Structural analyses of a channel-forming fragment of colicin E1 incorporated into lipid vesicles. Fourier-transform infrared and tryptophan fluorescence studies.

Structural changes upon binding to the membrane of a COOH-terminal channel-forming thermolytic fragment of colicin E1 have been studied by means of a variety of spectroscopic techniques. Circular dichroism measurements show that the thermolytic fragment predominantly takes a helical structure in aqueous and detergent solutions. Fourier transform infrared spectroscopic measurements indicate that the content of the beta-structure is significantly increased when the thermolytic fragment is bound to vesicles. On the basis of the result of tryptophan fluorescence measurements, we have concluded that each of the three tryptophan residues of the thermolytic fragment exists in different environments, i.e. one is buried in the lipid bilayer, one exists on the cis side of the vesicles, and one exists near the surface of the lipid bilayer. The Fourier transform infrared and fluorescence data have been used along with the crystal structure of colicin A, which is highly homologous to colicin E1 in structure and function, to propose a model of the thermolytic fragment bound to the lipid vesicles.     

Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG.

Cellular receptors for IgG (Fc gamma R) mediate important protective functions. By using site-specific mutants of a chimeric antibody (mouse V H domain and L chain; human IgG3 C H domains), we have demonstrated that human Fc gamma RI interacts with a site in the lower hinge of human IgG (residues 234 to 237) and that this interaction dictates Fc gamma RI-mediated superoxide generation. Mutations at position 235 resulted in the most profound reductions in Fc gamma RI recognition. We have also mapped an interaction site for Fc gamma RII to the same region; however, mutations at position 234 and 237 resulted in the greatest reductions in Fc gamma RII recognition. The two receptors appear to recognize overlapping but nonidentical sites on the lower hinge of IgG. Deviations from the optimal motif 234-Leu-Leu-Gly-Gly-237 may then explain the human IgG subclass specificity profile for human Fc gamma RI and Fc gamma RII.     

Chemical modification and 1H-NMR studies on the receptor-binding region of human interleukin 6

Oxidation of the Met residues of human interleukin 6 (IL-6) molecule has been performed. Reactivity of Met for the oxidation reaction was found to decrease in the order of Met50, Met118, Met185, Met162, and Met68. Chemical modifications involving oxidation and carboxypeptidase A digestion of IL-6 have led to the assignments of the methyl proton resonances of Met162 and Met185, respectively. The hydroxynitrobenzyl chromophore attached to Trp158 in the IL-6 molecule showed a different absorption spectrum when the labeled IL-6 was bound to the soluble IL-6 receptor. This result indicates that Trp158 is near the receptor-binding region in IL-6. On the basis of the 1H-NMR and chemical modification data, it has been concluded that Trp158 is in spatial proximity to Met162, His165 and Met185. The receptor-binding activity decreased with an increase in the number of oxidized Met residues. Of these five Met residues, Met162 was the residue in which the receptor-binding activity decreased in the most parallel degree with that of the oxidation reaction.     

Two Types of Channels Involved in the Malate Ion Transport across the Tonoplast of a Crassulacean Acid Metabolism Plant

Ion channels in tonoplast of leaf cells of a Crassulacean acid metabolism plant, Graptopetalum paraguayense, using the patch clamp technique were investigated. Results showed the existence of two types of channels involved in the malate ion transport across the tonoplast. One type corresponded to the slow-activating vacuolar-type (R Hedrich, E Neher [1987] Nature 329: 833-836), probably taking part in the malate efflux from vacuoles. Another showed the membrane potential-dependent channel current of malate flux over a wide range of cytoplasmic free Ca²⁺ concentration (10⁻⁸-10⁻⁵ molar), a property favoring the malate uptake. This type seems to be different from the fast-activating vacuolar-type.     

Structure and biosynthesis of the xylose-containing carbohydrate moiety of rice alpha-amylase

Suspension-cultured cells of rice secrete alpha-amylase into the culture medium. It has been shown that the mature form of the alpha-amylase contains xylose-bearing N-linked oligosaccharide: (formula; see text) We demonstrate that suspension-cultured cells of rice secrete alpha-amylase containing oligomannose-type oligosaccharides in the presence of 1-deoxymannojirimycin or tris(hydroxymethyl)aminomethane. On the other hand, alpha-amylase purified from germinated rice seedlings contains several kinds of oligomannose-type and N-acetyllactosamine-type oligosaccharides. The processing pathway of oligosaccharide moieties in rice cells is discussed on the basis of a comparison of these oligosaccharides structures.     

Ca2(+)-independent secretory mechanism of thyrotropin-releasing hormone (TRH) involves protein kinase C in rat pituitary cells

Thyrotropin-releasing hormone (TRH) stimulates biphasic prolactin (PRL) secretion from rat pituitary GH3 cells. The pretreatment of cells with EGTA (100 microM) plus arachidonic acid (15 microM), a condition which decreased TRH-responsive intracellular Ca2+ pools, eliminated the activity of TRH on burst PRL secretion (2 min) but did not alter that on sustained PRL secretion (30 min). However, the treatment of cells with EGTA, arachidonic acid and H-7 (300 microM), a potent inhibitor of protein kinase C (PKC), almost completely suppressed the activity of TRH for sustained PRL secretion. In cells down-modulated for PKC, TRH abolished this Ca2(+)-independent sustained PRL secretion. These results suggest that TRH acts through a separate, Ca2(+)-independent secretory mechanism, besides by modulating the Ca2(+)-dependent mechanism and that PKC is involved in this Ca2(+)-independent secretory pathway.     

Effect of short — chain fatty acids on electrical activity of the small intestinal mucosa of rat

Intravenous administration of short-chain fatty acid (SCFA), such as propionate, butyrate, valerate and caproate, caused a transient increase in transmural potential difference (p.d.) across the small intestine of rat $\text{in vivo}$. There was a sigmoid relationship between the change in the p.d. and the logarithm of the dose of SCFA. The median effective dose of propionate, n-butyrate, n-valerate and n-caproate, which was calculated from the each dose-response curve obtained from the terminal ileum, 1.31, 1.43, 0.83 and 0.81 μmole, respectively. Repeated administrations of the same dose of propionate evoked progressively smaller response. The dose-response curve of propionate was shifted to the left by neostigmine and to the right by atropine, suggesting that the action of SCFA may be mediated by acetylcholine, which was released from a nerve ending.     

Kinetics of photoinactivation and photooxidation of mitomycin C in the presence of riboflavin

The kinetic relation between the photoinactivation and photooxidation of mitomycin C in the presence of riboflavin was investigated. The photoinactivation was tested for lambda-phage induction in Escherichia coli K-12 (lambda) cells and colony formation of E. coli Bs-1 cells. Mitomycin C lost its phage-inducing and antibiotic activities when the antibiotic was irradiated in vitro with visible light in the presence of riboflavin. The loss of phage-inducing activity followed a Stern-Volmer type equation with respect to the dose of irradiation, and the inactivation constant was evaluated to be 0.96X10(-4)m2/J. The initial rate of decay of mitomycin C by photooxidation in the presence of riboflavin obeyed first order kinetics, and its cross section was estimated to be 2.51X10(-6)m/J independent of the intensity of incident light. The cross section for photooxidation was found to be proportional to the inactivation constant. These results suggest that the photoinactivation of mitomycin C is caused by its photooxidation. In order to rationalize this conclusion, a mechanism of photooxidation was proposed and reactions in vivo of the photoproduct were discussed in relation to the inactivation.     

Sampling Strategies in Antimicrobial Resistance Monitoring: Evaluating How Precision and Sensitivity Vary with the Number of Animals Sampled per Farm

Because antimicrobial resistance in food-producing animals is a major public health concern, many countries have implemented antimicrobial monitoring systems at a national level. When designing a sampling scheme for antimicrobial resistance monitoring, it is necessary to consider both cost effectiveness and statistical plausibility. In this study, we examined how sampling scheme precision and sensitivity can vary with the number of animals sampled from each farm, while keeping the overall sample size constant to avoid additional sampling costs. Five sampling strategies were investigated. These employed 1, 2, 3, 4 or 6 animal samples per farm, with a total of 12 animals sampled in each strategy. A total of 1,500 Escherichia coli isolates from 300 fattening pigs on 30 farms were tested for resistance against 12 antimicrobials. The performance of each sampling strategy was evaluated by bootstrap resampling from the observational data. In the bootstrapping procedure, farms, animals, and isolates were selected randomly with replacement, and a total of 10,000 replications were conducted. For each antimicrobial, we observed that the standard deviation and 2.5–97.5 percentile interval of resistance prevalence were smallest in the sampling strategy that employed 1 animal per farm. The proportion of bootstrap samples that included at least 1 isolate with resistance was also evaluated as an indicator of the sensitivity of the sampling strategy to previously unidentified antimicrobial resistance. The proportion was greatest with 1 sample per farm and decreased with larger samples per farm. We concluded that when the total number of samples is pre-specified, the most precise and sensitive sampling strategy involves collecting 1 sample per farm.