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Biomedical subjects

R H Griffey

Publications and source records attributed to R H Griffey.

At least 37 records · Page 2Linked to original sources
PubMed1997

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.

DNA↗
PubMed1996

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.

Exonucleases↗
PubMed1996

Hydration of single-stranded phosphodiester and phosphorothioate oligodeoxyribonucleotides.

Infrared spectroscopy was used to identify hydration-sensitive structural differences between single- stranded phosphorothioate (PS) and phosphodiester (PO) oligodeoxyribonucleotides. Spectra were recorded in the mid-infrared region, 500-1800 cm-1, at relative humidities between 0 and 98%; the PS and PO spectra are substantially different. The hydration effects on spectral bands in these single-stranded oligodeoxyribonucleotides is markedly different from such behavior in double- and triple-stranded oligodeoxyribonucleotides. A strong absorption occurs at 656 cm-1 in the phosphorothioate sample which is completely absent from the PO spectra. Gravimetric measurements were carried out on one PS and one PO sample to monitor and confirm hydration. The calculated BET adsorption constants [Brunauer, S., Emmett, RH. and Teller, E. (1938) J. Am. Chem. Soc., 60, 309-319] are 1.2 and 1.4 water molecules per nucleotide in the first hydration layer of PS and PO respectively. While the gravimetric data indicate that the single-stranded oligodeoxyribonucleotides hydrate very similarly to duplex DNA, the mid-infrared conformational marker bands are strikingly different from those observed for duplex DNA. In particular, the Vas of the phosphate group (PO2) at 1222 cm-1 in the single-stranded PO spectra is independent of relative humidity.

Base Sequence↗
PubMed1996

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.

Electrochemistry↗
PubMed1996

Negative ionization micro electrospray mass spectrometry of oligonucleotides and their complexes.

The utility of negative ionization micro electrospray (microspray) mass spectrometry is demonstrated for detection of oligonucleotides and their non-covalent complexes. A simple microspray ionization source is fabricated from an outer stainless-steel needle and an inner fused-silica capillary. Under these conditions, the liquid flow rate can be reduced 15-fold from 7.5 microL/min to 0.5 microL/min. Studies of a 14-mer DNA oligonucleotide show no change in the charge-state distribution and quantity of adducted salt ions during the microspray process compared to pneumatically assisted electrospray mass spectrometry. The microspray ion source is less sensitive to the presence of solution buffers, and an 11-fold increase in integrated ion abundance from oligonucleotide analyte is observed with a 10 mM concentration of ammonium acetate, compared to pneumatically assisted nebulization (PAN). A > 100-fold increase in the duplex:single strand ratio for a 14-mer oligodeoxynucleotide and its complementary strand is observed using the microspray ion source relative to experiments performed with PAN. Studies of duplexes between DNA and a peptide nucleic acid suggest that this effect may be related to the degree of adduction of counterions to the DNA during ionization.

Acetates↗
PubMed1996

Structure-function relationships within peripheral nerves in diabetic neuropathy: the hydration hypothesis.

To define the quantitative relationship between peripheral nerve structure and function imposed by endoneurial oedema in the diabetic state, we determined values for sural nerve hydration structure as measured by magnetic resonance spectroscopy, and for neurological function with scores for nerve conduction properties (NCV-score), neuropathic symptoms (NS-score), and examination signs (NE-score). The coefficient of sural nerve hydration was elevated to 30 +/- 6% (p < 0.05) in 79 symptomatic neuropathic diabetic subjects with an average of 15 years of diabetes mellitus, compared to a value of 25 +/- 3% in 72 non-diabetic control subjects. In contrast, in 75 asymptomatic diabetic subjects with an average of 6 additional years of diabetes, the mean hydration coefficient was only 28 +/- 5% (p < 0.05). A nerve hyperhydration state was identified with a prevalence of 25% within the asymptomatic group characterized by nerve hydration greater than the 95th percentile, early changes in nerve electrophysiology and neurological examination, but with no symptomatology of neuropathy. Stratification of the symptomatic neuropathic group by worsening nerve electrophysiology, demonstrates a coincident deterioration in neurological examination (RR = 5.39 at maximum NCV-score), and neuropathy symptomatology (RR = 4.80 at maximum NE-score). The present data are consistent with the hypothesis that endoneurial oedema initiates deterioration sequentially in nerve electrophysiology, followed by abnormal findings on neurological examination, preceding the patient's final perception of symptomatic stocking glove peripheral diabetic neuropathy.

Adult↗
PubMed1996

Pharmacokinetic properties of several novel oligonucleotide analogs in mice.

Biophysical and pharmacokinetic properties of five analogs of ISIS 3082, a 20-mer phosphorothioate oligodeoxynucleotide that inhibits the expression of mouse intercellular adhesion molecule 1, were evaluated. Compared to the parent compound, ISIS 3082, the 2'-propoxy modified phosphodiester, ISIS 9044 and the 2'-propoxy phosphorothioate, ISIS 9045, had greater affinity for complementary RNA and were more lipophilic. A chimeric oligonucleotide comprised of 2'-propoxy diester wings and a phosphorothioate deoxy center (ISIS 9046) had equal affinity. It was also more lipophilic than ISIS 3082, but less so than the other 2'-propoxy modified analogs. The two analogs with 5'-lipophilic conjugates, ISIS 9047 (5'-octadecylamine) and ISIS 8005 (5'-(2'-O-hexylamino-carbonyl-oxycholesterol) were more lipophilic than ISIS 3082 (3- and 7-fold, respectively) but had similar affinity for complementary RNA. Binding of ISIS 3082 to bovine serum albumin was salt-dependent and, at physiological concentration (320 mOsmol), the dissociation constant (Kd) was 140 microM. Similarly, the 2'-propoxy phosphodiester, ISIS 9044, displayed salt-dependent bovine serum albumin binding, but not binding was measurable at physiological salt conditions. In contrast, the more lipophilic phosphorothioate analogs displayed much higher affinity to bovine serum albumin at 320 mOsmol than ISIS 3082. After bolus injection to mice, the initial volumes of distribution of the more lipophilic phosphorothioate analogs, ISIS 9045, ISIS 9047 and ISIS 8005, were less and the initial clearance from plasma was slower than ISIS 3082. The pharmacokinetics of the other analogs was similar to ISIS 3082. Distribution of ISIS 3082 into peripheral tissues was similar to that reported for other phosphorothioates with liver and kidney accumulating the highest fraction of the dose. The only modification to markedly influence distribution was the very lipophilic cholesterol conjugate (ISIS 8005), which increased substantially the fraction of the dose accumulated by the liver. Little intact drug was found in urine or feces for any analog, and the patterns of metabolites suggested that for all analogs the principal metabolic pathway was due to 3'-exonuclease activity. The metabolism of ISIS 3082 was similar to that reported for other phosphorothioates. After 2 hr, most of the radioactivity in plasma represented metabolites but, in tissues, intact ISIS 3082 was present for much longer periods of time and metabolites accumulated more slowly. The 24-hr exposure to ISIS 3082 of liver and kidney was 20.7 and 67.9 microM/hr, respectively. The rates of metabolism in plasma, liver and kidney of the two 5'-conjugates, ISIS 9047 and ISIS 8005, were similar to ISIS 3082, as was the pattern of metabolism. The rate of metabolism of ISIS 9044 (2'-propoxy phosphodiester oligonucleotide) was much more rapid in liver and plasma, but surprisingly much slower in the kidney. ISIS 9045 (full 2-propoxy phosphorothioate) was much more stable than ISIS in all tissues, the enhanced stability of ISIS 9045 resulted in increased exposure of liver and kidney to the drug, whereas the exposure of the liver to the two more lipophilic analogs, ISIS 9047 and ISIS 8005, was greater because a higher fraction of the dose was distributed to the liver. The exposure of the kidney to ISIS 9044 was also greater than that to ISIS 3082 due to the surprising stability of the drug in the kidney.

Animals↗
PubMed1995

Spin-spin relaxation of brain tissues in systemic lupus erythematosus. A method for increasing the sensitivity of magnetic resonance imaging for neuropsychiatric lupus.

OBJECTIVE: To correlate the spin-spin relaxation time (T2) of brain tissue in neuropsychiatric systemic lupus erythematosus (NPSLE) with the patient's clinical condition. METHODS: T2 values were determined in 54 SLE patients and 45 non-SLE controls at 1.5 Tesla, using intensity from multi-echo magnetic resonance (MR) images fitted to an exponential decay curve with rate-constant T2. RESULTS: The T2 of white matter was increased in SLE patients compared with controls (P = 0.01) and was increased in those patients who had previously experienced major NPSLE: Patients with acute diffuse neurologic manifestations (seizures, psychosis, coma) demonstrated a longer T2 of the gray matter (mean +/- SD 92.75 +/- 6.35 ms, n = 10) than did other SLE patients (mean +/- SD 79.61 +/- 5.04 ms, n = 44) (P = 0.02 by t-test), which suggests acute cerebral edema. The mean T2 values of reversible and nonreversible focal lesions were significantly different (P < 0.02), indicating different microenvironments and micropathology. CONCLUSION: Quantitative T2 measurement extends the utility and sensitivity of conventional MR imaging for evaluating NPSLE:

Adolescent↗
PubMed1995

Utility of organic bases for improved electrospray mass spectrometry of oligonucleotides.

The sensitivity and accuracy of the mass spectrometric analysis of oligonucleotides using electrospray ionization can be compromised when the oligomer is adducted in the gas phase to cations such as sodium or potassium. We have evaluated the addition of mM concentrations of a series of organic bases with solution pKb values ranging from 11.5 to 5.5 and gas-phase proton affinities ranging from 213 to 232 kcal/mol as a method for suppression of signals from alkali-adducted ions. Stronger bases such as triethylamine and piperidine reduce the signals from bound sodium most effectively, but also decrease the total ion current from oligonucleotide. Imidazole, with a solution pH of approximately 8.0, provides modest suppression of sodium/potassium adduct ions, but up to a four-fold improvement in sensitivity. Co-addition of imidazole and triethylamine or piperidine produces high ion abundance and good suppression of cation-adducted species for samples of phosphodiester or phosphorothioate oligomers which have not been desalted via preliminary precipitation or by high-performance liquid chromatography. Addition of high concentrations of imidazole generates a bimodal distribution of charge states, which may reflect different gas-phase conformations for single-stranded oligomers.

Alkalies↗
PubMed1994

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.

Aldehyde Reductase↗
PubMed1994

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.

Adolescent↗
PubMed1993

Sural nerve water in vivo in normal humans measured by magnetic resonance spectroscopy: relation to age, height, gender, and neurological profile.

To enable the quantitative assessment of peripheral nerve structure and function, we determined the normal values for sural nerve hydration structure as measured by magnetic resonance proton imaging, and for neurological function with scores for neuropathic symptoms, signs, and nerve conduction properties. Normal human sural nerves contain 24.8 +/- 3.4% water. The structural water content of the nerves did not vary systematically in relation to age, height, gender, sural nerve conduction, neurologic symptoms, or examination deficits. In contrast, the neurological function scores were significantly influenced by age and selectively by height. Both nerve structure and function were stable over a 1-year interval. Measurement of human sural nerve water content in vivo by magnetic resonance proton imaging, and quantitation of the neurological profile of symptoms, signs, and conduction velocity are useful, noninvasive tools for the investigation of diseases in which changes in nerve structure may be related to alterations in nerve function.

Adult↗
PubMed1992

Magnetization transfer contrast in fat-suppressed steady-state three-dimensional MR images.

We demonstrate that magnetization transfer contrast can be used to improve the diagnostic utility of fat-suppressed steady-state three-dimensional gradient-recalled images. Fat suppression is achieved using a "jump-return" pair of contiguous shaped pulses. No time interval exists between the pulses, and no RF echo is generated. The sequence normally produces images with "density" weighting. Preparation of the spin magnetization with off-resonance frequency-selective excitation creates magnetization transfer contrast which attenuates signal intensity in proportion to the exchange rate of magnetization from free water with magnetization from water bound to macromolecules or protons that have restricted mobility. The resulting images have excellent fat suppression with low sensitivity to motion since no subtraction is used. In addition, the mechanism of signal attenuation is independent of paramagnetic effects, and addition of Gd-DTPA produces signal enhancement from vascularized regions of tissue. Examples are presented for the knee and breast, where the observation of pathology with signal enhancement from Gd-DTPA is improved over conventional 3D fat-suppressed images.

Breast↗
PubMed1992

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.

Cell Adhesion↗
PubMed1992

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.

Animals↗
PubMed1992

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.

Aged↗