Tomato annexins p34 and p35 bind to F-actin and display nucleotide phosphodiesterase activity inhibited by phospholipid binding.

Annexins are a family of proteins found in a range of eukaryotic cell types. They share a characteristic amino acid sequence and a Ca(2+)-dependent affinity for specific phospholipids. In plants, proteins with common properties and significant homology with annexins have been identified in a number of species and implicated in diverse cellular functions known to be modulated by Ca2+. This study describes several novel biochemical properties of the tomato annexins p34 and p35 that are relevant to our understanding of their functions in the plant. First, the annexins were found to bind to actin in a calcium- and pH-dependent interaction that was specific for F-actin and not G-actin. Second, an enzyme activity defined as a nucleotide phosphodiesterase activity was found associated with the purified annexin preparation. Selective immunoprecipitation of p34 and p35 strongly suggests that the enzyme activity is a property of the annexins and constitutes 60% of the total soluble activity found in root extracts capable of hydrolyzing free ATP. The substrate specificity of the enzyme within in vitro assays is broad. ATP is the preferred substrate, but nearly identical rates of hydrolysis of GTP and substantial hydrolysis of other nucleotide tri- and diphosphates are observed. The enzyme activity was found to be a property of both p34 and p35, although the specific activity was routinely higher for p34. Third, the enzyme activity of the annexins was not affected by F-actin binding but could be abolished by the specific Ca(2+)-dependent interaction of the annexins with phospholipids. Our results showed that p34 and p35 account for substantial enzyme activity in tomato root cells. This activity was exhibited when the proteins were either in soluble form or attached to actin filaments. Enzyme activity was not exhibited when the annexins were bound to phospholipids. These properties suggest a role for the proteins in mediating Ca(2+)-dependent events involving interactions of the cytoskeleton and cellular membranes.     

Biomarker studies on microbial carbonates: Extractable lipids of a Calcifying Cyanobacterial mat (Everglades, USA)

Summary Biomarker investigations are applied to the free lipid fractions of a naturally grown freshwater microbial mat, constructed by calcifying cyanobacteria (Scytonema sp. andSchizothrix sp.). The absolute and relative concentrations of hydrocarbons, free alcohols and carboxylic acids are studied and their probable biological precursors are discussed. A significant signal of cyanobacterial lipids is recognized by the strong predominance ofn-heptadecane (C₁₇),n-heptadecene, two monomethyl-heptadecanes, and the pentacyclic triterpenoid diploptene. Their occurrences parallel the lipid distributions found in pure cultured cyanobacteria and in recent cyanobacterial mats grown in particular environments (hypersaline, lagoonal, hot spring). The observed compound signature appears to be a suitable reference for environments, where cyanobacteria are directly associated with theloci of carbonate precipitation and thus, rock formation. In the studied material, a significant contribution of organic matter from other sources, especially higher plants is characterized by the occurrence of several specific marker compounds, namely lup-20(29)-ene-3-ol, high molecular weightn-alkanes and carboxylic acids. Although these components comprise a notably high portion of the sample’s lipid inventory, they are shown to be distinguished easily from the signal left by the predominant mat building organisms.     

Patient-Reported Outcomes for Quality of Life Assessment in Atrial Fibrillation: A Systematic Review of Measurement Properties

BackgroundAtrial fibrillation is a large and growing burden across all types of healthcare. Both incidence and prevalence are expected to double in the next 20 years, with huge impact on hospital admissions, costs and patient quality of life. Patient wellbeing determines the management strategy for atrial fibrillation, including the use of rhythm control therapy and the clinical success of heart rate control. Hence, evaluation of quality of life is an emerging and important part of the assessment of patients with atrial fibrillation. Although a number of questionnaires to assess quality of life in atrial fibrillation are available, a comprehensive overview of their measurement properties is lacking.InterpretationGiven the low ratings for many measurement properties, no single questionnaire can be recommended, although AFEQT performed strongest. Further studies to robustly assess reliability, validity and responsiveness of AF-specific quality of life questionnaires are required. This review consolidates the current evidence for quality of life assessment in patients with atrial fibrillation and identifies priority areas for future research.     

Role of thyroxine and insulin on the development of the fetal mouse duodenum in organ culture

The role of thyroxine and insulin in the regulation of proliferation and differentiation of the immature duodenal epithelium of the fetal mouse was investigated using an organ culture method with a serum-free medium. Thyroxine (10 nM) stimulates specifically the activity of maltase. Insulin (125 mU/mL) remains without effect on the maturation of all hydrolytic functions studied. Each hormone significantly increases the percentages of brush border enzyme activities liberated in the medium and reduces the amount of glucose released in the medium. In the presence of dexamethasone (76 nM) the effect of thyroxine on maltase activity is still observed. Finally, thyroxine and insulin do not modify the labelling index in the duodenal crypts of the explants in the presence or absence of dexamethasone. These findings indicate that thyroxine and insulin can act directly on the development of the fetal mouse duodenum at the end of gestation. Nevertheless, their implication in prenatal development of the gut functions appears to be of minor importance.     

The effect of epidermal growth factor (EGF) on the differentiation of the rough endoplasmic reticulum in fetal mouse small intestine in organ culture

The differentiation of the rough endoplasmic reticulum (RER) of mouse duodenal absorptive cells located at the tip of the villi at 17 days of gestation was compared to that of absorptive cells in duodenal explants of 15-day-old mouse fetuses cultured for 72 hr 1) with Trowell T8 medium (without insulin) alone or supplemented 2) with epidermal growth factor (EGF; 100 ng/ml) or 3) with 25% bovine amniotic fluid (BAF). Glucose-6-phosphatase activity (G6Pase) was localized cytochemically to ensure a better identification of the RER. The intersections of a double lattice falling over and outside the RER were counted and the percentage of intersections over the RER was estimated. With this method, the extent of the RER is not statistically different when the absorptive cells in utero are compared to those of explants cultured with EGF. However, the extent of the RER in the absorptive cells cultured with Trowell T8 medium alone or supplemented with BAF is 50% lower than in the former two groups. It is concluded that EGF promotes the maturation of duodenal absorptive cells in organ culture.     

Voraussetzungen und derzeitige Grenzen der quantitativen elektronenmikroskopischen Autoradiographie bei Kinetikstudien an Drüsenzellen

Zusammenfassung Einer weiblichen Maus wurde 3 Tage post partum 750 C ³H-Leucin i. p. injiziert. Zu verschiedenen Zeiten nach der Leucinapplikation wurden dem leicht narkotisierten Tier Gewebeteile der Milchdrüse entnommen und zu elektronenmikroskopischen Autoradiogrammen verarbeitet. An Hand der dabei gewonnenen Ergebnisse wurde versucht, den zeitlichen Ablauf der Milcheiweißbildung rechnerisch zu erfassen. 5 und 15 min nach der ³H-Leucinapplikation kann die Aktivität über dem rauhen endoplasmatischen Retikulum, nach 30 min über dem Golgi-Feld, und nach 240 min zur Hauptsache über den Lumina der Ausführungsgänge beobachtet werden. Die Halbwertszeit von markierten Proteinen im Ergastoplasma errechnete sich zu etwa 22 min, diejenige im Golgi-Feld zu etwa 3 Std.Die Voraussetzungen und derzeitigen Grenzen einer quantitativen elektronenmikroskopischen Autoradiographie werden diskutiert. Wegen der vielen möglichen Fehlerquellen wird die Berechnung der Kinetik der Milcheiweißbildung lediglich als Modell gewertet.

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Summary A female mouse, 3 days post partum, was injected with ³H-leucine. After various intervals parts of the mammary gland were processed for electronmicroscopic autoradiograms, the results of which were mathematically evaluated in order to understand the temporal course of milk protein formation. After 5 and 15 minutes the leucine-activity is located mainly in the rough endoplasmic reticulum, after 30 minutes in the Golgi field and after 240 minutes in the lumina of the mammary ducts. The half-live time of labelled proteins in the rough endoplasmic reticulum is about 22 minutes, in the Golgi field about 3 hours.The preconditions and limitations of quantitative electronmicroscopic autoradiography are discussed. Because of the many possible sources of error, the calculations of the kinetics of protein synthesis and secretion in the mammary gland are merely regarded as a model.

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Ausgeführt mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

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Wesentliche Teile der Arbeit werden Von Ute Seitter der Medizinischen Fakultät der Universität Freiburg i. Br. als Inaugural-Dissertation vorgelegt.     

Impact of separating amino acids between plasma, extracellular and intracellular compartments on estimating protein synthesis in rodents

Summary. Three models representing different separations of amino acid sources were used to simulate experimental specific radioactivity data and to predict protein fractional synthesis rate (FSR). Data were from a pulse dose of ¹⁴C-U Leu given to a non-growing 20 g mouse and a flooding dose of ³H Phe given to a non-growing 200 g rat. Protein synthesis rates estimated using the combined extracellular and intracellular (Ec + Ic) source pool and extracellular and plasma (Ec + Pls) source pool mouse models were 78 and 120% d⁻¹ in liver, 14 and 16% d⁻¹ in brain and 15 and 14% d⁻¹ in muscle. Predicted protein synthesis rates using the Ec + Ic, Ec + Ic + Tr (combined extracellular, intracellular and aminoacyl tRNA source pool) and Ec + Pls rat models were 57, 3.4 and 57% d⁻¹ in gastrocnemius, 58, 71 and 62% d⁻¹ in gut, 8.3, 8.4 and 7.9% d⁻¹ in heart, 32, 23 and 25% d⁻¹ in kidney, 160, 90 and 80% d⁻¹ in liver, 57, 5.5 and 57% d⁻¹ in soleus and 56, 3.4 and 57% d⁻¹ in tibialis. The Ec + Ic + Tr model underestimated protein synthesis rates in mouse tissues (5.0, 27 and 2.5% d⁻¹ for brain, liver and muscle) and rat muscles (3.4, 5.5 and 3.4% d⁻¹ for gastrocnemius, soleus and tibialis). The Ec + Pls model predicted the mouse pulse dose data best and the Ec + Ic model predicted the rat flooding dose data best. Model predictions of FSR imply that identification and separation of the source specific radioactivity is critical to accurately estimate FSR. Received June 11, 2000 Accepted September 26, 2000     

Effects of blocking plasma lipid transfer protein activity in the rabbit

Plasma lipid transfer protein activity was completely blocked in rabbits for up to 48 h by infusion with goat antibody to rabbit lipid transfer protein. Lipid transfer protein activity in plasma of control animals, infused with antibody from a non-immune goat, decreased during the experiment but was never less than 50% of pre-infusion levels. During the period that lipid transfer protein activity was completely blocked, there were changes in high-density lipoprotein composition (expressed as % by weight) with a reduction in triacylglycerol from 8.4 +/- 2.4% to 1.0 +/- 0.2% (P less than 0.05) and an increase in esterified cholesterol from 10.7 +/- 1.7% to 14.5 +/- 0.3% (P less than 0.1). In conjunction with the observed changes in high-density lipoprotein composition, there was an increase in high-density lipoprotein particle size from a mean radius of 4.7 to 5.4 nm. The change in composition and particle size was not observed in high-density lipoproteins from control animals. There was a change in the distribution of plasma cholesterol in control animals, with a fall in the proportion of cholesterol in high-density lipoproteins (P less than 0.02) and consequently an increase in the proportion of cholesterol in low-density lipoproteins (P less than 0.02). However, the distribution of plasma cholesterol in animals in which lipid transfer protein activity was inhibited was maintained at original levels during the period of inhibition. Consequently, in these animals, there was a less atherogenic distribution of cholesterol during the period of lipid transfer protein inhibition when compared with control animals. The changes observed in lipoproteins, in the absence of lipid transfer protein activity, demonstrate that lipid transfer protein modifies lipoproteins in vivo and appears to contribute to a more atherogenic lipid profile.