The oligomerization of a family of four genetically clustered human gastrointestinal mucins

Mucins are synthesized and secreted by many epithelia. They are complex glycoproteins that offer cytoprotection. In their functional configuration, mucins form oligomers by a biosynthetic process that is poorly understood. A family of four human gastrointestinal mucin genes (MUC2, MUC5AC, MUC5B, and MUC6) is clustered to chromosome 11p15.5. To study oligomerization of these related mucins, we performed metabolic labeling experiments with [35S]amino acids in LS174T cells, and isolated mucin precursors by specific immunoprecipitations that were analyzed on SDS-PAGE. Each of the precursors of MUC2, MUC5AC, MUC5B, and MUC6 formed a single species of disulfide-linked homo-oligomer within 1 h after pulse labeling. Based on apparent molecular masses, these oligomeric precursors were most likely dimers. Inhibition of vesicular RER-to-Golgi transport, with brefeldin A and CCCP, did not affect the dimerization of MUC2 precursors, localizing dimerization to the RER. O-Glycosylation of MUC2 followed dimerization. Inhibition of N- glycosylation by tunicamycin retarded, but did not inhibit, dimerization, indicating that N-glycans play a role in efficient dimerization of MUC2 precursors. Based on sequence homology, the ability of MUC2, MUC5AC, MUC5B and MUC6 to dimerize most likely resides in their C-terminal domains. Thus, the RER-localized dimerization of secretory mucins likely proceeds by similar mechanisms, which is an essential step in the formation of the human gastrointestinal mucus- gels.      

Local antitumour treatment in carcinoma patients with bispecific-monoclonal-antibody-redirected T cells

In a pilot clinical study carcinoma patients with malignant ascites or pleural exudates have been treated locally with autologous lymphocytes activated ex vivo and redirected towards tumour cells with bispecific monoclonal antibodies. BIS-1, the bispecific monoclonal antibody used in this study, combines specificity against a tumour-associated antigen, AMOC-31, present on carcinomas, with a specificity against the CD3 complex on T lymphocytes. Patients selected for treatment had malignant pleural or peritoneal effusions. Treatment consisted of isolating autologous peripheral blood lymphocytes, ex vivo activation, incubation with bispecific monoclonal antibodies and injection at the effusion site of these BIS-1-redirected lymphocytes. To evaluate the effects of the bispecific monoclonal antibody, five patients received treatments with activated lymphocytes without bispecific antibodies. Effusion samples taken before and at various times after treatment were analysed by immunocytology and for the presence of the soluble factors carcinoembryonic antigen (CEA), interleukin-6 (IL-6), tumour necrosis factor (TNF), C-reactive protein and soluble CD8. In this way both immune activation and anti-tumour activity could be monitored. Conjugate formation between tumour cells and activated lymphocytes was seen as soon as 4 h after injection of BIS-1-redirected activated lymphocytes, followed by a disappearance or reduction of tumour cells after 24–48 h. In parallel with this, the soluble tumour marker CEA decreased in the effusion fluid following injection with the BIS-1-redirected lymphocytes. Furthermore, a steep increase in local granulocyte numbers was observed in the effusion fluid, which reached a maximum 24–48 h after the start of the treatment. Also levels of IL-6 and TNF were greatly elevated. The data suggest tha the treatment induces both antitumour activity and a strong local inflammatory reaction. This is accompanied by no or only minor local and systemic toxicity, i.e. mild fever, which disappeared as the local inflammatory reaction diminished 48–72 h after treatment.     

Patch size effects are more important than genetic diversity for plant–herbivore interactions in Brassica crops

1. Considerable evidence suggests that the diversity within plant communities may strongly affect the strength of species interactions, but the majority of studies only considered interspecific diversity. 2. This paper examines the effect of intraspecific genetic diversity within Brassica fields on two Brassica specialists, cabbage root fly, and diamondback moth, and on a parasitoid attacking diamondback moths. Genetic diversity was manipulated both in a replacement and an additive design. 3. Both herbivore densities and parasitism rates were higher in smaller plots, with limited responses to increased within‐plot diversity. All species showed variable densities across genotypes, and preference hierarchies were species specific. 4. Responses to plot size in root flies scaled with the diameter‐to‐area ratio, suggesting that patch detectability affected local density, whereas responses by diamondback moths and parasitoids deviated from this ratio. These species differences could be traced to differences in the residence time within patches, where diamondback moths typically spend longer and more variable time periods in patches than root flies. 5. The lack of response to genetic diversity by both herbivores suggests that egg‐laying rates are affected by decisions on the plant and not by attraction from a distance, neither to the plant itself nor the patch. Patterns of differential attack may then be due to different acceptability for studied genotypes. 6. Future theories on insect responses to spatial heterogeneity should focus on species traits and how traits interact with information landscapes in the field.     

Disturbance mediates the effects of nutrients on developing assemblages of epibiota

Local dynamics such as resource enhancement (e.g. nutrient supply) and stochastic events of destruction (disturbances that provide new space) are hypothesized to counteractively affect species diversity and composition. We tested the independent and interactive effects of nutrients and disturbance on the development of assemblages of epibiota attached to vertical surfaces in an oligotrophic system. Nutrient concentrations were manipulated at three levels (ambient, medium and high) while disturbance was manipulated by removing biomass at seven frequencies (0×, 2×, 3×, 4×, 5×, 7×, 12×). Nutrient and disturbance regimes had opposing effects on diversity such that species richness increased with resource enhancement (nutrients) and declined with disturbance. These results support the model that increased heterogeneity of distribution of limiting resources allows the coexistence of species with low and high resource requirements.     

Simultaneous inhibition of epidermal growth factor receptor (EGFR) signaling and enhanced activation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-mediated apoptosis induction by an scFv:sTRAIL fusion protein with specificity for human EGFR

Epidermal growth factor receptor (EGFR) signaling inhibition by monoclonal antibodies and EGFR-specific tyrosine kinase inhibitors has shown clinical efficacy in cancer by restoring susceptibility of tumor cells to therapeutic apoptosis induction. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent with tumor-selective apoptotic activity. Here we present a novel approach that combines EGFR-signaling inhibition with target cell-restricted apoptosis induction using a TRAIL fusion protein with engineered specificity for EGFR. This fusion protein, scFv425:sTRAIL, comprises the EGFR-blocking antibody fragment scFv425 genetically fused to soluble TRAIL (sTRAIL). Treatment with scFv425:sTRAIL resulted in the specific accretion to the cell surface of EGFR-positive cells only. EGFR-specific binding rapidly induced a dephosphorylation of EGFR and down-stream mitogenic signaling, which was accompanied by cFLIP(L) down-regulation and Bad dephosphorylation. EGFR-specific binding converted soluble scFv425:sTRAIL into a membrane-bound form of TRAIL that cross-linked agonistic TRAIL receptors in a paracrine manner, resulting in potent apoptosis induction in a series of EGFR-positive tumor cell lines. Co-treatment of EGFR-positive tumor cells with the EGFR-tyrosine kinase inhibitor Iressa resulted in a potent synergistic pro-apoptotic effect, caused by the specific down-regulation of c-FLIP. Furthermore, in mixed culture experiments binding (L)of scFv425:sTRAIL to EGFR-positive target cells conveyed a potent apoptotic effect toward EGFR-negative bystander tumor cells. The favorable characteristics of scFv425:sTRAIL, alone and in combination with Iressa, as well as its potent anti-tumor bystander activity indicate its potential value for treatment of EGFR-expressing cancers.     

FAS activation induces dephosphorylation of SR proteins; dependence on the de novo generation of ceramide and activation of protein phosphatase 1

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.     

Influence of single-level lumbar degenerative disc disease on the behavior of the adjacent segments—A finite element model study

The current study investigated mechanical predictors for the development of adjacent disc degeneration. A 3-D finite element model of a lumbar spine was modified to simulate two grades of degeneration at the L4–L5 disc. Degeneration was modeled by changes in geometry and material properties. All models were subjected to follower preloads of 800 N and moment loads in the three principal directions of motion using a hybrid protocol. Degeneration caused changes in the loading and motion patterns of the segments above and below the degenerated disc. At the level (L3–L4) above the degenerated disc, the motion increased due to moderate degeneration by 21% under lateral bending, 26% under axial rotation and 28% under flexion/extension. At the level (L5-S1) below the degenerated disc, motion increased only during lateral bending by 20% due to moderate degeneration. Both the L3–L4 and L5-S1 segment showed a monotonic increase in both the maximum von Mises stress and shear stress in the annulus as degeneration progressed for all loading directions, expect extension at L3–L4. The most significant increase in stress was observed at the L5-S1 level during axial rotation with nearly a ten-fold increase in the maximum shear stress and 103% increase in the maximum von Mises stress. The L5-S1 segment also showed a progressive increase in facet contact force for all loading directions with degeneration. Nucleus pressure did not increase significantly for any loading direction at either the caudal or cephalic adjacent segment. Results suggest that single-level degeneration can increase the risk for injury at the adjacent levels.     

Approaches to lung cancer treatment using the CD3E×GP-2-directed Bispecific Monoclonal Antibody BIS-1

 The bispecific monoclonal antibody (bsAb) BIS-1 combines a monoclonal-antibody(mAb)-defined specificity for the CD3 complex, as present on all T lymphocytes, with a mAb-defined specificity for the pancarcinoma/epithelium associated glycoprotein EGP-2. In vitro studies indicate that BIS-1 can direct T lymphocytes to kill EGP-2-positive tumour target cells. T cell pre-activation is necessary for this activity and can be obtained either via incubation of isolated peripheral blood mononuclear cells with CD3 mAb, followed by short culturing in recombinant interleukin-2-containing medium, or via costimulation with CD5- and CD28-based bsAb. Clinical application of BIS-1 was started in a pilot study in which carcinoma patients suffering from malignant ascites or intrapleural effusion were treated. In this study, ex vivo activated autologous lymphocytes were applied locally, i.e. intraperitoneally or intrapleurally, in the presence of BIS-1. Local inflammation and antitumour activity were observed, whereas no or only minor systemic toxicity was seen in these patients. Intravenous administration of BIS-1 F(ab′)2 in combination with subcutaneously given recombinant interleukin-2 (i.v. bsAb/rIL-2 treatment) induced transient but considerable toxicity including peripheral vasoconstriction, dyspnoea and fever with a maximal tolerated dose of 5–8 μg/kg. High plasma concentrations of the inflammatory cytokines tumor necrosis factor α and interferon γ were observed at this dose. Whereas bsAb-dictated antitumour activity could be demonstrated to be present in blood samples of these patients in an in vitro assay, no clear clinical responses were observed. In a rat model it was found that i.v. bsAb/rIL-2 treatment of EGP-2-positive tumours was effective when a low systemic tumour burden was present, suggesting that systemic bsAb/rIL-2 treatment might be effective in situations of minimal residual disease. Accepted: 14 October 1997