Project description:The medial nucleus of the trapezoid body (MNTB) contains a nearly homogeneous population of neurons, which are innervated by large glutamatergic nerve terminals called calyces of Held (CH). Key steps in maturation of CHs and MNTB neurons, including CH growth and competition, occur very quickly for most cells between postnatal days (P)2 and P6. Therefore, we characterized genome-wide changes in this system, with dense temporal sampling during the first postnatal week. We identified 541 genes whose expression changed significantly between P0-6 and clustered them into eight groups based on temporal expression profiles. Candidate genes from each of the eight profile groups were validated in separate samples by qPCR.
Project description:Malaria remains one the most infectious and destructive protozoan diseases worldwide. Plasmodium falciparum, a protozoan parasite with a complex life cycle and high genetic variability responsible for the difficulties in vaccine development, is implicated in most malaria-related deaths. In the course of study, we prepared a set of antigens based on P-proteins from P. falciparum and determined their immunogenicity in an in vivo assay on a mouse model. The pentameric complex P0-(P1-P2)2 was prepared along with individual P1, P2, and P0 antigens. We determined the level of cellular- and humoral-type immunological response followed by development of specific immunological memory. We have shown that the number of Tc cells increased significantly after the first immunization with P2 and after the second immunization with P1 and P0-(P1-P2)2, which highly correlated with the number of Th1 cells. P0 appeared as a poor inducer of cellular response. After the third boost with P1, P2, or P0-(P1-P2)2, the initially high cellular response dropped to the control level accompanied by elevation of the number of activated Treg cells and a high level of suppressive TGF-β. Subsequently, the humoral response against the examined antigens was activated. Although the titers of specific IgG were increasing during the course of immunization for all antigens used, P2 and P0-(P1-P2)2 were found to be significantly stronger than P1 and P0. A positive correlation between the Th2 cell abundance and the level of IL-10 was observed exclusively after immunization with P0-(P1-P2)2. An in vitro exposure of spleen lymphocytes from the immunized mice especially to the P1, P2, and P0-(P1-P2)2 protein caused 2-3-fold higher cell proliferation than that in the case of lymphocytes from the nonimmunized animals, suggesting development of immune memory. Our results demonstrate for the first time that the native-like P-protein pentameric complex represents much stronger immune potential than individual P-antigens.
Project description:BackgroundSec18p/N-ethylmaleimide-sensitive factor (NSF) is a conserved eukaryotic ATPase, which primarily functions in vesicle membrane fusion from yeast to human. However, the function of the OsSec18 gene, a homologue of NSF in rice, remains unknown.ResultsIn the present study, we investigated the function of OsSec18 in rice and found that OsSec18 complements the temperature-sensitive phenotype and interferes with vacuolar morphogenesis in yeast. Overexpression of OsSec18 in rice decreased the plant height and 1000-grain weight and altered the morphology of the protein bodies. Further examination revealed that OsSec18 presented as a 290-kDa complex in rice endosperm cells. Moreover, Os60sP0 was identified a component of this complex, demonstrating that the OsSec18 complex contains another complex of P0(P1-P2)2 in rice endosperm cells. Furthermore, we determined that the N-terminus of OsSec18 can interact with the N- and C-termini of Os60sP0, whereas the C-terminus of OsSec18 can only interact with the C-terminus of Os60sP0.ConclusionOur results revealed that the OsSec18 regulates vacuolar morphology in both yeast and rice endosperm cell and the OsSec18 interacts with P0(P1-P2)2 complex in rice endosperm cell.
Project description:The ribosomal stalk is formed by four acidic phosphoproteins in Saccharomyces cerevisiae, P1α, P1β, P2α and P2β, which form two heterodimers, P1α/P2β and P1β/P2α, that preferentially bind to sites A and B of the P0 protein, respectively. Using mutant strains carrying only one of the four possible P1/P2 combinations, we found a specific phenotype associated to each P1/P2 pair, indicating that not all acidic P proteins play the same role. The absence of one P1/P2 heterodimer reduced the rate of cell growth by varying degrees, depending on the proteins missing. Synthesis of the 60S ribosomal subunit also decreased, particularly in strains carrying the unusual P1α-P2α or P1β-P2β heterodimers, although the distinct P1/P2 dimers are bound with similar affinity to the mutant ribosome. While in wild-type strains the B site bound P1β/P2α in a highly specific manner and the A site bound the four P proteins similarly, both the A and B binding sites efficiently bound practically any P1/P2 pair in mutant strains expressing truncated P0 proteins. The reported results support that while most ribosomes contain a P1α/P2β-P0-P1β/P2α structure in normal conditions, the stalk assembly mechanism can generate alternative compositions, which have been previously detected in the cell.
Project description:We have determined the kinetic parameters for the hydrolysis by papain, cathepsin B and cathepsin L of internally quenched fluorescent peptides derived from the lead peptides Abz-AAFRSAQ-EDDnp [in which Abz and EDDnp stand for o-aminobenzoic acid and N-(2,4-dinitrophenyl)ethylenediamine respectively], to map the specificity of S(4) and S(3) subsites, and Abz-AFRSAAQ-EDDnp, to identify the specificity of S(2)' and S(3)'. Abz and EDDnp were the fluorescent quencher pair. These two series of peptides were cleaved at the Arg-Ser bond and systematic modifications at P(4), P(3), P(2)' and P(3)' were made. The S(4) to S(2)' subsites had a significant influence on the hydrolytic efficiencies of the three enzymes. Only papain activity was observed to be dependent on S(3)', indicating that its binding site is larger than those of cathepsins B and L. Hydrophobic amino acids were accepted at S(4), S(3), S(2)' and S(3)' of the three enzymes. The best substrates for cathepsins L and B had Trp and Asn at P(2)' respectively; variations at this position were less accepted by these enzymes. The best substrates for papain were peptides containing Trp, Tyr or Asn at P(3)'. Basic residues at P(3) and P(4) were well accepted by cathepsin L and papain. We also explored the susceptibility of substrates Abz-AFRSXAQ-EDDnp, modified at P(2)' (X), to human cathepsin B mutants from which one or two occluding loop contacts had been removed. The modifications at His(111) (H111A) and His(110) (H110A) of cathepsin B led to an increase in k(cat) values of one or two orders of magnitude. The hydrolytic efficiencies of these cathepsin B mutants became closer to those of papain or cathepsin L.
Project description:The aim of this study is to investigate the efficacy, safety, and survival benefit of etoposide plus cisplatin and irinotecan plus cisplatin in first-line therapy of non-primary pancreatic metastatic and/or unresectable gastrointestinal neuroendocrine tumor G3 type. In addition, the investigators will explore the resistance mechanisms of gastrointestinal neuroendocrine tumor G3, and screen out biomarkers that can predict the efficacy of chemotherapy.
Project description:Veliparib showed limited single agent cytotoxicity but selectively potentiated (≥50% reduction in IC50) cisplatin, carboplatin and etoposide in vitro in 5 of 9 SCLC cell lines. Veliparib with cisplatin, etoposide or with both cisplatin and etoposide showed greater delay in tumor growth than chemotherapy alone in H146 but not H128. The potentiating effect of veliparib was associated with in vitro cell line sensitivity to cisplatin (CC=0.672; p=0.048) and DNA-PKcs protein modulation. Gene expression profiling identified differential expression of a 5-gene panel (GLS, UBEC2, HACL1, MSI2 and LOC100129585) in cell lines with relatively greater sensitivity to platinum and veliparib combination. Veliparib potentiates standard cytotoxic agents against SCLC in a cell specific manner. This potentiation correlates with platinum sensitivity, DNA-PKcs expression and a 5-gene expression profile.