Simultaneous determination of two human urinary metabolites of N,N-dimethylformamide using gas chromatography–thermionic sensitive detection with mass spectrometric confirmation

Two human urinary metabolites of the industrial solvent N,N-dimethylformamide (DMF), N-hydroxymethyl-N-methylformamide (HMMF) and N-acetyl-S-(N-methylcarbamoyl)cysteine (AMCC), were assayed using a new analytical method (gas chromatography and thermionic sensitive detection). Clean-up of urine samples includes a liquid–liquid extraction step followed by a solid-phase extraction step to separate HMMF and AMCC from other urine components. During clean-up, AMCC is converted into ethyl-N-methylcarbamate (EMC), and during gas chromatography, HMMF is degraded in the injector to N-methylformamide (NMF). All the validation data necessary for a quantitative procedure are given. The method was applied to urine samples from workers exposed to DMF and from the general population. The results were confirmed by mass spectrometric determination. For this purpose a further liquid–liquid extraction step was introduced in the clean-up procedure. Background levels of AMCC in the general population were identified.     

Randomization Analysis of Incomplete Data in Some Basic Designs

The first two randomization moments of W = Tt.S.S./(Tt.S.S. + Error S.S.) are derived in case of (a) a completely randomized design with one observation missing using (I) Yates method of fitting constants and (II) the available observations only and in case of (b) a randomized block design in which one treatment is tested twice by mistake in a block and another treatment is not tested at all in that block using (I) all the available observations and (II) the data after deleting the observation due to the treatment tested by mistake in place of another treatment in a block. It is concluded that in each of the two cases for a completely randomized design, the F-test is unbiased whereas in each of the two cases for a randomized block design the F-test is in general not unbiased. However, if one treatment is tested twice by mistake in randomly chosen plots of some block, the F-test is unbiased in both cases for a randomized block design. For a completely randomized design the F-test is found to be a good approximation to the corresponding randomized test if N ≧ 80 in case (I) whereas in case (II) this approximation is of the same accuracy as that of case (I) if N ≧ 90. For a randomized block design it is seen that in case (I), the F-test provides a good approximation to the corresponding randomization test if v≧r≧7 and in case (II) this approximation is of the same accuracy if r≧6 and r(v-1)≧45. The analysis of several uniformity trial data shows that for values of “N” in the neighbourhood of 50 the agreement of the first two moments of “W” under the randomization theory and normal theory are reasonably close in all the four cases considered.     

Microscale magnetic microparticle‐based immunopurification of cytokinins from Arabidopsis root apex

Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmol g^?1 fresh weight). We have developed a simple microscale magnetic immunoaffinity‐based method for selective one‐step isolation of CKs from very small amounts of plant tissue (less than 0.1 mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one‐step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30–80% depending on individual CKs. Immunoaffinity extraction using group‐specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry‐based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N⁹‐glucosides) in 1.0‐mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue.     

Diagonalization in a mixed basis: A method to compute low-frequency normal modes for large macromolecules

Low-frequency collective motions in proteins are generally very important for their biological functions. To study such motions, harmonic dynamics proved most useful since it is a straightforward method; it consists of the diagonalization of the Hessian matrix of the potential energy, yielding the vibrational spectrum and the directions of internal motions. Unfortunately, the diagonalization of this matrix requires a large computer memory, which is a limiting factor when the protein contains several thousand atoms. To circumvent this limitation we have developed three methods that enable us to diagonalize large matrices using much less computer memory than the usual harmonic dynamics. The first method is approximate; it consists of diagonalizing small blocks of the Hessian matrix, followed by the coupling of the low-frequency modes obtained for each block. It yields the low-frequency vibrational spectrum with a maximum error of 20%. The second method consists, after diagonalizing small blocks, of coupling the high- and low-frequency modes using an iterative procedure. It yields the exact low-frequency normal modes, but requires a long computational time with convergence problems. The third method, DIMB (Diagonalization in a Mixed Basis), which has the best performance, consists of coupling the approximate low-frequency modes with the mass-weighted cartesian coordinates, also using an iterative procedure. It reduces significantly the required computer memory and converges rapidly. The eigenvalues and eigenvectors obtained by this method are without significant error in the chosen frequency range. Moreover, it is a general method applicable to any problem of diagonalization of a large matrix. We report the application of these methods to a deca-alanine helix, trypsin inhibitor, a neurotoxin, and lysozyme. © 1993 John Wiley & Sons, Inc.     

Magnetic correlates in electromagnetic consciousness

ABSTRACT

We examine the hypothesis that consciousness is a manifestation of the electromagnetic field, finding supportive factors not previously considered. It is not likely that traditional electrophysiological signaling modes can be readily transmitted throughout the brain to properly enable this field because of electric field screening arising from the ubiquitous distribution of high dielectric lipid membranes, a problem that vanishes for low-frequency magnetic fields. Many reports over the last few decades have provided evidence that living tissue is robustly sensitive to ultrasmall (1–100 nT) ELF magnetic fields overlapping the γ-frequency range often associated with awareness. An example taken from animal behavior (coherent bird flocking) lends support to the possibility of a disembodied electromagnetic consciousness. In contrast to quantum consciousness hypotheses, the present approach is open to experimental trial.     

Protein F: an adhesin of Streptococcus pyogenes binds fibronectin via two distinct domains

The binding of Streptococcus pyogenes to fibronectin (FN) enables the adherence of this pathogen to target epithelial cells, which is the first necessary step for initiation of infection. Binding is mediated by a bacterial surface protein termed protein F. Here we provide the complete structure of protein F and identify two domains responsible for binding to fibronectin. The first domain is located towards the C-terminal end of the molecule and is composed of five repeats of 37 amino acids that are completely repeated four times and a fifth time partially. The second domain is adjacent to the first domain and is located on the /V-terminal side of it. It is composed of a single stretch of 43 amino acids. Protein F expressed in Escherichia coli completely blocked the binding of fibronectin to S. pyogenes. However, mutant proteins that contained only one or the other of the two domains were only capable of partial blockage of binding. Complete blockage of binding of fibronectin could be achieved when a protein extract containing the N-terminal domain was mixed in a binding reaction with a protein extract containing the C-terminal domain. Similarly, a purified recombinant protein containing the two domains only, blocked the binding completely. In contrast, a purified recombinant protein containing just the C-terminal domain, blocked the binding partially. A clone exclusively expressing the C-terminal domain, completely blocked the binding of the 30 kDa N-terminal fragment of fibronectin to S. pyogenes, whereas a clone expressing the N-terminal domain failed to block the binding of this FN fragment. Thus, the two FN-binding domains of protein F are necessary for maximal bacterial binding and act in concert to enhance the binding to fibronectin. The possibility that the two domains bind to two different regions on the fibronectin molecule is discussed.     

Studies on cytochrome c. Part III. Synthesis of the protected heneicosapeptide (sequence 24–44) of Baker's yeast iso-1-cytochrome c

Syntheses are described for two N-benzyloxycarbonylpeptide tert-butoxycarbonylhydrazides which correspond to positions 24–34 and 35–44, respectively, of the primary structure of baker's yeast iso-1-cytochrome c. The two peptide derivatives were coupled via the azide procedure to form the N-benzyloxycarbonylheneicosapeptide tert-butoxycarbonylhydrazide (sequence 24–44).     

A first view on the unsuspected intragenus diversity of N‐glycans in Chlorella microalgae

Chlorella microalgae are increasingly used for various purposes such as fatty acid production, wastewater processing, or as health‐promoting food supplements. A mass spectrometry‐based survey of N‐glycan structures of strain collection specimens and 80 commercial Chlorella products revealed a hitherto unseen intragenus diversity of N‐glycan structures. Differing numbers of methyl groups, pentoses, deoxyhexoses, and N‐acetylglucosamine culminated in c. 100 different glycan masses. Thirteen clearly discernible glycan‐type groups were identified. Unexpected features included the occurrence of arabinose, of different and rare types of monosaccharide methylation (e.g. 4‐O‐methyl‐N‐acetylglucosamine), and substitution of the second N‐acetylglucosamine. Analysis of barcode ITS1–5.8S–ITS2 rDNA sequences established a phylogenetic tree that essentially went hand in hand with the grouping obtained by glycan patterns. This brief prelude to microalgal N‐glycans revealed a fabulous wealth of undescribed structural features that finely differentiated Chlorella‐like microalgae, which are notoriously poor in morphological attributes. In light of the almost identical N‐glycan structural features that exist within vertebrates or land plants, the herein discovered diversity is astonishing and argues for a selection pressure only explicable by a fundamental functional role of these glycans.     

Synthesis of Oligonucleotide Conjugates with the Aid of N-Chloroacetamidohexyl Phosphoramidite Reagent

Abstract

A novel phosphoramidite building block derived from N-chloroacetyl-6-aminohexanol is attached at the 5′-terminus on the last step of oligonucleotide synthesis. Postsynthetic treatment of support-bound modified oligonucleotides with a variety of amines and mercaptanes affords oligonucleotide conjugates in a high yield.     

Preparative resolutions of an α-methyl carboxylic acid through selective transformations of readily epimerizable intermediates

Two preparations of the enantiomers of 2 are described. The first makes use of the chromatographic separation of the diastereomeric amides 6a and 6b. Standard hydrolysis of these amides caused racemization, so a milder sequence was developed which utilized carbonyldiimidazole and 1 equivalent of 1 N LiOH. The second preparation involved classical resolution of 9 with (?)-cinchonidine. Subsequent transformations of this substrate involved ester formation, Friedel–Crafts acylation, and ester hydrolysis, all without racemization. The most notable of these reactions was the use of EtAlCl₂ in the Friedel–Crafts step, which provided a mild acylation of 10. This second preparation affords a high yield, mild process for the potential preparation of kilogram quantities of (?)-(R)-2b.     

Examination of the interaction between FtsZ and MinCN in E. coli suggests how MinC disrupts Z rings

In Escherichia coli the Min system prevents Z ring assembly at cell poles by topologically regulating the division inhibitor MinC. The MinC protein has two domains of equal size and both domains can target FtsZ and block cell division in the proper context. Recently, we have shown that, along with MinD, the C‐terminal domain of MinC (MinC^C) competes with FtsA, and to a lesser extent with ZipA, for interaction with the C‐terminal tail of FtsZ to block division. Here we explored the interaction between the N‐terminal domain of MinC (MinC^N) and FtsZ. A search for mutations in ftsZ that confer resistance to MinC^N identified an α‐helix at the interface of FtsZ subunits as being critical for the activity of MinC^N. Focusing on one such mutant FtsZ–N280D, we showed that it greatly reduced the FtsZ–MinC interaction and was resistant to MinC^N both in vivo and in vitro. With these results, an updated model for the action of MinC on FtsZ is proposed: MinC interacts with FtsZ to disrupt two interactions, FtsZ–FtsA/ZipA and FtsZ–FtsZ, both of which are essential for Z ring formation.     

Elucidation of the mechanism of N-demethylation catalyzed by cytochrome P450 monooxygenase is facilitated by exploiting nitrogen-15 heavy isotope effects

¹⁵N heavy isotope effects are especially useful when detail is sought pertaining to the reaction mechanism for the cleavage of a C–N bond. Their potential in assisting to describe the mechanism of N-demethylation of tertiary amines by the action of cytochrome P450 monooxygenase has been investigated. As a working model for the first step, oxidation of the N-methyl group to N-methoxyl, tropine and a cytochrome P450 monooxygenase reaction centre composed of a truncated heme with sulfhydryl as the axial ligand were used. It is apparent that this first step of the reaction proceeds via a hydrogen atom transfer mechanism. Transition states for this step are described for both the high spin (⁴TS_H) and low spin (²TS_H) pathways in both gas and solvation states. Hence, overall normal secondary ¹⁵N KIE could be calculated for the reaction path modeled in the low spin state, and inverse for the reaction modeled in the high spin state. This partial reaction has been identified as the probable rate limiting step. The model for the second step, fission of the C–N bond, consisted of N-methoxylnortropine and two molecules of water. A transition state described for this step, TS_CN, gives a strongly inverse overall theoretical ¹⁵N KIE.     

SYNTHESIS OF N 6-ALKYLATED ADENOSINE DERIVATIVES

Abstract

N ⁶-ablated adenosine can be synthesized by reduction of the corresponding carboxylic acid amides using Lm. Starting from 2′,3′-O-isopropylidene adenosine, N ⁶-ethyladenosine, N ⁶-propyladenosine, N ⁶-isobutyladenosine, N ⁶-benzyladenosine and N ⁶-furfuryladenosine were obtained in a three step procedure (acylation, reduction, deprotection).     

Molecular Dynamics Reveal Binding Mode of Glutathionylspermidine by Trypanothione Synthetase

The trypanothione synthetase (TryS) catalyses the two-step biosynthesis of trypanothione from spermidine and glutathione and is an attractive new drug target for the development of trypanocidal and antileishmanial drugs, especially since the structural information of TryS from Leishmania major has become available. Unfortunately, the TryS structure was solved without any of the substrates and lacks loop regions that are mechanistically important. This contribution describes docking and molecular dynamics simulations that led to further insights into trypanothione biosynthesis and, in particular, explains the binding modes of substrates for the second catalytic step. The structural model essentially confirm previously proposed binding sites for glutathione, ATP and two Mg²⁺ ions, which appear identical for both catalytic steps. The analysis of an unsolved loop region near the proposed spermidine binding site revealed a new pocket that was demonstrated to bind glutathionylspermidine in an inverted orientation. For the second step of trypanothione synthesis glutathionylspermidine is bound in a way that preferentially allows N¹-glutathionylation of N⁸-glutathionylspermidine, classifying N⁸-glutathionylspermidine as the favoured substrate. By inhibitor docking, the binding site for N⁸-glutathionylspermidine was characterised as druggable.     

Testing for Treatment Effects on Subsets of Endpoints

Multiple endpoints are tested to assess an overall treatment effect and also to identify which endpoints or subsets of endpoints contributed to treatment differences. The conventional p‐value adjustment methods, such as single‐step, step‐up, or step‐down procedures, sequentially identify each significant individual endpoint. Closed test procedures can also detect individual endpoints that have effects via a step‐by‐step closed strategy. This paper proposes a global‐based statistic for testing an a priori number, say, r of the k endpoints, as opposed to the conventional approach of testing one (r = 1) endpoint. The proposed test statistic is an extension of the single‐step p‐value‐based statistic based on the distribution of the smallest p‐value. The test maintains strong control of the FamilyWise Error (FWE) rate under the null hypothesis of no difference in any (sub)set of r endpoints among all possible combinations of the k endpoints. After rejecting the null hypothesis, the individual endpoints in the sets that are rejected can be tested further, using a univariate test statistic in a second step, if desired. However, the second step test only weakly controls the FWE. The proposed method is illustrated by application to a psychosis data set.     

Effect of side-chain hydrophobic bonding on the stability of homopolyamino acid alpha-helices: conformational studies of poly-l-leucine in water

The conformational properties of block copolymers of poly-L -leucine in water have been examined. The degree of polymerization of the poly-L -leucine block was 11 and 21, respectively, for samples prepared by the Merrifield procedure, and 56 for a sample prepared by the polymerization of leucine N-carboxyanhydride. The optical rotatory dispersion parameter b₀ was used to obtain the helix content θ_h at various temperatures. Application of the Lifson-Roig theory gave the following parameters for the transition of a residue from a coil to a helical state: v = 0.05–0.011, ΔH = +100 cal/mole, ΔS = +0.70–1.00 e. u. These parameters, as well as those for other polyamino acids, are accounted for by hydrophobic bonds involving the nonpolar side chains in the helical and randomly coiled forms. From the data for poly-L -alanine and theoretical values of the thermodynamic parameters for hydrophobic bond formation, the parameters for formation of a polyglycine helix are computed. By separating the contributions of the backbone, it is possible to obtain a set of thermodynamic parameters for the side-chain contributions of a number of polyamino acids. Increased size of the nonpolar side chain (with a larger contribution from hydrophobic bonding) makes a larger contribution to the stability of the α-helix which is reflected, among other ways, in a higher helix content at given temperature.     

Phonon dispersion curves and normal coordinate analysis of -poly-L-alanine

The vibrational frequencies of α-helical poly-L -alanine and its N-deuterated analog have been assigned by normal coordinate analyses. The phonon dispersion curves and frequency distribution of α-poly-L -alanine have been calculated by using a model which includes hydrogen bonding. The frequency distribution was used to interpret the inelastic neutron scattering data and to calculate the heat capacity. The low-frequency chain modes involving accordian-like motions of the whole helix have been calculated and their dispersion investigated by means of a simplified model.     

A simple autoradiographic technique for studying diffusion of water into seeds

Visual observations on rates and modes of water penetration into black bean seeds and into wheat and sorghum kernels during conditioning were accomplished by an autoradiographic procedure that eliminates a freezing microtome and liquid and stripping film emulsions. Seeds soaked in tritiated H₂O were hand sectioned before freeze-quenching in liquid N₂ and subsequent block autoradiography on nuclear medicine film.     

Characterization of microsatellite loci in sea urchins (Strongylocentrotus spp.)

We present six dinucleotide repeats that were developed and characterized in Strongylocentrotus droebachiensis and also tested in S. purpuratus. Four of these loci are polymorphic in S. droebachiensis (13–20 alleles, N= 100) and five are polymorphic in S. purpuratus (5–12 alleles, N= 10). We are currently using these markers to investigate the population substructure of shallow water populations of S. droebachiensis in the north Atlantic.     

Synthesis and Antiviral Evaluation of 2-Mercapto-5,6-dichlorobenzimidazole-β-D-ribofuranonucleoside Derivatives

Abstract

2-Mercapto-5,6-dichlorobenzimidazole β-D-ribofuranonucleoside derivatives 8–10 have been synthesized and their antiviral properties examined. According to the glycosylation procedure used, the β-D-N-1 isomer (and the N, N-bis-riboside) or the β-D-S²-isomer have been obtained. All the prepared compounds were tested for their activity against a variety of RNA and DNA viruses, but they did not show significant antiviral activity.