Precursors of both conventional and Ly-1 B cells can escape feedback inhibition of Ig gene rearrangement

Experiments with transgenic mice carrying rearranged Ig transgenes have shown that membrane bound Ig molecules cause feedback inhibition of endogenous Ig gene rearrangement. However, this inhibition is never complete. It has been postulated that escape from feedback may be a property of the Ly-1 B cell subset, whereas rearrangement of endogenous Ig genes may be completely inhibited in conventional B cells. This possibility was investigated in transgenic mice carrying a lambda transgene under the control of the H chain enhancer. It was found that kappa producing B cells in these lambda transgenic mice were for the most part, although not exclusively, of the conventional B cell phenotype. Examination of peritoneal exudate cells revealed that a large proportion of Ly-1 B cells also express kappa. Adoptive transfer of bone marrow from adult lambda transgenic mice, a source of conventional B cell precursors, resulted in the production of relatively high levels of serum kappa 2 to 3 mo after transfer into recipient SCID mice. A high proportion of donor B cells in the spleen produced endogenous kappa protein with or without co-production of lambda. It is concluded that precursors of both conventional and Ly-1 B cells can escape feedback inhibition of L chain gene rearrangement.     

Determination of antithrombocyte antibodies in the blood serum of patients with idiopathic thrombocytopenic purpura by an immunoenzyme method

Enzyme-linked immunosorbent assay (ELISA) was developed for determination of serum antiplatelet antibodies. Platelets obtained from healthy donors of blood group 0(1) were washed off plasma and sedimented on the bottom of microtest wells. After washing off unattached platelets and blocking of plastic with albumin platelets were incubated with sera under investigation and binding of serum antibodies was detected using antihuman immunoglobulin antibodies conjugated with peroxidase. Ten patients with idiopathic thrombocytopenic purpura (ITP). 1 patient with systemic lupus erythematosus. 1 patient with red blood cell aplasia and 9 healthy donors (negative control) were studied by ELISA. Serum antibodies which effectively bound to platelets were detected in 5 patients with ITP, in patient with lupus erythematosus and in patient with red blood cell aplasia.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Factors Affecting Competence for Transformation in Staphylococcus aureus

A chemically defined medium has been developed for isolation of amino acid-requiring mutants of Staphylococcus aureus strain 8325, and for use as a selective medium in transformation assays. Variables affecting transformation of both plasmid and chromosomal markers have been studied. The optimal pH and temperature for transformation are 6.75 to 7.0 and 30 C, respectively. Ca ions are required for transformation, and only cells lysogenic for the phage phi11 can be transformed. Superinfection of competent cells with phi11 does not increase the transformation frequency. Maximal number of transformants is obtained after 20 min of contact between cells and deoxyribonucleic acid. The transformation frequencies for the plasmid marker erythromycin resistance (ero) and the chromosomal markers trp, thy, and cyt are of the same order of magnitude, whereas the frequency for the chromosomal marker tyr is approximately one order of magnitude lower.     

A conserved region of the MSP-1 surface protein of Plasmodium falciparum contains a recognition sequence for erythrocyte spectrin.

The major surface protein MSP-1 of Plasmodium falciparum blood-stage malaria parasites contains notably conserved sequence blocks with unknown function. The recombinant protein 190L, which represents such a block, exhibits a high affinity for red blood cell membranes. We demonstrate that both 190L and native MSP-1 protein bind to the inner red blood cell membrane skeleton protein spectrin. By using overlapping peptides covering the 190L molecule, we show that the spectrin contact site of 190L is included in a linear sequence of 30 amino acid residues. Association of 190L with naturally occurring spectrin deficient red blood cells is drastically reduced. In the same cells parasite invasion is normal, but the intracellular parasite development arrests late in the trophozoite stage. A similar situation arises when synthetic peptides covering the spectrin recognition sequence of 190L are added to P.falciparum cultures. These data and the cellular localization of MSP-1 suggest the possibility that MSP-1 associates with spectrin under natural conditions.     

Transport of proteins across the mitochondrial outer membrane. A precursor form of the cytoplasmically made intermembrane enzyme cytochrome c peroxidase.

Cytochrome c peroxidase, a cytoplasmically made enzyme located between the inner and outer membrane of yeast mitochondria, is synthesized as larger precursor in a reticulocyte cell-free lysate as well as in pulsed yeast spheroplasts. When the pulsed spheroplasts are chased, the precursor is converted to the mature apoprotein. When the in vitro synthesized precursor is incubated with isolated yeast mitochondria in the absence of protein synthesis, it is cleaved to the mature form; the mature form co-sediments with the mitochondria and is resistant to externally added proteases. These results, in conjunction with those reported earlier (Maccecchini, M.-L., Rudin, Y., Blobel, G., and Schatz, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 343-347) suggest that the mechanism of protein transport into the mitochondrial intermembrane space is quite similar to that of protein transport into the matrix or the inner membrane.     

The second cell membrane: the basal lamina and the tissue cell theory of metazoa.

We suggest that the basal lamina is essentially a second plasma or cell membrane appearing at the next higher level of biological organization; that together with associated cell monolayers it creates a tissue level membrane which is used to form multicellular cells and that collections of these provide the essential structure of metazoa. Thus when the histological structure of multicellular organisms is viewed in a topologically simplified form such organisms appear to be sets of multicellular cells (m-cells) formed by a unit tissue membrane built around the basal lamina. Not only are m-cells in this way structurally isomorphous (homeomorphic) to unit or classical biological cells (u-cells) but the two cellular levels are also functionally isomorphous. This suggests a “General Principle of Hierarchical Isomorphism or Iteration”, i.e. that multicellular evolution recapitulates unicellular evolution. This principle of structural and functional isomorphic mappability of unicellular onto multicellular organisms then governs the organization of matter all the way from molecules to man. Just as cytoplasm precipitates the bimolecular plasma membrane to form u-cells for the purpose of achieving reaction sequestration, in turn, these u-cells precipitate a common basal lamina to form m-cells, the histologist's acini, to produce sequestered “tissue plasms”. Thus, the “generalized acinus” with its basal laminar complex seem to constitute a second level (multicellular) cell and cell membrane, respectively.Four operators, ultimately under genetic control, can generate both u and m-cells from planar configurations of their respective unit membranes therewith providing the essential structure of all cells, tissues, organs and organisms. These are the ply, permeability vector, topological and stratificational operators. They are collected into a set of “organ formulae”. Both the plasma membrane and the basal lamina act as covering membranes and, again, as membranes for subcells so that a complete multicellular organism is a tetrahierarchical cell in which the molecule is the element of the first two cellular domains and the cell is the element of the last two. The analysis identifies a new transport organ group which together with the classical endocrine and exocrine groups comprises nearly the whole of the soft tissue organs. In a major reduction, all these organs are continuously (topologically) transformable into each and into hollow spheres, cells or acini thus greatly simplifying the histology of metazoa. Given this emphasis on cellularization it would seem that life, i.e. the autonomous chemoservo, results from the cooperation of cellularization and replication operations on the catalyzation process. Through cellularization, the lipid bilayer and basal laminar membranes provide the essential catalytic reaction sequestration demanded by chemical reaction theory while through complementary base pairing the DNA double helix provides the essential memory which stores the patterns of the variations of the sequestered reactions.