Simple methods for determination of the release limits for drug products

Gradients of cellular activities are ubiquitous in embryonic development. It is widely believed that the inhomogeneous spatial distribution of a morphogen would be able to set up such gradients. But how then does the morphogen propagate in the first place? Straightforward molecular diffusion is often proposed as a possible mechanism. We first show that, surprisingly, the mere binding of the diffusing morphogen to its membrane receptors suffices to prevent the establishment of a concentration-based positional signalling system. Instead, a flat, saturated distribution of receptor-bound morphogen builds up. Because the distribution spreads gradually from the morphogen source, however, cells may still know their position if they are able to integrate the morphogen signal in time. The irregularities of diffusion in the complex extracellular medium would in fact be partially compensated for by such time summation. Another, non-exclusive possibility is that morphogen transport does not occur by simple diffusion only. We put forth a novel model of receptor-aided, directed diffusion that achieves a spatial distribution of morphogen. Our model is based, as an illustration, on the properties of members of the TGFbeta family of molecules. We show that two simple hypotheses regarding the kinetics of TGBbeta binding to its receptors suffice to establish a remarkable transfer mechanism whereby a morphogen such as activin could be both propagated along cell membranes, and transferred between cells that are in contact. The model predicts that morphogen propagation properties depend strongly on the closeness of cell-cell appositions, does not necessitate protein synthesis, accumulation or slow degradation (in contrast to the diffusion/time integration model), and that the morphogen is localised mostly on or close to cell membranes.     

Comparison of nested PCR with immunofluorescent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline

A partial 16S rRNA gene was amplified in Ehrlichia canis-infected cells by nested PCR. The assay was specific and did not amplify the closely related Ehrlichia chaffeensis, Ehrlichia muris, Neorickettsia helminthoeca, and SF agent 16S rRNA genes. The assay was as sensitive as Southern hybridization, detecting as little as 0.2 pg of E. canis DNA. By this method, all blood samples from four dogs experimentally infected with E. canis were positive as early as day 4 postinoculation, which was before or at the time of seroconversion. One hundred five blood samples from dogs from Arizona and Texas (areas of E. canis endemicity) and 30 blood samples from dogs from Ohio (area of E. canis nonendemicity) were examined by nested PCR and immunofluorescent-antibody (IFA) test. Approximately 84% of dogs from Arizona and Texas had been treated with doxycycline before submission of blood specimens. Among Arizona and Texas specimens, 46 samples were PCR positive (44%) and 80 were IFA positive (76%). Forty-three of 80 IFA-positive samples (54%) were PCR positive, and 22 of 25 IFA-negative samples (88%) were negative in the nested PCR. None of the Ohio specimens were IFA positive, but 5 specimens were PCR positive (17%). Our results indicate that the nested PCR is highly sensitive and specific for detection of E. canis and may be more useful in assessing the clearance of the organisms after antibiotic therapy than IFA, especially in areas in which E. canis is endemic.     

Distribution of genetic diversity in relation to chromosomal inversions in the malaria mosquito Anopheles gambiae

The epidemiology of malaria in Africa is complicated by the fact that its principal vector, the mosquito Anopheles gambiae, constitutes a complex of six sibling species. Each species is characterized by a unique array of paracentric inversions, as deduced by karyotypic analysis. In addition, most of the species carry a number of polymorphic inversions. In order to develop an understanding of the evolutionary histories of different parts of the genome, we compared the genetic variation of areas inside and outside inversions in two distinct inversion karyotypes of A. gambiae. Thirty-five cDNA clones were mapped on the five arms of the A. gambiae chromosomes with divisional probes. Sixteen of these clones, localized both inside and outside inversions of chromosome 2, were used as probes in order to determine the nucleotide diversity of different parts of the genome in the two inversion karyotypes. We observed that the sequence diversity inside the inversion is more than three-fold lower than in areas outside the inversion and that the degree of divergence increases gradually at loci at increasing distance from the inversion. To interpret the data we present a selectionist and a stochastic model, both of which point to a relatively recent origin of the studied inversion and may suggest differences between the evolutionary history of inversions in Anopheles and Drosophila species.