Morphological evidence for the existence of a more complex cytoskeleton in Amoeba proteus

Summary Various stabilization and extraction procedures were tested to demonstrate the ultrastructural organization of the cytoskeleton in normal, locomoting Amoeba proteus. Most reliable results were obtained after careful fixation in glutaraldehyde/lysine followed by prolonged extraction in a polyethylene glycol/Triton X-100 solution. Before dehydration in a graded series of ethanol and critical-point drying, the amoebae were split by the sandwich-technique, i.e., by mechanical cleavage of cells mounted between two poly-L-lysine-coated glass slides. Platinum-carbon replicas as well as thin sections prepared from such cell fragments revealed a cytoskeleton composed of at least four different types of filaments: (1) 5–7-nm filaments organized as a more or less ordered cortical network at the internal face of the plasma membrane and probably representing F-actin; (2) 10–12-nm filaments running separately or slightly aggregated through the cytoplasm and probably representing intermediate filaments; (3) 24–26-nm filaments forming a loose network and probably representing microtubules; and (4) 2–4-nm filaments as connecting elements between the other cytoskeleton constituents. Whereas microfilaments are responsible for protoplasmic streaming and other motile phenomena, the function of intermediate filaments and cytoplasmic microtubules in amoebae is still obscure.     

Production of Cricotopus ornatus (Meigen) (Diptera: Chironomidae) in Waldsea Lake,Saskatchewan

Cricotopus ornatus was the predominant chironomid in meromictic, saline Waldsea Lake. Annual production of C. ornatus larvae in the mixolimnion was estimated to be 107 mg m^-2 (dry weight) in 1974, 66.5 mg m^-2 in 1975 and 69.5 mg m^-2 in 1976. These estimates are similar to those for chironomids in Canadian arctic lakes and deep-water areas of the Great Lakes. Annual P/B ratios were 5.4 in 1974, 6.8 in 1975 and 6.8 in 1976. These ratios are in the middle of the range reported for chironomids. The major factors limiting chironomid production in Waldsea Lake appear to be: (1) restriction of the habitable zone because of meromixis with accompanying loss of mobile first and second instars that are swept out of the mixolimnion (2) the relatively narrow zone of good C. ornatus habitat, i.e. areas of dense macrophyte or benthic algal growth and (3) predation by nine-spine stickleback and damselfly naiads.     

Studies on the control of mitotic activity

Summary A tetraploid cell population was produced in the primary root meristem of Pisum sativum by one-half hour treatments with various concentrations of colchicine. The tetraploid population so produced was found to be reasonably synchronous in its passage through successive mitotic cycles with the degree of synchrony being more or less proportional to the concentration of colchicine used. The average time between mitoses appears to be of the order of 12 hours which agrees well with previous estimates. Treatments of roots containing tetraploid populations with 2.52 × 10^–5 M. actidione for 15 minutes were used to demonstrate the possibility of using the system for studies on the differential susceptibility of cells at different stages of the mitotic cycle.This work was carried out under contract number RG-4835 of the National Institutes of Health, United States Public Health Service, and an Institutional Grant from the American Cancer Society.Contribution number 59-26 of the Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan.Predoctoral Fellow (CF-9871) of the National Cancer Institute, United States Public Health Service     

Die Photosynthese von Meerespflanzen in ihrer Beziehung Zum Salzgehalt

Zusammenfassung Werden Blattstücke vonPosidonia oceanica oder Thallusteile vonUlva lactuca unter sonst optimalen Assimilationsbedingungen aus Meerwasser in Süßwasser, überführt, so sinkt hier die apparente Assimilation beiPosidonia bis zu Null oder gar zu negativen Werten ab, beiUlva bis zu etwa 23% der Leistung im Meerwasser. Bei Rückführung in Meerwasser wird die Assimilationsrate ebenso schnell wieder erhöht, die Ausgangswerte werden allerdings innerhalb der Versuchszeit — einige 20-min-Perioden — meist nicht in voller Höhe wieder erreicht.Folgt der Wechsel zwischen Meer- und Süßwasser im Wechsel von 20 min, so reagieren die Versuchspflanzen mit der gleichen Präzision wie auf das Aus- und Einschalten einer Lichtquelle.Wird anPosidonia die photosynthetische Rate in verschiedenen Stufen verdünnten Meerwassers (Verdünnung nicht mit dest. Wasser, sondern mit Leitungswasser von bekanntem Bicarbonatgehalt und p_H) bestimmt und in Prozenten der Leistung in Meerwasser ausgedrückt, so ergibt sich die in Abb. 3 dargestellte Kurve. Innerhalb der Versuchszeit scheint jeder Meerwasserkonzentration ein bestimmter Photosynthesewert zugeordnet zu sein, ein Verhalten, das auf das Vorhandensein von Gleichgewichtszuständen hindeutet, die zwischen dem photosynthetischen System und dem Zustand des Außenmediums bestehen.Infolge der überaus raschen Reaktion der Pflanzen auf Änderungen im Außenmedium wird als Arbeitshypothese die Vermutung ausgesprochen, daß an der Änderung der photosynthetischen Rate bei Übertragung in und Rückführung aus Süßwasser in Meerwasser nicht Zustandsveränderungen imZellinneren, sondern Zustände der Membranen verantwortlich sind, welche das Ausmaß der CO₂-Permeation bestimmen und damit einen begrenzenden Faktor der Photosynthese darstellen.Mit 4 Textabbildungen     

Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice

The distribution of human low density lipoprotein (LDL) receptors was studied by immunofluorescence and immunoelectron microscopy in epithelial cells of transgenic mice that express high levels of receptors under control of the metallothionein-I promoter. In hepatocytes and intestinal epithelial cells, the receptors were confined to the basal and basolateral surfaces, respectively. Very few LDL receptors were present in coated pits or intracellular vesicles. In striking contrast, in the epithelium of the renal tubule the receptors were present on the apical (lumenal) surface where they appeared to be concentrated at the base of microvilli and were abundant in vesicles of the endocytic recycling pathway. Intravenously administered LDL colloidal gold conjugates bound to the receptors on hepatocyte microvilli and were slowly internalized, apparently through slow migration into coated pits. We conclude that (a) sorting of LDL receptors to the surface of different epithelial cells varies with each tissue; and (b) in addition to a signal for clustering in coated pits, the LDL receptor may contain a signal for retention in noncoated membrane that is manifest in hepatocytes and intestinal epithelial cells, but not in renal epithelial cells or cultured human fibroblasts.     

Effect of the expression of a hepatocyte-specific MHC molecule in transgenic mice on T cell tolerance

A hybrid murine class I gene, Q10/L, was injected into C3H/HeJ fertilized ova to produce transgenic (TG) mice. This fusion gene contained 414 bp of Q10 promoter sequences which was sufficient to direct liver-specific expression in two lines of animals. Animals from these lines did not have Q10/L mRNA in 10 nonhepatic tissues examined including thymus, spleen, and bone marrow. The ontogeny of Q10/Ld expression in both liver and yolk sac paralleled expression of endogenous Q10. Analysis of liver cells from these lines by flow cytometry and immunofluorescence demonstrated the presence of the Q10/L Ag solely on hepatocytes. TG animals showed no signs of hepatic disease as evidenced by an absence of cellular infiltrates in the liver and a normal profile of serum enzymes that are elevated in association with hepatic disease. When spleen cells from TG animals were cocultured with splenocytes that express Ag cross-reactive with Q10/L, CTL were generated that recognized and lysed L cells which express Q10/L. However, the extent of lysis was less than that generated from non-TG control littermates. That these cross-reactive T cells were physiologically significant was demonstrated by adoptive transfer of in vivo primed T cell enriched spleen cells which produced a mononuclear infiltration of the liver of TG recipients. However, inoculation of Q10/L L cells or splenocytes expressing Q10/L cross-reactive Ag into TG mice did not induce cellular infiltration or overt hepatic disease. Whereas inoculation of normal C3H mice with these cells led to priming of Q10/L reactive CTL, anti-Q10/L CTL could not be primed in TG mice. This suggests that Ag expression solely on hepatocytes can lead to inactivation of specific CTL clones and thus account for the observed in vivo tolerance.     

Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human beta 2m: an animal model of HLA-B27-associated human disorders

Humans who have inherited the human class I major histocompatibility allele HLA-B27 have a markedly increased risk of developing the multi-organ system diseases termed spondyloarthropathies. To investigate the role of B27 in these disorders, we introduced the B27 and human beta 2-microglobulin genes into rats, a species known to be quite susceptible to experimentally induced inflammatory disease. Rats from one transgenic line spontaneously developed inflammatory disease involving the gastrointestinal tract, peripheral and vertebral joints, male genital tract, skin, nails, and heart. This pattern of organ system involvement showed a striking resemblance to the B27-associated human disorders. These results establish that B27 plays a central role in the pathogenesis of the multi-organ system processes of the spondyloarthropathies. Elucidation of the role of B27 should be facilitated by this transgenic model.     

Oxygen transport in the blood of arctic mammals: adaptation to local heterothermia

Summary The oxygen binding of whole blood from humans and two arctic mammals, reindeer and muskox, has been studied as a function of carbon dioxide and temperature. All bloods display a marked Bohr effect with Bohr coefficients in the range –0.44––0.73. The Bohr effect is more pronounced at 20°C. The temperature sensitivity of reindeer and muskox blood expressed by the apparent heat of oxygenation, H, is almost three times lower than that of human HbA under the same experimental conditions. This thermodynamic difference gives special benefits to arctic mammals with large heterothermy by safeguarding oxygen unloading at very low ambient temperatures.     

Sialyl Lewis(x)/E-selectin-mediated rolling in a cell-free system.

Selections mediate transient adhesion of neutrophils to stimulated endothelial cells at sites of inflammation by binding counter-receptors that present carbohydrates such as sialyl Lewis(x). We have developed a cell-free adhesion assay using sialyl Lewis(x)-coated microspheres and E-selection-IgG chimera-coated substrates to investigate the premise that rolling primarily results from functional properties of selection-carbohydrate bonds, whereas cellular morphology and signaling act as secondary effects. Sialyl Lewis(x)-coated microspheres attach to and roll over E-selectin-IgG chimera-coated substrates between the physiological wall shear stresses of 0.7 and 2 dynes/cm2. Rolling velocities vary with time and depend on E-selectin-IgG chimera site density and wall shear stress. Our results show that sialyl Lewis(x) is a minimal functional recognition element required for rolling on E-selectin under flow.     

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).