Tumor cell proliferation requires rapid synthesis of macromolecules including lipids, proteins, and nucleotides. Many tumor cells exhibit rapid glucose consumption, with most of the glucose-derived carbon being secreted as lactate despite abundant oxygen availability (the Warburg effect). Here, we used 13 C NMR spectroscopy to examine the metabolism of glioblastoma cells exhibiting aerobic glycolysis. In these cells, the tricarboxylic acid (TCA) cycle was active but was characterized by an efflux of substrates for use in biosynthetic pathways, particularly fatty acid synthesis. The success of this synthetic activity depends on activation of pathways to generate reductive power (NADPH) and to restore oxaloacetate for continued TCA cycle function (anaplerosis). Surprisingly, both these needs were met by a high rate of glutamine metabolism. First, conversion of glutamine to lactate (glutaminolysis) was rapid enough to produce sufficient NADPH to support fatty acid synthesis. Second, despite substantial mitochondrial pyruvate metabolism, pyruvate carboxylation was suppressed, and anaplerotic oxaloacetate was derived from glutamine. Glutamine catabolism was accompanied by secretion of alanine and ammonia, such that most of the amino groups from glutamine were lost from the cell rather than incorporated into other molecules. These data demonstrate that transformed cells exhibit a high rate of glutamine consumption that cannot be explained by the nitrogen demand imposed by nucleotide synthesis or maintenance of nonessential amino acid pools. Rather, glutamine metabolism provides a carbon source that facilitates the cell's ability to use glucose-derived carbon and TCA cycle intermediates as biosynthetic precursors.cancer ͉ glioblastoma ͉ Warburg effect ͉ glutaminolysis ͉ anaplerosis I n mammals, cell proliferation is controlled by signal transduction pathways stimulated by lineage-specific growth factors or, in tumors, constitutive activation of these pathways through oncogenic mutations. A proximal effect of signaling pathways is a robust increase in nutrient uptake (1-3). Cells must also allocate these molecules into appropriate metabolic pathways to produce and maintain pools of intermediates needed to synthesize macromolecules. Therefore, a complete understanding of the biology of cell proliferation will require a comprehensive understanding of the regulation of metabolic fluxes.Glucose and glutamine are two of the most abundant nutrients in plasma, and together they account for most carbon and nitrogen metabolism in mammalian cells. Rapid cell proliferation has been associated with a robust but apparently wasteful metabolism of glucose. In the 1920s, Otto Warburg demonstrated that ascites tumor cells had high rates of glucose consumption and lactate production despite availability of sufficient oxygen to oxidize glucose completely (4). The ''Warburg effect'' is taken to be a metabolic hallmark of aggressive tumors; however, the phenotype is also observed in nontransformed cells during rapid proliferation (2, 5). Gl...

In recent years, a variety of low molecular weight antibiotics have been isolated from diverse animal species. These agents, which include peptides, lipids, and alkaloids, exhibit antibiotic activity against environmental microbes and are thought to play a role in innate immunity. We report here the discovery of a broad-spectrum steroidal antibiotic isolated from tissues of the dogfish shark Squalus acanthias. This water-soluble antibiotic, which we have named squalamine, exhibits potent bactericidal activity against both Gram-negative and Gram-positive bacteria. In addition, squalamine is fungicidal and induces osmotic lysis of protozoa. The chemical structure of the antibiotic 313-N-1-{N-[3-(4-aminobutyl)]-1,3-diaminopropane}-7a,24'-dihydroxy-Sacholestane 24-sulfate has been determined by fast atom bombardment mass spectroscopy and NMR. Squalamine is a cationic steroid characterized by a condensation of an anionic bile salt intermediate with spermidine. The discovery of squalamine in the shark implicates a steroid as a potential host-defense agent in vertebrates and provides insights into the chemical design of a family of broad-spectrum antibiotics.Animals must defend themselves against environmental microbes if they are to survive. Multiple mechanisms of host defense against microbes have been described such as the array of humoral and cellular responses of the classical vertebrate immune system and less-specific physical and chemical barriers. Over the past several years, an increasing number of low molecular weight antibiotic substances, believed to play a role in defense against environmental microbes, have been isolated from diverse species of animals. These molecules include peptides (1-3), lipids (4, 5), and alkaloids (6-8).In the course of our studies exploring the diversity of antibiotics from animal sources, we have surveyed tissues from a number of animal species (9, 10). We focused our search for antibiotic substances on the gastrointestinal tract of various animals after the recent discovery of peptide antibiotics in the gut of frogs (11), pigs (12, 13), mice (14), and humans (34). In the course of our survey, we discovered that stomach extracts of the shark Squalus acanthias exhibited potent antimicrobial activity, prompting efforts to purify and identify the responsible molecule.In this report we describe the isolation, structural determination, and characterization of a water-soluble cationic steroid from the shark that exhibits potent antimicrobial activity against fungi, protozoa, and both Gram-negative and Gram-positive bacteria. This molecule is shown to be an unusual adduct of spermidine with an anionic bile salt intermediate that, to our knowledge, is without precedent in vertebrates. We have named the aminosterol "squalamine," derived from the genus Squalus and its chemical structure as an amine. MATERIALS AND METHODSPurification of Squlamine. Squalus acanthias sharks were captured off the New England coast. The shark stomach tissue (400 g) was frozen immediately after dissectio...

Abstract-Recent evidence indicates that the type of atherosclerotic plaque, rather than the degree of obstruction to flow, is an important determinant of the risk of cardiovascular complications. In previous work, the feasibility of using MRI for the characterization of plaque components was shown. This study extends the previous work to all the plaque components and shows the accuracy of this method. Twenty-two human carotid endarterectomy specimens underwent ex vivo MRI and histopathological examination. Sixty-six cross sections were matched between MRI and histopathology. In each cross section, the presence or absence of plaque components were prospectively identified on the MRI images. The overall sensitivity and specificity for each tissue component were very high. Calcification and fibrocellular tissue were readily identified. Lipid core was also identifiable. However, thrombus was the plaque component for which MRI had the lowest sensitivity. A semiautomated algorithm was created to identify all major atherosclerotic plaque components. MRI can characterize carotid artery plaques with a high level of sensitivity and specificity. isruption of atherosclerotic plaques is the most frequent underlying cause of the unpredictable onset of acute thromboembolic vascular events including sudden death, myocardial infarction, unstable angina, stroke, transient cerebral ischemia, and peripheral thromboemboli. 1,2 Although clinical risk factors for atherosclerosis help predict risk of these events, identification of patients with plaques vulnerable to disruption is not possible by angiography that only visualizes the lumen. There is therefore a need for an in vivo noninvasive method for characterizing atherosclerotic plaques and identifying the "vulnerable" plaque.Previous work has shown that MRI can characterize both ex vivo 3-7 and in vivo 8 -11 the composition of human atherosclerotic plaques. However, the sensitivity and specificity of MRI have not been determined.This study reports the development of high-resolution MRI criteria for the ex vivo tissue characterization of human carotid atherosclerotic plaques and their sensitivity and specificity in comparison with histopathology. Using these criteria, a semiautomatic segmentation algorithm is developed for characterizing the constituents of an atherosclerotic plaque. Methods SpecimensHuman carotid endarterectomy specimens were studied. Specimens were obtained fresh and intact from the operating room, washed in phosphate buffered saline, grossly described, and samples taken for routine surgical pathology. The remaining 1-to 2-cm-long segments were flash frozen at Ϫ80°C until imaged. On the day of imaging, the specimens were placed in saline and slowly warmed to 37°C in a water bath. The artery was placed in either a 10-or 12-mm MR tube (Wilmad Glass) using the smallest possible tube for a given specimen. Care was taken to remove any air bubbles. Previous studies have shown no change in the T1 and T2 of atheromatous plaques under these conditions of freezing and rewa...

Magnetic resonance imaging has previously demonstrated its potential for indirectly mapping myelin density, either by relaxometric detection of myelin water or magnetization transfer. Here, we investigated whether myelin can be detected and possibly quantified directly. We identified the spectrum of myelin in the spinal cord in situ as well as in myelin lipids extracted via a sucrose gradient method, and investigated its spectral properties. Highresolution solution NMR spectroscopy showed the extract composition to be in agreement with myelin's known chemical make-up. The 400-MHz 1 H spectrum of the myelin extract, at 20°C (room temperature) and 37°C, consists of a narrow water resonance superimposed on a broad envelope shifted ∼3.5 ppm upfield, suggestive of long-chain methylene protons. Superimposed on this signal are narrow components resulting from functional groups matching the chemical shifts of the constituents making up myelin lipids. The spectrum could be modeled as a sum of super-Lorentzians with a T 2 * distribution covering a wide range of values (0.008-26 ms). Overall, there was a high degree of similarity between the spectral properties of extracted myelin lipids and those found in neural tissue. The normalized difference spectrum had the hallmarks of membrane proteins, not present in the myelin extract. Using 3D radially ramp-sampled proton MRI, with a combination of adiabatic inversion and echo subtraction, the feasibility of direct myelin imaging in situ is demonstrated. Last, the integrated signal from myelin suspensions is shown, both spectroscopically and by imaging, to scale with concentration, suggesting the potential for quantitative determination of myelin density. myelin in situ | myelin NMR spectrum | super-Lorentzian fitting | ultrashort echo time

Two major products (adducts A and B) from the reaction of 2-deoxyguanosine (dGuo) with 13-hydroperoxylinoleic acid were detected by liquid chromatography/mass spectrometry (LC/MS). Adducts A and B were also the major products formed enzymatically when dGuo was incubated in the presence of linoleic acid and lipoxygenase. The mass spectral fragmentation patterns of adducts A and B suggested that unique modifications to the nucleoside had been introduced. This resulted in the characterization of a novel bifunctional electrophile, 4-oxo-2-nonenal, as the principal breakdown product of linoleic acid hydroperoxide. In subsequent studies, adduct A was found to be a substituted ethano dGuo adduct that was a mixture of three isomers (A(1)-A(3)) that all decomposed to form adduct B. Adduct A(1) was the hemiacetal form of 3-(2-deoxy-beta-D-erythropentafuranosyl)-3,5,6, 7-tetrahydro-6-hydroxy-7-(heptane-2-one)-9H-imidazo[1, 2-alpha]purine-9-one. Adducts A(2) and A(3) were the diastereomers of the open chain ketone form. Adduct B was the substituted etheno dGuo adduct, 3-(2-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(heptane-2 -one)-9-hydroxy[1,2-alpha]purine, the dehydration product of adducts A(1)-A(3). Identical covalent modifications to dGuo were observed when calf-thymus DNA was treated with 4-oxo-2-nonenal. These data illustrate the diversity of reactive electrophiles produced from the peroxidative decomposition of lipids and have implications in fully assessing the role of lipid peroxidation in mutagenesis and carcinogenesis.

Background-The pathogenesis of atherosclerosis is currently being investigated in genetically engineered small animals.Methods to follow the time course of the developing pathology and/or the responses to therapy in vivo are limited. Methods and Results-To address this problem, we developed a noninvasive MR microscopy technique to study in vivo atherosclerotic lesions (without a priori knowledge of the lesion location or lesion type) in live apolipoprotein E-knockout (apoE-KO) mice. The spatial resolution was 0.0012 to 0.005 mm 3 . The lumen and wall of the abdominal aorta and iliac arteries were identified on all images in apoE-KO (nϭ8) and wild-type (nϭ5) mice on chow diet. Images obtained with MR were compared with corresponding cross-sectional histopathology (nϭ58). MR accurately determined wall area in comparison to histopathology (slopeϭ1.0, rϭ0.86). In addition, atherosclerotic lesions were characterized in terms of lesion shape and type. Lesion type was graded by MR according to morphological appearance/severity and by histopathology according to the AHA classification. There was excellent agreement between MR and histopathology in grading of lesion shape and type (slopeϭ0.97, rϭ0.91 for lesion shape; slopeϭ0.64, rϭ0.90 for lesion type). Conclusions-The combination of high-resolution MR microscopy and genetically engineered animals is a powerful toolto investigate serially and noninvasively the progression and regression of atherosclerotic lesions in an intact animal model and should greatly enhance basic studies of atherosclerotic disease. (Circulation. 1998;98:1541-1547.)Key Words: atherosclerosis Ⅲ magnetic resonance imaging Ⅲ genes G enetically engineered animal models provide enormous potential for the study of the pathogenesis and treatment of numerous human diseases. The mouse is the most widely used animal for genetic studies, and techniques for genetic modification in vivo (transgenic and gene-targeting) are much more advanced in the mouse than any other mammal. 1,2For example, genetically engineered mice are being increasingly used as a model of atherosclerosis. Apolipoprotein E-knockout (apoE-KO) mice produced by gene-targeting technologies 3 spontaneously develop atherosclerotic lesions similar in morphology to those observed in humans.3,4 Most experimental designs are typically limited to in vitro and ex vivo examination. However, it would be advantageous to develop an in vivo technique for serial, noninvasive imaging to monitor progression or regression of the arterial lesions in this and other mouse models. Such a technique would allow the performance of repeated analyses in the same animal, rather than study of multiple experimental groups with larger number of animals killed at different time points. See p 1477MRI is a noninvasive, nondestructive, 3-dimensional imaging technique that differentiates tissue structure on the basis of proton magnetic properties, allowing a wide range of image contrast, ie, proton density-weighted (PDW), T1-, T2-weighted (T1W, T2W), etc.5 Currently, MRI is be...

Q-space imaging (QSI), a diffusion MRI technique, can provide quantitative tissue architecture information at cellular dimensions not amenable by conventional diffusion MRI. By exploiting regularities in molecular diffusion barriers, QSI can estimate the average barrier spacing such as the mean axon diameter in white matter (WM). In this work, we performed ex vivo QSI on cervical spinal cord sections from healthy C57BL/6 mice at 400MHz using a custom-designed uniaxial 50T/m gradient probe delivering a 0.6 µm displacement resolution capable of measuring axon diameters on the scale of 1 µm. After generating QSI-derived axon diameter maps, diameters were calculated using histology from seven WM tracts (dorsal corticospinal, gracilis, cuneatus, rubrospinal, spinothalamic, reticulospinal, and vestibulospinal tracts) each with different axon diameters. We found QSI-derived diameters from regions drawn in the seven WM tracts (1.1 to 2.1 µm) to be highly correlated (r 2 = 0.95) with those calculated from histology (0.8 to 1.8 µm). The QSI-derived values overestimated those obtained by histology by approximately 20%, which is likely due to the presence of extracellular signal. Finally, simulations on images of synthetic circular axons and axons from histology suggest that QSI-derived diameters are informative despite diameter and axon shape variation and the presence of intra-cellular and extra-cellular signal. QSI may be able to quantify nondestructively changes in WM axon architecture due to pathology or injury at the cellular level.

Anisotropy of water diffusion in axon tracts, as determined by diffusion-weighted MRI, has been assumed to reflect the restriction of water diffusion across axon membranes. Reduction in this anisotropy has been interpreted as degeneration of axons. These interpretations are based primarily on a priori reasoning that has had little empirical validation. We used the experimental advantages of the sea lamprey spinal cord, which contains several very large axons, to determine whether intraaxonal diffusion is isotropic and whether anisotropy is attributable to restriction of water mobility by axon surface membranes. Through the application of magnetic resonance microimaging, we were able to measure the purely intraaxonal diffusion characteristics of the giant reticulospinal axons (20 -40 m in diameter). The intraaxonal apparent diffusion coefficients of water parallel (longitudinal ADC, l-ADC) and perpendicular (transverse ADC, t-ADC) to the long axis were 0.98 ؎ 0.06 (10 ؊3 mm 2 ͞sec) and 0.97 ؎ 0.11 (10 ؊3 mm 2 ͞sec), respectively. In white matter regions that included multiple axons, l-ADCs were almost identical regardless of axon density in the sampled axon tract. By comparison, t-ADCs were reduced and varied inversely with the number of axons (and thus axolemmas) in a fixed cross-sectional area. Thus, diffusion was found to be isotropic when measured entirely within a single axon and anisotropic when measured in regions that included multiple axons. These findings support the hypothesis that the cell membrane is the primary source of diffusion anisotropy in fiber tracts of the central nervous system. MR microimaging ͉ diffusion-weighted imaging ͉ giant axon ͉ intraaxonal apparent diffusion coefficient ͉ axolemma D iffusion-weighted MRI (DWI) techniques have been widely applied in the white matter (WM) for detection of damage to axons (1-4) and more recently for determining the orientation of fiber tracks (5, 6) in the brain and spinal cord. These applications invoke diffusional anisotropy, a larger apparent diffusion coefficient (ADC) in longitudinal (l-ADC) orientation than in transverse (t-ADC) orientation, in the WM (7).Possible causes of the diffusional anisotropy have been subjects of many studies for over a decade (8-10). To date, although these inferences have been derived from simulation and experimental studies, there have been few studies (11,12) in which the diffusion characteristics of axons have been related directly to axonal anatomy or physiology. This is due largely to limitations in spatial resolution of magnetic resonance (MR) compared with histological methods. In short, it has been possible to obtain MR images of relatively large fields of view, which must be related to the small fields of view achieved with high-power microscopy. Bringing the spatial resolution of MR closer to that of microscopy for diffusion studies will improve correlations of ADCs with histological changes.The nervous system of the sea lamprey (Petromyzon marinus) is unmyelinated. In the spinal cord, the Mauthner and Müll...