Vasopressin Regulates Water Flow in a Rat Cortical Collecting Duct Cell Line Not Containing Known Aquaporins

Transepithelial water movements and arginine-vasopressin (AVP)-associated ones were studied in a renal cell line established from a rat cortical collecting duct (RCCD₁). Transepithelial net water fluxes (J _w ) were recorded every minute in RCCD₁ monolayers cultured on permeable supports. Spontaneous net water secretion was observed, which was inhibited by serosal bumetanide (10⁻⁵ m), apical glibenclamide (10⁻⁴ m) and apical BaCl₂ (5 × 10⁻³ m). RT-PCR, RNAse protection and/or immunoblotting experiments demonstrated that known renal aquaporins (AQP1, AQP2, AQP3, AQP4, AQP6 and AQP7) were not expressed in RCCD₁ cells. AVP stimulates cAMP production and sodium reabsorption in RCCD₁ cells. We have now observed that AVP significantly reduces the spontaneous water secretory flux. The amiloride-sensitive AVP-induced increase in short-circuit current (I _sc ) was paralleled by a simultaneous modification of the observed J _w : both responses had similar time courses and half-times (about 4 min). On the other hand, AVP did not modify the osmotically driven J _w induced by serosal hypertonicity. We can conclude that: (i) transepithelial J _w occurs in RCCD₁ cells in the absence of known renal aquaporins; (ii) the ``water secretory component' observed could be linked to Cl⁻ and K⁺ secretion; (iii) the natriferic response to AVP, preserved in RCCD₁ cells, was associated with a change in net water flux, which was even observed in absence of AQP2, AQP3 or AQP4 and (iv) the hydro-osmotic response to AVP was completely lost. Received: 30 December 1999/Revised: 12 October 2000     

Nanocarrier for the Transdermal Delivery of an Antiparkinsonian Drug

The purpose of the present study was to investigate the potential of nanoemulsions as nanodrug carrier systems for the percutaneous delivery of ropinirole. Nanoemulsions comprised Capryol 90 as the oil phase, Tween 20 as the surfactant, Carbitol as the cosurfactant, and water as an external phase. The effects of composition of nanoemulsion, including the ratio of surfactant and cosurfactant (S _mix) and their concentration on skin permeation, were evaluated. All the prepared nanoemulsions showed a significant increase in permeation parameters such as steady state flux (J _ss) and permeability coefficient (K _p) when compared to the control (p < 0.01). Nanoemulsion composition (NEL3) comprising ropinirole (0.5% w/w), Capryol 90 (5% w/w), S _mix 2:1 (35% w/w), and water (59.5% w/w) showed the highest flux (51.81 ± 5.03 μg/cm²/h) and was selected for formulation into nanoemulsion gel. The gel was further optimized with respect to oil concentration (Capryol 90), polymer concentration (Carbopol), and drug content by employing the Box–Behnken design, which statistically evaluated the effects of these components on ropinirole permeation. Oil and polymer concentrations were found to have a negative influence on permeation, while the drug content had a positive effect. Nanoemulsion gel showed a 7.5-fold increase in skin permeation rate when compared to the conventional hydrogel. In conclusion, the results of the present investigation suggested a promising role of nanoemulsions in enhancing the transdermal permeation of ropinirole.     

Electric‐induced mortality of newly transformed juvenile fishes in waters of different conductivity

In three experiments, each with three species of newly transformed juvenile fishes, the immediate mortality was determined after electrical exposure to 60 Hz pulsed DC in waters of different conductivity (C_w). With a constant applied power density (D_a; 1·0–4·9 mW cm^?3 depending on species) over a range of C_w(10–1020 μS cm^?1), the results predicted that the highest fish mortality would occur at C_w of 65 μS cm^?1 for bluegill Lepomis macrochirus, 74 μS cm^?1 for largemouth bass Micropterus salmoides and at 140–175 μS cm^?1 for channel catfish Ictalurus punctatus. In experiment 2, the voltage gradient (E) was maintained constant (2·5–8·0 peak V cm^?1 depending on species) over the same range of C_w, and fish mortality increased with current density (J) or D_a, which are directly related to C_w. In experiment 3, fish mortality did not differ when peak E(3 or 8 V cm^?1 depending on species) and mean J(0·09 or 0·24 mA cm^?2 depending on species) were held constant by changing pulse width in waters with different C_w(99, 165 or 495 μS cm^?1). Fish mortality in this experiment was not significantly related to peak or mean transferred power density, and the ‘power transfer theory for electrofishing’ was not useful for predicting electrofishing mortality. Overall, the results of the present study indicated that mortality caused by exposure to electricity can be predicted more accurately with the variables peak E and mean J than with models requiring determination of effective conductivity of the fish.     

Influence of water activity and temperature on in vitro growth of surface cultures of a Phoma sp. and production of the pharmaceutical metabolites, squalestatins S1 and S2

A Phoma sp., known to produce the pharmaceutically active metabolites squalestatin 1 (S1) and squalestatin 2 (S2), was cultured on malt-extract/agar (MEA) over a range of water activities (a _w, 0.995–0.90) and temperatures (10–35 °C) to investigate the influence on growth and metabolite production. Use of the ionic solute NaCl to adjust a _w resulted in significantly lower (P < 0.01) squalestatin yields than when the Phoma sp. was grown on MEA amended with the non-ionic solute glycerol. Water activity and temperature and their interactions were highly significant factors (P < 0.001) affecting growth of the Phoma sp., with optimum conditions of 0.998–0.980 a _w and 25 °C. Squalestatin production was similarly influenced by a _w, temperature, time and their interactions (P < 0.001). S1 and S2 production occurred over a narrower a _w and temperature range than growth, with a slightly lower optimum a _w range of 0.995–0.980 a _w. The optimum temperature for squalestatin production varied from 20 °C (S1) to 25 °C (S2) and yields of S2 were up to 1000 times lower than those of S1. The ratio of S1 and S2 produced by the Phoma sp. was influenced by a _w and temperature, with highest values at 0.99–0.98 a _w, and at 15 °C. Incubation times of 28 days gave highest yields of both S1 and S2. Up to 2000-fold increases in squalestatin yields were measured at optimum environmental conditions, compared to the unmodified MEA. This indicates the need to consider such factors in screening systems used to detect biologically active lead compounds produced by fungi. Received: 2 June 1997 / Received last revision: 6 November 1997 / Accepted: 7 November 1997     

Improved accuracy in measuring one-bond and two-bond <Superscript>15</Superscript>N,<Superscript>13</Superscript>C<Superscript>α</Superscript> coupling constants in proteins by double-inphase/antiphase (DIPAP) spectroscopy

An extension to HN(CO-α/β-N,C^α-J)-TROSY (Permi and Annila in J Biomol NMR 16:221–227, 2000) is proposed that permits the simultaneous determination of the four coupling constants ¹ J _{N′(i)Cα(i)}, ² J _HN(i)Cα(i), ² J _{Cα(i−1)N′(i)}, and ³ J _{Cα(i−1)HN(i)} in ¹⁵N,¹³C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the ² J _CαN′ coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both ² J _CαN′ and ¹ J _N′Cα (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size.     

Diurnal changes in chlorophylla fluorescence and carotenoid composition inOpuntia ficus-indica,a CAM plant,and in three C3 species in Portugal during summer

Summary Diurnal changes in chlorophylla fluorescence were determined in four species, differing in life form, in Portugal during the summer of 1989. These includedOpuntia ficus-indica, a CAM plant, andHelianthus annuus, Ficus carica andArbutus unedo, three C₃ species. Steady state fluorescence yield,F _S, and maximum fluorescence yield,F _M′, were determined at different times of the day. Using the model of Genty et al. (1989), the photon use efficiency of photosystem II electron transport,φ _e, was calculated from (F _M′−F _S)/F _M′. Diurnal changes in relative rate of non-cyclic electron transport through photosystem II,J _e, were derived by multiplyingφ _e by the incident photon flux density (PFD). WhenJ _e, determined for each species for various points in time throughout the day, was plotted against corresponding values of PFD, the light response curves obtained showed thatJ _e was linearly dependent on PFD in low light and approached saturation in high light. The highest values ofJ _e were observed inHelianthus annuus, followed byOpuntia ficus-indica, Ficus carica andArbutus unedo. The proportion of the xanthophyll zeaxanthin to total carotenoids, determined around noon, was inversely related to maximum rates ofJ _e.     

Organisation in the pelagic ecosystem

A continuous, steady-state theory has been developed for the abundance of organisms in the pelagic ecosystem as a function of their body weight. It is based on accepted relationships for the weight-dependence of metabolism and growth, in a context where individual organisms are assigned to one of a series of size classes for which the nominal weights increase in a geometric progression. Analysis of the biomass flow in such a representation leads to the conclusion that, in the steady state, the total biomass in any given size class decreases in a regular manner with increasing size. Explicitly,b(w ₂)/b(w ₁)~(w ₂/w ₁)^0.22, whereb(w ₂) andb(w ₁) are the total biomasses in the size classes characterised by weightsw ₂ andw ₁, respectively. The exponent (–0.22) represents a balance between catabolism and anabolism, based on published reviews concerning the revelant parameters. This result agrees favourably with data collected by other workers in the subtropical oceans. The theory can be used to draw conclusions about the functional dynamics of the pelagic ecosystem, such as community respiration and rate of biomass flow.     

Relationship between specific leaf area of aCryptomeria japonica foliage shoot segment and its diameter

Dependency of specific leaf area (SLA) on shoot diameter (x) was studied forCryptomeria japonica foliage shoot segments about 5 cm in length taken from 18 branches at different height levels on 3 trees in August 1987. The leaf area of a shoot segment (S) was defined as half the sum of the needle area (s) estimated by the allometric relationship betweens and needle length. The dry weight of woody tissue (W _w) and needles (W _n) of a segment and itsS were divided by the segment length (L) to give linear densities asW _w/L,W _n/L andS/L, respectively. The densities were related tox by power-form equations.S/L values tended to be constant around 1.2 [cm² cm⁻¹] within the discussed range ofx, whileW _w/L andW _n/L values clearly increased withx. The approximately reciprocal relationship between average needle area (s) and linear density of the number of needles supported the fact thatS/L values were roughly constant regardless ofx.SLA andSNA were defined asS/(W _w+W _n) andS/W _n, respectively. TheSLA-x relationship expected from the average value ofS/L and theW _w/L-x and theW _n/L-x relationships was well fitted to the observed decrease inSLA with increasingx. SNA also decreased asx increased. Variations inSLA andSNA among the shoot segments with similarx were not systematically related to their height levels. An empirical equation with a maximum value ofx (Xmax) was also proposed in order to formulate theSLA-x relationship.     

Comparative evaluation of quantum theory of nerve excitation

Deliberate evaluation of the quantum theory of nerve excitation is made by comparing it with Hill's theory in fitting the experimental data on threshold-frequency relation, optimum frequency (v₀) for nerve excitation and strength-duration relation. Decrease of v₀ and increase of all the time constants (Hill's λ andk, Wei'sT ₂ and spike durationw) with decreasing temperature are interpreted on the basis of the dipole relaxation timeT ₂ but inexplicable from Hill's theory or any other existing theory. The closeness ofk,T ₂ andw values is explained. A variety of experimental results obtained by others is discussed. Finally, a comparison is made between the Hodgkin-Huxley equations and the quantum theory. Most of the facts (electrical and non-electrical) tend to support the thesis that nerve excitation is a macroscopic expression of quantum transitions of dipoles between energy states.     

In vitro studies on calcium absorption from the gastrointestinal tract in␣small ruminants

Unidirectional flux rates of Ca²⁺ across gastrointestinal tissues from sheep and goats were measured in vitro by applying the Ussing-chamber technique. Except for the sheep duodenum, mucosal to serosal Ca²⁺ flux rates (J _ms) exceeded respective flux rates in the opposite direction (J _sm) in both species and in all segments of the intestinal tract. This resulted in net Ca²⁺ flux rates␣(J _net = J _ms − J _sm) ranging between −2 and 9 nmol · cm⁻² · h⁻¹ in sheep and between 10 and 15 nmol cm⁻² · h⁻¹ in goats. In sheep, only J _net in jejunum, and in goats, J _netin duodenum and jejunum were significantly different from zero. Using sheep rumen wall epithelia, significant J _net of Ca²⁺ of around 5 nmol · cm⁻² · h⁻¹ could be detected. Since the experiments were carried out in the absence of an electrochemical gradient, significant net Ca²⁺ absorption clearly indicates the presence of active mechanisms for Ca²⁺ transport. Dietary Ca depletion caused increased calcitriol plasma concentrations and induced significant stimulations of net Ca²⁺ absorption in goat rumen. J _net of Ca²⁺ across goat rumen epithelia was significantly reduced by 1 mmol · l ⁻¹ verapamil in the mucosal buffer solution. In conclusion, there is clear evidence for the rumen as a main site for active Ca²⁺ absorption in small ruminants. Stimulation of active Ca²⁺ absorption by increased plasma calcitriol levels and inhibition by mucosal verapamil suggest mechanistic and regulatory similarities to active Ca²⁺ transport as described for the upper small intestines of monogastric species. Accepted: 31 July 1996     

Salt tolerance potential of wild resources of the tribe Triticeae

The salt tolerance potential of variousAegilops species of different genome combinationsviz., Aegilops squarrosa, Ae. cylindrica, Ae. ovata. Ae. triuncialis, Ae. variabilis, Ae. bicornis, Ae. longissima, Ae. umbellulata, andAe. sharonensis was tested to identify the high salt-tolerant genotype(s). Screening was done in cement tanks filled with gravel and Hoagland nutrient solution. Salinity was created by mixing Na₂SO₄, CaCl₂, MgCl₂ and NaCl in the ratio of 10∶5∶1∶4 and induced by a stepwise increase in electrical conductivity number of tillers and number of leaves. Inter-and intragenomic variations for cation uptake were also significant. Species with DD and CD genome were found to be highly tolerant. Possible factors responsible for these observations have been discussed.     

Bidirectional Transepithelial Water Transport: Chloride-Dependent Mechanisms

We hypothesized that inhibition and activation of basolateral to luminal chloride transport mechanisms were associated with respective decreases and increases in basolateral to luminal water fluxes. The luminal to basolateral (J _W ^L→B ) and basolateral to luminal (J _W ^B→L ) water fluxes across ovine tracheal epithelia were measured simultaneously. The mean J _W ^L→B (6.5 μl/min/cm²) was larger than J _W ^B→L (6.1 μl/min/cm²). Furosemide reduced J _W ^B→L from 6.0 to 5.6 μl/min/cm². Diphenylamine-2-carboxylate (DPC) reduced J _W ^B→L from 7.9 to 7.3 μl/min/cm² and reduced the membrane potential difference by 38%. Furosemide together with DPC decreased J _W ^L→B by 30% and J _W ^B→L by 15%. Norepinephrine increased J _W ^B→L from 4.9 to 6.0 μl/min/cm². Neuropeptide Y in the presence of norepinephrine decreased J _W ^L→B (6.4 to 5.2 μl/min/cm²) and returned J _W ^B→L to its baseline value. Vasopressin increased J _W ^B→L from 4.1 to 5.1 μl/min/cm². Endothelin-1 induced a simultaneous increase in J _W ^B→L (7.0 to 7.7 μl/min/cm²) and decrease in J _W ^L→B (7.4 to 6.4 μl/min/cm²); and decreased the membrane resistance. These data indicate that in tracheal epithelia under homeostatic conditions J _W ^B→L has a ∼15% actively coupled component. Consistent with our hypothesis, inhibition and receptor-induced stimulation of chloride effluxes were associated with decreases and increases in J _W ^B→L , respectively. However, as inhibition of transcellular chloride transport always decreased J _W ^L→B more than J _W ^B→L , reducing transepithelial chloride transport did not result in less water being transported into the airway lumen. Received: 12 October 1999/Revised: 14 March 2000     

The effect of drought stress on chlorophyll fluorescence in Lolium-Festuca hybrids

The effects of drought on photochemical efficiency of PSII in leaves of 22 hybrids of Festuca pratensis × Lolium multiflorum and Festuca pratensis × Lolium perenne and of Festuca pratensis cv. Skra were investigated. A significant decrease of electron transport efficiency (about 25%) in PSII (Φ_PSII) was not found before 9 days of seedling growth in hydroponics with water potential (Ψ_w) equal to −0.8 MPa (simulated “soil drought”). The decrease of Φ_PSII was similarly related to that of excitation energy capture by open PSII reaction centre (Fv’/Fm’) and also to the decrease of the proportion of oxidized to reduced Q_A (photochemical fluorescence quenching, q_p). According to the drought prolongation, variation of all parameters of fluorescence between genotypes significantly increased. The seedlings of some genotypes were able to recover electron transport efficiency in PSII after increasing water potential in nutrient solution (removing the “soil drought”). When plants grew in containers with soil and 4 genotypes with the highest sensitivity of electron transport to drought (S) as well as 4 genotypes with the highest tolerance (T) were compared 17 days after watering ceased, Ψ_w in leaves considerably decreased, but the differences between S and T genotypes were often not significant in this respect. The differences between S and T genotypes, as values of Fv/Fm were concerned, also appeared small (about 5%), similarly as that of Fv’/Fm’ (5%), q_p (12%) and Φ_PSII (about 15%). Drought stress increased non-photochemical quenching of chlorophyll fluorescence (NPQ) 15 to 47% and this could protect the PSII reaction centres from damages because of energy excess. The increase of NPQ was not closely connected with drought resistance of plants because it was similar in some genotypes tolerant to dehydration as well as in sensitive ones. The results of the experiments suggest that resources of genetic variability in Festulolium may be sufficient for revealing differences between genotypes on the basis of measurement of chlorophyll a fluorescence, as far as their tolerance to soil drought is concerned. As the tolerance of PSII against drought is high, the determinations of fluorescence should be performed rather under severe stress. Such methods seem to be useful for selection of genotypes with high drought tolerance as well as with the ability to at least partial repairing of PSII after drought.     

Temporal patterns of water flux in trees and lianas in a Panamanian moist forest

Using constant heat sap flow sensors, xylem water fluxes in ten tree species and two liana species were monitored for 5–10 days during the beginning of the wet season in May, 1993. For a subset of the trees, a branch was also monitored at the top of the crown for 5 days. Xylem flux (J _S) was related diurnally in all plants to vapor pressure deficit (D) measured within the upper-third of the canopy, and to incoming shortwave radiation R _S above the canopy. Cross-correlation analysis was used to estimate time lags between diurnal patterns of J _S and D or R _S, and between J _S in stems and branches. The maximum correlation coefficient from cross-correlation of J _S with R _S (range=0.57–0.92) was often higher than the maximum of J _S with D (range=0.43–0.89), indicating that diurnal J _S was more dependent on R _S than D. Time lags (lag corresponding to maximum correlation) of J _S at stem-base with D was shorter (0–45 min) than with radiation (5–115 min), highly variable within a species, and uncorrelated to the height or exposure of tree crowns or liana in the canopy. On a stand level, not accounting for the diel lag between stem sap flux and canopy flux resulted in errors in estimated canopy transpiration of up to 30%. Received: 19 October 1998 / Accepted: 8 June 1999     

Time- and dose-dependent effects of protein kinase C on proximal bicarbonate transport

Summary Activation of protein kinase C has been shown to cause both stimulation and inhibition of transport processes in the brush-border membrane and renal tubule. This study was designed to examine the dose-response nature and time-dependent effect of 4 -phorbol-12-myristate-13-acetate (PMA) on the rates of bicarbonate absorption (J _HCO3) and fluid absorption (J _v) in the proximal convoluted tubule (PCT) of rat kidney. Bicarbonate flux was determined by total CO₂ changes between the collected fluid and the original perfusate as analyzed by microcalorimetry. Luminal perfusion of PMA (10^–10 10^–5 M) within 10 min caused a significant increase ofJ _HCO3 andJ _v. A peaked curve of the dose response was observed with maximal effect at 10^–8 M PMA on both bicarbonate and fluid reabsorption, which could be blocked completely by amiloride (10^–3 m) and EIPA (10^–5 M). On the other hand, with an increase of perfusion time beyond 15 min, PMA (10^–8 and 10^–6 M) could inhibitJ _HCO3 andJ _v. Amiloride (10^–3 M) or EIPA (10^–5 M) significantly inhibitsJ _HCO3 andJ _v, while there is no additive effect of PMA and amiloride or EIPA on PCT transport. An inactive phorbol-ester, 4-phorbol, that does not activate protein kinase C, had no effects onJ _HCO3 andJ _v. Capillary perfusion of PMA (10^–8 M) significantly stimulate bothJ _HCO3 andJ _v; however, PMA did not affect glucose transport from either the luminal side or basolateral side of the PCT. These results indicate that activation of endogenous protein kinase C by PMA could either stimulate or inhibit both bicarbonate and fluid reabsorption in the PCT dependent on time and dose, and these effects are through the modulation of Na⁺/H^– exchange mechanism.     

A chloroplast DNA hypervariable region in eucalypts

Eucalypt chloroplast DNA (cpDNA) provides useful markers for phylogenetic and population research including gene flow and maternity studies. All cpDNA studies in Eucalyptus to date have been based on the RFLP technique, which requires relatively large amounts of clean DNA. The objective of this study was to develop PCR-based cpDNA markers for Eucalyptus. The chloroplast genome of Eucalyptus, like that of most angiosperms, possesses inverted repeats (IR). The two junctions between the IRs and the large single copy (LSC) regions are defined as J_LA andJ_LB. The region surrounding the J_LA junction was sequenced from 26 Eucalyptus DNA samples (21 of E. globulus, plus 5 other species), andtheJ_LB region was sequenced using 5 of these samples. The samples were chosen to represent all major haplotypes identified in previous cpDNA RFLP studies. The J_LA products were 150–210 bp in size, while those fromJ_LB were approximately 500 bp in size. Eighteen mutations were scored in total. Extensive variation was found in the IR within the intergenic spacer between rpl2 and the IR/LSC junctions. Many of these characters were complex indels. One sample of E. globulus possessed a relatively large (38 bp) insertion which caused displacement of the IR/LSC junctions. This is the first report of intraspecific variation in the position of IR/LSC junctions; interspecific variation was also found. The LSC region near J_LB had a low rate of mutation and none were informative. The LSC region near J_LA possessed 2 informative mutations. The variation revealed from these sequences reflects the differentiation previously uncovered in an extensive RLFP analysis on the same samples. These results suggest that the J_LA region will provide very useful cpDNA polymorphisms for future studies in Eucalyptus. Received: 20 March 1999 / Accepted: 16 December 1999     

Improved accuracy of <Superscript>15</Superscript>N–<Superscript>1</Superscript>H scalar and residual dipolar couplings from gradient-enhanced IPAP-HSQC experiments on protonated proteins

The presence of dipole-dipole cross-correlated relaxation as well as unresolved E.COSY effects adversely impacts the accuracy of ¹ J _NH splittings measured from gradient-enhanced IPAP-HSQC spectra. For isotropic samples, the size of the systematic errors caused by these effects depends on the values of ² J _NHα , ³ J _NHβ and ³ J _HNHα . Insertion of band-selective ¹H decoupling pulses in the IPAP-HSQC experiment eliminates these systematic errors and for the protein GB3 yields ¹ J _NH splittings that agree to within a root-mean-square difference of 0.04 Hz with values measured for perdeuterated GB3. Accuracy of the method is also highlighted by a good fit to the GB3 structure of the ¹H-¹⁵N RDCs extracted from the minute differences in ¹J_NH splitting measured at 500 and 750 MHz ¹H frequencies, resulting from magnetic susceptibility anisotropy. A nearly complete set of ² J _NHα couplings was measured in GB3 in order to evaluate whether the impact of cross-correlated relaxation is dominated by the ¹⁵N–¹H ^α or ¹⁵N–¹H ^β dipolar interaction. As expected, we find that ² J _NHα  ≤ 2 Hz, with values in the α-helix (0.86 ± 0.52 Hz) slightly larger than in β-sheet (0.66 ± 0.26 Hz). Results indicate that under isotropic conditions, N–H^N/N–H ^β cross-correlated relaxation often dominates. Unresolved E.COSY effects under isotropic conditions involve ³ J _HNHα and J _NHα , but when weakly aligned any aliphatic proton proximate to both N and H^N can contribute. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Angiotensin AT2 receptor deficiency after myocardial infarction

Hearts of normotensive angiotensin II type 2 receptor (AT₂)-deficient mice do not develop fibrosis after angiotensin II-induced chronic hypertension. Thus, the goal of our study was to clarify whether AT₂ knockouts (KOs) are also characterized by altered left ventricular (LV) function and modified remodeling of the extracellular matrix (ECM) after induction of myocardial infarction (MI). MI was induced in 5-mo-old female AT₂-deficient mice and controls by occlusion of the left coronary artery. Time-matched sham-operated animals served as controls. After 48 h, the first sets of mice were hemodynamically characterized using a pressure-tip catheter (n=8/group). We also obtained pressure volume loops using a microconductance catheter in additional sets of animals 3 wk after induction of MI (n=7/group). Finally, the collagen index was illustrated by Sirius red staining and quantified by digital analysis. Whereas the LV function of sham-operated animals did not differ between both genotypes, the collagen index was 44% lower in KO animals. Forty-eight hours and 3 wk post-MI, systolic and diastolic LV function were impaired in both AT₂-deficient and wild-type (WT) animals to the same extent by approx 45%. No differences were found between the two genotypes with respect to LV hypertrophy and the fibrosis index in the infarcted and noninfarcted areas 3 wk post-MI. While AT₂-KO mice had less cardiac collagen content under basal conditions, the receptor deficiency had no significant influence on LV function at the two investigated time points after induction of MI or on the remodeling of ECM at the latter time point. Thus, hypetension-induced fibrosis is probably triggered by other control mechanisms than fibrosis induced by MI.     

Investigation of exchange couplings in [Fe3S4]+ clusters by electron spin-lattice relaxation

3 S₄]⁺, S=1/2, composed of three, antiferromagnetically coupled high-spin ferric ions) by continuous wave (CW) and pulsed EPR techniques: Azotobacter vinelandii ferredoxin I, Desulfovibrio gigas ferredoxin II, and the 3Fe forms of Pyrococcus furiosus ferredoxin and aconitase. The 35 GHz (Q-band) CW EPR signals are simulated to yield experimental g tensors, which either had not been reported, or had been reported only at X-band microwave frequency. Pulsed X- and Q-band EPR techniques are used to determine electron spin-lattice (T ₁, longitudinal) relaxation times at several positions on the samples' EPR envelope over the temperature range 2–4.2 K. The T ₁ values vary sharply across the EPR envelope, a reflection of the fact that the envelope results from a distribution in cluster properties, as seen earlier as a distribution in g ₃ values and in ⁵⁷ Fe hyperfine interactions, as detected by electron nuclear double resonance spectroscopy. The temperature dependence of 1/T ₁ is analyzed in terms of the Orbach mechanism, with relaxation dominated by resonant two-phonon transitions to a doublet excited state at ∼20 cm⁻¹ above the doublet ground state for all four of these 3Fe proteins. The experimental EPR data are combined with previously reported ⁵⁷Fe hyperfine data to determine electronic spin exchange-coupling within the clusters, following the model of Kent et al. Their model defines the coupling parameters as follows: J ₁₃=J, J ₁₂=J(1+ε′), J ₂₃=J(1+ε), where J _ij is the isotropic exchange coupling between ferric ions i and j, and ε and ε′ are measures of coupling inequivalence. We have extended their theory to include the effects of ε′≠0 and thus derived an exact expression for the energy of the doublet excited state for any ε, ε′. This excited state energy corresponds roughly to ε J and is in the range 5–10 cm⁻¹ for each of these four 3Fe proteins. This magnitude of the product ε J, determined by our time-domain relaxation studies in the temperature range 2–4 K, is the same as that obtained from three other distinct types of study: CW EPR studies of spin relaxation in the range 5.5–50 K, NMR studies in the range 293–303 K, and static susceptibility measurements in the range 1.8–200 K. We suggest that an apparent disagreement as to the individual values of J and ε be resolved in favor of the values obtained by susceptibility and NMR (J≳200 cm⁻¹ and ε≳0.02 cm⁻¹ ), as opposed to a smaller J and larger ε as suggested in CW EPR studies. However, we note that this resolution casts doubt on the accepted theoretical model for describing the distribution in magnetic properties of 3Fe clusters. Received: 23 December 1999 / Accepted: 8 March 2000     

Rapid solubilization of insoluble phosphate by a novel environmental stress-tolerant Burkholderia vietnamiensis M6 isolated from ginseng rhizospheric soil

We isolated and characterized novel insoluble phosphate (P)-solubilizing bacteria tolerant to environmental factors like high salt, low and high pHs, and low temperature. A bacterium M6 was isolated from a ginseng rhizospheric soil and confirmed to belong to Burkholderia vietnamiensis by BIOLOG system and 16S rRNA gene analysis. The optimal cultural conditions for the solubilization of P were 2.5% (w/v) glucose, 0.015% (w/v) urea, and 0.4% (w/v) MgCl₂·6H₂O along with initial pH 7.0 at 35°C. High-performance liquid chromatography analysis showed that B. vietnamiensis M6 produced gluconic and 2-ketogluconic acids. During the culture, the pH was reduced with increase in gluconic acid concentration and was inversely correlated with P solubilization. Insoluble P solubilization in the optimal medium was about 902 mg l⁻¹, which was approximately 1.6-fold higher than the yield in NBRIP medium (580 mg l⁻¹). B. vietnamiensis M6 showed resistance against different environmental stresses like 10–45°C, 1–5% (w/v) salt, and 2–11 pH range. The maximal concentration of soluble P produced by B. vietnamiensis M6 from Ca₃(PO₄)₂, CaHPO₄, and hydroxyapatite was 1,039, 2,132, and 1,754 mg l⁻¹, respectively. However, the strain M6 produced soluble P with 20 mg l⁻¹ from FePO₄ after 2 days and 100 mg l⁻¹ from AlPO₄ after 6 days, respectively. Our results indicate that B. vietnamiensis M6 could be a potential candidate for the development of biofertilizer applicable to environmentally stressed soil.