Effects of iron deficiency on iron binding and internalization into acidic vacuoles in Dunaliella salina

Uptake of iron in the halotolerant alga Dunaliella salina is mediated by a transferrin-like protein (TTf), which binds and internalizes Fe(3+) ions. Recently, we found that iron deficiency induces a large enhancement of iron binding, which is associated with accumulation of three other plasma membrane proteins that associate with TTf. In this study, we characterized the kinetic properties of iron binding and internalization and identified the site of iron internalization. Iron deficiency induces a 4-fold increase in Fe binding, but only 50% enhancement in the rate of iron uptake and also increases the affinity for iron and bicarbonate, a coligand for iron binding. These results indicate that iron deprivation leads to accumulation and modification of iron-binding sites. Iron uptake in iron-sufficient cells is preceded by an apparent time lag, resulting from prebound iron, which can be eliminated by unloading iron-binding sites. Iron is tightly bound to surface-exposed sites and hardly exchanges with medium iron. All bound iron is subsequently internalized. Accumulation of iron inhibits further iron binding and internalization. The vacuolar inhibitor bafilomycin inhibits iron uptake and internalization. Internalized iron was localized by electron microscopy within vacuolar structures that were identified as acidic vacuoles. Iron internalization is accompanied by endocytosis of surface proteins into these acidic vacuoles. A novel kinetic mechanism for iron uptake is proposed, which includes two pools of bound/compartmentalized iron separated by a rate-limiting internalization stage. The major parameter that is modulated by iron deficiency is the iron-binding capacity. We propose that excessive iron binding in iron-deficient cells serves as a temporary reservoir for iron that is subsequently internalized. This mechanism is particularly suitable for organisms that are exposed to large fluctuations in iron availability.     

A computational method for design of connected catalytic networks in proteins

Computational design of new active sites has generally proceeded by geometrically defining interactions between the reaction transition state(s) and surrounding side‐chain functional groups which maximize transition‐state stabilization, and then searching for sites in protein scaffolds where the specified side‐chain–transition‐state interactions can be realized. A limitation of this approach is that the interactions between the side chains themselves are not constrained. An extensive connected hydrogen bond network involving the catalytic residues was observed in a designed retroaldolase following directed evolution. Such connected networks could increase catalytic activity by preorganizing active site residues in catalytically competent orientations, and enabling concerted interactions between side chains during catalysis, for example, proton shuffling. We developed a method for designing active sites in which the catalytic side chains, in addition to making interactions with the transition state, are also involved in extensive hydrogen bond networks. Because of the added constraint of hydrogen‐bond connectivity between the catalytic side chains, to find solutions, a wider range of interactions between these side chains and the transition state must be considered. Our new method starts from a ChemDraw‐like two‐dimensional representation of the transition state with hydrogen‐bond donors, acceptors, and covalent interaction sites indicated, and all placements of side‐chain functional groups that make the indicated interactions with the transition state, and are fully connected in a single hydrogen‐bond network are systematically enumerated. The RosettaMatch method can then be used to identify realizations of these fully‐connected active sites in protein scaffolds. The method generates many fully‐connected active site solutions for a set of model reactions that are promising starting points for the design of fully‐preorganized enzyme catalysts.     

Root-Derived Short-Chain Suberin Diacids from Rice and Rape Seed in a Paddy Soil under Rice Cultivar Treatments

Suberin-derived substituted fatty acids have been shown to be potential biomarkers for plant-derived carbon (C) in soils across ecosystems. Analyzing root derived suberin compounds bound in soil could help to understand the root input into a soil organic carbon pool. In this study, bound lipids were extracted and identified in root and topsoil samples. Short-chain suberin diacids were quantified under rice (Oryza sativa L.) and rape (Brassica campestris) rotations with different cultivar combinations in a Chinese rice paddy. After removal of free lipids with sequential extraction, the residual bound lipids were obtained with saponification and derivatization before analysis using gas chromatography–mass spectrometry (GC-MS). Diacids C16 and C18 in bound lipids were detected both in rice and rape root samples, while diacids C20 and C22 were detected only in rape root samples. Accordingly, diacids were quantified in both rhizosphere and bulk soil (0–15 cm). The amount of total root-derived diacids in bulk soil varied in a range of 5.6–9.6 mg/kg across growth stages and crop seasons. After one year-round rice-rape rotation, root-derived suberin diacids were maintained at a level of 7–9 mg/kg in bulk soil; this was higher under a super rice cultivar LY than under a hybrid cultivar IIY. While concentrations of the analyzed diacids were generally higher in rhizosphere than in bulk soil, the total diacid (DA) concentration was higher at the time of rape harvest than at rice harvest, suggesting that rape roots made a major contribution to the preservation of diacids in the paddy. Moreover, the net change in the concentration and the ratios of C_16:0 DA to C_18:1 DA, and of C_16:0 DA to C_18:0 DA, over a whole growing season, were greater under LY than under IIY, though there was no difference between cultivars within a single growth stage. Overall, total concentration of root-derived suberin diacids was found to be positively correlated to soil organic carbon concentration both for bulk soil and rhizosphere. However, the turnover and preservation of the root suberin biomolecules with soil property and field conditions deserve further field studies.     

Pre-Conception Dyslipidemia Is Associated with Development of Preeclampsia and Gestational Diabetes Mellitus

IntroductionThe association between glucose intolerance, elevated blood pressure and abnormal lipid levels is well established and comprises the basis of metabolic syndrome pathophysiology. We hypothesize that abnormal preconception lipid levels are associated with the increased risk of severe pregnancy complications such as preeclampsia and gestational diabetes mellitus.MethodsWe included all singleton deliveries (n = 27,721) of women without known cardiovascular morbidity and preeclampsia and gestational diabetes mellitus during previous pregnancies. Association between preconception low high density lipoprotein cholesterol (HDLc level≤50 mg/dL), high triglycerides (level≥150 mg/dL) and the primary outcome (composite of gestational diabetes mellitus/or preeclampsia) was assessed using Generalized Estimation Equations.ResultsPrimary outcome of preeclampsia and/or gestational diabetes was observed in a total of 3,243 subjects (11.7%). Elevated triglycerides and low HDLc were independently associated with the primary outcome: with odds ratio (OR) of 1.61 (95% CI 1.29–2.01) and OR = 1.33 (95% CI 1.09–1.63), respectively, after adjusting for maternal age, weight, blood pressure, repeated abortions, fertility treatments and fasting glucose. There was an interaction between the effects of HDLc≤50 mg/dL and triglycerides≥150 mg/dL with an OR of 2.69 (95% CI 1.73–4.19).ConclusionsOur analysis showed an increased rate of preeclampsia and/or gestational diabetes in women with low HDLc and high triglycerides values prior to conception. In view of the severity of these pregnancy complications, we believe this finding warrants a routine screening for the abnormal lipid profile among women of a child-bearing age.     

High rabdosiin and rosmarinic acid production in Eritrichium sericeum callus cultures and the effect of the calli on masugi-nephritis in rats

During an investigation of plant cell cultures that might be useful in the treatment of renal disorders, we established a vigorously-growing E-4 callus culture of Eritrichium sericeum that produced large amounts of caffeic acid metabolites, (-)-rabdosiin (1.8% dry wt) and rosmarinic acid (4.6% dry wt). Elicitation of the calli by methyl jasmonate induced a 38% increase in total polyphenol production. The most efficient method of eliciting (-)-rabdosiin biosynthesis was through the treatment of E-4 calli with cuprum glycerate, which induced an increase in (-)-rabdosiin production of as much as 4.1% dry wt. Oral administration of E-4 callus biomass (100 mg/kg/d for 30 d) to rats with induced Masugi-nephritis caused an increase in diuresis and lowered creatinine excretion and proteinuria levels as compared with Masugi-nephritis untreated rats. While all of the Masugi-nephritis untreated rats began to suffer, near a quarter of the E-4 treated rats remained in good health. This result indicates that the E-4 culture has the potential to alleviate the symptoms associated with nephritis.     

The reactive metabolite target protein database (TPDB) – a web-accessible resource

Background  

The toxic effects of many simple organic compounds stem from their biotransformation to chemically reactive metabolites which bind covalently to cellular proteins. To understand the mechanisms of cytotoxic responses it may be important to know which proteins become adducted and whether some may be common targets of multiple toxins. The literature of this field is widely scattered but expanding rapidly, suggesting the need for a comprehensive, searchable database of reactive metabolite target proteins.     

Info-gap robust-satisficing model of foraging behavior: do foragers optimize or satisfice?

In this note we compare two mathematical models of foraging that reflect two competing theories of animal behavior: optimizing and robust satisficing. The optimal-foraging model is based on the marginal value theorem (MVT). The robust-satisficing model developed here is an application of info-gap decision theory. The info-gap robust-satisficing model relates to the same circumstances described by the MVT. We show how these two alternatives translate into specific predictions that at some points are quite disparate. We test these alternative predictions against available data collected in numerous field studies with a large number of species from diverse taxonomic groups. We show that a large majority of studies appear to support the robust-satisficing model and reject the optimal-foraging model.     

Kinetics of Choline Uptake into Isolated Rat Forebrain Microvessels: Evidence of Endocrine Modulation

The active uptake of [methyl-3H]choline into isolated rat brain microvessel suspension was studied as a likely guide to the transport of choline across the blood-brain barrier. The method consisted primarily of incubation of the suspension with a fixed concentration of labeled choline in the presence of increasing concentrations of unlabeled choline or any other inhibitor (I) of active uptake, defined as the difference in uptake at 37 degrees and 0 degrees C. From the linear regression of (1/V) against [I], the following values of Vmax (nmol g-1 min-1) and Km (microM) were obtained for choline: 2-month-old males, 10.6 +/- 3.8 and 6.1 +/- 0.9; 3-month old random females, 28.4 +/- 5.9 and 12.6 +/- 4.0; females at metaestrus, 17.8 +/- 10.3 and 8.3 +/- 5.0; at diestrus, 31.1 +/- 9.3 and 13.0 +/- 2.6; at proestrus, 54.9 +/- 2.2 and 14.0 +/- 1.5; at estrus, 19.2 +/- 2.2 and 2.6 +/- 1.7. The differences between males and random females (p less than 0.018) and between females at proestrus and estrus (p less than 0.005) are significant. It is suggested that these inter- and intrasex variations in choline uptake reflect a dynamic adjustment of supply in accordance with brain demand for choline at the time of assay. Hemicholinium-3 was an effective inhibitor of choline uptake, Ki = 14.0 +/- 8.5 microM; dimethylaminoethanol was much less effective; and imipramine had no measurable effect.