Theoretical stability diagram of solvent-containing black lipid films

Recently, we have developed an analytical, semi-microscopic theory for the macroscopic behavior of a solvent-containing black lipid film subjected to an electric cross film voltage, . Here we employ the theoretical expressions derived for the disjoining pressure, _D, the film elasticity, _F, and the film tension, _F, to construct the stability diagram of the film, in the _D-. Depending on its state (_D, ), the film is stable or is prone to squeezing or bending deformations. For a monooleate film we show how the destruction of the plane film due to a periodic thickness fluctuation (squeezing) is facilitated by two mechanisms: i) lowering of _D at fixed ; ii) lowering of at fixed _D, provided that the film is in a stable state characterized by _D<–7.03×10³ dyne/cm² and >0 mV. Bending of a low tension film (single interface tension _s 0.025 dyne/cm¹) can be achieved only for >170 mV and _D > –8.7 × 10⁴ dyne/cm². Finally, we demonstrate the existence of a marginal state ( _D ⁰ , ⁰) where the film is predicted to exhibit strong fluctuations both in the squeezing and in the bending mode.     

Protein dynamics studied by rotating frame 15N spin relaxation times

Summary Conformational rate processes in aqueous solutions of uniformly ¹⁵N-labeled pancreatic trypsin inhibitor (BPTI) at 36°C were investigated by measuring the rotating frame relaxation times of the backbone ¹⁵N spins as a function of the spin-lock power. Two different intramolecular exchange processes were identified. A first local rate process involved the residues Cys³⁸ and Arg³⁹, had a correlation time of about 1.3 ms, and was related to isomerization of the chirality of the disulfide bond Cys¹⁴-Cys³⁸. A second, faster motional mode was superimposed on the disulfide bond isomerization and was tentatively attributed to local segmental motions in the polypeptide sequence-Cys¹⁴-Ala¹⁵-Lys¹⁶-. The correlation time for the overall rotational tumbling of the protein was found to be 2 ns, using the assumption that relaxation is dominated by dipolar coupling and chemical shift anistropy modulated by isotropic molecular reorientation.Abbreviations BPTI basic pancreatic trypsin inhibitor - 2D two-dimensional - COSY 2D correlation spectroscopy - TOCSY 2D total correlation spectroscopy - RF radio frequency - CW continuous wave - TPPI time-proportional phase incrementation - CSA chemical shift anisotropy - T₁ longitudinal relaxation time - T₂ transverse relaxation time - T₁ relaxation time in the rotating frame , correlation time for overall rotational reorientation of the protein - _ex ^s , _ex ^f , correlation times for two conformational exchange processes (slow and fast).     

Kinetics of Δψ-Dependent Sucrose Transport in Plasma Membrane Vesicles from Higher Plant Cells

The inside-out vesicles of plasma membranes were isolated from pumpkin stem cells, and the kinetics of sucrose efflux induced by the K⁺-diffusion potential (_D) was studied by measuring light transmission. Two phases differing in their rates and duration were identified in _D-dependent changes of light transmission. The increase in _Delevated the rate and magnitude of the fast phase in light transmission changes but did not markedly affect the rate of the slow phase. These two phases also differed in their sensitivity to inhibitors and to changes in sucrose concentration in the external medium. Measurements of _Dduring sucrose transport by means of the fluorescence probe dis-C₃-(5) revealed differences in the magnitude of _Dand its stability in vesicles loaded with sucrose and mannitol, as well as under the action of inhibitors. The two-phase dependence of sucrose efflux from vesicles on the applied diffusion potential is discussed in the context of modern concepts on the functioning of sucrose carriers in the membranes.     

NMR exchange broadening arising from specific low affinity protein self-association: Analysis of nitrogen-15 nuclear relaxation for rat CD2 domain 1

Nuclear spin relaxation monitored by heteronuclear NMR provides a useful method to probe the overall and internal molecular motion for biological macromolecules over a variety of time scales. Nitrogen-15 NMR relaxation parameters have been recorded for the N-terminal domain of the rat T-cell antigen CD2 (CD2d1) in a dilution series from 1.20 mM to 40 M (pH 6.0, 25 °C). The data have been analysed within the framework of the model- free formalism of Lipari and Szabo to understand the molecular origin of severely enhanced transverse relaxation rates found for certain residues. These data revealed a strong dependence of the derived molecular correlation time _c upon the CD2d1 protein concentration. Moreover, a number of amide NH resonances exhibited exchange broadening and chemical shifts both strongly dependent on protein concentration. These amide groups cluster on the major -sheet surface of CD2d1 that coincides with a major lattice contact in the X-ray structure of the intact ectodomain of rat CD2. The complete set of relaxation data fit well to an equilibrium monomer–dimer exchange model, yielding estimates of exchange rate constants (k_ON=5000 M^-1 s^-1; k_OFF=7 s^-1) and a dissociation constant (K_D 3–6 mM) that is consistent with the difficulty in detecting the weak interactions for this molecule by alternative biophysical methods. The self-association of CD2d1 is essentially invariant to changes in buffer composition and ionic strength and the associated relaxation phenomena cannot be explained as a result of neglecting anisotropic rotational diffusion in the analysis. These observations highlight the necessity to consider low affinity protein self-association interactions as a source of residue specific exchange phenomena in NMR spectra of macromolecular biomolecules, before the assignment of more elaborate intramolecular conformational mechanisms.     

Synthesis of Aminoethyl Glycosides of the Ganglioside GM1 and Asialo-GM1 Oligosaccharide Chains

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of asialo-GM₁ ganglioside in 72% overall yield. Selective 3-O-glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,6-di-O-benzyl--D-galactopyranosyl)--D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero--D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)oate]-(2 3)-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM₃ ganglioside, in 79% yield. Its 4-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl) 1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-{[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)onate]-(2 3)}-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)-[-D-Neu5Ac-(2 3)]--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of GM₁ ganglioside.     

The role of lipid and antioxidant exchanges in cell division synchronization (mathematical model)

Cell-cycle synchronization of two diffusecoupled cells has been studied in the framework of the membrane model for the cell division cycle, proposed by Chernavskii et al. (1977). It has been shown semianalytically (using the averaging principle) and by computer stimulation that a) if the duration of theG1-phase (T _G1 ) for two identical cells is comparable with the duration of the remaining cycle (T _S+G2+M ), the lipid (L)-exchange results in a synchronization with phase difference =0. The antioxidant (A)-exchange leads to a phase-locking with =T ₀/2 (whereT ₀ is the cell cycle period; b) ifT _G1 T _S+G2+M (orT _G1 T _S+G2+M ) theL-exchange makes synchronization possible both with =0 and =T ₀/2 while theA-exchange results in phase-locking with confined to the region 0 toT ₀/2; c) for non-identical cells differing in the values of kinetic parameters, the locking band narrows as the population density increases (when some model parameters are close to the bifurcation thresholds). We expect that the cells selected artificially at a definite phase of cycle might maintain the synchronous division for a long time if the lipid exchange between cells were stimulated.     

Reaction centers from three herbicide resistant mutants of Rhodobacter sphaeroides 2.4.1: Kinetics of electron transfer reactions

Electron transfer rates were measured in RCs from three herbicide-resistant mutants with known amino acid changes to elucidate the structural requirements for last electron transfer. The three herbicide resistant mutants were IM(L229) (Ile-L229 Met), SP(L223) (Ser-L223 Pro) and YG(L222) (Tyr-L222 Gly). The electron transfer rate D⁺Q_A ^-Q_BD⁺Q_AQ_B (k _AB) is slowed 3 fold in the IM(L229) and YG(L222) RCs (pH 8). The stabilization of D⁺Q_AQ_B ^- with respect to D⁺Q_AQ_B ^- (pH 8) was found to be eliminated in the IM(L229) mutant RCs (G⁰ 0 meV), was partially reduced in the SP(L223) mutant RCs (G⁰=–30 meV), and was unaltered in the YG(L222) mutant RCs (G⁰=–60 meV), compared to that observed in the native RCs (G⁰=–60 meV). The pH dependences of the charge recombination rate D⁺Q_AQ_B ^-DQ_AQ_B (k _BD) and the electron transfer from Q_A ^- (k _QA ^-Q_A) suggest that the mutations do not affect the protonation state of Glu-L212 nor the electrostatic interactions of Q_B and Q_B ^- with Glu-L212. The binding affinities of UQ₁₀ for the Q_B site were found in order of decreasing values to be native IM(L229) > YG(L222) SP(L223). The altered properties of the mutant RCs are used to deduce possible structural changes caused by the mutations and are dicscussed in terms of photosynthetic efficiency of the herbicide resistant strains.Abbreviations Bchl bacteriochlorophyll - Bphe bacteriopheophytin - cholate 3,7,12-trihydroxycholanic acid - D donor (bacteriochlorophyll dimer) - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - PS II photosystem II - Q_A and Q_B primary and secondary quinone acceptors - RC bacterial reaction center - Tris tris(hydroxymethyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

Selectively 13C-enriched DNA: Evidence from 13C1′ relaxation rate measurements of an internal dynamics sequence effect in the lac operator

Summary In order to study some internal dynamic processes of the lac operator sequence, the ¹³C-labeled duplex 5d(C₀G₁C₂T₃C₄A₅C₆A₇A₈T₉T₁₀) · d(A₁₀A₉T₈T₇G₆T₅G₄A₃G₂C₁G₀)3 was used. The spreading of both the H1 and C1 resonances brought about an excellent dispersion of the ¹H1-¹³C1 correlations. The spinlattice relaxation parameters R(C_z), R(C_x,y) and R(H_zC_z) were measured for each residue of the two complementary strands, except for the 3-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387–7401; and (1990) J. Am. Chem. Soc., 112, 4989–4991]. A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C1-H1 fragments exhibited both slow (_s = 1.5) and fast (_f = 20 ps) restricted libration motions (S _inf2 ^sups =0.74 to 1.0 and S _inf2 ^supf =0.52 to 0.96). Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex. The possible role played by the special structure of the AATT sequence is discussed. No evident correlation was found between the amplitude motions of the complementary residues. The 5-terminal residues showed large internal motions (S²=0.5), which describe the fraying of the double helix. Global examination of the microdynamical parameters S _inf2 ^supf and S _inf2 ^sups along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (S _inf2 ^supf =0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin. Moreover, the fit of both R(C_z) and R(HzC_z) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(C_x,y) for the purine residues in the (53) G₄A₃ and A₁₀A₉T₈T₇ sequences. This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale. It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator [Boelens et al. (1987) J. Mol. Biol., 193, 213–216] and that these motions could be implied in the lac-operator-lac-repressor recognition process.     

TROSY experiment for refinement of backbone psi and phi by simultaneous measurements of cross-correlated relaxation rates and 3,4J(H alpha HN) coupling constants

The TROSY principle has been introduced into a HNCA experiment, which is designed for measurements of the intraresidual and sequential H-C/H^N-N dipole/dipole and H-C/N dipole/CSA cross-correlated relaxation rates. In addition, the new experiment provides values of the ^3,4 J _{H HN} coupling constants measured in an E.COSY manner. The conformational restraints for the and angles are obtained through the use of the cross-correlated relaxation rates together with the Karplus-type dependencies of the coupling constants. Improved signal-to-noise is achieved through preservation of all coherence transfer pathways and application of the TROSY principle. The application of the [¹⁵N,¹³C]-DQ/ZQ-[¹⁵N,¹H]-TROSY-E.COSY experiment to the 16 kDa apo-form of the E. coli Heme Chaperon protein CcmE is described. Overall good agreement is achieved between and angles measured with the new experiment and the average values determined from an ensemble of 20 NMR conformers.     

Analysis of gas exchange in seedlings of Acer saccharum: integration of field and laboratory studies

In the field, photosynthesis of Acer saccharum seedlings was rarely light saturated, even though light saturation occurs at about 100 mol quanta m^-2 s^-1 photosynthetic photon flux density (PPFD). PPFD during more than 75% of the daylight period was 50 mol m^-2 s^-1 or less. At these low PPFD's there is a marked interaction of PPFD with the initial slope (CE) of the CO₂ response. At PPFD-saturation CE was 0.018 mol m^-2 s^-1/(l/l). The apparent quantum efficiency (incident PPFD) at saturating CO₂ was 0.05–0.08 mol/mol. and PPFD-saturated CO₂ exchange was 6–8 mol m^-2 s^-1. The ratio of internal CO₂ concentration to external (C _i /C _a ) was 0.7 to 0.8 except during sunflecks when it decreased to 0.5. The decrease in C _i /C _a during sunflecks was the result of the slow response of stomates to increased PPFD compared to the response of net photosynthesis. An empirical model, which included the above parameters was used to simulate the measured CO₂ exchange rate for portions of two days. Parameter values for the model were determined in experiments separate from the daily time courses being sumulated. Analysis of the field data, partly through the use of simulations, indicate that the elimination of sunflecks would reduce net carbon gain by 5–10%.List of symbols A measured photosynthetic rate under any set of conditions (mol m^-2 s^-1) - A _m (atm) measured photosynthetic rate at saturating PPFD, 350 l/l CO₂ and 21% (v/v) O₂ (mol m^-2 s^-1) - C constant in equation of Smith (1937, 1938) - C _a CO₂ concentration in the air (l/l) - C _i CO₂ concentration in the intercellular air space (l/l) - C _i ^/* C _i corrected for CO₂ compensation point, i.e., C _i -I ^*, (l/l) - CE initial slope of the CO₂ response of photosynthesis (mol m^-2 s^-1/(l/l)) - CEM CE at PPFD saturation - E transpiration rate (mmol m^-2 s^-1) - F predicted photosynthetic rate (mol m^-2 s^-1) - G leaf conductance to H₂O (mol m^-2 s^-1) - I photosynthetic photon flux density (mol m^-2 s^-1) - N number of data points - P _m predicted photosynthetic rate at saturating CO₂ and given PPFD (mol m^-2 s^-1) - P _ml predicted photosynthetic rate at saturating CO₂ and PPFD (mol m^-2 s^-1) - R _d residual respiratory rate (mol m^-2 s^-1) - T _a air temperature (°C) - T _l leaf temperature (°C) - V reaction velocity in equation of Smith (1937, 1938) - V _max saturated reaction velocity in equation of Smith (1937, 1938) - VPA vapor pressure of water in the air (mbar/bar) - VPD vapor pressure difference between leaf and air (mbar/bar) - X substrate concentration in equation of Smith (1937, 1938) - initial slope of the PPFD response of photosynthesis at saturating CO₂ (mol CO₂/mol quanta) - (atm) initial slope of the PPFD response of photosynthesis at 340 l/l CO₂ and 21% (v/v) O₂ (mol CO₂/mol quanta) - I ^* CO₂ compensation point after correction for residual respiration (l/l) - PPFD compensation point (mol m^-2 s^-1)     

Body temperature responses of Savanna Brown goat to the harmattan and hot-dry season

Rectal and vaginal temperature responses of the Savanna Brown goat indigenous to the Nigerian guinea savanna were determined during the harmattan and the hot-dry season. Measurements were made at 06:00h and at 14:00h after 8h exposure to field conditions. At the 06:00h measurements during the harmattan, all animals were observed to shiver. A significant (P<0.01) positive correlation was found between rectal (T_re) and vaginal temperatures. During the harmattan, mean T_re was 38.2C at 06:00h and 39.7C at 14:00h; the mean difference, T_re was 1.5C. During the hot-dry season, T_re at 06:00h was 38.1C, and at 14:00h, 38.7; T_re was 0.6C. It is concluded that the harmattan is thermally more stressful than the hot-dry season and that passive thermolability may not be an important mechanism in the Savanna Brown goat in adaptation to thermal stress.     

Temperature-sensitive coupling and uncoupling of ATPase-mediated,nonradiative energy dissipation: Similarities between chloroplasts and leaves

The effects of temperature on the dark relaxation kinetics of nonradiative energy dissipation in photosystem II were compared in lettuce (Lactuca sativa L.) chloroplasts and leaves of Aegialitis annulata R. Br. After high levels of violaxanthin de-epoxidation in the light, Aegialitis leaves showed a marked delay in the dark relaxation of nonradiative dissipation, measured as non-photochemical quenching (NPQ) of photosystem II chlorophyll a fluorescence. Aegialitis leaves also maintained a moderately high adenylate energy charge at low temperatures during and after high-light exposure, presumably because of their limited carbon-fixation capacity. Similarly, dark-sustained NPQ could be induced in lettuce chloroplasts after de-epoxidizing violaxanthin and light-activating the ATP synthase. The duration and extent of dark-sustained NPQ were strongly enhanced by low temperatures in both chloroplasts and leaves. Further, the NPQ sustained at low temperatures was rapidly reversed upon warming. In lettuce chloroplasts, low temperatures sharply decreased the ATP-hydrolysis rate while increasing the duration and extent of the resultant trans-thylakoid proton gradient that elicits the NPQ. This was consistent with a higher degree of energy-coupling, presumably due to reduced proton diffusion through the thylakoid membrane at the lower temperatures. The chloroplast adenylate pool was in equilibrium with the adenylate kinase and therefore both ATP and ADP contributed to reverse coupling. The low-temperature-enhanced NPQ quenched the yields of the dark level (F_o) and the maximal (F_m) fluorescence proportionally in both chloroplasts and leaves. The extent of NPQ in the dark was inversely related to the efficiency of photosystem II, and very similar linear relationships were obtained over a wide temperature range in both chloroplasts and leaves. Likewise, the dark-sustained absorbance changes, caused by violaxanthin de-epoxidation (A_508nm) and energy-dependent light scattering (A_536nm) were strikingly similar in chloroplasts and leaves. Therefore, we conclude that the dark-sustained, low-temperature-stimulated NPQ in chloroplasts and leaves is apparently directly dependent on lumen acidification and chloroplastic ATP hydrolysis. In leaves, the ATP required for sustained NPQ is evidently provided by oxidative phosphorylation in the mitochondria. The functional significance of this quenching process and implications for measurements of photo-protection versus photodamage in leaves are discussed.Abbreviations and Symbols A antheraxanthin - Chl chlorophyll - DPS de-epoxidation state of the xanthophyll cycle, ([Z+A]/[V+A+Z]) - F, F steady-state fluorescence in the absence, presence of thylakoid energization - F_o, F_o dark fluorescence level in the absence, presence of thylakoid energization - F_m, F_m maximal fluorescence in absence, presence of thylakoid energization - NPQ nonphotochemical quenching (F_m/F_m)–1 - V violaxanthin - Z zeaxanthin - NRD nonradiative dissipation - PFD photon flux density - [2ATP+ADP] - pH trans-thylakoid proton gradient - S pH-dependent light scattering - _PSII (F_m–F)/F_m, photon yield of PSII photochemistry at the actual reduction state in the light or dark - [ATP+ADP+AMP] We thank Connie Shih for skillful assistance in growing plants and for conducting HPLC analyses. Support from an NSF/USDA/DOE postdoctoral training grant to A.G. is gratefully acknowledged. A.G. also wishes to thank Prof. Govindjee for valuable discussions. C.I.W.-D.P.B. Publication No. 1197.     

Two dimensional diffusion of small molecules on protein surfaces: an EPR study of the restricted translational diffusion of protein-bound spin labels

Heisenberg spin exchange rates and dipole-dipole spin lattice relaxation rates for deuterated ¹⁴N- and ¹⁵N-spin labels bound selectively to the histidine His15 and to the lysines Lys13, 96, 97 of the lysozyme molecule have been determined with the aid of electron spin resonance spectroscopy. The results can be interpreted in terms of a two dimensional translational diffusion of the nitroxide tips of the spin labels along the protein surface within restricted surface areas. The spin labels are regarded as models for long amino acid side chains and as probes for the dynamics of protein and water in the vicinity of the protein surface. The translational diffusion coefficient DP_II is reduced by a factor of between six and thirty compared to the value of D found for the spin labels in bulk water, its value for T = 295 K is given by (1.3±0.6)·10^–10m²s^–1 D (2.4±0.3) 10^–11 m²s^–1. Offprint requests to: H.-J. Steinhoff     

Protocol-dependence of equivalent circuit parameters of toad urinary bladder

Summary Determinations of current-voltage relationships are widely employed in the characterization of epithelial sodium transport. In order to determine the protocol dependence of transport parameters in the toad urinary bladder, studies were carried out in the presence and absence of amiloride, an inhibitor of active sodium transport. With symmetric positive and negative perturbations of the transepithelial electrical potential difference (0±100 mV) for 30 sec, the amiloride-sensitive current-voltage (i ^a -) relationship was near linear over the range –75+100 mV, indicating constancy of the conductance ^a and the apparent electromotive force E _Na, lumped parameters of the standard electrical equivalent circuit model of the active transport system. With a reverse protocol (±1000 mV) or 15 min perturbations thei ^a - relationships were highly nonlinear. Nonlinearity reflected voltage dependence of parameters: perturbations that increased active transport decreased E _Na and increased ^a, as evaluated from 10 sec perturbations of ; slowing of active transport produced the converse changes. These effects are usefully analyzed in both quasi-steady states and true steady states by means of a detailed equivalent circuit incorporating the significant ionic currents across each plasma membrane. Precise understanding of the significance of ^a and E _Na will require characterization of the partial ionic conductances on perturbation of .     

Comparison of [35S]GTPγS binding to plasma membranes and endomembranes prepared from soybean and rat

Summary Plasma membranes were prepared from soybean hypocotyls and roots by aqueous two-phase partitioning and subsequent free-flow electrophoresis. The highly purified plasma membranes bound [³⁵S]GTPS with a relatively high affinity (K_d10nM). The binding was saturable and specific as it was indicated by the displacement of bound [³⁵S]GTPS by unlabeled GTPS and GTP, but not by ATPS, ATP, UTP or CTP. ITP was intermediate in its ability to displace [³⁵S]GTPS. When soybean plasma membrane proteins were separated by SDS-PAGE and displayed by autoradiography, two major [³⁵S]GTPS binding proteins were revealed with apparent molecular weights of 24 and 28 kDa. Results with plasma membranes from soybean hypocotyls and roots were similar but differed from those with plasma membranes prepared from rat liver and adipocytes where only a single major [³⁵S]GTPS binding activity with a molecular weight of 28 kDa was observed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - G protein hetero-trimeric GTP binding protein with , , subunits - G_n protein GTP binding protein detected on nitrocellulose blots - GTPS guanosine 5-[-thio]triphosphate - IAA 3-indoleacetic acid - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Central nervous versus total body thermosensitivity of the duck

Ducks were chronically implanted with thermodes in the POAH region, the lower brainstem or the vertebral canal. At thermoneutral conditions, lowering the temperature of the spinal cord (T_vc) or the lower brainstem (T_mb) stimulated metabolic heat production (M) with a subsequent rise of core temperature (T_c). Lowering the temperature of the POAH region (T_hy) induced a fall of T_c due to paradoxical activation of heat defence and, thus, induced slight to moderate general hypothermia depending on the cooling intensity. When T_hy was normalized, the hypothermia temporarily stimulated metabolic heat production until T_c was normalized. Cold sensitivity of the entire body, as revealed by the metabolic response to the hypothermia induced by preceding POAH cooling, and cold sensitivity of the spinal cord and the lower brainstem, as revealed by the metabolic response to local cooling, were quantified by calculating the quotient M/T from the maximum metabolic response and the experimentally induced drop of T_c, T_mb and T_vc. With lower brainstem cooling M/T_mbdid not exceed –0.4 W/(kg · C). With spinal cord cooling, M/T_vc did not exceed –0.6 W/(kg · C). The mean value of M/T_c after hypothermia induced by POAH cooling was –4.02 W/(kg · C). The results indicate that the cold sensitivity residing in the CNS of ducks represents only a small fraction of the entire cold sensitivity of the body.Presented at the Eighth International Congress of Biometeorology, 9–14 September 1979, Shefayim, Israel.     

Amino acid sequence of Kalinowaski's Tinamou (Nothoprocta kalinowskii) hemoglobin and the rate of evolution of bird alphaD-globin

Two hemoglobin components are recognized in erythrocytes of the adult Tinamou. We determined the amino acid sequences of Tinamou ^D-, ^A-, and -globins from intact globin chains and several chemically cleaved fragments. A remarkable feature of Tinamou hemoglobin was a deletion in the ^D-globin chain. This has not been reported in the literature, except in pigeon embryonic ^D-globin. The amino acid sequences of Tinamou globin were highly similar to those of Ostrich and Rhea hemoglobin. Comparison between Tinamou, Ostrich, and Rhea that suggested the evolution speed of globin, ^D = ^A > , was related with the early appearance birds. The important residues in Tinamou hemoglobin as the heme contact and oxygen binding regions were highly conserved in other species.     

Electrogenic and electroneutral transport modes of renal Na/K ATPase reconstituted into proteoliposomes

Summary This paper describes measurements of electrical potentials generated by renal Na/K-ATPase reconstituted into proteoliposomes, utilizing the anionic dye, oxonol VI. Calibration of absorption changes with imposed diffusion potentials allows estimation of absolute values of electrogenic potentials.ATP-dependent Na_cyt/K_exc exchange in K-loaded vesicles generates large potentials, up to 250 mV. By comparing initial rates or steady-state potentials with ATP-dependent²²Na fluxes in different conditions, it is possible to infer whether coupling ratios are constant or variable. For concentrations of Na_cyt (2–50mm) and ATP (1–1000 m) and pH's (6.5–8.5), the classical 3Na_cyt/2K_exc coupling ratio is maintained. However, at low Na_cyt concentrations (<0.8mm), the coupling ratio is apparently less than 3Na_cyt/2K_exc.ATP-dependent Na_cyt/congener_exc exchange in vesicles loaded with Rb, Cs, Li and Na is electrogenic. In this mode congeners, including Na_exc, act as K_exc surrogates in an electrogenic 3Na_cyt/2congener_exc exchange. (ATP+Pi)-dependent K_cyt/K_exc exchange in K-loaded vesicles is electroneutral.ATP-dependent uncoupled Na flux into Na- and K-free vesicles is electroneutral at pH 6.5–7.0 but becomes progressively electrogenic as the pH is raised to 8.5. The²²Na flux shows no anion specificity. We propose that uncoupled Na flux is an electroneutral 3Na_cyt/3H_exc exchange at pH 6.5–7.0 but at higher pH's the coupling ratio changes progressively, reaching 3Na/no ions at pH 8.5. Slow passive pump-mediated net K uptake into Na- and K-free vesicles is electroneutral, and may also involve K_cyt/H_exc exchange.We propose the general hypothesis that coupling ratios are fixed when cation transport sites are saturated, but at low concentrations of transported cations, e.g., Na_cyt in Na/K exchange and H_exc in uncoupled Na flux, coupling ratios may change.     

Expression of a chimeric stilbene synthase gene in transgenic wheat lines

A chimeric stilbene synthase (sts)gene was transferred into wheat. Stilbene synthases play a role in the defence against fungal diseases in some plant species (e.g. groundnut or grapevine) by producing stilbenetype phytoalexins like resveratrol. Resveratrol is also claimed to have positive effects to human health. Embryogenic scutellar calli derived from immature embryos of the two commercial German spring wheat cultivars Combi and Hanno were used as target tissue for cotransformation by microprojectile delivery. The selectable marker/reporter gene constructs contained the bargene either driven by the ubiquitinpromoter from maize (pAHC 25, also containing the uidAgene driven by the ubiquitinpromoter), or by the actinpromoter (pDM 302) from rice. The cotransferred plasmid pStil 2 consisted of a grapevine stscoding region driven by the ubiquitin promoter. Eight transgenic Combi and one Hanno T_Oplant were obtained and, except one Combi T_Oplant, found to be cotransformants due to the integration of both the stsgene and the selectable marker or reporter genes. Expression of the stsgene was proven by RTPCR, and, for the first time, by detection of the stilbene synthase product resveratrol by HPLC and mass spectrometry. The stsgene was expressed in four of the seven transgenic Combi T_{_o}plants. Two of the respective T₁progenies segregated in a Mendelian manner were still expressing the gene. Investigations into methylation of the stsgene showed that in three nonexpressing progenies inactivation was paralleled by methylation.     

O-Glycosylhydrolases of Marine Filamentous Fungi: β-1,3-Glucanases of Trichoderma aureviride

The ability to produce extracellular O-glycosylhydrolases was studied in 14 strains of marine filamentous fungi sampled from the bottom sediments of the South China Sea. The following activities were detected in the culture liquids of the fungi: N-acetyl--D-glucosaminidase, -D-glucosidase, -D-galactosidase, -1,3-glucanase, amylase, and pustulanase. -1,3-Glucanases were isolated by ultrafiltration, hydrophobic interaction chromatography, and ion exchange chromatography, and their properties were studied. Data on products of enzymatic digestion of laminaran, absence of transglycosylation activity, and the pattern of action of natural inhibitors confirmed that -1,3-glucanase belonged to the exo type. Inhibitor analysis demonstrated the role of a thiol group and tryptophan and tyrosine residues in the catalytic activity.