Segmental distribution in potentials of lateral root budding and oxygen uptake of plant hairy roots

The positional distributions in potential of lateral root budding and oxygen uptake rate were examined using the segments of madder and horseradish hairy roots with a length of 5.0×10⁻³ m obtained at different mean distances from the root tips of l=7.5×10⁻³–47.5×10⁻³ m. The average rate of lateral root budding and oxygen uptake rate of the roots with smaller l values were higher and both the rates gradually decreased with increase in l value. Positive relations were observed between the rates of lateral root budding and oxygen uptake of both the hairy roots. The relation indicated that the potential of lateral root budding was suppressed at the oxygen uptake rates of 0.15×10⁻⁵ and 0.32×10⁻⁵ mol O₂/(h m) for madder and horseradish hairy roots, respectively.     

Apparent differences in tryptophan hydroxylation by cockroach (periplaneta americana) nervous tissue and rat brain

Tryptophan hydroxylation in cockroach (Periplaneta americana) nervous tissue was measured and compared to the hydroxylation of tryptophan in rat brain. Tryptophan hydroxylation in both tissues requires a pterine cofactor, and is inhibited by p-chlorophenylalanine. The molecular weight of the protein responsible for hydroxylation of tryptophan in cockroach nervous tissue obtained from gel filtration was estimated to be 54,000.The pH optima and enzyme kinetics differed greatly between the two hydroxylases. Hydroxylation of tryptophan by the enzyme obtained from cockroach tissues incubated with dimethyltetrahydropterine had a pH optimum of about 5.8–5.9 and a K_m in crude enzyme preparations of 2.6 × 10⁻⁶ M and is activity was substrate inhibited above 10⁻⁴ M tryptophan. Hydroxylation of tryptophan by the enzyme obtained from rat brain incubated with dimethyltetrahydropterine had a pH optimum of about 6.5–7.0, a K_m of about 6.7 × 10⁻⁴ M and exhibited no substrate inhibition at tryptophan concentrations up to 2 × 10⁻³ M.When incubated with biopterin, the presumed natural cofactor, the hydroxylase from cockroach tissues had a K_m of about 6.8 × 10⁻⁵ M and no substrate inhibition occurred at tryptophan concentrations up to 2 × 10⁻³ M. Under the same conditions rat hydroxylase had a K_m of 1.1 × 10⁻⁵M and substrate inhibition occurred above 10⁻⁴ M tryptophan.Unlike the mammalian situation, administration of tryptophan peripherally did not change the 5-hydroxytryptamine concentration in cockroach nervous tissue, but did increase tryptophan levels. The low V_max values of the cockroach hydroxylase and the inability of administered tryptophan to elevate 5-hydroxytryptamine levels suggest that in the cockroach hydroxylation of tryptophan itself may be the limiting factor in the biosynthesis of 5-hydroxytryptamine.     

Ouabain-resistant Na,K-ATPases and cardenolide tolerance in the large milkweed bug,Oncopeltus fasciatus

Oncopeltus fasciatus tolerated 1954× and 7288×, respectively, the LD₅₀ ouabain dose of Schistocerca gregaria and Periplaneta americana when ouabain was injected into the haemocoel of these insects. The maximal ouabain dose that could be injected into O. fasciatus (200 nmol) resulted in no mortality; this dose is higher than the lethal ouabain doses recorded for vertebrates and invertebrates. The ouabain concentration resulting in 50% inhibition (I₅₀) of Na,K-ATPase activity was determined in lyophilates of nervous tissue of O. fasciatus and brain and recta of S. gregaria and were 2.0 × 10⁻⁴, 2.0 × 10⁻⁶ and 1.0 × 10⁻⁶ M, respectively. The I₅₀ value for ouabain inhibition of Na,K-ATPase activity in the nervous tissue of O. fasciatus is higher than the I₅₀ values for nervous tissue in most other insects as well as many other invertebrate and vertebrate tissues. Thus, the presence of ouabain-resistant Na,K-ATPases appears to be a factor in the tolerance and sequestration of plant cardenolides in O. fasciatus.     

PROPERTIES OF TYROSINE HYDROXYLASE IN PERIPHERAL NERVES

Approximately 80 per cent of tyrosine hydroxylase activity in bovine mandibular nerve and rabbit sciatic nerve was soluble, and the rest of the activity was particle-bound. The soluble enzyme in bovine mandibular nerve was isolated by ammonium sulphate fractionation (25–35 per cent saturation). The enzyme had a pH optimum at 5·9 in Tris-acetate buffer, and at 6·5 in Tris-HCl or phosphate buffer. The enzyme required a tetrahydropteridine cofactor. K_m values toward various tetrahydropteridines such as l -erythro-tetrahydrobiopterin (a probable natural cofactor), 2-amino-4-hydroxy-6-methyltetrahydropteridine, and 2-amino-4-hydroxy-6,7-dimethyltetrahydropteridine were 2 × 10⁻⁵m , 5 × 10⁻⁵m and 4 × 10⁻⁴m , respectively. The K_m value for tyrosine at 1 × 10⁻³m -2-amino-4-hydroxy-6-methyltetrahydropteridine as a cofactor was 5 × 10⁻⁵m . The enzyme activity was markedly stimulated with Fe²⁺ or catalase, but Fe²⁺ gave higher activity. The activity was inhibited with α, α′-dipyridyl, l -α-methyl-p-tyrosine, and various catecholamines. Among catecholamines, dopamine was the most potent inhibitor. l -5-Hydroxytryptophan was an inhibitor as potent as dopamine. Neither d -5-hydroxytryptophan nor 5-hydroxytryptamine inhibited the enzyme. The inhibition by l -5-hydroxytryptophan was partially competitive with tetrahydrobiopterin at concentrations higher than 9 × 10⁻⁵m , and partially uncompetitive at concentrations lower than 9 × 10⁻⁵m . The addition of heparin or lysolecithin did not affect enzyme activity with tetrahydrobiopterin as cofactor.     

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.     

Inhibition by dibromothymoquinone of photosynthetic electron transfer in chloroplasts of differing ultrastructure

The effect of the plastoquinone antagonist, dibromothymoquinone, on the photoreduction of ferricyanide and plastocyanin by maize mesophyll, maize bundle-sheath and Euglena gracilis chloroplasts has been investigated. Maximum inhibition of FeCN and plastocyanin reduction by mesophyll chloroplasts was obtained at dibromothymoquinone concentrations of 5 × 10^?7m. At higher concentrations dibromothymoquinone acted as an electron shuttle, increasing the rate of reduction of both substrates. In contrast, little inhibition of FeCN and plastocyanin reduction by bundle-sheath chloroplasts occurred at 5 × 10?7 m dibromothymoquinone, and above this concentration of inhibitor, the extent of inhibition increased, with no shuttle effect being observed. Euglena chloroplasts showed a response intermediate between that of mesophyll and bundle-sheath chloroplasts.The presence of a shuttle effect caused by dibromothymoquinone appears to be directly related to the presence of a proton pump in the chloroplast preparations. Plastocyanin is reduced by photosystem 2 alone and shows some of the properties of a class III electron acceptor, although the rates of reduction observed were much lower than those observed with lipophilic class III acceptors.     

Follicle-stimulating hormone stimulated differentiation and progesterone production of bovine granulosa cells in culture

Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×10⁵) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO₂ in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml⁻¹ l-glutamine, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg⁻¹ DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg⁻¹ DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg⁻¹ DNA per 24 h for 0, 1, 10 and 100 μu ml⁻¹ FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml⁻¹ FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg⁻¹ DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.     

Inhibitory effects of cadmium on carbonic anhydrase activity and ionic regulation of the estuarine crab Chasmagnathus granulata (Decapoda,Grapsidae)

This work was aimed at evaluating the gill carbonic anhydrase (CA) activity of the estuarine crab Chasmagnathus granulata exposed in vivo to cadmium, at different salinities. The in vivo effect of the specific inhibitor acetazolamide (AZ) was also assayed. Besides, the inhibition of CA activity by different heavy metals (cadmium, copper, zinc) and AZ were evaluated under in vitro conditions. For the in vivo assays, adult males were acclimated to salinities of 2.5 or 30‰. The corresponding 96-h LC₅₀ of cadmium was 2.69 mg l⁻¹ at 2.5‰, and >50 mg l⁻¹ at 30‰. Cadmium only caused a significant lower CA activity than control at 2.5‰. EC₅₀ for CA inhibition was estimated to be 1.59 mg l⁻¹ at 2.5‰. Statistical differences in Na⁺ hemolymphatic levels (P<0.05) were only detected at 2.5‰, between 0 and 1.25 mg l⁻¹ of cadmium, but no statistical differences were observed for Cl⁻ levels at any assayed salinity. As CA inhibition registered at 2.5‰ was followed by only changes in Na⁺ concentration, it is likely that cadmium exposure could differentially affect ions permeability, among others factors. The concentrations that inhibited in vitro 50% of enzymatic activity (IC₅₀) were 2.15×10⁻⁵, 1.62×10⁻⁵, 3.75×10⁻⁶ and 4.4×10⁻¹⁰ M for cadmium, copper, zinc and AZ, respectively. The comparison with IC₅₀ values of other aquatic species, indicates a higher CA sensitivity for C. granulata to pollutants.     

Histological and pharmacological investigations of the two symmetrically situated giant neurons,RPeNLN and LPeNLN,identified on the anterior surface of the pedal ganglia of an african giant snail (achatina fulica férussac)

• 1.1. Morphological and pharmacological investigations were made of two giant neurons, RPeNLN (right pedal nerve large neuron) and LPeNLN (left pedal nerve large neuron), situated symmetrically on the anterior surface of the pedal ganglia of an African giant snail (Achatina fulica Férussac).
• 2.]2. The two neurons (about 250–300 μm in diameter) were the largest ones identified in the ganglia of the snail species. The axonal pathways of the two neurons were symmetrical; of their four main axonal branches, the three main branches innervated the ipsilateral pedal nerves, whereas the last main branch projected to the contralateral pedal nerves.
• 3.]3. The pharmacological features of the two neurons were very similar. Both were inhibited markedly by dopamine [minimum effective concentrations (MECs): 3 × 10⁻⁶-10⁻⁵M], dl-octopamine (MECs: 2 × 10⁻⁶-2 × 10⁻⁵M), 5-hydroxytryptamine (MEC: 3 × 10⁻⁶M), GABA (MEC: 3 × 10⁻⁵ M), l-homocysteic acid (MECs: 3 × 10⁻⁵-10-10⁻⁴M) and erythro-β-hydroxy-l-ghitanuc acid (MEC: 3× 10⁻⁵M). Acetylcholine showed varied effects, either excitatory or inhibitory, on the two neurons examined. No substances were found to have any marked excitatory effects on the neurons.

     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. V—Rabbit (Oryctolagus Cuniculus)

• 1.1. The diffusional water permeability (P_d) of rabbit red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulfonate (PCMBS).
• 2.2. The values of P_d were around 6.3 × 10⁻³ cm/sec at 15°C, 7.0 × 10⁻³cm/sec at 20°C, 8.0 × 10⁻³ cm/sec at 25°C, 9.1 × 10⁻³ cm/sec at 30°C and10.7 × 10⁻³ cm/sec at 37°C.
• 3.3. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 15 min at 37°C with 0.5 mM PCMBS.
• 4.4. The values of maximal inhibition were around 71–74% at all temperatures.
• 5.5. The basal permeability to water was estimated as 1.6 × 10⁻³cm/sec at 15°C, 2.0 × 10⁻³cm/sec at 20°C, 2.4 × 10⁻³cm/sec at 25°C, 2.6 × 10⁻³cm/sec at 30°C, and 3.1× 10^{−3 cm/secat 37°C}.
• 6.6. The activation energy of water diffusion was around 18 kJ/mol and increased to 27 kcal/mol after incubation with PCMBS in conditions of maximal inhibition of water diffusion.
• 7.7. The membrane polypeptide electrophoretic pattern of rabbit RBCs has been compared with its human counterpart.
• 8.8. The rabbit membrane contained a higher amount of spectrin (bands 1 and 2), while the band 6 (glyceraldehyde-3-phosphate dehydrogenase) was markedly less intense.
• 9.9. Considerable differences in the electrophoretic patterns of the two sources of RBC membranes appeared in the bands migrating in the band 4.5 region and in front of band 7, where some polypeptides were apparent in higher amounts in the rabbit RBC membrane.

     

Preparation and antithrombogenic activities of heparinoid from 6-O-(carboxymethyl)chitin

The highest antithrombogenic activity was achieved by the sulphation of partially N-deacetylated O-(carboxymethyl)chitin among variously modified chitin derivatives. It was also suggested that the distribution of N-sulphate and N-acetyl groups on the C-2 position might be essential to the selective binding by antithrombin-III to inhibit thrombin activity. Kinetic evaluations demonstrated the non-competitive inhibition on direct interaction with thrombin (K_i = 9.26 × 10⁻⁸m) and the competitive inhibition with antithrombin-III (K_i = 3.33 × 10⁻⁸) m as well as with heparin. 6-O-Carboxymethyl groups were found, from the data of intravenous injection in mice, to suppress the toxicity of chitin heparinoids.     

The use of glucose to regulate pH values of culture media and increase the production of baculovirus (BmNPV) and foreign protein (HBsAg)

When BmN-4 and M-BmN cells were grown in shake flasks, the pH initially dropped and later increased. The increase in pH signaled a ‘metabolic switch’ that was used here as an indicator for initiating a supplemental glucose and glutamine feed. Using the pH-based fed-batch culture method described, the maximum cell densities of BmN-4 cells and M-BmN cells were increased from 30×10⁵ cells ml⁻¹ to 43×10⁵ and 52×10⁵ cells ml⁻¹, respectively. Correspondingly, the production of polyhedra (4·5×10⁵ OBs ml⁻¹) and HBsAg (574 ng ml⁻¹), from the infection of BmN-4 and M-BmN by wild-type and recombinant BmNPV viruses, respectively, were both significantly enhanced 50% and 100%, respectively. This feeding strategy was implemented with no advanced instrumentation yet facilitated significantly increased yield in shake flasks. The technique should benefit those in research laboratories employing the baculovirus expression system as a rapid and efficient production system.     

Inhibiton and gamma-aminobutyric acid-induced conductance on locust (Schistocerca gregaria) extensor tibiae muscle fibres

• 1.1. Increases in membrane conductance (g_m) were induced by GABA in distal bundles 32, 33 and 34 of extensor tibiae muscles of the locust (Schistocerca gregaria).
• 2.2. Bath application of GABA (10⁻⁵−5 × 10⁻³ M) induced reductions in muscle fibre space constant (λ).
• 3.3. GABA (5 × 10⁻³ M) induced additional membrane conductance of 2.21 ± 0.03 × 10⁻⁶ S/mm, 0.38 ± 0.03 × 10⁻⁶ S/mm and 0.29 ± 0.06 × 10⁻⁶ S/mm on muscle bundles 34, 33 and 32 respectively. The greater sensitivity of muscle fibres in bundle 34 to GABA is due at least in part to a larger number of GABA receptors on bundle 34 muscle fibres.
• 4.4. The decrement of electrotonic potentials in the presence of GABA were measured over distances of both half fibre length and whole fibre length. Good agreement was obtained between changes in space constant produced by GABA using half fibre length and whole fibre length data.
• 5.5. By taking into account changes in space constant induced by GABA it was possible to demonstrate that presynaptic GABA receptors were involved in the inhibition of slow excitatory postsynaptic potentials by GABA.
• 6.6. “Slow” excitatory postsynaptic potentials recorded under current clamp were inhibited in a dose-dependent manner by GABA. This inhibition was not dependent on muscle-fibre GABA sensitivity and could not be completely accounted for by GABA-induced changes in the cable properties of the muscle fibres.

     

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.     

Calcium and cyclic AMP as possible mediators of neurohormone action in the hindgut of the cockroach, Leucophaea maderae

Adenosine 3′,5′-monophosphate (cyclic AMP) (10⁻⁵ g/ml) often caused a gradual increase in spotaneous contractile activity of the hindgut of the cockroach, Leucophaea maderae, and on rare occasions it would evoke a hormone-like response. However, aminophylline (2·5 × 10⁻⁴ g/ml) was capable of mimicking the neurohormone, and a concentration of 2·5 × 10⁻⁵ g/ml potentiated the contractile response evoked by the neurohormone: these responses were blocked by either the presence of 1 mM manganous ion or in a high potassium solution (162 mM). Propranolol (10⁻⁶ g/ml) and dopamine (10⁻⁴ g/ml) suppressed both spontaneous contractile events and neurohormone action. Dopamine (5 × 10⁻⁶ g/ml) also blocked action potential generation as did propranolol at 10⁻⁴ g/ml.These results lead us to suppose that cyclic AMP might serve as a mediator of neurohormone action by increasing calcium transport across the surface membrane of muscle fibres. Caffeine (2·5 × 10⁻⁴ g/ml), like aminophylline, caused a hormone-like response in normal hindguts. Even when the visceral muscles of the hindgut were depolarized in 162 mM potassium solution (without calcium), caffeine was still capable of inducing a phasic response. However, the addition of 2 mM calcium to such potassium-depolarized preparations caused a gradual increase in muscle tonus and substantially potentiated the response to caffeine.Such findings clearly implicate calcium as the mediator of excitation-contraction coupling in visceral muscle. While the interactions between the neurohormone, cyclic AMP, and calcium seem to be largely associated with the surface membrane and action potential generation.     

δ-Aminolevulinic acid synthase of Euglena gracilis: Physical and kinetic properties

Physical and kinetic properties of δ-aminolevulinic acid synthase from wild-type and aplastidic strains of Euglena gracilis have been determined. Michaelis constants for glycine, succinyl-CoA and pyridoxal phosphate are 8.5 × 10^?3m, 2.5 × 10^?5m, and 2.9 × 10^?6m, respectively. Optimum reaction pH is 7.8, and maximal product yield during a 30-min incubation occurs at 40 °C. Activity in frozen cell extracts remains constant for 5 days, then falls slowly to one-third of the initial value after 3 months. Enzyme activity rapidly declines irreversibly in the absence of pyridoxal phosphate. Agarose gel chromatography of the native enzyme yields a single band of activity at an elution volume corresponding to a molecular weight of 138,000. δ-Aminolevulinic acid synthase obtained from green wild-type strain Z cells is identical in its physical properties to that obtained from white aplastidic mutant strain W₁₄ ZNalL cells.     

Ligation of EcoRI endonuclease-generated DNA fragments into linear and circular structures.

Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules.Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10⁻² μm) is smaller than the random coil segment value b (7·17 × 10⁻² μm) was observed, while circular structures of the dimer of the same molecule (12 × 10⁻² μm) were detected.     

Exploitation of micellar medium for photochemical-spectrofluorimetric flow-injection determination of fenvalerate

A photochemical flow-injection methodology for the fluorimetric determination of fenvalerate, a nonfluorescent pyrethroid pesticide, is proposed. The sample was irradiated by ultraviolet light inside a reaction coil in a nonstop mode and the photodegradation products were monitored by spectrofluorimetry at λ_exc=277 nm, λ_em=329 nm. The exploitation of a micellar medium resulted in a pronounced fluorescence enhancement with a concomitant tenfold sensitivity improvement. Several surfactants were evaluated, and best results were obtained with sodium dodecylsulphate (SDS). The calibration plot was linear within 2.0×10⁻⁶ and 1.0×10⁻⁵ mol l⁻¹ fenvalerate. The proposed system is very stable, handles 30 samples h⁻¹ and requires only 60 μmol SDS per determination. The detection limit is 5×10⁻⁷ mol l⁻¹. The method was evaluated in the determination of fenvalerate in tap water.     

Transition state stabilization by deaminases: Rates of nonenzymatic hydrolysis of adenosine and cytidine

Nonenzymatic rates of hydrolytic deamination of adenosine and cytidine by acids and bases analogous to side chains of naturally occurring amino acids are compared with the rates of uncatalyzed deamination in water and with the rates of the hydroxide- and hydrogen ion-catalyzed reactions. For adenosine, hydroxide ion is an effective catalyst, with a second-order rate constant of 7.5 × 10⁻⁶ m⁻¹ s⁻¹ at 85°C and an energy of activation of 19.9 kcal/mol. Acid-catalyzed deamination of adenine proceeds with a second-order rate constant of 1.5 × 10⁻⁶ m⁻¹ s⁻¹ at 85°C. At concentrations of 1 m and at pH values corresponding to their respective pK_a values, dimethylamine, acetate, selenide, imidazole, phosphate, and zinc(II) do not enhance the rate of deamination of adenosine beyond that observed in water, and 2-mercaptoethanol produces only a modest rate enhancement. The uncatalyzed rate of adenosine deamination in water is 8.6 × 10⁻⁹ s⁻¹ at 85°C: extrapolation to 37°C and comparison with k_cat for rat hepatoma adenosine deaminase yield a rate enhancement by the enzyme of approximately 2 × 10¹²-fold. 1,6-Dimethyladenosine, the conjugate acid of which has a pK_a value much higher than that of adenosine, is not readily deaminated, suggesting that the uncatalyzed deamination of adenosine does not proceed by hydroxide ion attack on the rare protonated form of adenosine, but rather by attack on the neutral species. Deamination of cytidine is catalyzed most effectively by hydroxide ion, with a second-order rate constant of 4.5 × 10⁻⁴ m⁻¹ s⁻¹ at 85°C and an energy of activation of 28.5 kcal/mol. The uncatalyzed rate of deamination of cytidine in water, which also exhibits an energy of activation of 28.5 kcal/mol, is 8.8 × 10⁻⁸ s⁻¹ at 85°C. Comparison of the rate extrapolated to 25°C with k_cat for bacterial cytidine deaminase gives a rate enhancement for the enzyme of 4 × 10¹¹-fold. The C-5 proton of the pyrimidine ring of cytidine does not exchange with solvent during alkaline hydrolysis, suggesting that deamination under these conditions does not involve prior addition of water across the 5,6 double bond.