Enhancing water sampling of buried binding sites using nonequilibrium candidate Monte Carlo.

Water molecules can be found interacting with the surface and within cavities in proteins. However, water exchange between bulk and buried hydration sites can be slow compared to simulation timescales, thus leading to the inefficient sampling of the locations of water. This can pose problems for free energy calculations for computer-aided drug design. Here, we apply a hybrid method that combines nonequilibrium candidate Monte Carlo (NCMC) simulations and molecular dynamics (MD) to enhance sampling of water in specific areas of a system, such as the binding site of a protein. Our approach uses NCMC to gradually remove interactions between a selected water molecule and its environment, then translates the water to a new region, before turning the interactions back on. This approach of gradual removal of interactions, followed by a move and then reintroduction of interactions, allows the environment to relax in response to the proposed water translation, improving acceptance of moves and thereby accelerating water exchange and sampling. We validate this approach on several test systems including the ligand-bound MUP-1 and HSP90 proteins with buried crystallographic waters removed. We show that our BLUES (NCMC/MD) method enhances water sampling relative to normal MD when applied to these systems. Thus, this approach provides a strategy to improve water sampling in molecular simulations which may be useful in practical applications in drug discovery and biomolecular design.

Efficient Approximation of Ligand Rotational and Translational Entropy Changes upon Binding for Use in MM-PBSA Calculations.

A major uncertainty in binding free energy estimates for protein-ligand complexes by methods such as MM-PB(GB)SA or docking scores results from neglecting or approximating changes in the configurational entropies (ΔSconfig.) of the solutes. In MM/PB(GB)SA-type calculations, ΔSconfig. has usually been estimated in the rigid rotor, harmonic oscillator approximation. Here, we present the development of a computationally efficient method (termed BEERT) to approximate ΔSconfig. in terms of the reduction in translational and rotational freedom of the ligand upon protein-ligand binding (ΔSR/T), starting from the flexible molecule approach. We test the method successfully in binding affinity computations in connection with MM-PBSA effective energies describing changes in gas-phase interactions and solvation free energies. Compared to related work by Ruvinsky and co-workers, clustering bound ligand poses based on interactions with the protein rather than structural similarity of the poses, and an appropriate averaging over single entropies associated with an individual well of the energy landscape of the protein-ligand complex, were found to be crucial. Employing three data sets of protein-ligand complexes of pharmacologically relevant targets for validation, with up to 20, in part related ligands per data set, spanning binding free energies over a range of ≤7 kcal mol-1, reliable and predictive linear models to estimate binding affinities are obtained in all three cases (R2 = 0.54-0.72, p < 0.001, root mean squared error S = 0.78-1.44 kcal mol-1; q2 = 0.34-0.67, p < 0.05, root mean squared error sPRESS = 1.07-1.36 kcal mol-1). These models are markedly improved compared to considering MM-PBSA effective energies alone, scoring functions, and combinations with ΔSconfig. estimates based on the number of rotatable bonds, rigid rotor, harmonic oscillator approximation, or interaction entropy method. As a limitation, our method currently requires a target-specific training data set to identify appropriate scaling coefficients for the MM-PBSA effective energies and BEERT ΔSR/T. Still, our results suggest that the approach is a valuable, computationally more efficient complement to existing rigorous methods for estimating changes in binding free energy across structurally (weakly) related series of ligands binding to one target.

Native metabolomics identifies the rivulariapeptolide family of protease inhibitors.

The identity and biological activity of most metabolites still remain unknown. A bottleneck in the exploration of metabolite structures and pharmaceutical activities is the compound purification needed for bioactivity assignments and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we develop a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry and detection of protein binding via native mass spectrometry. A native metabolomics screen for protease inhibitors from an environmental cyanobacteria community reveals 30 chymotrypsin-binding cyclodepsipeptides. Guided by the native metabolomics results, we select and purify five of these compounds for full structure elucidation via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy as well as evaluation of their biological activities. These results identify rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as a promising approach for drug discovery, chemical ecology, and chemical biology studies.

Fast Equilibration of Water between Buried Sites and the Bulk by Molecular Dynamics with Parallel Monte Carlo Water Moves on Graphical Processing Units.

Molecular dynamics (MD) simulations of proteins are commonly used to sample from the Boltzmann distribution of conformational states, with wide-ranging applications spanning chemistry, biophysics, and drug discovery. However, MD can be inefficient at equilibrating water occupancy for buried cavities in proteins that are inaccessible to the surrounding solvent. Indeed, the time needed for water molecules to equilibrate between the bulk solvent and the binding site can be well beyond what is practical with standard MD, which typically ranges from hundreds of nanoseconds to a few microseconds. We recently introduced a hybrid Monte Carlo/MD (MC/MD) method, which speeds up the equilibration of water between buried cavities and the surrounding solvent, while sampling from the thermodynamically correct distribution of states. While the initial implementation of the MC functionality led to considerable slowing of the overall simulations, here we address this problem with a parallel MC algorithm implemented on graphical processing units. This results in speed-ups of 10-fold to 1000-fold over the original MC/MD algorithm, depending on the system and simulation parameters. The present method is available for use in the AMBER simulation software.

Accounting for the Central Role of Interfacial Water in Protein-Ligand Binding Free Energy Calculations.

Rigorous binding free energy methods in drug discovery are growing in popularity because of a combination of methodological advances, improvements in computer hardware, and workflow automation. These calculations typically use molecular dynamics (MD) to sample from the Boltzmann distribution of conformational states. However, when part or all of the binding sites is inaccessible to the bulk solvent, the time needed for water molecules to equilibrate between bulk solvent and the binding site can be well beyond what is practical with standard MD. This sampling limitation is problematic in relative binding free energy calculations, which compute the reversible work of converting ligand 1 to ligand 2 within the binding site. Thus, if ligand 1 is smaller and/or more polar than ligand 2, the perturbation may allow additional water molecules to occupy a region of the binding site. However, this change in hydration may not be captured by standard MD simulations and may therefore lead to errors in the computed free energy. We recently developed a hybrid Monte Carlo/MD (MC/MD) method, which speeds up the equilibration of water between bulk solvent and buried cavities, while sampling from the intended distribution of states. Here, we report on the use of this approach in the context of alchemical binding free energy calculations. We find that using MC/MD markedly improves the accuracy of the calculations and also reduces hysteresis between the forward and reverse perturbations, relative to matched calculations using only MD with or without the crystallographic water molecules. The present method is available for use in AMBER simulation software.

Prevention of vascular and neural dysfunction in diabetic rats by C-peptide.

C-peptide, a cleavage product from the processing of proinsulin to insulin, has been considered to possess little if any biological activity other than its participation in insulin synthesis. Injection of human C-peptide prevented or attenuated vascular and neural (electrophysiological) dysfunction and impaired Na+- and K+-dependent adenosine triphosphate activity in tissues of diabetic rats. Nonpolar amino acids in the midportion of the peptide were required for these biological effects. Synthetic reverse sequence (retro) and all-D-amino acid (enantio) C-peptides were equipotent to native C-peptide, which indicates that the effects of C-peptide on diabetic vascular and neural dysfunction were mediated by nonchiral interactions instead of stereospecific receptors or binding sites.

Simulating Water Exchange to Buried Binding Sites.

Traditional molecular dynamics (MD) simulations of proteins, which relies on integration of Newton's equations of motion, cannot efficiently equilibrate water occupancy for buried cavities in proteins. This leads to slow convergence of thermodynamic averages for such systems. We have addressed this challenge by efficiently integrating standard Metropolis Monte Carlo (MC) translational water moves with MD in the AMBER simulation package. The translational moves allow water to easily enter or exit buried sites in a thermodynamically correct way during a simulation. To maximize efficiency, the algorithm avoids moves that only interchange waters within the bulk around the protein instead focusing on moves that can transfer water between bulk and the protein interior. In addition, a steric grid allows avoidance of moves that would lead to obvious steric clashes, and a fast grid-based energy evaluation is used to reduce the number of expensive full energy calculations. The potential energy distribution produced using MC/MD was found to be statistically indistinguishable from that of control simulations using only MD, and the algorithm effectively equilibrated water across steric barriers and into binding pockets that are not accessible with pure MD. The MC/MD method introduced here should be of increasing utility for applications spanning protein folding, the elucidation of protein mechanisms, and free energy calculations for computer-aided drug design. It is available in version 18 release of the widely disseminated AMBER simulation package.

Vascular dysfunction induced by elevated glucose levels in rats is mediated by vascular endothelial growth factor.

The purpose of these experiments was to investigate a potential role for vascular endothelial growth factor (VEGF) in mediating vascular dysfunction induced by increased glucose flux via the sorbitol pathway. Skin chambers were mounted on the backs of Sprague-Dawley rats and 1 wk later, granulation tissue in the chamber was exposed twice daily for 7 d to 5 mM glucose, 30 mM glucose, or 1 mM sorbitol in the presence and absence of neutralizing VEGF antibodies. Albumin permeation and blood flow were increased two- to three-fold by 30 mM glucose and 1 mM sorbitol; these increases were prevented by coadministration of neutralizing VEGF antibodies. Blood flow and albumin permeation were increased approximately 2.5-fold 1 h after topical application of recombinant human VEGF and these effects were prevented by nitric oxide synthase (NOS) inhibitors (aminoguanidine and N(G)-monomethyl L-arginine). Topical application of a superoxide generating system increased albumin permeation and blood flow and these changes were markedly attenuated by VEGF antibody and NOS inhibitors. Application of sodium nitroprusside for 7 d or the single application of a calcium ionophore, A23187, mimicked effects of glucose, sorbitol, and VEGF on vascular dysfunction and the ionophore effect was prevented by coadministration of aminoguanidine. These observations suggest a potentially important role for VEGF in mediating vascular dysfunction induced by "hypoxia-like" cytosolic metabolic imbalances (reductive stress, increased superoxide, and nitric oxide production) linked to increased flux of glucose via the sorbitol pathway.

Rigidity Theory-Based Approximation of Vibrational Entropy Changes upon Binding to Biomolecules.

We introduce a computationally efficient approximation of vibrational entropy changes (ΔSvib) upon binding to biomolecules based on rigidity theory. From constraint network representations of the binding partners, ΔSvib is estimated from changes in the number of low frequency ("spongy") modes with respect to changes in the networks' coordination number. Compared to ΔSvib computed by normal-mode analysis (NMA), our approach yields significant and good to fair correlations for data sets of protein-protein and protein-ligand complexes. Our approach could be a valuable alternative to NMA-based ΔSvib computation in end-point (free) energy methods.

Acute regulation of fatty acid oxidation and amp-activated protein kinase in human umbilical vein endothelial cells.

It is generally accepted that endothelial cells generate most of their ATP by anaerobic glycolysis and that very little ATP is derived from the oxidation of fatty acids or glucose. Previously, we have reported that, in cultured human umbilical vein endothelial cells (HUVECs), activation of AMP-activated protein kinase (AMPK) by the cell-permeable activator 5-aminoimidazole-4-carboximide riboside (AICAR) is associated with an increase in the oxidation of (3)H-palmitate. In the present study, experiments carried out with cultured HUVECs revealed the following: (1) AICAR-induced increases in palmitate oxidation during a 2-hour incubation are associated with a decrease in the concentration of malonyl coenzyme A (CoA) (an inhibitor of carnitine palmitoyl transferase 1), which temporally parallels the increase in AMPK activity and a decrease in the activity of acetyl CoA carboxylase (ACC). (2) AICAR does not stimulate either palmitate oxidation when carnitine is omitted from the medium or oxidation of the medium-chain fatty acid octanoate. (3) When intracellular lipid pools are prelabeled with (3)H-palmitate, the measured rate of palmitate oxidation is 3-fold higher, and in the presence of AICAR, it accounts for nearly 40% of calculated ATP generation. (4) Incubation of HUVECs in a glucose-free medium for 2 hours causes the same changes in AMPK, ACC, malonyl CoA, and palmitate oxidation as does AICAR. (5) Under all conditions studied, the contribution of glucose oxidation to ATP production is minimal. The results indicate that the AMPK-ACC-malonyl CoA-carnitine palmitoyl transferase 1 mechanism plays a key role in the physiological regulation of fatty acid oxidation in HUVECs. They also indicate that HUVECs oxidize fatty acids from both intracellular and extracellular sources, and that when this is taken into account, fatty acids can be a major substrate for ATP generation. Finally, they suggest that AMPK is likely to be a major factor in modulating the response of the endothelium to stresses that alter its energy state.

Induction of embryonal carcinoma cell differentiation by deferoxamine, a potent therapeutic iron chelator.

We investigated the effects of deferoxamine on the differentiation of embryonal carcinoma F9 cells. Deferoxamine, a widely used therapeutic agent for thalassemia and iron overload, was found to induce F9 cell differentiation and to have some unique characteristics compared with other chelators, hinokitiol and dithizone, which were previously reported to induce differentiation of these cells. This hydrophilic agent induced reversible differentiation as did sodium butyrate, whereas other chelators did not. However, morphological features of the cells after deferoxamine-induced differentiation were similar to those of cells incubated with the other chelators. The differentiation-inducing activity of deferoxamine was abolished by preincubation with Fe3+ ions, similarly to the other chelators examined. Moreover, cell proliferation was inhibited by treatment with this agent, and the numbers of cells in the colonies were reduced by apoptosis. Based on these results, we conclude that deferoxamine induces differentiation and apoptosis of F9 cells via chelation of extracellular and/or intracellular Fe3+ ions.

Effects of long-term aldose reductase inhibition on development of experimental diabetic neuropathy. Ultrastructural and morphometric studies of sural nerve in streptozocin-induced diabetic rats.

There is controversy over the efficacy of aldose reductase inhibitors in preventing the development of peripheral nerve lesions in experimental diabetes. This study was designed to show whether long-term (28-wk) inhibition of aldose reductase by ponalrestat influences structural changes in peripheral sensory nerve in rats with chronic streptozocin-induced diabetes. Sciatic nerve levels of sorbitol and fructose were significantly reduced but not completely normalized by ponalrestat treatment. myo-Inositol levels, which tended to decrease in diabetic rats, were significantly increased by ponalrestat treatment and exceeded the level in nondiabetic control rats (P less than 0.01). Ponalrestat treatment significantly increased nerve conduction velocity over the 28 wk of treatment (P less than 0.05), but levels remained well below those of control rats. Structural analysis of sural nerve of diabetic rats disclosed significant preventive effects of ponalrestat on the reduction in myelinated nerve fiber size and fiber occupancy. Axon-fiber size ratio was also preserved in the ponalrestat-treated group. However, diffuse deposition of glycogen and increased glycogenosomes within axons were not influenced by ponalrestat treatment. In contrast to the effect on myelinated nerve fibers, morphometry of unmyelinated nerve fibers did not reveal a significant effect of ponalrestat treatment. These results suggest that chronic treatment with an aldose reductase inhibitor has beneficial effects on the peripheral sensory nerve of experimentally diabetic rats. The effects were primarily on myelinated rather than unmyelinated nerve fibers.

Renal effects of nicardipine, a calcium entry blocker, in hypertensive type II diabetic patients with nephropathy.

We studied the renal effects of nicardipine, a calcium entry blocker, in eight patients with essential hypertension (group A, WHO I or II), six hypertensive type II diabetics with mild-to-moderate nephropathy (group B, urinary albumin 200-789 mg/day), and six hypertensive type II diabetics with severe or advanced nephropathy (group C, urinary albumin 1,596-4,300 mg/day). The patients received an intravenous dose of nicardipine hydrochloride (0.5 mg) or saline placebo in a random order. Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured by means of thiosulfate sodium and p-aminohippurate, respectively, during the 30 min after the nicardipine or saline injection. Blood pressures were serially monitored during the study. Nicardipine reduced both systolic and diastolic blood pressures significantly (P less than .05 to .01) at all measurement periods in all study groups compared with the respective placebo. Nicardipine increased RBF (P less than .01), GFR (P less than .05), and urinary Na+ excretion (P less than .01) and decreased total renal vascular resistance (P less than .01) in groups A and B, but these parameters remain unchanged in group C. The filtration fraction remained unaltered in all groups. The results indicate that nicardipine has several favorable renal effects with a concomitant hypotensive action in hypertensive type II diabetics with mild-to-moderate nephropathy, as observed in patients with uncomplicated essential hypertension, and the renal pharmacological responsiveness appears to be related to the severity of nephropathy.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of sorbitol dehydrogenase. Effects on vascular and neural dysfunction in streptozocin-induced diabetic rats.

These experiments were undertaken to assess the role of sorbitol dehydrogenase in mediating sorbitol pathway-linked neural and vascular dysfunction in rats with streptozocin-induced diabetes. 2-methyl-4-[N,N-dimethylsulfamoyl-piperazino]-pyrimidine (S-0773), a putative inhibitor of sorbitol dehydrogenase, was given in the drinking water to control and diabetic rats. After 5 weeks of diabetes, glycosylated hemoglobin levels were increased twofold and were unaffected by S-0773. Sorbitol levels in diabetic rats were increased 11- to 14-fold in ocular tissues and sciatic nerve; S-0773 increased sorbitol levels another 4-fold or more in these same tissues but had much smaller effects in other tissues. Diabetes-associated increases in fructose levels and lactate:pyruvate ratios in retina and in sciatic nerve were markedly attenuated by S-0773. S-0773 also attenuated, but did not completely normalize, impaired caudal nerve conduction and vascular dysfunction in ocular tissues, sciatic nerve, and aorta in diabetic rats. These observations, together with other evidence, suggest that sorbitol pathway-linked vascular dysfunction (in ocular tissues, peripheral nerve, and aorta) and electrophysiological dysfunction (in peripheral nerve) induced by diabetes are more closely linked to increased oxidation of sorbitol to fructose than to putative osmotic effects of elevated sorbitol levels or redox and metabolic imbalances associated with reduction of glucose to sorbitol by aldose reductase.

Neural dysfunction and metabolic imbalances in diabetic rats. Prevention by acetyl-L-carnitine.

The rationale for these experiments is that administration of L-carnitine and/or short-chain acylcarnitines attenuates myocardial dysfunction 1) in hearts from diabetic animals (in which L-carnitine levels are decreased); 2) induced by ischemia-reperfusion in hearts from nondiabetic animals; and 3) in nondiabetic humans with ischemic heart disease. The objective of these studies was to investigate whether imbalances in carnitine metabolism play a role in the pathogenesis of diabetic peripheral neuropathy. The major findings in rats with streptozotocin-induced diabetes of 4-6 weeks duration were that 24-h urinary carnitine excretion was increased approximately twofold and L-carnitine levels were decreased in plasma (46%) and sciatic nerve endoneurium (31%). These changes in carnitine levels/excretion were associated with decreased caudal nerve conduction velocity (10-15%) and sciatic nerve changes in Na(+)-K(+)-ATPase activity (decreased 50%), Mg(2+)-ATPase (decreased 65%), 1,2-diacyl-sn-glycerol (DAG) (decreased 40%), vascular albumin permeation (increased 60%), and blood flow (increased 65%). Treatment with acetyl-L-carnitine normalized plasma and endoneurial L-carnitine levels and prevented all of these metabolic and functional changes except the increased blood flow, which was unaffected, and the reduction in DAG, which decreased another 40%. In conclusion, these observations 1) demonstrate a link between imbalances in carnitine metabolism and several metabolic and functional abnormalities associated with diabetic polyneuropathy and 2) indicate that decreased sciatic nerve endoneurial ATPase activity (ouabain-sensitive and insensitive) in this model of diabetes is associated with decreased DAG.

Hyperglycemic pseudohypoxia and diabetic complications.

Vasodilation and increased blood flow are characteristic early vascular responses to acute hyperglycemia and tissue hypoxia. In hypoxic tissues these vascular changes are linked to metabolic imbalances associated with impaired oxidation of NADH to NAD+ and the resulting increased ratio of NADH/NAD+. In hyperglycemic tissues these vascular changes also are linked to an increased ratio of NADH/NAD+, in this case because of an increased rate of reduction of NAD+ to NADH. Several lines of evidence support the likelihood that the increased cytosolic ratio of free NADH/NAD+ caused by hyperglycemia, referred to as pseudohypoxia because tissue partial pressure oxygen is normal, is a characteristic feature of poorly controlled diabetes that mimics the effects of true hypoxia on vascular and neural function and plays an important role in the pathogenesis of diabetic complications. These effects of hypoxia and hyperglycemia-induced pseudohypoxia on vascular and neural function are mediated by a branching cascade of imbalances in lipid metabolism, increased production of superoxide anion, and possibly increased nitric oxide formation.

Aminoguanidine, a novel inhibitor of nitric oxide formation, prevents diabetic vascular dysfunction.

Increased blood flow and vascular leakage of proteins preferentially affect tissues that are sites of diabetic complications in humans and animals. These vascular changes in diabetic rats are largely prevented by aminoguanidine. Glucose-induced vascular changes in nondiabetic rats are also prevented by aminoguanidine and by NG-monomethyl-L-arginine (NMMA), an established inhibitor of nitric oxide (NO.) formation from L-arginine. Aminoguanidine and NMMA are equipotent inhibitors of interleukin-1 beta-induced 1) nitrite formation (an oxidation product of NO.) and cGMP accumulation by the rat beta-cell insulinoma cell line RINm5F, and 2) inhibition of glucose-stimulated insulin secretion and formation of iron-nitrosyl complexes by islets of Langerhans. In contrast, NMMA is approximately 40 times more potent than aminoquanidine in elevating blood pressure in nondiabetic rats. These results demonstrate that aminoguanidine inhibits NO. production and suggest a role for NO. in the pathogenesis of diabetic vascular complications.

Effect of sorbitol dehydrogenase inhibition on experimental diabetic autonomic neuropathy.

The polyol pathway and its dependent biochemical pathways are thought to play a role in the pathogenesis of diabetic neuropathy. We have developed an animal model of diabetic autonomic neuropathy characterized by neuroaxonal dystrophy involving ileal mesenteric nerves and prevertebral sympathetic superior mesenteric ganglia (SMG) in chronic streptozocin-diabetic rats. Our previous studies have shown a salutary effect of aldose reductase inhibitors on experimental autonomic neuropathy, suggesting a role for the polyol pathway in its pathogenesis. In the current studies we have examined the effect of the sorbitol dehydrogenase inhibitor (SDI) CP-166,572, which interrupts the conversion of sorbitol to fructose (and reactions dependent on the second step of the polyol pathway) resulting in markedly increased levels of sorbitol in peripheral nerve. Fourteen weeks of treatment with CP-166,572 resulted in a dramatically increased frequency of neuroaxonal dystrophy in ileal mesenteric nerves and SMG. Although lesions developed prematurely and in greater numbers in SDI-treated diabetics than untreated diabetics did, their anatomic distribution and ultrastructural appearance were identical to that previously reported in long-term untreated diabetics. CP-166,572 treatment did not produce neuroaxonal dystrophy in control animals despite the fact that sciatic nerve sorbitol levels were markedly increased, reaching the same levels as untreated diabetic animals. Treatment of diabetic rats for 14 weeks with the aldose reductase inhibitor zopolrestat resulted in a significant decrease in the frequency of neuroaxonal dystrophy compared with untreated diabetics.

Inhibition of sorbitol dehydrogenase exacerbates autonomic neuropathy in rats with streptozotocin-induced diabetes.

We have developed an animal model of diabetic autonomic neuropathy that is characterized by neuroaxonal dystrophy (NAD) involving ileal mesenteric nerves and prevertebral sympathetic superior mesenteric ganglia (SMG) in chronic streptozotocin (STZ)-diabetic rats. Studies with the sorbitol dehydrogenase inhibitor SDI-158, which interrupts the conversion of sorbitol to fructose (and reactions dependent on the second step of the sorbitol pathway), have shown a dramatically increased frequency of NAD in ileal mesenteric nerves and SMG of SDI-treated versus untreated diabetics. Although lesions developed prematurely and in greater numbers in SDI-treated diabetics, their distinctive ultrastructural appearance was identical to that previously reported in long-term untreated diabetics. An SDI effect was first demonstrated in the SMG of rats that were diabetic for as little as 5 wk and was maintained for at least 7.5 months. As in untreated diabetic rats, rats treated with SDI i) showed involvement of lengthy ileal, but not shorter, jejunal mesenteric nerves; ii) demonstrated NAD in paravascular mesenteric nerves distributed to myenteric ganglia while sparing adjacent perivascular axons ramifying within the vascular adventitia; and, iii) failed to develop NAD in the superior cervical ganglia (SCG). After only 2 months of SDI-treatment, tyrosine hydroxylase immunolocalization demonstrated marked dilatation of postganglionic noradrenergic axons in paravascular ileal mesenteric nerves and within the gut wall versus those innervating extramural mesenteric vasculature. The effect of SDI on diabetic NAD in SMG was completely prevented by concomitant administration of the aldose reductase inhibitor Sorbinil. Treatment of diabetic rats with Sorbinil also prevented NAD in diabetic rats not treated with SDI. These findings indicate that sorbitol pathway-linked metabolic imbalances play a critical role in the development of NAD in this model of diabetic sympathetic autonomic neuropathy.

Induction of apoptosis by hinokitiol, a potent iron chelator, in teratocarcinoma F9 cells is mediated through the activation of caspase-3.

Hinokitiol, a potent iron chelator, has been reported to induce differentiation in teratocarcinoma F9 cells with a reduction of viable cells. In this study, we examined the steps leading to eventual cell death by hinokitiol during differentiation. Hinokitiol induced DNA fragmentation of F9 cells in a concentration- and time-dependent manner. This effect was also observed in a cell-free system using the nuclei from intact cells and the cytosols from hinokitiol-treated cells. In contrast, hinokitiol methyl ether and hinokitiol-Fe (III) complex, which are deficient in iron-chelating activity, showed no DNA fragmentation activity in both cell culture and cell-free systems. These results suggest that iron deprivation by hinokitiol may be involved in the induction of apoptosis of F9 cells. Caspase-3, one of the key enzymes in the apoptotic cascade, was specifically activated by hinokitiol treatment, but not by the other two derivatives. In addition, its specific inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, strongly blocked hinokitiol-induced DNA fragmentation. These results indicate that iron deprivation by hinokitiol can induce apoptosis of F9 cells through the activation of caspase-3.