Multiple origins of Southern Hemisphere Anemone (Ranunculaceae) based on plastid and nuclear sequence data

 Using two molecular data sets, the plastid atpB-rbcL intergenic spacer region and the nuclear ribosomal internal transcribed spacer regions (ITS), the taxonomic affinities of two newly available Anemone species from the Southern Hemisphere were tested. From previous work based on morphology and geographic distribution, it was assumed that A. tenuicaulis from New Zealand was most closely related to the Tasmanian A. crassifolia, whereas the affinity of A. antucensis from Chile and Argentina was regarded as uncertain. Analyses of molecular sequence data from these and 18 other species of Anemone s.lat. (with Clematis as outgroup) result in trees largely congruent with past analyses based on morphology and plastid restriction site data. They strongly support A. richardsonii and A. canadensis (with boreal distributions in the Northern Hemisphere) as paraphyletic to a well supported Southern Hemisphere clade consisting of A. antucensis and A. tenuicaulis. This group of four species is part of an otherwise predominantly Northern Hemisphere assemblage (subgenus Anemonidium s.lat., chromosome base number x=7), including A. narcissiflora, A. obtusiloba, A. keiskeana and A. (=Hepatica) americana. All other austral species included in the present sampling, A. crassifolia (Tasmania), A. knowltonia (=Knowltonia capensis), and A. caffra (both South African), form a separate clade, sister to A. (=Pulsatilla) occidentalis and other Northern Hemisphere anemones (subgenus Anemone s.lat., x=8). Possible phytogeographical links of the Southern Hemisphere species are discussed. Received April 23, 2001 Accepted October 4, 2001     

A method to study zhizosphere processes in thin soil layers of different proximity to roots

Frankia is the diverse bacterial genus that fixes nitrogen within root nodules of actinorhizal trees and shrubs. Systematic and ecological studies of Frankia have been hindered by the lack of morphological, biochemical, or other markers to readily distinguish strains. Recently, nucleotide sequence of 16 S RNA from the small ribosomal subunit has been used to classify and identify a variety of microorganisms. We report nucleotide sequences from portions of the 16 S ribosomal RNA from Frankia strains AcnI1 isolated from Alnus viridis ssp. crispa (Ait.) Turrill and PtI1 isolated from Purshia tridentata (Pursh) DC. The number of nucleotide base substitutions and gaps we find more than doubles the previously reported sequence diversity for the same variable regions within other strains of Frankia.     

Patterns and sources of variation in Daphnia phosphorus content in nature

It has recently been shown that Daphnia can vary in the phosphorus (P)-content of their body tissues, but the relative importance of genetic versus environmental causes for this variation is unexplored. We measured variation in P-content (as % body mass) of Daphnia from eight lakes and conducted experiments to contrast three sources of variation: interspecific variation, clonal variation and phenotypic plasticity. Daphnia P-content decreased with increasing seston C:P ratio across lakes. This relationship reflected both inter- and intraspecific variation. Daphnia parvula and D. dubia exhibited high P-content and were found in shallow lakes with low C:P seston, whereas D. pulicaria had low P-content and was found in deep, stratified lakes having high C:P seston. Populations of D. dentifera spanned this lake gradient and exhibited P-content that was negatively related to seston C:P. Evidence for phenotypic plasticity came from experiments with D. pulicaria and D. dentifera collected from a lake with P-deficient seston and fed a P-sufficient diet in the laboratory. In addition, populations of D. dentifera differed in P-content even after 7 d of feeding on P-sufficient resources, suggesting within-species clonal variation. However, mesocosm experiments revealed broad and surprisingly continuous variation in the P-content of individual clones of D. pulex (range 1.54–1.05%) and D. mendotae (1.51–1.07%) over a gradient in dietary C:P. The broad range in P-content exhibited by individual clones, acclimated for generations, suggests that variation in Daphnia P-content from laboratory experiments needs to be interpreted with caution. These results also show that phenotypic variation in response to environment can be a larger source of variation in P-content than genetic differences within or among species.     

Cardiolipin biosynthesis and remodeling enzymes are altered during development of heart failure

Cardiolipin (CL) is responsible for modulation of activities of various enzymes involved in oxidative phosphorylation. Although energy production decreases in heart failure (HF), regulation of cardiolipin during HF development is unknown. Enzymes involved in cardiac cardiolipin synthesis and remodeling were studied in spontaneously hypertensive HF (SHHF) rats, explanted hearts from human HF patients, and nonfailing Sprague Dawley (SD) rats. The biosynthetic enzymes cytidinediphosphatediacylglycerol synthetase (CDS), phosphatidylglycerolphosphate synthase (PGPS) and cardiolipin synthase (CLS) were investigated. Mitochondrial CDS activity and CDS-1 mRNA increased in HF whereas CDS-2 mRNA in SHHF and humans, not in SD rats, decreased. PGPS activity, but not mRNA, increased in SHHF. CLS activity and mRNA decreased in SHHF, but mRNA was not significantly altered in humans. Cardiolipin remodeling enzymes, monolysocardiolipin acyltransferase (MLCL AT) and tafazzin, showed variable changes during HF. MLCL AT activity increased in SHHF. Tafazzin mRNA decreased in SHHF and human HF, but not in SD rats. The gene expression of acyl-CoA: lysocardiolipin acyltransferase-1, an endoplasmic reticulum MLCL AT, remained unaltered in SHHF rats. The results provide mechanisms whereby both cardiolipin biosynthesis and remodeling are altered during HF. Increases in CDS-1, PGPS, and MLCL AT suggest compensatory mechanisms during the development of HF. Human and SD data imply that similar trends may occur in human HF, but not during nonpathological aging, consistent with previous cardiolipin studies.     

An Ecteola-cellulose chromatography assay for 3′-phosphoadenosine 5′-phosphosulfate: Phenol sulfotransferase

A new assay procedure for phenol sulfotransferase which employs [35S]-3'-phosphoadenosine-5'-phosphosulfate as a sulfate donor and a variety of phenols as sulfate acceptors was developed. The appearance of the 35S-sulfated products or the disappearance of the [35S]-3'-phosphoadenosine-5'-phosphosulfate are determined simultaneously by chromatography of the assay incubation mixtures on Ecteola-cellulose columns, eluting with an NH4HCO3 step gradient. Various acidic, neutral, and basic phenols can be employed as substrates for phenol sulfotransferase using this procedure.     

Hormones and liver mitochondria: Influence of growth hormone,thyroxine, testosterone,and insulin on thermotropic effects of respiration and fatty acid composition of membranes

The effects of hypophysectomy and subsequent administration of growth hormone, thyroxine, insulin, and testosterone were examined in rat liver for the relationship between the thermotropic effects on State 3 respiration (ADP induced) and fatty acid composition of the phospholipid fraction of intact mitochondria as well as of inner membrane vesicles. The Arrhenius profile for energy-linked (succinate) State 3 respiration of mitochondria from hypophysectomized rats lacked the discontinuity at 23.5 °C seen with mitochondria from normal rats. After injections of the hormones the discontinuity representing the transition temperature from gel to liquid crystalline state of lipids occurred at different temperatures: 18.5 °C for growth hormone, 26.0 °C for thyroxine, 19.5 °C for growth hormone + thyroxine, 27.6 °C for insulin, and 25.3 °C for testosterone. The energy of activation between 37.5 and 23.5 °C was 1.9 times greater for hypophysectomy than for controls. Growth hormone was the most effective in restoring the energy of activation to normal, above as well as below transition temperature. The effect of thyroxine appears to be due to a larger stimulation of the State 4 respiration than that of growth hormone, insulin, or testosterone, especially at higher temperatures. Phospholipids extracted from intact mitochondria or inner membrane vesicles of hypophysectomized rats contained less arachidonic acid (20:4) and more linoleic acid (18:2) than those of normal rats. In addition, the contents of some of the minor fatty acids were also changed. Calculated unsaturation index showed an 18.8 and 14.9% depletion in unsaturation in whole mitochondria and inner membranes, respectively. Among the different hormones used to treat the hypophysectomized rats, growth hormone was the most effective in restoring the transition temperature and fatty acid composition to normal levels and increasing the gain in body weight. Although the other hormones increased total unsaturation index to some extent, some of the individual fatty acids were affected differently. Good correlation exists between the unsaturation index of mitochondrial fatty acids and transition temperature of State 3 respiration. These results strongly suggest a role for the hormones, particularly growth hormone, in the control of mitochondrial membrane fluidity of hypophysectomized rat liver, through fatty acid composition of phospholipids.     

Synthesis of N-quinonyltaurines

Summary.  Both 1,4-benzoquinones and 1,4-naphthoquinones were attached to the non-proteinogenic amino acid taurine to form N-quinonyl taurine derivatives. The products were formed via the direct Michael-like addition or by substitution of a good leaving group. An attempt to bridge the two moieties via an ureido spacer resulted in the formation of a bis-quinonylamino isocyanurate derivative. Preliminary MO calculations provided internal ground-state geometries and orbital coefficients of the HOMO levels in two representing taurine conjugates. Received May 6, 2002 Accepted August 13, 2002 Published online December 18, 2002 Acknowledgments This research was supported by the Israel Science Foundation founded by the Academy of Science and Humanities. We wish to thank Ms. Ethel Solomon for skilled technical help. Authors' address: Prof. Shmuel Bittner, Department of Chemistry, Ben-Gurion University of the Negev, P.O. Box 653, Beer-Sheva 84105, Israel, Fax: (972)-8-6472943, E-mail: bittner@bgumail.bgu.ac.il     

Resistivity of Red Blood Cells Against High-Intensity, Short-Duration Electric Field Pulses Induced by Chelating Agents

The interaction of human red blood cells (RBCs) with diethylenetriamine-pentaacetic acid (DTPA) or its Gd-complex (Magnevist, a widely used clinical magnetic resonance contrast agent containing free DTPA ligands) led to the following, obviously interrelated phenomena. (i) Both compounds protected erythrocytes against electrohemolysis in isotonic solutions caused by a high-intensity DC electric field pulse. (ii) The inhibition of electrohemolysis was observed only when cells were electropulsed in low-conductivity solutions. (iii) The uptake of Gd-DTPA by electropulsed RBCs was relatively low. (iv) (Gd-) DTPA reduced markedly deformability of erythrocytes, as revealed by the electrodeformation experiments using high-frequency electric fields. Taken together, the results indicate that (Gd-) DTPA produce stiffer erythrocytes that are more resistant to electric field exposure. The observed effects of the chelating agents on the mechanical properties and the electropermeabilization of RBCs must have an origin in molecular changes of the bilayer or membrane-coupled cytoskeleton, which, in turn, appear to result from an alteration of the ionic equilibrium (e.g., Ca²⁺ sequestration) in the vicinity of the cell membrane. Received: 19 January 1999/Revised: 1 April 1999     

Single-step,rapid separation of acidic and basic isoenzymes from commercial horseradish peroxidase

A simple and sensitive high-performance liquid chromatography method with electrochemical detector is described for the determination of free 3-methoxy-4-hydroxyphenylacetic acid (HVA) in human plasma. The method does not involve any extraction, is specific and reproducible, and has the potential to measure serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) simultaneously. The plasma concentration of free HVA in eight normal, healthy adult volunteers was 10.9 ± 4.6 (mean ± SD). In a preliminary study, in one schizophrenic patient the plasma HVA increased twofold after neuroleptic treatment.     

Plasma catecholamines: effects of footshock level and hormonal modulators of memory storage

Plasma levels of norepinephrine (NE) and epinephrine (EPI) were measured in male Sprague-Dawley rats before and at several times after training injections of agents known to enhance or to impair later retention performance for a one-trial inhibitory (passive) avoidance task. Two days before testing, each animal was surgically prepared with a chronic tail artery catheter that allows for repeated blood sampling in unhandled rats. Exposure to a single, intense training footshock (3.0 mA, 2.0 sec duration) resulted in an immediate but transient increase in plasma levels of EPI and to a lesser extent NE. Plasma levels of both catecholamines did not differ between unshocked controls and animals that received a weak training footshock (0.6 mA, 0.5 sec duration). An injection of EPI at a dose that enhances retention performance (0.1 mg/kg, sc) resulted in increments in plasma EPI levels of 0.8-1.9 ng/ml from 5 to 40 min after injection. An injection of EPI (0.5 mg/kg, sc) at a dose that produces retrograde amnesia resulted in increments in plasma EPI ranging from 3.7 to 4.5 ng/ml during the 40 min after injection. Plasma NE levels were not significantly altered following an EPI injection. A single injection of adrenocorticotropin (ACTH, 0.3 or 3.0 IU per rat) did not alter the plasma catecholamine responses to training with a weak footshock. Similarly, the synthetic ACTH analog, Organon 2766 (125 or 250 mg/Kg) did not affect plasma catecholamines in untrained (unshocked) rats.These results demonstrate that significant increments in plasma levels of NE and EPI occur shortly after inhibitory avoidance training. Furthermore, an injection of EPI that enhances retention of an inhibitory avoidance task mimics the magnitude, though not the temporal characteristics, of the endogenous adrenal medullary response to a training footshock. Other hormonal treatments (ACTH and Organon 2766) which enhance memory storage do not affect plasma levels of NE and EPI.