3D-anatomy and systematics of cocculinid-like limpets (Gastropoda: Cocculiniformia): more data, some corrections, but still an enigma

New material and new methodologies substantially widen the anatomical knowledge on cocculinid limpets. We first provide 3D-anatomies of Fedikovella caymanensis and Teuthirostria cancellata based on serial sections. Both species differ in several major points (mainly the gill-type and several features of the alimentary tract) from typical cocculinids, accordingly they are classified in a new clade, Teuthirostriidae fam. nov. Specimens studied by McLean and Harasewych (LA County Mus Contrib Sci 453:1-33, 1995) under Fedikovella beanii probably represent another species new to science. Additional investigations of original (type) section series of Cocculina laevis Thiele, 1904 (type species of Paracocculina Haszprunar, 1987) and of Cocculina radiata Thiele, 1904 (type species of Coccocrater Haszprunar, 1987) imply some nomenclatorial revisions: Cocculina cervae Fleming, 1948 is designated as type species of Pedococculina gen. nov. Anatomical characters confirm the subsequent placement of Cocculina viminensis Rocchini, 1990 into Coccopigya Marshall, 1986, whereas the original generic status of the whale-fall inhabitant Cocculina craigsmithi McLean, 1992 is confirmed despite the unusual habitat. The latter species probably has symbiotic bacteria in the midgut gland;if so this might be due to the environmental and feeding conditions at whale cadavers or hydrothermal vents. Contrary to Lepetelloidea, the Cocculiniformia cannot be included in Vetigastropoda. Recent molecular data support a sistergroup relationship of Cocculiniformia with Neomphalida, and we add the phenotypic perspective on this so-called Neomphaliones-hypothesis. In particular, more phylogenomic data are needed to specify the position of Cocculinida among the rhipidoglossate Gastropoda

Temozolomide- and fotemustine-induced apoptosis in human malignant melanoma cells: response related to MGMT, MMR, DSBs, and p53

Malignant melanomas are highly resistant to chemotherapy. First-line chemotherapeutics used in melanoma therapy are the methylating agents dacarbazine (DTIC) and temozolomide (TMZ) and the chloroethylating agents BCNU and fotemustine. Here, we determined the mode of cell death in 11 melanoma cell lines upon exposure to TMZ and fotemustine. We show for the first time that TMZ induces apoptosis in melanoma cells, using therapeutic doses. For both TMZ and fotemustine apoptosis is the dominant mode of cell death. The contribution of necrosis to total cell death varied between 10 and 40%. The O6-methylguanine-DNA methyltransferase (MGMT) activity in the cell lines was between 0 and 1100 fmol mg−1 protein, and there was a correlation between MGMT activity and the level of resistance to TMZ and fotemustine. MGMT inactivation by O6-benzylguanine sensitized all melanoma cell lines expressing MGMT to TMZ and fotemustine-induced apoptosis, and MGMT transfection attenuated the apoptotic response. This supports that O6-alkylguanines are critical lesions involved in the initiation of programmed melanoma cell death. One of the cell lines (MZ7), derived from a patient subjected to DTIC therapy, exhibited a high level of resistance to TMZ without expressing MGMT. This was related to an impaired expression of MSH2 and MSH6. The cells were not cross-resistant to fotemustine. Although these data indicate that methylating drug resistance of melanoma cells can be acquired by down-regulation of mismatch repair, a correlation between MSH2 and MSH6 expression in the different lines and TMZ sensitivity was not found. Apoptosis in melanoma cells induced by TMZ and fotemustine was accompanied by double-strand break (DSB) formation (as determined by H2AX phosphorylation) and caspase-3 and -7 activation as well as PARP cleavage. For TMZ, DSBs correlated significantly with the apoptotic response, whereas for fotemustine a correlation was not found. Melanoma lines expressing p53 wild-type were more resistant to TMZ and fotemustine than p53 mutant melanoma lines, which is in marked contrast to previous data reported for glioma cells treated with TMZ. Overall, the findings are in line with the model that in melanoma cells TMZ-induced O6-methylguanine triggers the apoptotic (and necrotic) pathway through DSBs, whereas for chloroethylating agents apoptosis is triggered in a more complex manner

Crystallization and preliminary crystallographic analysis of trypanothione reductase from Trypanosoma cruzi, the causative agent of Chagas' disease

Trypanothione reductase from Trypanosoma cruzi is the most promising target molecule for the rational design of a specific drug against Chagas' disease. The recombinant protein was purified in a single Chromatographie step and crystallized. Two crystal forms suitable for X-ray diffraction analysis were obtained. Tetragonal crystals (a = b = 87.4 Å, c = 152.3 Å) were grown from 30% polyethylene glycol (average M r = 8,000) in the presence of 0.2% β-n-octylglucoside (space group either P42 with one dimer or P4222 with one monomer in the asymmetric unit). Monoclinic crystals (space group P2, a = 136.3 Å, b = 91.1 Å, c = 126.0 Å, β = 94°) were grown from 1.2 M sodium citrate in the presence of 2% octanoyl-N-methyl-glucamide. They contain two dimers of the enzyme in the asymmetric unit; both crystal forms diffract to 3 Å resolution

Spiroepoxytriazoles are fumagillin-like irreversible inhibitors of MetAP2 with potent cellular activityγ.

Methionine aminopeptidases (MetAPs) are responsible for the co-translational cleavage of initiator methionines from nascent proteins. The MetAP2 subtype is up-regulated in many cancers, and selective inhibition of MetAP2 suppresses both vascularization and growth of tumors in animal models. The natural product fumagillin is a selective and potent irreversible inhibitor of MetAP2, and semi-synthetic derivatives of fumagillin have shown promise in clinical studies for the treatment of cancer, and, more recently, for obesity. Further development of fumagillin derivatives has been complicated, however, by their generally poor pharmacokinetics. In an attempt to overcome these limitations, we developed an easily diversifiable synthesis of a novel class of MetAP2 inhibitors that were designed to mimic fumagillin's molecular scaffold but have improved pharmacological profiles. These substances were found to be potent and selective inhibitors of MetAP2, as demonstrated in biochemical enzymatic assays against three MetAP isoforms. Inhibitors with the same relative and absolute stereoconfiguration as fumagillin displayed significantly higher activity than their diastereomeric and enantiomeric isomers. X-ray crystallographic analysis revealed that the inhibitors covalently modify His231 in the MetAP2 active site via ring-opening of a spiroepoxide. Biochemically active substances inhibited the growth of endothelial cells and a MetAP2-sensitive cancer cell line, while closely related inactive isomers had little effect on the proliferation of either cell type. These effects correlated with altered N-terminal processing of the protein 14-3-3-γ. Finally, selected substances were found to have improved stabilities in mouse plasma and microsomes relative to the clinically-investigated fumagillin derivative beloranib

Chimeric antigen receptor T cells engineered to recognize the P329G-mutated Fc part of effector-silenced tumor antigen-targeting human IgG1 antibodies enable modular targeting of solid tumors.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy has proven its clinical utility in hematological malignancies. Optimization is still required for its application in solid tumors. Here, the lack of cancer-specific structures along with tumor heterogeneity represent a critical barrier to safety and efficacy. Modular CAR T cells indirectly binding the tumor antigen through CAR-adaptor molecules have the potential to reduce adverse events and to overcome antigen heterogeneity. We hypothesized that a platform utilizing unique traits of clinical grade antibodies for selective CAR targeting would come with significant advantages. Thus, we developed a P329G-directed CAR targeting the P329G mutation in the Fc part of tumor-targeting human antibodies containing P329G L234A/L235A (LALA) mutations for Fc silencing. METHODS: A single chain variable fragment-based second generation P329G-targeting CAR was retrovirally transduced into primary human T cells. These CAR T cells were combined with IgG1 antibodies carrying P329G LALA mutations in their Fc part targeting epidermal growth factor receptor (EGFR), mesothelin (MSLN) or HER2/neu. Mesothelioma, pancreatic and breast cancer cell lines expressing the respective antigens were used as target cell lines. Efficacy was evaluated in vitro and in vivo in xenograft mouse models. RESULTS: Unlike CD16-CAR T cells, which bind human IgG in a non-selective manner, P329G-targeting CAR T cells revealed specific effector functions only when combined with antibodies carrying P329G LALA mutations in their Fc part. P329G-targeting CAR T cells cannot be activated by an excess of human IgG. P329G-directed CAR T cells combined with a MSLN-targeting P329G-mutated antibody mediated pronounced in vitro and in vivo antitumor efficacy in mesothelioma and pancreatic cancer models. Combined with a HER2-targeting antibody, P329G-targeting CAR T cells showed substantial in vitro activation, proliferation, cytokine production and cytotoxicity against HER2-expressing breast cancer cell lines and induced complete tumor eradication in a breast cancer xenograft mouse model. The ability of the platform to target multiple antigens sequentially was shown in vitro and in vivo. CONCLUSIONS: P329G-targeting CAR T cells combined with antigen-binding human IgG1 antibodies containing the P329G Fc mutation mediate pronounced in vitro and in vivo effector functions in different solid tumor models, warranting further clinical translation of this concept