Showing papers by "University of Marburg published in 1997"
1,098 citations
788 citations
TL;DR: The structure of the N‐terminal adenylation subunit in a complex with AMP and L‐phenylalanine indicates a conserved mechanism of nucleotide binding and substrate activation and reveals the role of the invariant residues within the superfamily of adenYLate‐forming enzymes.
Abstract: The non-ribosomal synthesis of the cyclic peptide antibiotic gramicidin S is accomplished by two large multifunctional enzymes, the peptide synthetases 1 and 2. The enzyme complex contains five conserved subunits of approximately 60 kDa which carry out ATP-dependent activation of specific amino acids and share extensive regions of sequence similarity with adenylating enzymes such as firefly luciferases and acyl-CoA ligases. We have determined the crystal structure of the N-terminal adenylation subunit in a complex with AMP and L-phenylalanine to 1.9 A resolution. The 556 amino acid residue fragment is folded into two domains with the active site situated at their interface. Each domain of the enzyme has a similar topology to the corresponding domain of unliganded firefly luciferase, but a remarkable relative domain rotation of 94 degrees occurs. This conformation places the absolutely conserved Lys517 in a position to form electrostatic interactions with both ligands. The AMP is bound with the phosphate moiety interacting with Lys517 and the hydroxyl groups of the ribose forming hydrogen bonds with Asp413. The phenylalanine substrate binds in a hydrophobic pocket with the carboxylate group interacting with Lys517 and the alpha-amino group with Asp235. The structure reveals the role of the invariant residues within the superfamily of adenylate-forming enzymes and indicates a conserved mechanism of nucleotide binding and substrate activation.
673 citations
TL;DR: Using a set of receptor-ligand complexes it is shown that LIGSITE is able to identify the binding sites of small molecule ligands with high precision and is well suited for identification of pockets in large sets of proteins for comparative studies.
Abstract: LIGSITE is a new program for the automatic and time-efficient detection of pockets on the surface of proteins that may act as binding sites for small molecule ligands. Pockets are identified with a series of simple operations on a cubic grid. Using a set of receptor-ligand complexes we show that LIGSITE is able to identify the binding sites of small molecule ligands with high precision. The main advantage of LIGSITE is its speed. Typical search times are in the range of 5 to 20 s for medium-sized proteins. LIGSITE is therefore well suited for identification of pockets in large sets of proteins (e.g., protein families) for comparative studies. For graphical display LIGSITE produces VRML representations of the protein-ligand complex and the binding site for display with a VRML viewer such as WebSpace from SGI.
660 citations
TL;DR: An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p, and is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm.
Abstract: An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p. In the thermosensitive mex67-5 mutant, poly(A)+ RNA accumulates in intranuclear foci shortly after shift to the restrictive temperature, but NLS-mediated nuclear protein import is not inhibited. In vivo, Mex67p tagged with green fluorescent protein (GFP) is found at the nuclear pores, but mutant mex67-5-GFP accumulates in the cytoplasm. Upon purification of poly(A)+ RNA derived from of UV-irradiated yeast cells, Mex67p, but not nucleoporins Nup85p and Nup57p, was crosslinked to mRNA. In a two-hybrid screen, a putative RNA-binding protein with RNP consensus motifs was found to interact with the Mex67p carboxy-terminal domain. Thus, Mex67p is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm.
521 citations
TL;DR: Guisse et al. as mentioned in this paper showed that under various conditions, the appearance of the K-step and the following dip, as well as the lowered maximum fluorescence level (FM) attainable, can be explained by an imbalance between the electron flow leaving the RC to the acceptor side and the electron flux coming to the RC from the donor side, which leads to a stable oxidation of the secondary electron donor, the tyrosine Z (YZ), and possibly to the accumulation of P680+.
Abstract: The chlorophyll a fluorescence transient measured under high light shows a typical O-J-I-P polyphasic rise. However, under certain stress situations such as heat or drought stress, a rapid phase with a maximum around 300 µs has been observed and called K (Guisse et al. (1995a) Arch Sci Geneve 48: 147–160). Here, we show that under various conditions, the appearance of the K-step and the following dip, as well as the lowered maximum fluorescence level (FM) attainable, can be explained by an imbalance between the electron flow leaving the RC to the acceptor side and the electron flow coming to the RC from the donor side. This leads to a stable oxidation of the secondary electron donor, the tyrosine Z (YZ), and possibly to the accumulation of P680+. In the case of heat stress, we confirm that this situation is caused by an inhibition of electron donation to YZ, which is due to a damaged oxygen evolving complex (OEC). Finally, we present a model which includes the OEC, YZ, P680, QA and QB which is in good agreement with the experimental data. The appearance of the K-step, under natural conditions, can now be used as a convenient stress indicator and specifically attributed to a damage on the electron donor side.
435 citations
TL;DR: The localization of COX-2 suggests that this enzyme may be primarily involved in the regulation of renal perfusion and glomerular hemodynamics, and its expression in podocytes of the fetal kidney and its absence in adult glomeruli suggest that this isoform might be involved in glomerulogenesis.
Abstract: To gain insight into the roles of cyclooxygenase (COX)-1 and -2 in human kidney, we analyzed their expressions and localization in adult and fetal normal kidney. Immunohistology showed expression of COX-1 in collecting duct cells, interstitial cells, endothelial cells, and smooth muscle cells of pre- and postglomerular vessels. Expression of COX-2 immunoreactive protein could be localized to endothelial and smooth muscle cells of arteries and veins and intraglomerularly in podocytes. In contrast to the rat, COX isoforms were not detected in the macula densa. These data were confirmed by in situ mRNA analysis using digoxigenin-labeled riboprobes. In fetal kidney, COX-1 was primarily expressed in podocytes and collecting duct cells. Expression levels of COX-1 in both cell types increased markedly from subcapsular to juxtamedullary cortex. Glomerular staining of COX-2 was detectable in podocytes only at the endstage of renal development. In summary, the localization of COX-2 suggests that this enzyme may be primarily involved in the regulation of renal perfusion and glomerular hemodynamics. The expression of COX-1 in podocytes of the fetal kidney and its absence in adult glomeruli suggests that this isoform might be involved in glomerulogenesis.
384 citations
TL;DR: The cloning and sequencing of the entire tyrocidine biosynthesis operon containing the tycA, tycB, and tycC genes are reported and strongly support the functional integrities of single domains within multifunctional peptide synthetases.
Abstract: The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.
294 citations
TL;DR: The data assign an important function to Atm1p in mitochondrial iron homeostasis, a mutant in which ATM1 was disrupted, which may be causative of the oxidative damage of heme‐containing proteins in Δatm1 cells.
287 citations
TL;DR: It is shown that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis.
Abstract: Neurotoxicity induced by overstimulation of N-methyl-d-aspartate (NMDA) receptors is due, in part, to a sustained rise in intracellular Ca2+; however, little is known about the ensuing intracellular events that ultimately result in cell death. Here we show that overstimulation of NMDA receptors by relatively low concentrations of glutamate induces apoptosis of cultured cerebellar granule neurons (CGNs) and that CGNs do not require new RNA or protein synthesis. Glutamate-induced apoptosis of CGNs is, however, associated with a concentration- and time-dependent activation of the interleukin 1β-converting enzyme (ICE)/CED-3-related protease, CPP32/Yama/apopain (now designated caspase 3). Further, the time course of caspase 3 activation after glutamate exposure of CGNs parallels the development of apoptosis. Moreover, glutamate-induced apoptosis of CGNs is almost completely blocked by the selective cell permeable tetrapeptide inhibitor of caspase 3, Ac-DEVD-CHO but not by the ICE (caspase 1) inhibitor, Ac-YVAD-CHO. Western blots of cytosolic extracts from glutamate-exposed CGNs reveal both cleavage of the caspase 3 substrate, poly(ADP-ribose) polymerase, as well as proteolytic processing of pro-caspase 3 to active subunits. Our data demonstrate that glutamate-induced apoptosis of CGNs is mediated by a posttranslational activation of the ICE/CED-3-related cysteine protease caspase 3.
276 citations
TL;DR: Growing evidence suggests that proteins may contribute directly to the spliceosome's active sites.
TL;DR: The existence of a distinct biallelic variation of the 5-HTTLPR in rhesus monkeys but similar allele and genotype frequencies in this species and humans supports the notion that there may be a relationship between functional 5- HT transporter expression, anxiety-related traits, and the complexity of socialization in human and nonhuman primate populations.
Abstract: By conferring allele-specific transcriptional activity on the 5-HT transporter gene promoter in humans, the 5-HT transporter gene-linked polymorphic region (5-HTTLPR) influences a constellation of personality traits related to anxiety and increases the risk for neurodevelopmental, neurodegenerative, and psychiatric disorders. Here we have analyzed the presence and variability of the 5-HTTLPR in several species of primates including humans, and other mammals. PCR, Southern blot, and sequence analyses of the 5-HT transporter gene's 5′-flanking region in different mammalian species confirmed the presence of the 5-HTTLPR in platyrrhini and catarrhini (hominoids, cercopithecoids) but not in prosimian primates and other mammals. Since the 5-HTTLPR is unique to humans and simian primates, a progenitor 5-HTTLPR sequence may have been introduced into the genome some 40 Mio. years ago. In humans the majority of alleles are composed of either 14 or 16 repeat elements, while alleles with 18 or 20 repeat elements are rare. In contrast, great apes including orang-utan, gorilla, and chimpanzee display a high prevalence of alleles with 18 and 20 repeat elements. In hominoids all alleles originate from variation at a single locus (polymorphic locus 1). In the 5-HTTLPR of rhesus monkeys (rh5-HTTLPR) we found an alternative locus for length variation (polymorphic locus 2) generated by a 21 bp insertion/deletion event. The existence of a distinct biallelic variation of the 5-HTTLPR in rhesus monkeys but similar allele and genotype frequencies in this species and humans supports the notion that there may be a relationship between functional 5-HT transporter expression, anxiety-related traits, and the complexity of socialization in human and nonhuman primate populations.
TL;DR: The ReC4Re complex (η5-C5Me5) as discussed by the authors is a 50:50 diastereomer mixture of ReC5Re and C4Re.
Abstract: Reaction of (η5-C5Me5)Re(NO)(PPh3)(C⋮CH) and Cu(OAc)2 (1.5 equiv, 80 °C, pyridine) gives the orange-brown ReC4Re complex (η5-C5Me5)Re(NO)(PPh3)(C⋮CC⋮C)(Ph3P)(ON)Re(η5-C5Me5) (2, 88%) as a 50:50 diastereomer mixture. Crystallization affords (SS,RR)-2·2CH2Cl2 and solutions enriched in (SR,RS)-2 (meso). Addition of Ag+PF6- (≥2 equiv) yields deep blue (SS,RR)- and (SR,RS)-22+2PF6- (86%), which give two geometric isomers (62:38, 89:11; CD2Cl2, −93 °C) about the +ReCCCCRe+ linkages. Crystal structures of (SS,RR)-2·2CH2Cl2 and (SS,RR)-22+2PF6- show ReC4Re units with bond angles (169−178°) and lengths (C⋮C, 1.202(7); C−C 1.398(5); CC 1.260−1.305(10) A) near those of butadiyne or cumulenes. Natural bond order analysis and topological electron density calculations confirm the valence formulations. Reactions of (SS,RR)-2 with (SS,RR)-22+2PF6- or Ag+PF6- (1 equiv) give a green radical cation (SS,RR)-2•+PF6- (μ = 1.74 μB; Faraday method). E° data yield a Kc value of 1.1 × 109 (CH2Cl2, 22.5 °C) for the comproportionati...
TL;DR: Two‐dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37°C and after cold shock, suggesting that C SPs down‐regulate production of members from this protein family.
Abstract: Like other bacteria, Bacillus subtilis possesses a family of homologous small acidic proteins (CspB, CspC and CspD, identity >70%) that are strongly induced in response to cold shock. We show that deletion of cspC or cspD genes did not result in a detectable phenotype; in contrast, csp double mutants exhibited severe reduction in cellular growth at 15 degrees C as well as at 37 degrees C, including impairment of survival during the stationary phase. Two-dimensional gel analysis showed that protein synthesis was deregulated in csp double mutants and that the loss of one or two CSPs led to an increase in the synthesis of the remaining CSP(s) at 37 degrees C and after cold shock, suggesting that CSPs down-regulate production of members from this protein family. A cspB/C/D triple mutant (64BCDbt) could only be generated in the presence of cspB in trans on a plasmid that was not lost, in spite of lack of antibiotic pressure, indicating that a minimum of one csp gene is essential for viability of B. subtilis. After cold shock, synthesis of CspB in 64BCDbt was drastically lower than in wild-type cells accompanied by cessation in growth and strong reduction in general protein synthesis. As CspB, CspC and CspD are shown to bind to RNA in a co-operative and interactive manner, CSPs are suggested to function as RNA chaperones facilitating the initiation of translation under optimal and low temperatures.
TL;DR: A further mechanism of 'respiratory control' via allosteric inhibition of cytochrome-c oxidase by ATP, which binds to the matrix domain, of subunit IV is described.
Abstract: The activity of cytochrome-c oxidase, the terminal enzyme of the mitochondrial respiratory chain, is known to be regulated by the substrate pressure, i.e. the ferro-/ferricytochrome c ratio, by the oxygen concentration, and by the electrochemical proton gradient delta muH+ across the inner mitochondrial membrane. Here we describe a further mechanism of 'respiratory control' via allosteric inhibition of cytochrome-c oxidase by ATP, which binds to the matrix domain, of subunit IV. The cooperativity between cytochrome-c-binding sites in the dimeric enzyme complex is mediated by cardiolipin, which is essential for cooperativity of the enzyme within the lipid membrane.
TL;DR: In this paper, the HERMES experiment at HERA was used for a measurement of the neutron spin structure function g1n(x, Q2) in deep inelastic scattering using 27.5 GeV longitudinally polarized positrons incident on a polarized 3He internal gas target.
TL;DR: The genes encoding the bacitracin synthetases BA1, BA2 and BA3 are organized in an operon that reflects the modular architecture expected of peptide synthetase, and a putative thiazoline ring formation domain was identified in the BA1 gene.
TL;DR: Plasma leptin concentrations were determined during inpatient treatment of 23 adolescent females with AN using a sensitive radioimmunoassay and set into relationship to leptin levels of females matched for age, body mass index (BMI) and/or percent body fat.
Abstract: Circulating leptin concentrations are known to be low in acute anorexia nervosa (AN), which is characterized by low weight, amenorrhea and specific psychopathological features. In this study plasma leptin concentrations were determined during inpatient treatment of 23 adolescent females with AN using a sensitive radioimmunoassay (RIA) and set into relationship to leptin levels of females matched for age, body mass index (BMI; kg m-2) and/or percent body fat. At referral patients had leptin concentrations well below the female controls. Weight gains led to steep increases of leptin levels which peaked at values well in excess of those observed in controls matched for BMI. In patients who reached the final treatment stage and who were followed-up after discharge, levels subsequently fluctuated and finally dropped into or below the control range. The low leptin levels at referral are likely to be involved in the pathogenesis of amenorrhea and the reduced metabolic state of acutely ill patients. Peak leptin levels reached after weight gain are possibly the cause of increased energy expenditure during this stage of the disorder.
TL;DR: H. pylori represents an important factor for the progression of fundic gastritis and the development of argyrophil cell hyperplasia during long-term treatment with lansoprazole in patients with peptic disorders resistant to H2-blockers.
TL;DR: Using excipients, such as hydroxypropyl-β-cyclodextrin, l -arginine, or bovine serum albumin (BSA), a distinct reduction of the formation of EPO aggregates could be achieved.
TL;DR: An evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein that is strikingly homologous to the ribosomal elongation factor EF‐2 (ribosomal translocase) is characterized and the first evidence that a G domain‐containing protein plays an essential role in the pre‐mRNA splicing process is shown.
Abstract: The driving forces behind the many RNA conformational changes occurring in the spliceosome are not well understood. Here we characterize an evolutionarily conserved human U5 small nuclear ribonucleoprotein (snRNP) protein (U5‐116kD) that is strikingly homologous to the ribosomal elongation factor EF‐2 (ribosomal translocase). A 114 kDa protein (Snu114p) homologous to U5‐116kD was identified in Saccharomyces cerevisiae and was shown to be essential for yeast cell viability. Genetic depletion of Snu114p results in accumulation of unspliced pre‐mRNA, indicating that Snu114p is essential for splicing in vivo. Antibodies specific for U5‐116kD inhibit pre‐mRNA splicing in a HeLa nuclear extract in vitro . In HeLa cells, U5‐116kD is located in the nucleus and co‐localizes with snRNP‐containing subnuclear structures referred to as speckles. The G domain of U5‐116kD/Snu114p contains the consensus sequence elements G1–G5 important for binding and hydrolyzing GTP. Consistent with this, U5‐116kD can be cross‐linked specifically to GTP by UV irradiation of U5 snRNPs. Moreover, a single amino acid substitution in the G1 sequence motif of Snu114p, expected to abolish GTP‐binding activity, is lethal, suggesting that GTP binding and probably GTP hydrolysis is important for the function of U5‐116kD/Snu114p. This is to date the first evidence that a G domain‐containing protein plays an essential role in the pre‐mRNA splicing process.
TL;DR: Observations indicate that the receptor affinity of FPV HA is controlled by oligosaccharides adjacent to the receptor binding site, which indicates that release from cell receptors depends on the presence of the HA glycans at Asn123 and Asn149.
Abstract: The hemagglutinin (HA) of the fowl plague virus (FPV) strain of influenza A virus has two N-linked oligosaccharides attached to Asn123 and Asn149 in the vicinity of the receptor binding site. The effect of these carbohydrate side chains on the binding of HA to neuraminic acid-containing receptors has been analyzed. When the oligosaccharides were deleted by site-specific mutagenesis, HA expressed from a simian virus 40 vector showed enhanced hemadsorbing activity. Binding was so strong under these conditions that erythrocytes were no longer released by viral neuraminidase and that release was significantly reduced when neuraminidase from Vibrio cholerae was used. Similarly, when these oligosaccharides were removed selectively from purified viruses by N-glycosidase F, such virions were unable to elute from receptors, although they retained neuraminidase activity. Thus, release of FPV from cell receptors depends on the presence of the HA glycans at Asn123 and Asn149. On the other hand, receptor binding was abolished when these oligosaccharides were sialylated after expression in the absence of neuraminidase (M. Ohuchi, A. Feldmann, R. Ohuchi, and H.-D. Klenk, Virology 212:77-83, 1995). These observations indicate that the receptor affinity of FPV HA is controlled by oligosaccharides adjacent to the receptor binding site.
TL;DR: Facial dominance can be an evolutionarily stable honest signal of dominant behavior, a crucial dimension of the potential for high stastus in a male dominance hierarchy, and may apply also to civilian populations.
Abstract: For a cohort of military officers, graduates of the class of 1950 of the United States Military Academy at West Point, dominant facial appearance was a cosnistent and important predictor of rank attainment at the academy and - for those who graduated from staff college - for high final rank. For men performing below the average, however, dominant facial appearance was a handicap for promotion. High rank came with high fitness. Thus, facial dominance can be an evolutionarily stable honest signal of dominant behavior, a crucial dimension of the potential for high stastus in a male dominance hierarchy. These findings may apply also to civilian populations.
TL;DR: It is found that subject-verb agreement is more impaired than tense marking, and that all SLI subjects achieve low scores for subject- verb agreement, which will cause acquisition problems for SLI children.
TL;DR: The results indicate that E. coli oxidises propionate to pyruvate via the methylcitrate cycle known from yeast.
Abstract: Escherichia coli grew in a minimal medium on propionate as the sole carbon and energy source. Initially a lag phase of 4–7 days was observed. Cells adapted to propionate still required 1–2 days before growth commenced. Incorporation of (2-13C), (3-13C) or (2H3)propionate into alanine revealed by NMR that propionate was oxidized to pyruvate without randomisation of the carbon skeleton and excluded pathways in which the methyl group was transiently converted to a methylene group. Extracts of propionate-grown cells contained a specific enzyme that catalyses the condensation of propionyl-CoA with oxaloacetate, most probably to methylcitrate. The enzyme was purified and identified as the already-known citrate synthase II. By 2-D gel electrophoresis, the formation of a second propionate-specific enzyme with sequence similarities to isocitrate lyases was detected. The genes of both enzymes were located in a putative operon with high identities (at least 76% on the protein level) with the very recently discovered prp operon from Salmonella typhimurium. The results indicate that E. coli oxidises propionate to pyruvate via the methylcitrate cycle known from yeast. The 13C patterns of aspartate and glutamate are consistent with the further oxidation of pyruvate to acetyl-CoA. Oxaloacetate is predominantly generated via the glyoxylate cycle rather than by carboxylation of phosphoenolpyruvate.
TL;DR: The capacity of brain cells to synthesize different cytokine mRNAs in vivo is shown and the kinetics of their expression in several brain areas and in the periphery in parallel to the activation of a neuroendocrine pathway by endotoxin is defined.
Abstract: The regional distribution and inducibility of cytokines in the normal brain is still a matter of controversy. As an attempt to clarify this issue, we studied the constitutive and induced expression of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ mRNAs in the brain, pituitary, and spleen of mice using qualitative and semiquantitative reverse-transcription polymerase chain reaction. The contribution of nonbrain cells to the cytokine transcripts detected was considered. With the exception of IFN-γ mRNA, transcripts for the other cytokines were found to be constitutively present in the brain. Following i.p. injection of lipopolysaccharide (LPS) at a dose below those described to disrupt the blood-brain barrier (BBB), cytokine mRNA expression was increased in the spleen, the pituitary, and the brain. In the brain, the onset of transcription varied from 45 min (IL-1β, TNF-α) to 4 hr (IFN-γ), and the peak of mRNA accumulation was observed at different times depending on the cytokine and the brain region studied. IL-1 and IL-6 were highly expressed in the hypothalamus and hippocampus, while TNF-α expression was more marked in the thalamus-striatum. The cortex was the region in which cytokines were less inducible. The inducible expression of cytokine mRNAs in the brain was paralleled by stimulation of hypothalamus-pituitary-adrenal axis. These results show the capacity of brain cells to synthesize different cytokine mRNAs in vivo and define the kinetics of their expression in several brain areas and in the periphery in parallel to the activation of a neuroendocrine pathway by endotoxin. J. Neurosci. Res. 48:287–298, 1997. © 1997 Wiley-Liss, Inc.
TL;DR: The results suggest that the topography of slow ERPs reflects the relative activation/inactivation of distinct cortical cell assemblies while the absolute amplitude of the negative maximum seems to reflect how much a particular cell assembly is activated at a particular time.
TL;DR: It is shown that cyclin E/cdk2 phosphorylates p27 at a carboxy-terminal threonine residue (T187) in vitro; mutation of this residue to valine stabilises cyclin W/w/w complexes and suggests that cdk2 bound to p27 is catalytically active.
Abstract: Activation of Myc triggers a rapid induction of cyclin E/cdk2 kinase activity and degradation of p27. Overt degradation of p27 is preceded by a specific dissociation of p27 from cyclin E/cdk2, but not from cyclin D/cdk4 complexes. We now show that cyclin E/cdk2 phosphorylates p27 at a carboxy-terminal threonine residue (T187) in vitro; mutation of this residue to valine stabilises cyclin E/cdk2 complexes. This reaction is not significantly inhibited by high concentrations of p27, suggesting that cdk2 bound to p27 is catalytically active. In vivo, p27 bound to cyclins E and A, but not to D-type cyclins is phosphorylated. Myc-induced release of p27 from cdk2 requires cdk2 kinase activity and is delayed in a T187V mutant of p27. After induction of Myc, p27 phosphorylated at threonine 187 transiently accumulates in a non cdk2 bound form. Our data suggest a mechanism in which p27 is released from cyclin E/cdk2 upon phosphorylation; in Myc-transformed cells, release is efficient as phosphorylated p27 is transiently bound in a non-cdk2 containing complex and subsequently degraded.
TL;DR: The green fluorescent protein (GFP) was expressed as a fusion protein with the putative nuclear localization domain of the transcriptional activator stuA in nuclei of Aspergillus nidulans and visualized these organelles in living cells to study nuclear positioning and the role of ApsA.
Abstract: Nuclear migration and nuclear positioning are fundamental processes in all eukaryotic cells. They are easily monitored during hyphal growth of filamentous fungi. We expressed the green fluorescent protein (GFP) as a fusion protein with the putative nuclear localization domain of the transcriptional activator stuA in nuclei of Aspergillus nidulans and visualized these organelles in living cells. Nuclear staining was observed in interphase nuclei but not during mitosis. Nuclear division, nuclear migration, septum formation and branching were analysed with time-lapse video microscopy during hyphal extension. Hyphae elongated at 0.1–1.2 μm min−1 and nuclei moved with similar speeds towards the hyphal tip until they had reached a defined position. An individual regulation of nuclear mobility in a given hyphal compartment was observed. Some representative movies are available on the Internet (http://www.blackwell-science.com/products/journals/molextra.htm). Nuclear positioning was also studied at the molecular level. The ApsA protein, which regulates nuclear migration, was localized at the cytoplasmic membrane in germlings and hyphae by immunofluorescence and GFP tagging. A model of nuclear migration, nuclear positioning and the role of ApsA is presented.
TL;DR: In two GCPS cases, both of which did not exhibit obvious cytogenetic rearrangements, point mutations were identified in different domains of the protein, showing for the first time that Greig syndrome can be caused by GLI3 point mutations.
Abstract: Greig cephalopolysyndactyly syndrome (GCPS, MIM 175700) is a rare autosomal dominant developmental disorder characterized by craniofacial abnormalities and post-axial and pre-axial polydactyly as well as syndactyly of hands and feet. Human GLI3, located on chromosome 7p13, is a candidate gene for the syndrome because it is interrupted by translocation breakpoints associated with GCPS. Since hemizygosity of 7p13 resulting in complete loss of one copy of GLI3 causes GCPS as well, haploinsufficiency of this gene was implicated as a mechanism to cause this developmental malformation. To determine if point mutations within GLI3 could be responsible for GCPS we describe the genomic sequences at the boundaries of the 15 exons and primer pair sequences for mutation analysis with polymerase chain reaction-based assays of the entire GLI3 coding sequences. In two GCPS cases, both of which did not exhibit obvious cytogenetic rearrangements, point mutations were identified in different domains of the protein, showing for the first time that Greig syndrome can be caused by GLI3 point mutations. In one case a nonsense mutation in exon X generates a stop codon truncating the protein in the C-H link of the first zinc finger. In the second case a missense mutation in exon XIV causes a Pro-->Ser replacement at a position that is conserved among GLI genes from several species altering a potential phosphorylation site.