An NMR investigation of solution aggregation reactions preceding the misassembly of acid-denatured cold shock protein A into fibrils.

At pH 2.0, acid-denatured CspA undergoes a slow self-assembly process, which results in the formation of insoluble fibrils. 1H-15N HSQC, 3D HSQC-NOESY, and 15N T2 NMR experiments have been used to characterize the soluble components of this reaction. The kinetics of self-assembly show a lag phase followed by an exponential increase in polymerization. A single set of 1H-15N HSQC cross-peaks, corresponding to acid-denatured monomers, is observed during the entire course of the reaction. Under lag phase conditions, 15N resonances of residues that constitute the beta-strands of native CspA are selectively broadened with increasing protein concentration. The dependence of 15N T2 values on spin echo period duration demonstrates that line broadening is due to fast NMR exchange between acid-denatured monomers and soluble aggregates. Exchange contributions to T2 relaxation correlate with the squares of the chemical shift differences between native and acid-denatured CspA, and point to a stabilization of native-like structure upon aggregation. Time-dependent changes in 15N T2 relaxation accompanying the exponential phase of polymerization suggest that the first three beta-strands may be predominantly responsible for association interfaces that promote aggregate growth. CspA serves as a useful model system for exploring the conformational determinants of denatured protein misassembly.     

Identification of respiratory vagal feedback in awake normal subjects using pseudorandom unloading.

Evidence of the Hering-Breuer reflex has been found in humans during anesthesia and sleep but not during wakefulness. Cortical influences, present during wakefulness, may mask the effects of this reflex in awake humans. We hypothesized that, if lung volume were increased in awake subjects unaware of the stimulus, vagal feedback would modulate breathing on a breath-to-breath basis. To test this hypothesis, we employed proportional assist ventilation in a pseudorandom sequence to unload the respiratory system above and below the perceptual threshold in 17 normal subjects. Tidal volume, integrated respiratory muscle pressure per breath, and inspiratory time were recorded. Both sub- and suprathreshold stimulation evoked a significant increase in tidal volume and inspiratory flow rate, but a significant decrease in inspiratory time was present only during the application of a subthreshold stimulus. We conclude that vagal feedback modulates respiratory timing on a breath-by-breath basis in awake humans, as long as there is no awareness of the stimulus.     

Physiology of the ECL cells.

The enterochromaffin-like (ECL) cells of the oxyntic mucosa (fundus) of the stomach produce, store and secrete histamine, chromogranin A-derived peptides such as pancreastatin, and an unanticipated but as yet unidentified peptide hormone. The cells are stimulated by gastrin and pituitary adenylate cyclase activating peptide and suppressed by somatostatin and galanin. Choline esters and histamine seem to be without effect on ECL cell secretion. The existence of a gastrin-ECL cell axis not only explains how gastrin stimulates acid secretion but also may help to explore the functional significance of the ECL cells with respect to the nature and bioactivity of its peptide hormone. From the results of studies of gastrectomized/fundectomized and gastrin-treated rats, it has been speculated that the anticipated ECL-cell peptide hormone acts on bone metabolism.     

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Analysis of water in Autonomous Biological Systems (ABS) samples.

Several soluble components, peptidase and amino acids, and carbon isotopic ratio in the water retrieved from flight experiments of Autonomous Biological Systems (ABS) as well as ground control samples are analyzed to interpret the condition, dynamics, material balance of the ABS ecosystems. Organic carbons in flight samples were found to be more abundant compared with the control ones, which suggested the uniform ecosystems in low gravity might easily dissolve more soluble components. The Mir-1997 flight sample showed higher C/N ratio probably because of the dissolution of carbon-rich plant materials.     

The light organ symbiont Vibrio fischeri possesses two distinct secreted ADP-ribosyltransferases.

We have previously described the purification, cloning, and initial characterization of a secreted ADP-ribosyltransferase, halovibrin (gene designation hvn), from the luminescent light organ symbiont Vibrio fischeri. This report describes a strategy for overexpression of halovibrin, the production and refinement of antihalo-vibrin antisera, and the molecular biological construction of a V. fischeri halovibrin null strain. Biochemical analysis of this mutant revealed that V. fischeri hvn null still possessed ADP-ribosyltransferase activity and that this activity is immunologically, genetically, and structurally distinct from the previously described enzyme. This unusual finding, of two ADP-ribosyltransferase enzymes produced by a microorganism, is complemented by the details of the purification to apparent homogeneity and in vitro regulation of this new protein, halovibrin-beta.