Functional and morphological characterization of cultures of Kupffer cells and liver endothelial cells prepared by means of density separation in Percoll,and selective substrate adherence

Summary This paper presents a study on the structure and function of Kupffer cells (KC) and liver endothelial cells (LEC) isolated by a simple and rapid technique involving 1) perfusion of the liver with collagenase; 2) cell separation by means of density centrifugation in Percoll; and 3) cell culture, taking advantage of the fact that KC and LEC differ in their preferences for growth substrate. The KC, which attach and spread under serum-free conditions on surfaces of glass or plastic during the first 15 min in culture exhibit a typical macrophage-like morphology including membrane ruffling and a heterogenous content of vacuoles. Moreover, these cells express (a) Fc receptors (FcR) for binding and phagocytosis of erythrocytes covered with immune globulin G (E-IgG), and (b) complement receptors (CR) for binding and serum dependent phagocytosis of erythrocytes covered with either human C3b or mouse inactivated C3b (iC3b). The cells also bind fluid phase fluoresceinated C3b. Approximately 30% of the KC express immune response-associated (Ia)-antigens.The LEC attach and spread on fibronectin coated surfaces, but not on glass or plastic surfaces, during the first two hours in culture with or without serum, and are morphologically distinct from KC. Cultured LEC are well spread out with no membrane ruffling and with numerous large vesicles surrounding the regularly shaped nucleus. These cells bind, but do not ingest E-IgG via the FcR, but no binding of fluid phase C3b or particle fixed C3b or iC3b can be observed. Incubation of LEC with fluorescein amine conjugates of ovalbumin or formaldehyde treated serum albumin, but not with fluoresceinated native serum albumin, results in accumulation of fluorescence specifically localized in the large perinuclear vesicles. Neither KC nor any other cell types tested have the ability to accumulate fluorescence upon incubation with these compounds. Iaantigens are not present on the LEC.Cytochemical demonstration of unspecific esterase, acid phosphatase, and peroxidase reveals different patterns and intensities of staining in KC as compared to LEC.Abbreviations Used KC Kupffer cells - LEC Liver endothelial cells - C Complement - C3b Major fragment of C3 activation - iC3b C3b that has been cleaved by factor I (C3b inactivator), present in serum - meC3b C3b produced by treating purified human C3 with methyl amine - trC3b C3b produced by treating purified human C3 with trypsin - CR Complement receptors for C3b and iC3b - IgG Immune globulin G - IgM Immune globulin M - E Erythrocytes - E-IgG E covered with anti-E IgG - E-IgM E covered with anti-E IgM - E-C3b(h) E-IgM reacted with purified human C1, C4, oxidized C2 and C3 (E-IgMC14^xyC2C3b) - E-iC3b(m) E-IgM incubated with C5 deficient serum from AKR mice - FcR Receptors for the Fc portion of IgG - FITC Fluorescein isothiocyanate - FITC-meC3b FITC conjugated to meC3b - FITC-trC3b FITC conjugated to trC3b - FA Fluorescein amine - FA-OA Ovalbumin conjugated with FA - FA-SA Serum albumin conjugated with FA - FA-FSA Formaldehyde-treated serum albumin conjugated with FA - Ia Immune response-associated AcE Acid unspecific esterase acting on alpha naphtyl acetate - NASDAE Unspecific esterase acting on naphthol AS-D acetate - NASDCAE Unspecific esterase acting on napthol AS-D chloroacetate     

Growth,acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in Frankia strains Cc1.17 and Cp1.2

A comparative study was conducted on the effect of NH₄Cl on growth, vesicle formation and formation of nitrogenase of Frankia strains Cc1.17 and Cp1.2, derived from root nodules of Colletia cruciata and Comptonia peregrina, respectively. On a medium without combined nitrogen (P-N), both strains formed spherical cells, called vesicles, like many other Frankia strains. Data are presented on the number of vesicles per mg protein, after cultivation in media with sodium propionate as C-source without combined nitrogen (P-N) or with 0.2 g NH₄Cl/l (P+N). Strain Cp1.2 as may other Frankia strains, showed on P+N medium a very strong reduction of vesicle formation of 99% relative to the number of vesicles formed on P-N medium, after 11 days growth. However, in strain Cc11.17 this reduction was only 70%. The occurence of relatively large numbers of vesicles in P+N media has not yet been reported for other Frankia strains. No acetylene reduction activity was found in NH ₄ ⁺ -grown cells. The regulation of induction of nitrogenase in Frankia by NH₄Cl was tested by immuno-gelectrophoresis using antisera against nitrogenase of Rhizobium leguminosarum PRE. The component I of the enzyme showed crossreactivity while the component II had only a weak crossreaction. The experiments indicated that no nitrogenase was detectable in the NH ₄ ⁺ -grown cells. For the localization of nitrogenase, relative amounts of the enzyme were compared in whole cells and vesicle-enriched fractions. Western blots showed a significant enrichment of nitrogenase in the vesicle fractions, which indicated that most of the nitrogenase was localized in the vesicle.     

Some synchorological aspects of basiphilous pine forests in Fennoscandia

Basiphilous pine forests and related birch forests are herb-and grass-rich forests on calcareous substrate. These forests are complex communities with floristic/ecological elements from different vegetation types occurring in a subtle micromosaic. These elements are e.g. species from acidophilous conifer forests, thermophilous forest-rim communities, calcareous shallow-soil and steppe communities, eutrophic wet meadows and fens, and in northern Fennoscandia also species from alpine Dryas heaths. Four associations are recognized in Fennoscandia: Convallario-Pinetum, Melico-Piceetum pinetosum, Peucedano-Pinetum and Epipacto atrorubentis-Betuletum. The main association is the Convallario-Pinetum, a widespread community in Fennoscandia and Estonia with a considerable floristic variation between the different regions. Examples of the floristic variation along west-east profiles and south-north profiles in Fennoscandia are presented. The basiphilous pine forest complex can be divided into a number of ecological types along the moisture and nutritional gradients. A further subdivision into geographical types (races) is presented.Nomenclature follows Lid (1974) for vascular plants, Nyholm (1954–1969) for musci and Dahl & Krog (1973) for lichens.     

Differences in reactivity of confluent and nonconfluent cultures of human endothelial cells toward thrombin-stimulated platelets or heparinized salt solution

Summary This study examined whether nonconfluent endothelial cell cultures reacted differently than confluent ones toward thrombin-stimulated platelets or a heparinized salt solution. The adherence to the endothelial cell cultures of⁵¹Cr-labeled human platelets stimulated at different thrombin concentrations was studied. There was significantly higher adherence of stimulated platelets to nonconfluent cultures compared with confluent ones. This was confirmed by scanning electron microscopy, which also revealed a tendency for the platelets to adhere at the cell periphery. Electron microscopy also showed that thrombin-stimulated platelets induced endothelial cell contraction. Part of the peripheral endothelial cell surface toward the bottom of the culture dish was inverted, facing the lumen of the dish. This phenomenon was particularly seen in nonconfluent cultures. When⁵¹Cr-labeled endothelial cultures were incubated with a mildly injurious fluid as heparinized sodium acetate and 20% serum, at 20° C for 30 min, the nonconfluent cultures showed significantly more cell detachment and release of⁵¹Cr than the confluent ones. We conclude that under the conditions of the present experiments there are differences in the reactivity of confluent and nonconfluent endothelial cell cultures. These differences probably reflect biological dissimilarities. In experiments where properties of cultured endothelium are studied, care should be taken that the degree of confluency is standardized.     

Conjugates of colloidal gold with native and acetylated low density lipoproteins for ultrastructural investigations on receptor-mediated endocytosis by cultured human monocyte-derived macrophages

Summary The morphological aspects of the binding and internalization of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) by cultured human monocyte-derived macrophages were investigated. For this purpose, LDL and AcLDL were conjugated to 20 nm colloidal gold particles. After incubation of the cells with the conjugated lipoproteins at 4° C some LDL-or AcLDL-gold complexes were found to be attached to the cell surface, but without characteristic localization. However, after incubation of the cells at 8° C with either LDL-gold or AcLDL-gold, lipoprotein-gold complexes were present in clusters on the plasma membrane, often in coated pits. Cells incubated at 37° C for various time periods showed internalization of both LDL- and AcLDL-gold complexes via small coated and non-coated vesicles and processing of the complexes in smooth-walled endosomes. When the cells were pulse-chased with LDL- or AcLDL-gold for 30 min at 37° C, the gold conjugates occurred in dense bodies, probably lysosomes. The results suggest that although native and modified LDL are reported to be metabolized differently by macrophages, the morphological aspects of the endocytosis of LDL and AcLDL by cultured human monocyte-derived macrophages are similar.     

Time course localization of immunoglobulin M monoclonal antibody and its fragments in leukemic tumor-bearing mice

Summary In vivo localization of a mouse monoclonal antibody (F₂-10.23 IgM) binding leukemic L 1210 cells was studied in DBA/2 mice bearing an L 1210 tumor. F(ab)₂ fragments were prepared and their specific binding to L 1210 cells was analyzed by flow cytofluorometry. Radiolocalization studies were performed by using ¹²⁵I- or ¹³¹I-labeled IgM monoclonal antibody or its F(ab')₂ fragments to ascertain their capacity to visualize the L 1210 tumor. F(ab)₂ fragments were cleared more rapidly than the whole IgM; the clearance was as fast in healthy as in tumor-bearing mice. The tumor-to-muscle ratio observed 24 h after injection of ¹²⁵I-radiolabeled F(ab)₂ fragments and ¹²⁵I-radiolabeled IgM was 10; the radioactivity level in the blood with F(ab)₂ fragments was lower than with IgM, and so -camera imaging was workable with F(ab)₂ fragments without background substraction. The tumor localization was studied over a period of 5 days by recording the distribution of the iodinated fragments in the tumor-bearing leg compared with that in the normal leg, and by computer analysis of the region of interest. F(ab)₂ fragments gave better results than intact IgM in tumor visualization. Nevertheless, the rapid clearance of this antibody or its F(ab)₂ fragments make them hardly suitable as carriers of toxic drugs. Abbreviations used are: MEM Minimum essential medium; SDS sodium dodecylsulfate; PAGE polyacrylamide gel electrophoresis     

Properties of action potentials carried by divalent cations in identified leech neurons

Properties of divalent cation potentials carried by either Sr2+ or Ca2+ ions in Na+-free, TEA-Ringer solution were characterized in identified neurons of two species of leeches (Macrobdella and Haementeria). In Macrobdella, the overshoot of the potentials varied logarithmically with [Sr2+]0 (28.5 mV per 10-fold change). The overshoot, Vmax, and duration of the potentials increased with increasing divalent cation concentration and saturated at about 20 to 30 mM [Sr2+]0. The Vmax, amplitude, and duration of the potentials were reversibly blocked by Co2+ and Mn2+. The block by Mn2+ could be well-fitted by a reverse Langmuir-curve with an apparent KI of 100 micromolar. The local anesthetic procaine also reversibly inhibited the Vmax and duration of the potentials. The inhibition was greater at alkaline pH suggesting that procaine blocks the calcium channel from inside the membrane. The identified leech neurons examined in Macrobdella varied considerably in their ability to sustain somatic divalent cation potentials. Stimulation of T cells and most motoneurons produced no or only weak potentials, whereas stimulation of Retzius, N, Nut, and AP cells evoked overshooting potentials of several seconds' duration. Stimulation of the ALG cell of Haementeria in normal Ringer solution evoked a slowly-rising, purely Ca2+-dependent potential of approximately 100 ms duration. This response was TTX-resistant, unaffected by complete removal of Na+ from the Ringer solution, and abolished by 1 mM Mn2+. The overshoot varied logarithmically with a slope of 28 mV/decade change in [Ca2+]0.     

Chronobiological aspects of the host-parasite relationships between Biomphalaria glabrata and Schistosoma mansoni: cercarial production and infectivity, and growth kinetics of the host

In the Biomphalaria glabrata-Schistosoma mansoni association, variations in cercarial production, in cercarial infectivity, and in the growth of infected snails are rhythmic. These chronobiological aspects are correlated with the dynamics of the intramolluscan larval stages of the parasite during the course of the infection. Rhythmic variations in the growth kinetics of infected snails are interpreted in terms of host-parasite metabolic exchanges.     

State-structure models of microbial growth

A framework for modelling microbial growth is given in which the biological properties of the regulation of cell growth and proliferation are taken as a basis. Some stages on the way of formulating state-structure models are discussed. The resulting delay-differential equations are both comprehensible and suitable for a mathematical treatment.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).