The Aer protein and the serine chemoreceptor Tsr independently sense intracellular energy levels and transduce oxygen, redox, and energy signals for Escherichia coli behavior

We identified a protein, Aer, as a signal transducer that senses intracellular energy levels rather than the external environment and that transduces signals for aerotaxis (taxis to oxygen) and other energy-dependent behavioral responses in Escherichia coli. Domains in Aer are similar to the signaling domain in chemotaxis receptors and the putative oxygen-sensing domain of some transcriptional activators. A putative FAD-binding site in the N-terminal domain of Aer shares a consensus sequence with the NifL, Bat, and Wc-1 signal-transducing proteins that regulate gene expression in response to redox changes, oxygen, and blue light, respectively. A double mutant deficient in aer and tsr, which codes for the serine chemoreceptor, was negative for aerotaxis, redox taxis, and glycerol taxis, each of which requires the proton motive force and/or electron transport system for signaling. We propose that Aer and Tsr sense the proton motive force or cellular redox state and thereby integrate diverse signals that guide E. coli to environments where maximal energy is available for growth.

The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

The yeast nonchromosomal gene [URE3] is due to a prion form of the nitrogen regulatory protein Ure2p. It is a negative regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p. Ure2p residues 1–80 are necessary for prion generation and propagation. The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation. The crystal structure of this C-terminal fragment, Ure2p(97–354), at 2.3 Å resolution is described here. It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity. However, Ure2p(97–354) lacks a properly positioned catalytic residue that is required for S-transferase activity. Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed.

Behavioral responses of Escherichia coli to changes in redox potential.

Escherichia coli bacteria sensed the redox state in their surroundings and they swam to a niche that had a preferred reduction potential. In a spatial redox gradient of benzoquinone/benzoquinol, E. coli cells migrated to form a sharply defined band. Bacteria swimming out of either face of the band tumbled and returned to the preferred conditions at the site of the band. This behavioral response was named redox taxis. Redox molecules, such as substituted quinones, that elicited redox taxis, interact with the bacterial electron transport system, thereby altering electron transport and the proton motive force. The magnitude of the behavioral response was dependent on the reduction potential of the chemoeffector. The Tsr, Tar, Trg, Tap, and CheR proteins, which have a role in chemotaxis, were not essential for redox taxis. A cheB mutant had inverted responses in redox taxis, as previously demonstrated in aerotaxis. A model is proposed in which a redox effector molecule perturbs the electron transport system, and an unknown sensor in the membrane detects changes in the proton motive force or the redox status of the electron transport system, and transduces this information into a signal that regulates phosphorylation of the CheA protein. A similar mechanism has been proposed for aerotaxis. Redox taxis may play an important role in the distribution of bacterial species in natural environments.

Assignment of Rfp-Y to the chicken major histocompatibility complex/NOR microchromosome and evidence for high-frequency recombination associated with the nucleolar organizer region.

Rfp-Y is a second region in the genome of the chicken containing major histocompatibility complex (MHC) class I and II genes. Haplotypes of Rfp-Y assort independently from haplotypes of the B system, a region known to function as a MHC and to be located on chromosome 16 (a microchromosome) with the single nucleolar organizer region (NOR) in the chicken genome. Linkage mapping with reference populations failed to reveal the location of Rfp-Y, leaving Rfp-Y unlinked in a map containing >400 markers. A possible location of Rfp-Y became apparent in studies of chickens trisomic for chromosome 16 when it was noted that the intensity of restriction fragments associated with Rfp-Y increased with increasing copy number of chromosome 16. Further evidence that Rfp-Y might be located on chromosome 16 was obtained when individuals trisomic for chromosome 16 were found to transmit three Rfp-Y haplotypes. Finally, mapping of cosmid cluster III of the molecular map of chicken MHC genes (containing a MHC class II gene and two rRNA genes) to Rfp-Y validated the assignment of Rfp-Y to the MHC/NOR microchromosome. A genetic map can now be drawn for a portion of chicken chromosome 16 with Rfp-Y, encompassing two MHC class I and three MHC class II genes, separated from the B system by a region containing the NOR and exhibiting highly frequent recombination.

Endogenous c-src as a determinant of the tumorigenicity of src oncogenes.

We have compared the tumorigenicity of two src oncogenes, v-src and c-src(527), whose respective protein products pp60v-src and pp60c-src(527) show a different spectrum of amino acid substitutions vis-à-vis the c-src protooncogene-encoded product pp60c-src. Whereas the extent of primary tumor growth induced by c-src(527) was quite similar in the two chicken lines tested, the extent of v-src-induced tumor growth showed a marked line dependence. As examined with a line of chickens that shows immune-mediated regression of v-src-induced tumors, a weaker tumor immunity, as correlated with a greater level of primary tumor growth, resulted from inoculation of c-src(527) DNA than of v-src DNA. These observations indicated that the v-src-specific amino acid substitutions define a major tumor antigenicity. That a separate src-associated antigenicity is also targetable by the tumor immune response followed from the finding that the level of protective immunity against the growth of c-src(527) DNA-induced tumors was augmented under conditions of the prior regression of v-src DNA-induced tumors. As this latter antigenicity may include one or more c-src(527)-encoded peptides that are equivalent to c-src-encoded self peptides, these observations suggest that a host tolerance to pp60c-src can be broken so as to permit a tumor immune response based on recognition of self peptides of pp60c-src(527).

Ex vivo evaluation of a Taylor-Couette flow, immobilized heparinase I device for clinical application

Efficient and safe heparin anticoagulation has remained a problem for continuous renal replacement therapies and intermittent hemodialysis for patients with acute renal failure. To make heparin therapy safer for the patient with acute renal failure at high risk of bleeding, we have proposed regional heparinization of the circuit via an immobilized heparinase I filter. This study tested a device based on Taylor-Couette flow and simultaneous separation/reaction for efficacy and safety of heparin removal in a sheep model. Heparinase I was immobilized onto agarose beads via cyanogen bromide activation. The device, referred to as a vortex flow plasmapheretic reactor, consisted of two concentric cylinders, a priming volume of 45 ml, a microporous membrane for plasma separation, and an outer compartment where the immobilized heparinase I was fluidized separately from the blood cells. Manual white cell and platelet counts, hematocrit, total protein, and fibrinogen assays were performed. Heparin levels were indirectly measured via whole-blood recalcification times (WBRTs). The vortex flow plasmapheretic reactor maintained significantly higher heparin levels in the extracorporeal circuit than in the sheep (device inlet WBRTs were 1.5 times the device outlet WBRTs) with no hemolysis. The reactor treatment did not effect any physiologically significant changes in complete blood cell counts, platelets, and protein levels for up to 2 hr of operation. Furthermore, gross necropsy and histopathology did not show any significant abnormalities in the kidney, liver, heart, brain, and spleen.