Imino-proton resonances of yeast tRNAPhe studied by two-dimensional nuclear Overhauser enhancement spectroscopy

Application of two-dimensional nuclear Overhauser enhancement (NOE) spectroscopy to yeast tRNAPhe in H2O solution demonstrates that all imino-proton resonances, related to the secondary structure, and nearly all imino proton resonances, originating from the tertiary structure, can be assigned efficiently by this method. The results corroborate the assignments of the imino-proton resonances of this tRNA as established previously by one-dimensional NOE experiments (only the assignment of base pairs G1 X C72 and C2 X G71 should be reversed). The advantages of two-dimensional NOE spectroscopy over one-dimensional NOE spectroscopy for the assignments of imino-proton resonances and the structure elucidation of tRNA are illustrated and discussed. Furthermore, the use of non-exchangeable proton resonances as probes of the molecular structure is explored.     

Creatine uptake in brain and skeletal muscle of mice lacking guanidinoacetate methyltransferase assessed by magnetic resonance spectroscopy.

Creatine (Cr) levels in skeletal muscle and brain of a mouse model of Cr deficiency caused by guanidinoacetate methyltransferase absence (GAMT-/-) were studied after Cr supplementation with 2 g.kg body wt-1.day-1 Cr for 35 days. Localized 1H magnetic resonance spectroscopy (MRS) was performed in brain (cerebellum and thalamus/hippocampus) and in hind leg muscle of GAMT-/- mice before and after Cr supplementation and in control (Con) mice. As expected, a signal for Cr was hardly detectable in MR spectra of GAMT-/- mice before Cr supplementation. In the thalamus/hippocampus region of these mice, an increase in N-acetylasparate (NAA) was observed. During Cr administration, Cr levels increased faster in skeletal muscle compared with brain, but this occurred only during the first day of supplementation. Thereafter, Cr levels increased by 0.8 mM/day in all studied locations. After 35 days of Cr supplementation, Cr levels in all locations were higher compared with Con mice on a Cr-free diet and NAA levels normalized. Only because of the repeated MRS measurements performed in this longitudinal Cr supplementation study on GAMT-/- mice were we able to discover the initial faster uptake of Cr in skeletal muscle compared with brain, which may represent muscular Cr uptake independent of Cr transporter expression. Our results can provide the basis for additional experiments to optimize Cr supplementation in GAMT deficiency, as increases in brain Cr are slow in patients after Cr supplementation.     

31P saturation transfer spectroscopy predicts differential intracellular macromolecular association of ATP and ADP in skeletal muscle

The kinetics of phosphoryl exchange involving ATP and ADP have been investigated successfully by in vivo ³¹P magnetic resonance spectroscopy using magnetization transfer. However, magnetization transfer effects seen on the signals of ATP also could arise from intramolecular cross-relaxation. This relaxation process carries information on the association state of ATP in the cell. To disentangle contributions of chemical exchange and cross-relaxation to magnetization transfer effects seen in ³¹P magnetic resonance spectroscopy of skeletal muscle, we performed saturation transfer experiments on wild type and double-mutant mice lacking the cytosolic muscle creatine kinase and adenylate kinase isoforms. We find that cross-relaxation, observed as nuclear Overhauser effects (NOEs), is responsible for magnetization transfer between ATP phosphates both in wild type and in mutant mice. Analysis of ³¹P relaxation properties identifies these effects as transferred NOEs, i.e. underlying this process is an exchange between free cellular ATP and ATP bound to slowly rotating macromolecules. This explains the β-ATP signal decrease upon saturation of the γ-ATP resonance. Although this usually is attributed to β-ADP ↔ β-ATP phosphoryl exchange, we did not detect an effect of this exchange on the β-ATP signal as expected for free [ADP], derived from the creatine kinase equilibrium reaction. This indicates that in resting muscle, conditions prevail that prevent saturation of β-ADP spins and puts into question the derivation of free [ADP] from the creatine kinase equilibrium. We present a model, matching the experimental result, for ADP ↔ ATP exchange, in which ADP is only transiently present in the cytosol.     

Distinct Disease Phases in Muscles of Facioscapulohumeral Dystrophy Patients Identified by MR Detected Fat Infiltration

Facioscapulohumeral muscular dystrophy (FSHD) is an untreatable disease, characterized by asymmetric progressive weakness of skeletal muscle with fatty infiltration. Although the main genetic defect has been uncovered, the downstream mechanisms causing FSHD are not understood. The objective of this study was to determine natural disease state and progression in muscles of FSHD patients and to establish diagnostic biomarkers by quantitative MRI of fat infiltration and phosphorylated metabolites. MRI was performed at 3T with dedicated coils on legs of 41 patients (28 men/13 women, age 34–76 years), of which eleven were re-examined after four months of usual care. Muscular fat fraction was determined with multi spin-echo and T1 weighted MRI, edema by TIRM and phosphorylated metabolites by 3D ³¹P MR spectroscopic imaging. Fat fractions were compared to clinical severity, muscle force, age, edema and phosphocreatine (PCr)/ATP. Longitudinal intramuscular fat fraction variation was analyzed by linear regression. Increased intramuscular fat correlated with age (p<0.05), FSHD severity score (p<0.0001), inversely with muscle strength (p<0.0001), and also occurred sub-clinically. Muscles were nearly dichotomously divided in those with high and with low fat fraction, with only 13% having an intermediate fat fraction. The intramuscular fat fraction along the muscle’s length, increased from proximal to distal. This fat gradient was the steepest for intermediate fat infiltrated muscles (0.07±0.01/cm, p<0.001). Leg muscles in this intermediate phase showed a decreased PCr/ATP (p<0.05) and the fastest increase in fatty infiltration over time (0.18±0.15/year, p<0.001), which correlated with initial edema (p<0.01), if present. Thus, in the MR assessment of fat infiltration as biomarker for diseased muscles, the intramuscular fat distribution needs to be taken into account. Our results indicate that healthy individual leg muscles become diseased by entering a progressive phase with distal fat infiltration and altered energy metabolite levels. Fat replacement then relatively rapidly spreads over the whole muscle.     

Muscle glycogen recovery after exercise during glucose and fructose intake monitored by 13C-NMR

Van Den Bergh, Adrianus J., Sibrand Houtman, ArendHeerschap, Nancy J. Rehrer, Hendrikus J. Van Den Boogert, BerendOeseburg, and Maria T. E. Hopman. Muscle glycogen recovery afterexercise during glucose and fructose intake monitored by¹³C-NMR. J. Appl.Physiol. 81(4): 1495-1500, 1996.The purpose of this study was to examine muscle glycogen recovery with glucose feeding(GF) compared with fructose feeding (FF) during the first 8 h afterpartial glycogen depletion by using¹³C-nuclear magneticresonance (NMR) on a clinical 1.5-T NMR system. After measurement of the glycogen concentration of the vastus lateralis (VL) muscle in seven male subjects, glycogen stores of the VLwere depleted by bicycle exercise. During 8 h after completion ofexercise, subjects were orally given either GF or FF while the glycogencontent of the VL was monitored by¹³C-NMR spectroscopy every secondhour. The muscular glycogen concentration was expressed as a percentageof the glycogen concentration measured before exercise. The glycogenrecovery rate during GF (4.2 ± 0.2%/h) was significantly higher(P < 0.05) compared withvalues during FF (2.2 ± 0.3%/h). This study shows that1) muscle glycogen levels areperceptible by ¹³C-NMRspectroscopy at 1.5 T and 2) theglycogen restoration rate is higher after GF compared with after FF.

     

Lower force and impaired performance during high-intensity electrical stimulation in skeletal muscle of GAMT-deficient knockout mice

Force characteristics of skeletal muscle of knockout mice lacking creatine (Cr) due to a deletion of guanidinoacetate methyltransferase (GAMT) were studied in situ. Medial gastrocnemius muscles of anesthetized GAMT-deficient (GAMT^–/–) and control (Con) littermates were stimulated at optimum length via the sciatic nerve at different stimulation frequencies (60–250 Hz). GAMT^–/– mice showed reduced maximal tetanic and twitch force, reduced relative force at 60 Hz, and increased relaxation times. High-intensity fatigue protocols consisting of 30 successive isometric or dynamic contractions showed a strong reduction in force at the beginning of the series in GAMT^–/– mice, followed by a smaller reduction compared with Con littermates toward the end of the series. Cr supplementation for 2 days in GAMT^–/– animals (GAMT) resulted in normalization to Con values for relaxation times, relative force at lower stimulation frequencies, and relative force during 30 isometric contractions. Force per muscle mass, however, remained decreased. Furthermore,GAMT mice showed differences compared with both Con and unsupplemented animals in maximal rates of force rise and relaxation times during the isometric protocol as well as in force during the dynamic protocol. Our results show that the absence of Cr plays a direct role in relaxation times, maximal rate of force rise, and force production during high-intensity fatigue protocols. The lower force per muscle mass, however, is probably caused by other factors; i.e., high intracellular guanidinoacetate concentrations. energy metabolism; creatine; fatigue; force characteristics     

Magnetic resonance tracking of dendritic cells in melanoma patients for monitoring of cellular therapy

The success of cellular therapies will depend in part on accurate delivery of cells to target organs. In dendritic cell therapy, in particular, delivery and subsequent migration of cells to regional lymph nodes is essential for effective stimulation of the immune system. We show here that in vivo magnetic resonance tracking of magnetically labeled cells is feasible in humans for detecting very low numbers of dendritic cells in conjunction with detailed anatomical information. Autologous dendritic cells were labeled with a clinical superparamagnetic iron oxide formulation or (111)In-oxine and were co-injected intranodally in melanoma patients under ultrasound guidance. In contrast to scintigraphic imaging, magnetic resonance imaging (MRI) allowed assessment of the accuracy of dendritic cell delivery and of inter- and intra-nodal cell migration patterns. MRI cell tracking using iron oxides appears clinically safe and well suited to monitor cellular therapy in humans.     

Modulation of hypoxia in murine liver metastases of colon carcinoma by nicotinamide and carbogen

There is increasing evidence that modulation of tumor hypoxia may improve therapy outcome. However, most preclinical data are derived from subcutaneous rather than orthotopic tumor models. We investigated the effect of the hypoxia-modulating agents nicotinamide and carbogen on tumor hypoxia, tumor blood perfusion, and proliferative activity in liver metastases of the murine colon carcinoma line C26a. In untreated C26a liver metastases, we observed a considerable amount of hypoxia, similar to the amount in liver metastases of patients with colorectal cancer. Compared to untreated mice, we observed a significantly smaller hypoxic fraction in the liver metastases of mice treated with nicotinamide and carbogen breathing as single treatments or in combination. In the group of mice that underwent carbogen breathing, perfusion was significantly lower than in the untreated group, but the decrease was only marginal. The proliferative activity was similar in all groups. In C26a subcutaneous tumors, a similar effect on hypoxia has been observed that was, however, combined with a decrease in proliferative activity. The different effects of nicotinamide and carbogen on parameters of the tumor microenvironment in liver metastases and subcutaneous tumors suggest that the host tissue influences the mechanism by which nicotinamide and carbogen exert their effects. Since tumor hypoxia may be a clinical problem in colorectal liver metastases, our results open possibilities for further research on the effect of hypoxia modifiers on colorectal liver metastases to improve therapy outcome.     

Effects of Dual Targeting of Tumor Cells and Stroma in Human Glioblastoma Xenografts with a Tyrosine Kinase Inhibitor against c-MET and VEGFR2

Anti-angiogenic treatment of glioblastoma with Vascular Endothelial Growth Factor (VEGF)- or VEGF Receptor 2 (VEGFR2) inhibitors normalizes tumor vessels, resulting in a profound radiologic response and improved quality of life. This approach however does not halt tumor progression by diffuse infiltration, as this phenotype is less angiogenesis dependent. Combined inhibition of angiogenesis and diffuse infiltrative growth would therefore be a more effective treatment approach in these tumors. The HGF/c-MET axis is important in both angiogenesis and cell migration in several tumor types including glioma. We therefore analyzed the effects of the c-MET- and VEGFR2 tyrosine kinase inhibitor cabozantinib (XL184, Exelixis) on c-MET positive orthotopic E98 glioblastoma xenografts, which routinely present with angiogenesis-dependent areas of tumor growth, as well as diffuse infiltrative growth. In in vitro cultures of E98 cells, cabozantinib effectively inhibited c-MET phosphorylation, concomitant with inhibitory effects on AKT and ERK1/2 phosphorylation, and cell proliferation and migration. VEGFR2 activation in endothelial cells was also effectively inhibited in vitro. Treatment of BALB/c nu/nu mice carrying orthotopic E98 xenografts resulted in a significant increase in overall survival. Cabozantinib effectively inhibited angiogenesis, resulting in increased hypoxia in angiogenesis-dependent tumor areas, and induced vessel normalization. Yet, tumors ultimately escaped cabozantinib therapy by diffuse infiltrative outgrowth via vessel co-option. Of importance, in contrast to the results from in vitro experiments, in vivo blockade of c-MET activation was incomplete, possibly due to multiple factors including restoration of the blood-brain barrier resulting from cabozantinib-induced VEGFR2 inhibition. In conclusion, cabozantinib is a promising therapy for c-MET positive glioma, but improving delivery of the drug to the tumor and/or the surrounding tissue may be needed for full activity.