Some aspects of cooperativity in human hemoglobin

The cooperativity in hemoglobin can be described by the Hill parameter n, the free energy of interaction ΔF₁ and the allosteric free energy ΔF_A. By this latter is meant here the free energy change associated with the transition from the deoxy to the oxy conformation in hemoglobin. In this paper some general relations between n, ΔF₁ and ΔF_A are given. A method is presented by which ΔF_A can be calculated from oxygenation data.     

Effect of hyperoxia and hypercapnia on tissue oxygen and perfusion response in the normal liver and kidney

Objective

Inhalation of air with altered levels of oxygen and carbon dioxide to manipulate tissue oxygenation and perfusion has both therapeutic and diagnostic value. These physiological responses can be measured non-invasively with magnetic resonance (MR) relaxation times. However, interpreting MR measurements is not straight-forward in extra-cranial organs where gas challenge studies have only begun to emerge. Inconsistent results have been reported on MR, likely because different organs respond differently. The objective of this study was to elucidate organ-specific physiological responses to gas challenge underlying MR measurements by investigating oxygenation and perfusion changes in the normal liver and kidney cortex.

Materials and Methods

Gas challenges (100% O₂, 10% CO₂, and carbogen [90% O₂+10% CO₂]) interleaved with room air was delivered to rabbits to investigate their effect on tissue oxygenation and perfusion. Real-time fiber-optic measurements of absolute oxygen and relative blood flow were made in the liver and kidney cortex.

Results

Only the liver demonstrated a vasodilatory response to CO₂. Perfusion changes to other gases were minimal in both organs. Tissue oxygenation measurements showed the liver responding only when CO₂ was present and the kidney only when O₂ was present.

Conclusion

This study reveals distinct physiological response mechanisms to gas challenge in the liver and kidney. The detailed characterization of organ-specific responses is critical to improving our understanding and interpretation of MR measurements in various body organs, and will help broaden the application of MR for non-invasive studies of gas challenges.     

Adaptative response to enhanced basal oxidative damage in sod mutants from Saccharomyces cerevisiae

We investigated the adaptative response of S. cerevisiae in sod mutants (sod1Δ, sod2Δ and sod1Δsod2Δ) after H₂O₂ treatment in the stationary phase. sod2Δ and sod1Δsod2Δ demonstrated the highest levels of GSH in the control, suggesting that pathways which include GSH protect these double mutants against oxidative stress. In addition, sod1Δ and sod1Δsod2Δ had higher iron levels than the wild-type, independently of H₂O₂ stress. Fe levels were increased in sod2Δ following H₂O₂ In addition, the sod2Δ mutant was more sensitive to H₂O₂ treatment than the wild-type. These results suggest that sod2Δ sensibility may be associated with •OH production by the Fenton reaction. This increased iron demand in the sod2Δ mutant may be a reflection of the cells’ efforts to reconstitute proteins that are inactivated in conditions of excess superoxide. MDA levels were assayed by HPLC in these mutants. The highest MDA levels could be observed after 10mM H₂O₂ treatment in the sod1Δsod2Δ double mutant. After treatment with a GSH inhibitor, the MDA level was still higher in the same strain. Thus, both direct and indirect GSH pathways are involved in the protection of lipid membranes and proteins in these mutants and may constitute an adaptative response to enhanced basal oxidative damage produced by superoxide.     

Cytochrome c2 and reaction center of Rhodospeudomonas spheroides Ga. membranes. Extinction coefficients, content, half-reduction potentials, kinetics and electric field alterations

1. The reduced minus oxidized extinction coefficients (Δ^red-ox) of reaction center P605 when in the chromatophore is about 20% smaller than in the detergent-isolated state. Presumably the coupling of the reaction center protein to the antenna bacteriochlorophylls and carotenoids causes this hypochromism. The chromatophore values for P605 are 19.5 mM⁻¹ · cm⁻¹ with the spectrophotometer on single beam mode at 605 nm, and 29.8 mM⁻¹ · cm⁻¹ on dual wavelength mode set at 605 – 540 nm. Cytochrome c₂, which is not affected by detergent, has a Δ^red-ox value at 550-540 nm of 19.0 mM⁻¹ · cm⁻¹.2. The total bacteriochlorophyll to reaction center bacteriochlorophyll protein (P) ratio is about 100 : 1. The cytochrome c₂: reaction center protein ratio approaches 2. In current French press chromatophore preparations, about 70% of the reaction centers are each associated on a rapid kinetic basis with two cytochrome c₂ molecules (intact P-c₂ units). The remaining reaction center proteins are not associated with cytochrome c₂ on a kinetically viable basis and may be the result of damage incurred during mechanical rupture of the cells.3. The half-reduction potential of cytochrome c₂ in the isolated state is 345 mV. In the chromatophore, two electrochemical species of cytochrome c₂ are recognized. The majority has a value of approx. 295 mV and is identifiable with cytochrome c₂ in a reaction center protein-associated state (kinetically active, intact P-c₂ units); the remainder has an approx. 350 mV half-reduction potential and is probably cytochrome c₂ in the “free” or reaction center-dissociated state (possibly from damaged P-c₂ units). It appears that there is no exchange of cytochrome c₂ between the reaction center-associated and the reaction center-dissociated state.4. The half-reduction potential of cytochrome c₂ is pH independent (from pH 5 to 9) whether measured in the free state or when associated with the chromatophore membrane. This shows that a proton is not involved in the oxidation and reduction of cytochrome c₂ in the physiological pH range.5. The kinetics of the intact reaction center, P, and cytochrome c₂ units in chromatophores and whole cells of Rhodopseudomonas spheroides are described. The two cytochrome c₂ molecules which are associated with one P exhibit similar oxidation kinetics; both are biphasic. The fast phase is estimated to be 20–40 μs in half time. The second slower phase is variable depending on the ionic strength of the medium used for the preparation of the chromatophores; it varies from 0.3 to 8 ms.6. An equilibrium for cytochrome c₂ and the reaction center and/or the membrane is suggested. The two states of the equilibrium are described by a population of cytochrome c₂ functionally “close” to the P⁺, and a population functionally distant from the P⁺, which might be physically off the binding site, or orientated unfavorably to the P⁺. The former population is identified by the 20–40 μs oxidation rate; the latter variable and somewhat slower oxidation (0.3–8 ms) is that whose rate is governed by the diffusional processes of the equilibrium which brings the cytochrome to the close position.7. Carotenoid bandshifts are kinetically compatible (a) with the P oxidation which is too fast to measure, and (b) with the two phases of cytochrome c₂ oxidation. These are interpreted as arising from local electric field alterations occurring during the electron transfer events in the reaction center and cytochrome c₂.     

Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45°C in the absence and presence of Mg²⁺. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. ΔH⁰ and ΔS⁰ for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, ΔH⁰ ≈ 15–16 kcal/mol (63–67 kJ/mol) and ΔS⁰ ≈ 50–57 cal/K per mol (209–239 J/K per mol). The presence of Mg²⁺ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg²⁺, the ΔH⁰ and ΔS⁰ values are larger and temperature-dependent, with ΔH⁰ ≈ 22 kcal/mol (92 kJ/mol) and ΔS⁰ ≈ 72 cal/K per mol (approx. 300 J/K per mol) at 25°C, and ΔC_p⁰ 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.     

Cycloheximide reduces PGD2 or Δ-PGJ2 cytotoxicity on NCG cells

To study the precise mechanism of cytotoxic activity of PGD₂ or Δ¹²-PGJ₂ (a biological active metabolite of PGD₂), we examined the effect of various compounds on PGD₂ or Δ¹²-PGJ₂ cytottoxic, using a human neuroblastoma cell line (NCG). Cycloheximide (CHM) specifically protected PGD₂ cytotoxicity on NCG cells. When Δ¹²-PGJ₂ was tested, CHM exhibited a similar rescue effect. Puromycin, mitomycin C, and α-amanitin did not affect PGD₂ or Δ¹²-PGJ₂ cytotoxicity. Emetine showed a variable and no consistent rescue effect CHM may have been active at the primary site where PGD₂ or Δ¹²-PGJ₂ exerts its cytotoxicity. This is the first report indicating that CHM reduces the cytotoxicity induced by PGD₂ or Δ¹²-PGJ₂.     

Magnetic field-stimulated luminescence and a matrix model for energy transfer. A new method for determining the redox state of the first quinone acceptor in the reaction center of whole cells of Rhodospirillum rubrum

In whole cells of Rhodospirillum rubrum the light-induced absorbance difference spectrum of the reduction of the first quinone electron acceptor Q₁ was determined in order to relate the emission yield ф and the magnetic field-induced emission increase Δф to the redox state of Q₁. It was found that Δф/ф² is a linear function of the number of reaction centers, in which Q₁ is reduced, independent of the fraction of reaction centers in the oxidized state. The emission yield is a hyperbolic function of the fraction of reaction centers closed, either by reduction of the acceptor Q₁ or by oxidation of the primary electron donor P. Apparently, in whole cells of R. rubrum a matrix model for energy transfer between various photosynthetic units can be applied. A model is presented, which is a generalization of theoretical considerations reported before (Duysens, L.N.M. (1978) in Chlorophyll Organization and Energy Transfer in Photosynthesis, Ciba Found. Symp. 61 (New Series), pp. 323–340, Elsevier/North-Holland, Amsterdam) and which is in excellent agreement with the experiments. From simultaneous measurements of Δф and ф the redox state of the reaction center can relatively easily be determined. So far, this is the only method for simultaneously measuring the fractions P⁺ and Q⁻₁ in intact cells under steady-state conditions.     

High-yield expression and purification of soluble forms of the anti-apoptotic Bcl-x(L) and Bcl-2 as TolAIII-fusion proteins

A method is presented to produce large amounts of Bcl-2 and Bcl-x_L, two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x_L proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described. The method delivers at least 20 and 10 mg of more than 90% pure TolAIII-Bcl-x_LΔC and TolAIII-Bcl-2(2)ΔC proteins, respectively, per liter of E. coli cell culture. The proteins are released by proteolysis with thrombin providing >12 mg of Bcl-x_LΔC or >6 mg of Bcl-2(2)ΔC per liter of E. coli cell culture with a purity of more than 95%. Whereas Bcl-x_LΔC is soluble both before and after TolAIII removal, Triton X-100 can significantly increase the extraction of TolAIII- Bcl-2(2)ΔC from the bacterial cells and its subsequent solubility. Far-UV CD spectroscopy demonstrated that they both have an α-helical structure. Fluorescence spectroscopy was used to quantitatively analyze the binding of the respiratory inhibitor antimycin A to recombinant Bcl-2 and Bcl-x_L proteins as well as the displacement of this ligand from the hydrophobic pocket with BH3 Bad-derived peptide. Purified Bcl-x_LΔC and Bcl-2(2)ΔC both protect isolated mitochondria from Bax-induced release of cytochrome c. The ensemble of data shows that the expressed proteins are correctly folded and functional. Therefore, the TolAIII-fusion system provides a convenient tool for functional characterization and structural studies of anti-apoptotic proteins.     

Proton pumps of the plasmalemma of the yeast Rhodotorula gracilis Their coupling to fluxes of potassium and other ions

(1) Intact cells of the obligatory aerobic yeast Rhodotorula gracilis (glutinis) generate a difference of the electrochemical proton potential (Δμ_H⁺) across the plasmalemma. In the range from pH 4.0 to 7.0 its value remains close to 12 kJ/mol. At pH 4.0 it is composed of the pH difference (inside alkaline) alone, at pH 7.0 of the membrane potential alone. (2) Both components of Δμ_H⁺ are generated by an active process, as shown by their rapid dissipation under anaerobic conditions. (3) In order to find out by which type of mechanism Δμ_H⁺ is generated the effect of a number of inhibitors of transport-ATPases (among them ouabain, triphenyltin chloride, quercetin, oligomycin, venturicidin, dicyclohexylcarbodiimide, Dio-9) were tested both on the generation of the membrane potential and on the extrusion of protons either in the absence or the presence of potassium ions. We found that all three processes were inhibited by Dio-9 and dicyclohexylcarbodiimide, which are specific for H⁺-ATPases. Triphenyltin chloride inhibited the K⁺/H⁺-exchange without having any effect either on the extrusion of H⁺ alone or on the membrane potential. (4) Dicyclohexylcarbodiimide and Dio-9, but not triphenyltin chloride inhibited at pH 4.0 the active transport of sugars. This class of substrates has been shown earlier to be transported by an electrogenic H⁺ symport driven by Δμ_H⁺ across the cell membrane. (5) Neither the rate of respiration nor the intracellular level of ATP were significantly decreased by any of these inhibitors (except for venturicidin). (6) We conclude that in Rhodotorula gracilis the difference of the electrochemical potential of H⁺ is created by an electrogenic proton pump, presumably in ATPase. The extrusion of protons in exchange against potassium is catalyzed by a different energy-dependent but electroneutral system. This conclusion is based on the observation that the H⁺/K⁺ exchange does not work under conditions where the membrane potential is large, and vice versa.     

Wave intensity amplification and attenuation in non-linear flow: Implications for the calculation of local reflection coefficients

Local reflection coefficients (R) provide important insights into the influence of wave reflection on vascular haemodynamics. Using the relatively new time-domain method of wave intensity analysis, R has been calculated as the ratio of the peak intensities (R_PI) or areas (R_CI) of incident and reflected waves, or as the ratio of the changes in pressure caused by these waves (R_ΔP). While these methods have not yet been compared, it is likely that elastic non-linearities present in large arteries will lead to changes in the size of waves as they propagate and thus errors in the calculation of R_PI and R_CI. To test this proposition, R_PI, R_CI and R_ΔP were calculated in a non-linear computer model of a single vessel with various degrees of elastic non-linearity, determined by wave speed and pulse amplitude (ΔP₊), and a terminal admittance to produce reflections. Results obtained from this model demonstrated that under linear flow conditions (i.e. as ΔP₊→0), R_ΔP is equivalent to the square-root of R_PI and R_CI (denoted by R_PI^p and R_CI^p). However for non-linear flow, pressure-increasing (compression) waves undergo amplification while pressure-reducing (expansion) waves undergo attenuation as they propagate. Consequently, significant errors related to the degree of elastic non-linearity arise in R_PI and R_CI, and also R_PI^p and R_CI^p, with greater errors associated with larger reflections. Conversely, R_ΔP is unaffected by the degree of non-linearity and is thus more accurate than R_PI and R_CI.     

Evidence of α fluctuations in myoglobin's denaturation in the high temperature region: Average relaxation time from an Adam–Gibbs perspective

In this work, we derive an analytical expression for the relaxation time τ as a function of temperature T for myoglobin protein (Mb, PDB:1MBN) in the high temperature limit (T > T_g = 200 K). The method is based on a modified version of the Adam–Gibbs theory (AG theory) for the glass transition in supercooled liquids and an implementation of differential geometry techniques. This modified version of the AG theory takes into account that the entropic component in protein's denaturation has two major sources: a configurational contribution ΔS_c due to the unfolding of the highly ordered native state N and a hydration contribution ΔS^hyd arising from the exposure of non-polar residues to direct contact with solvent polar molecules. Our results show that the configurational contribution ΔS_c is temperature-independent and one order of magnitude smaller than its hydration counterpart ΔS^hyd in the temperature range considered. The profile obtained for log τ(T) from T = 200 K to T = 300 K exhibits a non-Arrhenius behavior characteristic of α relaxation mechanisms in hydrated proteins and glassy systems. This result is in agreement with recent dielectric spectroscopy data obtained for hydrated myoglobin, where at least two fast relaxation processes in the high temperature limit have been observed. The connection between the relaxation process calculated here and the experimental results is outlined.     

Arrhythmogenic substrate and its modification by nicorandil in a murine model of long QT type 3 syndrome

The gain-of-function Scn5a+/ΔKPQ mutation in the cardiac Na⁺ channel causes human long QT type 3 syndrome (LQT3) associated with ventricular arrhythmogenesis. The K_ATP channel-opener nicorandil (20 μM) significantly reduced arrhythmic incidence in Langendorff-perfused Scn5a+/Δ hearts during programmed electrical stimulation; wild-types (WTs) showed a total absence of arrhythmogenicity. These observations precisely correlated with alterations in recently established criteria for re-entrant excitation reflected in: (1) shortened left-ventricular epicardial but not endocardial monophasic action potential durations at 90% repolarization (APD₉₀) that (2) restored transmural repolarization gradients, ΔAPD₉₀. Scn5a+/Δ hearts showed longer epicardial but not endocardial APD₉₀s, giving shorter ΔAPD₉₀s than WT hearts. Nicorandil reduced epicardial APD₉₀ in both Scn5a+/Δ and WT hearts thereby increasing ΔAPD₉₀. (3) Reduced epicardial critical intervals for re-excitation; Scn5a+/Δ hearts showed greater differences between APD₉₀ and ventricular effective refractory period than WT hearts that were reduced by nicorandil. (4) Reduced APD₉₀ alternans. Scn5a+/Δ hearts showed greater epicardial and endocardial alternans than WTs, which increased with pacing rate. Nicorandil reduced these in Scn5a+/Δ hearts to levels indistinguishable from untreated WTs. (5) Flattened restitution curves. Scn5a+/Δ hearts showed larger epicardial and endocardial critical diastolic intervals than WT hearts. Nicorandil decreased these in Scn5a+/Δ and WT hearts. The presence or absence of arrhythmogenesis in Scn5a+/Δ and WT hearts thus agreed with previously established criteria for re-entrant excitation, and alterations in these precisely correlated with the corresponding antiarrhythmic effects of nicorandil. Together these findings implicate spatial and temporal re-entrant mechanisms in arrhythmogenesis in LQT3 and their reversal by nicorandil.     

Halogenated 2,5-pyrrolidinediones: synthesis, bacterial mutagenicity in Ames tester strain TA-100 and semi-empirical molecular orbital calculations

The chloroimide 3,3-dichloro-4-(dichloromethylene)-2,5-pyrrolidinedione, a tetrachloroitaconimide, is the principal mutagen produced by chlorination of simulated poultry chiller water. It is the second most potent mutagenic disinfection by-product of chlorination ever reported. Six of seven new synthetic analogs of this compound are direct-acting mutagens in Ames tester strain TA-100. Computed energies of the lowest unoccupied molecular orbital (E_LUMO) and of the radical anion stability (ΔH_f^rad−ΔH_f) from MNDO-PM3 for the chloroimides show a quantitative correlation with the Ames TA-100 bacterial mutagenicity values. The molar mutagenicities of these direct acting mutagenic imides having an exocyclic double bond fit the same linear correlation (ln M_m vs. E_LUMO; ln M_m vs. ΔH_f^rad−ΔH_f) as the chlorinated 2(5H)-furanones, including the potent mutagen MX, 3-chloro-4-(dichloro-methyl)-5-hydroxy-2(5H)-furanone, a by-product of water chlorination and paper bleaching with chlorine. Mutagenicity data for related haloimides having endocyclic double bonds are also given. For the same number of chlorine atoms, the imides with endocyclic double bonds have significantly higher Ames mutagenicity compared to their structural analogs with exocyclic double bonds, but do not follow the same E_LUMO or ΔH_f^rad−ΔH_f correlation as the exocyclic chloroimides and the chlorinated 2(5H)-furanones.     

Determination of free energies in reaction centers of Rb. sphaeroides

Magnetic field-dependent recombination measurements together with magnetic field-dependent triplet lifetimes (Chidsey, E.D., Takiff, L., Goldstein, R.A. and Boxer, S.G. (1985) Proc. Natl. Acad. Sci USA 82, 6850–6854) yield a free energy change ΔG(P⁺H⁻ − ³P*) = 0.165 eV ±0.008 at 290 K. This does not depend on whether nuclear spin relaxation in the state ³P* is assumed to be fast or slow compared to the lifetime of this state. This value, being (almost) temperature independent, indicates ΔG(P⁺H⁻ − ³P*) ΔH(P⁺H⁻ − ³P*) and is consistent with ΔG(¹P* − P⁺H⁻) and ΔH(¹P* − ³P*) from previous delayed fluorescence and phosphorescence data, implying ΔG ΔH for all combinations of these states.     

Limiting similarity and the form of the competition coefficient

The question of whether there is a limit to the similarity of competing species has previously been investigated by a number of authors. These studies have all used the Lotka-Volterra model of competition, and have assumed that the competition coefficient α_ij may be calculated using the expression, α_ij = ∝ U_i(R) U_j(R) dR/∝ (U_i(R))² dR. In this paper, the generality of this formula is questioned and two alternative expressions for α_ij are proposed. When these expressions are used in an analysis of limiting similarity, qualitatively different conclusions emerge regarding the existence and nature of this limit, using either deterministic or stochastic models. The relevance of these findings to theories of character convergence and similarity barriers is discussed. The available field evidence does not strongly support the validity of the formula for α_ij used in previous studies. Since a given method of calculating α_ij must be derived from a higher level model, it is suggested that the Lotka-Volterra model is not sufficient in an investigation of limiting similarity.     

A biotin-based protein tagging system

Biotin protein ligase (BPL) mediates covalent attachment of biotin to a specific lysine residue of biotin carboxyl carrier protein (BCCP) of biotin-dependent enzymes. We recently found that the biotinylation reaction from thermophilic archaeon Sulfolobus tokodaii has a unique characteristic that the enzyme BPL forms a tight complex with the product, biotinylated BCCP (169 amino acid residues). In the current work, we attempted to apply this characteristic to a novel protein tagging system. Thus, the N terminus of S. tokodaii BCCP was truncated and the interaction of the resulting BCCP, BCCPΔ100 and BCCPΔ17 (with 69 and 152 residues, respectively), with BPL was investigated by surface plasmon resonance (SPR). It was found that the binding of BPL to the biotinylated BCCPΔ100 is extremely tight with a dissociation constant (K_D) of 1.2 nM, whereas that to the unbiotinylated counterpart was moderate with a K_D of 3.3 μM. Furthermore, chimeric proteins of glutathione S-transferase (GST) and green fluorescence protein (GFP) with BCCPΔ100 fused to their C terminus were prepared. The resulting fusion proteins were successfully biotinylated and captured on the BPL-modified SPR sensor chip or BPL-modified magnetic beads. The function of GST and GFP was hardly impaired on fusion with BCCPΔ100 and biotinylation of the latter.     

PGJ2 and ΔPGJ2 inhibit cell growth accompanied with inhibition of phosphoinositide turnover in human astrocytoma cells

PGJ₂ and Δ¹²PGJ₂ (1 μM to 30 μm) inhibited the growth of human astrocytoma cells (1321N1) in a time-dependent manner within 48 hrs, determined by [³H]thymidine incorporation into acid-insoluble fraction or amounts of protein. The EC₅₀ values for PGJ₂ and Δ¹²PGJ₂ were approximately 8 μM and 6 μM, respectively. [³H]Thymidine incorporation to acid insoluble fraction was inhibited by these PGs within 1 hr, indicating that these PGs rapidly affect cell functions. Although it has been reported that an increase in cyclic AMP inhibits cell growth, PGJ₂ and Δ¹²PGJ₂, but not PGE₁, reduced isoproterenol (10 μM)-induced accumulation of cyclic AMP, suggesting that PGJ₂ and Δ¹²PGJ₂ may disturb adenylate cyclase system, which might be independent on cell growth. On the other hand, these PGs inhibited the incorporation of [³H]inositol into phospholipid fraction within 6 hrs. Furthermore, PGJ₂ and Δ¹²PGJ₂ inhibited carbachol- and/or histamine-induced accumulation of inositol phosphates with a similar dose-dependency to their inhibitions of cell growth. In membrane preparations, however, PGJ₂ and Δ¹²PGJ₂ failed to inhibit GTPγS (10 μM)- nor Ca²⁺ (1mM)-induced accumulation of inositol phosphate. The site of PGJ₂ or Δ¹²PGJ₂ in inhibition of inositol phosphate accumulation would not be phospholipase C nor a putative GTP binding protein involved in activation of phospholipase C. The present results indicate that PGJ₂ and Δ¹²PGJ₂ inhibit cell growth in human astrocytoma cells and the inhibition of phosphoinositide turnover by these PGs might be involved in the inhibition of cell growth.     

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1–5) have high affinity (K_i=<20 nM) for the CB₂ receptor. Four of them (2, n=1–4) also have little affinity for the CB₁ receptor (K_i=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K_i=3.4±1.0 nM) and little affinity for the CB₁ receptor (K_i=677±132 nM).

Scheme 3. (a) (C₆H₅)₃PCH₃⁺ Br⁻, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, −78 to 25°C then H₂O₂/NaOH.     

Mechanism of perception of changes in the magnetic field by ampullae of lorenzini of elasmobranchs

The mechanisms of reception of changes in the magnetic field by electroreceptor formations were investigated in experiments on Black Sea rays in which spike activity was recorded from single nerve fibers connected with the ampullae of Lorenzini. The responses of the ampullae of Lorenzini to magnetic stimulation were shown to be due to induced electric currents creating potential gradients in the body tissues of the fish and the sea water. On the basis of differences in responses of different ampullae to magnetic stimulation, it is possible to distinguish between magnetic stimuli and other stimuli acting on the electroreceptor system. Potentiation of the receptor response to magnetic stimulation was found as the fish came closer to the "shore." The mechanisms and biological importance of reception of changes in the magnetic field by the ampullae of Lorenzini are discussed.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 10, No. 1, 75–83, January–February, 1978.