Detection of antibodies reactive with Ehrlichia chaffeensis in the raccoon

Antibodies reactive with Ehrlichia chaffeensis were detected in raccoon (Procyon lotor) serum samples by using an indirect immunofluorescence assay. Samples from 411 raccoons trapped in the southeastern United States from 1977 to 1999 were tested. Serologically reactive samples with reciprocal titers of > or =16 were detected from 83 raccoons (20%) from 13 of 16 counties in eight states, indicating that raccoons are commonly exposed to E. chaffeensis. Samples collected as early as 1977 were positive. A polymerase chain reaction assay specific for E. chaffeensis failed to detect the presence of ehrlichial DNA in serum samples from 20 representative seroreactive raccoons. Because of serologic cross-reactivity among antigens derived from different Ehrlichia spp., additional immunologic, molecular, or culture-based studies will be required to confirm E. chaffeensis infections of raccoons in the southeastern United States.     

Identification of the proton pathway in bacterial reaction centers: cooperation between Asp-M17 and Asp-L210 facilitates proton transfer to the secondary quinone (QB)

The reaction center (RC) from Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, Q(B) (the secondary quinone electron acceptor), to form quinol, Q(B)H2. Asp-L210 and Asp-M17 have been proposed to be components of the pathway for proton transfer [Axelrod, H. L., Abresch, E. C., Paddock, M. L., Okamura, M. Y., and Feher, G. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1542-1547]. To test the importance of these residues for efficient proton transfer, the rates of the proton-coupled electron-transfer reaction k(AB)(2) (Q(A-*)Q(B-*) + H+ <==>Q(A-*)Q(B)H* --> Q(A)Q(B)H-) and its associated proton uptake were measured in native and mutant RCs, lacking one or both Asp residues. In the double mutant RCs, the k(AB)(2) reaction and its associated proton uptake were approximately 300-fold slower than in native RCs (pH 8). In contrast, single mutant RCs displayed reaction rates that were < or =3-fold slower than native (pH 8). In addition, the rate-limiting step of k(AB)(2) was changed from electron transfer (native and single mutants) to proton transfer (double mutant) as shown from the lack of a dependence of the observed rate on the driving force for electron transfer in the double mutant RCs compared to the native or single mutants. This implies that the rate of the proton-transfer step was reduced (> or =10(3)-fold) upon replacement of both Asp-L210 and Asp-M17 with Asn. Similar, but less drastic, differences were observed for k(AB)(1), which at pH > or =8 is coupled to the protonation of Glu-L212 [(Q(A-*)Q(B))-Glu- + H+ --> (Q(A)Q(B-*)-GluH]. These results show that the pathway for proton transfer from solution to reduced Q(B) involves both Asp-L210 and Asp-M17, which provide parallel branches to the proton-transfer pathway and through their electrostatic interaction have a cooperative effect on the proton-transfer rate. A possible mechanism for the cooperativity is discussed.     

Oppositional effects of acetylcholine and isoproterenol on isometric tension and cyclic nucleotide concentrations in rabbit atria.

The effects of acetylcholine chloride and isoproterenol on myocardiial cyclic GMP, cyclic AMP and on isometric tension were studied in isolated electrically driven rabbit atria. Acetylcholine (0.5 muM) produced a significant decrease in isometric force that was associated with a significant elevation in atrial cyclic GMP. Cyclic AMP was significantly lowered at 15 seconds after the addition of acetylcholine, but was only slightly decreased at earlier time periods. Both the negative inotropic action and increase in cyclic GMP after addition of acetylcholine were blocked by atropine. Isoproterenol (0.1 muM) produced a significant increase in isometric tension that was associated with a significant elevation in atrial cyclic AMP levels, whereas cyclic GMP levels were not changed. These effects were blocked by practolol. The increases in atrial cyclic GMP and cyclic AMP following addition of acetylcholine and isoproterenol, respectively, preceded the changes in isometric tension in response to these agents. These data support the hypothesis that changes in intracellular levels of cyclic AMP and cyclic GMP may mediate the positive and negative inotropic effects of adrenergic and cholinergic agents.     

Membrane Potential Mediates H+-ATPase Dependence of ``Degradative Pathway' Endosomal Fusion

In some epithelial cell lines, the uptake and degradation of proteins is so pronounced as to be regarded as a specialized function known as ``degradative endocytosis.' The endosomal pathways of the renal proximal tubule and the visceral yolk sac share highly specialized structures for ``degradative endocytosis.' These endosomal pathways also have a unique distribution of their H⁺-ATPase, predominantly in the subapical endosomal pathway. Previous studies provide only indirect evidence that H⁺-ATPases participate in endosomal fusion events: formation of vesicular intermediates between early and late endosomes is H⁺-ATPase dependent in baby hamster kidney cells, and H⁺-ATPase subunits bind fusion complex proteins in detergent extracts of fresh rat brain. To determine directly whether homotypic endosomal fusion is H⁺-ATPase dependent, we inhibited v-type H⁺-ATPase during flow cytometry and cuvette-based fusion assays reconstituting endosomal fusion in vitro. We report that homotypic fusion in subapical endosomes derived from rat renal cortex, and immortalized visceral yolk sac cells in culture, is inhibited by the v-type H⁺-ATPase specific inhibitor bafilomycin A1. Inhibition of fusion by H⁺-ATPase is mediated by the membrane potential as collapsing the pH gradient with nigericin had no effect on homotypic endosomal fusion, while collapsing the membrane potential with valinomycin inhibited endosomal fusion. Utilizing an in vitro reconstitution assay this data provides the first direct evidence for a role of v-type H⁺-ATPase in mammalian homotypic endosomal fusion. Received: 29 October 1996/Revised: 8 December 1997     

Phylogeny and biogeography of ratite birds inferred from DNA sequences of the mitochondrial ribosomal genes

The origin of the flightless ratite birds of the southern continents has been debated for over a century. Whether dispersal or vicariance (continental breakup) best explains their origin depends largely on their phylogenetic relationships. No consensus has been reached on this issue despite many morphological and molecular studies. To address this question further we sequenced a 2.8-kb region of mitochondrial DNA containing the ribosomal genes in representative ratites and a tinamou. Phylogenetic analyses indicate that Struthio (Africa) is basal and Rhea (South America) clusters with living Australasian ratites. This phylogeny agrees with transferrin and DNA hybridization studies but not with sequence analyses of some protein-coding genes. These results also require reevaluation of the phylogenetic position of the extinct moas of New Zealand. We propose a new hypothesis for the origin of ratites that combines elements of dispersal and vicariance.      

Torulopsis petrophilum and Surface Activity

Torulopsis petrophilum can synthesize either a glycolipid surfactant or a protein emulsifier depending on the substrate used. These compounds were not produced to facilitate the uptake of an insoluble carbon source. The glycolipids produced were identical to the mixture isolated from T. bombicola.