The Polymeric State of Actin in the Human Erythrocyte Cytoskeleton

Reports on the polymeric state of actin in the red cell have been diverse. We have used phalloidin to stabilize the actin in erythrocyte ghosts prior to extraction in low ionic strength media. A mild proteolytic digestion and Sepharose 4B gel filtration enable an F-actin polymer to be isolated in pure form [1]. Detailed size analysis of this polymer in a range of experiments suggests that actin exists in the erythrocyte principally as a polymer of 100 nm length composed of 30 monomers in a double helical chain 15 monomers long with an estimated molecular weight of 1.3 × 10⁶ daltons.     

Digitization of sedimentation equilibrium and velocity data for analysis by minicomputer

A procedure is described whereby phosphorylated seryl residues may be unequivocally identified during the sequential degradation of a polypeptide chain by the Edman technique. The phosphoseryl residue, Ser(P), was first converted by treatment with methylamine in dilute alkali to a β-methylaminoalanyl residue which was split from the polypeptide by the degradative procedure as the derived phenylthiohydantoin. This was identified by high-performance liquid chromatography. The procedure was highly effective when the Ser(P) occupied an isolated position in a polypeptide chain but was less so when grouped consecutively with other Ser(P).     

Equilibrium constants under physiological conditions for the reactions of L-phosphoserine phosphatase and pyrophosphate: L-serine phosphotransferase

The observed equilibrium constants (K_obs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg²⁺]. Using Σ and square brackets to indicate total concentrations $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{Σ L-serine][Σ P}{}_{\mathrm{i}}\text{]}\text{Σ L-phosphoserine]H}{}_2\text{O]}\text{, K =}\text{L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O]}$. The value of K_obs has been found to be relatively sensitive to pH. At 38 °C, K⁺] = 0.2 m and free [Mg²⁺] = 0; K_obs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔG_obs⁰ = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H₂O] = 1). The effect of the free [Mg²⁺] on K_obs was relatively slight; at pH 7.0 ([K⁺] = 0.2 m) K_obs = 52.0 m at free [Mg²⁺] = 10^?3, m and 47.8 m at free [Mg²⁺] = 10^?2, m. K_obs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔG_obs⁰ = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H₂O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 10⁴, m [ΔG_obs⁰ = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}\text{[}\text{Σ L-phosphoserine}\text{][}\text{H}{}_2\text{O}\text{]}\text{, K =}\text{[L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O}$. Values of K_obs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg²⁺], being calculated to be 668 [ΔG_obs⁰ = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg²⁺] = 0; 111 [ΔG_obs⁰ = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg²⁺] = 10^?3, m; and 9.1 [ΔG_obs⁰ = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg²⁺] = 10^?2, m). K_obs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of K_obs are 4000 [ΔG_obs⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg²⁺] = 0; and 97.4 [ΔG_obs⁰ = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg²⁺] = 10^?3, m. Combining K_obs for the l-phosphoserine phosphatase reaction with K_obs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of K_obs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ NADH}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{][}\text{Σ}\text{α-}\text{ketoglutarate}\text{]}\text{[Σ d-3-}\text{phosphoglycerate}\text{][Σ NAD}{}^{\mathrm{+}}\text{][Σ L-glutamat0]}$ The value of K_obs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K⁺ as the monovalent cation) is 1.34 × 10^?2, m at free [Mg²⁺] = 0 and 1.27 × 10^?2, m at free [Mg²⁺] = 10^?3, m.     

Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro

Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.     

A plate assay method for the detection of fungal polygalacturonase secretion

Abstract A method for the detection of polygalacturonase activity has been developed using ruthenium red staining of fungal colonies on polygalacturonate- agarose plates. Ruthenium red was shown to penetrate beneath the surface layers of the gel, in the regions surrounding a fungal colony where degradation of polygalacturonate had occurred. Without degradation of polygalacturonate ruthenium red did not penetrate the medium, was restricted to binding to the surface layers and was easily washed off. The medium containing undegraded polygalacturonate was a colourless clear background and areas of polygalacturonate degradation around the colonies were visualised as an intense purple-red halo. The method has been used to screen yeasts and filamentous fungi for polygalacturonase secretion.     

The effect of defoliation intensity and history on photosynthesis, growth and carbon reserves of two conifers with contrasting leaf lifespans and growth habits

The effects of partial defoliation on photosynthesis, whole-seedling carbon allocation, partitioning and growth were studied for two species with contrasting foliar traits. Field-grown seedlings of deciduous Japanese larch ( Larix leptolepis ) and evergreen red pine ( Pinus resinosa ) were defoliated by hand in early summer for 2 consecutive years. In the first year (1990), seedlings were defoliated by removing the distal 0, 25, 50 or 75% of each needle. In the second year (1991), seedlings were defoliated either 0 or 50%, regardless of previous defoliation treatments. Defoliation had little effect on photosynthesis and starch concentration in whole seedlings of either species in the first year. In the second year, photosynthesis increased in both species in response to the 1991 defoliation treatment, and in red pine also increased in response to the 1990 defoliation treatment. Further, in 1991 both larch and pine had decreased whole-seedling total non-structural carbohydrate concentrations in all seedlings that were defoliated at least once over the 2-yr period. This decrease was noted mostly in the starch component of the non-structural carbohydrates, and was similar in both species. In 1991, biomass was similarly decreased in both species in response to 1991 defoliation. Both species showed overcompensation in total and component biomass in seedlings defoliated by 25% in 1990. Overall, the results do not support the widely held belief that evergreen trees are substantially more affected than deciduous trees by defoliation.     

Red oak litter promotes a microarthropod functional group that accelerates its decomposition

The contribution of microarthropod activity to litter decomposition varies widely but can be substantial. Oribatid mites are the most diverse and abundant of the microarthropod groups in forest litter. This experiment was designed to examine the effect of litter type and complexity on the diversity and species composition of oribatid mites, and to test whether alterations in species composition due to litter type affected litter decomposition. In an array of plots on a mixed-hardwood site in the mountains of North Carolina, I exposed microarthropod assemblages to a range of litter types: yellow birch, sugar maple, red oak and two mixed litters. Over several years, the litter types selected oribatid mite assemblages of different species composition. By comparing the decomposition of consecutive cohorts of litter, it was possible to detect differences in decomposition accompanying the shifts in the assemblage. A comparison of the mass loss rates between the two litter cohorts over eighteen months reveals similar trajectories for four litter types. In the oak litter, however, the second cohort disappeared significantly faster than the first. In both years, the litters came from the same trees and were nearly identical in initial carbon and nitrogen contents. Since the response was specific to oak litter, it is unlikely that differences in environmental factors are responsible for the faster mass loss of oak. A significant increase of endophagous oribatid mites, those that burrow into plant material, in the second cohort of oak may account for its accelerated decomposition. The woody petioles and thick leaf-planes of oak leaves provide microhabitats for burrowing mites. Endophage activity can accelerate the litter decomposition both through direct comminution of leaf material and by facilitating microbial growth. Because of their low population growth rates, oribatid populations that are reduced by disturbance are slow to recover and by disrupting these non-resilient populations, disturbance may have long-term repercussions for decomposition. This revised version was published online in June 2006 with corrections to the Cover Date.     

Rapid and selective removal of larval erythrocytes from systemic circulation during metamorphosis of the bullfrog, Rana catesbeiana

Mechanisms of hemoglobin transition during bullfrog metamorphosis were investigated by labeling red blood cells from larvae (L-RBC) and from froglets (A-RBC) with a fluorescent dye, PKH26. The life span of the labeled L-RBC in systemic circulation was significantly shorter when they were injected into the animals at the metamorphic climax, compared to injection into pre- or postmetamorphic animals. The A-RBC had a long life span regardless of the metamorphic stage of the recipient animal. Therefore, L-RBC were selectively removed from the systemic circulation at the time of metamorphic climax. During climax, the labeled L-RBC were ingested by hepatic and splenic macrophages, indicating that macrophages are involved in the specific elimination of L-RBC.     

Differential response of four tree species to ozone-induced acceleration of foliar senescence

Ozone (O₃)-induced accelerated senescence of leaves was measured in four tree species: black cherry ( Prunus serotina ), hybrid poplar ( Populus maximowizii x trichocarpa , clone 245), northern red oak ( Quercus rubra ) and sugar maple ( Acer saccharum ). Seedlings or ramets of the four species were subjected to chronic O₃ exposures and designated leaves harvested periodically from emergence to senescence. Gas exchange was analysed, and concentrations of total soluble protein and ribulose-1,5-bisphosphate carboxylase/oxygenase were measured as indicators of leaf senescence. Total antioxidant potential and ascorbate peroxidase and glutathione reductase activities also were determined. Black cherry and hybrid poplar exhibited O₃-induced accelerated leaf senescence, whereas sugar maple and northern red oak did not. When the O₃ effects were related to cumulative uptake of the gas, black cherry was the most sensitive of the four species. Although hybrid poplar exhibited similar symptoms of O₃-induced accelerated senescence after the same exposure period as did black cherry, this species took up much greater quantities of O₃ to achieve the same response. The O₃-induced increase in glutathione reductase activity in hybrid poplar was consistent with the capacity of this species to take up high concentrations of the gas. Relative tolerance of northern red oak and sugar maple could be explained only in part by lower cumulative O₃ uptake and lower rate of uptake. Sugar maple had the highest antioxidant potential of all four species, which may have contributed to O₃ tolerance of this species. Ascorbate peroxidase activity, when expressed on a fresh weight basis, could not account for differential sensitivity among the four species.