Desensitization by covalent modification of the chemoreceptor of Escherichia coli

Chemoreceptors in Escherichia coli were studied in situ in chemotactic mutants, deficient in the ability to modify the receptors, by using membrane vesicles prepared from the mutants. The affinity of the receptors for the ligands is related to the level of modification of the receptors. Unmodified serine receptor had a dissociation constant of 0.8 microM, while modified receptor had a dissociation constant that was at least 100-times higher. The results are discussed in relation to the two-state model of the chemoreceptor.     

Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.

Clostridial neurotoxins inhibit neurotransmitter release by selective and specific intracellular proteolysis of synaptobrevin/VAMP, synaptosomal-associated protein of 25 kDa (SNAP-25) or syntaxin. Here we show that in binary reactions synaptobrevin binds weakly to both SNAP-25 and syntaxin, and SNAP-25 binds to syntaxin. In the presence of all three components, a dramatic increase in the interaction strengths occurs and a stable sodium dodecyl sulfate-resistant complex forms. Mapping of the interacting sequences reveals that complex formation correlates with the presence of predicted alpha-helical structures, suggesting that membrane fusion involves intermolecular interactions via coiled-coil structures. Most toxins only attack the free, and not the complexed, proteins, and proteolysis of the proteins by different clostridial neurotoxins has distinct inhibitory effects on the formation of synaptobrevin-syntaxin-SNAP-25 complexes. Our data suggest that synaptobrevin, syntaxin and SNAP-25 associate into a unique stable complex that functions in synaptic vesicle exocytosis.     

Rabbit thymus gangliosides: species- and organ-specific distribution, age-dependence and their cell biological significance

Gangliosides of thymuses from rabbit, mouse, rat, calf, and man were analyzed. The ganglioside compositions of the thymuses showed species specificities, and the compositions of the species other than the rabbit were found to be markedly different from that of the rabbit, which contained characteristically substantial amounts of IV3NeuGc-nLc4Cer and VI3NeuGc-nLc6Cer (Iwamori, M. & Nagai, Y. (1981) Biochim. Biophys. Acta 665, 214-220). The inter-species differences in the thymus ganglioside compositions were not remarkable in the 8 mouse and 2 rabbit strains examined. Rabbit thymocytes, but not those of mouse and rat, were lysed with human Hanganutziu-Deicher serum in the presence of guinea pig complement, reflecting the high content of gangliosides containing N-glycolylneuraminic acid in rabbit thymus. As to age-dependent changes of gangliosides in rabbit thymus and spleen, the concentrations gradually decreased with age, while the molar ratio of total gangliosides to total phospholipids was constant in the spleen throughout life and in the thymus at 3, 4, and 6 weeks of age. It was noted that old (180 weeks of age) rabbit thymus, which is occupied largely by fat tissue, still contained a significant amount of neolactoseries gangliosides.     

Fatty acid ethyl esters in the liverwort Conocephalum conicum

A series of fatty acid ethyl esters ranging from C₁₄ to C₂₄ was isolated from a hexane extract of the liverwort Conocephalum conicum, these esters accounted for 77% of the extract. The ethyl esters consisting of even-numbered fatty acids were predominant and ethyl palmitate was the major constituent.     

Activation process of pepsinogen.

The activation process of pepsinogen was analyzed by a combination of computer simulation and experiment. In order to investigate in detail the behavior of the basic schemes proposed in the previous study, further computer simulations were conducted. Some experiments were performed based on the information obtained. The changes in the UV difference spectrum in the early stage was measured by the stopped-flow technique and the conversion of pepsinogen to pepsin [EC 3.4.23.1] was followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, on the basis of the experimental results, the most reasonable scheme was selected and modified. As a result, a scheme for the activation process of pepsinogen was obtained (Scheme 8). On the basis of the above analyses, it was assumed that the first step and the third step are pH-dependent based on the change in the UV spectrum, that the second step is a nonlinear reaction containing a looped reaction with a dimeric intermediate (in this step, peptide fragments are released and pepsinogen is converted to a pepsin-like molecule), and that the third step is an equilibrium reaction involving proton binding.     

Spermidine-dependent proteins are involved in the fusion of mouse alveolar macrophages induced by 1 alpha,25-dihydroxyvitamin D3 and interleukin 4

We have reported that 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] directly induces fusion of mouse alveolar macrophages by a mechanism involving protein synthesis (H. Tanaka et al., 1984, FEBS Lett. 174, 61). While examining further the mechanism of the fusion, we found that polyamines, most likely spermidine, are involved as an important intracellular mediator of the 1 alpha,25(OH)2D3 action in inducing protein synthesis, which in turn induces fusion of macrophages (T. Hayashi et al., 1986, J. Bone Miner. Res. 1, 235). In this study, spermidine-dependent proteins responsible for inducing fusion were examined by electrophoresis of [35S]methionine-labeled proteins. 1 alpha,25(OH)2D3 increased synthesis of 14 proteins at 24 h after the addition, before it initiated fusion at 36 h. When spermidine synthesis was inhibited by adding methylglyoxal bis(guanylhydrazone) (MGBG), the enhanced synthesis in 9 of the 14 proteins induced by 1 alpha,25(OH)2D3 was greatly diminished with a concomitant inhibition of fusion. Further addition of spermidine restored the synthesis of these 9 proteins and the fusion as well. The synthesis of 3 of the 9 proteins was similarly induced by interferon-gamma, retinoic acid, or lipopolysaccharides, which induced activation but not fusion of macrophages. The apparent molecular weights of the remaining 6 proteins were 142K, 98K, 78K, 60K, 50K, and 42K. Recombinant mouse interleukin 4 (IL-4) also induced fusion of alveolar macrophages by a spermidine-dependent mechanism, and it increased the synthesis of 5 proteins (172K, 98K, 78K, 53K, and 50K). These results suggest that 3 spermidine-dependent proteins (98K, 78K, and 50K) are involved in the fusion of mouse alveolar macrophages induced by 1 alpha,25(OH)2D3 and IL-4.     

The role of an invariant tryptophan residue in alpha-bungarotoxin and cobrotoxin. Investigation of active derivatives with the invariant tryptophan replaced by kynurenine

Ozone oxidation converted the single, invariant, tryptophan residue to N2-formylkynurenine in alpha-bungarotoxin and cobrotoxin. Upon this modification, the lethal toxicity was significantly reduced in cobrotoxin but mostly retained in alpha-bungarotoxin. Each neurotoxin containing kynurenine instead of tryptophan retained the same antigenicity as the native toxin. Fluorescence and CD spectroscopy revealed that, although the environment and state of the kynurenine residue were similar, [Kyn29]cobrotoxin was much more sensitive to pH change than alpha-[Kyn28]bungarotoxin. In terms of lethal toxicity and conformational stability, the invariant tryptophan residue appears to play a more important role in cobrotoxin, imparting a higher lethal toxicity than that in alpha-bungarotoxin, which has a disulfide bond at Cys29-Cys33.     

Studies on peroxisomes. VI. Relationship between the peroxisomal core and urate oxidase.

The peroxisomal core from the liver of rats was purified 450-fold as a marker of urate oxidase [EC 1.7.3.3.] activity. This preparation has a high specific activity of urate oxidase but not of other peroxisomal enzymes: D-amino acid oxidase [EC 1.4.3.3.], L-alpha-hydroxy acid oxidase [EC 1.1.3.15], or catalase [EC 1.11.1.6]. No activity of marker enzymes for other subcellular particles; cytochrome c oxidase [EC1.9.3.1] (mitochondria), acid phosphatase [EC 3.1.3.2] (lysosomes), or glucose-6-phosphatase [EC 3.1.3.9] (microsomes), was detected in this preparation. The core obtained showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the position of the band was found to correspond to a molecular weight 35,000. When the peroxisomal core was subjected to treatment at various pH's with 0.1 M carbonate buffer, urate oxidase was almost completely solubulized at pH 11.0, although approximately 35% of the core protein still remained in the pellet After solubilization of the core at pH 11.0, the specific activity of urate oxidase in the supernatant increased about 1.6 times; the density of the insoluble protein remaining in the pellet was identical with the that of the original core on sucrose density gradient centrifugation.     

Human cases of infection with canine whipworms, Trichuris vulpis (Froelich, 1789), in Japan

The eggs of Trichuris, detected in the feces of 19 persons who had stayed in institutions for mentally retarded and/or multi-handicapped patients, were identified as those of T. vulpis (Froelich, 1789) on the basis of morphological features. This is the first record of human infection with the canine whipworm in Japan.