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S Freund

Publications and source records attributed to S Freund.

At least 19 recordsLinked to original sources
PubMed2000

RNA recognition by a Staufen double-stranded RNA-binding domain.

The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem-loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.

Amino Acid Sequence↗
PubMed2000

Hand response differences in a self-face identification task.

Evidence has indicated that the right frontal cortex is preferentially involved in self-face recognition. To test this further, we employed a face identification task and examined hand response differences (N=10). Pictures of famous faces were combined with pictures of the participants' faces (self) and their co-workers' faces (familiar). These images were presented as a 'movie' in which one face transformed into another. Under the first instruction set, the movies began with either the participant's face or a co-worker's face, and the sequences gradually morphed into a famous face. When told to stop the movie when the face in the sequence became famous, a significantly later 'frame' was identified when the movies were composed of self-faces and the participants responded with their left hand. When the movies started with the famous faces and participants had to stop the movie when it became their own or their familiar co-worker's image (Instruction set 2), a significantly earlier frame was identified in the 'Self: Left hand' condition. The data suggest that participants are inclined to identify images as their own when the right hemisphere is preferentially accessed.

Adult↗
PubMed1999

Left hand advantage in a self-face recognition task.

Subjects were exposed to pictures of self and others (e.g., friend, stranger, and famous people) to determine if there was an advantage in reaction time and accuracy in identifying the self. It was found that upright and inverted self-faces were identified more rapidly than non-self faces when subjects responded with their left hand, which in other tasks has corresponded with contralateral hemispheric dominance. These data suggest that self-recognition may be correlated with neural activity in the right hemisphere, and that the differences observed may not be unique to self-face recognition. These results are in agreement with previous research indicating that self-directed awareness is correlated with right prefrontal activity.

Adult↗
PubMed1996

Protein kinase cross-talk: membrane targeting of the beta-adrenergic receptor kinase by protein kinase C.

The beta-adrenergic receptor kinase (betaARK) is the prototypical member of the family of cytosolic kinases that phosphorylate guanine nucleotide binding-protein-coupled receptors and thereby trigger uncoupling between receptors and guanine nucleotide binding proteins. Herein we show that this kinase is subject to phosphorylation and regulation by protein kinase C (PKC). In cell lines stably expressing alpha1B- adrenergic receptors, activation of these receptors by epinephrine resulted in an activation of cytosolic betaARK. Similar data were obtained in 293 cells transiently coexpressing alpha1B- adrenergic receptors and betaARK-1. Direct activation of PKC with phorbol esters in these cells caused not only an activation of cytosolic betaARK-1 but also a translocation of betaARK immunoreactivity from the cytosol to the membrane fraction. A PKC preparation purified from rat brain phospborylated purified recombinant betaARK-1 to a stoichiometry of 0.86 phosphate per betaARK-1. This phosphorylation resulted in an increased activity of betaARK-1 when membrane-bound rhodopsin served as its substrate but in no increase of its activity toward a soluble peptide substrate. The site of phosphorylation was mapped to the C terminus of betaARK-1. We conclude that PKC activates betaARK by enhancing its translocation to the plasma membrane.

Amino Acid Sequence↗
PubMed1995

Post-translational heterocyclic backbone modifications in the 43-peptide antibiotic microcin B17. Structure elucidation and NMR study of a 13C,15N-labelled gyrase inhibitor.

Microcin B17 (McB17), the first known gyrase inhibitor of peptidic nature, is produced by ribosomal synthesis and post-translational modification of the 69-residue precursor protein by an Escherichia coli strain. To elucidate the chemical structure of the mature 43-residue peptide antibiotic, fermentation and purification protocols were established and optimized which allowed the isolation and purification of substantial amounts of highly pure McB17 (non-labelled, 15N-labelled and 13C/15N-labelled peptide. By ultraviolet-absorption spectroscopy. HPLC-electrospray mass spectrometry and GC-mass spectrometry, amino acid analysis, protein sequencing, and, in particular, multidimensional NMR, we could demonstrate and unequivocally prove that the enzymic modification of the precursor backbone at Gly-Cys and Gly-Ser segments leads to the formation of 2-aminomethylthiazole-4-carboxylic acid and 2-aminomethyloxazole-4-carboxylic acid, respectively. In addition, two bicyclic modifications 2-(2-aminomethyloxazolyl)thiazole-4-carboxylic acid and 2-(2-aminomethylthiazolyl)oxazole-4-carboxylic acid were found that consist of directly linked thiazole and oxazole rings derived from one Gly-Ser-Cys and one Gly-Cys-Ser segment. Analogous to the thiazole and oxazole rings found in antitumor peptides of microbial and marine origin, these heteroaromatic ring systems of McB17 presumably play an important role in its gyrase-inhibiting activity, e.g. interacting with the DNA to trap the covalent protein-DNA intermediate of the breakage-reunion reaction of the gyrase.

Amino Acid Sequence↗
PubMed1995

The plastid aldolase gene from Chlamydomonas reinhardtii: intron/exon organization, evolution, and promoter structure.

Genomic clones encoding the plastidic fructose-1,6-bisphosphate aldolase of Chlamydomonas reinhardtii were isolated and sequenced. The gene contains three introns which are located within the coding sequence for the mature protein. No introns are located within or near the sequence encoding the transit-peptide, in contrast to the genes for plastidic aldolases of higher plants. Neither the number nor the positions of the three introns of the C. reinhardtii aldolase gene are conserved in the plastidic or cytosolic aldolase genes of higher plants and animals. The 5' border sequences of introns in the aldolase gene of C. reinhardtii exhibit the conserved plant consensus sequence. The 3' acceptor splice sites for introns 1 and 3 show much less similarity to the eukaryotic consensus sequences than do those of intron 2. The plastidic aldolase gene has two tandemly repeated CAAT box motifs in the promoter region. Genomic Southern blots indicate that the gene is encoded by a single locus in the C. reinhardtii genome.

Amino Acid Sequence↗
PubMed1995

Biotransformation of CI-937 in primary cultures of rat hepatocytes. Formation of glutathione conjugates.

The anticancer drug 7,10-dihydroxy-2-[2-[(2-hydroxyethyl)amino]- ethyl]-5-[[2-(methylamino)ethyl]amino]anthra[1,9-c,d]pyrazole- 6(2H)-one dihydrochloride (CI-937) is 1 of 3 anthrapyrazole derivatives chosen for phase I and phase II clinical trials. Although the chemical structure of CI-937 signals a contribution of redox reactions in the pharmacology of the drug, a study concerning the biotransformation of CI-937 is still missing. Incubations of primary cultures of rat hepatocytes with CI-937 result in the formation of three glutathione conjugates and a glucuronic acid conjugate. The structures of the glutathione conjugates have been established by reference synthesis with activated horseradish peroxidase and HPLC-MS-MS and two-dimensional NMR measurements. The glucuronic acid derivative of CI-937 has been identified by MS. The formation of the glutathione conjugates in cells establishes the ability of the drug to form covalent bonding to intracellular nucleophilic targets. The conjugation with glutathione has been rationalized by oxidative activation of CI-937, yielding an electrophilic intermediate that finally reacts with glutathione.

Animals↗
PubMed1994

Expression and sequence of the only detectable aldolase in Chlamydomonas reinhardtii.

Only one aldolase can be resolved from Chlamydomonas reinhardtii by anion-exchange chromatography on DEAE-cellulose. Western blots with specific antisera against plastid and cytosol aldolase of spinach and Northern blots with the respective cDNAs from spinach indicate that only one aldolase, the plastid enzyme, is expressed. Full-length cDNA clones for the plastidic aldolase were isolated from cDNA library constructed from poly(A)+ mRNA of C. reinhardtii with plastid-aldolase-specific probes from spinach. The clones contained a 1.7-kb insert with an open reading frame for 374 amino acid residues covering the mature chloroplast protein of 347 amino acids and a N-terminal transit peptide of 27 amino acids for chloroplast import. The calculated molecular mass of the mature protein is 37.6 kDa and that of the precursor protein is 40.3 kDa. The aldolase of C. reinhardtii shows amino acid similarity of 62 to 67% with the chloroplast aldolase and of 47 to 52% with the cytosolic enzymes of higher plants, respectively. An evolutionary tree with all known class I aldolases shows separate clusters for the chloroplast and for the cytosol aldolases in higher plants and green algae. The aldolase of C. reinhardtii connects at a basal position with the chloroplast aldolases of higher plants.

Amino Acid Sequence↗
PubMed1994

A1 adenosine receptors expressed in CHO-cells couple to adenylyl cyclase and to phospholipase C.

A1 adenosine receptors are in general coupled to inhibition of adenylyl cyclase, but have more recently been reported to be capable of also activating phospholipase C. The present study was done in order to investigate whether these different effects can be elicited by a single A1 receptor, or whether A1 receptor subtypes have to be invoked. The cDNA of a rat brain A1 adenosine receptor was stably expressed in CHO-cells, resulting in clones with varying receptor densities; a clone expressing 1.9 pmol receptors/mg membrane protein was used for further characterization. The ligand binding properties of the expressed receptors were typical for the rat A1 adenosine receptor. A1 receptor agonists caused a concentration-dependent inhibition of adenylyl cyclase activity in the membranes, with maximal inhibition by 70%. A1 receptor stimulation also caused concentration-dependent stimulation of inositol phosphate generation in these cells, with maximal effects of 300%. Both adenylyl cyclase inhibition and enhancement of inositol phosphate generation were essentially abolished after pretreatment of the cells with pertussis toxin. These results indicate that a single A1 adenosine receptor can couple to two effector pathways, and that both effectors are activated via pertussis toxin sensitive G proteins.

Adenylate Cyclase Toxin↗
PubMed1994

Synthesis and structural elucidation of biliary excreted thioether derivatives of mitoxantrone.

1. Hplc-MS coupling has been used for the identification of thioether derivatives of the anticancer agent mitoxantrone in the bile of pig. 2. Three biologically relevant new thioether derivatives of mitoxantrone have been synthesized by a horseradish peroxidase-catalysed reaction. 3. The thioether derivatives have been characterized by means of ion-spray tandem mass spectrometry and nmr spectrometry including two-dimensional techniques. 4. The carbon-sulphur bond formation takes place at the hydroquinone moiety of the anthraquinone skeleton pointing to the importance of a tautomeric equilibrium between different species of the oxidized drug. 5. The occurrence of the synthesized compounds in biological systems suggests a metabolic pathway that may be relevant for the cytotoxicity of mitoxantrone (oxidative activation).

Animals↗
PubMed1993

Solution structures of the lantibiotics duramycin B and C.

The solution structures of the lantibiotics duramycin B in H2O/2H2O (9:1) and duramycin C in (2H3)acetonitrile/H2O (1:1) have been determined by NMR followed by distance-geometry and restrained-molecular-mechanics calculations. The constitution and location of three thioether bridges and a lysinoalanine ring system could be established by unambiguously assigned NOE contacts between the bridging side chains. Model building based on NMR constraints resulted in a U-shaped topology of the tetracyclic 19-peptides with a turn around Pro9 and a kink along a virtual line from residues 5 to 13. This clamp-like conformation is stabilized by the thioether bridges and is additionally supported by an antiparallel beta-strand-like structure of the N-termini and C-termini and the inherent amphiphilicity of duramycin-type lantibiotics. The duramycins B and C differ mainly in the relative mobilities of their rings A, C and D. Duramycin B is closely related to cinnamycin with an exchange of Phe10 to leucine, whereas duramycin C differs from duramycin B by three conserved and two non-conserved amino-acid exchanges.

Amino Acid Sequence↗
PubMed1993

Ornibactins--a new family of siderophores from Pseudomonas.

Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins. The ornibactins represent modified tetrapeptide siderophores, possessing the sequence L-Orn1(N delta-OH, N delta-acyl)-D-threo-Asp(beta-OH)-L-Ser-L-Orn4(N delta-OH, N delta-formyl)-1,4-diaminobutane. The N delta-acyl groups of Orn1(N delta-OH, N delta-acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8. Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u. higher mass. The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques.

Amino Acid Sequence↗
PubMed1993

Purification and chemical characterization of staphyloferrin B, a hydrophilic siderophore from staphylococci.

This paper describes the chemical characterization of staphyloferrin B, a new complexone type siderophore isolated from low iron cultures of Staphylococcus hyicus DSM 20459. Purification of the very hydrophilic metabolite was achieved by anion exchange high performance liquid chromatography HPLC. Mass spectrometry showed a molecular mass of 448 amu. Hydrolysis with 8 M HCl revealed the presence of L-2,3-diaminopropionic acid, citrate, ethylenediamine and succinic semialdehyde. The connections between the four building blocks were determined by two-dimensional nuclear magnetic resonance measurements. UV/Vis and circular dichroism spectra are consistent with the proposed structure, which could also be confirmed by precursor feeding. The siderophore activity of staphyloferrin B was demonstrated by iron transport measurements.

Citrates↗
PubMed1993

Germicidin, an autoregulative germination inhibitor of Streptomyces viridochromogenes NRRL B-1551.

During germination spores of Streptomyces viridochromogenes NRRL B-1551 excrete a compound, germicidin, which has an inhibitory effect on the germination of its own arthrospores at a concentration as low as 200 pM (40 pg/ml). At higher concentrations germicidin inhibits porcine Na+/K(+)-activated ATPase and retards the germination of the cress Lepidium sativum. Germicidin is the first known autoregulative inhibitor of spore germination in the genus Streptomyces and was isolated from the supernatant of germinated spores, but also from the supernatant of the submerged culture. Spectroscopic analysis and derivatization reactions revealed germicidin to be 6-(2-butyl)-3-ethyl-4-hydroxy-2-pyrone (C11H16O3). Crude isolates of germicidin from the supernatant of submerged culture, but not from the spores, contained a second, structurally very similar compound (C10H14O3), in which in contrast to germicidin a 2-propyl instead of the 2-butyl chain was bound to C-6 and which did not show any activity in the germination and ATPase assay. The germination assay was evaluated as a new screening model for specifically active compounds.

Fermentation↗
PubMed1991

The solution structure of the lantibiotic gallidermin.

The 21-peptide amide antibiotic gallidermin is a potential therapeutic against acne disease. It belongs to the class of polycyclic lanthionine and alpha,beta-didehydroamino acids containing polypeptides, which were named "lantibiotics." The structural gene of the recently elucidated lantibiotic gallidermin encodes a precursor peptide containing Ser, Thr, and Cys residues in the C-terminal prolantibiotic part, and an unusually hydrophilic leader peptide. The ribosomally synthesized pregallidermin is posttranslationally modified and processed to a complex peptide antibiotic with four sulfide rings and two unsaturated residues. The complete solution structure of gallidermin was determined in trifluoroethanol: water (95:5) and dimethylsulfoxide by two-dimensional 1H-nmr at 500 MHz, using a combination of double quantum filtered correlated spectroscopy, homonuclear Hartman-Hahn, and nuclear Overhauser enhancement spectroscopy experiments. Using a total number of 152 distance constraints from NOEs and 14 torsional constraints, derived from coupling constants, we obtained a screwlike solution structure of gallidermin. Restrained molecular dynamics simulations yielded a set of five converging structures with an atomic rms difference of 1.7 A for the backbone atoms, not dependent on the starting structure. The spatial structure model is in excellent agreement with the amphiphilic and channel-forming properties of gallidermin on membranes and its tryptic cleavage at the exposed site between residues 13 and 14.

Anti-Bacterial Agents↗
PubMed1989

Molecular dynamics of the alpha-helical epitope of a novel synthetic lipopeptide foot-and-mouth disease virus vaccine.

A novel synthetic foot-and-mouth disease virus (FMDV) peptide vaccine consisting of a synthetic B-cell and macrophage activator covalently linked to an amphiphilic alpha-helical T-cell epitope was developed. The low molecular weight vaccine of 3400 daltons is composed of virus VP1 antigenic determinant and the immunologically active lipotripeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P3CSS) as built-in adjuvant. The vaccine, tripalmitoyl-S-glyceryl-cysteinyl-seryl-seryl-FMDV-VP1 (VP1 = serotype O1K 135-154) induces protection against homologous challenge and serotype-specific virus neutralizing antibodies in guinea pigs after single administration without further adjuvants or carriers. A P3CSS conjugate with the FMDV-VP1 segment 135-154 of strain O Wuppertal produced only poor cross-protection against challenge with O1K virus. The antigenic determinant VP1(135-154) is an amphiphilic alpha-helix, as shown by CD. Molecular dynamics simulations (MDS) carried out using the highly homologous alpha-helical alcohol dehydrogenase (ADH) segment H3 as starting conformation for VP1(138-149) suggest that the FMDV segment 138-149 may adopt alpha-helical conformation during binding to its T-cell receptor, and that the development of the system during MDS may be considered as the dissociation step of the complex.

Amino Acid Sequence↗
PubMed1987

Terminal ileitis associated with Mycobacterium avium-intracellulare infection in a homosexual man with acquired immune deficiency syndrome.

A 38-yr-old homosexual man developed fever, diarrhea, and weight loss. An upper gastrointestinal examination revealed terminal ileitis, and stains of stool revealed acid-fast bacilli that were subsequently identified as Mycobacterium avium-intracellulare. Antimycobacterial therapy was associated with weight gain and loss of fever and diarrhea. Several months later, cutaneous Kaposi's sarcoma was observed. When the patient developed strictures in the terminal ileum, a surgical resection was performed. Numerous granulomas and acid-fast bacilli, later identified as M. avium-intracellulare, were present in the resected terminal ileum. This report demonstrates that infection of the terminal ileum with M. avium-intracellulare in a patient with acquired immune deficiency syndrome can present with a clinical and radiologic picture resembling Crohn's disease. It also demonstrates symptomatic improvement of this infection temporally related to the administration of antimycobacterial therapy and the ability of an acquired immune deficiency syndrome patient to tolerate major abdominal surgery.

Acquired Immunodeficiency Syndrome↗