Deletion of the gliP gene of Aspergillus fumigatus results in loss of gliotoxin production but has no effect on virulence of the fungus in a low-dose mouse infection model

Gliotoxin is a secondary metabolite produced by several fungi including the opportunistic human pathogen Aspergillus fumigatus. As gliotoxin exerts immunosuppressive effects in vitro and in vivo, a role as a virulence determinant in invasive aspergillosis has been discussed for a long time but evidence has not been provided until now. Here, by the use of different selection marker genes A. fumigatus knock-out strains were generated that are deficient for the non-ribosomal peptide synthetase GliP, the putative key enzyme of the gliotoxin biosynthesis. Deletion of the gliP gene resulted in loss of gliotoxin production, as analysed by high performance liquid chromatography and tandem mass spectrometry. No differences in morphology or growth kinetics between wild-type and gliP-deletion strains were observed. In vitro, the culture supernatant of the gliP-deficient strains showed a reduced cytotoxic effect on both macrophage-like cells and T cell lines. In a low-dose murine infection model of invasive aspergillosis, gliotoxin was detected in the lung and absent when mice were infected with the gliP deletion strain. However, gliP deletion strains showed no difference in virulence compared with the corresponding wild-type strains. Taken together, the non-ribosomal peptide synthetase GliP is essential for gliotoxin production in A. fumigatus. Gliotoxin is not required for pathogenicity of the fungus in immunocompromised mice, despite the fact that a reduced cytotoxicity of the culture supernatant of gliP deletion strains was demonstrated.     

Chromomycin dimer-DNA oligomer complexes. Sequence selectivity and divalent cation specificity

This paper reports on a solution NMR characterization of the sequence selectivity and metal ion specificity in chromomycin-DNA oligomer complexes in the presence of divalent cations. The sequence selectivity studies have focused on chromomycin complexes with the self-complementary d(A1-A2-G3-G4-C5-C6-T7-T8) duplex containing a pair of adjacent (G3-G4).(C5-C6) steps and the self-complementary d(A1-G2-G3-A4-T5-C6-C7-T8) duplex containing a pair of separated (G2-G3).(C6-C7) steps in aqueous solution. The antitumor agent (chromomycin) and nucleic acid protons have been assigned following analysis of distance connectivities in NOESY spectra and coupling connectivities in DQF-COSY spectra for both complexes in H2O and D2O solution. The observed intermolecular NOEs establish that chromomycin binds as a Mg(II)-coordinated dimer [1 Mg(II) per complex] and contacts the minor-groove edge with retention of 2-fold symmetry centered about the (G3-G4-C5-C6).(G3-G4-C5-C6) segment of the d(A2G2C2T2) duplex. By contrast, complex formation is centered about the (G2-G3-A4-T5).(A4-T5-C6-C7) segment and results in removal of the two fold symmetry of the d(AG2ATC2T) duplex. Thus, the binding of one subunit of the chromomycin dimer at its preferred (G-G).(C-C) site assists in the binding of the second subunit to the less preferred adjacent (A-T).(A-T) site. These observations suggest a hierarchy of chromomycin binding sites, with a strong site detected at the (G-G) step due to the hydrogen-bonding potential of acceptor N3 and donor NH2 groups of guanosine that line the minor groove. The divalent cation specificity has been investigated by studies on the symmetric chromomycin-d(A2G2C2T2) complex in the presence of diamagnetic Mg(II), Zn(II), and Cd(II) cations and paramagnetic Ni(II) and Co(II) cations. A comparative NOESY study of the Mg(II) and Ni(II) symmetric complexes suggests that a single tightly bound divalent cation aligns the two chromomycins in the dimer through coordination to the C1 carbonyl and C9 enolate ions on the hydrophilic edge of each aglycon ring. Secondary divalent cation binding sites involve coordination to the major-groove N7 atoms on adjacent guanosines in G-G steps. This coordination is perturbed on lowering the pH below 6.0, presumably due to protonation of the N7 atoms. The midpoint of the thermal dissociation of the symmetric complex is dependent on the divalent cation with the stability for reversible transitions decreasing in the order Mg(II) greater than Zn(II) greater than Cd(II) complexes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sensory innervation of the canine esophagus, stomach, and duodenum.

The sensory innervation of the postpharyngeal foregut was investigated by injecting the enzyme horseradish peroxidase (HRP) into the walls of the esophagus, stomach, or duodenum. The transported HRP was identified histochemically, labeled neurons in the spinal and vagal ganglia were counted, and the results were plotted using an SAS statistical program. The spinal sensory fields of each viscus were defined using three determinations: craniocaudal extent, principal innervation field, and peak innervation field. The data revealed that innervation fields are craniocaudally extensive, the sensory field of each viscus overlaps significantly with its neighbor, yet each viscus can be characterized by a field of peak innervation density. Craniocaudal innervation of the esophagus spans as many as 22-23 paired spinal ganglia (C1-L2). There are two peak innervation fields for the cervical (C2-C6 and T2-T4) and for the thoracic (T2-T4 and T8-T12) sectors of the esophagus. The sensory innervation of the stomach extends craniocaudally over as many as 25 paired spinal ganglia (C2-L5). The peak innervation field of the stomach spans a large area comprising the cranial, middle, and the immediately adjoining caudal thoracic ganglia (T2-T10). The duodenum is innervated craniocaudally by as many as 15 paired thoracolumbar ganglia (T2-L3). Peak innervation originates in the middle and caudal thoracic ganglia and cranial lumbar (T6-L1) ganglia. There is a recognizable viscerotopic organization in the sensory innervation of the postpharyngeal foregut; successively more caudal sectors of this region of the alimentary canal are supplied with sensory fibers from successively more caudal spinal dorsal root ganglia. Vagal afferent innervation of the esophagus, stomach, and duodenum is bilateral and originates predominantly, but not exclusively, from vast numbers of neurons in the nodose (distal) ganglia. The esophagus is innervated bilaterally and more abundantly by jugular (proximal) ganglia neurons than is either the stomach or duodenum. The physiological significance of the findings are discussed in relation to the phenomena of visceral pain and referred pain.     

Internal mobility in a double-stranded B DNA hexamer and undecamer. A time-dependent proton-proton nuclear Overhauser enhancement study

The internal mobility of the deoxyribose H2'-H2" and base C(H5)-C(H6) and T(CH3)-T(H6) vectors has been investigated by means of time-dependent nuclear Overhauser enhancement (NOE) measurements in a B DNA hexamer and undecamer. Cross-relaxation rates between these proton pairs are determined from the initial slopes of the time development of the NOEs, and, as the interproton distances between these proton pairs are fixed, apparent correlation times for the 3 interproton vectors are calculated from the cross-relaxation rate data. It is shown that there is little residue to residue variation in the cross-relaxation rates of the interproton vectors within each oligonucleotide, that the mean apparent correlation times of the C(H5)-C(H6) and T(CH3)-T(H6) vectors are approximately equal and significantly greater than that of the H2'-H2" vectors, and that the data for the H2'-H2" vectors of both oligonucleotides and the C(H5)-C(H6) and T(CH3)-T(H6) vectors of the undecamer cannot be accounted for by isotropic tumbling alone. The data are analysed in terms of a two motion model with isotropic tumbling and a single internal motion. The relaxation time of the internal motion at 23 degrees C is less than or equal to 1 ns for the H2'-H2" vectors of both oligonucleotides and less than or equal to 3 ns for the C(H5)-C(H6) and T(CH3)-T(H6) vectors of the undecamer. In the case of the H2'-H2" vectors, however, the amplitude of the internal motion is found to be too large to be compatible with the known stereochemistry of DNA. This finding can only be explained by invoking additional degrees of internal freedom with a larger number of internal motions of small amplitude of the type deduced from the analysis of crystallographic thermal factors [(1984) J. Mol. Biol. 173, 361-388].     

胶霉毒素的研究进展

胶霉毒素(gliotoxin,GT)是一个分子量为326 Da的小分子化合物,其骨架是由非核糖体肽合成酶Gli P催化苯丙氨酸和丝氨酸缩合成的环二肽,属于表聚硫代哌嗪二酮类化合物,是一种重要的真菌次级代谢产物。体内外研究已经表明,GT对动植物产生多种效应,不仅具有免疫抑制功能和诱导细胞凋亡作用,在生物防治方面也具有潜在的应用价值。本文将对有关GT生物合成、诱导宿主效应机制及其潜在应用价值进行综述。     

Molecular recognition in noncovalent antitumor agent-DNA complexes: NMR studies of the base and sequence dependent recognition of the DNA minor groove by netropsin

We have investigated intermolecular interactions and conformational features of the netropsin complexes with d(G1-G2-A3-A4-T5-T6-C7-C8) duplex (AATT 8-mer) and the d(G1-G2-T3-A4-T5-A6-C7-C8) duplex (TATA 8-mer) by one and two-dimensional NMR studies in solution. We have assigned the amide, pyrrole and methylene protons of netropsin and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. The directionality of the observed distance-dependent NOEs demonstrates that the 8-mer helices remain right-handed and that the arrangement of concave and convex face protons of netropsin are retained in the complexes. The observed changes in NOE patterns and chemical shift changes on complex formation suggest small conformational changes in the nucleic acid at the AATT and TATA antibiotic binding sites and possibly the flanking G.C base pairs. We observe intermolecular NOEs between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4.T5 base pairs of the AATT 8-mer and TATA 8-mer duplexes. The concave face pyrrole protons of the antibiotic also exhibit NOEs to the sugar H1' protons of residues 5 and 6 in the AATT and TATA 8-mer complexes. We also detect intermolecular NOEs between the guanidino and propioamidino methylene protons at either end of netropsin and the adenosine H2 proton of the two flanking A3.T6 base pairs in the AATT 8-mer and T3.A6 base pairs in the TATA 8-mer duplexes. These studies establish a set of nine contacts between the concave face of the antibiotic and the minor groove AATT segment and TATA segment of the 8-mer duplexes in solution. The observed magnitude of the NOEs require that there be no intervening water molecules sandwiched between the concave face of the antibiotic and the minor groove of the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation. The observed differences in the netropsin amide proton chemical shifts in the AATT 8-mer and TATA 8-mer complexes suggest differences in the strength and/or type of intermolecular hydrogen bonds at the AATT and TATA binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution structure of the nogalamycin-DNA complex

The nogalamycin-d(A-G-C-A-T-G-C-T) complex (two drugs per duplex) has been generated in aqueous solution and its structure characterized by a combined application of two-dimensional NMR experiments and molecular dynamics calculations. Two equivalents of nogalamycin binds to the self-complementary octanucleotide duplex with retention of 2-fold symmetry in solution. We have assigned the proton resonances of nogalamycin and the d(A1-G2-C3-A4-T5-G6-C7-T8) duplex in the complex and identified the intermolecular proton-proton NOEs that define the alignment of the antitumor agent at its binding site on duplex DNA. The analysis was greatly aided by a large number of intermolecular NOEs involving exchangeable protons on both the nogalamycin and the DNA in the complex. The molecular dynamics calculations were guided by 274 intramolecular nucleic acid distance constraints, 90 intramolecular nogalamycin distance constraints, and 104 intermolecular distance constraints between nogalamycin and the nucleic acid protons in the complex. The aglycon chromophore intercalates at (C-A).(T-G) steps with the long axis of the aglycon approximately perpendicular to the long axis of the flanking C3.G6 and A4.T5 base pairs. The aglycon selectively stacks over T5 and G6 on the T5-G6-containing strand with the aglycon edge containing OH-4 and OH-6 substituents directed toward the C3-A4-containing strand. The C3.G6 and A4.T5 base pairs are intact but buckled at the intercalation site with a wedge-shaped alignment of C3 and A4 on the C3-A4 strand compared to the parallel alignment of T5 and G6 on the T5-G6 strand in the complex. The nogalose sugar in a chair conformation, the aglycon ring A in a half-chair conformation, and the COOCH3-10 side chain form a continuous domain that is sandwiched within the walls of the minor groove and spans the three base pair (G2-C3-A4).(T5-G6-C7) segment. The nogalose ring is positioned in the minor groove such that its nonpolar face is directed toward the G6-C7 sugar-phosphate backbone while its polar face containing OCH3 groups is directed toward the G2-C3 sugar-phosphate backbone in the complex. The intermolecular contacts include a nonpolar patch of aglycon (CH3-9) and nogalose (CH3-3') methyl groups forming van der Waals contacts with the base-sugar residues in the minor groove and intermolecular hydrogen bonds involving the amino groups of G2 and G6 with the ether oxygens OCH3-3' and O7, respectively, on the nogalose sugar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis, 3-D structure, and pharmacology of a reticulated chimeric peptide derived from maurotoxin and Tsk scorpion toxins

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulfide bridges that acts on both Ca(2+)-activated (SK) and voltage-gated (Kv) K(+) channels. A 38-mer chimera of MTX, Tsk-MTX, has been synthesized by the solid-phase method. It encompasses residues from 1 to 6 of Tsk at N-terminal, and residues from 3 to 34 of MTX at C-terminal. As established by enzyme cleavage, Tsk-MTX displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7 and C4-C8 which, contrary to MTX, correspond to a disulfide bridge pattern common to known scorpion toxins. The 3-D structure of Tsk-MTX, solved by (1)H NMR, demonstrates that it adopts the alpha/beta scaffold of scorpion toxins. In vivo, Tsk-MTX is lethal by intracerebroventricular injection in mice (LD(50) value of 0.2 microg/mouse). In vitro, Tsk-MTX is as potent as MTX, or Tsk, to interact with apamin-sensitive SK channels of rat brain synaptosomes (IC(50) value of 2.5 nM). It also blocks voltage-gated K(+) channels expressed in Xenopus oocytes, but is inactive on rat Kv1.3 contrary to MTX.     

Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation.     

Synthesis, 1H NMR structure, and activity of a three-disulfide-bridged maurotoxin analog designed to restore the consensus motif of scorpion toxins

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus. The toxin displays an exceptionally wide range of pharmacological activity since it binds onto small conductance Ca(2+)-activated K(+) channels and also blocks Kv channels (Shaker, Kv1.2 and Kv1.3). MTX possesses 53-68% sequence identity with HsTx1 and Pi1, two other K(+) channel short chain scorpion toxins cross-linked by four disulfide bridges. These three toxins differ from other K(+)/Cl(-)/Na(+) channel scorpion toxins cross-linked by either three or four disulfide bridges by the presence of an extra half-cystine residue in the middle of a consensus sequence generally associated with the formation of an alpha/beta scaffold (an alpha-helix connected to an antiparallel beta-sheet by two disulfide bridges). Because MTX exhibits an uncommon disulfide bridge organization among known scorpion toxins (C1-C5, C2-C6, C3-C4, and C7-C8 instead of C1-C4, C2-C5, and C3-C6 for three-disulfide-bridged toxins or C1-C5, C2-C6, C3-C7, and C4-C8 for four-disulfide-bridged toxins), we designed and chemically synthesized an MTX analog with three instead of four disulfide bridges ([Abu(19),Abu(34)]MTX) and in which the entire consensus motif of scorpion toxins was restored by the substitution of the two half-cystines in positions 19 and 34 (corresponding to C4 and C8) by two isosteric alpha-aminobutyrate (Abu) derivatives. The three-dimensional structure of [Abu(19), Abu(34)]MTX in solution was solved by (1)H NMR. This analog adopts the alpha/beta scaffold with now conventional half-cystine pairings connecting C1-C5, C2-C6, and C3-C7 (with C4 and C8 replaced by Abu derivatives). This novel arrangement in half-cystine pairings that concerns the last disulfide bridge results mainly in a reorientation of the alpha-helix regarding the beta-sheet structure. In vivo, [Abu(19),Abu(34)]MTX remains lethal in mice as assessed by intracerebroventricular injection of the peptide (LD(50) value of 0. 25 microg/mouse). The structural variations are also accompanied by changes in the pharmacological selectivity of the peptide, suggesting that the organization pattern of disulfide bridges should affect the three-dimensional presentation of certain key residues critical to the blockage of K(+) channel subtypes.     

Structural studies of the O6meG.T interaction in the d(C-G-T-G-A-A-T-T-C-O6meG-C-G) duplex

High-resolution proton and phosphorus NMR studies are reported on the self-complementary d(C1-G2-T3-G4-A5-A6-T7-T8-C9-O6meG10-C11-G12) duplex (henceforth called O6meG.T 12-mer), which contains T3.O6meG10 interactions in the interior of the helix. The imino proton of T3 is observed at 9.0 ppm, exhibits a temperature-independent chemical shift in the premelting transition range, and broadens out at the same temperature as the imino proton of the adjacent G2.C11 toward the end of the helix at pH 6.8. We observed inter base pair nuclear Overhauser effects (NOEs) between the base protons at the T3.O6meG10 modification site and the protons of flanking G2.C11 and G4.C9 base pairs, indicative of the stacking of the T3 and O6meG10 bases into the helix. Two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) studies have permitted assignment of the base and sugar H1', H2', and H2' nonexchangeable protons in the O6meG.T 12-mer duplex. The observed NOEs demonstrate an anti conformation about all the glycosidic bonds, and their directionality supports formation of a right-handed helix in solution. The observed NOEs between the T3.O6meG10 interaction and the adjacent G2.C11 and G4.C9 base pairs at the modification site exhibit small departures from patterns for a regular helix in the O6.meG.T 12-mer duplex. The phosphorus resonances exhibit a 0.5 ppm spectral dispersion indicative of an unperturbed phosphodiester backbone for the O6meG.T 12-mer duplex. We propose a model for pairing of T3 and O6meG10 at the modification site in the O6meG.T 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical synthesis and 1H-NMR 3D structure determination of AgTx2-MTX chimera, a new potential blocker for Kv1.2 channel, derived from MTX and AgTx2 scorpion toxins

Agitoxin 2 (AgTx2) is a 38-residue scorpion toxin, cross-linked by three disulfide bridges, which acts on voltage-gated K(+) (Kv) channels. Maurotoxin (MTX) is a 34-residue scorpion toxin with an uncommon four-disulfide bridge reticulation, acting on both Ca(2+)-activated and Kv channels. A 39-mer chimeric peptide, named AgTx2-MTX, was designed from the sequence of the two toxins and chemically synthesized. It encompasses residues 1-5 of AgTx2, followed by the complete sequence of MTX. As established by enzyme cleavage, the new AgTx2-MTX molecule displays half-cystine pairings of the type C1-C5, C2-C6, C3-C7, and C4-C8, which is different from that of MTX. The 3D structure of AgTx2-MTX solved by (1)H-NMR, revealed both alpha-helical and beta-sheet structures, consistent with a common alpha/beta scaffold of scorpion toxins. Pharmacological assays of AgTx2-MTX revealed that this new molecule is more potent than both original toxins in blocking rat Kv1.2 channel. Docking simulations, performed with the 3D structure of AgTx2-MTX, confirmed this result and demonstrated the participation of the N-terminal domain of AgTx2 in its increased affinity for Kv1.2 through additional molecular contacts. Altogether, the data indicated that replacement of the N-terminal domain of MTX by the one of AgTx2 in the AgTx2-MTX chimera results in a reorganization of the disulfide bridge arrangement and an increase of affinity to the Kv1.2 channel.     

Genetic association of Glutathione peroxidase-1 (GPx-1) and NAD(P)H:Quinone Oxidoreductase 1(NQO1) variants and their association of CAD in patients with type-2 diabetes

Coronary artery disease (CAD) is a major health concern and the leading cause of death in individuals with type-2 diabetes mellitus (T2DM). Glutathione peroxidase-1 (GPx-1) and NAD(P)H: quinone oxidoreductase (NQO1) are known for its broad range of detoxification. The role of functional variants of these genes in the development of various disorders is proven. Hereby, we investigated the possible role of these variants in the development of CAD in T2DM patients of South Indian population. In this case-control study, a total of 539 patients (T2DM = 241; T2DM-CAD = 298) and 285 controls were included. The C198T GPx-1 and C609T NQO1 single-nucleotide polymorphisms were analyzed by PCR-RFLP. Further, these genotypes were correlated with blood lipid profile. Regression analysis showed that GPx1-C/T genotype is associated with a 1.35-fold increase (95% CI = 1.000-1.824; P = 0.048) and GPx1-T/T genotype is associated with a 1.76-fold increase (95% CI = 1.011 to 3.066; P = 0.046) to the T2DM development. Increased odds ratio showed that NQO1-T/T genotype had a higher occurrence of CAD in diabetic patients with CAD (95% CI = 1.003-2.674, P = 0.049) than T2DM patients without CAD. The level of triglycerides alone showed significant increase for GPx-1-C/T and -T/T genotypes in Tukey's Post hoc analysis (177.1 ± 19.2 vs. 184 ± 23.5; P = 0.039 and 177.1 ± 19.2 vs. 190 ± 22.4; P = 0.006) among the patients with T2DM-CAD. Our work concludes that GPx-1 variants might contribute to the development of diabetes and both GPx-1 and NQO1 variants confirm the association of CAD in people with T2DM of South Indian population.     

The synthesis and biological testing of bacilysin analogues

A series of compounds based on the structure of bacilysin were synthesised and tested for antibacterial activity. The key steps in the syntheses are the coupling of an iodide to a diketopiperazine (DKP) and mono-lactim ether scaffold, respectively. The diastereoselectivity of the coupling reactions was dependant on the scaffold, with selectivity for DKP of about 4:1 and mono-lactim ether exceeding 98:2. Subsequent elaboration of the compounds to give open chain dipeptides and DKPs that mimic the structure of bacilysin but substitute the epoxy ketone for a saturated or unsaturated ketone is described. Overall yield from coupling to final product was between 5 and 21 %, with the yield of the saturated products notably higher. The open chain dipeptides demonstrated moderate antibacterial and antifungal activity.     

Polypeptide GalNAc-transferases, ST6GalNAc-transferase I, and ST3Gal-transferase I expression in gastric carcinoma cell lines.

Mucin O-glycosylation in cancer is characterized by aberrant expression of immature carbohydrate structures leading to exposure of simple mucin-type carbohydrate antigens and peptide epitopes. Glycosyltransferases controlling the initial steps of mucin O-glycosylation are responsible for the altered glycosylation observed in cancer. We studied the expression in gastric cell lines of six UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (GalNAc-T1, T2, T3, T4, T6, T11) that catalyze the initial key step in the regulation of mucin O-glycosylation, the transfer of GalNAc from UDP-GalNAc to serine and threonine residues. We also studied the expression of ST6GalNAc-I, the enzyme responsible for the synthesis of Sialyl-Tn antigen (NeuAcalpha2,6GalNAc) and the ST3Gal-I, the enzyme responsible for the synthesis of Sialyl-T antigen (NeuAcalpha2,3Galbeta1,3GalNAc). This study was done using specific monoclonal antibodies, enzymatic assays, and RT-PCR. Our results showed that GalNAc-T1, -T2, and -T3 have an ubiquitous expression in all gastric cell lines, whereas GalNAc-T4, -T6, and -T11 show a restricted expression pattern. The immunoreactivity with MAb VU-2-G7 suggests that, apart from GalNAc-T4, another GalNAc transferase is involved in the glycosylation of the Thr in the PDTR region of the MUC1 tandem repeat. The expression of ST3Gal-I correlates with the expression of the Sialyl-T antigen in gastric cell lines and in the control cell lines studied. The expression of ST6GalNAc-I is low in gastric cell lines, in accordance with the low/absent expression of the Sialyl-Tn antigen.     

NMR studies of echinomycin bisintercalation complexes with d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution: sequence-dependent formation of Hoogsteen A1.T4 and Watson--Crick T1.A4 base pairs flanking the bisintercalation site

We report on two-dimensional proton NMR studies of echinomycin complexes with the self-complementary d(A1-C2-G3-T4) and d(T1-C2-G3-A4) duplexes in aqueous solution. The exchangeable and nonexchangeable antibiotic and nucleic acid protons in the 1 echinomycin per tetranucleotide duplex complexes have been assigned from analyses of scalar coupling and distance connectivities in two-dimensional data sets recorded in H2O and D2O solution. An analysis of the intermolecular NOE patterns for both complexes combined with large upfield imino proton and large downfield phosphorus complexation chemical shift changes demonstrates that the two quinoxaline chromophores of echinomycin bisintercalate into the minor groove surrounding the dC-dG step of each tetranucleotide duplex. Further, the quinoxaline rings selectively stack between A1 and C2 bases in the d(ACGT) complex and between T1 and C2 bases in the d(TCGA) complex. The intermolecular NOE patterns and the base and sugar proton chemical shifts for residues C2 and G3 are virtually identical for the d(ACGT) and d(TCGA) complexes. A change in sugar pucker from the C2'-endo range to the C3'-endo range is detected at C2 on formation of the d(ACGT) and d(TCGA) complexes. In addition, the sugar ring protons of C2 exhibit upfield shifts and a large 1 ppm separation between the H2' and H2" protons for both complexes. The L-Ala amide protons undergo large downfield complexation shifts consistent with their participation in intermolecular hydrogen bonds for both tetranucleotide complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of two supplementation levels of linseed combined with CLA or tallow on meat quality traits and fatty acid profile of adipose and different muscle tissues in slaughter pigs

Dietary linseed supply efficiently elevates the linolenic acid concentration of pork. The main problem of increasing the n-3 fatty acid tissue levels arises from a higher susceptibility to lipid oxidation. Increasing the saturation level of tissue lipids by the dietary inclusion of conjugated linoleic acids (CLA) or tallow might prevent oxidation. Thus, the aim of the study was to evaluate the impact of dietary CLA or tallow supplementation combined with extruded linseed on the growth performance, carcass characteristics and fatty acid profile of muscles (longissimus, semimembranosus, biceps femoris) and subcutaneous fat (SF). The enzyme activity of the de novo lipogenesis and stearoyl-CoA desaturase in the SF was also assessed. From 18 to 104 kg BW, 32 Swiss Large White barrows were fed a diet supplemented with either: (1) 2% linseed (L2); (2) 3% linseed (L3); (3) 2% linseed + 1% CLA (L2-C) or (4) 2% linseed + 1% tallow (L2-T). The linolenic and eicosatrienoic acid concentrations were higher (P < 0.01) and the ∑n-6/∑n-3 ratio was lower (P < 0.01) in all tissues of L3 than L2 and L2-T barrows. Only in the SF the docosapentaenoic acid concentration was increased (P < 0.01) in L3 barrows. Compared with the other three diets, feeding the L2-C diets increased (P < 0.01) the amount of myristic, palmitic, stearic and palmitoleic acid at the expense of the oleic and eicosenoic acid content in the intramuscular and SF lipids. Except for the lower (P < 0.05) eicosadienoic acid concentration in the muscles, feeding the L2-C treatment resulted in similar polyunsaturated fatty acid concentrations and ∑n-6/∑n-3 ratio than feeding L2 or L2-T diets. Both the c9,t11- and t10,c12-CLA isomers found in the CLA-supplemented diet were also detected in the tissues, but the c9,t11-isomer was more abundant than the t10,c12-isomer. De novo lipogenesis was not (P > 0.05) affected by the dietary fats, whereas Δ9-desaturase activity was depressed (P < 0.05) by CLA inclusion (L2-C). Only when oxidation was challenged by cooking and subsequent storage for 4 days at 4°C values of thiobarbituric acid-reactive substances were lower (P < 0.05) in longissimus muscle chops of L2-C compared with L2, L3 and L2-T barrows. The present findings revealed that CLA, but not tallow, combined with extruded linseed enhanced the oxidative stability of pork probably by lowering the degree of unsaturation of the lipids without affecting the improved ∑n-6/∑n-3 ratio.     

Systematic and quantitative analysis of protein-protein recognition between nonribosomal peptide synthetases investigated in the tyrocidine biosynthetic template

We present a systematic and quantitative study of the protein-protein recognition between the three tyrocidine synthetases TycA, TycB, and TycC investigated with two artificial in trans assay systems, which had been previously developed: the "DKP assay system" for the interaction of TycA with TycB and the "L/D-Phe-L-Asn assay system" for the interaction of TycB with TycC. TycA-A(Phe)TE and TycB(3)-A(Phe)TE, which are used as donor enzymes, both provide D-Phe-S-Ppant, so that no substrate specificities interfered with the quantification of protein-protein recognition. We tested all donor/acceptor enzyme combinations between the two artificial assay systems for product formation activities as well as two hybrid enzymes, where the E-domains of TycA and TycB(3) had been exchanged against each other. Furthermore, four donor/acceptor protein fusions were constructed on gene level, resulting in dimodular proteins. We were able to show that the E-domains mediate protein-protein recognition in trans. Product formation of the different donor assayed with the two acceptor enzymes TycB(1)-CA(Pro)T and TycC(1)-CA(Asn)T/Te in trans was only obtained if the donor enzyme harbored the cognate E-domain. Interestingly, all in cis fusions (dimodular proteins) were active, giving strong evidence that unnatural protein-protein interactions can be "forced" by fusion of the distinct enzymes. Finally, we were able to detect product formation in the "DKP system" with engineered hybrid proteins where the A-domain of TycA had been exchanged against the isoleucine-activating A-domain of BacA(1) and the valine-activating A-domain of TycC(4), respectively. All of these findings are of high relevance for future NRPS engineering approaches.