Biochemical genetics of methylglyoxal dehydrogenases in the laboratory rat (Rattus norvegicus)

A genetic locus controlling the electrophoretic mobility of a methylglyoxal dehydrogenase (EC 1.2.1.23) in the rat is described. The locus, designatedMgd1, is expressed in liver and kidney. Inbred rat strains have fixed either alleleMgd1 ^a or alleleMgd1 ^b . Codominant expression is observed in heterozygotes, providing evidence for a tetrameric enzyme structure. Backcross progenies showed the expected 1:1 segregation ratio, and there is evidence thatMgd1 is linked toPep3 andFh1 on chromosome 13. There is also evidence for two additional methylglyoxal dehydrogenases:Mgd2, present in liver and kidney, andMgd3, present only in heart.Supported by the Deutsche Forschungsgemeinschaft (Grant Be 352/18-1).     

HIV-1 integrase blocks infection of bacteria by single-stranded DNA and RNA bacteriophages

Expression of human immunodeficiency virus-1 integrase in Escherichia coli, at levels that had no effect on bacterial cell growth, blocked plaque formation by bacteriophages having single-stranded genomic DNA (M13) or RNA (R17, Q, PRR1). Plaque formation by phages having double-stranded genomic DNA (T4, PR4) was unaffected. Integrase also inhibited infection by the phagemid M13KO7, but it had no effect on production of phage once infection by M13KO7 was established. This result indicated that integrase affects an early stage in infection. Integrase also inhibited phage production following transfection by either single-stranded or double-stranded (replicative form) M13 DNA, it blocked M13 DNA replication, as assayed by incorporation of radioactive nucleotides into DNA, and it failed to affect bacterial pilus function. These data suggest that integrase interacts in vivo with phage nucleic acid, a conclusion supported by studies in which integrase was shown to have a DNA-binding activity in its C-terminal portion. This portion of integrase was both necessary and sufficient for interference of plaque formation by M13 in the present study. Expression of the N-terminal portion of integrase at the same level as intact integrase had little effect on phage growth, indicating that expression of foreign protein in general was not responsible for the inhibitory effect. The simple bacteriophage assay described is potentially useful for identifying integrase mutants that lack single-stranded DNA binding activity.     

Phenyllactic acid but not tropic acid is an intermediate in the biosynthesis of tropane alkaloids in Datura and Brugmansia transformed root cultures

(S)-(-)-Tropic acid is the acidic moiety of the tropane ester alkaloids, hyoscyamine and scopolamine (hyoscine). When tropic acid is fed to transformed root cultures of Datura stramonium L. or a Brugmansia (Datura) Candida x B. aurea hybrid, the formation of these alkaloids is inhibited. Phenyllactic acid, from which the tropoyl moiety is derived, is considerably less inhibitory. Label from (RS)-phenyl[1,3-¹³C₂]lactic acid is incorporated at high levels into apoatropine, littorine, aposcopolamine, hyoscyamine, 7-hydroxyapoatropine, scopolamine and 7-hydroxyhyoscyamine when fed to these cultures. The presence of an excess concentration of unlabelled tropic acid has little influence on the specific incorporation into these products. It is concluded that free tropic acid is not an intermediate in hyoscyamine biosynthesis but rather that the rearrangement of phenyllactic acid occurs subsequent to its esterification.Abbreviations FM fresh mass - NMR nuclear magnetic resonance spectroscopy We are grateful to Drs. N.J. Walton, A.J. Parr, M.J.C. Rhodes (Institue of Food Research, Norwich) and B. Dräger (Münster, Germany) for helpful and critical discussions. We also wish to thank Dr. P. Bachmann (Braunschweig, Germany) for suggesting the use of the DB-17 column to separate littorine from hyoscyamine and for the modified Excel programme used to calculate the specific incorporations, Yannick Ford (AFRC Co-operative Award Studentship, University of Oxford) and Abigael Peerless for their able assistance, Dr. I. Colquhoun for assistance with some of the NMR spectroscopy and Drs. K. Shimomura (Tsukuba, Japan) and T. Hashimoto (Kyoto, Japan) for pure samples of 7-hydroxyhyoscyamine. J.G.W, gratefully acknowledges support from the Nuffield Foundation under the Small Grants Scheme to promote collaborative experimentation and M.A. is grateful to the Ministry of Education, Iran for a research scholarship.     

Modulation of fibroblast response to maitotoxin along the cell division cycle

Maitotoxin (MTX) induces an increase of [Ca²⁺]_i and of phosphoinositide breakdown in various cell types. The [Ca²⁺]_i increase followed with fluorescent probes on cell suspensions has been described as slow and lasting, in contrast to the signal induced by calcium ionophores such as ionomycin. MTX effects have been studied on two fibroblastic cell lines, BHK21 C13 and FR 3T3, synchronized by serum deprivation treatment performed in an isoleucine-free medium for BHK21 C13 cells. In BHK21 C13 cells, flow cytometry analysis showed that two stages, G1/S and G2/M, were particularly susceptible to MTX treatment. Scanning laser cytometry demonstrated that calcium response of FR 3T3 fibroblasts followed with Indo-1 varied during the cell division cycle. The [Ca²⁺]_i increase was almost always vertical, but its delay after MTX addition lasted from zero (S and G2/M transition) to 10–20 min (G1) or more (G2). No [Ca²⁺]_i change could be detected during mitosis. The [Ca²⁺]_i response at the S phase was biphasic. These observations suggest that (1) the lasting response described in the literature represents a global cell population effect, and (2) cells are more sensitive to MTX at specific stages of the cell division cycle, which could correspond to periods when calcium signals have been detected in different cell types.Abbreviations MTX maitotoxin - [Ca²⁺]_i intracellular calcium concentration - IP₃ inositol triphosphate     

Effect of disulfide bridge formation on the NMR spectrum of a protein: Studies on oxidized and reduced Escherichia coli thioredoxin

Summary As a prelude to complete structure calculations of both the oxidized and reduced forms of Escherchia coli thioredoxin (M_r 11 700), we have analyzed the NMR data obtained for the two proteins under identical conditions. The complete aliphatic ¹³C assignments for both oxidized and reduced thioredoxin are reported. Correlations previously noted between ¹³C chemical shifts and secondary structure are confirmed in this work, and significant differences are observed in the C and C shifts between cis- and trans-proline, consistent with previous work that identifies this as a simple and unambiguous method of identifying cis-proline residues in proteins. Reduction of the disulfide bond in the active-site Cys³²-Gly-Pro-Cys³⁵ sequence causes changes in the ¹H, ¹⁵N and ¹³C chemical shifts of residues close to the active site, some of them quite far distant in the amino acid sequence. Coupling constants, both backbone and side chain, show some differences between the two proteins, and the NOE connectivities and chemical shifts are consistent with small changes in the positions of several side chains, including the two tryptophan rings (Trp²⁸ and Trp³¹). These results show that, consistent with the biochemical behavior of thioredoxin, there are minimal differences in backbone configuration between the oxidized and reduced forms of the protein.     

Solution structure by 1H and dynamics by natural abundance 13C NMR of a receptor recognising peptide derived from a C-terminal fragment of neuropeptide Y

Summary A peptide consisting of 20 amino acid residues, derived from a C-terminal fragment of neuropeptide Y (NPY) and showing high affinity to NPY receptors, was synthesised. Its sequence is PAADLARYRHYIN-LITRQRY-NH₂, and the solution structure was calculated from NMR-derived distance and torsion angle restraints, obtained at 15°C in a solvent mixture of water and 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol, by using DIANA and restrained energy minimisation. The structure was found to consist of a well-defined -helix in the centre, with a few residues at the termini having less well defined conformations. The spinlattice and spin-spin relaxation rates of -carbons have been determined on ¹³C at natural abundance. From 1D experiments the global rotational correlation time was determined and from 2D experiments the dynamics of each individual residue was obtained. The results demonstrate that the C-H vectors in the -helix essentially follow the global motion. Towards the termini, contributions from local dynamics increase. This tendency is correlated to the increasing uncertainty of the structure towards the peptide ends. An effective molecular volume was calculated from the temperature dependence of the global rotational correlation time. This is well compatible with a monomeric peptide, solvated by water and 1,1,1,3,3,3-hexafluoro-2-propanol. The presence of peptide dimers was ruled out as being inconsistent with the relaxation data.Supplementary material available from the authors: Two data tables and 10 PDB coordinate files of the calculated NMR structures of P7. One data table contains all detected and integrated NOE intensities; the other connects each proton and pseudoatom to an atom number used in the NOE table. The table contents served as input data files for CALIBA.Currently on leave from the Institute of Chemical Physics and Biophysics, Tallinn, Estonia.     

Selectively 13C-enriched DNA: 13C and 1H assignments of a triple helix by two-dimensional relayed HMQC experiments

Summary We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homopyrimidine strands, the carbon C1 of the pyrimidines were selectively ¹³C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the ¹H1–¹³C1 connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

Identification of N-terminal helix capping boxes by means of 13C chemical shifts

Summary We have examined the ¹³C and ¹³C chemical shifts of a number of proteins and found that their values at the N-terminal end of a helix provide a good predictor for the presence of a capping box. A capping box consists of a hydrogen-bonded cycle of four amino acids in which the side chain of the N-cap residue forms a hydrogen bond with the backbone amide of the N3 residue, whose side chain in turn may accept a hydrogen bond from the amide of the N-cap residue. The N-cap residue exhibits characteristic values for its backbone torsion angles, with and clustering around 94±15° and 167±5°, respectively. This is manifested by a 1–2 ppm upfield shift of the ¹³C resonance and a 1–4 ppm downfield shift of the ¹³C resonance, relative to their random coil values, and is mainly associated with the unusually large value of . The residues following the N-cap residue exhibit downfield shifts of 1–3 ppm for the ¹³C resonances and small upfield shifts for the ¹³C ones, typical of an -helix.     

Carbon distribution in grass (Festuca pratensis L.) during regrowth after cutting—utilization of stored and newly assimilated carbon

Distribution of net assimilated C in meadow fescue (Fectuca pratensi L.) was followed before and after cutting of the shoots. Plants were continuously labelled in a growth chamber with ¹⁴C-labelled CO₂ in the atmosphere from seedling to cutting and with ¹³C-labelled CO₂ in the atmosphere during regrowth after the cutting. Labelled C, both ¹⁴C and ¹³C, was determined at the end of the two growth periods in shoots, crowns, roots, soil and rhizosphere respiration. Distribution of net assimilated C followed almost the same pattern at the end of the two growth periods, i.e. at the end of the ¹⁴C- and the ¹³C-labelling periods. Shoots retained 71–73% of net assimilated C while 9% was detected in the roots and 11–14% was released from the roots, determined as labelled C in soil and as rhizosphere respiration. At the end of the 2nd growth period, after cutting and regrowth, 21% of the residual plant ¹⁴C at cutting (¹⁴C in crowns and roots) was found in the new shoot biomass. A minor part of the residual plant ¹⁴C, 12%, was lost from the plants. The decreases in ¹⁴C in crowns and roots during the regrowth period suggest that ¹⁴C in both crowns and roots was translocated to new shoot tissue. Approximately half of the total root C at the end of the regrowth period after cutting was ¹³C-labelled C and thus represents new root growth. Root death after cutting could not be determined in this experiment, since the decline in root ¹⁴C during the regrowth period may also be assigned to root respiration, root exudation and translocation to the shoots. ei]{gnH}{fnLambers} ei]{gnA C}{fnBorstlap}