Effects of microbial community interactions on transformation rates of xenobiotic chemicals.

The effects of culture filtrates, mixed populations, and common microbial exudates on bacterial transformations of three agricultural and industrial chemicals were investigated. Test chemicals included methyl parathion, diethyl phthalate, and 2,4-dichlorophenoxyacetic acid butoxyethyl ester. The presence of various cultures, filtrates, or exudates of algae, fungi, or other bacteria either stimulated or inhibited bacterial transformation rates. Inhibition resulted from treatments that lowered the pH, and stimulation resulted from an increase in cell biomass (based on plate counts) and from a different process whereby rates of transformation per bacterial cell rapidly increased as much as 10-fold.     

Effects of Adaptation on Biodegradation Rates in Sediment/Water Cores from Estuarine and Freshwater Environments

Experiments were devised to determine whether exposure to xenobiotics would cause microbial populations to degrade the compounds more rapidly during subsequent exposures. Studies were done with water/sediment systems (ecocores) taken from a salt marsh and a river. Systems were tested for adaptation to the model compounds methyl parathion and p-nitrophenol. ¹⁴CO₂ released from radioactive parent compounds was used as a measure of mineralization. River populations preexposed to p-nitrophenol at concentrations as low as 60 μg/liter degraded the nitrophenol much faster than did control populations. River populations preexposed to methyl parathion also adapted to degrade the pesticide more rapidly, but higher concentrations were required. Salt marsh populations did not adapt to degrade methyl parathion. p-Nitrophenol-degrading bacteria were isolated from river samples but not from salt marsh samples. Numbers of nitrophenol-degrading bacteria increased 4 to 5 orders of magnitude during adaptation. Results indicate that the ability of populations to adapt depends on the presence of specific microorganisms. Biodegradation rates in laboratory systems can be affected by concentration and prior exposure; therefore, adaptation must be considered when such systems are used to predict the fate of xenobiotics in the environment.     

Potassium and the Recovery of Arterial Smooth Muscle after Cold Storage

The influence of K on the performance of vascular smooth muscle was studied by observing the mechanical performance of the muscle under conditions in which the magnitudes of [K_i] and of the [K_i]:[K_o] ratio varied in opposite directions. During prolonged storage at 4°C the artery strips lost K and their ability to respond to stimuli. Subsequently they were transferred to recovery solutions of various [K_o] at 38°C. The initial rate of K_i reaccumulation and steady state [K_i] were greater in solutions of higher [K_o]. Conversely for any time during recovery, the greater [K_o], the smaller the [K_i]:[K_o] ratio. When the strip was placed in the warm recovery solution it first contracted and then relaxed. The initial contraction was not relatable to [K_o] of the recovery solution but the subsequent relaxation was greater in rate and magnitude as [K_o] was greater. As the muscles recovered further they went into tonic contracture. As the [K_o] in the recovery solutions was greater these contractures occurred after shorter recovery times, and attained greater amplitude at a faster rate. Solution-switching experiments indicated a dependence of responses to electrical shocks on both the [K_i]:[K_o] ratio and [K_i]. Conclusions drawn were: (a) increased [K_i] increases contractility, (b) increased [K_i] increases the rate of relaxation, (c) excitability is decreased by too high or low a [K_i]: [K_o] ratio, and (d) the extent of tonic shortening depends on the [K_i]:[K_o] ratio.     

Mineralization of Parathion in the Rice Rhizosphere

We studied ¹⁴CO₂ evolution from ring-labeled [2,6-¹⁴C]parathion (O,O-diethyl-O-p-nitrophenyl phosphorothioate) in the rhizosphere of rice seedlings. The soil samples (nonflooded [60% water-holding capacity] and flooded) were treated first with technical parathion (20 μg/g) and then after 10 days with ring-labeled [¹⁴C]parathion. In unplanted soil, less than 5.5% of the ¹⁴C in the parathion was evolved as ¹⁴CO₂ in 15 days under both flooded and nonflooded conditions. In soil planted with rice, 9.2% of the radiocarbon was evolved as ¹⁴CO₂ under nonflooded conditions, and 22.6% was evolved under flooded conditions. These results suggest that soil planted with rice permits significant ring cleavage, especially under flooded conditions.     

Cell Swelling Activates the K+ Conductance and Inhibits the Cl? Conductance of the Basolateral Membrane of Cells from a Leaky Epithelium

Necturus gallbladder epithelial cells bathed in 10 mM HCO₃/1% CO₂ display sizable basolateral membrane conductances for Cl⁻ (G_Cl ^b) and K ⁺ (G_K ^b). Lowering the osmolality of the apical bathing solution hyperpolarized both apical and basolateral membranes and increased the K ⁺/Cl⁻ selectivity of the basolateral membrane. Hyperosmotic solutions had the opposite effects. Intracellular free-calcium concentration ([Ca²⁺]_i) increased transiently during hyposmotic swelling (peak at ∼30 s, return to baseline within ∼90 s), but chelation of cell Ca²⁺ did not prevent the membrane hyperpolarization elicited by the hyposmotic solution. Cable analysis experiments showed that the electrical resistance of the basolateral membrane decreased during hyposmotic swelling and increased during hyperosmotic shrinkage, whereas the apical membrane resistance was unchanged in hyposmotic solution and decreased in hyperosmotic solution. We assessed changes in cell volume in the epithelium by measuring changes in the intracellular concentration of an impermeant cation (tetramethylammonium), and in isolated polarized cells measuring changes in intracellular calcein fluorescence, and observed that these epithelial cells do not undergo measurable volume regulation over 10–12 min after osmotic swelling. Depolarization of the basolateral membrane voltage (V_cs) produced a significant increase in the change in V_cs elicited by lowering basolateral solution [Cl⁻], whereas hyperpolarization of V_cs had the opposite effect. These results suggest that: (a) Hyposmotic swelling increases G_K ^b and decreases G _Cl ^b. These two effects appear to be linked, i.e., the increase in G _K ^b produces membrane hyperpolarization, which in turn reduces G _Cl ^b. ( b) Hyperosmotic shrinkage has the opposite effects on G_K ^b and G _Cl ^b. ( c) Cell swelling causes a transient increase in [Ca²⁺]_i, but this response may not be necessary for the increase in G_K ^b during cell swelling.     

The rhodopsin system of the squid

Squid rhodopsin (λ_max 493 mµ)—like vertebrate rhodopsins—contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (λ_max 500 mµ) near neutrality, yellow (λ_max 380 mµ) in alkaline solution. The two forms—acid and alkaline metarhodopsin—are interconverted according to the equation, Alkaline metarhodopsin + H⁺ acid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.     

Photosynthetic Adaptation by Synechococcus leopoliensis in Response to Exogenous Dissolved Inorganic Carbon

Synechococcus leopoliensis was grown over a wide range of dissolved inorganic carbon (DIC) concentrations (4-25,000 micromolar) which were obtained by varying culture pH (6.2-9.6) and the CO₂ concentration of the gas stream (36-50,000 microliters per liter). The [DIC] required to half-saturate photosynthesis (K_½^DIC) was found to vary depending upon the ambient DIC concentration at which the cells were grown. Low [DIC] grown cells exhibited low values of K_½^DIC (4.7 micromolar) whereas cells grown at high [DIC] exhibited high values of K_½^DIC (1-2.5 millimolar). Intermediate concentrations of DIC produced intermediate values. Changes in K_½^DIC appeared to be solely a function of [DIC] and were independent of both culture pH and CO₂ concentration. As changes in K_½^DIC occur in response to DIC concentrations commonly found in natural systems we suggest this adaptation may be of ecological significance.     

Molecular insights into substrate recognition and catalysis by phthalate dioxygenase from Comamonas testosteroni

Phthalate, a plasticizer, endocrine disruptor, and potential carcinogen, is degraded by a variety of bacteria. This degradation is initiated by phthalate dioxygenase (PDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of phthalate to a dihydrodiol. PDO has long served as a model for understanding ROs despite a lack of structural data. Here we purified PDO_KF1 from Comamonas testosteroni KF1 and found that it had an apparent k_cat/K_m for phthalate of 0.58 ± 0.09 μM⁻¹s⁻¹, over 25-fold greater than for terephthalate. The crystal structure of the enzyme at 2.1 Å resolution revealed that it is a hexamer comprising two stacked α₃ trimers, a configuration not previously observed in RO crystal structures. We show that within each trimer, the protomers adopt a head-to-tail configuration typical of ROs. The stacking of the trimers is stabilized by two extended helices, which make the catalytic domain of PDO_KF1 larger than that of other characterized ROs. Complexes of PDO_KF1 with phthalate and terephthalate revealed that Arg207 and Arg244, two residues on one face of the active site, position these substrates for regiospecific hydroxylation. Consistent with their roles as determinants of substrate specificity, substitution of either residue with alanine yielded variants that did not detectably turnover phthalate. Together, these results provide critical insights into a pollutant-degrading enzyme that has served as a paradigm for ROs and facilitate the engineering of this enzyme for bioremediation and biocatalytic applications.     

Initial Test of the Benchmark Chemical Approach for Predicting Microbial Transformation Rates in Aquatic Environments

Using 2,4-dichlorophenoxyacetic acid methyl ester (2,4-DME) as a benchmark chemical, we determined relative pseudo-first-order rate coefficients for the butoxyethyl ester of 2,4-dichlorophenoxyacetic acid (2,4-DBE), methyl parathion, and methyl-3-chlorobenzoate in a diversity of microbial samples, including water, sediment, biofilm, and floating microbial mats collected from a laboratory mesocosm as well as from streams, lakes, and wetlands in Georgia and Florida. The decreasing order of reactivity for relative microbial transformation rates was 2,4-DBE > 2,4-DME > methyl-3-chlorobenzoate > methyl parathion. Half-lives of the chemicals varied about 60-fold depending on the chemical and microbial sample. Relative rate coefficients, however, typically varied only about threefold for field-collected samples. Relative rate coefficients determined with samples from a laboratory mesocosm were consistently low compared with the field sample data. Overall, the data indicated that microbial transformation rates of a chemical can be satisfactorily inferred for a wide variety of microbial habitats—such as water, biofilm, or a sediment—on the basis of its transformation rate relative to that of an appropriate benchmark chemical by using a single type of microbial sample.     

A comparison of acylation, phosphorylation and binding in related substrates and inhibitors of serum cholinesterase

1. The K_m and catalytic-centre activities for human serum cholinesterase and methyl, ethyl, n-propyl and n-butyl butyrate substrates were determined and compared with the related inhibition constants of a similarly substituted organophosphate inhibitor series based on malaoxon. The results indicated that the catalytic-centre activities approximated to k_+2(a), the acylation rate constant, and that K_m approximated to the equilibrium binding constant. The inhibition constants measured were K_a, the equilibrium binding constant, and k_+2(p), the phosphorylation rate constant. 2. The effects of the alkyl substituents on k_+2(p) and k_+2(a) were closely parallel, and the decreasing order in each case was: n-butyl; methyl; n-propyl; ethyl. The Taft constants did not follow this order, suggesting that alkyl substituents did not primarily effect acylation or phosphorylation by electron induction. 3. For comparable homologues, the k_+2(a) values were on average 435 times the k_+2(p) values. The k_+2(p) values at 25° and pH7·6 ranged from 6·6min.⁻¹ for the diethyl member to 22·6min.⁻¹ for the di-n-butyl member. 4. The effect of the alkyl substituents on K_a and K_m were closely paralleled. The increasing order in each case was: n-butyl; n-propyl; ethyl; methyl. The K_a values were about 100 times less than the comparable K_m values. 5. Consideration of the binding energies suggested that only one of the two alkyl groups on the malaoxon homologues bound to the active site. 6. The possibility that malaoxon acted as a substrate as well as an inhibitor for cholinesterase was also investigated, but no evidence of a substrate reaction was found.     

Thiophene bioisosteres of GluN2B selective NMDA receptor antagonists: Synthesis and pharmacological evaluation of [7]annuleno[b]thiophen-6-amines

Thiophene bioisosteres of potent GluN2B receptor negative allosteric modulators were prepared and evaluated pharmacologically. The five-step synthesis of 4,5,7,8-tetrahydro[7]annuleno[b]thiophen-6-one (10) was considerably improved by carboxylation of thiophene-3-carboxylic acid (8) in the first reaction step. Reductive amination and alkylation led to three homologous series of secondary and tertiary phenylalkylamines 5, 11 and 12. Metalation, reaction with 1-formylpiperidine and subsequent reduction provided hydroxymethyl derivatives 15 and 16, which had been designed as bioisosteres of phenols. 2-Bromo derivatives 18 were obtained by bromination of ketone 10 with NBS and subsequent reductive amination. High GluN2B affinity was achieved with [7]annuleno[b]thiophenes bearing a 3-phenylpropylamino or 4-phenylbutylamino moiety (e.g. 5c: K_i = 5.9 nM; 11d: K_i = 9.0 nM). Tertiary ethylamines 12 showed lower GluN2B affinity than tertiary methylamines 11 or secondary amines 5 (e.g. 5c: K_i = 5.9 nM; 11c: K_i = 6.0; 12c: K_i = 51 nM). A Br-atom or a hydroxymethyl moiety in 2-position were less tolerated by the GluN2B receptor. Very similar relationships between the structure and GluN2B affinity and structure and σ affinity, in particular σ₂ affinity, were detected. A slight preference for the ifenprodil binding site of GluN2B receptors over σ₁ and σ₂ receptors was found for methylamines 11c (≈2-fold) and 11d (≈1.5–2-fold) as well as for bromo derivative 18c (≈3-fold).     

DNA-binding and photocleavage properties of cationic porphyrin-anthraquinone hybrids with different lengths of links

Four cationic porphyrin–anthraquinone (Por–AQ) hybrids differing in lengths of flexible alkyl linkage, 5-[4-(1-N-anthraquinonon-yl)-l-oxophenyl]-10,15,20-tris(N-methylpyridinium-4-yl)porphyrin triiodide, (l = acetyl, pentanoyl, octanoyl, undecanoyl, designed as [AQATMPyP]I₃, [AQPTMPyP]I₃, [AQOTMPyP]I₃ and [AQUTMPyP]I₃, respectively, see Fig. 1), were synthesized and their interactions with DNA were investigated. The results of spectroscopic, denaturation and viscosity measurements suggest that [AQATMPyP]I₃ binds to DNA through non-intercalative mode while the other three hybrids with longer links bind via bis-intercalative mode. Ethidium bromide (EB) competition experiment was carried out to determine the binding constants (K_b) of these compounds for CT DNA, and [AQPTMPyP]I₃ shows the largest K_b among these hybrids. The photocleavage mechanism and wavelength-dependent cleaving abilities of these hybrids to pBR322 plasmid DNA were also comparably investigated.     

Genetic Variation at a Locus (TAM-1) for Submaxillary Gland Protease in the Mouse and Its Location on Chromosome 7

Electrophoretic and activity variants for a testosterone-induced esteroprotease have been discovered in submaxillary glands from inbred strains of mice. The enzyme is tentatively designated tamase (TAM-1) and the variant genetic locus is Tam-1. The alleles Tam-1^a and Tam-1^b determine electrophoretically distinct zones of tamase activity, while Tam-1^c produces no detectable enzyme activity. Data from recombinant inbred strains and B6AF₁ x B6 and B6D2F₁ x B6 backcrosses established linkage of Tam-1 to glucose phosphate isomerase (Gpi-1), pink-eyed dilution (p) and β-hemoglobin (Hbb) on chromosome 7. The gene order is Gpi-1—Tam-1—p—Hbb. Analysis of congenic resistant strains indicates that Tam-1 is closely linked to the minor histocompatibility locus, H-4. TAM-1 was not cross-reactive with antisera to mouse nerve growth factor, submaxillary renin, or tamases A and D.     

Metabolism of gibberellin a(12)-7-aldehyde by soybean cotyledons and its use in identifying gibberellin a(7) as an endogenous gibberellin

The level of gibberellin(GA)-like material in cotyledons of soybean (Glycine max L.) was highest at mid-pod fill—about 10 nanograms GA₃ equivalents per gram fresh weight of tissue, assayed in the immersion dwarf rice bioassay. This amount is about 1000-fold less than levels in Pisum and Phaseolus seed, other legume species whose spectrum of endogenous gibberellins (GAs) is well known. The metabolism of [¹⁴C]-GA₁₂-7-aldehyde (GA₁₂ald)—the universal GA precursor—by intact, mid-pod-fill, soybean cotyledons and their cell-free extracts was investigated. In 4 hours, extracts converted GA₁₂ald to two products—[¹⁴C]GA₁₂ (42% yield) and [¹⁴C]GA₁₅ (7%). Within 5 minutes, intact embryos converted GA₁₂ald to [¹⁴C]GA₁₂ and [¹⁴C]GA₁₅ in 15% yield; 4 hour incubations afforded at least 22 products (96% total yield). The putative [¹⁴C]GA₁₂ was identified as a product of [¹⁴C]GA₁₂ald metabolism on the basis of co-chromatography with authentic GA₁₂ on a series of reversed and normal phase high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC) systems, and by a dual feed of the putative [¹⁴C]GA₁₂ and authentic [¹⁴C]GA₁₂ to cotyledons of both peas and soybeans. The [¹⁴C]GA₁₅ was identified as a metabolite of [¹⁴C]GA₁₂ald by capillary gas chromatography (GC)-mass-spectrometry-selected ion monitoring, GC-radiocounting, HPLC, and TLC. By adding the [¹⁴C] metabolites of [¹⁴C]GA₁₂ald to a different and larger extract (about 0.2 kg fresh weight of soybean reproductive tissue) and purifying endogenous substances co-chromatographing with these metabolites, at least two GA-like substances were obtained and one identified as GA₇ by GC-mass spectrometry. Since [¹⁴C]GA₉ was not found as a [¹⁴C]metabolite of [¹⁴C]GA₁₂ald, soybean embryos might have a pathway for biosynthesis of active, C-19 gibberellins like that of the cucurbits; GA₁₂ald → GA₁₂ → GA₁₅ → GA₂₄ → GA₃₆ → GA₄ → GA₇.     

Susceptibility Test of Two Ca2+-ATPase Conformers to Denaturants and Polyols to Outline Their Structural Difference

To determine the effect of denaturants [guanidine hydrochloride (GdnHCl) and urea] and polyols [with various molecular masses (62.1–600)] on calcium binding at the two hypothesized conformers (A and B forms) of the chemically equivalent sarcoplasmic reticulum Ca²⁺-ATPase, which bind two calcium ions in different manners, we examined the effect of these reagents on the calcium dependence of ATP-supported phosphorylation of the ATPase molecules and of their calcium-activated, acetyl phosphatate hydrolytic activity. (1) GdnHCl (~0.05 M) and urea (~0.5 M) increased the apparent calcium affinity (K _0.5) of 2–6 μM of noncooperative binding [Hill coefficient (n _H) ~ 1] of the A form to 10–40 μM. (2) The employed polyols transformed the binding of the A form into cooperative binding (n _H ~ 2), accompanying the approach of its K _0.5 value to that (K _0.5 = 0.04–0.2 μM) of the cooperative binding (n _H ~ 2) of the B form; the transition concentration (0.025–2 M) of the polyols, above which such transformation occurs, was in inverse relation to their molecular mass. (3) The binding of the B form was resistant to these denaturants and polyols. Based on these data, a structural model of the two forms, calcium-binding domains of which are loosely and compactly folded, is presented.     

FURTHER OBSERVATIONS ON THE MECHANISM OF PHAGE ACTION

1. The reaction between an antistaphlycoccal phage and the homologous bacterium has been studied, applying the following experimental technics not used in earlier work reported from this laboratory: (a) Both the activity assay and the plaque count were utilized for determining [phage]. (b) Sampling was done at short intervals; i.e., every 0.1 hour. (c) Extracellular phage was separated from the cell-bound fraction by a filtration procedure permitting passage of < 95 per cent of free phage. 2. Using these technics, the reaction was followed: (a) with pH maintained at 6.10 and temperature at 28°C. to slow the process; (b) with pH maintained at 7.2 and temperature at 36°C. 3. In addition separate experiments were performed on the sorption of phage by bacteria at 30°, 23°, and 0°C. 4. At pH 6.10 and 28°C. the phage-bacterium reaction proceeds in the following sequence: (a) There is an initial phase of rapid logarithmic sorption of phage to susceptible cells, during which the total phage activity and the plaque numbers in the mixtures remain constant. (b) When 90 per cent of the phage has been bound, there is a sudden very rapid increase in phage activity not paralleled by an increase in plaques; i.e., phage is formed intracellularly, but is retained within cellular confines. (c) After a further drop in the extracellular phage fraction there occurs a pronounced increase in the total phage plaque count not accompanied by any increase in total activity. This indicates a redistribution of phage formed intracellularly. At the same time there is a rise in the extracellular phage curves (both activity and plaque). (d) With the concentrations of phage and bacteria used in the experiment carried out at pH 6.1 and 28°C. there are two further increments in [phage]_act. before massive lysis begins. (e) During terminal lysis there are sharp rises in the curves for [total phage]_plaq., [extracellular phage]_act., and [extracellular phage]_plaq.. (f) Immediately after the completion of lysis there is a considerable disparity between measurements of total phage and extracellular phage, probably occasioned by the association of phage molecules with cellular debris, the latter being of sufficient size to be removed by the super-cel filters. 5. At pH 7.2 and 36°C. the steps in the phage production curve as determined by activity assay and plaque count are much less prominent than those observed at pH 6.1 and 28°C. However, the plateaus described by Ellis and Delbrück (10) for B. coli and coli phage can be detected also in the present case if frequent samples are taken. 6. The sorption experiments show a significant rise in the rate of phage uptake with increase in temperature, again supporting the view that the reaction involves more than a purely physical adsorption. 7. Delbrück''s objections to: (a) the use of the activity assay for determining [total phage] in mixtures of phage and susceptible cells, and (b), to the demonstration of phage precursor in "activated" bacteria have been analyzed. 8. The activity assay has been demonstrated to be an accurate procedure for determining either phage free in solution or phage bound to living susceptible cells, under the conditions of the experiments reported here and in earlier work. 9. The titration values obtained in the experiments designed to exhibit intracellular phage precursor are not the result of artifacts as Delbrück has inferred. The data can be interpreted in terms of the precursor theory, although other explanations are not ruled out.     

Synthesis of carbon-11-labeled 4-(phenylamino)-pyrrolo[2,1-f][1,2,4]triazine derivatives as new potential PET tracers for imaging of p38α mitogen-activated protein kinase

The reference standards methyl 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (10a), methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate (10b) and corresponding precursors 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylic acid (11a), methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylic acid (11b) were synthesized from methyl crotonate and 3-amino-4-methylbenzoic acid in multiple steps with moderate to excellent yields. The target tracer [¹¹C]methyl 4-(2-methyl-5-(methoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate ([¹¹C]10a) and [¹¹C]methyl 4-(2-methyl-5-(ethoxycarbamoyl)phenylamino)-5-methylpyrrolo[2,1-f][1,2,4]triazine-6-carboxylate ([¹¹C]10b) were prepared from their corresponding precursors with [¹¹C]CH₃OTf under basic condition through O-[¹¹C]methylation and isolated by a simplified solid-phase extraction (SPE) method in 50–60% radiochemical yields at end of bombardment (EOB) with 185–555 GBq/μmol specific activity at end of synthesis (EOS).     

Evolution of enzyme catalytic power. Characteristics of optimal catalysis evaluated for the simplest plausible kinetic model

1. Evolutionary changes in the structure of an enzyme that provide an increase in its K_m value are considered. Provided that K_m increases as a result of increases in the forward rate constants of the catalysis relative to the reverse rate constants, the enzyme catalyses the conversion of a fixed concentration of its substrate more rapidly when its structure provides that K_m>[S] than when K_m<[S]. 2. Catalytic efficiency of enzymes is discussed in terms of the simplest plausible model, the Haldane [(1930) Enzymes, Longmans, London] reversible three-step model: [Formula: see text] The rate equation for the forward reaction of this model (formation of P) may be written in the simple form: [Formula: see text] K_eq. is the equilibrium constant (=[P]_eq./[S]_eq.), and k_cat.=V/[E]_T, where [E]_T is the total enzyme concentration. 3. To assess the effectiveness of an enzyme, it is necessary only to determine the extent to which the constraints of a particular kinetic mechanism permit v₂ (v when K_m»[S]) to approach v_d (the diffusion-limited rate). 4. The value of the optimal rate of catalysis (v_opt., the maximal value of v₂) is dictated by the equilibrium constant for the reaction, K_eq.; v₂=v_d/a, where [Formula: see text] when k₊₁ is assumed equal to k₋₃, and v_opt.=v_d/a_min.. When K_eq.≥1, it is necessary that k₊₂»k₋₁ for a to take its minimum value, a_min.; when K_eq.«1, it is necessary only that k₊₂»K_eq.·k₋₁, i.e. a can equal a_min. even if k₊₂<k₋₁. When K_eq.»1, v_opt.=v_d; when K_eq.=1, v_opt.=v_d/2, and when K_eq.«1, v_opt.=K_eq.·v_d. 5. The analysis, together with predicted effects of evolutionary pressure, suggests that in practice the rates of the fastest enzyme-catalysed freely reversible reactions might be expected to be lower than the value of k₊₁[E]_T[S] by about an order of magnitude, particularly if K_eq.<1. 6. The existing literature suggests that, in general, appropriate values of K_m have evolved for the provision of high rates of catalysis but that many values of k_cat. are not large enough to provide optimal rates of catalysis unless the value of k₊₁ in vivo is lower than its value in free solution.     

Novel synthesis of methyl 4,6-O-benzylidenespiro[2-deoxy-α-d-arabino-hexopyranoside-2,2′-imidazolidine] and its homologue and sugar-γ-butyrolactam derivatives from methyl 4,6-O-benzylidene-α-d-arabino-hexopyranosid-2-ulose

Novel methyl 4,6-O-benzylidenespiro[2-deoxy-α-d-arabino-hexopyranoside-2,2′-imidazolidine] and its homologue methyl 4,6-O-benzylidene-3′,4′,5′,6′-tetrahydro-1′H-spiro[2-deoxy-α-d-arabino-hexopyranoside-2,2′-pyrimidine] have been synthesized in good yields by reaction of methyl 4,6-O-benzylidene-α-d-arabino-hexopyranosid-2-ulose with 1,2-diaminoethane and 1,3-diaminopropane. The results are completely different from the reaction with arylamines or alkylamines. One-pot synthesis of novel (E)-methyl 4-[hydroxy (methoxy)methylene]-5-oxo-1-alkyl-(4,6-O-benzylidene-2-deoxy-α-d-glucopyranosido)[3,2-b]pyrrolidines has been achieved by the reaction of alkylamines with the butenolide-containing sugar, derived from the aldol condensation of methyl 4,6-O-benzylidene-α-d-arabino-hexopyranosid-2-ulose with diethyl malonate. These sugar-γ-butyrolactam derivatives are potential GABA receptor ligands.     

Reductive transformation of parathion and methyl parathion by Bacillus sp.

Based on the results of phenotypic features, phylogenetic similarity of 16S rRNA gene sequences and BIOLOG test, a soil bacterium was identified as Bacillus sp. DM-1. Using either growing cells or a cell-free extract, it transformed parathion and methyl parathion to amino derivatives by reducing the nitro group. Pesticide transformation by a cell-free extract was specifically inhibited by three nitroreductase inhibitors, indicating the presence of nitroreductase activity. The nitroreductase activity was NAD(P)H-dependent, O₂-insensitive, and exhibited the substrate specificity for parathion and methyl parathion. Reductive transformation significantly decreased the toxicity of pesticides.