Using affinity capillary electrophoresis to study the interaction of the extracellular binding domain of erythropoietin receptor with peptides.

We have shown that affinity capillary electrophoresis (ACE) can be utilized to screen peptides that bind to the extracellular binding domain of the erythropoietin receptor (EBP). The comparison of the cyclic peptides GGTYSCHFGPLTWVCKPQGG (EMP1) GGTYSCHFGPLTAVCKPQGG (EMP13), and LGRKYSCHFGPLTWVCQPAKKD (EMP37) with the linear peptides HFGPLTWV (EMP26) and FMRF as ACE buffer additives were investigated. When EMP1 and EMP37 were the buffer additives, an abrupt change in the electrophoretic mobility of EBP was observed in the electropherogram. When EMP13, EMP26, and FMRF were examined under identical ACE conditions as EMP1 and EMP37, no significant change in the electrophoretic mobility of EBP was observed. These results correlate well with previously reported IC50 competitive binding data; that is, EMP1 and EMP37 bind to EBP while EMP13 and EMP26 bind very weakly. These observations strongly infer that peptide.EBP dimerization were induced by EMP1, and EMP37 but not by EMP13, EMP26 or FMRF. This ACE method provides a rapid tool for the detection of small peptides or drugs that bind to EBP.     

Behavior ofPseudomonas fluorescens within the hydrodynamic boundary layers of surface microenvironments

Phase, darkfield, and computer-enhanced microscopy were used to observe the surface microenvironment of flow cells during bacterial colonization. Microbial behavior was consistent with the assumptions used previously to derive surface colonization kinetics and to calculate surface growth and attachment rates from cell number and distribution. Surface microcolonies consisted of closely packed cells. Each colony contained 2ⁿ cells, where n is the number of cell divisions following attachment. Initially, cells were freely motile while attached, performing circular looping movements within the plane of the solid-liquid interface. Subsequently, cells attached apically, maintained a fixed position on the surface, and rotated. This type of attachment was reversible and did not necessarily lead to the formation of microcolonies. Cells became irreversibly attached by progressing from apical to longitudinal attachment. Longitudinally attached cells increased in length, then divided, separated, moved apart laterally, and slid next to one another. This resulted in tight cell packing and permitted simultaneous growth and adherence. After approximately 4 generations, individual cells emigrated from developing microcolonies to recolonize the surface at new locations. Surface colonization byPseudomonas fluorescens can thus be subdivided into the following sequential colonization phases: motile attachment phase, reversible attachment phase, irreversible attachment phase, growth phase, and recolonization phase.     

Temporal expression of different pathways of 1-arginine metabolism in healing wounds

Arginine can be metabolized by inflammatory cells through at least two pathways. One is an oxidative l-arginine deiminase (OAD) that results in the formation of citrulline and reactive nitrogen intermediates. The other is arginase, which determines the production of ornithine and urea. The temporal expression of these pathways in an experimental wound model (s.c. implanted polyvinyl alcohol sponges in the rat) was investigated by examining the concentrations of amino acids and of nitrite in fluids obtained from the sponges 6 h to 15 day after implantation. These analyses revealed two distinct periods during which the arginine concentration in the fluids was markedly below plasma levels. During the early period (less than 3 days after sponge implantation) wound fluid contained more citrulline and nitrite than at any other time, suggesting OAD activity. In contrast, ornithine accumulated in the fluids during the late decrease in arginine concentration that extended beyond day 3, during which time the wound fluid also contained a high arginase activity. This time-dependent expression of different pathways of arginine metabolism in wounds was confirmed in sponge cultures containing [guanido-14C]-l-arginine. Cells contained in sponges harvested less than 48 h after implantation metabolized labeled arginine mainly to labeled citrulline, whereas labeled urea was produced during culture of sponges harvested after this time. The low arginine content of wound fluid did not appear to be rate limiting for the expression of OAD in late sponges because no OAD activity was evidenced when 4 mM arginine was added to the cultures. These results indicate that the OAD pathway is expressed in this model predominantly during the early, polymorphonuclear leukocyte-predominant, phase of repair. At this time, the reactive nitrogen intermediates resulting from the metabolism of arginine may mediate some of the events characteristic of early inflammation, including microbiostasis, vasodilation, and inhibition/reversal of platelet aggregation. In turn, the late suppression of this pathway and the catabolism of arginine through arginase may promote macrophage function within wounds.     

Bioavailability of organic and inorganic phosphates adsorbed on short-range ordered aluminum precipitate

A nonreductive community-level study of P availability was conducted using various forms of adsorbed P. Orthophosphate (Pi), inositol hexaphosphate (IHP), and glucose 6-phosphate (G6P) were adsorbed to a short-range ordered Al precipitate. These bound phosphates provided a P source sufficient to support the growth of microbial communities from acidic Brazilian soils (oxisols). Adsorbed IHP, the most abundant form of organic phosphate in most soils, had the lowest bioavailability among the three phosphates studied. Adsorbed G6P and Pi were almost equally available. The amount of adsorbed Pi (1 cmol P kg^–1) required to support microbial growth was at least 30 times less than that of IHP (30 cmol P kg^–1). With increased surface coverage, adsorbed IHP became more bioavailable. This availability was attributed to a change in the structure of surface complexes and presumably resulted from the decreased number of high-affinity surface sites remaining at high levels of coverage. It thus appears that the bioavailability of various forms of adsorbed phosphate was determined primarily by the stability of the phosphate-surface complexes that they formed, rather than by the total amount of phosphate adsorbed. IHP, having the potential to form stable multiple-ring complexes, had the highest surface affinity and the lowest bioavailability. Bioaggregates consisting of bacteria and Al precipitate were observed and may be necessary for effective release of adsorbed P. Bacteria in the genera Enterobacter and Pseudomonas were the predominate organisms selected during these P-limited enrichments. Correspondence to: C. Shang     

IL-1α and TNFα act synergistically to stimulate production of myeloid colony-stimulating factors by cultured human bone marrow stromal cells and cloned stromal cell strains

Human bone marrow stromal cells repond to stimulation by the monokines IL-1 and TNF by producing colony-stimulating factors such as GM-CSF and G-CSF. In this study we show that IL-1α and TNFα act synergistically to stimulate GM-CSF and G-CSF production by cultured marrow stromal cells. We further show that IL-1α and TNFα synergistically stimulate production of GM-CSF and G-CSF by a clonal stroma-derived cell strain. Although IL-1 and TNF share many of the same biological activities, we show that IL-1α and TNFα have an unequal ability to induce myeloid-CSF production by both cultures, with IL-1α being the more potent inducer. We found that induction by IL-1α and TNFα was independent of cell proliferation. The effect of IL-1α and TNFα on production of the two myeloid-CSFs by the clonal cells was significantly greater than the unfractionated passaged stromal cultures, having the greater effect on G-CSF production. The clonally derived stromal cells constitutively produced colony-stimulating activity, in particular GM-CSF, at levels easily detected by ELISA. These findings show that, in addition to the overlapping and additive activities of IL-1α and TNFα, they can interact synergistically. Our findings further suggest that a small subpopulation of stroma cells may be the major producer of G-CSF in the marrow microenvironment during immune response. © 1994 wiley-Liss, Inc.     

Ultraviolet-B Radiation-induced Inhibition of Leaf Expansion and Promotion of Anthocyanin Production: Lack of Involvement of the Low Irradiance Phytochrome System

Leaf discs from expanding leaves of Rumex patientia L. were exposed to 7 hours of visible plus different levels of ultraviolet radiation in the 290 to 315 nm waveband (UV-B) and then placed in darkness. Leaf disc expansion was reduced and anthocyanin production was increased in discs exposed to moderate or high levels of UV-B radiation when compared to control discs. The possibility that the inhibition of leaf expansion by UV-B radiation might be at least partially phytochrome-mediated was examined by giving discs brief red or far red irradiation following exposure to UV-B radiation. Brief red radiation (R) following treatment with moderate or high UV-B radiation did not alter the pattern of growth or anthocyanin production compared to discs placed in darkness following UV-B treatment. However, a posttreatment with far red radiation (FR) reduced the growth of discs subjected previously to either moderate UV-B or no UV-B irradiation to the level of growth of discs given high UV-B. FR posttreatment also decreased anthocyanin production in discs in moderate and high UV-B treatments. Effects of FR and UV-B radiation apparently do not involve the same mechanism. This was demonstrated by experiments in which FR following the UV-B treatments was in turn followed by R, which reversed the effects of the FR but did not alter the growth inhibition or increased anthocyanin production induced by moderate or high levels of UV-B radiation.     

Carbon isotope ratios of soil organic matter and their use in assessing community composition changes in Curlew Valley,Utah

Summary Stable carbon isotope ratios of roots and soil organic matter were measured in Curlew Valley, Utah to determine if changes in the relative dominance of two shrub species had occurred in this salt-desert community. Measurements were made on soil cores along transects stretching from monospecific stands of Ceratoides lanata, a C₃ shrub, to monospecific stands of Atriplex confertifolia, a C₄ shrub. ¹³C values of roots and soil organic matter under Ceratoides cover appeared to be in equilibrium with the current plant community. By contrast, ¹³C values of roots and soils under Atriplex portions of the transects were more negative than would be expected for a C₄-dominated community. These results indicate that a change in relative C₃/C₄ dominance has occurred, and suggest that the C₄ shrub Atriplex confertifolia is increasing in importance in this salt-desert community.     

Induction of microvillous fusion and pinching-off by concanavalin A

Concanavalin A (Con A) caused dramatic changes in the structure and function of rat colonic epithelium. The morphological effect was a pronounced, permanent alteration of the microvilli, including fusion and blebbing. The functional change involved an increased permeability to passively transported hydrophilic marker compounds. The functional change was transient in nature. Since coadministration of glucose or mannose inhibited the effect of Con A, the mechanistic effect of Con A probably involves binding to saccharide components of the membrane surface.