Coccolith crystals ofPleurochrysis carterae: Crystallographic faces, organization, and development

Summary The crystallographic and morphological configuration of the mineral ring associated with the coccoliths ofPleurochrysis carterae was determined by transmission electron microscopy and electron diffraction. Mature Pleurochrysis coccoliths consist of an oval organic base plate, a distal rim of interlocking calcite crystals, and a narrow ribbon of organic material which tethers the mineral ring to the base plate. Crystals of two distinct forms (R and V units) alternate about the rim in a quasi regular manner; their crystallographicc-axes are aligned parallel to and inclined about 63° to the coccolith plane, respectively. The mineral ring has four platelike elements: the distal-shield and outer-tube elements which form the V unit, and the proximal-shield and inner-tube elements which form the R units. The platy surfaces of both tube elements correspond to the common (10 4) rhombohedral faces of calcite, and the plates of the proximal-shield element are prismatic (2 0) faces. The plates of the distal-shield element are rather curved and their orientation does not correspond to a favorable calcite face; however, for convenience they are described as approximately ( 108) faces, faces which rarely, if ever, develop in inorganic sources of calcite. During coccolith development the earliest habits observed for both V and R units correspond to rectangular parallelepipeds. Outgrowth from the initial V unit begins by expansion of (10 4) faces which form the platy surfaces of the outer-tube element. Throughout this period of development the mineral ring is flexible, at least in an isolated state. Subsequent outgrowth of the inner-tube and proximal-shield elements from the initial R unit produces a rigid interlocking ring. The unusual ( 108) faces of the distal-shield element develop after the crystals are locked in place. Organic structures in intimate association with the mineral phase during its nucleation and growth include the coccolith ribbon, the calcium-polyanion particles, and the membrane of the coccolith vesicle. These structures are described in reference to their putative functions in regulating the development of V and R units.Abbreviation PS polysaccharide     

Electrochemical and kinetic response of heterobinuclear Ni(II)Zn(II) complexes derived from unsymmetrical lateral macrobicyclic tricompartmental ligands

Using the precursor compound 3,4:10,11-dibenzo-1,13[N,N′-bis{(3-formyl-2-hydroxy-5-methyl)benzyl}diaza]-5,9-dioxocyclopentadecane, a series of macrobicyclic heterobinuclear Ni(II)Zn(II) complexes have been synthesized from the corresponding mononuclear nickel(II) complexes via a template method by Schiff’s base condensation. Electrochemical and kinetic studies of the complexes have been carried out on the basis of macrocyclic ring size. Cyclic voltammetry and controlled electrolysis studies indicate that the nickel(II) metal ion in the heterobinuclear complexes undergo quasireversible one electron reduction and oxidation, whereas the zinc(II) metal ion does not undergo any reduction and oxidation. All the heterobinuclear Ni(II)Zn(II) complexes are ESR inactive and diamagnetic in nature. The kinetics of hydrolysis of 4-nitrophenyl phosphate explores that the catalytic activities of the complexes are found to increase with macrocyclic ring size of the complexes. As the macrocyclic ring size increases, the spectral, electrochemical and catalytic studies of the complexes show variation due to distortion in the geometry of metal centre.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-Stranded Polydeoxyribonucleotides. 1. Topotecan Dimerization in Solution

Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5–11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10^–5M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10^–6M) to 7.1 ([TPT] = 10^–4M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 ± 0.7)·10³M^–1.     

Degradation of the pterocarpan phytoalexin medicarpin by Ascochyta rabiei

Four strains of Ascochyta rabiei pathogenic to chickpea (Cicer arietinum L.) were shown to efficiently degrade medicarpin (3-hydroxy-9-methoxypterocarpan), the main phytoalexin of this plant. Degradative studies were performed with mycelium preparations or with crude protein extracts of the fungus. Isolation and structural elucidation of 10 catabolites by chromatographic and spectroscopic techniques revealed that medicarpin degradation involves 1. reductive conversion to a 2-hydroxyisoflavan, 2. O-demethylation, 3. aromatic hydroxylation in ring A and 4. formation of a 1a-hydroxy-pterocarp-1,4-diene-3-one. As terminal aromatic catabolite 2,4-dihydroxybenzoic acid was found. A catabolic sequence for medicarpin is postulated and the results are discussed with regard to pterocarpan dissimilation by other phytopathogenic fungi.     

Free radicals generated during oxidation of green tea polyphenols: Electron paramagnetic resonance spectroscopy combined with density functional theory calculations

Electron paramagnetic resonance spectroscopy and density functional theory calculations have been used to investigate the redox properties of the green tea polyphenols (GTPs) (?)-epigallocatechin gallate (EGCG), (?)-epigallocatechin (EGC), and (?)-epicatechin gallate (ECG). Aqueous extracts of green tea and these individual phenols were autoxidized at alkaline pH and oxidized by superoxide anion (O₂^?) radicals in dimethyl sulfoxide. Several new aspects of the free radical chemistry of GTPs were revealed. EGCG can be oxidized on both the B and the D ring. The B ring was the main oxidation site during autoxidation, but the D ring was the preferred site for O₂^? oxidation. Oxidation of the D ring was followed by structural degradation, leading to generation of a radical identical to that of oxidized gallic acid. Alkaline autoxidation of green tea extracts produced four radicals that were related to products of the oxidation of EGCG, EGC, ECG, and gallic acid, whereas the spectra from O₂^? oxidation could be explained solely by radicals generated from EGCG. Assignments of hyperfine coupling constants were made by DFT calculations, allowing the identities of the radicals observed to be confirmed.     

Molecular docking,molecular modeling,and molecular dynamics studies of azaisoflavone as dual COX-2 inhibitors and TP receptor antagonists

Designed multi-target ligand (DML) is an emerging strategy for the development of new drugs and involves the engagement of multiple targets with the same moiety. In the context of NSAIDs it has been suggested that targeting the thromboxane prostanoid (TP) receptor along with cyclooxygenase-2 (COX-2) may help to overcome cardiovascular (CVS) complications associated with COXIBs. In the present work, azaisoflavones were studied for their COX-2 and TP receptor binding activities using structure based drug design (SBDD) techniques. Flavonoids were selected as a starting point based on their known COX-2 inhibitory and TP receptor antagonist activity. Iterative design and docking studies resulted in the evolution of a new class scaffold replacing the benzopyran-4-one ring of flavonoids with quinolin-4-one. The docking and binding parameters of these new compounds are found to be promising in comparison to those of selective COX-2 inhibitors, such as SC-558 and celecoxib. Owing to the lack of structural information, a model for the TP receptor was generated using a threading base alignment method with loop optimization performed using an ab initio method. The model generated was validated against known antagonists for TP receptor using docking/MMGBSA. Finally, the molecules that were designed for selective COX-2 inhibition were docked into the active site of the TP receptor. Iterative structural modifications and docking on these molecules generated a series which displays optimum docking scores and binding interaction for both targets. Molecular dynamics studies on a known TP receptor antagonist and a designed molecule show that both molecules remain in contact with protein throughout the simulation and interact in similar binding modes.

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Graphical abstract ?

     

Climate control on ring width and intra-annual density fluctuations in <Emphasis Type="Italic">Pinus kesiya</Emphasis> growing in a sub-tropical forest of Manipur,Northeast India

Key message

Growth ring study of Pinus kesiya (khasi pine) growing in sub-tropical forest in Manipur, northeast India was performed to understand climate signatures in ring widths and intra-annual density fluctuations.

Abstract

The growth rings in khasi pine (Pinus kesiya Royle ex Gordon) growing in sub-tropical Reserve Forest in Imphal, Manipur, northeast India were analysed to understand environmental signals present in ring-width series and intra-annual density fluctuations (IADFs). For this the growth ring sequences in increment core samples collected from 28 trees were precisely dated and a ring-width chronology spanning AD 1958–2014 developed. The correlation analyses between ring-width chronology and weather data of Imphal revealed that a cool April–May–June favour tree growth. The wood anatomical features of growth rings revealed the occurrence of IADFs in early- and latewoods. The IADFs in earlywood were found to be associated with reduced precipitation in months from April to July. However, the wetter conditions in late growing season, especially August/September triggered the formation of IADFs in latewood. Our findings endorse that the IADF chronologies of khasi pine could emerge as an important proxy of summer monsoon rainfall in long-term perspective in data scarce region of northeast India.

     

Double ring structure around the constricting neck of dividing plastids ofAvena sativa

Summary Ultrastructure of the constricting neck of dividing proplastids and young chloroplasts in the first leaves ofAvena sativa was examined by electron microscopy. An electron-dense, double ring structure (plastid-dividing ring doublet; PD ring doublet) with a width of 15–40 nm was revealed around the narrow neck of the constricted and dividing plastids by serial section technique. The inner and outer ring of the doublet coated the inside (stromal side) of the inner envelope membrane and the outside (cytoplasmic side) of the outer envelope membrane, respectively. However, electron-dense materials were not observed within the lumen between the outer and inner envelope membranes.Although the PD ring doublet was commonly observed in the constricted plastids with a 70–140 nm wide neck, they could be scarcely observed in the constricted plastids with a 160 or more nm wide neck. The components of the PD ring were assumed not to be concentrated enough to identify by electron microscopy in the early stage of constriction and the PD ring may be formed and recognized at the final stage.The significance of the formation of the PD ring and its role in plastokinesis (plastid kinesis) were discussed.     

Concentration-dependent palladium(II)-indole bond formation in complexes with a 2N-donor ligand containing an indole moiety: Synthesis, characterization, and reaction analysis

The Pd(II) complexes of a 2N-donor ligand containing a pendent indole, 3-(2-pyridylmethylamino)ethylindole (L), were synthesized and characterized. Reaction of the ligand with [PdCl₂(CH₃CN)₂] at room temperature gave [Pd(L)Cl₂] (1) as pale yellow crystals. The X-ray crystal structure analysis and ¹H NMR spectrum of 1 revealed that the complex has a 2N2Cl-donor set in a square-planar geometry and that the pendent indole ring has no characteristic intramolecular interaction with the Pd(II) ion and the coordinated pyridine moiety. Refluxing a solution of 1 in CH₂Cl₂/DMF for a few hours under basic conditions gave yellow crystals, which were shown to be an indole-C2 binding complex [Pd(L)Cl] (2) by X-ray analysis. Conversion of complex 1 to 2 in DMSO was observed upon dilution of the solution of complex 1. From solution equilibrium and kinetic studies the initial step of the conversion by dilution has been assigned to the replacement of a coordinated Cl⁻ ion with the DMSO molecule. The ligand replacement easily occurred at low concentrations of 1. The complex with a coordinated solvent molecule exhibited a high reactivity and formed a stable Pd-C bond with the indole ring located close to the Pd(II) center. We discussed the concentration dependent formation of the indole-C2 binding complex 2 and its detailed mechanism.     

Does “ring syndrome” exist? An analysis of 207 case reports on patients with a ring autosome

Summary Analysis of 207 case reports on patients with ring autosome showed that: (1) Forty patients, a fifth of the total, had extreme growth failure together with an otherwise almost-normal appearance, viz. no major malformation, no specific deletion syndrome, no or only a few unspecific minor anomalies. This phenotype may be regarded as the ring syndrome, a term proposed by Cote et al. (1981) since it is independent of what chromosome is involved. (2) Severe growth failure, the sole major physical abnormality in the ring syndrome, was seen significantly more often among patients with ring of larger chromosomes than among patients with a smaller ring, indicating that the greater the chromosome involved in ring formation, the higher is the probability of severe growth failure. (3) Larger ring chromosomes showed significantly more often instability than smaller rings, suggesting that there may be a correlation between ring instability and the size of the chromosome involved. (4) Growth failure was present in significantly more patients with a labile ring than with a stable ring, indicating that a correlation may exist between ring instability and growth failure. It is suggested that the ring syndrome observed in many cases with ring autosome may result from end-to-end fusion of chromosome ends, an event not involving deletion in the genetic sense. It is also suggested that the ring syndrome is caused by a continuous generation of secondary aneuploid cells with increased mortality, i.e. structural ring instability which seems to be a function of the size of the chromosome involved. Thus, formation of a ring chromosome in certain cases might be regarded as a structural mutation, i.e. an alteration in the structure of the genetic material per se, rather than a loss or gain of genetic dosages.     

A rat parotid gland cell line, Par-C10, exhibits neurotransmitter-regulated transepithelial anion secretion

Because of thelack of salivary gland cell lines suitable for Ussing chamber studies,a recently established rat parotid acinar cell line, Par-C10, was grownon permeable supports and evaluated for development of transcellularresistance, polarization, and changes in short-circuit current(I_sc) inresponse to relevant receptor agonists. Par-C10 cultures reachedconfluence in 3-4 days and developed transcellular resistancevalues of 2,000 · cm².Morphological examination revealed that Par-C10 cells grew as polarizedmonolayers exhibiting tripartite junctional complexes and the acinarcell-specific characteristic of secretory canaliculi. Par-C10I_sc was increasedin response to muscarinic cholinergic and - and -adrenergicagonists on the basolateral aspect of the cultures and to ATP and UTP(through P2Y₂ nucleotidereceptors) applied apically. Ion replacement and inhibitor studiesindicated that anion secretion was the primary factor inagonist-stimulated I_sc. RT-PCR,which confirmed the presence ofP2Y₂ nucleotide receptor mRNA inPar-C10 cells, also revealed the presence of mRNA for the cysticfibrosis transmembrane conductance regulator and ClC-2 Cl channel proteins. These findingsestablish Par-C10 cells as the first cell line of salivary gland originuseful in transcellular ion secretion studies in Ussing chambers.

     

A possible role for actin dots in the formation of the contractile ring in the ultra-micro algaCyanidium caldarium RK-1

Summary In the primitive red algaCyanidium caldarium RK-1, cytokinesis is controlled by a simple contractile ring, as in animal cells. To clarify the mechanism of formation of the contractile ring, we isolated actin genes and performed an immunocytological study.C. caldarium RK-1 has two actin genes encoding proteins with the same sequence of 377 amino acids. The primary structure indicated that the actin molecules ofC. caldarium RK-1 are typical, despite the fact that the organism is considered to be phylogenetically primitive. We prepared antiserum against aC. caldarium RK-1 actin fusion protein and indirect immunofluorescence staining was performed. In interphase cells, many actin dots were observed in the cytoplasm but none at the future cleavage plane. Prior to cytokinesis, some of these dots appeared and became aligned along the equatorial plane. At the same time, a thin immature contractile ring was observed to appear to be formed by connection of the aligned actin dots. This immature contractile ring thickened to nearly its maximum size by the time cytokinesis began. The formation of the contractile ring seemed to be a result of de novo assembly of actin monomers, rather than a result of the accumulation and bundling of pre-existing actin filaments. During the constriction of the contractile ring, no actin dots were observed in the cytoplasm. These observations suggest that actin dots are responsible for the formation of the contractile ring, but are not necessary for its disintegration. Furthermore, immunogold localization specific for actin revealed at electron microscopy level that fine filaments running just beneath the cleavage furrow are, in fact, actin filaments.Abbreviations ORF open reading frame - IPTG isopropyl--D(–)-thiogalactopyranoside - SDS-PAGE sodium dodecyl sulphate-poly-acrylamide gel electrophoresis - DAPI 4,6-diamidino-2-phenylindole     

3D-SIM Super-resolution of FtsZ and Its Membrane Tethers in Escherichia coli Cells

FtsZ, a bacterial homolog of eukaryotic tubulin, assembles into the Z ring required for cytokinesis. In Escherichia coli, FtsZ interacts directly with FtsA and ZipA, which tether the Z ring to the membrane. We used three-dimensional structured illumination microscopy to compare the localization patterns of FtsZ, FtsA, and ZipA at high resolution in Escherichia coli cells. We found that FtsZ localizes in patches within a ring structure, similar to the pattern observed in other species, and discovered that FtsA and ZipA mostly colocalize in similar patches. Finally, we observed similar punctate and short polymeric structures of FtsZ distributed throughout the cell after Z rings were disassembled, either as a consequence of normal cytokinesis or upon induction of an endogenous cell division inhibitor.The assembly of the bacterial tubulin FtsZ has been well studied in vitro, but the fine structure of the cytokinetic Z ring it forms in vivo is not well defined. Super-resolution microscopy methods including photoactivated localization microscopy (PALM) and three-dimensional-structured illumination microscopy (3D-SIM) have recently provided a more detailed view of Z-ring structures. Two-dimensional PALM showed that Z rings in Escherichia coli are likely composed of loosely-bundled dynamic protofilaments (1,2). Three-dimensional PALM studies of Caulobacter crescentus initially showed that Z rings were comprised of loosely bundled protofilaments forming a continuous but dynamic ring (1–3). However, a more recent high-throughput study showed that the Z rings of this bacterium are patchy or discontinuous (4), similar to Z rings of Bacillus subtilis and Staphylococcus aureus using 3D-SIM (5). Strauss et al. (5) also demonstrated that the patches in B. subtilis Z rings are highly dynamic.Assembly of the Z ring is modulated by several proteins that interact directly with FtsZ and enhance assembly or disassembly (6). For example, FtsA and ZipA promote ring assembly in E. coli by tethering it to the cytoplasmic membrane (7,8). SulA is an inhibitor of FtsZ assembly, induced only after DNA damage, which sequesters monomers of FtsZ to prevent its assembly into a Z ring (9). Our initial goals were to visualize Z rings in E. coli using 3D-SIM, and then examine whether any FtsZ polymeric structures remain after SulA induction. We also asked whether FtsA and ZipA localized in patchy patterns similar to those of FtsZ.We used a DeltaVision OMX V4 Blaze microscope (Applied Precision, GE Healthcare, Issaquah, WA) to view the high-resolution localization patterns of FtsZ in E. coli cells producing FtsZ-GFP (Fig. 1). Three-dimensional views were reconstructed using softWoRx software (Applied Precision). To rule out GFP artifacts, we also visualized native FtsZ from a wild-type strain (WM1074) by immunofluorescence (IF).Open in a separate windowFigure 1Localization of FtsZ in E. coli. (A) Cell with a Z ring labeled with FtsZ-GFP. (B) Rotated view of Z ring in panel A. (C) Cell with a Z ring labeled with DyLight 550 (Thermo Fisher Scientific, Waltham, MA). (D) Rotated view of Z ring in panel C. (B1 and D1) Three-dimensional surface intensity plots of Z rings in panels B and D, respectively. (E) A dividing cell producing FtsZ-GFP. The cell outline is shown in the schematic. (Asterisk) Focus of FtsZ localization; (open dashed ovals) filamentous structures of FtsZ. Three-dimensional surface intensity plots were created using the software ImageJ (19). Scale bars, 1 μm.Both FtsZ-GFP (Fig. 1, A, B, and B1) and IF staining for FtsZ (Fig. 1, C, D, and D1) consistently localized to patches around the ring circumference, similar to the B. subtilis and C. crescentus FtsZ patterns (4,5). Analysis of fluorescence intensities (see Fig. S1, A and B, in the Supporting Material) revealed that the majority of Z rings contain one or more gaps in which intensity decreases to background levels (82% for FtsZ-GFP and 69% for IF). Most rings had 3–5 areas of lower intensity, but only a small percentage of these areas had fluorescence below background intensity (34% for FtsZ-GFP and 21% for IF), indicating that the majority of areas with lower intensity contain at least some FtsZ.To elucidate how FtsZ transitions from a disassembled ring to a new ring, we imaged a few dividing daughter cells before they were able to form new Z rings (Fig. 1 E). Previous conventional microscopy had revealed dynamic FtsZ helical structures (10), but the resolution had been insufficient to see further details. Here, FtsZ visualized in dividing cells by 3D-SIM localized throughout as a mixture of patches and randomly-oriented short filaments (asterisk and dashed oval in Fig. 1, respectively). These structures may represent oligomeric precursors of Z ring assembly.To visualize FtsZ after Z-ring disassembly another way, we overproduced SulA, a protein that blocks FtsZ assembly. We examined E. coli cells producing FtsZ-GFP after induction of sulA expression from a pBAD33-sulA plasmid (pWM1736) with 0.2% arabinose. After 30 min of sulA induction, Z rings remained intact in most cells (Fig. 2 A and data not shown). The proportion of cellular FtsZ-GFP in the ring before and after induction of sulA was consistent with previous data (data not shown) (1,11).Open in a separate windowFigure 2Localization of FtsZ after overproduction of SulA. (A) Cell producing FtsZ-GFP after 0.2% arabinose induction of SulA for 30 min. (B) After 45 min. (B1) Magnified cell shown in panel B. (C) Cell producing native FtsZ labeled with AlexaFluor 488 (Life Technologies, Carlsbad, CA) 30 min after induction; (D) 45 min after induction. (D1) Magnified cell shown in panel D. Scale bars, 1 μm. (Asterisk) Focus of FtsZ localization; (open dashed ovals) filamentous structures of FtsZ.Notably, after 45 min of sulA induction, Z rings were gone (Fig. 2, B and B1), replaced by numerous patches and randomly-oriented short filaments (asterisk and dashed ovals in Fig. 2), similar to those observed in a dividing cell. FtsZ normally rapidly recycles from free monomers to ring-bound polymers (11), but a critical concentration of SulA reduces the pool of available FtsZ monomers, resulting in breakdown of the Z ring (9). The observed FtsZ-GFP patches and filaments are likely FtsZ polymers that disassemble before they can organize into a ring.We confirmed this result by overproducing SulA in wild-type cells and detecting FtsZ localization by IF (Fig. 2, C, D, and D1). The overall fluorescence patterns in cells producing FtsZ-GFP versus cells producing only native FtsZ were similar (Fig. 2, B1 and D1), although we observed fewer filaments with IF, perhaps because FtsZ-GFP confers slight resistance to SulA, or because the increased amount of FtsZ in FtsZ-GFP producing cells might titrate the SulA more effectively.Additionally, we wanted to observe the localization patterns of the membrane tethers FtsA and ZipA. Inasmuch as both proteins bind to the same C-terminal conserved tail of FtsZ (12–14), they would be expected to colocalize with the circumferential FtsZ patches in the Z ring. We visualized FtsA using protein fusions to mCherry and GFP (data not shown) as well as IF using a wild-type strain (WM1074) (Fig. 3 A). We found that the patchy ring pattern of FtsA localization was similar to the FtsZ pattern. ZipA also displayed a similar patchy localization in WM1074 by IF (Fig. 3 B).Open in a separate windowFigure 3Localization of FtsA (A) and ZipA (B) by IF using AlexaFluor 488. (C) FtsA-GFP ring. (D) Same cell shown in panel C with ZipA labeled with DyLight 550. (C1 and D1) Three-dimensional surface intensity plots of FtsA ring from panel C or ZipA ring from panel D, respectively. (E) Merged image of FtsA (green) and ZipA (red) from the ring shown in panels C and D. (F) Intensity plot of FtsA (green) and ZipA (red) of ring shown in panel E. The plot represents intensity across a line drawn counterclockwise from the top of the ring around the circumference, then into its lumen. Red/green intensity plot and three-dimensional surface intensity plots were created using the software ImageJ (19). Scale bar, 1 μm.To determine whether FtsA and ZipA colocalized to these patches, we used a strain producing FtsA-GFP (WM4679) for IF staining of ZipA using a red secondary antibody. FtsA-GFP (Fig. 3 C) and ZipA (Fig. 3 D) had similar patterns of fluorescence, although the three-dimensional intensity profiles (Fig. 3, C1 and D1) reveal slight differences in intensity that are also visible in a merged image (Fig. 3 E). Quantitation of fluorescence intensities around the circumference of the rings revealed that FtsA and ZipA colocalized almost completely in approximately half of the rings analyzed (Fig. 3 F, and see Fig. S2 A), whereas in the other rings there were significant differences in localization in one or more areas (see Fig. S2 B). FtsA and ZipA bind to the same C-terminal peptide of FtsZ and may compete for binding. Cooperative self-assembly of FtsA or ZipA might result in large-scale differential localization visible by 3D-SIM.In conclusion, our 3D-SIM analysis shows that the patchy localization of FtsZ is conserved in E. coli and suggests that it may be widespread among bacteria. After disassembly of the Z ring either in dividing cells or by excess levels of the cell division inhibitor SulA, FtsZ persisted as patches and short filamentous structures. This is consistent with a highly dynamic population of FtsZ monomers and oligomers outside the ring, originally observed as mobile helices in E. coli by conventional fluorescence microscopy (10) and by photoactivation single-molecule tracking (15). FtsA and ZipA, which bind to the same segment of FtsZ and tether it to the cytoplasmic membrane, usually display a similar localization pattern to FtsZ and each other, although in addition to the differences we detect by 3D-SIM, there are also likely differences that are beyond its ∼100-nm resolution limit in the X,Y plane.As proposed previously (16), gaps between FtsZ patches may be needed to accommodate a switch from a sparse Z ring to a more condensed ring, which would provide force to drive ring constriction (17). If this model is correct, the gaps should close upon ring constriction, although this may be beyond the resolution of 3D-SIM in constricted rings. Another role for patches could be to force molecular crowding of low-abundance septum synthesis proteins such as FtsI, which depend on FtsZ/FtsA/ZipA for their recruitment, into a few mobile supercomplexes.How are FtsZ polymers organized within the Z-ring patches? Recent polarized fluorescence data suggest that FtsZ polymers are oriented both axially and circumferentially within the Z ring in E. coli (18). The seemingly random orientation of the non-ring FtsZ polymeric structures we observe here supports the idea that there is no strong constraint requiring FtsZ oligomers to follow a circumferential path around the cell cylinder. The patches of FtsZ in the unperturbed E. coli Z ring likely represent randomly oriented clusters of FtsZ filaments that are associated with ZipA, FtsA, and essential septum synthesis proteins. New super-resolution microscopy methods should continue to shed light on the in vivo organization of these protein assemblies.     

Synthesis of {15-benzyloxy-3,7,11,17-tetraazabicyclo[11.3.1]heptadeca-1(17),13,15-triene}nickel(II) perchlorate and its analogs, and their catalytic behavior in reductive debromination of 1-bromo-4-tert-butylbenzene

New nickel(II) complexes with macrocyclic ligands bearing benzyloxy [(5), (9)], 2-methylbenzyloxy (7), 3-methylbenzyloxy (8), and hydroxy (6) groups on the pyridine ring have been synthesized. Structures of the hydroxy substituted macrocyclic ligand (L-OH·3HCl·H₂O), and the benzyloxy substituted ligand (L-OBn·3HCl) and its nickel(II) complex (5), as well as an analogous Ni(II) complex (8), have been revealed by X-ray crystallography. Their catalytic capabilities in the reductive debromination of 1-bromo-4-tert-butylbenzene have been elucidated, which has revealed that the pyridine ring can be a suitable position for the introduction of functional groups while maintaining the catalytic capabilities of the nickel(II) complexes.     

Isolation, purification, and characterization of glutathione S-transferase from oat (Avena sativa) seedlings

Glutathione S-transferase (GST) from oat seedlings was purified by ammonium sulfate precipitation and glutathione (GSH) affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of two major protein subunits with molecular masses of 29 and 31 kDa, respectively. Isoelectric focusing revealed a major band with pI of 3.43 and a minor band with pI of 7.42. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene (CDNB) as substrate revealed a K _m of 1.18 mM and V _max of 0.94 mol/min and a specific activity of 17.96 mol/min/mg. Inhibition studies indicated that oat GST is strongly inhibited by chlorophyllin, hemin, and anthocyanin and only weakly by bilirubin and biliverdin.     

First report of a planar and a quasi-planar Al<Subscript>13</Subscript><Superscript>+</Superscript> cluster having localized antiaromatic deltas within an aromatic sea: NICS,ELF, AIM,and AdNDP bonding analysis

A perfectly planar Al₁₃⁺ cluster (CI) and a quasi-planar Al₁₃⁺ cluster (CII) have been found for the first time. Both clusters have a triangular core surrounded by a set of ten Al atoms in the form of a ring. These cationic clusters have substantial aromatic character. The planar CI cluster has local antiaromatic patches within global aromatic sea. It is doubly aromatic having both σ and π aromatic character. The quasi-planar CII cluster is also aromatic but it has more σ-delocalization.

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Graphical abstract Planar and quasi-planar Al₁₃⁺ clusters with triangular core surrounded by a ring of ten atoms.

     

Analogues of taurine as inhibitors of the phosphorylation of an ≈20K molecular weight protein present in a mitochondrial fraction of the rat retina

Summary It has been previously demonstrated that taurine inhibits the phosphorylation of an 20K apparent molecular weight protein present in the mitochondrial fraction of the rat retina (Lombardini, 1991). In the present studies, various analogues of taurine were tested for their inhibitory activity on the phosphorylation of this 20K protein. The most potent analogues were (±)trans-2-aminocyclopentanesulfonic acid (TAPS) and 1,2,3,4-tetrahydroquinoline-8-sulfonic acid (THQS) which were approximately 21 and 7 times more potent than the parent compound, taurine. Median-effect plots were used to calculate the inhibitory median effect and combination index values for the combined effects of taurine and taurine analogues. From these studies, it was determined that the inhibitory taurine analogues were antagonistic to taurine when used in combination with taurine to inhibit the phosphorylation of the 20K apparent molecular weight protein. It was also concluded that: 1) the distance between the nitrogen and sulfur atoms in the taurine structure was important for inhibitory activity; 2) if the nitrogen atom is either within or attached to an unsaturated ring structure the inhibitory potency was significantly decreased, and 3) if both the sulfur and nitrogen atoms are present within the ring structure the analogue has no activity.     

Modelling variability of wood density in beech as affected by ring age,radial growth and climate

Although it has been recognized as a key parameter of wood quality and a good source of information on growth, annual wood density has been little studied within diffuse-porous trees such as beech ( Fagus sylvatica Liebl.). In this paper we examine the variability encountered in beech ring density series and analyze the influences of ring age, ring width, climate and between-tree variability on density. Thirty ring sequences were sampled from 55-year- old dominant beech trees growing within the same stand; ring density and width were measured using radiography. Ring density proved to be less variable through time than ring width. The relationship between these two variables was less than observed in ring-porous trees and it showed great variation between trees. The sensitivity of ring width and density to climate was also different; width was strongly linked to soil water deficit whereas density was correlated to temperature and August rainfall. Unlike ring width, wood density showed sensitivity towards climatic characteristics of the late growing season. A large part of annual density variability remains unexplained, even using advanced modelled water balance variables. We hypothesize that a significant part of the tree ring is under internal control. We also demonstrated great inter-tree variability (the tree effect) in ring density, which has an influence on density but not on trees response to climate.     

Waves in a simple,excitable or oscillatory,reaction-diffusion model

A simple one variable caricature for oscillating and excitable reaction-diffusion systems is introduced. It is shown that as a parameter, , varies the system dynamics change from oscillatory ( > ₀) to excitable ( < ₀) and the frequency of the oscillation vanishes as for ₀. When such dynamics are coupled by continuous diffusion in a ring geometry (1-space dimension), propagating wave trains may be found. On an infinite ring excitable devices lead to unique solitary waves which are analogous to pulse waves. A solvable example is presented, illustrating properties of dispersion, excitability, and waves. Finally it is shown that the caricature arises in a natural way from more general excitable/oscillatory systems.     

Mitochondria-cytoskeleton associations in mammalian cytokinesis

Background

The role of the cytoskeleton in regulating mitochondrial distribution in dividing mammalian cells is poorly understood. We previously demonstrated that mitochondria are transported to the cleavage furrow during cytokinesis in a microtubule-dependent manner. However, the exact subset of spindle microtubules and molecular machinery involved remains unknown.

Methods

We employed quantitative imaging techniques and structured illumination microscopy to analyse the spatial and temporal relationship of mitochondria with microtubules and actin of the contractile ring during cytokinesis in HeLa cells.

Results

Superresolution microscopy revealed that mitochondria were associated with astral microtubules of the mitotic spindle in cytokinetic cells. Dominant-negative mutants of KIF5B, the heavy chain of kinesin-1 motor, and of Miro-1 disrupted mitochondrial transport to the furrow. Live imaging revealed that mitochondrial enrichment at the cell equator occurred simultaneously with the appearance of the contractile ring in cytokinesis. Inhibiting RhoA activity and contractile ring assembly with C3 transferase, caused mitochondrial mislocalisation during division.

Conclusions

Taken together, the data suggest a model in which mitochondria are transported by a microtubule-mediated mechanism involving equatorial astral microtubules, Miro-1, and KIF5B to the nascent actomyosin contractile ring in cytokinesis.