Resveratrol acts as a mixed agonist/antagonist for estrogen receptors alpha and beta.

Epidemiological evidence indicates that phytoestrogens inhibit cancer formation and growth, reduce cholesterol levels, and show benefits in treating osteoporosis. At least some of these activities are mediated through the interaction of phytoestrogens with estrogen receptors alpha and beta (ERalpha and ERbeta). Resveratrol, trans-3,5,4'-trihydroxystilbene, is a phytoestrogen in grapes that is present in red wine. Resveratrol was shown to bind ER in cytosolic extracts from MCF-7 and rat uteri. However, the contribution of ERalpha vs. ERbeta in this binding is unknown. Here we report that resveratrol binds ERbeta and ERalpha with comparable affinity, but with 7,000-fold lower affinity than estradiol (E2). Thus, resveratrol differs from other phytoestrogens that bind ERbeta with higher affinity than ERalpha. Resveratrol acts as an estrogen agonist and stimulates ERE-driven reporter gene activity in CHO-K1 cells expressing either ERalpha or ERbeta. The estrogen agonist activity of resveratrol depends on the ERE sequence and the type of ER. Resveratrol-liganded ERbeta has higher transcriptional activity than E2-liganded ERbeta at a single palindromic ERE. This indicates that those tissues that uniquely express ERbeta or that express higher levels of ERbeta than ERalpha may be more sensitive to resveratrol's estrogen agonist activity. For the natural, imperfect EREs from the human c-fos, pS2, and progesterone receptor (PR) genes, resveratrol shows activity comparable to that induced by E2. We report that resveratrol exhibits E2 antagonist activity for ERalpha with select EREs. In contrast, resveratrol shows no E2 antagonist activity with ERbeta. These data indicate that resveratrol differentially affects the transcriptional activity of ERalpha and ERbeta in an ERE sequence-dependent manner.

Hepatic utilization of exogenous nucleotide precursors for restoration of ATP after cold ischemia in rats.

The study objective was to assess hepatic utilization of exogenous adenosine or adenine to enhance ATP recovery in rat liver after cold ischemia. ATP was measured noninvasively by 31P nuclear magnetic resonance (NMR) in perfused livers before and after 18 h of cold ischemia. The hepatocellular concentration of ATP during the initial postischemic reperfusion without adenosine or adenine coinfusion was 60% of that in fresh liver. The ATP increased significantly (P < 0.001) to 139% and 82% of baseline in postischemic livers coinfused for 90 min with adenosine or adenine (final concentration, 1 mmol/L), respectively. Less than 0.5% of the excess adenosine was catabolized to uric acid. In conclusion, adenosine and, to a lesser extent, adenine are salvaged by liver after extended cold ischemia to enhance ATP restoration. Provision of these ATP precursors, as components of an enteral formulation may facilitate the repletion of liver ATP and foster early resumption of liver function after an ischemic insult.

31P nuclear magnetic resonance spectroscopic evaluation of liver preservation solutions.

The individual effects of verapamil and 2 antioxidants on perfused rat liver nucleoside triphosphate (NTP) recovery following cold storage in University of Wisconsin (UW) solution was assessed using 31P nuclear magnetic resonance spectroscopy. The pharmacological agents were added to UW solution and were present only during organ storage. NTP recovery was significantly higher for the 24-hr UW + 40 microM verapamil group as compared with the 24-hr UW group. None of the other pharmacological agents caused a significant increase in NTP recovery. These findings suggest that addition of verapamil to UW organ preservation solution may result in better poststorage liver function.

Hepatic microcirculatory changes after reperfusion in fatty and normal liver transplantation in the rat.

The hepatic microcirculation in fatty and normal liver grafts in ACI rats was investigated using in vivo microscopy. Six groups were studied. They were: normal and fatty control livers (sham operated), 6-hr cold University of Wisconsin solution (UW)-preserved fatty and normal liver grafts (survival conditions, fatty and normal liver grafts), 18-hr cold UW-preserved fatty livers (nonsurvival conditions, fatty liver graft), and 24-hr cold UW-preserved normal livers (nonsurvival conditions, normal liver grafts). Fatty livers in all groups were found to have narrow and irregular sinusoids with blood cell adhesions to endothelial cells. The number of adhesions increased as the preservation time increased. Sinusoidal blood flow area decreased as the preservation time increased and was correlated with survival in both normal and fatty liver grafts. The phagocytic activity of Kupffer cells (corrected for flow) increased as the preservation time increased. The phagocytic Kupffer cell activity of the 18-hr preserved fatty liver group was greater than the activity of any other group. These features may cause liver cell death and contribute to primary graft nonfunction after transplantation of a fatty liver.

In vivo microscopic assessment of hepatic microcirculation during liver allograft rejection in the rat.

The hemodynamic alterations in hepatic microvasculature during acute rejection of rat liver allografts was studied using in vivo fluorescence microscopy. ACI rat livers were transplanted into Lewis (allograft) or ACI (isograft) recipients. Microscopy was performed on days 3 (n = 7) and 6 (n = 7) in allografts, and on day 6 (n = 7) in isografts. Naive ACI livers (n = 7) served as nontransplant controls. Changes in sinusoidal blood perfusion, microvascular structure, and leukocyte-endothelial interactions were observed and quantitated. Six days after transplantation, acinar perfusion was markedly impaired in allografts and was accompanied by a lower percentage of perfused sinusoids (59 +/- 8%, mean +/- SEM, P < 0.01) relative to isografts (89 +/- 3%) and nontransplant controls (100 +/- 0%). The hepatic cord width in allografts was significantly greater than in isografts or in nontransplant controls, indicating swelling of parenchymal and sinusoidal lining cells. Furthermore, the number of leukocytes adhering to the sinusoidal endothelium significantly increased in allografts. Adherence to postsinusoidal venules was more prominent in allograft livers (4025 +/- 1400/mm2 of vascular endothelial surface) compared with that in isografts (574 +/- 77/mm2) and nontransplant controls (185 +/- 28/mm2). These microcirculatory alterations in allografts were significant even on day 3. The results show extensive abnormalities of the microcirculatory hemodynamics in rejecting liver allografts which were associated with increased leukocyte adherence to microvascular endothelium. Our findings may provide strategic information for the prevention and treatment of allograft rejection.

31P NMR assessment of orthotopic rat liver transplant viability. The effect of warm ischemia.

The relationship between NMR visible high energy phosphates and transplant outcome for the case of liver damage by warm ischemia was investigated. In vivo 31P nuclear magnetic resonance (NMR) spectroscopy of rat liver was performed before the induction of warm ischemia in the donor and 20 min after reestablishment of portal blood flow in the recipient. Pretransplant damage was varied by subjecting the livers to 0, 15, 30, or 60 min of warm ischemia prior to harvesting. In the controls (0 min warm ischemia), 4 of 4 rats survived transplantation (one week survival end-point) and the mean NTP recovery was 94 +/- 8%; 3 of 6 rats survived in the 15 min warm ischemia group. Mean NTP recovery was 77 +/- 20% in the 15 min survival subgroup and 32 +/- 20% in the nonsurvival subgroup. Of 6 rats, 1 survived in the 30 min group. NTP recovery was 44% for the 30 min survivor and 37 +/- 5% in the nonsurvival subgroup. Of 4 rats, 1 survived in the 60 min warm ischemia group. NTP recovery was 56% for the 60 min survivor and 28 +/- 7% in the nonsurvival subgroup. Overall, there was a significant difference between the mean NTP recovery of the survival and nonsurvival subgroups (78 +/- 21% versus 31 +/- 18%, P < 0.001). The dividing line between the survival and nonsurvival groups was approximately 50% NTP recovery. Of 9 rats with liver NTP recovery greater than 50%, 8 survived while 10 of 11 rats with less than 50% recovery died. NMR visible NTP recovery 20 min after the reestablishment of portal blood flow was a good indicator of transplant outcome in the case of rat liver damage by warm ischemia.

A rat fatty liver transplant model.

A rat model of fatty liver transplantation has been developed to study primary nonfunction in fatty liver grafts. ACI rats were fed with a diet deficient in choline and methionine for 7, 14, 28, and 42 days. Fat content in the pretransplant livers was examined by gas chromatography and histology. The main constituent of the fatty droplets was determined to be triglyceride. The triglyceride concentration reached a maximum by day 14 and remained constant for an additional 28 days. Histology revealed an absence of necrosis in 14- and 28-day fatty livers but scattered hepatocytic necrosis and inflammation in 42-day fatty livers. After being given cold (UW stored, 4 degrees C) or warm (37 degrees C) ischemia, the fatty liver was orthotopically transplanted into normal ACI rats. The one-week survival of fatty liver grafts after 6, 12, 18, and 24 hr cold preservation was 5/5, 5/6, 3/8, 0/6 for 14-day fatty liver and 5/5, 4/6, 0/8, 0/6 for 42-day fatty livers. The survival of normal liver grafts was 5/5, 6/6, 5/9, 2/8, respectively. Increased survival rate was correlated with the absence of hepatocytic necrosis. The survival after 15 and 30 min warm ischemia prior to transplant was 5/5, 2/6 for normal liver grafts and 4/7, 0/6 for 28-day fatty liver graft, respectively. Fatty livers were less resistant to damage induced by cold or warm ischemia.

The aryl hydrocarbon receptor (AHR)/AHR nuclear translocator (ARNT) heterodimer interacts with naturally occurring estrogen response elements.

To determine the molecular mechanisms underlying the "cross talk" between the activity of 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), which binds to arylhydrocarbon receptor (AHR) and estradiol (E2)-liganded estrogen receptor (ER), we first examined the initial step of estrogen action, ligand binding to ER. None of the AHR ligands tested, i.e. TCDD, benzo[a]pyrene, 3,3',4,4',5-pentachlorobiphenyl, beta-naphthoflavone, or alpha-naphthoflavone, bound to ER alpha. We report the first examination of TCDD interaction with ER beta: TCDD did not displace E2 from ER beta. We then examined a second possible mechanism, i.e. direct inhibition of ER alpha binding to estrogen response elements (EREs) by the AHR/AHR nuclear translocator (ARNT) complex. The AHR/ARNT heterodimer did not bind either a full or half-site ERE. However, AHR/ARNT bound specifically to oligomers containing naturally occurring EREs derived from the human c-fos, pS2, and progesterone receptor (PR) gene promoters that include xenobiotic response element (XRE)-like sequences. In contrast, neither purified E2-liganded-ER from calf uterus or recombinant human ER alpha bound a consensus XRE. TCDD inhibited E2-activated reporter gene activity from a consensus ERE and from EREs in the pS2, PR, and Fos genes in transiently transfected MCF-7 human breast cancer cells. However, this inhibition was not reciprocal since E2 did not inhibit TCDD-stimulated luciferase activity from the CYP1A1 promoter in transiently transfected MCF-7 or human endometrial carcinoma HEC-1A cells. We propose that at least part of the mechanism by which the AHR/ARNT complex inhibits estrogen action is by competitively inhibiting ER alpha binding to imperfect ERE sites, adjacent to or overlapping XREs.

Response to and control of destructive energy by magnetic resonance.

Magnetic resonance imaging techniques can be used to control and monitor the deposition of destructive energy. The authors evaluated the feasibility of phosphorus-31 magnetic resonance spectroscopy for the control, monitoring, and prediction of the three-dimensional extent of tissue destruction during interstitial laser surgery. Characteristic metabolic changes were demonstrated within the lesion and in the adjacent normal tissue during the deposition of thermal energy.

Multiecho approaches to spectroscopic imaging of the brain.

Spectroscopic imaging (SI) with nuclear magnetic resonance (NMR) is one of the most powerful tools available for studying brain chemistry in vivo. Both proton (1H) and phosphorus (31P) NMR offer valuable biochemical information that can in principle be mapped throughout the entire brain, thereby enhancing our understanding of brain function. With the exception of protons from tissue water and the triglycerides of adipose tissue, however, nuclei contributing to the NMR signals of living tissue are in relatively small (millimolar) concentrations. The low concentration of metabolite nuclei reduces the overall sensitivity of conventional SI techniques, making high-quality metabolite mapping a lengthy procedure. This problem has led to the development and testing of nonconventional methods for reducing SI scan times, including techniques based on the collection of multiple spin-echoes. The extent to which multiecho methods can be used to decrease SI scan times and maintain high-quality metabolite mapping depends on several factors. These include the spectral transverse relaxation times, the spectral resolution required, and J-coupling interactions. We have discussed these various technical aspects of multiecho SI methods as applied to 1H and 31P spectroscopic imaging of the living brain.

Development of analytical and preparative chromatographic separations of novel growth hormone secretagogue compounds.

Chromatographic separations of new growth hormone secretagogue compounds were developed to support structure-activity relationship (SAR) studies in conjunction with lead optimization. These new compounds differed from Merck's MK-677 by having two chiral centers and thus diastereomeric mixtures were generated. Separation of initial compounds in the SAR was achieved on a Kromasil C18 column using an ammonium acetate buffer and acetonitrile. However, additional candidates were not separable on C18 columns and a chiral Kromasil CHI-DMB column was used to resolve the diastereomeric compounds. The Kromasil CHI-DMB packing was also used in a preparative chromatographic system to resolve multigram quantities of secretagogue candidates for testing. Chiral separations of different intermediates were also developed in support of evolution of an asymmetric synthetic route. This report summarizes development of the preparative chromatographic system used to purify diastereomeric mixtures and chiral separations of intermediates in the synthesis.

Triexponential decomposition of 1H spin-lattice relaxation decay curves of paramagnetically doped red cell suspensions at 7 T.

The spin-lattice relaxation behaviour of protons in paramagnetically doped red cell suspensions at two haematocrit levels has been studied using high-field (7 T) inversion recovery data. The resulting relaxation decay curves were found to be best characterized by triexponential functions. The two-site exchange model, generally applied to biexponential relaxation data in calculating red cell water exchange parameters, was applied to the two fastest-relaxing fractions of the triexponential fits. The intracellular to extracellular water exchange rates so obtained were in good agreement with literature values. Additional exchange parameters including intracellular and extracellular water volume fractions and extracellular relaxation rates were also calculated directly from the relaxation data and found to be consistent with sample haematocrits and with independent relaxation rate measurements of the resuspension medium. The small-amplitude, slowly relaxing third component recovered from the triexponential fits of the relaxation data is attributed to intracellular haemoglobin protons.

In vivo bone marrow lipid characterization with line scan Carr-Purcell-Meiboom-Gill proton spectroscopic imaging.

Line scan Carr-Purcell-Meiboom-Gill spectroscopic imaging sequences have been used to extract lipid chemical composition indices in healthy adult bone marrow in the knee at 1.5 T. Since several spectroscopic echo readouts follow each excitation, the information acquired reflects a balance between spectral T2 decay processes and spectral resolution. To examine this balance in detail, data sets with two different echo spacings and spectral resolutions have been acquired to compare the information available from each in studies of bone marrow. Oils for which high field (7 T) proton spectra were recorded were used to evaluate the accuracy of lipid chemical composition indices extracted from the line scan Carr-Purcell-Meiboom-Gill spectroscopic imaging methods at 1.5 T. The extension of the method to fast spectroscopic imaging of bone marrow with multiple echoes is demonstrated.

1-naphthyl phosphate: specifications for high-quality substrate for measuring prostatic acid phosphatase activity.

Ten lots of sodium 1-naphthyl phosphate were compared by spectrophotometry, high-performance liquid chromatography, and kinetic measurements of the activity of human prostatic acid phosphatase (EC 3.1.3.2) in serum. Two lots were readily identified as the 2-naphthyl phosphate salt by spectrophotometry and liquid chromatography. Four of the remaining eight lots of sodium 1-naphthyl phosphate met the following specifications: (a) sodium 1-naphthyl phosphate content greater than 80% by A286nm measurements (epsilon286nm1-NP = 5630 L.mol-1.cm-1) and by enzymic conversion to 1-naphthol (epsilon332nm1-N = 7560 L.mol-1.cm-1), (b) free 1-naphthol less than 3 mmol/mol, (c) inorganic phosphate less than 10 mmol/mol, and (d) the catalytic activity concentration greater than 98% maximum by absorbance assay and greater than 90% maximum by spectrofluorometric assay during simultaneous comparisons of several substrates. The need for detailed specifications and the testing of each batch of sodium 1-naphthyl phosphate is readily demonstrated by this study.

High-resolution proton and carbon-13 NMR of membranes: why sonicate?

We have obtained high-field (11.7-T) proton and carbon-13 Fourier transform (FT) nuclear magnetic resonance (NMR) spectra of egg lecithin and egg lecithin-cholesterol (1:1) multibilayers, using "magic-angle" sample spinning (MASS) techniques, and sonicated egg lecithin and egg lecithin-cholesterol (1:1) vesicles, using conventional FT NMR methods. Resolution of the proton and carbon-13 MASS NMR spectra of the pure egg lecithin samples is essentially identical with that of sonicated samples, but spectra of the unsonicated lipid, using MASS, can be obtained very much faster than with the more dilute, sonicated systems. With the 1:1 lecithin-cholesterol systems, proton MASS NMR spectra are virtually identical with conventional FT spectra of sonicated samples, while with 13C NMR, we demonstrate that most 13C nuclei in the cholesterol moiety can be monitored, even though these same nuclei are essentially invisible, i.e., are severely broadened, in the corresponding sonicated systems. In addition, 13C MASS NMR, spectra can again be recorded much faster than with sonicated samples, due to concentration effects. Taken together, these results strongly suggest there will seldom be need in the future to resort to ultrasonic disruption of lipid bilayer membranes in order to obtain high-resolution proton or carbon-13 NMR spectra.

Density-matrix calculations of the 1.5 T citrate signal acquired with volume-localized STEAM sequences.

Citrate detection and quantitation with proton spectroscopic methods are of current interest as potential tools in the diagnosis and staging of prostate cancer. The stimulated echo acquisition mode (STEAM) sequence is a commonly used volume-localization method for detecting citrate signal. Since the 1H citrate resonance at clinically available field strengths arises from a strongly coupled two-spin system, the 90 degrees RF pulses and localizing gradients used in STEAM sequences result in a complicated dependence of signal intensity on timing intervals and gradient amplitudes. The density-matrix formalism has been applied to arrive at a general solution to this problem. Citrate-signal properties at 1.5 T for different gradient localization schemes are examined with the solution. Optimal interpulse delays, deleterious gradient balances, zero-quantum oscillations with mixing time, and a low-frequency, large-amplitude oscillation with echo time are identified for signals acquired with the standard disposition of gradients in STEAM. The generality of the solution also allows for an examination of non-standard gradient disposition schemes for enhancing citrate signal and for quantifying the sensitivity of such approaches to both field inhomogeneities and off-resonance effects.