Rivaroxaban attenuates neutrophil maturation in the bone marrow niche.

Pharmacological inhibition of factor Xa by rivaroxaban has been shown to mediate cardioprotection and is frequently used in patients with, e.g., atrial fibrillation. Rivaroxaban's anti-inflammatory actions are well known, but the underlying mechanisms are still incompletely understood. To date, no study has focused on the effects of rivaroxaban on the bone marrow (BM), despite growing evidence that the BM and its activation are of major importance in the development/progression of cardiovascular disease. Thus, we examined the impact of rivaroxaban on BM composition under homeostatic conditions and in response to a major cardiovascular event. Rivaroxaban treatment of mice for 7 days markedly diminished mature leukocytes in the BM. While apoptosis of BM-derived mature myeloid leukocytes was unaffected, lineage-negative BM cells exhibited a differentiation arrest at the level of granulocyte-monocyte progenitors, specifically affecting neutrophil maturation via downregulation of the transcription factors Spi1 and Csfr1. To assess whether this persists also in situations of increased leukocyte demand, mice were subjected to cardiac ischemia/reperfusion injury (I/R): 7 d pretreatment with rivaroxaban led to reduced cardiac inflammation 72 h after I/R and lowered circulating leukocyte numbers. However, BM myelopoiesis showed a rescue of the leukocyte differentiation arrest, indicating that rivaroxaban's inhibitory effects are restricted to homeostatic conditions and are mainly abolished during emergency hematopoiesis. In translation, ST-elevation MI patients treated with rivaroxaban also exhibited reduced circulating leukocyte numbers. In conclusion, we demonstrate that rivaroxaban attenuates neutrophil maturation in the BM, which may offer a therapeutic option to limit overshooting of the immune response after I/R.

Regional analysis of inflammation and contractile function in reperfused acute myocardial infarction by in vivo 19F cardiovascular magnetic resonance in pigs.

Inflammatory cell infiltration is central to healing after acute myocardial infarction (AMI). The relation of regional inflammation to edema, infarct size (IS), microvascular obstruction (MVO), intramyocardial hemorrhage (IMH), and regional and global LV function is not clear. Here we noninvasively characterized regional inflammation and contractile function in reperfused AMI in pigs using fluorine (19F) cardiovascular magnetic resonance (CMR). Adult anesthetized pigs underwent left anterior descending coronary artery instrumentation with either 90 min occlusion (n = 17) or without occlusion (sham, n = 5). After 3 days, in surviving animals a perfluorooctyl bromide nanoemulsion was infused intravenously to label monocytes/macrophages. At day 6, in vivo 1H-CMR was performed with cine, T2 and T2* weighted imaging, T2 and T1 mapping, perfusion and late gadolinium enhancement followed by 19F-CMR. Pigs were sacrificed for subsequent ex vivo scans and histology. Edema extent was 35 ± 8% and IS was 22 ± 6% of LV mass. Six of ten surviving AMI animals displayed both MVO and IMH (3.3 ± 1.6% and 1.9 ± 0.8% of LV mass). The 19F signal, reflecting the presence and density of monocytes/macrophages, was consistently smaller than edema volume or IS and not apparent in remote areas. The 19F signal-to-noise ratio (SNR) > 8 in the infarct border zone was associated with impaired remote systolic wall thickening. A whole heart value of 19F integral (19F SNR × milliliter) > 200 was related to initial LV remodeling independently of edema, IS, MVO, and IMH. Thus, 19F-CMR quantitatively characterizes regional inflammation after AMI and its relation to edema, IS, MVO, IMH and regional and global LV function and remodeling.

Rational manufacturing of functionalized, long-term stable perfluorocarbon-nanoemulsions for site-specific 19F magnetic resonance imaging.

BACKGROUND: Perfluorocarbon (PFC)-nanoemulsions (NE) are a convenient tool for 19F magnetic resonance imaging in cell and animal experiments. Typical preparation methods, like high-pressure homogenization or microfluidization, produce nanoemulsions in mL-scale. However, experiments usually require only miniscule amounts of PFC-NE, several 100 µL. For site-specific imaging tissue-specific ligands, e.g. peptides or antibodies, are covalently bound to the NE surface. This requires the use of expensive functionalized phospholipids containing reactive groups (e.g. maleimide), which often deteriorate quickly in liquid storage, rendering the manufacturing process highly cost-inefficient. A technique to manufacture storage stable NE that maintain their functionality for coupling of various ligands is desired. METHODS AND RESULTS: Different PFC-NE formulations and preparation techniques were compared and the most suitable of these was tested in short-, as well as long-term stability tests. Droplet size stability was investigated by dynamic light scattering and cryogenic transmission electron microscopy over 1.5 a. Surface modifiability was assessed by a fluorescence assay. The utility of these NE was proven in an in vitro model. CONCLUSION: The established PFC-NE platform offers a cost-efficient way to produce larger amounts of long-term storable imaging agents, which can be surface-modified on demand for application in targeted 19F MRI.

Genetic immunization with mouse thyrotrophin hormone receptor plasmid breaks self-tolerance for a murine model of autoimmune thyroid disease and Graves' orbitopathy.

Experimental models of Graves' hyperthyroid disease accompanied by Graves' orbitopathy (GO) can be induced efficiently in susceptible inbred strains of mice by immunization by electroporation of heterologous human TSH receptor (TSHR) A-subunit plasmid. In this study, we report on the development of a bona fide murine model of autoimmune Graves' disease induced with homologous mouse TSHR A-subunit plasmid. Autoimmune thyroid disease in the self-antigen model was accompanied by GO and characterized by histopathology of hyperplastic glands with large thyroid follicular cells. Examination of orbital tissues showed significant inflammation in extra-ocular muscle with accumulation of T cells and macrophages together with substantial deposition of adipose tissue. Notably, increased levels of brown adipose tissue were present in the orbital tissue of animals undergoing experimental GO. Further analysis of inflammatory loci by 19 F-magnetic resonance imaging showed inflammation to be confined to orbital muscle and optic nerve, but orbital fat showed no difference in inflammatory signs in comparison to control ß-Gal-immunized animals. Pathogenic antibodies induced to mouse TSHR were specific for the self-antigen, with minimal cross-reactivity to human TSHR. Moreover, compared to other self-antigen models of murine Graves' disease induced in TSHR knock-out mice, the repertoire of autoantibodies to mouse TSHR generated following the breakdown of thymic self-tolerance is different to those that arise when tolerance is not breached immunologically, as in the knock-out models. Overall, we show that mouse TSHR A-subunit plasmid immunization by electroporation overcomes tolerance to self-antigen to provide a faithful model of Graves' disease and GO.

Monocyte imaging after myocardial infarction with 19F MRI at 3 T: a pilot study in explanted porcine hearts.

AIM: Inflammation is a hallmark of cardiac healing after myocardial infarction and it determines subsequent cardiovascular morbidity and mortality. The aim of the present study was to explore whether inflammation imaging with two perfluorocarbon (PFC) nanoemulsions and fluorine magnetic resonance imaging ((19)F MRI) is feasible at 3.0 T with sufficient signal-to-noise ratio (SNR) using explanted hearts, an (19)F surface coil and dedicated MR sequences. METHODS AND RESULTS: Acute myocardial infarction (AMI) was induced by balloon angioplasty (50 min) of the distal left anterior descending artery in 12 pigs. One day thereafter, PFCs were injected intravenously to label circulating monocytes. Either emulsified perfluoro-15-crown-5 ether or already clinically applied perfluorooctyl bromide (PFOB) was applied. Four days after AMI and immediately after gadolinium administration, hearts were explanted and imaged with a 3.0 T Achieva MRI scanner. (19)F MRI could be acquired with an SNR of >15 using an in-plane resolution of 2 × 2 mm(2) within <20 min for both agents. Combined late gadolinium enhancement (LGE) and (19)F MRI revealed that (19)F signal was inhomogenously distributed across LGE myocardium reflecting patchy macrophage infiltration as confirmed by histology. In whole hearts, we found an apico-basal (19)F gradient within LGE-positive myocardium. The (19)F-positive volume was always smaller than LGE volume. Ex vivo experiments on isolated monocytes revealed that pig and human cells phagocytize PFCs even more avidly than mouse monocytes. CONCLUSION: This pilot study demonstrates that (19)F MRI at 3.0 T with clinically applicable PFOB is feasible, thus highlighting the potential of (19)F MRI to monitor the inflammatory response after AMI.

Noninvasive detection of graft rejection by in vivo (19) F MRI in the early stage.

Diagnosis of transplant rejection requires tissue biopsy and entails risks. Here, we describe a new (19) F MRI approach for noninvasive visualization of organ rejection via the macrophage host response. For this, we employed biochemically inert emulsified perfluorocarbons (PFCs), known to be preferentially phagocytized by monocytes and macrophages. Isografts from C57BL/6 or allografts from C57B10.A mice were heterotopically transplanted into C57BL/6 recipients. PFCs were applied intravenously followed by (1) H/(19) F MRI at 9.4 T 24 h after injection. (1) H images showed a similar position and anatomy of the graft in the abdomen for both cases. However, corresponding (19) F signals were only observed in allogenic tissue. (1) H/(19) F MRI enabled us to detect the initial immune response not later than 3 days after surgery, when conventional parameters did not reveal any signs of rejection. In allografts, the observed (19) F signal strongly increased with time and correlated with the extent of rejection. In separate experiments, rapamycin was used to demonstrate the ability of (19) F MRI to monitor immunosuppressive therapy. Thus, PFCs can serve as positive contrast agent for the early detection of transplant rejection by (19) F MRI with high spatial resolution and an excellent degree of specificity due to lack of any (19) F background.

Unmasking the Janus face of myoglobin in health and disease.

For more than 100 years, myoglobin has been among the most extensively studied proteins. Since the first comprehensive review on myoglobin function as a dioxygen store by Millikan in 1939 and the discovery of its structure 50 years ago, multiple studies have extended our understanding of its occurrence, properties and functions. Beyond the two major roles, the storage and the facilitation of dioxygen diffusion, recent physiological studies have revealed that myoglobin acts as a potent scavenger of nitric oxide (NO(*)) representing a control system that preserves mitochondrial respiration. In addition, myoglobin may also protect the heart against reactive oxygen species (ROS), and, under hypoxic conditions, deoxygenated myoglobin is able to reduce nitrite to NO(*) leading to a downregulation of the cardiac energy status and to a decreased heart injury after reoxygenation. Thus, by controlling the NO(*) bioavailability via scavenging or formation, myoglobin serves as part of a sensitive dioxygen sensory system. In this review, the physiological relevance of these recent findings are delineated for pathological states where NO(*) and ROS bioavailability are known to be critical determinants for the outcome of the disease, e.g. ischemia/reperfusion injury. Detrimental and beneficial effects of the presence of myoglobin are discussed for various states of tissue oxygen tension within the heart and skeletal muscle. Furthermore, the impact of myoglobin on parasite infection, rhabdomyolysis, hindlimb and liver ischemia, angiogenesis and tumor growth are considered.

Targeting murine heart and brain: visualisation conditions for multi-pinhole SPECT with (99m)Tc- and (123)I-labelled probes.

PURPOSE: The study serves to optimise conditions for multi-pinhole SPECT small animal imaging of (123)I- and (99m)Tc-labelled radiopharmaceuticals with different distributions in murine heart and brain and to investigate detection and dose range thresholds for verification of differences in tracer uptake. METHODS: A Triad 88/Trionix system with three 6-pinhole collimators was used for investigation of dose requirements for imaging of the dopamine D(2) receptor ligand [(123)I]IBZM and the cerebral perfusion tracer [(99m)Tc]HMPAO (1.2-0.4 MBq/g body weight) in healthy mice. The fatty acid [(123)I]IPPA (0.94 +/- 0.05 MBq/g body weight) and the perfusion tracer [(99m)Tc]sestamibi (3.8 +/- 0.45 MBq/g body weight) were applied to cardiomyopathic mice overexpressing the prostaglandin EP(3) receptor. RESULTS: In vivo imaging and in vitro data revealed 45 kBq total cerebral uptake and 201 kBq cardiac uptake as thresholds for visualisation of striatal [(123)I]IBZM and of cardiac [(99m)Tc]sestamibi using 100 and 150 s acquisition time, respectively. Alterations of maximal cerebral uptake of [(123)I]IBZM by >20% (116 kBq) were verified with the prerequisite of 50% striatal of total uptake. The labelling with [(99m)Tc]sestamibi revealed a 30% lower uptake in cardiomyopathic hearts compared to wild types. [(123)I]IPPA uptake could be visualised at activity doses of 0.8 MBq/g body weight. CONCLUSION: Multi-pinhole SPECT enables detection of alterations of the cerebral uptake of (123)I- and (99m)Tc-labelled tracers in an appropriate dose range in murine models targeting physiological processes in brain and heart. The thresholds of detection for differences in the tracer uptake determined under the conditions of our experiments well reflect distinctions in molar activity and uptake characteristics of the tracers.

Myoglobin: A scavenger of bioactive NO.

The present study explored the role of myoglobin (Mb) in cardiac NO homeostasis and its functional relevance by employing isolated hearts of wild-type (WT) and myoglobin knockout mice. (1)H NMR spectroscopy was used to measure directly the conversion of oxygenated Mb (MbO(2)) to metmyoglobin (metMb) by reaction with NO. NO was applied intracoronarily (5 nM to 25 microM), or its endogenous production was stimulated with bradykinin (Bk; 10 nM to 2 microM). We found that infusion of authentic NO solutions dose-dependently (>/= 2.5 microM NO) increased metMb formation in WT hearts that was rapidly reversible on cessation of NO infusion. Likewise, Bk-induced release of NO was associated with significant metMb formation in the WT (>/=1 microM Bk). Hearts lacking Mb reacted more sensitively to infused NO in that vasodilatation and the cardiodepressant actions of NO were more pronounced. Similar results were obtained with Bk. The lower sensitivity of WT hearts to changes in NO concentration fits well with the hypothesis that in the presence of Mb, a continuous degradation of NO takes place by reaction of MbO(2) + NO to metMb + NO(3)(-), thereby effectively reducing cytosolic NO concentration. This breakdown protects myocytic cytochromes against transient rises in cytosolic NO. Regeneration of metMb by metMb reductase to Mb and subsequent association with O(2) leads to reformation of MbO(2) available for another NO degradation cycle. Our data indicate that this cycle is crucial in the breakdown of NO and substantially determines the dose-response curve of the NO effects on coronary blood flow and cardiac contractility.

Effects of ammonia exposition on glioma cells: changes in cell volume and organic osmolytes studied by diffusion-weighted and high-resolution NMR spectroscopy.

NH(4)Cl (10 mM) caused a sustained increase in the cell volume in immobilized, perfused F98 glioma cells to approx. 125% of control after 3 h, as measured by diffusion-weighted (1)H NMR spectroscopy. Concomitantly, the glutamine (Gln) concentration increased by 130%, accompanied by a marked decrease in cytosolic osmolytes, i.e. myo-inositol and taurine, determined from (1)H NMR spectra of PCA extracts. Inhibition of Gln synthetase partially prevented the increase in water content. While losses of organic osmolytes are also observed under hypotonic conditions, the rapid cell swelling is followed by the regulatory cell volume decrease (RVD), and is accompanied by decreased cytosolic Gln. We suggest that the rise in intracellular osmolarity, which is attributed to NH(4)Cl metabolism to Gln, but also to alanine (Ala), is not compensated by the release of other osmolytes, and causes cell swelling without RVD.

Disruption of myoglobin in mice induces multiple compensatory mechanisms.

Myoglobin may serve a variety of functions in muscular oxygen supply, such as O(2) storage, facilitated O(2) diffusion, and myoglobin-mediated oxidative phosphorylation. We studied the functional consequences of a myoglobin deficiency on cardiac function by producing myoglobin-knockout (myo(-/-)) mice. To genetically inactivate the myoglobin gene, exon 2 encoding the heme binding site was deleted in embryonic stem cells via homologous recombination. Myo(-/-) mice are viable, fertile, and without any obvious signs of functional limitations. Hemoglobin concentrations were significantly elevated in myo(-/-) mice. Cardiac function and energetics were analyzed in isolated perfused hearts under resting conditions and during beta-adrenergic stimulation with dobutamine. Myo(-/-) hearts showed no alteration in contractile parameters either under basal conditions or after maximal beta-adrenergic stimulation (200 nM dobutamine). Tissue levels of ATP, phosphocreatine ((31)P-NMR), and myocardial O(2) consumption were not altered. However, coronary flow [6.4 +/- 1.3 ml.min(-1).g(-1) [wild-type (WT)] vs. 8.5 +/- 2.4 ml.min(-1).g(-1) [myo(-/-)] [and coronary reserve [17.1 +/- 2.1 (WT) vs. 20.8 +/- 1.1 (myo(-/-) ml. min(-1).g(-1) were significantly elevated in myo(-/-) hearts. Histological examination revealed that capillary density also was increased in myo(-/-) hearts [3,111 +/- 400 mm(-2) (WT) vs. 4,140 +/- 140 mm(-2) (Myo(-/-)]. These data demonstrate that disruption of myoglobin results in the activation of multiple compensatory mechanisms that steepen the pO(2) gradient and reduce the diffusion path length for O(2) between capillary and the mitochondria; this suggests that myoglobin normally is important for the delivery of oxygen.

Contribution of NO to ischemia-reperfusion injury in the saline-perfused heart: a study in endothelial NO synthase knockout mice.

The contribution of endogenous NO to ischemia-reperfusion injury was studied in isolated perfused hearts of wild-type (WT) and endothelial NO synthase knockout (eNOS-) mice. The hearts were subjected to a 16-min period of global no-flow ischemia and were subsequently reperfused for 1 h. Cardiac contractile function was evaluated and 31P-NMR spectroscopy was used to monitor myocardial energy status and the intracellular pH. During both baseline and ischemia, there were neither significant differences in mechanical function nor in energetic parameters between the two groups, for example at baseline left ventricular developed pressure (LVDP) was 56.5+/-5.4 mmHg in WT and 58.7+/-5.2 mmHg in eNOS-and phosphocreatine (PCr) level was 12.9+/-1.3 m m in WT and 12.7+/-1.7 m m in eNOS-. In reperfusion, however, a significant improvement of the post-ischemic functional and metabolic recovery became apparent in the eNOS-hearts. While in the WT group, LVDP recovered only to 38. 4+/-5.3 mmHg, LVDP in the eNOS-group attained 49.4+/-5.5 mmHg at the end of 60 min reperfusion (P<0.05, n=8). Similarly, the recovery of PCr was significantly enhanced in the transgenic hearts as compared to WT (10.4+/-1.6 vs 8.1+/-1.3 m m, P<0.05). eNOS-hearts also showed a better restoration of dP/d t and a significant lower left ventricular enddiastolic pressure. In an additional series of wild-type hearts, the NO synthase inhibitor NG-monomethyl-L-arginine methyl ester (100 microm) also tended to improve the recovery of both LVDP (43.8+/-6.8 mmHg) and PCr (9.5+/-1.6 m m) in reperfusion (1 h), but the restoration of functional and metabolic parameters was less pronounced when compared with eNOS-. The results provide clear evidence that endogenously formed NO significantly contributes to ischemia-reperfusion injury in the saline-perfused mouse heart, most likely by peroxynitrite formation from NO.

Rat brain primary neurons immobilized in basement membrane gel threads: an improved method for on-line 13C NMR spectroscopy of live cells.

In vivo MRS studies on intact brain reflect the metabolism of all cells present, but do not distinguish between different cell types. NMR studies of immobilized cultured primary cells, such as neurons and astrocytes, are a useful model to monitor the specific differences in metabolism of the various cell types in the brain. The present study shows that primary rat neuronal cells can be cultured in basement membrane gel threads. After 4 days of incubation the threads are filled with viable cells, and represent a population of morphologically differentiated neuronal cells with less than 5% of non-neuronal cells, i.e., astrocytes. These threads were placed into a NMR tube and used for on-line monitoring of neuronal metabolism. Under these conditions cells remained viable and metabolically active for several days. The energy status was monitored by using 31P NMR spectroscopy. To study neuronal glucose metabolism [1-13C]glucose was added to the perfusion medium and 30 min later 13C-labeled metabolites were detectable by 13C NMR spectroscopy. Immobilized neurons synthesized glycolytic products such as [3-13C]lactate and [3-13C]alanine, as well as several tricarboxylic acid cycle products, i.e., [2-13C]glutamate, [3-13C]glutamate, [4-13C]glutamate, [2-13C]aspartate, and [3-13C]aspartate. In summary, 31P and 13C NMR spectra can be recorded from live neuronal cells for up to 24 h using the newly designed procedure described in the present communication.

Expression of aquaporins in Xenopus laevis oocytes and glial cells as detected by diffusion-weighted 1H NMR spectroscopy and photometric swelling assay.

Expression of aquaporins (AQP) and water permeability were studied in Xenopus laevis oocytes and immobilized glial cells by a pulsed-field gradient spin echo NMR technique and a photometric swelling assay. Oocytes injected with poly(A) RNA from C6-BU-1 cells showed increased swelling behavior under hypoosmotic stress due to expressed water channels as compared to control oocytes. The swelling could be reversibly inhibited by HgCl2. Furthermore, the intracellular relaxation time and the apparent intracellular diffusion coefficient of water in oocytes were determined by diffusion-weighted 1H NMR experiments to be T2=36 ms and Dapp, intra=0.18x10-3 mm2/s. In immobilized C6 and F98 cells the mean exchange time of intracellular water was found to be 51 ms which increased to 75 ms upon chronic treatment (4 days) in hypertonic medium. Additional hybrid depletion experiments with antisense oligonucleotides directed against AQP1 were performed on oocytes and C6 cells. Moreover, different water channel subtypes of glial cells were assessed by a reverse transcriptase polymerase chain reaction assay. With this, the mRNA encoding AQP1 could be detected in primary cultures and glial cell lines, whereas AQP4 mRNA was found in astroglia-rich primary cultures, but not in F98 and C6 cells. Our results show that water permeability in glial cells is mainly mediated by water channels which play an important role in the regulation of water flow in brain under normal and pathological conditions.

Alterations in glial cell metabolism during recovery from chronic osmotic stress.

NMR spectroscopy of F98 glioma cell extracts showed that chronic hypertonic conditions largely increased the intracellular content of small, osmotically active molecules. Moreover, hypertonic stress decreased the incorporation of 13C-labeled amino acids into the cellular proteins albeit their cytosolic concentrations were increased, which reflects an inhibition of protein synthesis under these conditions. Reincubation with isotonic medium restored almost completely the control values for the cytosolic metabolites but not for amino acid incorporation into the protein. An increased amount of 13C label was found in the phospholipids, which indicates stimulation of membrane synthesis processes due to the recovery-induced cell swelling. On the other hand, chronic hypotonic conditions largely decreased the steady state concentration and synthesis of small, cytosolic molecules, whereas the effect on the incorporation of 13C-labeled amino acids into the cellular proteins was variable. Reincubation with isotonic medium partially restored the depressed cytosolic metabolite content and also the incorporation of labeled amino acids into cellular protein, but induced an inhibition of phospholipid synthesis. The results verify that 'readaptation' of glial cell metabolism during recovery from chronic osmotic stress is impaired or at least seriously retarded.

Monitoring of cell volume and water exchange time in perfused cells by diffusion-weighted 1H NMR spectroscopy.

Diffusion of intracellular water was measured in perfused cells embedded in basement membrane gel threads. F98 glioma cells, primary astrocytes, and epithelial KB cells were used and were exposed to osmotic stress, immunosuppressiva, the water channel blocker p-chloromercuriobenzenesulfonate (pCMBS), and apoptotic conditions. With diffusion-weighted 1H NMR spectroscopy changes in the intracellular signal could be monitored and quantified with single signal (ss), constant diffusion time (ct), and constant gradient strength (cg) experiments. The temporal resolution of the ss monitoring was 3.5 s with a standard deviation of 0.5% of the signal intensity and 32 s (3%) with ct monitoring, respectively. A mean intracellular residence time of water was determined with the cg experiment to about 50 ms. Changes of this exchange time from (51.9 +/- 1.0) to (59.0 +/- 1.1) ms were observed during treatment with pCMBS. The changes in the diffusion attenuated signal could be simulated analytically varying the intracellular volume fraction and exchange time by combination of restricted diffusion (Tanner model) and water exchange (Kärger model). This sensitive and noninvasive NMR method on perfused cells allows to determine changes in the intracellular volume and residence time in a simple and accurate manner. Many further applications as anoxia, volume regulation, ischemia and treatment with various pharmaceuticals are conceiveable to follow up their effect on the cell volume and the exchange time of intracellular water.

Restricted diffusion and exchange of intracellular water: theoretical modelling and diffusion time dependence of 1H NMR measurements on perfused glial cells.

Intracellular diffusion properties of water in F98 glioma cells immobilized in basement membrane gel threads, are investigated with a pulsed-field-gradient spin-echo NMR technique at diffusion times from 6 to 2000 ms and at different temperatures. In extended model calculations the concept of 'restricted intracellular diffusion at permeable boundaries' is described by a combined Tanner-Kärger formula. Signal components in a series of ct experiments (constant diffusion time) are separated due to different diffusion properties (Gaussian and restricted diffusion), and physiological as well as morphological cell parameters are extracted from the experimental data. The intracellular apparent diffusion coefficients strongly depend on the diffusion time and are up to two orders of magnitude smaller than the self diffusion constant of water. Propagation lengths are found to be in the range of 4-7 microns. Hereby intracellular signals of compartments with a characteristic diameter could be selected by an appropriate gradient strength. With cg experiments (constant gradient) a mean intracellular residence time for water is determined to be about 50 ms, and the intrinsic intracellular diffusion constant is estimated to 1 x 10(-3)mm2/s. Studying the water diffusion in glial cells provides basic understanding of the intracellular situation in brain tissue and may elucidate possible influences on the changes in the diffusion contrast during ischemic conditions.

A 1H/13C inverse 2D method for the analysis of the polyamines putrescine, spermidine and spermine in cell extracts and biofluids.

The polyamines putrescine, spermidine and spermine are involved in the regulation of various metabolic processes. It is therefore desirable to detect and quantify the polyamines with NMR. We present the proton and carbon assignments for all polyamine signals obtained from PCA extracts of F98 glioma cells with high resolution using a semi-selective HSQC 2D-experiment. The biosynthesis of the polyamines in cell culture was examined using the labeled substrates [U-13C]glucose and [U-13C]glutamate. In such studies the high resolution of the semi-selective HSQC experiment at very high magnetic fields (14-19 T) allows the analysis of carbon-carbon couplings, and isotopomer patterns. The different effects of osmotic stress on the concentrations of polyamines and amino acids are also reported.