The presence and survival of pathogens inside the gut of leeches were studied by means of light and electron microscopy. In African leeches from Cameroon, blood was serologically positive for human immunodeficiency virus (HIV) and hepatitis B; blood of Hirudo medicinalis bought in German pharmacies contained up to 11 different species of bacteria. In experiments done at low (3 degrees C) and high (22 degrees, 32 degrees C) temperatures, it was shown that ingested red and white blood cells survive for long periods. The time was prolonged to at least 6 months in cases in which the leeches were stored at 3 degrees C. The same effect occurred with pathogens. Bacteriophages (viruses of bacteria) and bacteria persisted in large numbers for at least 6 months in the gut of experimentally infected leeches. Protozoan parasites such as Toxoplasma gondii, Trypanosoma brucei brucei, or Plasmodium berghei were even capable of reproducing inside the gut of the leech. In the case of Plasmodium parasites, this proceeded at low (3 degrees C) and high (22 degrees C) temperatures until all erythrocytes were used up. These parasites survived as long as the erythrocytes and lymphocytes were of good shape, i.e., around 5-6 weeks p.i. Single stages survived longer, especially at low temperatures. However, electron microscopy studies gave no hint of penetration of such pathogens into the unicellular salivary glands, which would initiate a direct transmission. Such transmission, however, is possible--many fish leeches directly transmit several blood parasites--when the leeches are squeezed during skin attachment or when they are manipulated by dropping salt solution on their backs while they are sucking. Consequently, the leech is a potential vector of many pathogens, especially in regions with an endemic spread of human and/or animal pathogens.

We have analyzed the functional domain structure of vinculin, a protein involved in linking microfilaments to the cytoplasmic face of cell membranes in animal cells. For this purpose, we used several monoclonal antibodies raised against chicken gizzard vinculin whose epitopes could be assigned to discrete regions in the vinculin sequence by immunoblotting of proteolytic fragments combined with N‐terminal amino acid sequencing. Two of these antibodies induced the disruption of stress fibers and changed the number of morphology of focal contacts after microinjection in chicken embryo fibroblasts. Based on the location of its epitope in comparison with vinculin domains previously identified by other groups, we propose that one of these antibodies (15B7) interferes with the binding of vinculin to talin, the most peripheral of the microfilament proteins. The second antibody (14C10) binds within a region comprising three internal repeats and might therefore distort the inner architecture of vinculin. A third antibody (As3) inhibited the binding of F‐actin to vinculin in an in vitro assay but had no effect on the microfilament system in cells. These data emphasize the role of vinculin as a key protein in microfilament‐membrane linkage and support previous work on a direct interaction between vinculin and actin.

The decrease of the rate of actin polymerization by tropomyosin molecules which bind near the ends of actin filaments was analyzed in terms of the rate of binding of tropomyosin to actin filaments. Monomeric actin was polymerized onto actin filaments in the presence of various concentrations of tropomyosin. At high concentrations of monomeric actin (c1) and low tropomyosin concentrations (ct) (c1/ct greater than 10), actin polymerization was not retarded by tropomyosin because actin polymerization was faster than binding of tropomyosin to actin filaments. At low actin concentrations and high tropomyosin concentrations (c1/ct less than 5), the rate of elongation of actin filaments was decreased because actin polymerization was slower than binding of tropomyosin at the ends of actin filaments. The results were quantitatively analyzed by a model in which it was assumed that actin-bound tropomyosin molecules which extend beyond the ends of actin filaments retard association of actin monomers with filament ends. Under the experimental conditions (100 mM KCl, 1 mM MgCl2, pH 7.5, 25 degrees C), the rate constant for binding of tropomyosin to actin filaments turned out to be about 2.5 X 10(6) to 4 X 10(6) M-1 S-1.

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The interaction of vinculin with actin filaments was investigated by methods which exclude interference by contaminating proteins which may occur in vinculin preparations. Vinculin which was blotted from SDS-polyacrylamide gels onto nitrocellulose, was stained specifically by fluorescently labeled polymeric actin (100 mM KCl, 2 mM MgCl2). Vinculin which was purified from alpha-actinin and an actin polymerization-inhibiting protein (HA1), was found to be cosedimented with polymeric actin. Maximally one vinculin molecule was cosedimented per one hundred actin filament subunits. Half maximal binding of vinculin was observed at about 0.25 microM free vinculin. Vinculin could be replaced from actin by the addition of tropomyosin.