Cytotoxicity of a hybrid prepared by coupling diphtheria toxin A-chain with immunoglobulin Fab′ with N,N′-o-phenylenedimaleimide

A hybrid protein was prepared by coupling the A-chain of diphtheria toxin with the Fab′ fragment of immunoglobulin with $\text{N}\text{,}\text{N′}\text{-}\text{o}$-phenylenedimaleimide (PDM). Although in this hybrid, the two components were linked with each other with bonds which could not be reductively cleaved with 2-mercaptoethanol as in a hybrid cross-linked with a disulfide bond (e.g. Fab′-S-S-A-chain), it exhibited a potent cytotoxicity $\text{in}\text{vitro}$, one-third of that of Fab′-S-S-A-chain, against the target L1210 cells.     

Intermolecular nuclear Overhauser effect and atomic pair potential approaches to wheat germ agglutinin-sugar binding

The detailed binding mechanism of wheat germ agglutinin (WGA) with N-acetylglucosamine (GlcNAc) was investigated using intermolecular 1H-1H nuclear Overhauser effect (NOE) and atomic pair potential (APP) calculations. Negative NOE was observed on the 1H spectrum of 1-O-methyl derivative of GlcNAc in a solution containing WGA, when the aromatic region of the WGA spectrum was irradiated. Analyses of the time dependence of NOE revealed that H2 and the N-acetyl methyl protons of the sugar are in close proximity to the aromatic protons of WGA in the bound state. This was confirmed and further elucidated by the APP calculations. According to the calculation, the major binding force comes from a hydrogen-bonding between C3-OH of sugar and an acidic residue present in each of the two binding sites of WGA: Glu115 in site 1 and Asp29 in site 2. The binding is further assisted by the N-acetyl group which interacts with a few more polar amino acid residues in the binding sites. The optimized binding mode suggested by the APP calculations supports the NMR results in that H2 and a part of the N-acetyl methyl protons are within 4.5 A distance from protons of both Tyr64 and Tyr73 in site 1 and of Tyr159 in site 2.     

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.     

An inhibitory sex pheromone tastes bitter for Drosophila males

Sexual behavior requires animals to distinguish between the sexes and to respond appropriately to each of them. In Drosophila melanogaster, as in many insects, cuticular hydrocarbons are thought to be involved in sex recognition and in mating behavior, but there is no direct neuronal evidence of their pheromonal effect. Using behavioral and electrophysiological measures of responses to natural and synthetic compounds, we show that Z-7-tricosene, a Drosophila male cuticular hydrocarbon, acts as a sex pheromone and inhibits male-male courtship. These data provide the first direct demonstration that an insect cuticular hydrocarbon is detected as a sex pheromone. Intriguingly, we show that a particular type of gustatory neurons of the labial palps respond both to Z-7-tricosene and to bitter stimuli. Cross-adaptation between Z-7-tricosene and bitter stimuli further indicates that these two very different substances are processed by the same neural pathways. Furthermore, the two substances induced similar behavioral responses both in courtship and feeding tests. We conclude that the inhibitory pheromone tastes bitter to the fly.     

Inhibition of intestinal cholesterol absorption decreases atherosclerosis but not adipose tissue inflammation

Adipose tissue inflammation is associated with insulin resistance and increased cardiovascular disease risk in obesity. We previously showed that addition of cholesterol to a diet rich in saturated fat and refined carbohydrate significantly worsens dyslipidemia, insulin resistance, adipose tissue macrophage accumulation, systemic inflammation, and atherosclerosis in LDL receptor-deficient (Ldlr^−/−) mice. To test whether inhibition of intestinal cholesterol absorption would improve metabolic abnormalities and adipose tissue inflammation in obesity, we administered ezetimibe, a dietary and endogenous cholesterol absorption inhibitor, to Ldlr^−/− mice fed chow or high-fat, high-sucrose (HFHS) diets without or with 0.15% cholesterol (HFHS+C). Ezetimibe blunted weight gain and markedly reduced plasma lipids in the HFHS+C group. Ezetimibe had no effect on glucose homeostasis or visceral adipose tissue macrophage gene expression in the HFHS+C fed mice, although circulating inflammatory markers serum amyloid A (SSA) and serum amyloid P (SSP) levels decreased. Nevertheless, ezetimibe treatment led to a striking (>85%) reduction in atherosclerotic lesion area with reduced lesion lipid and macrophage content in the HFHS+C group. Thus, in the presence of dietary cholesterol, ezetimibe did not improve adipose tissue inflammation in obese Ldlr^−/− mice, but it led to a major reduction in atherosclerotic lesions associated with improved plasma lipids and lipoproteins.     

Comparative effects of pitavastatin and probucol on oxidative stress, Cu/Zn superoxide dismutase, PPAR-gamma, and aortic stiffness in hypercholesterolemia

Reactive oxygen species-scavenging enzyme Cu/Zn superoxide dismutase (SOD) regulated by peroxisome proliferator-activated receptors (PPARs) plays an important role in vascular responsiveness. However, it remains unknown whether statin restores vascular dysfunction through the activation of reactive oxygen species-scavenging enzymes in vivo. We hypothesized that pitavastatin restores vascular function by modulating oxidative stress through the activation of Cu/ZnSOD and PPAR-gamma in hypercholesterolemia. New Zealand White male rabbits were fed either normal chow or a 1% cholesterol (CHO) diet for 14 wk. After the first 7 wk, the CHO-fed rabbits were further divided into three groups: those fed with CHO feed only (HC), those additionally given pitavastatin, and those additionally given an antioxidant, probucol. The extent of atherosclerosis was assessed by examining aortic stiffness. When compared with the HC group, both the pitavastatin and probucol groups showed improved aortic stiffness by reducing aortic levels of reactive oxidative stress, nitrotyrosine, and collagen, without affecting serum cholesterol or blood pressure levels. Pitavastatin restored both Cu/ZnSOD activity (P < 0.005) and PPAR-gamma expression and activity (P < 0.01) and inhibited NAD(P)H oxidase activity (P < 0.0001) in the aorta, whereas probucol inhibited NAD(P)H oxidase activity more than did pitavastatin (P < 0.0005) without affecting Cu/ZnSOD activity or PPAR-gamma expression and activity. Importantly, Cu/ZnSOD activity was positively correlated with the PPAR-gamma activity in the aorta (P < 0.005), both of which were negatively correlated with aortic stiffness (P < 0.05). Vascular Cu/ZnSOD and PPAR-gamma may play a crucial role in the antiatherogenic effects of pitavastatin in hypercholesterolemia in vivo.     

Tyrosine phosphorylation of vinexin in v-Src-transformed cells attenuates the affinity for vinculin

Vinexin is an adaptor-type focal adhesion protein that interacts with vinculin. Here, we report the tyrosine phosphorylation of vinexin α in v-Src-transformed NIH3T3 cells. Point mutational analysis of vinexin α clarified that three tyrosine residues in vinexin α were phosphorylated. A non-phosphorylatable mutant of vinexin α had higher binding affinity for vinculin than its wild-type counterpart. In conclusion, vinexin α is tyrosine phosphorylated in v-Src-transformed cells, and this tyrosine phosphorylation of vinexin α attenuates the association of vinexin α with vinculin.     

Conformational differences in liganded and unliganded states of Galectin-3

The conformation of the carbohydrate recognition domain of Galectin-3, a lectin known to bind galactose containing oligosaccharides in mammalian systems, has been investigated in the absence of ligand and in the presence of N-acetylactosamine. A new methodology based on the measurement of residual dipolar couplings from NMR spectra has been used to characterize differences in protein structure along the backbone in the presence and absence of ligand, as well as the binding geometry of the ligand itself. The data on the ligand are consistent with the ligand binding geometry found in a crystal structure of the complexed state. However, a significant rearrangement of backbone loops near the binding site appears to occur in the absence of ligand. The implications for ligand specificity and protein functionality are discussed.     

Binding of Host-Associated Treponeme Proteins to Collagens and Laminin: A Possible Mechanism of Spirochetal Adherence to Host Tissues

The polypeptides of seven strains of human treponemes were investigated by immunoblot analysis for their binding to the human placental collagens and laminin. Of the treponemal polypeptides, eleven polypeptides, 45-kDa, 49-kDa, and 62-kDa polypeptides from T. pallidum ATCC 27087, a 48-kDa polypeptide from T. phagedenis biotype Reiter, 51-kDa and 53-kDa polypeptides from T. vincentii ATCC 35580, 30-kDa, 53-kDa and 63-kDa polypeptides from T. socranskii subsp. buccale ATCC 35534, a 52-kDa polypeptide from T. denticola ATCC 35405, and a 53-kDa polypeptide from T. denticola ATCC 33520 possessed an ability to bind to the laminin, type I, III, IV, or V collagen. An intermediate-sized human oral isolate strain G7201 did not possess any laminin- or collagen-binding polypeptides. Immunoelectron microscopy using intact treponemal cells with a single collagen-binding polypeptide and the corresponding antisera demonstrated that the 51-kDa and 53-kDa polypeptides from T. vincentii, the 53-kDa polypeptide from T. socranskii subsp. buccale ATCC 35534 and the 52-kDa polypeptide from T. denticola ATCC 35405, were outer envelope proteins.