Multiline varieties and disease control

Summary A general model for the evolution of pathogen populations on mixtures or multilines is developed. This model is used to extend previous analyses of the effects of the widespread cultivation of multilines on the evolution of virulence in obligate parasites to mixtures of lines carrying different numbers of resistance genes. It is concluded that the composition of an equilibrium pathogen population growing on a multiline may vary within wide limits and the prinicipal determinant of its composition is the number of components in the multiline and the resistance genes they carry. Other factors of importance are (i) the relative contribution made by each host class (with different numbers of resistance genes) to the pathogen spore pool each generation; (ii) the levels of stabilizing selection against unnecessary virulence genes; and (iii) the way in which unnecessary genes for virulence combine to reduce pathogen fitness. Conditions for the fixation of avirulent biotypes in the pathogen population and the evolution of a pathogen superrace are given for multilines of various compositions.Paper No. 9246 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina. This investigation was supported in part by NIH Research Grant No. GM 11546 from the National Institute of General Medical Sciences     

Localization of Tamm-Horsfall-glycoprotein-like immunoreactivity in cultured baby-hamster kidney cells, shown by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques.

Tamm-Horsfall glycoprotein was isolated from hamster urine, and antiserum against it was produced in rabbits. IgG was isolated from the antiserum. Immunocytochemical methods were used to localize Tamm-Horsfall-like immunoreactivity in three substrains of baby-hamster kidney (BHK) cells. Indirect immunofluorescence techniques showed that, in two substrains (BHK-21/C13/2P and BHK-21/C13/3P), a proportion of the cells fluoresced brilliantly, whereas those of the third substrain (BHK-21/ICRF) were totally negative. Related findings were obtained by the immunoperoxidase optical-microscopic technique. From the results of immunoperoxidase techniques using the electron microscope, it was concluded that the substance was present in association with the plasma membranes of the reacting cells. Our data suggest that the line of baby-hamster kidney cells, BHK-21/C13, may contain cells of renal-tubular epithelial origin, and that the proportion of these may be variable from one subculture to another.     

Platelet immune complex interaction in pathogenesis of Kawasaki disease and childhood polyarteritis.

The role of platelets in the pathogenesis of vasculitis and the formation of coronary artery aneurysms was studied in 19 children with Kawasaki disease and five with polyarteritis. All patients with Kawasaki disease developed thrombocytosis in the third week of illness. The peak platelet count was significantly correlated (p less than 0.005) with the subsequent development of coronary artery aneurysms. The rise in platelet count was associated with the appearance in the circulation of a factor that induced aggregation and serotonin release in normal platelets. This factor was shown to be of high molecular weight, and its activity was lost at low pH--features suggestive of an immune complex. Immune complexes, detected by precipitation with polyethylene glycol, also appeared in the circulation as the platelet count increased. These complexes induced platelet aggregation, and there was a significant correlation (p less than 0.001) between the concentrations of IgG and IgA in the polyethylene glycol precipitated material and the platelet aggregating activity. Similar platelet aggregating activity was also detected in patients with polyarteritis but followed a different time course, persisting in the circulation for several months in association with continued disease activity. These findings imply that different mechanisms have a role in distinct phases of Kawasaki disease. The initial feverish phase (probably infective) is probably followed by an immune complex vasculitis that occurs when antibodies to the initiating agent appear in the circulation. The immune complexes aggregate platelets and induce release of serotonin. Platelet derived vasoactive mediators may increase vascular permeability and facilitate further deposition of complexes in the tissues.     

Effects of the removal of adherent and phagocytic cells on the spleen cell lymphoproliferative response of tumor-bearing mice

Summary Cell-mediated immunity was investigated in two BALB/c mouse tumor systems using the lymphoblastogenesis test with phytohemagglutinin as the mitogen. This lymphoproliferative response was quantitated using the Stimulation Index (SI). There was little evidence for suppressor cell activity in cell mixing experiments in which spleen cells from #51 cell-injected mice were mixed with spleen cells from normal mice. Following macrophage removal by Sephadex G-10 columns and carbonyl iron ingestion, there were no significant changes in the SI values for spleen cells from the #51 cell-injected mice. In contrast, spleen cells from mice injected with H238 cells, a herpes virus-transformed cell line, had a significantly lower SI value than that of normal mice. Suppressor cell activity was demonstrated in cell mixing experiments in which spleen cells from H238 cell-injected mice were mixed with normal spleen cells. Removal of adherent cells from spleen cells from H238 cell-injected mice by Sephadex G-10 columns restored the SI value to that of normal mice. An increased SI value was also seen after removal of phagocytic cells by carbonyl iron. These results suggested that cells with the functional properties of macrophages played an important part in the immunosuppression observed in the H238 tumor system. Comparison of the two macrophage depletion methods suggested that another cell population was also involved in the suppressive effect. Results of immunofluorescent techniques with anti-Lyt-1 and anti-Lyt-2 monoclonal antibodies show these cells to be Ly 1^–, Ly 2,3⁺ phenotypes of T-lymphocytes.     

Base pairing in wheat germ ribosomal 5S RNA as measured by ultraviolet absorption, circular dichroism, and Fourier-transform infrared spectrometry

Ultraviolet absorption (UV) and circular dichroism (CD) spectra of wheat germ 5S RNA, when compared to tRNAPhe, indicate a largely base-paired and base-stacked helical structure, containing up to 36 base pairs. Fourier-transform infrared (FT-IR) spectra of tRNAPhe and wheat germ ribosomal 5S RNA have been acquired at 30 and 90 degrees C. From the difference of the FT-IR spectra between 90 and 30 degrees C, the number of base pairs in both RNAs was determined by modification of a previously published procedure [Burkey, K. O., Marshall, A. G., & Alben, J. O. (1983) Biochemistry 22, 4223-4229]. The base-pair composition and total base-pair number from FT-IR data are now consistent for the first time with optical (UV, CD, Raman) and NMR results for ribosomal 5S RNA. Without added Mg2+, tRNAPhe gave 18 +/- 2 base pairs [7 A-U and 11 G-C], in good agreement with the number of secondary base pairs from X-ray crystallography [8 A-U, 12 G-C, and 1 G-U]. Within the 10% precision of the FT-IR method, wheat germ 5S RNA exhibits essentially the same number of base pairs [14 A-U, 17 G-C, and 5 G-U; for a total of 36] in the absence of Mg2+ as in the presence of Mg2+ [14 A-U, 18 G-C, and 3 G-U; for a total of 35], in agreement with the UV hyperchromism estimate of G-C/(A-U + G-C) = 0.58.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stable isotope ratio as a tracer of mangrove carbon in Malaysian ecosystems

Summary The ratio of stable carbon isotopes (¹³C) in plants and animals from Malaysian mangrove swamps, coastal inlets, and offshore waters was determined. Vascular plants of the swamps were isotopically distinct ( x±s.d.=-27.1±1.2) from plankton (-21.0±0.3) and other algae (-18.7±2.2). Animals from the swamps (-20.9±4.1) and inlets (-19.8±2.5) had a wide range of isotope ratios (-28.6 to-15.4), indicating consumption of both mangrove and algal carbon. Several commercially important species of bivalves, shrimp, crabs, and fish obtained carbon from mangrove trees. Mangrove carbon was carried offshore as detritus and was isotopically distinguishable in suspended particulate matter and sediments. Animals collected from 2 to 18 km offshore, however, showed no isotopic evidence of mangrove carbon assimilation, with ratios (-16.5±1.1, range-19.1 to-13.1) virtually identical to those reported for similar animals from other plankton-based ecosystems. Within groups of animals, isotope ratios reflected intergencric and interspecific differences in feeding and trophic position. In particular, there was a trend to less negative ratios with increasing trophic level.     

Characterization and partial purification of keratinocyte growth factor from the hypothalamus

An extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum-free culture system were performed. The keratinocyte growth-promoting activity of the hypothalamic extract was unaffected by heating (100 degrees C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G-25 column, and an approximate pl of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150-300 micrograms/ml) increased keratinocyte growth approximately 50-fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat-treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum-free culture system. Keratinocyte growth-promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.     

Exclusion of erythrocyte-specific membrane proteins from clathrin- coated pits during differentiation of human erythroleukemic cells

When human erythroleukemic cells are induced to differentiate, they produce globin and redistribute glycophorin and spectrin to one pole of the cell. This process was accompanied by an alteration in the clathrin-coated pits at the cell surface. In nondifferentiating cells, receptors for Concanavalin A have been shown, using electron microscopy, to be concentrated into coated pits and rapidly internalized. Glycophorin was also internalized via coated pits, but was not greatly concentrated into these portions of the surface membrane. Ligands attached to glycophorin were, therefore, cleared from the cell surface more slowly than Concanavalin A. In nondifferentiating cells, immunoelectron microscopy showed that spectrin is largely excluded from coated pits. After erythroid differentiation proceeded for several days, glycophorin was totally excluded from the coated pits along with spectrin. This did not reflect a general cessation of endocytosis, however, because Concanavalin A receptors continued to be internalized. It is possible that the specific exclusion of glycophorin from coated pits is part of the remodeling process that occurs when the precursor cell membrane differentiates into that of the mature erythrocyte.     

High-speed preparative-scale separation and purification of ribosomal 5S and 5.8S RNA's via Sephacryl S-300 gel filtration chromatography

In this work we present a rapid and economical alternative to Sephadex and high performance size exclusion chromatography (HPSEC) for preparative-scale separation and purification of low molecular weight RNA's: 5.8S RNA, 5S RNA, and tRNA's. These three RNA species can be well resolved from each other and from higher molecular weight RNA species via Sephacryl S-300 gel filtration chromatography under mild eluting conditions: 10 mM Tris-HCl buffer, pH 7.5, containing 1.0 M NaCl. For a sample load of about 250 mg, the resolving power of a Sephacryl S-300 column (78 X 3.2 cm) is comparable to that of a 4.5 times larger Sephadex G-75 column (144 X 5 cm). Moreover, the total separation period is 2.5 times shorter for the Sephacryl method. Up to 500 mg or more of crude ribosomal RNA mixtures could be separated via two Sephacryl S-300 columns operated in tandem.