Variant HeLa cells selected for their resistance to ouabain

The cardiac glycoside, ouabain, normally kills HeLa cells at concentrations of about 10⁻⁷ m or greater. By treating a population of HeLa cells with increasingly higher concentrations of the drug, a variant population was obtained of HeLa cells capable of growing in medium containing 10⁻⁴ M ouabain. Inhibition of volume regulation of cells subjected to hypotonic shock was used as a measure of inhibition of active transport of Na across the plasma membrane. In that way dose-response curves for the rapid effects of ouabain and other inhibitors of active Na transport were obtained with both the original, ouabain-sensitive (OS) and the variant, ouabain-resistant (OR) cells. Three other cardiac glycosides (digoxin, digitoxin and hellebrin) and two aglycones (digitoxigenin and strophanthidjn) were found to be equally as effective as ouabain in inhibiting volume regulation of the OS cells; the concentration which produced half-maximum inhibition, I(max/2), was about 6 × 10⁻⁷ M in each case. Similar inhibition of the OR population by ouabain was observed only when the concentration exceeded 10⁻⁴ m [I(max/2)∼2.5 × 10⁻⁴ m], and the other steroid compounds had no effect on the variant cells at the highest concentrations tested (∼2 × 10⁻⁵ m). OR and OS cells differed also in their sensitivities to the cardioactive erythrophleum alkaloid, coumingine; I(max/2) for OS and OR cells was 5 × 10⁻⁸ m and 6 × 10⁻⁷ M, respectively. These results, in addition to results of ouabain binding experiments and measurements of the rates of reversal of inhibition of volume regulation, suggest that a major reason for the differential sensitivities of the two phenotypes to these drugs is different affinities of their sodium pumps for inhibitors of active transport.     

A comparison of the 8-hydroxydeoxyguanosine,chromosome aberrations and micronucleus techniques for the assessment of the genotoxicity of mercury compounds in human blood lymphocytes

We compared the mechanism of action of micronuclei (MN), unstable chromosome aberrations, and 8-hydroxydeoxyguanosine (8-OHdG) levels to evaluate the genotoxicity of methyl mercuric chloride (CH₃HgCl) and mercuric chloride (HgCl₂) in human peripheral lymphocytes. The chromosome aberrations in human peripheral lymphocytes exposed to various concentrations of CH₃HgCl or HgCl₂ increased in a concentration-dependent manner and were significantly higher than the control when the cells were incubated with 1 × 10⁻⁵ M (HgCl₂) or 2 × 10⁻⁶ M (CH₃HgCl). The increase in the incidence of micronucleated lymphocytes was significant among the exposed groups, being 2 × 10⁻⁵ M (HgCl₂) and 5 × 10⁻⁶ M (CH₃HgCl) compared with the control. CH₃HgCl was about 4-fold more potent than HgCl₂. We determined the 8-OHdG levels in human peripheral blood mononuclear cells(PBMC) and found that they were significantly higher in the exposed groups at 1 × 10⁻⁵ M (HgCl₂) and 5 × 10⁻⁶ M (CH₃HgCl) compared with the control. A detectable (p < 0.05) increase in the level of 8-OHdG was induced by CH₃HgCl at a concentration that was about 50% of the amount of HgCl₂ required to produce a similar response. The data confirmed the value of the MN and/or chromosome aberration assays for assessing of HgCl₂- and/or CH₃HgCl-induced genotoxicity, and indicated that they are about the same concentration as the 8-OHdG assay. The presence of genotoxic effects in peripheral blood lymphocytes exposed to the mercuric compounds indicated by the chromosome aberrations and the MN assays could be partly due either to the disturbance of the spindle mechanism, or to the elevated level of 8-OHdG brought by the generation of reactive oxygen species.     

Microbial Ecosystem in Sunderban Mangrove Forest Sediment,North-East Coast of Bay of Bengal,India

This is the first documentation of seasonal and spatial fluctuation of the culturable microbial population collected from different zones in the sediment of the Sunderban mangrove forest. The population of cellulose degrading bacteria, [mean value of CFU 6.189 ± 1.025 × 10⁶ (g dry weight of sediment)^?1] was found to be maximum during post monsoon in the deep forest region, whereas, the fungal population [mean value of CFU 3.424 ± 0.886 × 10⁶ (g dry weight of sediment)^?1] was found to be maximum during pre-monsoon in the rooted region. The abundances of microbes, in decreasing order, studied from different zones are nitrifying bacteria [mean value of CFU 1.125 ± 0.359 × 10⁶ (g dry weight of sediment)^?1], phosphorous solubilizing bacteria (PSB) [mean value of CFU 0.805 ± 0.322 × 10⁶ (g dry weight of sediment)^?1], free living nitrogen fixing bacteria [mean value of CFU 0.417 ± 0.120 × 10⁶ (g dry weight of sediment)^?1] and sulfur reducing bacteria (SRB) [mean value of CFU 0.356 ± 0.125 × 10⁶ (g dry weight of sediment)^?1]. The content of organic carbon in the soil decreased from the deep forest region to the rooted and unrooted region but a reverse profile was found for soil salinity and soil silicate concentration. The results from the present study indicate that the monsoon cycle has a pronounced effect on the microbially dominated biogeochemistry in the sediment and consequently on the ecology of the Sundarban mangrove forest.     

Characterization of azadirachtin binding to Sf9 nuclei in vitro

[22,23-³H₂]dihydroazadirachtin was incorporated by Sf9 cells in culture and was bound specifically to the nuclear fraction. The observed association constant of the binding of the radioligand to a purified nuclear fraction was determined to be 0.037 ± 0.008 min ¹ using a one-phase exponential association equation, and binding appeared to be to a single population of sites. The binding was essentially irreversible, and the dissociation constant was estimated to be 0.00065 ± 0.00013 min ¹. An association rate constant of 7.3 × 10⁶ M ¹ min ¹ was calculated from these data. Binding was saturable, and the receptor number and affinity were determined as B_max = 23.87 ± 1.15 pmol/mg protein, K_d = 18.1 ± 2.1 nM. The order of potency of semisynthetic azadirachtin analogues for competition for the binding site was as follows (IC₃₀ in parentheses): azadirachtin (1.55 × 10⁻⁸ M) > dihydroazadirachtin (3.16 × 10⁻⁸ M) > dansyl dihydroazadirachtin (7.40 × 10⁻⁸ M) > DNP-azadirachtin (7.50 × 10⁻⁸ M) > biotin dihydroazadirachtin (1.27 × 10⁻⁷ M) ≫ 11-methoxy 22,23-dihydroazadirachtin (6.67 × 10⁻⁷ M). Arch. Insect Biochem. Physiol. 34:461–473, 1997. © 1997 Wiley-Liss, Inc.     

THE MARINE MIXOTROPH DINOBRYON BALTICUM (CHRYSOPHYCEAE): PHAGOTROPHY AND SURVIVAL IN A COLD OCEAN1

The marine chrysophyte Dinobryon balticum (Schzütt) Lemm. was one of the dominant members of the phytoplankton community (1.8×10³ cells-L⁻¹) in June and July in Conception Bay, Newfoundland. Dinobryon balticum colonies were common only in samples from June and July. The cells were concentrated at 5 m (X±SD=1.11±4 × 10⁵ cells.L⁻¹) and at 40 m (3.32±2×10⁴.L⁻¹) depths. Colonies were composed of up to 560 cells with a mean (±SD) colony size of 10 ± 1 cells at 5 m and 40 ± 8 cells at 40 m. Fluorescent latex bead-uptake experiments conducted with field samples indicated that this marine species was capable of phagotrophy and that twice as many Dinobryon cells were ingesting beads at 40 m than at 5 m, although the ingestion rates for those cells actively ingesting beads were similar at both depths. This chrysophyte was found in association with bacteria-and nutrient-rich microhabitats of microaggregates and fecal pellets. The cells and colonies observed in this study appeared to be healthy, as demonstrated by their appearance and their ability to ingest beads.     

Adenosine Triphosphate Acceleration of the Nephridial Apparatus of Paramecium multimicronucleatum*

SYNOPSIS. Paramecium multimicronucleatum was exposed to various external concentrations of adenosine triphosphate (Na₂ATP) to determine the effects thereof on the cycling rate of the nephridial apparatus. Normal rate was found to vary from 3.46 to 4.28 cycles/min with a mean rate of 3.85 cycles/min at 20 C. Concentrations of ATP of less than 5 × 10⁻⁴ M caused only slight, very temporary acceleration of the cycling rate. At 5 × 10⁻⁴ M the cycling rate was accelerated less than 15%. At 3 × 10⁻³ M cycling rate was accelerated, varying from 5.35 to 7.24 cycles/min, with a mean accelerated rate of 6.25 cycles/min, a mean aoceleration of 88.3%. Changes in rate after addition of 5 × 10⁻³ M ATP ranged from a decrease of 6.2% to an increase of 1.8%, with the nephridial apparatus ultimately stopping. At higher concentrations, stoppage was almost immediate. Paramecium is rapidly dehydrated by the ATP-accelerated cycling of its nephridial apparatuses, with a net loss of 27% of its volume in 6 minutes in the 3 × 10⁻³ M ATP.     

Protein kinase C β1 overexpression augments phorbol ester-induced increase in endothelial permeability

We studied the postulated involvement of the protein kinase C β₁ (PKCβ₁) isoform in the regulation of endothelial permeability using human dermal microvascular endothelial cell line (HMEC-1). We overexpressed the recombinant PKCβ₁ gene via retroviral-mediated transduction in these cells. PKCβ₁ gene transfer was stable, and PKCβ₁ protein production was persistent for at least 1 month posttransduction. Addition of 2 × 10⁻⁹ M and 2 × 10⁻⁸ M phorbol 12-myristate 13-acetate (PMA) to the control (nontransduced) HMEC-1 cells increased the transendothelial ¹²⁵I-albumin clearance rate (an index of endothelial permeability) from 2.5 ± 0.2 × 10⁻² μl/min to 5.4 ± 1.2 × 10⁻² μl/min and 16.8 ± 3.1 × 10⁻² μl/min, respectively. However, addition of 2 × 10⁻⁹ M PMA to PKCβ₁-overexpressing HMEC-1 cells produced a maximal increase in the transendothelial ¹²⁵I-albumin clearance rate of 15.9 ± 2.0 × 10⁻² μl/min. Challenge of these cells with 2 × 10 ⁻⁸ M PMA did not further augment the increase in permeability. Activation with PMA was associated with the translocation of the PKCβ₁ from the cytosol to the membrane. These data show that PKCβ₁ overexpression augments the increase in endothelial permeability in response to PKC activation, suggesting an important function for the PKCβ₁ isoform in the regulation of endothelial barrier. © 1996 Wiley-Liss, Inc.     

Effects of leucomyosuppressin on the excitation-concentration coupling of insect Leucophaea maderae visceral muscle

1. Leucomyosuppressin (LMS) inhibited neurally evoked contractions of the hindgut of the cockroach Leucophaea maderae. The threshold for this inhibition of LMS was in the range of 1 × 10⁻¹⁰ M.2. LMS caused a sharp reduction in both l-glutamate and proctolin induced contractions. Dose-response profiles of the effect of LMS (held constant at 10⁻⁸M) on variable amounts of proctolin showed an inhibitory effect at 10⁻⁹ M proctolin and below, but at 5 × 10⁻⁹ M proctolin and above, LMS caused no inhibition.3. Potassium (158 mM) depolarized hindguts treated with LMS (10⁻⁸ M) showed a marked reduction (76% ± 2.1) in the proctolin (10⁻⁸ M) response.4. When calcium depleted preparations were returned to normal calcium levels (2 mM) in the presence of proctolin (10 ⁻⁸ M) a contraction occurred that was 45% ± 4 of the maximum in normal saline solution. However, LMS (10⁻⁸ M) reduced this response to only 28% ± 2 of the maximum.5. Proctolin (10⁻⁸ M) induced contractions in the presence of the manganous ions (2mM) fell to 63% ± 4 of the maximum but on the addition of LMS (10⁻⁸M), such responses fell to only 16% ± 5 of the maximum.6. These results offer evidence for a non-synaptic site of action for LMS and a perturbation of key calcium dependent events in the excitation-contraction coupling sequence of visceral muscle by this peptide.     

Trypanosoma congolense: Thrombocyte survival in infected steers

Charolais steers infected with Trypanosoma congolense developed a thrombocytopenia that was first demonstrated shortly before the onset of parasitemia. The thrombocyte count progressively decreased from a level of 6 × 10⁵/mm³ on the 3rd day postinfection to l × 10⁵/mm³, its most depressed level, on the 11th day postinfection. The mean of the thrombocyte level of five infected bovines at the time of thrombocyte survival analysis was 195.6 ± 83.5 × 10³(± 2 SE) as compared to 998 ± 245.9 × 10³ in the control group. In parallel with depressed thrombocyte levels, the mean of the apparent half-lives of ⁵¹Cr-labeled thrombocytes was 1.3 ± 0.5 days as compared to 3.7 ± 0.5 days in control animals. A similar survival was noted in the apparent half-lives of ⁵¹Cr-labeled autologous and heterologous thrombocytes transfused to normal recipients.     

Myometrial LH/hCG receptors during the estrous cycle and pregnancy in pigs

Receptors for luteinizing hormone/human chorionic gonadotropin (LH/hCG) have been identified in porcine, rabbit, rat, and human myometrium. To determine the estrous cycle and pregnancy related changes in the receptor capacity and affinity, radioreceptor assays were performed with membrane homogenates of porcine uterine tissues. Cycling gilts were divided into four experimental groups: I (n=6), day 1–2; II (n=5), day 6–7; III (n=5), day 11–12; and IV (n=6), day 18–20 of the estrous cycle. Pregnant pigs were divided into three experimental groups: I (n=5), day 35–40; II (n=5), day 65–70; and III (n=4), day 95–105 of pregnancy. The concentrations [femtomoles/mg protein (fmol/mg protein)] and affinities of unoccupied LH/hCG binding sites were characterized in all samples of myometrium. Receptor concentrations were highest (P<0.01) in groups II and III (19.3±2.5 and 35.8±2.1 fmol/mg protein, respectively), and was lowest in groups I and IV (5.3±1.4 and 7.5±0.7 fmol/mg protein, respectively). Receptor affinity constants (K_a) were consistent (P>0.05) throughout the estrous cycle [I, (5.1±1.5)×10⁹; II, (3.0±0.8)×10⁹; III, (3.2±0.9)×10⁹; IV, 5.5±0.7×10⁹ lm⁻¹]. Plasma hormone concentrations of progesterone, estrogen and LH were typical of values noted at these times. During pregnancy, receptor concentrations were greatest (P<0.05) in group II (85.4±18.5 fmol/mg protein). In groups I and III receptor numbers were 10.8±2.3 and 26.7±6.6 fmol/mg protein, respectively. The K_a in group I was 10 times greater (P<0.05) than K_a in groups II and III, (I, 3.1±0.9×10¹⁰ lm⁻¹; II, 3.4±0.3×10⁹ lm⁻¹; III, 3.3±1.1×10⁹ lm⁻¹). Plasma hormone concentrations typically found during pregnancy were noted. The function of these LH/hCG binding sites remains unknown; however, changes in receptor capacity during the estrous cycle and pregnancy support a role for modulation of the receptor by hormonal factors.     

The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated and occupationally exposed people

Peripheral blood lymphocytes from unirradiated control subjects and workers exposed within permitted limits to γ-radiation, have been examined for the incidence of dicentric and acentric chromosome aberrations. The results are compared with a review of data published elsewhere. Background levels show inter-laboratory variation and possible reasons for this are discussed. By combining the present data with those from the literature the spontaneous incidence of dicentric aberrations is approx. 0.55 × 10^?3 and for acentrics is 3.7 × 10^?3. In occupationally exposed subjects a significantly higher incidence of aberrations was found. When allowance was made for the turnover of lymphocytes for the period over which each man had worked with radiation a linear dose-effect relationship was apparent. The incidence of dicentrics was 2.22 ± 0.94 × 10^?4 rad^?1 and for all unstable aberrations 8.24 ± 2.8 × 10^?4 rad^?1. These are in reasonable agreement with dose-response data obtained in vitro.     

Stabilities,solubility, and kinetics and mechanism for formation and hydrolysis of some palladium(II)and platinum(II) iodo complexes in aqueous solution

Complex formation between Pd(II), Pt(II) and iodide has been studied at 25 °C for an aqueous 1.00 M perchloric acid medium. Measurements of the solubility of PdI₂(s) in aqueous mercury(II) perchlorate and of AgI(s) and PdI₂(s) in aqueous solutions of Pd²⁺(aq) and Ag⁺(aq) gave the solubility product of PdI₂(s) as K_s^o=(7±3) × 10⁻³² M³, which is much smaller than previous literature values.The stability constants β₁=[MI(H₂O)₃⁺]/([M(H₂O)₄²⁺][I⁻]) for the two systems were obtained as the ratio between rate constants for the forward and reverse reactions of (i).

The following values of k₁ (s⁻¹ M⁻¹), k₋₁ (s⁻¹) and β₁ (M⁻¹) were obtained at 25 °C: (1.14±0.11) × 10⁶, (0.92±0.18), (12±4) × 10⁵ for MPd, and (7.7±0.4), (8.0±0.7) × 10⁻⁵, (9.6±1.3) × 10⁴ for MPt. Combination with previous literature data gives the following values of log(β₁ (M⁻¹)) to log(β₄ (M⁻⁴)): 6.08, ∼22, 25.8 and 28.3 for MPd, and 4.98, ∼25, ∼28, and ∼30 for MPt. The present results show that the large overall stability constants β₄ observed for the M²⁺I⁻ systems are most likely due to a very large stability of the second complex MI₂(H₂O)₂, which is probably a cis-isomer. A distinct plateau in the formation curve for mean ligand number 2 is obtained both for MPd and Pt. The other iodo complexes are not especially stable compared to those of chloride and bromide.ΔH^≠ (kJ mol⁻¹) and ΔS^≠ (JK⁻¹ mol⁻¹) for the forward reaction of (i), MPd, are (17.3±1.7) and (−71±5), and for the reverse reaction of (i) MPd, (45±3) and (−95±6), respectively. The kinetics are compatible with associative activation (I_a). The contribution from bond-breaking in the formation of the transition state seems to be less important for Pd than for Pt.     

Atrial natriuretic factor receptors and stimulation of cyclic GMP formation in normal and malignant osteoblasts

Synthetic rat atrial natriuretic factor (Ile-ANF-26) stimulated cyclic GMP formation by up to several hundred-fold in osteoblast-rich cultures from newborn rat calvaria and in clonal osteogenic sarcoma cells (UMR 106-01) which are phenotypically osteoblast. ANF had no effect on the cyclic AMP response to parathyroid hormone in the same cells. Specific, high-affinity binding sites for ANF were identified in both cell types, with K_d and receptor numbers in normal osteoblasts of 1.2 ± 0.1 × 10⁻¹⁰ M and 42 ± 4 × 10³ per cell, and in UMR 106-01 cells of 1.4 ± 0.1 × 10⁻¹⁰M and 22 ± 4 × 10³per cell.     

Micronucleus assay in bovine lymphocytes after exposure to bisphenol A in vitro

Bisphenol A (BPA) [2,2-bis-(4-hydroxyphenyl) propane] is an important industrial agent, made by combining acetone and phenol, that is used extensively as a monomer in the production of polycarbonate plastics and as a precursor of epoxy resins. Micronucleus assays have served as an index of cytogenetic damage in in vivo and in vitro studies. We studied the genotoxic and cytotoxic effects of BPA on bovine peripheral lymphocytes in vitro. Lymphocyte cultures from two donors were exposed to four different concentrations of BPA (1?×?10^?4, 1?×?10^?5, 1?×?10^?6, and 1?×?10^?7 mol.L^?1) for 48 h. The highest concentration of BPA (1?×?10^?4 mol.L^?1) resulted in a significant increase in the number of micronuclei in comparison with the negative control (67.50?±?2.121/1,000 binucleated cells versus 36.0?±?5.657/1,000 binucleated cells in the DMSO control, P??=??0.018). BPA did not affect the nuclear division index at any treatment concentrations. The present results thus demonstrate a significant genotoxic effect by BPA on bovine peripheral lymphocytes in vitro, only at the highest concentration.     

A genome-wide association study identifies a new variant associated with word reading fluency in Chinese children

Reading disability exhibited defects in different cognitive domains, including word reading fluency, word reading accuracy, phonological awareness, rapid automatized naming and morphological awareness. To identify the genetic basis of Chinese reading disability, we conducted a genome-wide association study (GWAS) of the cognitive traits related to Chinese reading disability in 2284 unrelated Chinese children. Among the traits analyzed in the present GWAS, we detected one genome-wide significant association (p < 5 × 10⁻⁸) on word reading fluency for one SNP on 4p16.2, within EVC genes (rs6446395, p = 7.33 × 10⁻¹⁰). Rs6446395 also showed significant association with Chinese character reading accuracy (p = 2.95 × 10⁻⁴), phonological awareness (p = 7.11 × 10⁻³) and rapid automatized naming (p = 4.71 × 10⁻³), implying multiple effects of this variant. The eQTL data showed that rs6446395 affected EVC expression in the cerebellum. Gene-based analyses identified a gene (PRDM10) to be associated with word reading fluency at the genome-wide level. Our study discovered a new candidate susceptibility variant for reading ability and provided new insights into the genetics of developmental dyslexia in Chinese children.     

Purine salvage pathway enzyme activities in human T-, B-, and null lymphocyte populations

Deficiencies of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) result in distinctly different immunodeficient states. In order to better understand the roles of these two purine salvage pathway enzymes in normal immunity we have characterized their activities in peripheral blood T-, B-, and null (non-T, non-B) cell populations. We have found that T lymphocytes have significantly higher ADA activity than B or null cells (P < 0.001) only when expressed as a function of cell protein. Contrary to other reports B lymphocytes cannot be distinguished from T lymphocytes on the basis of PNP activity. When the enzyme activities are expressed per cell number null cells have a much higher PNP activity (mean ± SD = 461 ± 174 ng/hr/10⁶ cells) than either T (57 ± 10) or B (93 ± 51) lymphocytes (P < 0.001 and 0.002, respectively). Null cells also have a significantly greater protein content per cell (55.0 ± 9.6 μg/10⁶ cells) than T (13.3 ± 4.6) or B (18.7 ± 13.8) lymphocytes. When expressed as a function of cell number null cells have a higher mean, though not statistically significant, ADA activity than T and B lymphocytes. These results indicate that normal human peripheral blood T, B, and null cells can be distinguished on the basis of their ADA and PNP activities and that the method of expression of the activities is critical. The significantly higher PNP activity and protein content per cell in null lymphocytes is compatible with a less differentiated cell population which may include B- as well as T-cell precursors.     

Electron microscopy of human interphase nuclei

Quantitative electron microscopy was used to analyze surface-spread, critical-point-dried human interphase nuclei and chromatin. The following information is presented: (1) Unstimulated interphase nuclei of lymphocytes from peripheral blood have a mean dry mass of 50.30×10^?12 g. The mean dry mass of stimulated nuclei of lymphocytes was determined to be 59.34×10^?12 g, a significant statistical difference from the unstimulated ones. (2) Mean diameter of chromatin fibers and mean fiber mass per micron were 199ű15% coefficient of variation (C.V.) and 5.95×10^?16g×29% C.V., respectively. (3) A line of regression of fiber mass on fiber diameter for 83 fibers indicated that a 200-Å fiber has a mass of 5.86×10^?16g/μ, or almost the same as the mean fiber mass of 5.95× 10^?16g/μ. (4) With the value 7×10^?12g for the DNA content of an unstimulated lymphocyte nucleus, a total length of 215 cm is calculated for the DNA double helix. When this length is compared to the mean length of chromatin fiber per nucleus (7.59 cm), a ratio of 28.3 to 1 results, which is called the DNA-packing ratio. (5) This DNA-packing ratio of 28.3 is reasonably close to the packing ratio of 26.9 suggested from model calculations for the second DNA supercoil in a 200-Å chromatin fiber.     

The kinetics and mechanisms of the reactions of chromium(III) with pentane-2,4-dione in aqueous solution

The equilibrium, kinetics and mechanism of the reaction of chromium(III) with pentane-2,4-dione (Hpd) have been investigated in aqueous solution at 55°C and ionic strength 0.5 mol dm⁻³ NaClO₄. The equilibrium constant (log β₁) is 10.08(±0.01) while the pK of Hpd is 8.69(±0.01). The kinetics are consistent with a mechanism in which [Cr(H₂0)₆]³⁺ and [Cr(H₂0)₅(OH)]²⁺ react with the enol tautomer of Hpd with rate constants of 1.05(±0.26) × 10⁻² and 2.78(±0.08) × 10⁻¹ dm³ mol⁻¹ s⁻¹ respectively. These rate constants are considerably more rapid than those predicted by the Eigen-Wilkins mechanism. These data are compared with literature values.