Inside-out membrane vesicles isolated from spinach thylakoids

Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The separation was obtained by partition in an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.     

Catalytic properties and stability of three common variants of placental alkaline phosphatase

Three placental alkaline phosphatases purified to homogeneity, i.e., the F, I, and S variants, were investigated for catalytic and stability properties. All three forms of the enzyme were found to have almost identical pH optima (10.7–10.8), similar sensitivity to the uncompetitive inhibitors L-phenylalanine (70%) and L-leucine (30%), and identical K_m values against p-nitrophenylphosphate, -glycerophosphate, and -naphthylphosphate. Significant differences among the three types were observed in thermal stability. The F variant was found to be most stable and the I variant most labile at 79 C. At 70 C all three forms were stable.This investigation was supported by grants from the Swedish Medical Research Council (Projects No. 4217 and 03X-2725), from the Medical Faculty, University of Umeå, and Jubileumsklinikens i Umeå forskningsfond.     

Isoelectric points of spinach thylakoid membrane surfaces as determined by cross partition

The isoelectric points of unbroken chloroplast lamellae and various subchloroplast fractions, including a preparation of inside-out thylakoids, have been determined using aqueous two-phase systems containing dextran and charged polyethylene glycol. When the amounts of material in the top phase in a phase system with the positively charged trimethylamino polyethylene glycol are plotted against pH the curve intersects the corresponding curve obtained from phase systems with the negatively charged polyethylene glycol sulfonate. This cross-point can be correlated with the isoelectric point of the material.The cross-point for unbroken chloroplast lamellae was found to be around pH 4.7. Mechanical disintegration lowered the cross-point to around pH 4.4, probably because of exposure of new membrane surfaces. The disintegrated chloroplasts were fractionated by differential centrifugation to separate the grana and stroma lamellae. The stroma lamellae vesicles showed the same isoelectric point as the unbroken lamellae, while a cross-point at pH 4.3 was obtained for the grana-enriched fraction. For thylakoid membranes destacked under low salt conditions the cross-point was 0.3 pH unit lower than for membranes originating exclusively from the stroma lamellae. The most acidic cross-point (pH 4.1) was observed for the fraction enriched in inside-out grana thylakoids. It is suggested that the differences in isoelectric point between various subchloroplast fractions reflect a heterogeneous arrangement of surface charge along and across the thylakoid membrane.     

Effect of cations on the temperature sensitivity of Ca2+ transport in rat-liver mitochondria and safranine uptake by liposomes

The activation energy of mitochondrial Ca²⁺ transport has been studied in various conditions by Arrhenius plots in the temperature range 6–20°C. In the presence of Mg²⁺ the activation energy is decreased to 18 kJ/mole from that of 40 kJ/mole found in a sucrose medium. In the presence of the polyamine spermine the activation energy is practically 0 kJ/mole. A lanthanide Eu³⁺, which is a potent inhibitor of Ca²⁺ transport, has no significant effect on the activation energy. In a KCl medium the activation energy is increased to 70 kJ/mole. When both K⁺ and Mg⁺ are present the activation energy is nonlinear between 11 and 18°C. In the presence of K⁺ and spermine it is about 0 kJ/mole between 6 and 13°C and at higher temperatures 68 kJ/mole. Neither Mg²⁺ nor spermine affect the slope of the Arrhenius plot for state 4 respiration. Spermine decreases slightly the activation energy of Ca²⁺-stimulated respiration. Spermine also decreases the activation energy of valinomycin- or gramicidin-induced safranine uptake by liposomes from 68 to almost 0 kJ/mole between 17 and 30°C. The results indicate that Ca²⁺ binding to the polar head groups of the phospholipids at the membrane surface is the rate-limiting step of mitochondrial Ca²⁺ transport, because agents that inhibit Ca²⁺ binding to these sites (Mg²⁺, spermine, K⁺) have the most marked effect, whereas Eu³⁺, which, because of the small concentration used, ought to interact mainly with the mitochondrial Ca²⁺ transport system, has no significant effect on the temperature sensitivity of mitochondrial Ca²⁺ transport.     

Colpoda spiralis sp. n., a Colpodid Ciliate Found Inhabiting Treeholes (Colpodida,Ciliophora)*

SYNOPSIS A new species of the family Colpodidae, genus Colpoda O. F. Müller, Colpoda spiralis sp. n. is described. This organism has been found only in treehole fluid. Its species status is based on the possession of a unique and prominent preoral protuberance, multimicronuclearity, and the preference for a treehole habitat. Its range is known to include the tri-state area of Arizona, New Mexico and Utah, and the mixed-deciduous forests of Maryland. This species prefers treehole fluids with a pH > 7 and is naturally limited to tree species which possess these alkaline treeholes. It encysts as a treehole dries and excysts when the treehole is again filled by fluids from the tree, or rainwater.     

Purification and identification of the violaxanthin deepoxidase as a 43 kDa protein

Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH₄)₂SO₄ fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 mol g^–1 s^–1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with -mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b ₆ and an internal sequence of polyphenol oxidase.Abbreviations DMSO dimethylsulfoxid - HIC hydrophobic interaction chromatography - MGDG monogalactosyl diacylglycerol - VDE violaxanthin deepoxidase A preliminary report of these results was presented at the Xth Int. Congress on Photosynthesis, Montpellier, France, 1995.     

Effects of transforming growth factor β, tumor necrosis factor α and interferon γ on pancreatic islet β-cell responsiveness to transforming growth factor α

The insulin-producing pancreatic islet -cell, characterized by low proliferative potential, is normally not responsive to the polypeptide epidermal growth factor (EGF) or its homolog transforming growth factor (TGF-). Since EGF receptors in other tissues can be up-regulated by other growth factors and by cytokines, we have in this paper investigated whether such a -cell responsiveness to TGF-, or EGF, can be conferred by co-culture with interferon (IFN-), tumor necrosis factor (TNF-) or transforming growth factor (TGF-) in various combinations. To this end, fetal rat pancreatic islets enriched in -cells were isolated and cultured for 3 days with or without 200 pM or 20 nM TGF-. It was found that neither of these TGF- concentrations affected -cell mitogenesis, insulin content or insulin secretion. However, IFN- (1000 U/ml) evoked a modest stimulation of -cell replication, while suppressing insulin secretion and leaving the islet insulin content unaltered. TNF- (1000 U/ml), on the other hand, affected none of these parameters either alone or in any combination with TGF- or IFN-. However, when TNF- or IFN-, either alone or in combination, were combined with the cytokine interleukin-1, this resulted in islet disintegration, whereas the latter cytokine alone did not exert any gross necrotic changes evident by light microscopy. TGF- (500 pM) stimulated insulin secretion but did not influence islet insulin content or -cell mitogenesis either alone or in combination with TGF- (200 pM or 20 nM). In no instance could any mitogenic or secretory response to low or high concentrations of TGF- be conferred by IFN-, TNF- or TGF- whether used alone or in combinations. Hence, responsiveness to TGF- or EGF in the -cell obviously cannot be achieved by any of these peptides.Abbreviations EGF epidermal growth factor - IFN- interferon - TGF- transforming growth factor - TGF- transforming growth factor - TNF- tumor necrosis factor      

Plant defence and stochastic risk of herbivory

Summary The influence of risk of herbivory and its variation in time on the optimal defence strategy in plants is analysed by a simple optimization model. We contrast two possible defence strategies; a constitutive defence with an invariant defence level in time and an idealized induced defence, that is, a strategy that adjusts the defence level to the prevailing risk of herbivory. We also take into account effects of the efficiency of the defence. If there is no variation in risk of herbivory over years, constitutive and induced defence should have the same expected optimal defence level and both strategies are equally fit. The optimal defence level increases as the maximum fecundity and the adult to juvenile survival ratio of the plants both increase. If the risk of herbivory varies stochastically, the expected optimal level of the constitutive defence is either increased or unaffected by the variation, whereas the induced defence strategy may result in both higher or lower expected optimal defence levels as variance increases. This outcome is dependent on the mean risk of herbivory. It also depends on the defence efficiency, i.e. the shape (convex, concave or linear) of the defence function that relates the probability of survival if encountered by a herbivore to defence level. Thus, the defence level of plants interacting with variable herbivore populations cannot be unambiguously predicted unless the defence strategy (constitutive or induced), mean risk of herbivory, the form of the defence function and plant life history are known.     

Inhibition of lactoferrin and vitronectin binding toStaphylococcus aureus by heparin

Cells ofStaphylococcus aureus strain ISP 546, previously shown to express high binding of vitronectin (Vn), were shown also to bind radioactive iodine-labeled human lactoferrin (Lf). The binding was specific, and heat treatment ofS. aureus cells abolished Lf binding. When cells were preincubated with heparin (2.5 g/ml), binding of Vn and Lf was inhibited by more than 90%. Two other sulfated carbohydrate polymers, fucoidan and dextran sulfate (mol weights 5000 and 8000) inhibited binding of both iodine-labeled Vn and Lf by about 75% each. No synergistic inhibitory effect between heparin and fuocidan on Lf binding byS. aureus was detected. Mannose, mannoseamine and poly--sialic acid (colominic acid) inhibited Vn binding by about 40% but had no effect on Lf binding. A cell surface extract of ISP 546 inhibited Vn binding but did not affect Lf binding. Lf binding toS. aureus was inhibited by preincubation of the cells with Vn, and Vn binding toS. aureus was inhibited by preincubation with Lf. Our data indicate that Vn and Lf bind to different molecules on theS. aureus cell surface, involving heparin and other carbohydrate interactions.     

Winter Roosting Ecology of Eastern Red Bats in Southwest Missouri

Abstract: Eastern red bats (Lasiurus borealis) have been found to overwinter in areas that can experience severe fluctuations in temperature. We examined the red bat's use of winter roosts in southwest Missouri, USA, for 2 winters (2003–2005). We found tree roosts in eastern red cedars (Juniperus virginiana) and hardwoods. Tree roost sites were located on the south side of trees, and we found roost trees on south-facing slopes. Roost sites occurred more frequently in the location with least canopy cover. Bats switched from tree roosts to leaf litter roosts when ambient temperatures approached or fell below freezing. We found habitat characteristics and aspect to be determining factors in the selection of leaflitter roosts. Management of overwintering red bats requires a diverse forest structure, including canopy gaps, stand-density variation, and leaf-bearing trees, including oaks (Quercus spp.).