Pseudobase Formation Research Articles

Fluorescence labeling of mitochondria in living cells by the cationic photosensitizer ZnTM2,3PyPz, and the possible roles of redox processes and pseudobase formation in facilitating dye uptake

ABSTRACT The study of labeling selectivity and mechanisms of fluorescent organelle probes in living cells is of continuing interest in biomedical sciences. The tetracationic phthalocyanine-like ZnTM2,3PyPz photosensitizing dye induces a selective violet fluorescence in mitochondria of living HeLa cells under UV excitation that is due to co-localization of the red signal of the dye with NAD(P)H blue autofluorescence. Both red and blue signals co-localize with the green emission of the mitochondria probe, rhodamine 123. Microscopic observation of mitochondria was improved using image processing and analysis methods. High dye concentration and prolonged incubation time were required to achieve optimal mitochondrial labeling. ZnTM2,3PyPz is a highly cationic, hydrophilic dye, which makes ready entry into living cells unlikely. Redox color changes in solutions of the dye indicate that colorless products are formed by reduction. Spectroscopic studies of dye solutions showed that cycles of alkaline titration from pH 7 to 8.5 followed by acidification to pH 7 first lower, then restore the 640 nm absorption peak by approximately 90%, which can be explained by formation of pseudobases. Both reduction and pseudobase formation result in formation of less highly charged and more lipophilic (cell permeant) derivatives in equilibrium with the parent dye. Some of these are predicted to be lipophilic and therefore membrane-permeant; consequently, low concentrations of such species could be responsible for slow uptake and accumulation in mitochondria of living cells. We discuss the wider implications of such phenomena for uptake of hydrophilic fluorescent probes into living cells.

Synthesis and properties of differently charged chemiluminescent acridinium ester labels

Chemiluminescent acridinium dimethylphenyl esters containing N-sulfopropyl groups in the acridinium ring are highly sensitive, hydrophilic labels that are used in automated immunoassays for clinical diagnostics. Light emission from these labels is triggered with alkaline peroxide in the presence of a cationic surfactant. At physiological pH, N-sulfopropyl acridinium esters exist as water adducts that are commonly referred to as pseudobases. Pseudobase formation, which results from addition of water to the zwitterionic N-sulfopropyl acridinium ring, neutralizes the positive charge on the acridinium nitrogen and imparts a net negative charge to the label due to the sulfonate moiety. As a consequence, N-sulfopropyl acridinium ester conjugates of small molecule haptens as well as large molecules such as proteins gain negative charges at neutral pH. In the current study, we describe the synthesis and properties of two new hydrophilic acridinium dimethylphenyl ester labels where the net charge in the labels was altered. In one label, the structure of the hydrophilic N-alkyl group attached to the acridinium ring was changed so that the pseudobase of the label contains no net charge. In the second acridinium ester, two additional negative charges in the form of sulfopropyl groups were added to the acridinium ring to make this label's pseudobase strongly anionic. Chemiluminescence measurements of these labels, as well as their conjugates of an antibody with a neutral pI, indicate that acridinium ester charge while having a modest effect on emission kinetics has little influence on light output. However, our results demonstrate that acridinium ester charge can affect protein pI, apparent chemiluminescence stability and non-specific binding of protein conjugates to microparticles. These results emphasize the need for careful consideration of acridinium ester charge in order to optimize reagent stability and performance in immunoassays. In the current study, we observed that for a neutral protein, an acridinium ester with a hydrophilic but charge-neutral N-alkyl group afforded faster light emission, lower non-specific binding and better chemiluminescence stability than an analogous label with an anionic N-alkyl group.

Pseudo-base formation in the attempted synthesis of a conjugatively coupled bis(nitrosylruthenium) complex and spectroelectrochemistry of bipyrimidine-bridged dinuclear Ru(terpy)X precursor compounds (X = Cl, NO2)

Reaction of {(mu-bpym)[RuCl(terpy)]2}(PF6)2, bpym = 2,2'-bipyrimidine and terpy = 2,2':6',2''-terpyridine, with NaNO2 yields {(mu-bpym)[Ru(NO2)(terpy)]2}(PF6)2. In CH3CN/0.1 M Bu4NPF6 both dinuclear complexes can undergo two reversible bpym-centered one-electron reduction processes and two metal-centered one-electron oxidation steps, the latter involving mixed-valent intermediates with weak intermetallic coupling. Acidification of {(mu-bpym)[Ru(NO2)(terpy)]2}(PF6)2 does not lead to the expected {(mu-bpym)[Ru(NO)(terpy)]2}6+ but, probably because of the high charge, to the insoluble but structurally and IR-spectroscopically characterised pseudo-base product syn-{(mu-bpym-(4-OH))[Ru(NO)(terpy)]2}(PF6)5. The addition of one hydroxide to one of the 4-positions of bis-chelating bpym interrupts the aromatic pi conjugation and is accompanied by corresponding intra-pyrimidine bond length variations, however, the effect on the electronic interaction of the two different syn positioned {RuNO}6 moieties remains small, possibly due to their situation within the central molecular pi plane.

Cytotoxicity of Benzo[c]phenanthridinium Alkaloids in Isolated Rat Hepatocytes

Sanguinarine, chelerythrine and fagaronine were examined for hepatotoxic effect. Administration of sanguinarine and chelerythrine (10 mg/kg/day; i.p.) produced marked parenchymal injury not found in the livers of fagaronine treated rats. In chronic administration of sanguinarine (0.2 mg/kg/56 days; i.p.) degeneration and necrosis were not observed in the liver. The effect of the alkaloids was tested by enzyme leakage (LDH and ASAT) and changes in glutathione levels in isolated rat hepatocytes. Sanguinarine produced the greatest toxicity followed by lesser toxicity in the case of chelerythrine while no toxicity was displayed with fagaronine. These effects correlate with pK R values for pseudobase formation. Finally, no increased lipid peroxidation or mitochondrial membrane damage were found.

Cytotoxicity of Benzo[c]phenanthridinium Alkaloids in Isolated Rat Hepatocytes

Sanguinarine, chelerythrine and fagaronine were examined for hepatotoxic effect. Administration of sanguinarine and chelerythrine (10 mg/kg/day; i.p.) produced marked parenchymal injury not found in the livers of fagaronine treated rats. In chronic administration of sanguinarine (0.2 mg/kg/56 days; i.p.) degeneration and necrosis were not observed in the liver. The effect of the alkaloids was tested by enzyme leakage (LDH and ASAT) and changes in glutathione levels in isolated rat hepatocytes. Sanguinarine produced the greatest toxicity followed by lesser toxicity in the case of chelerythrine while no toxicity was displayed with fagaronine. These effects correlate with pKR+ values for pseudobase formation. Finally, no increased lipid peroxidation or mitochondrial membrane damage were found.

Kinetics and mechanism of covalent addition of S(IV) species to the acridinium cation

The reaction of acridine with S(IV) species (SO2•H2O, HSO3−, and SO32−) to form the adduct acridine–S(IV) has been studied spectrophotometrically throughout the pH range 2.6–8 in aqueous solutions. The observed pseudo-first-order rate constants, kobs, were determined at 25 °C and ionic strength I = 0.11 M, and the pH profile of the rate reached a maximum at pH ≈ 6.1. At constant pH the kobs values were a linear function of the total S(IV) concentration with slopes that increased significantly with pH. These data are consistent with the rate-determining attack of SO3H− and SO32− upon the C-9 of the acridinium cation. A nonlinear least-squares fitting of the experimental values to the model equation, within the overall pH region studied, yields the pH-independent rate constants k1 = 3.7 ± 0.1 and k2 = (6.24 ± 0.04) × 104 M−1 s−1 for the attack of these two species, respectively. The experimental results agree very well with the kinetic model. Due to the experimental conditions used we did not detect any possible pseudobase formation in the pH range studied. The reactivity of the S(IV) species with acridine follows the order: [Formula: see text] The value obtained for the ratio k1/k2 is similar to the results given for other addition reactions of S(IV) species to the double bond of carbonyl compounds such as benzaldehyde and formaldehyde. Key words: covalent addition, acridine, acridine – S(IV) adducts, kinetics and mechanism.

Antimicrotubule properties of benzophenanthridine alkaloids.

Chelidonine, sanguinarine, and chelerythrine are natural benzophenanthridine alkaloids that inhibit taxol-mediated polymerization of rat brain tubulin in the micromolar range. Chelidonine is a weak, competitive inhibitor of colchicine binding to tubulin but does not inhibit podophyllotoxin binding. On the other hand, sanguinarine inhibits both colchicine and podophyllotoxin binding to tubulin with I50 values of 32 and 46 microM, respectively, and chelerythrine inhibits with I50 values of 55 and 60 microM, respectively. The inhibition by these two agents is of the mixed type. Tubulin forms an acid-reversible pseudobase with the imminium ion of sanguinarine, probably through several of its sulfhydryl groups, as shown by the loss of the yellow color of sanguinarine and its 596-nm fluorescence emission peak. Chelidonine, on the other hand, cannot undergo such pseudobase formation, and we conclude that it acts by a different mechanism. A number of previously described pharmacologic effects of these agents may be due to their inhibition of microtubule function.

Kinetics and mechanism of ring cleavage of 1-Benzyl-4-phenyl-1,2,4-triazolium Chloride

1-Benzyl-4-phenyl-1,2,4-triazolium chloride (1) is cleaved by aqueous sodium hydroxide to form 4-benzyl-1-formyl-1-phenyl-amidrazone (5), the structure of which is proved by mass spectroscopy. The kinetics of the cleavage reaction are studied by UV spectroscopy in methanol/water 1:1 (v/v) in the presence of Na2CO3/HCl buffer at pH values from 11.64 to 12.61. The reaction obeys a first-order kinetic law. The rate constants do not depend linearly on the hydroxide concentration. This is in coincidence with fast formation of the pseudobase from 1, followed by fast deprotonation to the pseudobase anion, and slow decay to the amidrazone. The pseudobase anion is proved spectroscopically, and its spectroscopically determined pKQOH = 12.7 coincides with that from the kinetic experiments. The equilibrium constant of pseudobase formation pKA = 10.99 is two orders of magnitude higher than that from the azocoupling of 1 [1] which shows that in the azocoupling not the pseudobase but its methyl ether (pKQOMe = 8.7) is active. 4-Benzyl-1-formyl-1-phenyl-amidrazone (5) re-cyclizes in the presence of HCl in MeOH/H2O to form 1. The kinetics of this reaction are also studied.

PK R+ values for pyridinium cations

Literature data for nucleophilic addition at C-4 of C-3 substituted 1-methylpyridinium cations are evaluated. It is shown that sufficient data are available to allow the confident evaluation of a linear free energy relationship for the equilibration of these cations with their C-4 hydroxide adducts (pseudobases) in aqueous solution: pK R+ = -8.5 (±0.4) σ-p + 20.5 (± 0.5) This allows the prediction of pKR+ = 20.5 for pseudobase formation at C-4 of the 1-methylpyridinium cation. A less confident estimate of pK R+ = 18.2 at C-2 of this cation is also made.

Kinetics of the reduction of 1-methylquinolinium cations by 1-benzyl-1,4-dihydronicotinamide

The reduction of a series of 3-W-1-methylquinolinium cations (1: W = H, Br, CONH2, CO2CH3, CN, NO2) by 1-benzyl-1,4-dihydronicotinamide has been investigated. In all cases the kinetically controlled product from these reactions is the appropriate 3-W-1,4-dihydro-1-methylquinoline. Only for W = Br is any significant amount of the 1,2-dihydro isomer obtained (15% in this case). This kinetic preference for C-4 attack over C-2 attack in dihydronicotinamide reductions contrasts with the kinetically preferred attack at C-2 by hydroxide ion and in borohydride reductions. Rates of reduction were measured for each 1 and also 1,2-dimethyl- and 1,4-dimethylquinolinium cations in 20% CH3CN – 80% H2O, ionic strength 1.0 at 25 °C, under pseudo-first-order conditions. Kinetic saturation due to nonproductive 1:1 complex formation was observed for several cations at high concentrations (> 0.1 M). Second-order rate constants [Formula: see text] were evaluated for each W, and also kinetic isotope effects from second-order rate constants [Formula: see text] for reduction by 1-benzyl-4,4-dideuterio-1,4-dihydronicotinamide. Second-order rate constants are correlated with σp− for W with ρ = 4.5, and are also closely correlated with [Formula: see text] for pseudobase formation at C-4 of these quinolinium cations by: [Formula: see text]. Values of [Formula: see text] vs. [Formula: see text] describe a Westheimer curve reaching a maximum of 5.8 for W = Br and falling to 1.5 for W = NO2 and 4.2 for W = H. These data are consistent with an intrinsic barrier of 2.9 ± 0.5 kcal/mol for hydride transfer between this 1,4-dihydronicotinamide and quinolinium cations. However, quinolinium cations display a dramatically enhanced rate of dihydronicotinamide reduction relative to hydroxide ion attack when compared with isoquinolinium cations. This observation, and the predominance of C-4 rather than C-2 reduction, suggests that these reactions may not be simple one-step hydride transfer processes.

Kinetic and thermodynamic control of pseudobase formation from C-3 substituted 1-methylquinolinium cations

The kinetic and thermodynamic control of pseudobase formation from 3-W-1-methylquinolinium cations has been studied for a variety of substituents (W). Spectral data indicate that, in both aqueous and methanolic solution, the C-2 pseudobases predominate at equilibrium for W = H and Br, while the C-4 pseudobases are the thermodynamically preferred species for W = CONH2, CO2CH3, CN, and NO2. Stopped-flow studies indicate that in all cases the C-2 pseudobases are the kineticallycontrolled products upon basification of the aqueous solutions of these cations. Equilibrium constants (pKR+) have been measured for pseudobase formation at both C-2 and C-4 for each W in all cases where they are experimentally accessible. Substituent effects upon [Formula: see text] correlate with σm for W, while [Formula: see text] depends upon σp−. These substituent effects allow the prediction of [Formula: see text] and [Formula: see text] for the 1-methylquinolinium cation. Rates of C-2 to C-4 pseudobase equilibration have been measured in all cases where the latter species is thermodynamically more stable. These kinetic data allow the evaluation of rate constants for C-4 pseudobase equilibration with each cation. In all cases except W = CN, C-2 pseudobase formation is complete within the mixing time of the stopped-flow instrument.

Kinetics of the reduction of C-9 substituted acridinium cations by 1,4-dihydronicotinamides

Second-order rate constants ( k 2) are reported for the reduction of 9-R-10-methylacridinium cations ( 5:R = H, CH 3, CH 3CH 2, C 6H 5CH 2, (CH 3) 2CH, C 6H 5, 4-(CH 3) 2NC 6H 4) by 1-benzyl-1,4-dihydronicotinamide ( 2:R = C 6H 5CH 2) in 20% CH 3CN-80% H 2O at 25°C. All 5:R ≠ H are reduced in the range 20- to 140-fold more slowly than 5:R = H. However, there is no simple relationship between k 2 and the nature of R, nor between k 2 and the second-order rate constant for hydroxide ion attack at C-9 of these cations in pseudobase formation. Rates of reduction of 5 by 1-benzyl-4,4-dideuterio-1,4-dihydronicotinamide allow the calculation of the following kinetic isotope effects in this solvent medium: 5:R, k H k D : H , 1.56; C 6H 5CH 2, 2.7; C 6H 5, 5.4. Substituent effects upon k 2 were evaluated for the reduction of 5 by 1-(X-benzyl)-1,4-dihydronicotinamides, and lead to the following Hammett ϱ parameters: 5:R, ϱ: H, −0.68; C 6H 5CH 2, −0.92; C 6H 5, −0.96. The latter two values require essentially complete unit positive charge generation on the nicotinamide moiety in the rate-determining transition state. It is shown that these Hammett ϱ values and the above isotope effects can only be rationalized by a two-step e − + H • mechanism for hydride transfer from 2 to 5 in this solvent system. This result contrasts with our earlier conclusion of direct, one-step hydride transfer in the reduction of isoquinolinium cations by 2, but is consistent with our observation that acridinium cations are reduced 37500-fold faster by 2 than predicted on the basis of the relative rates of nucleophilic attack (hydroxide ion) on acridinium and isoquinolinium cations. It is suggested that the availability of both Hammett ϱ values and primary kinetic isotope effects will generally allow the establishment of the mechanism of hydride transfer in these systems. Application of these ideas to literature data suggests that 5:R = H is reduced by direct hydride transfer in acetonitrile solution, in contrast to the above result in predominantly aqueous solution. The ready formation of acridanyl radicals by electron transfer to acridinium cations is demonstrated by the formation of Wurster's Blue radical cation upon mixing solutions of acridinium cations with N,N,N′,N′-tetramethyl- p-phenylenediamine.

Pseudobase formation from 9-substituted 10-methylacridinium cations in aqueous solution

Rates of cation–pseudobase equilibration have been measured for a series of C-9 substituted (CH3, CH3CH2, C6H5CH2, (CH3)2CH, C6H5, 4-(CH3)2NC6H4) 10-methylacridinium cations in aqueous solution at 25 °C over the pH range of approximately 9–13. Separation of the rate constants for formation (kOH) and decomposition (k2) of each of these pseudobases allowed the calculation of the [Formula: see text] value for each cation. The presence of a C-9 isopropyl substituent enhances the stability of the pseudobase relative to the cation, whereas all other C-9 substituted cations have [Formula: see text] values greater than the 10-methylacridinium cation. There is no simple quantitative relationship between [Formula: see text] and the size of the C-9 substituent. Rate constants (kOH) for hydroxide ion attack on these C-9 substituted cations are in the order: H > primary alkyl > secondary alkyl > aryl, while rate constants (k2) for pseudobase decomposition are less predictable but generally follow the order: H > primary alkyl > aryl > secondary alkyl. These phenomena are shown to be consistent with a competition between destabilization of the cation by peri interactions between the C-9 substituent and H(1) and H(8) and reduced pseudobase solvation for large C-9 substituents. Resonance interactions of 9-aryl substituents with the acridine moiety of these cations are shown to be quite small. 9-Aryl-10-methylacridinium cations are approximately 30-fold more reactive towards hydroxide ion attack than are their triphenylmethyl carbocation analogues.

The Use of 18-Crown-6 As Phase Transfer Catalyst in the Reissert Reaction

Abstract Solid-liquid phase transfer of cyanide ion by 18-cro'wn-6 increases the yield of the Reissert reaction and eliminates the undesirable pseudo-base formation.

Hydride transfer reactions. Substituent effects in oxidation of N-benzylacridans by π acceptors

The kinetics of oxidation of a series of eight N-(substituted benzyl)acridans (3, NBA's) by 1,4-benzoquinone (BQ), p-chloranil (CA), 2,3-dicyano-1,4-benzoquinone (DCBQ), 7,7,8,8-tetracyanoquinodimethane (TCNQ), and tetracyanoethylene (TCNE) in acetonitrile (AN) and by BQ in 50:50 (v/v) AN-water were measured at 25 °C. Equilibrium constants for pseudobase formation, [Formula: see text], from the corresponding acridinium ions (4) were measured in water at 25 °C. Hammett correlations of the second-order rate constants for reaction of the NBA's without ortho substitutents (3a–e) led to ρ values of −0.29 (BQ, AN), −0.55 (CA), −0.56 (DCBQ), −0.64 (TCNQ), −0.41 (TCNE), and −0.47 (BQ, 50:50 AN–water). The second-order rate constants for 3a–e also give good linear free energy correlations with the [Formula: see text] values of 4a–e. These correlations and the [Formula: see text] values for the ortho-substituted acridinium ions (4f, g, h) are used to calculate rate constants for oxidation of the corresponding ortho-substituted NBA's (3f, g, h). The rate constants calculated in this way are 4.7 to 6.6 times and 6.4 to 12 times larger, respectively, than the observed rate constants for N-(2,4,6-trimethylbenzyl) and N-(2,6-dichlorobenzyl)acridan (3g and h). The variations in ρ values are attributed mainly to differences in the amount of electrostatic stabilization in the transition state resulting from differences in the separation of donor and acceptor and the degree of delocalization of the negative charge. The rate retarding effect of a pair of ortho substituents is attributed to sterie effects in a preferred face-to-face transition state.

Covalent hydration and pseudobase formation in transition metal polypyridyl complexes: Reality or myth?

Covalent hydration and pseudobase formation in transition metal polypyridyl complexes: Reality or myth?

Destruction of leishmania mexicana amazonensis amastigotes within macrophages in culture by phenazine methosulfate and other electron carriers

Exposure of macrophages infected with Leishmania mexicana amazonensis to phenazine methosulfate (PMS) resulted in rapid damage and disappearance of the intracellular amastigotes without obvious ill effects to the host cells. The reduction of the percent infection was related to the concentration of PMS and to the duration of the pulse. Most Leishmania disappeared within 2 h of a 2-h pulse with 10 muM of the drug. In contrast, pretreatment of the macrophages with PMS followed by removal of the drug before infection did not result in disappearance of the parasites. The pH of the PMS medium markedly influenced the disappearance of Leishmania: maximum effect was observed at pH 8.0, while the effect was negligible at pH 6.3. The pH effect may be related to pseudobase formation by the PMS cation. Dose-response curves for PMS were similar for resident, elicited, or activated macrophages. Observations by time-lapse cinemicrography documented the explosion-like fragmentation of the amastigotes within 1-2 h of exposure of infected macrophages to the drug. Parasite-derived granules and vacuoles were seen to scatter within the parasitophorous vacuoles. This early damage to the parasites was confirmed by transmission electron microscopic observations. Infected macrophages incubated with PMS displayed detectable vacuolar fluorescence, indicating that PMS or a metabolite of PMS had access to the vacuoles. A series of other electron carriers, including phenyl methanes, phenazines, oxazines, a xanthene, and a naphthoquinone, given continuously for 18 h, also induced the disappearance of the Leishmania. The most potent was crystal violet, active at 70 nM. The presence of apolar substituents enhanced activity and this is probably related to increased permeation of the dyes. Finally, PMS, as well as other electron carriers examined, also reduced the growth of Leishmania promastigotes in culture. The results are compatible with a direct effect of the drugs on the intracellular amastigotes, involving only a permissive participation of the macrophages. We propose that the diverse agents destroy the amastigotes by redox-cycling generation of active oxygen metabolites at or near the parasites. Alternatively, the effect of the drugs could be mediated by toxic free radical reduction species of the drugs or by interference with electron flow or with the intermediary metabolism of Leishmania.

Equilibria in complexes ofN-heterocyclic molecules. Part XXXII. Dissociation oftris-(5-halo-1,10-phenanthroline)iron(III) ions in acid solution

The kinetics of reaction of thetris-(5-X-1,10-phenanthroline) iron(III) ions where X=Br or Cl have been studied in detail in 0–100% sulphuric acid solutions. Relationships between the rates of reactions and the activity of water have been examined. No reaction occurs without water present. The reactions are interpreted in terms of covalent hydrate and pseudo-base formation. By taking into account previous studies, rates and magnitudes of hydrate formation for these and some other related complexes can be estimated.

Pseudobase formation in 2-methylpapaverinium cations and their biotransformation by enzymes of rat liver homogenates in vitro

2-Methylpapaverinium iodide (I), 2'-hydroxymethyl-2-methylpapaverinium iodide (IX), and 2-methyl-3,4-dihydropapaverinium iodide (X. CH3I) form pseudobase by addition of hydroxide ions to the C(1)=N(+) bond. 2'-Hydroxymethyl-2-methyl-3,4-dihydropapaverinium iodide (XV) and 2'-hydroxymethyl-2-methyl-9-oxo-3,4-dihydropapaverinium iodide (XVI) react with hydroxide ions in aqueous medium under formation of cyclic pseudobases XVII and XVIII. The equilibrium constants (KR+) of pseudobase formation have been measured in aqueous ethanol (1 : 1 w/w, 25°C, ionic strength 0.1). The quaternary papaverinium derivatives are metabolized to isoquinilones and carbonyl compounds by means of rat liver enzymes. The role of pseudobases in these biotransformations has been discussed and biogenetic conclusions have been drawn.

ChemInform Abstract: EQUILIBRIA IN COMPLEXES OF N‐HETEROCYCLIC MOLECULES. 28. RATES OF INTERMEDIATE PSEUDOBASE FORMATION BETWEEN HYDROXIDE AND TRIS(5‐NITRO‐1,10‐PHENANTHROLINE)‐M(II) IONS, M = COBALT, NICKEL, OR COPPER

Chemischer InformationsdienstVolume 10, Issue 48 Organoelement Compounds ChemInform Abstract: EQUILIBRIA IN COMPLEXES OF N-HETEROCYCLIC MOLECULES. 28. RATES OF INTERMEDIATE PSEUDOBASE FORMATION BETWEEN HYDROXIDE AND TRIS(5-NITRO-1,10-PHENANTHROLINE)-M(II) IONS, M = COBALT, NICKEL, OR COPPER J. A. A. + SAGUEES, J. A. A. + SAGUEESSearch for more papers by this authorR. D. GILLARD, R. D. GILLARDSearch for more papers by this authorP. A. WILLIAMS, P. A. WILLIAMSSearch for more papers by this author J. A. A. + SAGUEES, J. A. A. + SAGUEESSearch for more papers by this authorR. D. GILLARD, R. D. GILLARDSearch for more papers by this authorP. A. WILLIAMS, P. A. WILLIAMSSearch for more papers by this author First published: November 27, 1979 https://doi.org/10.1002/chin.197948319Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume10, Issue48November 27, 1979 RelatedInformation