Syntheses, crystal structures and antimicrobial activities of monomeric 8-coordinate, and dimeric and monomeric 7-coordinate bismuth(III) complexes with tridentate and pentadentate thiosemicarbazones and pentadentate semicarbazone ligands

Novel bismuth(III) complexes 1-4 with the tridentate thiosemicarbazone ligand of 2N1S donor atoms [Hmtsc (L1); 2-acetylpyridine (4N-morpholyl thiosemicarbazone)], the pentadentate double-armed thiosemicarbazone ligand of 3N2S donor atoms [H2dmtsc (L3); 2,6-diacetylpyridine bis(4N-morpholyl thiosemicarbazone)] and the pentadentate double-armed semicarbazone ligand of 3N2O donor atoms [H2dasc (L4b); 2,6-diacetylpyridine bis(semicarbazone)], were prepared by reactions of bismuth(III) nitrate or bismuth(III) chloride and characterized by elemental analysis, thermogravimetric and differential thermal analysis (TG/DTA), FTIR and NMR (1H and 13C) spectroscopy. The crystal and molecular structures of complexes 1, 2a, 2b and 4b, and the "free" ligand L1 were determined by single-crystal X-ray structure analysis. The dimeric 7-coordinate bismuth(III) complex [Bi(dmtsc)(NO3)]2, 1, and the monomeric 7-coordinate complexes [Bi(Hdasc)(H2O)](NO3)2.H2O (major product), 2a, and [Bi(dasc)(H2O)]NO3.H2O (minor product), 2b, all with pentagonal bipyramidal bismuth(III) centers, are depicted with one electron pair (6s2) of the bismuth(III) atom, deprotonated forms of multidentate thiosemicarbazone or semicarbazone ligands, and monodentate NO3 or H2O ligands, respectively. These complexes are related to the positional isomers of one electron pair of the bismuth(III) atom; 1 has an electron pair positioned in the pentagonal plane (basal position), while 2a and 2b have an electron pair in the apical position. The monomeric 8-coordinate complex [Bi(mtsc)2(NO3)], 4b, which was obtained by slow evaporation in MeOH of the 1.5 hydrates 4a, was depicted with one electron pair of the bismuth(III) atom, two deprotonated mtsc- ligand and one nitrate ion. On the other hand, crystals of the complex "[Bi(mtsc)Cl2]", 3, prepared by a reaction of BiCl3 with L1 showed several polymorphs (3a, 3b, 3c and 3d) due to coordination and/or solvation of dimethyl sulfoxide (DMSO) used in the crystallization. Bismuth(III) complexes 1 and 4a showed selective and effective antibacterial activities against Gram-positive bacteria. The structure-activity relationship was discussed.     

Isα-endorphin an uremic toxin?α-Endorphin isolated from filtrate of uremic patients with carbohydrate intolerance

Summary A 16-residue peptide was isolated from filtrate of uremic patients with carbohydrate intolerance by ultrafiltration with an Amicon Centriflo DM-5 membrane, followed by gel-filtration on Sephadex G-50 and Sephadex G-25, droplet countercurrent chromatography and high performance liquid chromatography. The hexadecapeptide thus obtained was identical as the entire amino acid sequence of-endorphin by amino acid analysis, application of the Edman degradation analysis and measurement of physical constants and analytical data of the synthetic hexadecapeptide. This result seems to suggest that an accumulation of-endorphin in uremic patients might cause carbohydrate intolerance.     

Linkage maps for the Pacific abalone (genus Haliotis) based on microsatellite DNA markers

This study presents linkage maps for the Pacific abalone (Haliotis discus hannai) based on 180 microsatellite DNA markers. Linkage mapping was performed using three F1 outbred families, and a composite linkage map for each sex was generated by incorporating map information from the multiple families. A total of 160 markers are placed on the consolidated female map and 167 markers on the male map. The numbers of linkage groups in the composite female and male maps are 19 and 18, respectively; however, by aligning the two maps, 18 linkage groups are formed, which are consistent with the haploid chromosome number of H. discus hannai. The female map spans 888.1 cM (Kosambi) with an average spacing of 6.3 cM; the male map spans 702.4 cM with an average spacing of 4.7 cM. However, we encountered several linkage groups that show a high level of heterogeneity in recombination rate between families even within the same sex, which reduces the precision of the consolidated maps. Nevertheless, we suggest that the composite maps are of significant potential use as a scaffold to further extend the coverage of the H. discus hannai genome with additional markers.     

Many faces of drebrin: from building dendritic spines and stabilizing gap junctions to shaping neurite-like cell processes

In this review we consider the multiple functions of developmentally regulated brain protein (drebrin), an actin-binding protein, in the formation of cellular polarity in different cell types. Drebrin has a well-established role in the morphogenesis, patterning and maintenance of dendritic spines in neurons. We have recently shown that drebrin also stabilizes Connexin-43 containing gap junctions at the plasma membrane. The latest literature and our own data suggest that drebrin may be broadly involved in shaping cell processes and in the formation of stabilized plasma membrane domains, an effect that is likely to be of crucial significance for formation of cell polarity in both neuronal and non-neuronal types. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional conformation changes in the TF(1)-ATPase beta subunit probed by 12 tyrosine residues.

The effect of nucleotide binding on the structure of the F(1)-ATPase beta subunit from thermophilic bacillus PS-3 (TF(1)beta) was investigated by monitoring the NMR signals of the 12 tyrosine residues. The 3,5-proton resonances of 12 tyrosine residues could be observed for the specifically deuterated beta subunit. The assignment of 3,5-proton resonances of all of the tyrosine residues was accomplished using 14 mutant proteins, in each of which one or two tyrosine residues were replaced by phenylalanine. Binding of Mg. ATP induced an upfield shift of Tyr(341) resonance, suggesting that their aromatic rings are stacked to each other. Besides Tyr(341), the signal shift observed on Mg.ATP binding was restricted to the resonances of Tyr(148), Tyr(199), Tyr(238), and Tyr(307), suggesting that Mg.ATP induces a conformational change in the hinge region. This can be correlated to the change from the open to closed conformations as implicated in the crystal structure. Mg.ADP induced a similar but distinctly different conformational change. Therefore, the intrinsic conformational change in the beta subunit induced by the nucleotide binding is proposed to be one of the essential driving forces for the F(1) rotation. Reconstitution experiments showed that Tyr(277), one of the four conserved tyrosines, is essential to the formation of the alpha(3)beta(3)gamma complex.     

Nicotine increases cancer stem cell population in MCF-7 cells

Epidemiological studies have suggested that cigarette smoking is related to increased breast cancer risk. Nicotine is most likely related to the risk in cigarette smoking. However, the mechanisms by which nicotine promotes cancer development are not fully understood. It has recently been suggested that development of breast cancer are originated from cancer stem cells, which are a minor population of breast cancer. In the present study, we investigated the effects of nicotine on the population of cancer stem cells in MCF-7 human breast cancer cells, using flow cytometry with a cancer stem cell marker aldehyde dehydrogenase (ALDH). We found that nicotine increased ALDH-positive cell population in a dose-dependent manner. We further demonstrated that a PKC-Notch pathway is involved in the effect of nicotine. In addition, the effect of nicotine was blocked by treatment with the α7 subunit-selective antagonist of nicotinic acetylcholine receptors (nAChR) α-Bungarotoxin. These data suggest that nicotine increases the stem cell population via α7-nAChR and the PKC-Notch dependent pathway in MCF-7 cells. These findings reveal a relationship between nicotine and the cancer stem cells in human breast cancer.     

Accumulation and Utilization of Dissolved Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi

The mechanism for utilization of dissolved inorganic carbon(DIC) was investigated in the marine unicellular calcareousalga Emiliania huxleyi, grown with constant aeration. The apparentK_0.5 (DIC), the concentration of DIC which attains one-halfof the maximum velocity of apparent photosynthesis, for photosyntheticevolution of O₂, measured under saturating light, was 5.5 mM(55 µM for CO₂) at pH 8.0 and 25°C. The value of K_0.5was not affected by inhibitors of carbonic anhydrase (CA), andan electrometric assay of CA showed that the enzyme was notinvolved in photosynthesis in this alga. The rate of photosyntheticfixation of ¹⁴C-DIC into acid-stable products was about 20 timeshigher than that into CaCO₃, irrespective of the external concentrationof DIC. In short-term experiments, ¹⁴C-DIC was usually incorporatedinto the internal pool of DIC (IIC) to concentrations up to13 to 16 times higher than that of the external DIC. CO₂ addedexternally was utilized mainly for fixation of CO₂ and accumulationof IIC. By contrast, HCO^-₃ was utilized mainly for productionof CaCO₃ and accumulation of IIC. Incorporation of ¹⁴C intoIIC was partially suppressed by DCMU or in darkness but itstransfer to CaCO₃ was unaffected. These results suggest thataccumulation of IIC in this alga, even under ordinary circumstances,is only partially responsible for increasing the efficiencyof utilization of DIC by photosynthetic fixation but may bemost useful for the production of CaCO₃. (Hydroxyethylidene) bisphosphonic acid, an inhibitor of thegrowth of CaCO₃ crystals, completely suppressed production ofCaCO₃. The accumulation of IIC was also partially suppressed,but photosynthetic fixation of CO₂ was enhanced. In a pulse-chaseexperiment with ¹⁴CDIC, ¹⁴C incorporated into IIC and CaCO₃in darkness was transferred to acid-stable products of photosynthesisin the light. These results suggest that ¹⁴C-DIC in IIC andpre-formed CaCO₃ may be useful sources of carbon for fixationof CO₂. (Received July 2, 1993; Accepted January 10, 1994)     

Role of Coccoliths in the Utilization of Inorganic Carbon by a Marine Unicellular Coccolithophorid, Emiliania huxleyi: a Survey Using Intact Cells and Protoplasts

The utilization of inorganic carbon and role of the coccolithswere investigated in intact cells and protoplasts of a marineunicellular calcareous alga, Emiliania huxleyi. Protoplastswith high photosynthetic activity were obtained by artificialdecalcification with 50 mM MES-NaOH (pH5.5). (1) The kineticsof the photosynthetic evolution of O₂ at various concentrationsof externally added NaHCO₃ were the same for intact cells andprotoplasts, indicating that the kinetic properties with respectto dissolved inorganic carbon (DIC) were not affected by thepresence or absence of the coccoliths on the cell surface. Double-reciprocalplots and plots of the concentration of substrate divided byvelocity (s/v) against the concentration of substrate (s) werebiphasic in the case of both intact cells and protoplasts. TheCO₂-utilization reaction was, therefore, considered to involvetwo processes with different values of K_m and V_max. From thekinetic analyses, K_m and V_max [µmoles O₂ (ml PCV)^–1h^–1] were deduced to be 92 µM and 76.3 for a "low-K_m"reaction and 4.1 mM and 252 for a "high-K_m" reaction, respectively.(2) In short-term (40-min) experiments, time courses of thetotal uptake of ¹⁴C-DIC and the incorporation of ¹⁴C into acid-stableproducts of photosynthesis and the internal pool of DIC, determinedas acid-labile compounds, under CO₂-limiting conditions (80µM) were very similar for intact cells and protoplasts.However, incorporation of ¹⁴C into CaCO₃ apparently occurredmore slowly in protoplasts than in intact cells. (3) In longterm (24-h) experiments, patterns of incorporation of ¹⁴C werealmost same for intact cells and protoplasts, with the exceptionthat the amount of ¹⁴C incorporated into CaCO₃ was much smallerin the former than the latter. The production of Ca¹⁴CO₃ increasedduring the course of 10 h after a 4-h lag. However, after 10h the level of Ca¹⁴CCO₃ started to decrease. The decrease wasaccompanied by an increase in ¹⁴C in the products of photosynthesis,suggesting that CaCO₃ was reutilized for the photosyntheticfixation of CO₂ and, therefore, that the coccoliths functionas sites of storage of DIC. However, the internal level of DICremained at the same level even after the supply of externalDIC has been almost completely depleted. (Received July 25, 1995; Accepted December 11, 1995)     

Genetic Differences Between Hatchery Stocks and Natural Populations in Pacific Abalone (<Emphasis Type="Italic">Haliotis discus</Emphasis>) Estimated Using Microsatellite DNA Markers

Genetic variations within and between nine hatchery stocks and seven natural populations of abalone including Ezo-abalone (Haliotis discus hannai) and Kuro-abalone (H. d. discus) were assayed with nine microsatellite markers. Marked reductions of genetic variability in the hatchery stocks were recognized in the allelic diversity and mean heterozygosity compared with the natural populations. Thirteen of 16 significant HWE deviations in hatchery stocks revealed heterozygotes excess, while all natural populations did not show such a tendency. Highly significant F _ST values were observed for all cases between the hatchery stocks, and between the hatchery stocks and natural populations. Genetic distance (D _A) between each hatchery stock and the geographically proximal population (mean ± SD, 0.108 ± 0.035) were similar to those estimated for between the natural Ezo-abalone and Kuro-abalone (0.101 ± 0.021). The self-assignment test, which allocated individuals to their own stock with a high success rate, provided evidence of solid genetic differences among the nine hatchery stocks. These results suggests that the allelic composition and diversity in the natural populations was not necessarily reflected in the hatchery stocks owing to population bottleneck and genetic drift through seedling process, and thus the seedling and stocking practice of these hatchery stocks should take much notice of the results to conserve the genetic diversity of natural populations.     

Role of actin cytoskeleton in dendritic spine morphogenesis

Dendritic spines are the postsynaptic receptive regions of most excitatory synapses, and their morphological plasticity play a pivotal role in higher brain functions, such as learning and memory. The dynamics of spine morphology is due to the actin cytoskeleton concentrated highly in spines. Filopodia, which are thin and headless protrusions, are thought to be precursors of dendritic spines. Drebrin, a spine-resident side-binding protein of filamentous actin (F-actin), is responsible for recruiting F-actin and PSD-95 into filopodia, and is suggested to govern spine morphogenesis. Interestingly, some recent studies on neurological disorders accompanied by cognitive deficits suggested that the loss of drebrin from dendritic spines is a common pathognomonic feature of synaptic dysfunction. In this review, to understand the importance of actin-binding proteins in spine morphogenesis, we first outline the well-established knowledge pertaining to the actin cytoskeleton in non-neuronal cells, such as the mechanism of regulation by small GTPases, the equilibrium between globular actin (G-actin) and F-actin, and the distinct roles of various actin-binding proteins. Then, we review the dynamic changes in the localization of drebrin during synaptogenesis and in response to glutamate receptor activation. Because side-binding proteins are located upstream of the regulatory pathway for actin organization via other actin-binding proteins, we discuss the significance of drebrin in the regulatory mechanism of spine morphology through the reorganization of the actin cytoskeleton. In addition, we discuss the possible involvement of an actin-myosin interaction in the morphological plasticity of spines.