Arabidopsis semidwarfs evolved from independent mutations in GA20ox1, ortholog to green revolution dwarf alleles in rice and barley

Understanding the genetic bases of natural variation for developmental and stress-related traits is a major goal of current plant biology. Variation in plant hormone levels and signaling might underlie such phenotypic variation occurring even within the same species. Here we report the genetic and molecular basis of semidwarf individuals found in natural Arabidopsis thaliana populations. Allelism tests demonstrate that independent loss-of-function mutations at GA locus 5 (GA5), which encodes gibberellin 20-oxidase 1 (GA20ox1) involved in the last steps of gibberellin biosynthesis, are found in different populations from southern, western, and northern Europe; central Asia; and Japan. Sequencing of GA5 identified 21 different loss-of-function alleles causing semidwarfness without any obvious general tradeoff affecting plant performance traits. GA5 shows signatures of purifying selection, whereas GA5 loss-of-function alleles can also exhibit patterns of positive selection in specific populations as shown by Fay and Wu’s H statistics. These results suggest that antagonistic pleiotropy might underlie the occurrence of GA5 loss-of-function mutations in nature. Furthermore, because GA5 is the ortholog of rice SD1 and barley Sdw1/Denso green revolution genes, this study illustrates the occurrence of conserved adaptive evolution between wild A.thaliana and domesticated plants.Bioactive gibberellins (GAs) are plant growth regulators involved in important traits such as seed germination, flowering time, flower development, and elongation growth (1). GA biosynthesis and signaling pathways are well defined (1, 2) and have been targeted in crop breeding. Modification of GA pathways was crucial in the green revolution because it conferred semidwarfness, thus reducing lodging and increasing crop yields (3–6). Green revolution semidwarf varieties in wheat are due to mutations in DELLA genes, whereas many short straw rice varieties carry a mutation in the Semi-Dwarf-1 (SD1) locus. This locus codes for GA 20-oxidase-2, a GA biosynthesis gene that is also mutated in most modern barley varieties in which the gene was called Denso or Semi-dwarf 1 (Sdw1) (7).GA 20-oxidases are involved in the later steps of GA biosynthesis and belong to the group of 2-oxoglutarate–dependent dioxygenases that, together with GA 3-oxidases, form biologically active GA (8). Arabidopsis thaliana has five GA20ox paralogous genes. AtGA20ox-1, AtGA20ox-2, AtGA20ox-3, and AtGA20ox-4 can catalyze the in vitro conversion of GA₁₂ to GA₉. Therefore, GA20ox paralogs might have partial redundant functions (9). However, among paralog genes, only AtGA20ox-1 (GA5), which was cloned on the basis of the ga5 mutant (10), affected plant height (8).Natural variation for GA biosynthesis has been previously described in A. thaliana because the Bur-0 accession carries a loss-of-function allele at GA20ox4 (9), which does not result in a semidwarf phenotype. In addition, genetic variation in GA1 has been associated with variation in floral morphology (11). Furthermore, the semidwarf phenotype (here defined as a plant height shorter than half the size of genetically related individuals) observed in the Kas-2 accession is due to a recessive allele at the GA5 locus (12). The latter finding led to the questions of whether green revolution alleles, artificially selected in cereals, could also occur in natural populations of the wild species A. thaliana, and if so, how many different GA5 loss-of-function alleles exist, how they are distributed, and why they occur in some populations.     

Ferricenium complexes: A new type of water-soluble antitumor agent

Summary The antitumor activity of a series of iron complexes, i.e., of ferrocene [Cp₂Fe], of tetrachloroferrates(III) [R₄N]⁺[FeCl₄]^-, and of ferricenium complexes [Cp₂Fe]⁺X^- (X^-=[FeCl₄]^-, 1/2[Cl₃FeOFeCl₃]^2-, [H₅Mo₇O₂₄]^-·2H₂O, [2,4,6-(NO₂)₃C₆H₂O]^-, or [CCl₃COO]^-·2 CCl₃COOH) was investigated against EAT in CF1 mice. Whereas ferrocene and the ammonium tetrachloroferrates(III) did not show recognizable tumor-inhibiting activity, such activity was exhibited by the water-soluble, salt-like ferricenium complexes; the best antineoplastic properties, with optimum cure rates of 100%, were found for ferricenium picrate and ferricenium trichloroacetate.The ferricenium compounds are the first iron complexes for which antineoplastic activity has now been shown. They represent a new type of antitumor agent insofar as they differ fundamentally from known inorganic and organometallic antitumor agents (a) by their ionic, salt-like character, which is responsible for their high water solubility, and (b) by the absence of a cis-dihalometal moiety; this moiety has been recognized as important for the intracellular action of other known inorganic cytostatics.Abbreviations Cp C₅H₅, cyclopentadienyl ring ligand - EAT Ehrlich ascites tumor - IP intraperitoneal(ly) - p.t.t. post transplantationem tumoris Supported by the Fonds der Chemischen Industrie and by Council, University of the Witwatersrand     

Synthetic analogues of [Fe4S4(Cys)3(His)] in hydrogenases and [Fe4S4(Cys)4] in HiPIP derived from all-ferric [Fe4S4{N(SiMe3)2}4]

The all-ferric [Fe₄S₄]⁴⁺ cluster [Fe₄S₄{N(SiMe₃)₂}₄] 1 and its one-electron reduced form [1]^- serve as convenient precursors for the synthesis of 3∶1-site differentiated [Fe₄S₄] clusters and high-potential iron-sulfur protein (HiPIP) model clusters. The reaction of 1 with four equivalents (equiv) of the bulky thiol HSDmp (Dmp = 2,6-(mesityl)₂C₆H₃, mesityl = 2,4,6-Me₃C₆H₂) followed by treatment with tetrahydrofuran (THF) resulted in the isolation of [Fe₄S₄(SDmp)₃(THF)₃] 2. Cluster 2 contains an octahedral iron atom with three THF ligands, and its Fe(S)₃(O)₃ coordination environment is relevant to that in the active site of substrate-bound aconitase. An analogous reaction of [1]^- with four equiv of HSDmp gave [Fe₄S₄(SDmp)₄]^- 3, which models the oxidized form of HiPIP. The THF ligands in 2 can be replaced by tetramethyl-imidazole (Me₄Im) to give [Fe₄S₄(SDmp)₃(Me₄Im)] 4 modeling the [Fe₄S₄(Cys)₃(His)] cluster in hydrogenases, and its one-electron reduced form [4]^- was synthesized from the reaction of 3 with Me₄Im. The reversible redox couple between 3 and [3]^- was observed at E_1/2 = -820 mV vs. Ag/Ag⁺, and the corresponding reversible couple for 4 and [4]^- is positively shifted by +440 mV. The cyclic voltammogram of 3 also exhibited a reversible oxidation couple, which indicates generation of the all-ferric [Fe₄S₄]⁴⁺ cluster, [Fe₄S₄(SDmp)₄].     

Monitoring tumor cell death in murine tumor models using deuterium magnetic resonance spectroscopy and spectroscopic imaging

²H magnetic resonance spectroscopic imaging has been shown recently to be a viable technique for metabolic imaging in the clinic. We show here that ²H MR spectroscopy and spectroscopic imaging measurements of [2,3-²H₂]malate production from [2,3-²H₂]fumarate can be used to detect tumor cell death in vivo via the production of labeled malate. Production of [2,3-²H₂]malate, following injection of [2,3-²H₂]fumarate (1 g/kg) into tumor-bearing mice, was measured in a murine lymphoma (EL4) treated with etoposide, and in human breast (MDA-MB-231) and colorectal (Colo205) xenografts treated with a TRAILR2 agonist, using surface-coil localized ²H MR spectroscopy at 7 T. Malate production was also imaged in EL4 tumors using a fast ²H chemical shift imaging sequence. The malate/fumarate ratio increased from 0.016 ± 0.02 to 0.16 ± 0.14 in EL4 tumors 48 h after drug treatment (P = 0.0024, n = 3), and from 0.019 ± 0.03 to 0.25 ± 0.23 in MDA-MB-231 tumors (P = 0.0001, n = 5) and from 0.016 ± 0.04 to 0.28 ± 0.26 in Colo205 tumors (P = 0.0002, n = 5) 24 h after drug treatment. These increases were correlated with increased levels of cell death measured in excised tumor sections obtained immediately after imaging. ²H MR measurements of [2,3-²H₂]malate production from [2,3-²H₂]fumarate provide a potentially less expensive and more sensitive method for detecting cell death in vivo than ¹³C MR measurements of hyperpolarized [1,4-¹³C₂]fumarate metabolism, which have been used previously for this purpose.

Currently, the response of solid tumors to treatment is assessed mainly on the basis of changes in tumor size [Response Evaluation Criteria in Solid Tumors (RECIST) (1)]. However, changes in size may take weeks to appear after the initiation of treatment and, in some cases, may not appear at all, for example, in the case of treatments that inhibit tumor growth but do not result in tumor regression (2, 3). Changes in metabolism can give an earlier indication of treatment response, for example, assessment of glycolytic activity using positron emission tomography (PET) measurements of 2-[¹⁸F]-fluoro-2-deoxy D-glucose uptake (FDG-PET). PET Response Criteria in Solid Tumors (PERCIST) was introduced as a potentially more sensitive method of assessing treatment response when compared to assessment based on changes in tumor size alone (4), particularly with therapies that stabilize disease. Imaging with hyperpolarized [1-¹³C]pyruvate, like FDG-PET, can also be used to detect drug target engagement as well as subsequent tumor cell death (5, 6). We have shown recently that imaging hyperpolarized [1-¹³C]pyruvate metabolism can be more sensitive than FDG-PET in detecting reductions in glycolytic flux associated with tumor cell death posttreatment (7).While metabolic imaging may indicate drug target engagement, and in some cases tumor cell death, there is a need for imaging methods that detect tumor cell death more directly posttreatment and that can give an indication of longer-term treatment outcomes (8). Fumarate is hydrated in the reaction catalyzed by the intracellular enzyme fumarase to produce malate. Previous ¹³C magnetic resonance spectroscopic imaging (MRSI) studies with hyperpolarized [1,4-¹³C₂]fumarate in tumor models and in models of myocardial infarction and acute kidney necrosis (9–16) have demonstrated that the production of labeled malate can be used to image cell death in vivo. The increased production of malate was attributed to loss of the plasma membrane permeability barrier in necrotic cells and increased access of hyperpolarized [1,4-¹³C₂]fumarate to fumarase. However, imaging with hyperpolarized ¹³C-labeled substrates is limited both by the transient nature of the hyperpolarization, which restricts its application to relatively fast metabolic processes, and the requirement for relatively large amounts of ¹³C-labeled compounds and access to clinical hyperpolarizers, which are expensive. The recent demonstration by De Feyter et al. that ²H MRSI can be used to image the metabolism of ²H-labeled substrates in vivo, including in human subjects (17), has provided a potentially lower-cost alternative for clinical metabolic imaging. The relatively low sensitivity of ²H detection is compensated by its very short T₁, which means that signal can be acquired rapidly without saturation. The main limitation is the narrow frequency range, which requires the use of relatively high magnetic field strengths. Nevertheless, by collecting a series of rapidly acquired images, the technique is capable of generating quantitative images of metabolic flux (18). We show here that ²H magnetic resonance spectroscopy (MRS) and MRSI measurements of [2,3-²H₂]fumarate conversion to ²H-labeled malate can be used to detect tumor cell death in vivo and that this is potentially a more sensitive method for detecting cell death than ¹³C MRSI with either hyperpolarized [1-¹³C]pyruvate or [1,4-¹³C₂]fumarate.     

EDTA-Reduction of Water to Molecular Hydrogen Catalyzed by Visible-Light-Response TiO2-Based Materials Sensitized by Dawson- and Keggin-Type Rhenium(V)-Containing Polyoxotungstates

The synthesis and characterization of a Keggin-type mono-rhenium(V)- substituted polyoxotungstate are described. The dimethylammonium salt [Me₂NH₂]₄[PW₁₁Re^VO₄₀] was obtained as analytically pure homogeneous black-purple crystals by reacting mono-lacunary Keggin polyoxotungstate with [Re^IVCl₆]^2- in water, followed by crystallization from acetone at ca. 5 °C. Single-crystal X-ray structural analysis of [PW₁₁Re^VO₄₀]^4- revealed a monomeric structure with overall T_d symmetry. Characterization of [Me₂NH₂]₄[PW₁₁Re^VO₄₀] was also accomplished by elemental analysis, magnetic susceptibility, TG/DTA, FTIR, UV-vis, diffuse reflectance (DR) UV-vis, and solution ³¹P-NMR spectroscopy. Furthermore, [PW₁₁Re^VO₄₀]^4- and the Dawson-type dirhenium(V)-oxido-bridged polyoxotungstate [O{Re^V(OH)(α₂-P₂W₁₇O₆₁)}₂]^14- were supported onto anatase TiO₂ surface by the precipitation methods using CsCl and Pt(NH₃)₄Cl₂. With these materials, hydrogen evolution from water in the presence of EDTA·2Na (ethylenediamine tetraacetic acid disodium salt) under visible light irradiation (≥400 nm) was achieved.     

Platinum(II) Complexes with Bulky Disubstitute Triazolopyrimidines as Promising Materials for Anticancer Agents

Herein, we present dicarboxylate platinum(II) complexes of the general formula [Pt(mal)(DMSO)(L)] and [Pt(CBDC)(DMSO)(L)], where L is dbtp 5,7-ditertbutyl-1,2,4-triazolo[1,5-a]pyrimidine) or ibmtp (7-isobutyl-5-methyl-1,2,4- triazolo[1,5-a]pyrimidine), as prospective prodrugs. The platinum(II) complexes were synthesized in a one-pot reaction between cis-[PtCl₂(DMSO)₂], silver malonate or silver cyclobutane-1,1-dicarboxylate and triazolopyrimidines. All platinum(II) compounds were characterized by FT-IR, and ¹H, ¹³C, ¹⁵N and ¹⁹⁵Pt NMR; and their square planar geometries with one monodentate N(3)-bonded 5,7-disubstituted-1,2,4-triazolo[1,5-a]pyrimidine, one S-bonded molecule of dimethyl sulfoxide and one O,O-chelating malonato (1, 2) or O,O-chelating cyclobutane-1,1-dicarboxylato (3, 4) was determined. Additionally, [Pt(CBDC)(dbtp)(DMSO)] (3) exhibited (i) substantial in vitro cytotoxicity against the lung adenocarcinoma epithelial cell line (A549) (IC₅₀ = 5.00 µM) and the cisplatin-resistant human ductal breast epithelial tumor cell line (T47D) (IC₅₀ = 6.60 µM); and (ii) definitely exhibited low toxicity against normal murine embryonic fibroblast cells (BALB/3T3).     

A link between polymorphonuclear leukocyte intracellular calcium,plasma insulin,and essential hypertension

BackgroundIntracellular ionized calcium ([Ca²⁺]_i) is a key mediator in the activation and oxidant production by peripheral polymorphonuclear leukocytes (PMN). Primed PMN contribute to oxidative stress (OS) and inflammation in essential hypertension (EH). Elevated [Ca²⁺]_i has been described in insulin-resistant states and in various cell types in EH but not in EH PMN. The aim of this study was to evaluate the levels of [Ca²⁺]_i in peripheral EH PMN in relation to plasma insulin levels and blood pressure (BP).MethodsThe PMN were separated from blood of 20 nonsmoking, nonobese untreated EH patients, age range 20 to 60 years and from 20 age- and gender-matched healthy individuals (NC). Plasma glucose and insulin levels 2 h after a 75-g oral glucose load, reflected insulin resistance. PMN [Ca²⁺]_i was measured by flow cytometry in isolated cells stained with Fluo-3.ResultsThe EH PMNs showed significantly increased [Ca²⁺]_i compared to NC PMN. Eighty percent of EH patients showed significantly higher plasma insulin levels after glucose load. Linear regression analysis showed significant correlation between 1) PMN [Ca²⁺]_i and mean arterial pressure (MAP) (r = 0.5, P < .006); 2) PMN [Ca²⁺]_i and fasting plasma insulin (r = 0.7, P < .005); and 3) fasting plasma insulin and MAP (r = 0.4, P < .04).ConclusionsThis study adds PMN to previously described cells exhibiting elevated [Ca²⁺]_i, contributing to OS and inflammation. The correlation of individual BP with both PMN [Ca²⁺]_i and plasma insulin levels, together with the fact that elevated [Ca²⁺]_i mediates PMN priming, suggest that elevated [Ca²⁺]_i and insulin are involved in the pathogenesis of hypertension-induced vascular injury in EH.     

Cellular resting and diastolic potentials in canine cardiac Purkinje cells: effects of external (K+) and repetitive excitation

Cellular potentials were measured in canine cardiac Purkinje fibers bathed in physiological solution in which the concentration of potassium, [K⁺]_o, was varied systematically from 0.5 mm to 16 mm. Resting potentials, E_C, were measured in quiescent preparations, and maximum diastolic potentials, E_D, were measured in spontaneously-active preparations. In [K⁺]_o of 1 mm and 2 mm, all preparations underwent spontaneous excitation; in [K⁺]_o of 0.5 mm and 4 mm, some preparations were spontaneously active, and some were quiescent; in [K⁺]_o of 8 mm and 16 mm, all preparations were quiescent. E_c increased as [K⁺]_o was reduced from 16 mm to 4 mm, but the potential increased along a slope less than and deviating continuously from that predicted by the Nernst equation for a K⁺ electrode. E_D increased as [K⁺]_o was reduced from 4 mm to 2 mm, but it decreased again as [K⁺]_o was reduced from 2 mm to 0.5 mm. All preparations subsequently were driven by repetitive stimuli for 5 min at a rate of 3.5 Hz, producing maximum hyperpolarization (increase of E_D). Under these conditions, E_D increased as [K⁺]_o was reduced from 16 mm to 2 mm, along a slope closer to that predicted by the Nernst equation, yet diverging continuously. The potential decreased again as [K⁺]_o was reduced from 2 mm to 0.5 mm. Possible explanations for the observed changes of potential with change of [K⁺]_o from high levels to low levels include, according to conventional Membrane Theory, (i) the effects of other ionic gradients, (ii) inward-going rectification, and (iii) active transport of ions, and, according to the Association-Induction Hypothesis, relative reduction of the preferential surface adsorption of K⁺ over Na⁺.     

Radioimmunoassay for leukotriene B4

A rabbit immunized with leukotriene B₄ [LTB₄; (5S,12R)-6, 14-cis-8, 10-trans-icosatetraenoic acid] coupled to bovine serum albumin via the 12-oxy function of the lipid produced antibodies having an average association constant (K_a) for [14,15-³H]LTB₄ of 3.2 × 10⁹ M^-1 at 37°C and in a concentration of 0.37 μg/ml of the immune plasma. When 10 μl of anti-LTB₄ and 3.9 nCi of [14,15-³H]LTB₄ (28 Ci/mmol; 1 Ci = 3.7 × 10¹⁰ becquerels) were incubated in a volume of 250 μl, 50% inhibition of radioligand binding was achieved with 0.31 ng of LTB₄ and with 1.95 ng of (5S,12S)-6-trans-8-cis-LTB₄. The sulfidopeptide leukotrienes, LTC₄ and LTD₄, displaced the radioligand from this antibody with less than 1/100th the activity of LTB₄, and the diastereoisomers of 6-trans-LTB₄, 5-L-hydroxy-6-trans-8,11,14-cis-icosatetraenoic acid (5-HETE), and three prostaglandins were minimally effective. The specificity of this radioimmunoassay was further shown by assessment of the immunoreactive products generated from calcium ionophore (A23187)-activated rat serosal mast cells and human neutrophils after reversed-phase HPLC. Resolution of the supernatants from each cell type yielded a single immunoreactive peak that coeluted with synthetic LTB₄ and quantitatively correlated with the physical measurement by integrated A₂₆₉ in that peak; UV-absorbing peaks eluting at other retention times were not immunoreactive. The immunoreactive LTB₄ generated averaged 4.6 ng per 10⁶ rat mast cells and resolution of the supernatants by reversed-phase HPLC without a prior extraction step gave a recovery of 54%, validating the direct applicability of this sensitive and specific assay for LTB₄, a highly potent chemotactic factor, to unfractionated biologic fluids.     

Metabolism of primed, constant infusions of [1,2-¹³C₂] glycine and [1-¹³C₁] phenylalanine to urinary oxalate

Experiments in humans and rodents using oral doses of glycine and phenylalanine have suggested that the metabolism of these amino acids contributes to urinary oxalate excretion. To better define this contribution, we have examined the primed, constant infusion of [1-¹³C₁] phenylalanine and [1,2-¹³C₂] glycine in the postabsorptive state in healthy adults. Subjects were infused for 5 hours, hourly urines were collected, and blood was drawn every 30 minutes. Ion chromatography/mass spectrometry was used to measure [¹³C] enrichment in urinary oxalate, glycolate, and hippurate; and the enrichment of ¹³C-amino acids in plasma samples was measured by gas chromatography/mass spectrometry. Following infusion with either 6 μmol/(kg h) [1-¹³C₁] phenylalanine or 6 μmol/(kg h) [1,2-¹³C₂] glycine, no isotopic glycolate or oxalate was detected in urine. Based on the limits of detection of our ion chromatography/mass spectroscopy method, these data indicate that less than 0.7% of the urinary oxalate could be derived from phenylalanine catabolism and less than 5% from glycine catabolism. Infusions with high levels of [1,2-¹³C₂] glycine, 60 μmol/(kg h), increased mean plasma glycine by 29% and the whole-body flux of glycine by 72%. Under these conditions, glycine contributed 16.0% ± 1.6% and 16.6% ± 3.2% to urinary oxalate and glycolate excretion, respectively. Experiments using cultured hepatoma cells demonstrated that only at supraphysiological levels (>1 mmol/L) did glycine and phenylalanine metabolism increase oxalate synthesis. These data suggest that glycine and phenylalanine metabolism make only minor contributions to oxalate synthesis and urinary oxalate excretion.     

Dysfunction of diastolic [Ca²⁺] in cardiomyocytes isolated from chagasic patients

Introduction and objectives

Chagas is an endemic disease in Latin America, caused by the parasite Trypanosoma cruzi, which usually affects the functioning of the heart. We have studied the regulation of intracellular calcium in cardiomyocytes isolated from chagasic patients with different degrees of heart dysfunction.

Methods

Calcium selective microelectrodes were used to simultaneously measure diastolic calcium concentration ([Ca²⁺]_d) and resting membrane potential in endomyocardial biopsies obtained from chagasic patients and controls.

Results

The [Ca²⁺]_d increased by 123%, 295%, and 738% in chagasic patients in functional class I, II, and III, respectively, in relation to controls. Membrane potential showed a partial depolarization of 6% in functional class I, 10% in functional class II, and 22% in functional class III, compared to control values. Alteration in the [Ca²⁺]_d was partially reverted by 1-[6-[[(17ß)-3-metoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), a β-phospholipase C antagonist, and by 2-aminoethoxydiphenyl-borate (2-APB), an inositol 1,4,5-trisphosphate receptor blocker. Phenylephrine, an agent that induces a rapid transient increase in 1,4,5-trisphosphate intracellular content, produced a rise in [Ca²⁺]_d, higher in chagasic cardiomyocytes than in controls, and its effect was fully inhibited by 2-APB.

Conclusions

In cardiomyocytes from chagasic patients there is a dysfunction of the regulation of the [Ca²⁺]_d, which correlates with the cardiac abnormalities observed in the different stages of the disease. This disturbance in the regulation of intracellular calcium appears to be associated with alterations in the regulation of intracellular messenger inositol 1,4,5-trisphosphate.Full English text available from: www.revespcardiol.org     

Differential biosynthesis and cellular permeability explain longitudinal gibberellin gradients in growing roots

Control over cell growth by mobile regulators underlies much of eukaryotic morphogenesis. In plant roots, cell division and elongation are separated into distinct longitudinal zones and both division and elongation are influenced by the growth regulatory hormone gibberellin (GA). Previously, a multicellular mathematical model predicted a GA maximum at the border of the meristematic and elongation zones. However, GA in roots was recently measured using a genetically encoded fluorescent biosensor, nlsGPS1, and found to be low in the meristematic zone grading to a maximum at the end of the elongation zone. Furthermore, the accumulation rate of exogenous GA was also found to be higher in the elongation zone. It was still unknown which biochemical activities were responsible for these mobile small molecule gradients and whether the spatiotemporal correlation between GA levels and cell length is important for root cell division and elongation patterns. Using a mathematical modeling approach in combination with high-resolution GA measurements in vivo, we now show how differentials in several biosynthetic enzyme steps contribute to the endogenous GA gradient and how differential cellular permeability contributes to an accumulation gradient of exogenous GA. We also analyzed the effects of altered GA distribution in roots and did not find significant phenotypes resulting from increased GA levels or signaling. We did find a substantial temporal delay between complementation of GA distribution and cell division and elongation phenotypes in a GA deficient mutant. Together, our results provide models of how GA gradients are directed and in turn direct root growth.

The location of cell division and expansion guided by mobile growth regulators is fundamental to morphogenesis in multicellular organisms. How such regulators are controlled and the extent to which regulator patterns influence cell growth patterns has received considerable interest. In plants, the hormone gibberellin (GA) is a mobile growth regulator that plays an essential role during multiple stages of the plant life cycle (from seed germination to reproductive development) and acts through the destabilization of growth repressive DELLA proteins (1–9). In Arabidopsis thaliana roots, where cell division and expansion are separated in distinct longitudinal zones, GA plays a key role in regulating both cell division in the meristematic zone (10) and cell elongation in the elongation zone (11). Deficiencies in GA biosynthesis indeed result in reduced length of the meristematic zone and in reduced length of elongated mature cells (12). Using a genetically encoded fluorescent biosensor, nlsGPS1, we recently found that GA levels correlate with cell length in growing roots (13). Nonetheless, how this GA distribution is generated and the quantitative relationship between GA distribution and cell growth patterning remained unclear.Plant hormones often coordinate the signal integration of environmental conditions into plant developmental programs and can be synthesized locally as well as distally from the site of action. Within cells, biosynthetic and catabolic enzymes in combination with transport activities control GA levels, and thus the differential spatiotemporal distribution of enzymatic and transport activities can all contribute to GA distributions that occur during plant development. GA biosynthesis proceeds in several steps with the final steps occurring in the cytoplasm, where the precursor GA₁₂ is converted to GA₉ by gibberellin 20-oxidase (GA20ox) enzymes and GA₉ is converted into bioactive GA₄ by gibberellin 3-oxidase (GA3ox) enzymes (9, 14). Previous works highlighted how the expression pattern of isozymes of GA20ox and GA3ox families, which partly overlapped in different cell types, organs, and developmental stages, might generate GA patterns (15, 16). GA can be disabled by enzymes belonging to gibberellin 2-oxidase (GA2ox), GA methyltransferase and CYP714A families (15, 17, 18). As with Arabidopsis GA biosynthetic genes, GA catabolic genes present partly overlapping expression patterns in various organs and developmental stages, likely influencing the GA distributions initiated by GA biosynthesis.In recent years, several transmembrane transporters belonging to the nitrate transporter (NRT) 1/peptide transporter family (NPF) and two sugar transporters SWEET13 and SWEET14 have been identified as GA importers (19–22), but as yet no GA exporters have been identified. Because GA is a weak acid, an acid-trap mechanism might also accomplish GA import from the low pH apoplasm into higher pH cytoplasm separate from transmembrane transporter activities (23).To understand in a quantitative manner the factors controlling longitudinal GA distribution in roots, we previously developed a multicellular mathematical model that predicted GA dynamics in the root elongation zone (12). The model revealed that the rapid cell expansion as cells traverse this zone results in hormone dilution that significantly affects cellular GA concentration. Assuming that GA biosynthesis occurs only in the meristem (and not the elongation zone) based on pAtGA20ox1-GUS staining (12), the model predicted that the GA dilution caused a reducing gradient of GA along the elongation zone (with high GA levels in the meristem grading to lower GA levels in the mature zone). These results did not agree with subsequent measurements using nlsGPS1, in which endogenous GA was found to grade from lower levels in the smaller cells of the meristematic zone to higher levels at the end of the elongation zone (13). Furthermore, treatment with exogenous GA showed faster accumulation of GA in the elongation zone compared to the meristematic zone, suggesting the presence of an accumulation gradient of exogenous GA, hereafter referred to as an exogenous-GA-generated gradient, that is independent of GA biosynthesis. This exogenous-GA-generated gradient was consistent with measurements of roots treated with exogenous fluorescein-labeled GAs, where the fluorescence of labeled GAs accumulated preferentially in the elongation zone (24).By using nlsGPS1 live-imaging, genetic and biochemical perturbations, and multiscale modeling, we now show that a longitudinal differential in GA biosynthesis in Arabidopsis roots is responsible for shaping endogenous GA distribution and that a longitudinal differential in cellular permeability for GA is responsible for the exogenous-GA-generated gradient. We also used genetic and biochemical perturbations to investigate the functional relationship between GA gradients and cell elongation gradients. Interestingly, increasing GA levels or GA signaling does not affect cell growth patterning in Arabidopsis roots, indicating that a local dose–response relationship is unlikely. Thus, we propose a cooperative local dose–response relationship in which increasing GA concentrations in the elongation zone act in concert with other signals to direct cell elongation. However, we also demonstrate that complementation of GA deficiency phenotypes is substantially delayed compared with complementation of GA levels. Thus, a hysteretic relationship could instead be functioning in which GA influences cell elongation via prior signaling in the meristematic zone.     

Endogenous gibberellins in Arabidopsis thaliana and possible steps blocked in the biosynthetic pathways of the semidwarf ga4 and ga5 mutants.

Twenty gibberellins (GAs) have been identified in extracts from shoots of the Landsberg erecta line of Arabidopsis thaliana by full-scan gas chromatography-mass spectrometry and Kovats retention indices. Eight of them are members of the early-13-hydroxylation pathway (GA53, GA44, GA19, GA17, GA20, GA1, GA29, and GA8), six are members of the early-3-hydroxylation pathway (GA37, GA27, GA36, GA13, GA4, and GA34), and the remaining six are members of the non-3,13-hydroxylation pathway (GA12, GA15, GA24, GA25, GA9, and GA51). Seven of these GAs were quantified in the Landsberg erecta line of Arabidopsis and in the semidwarf ga4 and ga5 mutants by gas chromatography-selected ion monitoring (SIM) using internal standards. The relative levels of the remaining 13 GAs were compared by the use of ion intensities only. In comparison with the Landsberg erecta line, the ga4 mutant had reduced levels of the 3-hydroxy- and 3,13-dihydroxy-GAs, and it accumulated the 13-hydroxy-GAs, except GA53, and the non-3,13-hydroxy-GAs, except GA12. The GA4 gene encodes, therefore, a protein with 3 beta-hydroxylation activity. The ga5 mutant had reduced levels of the C19-GAs, which indicates that the product of the GA5 gene catalyzes the elimination of C-20 at the aldehyde level. The ga5 mutant also had increased levels of certain C20-GAs, which indicates existence of an additional control, possibly hydroxylation of C-20. The growth-response data, as well as the accumulation of GA9 in the ga4 mutant, indicate that GA9 is not active in Arabidopsis, but it must be 3 beta-hydroxylated to GA4 to become bioactive. It is concluded that the reduced levels of the 3 beta-hydroxy-GAs, GA1 and GA4, are the cause of the semidwarf growth habit of both mutants.     

Steroid biosynthesis by the ovary of the European eel, Anguilla anguilla L., at the silver stage.

The ovary of the European eel, Anguilla anguilla, at the silver stage, was incubated either as an intact tissue preparation or as a homogenate with and without cofactors in the presence of [4-¹⁴C] pregnenolone and [4-¹⁴C]progesterone. Intact tissue incubates displayed a more complex metabolite profile than reinforced homogenates, and deprivation of exogenous cofactors reduced the profile even further. Among the metabolites derived from pregnenolone, the following steroids were identified by their isopolarity and isomorphicity with standard compounds: 17α-hydroxypregnenolone; dehydroepiandrosterone; progesterone; 17α-hydroxyprogesterone; and androstenedione. The last three steroids plus testosterone, 17β-hydroxyandrostenedione, and adrenosterone were identified using progesterone as a precursor. Metopirone inhibited the formation of 11-oxygenated androgens. 11-Deoxycorticosteroids were not found, indicating the absence of steroid 21-hydroxylase activity in the eel ovary. Integration of the product yield-time curves demonstrates that in vitro the activities of the enzymes 3β-, 17β-, and 11β-hydroxysteroid dehydrogenases were less apparent than those of steroid 17α,20-C₂₁-desmolase, and 17α-, and to a lesser extent 11β-hydroxylase. Irrespective of the incubation conditions, pregnenolone produced more Δ⁵-3β-hydroxy-thanΔ⁴-3-ketosteroids, suggesting a predominance of the former biosynthetic pathway. Among the unidentified metabolites, water-soluble compounds were formed from both precursors in intact tissue incubates.     

Crystal engineering of lattice metrics of perhalometallate salts and MOFs

The synthesis of the salt 3 and metallo-organic framework (MOF) [{(4,4^′-bipy)CoBr₂}_n] 4 by a range of solid state (mechanochemical and thermochemical) and solution methods is reported; they are isostructural with their respective chloride analogues 1 and 2. 3 and 4 can be interconverted by means of HBr elimination and absorption. Single phases of controlled composition and general formula [4,4^′-H₂bipy][CoBr_4-xCl_x] 5_x may be prepared from 2 and 4 by solid—gas reactions involving HBr or HCl respectively. Crystalline single phase samples of 5_x and [{(4,4^′-bipy)CoBr_2-xCl_x}_n] 6_x were prepared by solid-state mechanochemical routes, allowing fine control over the composition and unit cell volume of the product. Collectively these methods enable continuous variation of the unit cell dimensions of the salts [4,4^′-H₂bipy][CoBr_4-xCl_x] (5_x) and the MOFs [{(4,4^′-bipy)CoBr_2-xCl_x}_n] (6_x) by varying the bromide to chloride ratio and establish a means of controlling MOF composition and the lattice metrics, and so the physical and chemical properties that derive from it.     

Determination of retinal Schiff base configuration in bacteriorhodopsin

Resonance Raman spectra of the BR₅₆₈, BR₅₄₈, K₆₂₅, and L₅₅₀ intermediates of the bacteriorhodopsin photocycle have been obtained in ¹H₂O and ²H₂O by using native purple membrane as well as purple membrane regenerated with 14,15-¹³C₂ and 12,14-²H₂ isotopic derivatives of retinal. These derivatives were selected to determine the contribution of the C₁₄—C₁₅ stretch to the normal modes in the 1100- to 1400-cm^-1 fingerprint region and to characterize the coupling of the C₁₄—C₁₅ stretch with the NH rock. Normal mode calculations demonstrate that when the retinal Schiff base is in the C[unk]N cis configuration the C₁₄—C₁₅ stretch and the NH rock are strongly coupled, resulting in a large (≈50-cm^-1) upshift of the C₁₄—C₁₅ stretch upon deuteration of the Schiff base nitrogen. In the C[unk]N trans geometry these vibrations are weakly coupled and only a slight (<5-cm^-1) upshift of the C₁₄—C₁₅ stretch is predicted upon N-deuteration. In BR₅₆₈, the insensitivity of the 1201-cm^-1 C₁₄—C₁₅ stretch to N-deuteration demonstrates that its retinal C[unk]N configuration is trans. The C₁₄—C₁₅ stretch in BR₅₄₈, however, shifts up from 1167 cm^-1 in ¹H₂O to 1208 cm^-1 in ²H₂O, indicating that BR₅₄₈ contains a C[unk]N cis chromophore. Thus, the conversion of BR₅₆₈ to BR₅₄₈ (dark adaptation) involves isomerization about the C[unk]N bond in addition to isomerization about the C₁₃[unk]C₁₄ bond. The insensitivity of the native, [14,15-¹³C₂]-, and [12,14-²H₂]K₆₂₅ and L₅₅₀ spectra to N-deuteration argues that these intermediates have a C[unk]N trans configuration. Thus, the primary photochemical step in bacteriorhodopsin (BR₅₆₈ → K₆₂₅) involves isomerization about the C₁₃[unk]C₁₄ bond alone. The significance of these results for the mechanism of proton-pumping by bacteriorhodopsin is discussed.     

Stereochemical Control of Valence and Its Application to the Reduction of Coordinated NO and N(2)

The electronic structures of transition metal complexes of NO are controlled by the stereochemistry about the metal atom (stereochemical control of valence). The six-coordinate complex, trans-[CoNO(NCS)-(C₆H₄ [As(CH₃)₂]₂)₂]⁺, consists of [Co(III)-(N=O^-)]²⁺ (angle_Co-N-O = 135°), while the pentacoordinate trigonal bipyramidal complex, [CoNO(C₆H₄[As(CH₃)₂]₂)₂] ²⁺, is best formulated as [Co(I)-(NO⁺]²⁺ (angle_Co-N-O = 179°). Evidence indicates that complexes of (NO)⁺ and N₂ are electronically similar. Hence, the principles of stereochemical control of valence may be applied to metal complexes of N₂. In a linearly coordinated Mⁿ˜m(NN) complex, valence electrons can be transferred from the metal to the N₂ ligand producing a bent, protonated, and/or metallated Mⁿ⁺²-(N=N^2-) complex. This reduction of N₂ can be effected by the addition of an appropriate ligand to M or by a change in the coordination geometry about M. Stereo-chemical control of valence leads to the rejection of one of the previously proposed mechanisms for reduction by nitrogenase.     

The conversion of phenylalanine to tyrosine in man. Direct measurement by continuous intravenous tracer infusions of L-[ring-2H5]phenylalanine and L-[1-13C]tyrosine in the postabsorptive state

Steady state phenylalanine and tyrosine turnover and the rate of conversion of phenylalanine to tyrosine in vivo were determined in 6 healthy postabsorptive adult volunteers. Continuous infusions of tracer amounts of L-[ring-²H₅]phenylalanine were administered intravenously for 13–14 hr. After 9–10 hr, a priming dose followed by a continuous infusion of L-[1-¹³C]tyrosine was added and maintained, along with the [²H₅]phenylalanine infusion, for 4 hr. Venous plasma samples were obtained before the initiation of each infusion and every 30 min during the course of the combined [²H₅]phenylalanine and [¹³C]tyrosine infusion for determination of isotopic enrichments of [²H₅]phenylalanine, [¹³C]tyrosine, and [²H₄[tyrosine by gas chromatograph-mass spectrometric analysis of the N-trifluoroacetyl-, methyl ester derivatives of the amino acids. Calculated from the observed enrichments, free phenylalanine and tyrosine turnover rates were 36.1 ± 5.1 μmole · kg^?1 · h^?1 and 39.8 ± 3.5 μmole · kg^?1 · h^?1, respectively. Phenylalanine was converted to tyrosine at the rate of 5.83 ± 0.59 μmole · kg^?1 · h^?1, accounting for approximately 16% of either the phenylalanine or the tyrosine flux. The results indicate that the normal basal steady state phenylalanine hydroxylase activity in vivo in man is lower than that obtained from phenylalanine loading studies. This supports the existence of some type of substrate activation of the enzyme as reflected in the previously reported exponential relationship between phenylalanine concentration and phenylalanine hydroxylase activity in vitro. The use of continuous simultaneous infusions of tracer amounts of stable isotope-labeled phenylalanine and tyrosine provides a direct means for studying physiological regulation of phenylalanine hydroxylase activity in vivo.     

The dwarf-1 (dt) Mutant of Zea mays blocks three steps in the gibberellin-biosynthetic pathway

In plants, gibberellin (GA)-responding mutants have been used as tools to identify the genes that control specific steps in the GA-biosynthetic pathway. They have also been used to determine which native GAs are active per se, i.e., further metabolism is not necessary for bioactivity. We present metabolic evidence that the D1 gene of maize (Zea mays L.) controls the three biosynthetic steps: GA20 to GA1, Ga20 to GA5, and GA5 to GA3. We also present evidence that three gibberellins, GA1, GA5, and GA3, have per se activity in stimulating shoot elongation in maize. The metabolic evidence comes from the injection of [17-13C,3H]GA20 and [17-13C,3H]GA5 into seedlings of d1 and controls (normal and d5), followed by isolation and identification of the 13C-labeled metabolites by full-scan GC-MS and Kovats retention index. For the controls, GA20 was metabolized to GA1,GA3, and GA5; GA5 was metabolized to GA3. For the d1 mutant, GA20 was not metabolized to GA1, GA3, or to GA5, and GA5 was not metabolized to GA3. The bioassay evidence is based on dosage response curves using d1 seedlings for assay. GA1, GA3, and GA5 had similar bioactivities, and they were 10-times more active than GA20.     

The effects of calcium antagonists on extracellular potassium accumulation during global ischaemia in isolated perfused rat hearts

Summary The effects of equipotent concentrations of diltiazem, verapamil, and nifedipine upon the accumulation of extracellular potassium [K⁺]_out and the left ventricular pressure (LVP) were studied during global ischemia in isolated perfused rat hearts. Measurement of [K⁺]_out and LVP were performed in two series of experiments. Diltiazem (2×10^-6, 3×10^-6, and 10^-5M), verapamil (3×10^-8, 10^-7, and 3×10^-7 M), and nifedipine (3×10^-8, 10^-7, and 1.5×10^-7 M) were able to slow, in a concentration-dependent manner, the initial rate of rise of [K⁺]_out without affecting the final plateau value of [K⁺]_out reached at t=5 to t=10 minutes. Notably, at the lowest concentrations, which slightly influenced LVP diltiazem, verapamil, and to a lesser degree nifedipine, were still able to slow the rise in [K⁺]_out. In addition, after preper-fusion with low-calcium media ([Ca²⁺] from 1.8 to 1.3 or 0.9 mM), inducing similar negative inotropic effects as those of the calcium antagonists, the rise in [K⁺]_out was not significantly influenced. Our data indicate that the ability to slow the rise in [K⁺]_out is a specific characteristic of calcium antagonists that is independent of their negative inotropic effects.