Jeewoo Lee

Lovastatin enhances Aβ production and senile plaque deposition in female Tg2576 mice

A recent clinical study showed that statins, which are inhibitors of cholesterol biosynthesis pathway, reduced the prevalence of Alzheimers disease (AD). Animal studies that have employed high cholesterol diet indicate significant relationship between cholesterol level and senile plaque deposition. Here, we investigated the effects of lovastatin on beta-amyloid production and senile plaque deposition in an animal model of AD (Tg2576 mice). As expected, lovastatin treatment reduced plasma cholesterol level in both male and female mice. However, lovastatin enhanced the amounts of beta-amyloid and other beta-secretase derived peptides in females, but not in males. Likewise, lovastatin increased the number of plaques in the hippocampus and cortex of females, but not in males. Lovastatin did not change the amounts of full-length or alpha-secretase processed amyloid precursor protein (APP), or presenilin 1 (PS1) in either sex. Thus, lovastatin lowers cholesterol level in both genders, but enhances beta-amyloid production and senile plaque deposition only in brains of female Tg2576 mice. Our results suggest that low plasma cholesterol levels might be a risk factor for AD in females.

Distinct structure-activity relations for stimulation of 45Ca uptake and for high affinity binding in cultured rat dorsal root ganglion neurons and dorsal root ganglion membranes

The [3H]resiniferatoxin (RTX) binding assay using membrane preparations has been used to identify and characterize the vanilloid receptors in the central and peripheral nervous system of different species. In the present study, using cultured adult rat dorsal root ganglion neurons either in suspension or attached to the tissue culture plates, we developed an assay to measure specific [3H]RTX binding by the intact cells. We were able to characterize the vanilloid binding characteristics of the neurons and compared those to the properties of vanilloid binding sites present in rat dorsal root ganglia membrane preparations. We found that [3H]RTX bound with similar affinity and positive cooperativity to attached neurons (cultured for 5 days before being assayed), neurons in suspension (using a filtration assay) and dorsal root ganglion membrane preparations. Dissociation constants obtained in the three assays were 47.6 +/- 3.5 pM, 38.4 +/- 3.1 pM and 42.6 +/- 3.1 pM, respectively. The cooperativity indexes determined by fitting the data to the Hill equation were 1.73 +/- 0.11, 1.78 +/- 0.12 and 1.78 +/- 0.09, respectively. The maximal binding capacity was 0.218 +/- 0.026 fmol/10(3) cells and 0.196 +/- 0.021 fmol/10(3) cells in the case of the attached cells and cells in suspension, respectively. Nonradioactive RTX, capsaicin, capsazepine and resiniferonol 20-homovanillylamide fully displaced specifically bound [3H]RTX from cells in suspension with Ki and Hill coefficient values of 42.5 +/- 5.3 pM, 2.06 +/- 0.16 microM, 3.16 +/- 0.21 microM and 32.4 +/- 4.1 nM and 1.79 +/- 0.17, 1.68 +/- 0.06, 1.72 +/- 0.11 and 1.81 +/- 0.12, respectively. Structure-activity analysis of different vanilloid derivatives revealed that the various compounds have distinct potencies for receptor binding and inducing 45Ca uptake in rat dorsal root ganglion neurons. Affinities for receptor binding and stimulation of 45Ca uptake of RTX, resiniferonol 20-homovanillylamide, RTX-thiourea, tinyatoxin, phorbol 12,13-dibenzoate 20-homovanillylamide and capsaicin were 38.5 +/- 2.9 pM, 25.7 +/- 3.0 nM, 68.5 +/- 3.8 nM, 173 +/- 25 pM, 7.98 +/- 0.83 microM and 4.93 +/- 0.35 microM as compared to 0.94 +/- 0.12 nM, 26.5 +/- 3.5 nM, 149 +/- 30 nM, 1.46 +/- 0.25 nM, 1.41 +/- 0.48 microM and 340 +/- 57 nM. Computer fitting of the data yielded Hill coefficient values indicating positive cooperativity of receptor binding; however, stimulation of 45Ca uptake appeared to follow a non-cooperative mechanism of action. The competitive capsaicin antagonist capsazepine inhibited specific binding of [3H]RTX by rat dorsal root ganglion membrane preparations with Ki and Hill coefficient values of 3.89 +/- 0.38 microM and 1.74 +/- 0.11. On the other hand it inhibited the induction of 45Ca uptake into the cells induced by capsaicin and RTX in a non-cooperative fashion with Ki values of 271 +/- 29 nM and 325 +/- 47 nM. Our results show that the membrane binding assay relates to the reality of receptor function in the intact, cultured neurons, both in terms of affinity and positive cooperativity. However the different vanilloid derivatives displayed markedly distinct structure-activity relations for high affinity receptor binding and stimulation of 45Ca uptake into rat dorsal root ganglion neurons. Among various explanations for this discrepancy, we favor the possibility that the two assays detect distinct classes of the vanilloid (capsaicin) receptor present in primary sensory neurons.

Uric acid induces endothelial dysfunction by vascular insulin resistance associated with the impairment of nitric oxide synthesis

Endothelial dysfunction is defined as impairment of the balance between endothelium‐dependent vasodilation and constriction. Despite evidence of uric acid‐induced endothelial dysfunction, a relationship with insulin resistance has not been clearly established. In this study, we investigated the role of vascular insulin resistance in uric acid‐induced endothelial dysfunction. Uric acid inhibited insulin‐induced endothelial nitric oxide synthase (eNOS) phosphorylation and NO production more substantially than endothelin‐1 expression in HUVECs, with IC50 of 51.0, 73.6, and 184.2, respectively. Suppression of eNOS phosphorylation and NO production by uric acid was PI3K/Akt‐dependent, as verified by the transfection with p110. Treatment of rats with the uricase inhibitor allantoxanamide induced mild hyperuricemia and increased mean arterial pressure by 25%. While hyperuricemic rats did not show systemic insulin resistance, they showed impaired vasorelaxation induced by insulin by 56%. A compromised insulin response in terms of the Akt/eNOS pathway was observed in the aortic ring of hyperuricemic rats. Coadministration with allopurinol reduced serum uric acid levels and blood pressure and restored the effect of insulin on Akt‐eNOS pathway and vasorelaxation. Taken together, uric acid induced endothelial dysfunction by contributing to vascular insulin resistance in terms of insulin‐induced NO production, potentially leading to the development of hypertension.—Choi, Y.‐J., Yoon, Y., Lee, K.‐Y., Hien, T. T., Kang, K. W., Kim, K.‐C., Lee, J., Lee, M.‐Y., Lee, S. M., Kang, D.‐H., Lee, B.‐H. Uric acid induces endothelial dysfunction by vascular insulin resistance associated with the impairment of nitric oxide synthesis. FASEB J. 28, 3197–3204 (2014). www.fasebj.org

Structural insights into transient receptor potential vanilloid type 1 (TRPV1) from homology modeling, flexible docking, and mutational studies

The transient receptor potential vanilloid subtype 1 (TRPV1) is a non-selective cation channel composed of four monomers with six transmembrane helices (TM1–TM6). TRPV1 is found in the central and peripheral nervous system, and it is an important therapeutic target for pain relief. We describe here the construction of a tetrameric homology model of rat TRPV1 (rTRPV1). We experimentally evaluated by mutational analysis the contribution of residues of rTRPV1 contributing to ligand binding by the prototypical TRPV1 agonists, capsaicin and resiniferatoxin (RTX). We then performed docking analysis using our homology model. The docking results with capsaicin and RTX showed that our homology model was reliable, affording good agreement with our mutation data. Additionally, the binding mode of a simplified RTX (sRTX) ligand as predicted by the modeling agreed well with those of capsaicin and RTX, accounting for the high binding affinity of the sRTX ligand for TRPV1. Through the homology modeling, docking and mutational studies, we obtained important insights into the ligand-receptor interactions at the molecular level which should prove of value in the design of novel TRPV1 ligands.

Inhibition of mouse skin tumor promotion by anti-inflammatory diarylheptanoids derived from Alpinia oxyphylla Miquel (Zingiberaceae)

Alpinia oxphylla Miquel, which belongs to the ginger family (Zingiberaceae), has been used in Oriental herbal medicine. Our recent studies have revealed that the methanolic extract of A. oxyphylla suppresses mouse skin tumor promotion and induces apoptosis in cultured human promyelocytic leukemia cells. In the present work, we have assessed effects of yakuchinone A and yakuchinone B, phenolic diarylheptanoids derived from A. oxyphylla, on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation and epidermal ornithine decarboxylase (ODC) activity as well as on skin tumor promotion in female ICR mice. Thus, topical application of 2 or 6 micromol of the diarylheptanoids prior to each topical dose of TPA significantly ameliorated 7,12-dimethylbenz[a]anthracene-initiated mouse skin tumor formation. In parallel with suppression of tumor promotion, topically applied yakuchinone A and B markedly inhibited TPA-induced epidermal ODC activity and ODC mRNA expression. In another experiment, yakuchinone A and B reduced production of tumor necrosis factor-alpha in TPA-stimulated mouse skin. Furthermore, both compounds inhibited the TPA-induced expression of cyclooxygenase-2 at both transcriptional and translational levels. These findings indicate that pungent diarylheptanoids from A. oxyphylla Miquel have an antitumor promotional activity that might be related to their anti-inflammatory properties.

Migration of neutrophils targeting amyloid plaques in Alzheimer's disease mouse model

Immune responses in the brain are thought to play a role in disorders of the central nervous system, but an understanding of the process underlying how immune cells get into the brain and their fate there remains unclear. In this study, we used a 2-photon microscopy to reveal that neutrophils infiltrate brain and migrate toward amyloid plaques in a mouse model of Alzheimers disease. These findings suggest a new molecular process underlying the pathophysiology of Alzheimers disease.

N-Alkoxysulfamide, N-hydroxysulfamide, and sulfamate analogues of methionyl and isoleucyl adenylates as inhibitors of methionyl-tRNA and isoleucyl-tRNA synthetases.

A series of sulfamate surrogates of methionyl and isoleucyl adenylate have been investigated as MetRS and IleRS inhibitors by modifications of the sulfamate linker and adenine moieties. The discovery of 2-iodo Ile-NHSO(2)-AMP (58) as a potent Escherichia coli IleRS inhibitor revealed that a significant hydrophobic interaction between the 2-substituent of Ile-NHSO(2)-AMP and the adenine binding site of IleRS provided its high potency to the enzyme.

Transient receptor potential vanilloid type 1 antagonists: a patent review (2011 – 2014)

Introduction: Transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel that can be activated by noxious heat, low pH and vanilloid compounds such as capsaicin. Since TRPV1 acts as an integrator of painful stimuli, TRPV1 antagonists can be used as promising therapeutics for new types of analgesics. Areas covered: This review article covers the patents that claim TRPV1 antagonists and were published during 2011 – 2014. The patent evaluation is organized according to the applicant companies, and the representative chemical entities with important in vitro and in vivo data are summarized. Expert opinion: Many pharmaceutical companies showed promising results in the discovery of potent small molecule TRPV1 antagonists, and recently, a number of small molecule TRPV1 antagonists have been advanced into clinical trials. Unfortunately, several candidate molecules showed critical side effects such as hyperthermia and impaired noxious heat sensation in humans, leading to their withdrawal from clinical trials. Some TRPV1 antagonists patented in recent years (2011 – 2014) overcame these undesirable side effects, making the development of TRPV1 antagonists much more promising.

Transient Receptor Potential Vanilloid-1 (TRPV1) Is a Mediator of Lung Toxicity for Coal Fly Ash Particulate Material

Environmental particulate matter (PM) pollutants adversely affect human health, but the molecular basis is poorly understood. The ion channel transient receptor potential vanilloid-1 (TRPV1) has been implicated as a sensor for environmental PM and a mediator of adverse events in the respiratory tract. The objectives of this study were to determine whether TRPV1 can distinguish chemically and physically unique PM that represents important sources of air pollution; to elucidate the molecular basis of TRPV1 activation by PM; and to ascertain the contributions of TRPV1 to human lung cell and mouse lung tissue responses exposed to an insoluble PM agonist, coal fly ash (CFA1). The major findings of this study are that TRPV1 is activated by some, but not all of the prototype PM materials evaluated, with rank-ordered responses of CFA1 > diesel exhaust PM > crystalline silica; TRP melastatin-8 is also robustly activated by CFA1, whereas other TRP channels expressed by airway sensory neurons and lung epithelial cells that may also be activated by CFA1, including TRPs ankyrin 1 (A1), canonical 4α (C4α), M2, V2, V3, and V4, were either slightly (TRPA1) or not activated by CFA1; activation of TRPV1 by CFA1 occurs via cell surface interactions between the solid components of CFA1 and specific amino acid residues of TRPV1 that are localized in the putative pore-loop region; and activation of TRPV1 by CFA1 is not exclusive in mouse lungs but represents a pathway by which CFA1 affects the expression of selected genes in lung epithelial cells and airway tissue.

Intracellular Amyloid-β Accumulation in Calcium-Binding Protein-Deficient Neurons Leads to Amyloid-β Plaque Formation in Animal Model of Alzheimer's Disease

One of the major hallmarks of Alzheimers disease (AD) is the extracellular deposition of amyloid-β (Aβ) as senile plaques in specific brain regions. Clearly, an understanding of the cellular processes underlying Aβ deposition is a crucial issue in the field of AD research. Recent studies have found that accumulation of intraneuronal Aβ (iAβ) is associated with synaptic deficits, neuronal death, and cognitive dysfunction in AD patients. In this study, we found that Aβ deposits had several shapes and sizes, and that iAβ occurred before the formation of extracellular amyloid plaques in the subiculum of 5XFAD mice, an animal model of AD. We also observed pyroglutamate-modified Aβ (N3pE-Aβ), which has been suggested to be a seeding molecule for senile plaques, inside the Aβ plaques only after iAβ accumulation, which argues against its seeding role. In addition, we found that iAβ accumulates in calcium-binding protein (CBP)-free neurons, induces neuronal death, and then develops into senile plaques in 2-4-month-old 5XFAD mice. These findings suggest that N3pE-Aβ-independent accumulation of Aβ in CBP-free neurons might be an early process that triggers neuronal damage and senile plaque formation in AD patients. Our results provide new insights into several long-standing gaps in AD research, namely how Aβ plaques are formed, what happens to iAβ and how Aβ causes selective neuronal loss in AD patients.