Avocado fruit protoplasts: a cellular model system for ripening studies

Summary Mesocarp protoplasts were isolated from mature avocado fruits (Persea americana cv. Hass) at varying stages of propylene-induced ripening. Qualitative changes in the pattern of radiolabel incorporation into polypeptides were observed in cells derived from fruit at the different stages. Many of these differences correlate with those observed during radiolabeling of polypeptides from fresh tissue slices prepared from unripe and ripe fruit. Protoplasts isolated from fruit treated with propylene for one day or more were shown to synthesize cellulase (endo-ß-1,4-glucanase) antigen, similar to the intact propylene-treated fruit. These results suggest that the isolated protoplasts retain at least some biochemical characteristics of the parent tissue. The cells may also be used in transient gene expression assays. Protoplasts isolated from preclimacteric and climacteric fruit were equally competent in expressing a chimeric test gene, composed of the CaMV 35S RNA promoter fused to the bacterial chloramphenicol acetyltransferase gene, which was introduced by electroporation.Abbreviations PCM Murashige and Skoog salts and growth factors, supplemented with 3% sucrose, 0.3 % glucose, 0.3% enzymatic casein hydrolysate, 0.5 M mannitol, and 5 mM CaCl₂ - CAT chloramphenicol acetyltransferase     

Endothelial and lipoprotein lipases in human and mouse placenta

Placenta expresses various lipase activities. However, a detailed characterization of the involved genes and proteins is lacking. In this study, we compared the expression of endothelial lipase (EL) and LPL in human term placenta. When placental protein extracts were separated by heparin-Sepharose affinity chromatography, the EL protein eluted as a single peak without detectable phospholipid or triglyceride (TG) lipase activity. The major portion of LPL protein eluted slightly after EL. This peak also had no lipase activity and most likely contained monomeric LPL. Fractions eluting at a higher NaCl concentration contained small amounts of LPL protein (most likely dimeric LPL) and had substantial TG lipase activity. In situ hybridization studies showed EL mRNA expression in syncytiotrophoblasts and endothelial cells and LPL mRNA in syncytiotrophoblasts. In contrast, immunohistochemistry showed EL and LPL protein associated with both cell types. In mouse placentas, lack of LPL expression resulted in increased EL mRNA expression. These results suggest that the cellular expression of EL and LPL in human placenta is different. Nevertheless, the two lipases might have overlapping functions in the mouse placenta. Our data also suggest that the major portions of both proteins are stored in an inactive form in human term placenta.     

The Apolipoprotein M/S1P Axis Controls Triglyceride Metabolism and Brown Fat Activity

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Specific growth rates of heterotrophic plankton organisms in a eutrophic lake during a spring bloom

The in situ growth of the dominating pelagic organisms at severaltrophic levels was investigated during a spring bloom characterizedby well-mixed cold water. The study includes primary productionand the carbon flow through the nano-, micro- and mesozooplanktonpopulations based on population dynamics and specific growthrates. The phytoplankton biomass and production were totallydominated by small algae <20 µm. of which {small tilde}5%were <3µm. potentially a food source for the nano-and microzooplankton. The mean carbon-specific primary productionwas 0.15 day^–1 and was regulated solely by light. Themean volume-based specific growth rate of bacterioplankton wasmodest. 0.1 day^–1. and probably controlled by the lowtemperature. The volume-based specific growth rates of heterotrophicnanoflagellates. ciliates. rotifers and copepods were 0.35.0.13. 0.16 and 0.03 day^–1, respectively. The observedgrowth of the heterotrophic plankton was generally not foodlimited, but was controlled by temperature. The stable temperatureduring the experiment therefore allows a cross-taxonomic comparisonof specific growth rates. The b exponent in the allometric relationship(G = aV^th) between volume-specific growth rate (G) and individualbody size (V) was –0.15 ± 0.03 for all filtratingzooplankton. indicating an in situ scaling not far from thephysiological principles onginally demonstrated for laboratorypopulations.     

The relationship between autophagy and the intracellular degradation of asialoglycoproteins in cultured rat hepatocytes

The relationship between autophagy and the intracellular distribution of endocytosed asialoorosomucoid was studied in cultured rat hepatocytes. Overt autophagy was induced by shifting the cells to a minimal salt medium. Incubation in minimal salt medium led to the formation of buoyant lysosomes at the expense of denser lysosomes manifested as a dual distribution of these organelles in Nycodenz gradients. Asialoorosomucoid was labeled with 125I-tyramine cellobiose. The labeled degradation products formed from this ligand are trapped at the site of degradation and may therefore serve as markers for the subgroup of lysosomes involved in the degradation. In control cells the degradation of the ligand was initiated in a light prelysosomal compartment and continued in denser lysosomes. In cells with high autophagic activity, the degradation of labeled asialoorosomucoid took place exclusively in a buoyant group of lysosomes. These results suggest that degradation of endocytosed ligand takes place in the same secondary lysosomes as substrate sequestered by autophagic mechanisms. These light lysosomes represent a subgroup of active lysosomes which are gradually recruited from dense bodies. Data are also presented that indicate that insulin may prevent the change in buoyant density brought about by incubation in deficient medium.     

Amiloride inhibits constitutive internalization and increases the surface number of epidermal growth factor receptors in intact rat hepatocytes

In previous experiments the surface expression of epidermal growth factor (EGF) receptors in freshly isolated rat hepatocytes varied temperature- and time-dependently and was depleted by monensin and cycloheximide in a way suggesting that a subpopulation of these receptors are subject to constitutive cycling (Gladhaug and Christoffersen; 1988). We here report the finding that pretreatment of the hepatocytes with amiloride exerts marked effects on cellular EGF receptor movements. After 2 h incubation with 1 mM amiloride, the receptor level was approximately 270,000 sites/cell surface vs. 140,000 in the untreated cell, with no change in receptor affinity. Amiloride thus stabilized the surface EGF receptor pool at an elevated level. In cells pretreated with amiloride for 60 min, the relative endocytosis decreased from about 2.6 EGF molecules internalized per receptor during 15 min endocytosis in untreated cells to about 1.5 molecules/receptor in amiloride-treated cells. These results suggest that amiloride causes an accumulation of EGF receptors at the hepatocyte surface due to inhibition of constitutive receptor internalization. In addition, it was found that in amiloride-treated hepatocytes the phorbol ester TPA strongly inhibited high-affinity EGF binding without affecting the total surface receptor number. In control cells, TPA did not consistently affect binding. Pretreatment with amiloride prevented surface EGF receptor depletion induced by cycloheximide and puromycin, but it did not significantly inhibit surface receptor depletion caused by monensin. Although the underlying mechanism of the amiloride effect on intracellular receptor trafficking is not clear, the results provide further evidence for a continuous, ligand-independent EGF receptor cycling pathway in hepatocytes.     

Pelagic food web processes in an oligotrophic lake

Major pelagic carbon pathways, including primary production, release of extracellular products (EOC), bacterial production and zooplankton grazing were measured in oligotrophic Lake Almind (Denmark) and in enclosures (7 m³) subjected to artificial eutrophication. Simultaneous measurements at three days interval of carbon exchange rates and pools allowed the construction of carbon flow scenarios over a nineteen day experimental period.The flow of organic carbon was dominated by phytoplankton EOC release, which amounted from 44 to 58% of the net fixation of inorganic carbon. Gross bacterial production accounted for 33 to 75% of the primary production. The lower values of EOC release (44%) and bacterial production (33%) were found in the enclosures with added nutrients. The release of recently fixed photosynthetic products was the most important source of organic carbon to the bacterioplankton. Uptake of dissolved free amino acids was responsible for 52 to 62% of the gross bacterial production. Thus, amino acids constituted a significant proportion of the EOC. Zooplankton (< 50 µm) grazing on algae and bacteria accounted only for a minor proportion of the particulate production in May. Circumstantial evidence is presented that suggests the chrysophycean alga Dinobryon was the most important bacterial remover.The results clearly demonstrated EOC release and bacterial metabolism to be key processes in pelagic carbon cycling in this oligotrophic lake.     

Changes in hormone responsiveness and cyclic AMP metabolism in rat hepatocytes during primary culture and effects of supplementing the medium with insulin and dexamethasone

Primary monolayer cultures of rat hepatocytes were used for studies of long-term and acute effects of hormones on the cyclic AMP system. When hepatocyte lysates were assayed at various times after plating of the cells three major changes in the metabolism of cyclic AMP and its regulation were observed: Glucagon-sensitive adenylate cyclase activity gradually declined in culture. In contrast, catecholamine-sensitive activity, being very low in normal adult male rat liver and freshly isolated hepatocytes, showed a strong and rapid increase after seeding of the cells. Concomitantly, there was an early elevation (peak approximately equal to 6 h) and a subsequent decrease in activity of both high-Km and low-Km cyclic AMP phosphodiesterase. These enzymic changes probably explained the finding that in intact cultured cells the cyclic AMP response to glucagon was diminished for 2-24 h after seeding, followed by an increase in the responsiveness to glucagon as well as to adrenergic agents up to 48 h of culture. Supplementation of the culture media with dexamethasone and/or insulin influenced the formation and breakdown of cyclic AMP in the hepatocytes. Insulin added at the time of plating moderately increased the adenylate cyclase activity assayed at 48 h, while dexamethasone had no significant effect. In the presence of dexamethasone, insulin exerted a stronger, and dose-dependent (1 pM - 1 microM), elevation of the adenylate cyclase activity in the lysates, particularly of the glucagon responsiveness. Thus, insulin plus dexamethasone counteracted the loss of glucagon-sensitive adenylate cyclase activity occurring in vitro. Kinetic plots of the cyclic AMP phosphodiesterase activity showed three affinity regions for the substrate. Of these, the two with high and intermediate substrate affinity (Km approximately equal to 1 and approximately equal to 10 microM) were decreased in the dexamethasone-treated cells. Insulin partly prevented this effect of dexamethasone. Accumulation of cyclic AMP in intact cells in response to glucagon or beta-adrenergic agents was strongly increased in cultures pretreated with dexamethasone. The results suggest that insulin and glucocorticoids modulate the effects of glucagon and epinephrine on hepatocytes by exerting long-term influences on the cyclic AMP system.