A simple method for measuring signs of <Superscript>1</Superscript>H<Superscript>N</Superscript> chemical shift differences between ground and excited protein states

NMR relaxation dispersion spectroscopy is a powerful method for studying protein conformational dynamics whereby visible, ground and invisible, excited conformers interconvert on the millisecond time-scale. In addition to providing kinetics and thermodynamics parameters of the exchange process, the CPMG dispersion experiment also allows extraction of the absolute values of the chemical shift differences between interconverting states, | \Updelta [(w)\tilde] | \left| {\Updelta \tilde{\omega }} \right| , opening the way for structure determination of excited state conformers. Central to the goal of structural analysis is the availability of the chemical shifts of the excited state that can only be obtained once the signs of \Updelta [(w)\tilde] \Updelta \tilde{\omega } are known. Herein we describe a very simple method for determining the signs of ¹H^N \Updelta [(w)\tilde] \Updelta \tilde{\omega } values based on a comparison of peak positions in the directly detected dimensions of a pair of ¹H^N–¹⁵N correlation maps recorded at different static magnetic fields. The utility of the approach is demonstrated for three proteins that undergo millisecond time-scale conformational rearrangements. Although the method provides fewer signs than previously published techniques it does have a number of strengths: (1) Data sets needed for analysis are typically available from other experiments, such as those required for measuring signs of ¹⁵N \Updelta [(w)\tilde] \Updelta \tilde{\omega } values, thus requiring no additional experimental time, (2) acquisition times in the critical detection dimension can be as long as necessary and (3) the signs obtained can be used to cross-validate those from other approaches.     

Finite element–based injury metrics for pulmonary contusion via concurrent model optimization

This study explores the relationship between impact severity and resulting pulmonary contusion (PC) for four impact conditions using a rat model of the injury. The force–deflection response from a Finite Element (FE) model of the lung was simultaneously matched to experimental data from distinct impacts via a genetic algorithm optimization. Sprague-Dawley rats underwent right-side thoracotomy prior to impact. Insults were applied directly to the lung via an instrumented piston. Five cohorts were tested: a sham group and four groups experiencing lung insults of varying degrees of severity. The values for impact velocity (V) and penetration depth (D) of the cohorts were Group 1, (V = 6.0 m · s⁻¹, D = 5.0 mm), Group 2, (V = 1.5 m · s⁻¹,D = 5.0 mm), Group 3, (V = 6 m · s⁻¹, D = 2.0 mm), and Group 4, (V = 1.5 m · s⁻¹, D = 2.0 mm). CT scans were acquired at 24 h, 48 h, and 1 week post-insult. Contusion volume was determined through segmentation. FE-based injury metrics for PC were determined at 24 h and 1 week post-impact, based on the observed volume of contusion and first principal strain. At 24 h post-impact, the volume of high radiopacity lung (HRL) was greatest for the severe impact group (mean HRL = 9.21 ± 4.89) and was significantly greater than all other cohorts but Group 3. The concurrent optimization matched simulated and observed impact energy within one standard deviation for Group 1 (energy = 3.88 ± 0.883 mJ, observed vs. 4.47 mJ, simulated) and Group 2 (energy = 1.46 ± 0.403 mJ, observed vs. 1.50 mJ, simulated) impacts. Statistically significant relationships between HRL and impact energy are presented. The FEA-based injury metrics at 24 h post-contusion are e_max·[(e)\dot]_max{\varepsilon_{\max}\cdot \dot {\varepsilon}_{\max}} exceeding 94.5 s⁻¹, ε _max exceeding 0.284 and [(e)\dot]_max{\dot{\varepsilon}_{\max}} exceeding 470 s⁻¹. Thresholds for injury to the lung still present at 1 week post-impact were also determined. They are e_max·[(e)\dot]_max{\varepsilon_{\max}\cdot \dot {\varepsilon}_{\max}} exceeding 149 s⁻¹, ε _max exceeding 0.343 and [(e)\dot]_max{\dot{\varepsilon}_{\max}} exceeding 573 s⁻¹. A mesh sensitivity study found that thresholds based on strain rate were more sensitive to changes to mesh density than the threshold based on strain only.     

Minichromosome stability induced by partial genome duplication in Arabidopsis thaliana

Two partially reconstructed karyotypes (RK1 and RK2) of Arabidopsis thaliana have been established from a transformant, in which four structurally changed chromosomes (α, β, γ, and δ) were involved. Both karyotypes are composed of 12 chromosomes, 2n = 1￠￠+ 3￠￠+ 4￠￠+ 5￠￠+ a￠￠+ g￠￠ = 12 {2}n = {1}\prime \prime + {3}\prime \prime + {4}\prime \prime + {5}\prime \prime + \alpha \prime \prime + \gamma \prime \prime = {12} for RK1 and 2n = 3￠￠+ 4￠￠+ 5￠￠+ a￠￠+ b￠￠+ g￠￠ = 12 {2}n = {3}\prime \prime + {4}\prime \prime + {5}\prime \prime + \alpha \prime \prime + \beta \prime \prime + \gamma \prime \prime = {12} for RK2, and these chromosome constitutions were relatively stable at least for three generations. Pairing at meiosis was limited to the homologues (1, 3, 4, 5, α, β, or γ), and no pairing occurred among non-homologous chromosomes in both karyotypes. For minichromosome α (mini α), precocious separation at metaphase I was frequently observed in RK2, as found for other minichromosomes, but was rare in RK1. This stable paring of mini α was possibly caused by duplication of the terminal tip of chromosome 1 that is characteristic of RK1.     

Web-FACE: a new canopy free-air CO<Subscript>2</Subscript> enrichment system for tall trees in mature forests

The long-term responses of forests to atmospheric CO₂ enrichment have been difficult to determine experimentally given the large scale and complex structure of their canopy. We have developed a CO₂ exposure system that uses the free-air CO₂ enrichment (FACE) approach but was designed for tall canopy trees. The system consists of a CO₂-release system installed within the crown of adult trees using a 45-m tower crane, a CO₂ monitoring system and an automated regulation system. Pure CO₂ gas is released from a network of small tubes woven into the forest canopy (web-FACE), and CO₂ is emitted from small laser-punched holes. The set point CO₂ concentration ([CO₂]) of 500 µmol mol^-1 is controlled by a pulse-width modulation routine that adjusts the rate of CO₂ injection as a function of measured [CO₂] in the canopy. CO₂ consumption for the enrichment of 14 tall canopy trees was about 2 tons per day over the whole growing season. The seasonal daytime mean CO₂ concentration was 520 µmol mol^-1. One-minute averages of CO₂ measurements conducted at canopy height in the center of the CO₂-enriched zone were within ᆨ% and ᆞ% of the target concentration for 76% and 47% of the exposure time, respectively. Despite the size of the canopy and the windy site conditions, performance values correspond to about 75% of that reported for conventional forest FACE with the added advantage of a much simpler and less intrusive infrastructure. Stable carbon isotope signals captured by 80 Bermuda grass (Cynodon dactylon) seedlings distributed within the canopy of treated and control tree districts showed a clearly delineated area, with some nearby individuals having been exposed to a gradient of [CO₂], which is seen as added value. Time-integrated values of [CO₂] derived from the C isotope composition of C. dactylon leaves indicated a mean (-SD) concentration of 513ᇓ µmol mol^-1 in the web-FACE canopy area. In view of the size of the forest and the rough natural canopy, web-FACE is a most promising avenue towards natural forest experiments, which are greatly needed.     

Studying Genetic Code by a Matrix Approach

Following Petoukhov and his collaborators, we use two length n zero-one sequences, α and β, to represent a length n genetic sequence ((a) || (b)){\alpha\choose\beta} so that the columns of ((a) || (b)){\alpha\choose\beta} have the following correspondence with the nucleotides: C ~ (0 || 0)C\sim{0\choose0} , U ~ (1 || 0)U\sim{1\choose0} , G ~ (1 || 1)G\sim{1\choose1} , A ~ (0 || 1)A\sim{0\choose1} . Using the Gray code ordering to arrange α and β, we build a 2 ⁿ ×2 ⁿ matrix C _n including all the 4 ⁿ length n genetic sequences. Furthermore, we use the Hamming distance of α and β to construct a 2 ⁿ ×2 ⁿ matrix D _n . We explore structures of these matrices, refine the results in earlier papers, and propose new directions for further research.     

In situ oxygen microelectrode measurements of bottom-ice algal production in McMurdo Sound, Antarctica

In-situ estimates of fast-ice algal productivity at Cape Evans, McMurdo Sound, in 1999 were lower than at the same site in previous years. Under-ice irradiance was between 0 and 8 µmol photons m^-2 s^-1; the ice was between 1.9 and 2.0 m thick and the algal biomass averaged 150 mg chl a m^-2, although values as high as 378 mg chl a m^-2 were recorded. Production on 11 and 12 November was between 0.053 and 1.474 mg C m^-2 h^-1. When the data from 11 November were fitted to a hyperbolic tangent function, a multilinear regression gave estimates for P_max of 0.571 nmol O₂ cm^-2 s^-1, an ! of 0.167 nmol O₂ cm^-2 s^-1 µmol^-1 photons m^-2 s^-1 and an E_k of 3.419 µmol photons m^-2 s^-1. A P_max of 2.674 nmol O₂ cm^-2 s^-1, an ! of 0.275 nmol O₂ cm^-2 s^-1 µmol^-1 photons m^-2 s^-1, r of 0.305 nmol O₂ cm^-2 s^-1 and an E_k of 9.724 µmol^-1 photons m^-2 s^-1 were estimated from the 12 November data. The sea-ice algal community was principally comprised of Nitzschia stellata, Entomoneis kjellmanii and Berkeleya adeliensis. Other taxa present included N. lecointei, Fragilariopsis spp., Navicula glaciei, Pleurosigma spp. and Amphora spp. Variations in the method for estimating the thickness of the diffusive boundary layer were not found to significantly affect the measurements of oxygen flux. However, the inability to accurately measure fine-scale variations in biomass is thought to contribute to the scatter of the P versus E data.     

The Composition of Phloem Exudate and Xylem Sap from Broccoli (Brassica oleracea var. italica) Supplied with NH4+, NO""3 or NH4NO3

Shelp, B. J. 1987. The composition of phloem exudate and xylemsap from broccoli (Brassica oleracea var. italica) suppliedwith NH⁺₄, NO₃ or NH₄NO₃.—J. exp. Bot. 38: 1619–1636. The detailed composition of xylem sap and exudate from stemincisions of attached inflorescences of broccoli (Brassica oleraceavar. italica) was compared in plants supplied with NH⁺₄, NO₃or NH₄NO₃. A phloem origin for the exudate was suggested fromthe high levels of sugars (71–133 mg cm^-3), amino acids(8·1-26·7 mg cm³) and K. (2·3–3·8mg cm³), the low levels of NO₃ and Ca, the high C: N (w/w) ratios(8·3–33), and the alkaline pH (7·2–7·3).In contrast, the xylem sap was mildly acidic (pH 5·6–6·0),and possessed lower levels of all organic and inorganic solutesbut NO₃ and Ca, and lower ratios of K: Ca, Mg: Ca and C: N (0·6–4·4). Glutamine was the predominant o-phthalaldehyde-reactive aminocompound in both transport fluids with the next most abundantamino acids dependent on sap type and N-form. Together witharginine, -aminobutyric acid, which was found only in the xylemstream, was enhanced by NH⁺₄compared to NO₃ -nutrition suggestingthat glutamate metabolism was stimulated in the roots. Underlimiting N the amino acid concentrations in the transport fluidswere greater with NH⁺₄ than with NO₃. NO₃ reduction occurredin both the root and shoot with the latter site predominatingover the entire N range (0-300 mol m³). Even though the compositionof nitrogenous solutes in the xylem was dependent on cultivarand N source, the composition of the phloem streams supplyingthe developing inflorescence was relatively unaffected. The data on the element composition of organs and phloem sapare interpreted to suggest that, in spite of the restrictedmobility of some elements such as B and Mn, a significant proportionof their total supply to developing sinks is carried in thephloem stream. Key words: Transport fluid composition, plant nutrition, phloem mobility.     

脱氧鬼臼毒素对美洲大蠊的毒力及几种酶系的影响

采用药膜法测试了脱氧鬼臼毒素对美洲大蠊Periplaneta americana初孵若虫的触杀活性,并测定了其对成虫中枢神经系统乙酰胆碱酯酶（AChE）和腺苷三磷酸酶(ATPase)离体活性的影响。结果表明：脱氧鬼臼毒素对美洲大蠊初孵若虫具有较强的毒杀活性,在接触时间24、48、72和96 h 的LC₅₀分别为26.26、4.68、1.51和0.62 μg/cm²;其对AChE没有明显影响; 对Na⁺ -K⁺ -ATPase有明显抑制作用,并存在浓度 效应关系,IC₅₀为44.9 μmol/L; 对Ca²⁺-Mg²⁺-ATPase表现出低剂量激活,高剂量抑制的现象。结果提示美洲大蠊的AChE不是脱氧鬼臼毒素靶标,而ATPase可能是脱氧鬼臼毒素的重要靶标之一。     

Effects of Elevated Carbon Dioxide Concentration in the Dark on the Growth of Soybean Seedlings

Previous work has shown that elevated carbon dioxide (CO₂) concentrationsin the dark reversibly reduce the rate of CO₂ efflux from soybeans.Experiments were performed exposing soybean plants continuallyto concentrations of 350 or 700 cm³ m^-3 for 24 h d^-1, or to350 during the day and 700 cm³ m^-3 at night, in order to determinethe importance of the reduced rate of dark CO₂ efflux for plantgrowth. High CO₂ applied only at night conserved carbon andincreased dry mass during initial growth compared with the constant350 cm³ m^-3 treatment. Long-term net assimilation rate was increasedby high CO₂ in the dark, without any increase in daytime leafphotosynthesis. However, leaf area ratio was reduced by thedark CO₂ treatment to values equal to those of plants continuallyexposed to the higher concentration. From days 14-21, leaf areawas less for the elevated night-time CO₂ treatment than foreither the constant 350 or 700 cm³ m^-3 treatments. For the days7-21-period, relative growth rate was significantly reducedby the high night CO₂ treatment compared with the 350 cm³ m^-3continuous treatment. The results indicate that some functionallysignificant component of respiration was reduced by the elevatedCO₂ concentration in the dark.Copyright 1995, 1999 AcademicPress Glycine max L. (Merr.), carbon dioxide, plant growth, respiration     

Spectroscopic characterization of Co(II)-, Ni(II)-, and Cd(II)-substituted wild-type and non-native retroviral-type zinc finger peptides

The nucleocapsid protein (NCP) from Mason-Pfizer monkey virus (MPMV) contains two evolutionary invariant Cys-X₂-Cys-X₄-His-X₄-Cys retroviral-type zinc finger structures, where the Cys and His residues provide ligands to a tetrahedrally coordinated Zn(II) ion. The N-terminal zinc finger (F1) of NCP from MPMV contains an immediately contiguous Cys in the -1 position relative to the start of this conserved motif: Cys-Cys-X₂-Cys-X₄-His-X₄-Cys. Metal complexes of 18-amino acid peptides which model the native zinc finger sequence, SER-Cys-X₂-Cys-X₄-His-X₄-Cys (F1_SC), and non-native Cys-SER-X₂-Cys-X₄-His-X₄-Cys (F1_CS) and SER-SER-X₂-Cys-X₄-His-X₄-Cys (F1_SS) sequences have been spectroscopically characterized and compared to the native two-zinc-finger protein fragment, MPMV NCP 21-80. All Co(II)-substituted peptide complexes adopt tetrahedral ligand geometries and have S^-MCo(II) ligand-to-metal charge-transfer (LMCT) transition intensities consistent with three Co(II)-S bonds for F1_SC and F1_CS. The non-native F1_CS peptide binds Co(II) with K_Co=1.5᎒⁶ M^-1, comparable to that of the native complex, and 걄-fold tighter than F1_SS. Like the Co(II) derivative, the absorption spectrum of Ni(II)-substituted NCP 21-80 is most consistent with tetrahedral Ni(II) complexes with multiple thiolate donors. In contrast, Ni(II) complexes of F1_SC and F1_CS exhibit a single absorption band in the 400-550 nm region ()겨-300 M^-1 cm^-1), distinct in the two complexes, assignable to a degenerate d-d transition envelope characteristic of non-native square-planar coordination geometry, and an intense LMCT transition in the UV ()₂₅₅ᄾ,000 M^-1 cm^-1). Cd(II) complexes have intense absorption in the UV (5_max=233 nm), with absolute intensities consistent with 񬩈 M^-1 cm^-1 per Cd(II)-S bond. ¹¹³Cd NMR spectroscopy of ¹¹³Cd MPMV NCP gives '=649 ppm, consistent with S₃N coordination. Co(II) and Cd(II) complexes of non-native F1_CS peptides are more sensitive to oxidation by O₂, relative to F1_SC, suggestive of a higher lability in the non-native chelate. The implications of these findings for the evolutionary conservation of this motif are discussed.     

Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt

Zusammenfassung Es wurden analysenreine Proben der Romanowsky-Farbstoffe Eosin Y, Erythrosin B und Tetrachlorfluoreszein hergestellt.Im DC der Farbstoffproben konnten keine Verunreinigungen nachgewiesen werden. Die Absorptionsspektren der Farbstoffdianionen in wäßriger alkalischer Lösung und der Farbstoffsäuren in 95%igem Ethanol wurden bei sehr kleinen Farbstoffkonzentrationen gemessen und der molare Extinktionskoeffizient der längstwelligen Absorptionsbande der monomeren Farbstoffspezies bestimmt (Tabelle 1). Die Extinktionskoeffizienten können zur Standardisierung von Farbstoffproben verwendet werden. Die Absorptionsspektren von Eosin Y hängen in wäßriger Lösung von der Farbstoffkonzentration ab. Aus der Konzentrationsabhängigkeit wurden mit einem neuen, sehr empfindlichen Verfahren zwei Assoziationsgleichgewichte ermittelt. Bereits in sehr verdünnter Lösung bilden sich Dimere, bei erhöhter Konzentration Tetramere, Die Dissoziationskonstante der DimerenD in MonomereM beträgt bei pH=12, 293K:K ₂₁=2,9 × 10^–5 M; der TetramerenQ in DimereD:K ₄₂=2,4 × 10^–3 M. Aus den gemessenen Spektren von Eosinlösungen verschiedener Konzentration, pH=12, und den GleichgewichtskonstantenK ₂₁,K ₄₂ haben wir die Spektren der reinen Monomeren, Dimeren und Tetrameren bestimmt.M hat eine langwellige Absorptionsbande: , _M =1,03 x 10₅ M_-1 cm_-1;D eine Bande: , _D =1,74 x 10₅ M_-1 cm_-1;Q zwei Banden: , , _Q1=1,65 x 10⁵, _Q2=1,96 x 10⁵ M^-1 cm^-1. Das Absorptionsspektrum der Dimeren wird quantenmechanisch interpretiert.

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Romanowsky dyes and Romanowsky-Giemsa effect. 2. Eosin Y, Erythrosin B, tetrachlorofluorescein, Spectroscopic characterization of pure dyes, association of Eosin Y

Summary Analytically pure smaples of the Romanowsky dyes eosin y, erythrosin b and tetrachlorofluorescein are prepared. DC of the dye samples shows no contaminations. We measured the absorption spectra of the dye dianions in alkaline aqueous solution and of the dye acids in 95% ethanol at very low dye concentrations. The molar extinction coefficients of the long wavelength absorption of the monomeric dye species are determined (Table 1). The extinction coefficients may be used for standardisation of dye samples. The absorption spectra of eosin y in aqueous solution are dependend on concentration. Using a new very sensitive method it was possible to identify two association equilibria from the concentration dependency of the spectra. Dimers are formed even in very dilute solutions, at higher concentrations tetramers. The dissociation constant of the dimersD in monomersM at 293 K, pH=12, isK ₂₁=2,9×10^–5 M; of the tetramersQ in dimersDK ₄₂=2,4×10^–3 M. From the experimental spectra of eosin solutions at various concentrations, pH=12, and the equilibrium constantsK ₂₁,K ₄₂ the absorption spectra of the pure monomers, dimers and tetramers are calculated. M has one long wavelength absorption band, , _M =1,03 x 10₅ M_-1 cm_-1;D also one absorption band, , _D =1,74 x 10₅ M_-1 cm_-1;Q two absorption bands, , , _Q1=1,65 x 10⁵, _Q2=1,96 x 10⁵ M^-1 cm^-1. The absorption spectrum of the dimers is discussed by quantum mechanics.

     

禾谷缢管蚜和麦长管蚜玻璃管药膜法敏感毒力基线的建立

【目的】建立禾谷缢管蚜Rhopalosiphum padi（Linnaeus）和麦长管蚜Sitobion avenae（Fabricius）对常用杀虫剂的相对敏感基线。【方法】从田间采集麦蚜在实验室内饲养30代以上,利用玻璃管药膜法测定其对杀虫剂的敏感度,每条毒力基线为2次以上独立测定数据合并后的计算结果。【结果】用玻璃管药膜法建立了包括新烟碱类、吡啶类、氨基甲酸酯类、有机磷类和拟除虫菊酯类共22个药剂品种对禾谷缢管蚜和麦长管蚜3 h的敏感毒力基线。禾谷缢管蚜对新烟碱类药剂吡虫啉和啶虫脒的LC₅₀值分别为0.02和0.007 μg/cm²;对吡啶类药剂吡蚜酮的LC₅₀值为0.124 μg/cm²;对氨基甲酸酯类药剂丁硫克百威、硫双灭多威、灭多威、抗蚜威、西维因的LC₅₀值为0.0026~0.70 μg/cm²;对有机磷类药剂三唑磷、丙溴磷、氧乐果、乐果、马拉硫磷、辛硫磷、敌敌畏、毒死蜱的LC₅₀值为0.005~0.065 μg/cm²;对拟除虫菊酯类药剂三氟氯氰菊酯、高效氯氰菊酯、溴氰菊酯、联苯菊酯、氰戊菊酯、氯氰菊酯的LC₅₀值为0.033~0.240 μg/cm²。麦长管蚜对新烟碱类药剂吡虫啉和啶虫脒的LC₅₀值分别为0.15和0.12 μg/cm²;对吡啶类药剂吡蚜酮的LC₅₀值为0.41 μg/cm²;对氨基甲酸酯类药剂丁硫克百威、硫双灭多威、灭多威、抗蚜威、西维因的LC50值为0.005~0.76 μg/cm²;对有机磷类药剂三唑磷、丙溴磷、氧乐果、乐果、马拉硫磷、辛硫磷、敌敌畏、毒死蜱的LC₅₀值为0.018~0.36 μg/cm²;对拟除虫菊酯类药剂三氟氯氰菊酯、高效氯氰菊酯、溴氰菊酯、联苯菊酯、氰戊菊酯、氯氰菊酯的LC50值为0.20~2.94 μg/cm²。【结论】建立的两种麦蚜对22种杀虫药剂的相对敏感基线,包括当前所有可能用于防治麦蚜的药剂,可以用于以后麦蚜抗药性监测或其他相关研究的参照;禾谷缢管蚜对药剂的敏感度高于麦长管蚜。     

ENaC- and CFTR-dependent ion and fluid transport in mammary epithelia

Mammary epithelial 31EG4 cells (MEC) were grown as monolayers onfilters to analyze the apical membrane mechanisms that help mediate ionand fluid transport across the epithelium. RT-PCR showed the presenceof cystic fibrosis transmembrane conductance regulator (CFTR) andepithelial Na⁺ channel (ENaC) message, and immunomicroscopyshowed apical membrane staining for both proteins. CFTR was alsolocalized to the apical membrane of native human mammary ductepithelium. In control conditions, mean values of transepithelialpotential (apical-side negative) and resistance(R_T) are 5.9 mV and 829  · cm², respectively. The apical membranepotential (V_A) is 40.7 mV, and the mean ratioof apical to basolateral membrane resistance (R_A/R_B) is 2.8. Apicalamiloride hyperpolarized V_A by 19.7 mV andtripled R_A/R_B. AcAMP-elevating cocktail depolarized V_A by 17.6 mV, decreased R_A/R_B by60%, increased short-circuit current by 6 µA/cm²,decreased R_T by 155  · cm², and largely eliminated responses toamiloride. Whole cell patch-clamp measurements demonstratedamiloride-inhibited Na⁺ currents [linear current-voltage(I-V) relation] and forskolin-stimulated Clcurrents (linear I-V relation). A capacitance probe methodshowed that in the control state, MEC monolayers either absorbed orsecreted fluid (2-4µl · cm² · h¹). Fluidsecretion was stimulated either by activating CFTR (cAMP) or blockingENaC (amiloride). These data plus equivalent circuit analysis showedthat 1) fluid absorption across MEC is mediated byNa⁺ transport via apical membrane ENaC, and fluid secretionis mediated, in part, by Cl transport via apicalCFTR; 2) in both cases, appropriate counterions move throughtight junctions to maintain electroneutrality; and 3)interactions among CFTR, ENaC, and tight junctions allow MEC to eitherabsorb or secrete fluid and, in situ, may help control luminal[Na⁺] and [Cl].

     

多炔类化合物对美洲大蠊的触杀活性及对乙酰胆碱酯酶和腺苷三磷酸酶活性的影响

采用药膜法测定了人工合成的11个多炔类化合物对美洲大蠊Periplaneta americana初孵若虫的触杀活性。结果表明,当化合物处理浓度为20 μg/cm²时,致死率达70%以上的有: 化合物2 (1-叔丁基-4-羟甲基-丁二炔)、化合物9 (1-苯甲基-4-甲基-丁二炔)和化合物10 (O-炔丙基硫代磷酸二乙酯)。经毒力测定,化合物9和化合物10的LC₅₀分别为3.91 μg/cm²和1.50 μg/cm²。化合物2、化合物7 (1-苯基-4-邻硝基苯基-丁二炔)和化合物10对美洲大蠊乙酰胆碱酯酶(AChE)具有抑制活性,抑制率分别为12.00%、27.24%和62.22%。化合物2和化合物10对Na⁺-K⁺-ATPase具有抑制活性,抑制率分别为44.55%和31.44%;而化合物5 (1-苯基-4-（3,4-亚甲基二氧）苯基-丁二炔)和化合物6 (1-苯基-4-间硝基苯基-丁二炔)对该酶具有激活活性,激活率分别为24.98%和20.99%。化合物2、化合物4 （1-苯基-4-对甲氧基苯基-丁二炔）和化合物7对Ca²⁺-Mg²⁺-ATPase具有抑制活性,抑制率分别为49.02%、38.53%和35.32%, 其他化合物对该酶具有激活活性,其中激活活性最高的为化合物5,激活率达81.12%。     

Ovine male genital duct epithelial cells differentiate in vitro and express functional CFTR and ENaC

To investigate the biology of the malegenital duct epithelium, we have established cell cultures from theovine vas deferens and epididymis epithelium. These cells develop tightjunctions, high transepithelial electrical resistance, and alumen-negative transepithelial potential difference as a sign of activetransepithelial ion transport. In epididymis cultures the equivalentshort-circuit current (I_sc) averaged 20.8 ± 0.7 µA/cm² (n = 150) and was partially inhibited byapical application of amiloride with an inhibitor concentration of 0.64 µM. In vas deferens cultures, I_sc averaged 14.4 ± 1.1 µA/cm² (n = 18) and was also inhibited byapical application of amiloride with a half-maximal inhibitorconcentration (K_i) of 0.68 µM. The remainingamiloride-insensitive I_sc component in epididymisand vas deferens cells was partially inhibited by apical application ofthe Cl channel blocker diphenylamine-2-carboxylicacid (1 mM). It was largely dependent on extracellularCl and, to a lesser extent, on extracellularHCO₃. It was further stimulated bybasolateral application of forskolin (10⁵ M), which increasedI_sc by 3.1 ± 0.3 µA/cm² (n=65) in epididymis and 0.9 ± 0.1 µA/cm² (n =11) in vas deferens. These findings suggest that cultured ovine vasdeferens and epididymis cells absorb Na⁺ viaamiloride-sensitive epithelial Na⁺ channels (ENaC) andsecrete Cl and HCO₃via apical cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. This interpretation is supported byRT-PCR data showing that vas deferens and epididymis cells express CFTRand ENaC mRNA.

     

Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements

Members of the SLC20 family or type III Na⁺-coupled P_i cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular P_i homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic P_i cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na⁺-dependent ³²P_i uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li⁺ substituted for Na⁺. The dependence of the P_i-induced current on P_i concentration was Michaelian, and the dependence on Na⁺ concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent P_i affinity constant showed a minimum in the pH range 6.2–6.8 of 0.05 mM and increased to 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual ²²Na-³²P_i uptake and suggested a 2:1 Na⁺:P_i stoichiometry. A correlation of ³²P_i uptake and net charge movement indicated one charge translocation per P_i. Changes in oocyte surface pH were consistent with transport of monovalent P_i. On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na⁺:H₂PO₄^–:Na⁺. Significantly, in contrast to type II Na⁺-P_i cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity. Na⁺-P_i cotransport; two-electrode voltage clamp; surface pH electrode; SLC20; retroviral receptor     

The effects of Cu on the adenylate energy charge of open ocean phytoplankton

The effects of short-term, acute Cu exposure (6 h) on the adenylateenergy charge (EC_A) of open-ocean phytoplankton populations(northeastern equatorial Pacific) were investigated. Energycharge remained at {small tilde}0.77 over the range of Cu additions(0.025 – 5.µg l^–1), even though ¹⁴C uptakeand total adenylate levels (ATP + ADP + AMP) were reduced byas much as 60%. These findings suggest that EC_A alone is nota sensitive indicator of acute sublethal metal effects on phytoplankton. ¹This research was supported by the NSF Biological OceanographyProgram grant #OCE 81-17286.     

Ouabain potentiates the activation of ERK1/2 by carbachol in parotid gland epithelial cells; inhibition of ERK1/2 reduces Na(+)-K(+)-ATPase activity

The Na⁺-K⁺-ATPase and the ERK1/2 pathway appear to be linked in some fashion in a variety of cells. The Na⁺-K⁺-ATPase inhibitor ouabain can promote ERK1/2 activation. This activation involves Src, intracellular Ca²⁺ concentration ([Ca²⁺]_i) elevation, reactive oxygen species (ROS) generation, and EGF receptor (EGFR) transactivation. In contrast, ERK1/2 can mediate changes in Na⁺-K⁺-ATPase activity and/or expression. Thus signaling between ERK1/2 and Na⁺-K⁺-ATPase can occur from either direction. Whether such bidirectionality can occur within the same cell has not been reported. In the present study, we have demonstrated that while ouabain (1 mM) produces only a small (50%) increase in ERK1/2 phosphorylation in freshly isolated rat salivary (parotid acinar) epithelial cells, it potentiates the phosphorylation of ERK1/2 by submaximal concentrations of carbachol, a muscarinic receptor ligand that initiates fluid secretion. Although ERK1/2 is only modestly phosphorylated when cells are exposed to 1 mM ouabain or 10^–6 M carbachol, the combination of these agents promotes ERK1/2 phosphorylation to near-maximal levels achieved by a log order carbachol concentration. These effects of ouabain are distinct from Na⁺-K⁺-ATPase inhibition by lowering extracellular K⁺, which promotes a rapid and large increase in ERK1/2 phosphorylation. ERK1/2 potentiation by ouabain (EC₅₀ 100 µM) involves PKC, Src, and alterations in [Ca²⁺]_i but not ROS generation or EGFR transactivation. In addition, inhibition of ERK1/2 reduces Na⁺-K⁺-ATPase activity (measured as stimulation of QO₂ by carbachol and the cationophore nystatin). These results suggest that ERK1/2 and Na⁺-K⁺-ATPase may signal to each other in each direction under defined conditions in a single cell type. protein kinase C; intracellular Ca²⁺ concentration; muscarinic receptor; ₁-subunit; potassium removal     

Structure of the O-specific polysaccharide of the marine bacterium Arenibacter palladensis KMM 3961T containing 2-acetamido-2-deoxy-L-galacturonic acid

The O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide of the marine bacterium Arenibacter palladensis type strain KMM 3961^T and studied by chemical methods and ¹H and ¹³C NMR spectroscopy including 2D COSY, TOCSY, ¹H,¹³C HSQC, and HMBC experiments. The polysaccharide was shown to consist of tetrasaccharide repeating units containing two mannose residues (Man), one 2-acetamido-2-deoxy-D-galactose residue (D-GalNAc), and one 2-acetamido-2-deoxy-L-galacturonic acid residue (L-GalNAcA) and having the following structure: ? 2) - a- D - Manp - (1 ? 6) - a- D - Manp - (1 ? 4) - a- L - GalpNAcA - (1 ? 3) - b- D - GalpNAc - (1 ?\to 2) - \alpha - D - Manp - (1 \to 6) - \alpha - D - Manp - (1 \to 4) - \alpha - L - GalpNAcA - (1 \to 3) - \beta - D - GalpNAc - (1 \to.     

Life history of the nonnative convict cichlid <Emphasis Type="Italic">Amatitlania nigrofasciata</Emphasis> in the Haebaru Reservoir on Okinawa-jima Island,Japan

Life history parameters associated with reproductive biology, age, and growth of the convict cichlid (also known as the zebra cichlid) Amatitlania nigrofasciata, which was introduced into the Haebaru Reservoir on Okinawa-jima Island, were estimated using 437 specimens that ranged from 13.7 to 82.9 mm standard length (SL). Lengths of females at first maturity (SL) and 50% maturity (L ₅₀) were estimated to be 32.2 and 37.3 mm SL, respectively. The spawning period continued throughout the year, with a peak spawning cycle from March to May 2006–2007. Observations of postovulatory follicles and tertiary yolk stage oocytes indicate that convict cichlids spawn multiple times within a year. Female cichlids that hatched during the peak spawning seasons matured after October of the same year. Batch fecundity of females (32.2–61.2 mm SL) ranged from 65 to 345 (mean ± SD = 155 ± 63). Opaque zones along the outer margins of otoliths formed annually. The maximum age of male and female cichlids was 3 years. The von Bertalanffy growth formulae (VBGF) were expressed as L_t = 57.4( 1 - e ^{- 0.78( t + 0.91 )} ) {{\hbox{L}}_{\rm{t}}}{ = 57}{.4}\left( {1 - {e^{ - 0.78\left( {t + 0.91} \right)}}} \right) for females and L_t = 69.5( 1 - e ^{- 1.07( t + 0.24 )} ) {{\hbox{L}}_{\rm{t}}}{ = 69}{.5}\left( {1 - {e^{ - 1.07\left( {t + 0.24} \right)}}} \right) for males. Males grew larger than females beginning from the first year. Certain life history characteristics, such as year-round spawning and early maturation, probably contributed to the successful establishment of the convict cichlid, and this species in particular is thought to adapt and become established quickly upon introduction to freshwater systems on Okinawa-jima Island.