Michael G. Irving

Water signal elimination in vivo, using suppression by mistimed echo and repetitive gradient episodes

A number of techniques (I) are available for the elimination of an unwanted signal (usually the ‘H signal from water) from a spectrum. Unless the unwanted signal is removed before the preamplifier and ADC are encountered in the signal detection pathway, difficulties are experienced in the observation of very weak signals because of dynamic range problems. O f the available techniques the most efficient appears to be the 1331 method developed by Hore (I, 2); however, as Hore points out, this technique is only of limited value if the water resonance is broad. This is the situation often encountered in vivo where very broad water signals are observed, the broadness usually being the result of magnetic field inhomogeneity. In general, for signal elimination the most successful methods have in the past relied on overall nonexcitation of the unwanted resonance, the aim being to leave the unwanted spin coherence along the z axis and away from the detection plane. The alternative procedure would be to destroy selectively the unwanted spin coherence and it is this strategy which has led to the application of reverse polarization transfer methods (3) as probably the most efficient water signal elimination method yet devised. In this paper we develop a method for selectively destroying the water signal, applicable to in vivo NMR studies where broad water signals are often encountered. We call the technique SUBMERGE. The method eliminates transverse water spin coherence over a wide but controllable frequency band. The significant point is that the water spin coherence is destroyed, thus eliminating the signal from the preamplifier and ADC, allowing the efficient detection of weak signals. As a test, we attempt to observe the methyl signal from lactic acid at a concentration of 10 mM in neat HzO. The water signal is thus about 3700 times more intense than the signal of interest. A 250 ml round-bottom flask was used as the sample container. A water signal having a half-height width of 10 Hz was typically detected in a single pulse experiment. In Fig. 1 is shown the experimentally determined excitation profile from a 10 ms 7r/2 sine pulse. The excitation profile from a 10 ms 7r/2 gaussian pulse is also shown for comparison. At a ‘H resonance frequency of 100 MHz (field strength 2.4 T) the chemical-shift separation between the two resonances of interest is about 345 Hz. Thus, by use of such shaped pulses, it is possible to excite the water signal but not the

A simple procedure for selective inversion of NMR resonances for spin transfer enzyme kinetic measurements

Abstract The unidirectional fluxes through enzyme-catalyzed reactions operating at equilibrium can be measured using saturation or inversion transfer NMR spectroscopy. The procedure requires selective saturation or inversion of the magnetization of one species (A) which is exchanging with others (B). To date selective excitation has been via a separate frequency synthesizer channel or multiple-pulse sequences such as DANTE. We apply here a far simpler pulse sequence for selective inversion of spins which generates the so-called d-ordered state. The pulse sequence is π 2 x (η A )-τ 1 - π 2 ±x -τ 2 - π 2 ±x,±y - acquisition , where τ1 = 1 (2|;η A − η B | ), the subscripted ± x and ± y refer to rotating frame axes along which the rf irradiation is applied and τ 2 is a variable delay time. The application of this procedure to measurement of the rate of phosphoryl group transfer catalyzed by phosphoglyceromutase is demonstrated.

Purification and molecular properties of alcohol dehydrogenase from Drosophila melanogaster: Evidence from NMR and kinetic studies for function as an aldehyde dehydrogenase

Abstract 1. 1. Alcohol dehydrogenase (ADH) allozymes (FF, SF, SS) were purified to homogeneity from strains of Drosophila melanogaster using a new procedure. 2. 2. All allozymes displayed aldehyde dehydrogenase activity on cellulose (ALDH) acetate zymograms, corresponding to the ADH activity zones. 3. 3. Kinetic analyses with acetaldehyde as substrate revealed non-linear, biphasic Lineweaver-Burk plots giving two apparent Michaelis constants ( K m ) ranges for the allozymes of 1–10 μM and 0.1–1 mM. 4. 4. In contrast, kinetic analyses with ethanol as substrate gave results consistent with a single K m value of approximately 2 mM. 5. 5. At approx. 3 μM substrate concentration, the enzymes exhibited equivalent rates of dehydrogenase activity with either ethanol or acetaldehyde; whereas at a concentration of 10 mM, the ADHs exhibited approx. a 10-fold higher activity with ethanol as substrate. 6. 6. The specific activity of ADH-FF was 2–3 times higher than ADH-SS with both ethanol and acetaldehyde as dehydrogenase substrates. ADH and ALDH activities were inhibited by pyrazole, disulphiram and p -hydroxymercuribenzoate. 7. 7. Atomic absorption spectrometry confirmed the absence of zinc. 8. 8. The oxidation of 2-[ 13 C]-labelled ethanol by ADH allozymes in vitro was studied using nuclear magnetic resonance spectrometry. 9. 9. Acetaldehyde, its diol and acetate were detected within 20 min and monitored for 10 hr. 10. 10. The significance of these results for studies on ethanol metabolism in D. melanogaster is discussed.

Oxygen consumption and ammonia excretion of the brown tiger prawn Penaeus esculentus fed diets of varying protein content.

Abstract 1. 1. Diets containing 30, 40 or 50% protein were assessed for their effect on the pre- and postfeecling oxygen consumption and ammonia production of juvenile Penaeus esculentus. 2. 2. Prawns fed all diets had significantly higher respiration rates after feecling due to the SDA (Specific Dynamic Action). 3. 3. There was a significant difference between pre- and postfeecling ammonia production for prawns fed the 30 and 50% protein diets. 4. 4. Prawns maintained on the 40% protein diet had the lowest levels of pre- and postfeecling respiration and the smallest SDA. 5. 5. Prawns fed the 40% diet were metabolizing at an optimal dietary protein level or protein/energy ratio.

Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

BackgroundMatrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown.MethodsTo investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential.ResultsCompetitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed.ConclusionWe have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

Application of the reverse dept polarization-transfer pulse sequence to monitor in vitro and in vivo metabolism of 13C-ethanol by 1H-NMR spectroscopy

Using the reverse 13C----1H DEPT polarization-transfer pulse sequence the metabolism of 13C ethanol in vitro and in vivo has been monitored by 1H-NMR spectroscopy. Using yeast alcohol dehydrogenase, acetaldehyde, the hydrated form of acetaldehyde and acetate were identified as metabolites of [2-13C]-ethanol. The ratio of hydrated to free acetaldehyde was dependent upon the protein concentration of the reaction mixture. Binding of acetaldehyde in an irreversible Schiffs base resulted in optimal enzyme activity. Hepatocytes from rats fasted for 20 h, metabolised [1-13C] and [2-13C]ethanol in a linear fashion, but no [13C]acetaldehyde was detected. Metabolic integrity of the hepatocytes was confirmed with [2-13C]acetate. The addition of disulfiram (50 micron) to hepatocyte suspensions which had been incubated with [1-13C]ethanol, resulted in the resynthesis of [13C]ethanol. The amount of [13C]ethanol resynthesized under these conditions represents intracellular acetaldehyde whose concentration was in the range of 400-800 mumol/g wet weight of hepatocytes when 50 mM ethanol had been originally incubated with the hepatocyte suspension. These studies show how NMR-polarization transfer pulse sequences can be used to monitor the metabolism of 13C-ethanol in vivo, and provide a unique tool to measure in vivo concentrations of acetaldehyde. The studies also suggest that cytoplasmic aldehyde dehydrogenase may play a major role in hepatic ethanol metabolism.

Preliminary studies on the potential of in vivo deuterium NMR spectroscopy

Natural abundance deuterium NMR spectroscopy can be used to characterise in vivo 2H signals arising from water and fat in mice, with acquisition times of less than two minutes. Administration of D(2)0 (10% V/V) in the drinking water enhances these signals so that excellent spectra can be obtained with one scan. Using these procedures the in vivo turnover of 2H in water and fat in mice has been determined. This procedure may be of particular importance in studies of fat turnover in obesity.

Collagen biosynthesis in cultured rat testicular sertoli and peritubular myoid cells

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.

In vitro and in vivo MMP gene expression localisation by In Situ-RT-PCR in cell culture and paraffin embedded human breast cancer cell line xenografts.

BackgroundMembers of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14, membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo. As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult.MethodsWe utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature and provide a reproducible in vivo model system correlated to the human disease state.ResultsIn vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231, MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma.ConclusionWe have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.

In vivo determination of 31P spin relaxation times (T1, T2, T1ϱ) in rat leg muscle. Use of an off-axis solenoid coil

A probe using a solenoid coil tilted 45 degrees off-axis has been used to study the 31P NMR relaxation characteristics of the resonances arising from phosphorus metabolites in rats in vivo. T1, T1 rho and T2 values have been determined for phosphocreatine and ATP in leg muscle. The ratio of 31P T1(1700ms) to T2(12ms) for ATP was in excess of 200:1 compared with a ratio of 5:1 for 1H T1:T2. Of major significance was the observation that T2 values for phosphocreatine (230ms) were markedly longer than T2 values for ATP (12ms). Thus by use of appropriate delay times in spin echo sequences ATP signals can be nulled, and discrete 31P imaging of phosphocreatine in muscle may be possible provided the overall signal-to-noise is satisfactory.