The location of proteins labeled by the 125I-lactoperoxidase system in the NIL 8 hamster fibroblast.

NIL 8 hamster fibroblast cells were labeled by lactoperoxidase-catalyzed iodination. Their membranes were fractionated by sedimentation-rate and isopycnic zonal centrifugation. All the iodinated proteins except the very prominently labeled high molecular weight protein (greater than 200,000 daltons) were located in a fraction identified enzymically and compositionally as plasma membrane. The high molecular weight protein that was previously shown to be sensitive to virus transformation (Hynes, 1973) is concentrated in a very high density particle (rho equals 1.253-1.259) which contains mainly carbohydrate and protein and very low levels of lipid. 5'-nucleotidase was the only enzyme reproducibly demonstrated in this fraction, and electron micrographs revealed a predominantly amorphous morphology together with a few membraneous structures. The iodine label in this fraction was very sensitive to trypsinization prior to homogenization. All the available evidence indicates that this fraction is derived from the surface coat. Mitochondria, nuclei, and soluble protein were labeled to an insignificant extent. The presence of the iodinated surface proteins associated with the endoplasmic reticulum fraction is discussed in the light of these results.     

Proteolytic activity within lysosomes and turnover of pinocytic vesicles a kinetic analysis

In previous studies, in vitro digestion of [^{1 2 5}I] ribonuclease by lysosomes of mouse kidney was limited because breakdown, which was rapid at first slowed markedly so that most of the labeled protein escaped degradation. We now describe incubation conditions which allow digestion to proceed until approximately 70% of the exogenous protein label is released in acid-soluble from, after 30–45 min at 37°C. Such activity is seen with either the addition of EDTA or incubation of concentrated cell particle suspensions. EDTA is effective in low concentrations and shows the same stimulation of digestion over a range of approximately 10⁻⁶−10⁻³ M. Other chelating agents have similar effects; dipyridyl and hydroxquinoline are as effective as EDTA, o-phenanthroline and diethyldithiocarbamate are slightly less effective. When the incubation medium had been treated with a chelating resin, Chelex 100, dilute suspensions of lysosomes were as active as those in EDTA. These results lead to the conclusion that metal ions, present as contaminants in very small concentraions, inhibit the activity of mouse kidney lysosomes.The effect of the metal ions is to diminish lysosomal stability, leading to release of intact labeled ribonuclease in non-sedimentable form. Interaction between lysosomes and metal, leading to inhibition of digestion upon heating occurs at low temperature, but breakdown requires incubation at 37°C and may be autolytic. In contrast to chelators, mercaptoethanol is without marked effect on stability; the stimulation in digestion rate caused by this agent is due either to a direct effect on the lysosomal enzymes or to a non-destructive influence on the lysosomal structure.     

A mechanism of abiotic immobilization of nitrate in forest ecosystems: the ferrous wheel hypothesis

Forest soils, rather than woody biomass, are the dominant long‐term sink for N in forest fertilization studies and, by inference, for N from atmospheric deposition. Recent evidence of significant abiotic immobilization of inorganic‐N in forest humus layers challenges a previously widely held view that microbial processes are the dominant pathways for N immobilization in soil. Understanding the plant, microbial, and abiotic mechanisms of N immobilization in forest soils has important implications for understanding current and future carbon budgets. Abiotic immobilization of nitrate is particularly perplexing because the thermodynamics of nitrate reduction in soils are not generally favorable under oxic conditions. Here we present preliminary evidence for a testable hypothesis that explains abiotic immobilization of nitrate in forest soils. Because iron (and perhaps manganese) plays a key role as a catalyst, with Fe(II) reducing nitrate and reduced forms of carbon then regenerating Fe(II), we call this ‘the ferrous wheel hypothesis’. After nitrate is reduced to nitrite, we hypothesize that nitrite reacts with dissolved organic matter through nitration and nitrosation of aromatic ring structures, thus producing dissolved organic nitrogen (DON). In addition to ignorance about mechanisms of DON production, little is known about DON dynamics in soil and its fate within ecosystems. Evidence from leaching and watershed studies suggests that DON production and consumption may be largely uncoupled from seasonal biological processes, although biological processes ultimately produce the DOC and reducing power that affect DON formation and the entire N cycle. The ferrous wheel hypothesis includes both biological and abiological processes, but the reducing power of plant‐derived organic matter may build up over seasons and years while the abiotic reduction of nitrate and reaction of organic matter with nitrite may occur in a matter of seconds after nitrate enters the soil solution.     

Photosynthetic responses of Eucalyptus nitens (Deane and Maiden) Maiden to green pruning

 Three-year-old Eucalyptus nitens (Deane and Maiden) Maiden trees and 1-year-old ramets of a single clone of E. nitens were pruned to remove 0, 50% or 70% of the green crown length. This was equivalent to removal of 0, 55% or 88% of foliage area of trees, and 0, 77% or 94% of foliage area of ramets. CO₂ assimilation (A) and stomatal conductance (g_s) were measured at constant illumination in five height zones and three foliage-age classes of trees over a 16-month period following pruning. Foliar nitrogen (N) and phosphorus (P) concentrations were determined for each measurement time during the first 12 months of the experiment. In ramets A and g_s were measured in four height zones and two foliage-age classes over a six-week period, and N and P concentrations were measured only once, at the end of the experiment. Rates of A increased by up to 175% following pruning. This response occurred throughout the canopy irrespective of position in the crown or foliage age. The magnitude of the response was generally greater in ramets than in trees, and increased with increasing severity of pruning. The initiation of the response was later, and the duration of the response was longer, in trees than ramets. In the lower crown of trees there was evidence of delayed senescence following pruning. Photosynthetic enhancement was not related to changes in foliar N concentrations. The ratio of A/N increased in many zones following pruning, especially after more severe defoliation. There was no evidence that changes in P concentrations were responsible for the result. The increases in A may have been related to changes in g_s, as maximum values of g_s were greater, and the ratio of A/g_s was generally lower, in pruned than unpruned plants. Received: 31 December 1996 / Accepted: 19 August 1997     

Methods for simultaneous measurement of apoptosis and cell surface phenotype of epithelial cells in effusions by flow cytometry

We describe here a protocol for the detection of epithelial cells in effusions combined with quantification of apoptosis by flow cytometry (FCM). The procedure described consists of the following stages: culturing and induction of apoptosis by staurosporine in control ovarian carcinoma cell lines (SKOV-3 and OVCAR-8); preparation of effusion specimens and cell lines for staining; staining of cancer cells in effusions and cell lines for cell surface markers (Ber-EP4, EpCAM and CD45) and intracellular/nuclear markers of apoptosis (cleaved caspase-3 and caspase-8, and incorporated deoxyuridine triphosphates); and FCM analysis of stained cell lines and effusions. This protocol identifies a specific cell population in cytologically heterogeneous clinical specimens and applies two methods to measure different aspects of apoptosis in the cell population of interest. The cleaved caspase and deoxyuridine triphosphate incorporation FCM assays are run in parallel and require (including sample preparation, staining, instrument adjustment and data acquisition) 8 h. The culturing of cell lines requires 2-3 days and induction of apoptosis requires 16 h.     

The age of fine-root carbon in three forests of the eastern United States measured by radiocarbon

Using a new approach involving one-time measurements of radiocarbon (¹⁴C) in fine (<2 mm diameter) root tissues we have directly measured the mean age of fine-root carbon. We find that the carbon making up the standing stock of fine roots in deciduous and coniferous forests of the eastern United States has a mean age of 3-18 years for live fine roots, 10-18 years for dead fine roots, and 3-18 years for mixed live+dead fine roots. These ¹⁴C-derived mean ages represent the time C was stored in the plant before being allocated for root growth, plus the average lifespan (for live roots), plus the average time for the root to decompose (for dead roots and mixtures). Comparison of the ¹⁴C content of roots known to have grown within 1 year with the ¹⁴C of atmospheric CO₂ for the same period shows that root tissues are derived from recently fixed carbon, and the storage time prior to allocation is <2 years and likely <1 year. Fine-root mean ages tend to increase with depth in the soil. Live roots in the organic horizons are made of C fixed 3-8 years ago compared with 11-18 years in the mineral B horizons. The mean age of C in roots increases with root diameter and also is related to branching order. Our results differ dramatically from previous estimates of fine-root mean ages made using mass balance approaches and root-viewing cameras, which generally report life spans (mean ages for live roots) of a few months to 1-2 years. Each method for estimating fine-root dynamics, including this new radiocarbon method, has biases. Root-viewing approaches tend to emphasize more rapidly cycling roots, while radiocarbon ages tend to reflect those components that persist longest in the soil. Our ¹⁴C-derived estimates of long mean ages can be reconciled with faster estimates only if fine-root populations have varying rates of root mortality and decomposition. Our results indicate that a standard definition of fine roots, as those with diameters of <2 mm, is inadequate to determine the most dynamic portion of the root population. Recognition of the variability in fine-root dynamics is necessary to obtain better estimates of belowground C inputs.     

The antiglucocorticoid, cortexolone, fails to promote in vitro activation of cytoplasmic glucocorticoid receptors from the human leukemic cell line CEM-C7

Cortexolone functions as an antiglucocorticoid in the human leukemic cell line CEM-C7, since it blocks the growth inhibition and cell lysis mediated by the potent agonist triamcinolone acetonide (TA). At high concentrations (10(-5) M) cortexolone alone is inactive. The ability of cortexolone to block the TA-mediated biological effects is reflected in its ability (1000-fold molar excess) to effectively block the binding of [3H]TA to the cytoplasmic unactivated form of the receptors eluted from DEAE-cellulose at approx. 180 mM potassium phosphate (KP). Likewise a 1000-fold molar excess of TA inhibits the specific binding of [3H]cortexolone to the unactivated receptors and to a peak which elutes at low salt concentration (35 mM KP) but does not appear to represent activated [3H]cortexolone-receptor complexes. Thermal activation/transformation (25 degrees C for 30 min +/- 10 mM ATP) of the [3H]TA-receptor complexes significantly enhances the subsequent DNA-cellulose binding capacity of these complexes and also results in their elution from DEAE-cellulose at the low salt (50 mM KP) activated position. In contrast, exposure of the cytoplasmic [3H]cortexolone-receptor complexes to identical in vitro activating (transforming) conditions fails to enhance subsequent DNA-cellulose binding capacity or to result in the appropriate shift in DEAE-cellulose elution profile. This inability of [3H]cortexolone to facilitate activation/transformation of receptors was also verified using cytosol prepared from the glucocorticoid-resistant 'activation-labile' mutant, 3R7. Taken collectively the data suggest that cortexolone, unlike an agonist such as TA, fails to promote in vitro activation/transformation, a conformational change which also occurs in vivo under physiological conditions and is a prerequisite for nuclear binding.     

Molecular mechanisms for environmentally induced and evolutionarily rapid redistribution (plasticity) of meiotic recombination

It has long been known (circa 1917) that environmental conditions, as well as speciation, can affect dramatically the frequency distribution of Spo11/Rec12-dependent meiotic recombination. Here, by analyzing DNA sequence-dependent meiotic recombination hotspots in the fission yeast Schizosaccharomyces pombe, we reveal a molecular basis for these phenomena. The impacts of changing environmental conditions (temperature, nutrients, and osmolarity) on local rates of recombination are mediated directly by DNA site-dependent hotspots (M26, CCAAT, and Oligo-C). This control is exerted through environmental condition-responsive signal transduction networks (involving Atf1, Pcr1, Php2, Php3, Php5, and Rst2). Strikingly, individual hotspots modulate rates of recombination over a very broad dynamic range in response to changing conditions. They can range from being quiescent to being highly proficient at promoting activity of the basal recombination machinery (Spo11/Rec12 complex). Moreover, each different class of hotspot functions as an independently controlled rheostat; a condition that increases the activity of one class can decrease the activity of another class. Together, the independent modulation of recombination rates by each different class of DNA site-dependent hotspots (of which there are many) provides a molecular mechanism for highly dynamic, large-scale changes in the global frequency distribution of meiotic recombination. Because hotspot-activating DNA sites discovered in fission yeast are conserved functionally in other species, this process can also explain the previously enigmatic, Prdm9-independent, evolutionarily rapid changes in hotspot usage between closely related species, subspecies, and isolated populations of the same species.