Chain-length dependence for secondary structure formation of homo-oligopep tides fromε-tert.-butyloxycarbonyl-L-lysine with a lipophilic C-terminal group

A solid-state and solution analysis of the homo-oligopeptides from ε-tert.-butyloxycarbonyl-L-lysine with p-oxymethylbenzylcholestan-3β-yl succinate as C-terminal group, using infrared absorption and circular dichroism, is described. The occurrence of intermolecular β-structure is seen in the solid state and in solvents of low polarity, e.g. methylene chloride, for peptides of intermediate size (from pentamer to decamer). Conversely, the eicosapeptide exhibits a high percentage of α-helical structure both in the solid state and in 2, 2, 2-trifluoroethanol. The influence of the C-terminal group on the conformational preferences of the ε-blocked homo-oligolysines in the solid state and in organic solvents appears negligible.     

Induction of rifampicin metabolism during treatment of tuberculous patients with daily and fully intermittent regimens containing the drug

Self-induction of rifampicin metabolism during daily and intermittent chemotherapy was studied by monitoring the changes in the serum half-life of the drug over a 4-week period in patients with pulmonary tuberculosis. Rifampicin 450 mg was administered to 8 patients who received treatment daily, 7 on thrice-weekly and 7 others on twice-weekly treatment. Serum half-life was computed from concentrations of the drug determined at 3, 4 1/2 and 6 hours after drug administration, on admission and at 1, 2 and 4 weeks after start of treatment. In the daily series, the mean serum half-life decreased from 4.9 hours on admission to 3.6 hours at 1 week (P = 0.02), and treatment beyond this had no further effect. In the thrice-weekly series, maximal induction was observed at the 2nd week, the mean values on admission and at 2 weeks being 5.8 and 3.7 hours, respectively (P less than 0.01). In the twice-weekly series, maximal induction was observed only at the 4th week, the mean values on admission and at 4 weeks being 4.9 and 3.7 hours, respectively (P less than 0.01). Serum activity of gamma glutamyl transferase was not found to be a suitable in vivo marker to monitor induction of the hepatic microsomal enzymes as no significant changes were observed in the activity of this enzyme in any of the 3 series during the 4-week period.     

T cell division and death are segregated by mutation of TCRbeta chain constant domains

We have studied the role of the T cell receptor (TCR) beta chain transmembrane and cytoplasmic domains (betaTM/Cyto) in T cell signaling. Upon antigen stimulation, T lymphocytes expressing a TCR with mutant and betaTM and Cyto domains accumulate in large numbers and are specifically defective in undergoing activation-induced cell death (AICD). The mutant TCR poorly recruits the protein adaptor Carma-1 and is subsequently impaired in activating NF-kappaB. This signaling defect leads to a reduced expression of Fas ligand (FasL) and to a reduction in AICD. These beta chain domains are involved in discriminating cell division and apoptosis.     

Heparanase powers a chronic inflammatory circuit that promotes colitis-associated tumorigenesis in mice

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is closely associated with colon cancer. Expression of the enzyme heparanase is clearly linked to colon carcinoma progression, but its role in UC is unknown. Here we demonstrate for what we believe to be the first time the importance of heparanase in sustaining the immune-epithelial crosstalk underlying colitis-associated tumorigenesis. Using histological specimens from UC patients and a mouse model of dextran sodium sulfate-induced colitis, we found that heparanase was constantly overexpressed and activated throughout the disease. We demonstrate, using heparanase-overexpressing transgenic mice, that heparanase overexpression markedly increased the incidence and severity of colitis-associated colonic tumors. We found that highly coordinated interactions between the epithelial compartment (contributing heparanase) and mucosal macrophages preserved chronic inflammatory conditions and created a tumor-promoting microenvironment characterized by enhanced NF-κB signaling and induction of STAT3. Our results indicate that heparanase generates a vicious cycle that powers colitis and the associated tumorigenesis: heparanase, acting synergistically with the intestinal flora, stimulates macrophage activation, while macrophages induce production (via TNF-α-dependent mechanisms) and activation (via secretion of cathepsin L) of heparanase contributed by the colon epithelium. Thus, disruption of the heparanase-driven chronic inflammatory circuit is highly relevant to the design of therapeutic interventions in colitis and the associated cancer.     

Combining MHC tetramer and intracellular cytokine staining for CD8(+) T cells to reveal antigenic epitopes naturally presented on tumor cells

As more tumor antigens are discovered and as computer-guided T cell epitope prediction programs become more sophisticated, many potential T cell epitopes are synthesized and demonstrated to be antigenic in vitro. However, it is estimated that about 50% of such tumor antigen-specific T cells have not been demonstrated to recognize the naturally presented epitopes due to either technical difficulties, such as T cell cloning which is still challenging for many laboratories; or the predicted T cell epitopes are not generated or not generated in sufficient amounts by the antigen processing machinery. However, to potentially identify clinically relevant vaccine candidate epitopes, it is essential to demonstrate natural antigen presentation. Here we combine the advantages of MHC tetramer and intracellular cytokine staining to sensitively detect natural antigen presentation by tumor cells for epitopes of interest. The novel method does not require T cell cloning or long-term T cell culture. Because the antigen-specific T cells are positively identified, this method is much less influenced by IFNγ producing cells with unknown specificities and should be widely applicable.     

Loss of Hemolectin reduces the survival of Drosophila larvae after wounding

Coagulation involving both hemocytes and humoral factors is important for insect survival and immune defense. Hemolectin is a major larval clotting factor in Drosophila, and hemolymph from hml mutants does not clot ex vivo. Yet surprisingly third instar hml larvae survived injury as well as controls. The number of hemocytes in circulation changes during larval development. Reasoning that this could affect coagulation, we studied larval survival after injury at different stages. We found that hml larvae survived less than controls when injured during the feeding stage with fewer hemocytes. This important in vivo result reinforces the role of Hemolectin in larval hemostasis. A subtle effect of hml on immunity was found in adults. Similar experiments on hml mutant larvae gave different results, but feeding stage hml larvae were differentially sensitive to infections with different strains of Serratia marcescens.     

The absence of Complexin 3 and Complexin 4 differentially impacts the ON and OFF pathways in mouse retina

Complexins (Cplxs) regulate the speed and Ca₂₊‐sensitivity of synaptic vesicle fusion. It has been shown that all four known Cplxs are present at mouse retinal synapses – at conventional amacrine cell synapses (Cplx 1 to Cplx 3) and at photoreceptor and bipolar cell ribbon synapses (Cplx 3 and Cplx 4) [ K. Reim et al. (2005) J. Cell Biol., 169 , 669‐680]. Electroretinographic recordings in Cplx 3/Cplx 4 double‐knockout (DKO) mice showed perturbed transmission in the outer plexiform layer, and possible changes in the inner plexiform layer [ K. Reim et al. (2009) J. Cell Sci., 122 , 1352–1361]. In the present study, we examined the effects of the absence of Cplx 3 and Cplx 4 on ganglion cell responses. We report that the lack of Cplx 3 and Cplx 4 differentially impacts the ON and OFF pathways. Under photopic conditions, the responses in the cone OFF pathway are largely unaffected, whereas the responses in the cone ON pathway are diminished in Cplx 3/Cplx 4 DKO mice. Under scotopic conditions, both ON and OFF response rates are reduced and high‐sensitivity OFF responses are missing in Cplx 3/Cplx 4 DKO mice. The electrophysiological findings are corroborated by new immunocytochemical findings. We now show that rod spherules contain only Cplx 4. However, both Cplx 3 and Cplx 4 co‐localize in cone pedicles. In the inner plexiform layer, Cplx 3 is present in rod bipolar cell terminals and in amacrine cell processes. Most importantly, Cplx 3 is localized in the lobular appendages of AII amacrine cells, the sites of signal transmission from the primary rod pathway into the OFF pathway in the inner plexiform layer.