Identification of a highly immunogenic mouse breast cancer sub cell line, 4T1-S

Cancer vaccines serve as a promising clinical immunotherapeutic strategy that help to trigger an effective and specific antitumor immune response compared to conventional therapies. However, poor immunogenicity of tumor cells remains a major obstacle for clinical application, and developing new methods to modify the immunogenicity of tumor cells may help to improve the clinical outcome of cancer vaccines. 4T1 mouse breast cancer cell line has been known as poorly immunogenic and highly metastatic cell line. Using this model, we identified a sub cell line of 4T1—designated as 4T1-Sapporo (4T1-S)—which shows immunogenic properties when used as a vaccine against the same line. In 4T1-S-vaccinated mice, subcutaneous injection of 4T1-S resulted in an antitumor inflammatory response represented by significant enlargement of draining lymph nodes, accompanied with increased frequencies of activated CD8 T cells and a subpopulation of myeloid cells. Additionally, 4T1-S vaccine was ineffective to induce tumor rejection in nude mice, which importantly indicate that 4T1-S vaccine rely on T cell response to induce tumor rejection. Further analysis to identify mechanisms that control tumor immunogenicity in this model may help to develop new methods for improving the efficacies of clinical cancer vaccines.     

Establishment and characterization of a clear cell carcinoma cell line,designated NOCC,derived from human ovary

A cell line, designated NOCC, was established from the ascites of a patient with clear cell adenocarcinoma of the ovary. The cell line has been grown without interruption and continuously propagated by serial passaging (more than 76 times) over 7 years. The cells are spherical to polygonal-shaped, display neoplastic, and pleomorphic features, and grow in a jigsaw puzzle-like pattern while forming monolayers without contact inhibition. The cells proliferate rapidly, but are easily floated as a cell sheet. The population doubling time is about 29 h. The number of chromosomes ranges from 60 to 83. The modal number of chromosomes is 70–74 at the 30th passage. NOCC cells secreted 750.5 ng/ml of VEGF over 3 days of culture. Hypoxia inducible factor-1α (HIF-1α) is a primary regulator of VEGF under hypoxic conditions. NOCC cells were not sensitive to the anticancer drugs BEV, DOX, GEM, ETP, CDDP, or TXT. The graft of NOCC cells to a scid mouse displayed similar histological aspects to the original tumor. Both the NOCC cells and the graft of the NOCC cells gave a positive PAS reaction.     

4-Aryl-5-carbamoyl-3-isoxazolols as competitive antagonists of insect GABA receptors: Synthesis,biological activity,and molecular docking studies

Competitive antagonists (CAs) of ionotropic GABA receptors (GABARs) reportedly exhibit insecticidal activity and have potential for development as novel insecticides for overcoming emerging resistance to traditional GABAR-targeting insecticides. Our previous studies demonstrated that 4,5-disubstituted 3-isoxazolols or 3-isothiazolols are an important class of insect GABAR CAs. In the present study, we synthesized a series of 4-aryl-5-carbamoyl-3-isoxazolols and examined their antagonism of insect GABARs expressed in Xenopus oocytes. Several of these 3-isoxazolols exhibited potent antagonistic activities against housefly and common cutworm GABARs, with IC₅₀ values in the low-micromolar range in both receptors. 4-(3-Amino-4-methylphenyl)-5-carbamoyl-3-isoxazolol (3u) displayed the highest antagonism, with IC₅₀ values of 2.0 and 0.9?μM in housefly and common cutworm GABARs, respectively. Most of the synthesized 3-isoxazolols showed moderate larvicidal activities against common cutworms, with more than 50% mortality at 100?μg/g. These results indicate that 4-monocyclic aryl-5-carbamoyl-3-isoxazolol is a promising scaffold for insect GABAR CA discovery and provide important information for the design and development of GABAR-targeting insecticides with a novel mode of action.     

Phosphorylation of adult type Sept5 (CDCrel-1) by cyclin-dependent kinase 5 inhibits interaction with syntaxin-1

Increasing evidence implicates cyclin-dependent kinase 5 (Cdk5) in neuronal synaptic function. We searched for Cdk5 substrates in synaptosomal fractions prepared from mouse brains. Mass spectrometric analysis after two-dimensional SDS-PAGE identified several synaptic proteins phosphorylated by Cdk5-p35; one protein identified was Sept5 (CDCrel-1). Although septins were isolated originally as cell division-related proteins in yeast, Sept5 is expressed predominantly in neurons and is implicated in exocytosis. We confirmed that Sept5 is phosphorylated by Cdk5-p35 in vitro and identified Ser17 of adult type Sept5 (Sept5_v1) as a major phosphorylation site. We found that Ser17 of Sept5_v1 is phosphorylated in mouse brains. Coimmunoprecipitation from synaptosomal fractions and glutathione S-transferase-syntaxin-1A pulldown assays of Sept5_v1 expressed in COS-7 cells showed that phosphorylation of Sept5_v1 by Cdk5-p35 decreases the binding to syntaxin-1. These results indicate that the interaction of Sept5 with syntaxin-1 is regulated by the phosphorylation of Sept5_v1 at Ser17 by Cdk5-p35.     

Hydrogen production by termite gut protists: characterization of iron hydrogenases of Parabasalian symbionts of the termite Coptotermes formosanus

Cellulolytic flagellated protists in the guts of termites produce molecular hydrogen (H(2)) that is emitted by the termites; however, little is known about the physiology and biochemistry of H(2) production from cellulose in the gut symbiotic protists due to their formidable unculturability. In order to understand the molecular basis for H(2) production, we here identified two genes encoding proteins homologous to iron-only hydrogenases (Fe hydrogenases) in Pseudotrichonympha grassii, a large cellulolytic symbiont in the phylum Parabasalia, in the gut of the termite Coptotermes formosanus. The two Fe hydrogenases were phylogenetically distinct and had different N-terminal accessory domains. The long-form protein represented a phylogenetic lineage unique among eukaryotic Fe hydrogenases, whereas the short form was monophyletic with those of other parabasalids. Active recombinant enzyme forms of these two Fe hydrogenases were successfully obtained without the specific auxiliary maturases. Although they differed in their extent of specific activity and optimal pH, both enzymes preferentially catalyzed H(2) evolution rather than H(2) uptake. H(2) evolution, at least that associated with the short-form enzyme, was still active even under high hydrogen partial pressure. H(2) evolution activity was detected in the hydrogenosomal fraction of P. grassii cells; however, the vigorous H(2) uptake activity of the endosymbiotic bacteria compensated for the strong H(2) evolution activity of the host protists. The results suggest that termite gut symbionts are a rich reservoir of novel Fe hydrogenases whose properties are adapted to the gut environment and that the potential of H(2) production in termite guts has been largely underestimated.     

Placental Mammals Acquired Functional Sequences in NRK for Regulating the CK2–PTEN–AKT Pathway and Placental Cell Proliferation

The molecular evolution processes underlying the acquisition of the placenta in eutherian ancestors are not fully understood. Mouse NCK-interacting kinase (NIK)-related kinase (NRK) is expressed highly in the placenta and plays a role in preventing placental hyperplasia. Here, we show the molecular evolution of NRK, which confers its function for inhibiting placental cell proliferation. Comparative genome analysis identified NRK orthologs across vertebrates, which share the kinase and citron homology (CNH) domains. Evolutionary analysis revealed that NRK underwent extensive amino acid substitutions in the ancestor of placental mammals and has been since conserved. Biochemical analysis of mouse NRK revealed that the CNH domain binds to phospholipids, and a region in NRK binds to and inhibits casein kinase-2 (CK2), which we named the CK2-inhibitory region (CIR). Cell culture experiments suggest the following: 1) Mouse NRK is localized at the plasma membrane via the CNH domain, where the CIR inhibits CK2. 2) This mitigates CK2-dependent phosphorylation and inhibition of PTEN and 3) leads to the inhibition of AKT signaling and cell proliferation. Nrk deficiency increased phosphorylation levels of PTEN and AKT in mouse placenta, supporting our hypothesis. Unlike mouse NRK, chicken NRK did not bind to phospholipids and CK2, decrease phosphorylation of AKT, or inhibit cell proliferation. Both the CNH domain and CIR have evolved under purifying selection in placental mammals. Taken together, our study suggests that placental mammals acquired the phospholipid-binding CNH domain and CIR in NRK for regulating the CK2–PTEN–AKT pathway and placental cell proliferation.     

Opposing pharmacological actions of cepharanthin on lipopolysaccharide-induced histidine decarboxylase activity in mice spleens

A biscoclaurin alkaloid preparation, cepharanthin (Ceph), is reported to have opposing pharmacological effects, enhancement or depression, on several cells and tissues, although detailed mechanisms remain unclear. Previously, we reported that Ceph enhanced lipopolysaccharide (LPS)-induced histidine decarboxylase (HDC) activity in mice spleens by consecutive pre-administration. In this study, we examined the pharmacological effects on HDC activity of a single Ceph pre-administration to test the influence of the administration method. Consequently, HDC activities were decreased by a single administration 15 minutes before LPS challenge in ddY and ICR mice spleens. Moreover, to further examine this suppressing effect, we employed genetically mast cell-deficient WBB6F1 W/Wv (W/Wv) mice to avoid the influence of mast cells. In W/Wv mice, HDC activity was enhanced, but not in the congenic WBB6F1 +/+ mice. These findings suggest that mast cells influence the depressant effect on HDC activity by a Ceph single administration in mast cell sufficient mice.