Glucuronidation and Methylation of Procyanidin Dimers B2 and 3,3″-Di-O-Galloyl-B2 and Corresponding Monomers Epicatechin and 3-O-Galloyl-Epicatechin in Mouse Liver

Purpose  

The 3,3″-di-O-galloyl ester of procyanidin B2 (B2G2) is a component of grape seed extract that inhibits growth of human prostate carcinoma cell lines. In preparation for studies in mice, its hepatic metabolism was examined in vitro and compared to B2 and the corresponding monomers, epicatechin (EC) and 3-O-galloyl-epicatechin (ECG).     

β1- and β2-Adrenoceptor polymorphisms and cardiovascular diseases

β₁- and β₂-Adrenoceptors (AR) play a pivotal role in the regulation of cardiovascular function. Both β-AR subtypes are polymorphic: two single nucleotide polymorphisms (SNPs) have been described for the β₁- (Ser49Gly, Arg389Gly) and four for the β₂-AR (Arg-19Cys, Arg16Gly, Gln27Glu, Thr164Ile), and they are possibly of functional relevance. In recombinant cell systems, Gly49-β₁-AR are more susceptible to agonist-promoted down-regulation than Ser49-β₁-AR, whereas Arg389-β₁-AR are three to four times more responsive to agonist-evoked stimulation than Gly389-β₁-AR. With respect to β₂-AR, the Cys-19 variant is associated with greater β₂-AR expression than the Arg-19 variant; Gly16-β₂-AR are more susceptible, whereas Glu27-β₂-AR are almost resistant to agonist-promoted down-regulation; Thr164-β₂-AR are three to four times more responsive to agonist-evoked stimulation than Ile164-β₂-AR. Several studies addressed potential phenotypic consequences of these SNPs in vivo by influencing and/or contributing to the pathophysiology of cardiovascular/pulmonary diseases such as hypertension, congestive heart failure, arrhythmias or asthma. At present, it appears that these β-AR SNPs are very likely not disease-causing genes but possibly predictive for the responsiveness to agonists and antagonists. Patients carrying one or two alleles of the Gly389-β₁-AR are poor or non-responders to agonists and antagonists, whereas patients homozygous for the Arg389-β₁-AR are good responders. Subjects carrying the Ile164-β₂-AR exhibit blunted responses to β₂-AR stimulation. Asthma patients carrying the Arg16-Gln27-Thr164-β₂-AR haplotype who receive regularly short- or long-acting β₂-AR agonists are rather susceptible to agonist-induced desensitization and in consequence exhibit reduced bronchodilating and -protective effects and/or increased asthma exacerbations. The clinical relevance of these findings is still under debate.     

Ochratoxin A and β2-microglobulin in BEN patients and controls

Ochratoxin A (OTA) is a mycotoxin naturally occurring in different foods. OTA is arguably a risk factor for Balkan endemic nephropathy (BEN). The aims of this study are to (1) test the OTA-BEN association in BEN-groups and controls and (2) determine whether urine β2-microglobulin, a marker of impaired ability of the kidneys to re-absorb, is related to OTA. BEN patients had significantly higher OTA serum levels. Within the offspring, OTA was significantly related to higher β2-microglobulin excretion. OTA (2005/2006) was related to a higher incidence of BEN after 2008, providing further evidence that OTA is a risk factor for BEN.     

Dalargin and [Cys-(O2NH2)]2 analogues of enkephalins and their selectivity for μ opioid receptors

• 1.1. Effects of the enkephalins Met-enk (M) and Leu-enk (L), of two newly synthesized analogues—[Cys-(O₂NH₂)]²-Met-enk (CM) and [Cys-(O₂ NH₂)]²-Leu-enk (CL)-and of a hexapeptide—d-Ala²-Leu⁵-Arg⁶ (Dalargin; DL) on the spontaneous and electrically stimulated activity were examined with respect to their selectivity for the μ opioid receptors in the longitudinal layer of guinea pig ileum.
• 2.2. M and CM exerted relaxing and contractile effects on the spontaneous contractile activity while L, CL and DL produced only relaxation. The order of potency towards the relaxatory phase was DL > M > CM > L > CL and towards the contractile phase CM > M.
• 3.3. The effects of enkephalins on the spontaneous activity were naloxone and TTX sensitive except for the contractile phase of M and CM which persisted in the presence of TTX. NO was not involved in the neurotransmission of the relaxatory responses, while the blockade of α and β adrenoceptors showed the participation of adrenergic mechanisms. Relaxation and contraction induced by enkephalins could not be directly attributed to cholinergic neurotransmission.
• 4.4. The naloxone-sensitive and concentration-dependent inhibitory effects of enkephalins and their analogues on the electrically stimulated cholinergic contractions were established. The order of the relative potency of opioids was: DL—3.8; M—1.0; L-0.4; CM-0.01; CL—0.005.
• 5.5. These data indicated that the D-Ala² substitution and lengthening of the peptide chain by Arg⁶ in the molecule of L increased the potency at the μ opiate receptors, while the substitution in position 2 with Cys-(O₂NH₂) in the molecule of M and L yielded a less potent and selective μ agonists.

     

Antagonists that differentiate between α2A- and α2D-adrenoceptors

Four antagonists were examined for their ability to differentiate _2A from the orthologous _2Dadrenoceptors. The antagonists were (2S,12bS) 1, 3-di-methylspiro(1, 3, 4, 5, 6, 6, 7,12b-octahydro-2H-benzo[b]furo[2,3-a]quinolizine)-2,4-pyrimidin-2-one (MK 912), 2-[2-(methoxy-1, 4-benzodioxanyl)imidazoline (RX 821002), efaroxan and benoxathian. The ₂-autoreceptors in rabbit brain cortex were chosen as _2A- and the a₂-autoreceptors in guinea-pig brain cortex as _2D-adrenoceptors. Slices of the brain cortex were preincubated with ³H-noradrenaline and then superfused and stimulated electrically by brief pulse trains (4 pulses, 100 Hz) that led to little, if any, ₂-autoinhibition. 5-Bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK 14,304) was used as an ₂-adrenoceptor agonist.UK 14, 304 decreased the stimulation-evoked overflow of tritium. The antagonists shifted the concentration-inhibition curve of UK 14, 304 to the right in an apparently competitive manner. Dissociation constants of the antagonists were calculated from the shifts. MK 912, RX 821002 and efaroxan had markedly higher affinity for (guinea-pig) _2D-adrenoceptors (pK _d values 10.0, 9.7 and 9.1, respectively) than for (rabbit) _2A-adrenoceptors (pK _d 8.9, 8.2 and 7.6, respectively). Benoxathian had higher affinity for _2A- (pK _d 7.4) than for _2D-adrenoceptors (pK _d 6.9). Ratios calculated from the K _d values of the four compounds differentiated between _2A and _2D up to 100 fold. It is concluded that MK 912, RX 821002, efaroxan and benoxathian are antagonists with high power to differentiate _2A- from _2D-adrenoceptors.     

Involvement of α2- and β2-adrenoceptors on breast cancer cell proliferation and tumour growth regulation

BACKGROUND AND PURPOSE

β-Adrenoceptors are expressed in human and experimental animal breast cancer cells. However, the effect of the agonists and antagonists reported on cell proliferation and tumour growth was paradoxical, precluding their utilization as possible adjuvant therapy, mainly in the cases of refractory tumours.

EXPERIMENTAL APPROACH

β-Adrenoceptor expression was analysed by immunofluorescence and RT-PCR. Cell proliferation was assessed by [³H]-thymidine incorporation, tumour growth by measuring with a calliper and ERK 1/2 phosphorylation by Western blotting.

KEY RESULTS

β₂-Adrenoceptor expression was confirmed in the mouse and human cells tested. Cell proliferation was increased by adrenaline (by α₂-adrenoceptor action) and decreased in every tested cell line by the β-adrenoceptor agonist isoprenaline and the β₂-adrenoceptor agonist salbutamol. Isoprenaline and salbutamol reduced tumour growth in every tumour tested (mouse C4-HD and CC4-3-HI and human IBH-4, IBH-6 and MDA-MB-231 cell lines growing as xenografts in nude mice). These effects were reversed by the β-adrenoceptor antagonist propranolol. The α₂-adrenoceptor antagonist rauwolscine and the β₂-adrenoceptor agonist salbutamol were equally effective in diminishing tumour growth. ERK 1/2 activation analysed in IBH-4 tumours correlated with tumour growth, with the β-adrenoceptor agonists decreasing its activation. Inhibition of ERK 1/2 phosphorylation in vitro was mainly mediated by the PKA pathway.

CONCLUSIONS AND IMPLICATIONS

In our experimental models, the β-adrenoceptor agonists inhibited breast cancer cell proliferation and tumour growth, probably mediated by inhibition of ERK 1/2 phosphorylation. The β-adrenoceptor agonists were as effective as the α₂-adrenoceptor antagonist rauwolscine, providing possible novel adjuvant treatments for breast cancer.     

Are the pharmacology and physiology of α2adrenoceptors determined by α2-heteroreceptors and autoreceptors respectively?

α(2)-Adrenoceptors are important mediators of physiological responses to the endogenous catecholamines noradrenaline and adrenaline. In addition, α(2)-adrenoceptors are pharmacological targets for the treatment of hypertension, sympathetic overactivity and glaucoma. α(2)-Adrenoceptors are also targeted to induce sedation and analgesia in anaesthesia and intensive care. α(2)-Adrenoceptors were first described as presynaptic receptors inhibiting the release of various transmitters from neurons in the central and peripheral nervous systems. In addition to these presynaptic neuronal receptors, α(2)-adrenoceptors were also identified in many non-neuronal cell types of the body. Gene-targeting in mice provided a comprehensive assignment of the physiological and pharmacological functions of these receptors to specific α(2A)-, α(2B) - and α(2C)-adrenoceptor subtypes. However, the specific cell types and signalling pathways involved in these subtype-specific α(2)-adrenoceptor functions were largely unexplored until recently. This review summarizes recent findings from transgenic mouse models, which were generated to define the role of α(2)-adrenoceptors in adrenergic neurons, that is, α(2)-autoreceptors, versus α(2)-adrenoceptors in non-adrenergic neurons, termed α(2)-heteroreceptors. α(2)-Autoreceptors are primarily required to limit release of noradrenaline from sympathetic nerves and adrenaline from adrenal chromaffin cells at rest. These receptors are desensitized upon chronic activation as it may for instance occur due to enhanced sympathetic activity during chronic heart failure. In contrast, pharmacological effects of acutely administered α(2)-adrenoceptor agonist drugs essentially require α(2)-heteroreceptors in non-adrenergic neurons, including analgesia, sedation, hypothermia and anaesthetic-sparing as well as bradycardia and hypotension. Thus a clear picture has emerged of the significance of auto- versus heteroreceptors in mediating the physiological functions of α(2)-adrenoceptors and the pharmacological functions of α(2)-adrenoceptor agonist drugs respectively.     

Crosstalk between CYP2E1 and PPARα substrates and agonists modulate adipose browning and obesity

Although the functions of metabolic enzymes and nuclear receptors in controlling physiological homeostasis have been established, their crosstalk in modulating metabolic disease has not been explored. Genetic ablation of the xenobiotic-metabolizing cytochrome P450 enzyme CYP2E1 in mice markedly induced adipose browning and increased energy expenditure to improve obesity. CYP2E1 deficiency activated the expression of hepatic peroxisome proliferator-activated receptor alpha (PPARα) target genes, including fibroblast growth factor (FGF) 21, that upon release from the liver, enhanced adipose browning and energy expenditure to decrease obesity. Nineteen metabolites were increased in Cyp2e1-null mice as revealed by global untargeted metabolomics, among which four compounds, lysophosphatidylcholine and three polyunsaturated fatty acids were found to be directly metabolized by CYP2E1 and to serve as PPARα agonists, thus explaining how CYP2E1 deficiency causes hepatic PPARα activation through increasing cellular levels of endogenous PPARα agonists. Translationally, a CYP2E1 inhibitor was found to activate the PPARα–FGF21–beige adipose axis and decrease obesity in wild-type mice, but not in liver-specific Ppara-null mice. The present results establish a metabolic crosstalk between PPARα and CYP2E1 that supports the potential for a novel anti-obesity strategy of activating adipose tissue browning by targeting the CYP2E1 to modulate endogenous metabolites beyond its canonical role in xenobiotic-metabolism.     

Synthesis and SAR of Benzisothiazole- and Indolizine-β-d-glucopyranoside Inhibitors of SGLT2

d-glucopyranoside inhibitors of human SGLT2 are described. The synthesis of the C-linked heterocyclic glucosides took advantage of a palladium-catalyzed cross-coupling reaction between a glucal boronate and the corresponding bromo heterocycle. The compounds have been evaluated for their human SGLT2 inhibition potential using cell-based functional transporter assays, and their structure−activity relationships have been described. Benzisothiazole-C-glucoside 16d was found to be an inhibitor of SGLT2 with an IC₅₀ of 10 nM.     

Pharmacokinetics of flutamide and its metabolite 2-hydroxyflutamidein normal and hepatic injury rats

目的：建立一新的高压液相色谱法用来研究氟他胺（Ｆｌｕ）及其活性代谢产物２羟基氟他胺（ＨＦ）的药物动力学．方法：正常及肝损伤大鼠灌胃Ｆｌｕ５０ｍｇ·ｋｇ－１．采用反相高压液相色谱法，以甲基睾丸素为内标，流动相为甲醇∶乙腈∶水∶乙醚＝４０∶２０∶３５∶１（体积比），检测波长为２３４ｎｍ．结果：Ｆｌｕ的Ｋ与Ｃｌ分别由０６２±０１６ｈ－１及６０±１０Ｌ·ｋｇ－１·ｈ－１减小到０１６±００３ｈ－１及０６３±０２９Ｌ·ｋｇ－１·ｈ－１（Ｐ＜００１），ＡＵＣ与Ｃｍａｘ分别由８６±１３ｍｇ·Ｌ－１·ｈ及２４±０７ｍｇ·Ｌ－１增加到１００±４４ｍｇ·ｋｇ－１·ｈ及６７±２８ｍｇ·Ｌ－１（Ｐ＜００１）．ＨＦ的Ｋ（ｍ）由００７±００１ｈ－１减小到００５±００１ｈ－１（Ｐ＜００１）．结论：在肝损伤大鼠，Ｆｌｕ与ＨＦ消除受到显著抑制．     

Analgesic synergy between opioid and α2-adrenoceptors

Opioid and α₂-adrenoceptor agonists are potent analgesic drugs and their analgesic effects can synergize when co-administered. These supra-additive interactions are potentially beneficial clinically; by increasing efficacy and/or reducing the total drug required to produce sufficient pain relief, undesired side effects can be minimized. However, combination therapies of opioids and α₂-adrenoceptor agonists remain underutilized clinically, in spite of a large body of preclinical evidence describing their synergistic interaction. One possible obstacle to the translation of preclinical findings to clinical applications is a lack of understanding of the mechanisms underlying the synergistic interactions between these two drug classes. In this review, we provide a detailed overview of the interactions between different opioid and α₂-adrenoceptor agonist combinations in preclinical studies. These studies have identified the spinal cord as an important site of action of synergistic interactions, provided insights into which receptors mediate these interactions and explored downstream signalling events enabling synergy. It is now well documented that the activation of both μ and δ opioid receptors can produce synergy with α₂-adrenoceptor agonists and that α₂-adrenoceptor agonists can mediate synergy through either the α_2A or the α_2C adrenoceptor subtypes. Current hypotheses surrounding the cellular mechanisms mediating opioid–adrenoceptor synergy, including PKC signalling and receptor oligomerization, and the evidence supporting them are presented. Finally, the implications of these findings for clinical applications and drug discovery are discussed.

LINKED ARTICLES

This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2     

Physical and functional interaction between CB1 cannabinoid receptors and ��2-adrenoceptors

Background and purpose:

The CB₁ cannabinoid receptor and the β₂-adrenoceptor are G protein-coupled receptors (GPCRs) co-expressed in many tissues. The present study examined physical and functional interactions between these receptors in a heterologous expression system and in primary human ocular cells.

Experimental approach:

Physical interactions between CB₁ receptors and β₂-adrenoceptors were assessed using bioluminescence resonance energy transfer (BRET). Functional interactions between these receptors were evaluated by examining receptor trafficking, as well as extracellular signal-regulated kinase (ERK) and cyclic AMP response element binding protein (CREB) signalling.

Key results:

Physical interactions between CB₁ receptors and β₂-adrenoceptors were demonstrated using BRET. In human embryonic kidney (HEK) 293H cells, co-expression of β₂-adrenoceptors tempered the constitutive activity and increased cell surface expression of CB₁ receptors. Co-expression altered the signalling properties of CB₁receptors, resulting in increased Gα_i-dependent ERK phosphorylation, but decreased non-Gα_i-mediated CREB phosphorylation. The CB₁ receptor inverse agonist AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide) attenuated β₂-adrenoceptor-pERK signalling in cells expressing both receptors, while the CB₁ receptor neutral antagonist O-2050 ((6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran) did not. The actions of AM251 and O-2050 were further examined in primary human trabecular meshwork (HTM) cells, which are ocular cells endogenously co-expressing CB₁ receptors and β₂-adrenoceptors. In HTM cells, as in HEK 293H cells, AM251 but not O-2050, altered the β₂-adrenoceptor–pERK response.

Conclusion and implications:

A complex interaction was demonstrated between CB₁ receptors and β₂-adrenoceptors in HEK 293H cells. As similar functional interactions were also observed in HTM cells, such interactions may affect the pharmacology of these receptors in tissues where they are endogenously co-expressed.This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x     

Ablation of Akt2 and AMPKα2 rescues high fat diet-induced obesity and hepatic steatosis through Parkin-mediated mitophagy

Given the opposing effects of Akt and AMP-activated protein kinase (AMPK) on metabolic homeostasis, this study examined the effects of deletion of Akt2 and AMPKα2 on fat diet-induced hepatic steatosis. Akt2–Ampkα2 double knockout (DKO) mice were placed on high fat diet for 5 months. Glucose metabolism, energy homeostasis, cardiac function, lipid accumulation, and hepatic steatosis were examined. DKO mice were lean without anthropometric defects. High fat intake led to adiposity and decreased respiratory exchange ratio (RER) in wild-type (WT) mice, which were ablated in DKO but not Akt2^−/− and Ampkα2^−/− mice. High fat intake increased blood and hepatic triglycerides and cholesterol, promoted hepatic steatosis and injury in WT mice. These effects were eliminated in DKO but not Akt2^−/− and Ampkα2^−/− mice. Fat diet promoted fat accumulation, and enlarged adipocyte size, the effect was negated in DKO mice. Fat intake elevated fatty acid synthase (FAS), carbohydrate-responsive element-binding protein (CHREBP), sterol regulatory element-binding protein 1 (SREBP1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), peroxisome proliferator-activated receptor-α (PPARα), PPARγ, stearoyl-CoA desaturase 1 (SCD-1), phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6Pase), and diglyceride O-acyltransferase 1 (DGAT1), the effect was absent in DKO but not Akt2^−/− and Ampkα2^−/− mice. Fat diet dampened mitophagy, promoted inflammation and phosphorylation of forkhead box protein O1 (FoxO1) and AMPKα1 (Ser⁴⁸⁵), the effects were eradicated by DKO. Deletion of Parkin effectively nullified DKO-induced metabolic benefits against high fat intake. Liver samples from obese humans displayed lowered microtubule-associated proteins 1A/1B light chain 3B (LC3B), Pink1, Parkin, as well as enhanced phosphorylation of Akt, AMPK (Ser⁴⁸⁵), and FoxO1, which were consolidated by RNA sequencing (RNAseq) and mass spectrometry analyses from rodent and human livers. These data suggest that concurrent deletion of Akt2 and AMPKα2 offers resilience to fat diet-induced obesity and hepatic steatosis, possibly through preservation of Parkin-mediated mitophagy and lipid metabolism.     

Characterization of sensory neurotransmission and its inhibition via α2B-adrenoceptors and via non-α2-receptors in rabbit iris

Summary To find out whether, and which type of, adrenoceptors mediate prejunctional inhibition of sensory neurotransmitter release from trigeminal fibres, the modulation of twitch response to electrical field stimulation on rabbit isolated iris was investigated. Evoked iris sphincter contractions consisted of a minor fast cholinergic and a large slow component. The latter was unaffected by atropine and guanethidine, hence nonadrenergic noncholinergic in nature (NANC), but nearly completely abolished by capsaicin pretreatment and by the neurokinin receptor antagonist spantide. The response was probably not mediated by NK₂ receptors as SR 48,968, an NK₂ selective nonpeptide antagonist, failed to reduce the response to the release of the endogenous neurokinin(s) (and exogenous substance P), but in part due to NK₁ receptor activation as shown by a reduction of response by CP 96,345, an NK₁ selective non-peptide antagonist, and in part perhaps mediated by NK₃ receptors. A small neurokinin receptor antagonist- and capsaicin-insensitive NANC contraction is probably not mediated by CGRP receptors.The ₂-adrenoceptor agonist oxymetazoline inhibited the evoked NANC response (22 nmol/1, IC₂₀; about 40%, maximum inhibition) without affecting the cholinergic response (up to 1 mol/1) or the postjunctional iris sensitivity to exogenous substance P. The inhibition was antagonized by rauwolscine (apparent -log K_B 8.04) and by the relatively _2B-adrenoceptor selective antagonist ARC-239 (-log K_B 8.51).The ₂- and imidazoline receptor agonist aganodine inhibited the evoked NANC response (0.25 mol/l, IC₂₀; about 30%, maximum inhibition) without affecting the postjunctional substance P responses. Rauwolscine 0.3 mol/l failed to antagonize this effect.It is concluded that the release of sensory neurotransmitter(s) from trigeminal fibres in the rabbit eye may be inhibited by _2B-adrenoceptors and by a non-₂-receptor, perhaps an imidazoline receptor.This study was supported by the Deutsche Forschungsgemeinschaft (Fu 163/3)Correspondence to H. Fuder at the above address     

Effects of Sr2+ and Mg2+ on the phospholipase A and the presynaptic neuromuscular blocking actions of β-bungarotoxin,crotoxin and taipoxin

1.beta-Bungarotoxin, crotoxin and taipoxin, presynaptic neurotoxins of snake venom origin, have about the same phospholipid-splitting activities as a much less toxic cobra phospholipase A2 in the presence of Ca2+ and deoxycholate. 2. Sr2+ was a much less effective activator of the enzymes than is Ca2+, the activation by Sr2+ being only 3-6% for beta-bungarotoxin and crotoxin and 12% for taipoxin. 3. Sr2+ also inhibited the Ca2+ -activated enzymes by 80% in the cases of beta-bungarotoxin and crotoxin, but only 16% in the case of taipoxin. 4. Mg2" had no significant effect on beta-bungarotoxin or crotoxin, but activated taipoxin in the presence or absence of Ca2". 5. In Sr2+ -Tyrode lacking Ca2+ all three toxins exhibited the same immediate depression followed by facilitation in the rat and mouse diaphragms, but the final blocking activity was only 3-10% with beta-bungarotoxin and crotoxin and was 30% with taipoxin. 6. In Sr2+ -Tyrode, increasing in the rate of nerve stimulation had less accelerating effect on the development of neuromuscular block than in Ca2+ -Tyrode for any of the toxins. 7. Removal of Mg2+ from Sr2+ -Tyrode did not diminish the potency of taipoxin in blocking neuromuscular transmission, suggesting that enzyme activity at the outer surface of the axolemma does not contribute to the neuromuscular blocking action. 8. All of the results indicate that there are close correlations between the presynaptic activities of these toxins and their phospholipid-splitting activities in the cationic environment prevailing in the axoplasm. Apparently the final blocking effect of these toxins is due to phospholipase A action within the nerve terminal.     

COX-2 and Alzheimer’s disease: potential roles in inflammation and neurodegeneration

Epidemiological and clinical data suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) are beneficial in the treatment and prevention of Alzheimer’s disease (AD). NSAIDs act by inhibiting cyclooxygenase, an enzyme that occurs in constitutive and inducible isoforms, known respectively as COX-1 and COX-2. Recognition that COX-2 plays a key role in inflammation led to the hypothesis that COX-2 might represent the primary target for NSAIDs in AD, consistent with inflammatory processes occurring in AD brain. This review highlights recently gathered evidence leading to a more complex view of the role of COX-2 in AD, including evidence that COX-2 directly contributes to neuronal vulnerability. Consideration of these roles is critical for the rational implementation of NSAID therapy in AD.     

Solubility and stability of melatonin in propylene glycol and 2-hydroxypropyl-β-cyclodextrin vehicles

The physicochemical properties of melatonin (MT) in propylene glycol (PG) and 2-hydroxypropyl-beta-cyclodextrin (2-HPbetaCD) vehicles were characterized. MT was endothermally decomposed as determined by differential scanning calorimetry (DSC). Melting point and heat of fusion obtained were 116.9+/-0.24 degrees C and 7249+/-217 cal/mol, respectively. MT as received from a manufacture was very pure, at least 99.9%. The solubility of MT in PG solution increased slowly until reaching 40% PG and then steeply increased. Solubility of MT increased linearly as concentration of 2-HPbetaCD without PG increased (R(2)=0.993). MT solubility in the mixtures of PG and 2-HPbetaCD also increased linearly but was less than the sum of its solubility in 2-HPbetaCD and PG individually. The MT solubility was low in water, simulated gastric or intestinal fluid but the highest in the mixture of PG (40 v/v%) and 2-HPbetaCD (30 w/v%) although efficiency of MT solubilization in 2-HPbetaCD decreased as the concentration of PG increased. MT was degraded in a fashion of the first order kinetics (r(2)>0.90). MT was unstable in strong acidic solution (HCl-NaCl buffer, pH 1.4) but relatively stable in other pH values of 4 approximately 10 at 70 degrees C. In HCl-NaCl buffer, MT in 10% PG was more quickly degraded and then slowed down at a higher concentration. However, the degradation rate constant of MT in 2-HPbetaCD was not changed significantly when compared to the water. The current studies can be applied to the dosage formulations for the purpose of enhancing percutaneous absorption or bioavailability of MT.     

Antitumor cell and antimetabolic effects of 5-ethyl-2′-deoxyuridine and 5′-substituted 5-ethyl-2′-deoxyuridine derivatives

Summary A series of forty 5-ester derivatives of 5-ethyl-2-deoxyuridine (EDU) have been evaluated for their inhibitory effects on the growth and metabolism of murine leukemia L1210 cells. Several EDU esters proved as potent as EDU in their inhibitory effects on L1210 cell growth (inhibitory dose-50: 5–10 g/ml), suggesting that these esters were readily hydrolyzed to release the parent compound EDU. That the EDU esters had to be hydrolyzed first to EDU was further suggested by the dependence of their antiproliferative action on the thymidine kinase activity of the cells. It was further ascertained that EDU and its esters acquired their antiproliferative effects by an interaction with dCTP biosynthesis, possibly at the CDP ribonucleotide reductase step. Under conditions where thymidine was readily incorporated, we were unable to demonstrate any incorporation of EDU into L1210 cell DNA.     

Natural killer group 2A (NKG2A) and natural killer group 2C (NKG2C) bind to sulfated glycans and α2,3-NeuAc-containing glycoproteins

Killer lectin-like receptors on natural killer (NK) cells mediate cytotoxicity through glycans on target cells. We prepared recombinant glutathione S-transferase-fused extracellular lectin-like domains (AA 94-231) of natural killer group 2A (NKG2A) (rGST-NKG2A) and NKG2C (rGST-NKG2C) and determined the binding of these receptors to plates coated with heparin-conjugated bovine serum albumin (heparin-BSA) and glycoproteins. rGST-NKG2A and rGST-NKG2C directly bound to heparin-BSA with K(d) values of 20 and 40 nM, respectively. Binding of rGST-NKG2A and rGST-NKG2C to heparin-BSA was suppressed in the presence of soluble heparin, heparan sulfate, fucoidan, λ-carrageenan, and dextran sulfate. 2-O-Sulfate residues in heparin were essential for the binding of rGST-NKG2A and rGST-NKG2C. Moreover, rGST-NKG2A and rGST-NKG2C bound to multimeric sialyl Lewis X expressing transferrin secreted by HepG2 cells with K(d) values of 80 and 114 nM, respectively. This is the first report showing that NKG2A and NKG2C bind to heparin and α2,3-NeuAc-containing glycoproteins.