Paramagnetic spin catalysis of a radical recombination reaction

Recombination of the triplet state radical pair consisting of two hydrogen atoms catalysed by molecular oxygen is considered as a simulating example of a paramagnetic-exchange catalytic process. Intermolecular exchange interaction in the collision complex between the H₂ and O₂ molecules is calculatedab initio in STO-6G and 6–31 G^* basis sets with complete active space configuration interaction. Calculations are done at a fixed O–H distance (3 Å), scanning the H–H bond length from 0.6 till 12 Å at the linear geometry of collision. The mixture of the triplet (T)³ _u ⁺ and singlet (S)¹ _g ⁺ states of the hydrogen moiety is possible because both states have the same triplet symmetry in the collision complex with O₂ (³ _g ^– ). A strong mixture of the S (¹ _g ⁺ , H₂ +³ _g ^– , O₂) and T) and T (³ _u ⁺ , H₂ +³ _g ^– , O₂) states is actually obtained even at large H–H distances. The quintet and singlet states^5,1(³ _u ⁺ , H₂ +³ _g ^– , O₂) are also considered for comparison of the exchange potentials. Atr(H–H)4.4 Å the S-T splitting is approximately constant (12 cm^-1 in the STO-6G basis set; 55.5 cm^-1 in the 6–31 G^* basis set) and is determined by the exchange interaction between O₂ and the nearest hydrogen atom in the O–O...H fragment. The paramagnetic catalyst can accelerate radical recombination through the triplet-singlet nonadiabatic transition to the lowest S reactive state when the radical encounter takes place in the vicinity of the catalyst. Though we do not consider the radical dynamics in a real solvent, which modulates the exchange potentials and the T-S transitions, the nature of this mechanism of spin catalysis is obvious. The electric polarization and charge transfer are important in the analysis of the exchange interaction and radical recombination potentials for all multiplets. In accordance with the concept of spin catalysis, the electronic spin-uncoupling mechanism, induced by O₂ perturbation, has the same nature as other known catalytic processes of paramagnetic-exchange type.     

Laboratory culture studies of Trichodesmium isolated from the Great Barrier Reef Lagoon, Australia

Cultures of Trichodesmium from the Northern and Southern Great Barrier Reef Lagoon (GBRL) have been established in enriched seawater and artificial seawater media. Some cultures have been maintained with active growth for over 6years. Actively growing cultures in an artificial seawater medium containing organic phosphorus (glycerophosphate) as the principal source of phosphorus have also been established. Key factors that contributed to the successful establishment of cultures were firstly, the seed samples were collected from depth, secondly, samples were thoroughly washed and thirdly, incubations were conducted under relatively low light intensities (PAR 40–50molquantam^–2s^–1). N₂ fixation rates of the cultured Trichodesmium were found to be similar to those measured in the GBRL. Specific growth rates of the cultures during the exponential growth phase in all enriched media were in the range 0.2–0.3day^–1 and growth during this phase was characterised by individual trichomes (filaments) or small aggregations of two to three trichomes. Characteristic bundle formation tended to occur following the exponential growth phase, which suggests that the bundle formation was induced by a lack of a necessary nutrient e.g. Fe. Results from some exploratory studies showed that filament-dominated cultures of Trichodesmium grew over a range of relatively low irradiances (PAR 5–120molquantam^–2s^–1) with the maximum growth occurring at 40–50molquantam^–2s^–1. These results suggest that filaments of the tested strain are well adapted for growth at depth in marine waters. Other studies showed that growth yields were dependent on salinity, with maximum growth occurring between 30 and 37psu. Also the cell yields decreased by an order of magnitude with the reduction of Fe additions from 450 to 45nM. No active growth was observed with the 4.5nM Fe addition.     

Reconstitution of a calcium-activated potassium channel in basolateral membranes of rabbit colonocytes into planar lipid bilayers

Summary A highly enriched preparation of basolateral membrane vesicles was isolated from rabbit distal colon surface epithelial cells employing the method described by Wiener, Turnheim and van Os (Weiner, H., Turnheim, K., van Os, C.H. (1989)J. Membrane Biol.110:147–162) and incorporated into planar lipid bilayers. With very few exceptions, the channel activity observed was that of a high conductance, Ca²⁺-activated K⁺ channel. This channel is highly selective for K⁺ over Na⁺ and Cl^–, displays voltage-gating similar to maxi K(Ca) channels found in other cell membranes, and kinetic analyses are consistent with the notion that K⁺ diffusion through the channel involves either the binding of a single K⁺ ion to a site within the channel or single-filling (multi-ion occupancy). Channel activity is inhibited by the venom from the scorpionLeiurus quinquestriatus, Ba²⁺, quinine, and trifluoperazine. The possible role of this channel in the function of these cells is discussed.     

Template-directed reactions of aminonucleosides

Summary We have studied the reactions between adenosine 5-phosphorimidazolide and 9-(2-amino-2-deoxyxylofuranosyl) adenine (I) or 3-methylamino-3-deoxyadenosine (II), both with and without a poly (U) template. We find that both amino compounds react much more rapidly than does adenosine, in the absence of a template. The rate of reaction is greatly enhanced by a poly (U) template in the case of I, but the enhancement is slight in the case of II.Abbreviations A adenosine - xylo A_NH2 9-(2-amino-2-deoxy--D-xylofuranosyl) adenine - A_NHMe 3-methylamino-3-deoxyadenosine - ImpA adenosine 5-phosphorimidazolide - A³ pA adenylyl-[35]-adenosine - A² pA adenylyl-[25]-adenosine - U_NPA adenylyl-[52]-2-amino-2-deoxyuridine - xylo A_NPA 9-[adenylyl-(52)-2-amino-2-deoxy--D-xylofuranosyl]adenine - A_(NMe)pA adenylyl-[53]-3-methylamino-3-deoxyadenosine - pA adenosine 5phosphate - AppA P₁, P₂-diadenosine 5pyrophosphate - (pA)_n n = 2, 3 [2-5]-linked oligomers of pA - A² pA² pA [2-5]-linked trinucleoside diphosphate of A - poly (U) polyuridylic acid     

Conformational energy of the 5-membered ring. Implication of geometric and energetic properties in the conformational characteristics

In a previous article (8) a geometrical study of the five-membered ring showed that: a) for the case of the 20 symmetrical C₂ and C_s conformations, the pseudorotation formulae for the torsion angles are a geometrical property of the ring; b) geometrical considerations alone are unable to define the puckering amplitude, the bond angle values, and the pathway between two symmetrical conformations. Here we examine how the energy equations enable us to define the deformation amplitude _m, establish the bond angles expressions and check the energy invariability along the pseudorotation circuit. The problem is next developed fully in the case where the bond and torsional energy only are considered: the literal expression¹ of _m is then given as a function of the bond angle which cancels out the bond angle energy. A numerical application is carried out on cyclopentane and the values of the parameters K^t, K¹ and used in the Conformational energy calculations are considered.Notations used 1 _i bond lengths 1 in the case of the regular ring - _i torsional angles - _i bond angles - 3/5 = 108 - 4/5 = 144 - , _{i i} – = complement to the 108 bond angle _i - _T - E Conformational energy of the 5-membered ring - E Conformational energy difference between planar and deformed ring - A _n Coefficients of the energy development in terms of - E _i ^l Bond energy relative to atom i (associated with angle _i) - K _i ^l Bond constant relative to atom i (associated with angle _i) - E _i ^l Torsional energy relative to the i ^th bond (associated with angle _i) - k _i ^l Torsional constant relative to the i ^th bond (associated with angle _i) - _i Angle _i value corresponding to zero bond energy E _i ^l (when the 5 atoms of the ring are identical, _i ) - r _ij Distance between atoms i and j - q _i Charge carried by atom i - e Constant of proportionality including the effective dielectric constant - A _ij, B_ij, d_ij Coefficients dependent on the nature of the atoms i and j and accounted for in the Van der Waals energy and hydrogen bond expressions - S (r _ij) Electrostatic contribution to the hydrogen bond energy - P Pseudorotation phase angle - _m Maximum torsional angle value characterising the deformation amplitudeM     

Patterned disjunction in Drosophila melanogaster. I. Assortment of SPA pol /+ and X,Y in the stock N1

N1 (= Nijmegen 1) D. melanogaster heterozygous for sparkling poliert (4) (= pol, here) were backcrossed as single pairs. When were not selected for departure from 1/1, pol/pol ⁺, many exceptional ratios were observed even though the net for all 67 pairs was approximately one-to-one; in the same experiment a net excess of was observed. In a second experiment were selected for departure from 1/1, pol/pol ⁺ratios. The net pol/pol ⁺ratios became significantly different from the 1/1 expected but the sex ratio approached normal. Lineage of the males in the second experiment were recorded and displayed as pedigrees. These together with tabulated data suggest that in some pairs, one of the four categories pol , pol , pol ⁺, pol ⁺ may be significantly greater or less than 1/4 of the total offspring recovered.     

Diagnosing lactic acid fermentation based on specific rates of growth,substrate consumption and product formation

The on-line calculated specific rates of growth, substrate consumption and product formation were used to diagnose microbial activities during a lactic acid fermentation. The specific rates were calculated from on-line measured cell mass, and substrate and product concentrations. The specific rates were more sensitive indicators of slight changes in fermentation conditions than such monitored data as cell mass or product concentrations.List of Symbols 1/h specific rate of cell growth - 1/h specific rate of substrate consumption - 1/h specific rate of product formation - ^* dimensionless specific rate of cell growth - ^* dimensionless specific rate of substrate consumption - ^* dimensionless specific rate of product formation - _max 1/h maximum specific rate of cell growth - _max 1/h maximum specific rate of substrate consumption - _max 1/h maximum specific rate of product formation - X g/l cell mass concentration - S g/l substrate concentration - S ^* dimensionless substrate concentration - S ₀ g/l initial substrate concentration - P g/l product concentration     

A K+-selective,three-state channel from fragmented sarcoplasmic reticulum of frog leg muscle

Summary Sarcoplasmic reticulum (SR) vesicles from frog leg muscle were fused with a planar phospholipid bilayer by a method described previously for rabbit SR. As a result of the fusion, K⁺-selective conduction channels are inserted into the bilayer. Unlike the two-state rabbit channel, the frog channel displays three states: a nonconducting (closed) state and two conducting states and . In 0.1m K⁺ the single-channel conductances are 50 and 150 pS for and , respectively. The probabilities of appearearance of the three states are voltage-dependent, and transitions between the closed and states proceed through the state. Both open states follow a quantitatively identical selectivity sequence in channel conductance: K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Li⁺>Cs⁺. Both open states are blocked by Cs⁺ asymmetrically in a voltage-dependent manner. The zero-voltage dissociation constant for blocking is the same for both open states, but the voltage-dependences of the Cs⁺ block for the two states differ in a way suggesting that the Cs⁺ blocking site is located more deeply inside the membrane in the than in the state.     

De novo induction of adventitious roots in excised shoots of tomatoes by fumonisin B1, a metabolite of Fusarium moniliforme

The de novo induction of roots in tomatoes (Lycopersicon esculentum) Mill. cvs. Earlypak-7, Ace, Better Boy, Roma, and Parks' Whopper by fumonisin B₁, a mycotoxin produced by Fusarium moniliforme J. Sheld., was studied. In graded dosages of fumonisin B₁, detached stems of the cultivars Ace, Better Boy, and Roma were induced to produce calluses and roots earlier than controls. The cultivar Ace was especially responsive to this mycotoxin, and following a single application, callus initiation was observed to occur within a 24–48-h period and roots were produced as early as 72 h with 10 g/shoot or as late as 96 h with low dosages. The control plants of all cultivars were completely negative for a rooting response during this time. Some cultivars treated with fumonisin B₁ showed either no response or developed signs of phytotoxicity. Those cultivars that were stimulated to produce roots did not show signs of phytotoxicity, except at dosages of 0.5 mg/plant and higher. One cultivar did not show any signs of phytotoxicity nor was it induced to root. The ability of fumonisin B₁ to affect the accumulation of calcium in other systems, and its structural similarity to sphingosine suggest that the induction of adventitious roots may be a calcium-dependent process.     

Distribution of carbon isotope composition of modern soils on the Qinghai-Tibetan Plateau

This paper presents a large data set on carbon isotope composition (¹³C) of modern soils which were collected under the main vegetation communities along an altitude of 1250–5500m above sea level in the Qinghai-Tibetan Plateau. The ¹³C values of 198 samples range from –28.6 to –15.1 versus PDB and exhibit a clean relation to different vegetation communities from forest (–25.9±1.2) to shrub (–24.7±1.4), steppe (–23.1±1.3), alpine meadow (–23.6±0.7), alpine desert steppe (–21.3±1.6), and alpine desert (–18.9±2.5). We attributed the observed variability in ¹³C values to that the mean annual precipitation (MAP) and the mean annual temperature (MAT) are the main factors controlling the distribution of vegetation types in the Tibetan Plateau, which causes the change in carbon isotope composition of modern soils at any given altitude. The result of both linear and nonlinear regression analyses also confirms that MAP and MAT are the major factors affecting the ¹³C values of surface soils. In the absence of favorable moisture and temperature conditions, low pCO₂ alone is not sufficient to cause the distinct changes in carbon isotope composition of modern soils in the Tibetan Plateau. This study provides some fundamental information on the carbon isotope composition of terrestrial carbon pools and bears some practical significance for the use of carbon isotope data to document vegetation changes and environmental conditions of the high plateau in the past.     

Contribution of methanol to the production of methane and its <Superscript>13</Superscript>C-isotopic signature in anoxic rice field soil

Conversion of methanol to CH₄ has a large isotope effect so that a small contribution of methanol-dependent CH₄ production may decrease the ¹³CH₄ of total CH₄ production. Therefore, we investigated the role of methanol for CH₄ production. Methanol was not detectable above 10 M in anoxic methanogenic rice field soil. Nevertheless, addition of ¹³C-labeled methanol (99% enriched) resulted in immediate accumulation of ¹³CH₄. Addition of 0.1 M ¹³C-methanol resulted in increase of the ¹³CH₄ from –47 to –6 within 2 h, followed by a slow decrease. Addition of 1 M ¹³C-methanol increased ¹³CH₄ to +500 within 4 h, whereas 10 M increased ¹³CH₄ to +2500 and continued to increase. These results indicate that the methanol concentrations in situ, which diluted the ¹³C-methanol added, were 0.1 M and that the turnover of methanol contributed only about 2% to total CH₄ production at 0.1 M. However, contribution increased up to 5 and 17% when 1 and 10 M methanol were added, respectively. Anoxic rice soil that was incubated at different temperatures between 10 and 37 °C exhibited maximally 2–6% methanol-dependent methanogenesis about 1–2 h after addition of 1 M ¹³C-methanol. Only at 50 °C, contribution of methanol to CH₄ production reached a maximum of 10%. After longer (7–10 h) incubation, however, contribution generally was only 2–4%. Methanol accumulated in the soil when CH₄ production was inhibited by chloroform. However, the accumulated methanol accounted for only up to 0.7 and 1.2% of total CH₄ production at 37 and 50 °C, respectively. Collectively, our results show that methanol-dependent methanogenesis was operating in anoxic rice field soil but contributed only marginally to total CH₄ production and the isotope effect observed at both low and high temperature.     

Carcinogenic potential and genomic instability of beryllium sulphate in BALB/c‐3T3 cells

Occupational exposure to beryllium (Be) and Be compounds occurs in a wide range of industrial processes. A large number of workers are potentially exposed to this metal during manufacturing and processing, so there is a concern regarding the potential carcinogenic hazard of Be. Studies were performed to determine the carcinogenic potential of beryllium sulfate (BeSO₄) in cultured mammalian cells. BALB/c3T3 cells were treated with varying concentrations of BeSO₄ for 72 h and the transformation frequency was determined after 4 weeks of culturing. Concentrations from 50–200 g BeSO₄/ml, caused a concentrationdependent increase (9–41 fold) in transformation frequency. Nontransformed BALB/c3T3 cells and cells from transformed foci induced by BeSO₄ were injected into both axillary regions of nude mice. All ten Beinduced transformed cell lines injected into nude mice produced fibrosarcomas within 50 days after cell injection. No tumors were found in nude mice receiving nontransformed BALB/c3T3 cells 90 days postinjection. Gene amplification was investigated in Kras, cmyc, cfos, cjun, csis, erbB2 and p53 using differential PCR while random amplified polymorphic DNA fingerprinting was employed to detect genomic instability. Gene amplification was found in Kras and cjun, however no change in gene expression or protein level was observed in any of the genes by Western blotting. Five of the 10 transformed cell lines showed genetic instability using different random primers. In conclusion, these results indicate that BeSO₄ is capable of inducing morphological cell transformation in mammalian cells and that transformed cells induced by BeSO₄ are potentially tumorigenic. Also, cell transformation induced by BeSO₄ may be attributed, in part, to the gene amplification of Kras and cjun and some BeSO₄induced transformed cells possess neoplastic potential resulting from genomic instability.     

Existence and uniqueness of a sharp travelling wave in degenerate non-linear diffusion Fisher-KPP equations

In this paper we use a dynamical systems approach to prove the existence of a unique critical value c ^* of the speed c for which the degenerate density-dependent diffusion equation u _ct = [D(u)u _x ] _x + g(u) has: 1. no travelling wave solutions for 0 < c < c ^*, 2. a travelling wave solution u(x, t) = (x - c ^* t) of sharp type satisfying (– ) = 1, () = 0 ^*; '(^*–) = – c ^*/D'(0), '(^*+) = 0 and 3. a continuum of travelling wave solutions of monotone decreasing front type for each c > c ^*. These fronts satisfy the boundary conditions (– ) = 1, '(– ) = (+ ) = '(+ ) = 0. We illustrate our analytical results with some numerical solutions.     

Light and GTP dependence of transducin solubility in retinal rods

The physical origin and functional significance of the near infra-red light scattering changes observable upon flash illumination of diluted suspensions of magnetically oriented, permeabilised frog retinal rods has been reinvestigated with particular attention paid to the degree with which transducin remains attached to the membrane. In the absence of GTP, the so called binding signal is shown to include two components of distinctive origins, widely different kinetics, and whose relative amplitudes depend on the dilution of the suspension and resulting detachment of transducin from the disc membrane. The fast component is a consequence of the fast interaction between photoexcited rhodopsin (R^*) and the transducin remaining on the membrane. Its kinetics monitors a structural modification of the discs caused by a change in electrostatic interaction between closely packed membranes upon the formation of R^*-T complexes. The slow component monitors the slow rebinding to the membrane and possible subsequent interaction with excess R^* of T-GDP which, in spite of its low solubility, had eluted into solution given the high dilution of the permeated rods. In the presence of GTP, the so called dissociation signal includes a fast, anisotropic release component that specifically monitors the release into the interdiscal space of T -GTP formed from the membrane-bound pool, and a slower isotropic loss component monitoring the leakage from the permeated rod of the excess T -GTP which did not interact with the cGMP phosphodiesterase. The amplitudes of both components depend exclusively on the membrane bound T-GDP pool. The kinetics of the loss component is limited by the size and degree of permeation of the rod fragments, rather than by the dissociation rate of T -GTP from the membrane.Abbreviations ROS rod outer segment - R rhodopsin - R^* photoactivated rhodopsin - T, T-GDP, T -GDP, T -GTP, T transducin and its various forms - T _mb, T _sol: T bound to membrane or soluble - PDE cGMP-phosphodiesterase - GTP guanosine 5-triphosphate - GDP guanosine 5-diphosphate - GDP S guanosine 5-O-(2-thiodiphosphate) - cGMP guanosine-3-5 cyclic-monophosphate - DTT dithiothreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - TRIS Tris (hydroxymethyl)aminomethane - SDS sodium dodecyl sulfate     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Modification of domains of α and β subunits of F1-ATPase from the thermophylic bacterium PS3, in their isolated and associated forms,by 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP)

Photoaffinity labeling by 3-O-(4-benzoyl)benzoyl adenosine 5-triphosphate (BzATP) of the adenine nucleotide binding site(s) on isolated and complexed and subunits of F₁-ATPase from the thermophilic bacterium PS3 (TF₁) is described. BzATP binds to both isolated and subunits, to complexed subunit but not to complexed subunit. Amino acid sequence determination of radiolabeled peptides obtained by proteolytic digestion of [-³²P]BzATP-labeled subunit indicates that residues on both the amino-terminal (residues A41-E67) and carboxy-terminal (residues Q422–Q476) were modified by BzATP. One of the residues in the carboxy-terminal modified by BzATP is most probably Q422. Although the binding stoichiometry of 1 mol of BzATP incorporated by either isolated or complexed subunit was maintained, the spatial conformation of the polypeptide determines which amino acid residue(s) is more accessible to the reactive radical. CNBr derived fragments G10-M64, E75-M233, and D390-M469 were labeled with the isolated subunit. With complexed subunit the label was found only in CNBr fragments: E75-M233 and G339-M389. The locations where the covalently bound BzATP was found, in the soluble and assembled subunits, indicate that different conformational states exist. In the isolated form of the and subunits the amino- and carboxy-termini can fold and reach the central domain of the polypeptide, the domain containing the adenine nucleotide binding site. When combines with to form the ₃₃ core complex the new conformation of the subunits is such that covalent labeling by BzATP of and of the amino terminal of subunit is excluded.     

Secondary structural effects on protein NMR chemical shifts

For an amino acid in protein, its chemical shift, (, )_s, is expressed as a function of its backbone torsion angles ( and ) and secondary state (s): (, )_{s=, )_coil+(, )_s}, where (, )_coil represents its chemical shift at coil state (s=coil); (, )_s (s=sheet or helix) is herein defined as secondary structural effect correction factor, which are quantitatively determined from Residue-specific Secondary Structure Shielding Surface (RSS) for ¹³CO, ¹³C, ¹³C,¹H, ¹⁵N, and ¹HN nuclei. The secondary structural effect correction factors defined in this study differ from those in earlier investigations by separating out the backbone conformational effects. As a consequence, their magnitudes are significantly smaller than those earlier reported. The present (, )_sheet and (, )_helix were found varying little with backbone conformation and the 20 amino acids, specifically for ¹³CO, ¹³C, and ¹H nuclei. This study also carries out some useful investigations on other chemical shift prediction approaches – the traditional shielding surfaces, SHIFTS, SHIFTX, PROSHIFT, and identifies some unexpected shortcomings with these methods. It provides some useful insights into understanding protein chemical shifts and suggests a new route to improving chemical shifts prediction. The RSS surfaces were incorporated into the program PRSI [Wang and Jardetzky, J. Biomol. NMR, 28: 327–340 (2004)], which is available for academic users at http://www.pronmr.com or by sending email to the author (yunjunwang@yahoo.com).     

Polymorphism of human liver alcohol dehydrogenase: Identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing

Liver homogenate-supernatants from most Japanese exhibit an atypical pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the typical pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with ₂ subunits while typical livers contain isozymes with ₁ subunits, both produced by the ADH ₂ gene. Because it is difficult to differentiate the atypical ADH₂ 2-2 phenotype from the ADH₂ 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The isozymes in A1 and A2 livers were purified. Type A1 livers contained a single isozyme with an atypical pH-rate profile; it was designated ₂₂. Three isozymes were isolated from A2 livers, two of which corresponded to ₁₁ and ₂₂. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated ₂₁. Type A1 livers are, therefore, the homozygous ADH₂ 2-2 phenotype, and type A2 livers, the heterozygous ADH₂ 2-1 phenotype. The ADH₂ 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH₂ 2-1 phenotype, in 31%. Accordingly, the frequency of ADH ₂ ² was 0.68.This study was supported by U.S. Public Health Service Grant AA 02342.     

Chlorophyll interference with phytochrome measurement

Measurements of phytochrome by (A_{725–815 nm}) were completely suppressed at chlorophyll concentrations of the order of 20–40 g g^-1 f.wt. in vivo and 37 g cm^-3 in vitro, and the readings were reduced by 50% at only 12 g cm^-3 in vitro. At these concentrations of chlorophyll in aqueous methanol, the loss of phytochrome signal in vitro appeared to be due to failure of phytochrome photoconversion rather than to interference with A measuremebt by chlorophyll fluorescence in the 125/815 nm measuring beam.Abbreviations Chl chlorophyll - P phytochrome - P_r and P_fr phytochrome in red absorbing and far-red absorbing forms     

Effect of transmembrane Ca2+ gradient on the coupling of β-adrenergic receptors and adenylyl cyclase

In order to investigate the effect of transmembrane Ca²⁺ gradient on G_s mediated coupling of -AR and adenylyl cyclase, -AR from duck erythrocytes and G_s and adenylyl cyclase from bovine brain cortices were co-reconstituted into asolectin liposomes with different transmembrane Ca²⁺ gradient. These proteoliposomes were proven to be impermeable to water-soluble substances. The results obtained indicate that a physiological transmembrane Ca^2– gradient (1000-fold) is essential for higher stimulation of adenylyl cyclase by hormone-activated -AR via coupling to G_s and can be further enhanced by the decrease of such Ca²⁺ gradient within certain range (100 fold) following Ca²⁺ influx into cells during signal transduction. Fluorescence polarization of DPH revealed that transmembrane Ca²⁺ gradient modulates adenylyl cyclase and its stimulation by hormones through mediating a change in lipid fluidity. Correspondent conformational changes of -AR were also detected from the fluorescence spectra and quenching of Acrylodan-labelled -AR in those proteoliposomes. It is suggested that a proper transmembrane Ca²⁺ gradient is essential for the optimal fluidity of the phospholipid bilayer in the proteoliposomes, which favors the formation of a suitable conformation of the reconstituted -AR and thus promotes the stimulation of adenylyl cyclase activities by hormone-activated -AR via Gs.Abbreviations ATP adenosine triphosphate - -AR -adrenergic receptors - AC adenylyl cyclase - DHA dihydroalprenolol - DPH diphenylhexatriene - [Ca²⁺]_i Ca²⁺ concentration inside proteoliposomes - [Ca²⁺]_o Ca²⁺ concentration outside proteoliposomes - cAMP cyclic adenosine monophosphate - DTT Dithiothreitol - FS fluorescein sulfonate - G_s Stimulatory GTP-binding protein - GTP guanosine triphosphate - GTPS guanosine 5-O-(3-thiotriphosphate) - kDa kilodalton - SDS sodium dodecyl sulfate - Tris N-tris(hydroxymethyl)aminomethane