibited by Trypanosoma cruzi, the causative agent of Chagas' disease. It has previously been reported by us that, in infected cells, T. cruzi upregulates and exploits cFLIPL, a mammalian inhibitor of death receptor signaling. Here it is shown that ubiquitination of cFLIPL, leading to proteasomal degradation, is inhibited in parasite-infected cells. The extent of expression of Itch, a protein thought to be an ubiquitin ligase for cFLIPL, was found to be equivalent in T. cruzi-infected and in uninfected cells. However, co-immunoprecipitation analysis showed that the interaction between cFLIPL and Itch is strongly inhibited in T. cruzi-infected cells. This unique parasite strategy, which has not been reported in any other pathogen-infected cells, may allow the host cell to accumulate cFLIPL, eventually resulting in the inhibition of apoptosis of T. cruzi-infected cells. [ABSTRACT FROM AUTHOR]

Inhibin-A is a glycoprotein composed of an a subunit containing a glycosylation site and a βA subunit, whereas activin-A is a homodimer of two inhibin βA subunits. We examined the recognition of activin-A and inhibin-A by several antisera to the α or βA; subunit, and factors affecting the recognition. A total of six polyclonal antibodies to inhibin subunits, i.e., two antisera to a peptide fragment of the α subunit [α(l–19) and α(lα26)], and four antisera to the βA subunit [βA(l–10), βA(70–79), βA(87–99), and βA(94–105)], was generated. On Western blot analysis, the anti-βA(87–99) and βA(94-105) sera recognized recombinant human activin-A but not inhibin-A under non-reducing conditions. When inhibin-A was deglycosylated with N-glycosidase-F, inhibin-A could be recognized by the anti-βA(87–99) and βA(94–105) sera. In addition, when activin-A bound to a nitrocellulose membrane was p re-incubated with recombinant human follistatin, the recognition of activin-A by the anti-βA(87–99) and βA(94–105) sera was decreased. These results suggested that the lower affinity of follistatin to inhibin-A than to activin-A might be likely explained as reflecting a site associated with the glycosylation of inhibin-A. However, the exposure of amino acids 87–105 of the inhibin 0A subunit on the molecular surface through deglycosylation did not increase the affinity of inhibin-A for follistatin but rather resulted in poor binding with follistatin. The present data suggest that (1) amino acids 87–105 of the inhibin/activin βA subunit are located on the molecular surface, although this region of inhibin-A is concealed by the carbohydrate chain of the α subunit, (2) the region responsible for follistatin binding within the activin βA subunit is spanned by amino acids 87-105, and (3) the mode of binding of inhibin-A to follistatin is quite different from that of activin-A to follistatin, and the former may be influenced by glycosylation. [ABSTRACT FROM AUTHOR]

Malaria parasite transmission to humans is initiated by the inoculation of Plasmodium sporozoites into the skin by mosquitoes. Sporozoites develop within mosquito midgut oocysts, first invade the salivary glands of mosquitoes, and finally infect hepatocytes in mammals. The apical structure of sporozoites is conserved with the infective forms of other apicomplexan parasites that have secretory organelles, such as rhoptries and micronemes. Because some rhoptry proteins are crucial for Plasmodium merozoite infection of erythrocytes, we examined the roles of rhoptry proteins in sporozoites. Here, we demonstrate that rhoptry neck protein 2 (RON2) is also localized to rhoptries in sporozoites. To elucidate RON2 function in sporozoites, we applied a promoter swapping strategy to restrict ron2 transcription to the intraerythrocytic stage in the rodent malaria parasite, Plasmodium berghei. Ron2 knockdown sporozoites were severely impaired in their ability to invade salivary glands, via decreasing the attachment capacity to the substrate. This is the first rhoptry protein demonstrated to be involved in salivary gland invasion. In addition, ron2 knockdown sporozoites showed less infectivity to hepatocytes, possibly due to decreased attachment/gliding ability, indicating that parts of the parasite invasion machinery are conserved, but their contribution might differ among infective forms. Our sporozoite stage‐specific knockdown system will help to facilitate understanding the comprehensive molecular mechanisms of parasite invasion of target cells. [ABSTRACT FROM AUTHOR]