Cultural, molecular, and immunological characterization of the etiologic agent for atypical canine ehrlichiosis.

More than 100 cases of canine ehrlichiosis, with three fatalities, were serologically negative by the indirect immunofluorescent antibody (IFA) test with Ehrlichia canis or E. sennetsu antigen but were reactive at titers of 10 to 640 with E. risticii. Ehrlichia-like agents were isolated from three such cases. The agents isolated from those cases were morphologically indistinguishable from each other and from a prototype, E. risticii, the etiologic agent of equine monocytic ehrlichiosis, in terms of growth characteristics and by light or electron microscopy. The patterns of and products from PCR were identical to those of E. risticii. The 16S rRNA sequences were distinct from those of E. canis and E. ewingii but were identical to those of E. risticii. A PCR product corresponding to the 5' half of the 16S rRNA gene was obtained from amplification of DNA from E. risticii and both sources of the atypical canine ehrlichiosis agent but was not obtained from uninfected host cells. The entire sequence of 719 nucleotides was identical for all three sources. The percentages of relatedness of the partial 16S rRNA gene of the atypical canine ehrlichiosis agent to E. risticii, E. sennetsu, E. platys, E. equi, E. phagocytophila, E. canis, E. chaffeensis, and E. ewingii were 100.0, 98.9, 83.7, 83.0, 83.0, 82.2, 81.8, and 81.5, respectively. These data are consistent with the identity of these isolates as E. risticii. The caninotropic characteristics of naturally acquired infections due to E. risticii are herein described for the first time, and the epizootiological implications are discussed in relation to the host range of E. risticii, which may include dogs as reservoirs.

Transgenic embryo yield is increased by a simple, inexpensive micropipet treatment.

A method for the treatment of micropipet tips for injection of DNA into pronuclei of mouse embryos is described. The method requires only standard laboratory equipment, a magnetic stirrer and an inexpensive commercially available grinding powder. The method involves little extra time and no special skills. After introduction of the method, the time required for injections was reduced by about 30%, and the yield of surviving embryos was increased by 28%.

43K protein and acetylcholine receptors colocalize during the initial stages of neuromuscular synapse formation in vivo.

The 43K protein is a cytoplasmic peripheral membrane protein concentrated subsynaptically in skeletal muscle. Recombinant 43K has been shown to cause clustering of acetylcholine receptors (AChRs) in cultured cells. However, the role of 43K in vivo is disputed, because in some cases it appears only after AChRs have clustered. We therefore examined the expression and distribution of 43K and AChRs during synapse formation in embryonic mouse muscles. Messenger RNA for 43K was detected on Embryonic Day (E) 12, a day prior to the first AChR clusters. Immunofluorescence showed that both AChRs and 43K were colocalized in patches by E13, the stage at which intramuscular nerves were first detected. The AChR/43K patches were nerve associated, and more than 98% of AChR patches were accompanied by 43K. The precise colocalization of 43K and AChRs persisted through development. These results are consistent with 43K being involved in the nerve-induced clustering of AChRs during synapse formation.

Mapping of myogenin transcription during embryogenesis using transgenes linked to the myogenin control region.

During vertebrate embryogenesis, the muscle-specific helix-loop-helix protein myogenin is expressed in muscle cell precursors in the developing somite myotome and limb bud before muscle fiber formation and is further upregulated during myogenesis. We show that cis-acting DNA sequences within the 5' flanking region of the mouse myogenin gene are sufficient to direct appropriate temporal, spatial, and tissue-specific transcription of myogenin during mouse embryogenesis. Myogenin-lacZ transgenes trace the fate of embryonic cells that activate myogenin transcription and suggest that myogenic precursor cells that migrate from the somite myotome to the limb bud are committed to a myogenic fate in the absence of myogenin transcription. Activation of a myogenin-lacZ transgene can occur in limb bud explants in culture, indicating that signals required for activation of myogenin transcription are intrinsic to the limb bud and independent of other parts of the embryo. These results reveal multiple populations of myogenic precursor cells during development and suggest the existence of regulators other than myogenic helix-loop-helix proteins that maintain cells in the early limb bud in the myogenic lineage.

Role of Tannins in Defending Plants Against Ruminants: Reduction in Dry Matter Digestion?

Polyphenolic allelochemicals, such as tannins, are widely thought to reduce the digestibility of plants consumed by herbivores by binding to digestive enzymes and dietary proteins. While the apparent digestibility of protein and, therefore, cell solubles is reduced in mule deer (Odocoileus hemionus) and white-tailed deer (O. virginianus) consuming tanniferous forages, digestion of the plant cell wall is not reduced beyond that predicted from its content of lignin, cutin, and silica. The lack of a tannin effect on cell wall digestion in deer is in contrast to studies with domestic sheep and numerous in vitro studies. Herbivores adapted to consume tanniferous forages may defend against such allelochemicals by producing salivary proteins that bind tannins in a highly specific manner. These tannin-salivary protein complexes would reduce apparent digestibilities of protein and cell solubles and, if completely effective, would not reduce cell wall digestion. The occurrence of such proteins in ruminants is reported here for the first time. The saliva composition of mule deer (a mixed feeder that commonly consumes browse) and domestic cattle and sheep (predominant grazers) are compared, and the higher potential of the deer saliva to neutralize tannins is related to their feeding habits. Salivary proteins that preferentially bind tannins may minimize fecal nitrogen losses by maximizing the efficiency of tannin-binding per unit of protein and may reduce the absorption of hydrolyzable tannins and the potential for tannin toxicity.