Isolation and biochemical characterization of hemorrhagic toxin f from the venom of Crotalus atrox (western diamondback rattlesnake)

Hemorrhagic toxin f (HT-f) was isolated from Crotalus atrox (Western Diamondback Rattlesnake) venom by a five-step purification procedure. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing, and sodium dodecyl sulfate (SDS)-electrophoresis. HT-f has a molecular weight of 64,000 and contains 572 amino acid residues. It contains 1 mol of zinc per mol of protein. Zinc is essential for both hemorrhagic and proteolytic activities. HT-f possesses proteolytic activity hydrolyzing the Val-Asn, Gln-His, Leu-Cys, His-Leu, Ala-Leu, and Tyr-Leu bonds of oxidized insulin B chain. HT-f did not coagulate fibrinogen to fibrin, yet it did hydrolyze the gamma chain of fibrinogen without affecting either the A alpha or B beta chains. This is the first time that a hemorrhagic toxin was shown to have fibrinogenase activity. HT-f was shown to differ immunologically from other hemorrhagic toxins such as HT-a and HT-c. HT-f also possesses lethal toxicity. When zinc was removed the apo-HT-f lost its lethal toxicity. HT-f produced not only local hemorrhage in the skin and muscle, but also produced systemic hemorrhage in internal organs such as the intestine, kidney, lung, heart, and liver.     

Target-selective cytotoxicity of methotrexate conjugated with monoclonal anti-MM46 antibody

Summary In studies on antitumor antibody-cytotoxic drug conjugates as potential tumor-selective cytotoxic agents, methotrexate (MTX) was conjugated via its active ester derivative with a murine monoclonal antibody (aMM46) to a mouse mammary tumor antigen (MM antigen) on syngeneic, ascitic C3H/He mouse mammary tumor MM46 cells. The conjugate retained full antibody activity, as assayed by complement-dependent cytolysis. The target-selective cytotoxicity of aMM46-MTX was verified by the observations that this conjugate showed greater cytotoxicity than the corresponding normal mouse immunoglobulin (nIg) conjugate to MM46 cells, neither aMM46 nor nIg being cytotoxic, and that it showed less cytotoxicity to MM antigen negative mouse mammary tumor MM48 cells than to MM46 cells, its cytotoxicity to MM48 cells being similar to that of the nIg conjugate. From the results of assays of cell binding and uptake of ¹³¹I-labeled aMM46 and aMM46-³H-MTX, aMM46 and aMM46-MTX were internalized after their binding to MM46 cell surface antigen. Leupeptin, an inhibitor of the lysosomal endopeptidase cathepsin, decreased the cytotoxicity of aMM46-MTX, supporting the involvement of lysosomal degradation of the conjugate in its action.     

Proximate stimuli: An overlooked driving force for risk-induced trait responses affecting interactions in aquatic ecosystems

Inducible responses in prey to predation risk can influence species interaction strength, with significant ecological consequences. Much of the past research on interactions in aquatic ecosystems has focused on remote stimuli (e.g., diffusible chemicals emitted from predators and injured conspecifics, which easily propagate through environmental water), as cues triggering trait responses in prey, and has overlooked the importance of proximate stimuli (e.g., physical disturbance and less-diffusible chemicals), which occur in attack or direct contact to prey by predators. Proximate stimuli from predators as well as remote stimuli may induce significant responses in prey functional traits such as behavior, morphology, and life history and, therefore, act as an important mechanism of top-down effects in aquatic ecosystems. In this opinion paper, we argue that studying the effects of proximate stimuli is essential to better understanding of individual adaptation to predation risk in nature and ecological consequences of predator–prey interactions. Here, we propose research directions to examine the role of proximate stimuli for phenotypic plasticity and interaction systems.     

The olfactory receptor gene repertoires in secondary-adapted marine vertebrates: evidence for reduction of the functional proportions in cetaceans

An olfactory receptor (OR) multigene family is responsible for the well-developed sense of smell possessed by terrestrial tetrapods. Mammalian OR genes had diverged greatly in the terrestrial environment after the fish-tetrapod split, indicating their importance to land habitation. In this study, we analysed OR genes of marine tetrapods (minke whale Balaenoptera acutorostrata, dwarf sperm whale Kogia sima, Dall's porpoise Phocoenoides dalli, Steller's sea lion Eumetopias jubatus and loggerhead sea turtle Caretta caretta) and revealed that the pseudogene proportions of OR gene repertoires in whales were significantly higher than those in their terrestrial relative cattle and also in sea lion and sea turtle. On the other hand, the pseudogene proportion of OR sequences in sea lion was not significantly higher compared with that in their terrestrial relative (dog). It indicates that secondary perfectly adapted marine vertebrates (cetaceans) have lost large amount of their OR genes, whereas secondary-semi-adapted marine vertebrates (sea lions and sea turtles) still have maintained their OR genes, reflecting the importance of terrestrial environment for these animals.     

Synaptic plasticity deficits and mild memory impairments in mouse models of chronic granulomatous disease

Reactive oxygen species (ROS) are required in a number of critical cellular signaling events, including those underlying hippocampal synaptic plasticity and hippocampus-dependent memory; however, the source of ROS is unknown. We previously have shown that NADPH oxidase is required for N-methyl-D-aspartate (NMDA) receptor-dependent signal transduction in the hippocampus, suggesting that NADPH oxidase may be required for NMDA receptor-dependent long-term potentiation (LTP) and hippocampus-dependent memory. Herein we present the first evidence that NADPH oxidase is involved in hippocampal synaptic plasticity and memory. We have found that pharmacological inhibitors of NADPH oxidase block LTP. Moreover, mice that lack the NADPH oxidase proteins gp91(phox) and p47(phox), both of which are mouse models of human chronic granulomatous disease (CGD), also lack LTP. We also found that the gp91(phox) and p47(phox) mutant mice have mild impairments in hippocampus-dependent memory. The gp91(phox) mutant mice exhibited a spatial memory deficit in the Morris water maze, and the p47(phox) mutant mice exhibited impaired context-dependent fear memory. Taken together, our results are consistent with NADPH oxidase being required for hippocampal synaptic plasticity and memory and are consistent with reports of cognitive dysfunction in patients with CGD.     

Difructose anhydride III does not contribute to body energy accumulation in rats

We evaluated the body energy accumulation as fat and protein from ingestion of difructose anhydride III (DFAIII). Male Wistar rats were fed 0, 0.25, 0.5, 1.0, or 1.5 g per d of sucrose or DFAIII added to a 7 g of basal diet for 20 d. Supplements of DFAIII did not increase whole body or peripheral fat or total body energy, whereas sucrose increased them in a dose-dependent manner. Dose-dependent increases in body water were observed in both groups. The body protein was influenced by the dose of sugars. The estimated available energy value of DFAIII was 0.263 kcal per gram; this value is one-fifteenth that of sucrose. Ingestion of DFAIII dose-dependently increased the cecal SCFA pool. DFAIII was not detected in feces, showing complete degradation of DFAIII in the intestine. These results indicate that DFAIII is a fermentable saccharide with quite low available energy for fat accumulation.     

Effect of benzylic oxygen on the cytotoxic activity for colon 26 cell line of phenolic lignans

The cytotoxic activity for colon 26 cell line of matairesinol, oxidized matairesinol, 9,9'-epoxylignan and oxidized 9,9'-epoxylignan were examined. (-)-Matairesinol (Mat 1) showed greatest cytotoxic activity (LC(50)=9 microg/ml) of the lactone-type lignans. 7,7'-Oxomatairesinol having same steric configuration as that of (-)-matairesinol showed greater activity (LC(50)=25 microg/ml) than hydroxy or mono-oxomatairesinol. The activities of 9,9'-epoxylignan and 7,7'-oxo-9,9'-epoxylignan having same steric configurations as (-)-matairesinol were weaker than that of corresponding matairesinols. Different activity levels were observed between enantiomers.     

Novel carbohydrate-binding activity of bovine liver beta-glucuronidase toward lactose/N-acetyllactosamine sequences

Beta-glucuronidase is a lysosomal enzyme that plays an essential role in normal turnover of glycosaminoglycans and remodeling of the extracellular matrix components in both physiological and inflammatory states. The regulation mechanisms of enzyme activity and protein targeting of beta-glucuronidase have implications for the development of a variety of therapeutics. In this study, the effectiveness of various carbohydrate-immobilized adsorbents for the isolation of bovine liver beta-glucuronidase (BLG) from other glycosidases was tested. Beta-glucuronidase and contaminating glycosidases in commercial BLG preparations bound to and were coeluted from adsorbents immobilized with the substrate or an inhibitor of beta-glucuronidase, whereas beta-glucuronidase was found to bind exclusively with lactamyl-Sepharose among the adsorbents tested and to be effectively separated from other enzymes. Binding and elution studies demonstrated that the interaction of beta-glucuronidase with lactamyl-Sepharose is pH dependent and carbohydrate specific. BLG was purified to homogeneity by lactamyl affinity chromatography and subsequent anion-exchange high-performance liquid chromatography (HPLC). Lactose was found to activate beta-glucuronidase noncompetitively, indicating that the lactose-binding site is different from the substrate-binding site. Binding studies with biotinyl glycoproteins, lipids, and synthetic sugar probes revealed that beta-glucuronidase binds to N-acetyllactosamine/lactose-containing glycoconjugates at neutral pH. The results indicated the presence of N-acetyllactosamine/lactose-binding activity in BLG and provided an effective purification method utilizing the novel carbohydrate binding activity. The biological significance of the carbohydrate-specific interaction of beta-glucuronidase, which is different from the substrate recognition, is discussed.     

Interleukin-21 triggers both cellular and humoral immune responses leading to therapeutic antitumor effects against head and neck squamous cell carcinoma

BACKGROUND: Interleukin-21 (IL-21) plays important roles in the regulation of T, B, and natural killer (NK) cells. We hypothesized that the cytokine may provide a novel immunotherapy strategy for cancer by stimulating both Th1 and Th2 immune responses. In this context, antitumor immunity induced by IL-21 was examined in mice bearing subcutaneous head and neck squamous cell carcinomas (HNSCC). METHODS: A plasmid vector encoding murine IL-21 was injected intravenously into mice with pre-established HNSCC tumors, either alone or in combination with a vector construct expressing IL-15. Cytotoxic T lymphocyte (CTL) and NK killing activities were evaluated by chrome release assays, while HNSCC-specific antibody was examined by flow cytometry and ELISA. RESULTS: Significant antitumor effects were obtained by repeated transfection with either the IL-21 or the IL-15 gene. Co-administration of both cytokine genes resulted in increased suppression of tumor growth, significantly prolonging the survival periods of the animals. Thirty percent of the tumor-bearing mice that received the combination therapy survived for more than 300 days, completely rejecting rechallenge with the tumor at a distant site. IL-21 induced significant elevation of HNSCC-specific CTL activity, while IL-21 and IL-15 augmented NK activity in an additive manner. IL-21 gene transfer also promoted the production of tumor-specific IgG. CONCLUSIONS: In vivo transduction of the IL-21 gene elicits powerful antitumor immunity, including both humoral and cellular arms of the immune response, and results in significant suppression of pre-established HNSCC. Co-transfer of the IL-15 gene further improved the therapeutic outcome, mainly by augmenting NK tumoricidal activity. The biological effects of IL-21 may be in sharp contrast to those of conventional Th1 and Th2 cytokines, suggesting intriguing implications of this cytokine for the classical concept of Th1 vs. Th2 paradigm.