IS1237, a Repetitive Chromosomal Element from Clavibacter xyli subsp. cynodontis, Is Related to Insertion Sequences from Gram-Negative and Gram-Positive Bacteria

We describe here a repetitive chromosomal element, which appears to be an insertion sequence, isolated from Clavibacter xyli subsp. cynodontis, a gram-positive plant-associated bacterium. The element, IS1237, is 905 bp in size, is bounded by 19-bp perfect inverted repeats and 3-bp direct repeats, and appears at least 16 times in the genome. It contains three open reading frames which show similarity to open reading frames from various other insertion sequences. We have found that there are two groups of related mobile elements: one in which two open reading frames are read separately and the other in which these two open reading frames are fuse together to give one predicted protein product. Using one of these open reading frames to search amino acid sequence databases, we found two instances in which similar reading frames flank genes carried on plasmids. We believe therefore that these plasmid-borne genes may be parts of previously unidentified mobile elements. For IS1237, a frameshift in two of the open reading frames and a stop codon in the third may indicate that this particular copy of the element is no longer active in transposition. The similarity of IS1237 to other elements from both gram-negative and gram-positive bacteria provides further evidence that mobile elements have been transferred between these two bacterial groups.     

The kinetics of sucrose concentration in the phloem of individual vascular bundles of the Ricinus communis seedling measured by nuclear magnetic resonance microimaging

The sucrose concentration was measured at 70-min intervals in the phloem of individual bundles of the hypocotyl of Ricinus seedlings by ¹H nuclear magnetic resonance (NMR) spectroscopic imaging. The sucrose concentration stayed fairly constant in all bundles for more than 7 h if the cotyledons were embedded in the endosperm or excised and incubated in 100 mM sucrose. If, however, the sucrose solution was replaced by sucrose-free buffer solution, the sucrose levels in the phloem decreased with a kinetic depending on the seedling: in some cases there was a smooth decline, in some a decline followed by a slight recovery and in some cases a clear-cut oscillation. The sucrose concentration was often not identical in the phloem of the individual bundles. The oscillations were larger in the phloem at the apex of the hypocotyl than in the phloem at the base of the hypocotyl. Cutting the petiole of one cotyledon led to a decrease in sucrose not only in the four bundles directly connected to the severed petiole but in all eight bundles of the hypocotyl. Cutting the petiole and dividing the vascular ring at the cotyledonary node and at the root crown did not prevent the decline of sucrose in all eight bundles. Therefore, a functional equilibration of translocated solutes between the eight bundles may occur within the 1-h measuring interval by radial diffusion through the parenchyma of the hypocotyl. Received 4 July 1997 / Accepted: 4 October 1997     

Simulated annealing with restrained molecular dynamics using a flexible restraint potential: theory and evaluation with simulated NMR constraints.

A new functional representation of NMR-derived distance constraints, the flexible restraint potential, has been implemented in the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168) for molecular structure generation. In addition, flat-bottomed restraint potentials for representing dihedral angle and vicinal scalar coupling constraints have been introduced into CONGEN. An effective simulated annealing (SA) protocol that combines both weight annealing and temperature annealing is described. Calculations have been performed using ideal simulated NMR constraints, in order to evaluate the use of restrained molecular dynamics (MD) with these target functions as implemented in CONGEN. In this benchmark study, internuclear distance, dihedral angle, and vicinal coupling constant constraints were calculated from the energy-minimized X-ray crystal structure of the 46-amino acid polypeptide crambin (ICRN). Three-dimensional structures of crambin that satisfy these simulated NMR constraints were generated using restrained MD and SA. Polypeptide structures with extended backbone and side-chain conformations were used as starting conformations. Dynamical annealing calculations using extended starting conformations and assignments of initial velocities taken randomly from a Maxwellian distribution were found to adequately sample the conformational space consistent with the constraints. These calculations also show that loosened internuclear constraints can allow molecules to overcome local minima in the search for a global minimum with respect to both the NMR-derived constraints and conformational energy. This protocol and the modified version of the CONGEN program described here are shown to be reliable and robust, and are applicable generally for protein structure determination by dynamical simulated annealing using NMR data.     

Determination of the three-dimensional solution structure of ragweed allergen Amb t V by nuclear magnetic resonance spectroscopy.

Analysis of two-dimensional NMR experiments has afforded essentially complete assignment of all proton resonances in the allergenic protein Amb t V. Conformational constraints were obtained from the NMR data in three forms: interproton distances derived from NOE cross-peak intensities of NOESY spectra, torsion angle constraints derived from J-coupling constants of COSY and PE-COSY spectra, and hydrogen bond constraints derived from hydrogen-exchange experiments. Conformations of Amb t V with low constraint violations were generated using dynamic simulated annealing in the program XPLOR. The refined structures are comprised of a C-terminal alpha-helix, a short stretch of triple-stranded antiparallel beta-sheet, and several loops. In addition, the cystine partners of the four disulfide linkages (for which there are no biochemical data) have been assigned. The refined structures of Amb t V will allow us to suggest surface substructures for the Amb V allergens that are likely to participate in B cell epitopes and will assist us in defining the Ia/T cell epitopes that interact with the MHC class II (or Ia) molecule and the T cell receptor leading to the induction of the immune response to Amb t V.     

Effects of various inducers on diethylstilbestrol metabolism, drug-metabolizing enzyme activities and the aromatic hydrocarbon (Ah) receptor in male Syrian golden hamster liver

In order to elucidate the role of metabolic activation of the synthetic estrogen, diethylstilbestrol (DES), in the mechanism of liver tumor formation in male Syrian golden hamsters observed after combined treatment with DES and 7,8-benzoflavone (7,8-BF), the metabolism of DES and the concentrations and activities of various drug-metabolizing enzymes were studied in hamster liver microsomes after various pretreatments. The levels of the hepatic aromatic hydrocarbon (Ah) receptor were also determined. Pretreatment with 7,8-BF increased both P450 and cytochrome b5 levels, whereas phenobarbital (PB) and 3-methylcholanthrene (MC) induced P450 but not cytochrome b5. 7,8-BF pretreatment increased 7-ethoxyresorufin-O-deethylase (EROD) 3-fold and 7-pentoxyresorufin-O-dealkylase (PROD) 2.5-fold, whereas aromatic hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin-O-deethylase (ECOD) activities were only slightly induced by 7,8-BF. MC pretreatment increased EROD 8-fold and PROD activity 7-fold, whereas PB pretreatment enhanced AHH 4.5-fold and PROD activity 4-fold. In contrast to PB, pretreatment with 7,8-BF and MC reduced the oxidative metabolism of DES in hepatic microsomes, but the pattern of metabolites was identical with that in untreated controls. Treatment of hamsters with the inducers changed the hepatic Ah receptor level. PB and MC-pretreatment resulted in an increase of the receptor level 1.5-fold and 1.3-fold, respectively, whereas 7,8-BF-pretreatment leads to a 1.5-fold decrease. The dissociation constant Kd is 170 nM for the reaction of 7,8-BF with the hamster Ah receptor compared to 70 nM for 5,6-BF and 38 nM for 2,3,7,8-tetrachlorodibenzofuran (TCDF). The Kd-value is 3.6 nM for TCDF with the rat receptor protein. It is concluded from these data that metabolic activation of DES is not involved in the mechanism of hepatocarcinogenesis in this animal tumor model.     

Determining local conformational variations in DNA. Nuclear magnetic resonance structures of the DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC) generated using back-calculation of the nuclear Overhauser effect spectra, a distance geometry algorithm and constrained molecular dynamics

Two-dimensional nuclear magnetic resonance (n.m.r.) spectroscopy and a variety of computational techniques have been used to generate three-dimensional structures of the two DNA duplexes d(CGCCTAATCG) and d(CGTCACGCGC). The central six base-pairs in these two decamers contain all ten dinucleotide pairs in DNA and thus, represent a model system for investigating how the local structure of DNA varies with base sequence. Resonance assignments were made for the non-exchangeable base protons and most of the C-1'-C-4' sugar protons in both decamers. Three-dimensional structures were generated using a distance geometry algorithm and these initial structures were refined by optimizing the fit of back-calculated spectra against the experimental two-dimensional nuclear Overhauser effect (NOE) spectra. This back-calculation procedure consists of calculating NOE cross relaxation rates for a given structure by solution of the Bloch equations, and directly accounts for spin diffusion effects. Use of this refinement procedure eliminates some assumptions that have been invoked when generating structures of DNA oligomers from n.m.r. data. Constrained energy minimization and constrained quenched molecular dynamics calculation were also performed on both decamers to help generate energetically favorable structures consistent with the experimental data. Analysis of the local conformational parameters of helical twist, helical rise, propeller twist, displacement and the alpha, beta, gamma, epison and zeta backbone torsion angles in these structures shows that these parameters span a large range of values relative to the X-ray data of nucleic acids. However, the glycosidic and pseudorotation angles are quite well defined in these structures. The implications that these results have for determination of local structural variations of DNA in solution, such as those predicted by Callidine's rules, are discussed. Our results differ significantly from some previous studies on determining local conformations of nucleic acids and comparisons with these studies are made.     

Interaction of a 5-trans-vinylcarboxylic acid analogue of pyridoxal 5'-phosphate with apoaspartate aminotransferase. Covalent labeling of the enzyme.

The 5-trans-vinylcarboxylic acid analogue of pyridoxal 5'-phosphate has been prepared. Its pKa values were determined as 3.08, 4.10, and 7.33. The third pKa, that of the pyridinium nitrogen, is considerably lower than that of 8.2 observed for the corresponding saturated compound, 5'-carboxymethyl-5'-deoxypyridoxal. Absorption spectra of individual ionic forms have been resolved into component bands using lognormal distribution curves. The vinylcarboxylic acid analogue inactivates apoaspartate aminotransferase slowly at pH 8.3. An initial product absorbs at 26 kK (385 nm) and is converted slowly to a species with a narrow absorption band at 24.0 kK (417 nm). Meanwhile, the circular dichroism in the same region changes from positive to negative. At pH 5.2 the product abosrbs at 25.2 kK (397 nm). The 24.0-kK (417 nm) form is not reducible with sodium borohydride and the tightly bound chromophore is not released from the protein during denaturation by acid, base, or heat. L-Glutamate and erythro-beta-hydroxyaspartate both facilitate the formation of the 24.0-kK form. The reaction of the analogue with apoenzyme in the presence of erythro-beta-hydroxyaspartate is also accompanied by transient peaks, presumably representing quinonoid forms, at 19.0 kK (526 nm) and 20.3 kK (492 nm). The analogue reacts at basic pH with arginine, alpha-amino-gamma-guanidinobutyric acid, ornithine, cysteine, alpha, gamma-diaminobutyric acid, eh narrow absorption bands centered in the 24.0-24.4-kK (417-410 nm) region and resembling the product formed with the apoenzyme. Nuclear magnetic resonance and absorption spectroscopy indicate that the reaction with alpha- gamma-diaminobutyric acid proceeds via a hexahydropyrimidine derivative to a substituted tetrahydropyrimidine (a cyclic Schiff base) which is the final product. A similar reaction sequence with the apoenzyme is postulated and a structure with an unknown X group from the enzyme replacing the gamma-amino group of alpha, gamma-diaminobutyric acid is proposed for the 24.0-kK (417 nm) chromophore obtained with the apoenzyme. The proposed reactions are closely related to enzymatic and nonenzymatic reactions of pyridoxal 5'-sulfate (Yang, I. -Y., Khomutov, R. M., and Metzler, D. E. (1974), Biochemistry 13, 3877).     

Estimation of the Life Span of Red Blood Cells in the Growing Animal in Different Nutritional States

In this study the model of Shemin and Rittenberg for estimating the life span of red blood cells was extended so that non-steady-state conditions, exemplified by growth or changing physiological states, might be considered. The parameters were estimated by use of the modified Gauss-Newton method. The biological data that were used came from growing sheep in different physiological states with regard to copper. The model was extended to include changes in total blood hemin and changes in blood hemin synthesis that may occur with time. In the present study a linear function was taken as a first approximation. The model appeared to be a sufficiently good approximation in the study reported herein. It was found, however, that the parameters associated with changes in hemin should be estimated from ancillary measurements such as blood volume, Hb, body weight, etc., in order to obtain a good fit or definition of the model.     

In situ inactivation of animal viruses and a coliphage in nonaerated liquid and semiliquid animal wastes.

The persistence of five animal viruses, representing picorna-, rota-, parvo-, adeno-, and herpesviruses, and the coliphage f2 was determined in the field by exposing the viruses to different animal wastes and by adopting an established filter sandwich technique. This technique allows us to copy the natural state of viruses in the environment, where adsorption onto or incorporation into suspended solids may prolong virus survival. Using filter sandwiches either equipped with porous (15 nm in diameter) or poreless polycarbonate (PC) membranes, it was possible to differentiate between overall virus inactivation and the effect of virucidal agents that act through poreless PC membranes. Depending on ambient temperature, pH, and type of animal waste, values for time, in days, required for a 90% reduction of virus titer varied widely, ranging from less than 1 week for herpesvirus to more than 6 months for rotavirus. Virus inactivation progressed substantially faster in liquid cattle manure, a mixture of urine and water (pH > 8.0), than in semiliquid wastes that consisted of mixtures of feces, urine, water, and bedding materials (pH < 8.0). Hitherto unidentified virucidal agents that permeate poreless PC membranes contributed substantially to the overall inactivation. On the other hand, substances that protect rotavirus and possibly other viruses from inactivation may be present in animal wastes. Together, the study showed that viruses contained in manure may persist for prolonged periods of time if stored under nonaerated conditions. At times of land application, this may lead to environmental contamination with pathogens.