The RNA-binding domain of ribosomal protein L11 recognizes an rRNA tertiary structure stabilized by both thiostrepton and magnesium ion.

Antibiotics that inhibit ribosomal function may do so by one of several mechanisms, including the induction of incorrect RNA folding or prevention of protein and/or RNA conformational transitions. Thiostrepton, which binds to the 'GTPase center' of the large subunit, has been postulated to prevent conformational changes in either the L11 protein or rRNA to which it binds. Scintillation proximity assays designed to look at the binding of the L11 C-terminal RNA-binding domain to a 23S ribosomal RNA (rRNA) fragment, as well as the ability of thiostrepton to induce that binding, were used to demonstrate the role of Mg(2+), L11 and thio-strepton in the formation and maintenance of the rRNA fragment tertiary structure. Experiments using these assays with both an Escherichia coli rRNA fragment and a thermostable variant of that RNA show that Mg(2+), L11 and thiostrepton all induce the RNA to fold to an essentially identical tertiary structure.

Senescence-induced alteration in cell surface carbohydrates correlated using proton NMR spectroscopy and a lectin-based affinity-binding assay.

Changes in the cell surface oligosaccharides in human fetal lung fibroblasts (IMR-90) are studied as the cells progress to senescence using nuclear magnetic resonance spectroscopy (NMR) and a biochemical assay. A lectin-based affinity-binding technique is used which measures the organization of carbohydrates on the cell surface. Proton NMR studies of the water in samples of frozen cell suspensions of young and old cells provide information on the local dynamics of the cell surface by monitoring the motion of bound water. Changes in the lectin binding density and affinity class distribution correlate with a decrease in the water proton linewidth in frozen cells. These observations reflect alterations in the conformation or structure of the cell surface oligosaccharides and local constituent water.

Galactose neuropathy. Structural changes evaluated by nuclear magnetic resonance spectroscopy.

We examined the hypothesis that the polyol accumulation resulting from chronic galactose supplementation in the diet produces endoneurial edema that can be prevented by inhibition of aldose reductase. We explored the potential of nuclear magnetic resonance (NMR) spectroscopy to quantitate and characterize the water accumulation in the sciatic nerve in this "galactose neuropathy." The data demonstrate a 16% increase in gravimetrically determined total water content of nerve in the galactose-fed rat after 8 mo of this diet and a 50% increase in the T1 relaxation time for nerve water as determined by NMR spectroscopy. Prolongation of the T1 relaxation time reflects increased rotation of water in a magnetic field, consistent with an extracellular site of the additional water. Simultaneous feeding of sorbinil to inhibit aldose reductase resulted in normalization of both total nerve water and of the prolongation of T1 relaxation time. These data define the NMR-spectroscopic state of endoneurial edema in the galactose-fed rat and suggest specific application to the investigation of the role of aldose reductase in human diabetic neuropathy.

Cell surface oligosaccharide modulation during differentiation: VI. The effect of biomodulation on the senescent and neoplastic cell phenotype.

These investigations test the hypothesis developed previously, that there are biomolecules which control and integrate cellular differentiation. Our specific interest in cellular differentiation lies in the area of what we refer to as basal or primitive cellular differentiation mechanisms. These mechanisms are common to all cells, and are required for simple recognition and growth regulation. We have investigated two models, the IMR-90 human fetal lung fibroblast model as a representative of normal growth control, and the CG model, canine glioma cells, a transplantable growth transformed cell line. These two models represent normal, and aberrant cellular differentiation control. In previous studies we have shown that the arrangement of the cell surface oligosaccharide structure on these cell types are predictive of phenotypic transition. We have developed, and partially characterized a series of BIOMODULATORS (BM) which delay the onset of display of neoplastic cells. Three classes of BIOMODULATOR have been explored; (1) a large molecular weight natural product (25-35 kDa), PokeWeed Mitogen (PWM); (2) a small molecular weight natural product (500 Da) Cellular Activator and Differentiator (CAD) and a number of natural and synthetic analogs; and (3) an indolizidine alkaloid natural product, Swainsonine (Sw) which has a known cellular target (oligosaccharide biosynthesis). Preliminary data is presented which structurally links some of these BIOMODULATORS in terms of their effective stereochemistry. These BIOMODULATORS, when used before PDL 38, prevent the cell surface oligosaccharide display changes typical of morphological senescence and delay their onset to PDL 100 or more. These BIOMODULATORS also appear to have regulatory effects on the neoplastic cell models. This re-regulation results in increases in generation time and an increase in the ability of these cells to be recognized by cytotoxic lymphocytes. Proton NMR linewidth measurements of the fraction of 'bound' water associated with the cellular surface of treated and untreated cell populations showed induced physical changes in the cell surface related to the use of the BIOMODULATOR and correlated to the oligosaccharide display changes. These data were interpreted as indicating an increase in the organizational level of these cells. The data for normal and neoplastic cell populations are compared and contrasted in an effort to form the basis for an analytical approach to the control and integration of differentiation mechanisms.

Cell surface oligosaccharide modulation during differentiation: V. Partial characterization of the regulated surface during substrate adhesion and spreading.

In previous studies with IMR-90 human fetal lung fibroblasts, it was shown that modulation of a small finite number of unique cell surface oligosaccharide structural specificities (as defined by reaction with specific lectins and monoclonal antibodies) define the cellular senescence phenotype. The cell surface oligosaccharide display can be characterized by assessing the epitope density and conformational arrangement of three or four individual carbohydrate specificities. Development of the senescence morphological phenotype was preceded by specific alterations in the cell surface oligosaccharide display. The senescence-dependent changes in these displays are primarily related to the binding affinity (the Kij of the Scatchard analyses) rather than the epitope density (the binding capacity, Rij of the Scatchard analyses). These alterations involve rearrangements within specific carbohydrate classes. In the course of these studies, the observation was made that low-density and contact-inhibited growth-retarded Phase II cells showed similar surface modulation of the oligosaccharide display. This suggests a broader significance for the date in growth regulation. These data suggest a structural/functional relationship between cellular senescence mechanisms and growth control in general. In this study we have investigated the possible role of cell surface oligosaccharide regulation in substrate interactions. Trypsinized cells were studied during substrate adherence, spreading and initiation of growth on control and poly-L-ornithine-treated polystyrene substrate. The cell surface oligosaccharide display was characterized using both biological assays and NMR-based measurements of the mobility of water at the cell surface. Results show that trypsinization does not remove or diminish the oligosaccharide display. The initial adherence of the cells to unmodified substrate is mediated by oligosaccharides. Cellular spreading results in specific changes in the display. Initiation of growth corresponds to further specific changes in the display. These data suggest a mechanistic connection between the cell surface oligosaccharide display and growth control of these fibroblasts.

Glucose inhibition of human fibroblast proliferation and response to growth factors is prevented by inhibitors of aldose reductase.

Diabetes mellitus is associated with premature senescence of cultured dermal fibroblasts. The present study investigated the effect of elevated glucose concentrations on cultured human fibroblasts from normal donors. Mean population doubling times, population doublings until senescence, saturation density at confluence (cells/cm2), tritiated thymidine incorporation, and response to platelet-derived growth factor (PDGF) were inhibited with the increasing glucose concentrations (11.0, 22, 44, or 55 mM glucose) (P less than 0.05). Replicative life span was markedly diminished by multiple passages in high glucose medium (5.5 mM glucose: 62.4 +/- 7.9 population doublings; 22 mM glucose: 22.8 +/- 3.4 population doublings: P less than 0.05). Aldose reductase activity was present in the cultured fibroblasts (3.9 +/- 0.5 nmol/min per mg protein), and inhibitors of aldose reductase, including sorbinil (10(-4) M--10(-6) M) and tolrestat (10(-6) M--10(-8) M), completely prevented glucose-mediated inhibition of fibroblast proliferation, restored the response to PDGF, and allowed a normal replicative life span. Myo-inositol (11 microM--5.5 mM) also reversed the adverse effects of glucose. These in vitro data demonstrate that elevated concentrations of glucose inhibit cell growth and promote premature senescence, effects which can be prevented with inhibitors of aldose reductase or supplemental myo-inositol. These aldose reductase-related effects may explain the impaired growth and premature senescence of cultured connective tissue from diabetic patients.

Multivoxel 1H-MRS of stroke.

Proton nuclear magnetic resonance spectroscopy is a noninvasive technique allowing the localized, in vivo detection of proton-containing brain metabolites. We used this technique to study eight patients with cerebral infarction or ischemia. A stimulated echo-pulse sequence with chemical shift imaging was used to acquire spectra from multiple contiguous 4-cc volumes extending from the site of ischemia to the opposite hemisphere. Six patients had a reduction in the signal from N-acetyl groups (NAG) in the stroke area compared with controls, and those with the lowest NAG to phosphocreatine/creatine ratios had the least recovery of function. Lactate was observed within the infarcted region in two patients at 9 and 11 days after infarction and may have been present in other patients up to 15 weeks after stroke.

2'-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides.

Oligonucleotides containing 2'-O-aminopropyl-substituted RNA have been synthesized. The 2'-O-(aminopropyl)adenosine (APA), 2'-O-(aminopropyl)cytidine (APC), 2'-O-(aminopropyl)-guanosine (APG), and 2'-O-(aminopropyl)uridine (APU) have been prepared in high yield from the ribonucleoside, protected, and incorporated into an oligonucleotide using conventional phosphoramidite chemistry. Molecular dynamics studies of a dinucleotide in water demonstrates that a short alkylamine located off the 2'-oxygen of ribonucleotides alters the sugar pucker of the nucleoside but does not form a tight ion pair with the proximate phosphate. A 5-mer with the sequence ACTUC has been characterized using NMR. As predicted from the modeling results, the sugar pucker of the APU moiety is shifted toward a C3'-endo geometry. In addition, the primary amine rotates freely and is not bound electrostatically to any phosphate group, as evidenced by the different sign of the NOE between sugar proton resonances and the signals from the propylamine chain. Incorporation of aminopropyl nucleoside residues into point-substituted and fully modified oligomers does not decrease the affinity for complementary RNA compared to 2'-O-alkyl substituents of the same length. However, two APU residues placed at the 3'-terminus of an oligomer gives a 100-fold increase in resistance to exonuclease degradation, which is greater than observed for phosphorothioate oligomers. These structural and biophysical characteristics make the 2'-O-aminopropyl group a leading choice for incorporation into antisense therapeutics. A 20-mer phosphorothioate oligonucleotide capped with two phosphodiester aminopropyl nucleotides targeted against C-raf mRNA has been transfected into cells via electroporation. This oligonucleotide has 5-10-fold greater activity than the control phosphorothioate for reducing the abundance of C-raf mRNA and protein.

Polyol and water accumulation in muscle of galactose-fed rats.

Skeletal muscle contains high levels of aldose reductase that catalyzes the reduction of galactose to the polyol galactitol. Galactitol and water were measured in muscle of rats fed a high galactose diet with or without addition of the aldose reductase inhibitor sorbinil. Galactitol, measured in isolated samples of muscle by HPLC, reached steady-state levels (5.9 +/- 1.0 mg/g tissue) within 3 days. Muscle water, determined in vivo by magnetic resonance imaging, increased (51 +/- 5%, P < 0.02) to steady-state levels within 7 days. Both the increased galactitol and water remained constant for the 4-month duration of this study. Aldose reductase activity also remained constant. Sorbinil prevented both the increase in galactitol and the increase in water. These results suggest that the increase in water is due to the osmotic effects of galactitol accumulation and demonstrate that galactitol and water accumulation neither up-regulate nor down-regulate aldose reductase expression in skeletal muscle.

Carbon-13 magnetic resonance spectroscopy. Problems and promise.

Carbon-13 magnetic resonance spectroscopy (13C MRS) is being used to investigate metabolism in the brain, liver, heart, and skeletal muscle of animals. The tricarboxylic acid cycle, glycolysis, gluconeogenesis, hepatic ketogenesis, and metabolism of ethanol are some of the metabolic processes amenable to 13C MRS. Clinical investigation using 13C in humans will become widespread as improvements in sensitivity, localization, enrichment, and decoupling are realized. Carbon-13 MRS offers potential as a new investigative probe into organ physiology.

Tumor characterization using unconventional MR modalities.

This article reviews many of the promising new magnetic resonance (MR) modalities for the characterization of tumor structure and metabolism, excluding 31P magnetic resonance spectroscopy (MRS). The report focuses on recent work with localized proton MRS, MR imaging of diffusion and perfusion, and work on imaging bound pools of sodium ions. The technical problems associated with these methods are discussed, along with the unique information each provides. Many of these techniques have already been used in studies of human disease, or are of sufficient importance that clinical applications are imminent.

An efficient synthesis of mimetics of neamine for RNA recognition.

As mimetics of neamine, several 4-heterocyclic 2-deoxystreptamine derivatives were chemically synthesized for RNA recognition. Conversion of 4-methylthiomethyl-5,6-di-O-acetyl-diazido-2-deoxystreptamine to the 4-chloromethyl derivative followed by reactions with different nuclophilic reagents gave the 4-heterocyclic 2-deoxystreptamine derivatives in satisfactory yields.

The effect of backbone charge on the collision-induced dissociation of oligonucleotides.

Knowledge of the effects of structural changes in oligonucleotides on their dissociation reaction is important in the application of mass spectrometry to sequence determination. The effect of backbone charge on the collision-induced dissociation of multiply-charged oligonucleotides produced by electrospray was explored by examination of models in which the normal phosphodiester linkage was partially replaced with an uncharged methylphosphonate (MP) linkage. Three different MP-containing oligonucleotides were studied, designed to represent a concentration of charge on the 5'- and 3'-ends of the molecule and with an even distribution of charge along the backbone, compared with a control molecule containing only phosphodiester linkages. In all MP-containing oligonucleotides charging of over 90% of phosphate groups were observed, compared with typical charging patterns of about 60% in normal all-phosphodiester oligonucleotides. This unexpected effect is attributed to charge stabilization by interactions of charged sites with uncharged residues. Analysis of the collision-induced dissociation mass spectra showed that backbone cleavage occurred at every residue (w and a-base ion series), producing a full set of sequencing ions whether or not the linkage at that site was formally charged. It is concluded that under the multiple collision conditions of the quadrupole collision cell that backbone cleavage proceeds through two generic pathways, one involving base loss followed by cleavage of the adjacent C3'-CO bond and the other requiring neither base loss nor charged phosphate at the cleavage site. These results suggest that backbone cleavage reactions in conventional phosphodiester oligonucleotides can occur at non-ionized linkage sites, of which there are a high proportion in both electrospray- and MALDI-produced molecular ions.

Enhanced gas-phase hydrogen-deuterium exchange of oligonucleotide and protein ions stored in an external multipole ion reservoir.

Rapid gas-phase hydrogen-deuterium (H-D) exchange from D(2)O and ND(3) into oligonucleotide and protein ions was achieved during storage in a hexapole ion reservoir. Deuterated gas is introduced through a capillary line that discharges directly into the low-pressure region of the reservoir. Following exchange, the degree of H-D exchange is determined using Fourier transform ion cyclotron resonance mass spectrometry. Gas-phase H-D exchange experiments can be conducted more than 100 times faster than observed using conventional in-cell exchange protocols that require lower gas pressures and additional pump-down periods. The short experimental times facilitate the quantitation of the number of labile hydrogens for less reactive proteins and structured oligonucleotides. For ubiquitin, we observe approximately 65 H-D exchanges after 20 s. Exchange rates of > 250 hydrogens s(-1) are observed for oligonucleotide ions when D(2)O or ND(3) is admitted directly into the external ion reservoir owing to the high local pressure in the hexapole. Partially deuterated oligonucleotide ions have been fragmented in the reservoir using infrared multiphoton dissociation (IRMPD). The resulting fragment ions show that exchange predominates at charged sites on the 5'- and 3'-ends of the oligonucleotide, whereas exchange is slower in the core. This hardware configuration is independent of the mass detector and should be compatible with other mass spectrometric platforms including quadrupole ion trap and time-of-flight mass spectrometers.

Characterization of oligonucleotide metabolism in vivo via liquid chromatography/electrospray tandem mass spectrometry with a quadrupole ion trap mass spectrometer.

The pattern of nuclease degradation observed for an antisense phosphorothioate oligonucleotide in pig kidney was determined using liquid chromatography/electrospray mass spectrometry (LC/ESI-MS) and LC/ESI-MS/MS with a quadrupole ion trap mass spectrometer. Metabolites were separated by length using reversed-phase high-performance liquid chromatography with aqueous hexafluoropropan-2-ol-triethylamine and a methanol gradient. The individual masses of metabolites in each LC peak were determined via deconvolution and converted into potential nucleotide compositions. The nucleotide composition was used to locate metabolites within the known oligomer sequence. The identity of metabolites was confirmed using on-line LC/MS/MS to generate fragment ions suitable for sequence verification. A limited number of shorter oligonucleotide fragments were observed, suggesting that metabolism in vivo may be sequence dependent.

Analysis of cerebral structural changes in systemic lupus erythematosus by proton MR spectroscopy.

PURPOSE: To determine whether cerebral atrophy in systemic lupus erythematosus is associated with decreased levels of the neuronal marker N-acetyl-aspartic acid. METHODS: Two groups of patients with systemic lupus erythematosus were studied, those with significant atrophy (n = 11) and those without significant atrophy (n = 10), using proton MR spectroscopy on a 1.5-T imaging unit. The solvent-suppressed, short-echo, volume-localized proton spectroscopy technique showed typical brain metabolites, including N-acetylaspartate, creatine/phosphocreatine, and choline-containing compounds. RESULTS: The N-acetylaspartate-to-creatine/phosphocreatine ratio was smaller in those patients with significant cerebral atrophy (1.68 +/- 0.27) than in those patients with minimal or no atrophy (2.17 +/- .30). The degree of atrophy was negatively correlated with the N-acetylaspartate-to-creatine/phosphocreatine ratio. The choline-to-creatine/phosphocreatine ratio was not significantly altered in systemic lupus erythematosus patients with atrophy. CONCLUSION: These data suggest that cerebral atrophy in systemic lupus erythematosus is associated with neuronal dropout (or damage), which results in decreased N-acetylaspartate ratios. A change in choline ratios is not implicated in the biochemical changes associated with cerebral atrophy. Proton MR spectroscopy may be useful in correlating brain metabolites with cerebral structural changes in patients with autoimmune diseases.

The opening of a single base without perturbations of neighboring nucleotides: a study on crystal B-DNA duplex d(CGCGAATTCGCG)2.

In this work we explore the possibility of the opening of a single base without perturbation of its neighboring nucleotides. Low energy base opening into the grooves can be accomplished by rotation of the relevant backbone and glycosidic bond torsion angles. The pathway has been determined by identifying zeta torsion angle as the reaction coordinate together with the accompanying geometric requirement that guides the displacement of other torsion angles. Our study on Dickerson dodecamer duplex d(CGCGAATTCGCG)2 showed that all bases with normal equilibrium zeta can be rotated by approximately 30 degrees, corresponding to approximately 3.5A base displacement, towards the major groove. Such an opening extent is comparable with estimated amplitudes of local angular motions in DNA bases from NMR experiments, which might facilitate proton exchange. The computed base opening energy barrier is also comparable with measured base pair opening enthalpy. These results indicate possible relevance of the pathway studied in this work with experimentally observed base pair opening process. Our analysis also showed a preference for base opening along the major groove and an abnormal opening behavior for bases with unusual equilibrium zeta torsion angle.

Raman and infrared studies of nucleosides at high pressures: II. Cytidine.

Raman and mid-infrared (MIR) spectra have been recorded for crystalline cytidine at pressures up to 10 GPa at room temperature. Broadening and positive wavenumber shifts are observed for most of the Raman and MIR peaks with increasing pressure. However, some of the MIR peaks associated with hydrogen-stretching modes display a negative wavenumber shift as a result of charge transfer effects. Evidence of a phase transition near 4 GPa is presented and attributed to a change in the conformation of the five membered sugar ring.

Vibrational analysis of phosphorothioate DNA: II. The POS group in the model compound dimethyl phosphorothioate [(CH3O)2(POS)]-.

The results of Raman and Infrared (IR) spectroscopic investigations on the vibrational modes of dimethyl phosphorothioate (DMPS) anion, [(CH3O)2(POS)]-, are reported. Ab initio calculations of the vibrational modes, the IR and Raman spectra and the interatomic force constants of DMPS were performed. A normal mode calculation was performed and the results were used to calculate the potential energy distribution for the vibrational modes. This analysis shows that in DMPS the P-S stretching mode has a frequency of about 630 cm-1 and an angle bending mode involving the sulfur atom has a frequency of about 440 cm-1. The proposed vibrational mode assignments will serve as marker bands in the conformational studies of phosphorothioate oligonucleotides which play a central role in the novel antisense therapeutic paradigm.

Raman and infrared studies of nucleosides at high pressures: I. Adenosine.

Room temperature Raman and infrared (IR) spectra of crystalline adenosine at pressures between 1 atm and 10 GPa are reported. Vibrational modes were identified through assignments in the literature. Many modes were found to increase in frequency with pressure; however, some irregularities were observed. Discontinuities were observed in numerous Raman and IR modes near 2.5 GPa, indicating a phase transition. The modes associated with the glycosidic bond shift significantly down in frequency near this pressure, suggesting a weakening of the associated bond. The IR modes associated with hydrogen-stretching motions were found to decrease in frequency with pressure.