The large intracellular pool of asialoglycoprotein receptors functions during the endocytosis of asialoglycoproteins by isolated rat hepatocytes

The function of intracellular asialoglycoprotein receptors during the endocytosis of asialo-orosomucoid in isolated hepatocytes was assessed by following changes in the occupancy of intracellular receptors. Unoccupied total cellular (inside and surface) or surface receptors were quantified at 0 degrees C by the binding of 125I-asialo-orosomucoid in the presence or absence, respectively, of digitonin. Freshly isolated cells had about 17% of their total receptors on the surface. After incubation at 37 degrees C, the receptor distribution changed to 25 to 50% on the cell surface and 50 to 75% inside the cell. At 37 degrees C, the average total number of receptors/cell was 4.5 x 10(5). Dissociation constants, determined from equilibrium binding studies in the presence or absence of digitonin to assess total or surface receptors, were identical (5.4 +/- 1.4 and 5.6 +/- 1.1 x 10(-9) M, respectively). In the presence of asialo-orosomucoid at 37 degrees C, there was both a time- and a concentration-dependent decrease in surface and intracellular receptor activity. This receptor activity decrease was reversed by removing asialo-orosomucoid from the medium or by washing the digitonin-permeabilized cells with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid prior to quantification of receptor activity. Within 1 to 2 h in the presence of excess asialo-orosomucoid, a steady state was attained in which approximately 70% of the intracellular receptors were occupied. The kinetics of receptor activity recovery on the cell surface after internalization of a pulse of ligand is different than the rate of recovery of internal receptor activity. The results suggest that all of the internal asialoglycoprotein receptors are functional and participate during endocytosis. Internal receptors may be functionally equivalent to those on the surface or they may serve a reservoir or routing function for internalized ligand.     

Effects of α-fluoromethylhistidine (FMH), an irreversible inhibitor of histidine decarboxylase,on development of brain histamine and catecholamine systems in the neonatal rat

Daily administration of FMH to neonatal rats produced long-lasting inhibition of histidine decarboxylase in hypothalamus and cerebral cortex and led to depletion of histamine in both brain regions. The onset of depletion was more rapid in cerebral cortex, a region in which non-neurotransmitter pools of histamine predominate in early postnatal life, appearing as early as postnatal day 3; depletion in the hypothalamus, a region rich in histaminergic neuronal projections, appeared later. No effects were seen on body or brain growth, nor was development of other biogenic amine systems affected. FMH thus provides a selective probe for examining the role of histamine in brain development.     

Hormone-induced changes in the in vitro DNA-binding activity of the chicken progesterone receptor

Previous analyses have indicated that steroid hormone receptors undergo an allosteric change in structure upon binding by the steroid ligand. This structural change was envisioned as an intramolecular unmasking of the protein's DNA-binding domain, thus allowing the receptor to function in gene regulation. We report an analysis of the effect of hormone on the DNA-binding activity of the chicken progesterone receptor. Using an isocratic elution of DNA affinity columns we show that unliganded receptor (aporeceptor) can bind a 23-basepair progesterone response element with high affinity and a high degree of sequence preference. Hormone causes a 1.5-fold increase in affinity for the PRE sequence and a 2-fold decrease in affinity for non-specific DNA. Kinetic analysis of the off-rate of receptor-DNA complexes is consistent with this minor effect of hormone. In addition, gel retardation analysis of receptor-progesterone response element complexes further substantiates that hormone is not required for sequence-specific DNA binding. These results indicate that hormone is not necessary for the progesterone receptor to fold into a conformation that recognizes specific gene regulatory sequences.     

Regulatory elements controlling expression of the Drosophila homeotic gene fork head.

The region-specific homeotic gene fork head (fkh) is expressed and required in a variety of tissues of the developing Drosophila embryo. In order to identify the cis regulatory elements directing the complex spatio-temporal expression pattern of fkh, we have studied the subpatterns directed by defined fragments of fkh genomic DNA. These experiments enabled us to distinguish separate regulatory elements specific for the different expression domains of fkh. In addition, our analysis revealed several unexpected features such as the redundancy of regulatory elements and the overlap of regulatory elements with the transcribed regions of other genes. Moreover, the separation of normally contiguous elements effecting expression in the posterior terminal fkh domain appears to lead to novel expression domains which do not correspond to known developmental units in the embryo.     

Summer drought exposure,stand structure,and soil properties jointly control the growth of European beech along a steep precipitation gradient in northern Germany

Increasing exposure to climate warming-related drought and heat threatens forest vitality in many regions on earth, with the trees' vulnerability likely depending on local climatic aridity, recent climate trends, edaphic conditions, and the drought acclimatization and adaptation of populations. Studies exploring tree species' vulnerability to climate change often have a local focus or model the species' entire distribution range, which hampers the separation of climatic and edaphic drivers of drought and heat vulnerability. We compared recent radial growth trends and the sensitivity of growth to drought and heat in central populations of a widespread and naturally dominant tree species in Europe, European beech (Fagus sylvatica), at 30 forest sites across a steep precipitation gradient (500–850 mm year⁻¹) of short length to assess the species' adaptive potential. Size-standardized basal area increment remained more constant during the period of accelerated warming since the early 1980s in populations with >360 mm growing season precipitation (April–September), while growth trends were negative at sites with <360 mm. Climatic drought in June appeared as the most influential climatic factor affecting radial growth, with a stronger effect at drier sites. A decadal decrease in the climatic water balance of the summer was identified as the most important factor leading to growth decline, which is amplified by higher stem densities. Inter-annual growth variability has increased since the early 1980s, and variability is generally higher at drier and sandier sites. Similarly, within-population growth synchrony is higher at sandier sites and has increased with a decrease in the June climatic water balance. Our results caution against predicting the drought vulnerability of trees solely from climate projections, as soil properties emerged as an important modulating factor. We conclude that beech is facing recent growth decline at drier sites in the centre of its distribution range, driven by climate change-related climate aridification.     

Effects of temperature and nutrients on microscopic stages of the bull kelp (Nereocystis luetkeana,Phaeophyceae)

Warming ocean temperatures have been linked to kelp forest declines worldwide, and elevated temperatures can act synergistically with other local stressors to exacerbate kelp loss. The bull kelp Nereocystis luetkeana is the primary canopy-forming kelp species in the Salish Sea, where it is declining in areas with elevated summer water temperatures and low nutrient concentrations. To determine the interactive effects of these two stressors on microscopic stages of N. luetkeana, we cultured gametophytes and microscopic sporophytes from seven different Salish Sea populations across seven different temperatures (10–22°C) and two nitrogen concentrations. The thermal tolerance of microscopic gametophytes and sporophytes was similar across populations, and high temperatures were more stressful than low nitrogen levels. Additional nitrogen did not improve gametophyte or sporophyte survival at high temperatures. Gametophyte densities were highest between 10 and 16°C and declined sharply at 18°C, and temperatures of 20 and 22°C were lethal. The window for successful sporophyte production was narrower, peaking at 10–14°C. Across all populations, the warmest temperature at which sporophytes were produced was 16 or 18°C, but sporophyte densities were 78% lower at 16°C and 95% lower at 18°C compared to cooler temperatures. In the field, bottom temperatures revealed that the thermal limits of gametophyte growth (18°C) and sporophyte production (16–18°C) were reached during the summer at multiple sites. Prolonged exposure of bull kelp gametophytes to temperatures of 16°C and above could limit reproduction, and therefore recruitment, of adult kelp sporophytes.     

Effects of CO2 enrichment and intraspecific competition on biomass partitioning, nitrogen content and microbial biomass carbon in soil of perennial ryegrass and white clover

Seedlings of perennial ryegrass (Lolium perenne L. cv. Parcour)and white clover (Trifolium repens L. cv. Karina) grown at fivedifferent plant densities were exposed to ambient (390 ppm)and elevated (690 ppm) CO₂ concentrations. After 43 d the effectsof CO₂ enrichment and plant density on growth of shoot and root,nitrogen concentration of tissue, and microbial biomass carbon(C_mic) in soil were determined. CO₂ enrichment of Lolium perenneincreased shoot growth on average by 17% independent of plantdensity, while effects on root biomass ranged between -4% and+ 107% due to an interaction with plant density. Since tilernumber per plant was unaffected by elevated CO₂, the small responseof shoot growth to CO₂ enrichment was atributed to low sinkstrength. A significant correlation between nitrogen concentrationof total plant biomass and root fraction of total plant drymatter, which was not changed by CO₂ enrichment, indicates thatnitrogen status of the plant controls biomass partitioning andthe effect of CO₂ enrichment on root growth. Effects of elevatedCO₂ and plant density on shoot and root growth of Trifoliumrepens were not significantly interacting and mean CO₂-relatedincrease amounted to 29% and 66%, respectively. However, growthenhancement due to elevated CO₂ was strongest when leaf areaindex was lowest. Total amounts of nitrogen in shoots and rootswere bigger at 690 ppm than at 390 ppm CO₂. There was a significantincrease in C_mic in experiments with both species whereas plantdensity had no substantial effect. Key words: CO₂ enrichment, intraspecific competition, biomass partitioning, Lolium perenne, Trifolium repens, grassland     

Identification of a 175 kDa protein as the ligand-binding subunit of the rat liver sinusoidal endothelial cell hyaluronan receptor

The rat liver sinusoidal endothelial cell (LEC) hyaluronan (HA)receptor was previously identified using a photoaffinity HAderivative (J. BioL Chem., 267, 20451–20456, 1992). Twopolypeptides with M_r = 175,000 and 166,000, were consistentlycrosslinked, suggesting that the LEC HA receptor is an oligomer.Whether one or both subunits participate in HA binding, wasnot determined. Here we investigate the HA-subunit interactionsand the potential oligomeric nature of the LEC HA receptor.When Sephacryl-400 gel filtration chromatography was used toenrich the HA receptor, the 175 kDa polypeptide was the majorband seen by SDS-PAGE analysis. Little staining was seen at166 kDa, suggesting that the 175 kDa protein could be separatedfrom the 166 kDa protein and still retain HA-binding activity.A ligand blot assay was used to determine if each individualsubunit could bind HA. LEC proteins were separated by nonreducingSDS-PAGE, and then immobilized onto nitrocellulose. ¹²⁵I-HAbound to a 175 kDa polypeptide but not to the 166 kDa protein.A high molecular weight band of