A new regioselective method of macrobicyclic Schiff bases synthesis

A new regioselective method of di-gem-thio-substituted PNP-crown derivatives synthesis is presented. The geminalmercaptoethanolanetricyclophosphaza-PNP-lariat ether structure has been determined by X-ray crystallography and characterised by ab initio calculations. The 16-membered PNP-crown ether ring exists in unique conformation: ac⁻ap ac⁻sc⁺sc⁺ap sc⁺ap ap sc⁻ap sc⁻sc⁻ac⁺ap ac⁺. All the ether oxygen atoms are directed into the interior of the ring. All endocyclic P-N bond lengths are equal within experimental error with the mean value 1.578(2) Å. The P-S bond properties have been characterised in terms of natural bond orbital (NBO) analysis, and its interactions with other NBO have been described. The spirocyclisation mechanism at cyclophosphazene phosphorus atom has been proposed.     

Carbon-13 nuclear magnetic resonance spectra of carbohydrate oxirane derivatives

The ¹³C-chemical shifts and ¹J_C,H values of two series of carbohydrate oxirane derivatives, namely methyl 2,3-anhydro-ribo- and -lyxofuranosides and methyl 2,3-anhydro-4,6-O-benzylidene-manno- and -allopyranosides have been determined. The assignment of ¹³C resonances has been established mainly by the examination of the proton-coupled and the selective proton-decoupled spectra. The effect of the oxirane rings on the chemical shifts of β and γ carbon atoms (from the oxirane ring oxygen atom) has been observed. Large ¹J_C,H values associated with cis CH bonds adjacent to the oxirane rings relative to those of trans counterparts have been found.     

The preparation and properties of a stable mercury derivative of transfer RNAs from Escherichia coli

Further investigations into the properties of the mercury derivative formed by the reaction of 4-thiouridine-containing tRNAs and pentafluorophenylmercury chloride have been carried out. tRNA^fMet (which contains only one 4-thiouridine residue) has been isolated by a one-step column Chromatographic procedure from unfractionated Escherichia coli tRNA and has been shown to react with the mercury compound to give a derivative which has similar properties to those previously reported for the corresponding mercury derivative of tRNA^Tyr which contains two adjacent 4-thiouridine residues. The mercury derivative of tRNA^Tyr appears to be a competitive inhibitor of tRNA^Tyr in the aminoacylation reaction (tRNA^TyrK_m = 0.42 μM, mercury derivative of tRNA^TyrK_i = 0.11 μM). The mercury derivative of Tyr-tRNA^Tyr can be made, but only by the reaction of the mercury compound with the aminoacylated tRNA.     

The oxidative cleavage of folates. A critical study

Alkaline permanganate oxidation has been used to determine the chain length of naturally occurring pteroylpolyglutamates on the assumption that all forms of folates cleave at the C⁹N¹⁰ bond to produce the corresponding p-aminobenzoyl-polyglutamates. The chain length of the latter could be determined by cochromatography with synthetic markers. The products of alkalinc (ammonium bicarbonate buffer, pH 9.0) permanganate oxidation of a number of reduced and oxidized, one-carbon-substituted and unsubstituted folic acid derivatives have been identified, and their yields and stability to the oxidative treatment have been determined. Unsubstituted, oxidized and reduced folic acid and N⁵-formyl-tetrahydrofolic acid are cleaved at the C⁹N¹⁰ bond to produce p-aminobenzoylglutamic acid. N⁵, N¹⁰-methenyl-tetrahydrofolic acid, N⁵,N¹⁰-methylene-tetrahydrofolic acid, and N¹⁰-formyl-tetrahydrofolic acid are not cleaved but are oxidized to N¹⁰-formyl-folic acid which is completely stable to the oxidative treatment employed. N⁵-methyl-tetrahydrofolic acid is not cleaved either but is oxidized to N⁵-methyl-dihydrofolic acid which upon continued oxidation decomposes slowly to unidentified products. The γ-glutamyl peptide linkage is completely stable to oxidation. Using p-amino-[3,5-³H]benzoylglutamic acid, it is also shown that this product, previously thought to be stable to the oxidative treatment is decomposed by it. The significance of these findings in terms of the errors that may have been introduced in prior estimations of the chain length and pool sizes of the naturally occurring pteroylpolyglutamates is discussed. The possibility of developing a method for the chain length determination of noncleavable pools of one-carbon-substituted folates using [2-¹⁴C]folic acid to label the folates in vivo is presented.     

Specific pattern of instability of Escherichia coli HisG gene cloned in Bacillus subtilis via the Staphylococcus aureus plasmid pCS194

The plasmid pCS194, generated in vivo by recombination of two Staphylococcus aureus plasmids, pC194 and pS194, coding, respectively, for chloramphenicol (Cm) and streptomycin (Sm) resistance, can be replicated also in Bacillus subtilis in the presence of either of the two antibiotics. In their absence, no segregation of the individual components is observed, but the whole plasmid is lost at a rate of about 10% per generation. The unique EcoRI site of pCS194 is located in the Sm^R determinant. EcoRI-cleaved pCS194 has been joined to an EcoRI-linearized Escherichia coli replicon, the in vitro recombinant pHisG plasmid, composed of the vector pBR313 plus a BglII-segment of E. coli chromosomal DNA, containing a functional hisG gene. The ligation mixture has been used to transform either E. coli or B. subtilis. Following E. coli transformation and selection for Ap^R and Cm^R (the latter is expressed in E. coli by the pC194 determinant), two his⁺ clones were picked at random and the plasmids extracted. These appear identical and contain the original segments. Conversely, after transformation of B. subtilis and selection for Cm^R, only his^? clones have been obtained. From them, deleted plasmids have been extracted. They have lost part or, more frequently, all of the E. coli DNA insert. In the latter case also most of the bracketing pS194 sequence has been lost, and the resulting plasmids are almost identical to pC194, the Cm^R parent of pCS194. When the intact recombinant plasmids, isolated from his⁺ Ap^R Cm^RE. coli clones, have been used to transform B. subtilis cells for Cm^R, again deleted plasmids almost identical to pC194 have been obtained. The events causing these rearrangements occur after in vitro ligation, during either transformation or early propagation of the plasmids, and are probably caused by a translocatable element present in pCS194. A detailed physical map of pC194, carrying the restriction sites for HindIII, HaeIII, HpaII, MboII, AluI, HhaI, and BglI, is presented.     

Mitochondrial Genetics. VI the Petite Mutation in SACCHAROMYCES CEREVISIAE: Interrelations between the Loss of the ρ+ Factor and the Loss of the Drug Resistance Mitochondrial Genetic Markers

The survival of the ρ⁺ factor and of Drug^R mitochondrial genetic markers after exposure to ethidium bromide has been studied. A technique allowing the determination of Drug^R genetic markers among a great number of both grande and petite colonies has been developed. The results have been analyzed by the target theory. The survival of the ρ⁺ factor is always less than the survival of any Drug^R genetic marker. The survivals of C^R and E^R are similar to each other, while that of O^R is greater than that of the other two Drug^R markers. All possible combinations of Drug^R markers have been found among the ρ^- petite cells induced, while the only type found among the grande colonies is the preexisting one. The loss of the C^R and E^R genetic markers was found to be the most frequently concomitant, while the correlation between the loss of the O^R marker and the other two Drug^R markers is less strong. Similar results have been obtained after U.V. irradiation. Interpretations concerning the structure of the yeast mitochondrial genome are given and hypotheses on the mechanism of petite mutation discussed.     

Preparation of NG-monoethyl-l-arginine

N^G-Monoethyl-l-arginine, a putative in vivo product after administration of the potent hepatocarcinogen l-ethionine to rats, has been chemically synthesized by coupling N-ethyl, S-methylthiopseudouronium iodide with α-amino-blocked l-ornithine. The structure of the compound as N^G-monoethyl-l-arginine was confirmed by ¹³C NMR. Its elution time on an automatic amino acid analyzer, R_f values using thin-layer chromatography, and isoelectric point have been compared with those of N^G-monomethyl-l-arginine.     

Reversion from Suppression to Nonsuppression in SUQ5 [psi+] Strains of Yeast: The Classification of Mutations

Reversion from the suppressed to nonsuppressed phenotype in strains of genotype SUQ5 [psi⁺] ade2-1 his5-2 lys1-1 can1-100 ura3-1 has been induced by treatment with ethyl methanesulphonate, nitrosoguanidine or UV (254 nm) light. Spontaneously occurring revertants have also been selected by two different methods. Reversion has been shown to occur through a variety of nuclear mutations and through mutation of [psi⁺] to [psi^-]. Nuclear mutations included back-mutation of SUQ5, antisuppressor mutations that were recessive, semi-dominant or dominant, and dominant or recessive mutations of genes required for the maintenance of the [psi⁺] factor. Complementation tests by which the various kinds of mutations could be distinguished from one another were designed. The spectra of spontaneously occurring and induced mutations have been described.     

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes ¹. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions ^2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes ^{4, 5, 6}. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti ⁷ and Cx. quinquefasciatus ⁸, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates ⁹. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti ¹⁰, the accumulation of multiple chromosomal rearrangements in cell line chromosomes ¹¹ makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4^th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol ¹² is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.     

Control of Endosperm Proteins in TRITICUM AESTIVUM (Var. Chinese Spring) and AEGILOPS UMBELLULATA by Homoeologous Group 1 Chromosomes

The genetic control of major wheat endosperm proteins by homoeologous group 1 chromosomes has been studied by two-dimensional polyacrylamide gel electrophoresis. The control of at least 15 distinct protein subunits or groups of protein subunits has been allocated to chromosomes 1A, 1B and 1D of Chinese Spring wheat from the analysis of grains of aneuploid genotypes. In addition, six protein subunits have been shown to be controlled by chromosome 1C^u of the related species, Aegilops umbellulata, from studies of wheat lines carrying disomic substitutions of 1C^u chromosomes. On the basis of protein subunit patterns, chromosome 1C^u is more closely related to chromosome 1D of wheat than to chromosomes 1A or 1B.     

In vitro iodination of plant ribonucleic acids

The optimum conditions for in vitro iodination of RNAs have been established which yield specific radioactivities ranging from 10 × 10⁴ to 10 × 10⁶ cpm/μg. A nomogram has been constructed by correlating specific radioactivities of RNA with concentration of KI, RNA, and ¹²⁵I. This nomogram can be used to determine the conditions for the desired specific radioactivities for any unknown RNA. The in vitro iodinated RNA has been compared with in vivo labeled RNA for hybridization characteristics. Competition hybridization between ¹²⁵I-labeled chloroplast-rRNA and unlabeled pea (Pisum sativum) chloroplast-rRNA was identical to that found using [³²P]chloroplast-rRNA. Thermal stability of DNA-¹²⁵I-rRNA hybrids was similar to the thermal stability of DNA-[³²P]rRNA hybrids. The iodinated RNA was not found to have undergone any changes in its hydrogen-bonding properties.     

Low field electric birefringence study of monovalent cation-DNA interactions

The effects of monovalent cations on DNA have been studied using static and dynamic electric birefringence. Kerr's law is obeyed in a limited E range (<30 Vcm_?1) and the steady state birefringence values are close for the different cations. The birefringence kinetics have been analysed in terms of three relaxation times. On a semilogarithmice plot of Δn(t), the tail of the curve is linear over a wide range of time for Na⁺, K⁺, NH₄⁺ and Li⁺. Only for Cs⁺ solution is no linear part found and a much longer relaxation time is determined. This only contributes a small part of the total birefringence. With Cs⁺ this contribution is more field-dependent than for the other cations and we observe a larger molecular flexibility. On the other hand, with Li⁺ a greater stiffness of the DNA molecule appears. The electrical polarizabilities anisotropies decrease in the order: Cs⁺ >NH⁺₄ >K⁺ >Na⁺ >Li⁺. There are no significant differences in the optical anisotropy factors.     

Blockers of Ca2+ channels in the plasmalemma of perfused Characeae cells

Ionic currents in the plasmalemma of perfused Nitella syncarpa cells identified as currents through Ca²⁺ channels were registered for the first time. The effect of 1,4-dihydropyridine derivatives (nifedipine, nitredipine, riodipine) and phenylalkylamines (verapamil, D600) as well as the agonist CGP-28392 on the Ca²⁺ channels in the plasmelemma of perfused cells of Nitellopsis obtusa and Nitella syncarpa have been studied. A blocking effect of 1,4-dihydropyridine derivatives and phenylalkylamines on the plasmalemma Ca²⁺ channels has been detected. Phenylalkylamines have been found to block both inward and outward Ca²⁺ currents. The activating effect of the agonist CGP-28392 on the Ca²⁺ channels of plasmalemma has been shown.     

Cytochrome P450 3A Enzymes Catalyze the O6-Demethylation of Thebaine,a Key Step in Endogenous Mammalian Morphine Biosynthesis

Morphine, first characterized in opium from the poppy Papaver somniferum, is one of the strongest known analgesics. Endogenous morphine has been identified in several mammalian cells and tissues. The synthetic pathway of morphine in the opium poppy has been elucidated. The presence of common intermediates in plants and mammals suggests that biosynthesis occurs through similar pathways (beginning with the amino acid l-tyrosine), and the pathway has been completely delineated in plants. Some of the enzymes in the mammalian pathway have been identified and characterized. Two of the latter steps in the morphine biosynthesis pathway are demethylation of thebaine at the O³- and the O⁶-positions, the latter of which has been difficult to demonstrate. The plant enzymes responsible for both the O³-demethylation and the O⁶-demethylation are members of the Fe^II/α-ketoglutarate-dependent dioxygenase family. Previous studies showed that human cytochrome P450 (P450) 2D6 can catalyze thebaine O³-demethylation. We report that demethylation of thebaine at the O⁶-position is selectively catalyzed by human P450s 3A4 and 3A5, with the latter being more efficient, and rat P450 3A2. Our results do not support O⁶-demethylation of thebaine by an Fe^II/α-ketoglutarate-dependent dioxygenase. In rat brain microsomes, O⁶-demethylation was inhibited by ketoconazole, but not sulfaphenazole, suggesting that P450 3A enzymes are responsible for this activity in the brain. An alternate pathway to morphine, oripavine O⁶-demethylation, was not detected. The major enzymatic steps in mammalian morphine synthesis have now been identified.     

Substrate stereochemistry of isovaleryl-CoA dehydrogenase elimination of the 2-pro-R hydrogen in biotin-deficient rats

(2R)-[³H]Isovaleric acid and (2S)-[³H]isovaleric acid (ammonium salts) have been synthesized. These substances, mixed with [1-¹⁴C]isovalerate, have been administered to biotin-deficient rats, which accumulate β-hydroxyisovaleric acid in their urine, the metabolite being formed via isovaleryl-CoA and β-methylcrotonyl-CoA. The results show that most of the tritium from (2R)-[³H]isovalerate was lost, and most of the tritium from (2S)-[³H]isovalerate retained in the conversion to β-hydroxyisovalerate. The stereochemistry of the isovaleryl-CoA dehydrogenase reaction is compared with the stereochemistry of other short-chain acyl-CoA dehydrogenase reactions.     

Steady-state kinetics of electron transfer through cytochrome chain of uncoupled submitochondrial particles. II. Influence of pH on kinetics of electron transfer

pH Dependences of steady-state kinetic parameters of cytochrome chains of submitochondrial particles have been studied. It has been shown that the lifetimes of activated states (τ) of the pairs of cytochromes b → c₁ and a → a₃ have different pH dependences; those for the c₁ → c and c → a cytochrome pairs being similar. The rate constants for the non-activated state of the respiratory chains decreased for the b → c₁ pair and increased for the a → a₃ pair when the pH value was increased.The values of pK calculated from these dependences for the pairs b → c₁ and a → a₃ were 7.2 and 8.9, respectively. It has been supposed that the ratio of activated to non-activated electron carriers may be controlled by the local pH value in the mitochondrial membrane, the latter being dependent upon the rate of electron transfer. The kinetic model based on this assumption allows one to explain the experimental dependences on pH of the rate constants for cytochromes b → c, and a → a₃.The values of the diffusion rate constants for H⁺ and OH^? ions in the mitochondrial membrane estimated from these kinetic data obtained in this study weree 10⁴–10⁵ s^?1 and 10²–10³ s^?1, respectively.     

Nutritional control of xanthine dehydrogenase. II. Effects on xanthine dehydrogenase and aldehyde oxidase of culturing wild-type and mutant Drosophila on different levels of molybdenum

Two new mutants, deficient in aldehyde oxidase and xanthine dehydrogenase, have been isolated from a wild-type stock of Drosophila melanogaster and have been provisionally termed lxd ^c and lxd ^d, respectively, as both mutants appear to be allelic with lxd (low xanthine dehydrogenase). An analysis has been made of the effects of dietary molybdenum on lxd, lxd ^c, lxd^d, lao (low aldehyde oxidase), mal (maroon-like eye color), and pac (Pacific) wild-type flies. On the lower dietary levels of 10 ^?3 M and 10 ^?2 M molybdenum, increases in specific activity of both enzymes were observed only in lxd. Furthermore, two- to three-fold increases in specific activity of both enzymes occurred in all strains, except mal, when cultured on 5×10 ^?2 M molybdenum. The lxd and lxd ^c strains failed to survive on this high concentration of the ion. Similar concentrations of molybdenum had no effect in vitro. An extra electrophoretic band of xanthine dehydrogenase was observed on polyacrylamide gel from extracts of wild-type flies cultured on certain levels of molybdenum, but its appearance was not always correlated with the increases in specific activity.     

The lateral diffusion coefficient of lecithin molecules in single bilayer vesicles studied by 14N NMR

The ¹⁴N nuclear relaxation times T₁ and T₂ in egg yolk phosphatidylcholine have been observed in single bilayer vesicles dispersed in the media of different viscosities, ¹H₂O and ²H₂O. The lateral diffusion coefficient of lipid molecule D has been calculated according to the method reported earlier: D = 2.2 × 10^?8cm²s^?1 in ¹H₂O and 2.1 × 10^?8cm²s^?1 in ²H₂O at 20°C. They are in excellent agreement. This result gives a strong basis of usefulness of ¹⁴N NMR method in the evaluation of D without introducing any system perturbation.     

The self-association of jack bean urease and its modification by silver ions.

At low ionic strength urease has been found to dissociate at protein concentrations below 1 × 10⁸m. The inhibition of enzyme activity by Ag⁺ has been used to demonstrate this. The inhibition by Ag⁺ has been shown to be independent of dissociation but, at dilutions where dissociation occurs, silver ion modifies the process. Urease is aggregated by Ag⁺ at high Ag⁺:protein ratios. Such inactive aggregates can be solubilized and reactivated by dithiothreitol. Further evidence has been obtained indicating the similarity of the (8n) and (16n) forms of urease. The phenomena of inhibition and aggregation in the presence of the heavy metal ion have been shown to be separate processes.     

A comparison of the coordination of two linkage isomers of bis(1-methylthioimidazolyl)methane to zinc salts

A structural comparison of the coordination chemistry of the two linkage isomeric forms of bis(1-methylthioimidazolyl)methane, [H₂C(N-mt)₂] (L^S), and [H₂C(S-mt)₂] (L^N) to a number of zinc complexes has been carried out. The complexes ZnX₂L^S and ZnX₂L^N (where X = Cl, Br and I) have all been prepared in good to high yields and have been characterised by spectroscopic and analytical methods. X-ray crystallography studies were also carried out on all of the newly prepared compounds, revealing κ²-SS and κ²-NN coordination modes for L^S and L^N containing complexes, respectively.