Egfr signaling controls the size of the stem cell precursor pool in the Drosophila ovary

In many animals, germline progenitors are kept undifferentiated to give rise to germline stem cells (GSCs), enabling continuous production of gametes throughout animal life. In the Drosophila ovary, GSCs arise from a subset of primordial germ cells (PGCs) that stay undifferentiated even after gametogenesis has started. How a certain population of PGCs is protected against differentiation, and the significance of its regulatory mechanisms on GSC establishment remain elusive. Here we show that epidermal growth factor receptor (Egfr) signaling in somatic stromal intermingled cells (ICs), activated by its ligand produced in germ cells, controls the size of the PGC pool at the onset of gametogenesis. Egfr signaling in ICs limits the number of cells that express the heparan sulfate proteoglycan Dally, which is required for the movement and stability of the locally-produced stromal morphogen, Decapentaplegic (Dpp, a BMP2/4 homologue). Dpp is received by PGCs and maintains them in an undifferentiated state. Altering Egfr signaling levels changes the size of the PGC pool and affects the number of GSCs established during development. While excess GSC formation is compensated by the adult stage, insufficient GSC formation can lead to adult ovarioles that completely lack GSCs, suggesting that ensuring an absolute size of the PGC pool is crucial for the GSC system.     

The xanthophyll cycle pool size controls the kinetics of non-photochemical quenching in Arabidopsis thaliana

Arabidopsis plants overexpressing beta-carotene hydroxylase 1 accumulate over double the amount of zeaxanthin present in wild-type plants. The final amplitude of non-photochemical quenching (NPQ) was found to be the same in these plants, but the kinetics were different. The formation and relaxation of NPQ consistently correlated with the de-epoxidation state of the xanthophyll cycle pool and not the amount of zeaxanthin. These data indicate that zeaxanthin and violaxanthin antagonistically regulate the switch between the light harvesting and photoprotective modes of the light harvesting system and show that control of the xanthophyll cycle pool size is necessary to optimize the kinetics of NPQ.     

Monocyte kinetics: observations after pulse labeling

Because monocytes and their precursors cannot be recognized with certainty in tissues, an approach to the study of monocyte kinetics was made through examination of the peripheral blood. Injection of a single pulse of tritiated thymidine into rats resulted in the appearance of labeled monocytes identified as circulating peroxidase-positive mononuclear cells. The increase in the percent of labeled cells and in the mean grain count per cell followed a course described by a mathematical model with a generation time of 21 hours and a DNA synthesis time of 12.5 hours. The generation and synthesis times appear to be very uniform for the monocyte so that the phasing of cells represented by the uptake of label could be followed for more than two generations, a property not shared by neutrophils or lymphocytes. Monocytes appear in the circulation within eight hours of DNA synthesis.     

Variation in precursor pool size during the division cycle of Escherichia coli: further evidence for linear cell growth.

The magnitudes of several pools of radioactively labeled precursors for RNA and protein synthesis were determined as a function of cell age during the division cycle of Escherichia coli 15 THU. Uracil, histidine, and methionine pools increased from low initial values for cells at birth to maxima during midcycle and then subsided again. These pools were small or nonexistent at the beginning and the end of the cycle, and their average values during the cycle were less than 4% of the total cellular radioactivity. The results are consistent with a linear pattern of growth for cells during the division cycle and provide strong evidence against exponential or bilinear growth of E. coli cells.     

A one-pot analysis approach to simplify measurements of protein stability and folding kinetics

To achieve a good understanding of the characteristics of a protein, it is important to study its stability and folding kinetics. Investigations of protein stability have been recently applied to drug-target identification, drug screening, and proteomic studies. The efficiency of the experiments performed to study protein stability and folding kinetics is now a crucial factor that needs to be optimized for these potential applications. However, the standard procedures used to carry out these experiments are usually complicated and time consuming. Large number of measurements is the bottleneck that limits the application of protein folding to large-scale experiments. To overcome this limitation, we developed a method denoted as “one-pot analysis” which is based on taking a single measurement from a mixture of samples rather than from every sample. We combined one-pot analysis with pulse proteolysis to determine the effects of the binding of maltose to maltose-binding protein on the protein folding properties. After carrying out a simple optimization, we demonstrated that protein stability or unfolding kinetics could be measured accurately with just one detection measurement. We then further applied the optimized conditions to cellular thermal shift assay (CETSA). Combining one-pot analysis with CETSA led to a successful determination of the effects of the binding of methotrexate to dihydrofolate reductase in HCT116 cancer cells. Our results demonstrated the applicability of one-pot analysis to energetics-based methods for studying protein folding. We expect the combination of one-pot analysis and energetics-based methods to significantly benefit studies such as drug-target identification, proteomic investigations, and drug screening.     

Global combined precursor isotopic labeling and isobaric tagging (cPILOT) approach with selective MS3 acquisition

Recently, we reported a novel proteomics quantitation scheme termed “combined precursor isotopic labeling and isobaric tagging (cPILOT)” that allows for the identification and quantitation of nitrated peptides in as many as 12–16 samples in a single experiment. cPILOT offers enhanced multiplexing and posttranslational modification specificity, however excludes global quantitation for all peptides present in a mixture and underestimates reporter ion ratios similar to other isobaric tagging methods due to precursor co‐isolation. Here, we present a novel chemical workflow for cPILOT that can be used for global tagging of all peptides in a mixture. Specifically, through low pH precursor dimethylation of tryptic or LysC peptides followed by high pH tandem mass tags, the same reporter ion can be used twice in a single experiment. Also, to improve triple‐stage mass spectrometry (MS³) data acquisition, a selective MS³ method that focuses on product selection of the y₁ fragment of lysine‐terminated peptides is incorporated into the workflow. This novel cPILOT workflow has potential for global peptide quantitation that could lead to enhanced sample multiplexing and increase the number of quantifiable spectra obtained from MS³ acquisition methods.     

Cultured human fibroblasts contain a large pool of precursor beta 1-integrin but lack an intracellular pool of mature subunit

Previous work has shown the presence of an important intracellular pool of beta 1-integrin subunit in human skin fibroblasts as detected with monoclonal antibody DH12 [De Strooper, B., Van der Schueren, B., Jaspers, M., Saison, M., Spaepen, M., Van Leuven, F., Van den Berghe, H. & Cassiman, J. J. (1989) J. Histochem. Cytochem. 37,299-307]. To analyze this more quantitatively, a radioimmunoassay with radioiodinated monoclonal antibody was developed. The total amount of specific binding sites for monoclonal antibody DH12 on skin fibroblasts was between 0.8-1.5 x 10(6)/cell. After permeabilizing the cells with digitonin, a threefold increase in specific binding was observed, which suggested that about 60% of the total amount of beta 1-subunit was localized intracellularly. From pulse/chase experiments, it was deduced that an important pool of precursor subunit, as defined by its sensitivity to endoglycosidase treatment, existed in fibroblasts. Since in steady-state-labeling conditions, at least three to four times more precursor than mature subunit was immunoprecipitated with monoclonal antibody DH12, we suggested that the intracellular pool of beta 1-integrin subunit is mainly precursor pool. This precursor pool contains a degradation compartment and a maturation compartment. Other investigators have found evidence for a recirculating pool of mature integrin in Chinese hamster ovary cells. Therefore, the presence of a recirculating pool of integrin in human fibroblasts was also considered. The data obtained with mAb DH12 showed that less than 10% of the surface pool of integrin was internalized by endocytosis. Since, however, cross linking of beta 1-integrins with polyclonal antibodies leads to rapid endocytosis of most of the integrin, it remains possible that the quantitatively small effect was actually an artefact induced by the divalent mAb. We conclude that the intracellular pool of beta 1-integrins observed in our previous studies consists of precursor and that in skin fibroblasts no mature beta 1-integrin is available intracellularly for rapid quantitative modulations at the cell surface.     

Centrosome size: scaling without measuring

Centrosome size is controlled by a limiting component mechanism in which a fixed quantity of precursor protein is divided up among however many centrosomes are present. This simple scheme explains size control and scaling of centrosomes relative to cell volume.     

Species pool size and invasibility of island communities: a null model of sampling effects

The success of alien species on oceanic islands is considered to be one of the classic observed patterns in ecology. Explanations for this pattern are based on lower species richness on islands and the lower resistance of species‐poor communities to invaders, but this argument needs re‐examination. The important difference between islands and mainland is in the size of species pools, not in local species richness; invasibility of islands should therefore be addressed in terms of differences in species pools. Here I examine whether differences in species pools can affect invasibility in a lottery model with pools of identical native and exotic species. While in a neutral model with all species identical, invasibility does not depend on the species pool, a model with non‐zero variation in population growth rates predicts higher invasibility of communities of smaller pools. This is because of species sampling; drawing species from larger pools increases the probability that an assemblage will include fast growing species. Such assemblages are more likely to exclude random invaders. This constitutes a mechanism through which smaller species pools (such as those of isolated islands) can directly underlie differences in invasibility.     

Kinetics of labeling of the S-adenosylmethionine pool of Saccharomyces cerevisiae.

A method has been developed for studying the specific activity of the pool of S-adenosylmethionine in yeast. The pool reaches half-maximal specific activity within 30 s after the addition of [methyl-3H]methionine. After addition of an excess of nonradioactive methionine, the specific activity of S-adenosylmethionine is reduced by half within 20 s. During that period there is a substantial expansion of the pool. A logarithmically growing cell in synthetic medium contains about 2 X 10(6) molecules of S-adenosylmethionine, of which only 10% is used for the methylation of ribonucleic acid molecules.     

The kinetics of periodate oxidation of carbohydrates: a calorimetric approach

A calorimetric approach is described for analysing the kinetics of periodate oxidation on a series of monosaccharidic substrates. Rate constants at several temperatures were calculated from the calorimetric decay curves that are proportional to the rate of conversion. Arrhenius plots provided the activation parameters for the various carbohydrates and a linear correlation was found between the values of enthalpy and entropy of activation. The dependence of the values of kinetic rates on stereochemistry is interpreted in terms of conformational probability of the reactive state. The suitability of the calorimetric method to track the kinetic process of slow reactions is emphasised, in particular its ability to monitor, directly and continuously, the course of the reaction.     

Determination of antibody-hapten association kinetics: a simplified experimental approach.

A simplified method has been developed for the determination of antibody-hapten association kinetics that permits the study of high affinity interactions with second order forward rate constants of the order of 10-7 to 10-8 M-1 sec-1. Use of tritiated haptens of high specific activity and antibodies of high affinity allows reactions to be run at initial hapten and antibody concentrations of the order of 10-9 to 10-10M, well below the level at which mixing becomes the rate-limiting step. Separation of antibody-bound from free hapten by the use of dextran-coated charcoal can be carried out with sufficient rapidity (2 sec) that the systems under investigation are not appreciably disturbed. With this technique, the association of 3-H-ouabain with rabbit ouabain-specific antibody was found to occur with a rate constant of 0.8 times 10-7 M-1 sec-1, similar to association rates of dye haptens with antibodies of substantially lower affinity. The ratio of this association rate constant to the independently determined dissociation rate constant was 5.4 times 10-9 M-1, in satisfactory agreement with a ko value of 3.5 times 10-9 M-1 determined by Sips analysis of data obtained under equilibrium conditions. This approach should be applicable to the direct kinetic assessment of numerous high affinity antibody-hapten systems of current interest.     

Gas exchange measurements required to predict the newly fixed carbon pool size in a source leaf of corn (Zea mays L.)

Abstract. Steady-state photosynthesis (P_n), post-illumination CO₂ release rates (R), sucrose-phosphate synthase (SPS) activities, and levels of starch, sucrose and hexoses were measured in the source leaf of corn ( Zea mays L.) during a 16-h photoperiod at 800 μmol m ² s ¹. P_n and SPS activity remained constant. Carbohydrate pools increased at a linear rate, except the first and last hour of the photoperiod. Both the CO₂ evolution rate at the moment of light removal (R_max) and SPS activity decreased by one half after the onset of darkness (0 60 min). Sucrose diminished during this period by 40%, whereas the starch remained constant. Thereafter, starch mobilization began, followed by a gradual decline in leaf respiration. The average dark export rate was calculated to be 60% less than that during the day. Maintenance respiration (R_m) of an attached leaf after 48 h darkness was determined. Plants were illuminated for different intervals (e.g. 5, 10 or 20 min), each followed by dark periods sufficient for respiration to decline to R_m. The ratio of C assimilated in light to that released in dark was 6:1. After the 48-h dark period, the minimal period of illumination (T_min) required to restore P_n and R_max to the original level was determined. A mathematical analysis of the kinetics involved in the recovery of P_n and R_max provided an estimate of turnover time (0.22h) and pool size 9.15 mmol m ²) for the newly fixed carbon.     

Flow cytometry measurements of human chromosome kinetochore labeling

A method for the preparation and measurement of immunofluorescent human chromosome centromeres in suspension is described using CREST antibodies, which bind to the centromeric region of chromosomes. Fluorescein isothiocyanate (FITC)-conjugated antihuman antibodies provide the fluorescent label. Labeled chromosomes are examined on microscope slides and by flow cytometry. In both cases a dye which binds to DNA is added to provide identification of the chromosome groups. Sera from different CREST patients vary in their ability to bind to chromosome arms in addition to the centromeric region. Flow cytometry and microfluorimetry measurements have shown that with a given CREST serum the differences in kinetochore fluorescence between chromosomes are only minor. Flow cytometry experiments to relate the number of dicentric chromosomes, induced by in vitro radiation of peripheral blood cells to the slightly increased number of chromosomes with above-average kinetochore fluorescence did not produce decisive radiation dosimetry results.     

China's wetland soil organic carbon pool: New estimation on pool size,change, and trajectory

Robust estimates of wetland soil organic carbon (SOC) pools are critical to understanding wetland carbon dynamics in the global carbon cycle. However, previous estimates were highly variable and uncertain, due likely to the data sources and method used. Here we used machine learning method to estimate SOC storage and their changes over time in China's wetlands based on wetland SOC density database, associated geospatial environmental data, and recently published wetland maps. We built a database of wetland SOC density in China that contains 809 samples from 181 published studies collected over the last 20 years as presented in the published literature. All samples were extended and standardized to a 1-m depth, on the basis of the relationship between SOC density data from soil profiles of different depths. We used three different machine learning methods to evaluate their robustness in estimating wetland SOC storage and changes in China. The results indicated that random forest model achieved accurate wetland SOC estimation with R² being .65. The results showed that average SOC density of top 1 m in China's wetlands was 25.03 ± 3.11 kg C m⁻² in 2000 and 26.57 ± 3.73 kg C m⁻² in 2020, an increase of 6.15%. SOC storage change from 4.73 ± 0.58 Pg in 2000 to 4.35 ± 0.61 Pg in 2020, a decrease of 8.03%, due to 13.6% decreased in wetland area from 189.12 × 10³ to 162.8 × 10³ km² in 2020, despite the increase in SOC density during the same time period. The carbon accumulation rate was 107.5 ± 12.4 g C m⁻² year⁻¹ since 2000 in wetlands with no area changes. Climate change caused variations in wetland SOC density, and a future warming and drying climate would lead to decreases in wetland SOC storage. Estimates under Shared Socioeconomic Pathway 1-2.6 (low-carbon emissions) suggested that wetland SOC storage in China would not change significantly by 2100, but under Shared Socioeconomic Pathway 5-8.5 (high-carbon emissions), it would decrease significantly by approximately 5.77%. In this study, estimates of wetland SOC storage were optimized from three aspects, including sample database, wetland extent, and estimation method. Our study indicates the importance of using consistent SOC density and extent data in estimating and projecting wetland SOC storage.     

Regulation of the mitochondrial adenine nucleotide pool size

A mechanism by which normal adult rat liver mitochondria may regulate the matrix adenine nucleotide content was studied in vitro. If mitochondria were incubated with 1 mm ATP at 30 ° C in 225 mm sucrose, 2 mm K₂HPO₄, 5 mm MgCl₂, and 10 mm Tris-Cl (pH 7.4), the adenine nucleotide pool size increased at a rate of 0.44 ± 0.02 nmol/mg mitochondrial protein/min. The rate of adenine nucleotide accumulation under these conditions was concentration dependent and specific for ATP or ADP; AMP was not taken up. The rate of net ADP uptake was 50–75% slower than that for ATP. The K_m values for net uptake of ATP and ADP were 2.08 and 0.36 mm, respectively. Adenine nucleotide uptake was stoichiometrically dependent on Mg²⁺ and stimulated by inorganic phosphate. Net uptake was inhibited by n-ethylmaleimide, or mersalyl, but not by n-butylmalonate. Nigericin inhibited net uptake, but valinomycin did not. In the presence of uncouplers, net uptake was not only inhibited, but adenine nucleotide efflux was observed instead. Like uptake, uncoupler-induced efflux of adenine nucleotides was inhibited by mersalyl, indicating that a protein was required for net flux in either direction. Carboxyatractyloside, bongkrekic acid, or respiratory substrates reduced the rate of adenine nucleotide accumulation, however, this did not appear to be a direct inhibition of the transport process, but rather was probably related indirectly to an increase in the matrix $\text{ATP}\text{ADP}$ ratio. The collective properties of the transport mechanism(s) for adenine uptake and efflux were different from those which characterize any of the known transport systems. It is proposed that uptake and efflux operate to regulate the total matrix adenine nucleotide pool size: a constant pool size is maintained if the rates of uptake and efflux are equal. Transient alterations in the relative rates of uptake and efflux may occur in response to hormones or other metabolic signals, to bring about net changes in the pool size.     

Analysis of 3-aminopropionamide: a potential precursor of acrylamide

An analytical method for the analysis of 3-aminopropionamide (3-APA) based on derivatization with dansyl chloride and liquid chromatography/fluorescence detection was developed. We have analysed 3-APA formation in raw potatoes, grown and stored under different condition, green and roasted coffee beans and in freeze dried mixtures of asparagine with sucrose and glucose in molar ratio of 1:0.5, 1:1, and 1:1.5. In potatoes the 3-APA content varied depending on the potato variety. We detected 3-APA in potatoes up to 14 microg/g fresh weight. In the model experiment glucose had a stronger capacity to form 3-APA. The substance was formed at temperatures as low as 130 degrees C. However, in the model experiment with sucrose 3-APA was formed not below 150 degrees C. In heated mixtures with increasing molar ratio of sucrose at 170 degrees C we noticed a decrease of 3-APA and in the same mixtures at 150 degrees C we observed an increase of 3-APA. In coffee 3-APA was not formed, neither in green nor in roasted beans.     

Lipophorin as a yolk precursor in Hyalophora cecropia: uptake kinetics and competition with vitellogenin

Vitellogenic follicles of Hyalophora cecropia were incubated in metabolically radiolabeled, high-density lipophorin isolated from pharate adult hemolymph by KBr density gradient centrifugation. The follicles transferred this probe from the incubation medium to the cortical yolk spheres in the oocyte by an energy-dependent and saturable mechanism. Vitellogenin and high-density lipophorin competed with each other for uptake, and are therefore concentrated by the follicle with a common mechanism. Microvitellin and lipophorin, in contrast, did not compete for uptake. The K(uptake) for the accumulation of high-density lipophorin was substantially higher than the value estimated earlier for vitellogenin (133 microM vs. 18 microM). This relationship helps explain why the shared concentrating mechanism does not deplete the lipid transport capacity of the hemolymph, and how a low vitellogenin: lipophorin molar ratio in the hemolymph yields a high ratio in the mature egg.