Perspectives: structural biology. SRP--where the RNA and membrane worlds meet.
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Amino Acid Motifs↗
Biomedical subjects
U Schmitz
Publications and source records attributed to U Schmitz.
One-dimensional imaging with a palm-size probe.
A new portable magnetic resonance imaging device was built. Spatially resolved NMR was achieved by placing a gradient coil pair and a Helmholtz pair type radio-frequency probe in the gap between two antiparallel polarized permanent magnets. The flat face of the low-field (nu(proton) = 20 MHz) apparatus allowed for the study of arbitrarily large objects in situ. Relaxation time weighted 1D images were achieved over a 15-mm field of view by a single-point spin-echo sequence. A phase encoding time on the order of 200 micros permited the scanning of a wide range of heterogeneous materials.
Cost-Benefit Analysis↗
Angiotensin II-induced growth of vascular smooth muscle cells requires an Src-dependent activation of the epidermal growth factor receptor.
BACKGROUND: Angiotensin II (Ang II) is a potent stimulus of vascular smooth muscle cell (VSMC) growth. Activation of extracellular signal-regulated kinase (ERK), the archetypal mitogen-activated protein (MAP) kinase, and phosphatidylinositol 3 (PI3) kinase are critical steps in Ang II-induced mitogenic signaling. However, the mechanism involved in the activation of these kinases upon binding of Ang II to its receptor is poorly understood. METHODS: In the present study, we examined the role of the epidermal growth factor receptor (EGFR) in Ang II signaling in VSMCs employing immunoprecipitation, Western blot analysis, kinase immunocomplex assay, and [3H]-thymidine incorporation. RESULTS: A time-dependent tyrosine phosphorylation of the EGFR in response to Ang II was observed that was mediated by the Ang II type 1 receptor. This transactivation of the EGFR was blocked in the presence of PP1, an inhibitor of the intracellular Src-like tyrosine kinases. The tyrphostin AG 1478, a selective EGFR antagonist, inhibited both Ang II- and EGF-induced tyrosine phosphorylation of the EGFR. Furthermore, Ang II induced the binding of the adaptor protein Shc to the EGFR, leading to phosphorylation of Shc. In addition, the same nanomolar concentrations of AG 1478 that were effective in EGF signaling blocked the Ang II-induced activation of ERK and PI3 kinase in a dose-dependent manner. Proliferation of VSMCs, detected by measurements of DNA synthesis, following stimulation with Ang II was potently inhibited in the presence of AG 1478 or PP1. CONCLUSION: Our data suggest that EGFR serves as a role in mitogenic signaling following stimulation with Ang I through a ligand-independent and Src-dependent transactivation of the EGFR. Furthermore, we demonstrate this transactivation as a pivotal step in Ang II-induced activation of MAP kinase and PI3 kinase, as well as growth of VSMCs.
Angiotensin II↗
Effects of captopril on fibrinolytic function in healthy humans.
Several lines of evidence point to an interrelation of the renin angiotensin system (RAS) with the endogenous fibrinolytic system. In the present study, we have therefore investigated the effect of the ACE-inhibitor captopril on various parameters of the fibrinolytic system in healthy volunteer subjects. 10 male subjects aged 28-38 years were given captopril 25 mg b.i.d. over 2 weeks. Venous blood was drawn before and at the end of the treatment period at 09.00 AM, after the volunteers had received their last dose of captopril by 07. 30 AM. Blood samples were processed for the determination of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). Both parameters were determined with respect to their abundance (as antigen concentrations) and function (activity). In addition, the concentration and activity of the von Willebrand factor were also determined. Two weeks of captopril treatment had no significant effect on any of the above mentioned parameters. Our results thus show that short-term treatment with the ACE-inhibitor captopril, at least in healthy subjects on an unrestricted NaCl intake, does not affect the fibrinolytic balance between t-PA and PAI-1 or the von Willebrand factor.
Adult↗
[Reliability of functional x-ray analysis of cervical vertebrae flexion and extension].
Functional radiographic analysis of the cervical spine in flexion and extension position is increasingly used as screening method for the diagnosis of segmental functional disorders. The objective of this study was to prove Penning's evaluation method for the metrical recording of segmental angles in the sagittal plane for selectivity, reliability and usability. Passive functional radiographs of the cervical spine in flexion and extension were taken of 20 patients with painful limitation of mobility of the cervical spine and 20 subjects, similar in sex and age, without complaints. The radiographs were duplicated. Five physicians measured the angles of segmental mobility in a blind study. Statistical analysis was conducted using the t-test and calculating the correlation coefficient "r". The results of the study prove that the evaluation method by Penning shows a usable and, for segments C3/C4 to C6/C7, significant selectivity. The selectivity of p < or = 0.05 and p < or = 0.01 is sufficient to distinguish patients from healthy subjects. The correlation between the five reviewers showed good to very good results (0.6 < r < or = 0.8; r > 0.8). The measured values, however, have to be considered, in connection with the appropriate clinical symptoms, as still "normal" or "functionally disordered" in the context of segmental hypo-resp. hypermobility.
Adult↗
Lysophosphatidic acid stimulates protein kinase C isoforms alpha, beta, epsilon, and zeta in a pertussis toxin sensitive pathway in vascular smooth muscle cells.
The natural phospholipid lysophosphatidic acid (LPA) has been characterized as an important vascular smooth muscle cell (VSMC) mitogen whose effects are mainly mediated by pertussis toxin (PTX)-sensitive guanosine triphosphate (GTP)-binding protein (Gi-protein). Protein kinase C (PKC) isoforms play an important role in intracellular signaling cascades and in growth of VSMC. In the present study we investigated the effect of LPA on activation of PKC isoforms alpha, beta, epsilon, and zeta in VSMC by Western blot of cytosolic and membrane fractions. Furthermore, we examined the role of PKC activation on LPA-induced growth of VSMC using PKC inhibitor 19-27. Stimulation of VSMC by 5 microg/mL LPA for 10 min increased the amount of PKC alpha, beta, epsilon, and zeta in the particulate fraction by 689%, 285%, 424%, and 510%, respectively, and returned to control level after 30 min. Correspondingly, the amount of PKC alpha, beta, epsilon, and zeta in the cytosolic fraction decreased by 32%, 94%, 44%, and 95%, respectively, compared to control. Furthermore, we could show that LPA-induced activation of PKC alpha, beta, epsilon, and zeta isoforms was PTX sensitive. Incubation of VSMC with nonspecific PKC inhibitor 19-27 (10 micromol/L) for 24 h resulted in a 30% inhibition of LPA-induced DNA synthesis as measured by [3H]thymidine incorporation. In conclusion, in VSMC LPA stimulated translocation of PKC isoforms alpha, beta, epsilon, and zeta in a PTX-sensitive manner. Furthermore stimulation of PKC might be critically involved in LPA-induced mitogenesis in VSMC.
Animals↗
Structure of the phylogenetically most conserved domain of SRP RNA.
The signal recognition particle (SRP) is a phylogenetically conserved ribonucleoprotein required for cotranslational targeting of proteins to the membrane of the endoplasmic reticulum of the bacterial plasma membrane. Domain IV of SRP RNA consists of a short stem-loop structure with two internal loops that contain the most conserved nucleotides of the molecule. All known essential interactions of SRP occur in that moiety containing domain IV. The solution structure of a 43-nt RNA comprising the complete Escherichia coli domain IV was determined by multidimensional NMR and restrained molecular dynamics refinement. Our data confirm the previously determined rigid structure of a smaller subfragment containing the most conserved, symmetric internal loop A (Schmitz et al., Nat Struct Biol, 1999, 6:634-638), where all conserved nucleotides are involved in nucleotide-specific structural interactions. Asymmetric internal loop B provides a hinge in the RNA molecule; it is partially flexible, yet also uniquely structured. The longer strand of internal loop B extends the major groove by creating a ledge-like arrangement; for loop B however, there is no obvious structural role for the conserved nucleotides. The structure of domain IV suggests that loop A is the initial site for the RNA/protein interaction creating specificity, whereas loop B provides a secondary interaction site.
Base Sequence↗
Structure of the most conserved internal loop in SRP RNA.
The signal recognition particle (SRP) directs translating ribosomes to the protein translocation apparatus of endoplasmic reticulum (ER) membrane or the bacterial plasma membrane. The SRP is universally conserved, and in prokaryotes consists of two essential subunits, SRP RNA and SRP54, the latter of which binds to signal sequences on the nascent protein chains. Here we describe the solution NMR structure of a 28-mer RNA composing the most conserved part of SRP RNA to which SRP54 binds. Central to this function is a six-nucleotide internal loop that assumes a novel Mg2+-dependent structure with unusual cross-strand interactions; besides a cross-strand A/A stack, two guanines form hydrogen bonds with opposite-strand phosphates. The structure completely explains the phylogenetic conservation of the loop bases, underlining its importance for SRP54 binding and SRP function.
Base Sequence↗
Early intracellular signalling pathway of ethanol in vascular smooth muscle cells.
1. ERKs belong to MAP kinase family and are activated by several growth and stress factors. Although ethanol has been shown to modulate ERK1 and ERK2 (p44(mapk) and p42(mapk)) activity, it can also act as an antiproliferative agent in various mammalian cells. Since the nature of the antiproliferative effect of ethanol in VSMCs has not been defined, we examined its effects on growth and on early intracellular events normally induced by growth factors in VSMCs. 2. Measurement of cytosolic Ca(2+) and pH in cell monolayers was performed using fura-2/AM and BCECF/AM, respectively. The effect of ethanol on VSMCs growth was assessed by [(3)H]-thymidine incorporation, by cell counting and by determination of the caspase 3 activity. Stimulation of ERK1 and ERK2 was examined by the chemiluminescence Western blotting method. The expression of c-fos was quantitated by Northern blotting. Determination of inositolphosphates was performed after labelling of VSMCs with myo-[2-(3)H]-inositol and separation of inositolphosphates by HPLC. 3. Ethanol (0.3 - 1.0% v v(-1), 17 - 170 mM) induced a dose-dependent maximal stimulation of p44(mapk)/p42(mapk) at 30 min and expression of c-fos mRNA with a maximum at 120 min. Intracellular events upstream to MAP kinase, like an increase in [Ca(2+)](i), activation of the Na(+)/H(+) exchanger and formation of phosphoinositol metabolites were also markedly activated by ethanol. Treatment of VSMCs with ethanol for 3 - 5 min induced an increase in DNA synthesis whereas treatment of the cells for more than 30 min was toxic. Caspase 3 activity was not modulated by ethanol treatment of VSMCs. 4. We may postulate that the activation of these mitogenic signals including the elevation of DNA synthesis reflects a cell effort to protect itself against the toxic effects of ethanol.
Animals↗
Evidence that lipoproteins are carriers of bioactive factors.
We recently demonstrated that the mitogenic effect of LDL (100 microg/mL) as well as its early intracellular signaling pathway are mediated by a pertussis-toxin (PTX)-sensitive G(i) protein-coupled receptor that is independent from its classical receptor and involves activation of extracellular response kinases (ERK1/2) (also known as p44(mapk)/p42(mapk)). In the present study we examined whether LDL-adherent factors may be responsible for some of the effects of LDL. The term "signaling activity" is used to characterize fractions that cause an increase in intracellular free Ca(2+) concentration or stimulate ERK1/2 and c-fos mRNA expression. LDL, HDL, and VLDL stimulate ERK1/2 with the following order of potency: LDL>HDL>VLDL. After delipidation of LDL with chloroform/methanol/water mixtures a PTX-sensitive signaling activity was found in one fraction arbitrarily called LDL-F. After further analysis of LDL-F compounds by high pressure liquid chromatography, a PTX-sensitive signaling activity was detected only in the fraction with a retention time of 33 minutes (arbitrarily called LDL-F33). Similarly, after separation of sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPC) by high pressure liquid chromatography, a PTX-sensitive signaling activity was found in the fractions 33 and 33 to 35, respectively. These findings demonstrate that the effects of LDL-F33 are mimicked by similar fractions collected from SPP/SPC, hence suggesting that these LDL-adherent molecules are possibly closely related to SPP/SPC. A PTX-sensitive signaling activity was also detected in HDL and HDL-F33. Therefore, LDL and other lipoproteins may function as carriers for bioactive phospholipids thereby contributing to the development of coronary artery disease. Our findings support a new research concept that may contribute in elucidating cellular mechanisms promoting coronary artery disease.
Animals↗
Small structural ensembles for a 17-nucleotide mimic of the tRNA T psi C-loop via fitting dipolar relaxation rates with the quadratic programming algorithm.
Solution structures are typically average structures determined with the help of nmr-derived distance and torsion angle information. However, when a biomolecule populates significantly different conformations, the average structure might be prone to artifacts, and other refinement strategies are necessary. For example, when experimental restraints are used in molecular dynamics simulations in a time-averaged fashion (MDtar), the experimental structural information does no longer need to be satisfied at each step of the simulation; instead, the whole trajectory must agree with the restraints. However, the resulting structural ensembles are large and not unique and it is not trivial to extract the essential dynamic features for a system. Here we demonstrate that large MDtar ensembles can be simplified substantially by reducing the number of members to just a few on the basis of adjusting the individual probabilities of the members with the PDQPRO program [N. B. Ulyanov et al. Biophysical Journal (1995), Vol. 68, p. 13]. This algorithm finds the global minimum for a search function that represents the best match of a given ensemble with the experimental dipolar interproton relaxation rates. We have applied this strategy to a 17-residue RNA hairpin, whose loop exhibited considerable flexibility evident from nmr data. This 17mer is a mimic of the T psi C-loop of tRNA, where nucleotide 54 is usually a ribosylthymidine. The methylation of U54, which is completely buried in transfer ribonucleic acid, is administered by tRNA (m5 U54)-methyltransferase (RUMT). Since the 17mer is a good substrate for RUMT, we previously concluded that the flexibility of the 17mer's loop is a key to how RUMT gains access to the methylation site [L. J. Yao et al. Journal of Biomolecular NMR (1996) Vol. 9. p. 229]. Application of the PDQPRO algorithm to the previously acquired MDtar trajectories allowed us to reduce the number of conformations from several hundred to one major and five or six minor conformations with individual populations from approximately 5% to approximately 50% without any deterioration in the match with the experimental data. The major conformation exhibits a continuation of A-form helicity through part of the loop, involving C60 and U59. In this and most other conformations the methylation site in U54 is no longer buried.
Algorithms↗
Angiotensin II stimulates p21-activated kinase in vascular smooth muscle cells: role in activation of JNK.
Angiotensin II (Ang II) has been previously shown to stimulate the extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinase family members. Little is known regarding the upstream signaling molecules involved in Ang II-mediated JNK activation. Ang II has been shown to activate the Janus kinase/signal transducer(s) and activator(s) of transcription (JAK/STAT) pathway, suggesting similarities to cytokine signaling. In response to cytokines such as interleukin-1 and tumor necrosis factor-alpha, the p21-activated kinase (PAK) has been identified as an upstream component in JNK activation. Therefore, we hypothesized that PAK may be involved in JNK activation by Ang II in vascular smooth muscle cells (VSMCs). AlphaPAK activity was measured by myelin basic protein phosphorylation in rat aortic VSMCs. In response to Ang II, alphaPAK was rapidly stimulated within 1 minute, with a peak (5-fold increase) at 30 minutes. AlphaPAK stimulation preceded activation of JNK in VSMCs. Ang II-mediated activation of both alphaPAK and JNK was Ca2+ dependent and inhibited by downregulation of phorbol ester-sensitive protein kinase C isoforms (by pretreatment with phorbol 12,13-dibutyrate) but not by pretreatment with GF109203X. Activation of both PAK and JNK was partially inhibited by tyrosine kinase inhibitors but not by specific Src inhibitors, suggesting regulation by a tyrosine kinase other than c-Src. Finally, introduction of dominant negative PAK markedly reduced the JNK activation by Ang II in both Chinese hamster ovary and COS cells stably expressing the Ang II type 1 receptor (AT1R). Our data provide evidence for alphaPAK as an upstream mediator of JNK in Ang II signaling and extend the role of Ang II as a proinflammatory mediator for VSMCs.
Angiotensin II↗
Computer-assisted molecular design for the determination of structure-activity relationships for chemopreventive agents.
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Antineoplastic Agents↗
The dynamic NMR structure of the T psi C-loop: implications for the specificity of tRNA methylation.
tRNA (m5U54)-methyltransferase (RUMT) catalyzes the S-adenosylmethionine-dependent methylation of uridine-54 in the T psi C-loop of all transfer RNAs in E. coli to form the 54-ribosylthymine residue. However, in all tRNA structures, residue 54 is completely buried and the question arises as to how RUMT gains access to the methylation site. A 17-mer RNA hairpin consisting of nucleotides 49-65 of the T psi-loop is a substrate for RUMT. Homonuclear NMR methods in conjunction with restrained molecular dynamics (MD) methods were used to determine the solution structure of the 17-mer T-arm fragment. The loop of the hairpin exhibits enhanced flexibility which renders the conventional NMR average structure less useful compared to the more commonly found situation where a molecule exists in predominantly one major conformation. However, when resorting to softer refinement methods such as MD with time-averaged restraints, the conflicting restraints in the loop can be satisfied much better. The dynamic structure of the T-arm is represented as an ensemble of 10 time-clusters. In all of these, U54 is completely exposed. The flexibility of the T psi-loop in solution in conjunction with extensive binding studies of RUMT with the T psi C-loop and tRNA suggest that the specificity of the RUMT/ tRNA recognition is associated with tRNA tertiary structure elements. For the methylation, RUMT would simply have to break the tertiary interactions between the D- and T-loops, leading to a melting of the T-arm structure and making U54 available for methylation.
Base Sequence↗
Angiotensin II stimulates tyrosine phosphorylation of phospholipase C-gamma-associated proteins. Characterization of a c-Src-dependent 97-kD protein in vascular smooth muscle cells.
Stimulation of phospholipase C-gamma (PLC-gamma) is a critical event in angiotensin II (Ang II) signal transduction. We have previously shown that in rat aortic smooth muscle (RASM) cells Ang II stimulates tyrosine phosphorylation of PLC-gamma via activation of c-Src. Because we failed to demonstrate a direct association between c-Src and PLC-gamma, we hypothesized that a linker protein mediates the interaction between these molecules. To identify PLC-gamma-associated proteins, RASM cells were labeled with [32P]orthophosphate and stimulated with 100 nmol/L Ang II for 5 minutes. PLC-gamma was immunoprecipitated, and associated proteins were characterized by autoradiography and Western blotting with anti-phosphotyrosine antibodies. Ang II stimulated the phosphorylation of 47-, 60-, 84-, and 97-kD PLC-gamma-associated proteins. Because Ang II increased tyrosine phosphorylation of only the 97-kD protein, we characterized p97 further. An important role for Src in tyrosine phosphorylation of p97 was suggested by findings that p97 phosphorylation was inhibited by the selective Src-family kinase inhibitor CP-118,556, diminished in mouse aortic smooth muscle (MASM) cells from c-Src knockout mice compared with wild-type MASM cells, and increased in v-Src-transformed NIH-3T3 cells compared with wild-type NIH-3T3 cells. These studies are the first to define a PLC-gamma-associated protein that may be required for Ang II-mediated signal transduction.
Angiotensin II↗
An extended nomenclature of the canine karyotype.
In contrast to many other animals, knowledge about the canine karyotype is quite sparse. This is due in part to the rather difficult canine karyotypic pattern. Except for the X and the Y chromosome, there are only acrocentric chromosomes, which appear to be quite small and difficult to identify unambiguously. In previous reports, schematic representations of the canine karyotype have been described. However, a nomenclature comparable to that of the human karyotype or the karyotypes of sheep, cattle, or goats does not yet exist for the dog. Based on high-resolution banding of metaphase chromosomes from canine fibroblasts, we propose an ideogram of the canine karyotype with 460 numbered bands and characteristic landmarks. In addition, the centromere positions of the canine chromosomes are determined by a combined GTG-banding/FISH approach, and the R- and G-banding patterns are compared.
Animals↗
NMR studies of the most conserved RNA domain of the mammalian signal recognition particle (SRP).
Mammalian signal recognition particle (SRP) and its homologues exhibit a phylogenetically conserved RNA domain, whose predicted secondary structure exhibits a hairpin motif with two bulged regions. Two RNA fragments comprising one (24 nt) or two (43 nt) of the conserved bulges were studied. Each fragment binds specifically to the domain of the Escherichia coli homologue of the SRP54 protein, which is involved in signal sequence recognition. The SRP RNA fragments exhibited a pronounced structural stabilization in the presence of Mg2+. Assignments of all base, H1', H2', and most imino proton resonances in the presence of Mg2+ were obtained for the 24mer RNA via NOE spectroscopy and correlated homonuclear NMR methods. 2D NOE patterns permitted a coarse structural description, revealing a relatively compact A-type geometry for the 24mer without any indications of looped-out nucleotides, syn-oriented bases, or base triplets. The GGAA-loop is structurally very similar to that of the GCAA tetraloop [Heus HA, Pardi A, 1991, Science 253:191-194]. Mg2+ seems to stabilize the structure of the conserved bulged region, which involves G:A and C:A mismatch pairs. Deviations from ideal A-type helicity are found for a larger region than the predicted secondary structure implies. Although no explicit assignment effort has been dedicated to the 43mer yet, striking similarity in chemical shift changes upon addition of Mg2+ allowed some structural conclusions. The bulge present in both RNA fragments exhibits a similar, pronounced flexibility in the absence of Mg2+, indicating that the additional bulge in the 43mer does not stabilize the other bulge.
Binding Sites↗
[Orthostatic hypotension].
We report about a 67-year-old woman presenting with progressive orthostatic vertigo, urinary incontinence and clinical signs of Parkinson's disease. The Schellong test revealed deficient sympathetic orthostatic pressure response without an increase of plasma norepinephrine; therefore, a Shy-Drager syndrome was diagnosed. Because of inefficiency of the general measures (compressive pantyhose), the sympathomimetic agonists, and the centrally active alpha-2-antagonists, norepinephrine was administered via a miniature dosing pump. By this therapeutic regimen a marked improvement of orthostatic hypotension was achieved.
Aged↗