Computation of relaxation matrix elements from incomplete NOESY data sets

Summary The structural determination of biological molecules in solution by NMR relies on the determination of a set of interatomic distances obtained by measurement of intramolecular nuclear Overhauser effects (NOE). It is shown in this paper that it is possible to obtain the accurate relaxation rate (and hence the interatomic distance) from the direct measurement of a single NOE signal. The precise analysis of a NOESY peak evolution with respect to the mixing time allows the evaluation of the relaxation parameters for the pair of spins under consideration. This is done without any assumption on the relaxation of unmeasured spins, or on the movement of the molecule. The theoretical basis of this method is presented. In order to evaluate the proposed method, a simulated case on the protein BPTI is studied, which shows that the method performs very well even in the case of noisy data sets.     

Liquefaction of dulse (Palmaria palmata (L.) Kuntze) by a commercial enzyme preparation and a purified endo ,β-1,4-D-xylanase

Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo--1,4-D-xylanase was studied.The endo--1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl--D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a K_m of 2.2 and 2.8 mg ml^-1 and V_max of 3600 and 3900 nkat mg^-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater.     

Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase

The xcp genes are required for the secretion of most extracellular proteins by Pseudomonas aeruginosa. The products of these genes are essential for the transport of exoproteins across the outer membrane after they have reached the periplasm via a signal sequence-dependent pathway. To date, analysis of three xcp genes has suggested the conservation of this secretion pathway in many Gram-negative bacteria. Furthermore, the xcpA gene was shown to be identical to pilD, which encodes a peptidase involved in the processing of fimbrial (pili) subunits, suggesting a connection between pili biogenesis and protein secretion. Here the nucleotide sequences of seven other xcp genes, designated xcpR to -X, are presented. The N-termini of four of the encoded Xcp proteins display similarity to the N-termini of type IV pili, suggesting that XcpA is involved in the processing of these Xcp proteins. This could indeed be demonstrated in vivo. Furthermore, two other proteins, XcpR and XcpS, show similarity to the PilB and PilC proteins required for fimbriae assembly. Since XcpR and PilB display a canonical nucleotide-binding site, ATP hydrolysis may provide energy for both systems.     

A multi-recombination model for the mtDNA rearrangements seen in maize cmsT regenerated plants

Regeneration of plants from maize cytoplasmic male sterile type T (cmsT) callus tissue culture promotes, in some instances, genetic variability in their mitochondrial genomes. These mutations have been analyzed in various cmsT regenerated plants that have or have not regained the male fertile phenotype. A unique multi-recombination model explains the various mitochondrial genome rearrangements. First, recombination involving two different sets of direct repeats gives rise to subgenomic recombinant circles. Second, intermolecular recombination between some selected subgenomes gives rise to a new rearranged master chromosome. The consequence of these events is the formation of a new master chromosome containing sequence deletions and duplications when compared to the progenitor. This new mitochondrial genome seems stable, although it does not contain the entire genetic complexity of the progenitor.     

Immobilization of microsomes into alginate beads is a convenient method for producing glucuronides from drugs

Summary The production of glucuronides from drugs by immobilized microsomal uridine diphosphate (UDP)-glucuronosyltransferase has been investigated. Of all the immobilization methods used (covalent binding, adsorption by ionic or hydrophobic interactions), only entrapment of microsomes into alginate beads in the presence of polyethyleneimine was effective in producing high glucuronidation rates, thus leading to the formation of large amounts of metabolites. The performance of the bioreactor was optimized with the drug 3-azido-3-deoxythymidine (AZT), active against the human immunodeficiency virus, as a model substrate of UDP-glucuronosyltransferase. Calcium (12 mm) could optimally improve the stability of microsomes entrapped in alginate beads. Upon immobilization, enzyme activation occurred, leading to a fivefold increase in specific activity. The determination of apparent K _m and V _max revealed that AZT was a better substrate for the immobilized enzyme than free microsomes. The AZT-glucuronide production obtained after 6 h was threefold higher than that observed with free microsomes. This bioreactor was also efficient in production of glucuronides from structurally different compounds such as bilirubin, 4-nitrophenol, clofibric acid, pirprofen, dextrorphan or morphine, the corresponding glucuronide of which possesses pharmacological or toxicological interest. Offprint requests to: J. Magdalou     

Cytostatic product(s) released by activated macrophages, unrelated to interleukin 1, tumor necrosis factor alpha, and interferon-alpha/beta

Murine peritoneal macrophages activated for cytotoxicity by trehalose dimycolate in vivo and lipopolysaccharide in vitro released cytostatic factor(s) against EMT6 target cells, in 8-hr conditioned medium (CM). The cytostatic factor(s) completely blocked DNA synthesis by EMT6 cells within 16 hr. Other cell lines are less sensitive (P815 and R-L929) or resistant (KB and HT29) to the cytostatic effect of CM. The anti-proliferative activity of CM had a MW greater than 10,000 Da, as judged by ultrafiltration. It was destroyed by proteases and strongly inhibited by P815 cell product(s). Conditioned media from nonactivated macrophages were not cytostatic against EMT6 cells. No relationship was found between cytostatic factor(s) in CM and interleukin 1 (IL-1), tumor necrosis factor alpha (TNF-alpha), and interferon-alpha/beta (IFN-alpha/beta): the growth of EMT6 cells was unaffected by Hu.r.IL-is and Hu.r.TNF-alpha and was only slightly inhibited by IFN-alpha/beta. Furthermore, cytostatic CM contained low levels of TNF and IFN activities. Finally, antibodies raised against murine IFN-alpha/beta had no effect on the cytostatic activity of CM.     

Synthesis and biological activity of analogs of dynorphin-A(1-13) substituted in positions 2 and 4: design of [Ala2,Trp4]-Dyn-A(1-13) as a putative selective opioid antagonist

Mono- and di-substituted analogs of dynorphin-A(1-13) (Dyn-A(1-13)) were synthesized by the solid-phase procedure. The products were purified and analyzed for their ability to inhibit the electrically evoked contractions of the guinea pig ileum (GPI) and mouse vas deferens (MVD) and to compete with the binding of [3H]etorphine ([3H]ET) and [3H]ethylketocyclazocine ([3H]EKC) to homogenates of rat brain (mu-, delta-, kappa 2-receptors) and guinea pig cerebellum (kappa-receptor), respectively. Introduction of Ala in position 2 caused a drastic decrease in the activity of the peptide on the smooth muscle preparations (IC50 of 104 and 2.250 nM in the GPI and the MVD as compared with 0.7 and 21 nM for the parent peptide, respectively). Conversely, this analog retained much of the opioid binding activity of Dyn-A(1-13) (relative binding potencies of 15 and 72% for the displacement of [3H]ET and [3H]EKC, respectively). The replacement of Phe4 by Trp also caused drastic decreases in the activity of the peptide in the smooth muscle preparations (relative potencies of 0.8 and 8.8% on the GPI and MVD) while much of the binding potency to the opioid receptors was retained (31 and 67% for the displacement of [3H]ET and [3H]EKC, respectively). [Ala2,Trp4]-Dyn-A(1-13) was the least potent peptide tested in the smooth muscle assays (relative potencies: 0.1 and 0.6%). However, this latter analog still retained some opioid binding activity in the displacement of [3H]ET to rat brain homogenates (3%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative spinal analgesic action of dynorphin1-8, dynorphin1-13, and a kappa-receptor agonist U50,488

The effect of intrathecal injections of dynorphin1-8 (DYN1-8), dynorphin1-13 (DYN1-13), and a putative kappa agonist, U50,488 was tested in the rat tail-flick test. DYN1-8 and DYN1-13 (5, 10, 20 micrograms) produced a dose-related biphasic antinociceptive response consisting of an initial and a delayed response. Injection of U50,488 (20, 40 60 micrograms) produced a monophasic response. The antinociceptive effect of DYN1-8 (5, 10, 20 micrograms) and DYN1-13 (20 micrograms), was present 24 h postintrathecal injection. Pretreatment with systemic naloxone (2 mg/kg s.c.) attenuated the delayed response, but not the initial response induced by DYN1-8 and DYN1-13. The initial response was attenuated by pretreatment with intrathecal naloxone at a dose of 0.5 and 2.0 micrograms. The antinociceptive effect of U50,488 (20, 60 micrograms) was not affected by pretreatment with 2.0 micrograms intrathecal naloxone, but was significantly reduced by 4 micrograms of the antagonist. Both DYN1-8 and DYN1-13 (5 micrograms) augmented the antinociceptive effect of intrathecally administered morphine (5, 10 micrograms). Intrathecal injection of DYN1-8 (5, 10, 20 micrograms), DYN1-13 (5 micrograms), and morphine (10 micrograms) reduced the spontaneous output of urine measured at 2 and 24 h postintrathecal injection. A similar injection of U50,488 (20 micrograms) had no significant action on the urinary output. The results show that long and short dynorphin fragments have a comparable activity and the spinal antinociceptive actions of dynorphin are sensitive to low doses of intrathecal naloxone. The activity profile of spinally administered dynorphins differs from that of the kappa agonist U50,488.     

14-beta-Methyl-8-oxacyclorphan (BC-3016), a morphinan derivative with high affinity for kappa opioid receptor: comparison with dynorphin-A(1-13)

14-beta-Methyl-8-oxacyclorphan (BC-3016) was tested for its ability to depress the electrically evoked contractions of the guinea pig ileum (GPI) and of the mouse vas deferens (MVD) and to compete with the binding of prototype ligands selective for kappa-, mu-, or delta-opioid receptors in membrane preparations of rat brain and guinea pig cerebellum. BC-3016 was a very potent agonist in the GPI and MVD preparations, with ID50 of 0.7 and 31 nM, respectively. The activity of levorphanol, a standard alkaloid related to BC-3016, was much lower in both assays with ID50 values of 44 and 86 nM, respectively. Conversely, the activity of BC-3016 was quite comparable to that of dynorphin-A(1-13) in both preparations. In the GPI assay, a putative kappa-receptor antagonist, MR-2266, was 6.6 and 5.5 times more potent than naloxone in blocking the activity of BC-3016 and dynorphin-A(1-13), respectively. BC-3016 was also very potent in displacing bound [3H]ethylketocyclazocine ([3H]EKC) to membrane preparations of the guinea pig cerebellum, a brain component containing predominantly kappa-opioid receptors (Ki of 0.58 nM). Its potency in the displacement of the bound mu-ligand, 3H-labelled (D-Ala2,MePhe4,Gly-OH5)-enkephalin ([3H]DAGO), to rat brain homogenates was somewhat lower (Ki of 0.8 nM) but still high when compared with its ability to displace the delta-ligand, 3H-labelled (D-Ser2, Thr6)-Leu-enkephalin ([3H]DSLET) to rat brain homogenates (Ki of 4.45 nM). The affinity of BC-3016 for the opioid receptor was 2.1-fold higher than that of U-50488H, a selective kappa-opioid ligand.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hybridization of DNA from methanogenic bacteria with nitrogenase structural genes (nifHDK)

Summary Using the Southern hybridization technique, homologies were examined between restricted DNA of four methanogenic bacteria (Methanobacterium ivanovi, Methanobacterium thermoautotrophicum, Methanococcus voltae, Methanosarcina barkeri) and the nif (nitrogen fixation) genes of Klebsiella pneumoniae and Anabaena strain 7120. With K. pneumoniae probes, no hybridization was observed with nifA, nifNE, and nifJ but positive results were obtained with the nifHDK genes coding for nitrogenase. Homology was detected, in the four strains, with K. pneumoniae and Anabaena nifH probes. In M. voltae and M. ivanovi, the homology found with nifH was estimated to be about 70% and a weaker hybridization was observed also with nifD and nifK. In M. voltae, the sequence homologous to nifH was found on a 3.0 kbp HindIII fragment and sequences homologous to nifD and nifK on a 3.8 kbp HindIII fragment. The 3.0 kbp fragment was cloned and the region homologous to nifH was localized more precisely. When this fragment was used as a probe against other DNAs, it behaved as a K. pneumoniae and Anabaena nifH probe. The results suggest that the structural genes for nitrogenase may be present in archaebacteria and raise interesting questions regarding their evolution.