Dielectric dispersion of actin

The dielectric dispersion of G-actin was 7 X 10(6) Hz, and the low frequency reduced dielectric increment 0.99 +/- 0.15 ml/mg. The dielectric increment of F-actin in the range 10(5) to 10(8) Hz was very low, about 0.3.     

Studies on the interaction between protein A and immunoglobulin G. I. Effect of protein A on the functional activity of IgG.

Staphylococcal protein (A (PA) and IgG anti-Forssman immunoglobulin formed complexes that behaved functionally like IgM in their ability to lyse sheep erythrocytes (E) in the presence of whole guinea pig complement (GPC) and to fix purified guinea pig C1. Concanavalin A, a plant lectin that inhibited IgM but not IgG hemolytic activity, inhibited the hemolytic activity of IgG-protein A complexes that behaved like IgM but had no effect on complexes that behaved functionally like IgG. Since Con A is known to bind specifically to glucose and mannose residues, our results suggested that the interaction of protein A with the Fc region of IgG led to exposure of sugar moieties that may participate in complement (C) binding. The production of IgM-like complexes depended on the ratio of protein A to IgG and the empirical formula of these IgM-like complexes was found to be [(IgG)2PA]n. As the ratio of PA to IgG was increased, the resulting complexes tended to behave functionally like IgG but with reduced hemolytic activity and C1 fixing ability. Furthermore, the binding of C1 to EIgG was inhibited by PA and the binding of PA to EIgG was inhibited by C1 indicating that the binding sites for C1 and PA were located near each other or were identical. Our results offer a reasonable explanation for the reported effects of PA or mixtures of PA and IgG in vitro and in vivo.     

Evaluation of phosphorus in forest soils: Comparison of phosphorus uptake,extraction method and soil properties

Summary Phosphorus in soils from plantation of red pine (Pinus resinosa Ait.) was determined using six extractants: 0.002N H₂SO₄ (pH 3.0); 0.025N HCl+ +0.03N NH₄F; 0.5N NaHCO₃ (pH 8.5);N NH₄OAc (pH 4.8); anion exchange resin (Dower –2, Cl-form); H₂O. Correlations of extractable P with Al- and Al-+Fe-P indicated that these fractions are the dominant forms of inorganic P in most of the soils.Uptake of P by corn and Monterey pine seedlings grown in greenhouse culture was correlated with soil P extracted by the different methods. The most successful of the extractants for predicting P uptake was resin extractable P; the simple correlation coefficients were 0.811 and 0.609 for pine and corn respectively. P uptake by pine correlated significantly with 0.002N H₂SO₄ P (r=0.679),N NH₄OAc P (r=0.443), H₂O P (r=0.549) and Al-+Fe-P (r=0.532) while P uptake by corn correlated with 0.002N H₂SO₄ P (r=0.579), H₂O P (r=0.477) and organic P (r=0.460). Per cent P in pine seedling tops correlated significantly with 0.002N H₂SO₄, resin andN NH₄ OAc extractable P. Multiple regressions which included silt+clay and organic P improved correlations of some soil tests with P uptake in corn and pine seedlings respectively.Research supported by the School of Natural Resources, College of Agricultural and Life Sciences, Univ. of Wisconsin, Madison, the Wisconsin Department of Natural Resources and FAO Fellowship to the senior author.     

The role of trehalose in the osmoadaptation of Escherichia coli NCIB 9484: interaction of trehalose, K+ and glutamate during osmoadaptation in continuous culture.

Natural abundance 13C nuclear magnetic resonance spectroscopy identified the disaccharide trehalose as the major organic osmolyte synthesized by Escherichia coli grown in continuous culture under nitrogen limitation in the presence of 0.5 M-NaCl. Trehalose accumulation was dependent on both the growth phase of the culture and the osmolality of the growth medium, but independent of the solute used to increase the osmolality as long as the solute was non-penetrant. The penetrant solute glycerol did not induce trehalose synthesis indicating that the loss of cell turgor rather than increasing medium osmolality per se was the mechanism stimulating trehalose synthesis. Under conditions of either carbon or nitrogen limitation osmoadaptation was distinctly biphasic. The initial response consisted of a rapid (within 30 min) accumulation of K+ and a concurrent synthesis of the amino acid glutamate; trehalose synthesis occurred during the second slower phase of osmoadaption. Chloramphenicol severely inhibited trehalose accumulation indicating that the enzyme(s) involved in trehalose synthesis were inducible.     

Induction of vascular smooth muscle cell growth by selective activation of the thrombin receptor. Effect of heparin.

The synthetic peptide, SFLLRNPNDKYEPF, has been recently described as a peptide mimicking the new amino-terminus created by cleavage of the thrombin receptor, therefore acting as an agonist of the thrombin receptor. This peptide was a potent mitogen for rabbit arterial smooth muscle cells (SMC) and exhibited the same activity as that of native alpha-thrombin. Both compounds stimulated the proliferation of growth-arrested SMCs with half-maximum mitogenic responses at 1 nM. NAPAP, a synthetic inhibitor of the enzymatic activity of thrombin, specifically inhibited thrombin-induced SMC growth (IC50 = 0.35 +/- 0.04 microM) but was without effect on the mitogenic effect of the agonist peptide. These results therefore demonstrate that the mitogenic effect of alpha-thrombin for SMCs is intimately linked to its esterolytic activity. Heparin, which inhibited fetal calf serum-induced SMC growth, was without effect on thrombin-induced SMC growth but strongly reduced the mitogenic effect of the agonist peptide (IC50 = 32 +/- 5 micrograms/ml). This effect was not related to the anti-coagulant activity of heparin but was highly dependent on molecular mass and on the global charge of the molecule and was also observed for other sulphated polysaccharides such as pentosan polysulphate.     

Construction of Bordetella pertussis strains that overproduce genetically inactivated pertussis toxin.

Nontoxic analogs of pertussis toxin (PT), produced by in vitro mutagenesis of the tox operon, are immunogenic and protective against infection by Bordetella pertussis. The moderate levels of PT production by B. pertussis, however, make it the limiting antigen in the formulation of multicomponent, acellular, recombinant whooping cough vaccines. To increase production of the highly detoxified Lys9Gly129 PT analog by B. pertussis, additional copies of the mutated tox operon were integrated into the bacterial chromosome at the tox or fha locus by unmarked allelic exchange. Recombinant strains produced in this way secreted elevated levels of the PT analog proportional to gene dosage. The strains were stable during 10-liter fermentations, and yields of up to 80 mg of PT analog per liter were obtained under production-scale conditions. The nontoxic analog was purified and shown to be indistinguishable from material obtained from a B. pertussis strain that contained only a single copy of the toxLys9Gly129 operon. Such strains are therefore suitable for large-scale, industrial production of an acellular whooping cough vaccine containing a genetically detoxified PT analog.     

Critical fields in cell fusion and dielectrophoresis

It is increasingly recognized that exposure to electrical fields can reversibly increase the electrical permeability and conductivity of the cellular membrane. It is therefore worthwhile to determine under what circumstances the critical fields capable of affecting the cellular membrane can be reached. We have evaluated the field intensities E and the gradients of E² in the neighborhood of the more common field electrode shapes, the sphere-sphere and cylinder-cylinder electrodes, and put these in terms of the values useful for the practical electro-fusion and dielectrophoresis (DEP) of cells. The calculations were performed using the charge-image technique. From this it was observed that gross errors (underestimations of up to 1000-fold) of the field would be made if neglect of the electrode polarization were made as in simple electrostatic calculation. The dielectric forces (in terms of (E)²) show interesting spatial character.The electrical breakdown of cellular membranes affects the exchange of information and materials between the cell and its environment, and can be used to fuse cells of the same type or of differing types. It is moreover of importance in handling cells during DEP while obtaining their spectral characteristics or in cell-sorting. It is expected that the results here will aid in the more proper use of electrical fields for such ends.     

Membranes of the protoplast L-form of Proteus mirabilis

Isolated membranes of the cell wall-less stable protoplast L-form of Proteus mirabilis were characterized by density gradient centrifugation and by assay for their major chemical constituents, proteins, phospholipids and lipopolysaccharide, and for some specific marker enzymes of the cytoplasmic membrane. In most of the analyzed properties the L-form protoplast membrane resembled the bacterial cytoplasmic membrane, with some notable modifications. considerable amounts of lipopolysaccharide, normally an exclusive constituent of the outer membrane, were found. Furthermore, the L-form membranes contained the functions of the reduced nicotinamide adenine dinucleotide oxidase system, of d-lactate dehydrogenase (EC 1.1.1.28) and of succinate dehydrogenase (EC 1.3.99.1) at specific activities comparable to, or in some cases considerably higher than, those present in cytoplasmic membranes of the bacterial form. Of two peptidoglycan DD-carboxypetidase/transpeptidases (EC 3.4.17.8 and EC 2.3.2.10), which are normally present in the cytoplasmic membrane of the bacterial form of P. mirabilis, the membrane of the protoplast L-form contained only one. Electron microscopy of thin sectioned L-form protoplasts showed extensive heterogeneity of membraneous structures. In addition to the single membraneous integument, internal membrane-bounded vesicles and multiple stacks of membranes were present, as the result of unbalanced growth and membrane synthesis in the L-form state.