Fluorescence characteristics of pyrene and phosphatidylethanolamine-bound pyrene incorporated into lipid vesicles solubilized in media of differing NaCl concentrations

We used the excimer/monomer ratio of pyrene (PY) and N-(1-pyrenesulfonyl)dipalmitoyl-L-alpha-phosphatidylethanolamine (DPPE-PY) fluorescence intensities (IE/IM), and the polarity ratio I/III to investigate the state of the polar head group region of small, unilamellar phosphatidylcholine vesicles (SUV-PC) solubilized in media of differing NaCl concentrations. PY or DPPE-PY excimer formation resulting from vesicles' collisions is not affected by the presence of monovalent ions. In addition, the ionic strength does not alter the dielectric environment in the neighborhood of PY incorporated into SUV-PC. Since IE/IM of both PY and DPPE-PY is insensitive to variations in the ionic strength, we conclude that the probes' diffusion in SUV-PC, and consequently the membrane fluidity, are independent of NaCl concentration at least up to 0.5 M. The vesicles' concentration in the aqueous solution was the only factor which induced a rise of IE/IM. To explain the results in the context of the transient-fusion model developed previously (G.P. L'Heureux and M. Fragata, Biophys. Chem. 30 (1988) 293) and the hypothesis of repulsive hydration forces, we postulate a heterogeneous distribution of dehydrated domains, or contact areas, on the outer surfaces of colliding vesicles.     

Merocyanine 540, a fluorescent probe sensitive to lipid packing

Binding of the lipophilic probe merocyanine 540 to artificial bilayers was assessed by measuring the enhancement of fluorescence which results when dye enters the hydrophobic environment of the membrane. Titration of a constant amount of dye with increasing amounts of vesicles revealed that much more dye binds to multilamellar and 1000-Å, unilamellar vesicles which are in the fluid-phase state than to comparable vesicles which are in the gel-phase state. Incorporation of cholesterol into fluid-phase vesicles at levels of greater than 20 mol% reduced dye binding, whereas cholesterol had no effect at any concentration when incorporated into gel-phase vesicles. Sonicated 200–300-Å unilamellar gel-phase vesicles, which because of their reduced radius of curvature resemble fluid-phase bilayers in their more widely spaced exterior leaflet lipids, bound more dye than 1000-Å unilameilar gel-phase vesicles constructed from the same lipid. These results suggest that merocyanine 540 is able to sense the degree of lipid packing of bilayers and inserts preferentially into bilayers whose lipids are more widely spaced.     

Modulation of concentration fluctuations in phase-separated lipid membranes by polypeptide insertion

The lateral membrane organization and phase behavior of the binary lipid mixture DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) - DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine) without and with incorporated gramicidin D (GD) as a model biomembrane polypeptide was studied by small-angle neutron scattering, Fourier-transform infrared spectroscopy, and by two-photon excitation fluorescence microscopy on giant unilamellar vesicles. The small-angle neutron scattering method allows the detection of concentration fluctuations in the range from 1 to 200 nm. Fluorescence microscopy was used for direct visualization of the lateral lipid organization and domain shapes on a micrometer length scale including information of the lipid phase state. In the fluid-gel coexistence region of the pure binary lipid system, large-scale concentration fluctuations appear. Infrared spectral parameters were used to determine the peptide conformation adopted in the different lipid phases. The data show that the structure of the temperature-dependent lipid phases is significantly altered by the insertion of 2 to 5 mol% GD. At temperatures corresponding to the gel-fluid phase coexistence region the concentration fluctuations drastically decrease, and we observe domains in the giant unilamellar vesicles, which mainly disappear by the incorporation of 2 to 5 mol% GD. Further, the lipid matrix has the ability to modulate the conformation of the inserted polypeptide. The balance between double-helical and helical dimer structures of GD depends on the phospholipid chain length and phase state. A large hydrophobic mismatch, such as in gel phase one-component DSPC bilayers, leads to an increase in population of double-helical structures. Using an effective molecular sorting mechanism, a large hydrophobic mismatch can be avoided in the DMPC-DSPC lipid mixture, which leads to significant changes in the heterogeneous lipid structure and in polypeptide conformation.     

Effect of lysophosphatidylcholine on behavior and structure of phosphatidylcholine liposomes

Differential scanning calorimetry (DSC), fluorescence polarization and X-ray diffraction were per-formed to investigate the kinetics of the micellar to the lamellar phase transition of dipalmitoylphosphatidylcholine/1-palmitoylphosphatidylcholine (16:0 LPC/DPPC) liposomes at gel phase. With a 16:0 LPC concentration up to 27 mol% only the sharp main transition with relatively high enthalpy (△H) values of DPPC was observed. Increasing 16 : 0 LPC concentration, the phase transition was broadened and the transition enthalpy was decreased and finally totally disappeared. The fluorescence probes of 3AS, 9AS, 12AS, and 16AP were employed, respectively, to detect the mo-bility of various sites of carbon chains of DPPC or 16:0 LPC/DPPC liposomes. It was shown that DPPC liposomes formed in the absence of 16:0 LPC always had a fluidity gradient in both gel and liquid-crystalline phase, while in the presence of 14.1 mol% and 27.0 mol% 16:0 LPC in the mixtures, the fluidity gradient tended to disappear below 40℃:     

Perturbation of phospholipid bilayers by DDT

The localization of the effects of DDT (5–50 mol%) addition on the acyl chain dynamics in unilamellar vesicles of two phosphatidylcholines (DPPC and egg PC) has been investigated by steady-state fluorescence polarization of a series of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12 and 16) whose fluorophore is located at a graded series of depths from the surface to the centre of the bilayer. The results show that DDT is a fluidizer of DPPC and egg PC bilayers. The increase in microviscosity of DPPC bilayers at 23°C begins at the centre of the bilayer (5 mol% DDT) and proceeds outward to the surface with increasing concentration of DDT (17 mol%). This pattern of effects is not evident in fluid bilayers of DPPC at 54°C or egg PC at 23°C. DDT (33 mol%) also lowers the phase transition temperature of DPPC bilayers by approximately 2 Cdeg. DDT (17 mol%) had no effect on the mean excited fluorescence life-time of 2-AP and 12-AS in DPPC, DOPC and egg PC bilayers. No quenching of 2-AP fluorescence was evident.     

Intermediates in the refolding of urea-denatured dimeric arginine kinase from Stichopus japonicus

The refolding of urea-denatured dimeric AK was investigated by both equilibrium and kinetic measurements. Both studies indicated that the refolding of dimeric AK is a multiphasic process. The equilibrium studies, monitored by enzyme activity, intrinsic protein fluorescence, circular dichroism (CD), 1-anilinonaphtalene-8-sulfonate (ANS) binding, size-exclusion chromatography and glutaraldehyde cross-linking showed that there were at least two intermediates involved in this process: I₁ (existing in 1.8–1.4 M urea) and I₂ (existing in 0.8–0.4 M urea). I₁ was a monomeric intermediate and possessed characteristic similar to the globular folding intermediates described in the literature. I₂ was an active native-like intermediate. The kinetic studies suggested that the refolding of AK possessed a burst phase, fast phase and slow phase, which involved at least the burst phase intermediates (I_B). Comparison of the properties of these intermediates suggested that I_B in the kinetic process corresponded to I₁ in the equilibrium process. Based on these results, a scheme for refolding of urea-denatured AK was proposed.     

Effect of cholesterol on the formation of an interdigitated gel phase in lysophosphatidylcholine and phosphatidylcholine binary mixtures

We previously reported that 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) forms an interdigitated gel phase in the presence of 1-palmitoyl-sn-glycero-3-phosphocholine (16:0LPC) at concentrations below 30 mol%. In the present investigation, fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), X-ray diffraction, and differential scanning calorimetry (DSC) were used to investigate the effect of cholesterol on the phase behavior of 16:0LPC/DPPC binary mixtures. At 25 degrees C, 30 mol% 16:0LPC significantly decreases the DPH fluorescence intensity during the transition of DPPC from the L(beta') phase to the L(betaI) phase. However, the addition of cholesterol to 16:0LPC/DPPC mixtures results in a substantial increase in fluorescence intensity. The changes in DPH fluorescence intensity reflect the probe's redistribution from an orientation parallel to the acyl chain to the center of the bilayer, suggesting a bilayer structure transition from interdigitation to noninterdigitation. The normal repeat period of small angle X-ray diffraction patterns can be restored and a reflection appears at 0.42 nm with a broad shoulder around 0.41 nm in wide angle X-ray diffraction patterns when 10 mol% cholesterol is incorporated into 30 mol% 16:0LPC/DPPC vesicles, indicating that the mixtures are in the gel phase (L(beta')). Moreover, DSC results demonstrate that 10 mol% cholesterol is sufficient to significantly decrease the main enthalpy, cooperativity and lipid chain melting of 30 mol% 16:0LPC/DPPC binary mixtures, which are L(betaI), indicating that the transition of the interdigitated phase is more sensitive to cholesterol than that of the noninterdigitated phase. Our data imply that the interdigitated gel phase induced by 16:0LPC is prevented in the presence of 10 mol% cholesterol, but unlike ethanol, an increasing concentration of 16:0LPC is not able to restore the interdigitation structure of the lipid mixtures.     

Determination of closantel residues in plasma and tissues by high-performance liquid chromatography with fluorescence detection

The influence of the pH of the mobile phase with some modifiers on the chromatographic behavior and fluorescence properties of closantel have been investigated. At acidic pH values (2–6), the benzamide moiety of the closantel forms a six-membered ring by hydrogen bonding and possesses a native fluorescence. Using the fluorescence emission of closantel at λ_ex=335 nm, λ_em=510 nm, and pH 2.5 of the mobile phase, a linear calibration curve was estimated over a concentration range of about two orders of magnitude with a correlation coefficient larger than 0.992. The limit of the fluorescence detection was 10 μg/kg. This value was at least 10 times lower than that using UV detection. The method was applied to the determination of closantel in plasma and tissue samples, purified by a solid-phase extraction with C₁₈ cartridges.     

Structural characterization of cationic DODAB bilayers containing C24:1 β-glucosylceramide

The effect of 5 mol%, 9 mol%, and 16 mol% of C24:1 β-glucosylceramide (βGlcCer) on the structure of cationic DODAB bilayers was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR) spectroscopy and fluorescence microscopy. βGlcCer is completely miscible with DODAB at all fractions tested, since no domains were observed in fluorescence microscopy or ESR spectra. The latter showed that βGlcCer destabilized the gel phase of DODAB bilayers by decreasing the gel phase packing. As a consequence, βGlcCer induced a decrease in the phase transition temperature and cooperativity of DODAB bilayers, as seen in DSC thermograms. ESR spectra also showed that βGlcCer induced an increase in DODAB fluid phase order and/or rigidity. Despite their different structures, a similar effect of loosening the gel phase packing and turning the fluid phase more rigid/organized has also been observed when low molar fractions of cholesterol were incorporated in DODAB bilayers. The structural characterization of mixed membranes made of cationic lipids and glucosylceramides may be important for developing novel immunotherapeutic tools such as vaccine adjuvants.     

Composition of triglycerides from aphids of six different families and from different seasonal forms of Aphis evonymi

The triglyceride compositions of extracts and cornicle secretions of specimens from a range of aphid families were examined by mass spectrometry and found to support earlier evidence that there is little correlation between taxonomic position and chemical constitution.

Parasites developing within Myzus persicae preferentially consumed the typical aphid triglycerides which contain hexanoic (C₆), sorbic (C_6:2), myristic (C₁₄), and palmitic (C₁₆) acid moieties, so that when parasitism was advanced, only triglycerides with three long-chain fatty acids per molecule remained. The adult parasites themselves contained very little triglyceride.

Triglycerides of Aphis evonymi and Aphis fabae were similar and varied in composition regularly throughout the year. In the apterous and alate (summer) viviparae, there were equal amounts of myristoyl (C₁₄) and palmitoyl (C₁₆) triglycerides, while in fundatrices and fundatrigeniae (spring forms) myristoyl was predominant. Males collected in autumn resembled the spring forms. However, oviparae and eggs of these and other species differed considerably from the other seasonal forms, in having more hexenoic (C_6:1), sorbic (C_6:2), and myristoleic (C_14:1) acid moieties.     

Effect of calcium on phospholipid interaction with pulmonary surfactant protein C.

Porcine pulmonary surfactant-associated protein SP-C was incorporated into bilayers of chain-perdeuterated dipalmitoylphosphatidylglycerol (DPPG-d62) and chain-perdeuterated dipalmitoyl-phosphatidylcholine (DPPC-d62) and into bilayers containing 70 mol% dipalmitoyl-phosphatidylcholine (DPPC) and 30 mol% DPPG-d62 or 70 mol% DPPC-d62 and 30 mol% dipalmitoylphosphatidylglycerol (DPPG). The effect of SP-C on the phase behavior, lipid chain order, and dynamics in these bilayers was examined by using deuterium nuclear magnetic resonance. SP-C was found to have a similar effect on the chain order and phase behavior of DPPC-d62 and DPPG-d62 in bilayers with a single lipid component. In gel phase DPPC/DPPG (7:3) bilayers with one or the other lipid component chain-perdeuterated, SP-C was found to affect first spectral moment more strongly for DPPG-d62 than for DPPC-d62. This may indicate that SP-C induced a nonrandom lateral distribution in the mixed lipid bilayer. SP-C was also found to influence motions responsible for deuteron transverse relaxation in both the gel and liquid crystalline phases. The presence of 5 mM Ca2+ in the aqueous phase substantially altered the effect of SP-C on transverse relaxation in the bilayer.     

Perturbations induced by alpha- and beta-endosulfan in lipid membranes: a DSC and fluorescence polarization study.

The interaction of alpha- and beta-endosulfan isomers with lipid bilayers was searched by differential scanning calorimetry (DSC) and fluorescence polarization of 2-, 6- and 12-(9-anthroyloxy) stearic acids (2-AS, 6-AS and 12-AS) and 16-(9-anthroyloxy) palmitic acid (16-AP). Both endosulfan isomers, at insecticide/lipid molar ratios ranging from 1/40 to 1/1, shift the phase transition midpoint to lower temperature values and broaden the transition profile of dipalmitoylphosphatidylcholine (DPPC) bilayers. At insecticide/lipid molar ratios of 1/40, the isomers fully abolish the bilayer pretransition. Conversely to beta-endosulfan, alpha-endosulfan promotes a new phase transition, centered at 35.4 degrees C, in addition to the main phase transition of DPPC. Therefore, the alpha-isomer may undergo a heterogeneous distribution in separate domains in the plane of the membrane, whereas the beta-isomer may undergo a homogeneous distribution. Fluorescence polarization data indicate that alpha-endosulfan increases the lipid structural order in the regions probed by 2-AS and decreases it in the regions probed by 6-AS, 12-AS and 16-AP. On the other hand, the beta-isomer produces disordering effects in the upper regions of the bilayers, probed by 2-AS, and ordering in deeper regions, probed by 6-AS, 12-AS and 16-AP, mainly in the gel phase. The incorporation of cholesterol into DPPC bilayers progressively decreases the effects of beta-isomer which are vanished at 20 mol% cholesterol. However, this and higher cholesterol concentrations did not prevent alpha-endosulfan membrane interaction, as revealed by DSC and fluorescence polarization. The distinct effects promoted by alpha- and beta-endosulfan are discussed in terms of molecular orientation and positioning within the bilayer. Apparently, the alpha-isomer preferentially locates closer to the phospholipid headgroups whereas the beta-isomer distributes in deeper domains of the bilayer.     

Alterations in the organization of phosphatidylcholine/cholesterol bilayers by tetrahydrocannabinol

The interactions of delta 9-tetrahydrocannabinol (THC) with various phosphatidylcholines (PCs) was studied in model membranes by differential scanning calorimetry. THC present in PC bilayers above a certain concentration complexed stoichiometrically with phospholipids containing both saturated and unsaturated fatty acids. When the bilayer PCs were sufficiently dissimilar for phase separation to occur, THC preferentially associated with the lower melting point lipid. The presence of cholesterol below 20 mol% in dipalmitoylphosphatidylcholine bilayers enhanced THC X PC complex formation. Above 20 mol% cholesterol, there was no indication of THC X dipalmitoylphosphatidylcholine complex formation. This is in agreement with a phase rearrangement occurring in PC bilayers at concentrations of cholesterol of approximately 20 mol%. These studies suggest several possible mechanisms for the modulation of membrane activities by hydrophobic drugs such as THC.     

Calcium-induced lipid phase separations and interactions of phosphatidylcholine/anionic phospholipid vesicles. Fluorescence studies using carbazole-labeled and brominated phospholipids

A novel method that uses a carbazole-labeled fluorescent phosphatidylcholine, which partitions preferentially into liquid-crystalline lipid domains, to monitor the kinetics and the extents of thermotropic and ionotropic lateral phase separations in vesicles combining brominated and nonbrominated phosphatidylcholines (PCs), phosphatidic acids (PAs), and phosphatidylserines (PSs) is described. The calcium-induced segregation of several nonbrominated PA species in liquid-crystalline brominated PC bilayers behaves as a well-defined lateral phase separation; the residual solubility of the PA component in the PC-rich phase in the presence of calcium can vary severalfold depending on the PA acyl chain composition. PC/PS mixtures show a pronounced tendency to form metastable solutions in the presence of calcium, particularly when they contain less than equimolar proportions of PS. This metastability is not readily relaxed by repeated freeze-thawing of vesicles in the presence of calcium, by avidin-mediated contacts between PC/PS vesicles containing biotinylated lipids, or by calcium-induced lateral segregation of PA in the same vesicles. Different PS species exhibit different apparent residual solubilities in liquid-crystalline PC bilayers, ranging from less than 10 mol % for dimyristoyl-PS to ca. 45 mol% for dioleoyl-PS, after prolonged incubations of PC/PS multilamellar vesicles with excess calcium. Results are presented, obtained by using the above lipid-segregation assay and parallel assays of intervesicle lipid mixing, that raise questions concerning the relevance of the equilibrium behavior of calcium-treated PS/PC mixtures to the relatively rapid interactions (fusion and lipid mixing) of PC/PS vesicles that follow initial exposure to calcium.     

Iodide penetration into lipid bilayers as a probe of membrane lipid organization.

The quenching efficiency of iodide as a penetrating fluorescence quencher for a membrane-associated fluorophore was utilized to measure the molecular packing of lipid bilayers. The KI quenching efficiency of tryptophan-fluorescence from melittin incorporated in DMPC bilayer vesicles peaks at the phase transition temperature (24 degrees C) of DMPC, whereas acrylamide quenching efficiency does not depend on temperature. The ability of iodide to penetrate the hydrocarbon region of the bilayer was examined by measuring the fluorescence quenching of the pyrene-phosphatidylcholine incorporated into DMPC vesicles (pyrene was attached to the 10th carbon of the sn-2 chain). The quenching efficiency of pyrene by iodide again shows a maximum at the lipid phase transition. We conclude that iodide penetrates the membrane hydrocarbon region at phase transition through an increased number of bilayer defects. The magnitude of change in quenching efficiency of iodide during lipid phase transition provides a sensitive technique to probe the lipid organization in membranes.     

Effects of lindane on membrane fluidity: intramolecular excimerization of a pyrene derivative and polarization of diphenylhexatriene.

Fluorescence polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH) have been compared with the excimer/monomer fluorescence intensity ratio (I'/I) of 1,3-di(2-pyrenyl)propane, (2Py(3)2Py). This ratio permits evaluation of changes in fluidity of the outer regions of the bilayer, where 2Py(3)2Py preferentially distributes. On the other hand, fluorescence polarization of DPH reports the structural order of the bilayer core. In the fluid phase of DMPC bilayers, for lindane concentrations higher than 25 microM, the excimer/monomer fluorescence intensity ratio (I'/I) decreases, thus reflecting an order increase of the probe environment. However, in the same conditions, the fluorescence polarization of DPH is almost insensitive to any perturbation. Identical results have been obtained in other pure lipid bilayers, namely DPPC and DSPC. However, both probes detect disordering effects of lindane in the gel phase of these lipids. The pyrene probe, unlike DPH, is very sensitive to the pretransitions of DPPC and DSPC, removed in the presence of lindane. Both probes fail to detect any apparent effect of lindane in DMPC bilayers enriched with high cholesterol content (greater than 30 mol%). However, in DMPC bilayers with low cholesterol content (less than 30 mol%), for temperatures below the phase transition of DMPC, both probes detect fluidizing effects induced by lindane. Nevertheless, above the phase transition of DMPC, 2Py(3)2Py detects ordering effects of lindane, whereas DPH detects hardly any effect. These results in DMPC bilayers with low cholesterol content are qualitatively similar to those described for DMPC without cholesterol.     

许氏平鲉发育早期的氨基酸与脂肪酸组成及变化

为了研究许氏平鲉(Sebastes schlegelii)雌鱼体内受精后仔鱼开口前和仔鱼开口后两个阶段氨基酸和脂肪酸的组成变化规律, 采用生化常规方法定量测定并分析了许氏平鲉发育早期的受精卵(FE)、胚胎期(ES)、初产仔鱼(PL₁)、前仔鱼期(PL₂)、后仔鱼期(PL₃)和稚鱼期(J)6个阶段的氨基酸和脂肪酸组成特点及含量变动。结果表明: 总氨基酸含量从FE至PL₁显著下降, 至PL₃显著上升, 至J又显著下降(P<0.05); 游离氨基酸含量以FE最低(12.77 mg/g), 从FE到PL₁显著上升(P<0.05), 并在PL₁含量达到最高值(92.19 mg/g), PL₁发育到J呈现先下降后上升再下降的变化趋势(P<0.05), 游离氨基酸与总氨基酸的比值范围在2.37%—19.66%。在各发育阶段干样中检出碳链长度在C₁₄-C₂₄的29种脂肪酸, 分别为9种饱和脂肪酸(SFA)、9种单不饱和脂肪酸(MUFA)和11种多不饱和脂肪酸为(PUFA), 受精卵中主要脂肪酸依次为C_22:6n-3(DHA)、C_18:ln-9c、C_16:0和C_20:5n-3(EPA)。胚胎期(FE-ES)的脂肪酸利用率顺序为SFA、MUFA、n-6PUFA、n-3PUFA, 主要以C_18:3n-3、C_18:0、C_16:1n-7及C_20:5n-3(EPA)作为胚胎期的能量来源, C_22:6n-3(DHA)的实际利用率最低(9.71%), 被优先保存下来, C_16:0的实际利用量最高(10.94mg/g); 仔鱼内源营养阶段(ES-PL₁)脂肪酸利用率顺序为MUFA、n-6PUFA、SFA、n-3PUFA, 主要以C_16:1n-7、C_18:0、C_20:4n-6(ARA)及C_18:1n-9c作为开口前仔鱼的主要能量来源, 其中仔鱼对DHA实际利用量最高(18.23 mg/g)。PL₁-PL₃阶段DHA相对于EPA和ARA被选择性利用; PL₃至J阶段ARA相对于EPA和DHA被选择性利用。研究表明: 许氏平鲉仔鱼开口前阶段总氨基酸含量与游离氨基酸含量的变化趋势截然相反, 胚胎期与仔鱼内源营养阶段脂肪酸利用率和利用量均有所不同, 仔鱼期DHA优先被利用, 过渡至稚鱼期ARA优先被利用。建议在仔鱼开口后添加富含DHA生物性饵料, 仔鱼过渡到稚鱼期在配合饵料中添加ARA营养物质, 防止苗种营养不足, 保证成活率。     

Assessment of fatty acids in cardiac tissue as 9-anthryldiazomethane esters by high-performance liquid chromatography

A high-performance liquid chromatographic technique for the rapid assessment of fatty acids in cardiac tissue is described. A level of 50.4 ± 14.9 nmol fatty acids per g wet weight of rat myocardial tissue could be monitored. The content of the individual fatty acids C_14:0, C_16:0, C_16:1, C_18:0, C_18:1, C_18:2 and C_20:4 amounted to 1.9, 13.5, 0.6, 14.4, 6.1, 6.5 and 7.2 nmol/g wet weight, respectively. A comparison of this method with a well established gas chromatographic technique yielded good agreement. In contrast with time-consuming gas chromatographic techniques, there is no need to isolate (unesterified) fatty acids from the other lipid classes with column chromatography or thin-layer chromatography, because the derivatizing reagent 9-anthryldiazomethane reacts highly specifically with fatty acids.     

Deinococcus misasensis and Deinococcus roseus, novel members of the genus Deinococcus, isolated from a radioactive site in Japan

Two gamma- and UV-radiation resistant, Gram-positive, red- or pink-pigmented, rod-shaped, strictly aerobic, oxidase- and catalase-positive bacterial strains, TDMA-25^T and TDMA-uv51^T, were isolated from fresh water collected at Misasa, a radioactive site in Japan. Phylogenetic analysis based on 16S rRNA gene sequences placed both in a distinct lineage in the family Deinococcaceae, and the highest degrees of sequence similarity determined belonged to Deinococcus maricopensis LB-34^T (88.8–89.3%), Deinococcus pimensis KR-235^T (86.4–86.7%) and Deinococcus yavapaiensis KR-236^T (86.1%). The DNA G+C content of the strains was 53–58 mol%. The major respiratory quinone was MK-8. The predominant fatty acids were C_15:0 iso, C_16:0 iso, C_13:0 iso, C_17:0 iso, C_16:0, C_13:0 anteiso, C_15:0 and C_12:0 iso. The strains degraded gelatin, casein, starch and Tween 80. Unique physiological characteristics, differences in their fatty acid profiles, and genotypic and phylogenetic features, differentiated strains TDMA-25^T and TDMA-uv51^T from closely related Deinococcus species. Hence, the two strains are described as novel species of the genus Deinococcus. The names Deinococcus misasensis sp. nov. (type strain TDMA-25^T=JCM 14369=NBRC 102116=CCUG 53610) and Deinococcus roseus sp. nov. (type strain TDMA-uv51^T=JCM 14370=NBRC 102117=CCUG 53611) are proposed.     

Amino-functionalized zirconocene (C5H4CH(Me)NMe2)2ZrCl2, a catalyst for the dehydropolymerization of PhSiH3: Probing of peripheral substituent effects on catalyst dehydropolymerization activity

The amino-functionalized metallocene (C₅H₄CH(Me)NMe₂)₂ZrCl₂, [(Cp^N)₂ZrCl₂] was synthesized by salt metathesis of ZrCl₄ and 2 equiv. of C₅H₄CH(Me)NMe₂Li. The metallocene was obtained in good yield as a mixture of rac and meso diastereomers as established by NMR spectroscopy. The addition of 2 equiv. of n-BuLi to the metallocene (Cp^N)₂ZrCl₂ produced a co-catalyst system which was active, at a 1.0 mol% loading, in the dehydropolymerization of PhSiH₃ to poly(phenylsilane), PPSi. The PPSi was obtained as a 9:1 linear–cyclic mixture (M_w=3850, M_n=2300) as established by GPC analysis; ²⁹Si{¹H} NMR spectroscopy revealed an atactic polymer microstructure.