Synthesis and structure-activity relationship studies of CD4 down-modulating cyclotriazadisulfonamide (CADA) analogues

HIV attachment via the CD4 receptor is an important target for developing novel approaches to HIV chemotherapy. Cyclotriazadisulfonamide (CADA) inhibits HIV at submicromolar levels by specifically down-modulating cell-surface and intracellular CD4. An effective five-step synthesis of CADA in 30% overall yield is reported. This synthesis has also been modified to produce more than 50 analogues. Many tail-group analogues have been made by removing the benzyl tail of CADA and replacing it with various alkyl, acyl, alkoxycarbonyl and aminocarbonyl substituents. A series of sidearm analogues, including two unsymmetrical compounds, have also been prepared by modifying the CADA synthesis, replacing the toluenesulfonyl sidearms with other sulfonyl groups. Testing 30 of these compounds in MT-4 cells shows a wide range of CD4 down-modulation potency, which correlates with ability to inhibit HIV-1. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were constructed using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) approaches. The X-ray crystal structures of four compounds, including CADA, show the same major conformation of the central 12-membered ring. The solid-state structure of CADA was energy minimized and used to generate the remaining 29 structures, which were similarly minimized and aligned to produce the 3D-QSAR models. Both models indicate that steric bulk of the tail group, and, to a lesser extent, the sidearms mainly determine CD4 down-modulation potency in this series of compounds.

(-)Epicatechin stimulates ERK-dependent cyclic AMP response element activity and up-regulates GluR2 in cortical neurons

Emerging evidence suggests that the cellular actions of flavonoids relate not simply to their antioxidant potential but also to the modulation of protein kinase signalling pathways. We investigated in primary cortical neurons, the ability of the flavan-3-ol, (-)epicatechin, and its human metabolites at physiologically relevant concentrations, to stimulate phosphorylation of the transcription factor cAMP-response element binding protein (CREB), a regulator of neuronal viability and synaptic plasticity. (-)Epicatechin at 100-300 nmol/L stimulated a rapid, extracellular signal-regulated kinase (ERK)- and PI3K-dependent, increase in CREB phosphorylation. At micromolar concentrations, stimulation was no longer apparent and at the highest concentration tested (30 mu mol/L) (-)epicatechin was inhibitory. (-)Epicatechin also stimulated ERK and Akt phosphorylation with similar bell-shaped concentration-response characteristics. The human metabolite 3 '-O-methyl-(-)epicatechin was as effective as (-)epicatechin at stimulating ERK phosphorylation, but (-)epicatechin glucuronide was inactive. (-)Epicatechin and 3 '-O-methyl-(-)epicatechin treatments (100 nmol/L) increased CRE-luciferase activity in cortical neurons in a partially ERK-dependent manner, suggesting the potential to increase CREB-mediated gene expression. mRNA levels of the glutamate receptor subunit GluR2 increased by 60%, measured 18 h after a 15 min exposure to (-)epicatechin and this translated into an increase in GluR2 protein. Thus, (-)epicatechin has the potential to increase CREB-regulated gene expression and increase GluR2 levels and thus modulate neurotransmission, plasticity and synaptogenesis.

In vitro investigation into the potential prebiotic activity of honey oligosaccharides

The effect of honey oligosaccharides on the growth of fecal bacteria was studied using an in vitro fermentation system. Prior to treatment, glucose and fructose (31.73 and 21.41 g/100 g of product, respectively) present in honey, which would be digested in the upper gut, were removed to avoid any influence on bacterial populations in the fermentations. Nanofiltration, yeast (Saccharomyces cerevisiae) treatment, and adsorption onto activated charcoal were used to remove monosaccharides. Prebiotic (microbial fermentation) activities of the three honey oligosaccharide fractions and the honey sample were studied and compared with fructooligosaccharide (FOS), using 1% (w/v) fecal bacteria in an in vitro fermentation system (10 mg of carbohydrate, 1.0 mL of basal medium). A prebiotic index (PI) was calculated for each carbohydrate source. Honey oligosaccharides seem to present potential prebiotic activity (PI values between 3.38 and 4.24), increasing the populations of bifidobacteria and lactobacilli, although not to the levels of FOS (PI of 6.89).

Influence of plant and bacterial myrosinase activity on the metabolic fate of glucosinolates in gnotobiotic rats

The breakdown of glucosinolates, a group of thioglucoside compounds found in cruciferous plants, is catalysed by dietary or microbial myrosinase. This hydrolysis releases a range of breakdown products among which are the isothiocyanates, which have been implicated in the cancer-protective effects of cruciferous vegetables. The respective involvement of plant myrosinase and gut bacterial myrosinase in the conversion, in vivo, of glucosinolates into isothiocyanates was investigated in sixteen Fischer 344 rats. Glucosinolate hydrolysis in gnotobiotic rats harbouring a whole human faecal flora (Flora+) was compared with that in germ-free rats (Flora-). Rats were offered a diet where plant myrosinase was either active (Myro+) or inactive (Myro-). The conversion of prop-2-enyl glucosinolate and benzyl glucosinolate to their related isothiocyanates, allyl isothiocyanate and benzyl isothiocyanate, was estimated using urinary mercapturic acids, which are endproducts of isothiocyanate metabolism. The highest excretion of urinary mercapturic acids was found when only plant myrosinase was active (Flora-, Myro+ treatment). Lower excretion was observed when both plant and microbial myrosinases were active (Flora+, Myro+ treatment). Excretion of urinary mercapturic acids when only microbial myrosinase was active (Flora+, Myro- treatment) was low and comparable with the levels in the absence of myrosinase (Flora-, Myro- treatment). No intact glucosinolates were detected in the faeces of rats from the Flora+ treatments confirming the strong capacity of the microflora to break down glucosinolates. The results confirm that plant myrosinase can catalyse substantial release of isothiocyanates in vivo. The results also suggest that the human microflora may, in some circumstances, reduce the proportion of isothiocyanates available for intestinal absorption.

Exaggerated postprandial lipaemia and lower post-heparin lipoprotein lipase activity in middle-aged men

An exaggerated postprandial lipaemic response is thought to play a central role in the development of an atherogenic lipoprotein phenotype, a recognized lipid risk factor for coronary heart disease. A small number of limited studies have compared postprandial lipaemia in subjects of varying age, but have not investigated mechanisms underlying age-associated changes in postprandial lipaemia. In order to test the hypothesis that impaired lipaemia in older subjects is associated with loss of insulin sensitivity, the present study compared the postprandial lipaemic and hormone responses for 9 h following a standard mixed meal in normolipidaemic healthy young and middle-aged men. Lipoprotein lipase (LPL) and hepatic lipase (HL) activities were determined in post-heparin plasma 9 h postprandially and on another occasion under fasting conditions. Postprandial plasma glucose (P < 0.02), retinyl ester (indirect marker for chylomicron particles; P < 0.005) and triacylglycerol (TAG)-rich lipoprotein (density < 1.006 g/ml fraction of plasma) TAG (P < 0.05) and retinyl ester (P < 0.005) responses were higher in middle-aged men, whereas plasma insulin responses were lower in this group (P < 0.001). Fasting and 9 h postprandial LPL and HL activities were also significantly lower in the middle-aged men compared with the young men (P < 0.006). In conclusion, the higher incremental postprandial TAG response in middle-aged men than young men was attributed to the accumulation of dietary-derived TAG-rich lipoproteins (density < 1.006 g/ml fraction of plasma) and occurred in the absence of marked differences in fasting TAG levels between the two groups. Fasting and postprandial LPL and HL activities were markedly lower in middle-aged men, but lack of statistical associations between measures of insulin response and post-heparin lipase activities, as well as between insulin and measures of postprandial lipaemia, suggest that this lower activity cannot be attributed to lack of sensitivity of lipases to activation by insulin. Alternatively, post-heparin lipase activities may not be good markers for the insulin-sensitive component of lipase that is activated postprandially.

Effect of heat treatment on the antioxidant activity, color, and free phenolic acid profile of malt

Green malt was kilned at 95 degrees C following two regimens: a standard regimen (SKR) and a rapid regimen (RKR). Both resulting malts were treated further in a tray dryer heated to 120 degrees C, as was green malt previously dried to 65 degrees C (TDR). Each regimen was monitored by determining the color, antioxidant activity (by both ABTS(center dot+) and FRAP methods), and polyphenolic profile. SKR and RKR malts exhibited decreased L* and increased b* values above approximately 80 degrees C. TDR malts changed significantly less, and color did not develop until 110 degrees C, implying that different chemical reactions lead to color in those malts. Antioxidant activity increased progressively with each regimen, although with TDR malts this became significant only at 110-120 degrees C. The RKR malt ABTS(center dot+) values were higher than those of the SKR malt. The main phenolics, that is, ferulic, p-coumaric, and vanillic acids, were monitored throughout heating. Ferulic acid levels increased upon heating to 80 degrees C for SKR and to 70 degrees C for RKR, with subsequent decreases. However, the levels for TDR malts did not increase significantly. The increase in free phenolics early in kilning could be due to enzymatic release of bound phenolics and/or easier extractability due to changes in the matrix. The differences between the kilning regimens used suggest that further modification of the regimens could lead to greater release of bound phenolics with consequent beneficial effects on flavor stability in beer and, more generally, on human health.

Heat-inactivated peroxidases and the role of calcium abstraction as a cause of their enhanced lipid oxidation activity: potential effects on the flavour quality of heat-processed vegetables

Cationic swede and anionic turnip peroxidases were partially purified by ion-exchange and gel-filtration chromatography, respectively. Heat treatment of these enzymes and of a commercial high purity horseradish peroxidase (HRP) caused a loss of enzyme activity and a corresponding increase in linoleic acid hydroperoxide formation activity. The hydroperoxide levels in model systems increased only in the early stages of the oxidation reaction and then declined as degradation became more significant. The presence of a dialysed blend of cooked swede markedly lowered the hydroperoxide level formed. Analysis of volatile compounds formed showed that hexanal predominated in a buffer system and in a blend of cooked turnip. In dialysed blends of cooked swede, hexanol was the primary volatile compound generated. After inactivation under mild conditions in the presence of EDTA, the peroxidases showed hydroperoxide formation activity and patterns of volatile compounds from linoleic acid that were similar to those found on heat-inactivation. This suggested that calcium abstraction from the peroxidases was critical for the enhancement of lipid oxidation activity. (C) 2002 Elsevier Science Ltd. All rights reserved.

Comparison of the antidiabetic activity of Berberis lyceum root extract and berberine in alloxan-induced diabetic rats

Berberine has been shown to have hypoglycaemic activity in several in vitro and in vivo models, although the mechanism of action is not fully known. Berberis lyceum Royle root produces high concentrations of berberine, and in traditional medicine, the whole extract of this plant is used widely to treat diabetes. The antidiabetic activity of the ethanol root extract of Berberis lyceum was compared with pure berberine in normal and alloxan-diabetic rats using similar doses of each. The concentration of berberine in the extract was determined to be 80% dry weight with only trace amounts of other alkaloids present. The purpose of the study was to investigate the effects of berberine and a whole extract of Berberis lyceum on blood glucose and other parameters associated with diabetes, to compare the effects of the crude extract with those of pure berberine and thus validate its use as a therapeutic agent, and finally to identify any contribution of the other components of the extract to these effects. Oral administration of 50 mg/kg of Berberis extract and berberine to normal and experimental diabetic rats produced a significant (p < 0.05) reduction in blood glucose levels from days 3-7 days of treatment. Significant effects were also observed on the glucose tolerance, glycosylated haemoglobin, serum lipid profiles and body weight of experimental animals. Berberis extract and berberine demonstrated similar effects on all parameters measured, and although the extract was comparable in efficacy to berberine, it did not produce any effects additional to those shown by pure berberine. The results support the use of the extract in traditional medicine, and demonstrate that apart from being a highly cost-effective means of treating with berberine, the total extract does not appear to confer any additional benefits or disadvantages compared with the pure compound. Copyright (c) 2008 John Wiley & Sons, Ltd.

Dietary flavonoid (-)epicatechin stimulates phosphatidylinositol 3-kinase-dependent anti-oxidant response element activity and up-regulates glutathione in cortical astrocytes

Flavonoids are plant-derived polyphenolic compounds with neuroprotective properties. Recent work suggests that, in addition to acting as hydrogen donors, they activate protective signalling pathways. The anti-oxidant response element (ARE) promotes the expression of protective proteins including those required for glutathione synthesis (xCT cystine antiporter, gamma-glutamylcysteine synthetase and glutathione synthase). The use of a luciferase reporter (ARE-luc) assay showed that the dietary flavan-3-ol (-)epicatechin activates this pathway in primary cortical astrocytes but not neurones. We also examined the distribution of NF-E2-related factor-2 (Nrf2), a key transcription factor in ARE-mediated gene expression. We found, using immunocytochemistry, that Nrf2 accumulated in the nuclei of astrocytes following exposure to tert-butylhydroquinone (100 mu M) and (-)epicatechin (100 nM). (-)Epicatechin signalling via Nrf2 was inhibited by wortmannin implicating a phosphatidylinositol 3-kinase-dependent pathway. Finally, (-)epicatechin increased glutathione levels in astrocytes consistent with an up-regulation of ARE-mediated gene expression. Together, this suggests that flavonoids may be cytoprotective by increasing anti-oxidant gene expression.

Detection of neural correlates of self-paced motor activity using empirical mode decomposition phase locking analysis

Transient episodes of synchronisation of neuronal activity in particular frequency ranges are thought to underlie cognition. Empirical mode decomposition phase locking (EMDPL) analysis is a method for determining the frequency and timing of phase synchrony that is adaptive to intrinsic oscillations within data, alleviating the need for arbitrary bandpass filter cut-off selection. It is extended here to address the choice of reference electrode and removal of spurious synchrony resulting from volume conduction. Spline Laplacian transformation and independent component analysis (ICA) are performed as pre-processing steps, and preservation of phase synchrony between synthetic signals. combined using a simple forward model, is demonstrated. The method is contrasted with use of bandpass filtering following the same preprocessing steps, and filter cut-offs are shown to influence synchrony detection markedly. Furthermore, an approach to the assessment of multiple EEG trials using the method is introduced, and the assessment of statistical significance of phase locking episodes is extended to render it adaptive to local phase synchrony levels. EMDPL is validated in the analysis of real EEG data, during finger tapping. The time course of event-related (de)synchronisation (ERD/ERS) is shown to differ from that of longer range phase locking episodes, implying different roles for these different types of synchronisation. It is suggested that the increase in phase locking which occurs just prior to movement, coinciding with a reduction in power (or ERD) may result from selection of the neural assembly relevant to the particular movement. (C) 2009 Elsevier B.V. All rights reserved.

A comparative study on the relationship between acetylcholinesterase activity and acute toxicity in Daphnia magna exposed to anticholinesterase insecticides

Acetylcholinesterase (AChE) activity was measured in Daphnia magna that had been exposed to four organophosphates (OPs; parathion, chlorpyrifos, malathion, and acephate) and one carbamate (propoxur) for 48 h. These results were related to acute toxicity (median effective concentration [EC50] for immobility). For the four OPs, the EC50s were 7.03 pM, 3.17 pM, 10.56 pM, and 309.82 microM, respectively. The EC50 for propoxur was 449.90 pM. Reduction in AChE activity was directly related to an increase in immobility in all chemicals tested. However, the ratio between the EC50 and the AChE median inhibiting concentration ranged from 0.31 to 0.90. A 50% reduction in AChE activity generally was associated with detrimental effects on mobility. However, for acephate, high levels of AChE inhibition (70%) were observed in very low concentrations and were not associated with immobility. In addition, increasing the concentration of acephate further had a slight negative effect on AChE activity but a strong detrimental effect on mobility. Binding sites other than AChE possibly are involved in acephate toxicity to D. magna. Our findings demonstrate different associations between AChE inhibition and toxicity when different chemicals are compared. Therefore, the value of using AChE activity as a biomarker in D. magna will be dependent on the chemical tested.

Cardiac Na+/H+ exchanger during post-natal development in the rat: Changes in mRNA expression and sarcolemmal activity

We examined Na+–H+exchanger isoform 1 (NHE-1) mRNA expression in ventricular myocardium and its correlation with sarcolemmal NHE activity in isolated ventricular myocytes, during postnatal development in the rat. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA did not change in ventricular myocardium between 2 and 42 days after birth. Therefore, at seven time points within that age range, GAPDH expression was used to normalize NHE-1 mRNA levels, as determined by reverse transcription polymerase chain reaction analysis. There was a progressive five-fold reduction in NHE-1 mRNA expression in ventricular myocardium from 2 days to 42 days of age. As an index of NHE activity, acid efflux rates (JH) were determined in single neonatal (2–4-day-old) and adult (42-day-old) ventricular myocytes (n=16/group) loaded with the pH fluoroprobe carboxy-seminaphthorhodafluor-1. In HEPES-buffered medium, basal intracellular pH (pHi) was similar at 7.28±0.02 in neonatal and 7.31±0.02 in adult myocytes, but intrinsic buffering power was lower in the former age group. The rate at which pHirecovered from a similar acid load was significantly greater in neonatal than in adult myocytes (0.36±0.07v0.16±0.02 pH units/min at pHi=6.8). This was reflected by a significantly greaterJH(22±4v9±1 pmol/cm2/s at pHi=6.8), indicating greater sarcolemmal NHE activity in neonatal myocytes. The concomitant reductions in tissue NHE-1 mRNA expression and sarcolemmal NHE activity suggest that myocardial NHE-1 is subject to regulation at the mRNA level during postnatal development.

Cortisol levels in response to starting school in children at increased risk for social phobia

Background: Research on depression has identified hyperactivity of the HPA axis as a potential contributory factor to the intergenerational transmission of affective symptoms. However, this has not yet been examined in the context of social phobia. The current study compared HPA axis activity in response to a universal social stressor (starting school) in children of 2 groups of women: one with social phobia and one with no history of anxiety (comparison group). To determine specificity of effects of maternal social phobia, a third group of children were also examined whose mothers had generalised anxiety disorder (GAD). Method: Children provided salivary cortisol samples in the morning, afternoon and at bedtime across 3 time-blocks surrounding the school start: a month before starting school (baseline), the first week at school (stress response), and the end of the first school term (stress recovery). Child behavioural inhibition at 14 months was also assessed to explore the influence of early temperament on later stress responses. Results: All children displayed an elevation in morning and afternoon cortisol from baseline during the first week at school, which remained elevated until the end of the first term. Children in the social phobia group, however, also displayed an equivalent elevation in bedtime cortisol, which was not observed for comparison children or for children of mothers with GAD. Children in the social phobia group who were classified as 'inhibited' at 14 months displayed significantly higher afternoon cortisol levels overall. Summary: A persistent stress response to school in the morning and afternoon is typical for all children, but children of mothers with social phobia also display atypical elevations in evening cortisol levels when at school - signalling long-term disruption of the circadian rhythm in HPA axis activity. This is the first study to report HPA axis disruption in children at risk of developing social phobia, and future research should aim to determine whether this represents a pathway for symptom development, taking early temperament into account.

Relationship between dietary-induced changes in intestinal commensal microflora and duodenojejunal myoelectric activity monitored by radiotelemetry in the rat in vivo

Interdigestive intestinal motility, and especially phase III of the migrating myoelectric/motor complex (MMC), is responsible for intestinal clearance and plays an important role in prevention of bacterial overgrowth and translocation in the gut. Yet previous results from gnotobiotic rats have shown that intestinal microflora can themselves affect the characteristics of the myoelectric activity of the gut during the interdigestive state. Given that the composition of the intestinal microflora can be altered by dietary manipulations, we investigated the effect of supplementation of the diet with synbiotics on intestinal microflora structure and the duodenojejunal myoelectric activity in the rat. To reduce animal distress caused by restraint and handling, which can itself affect GI motility, we applied radiotelemetry for duodenojejunal EMG recordings in conscious, freely moving rats. Thirty 16-month-old Spraque-Dawley rats were used. The diet for 15 rats (E group) was supplemented with chicory inulin, Lactobacillus rhamnosus and Bifidobacterium lactis. The remaining 15 rats were fed control diet without supplements (C group). Three rats from each group were implanted with three bipolar electrodes positioned at 2, 14 and 28 cm distal to the pylorus. After recovery, two 6 h recordings of duodenojejunal EMG were carried out on each operated rat. Subsequently, group C rats received feed supplements and group E rats received only control diet for 1 week, and an additional two 6 h recordings were carried out on each of these rats. Non-operated C and E rats were killed and samples of GI tract were collected for microbiological analyses. Supplementation of the diet with the pro- and prebiotics mixture increased the number of bifidobacteria, whereas it decreased the number of enterobacteria in jejunum, ileum, caecum and colon. In both caecum and colon, the dietary supplementation increased the number of total anaerobes and lactobacilli. Treatment with synbiotics increased occurrence of phase III of the MMC at all three levels of the small intestine. The propagation velocity of phase III in the whole recording segment was also increased from 3.7 +/- 0.2 to 4.4 +/- 0.2 cm min(-1) by dietary treatment. Treatment with synbiotics increased the frequency of response potentials of the propagated phase III of the MMC at both levels of the jejunum, but not in the duodenum. In both parts of the jejunum, the supplementation of the diet significantly decreased the duration of phase II of the MMC, while it did not change the duration of phase I and phase III. Using the telemetry technique it was demonstrated that changes in the gastrointestinal microflora exhibited an intestinal motility response and, more importantly, that such changes can be initiated by the addition of synbiotics to the diet.

Elevated levels of corticotrophin-releasing factor binding protein in the blood of patients suffering from arthritis and septicaemia and the presence of novel ligands in synovial fluid.

In view of the reported inflammatory effects of corticotrophin-releasing factor (CRF) and the associated regulatory elements in the gene of its binding protein (BP), we postulate that both BP as well as novel BP-ligands other than CRF may be involved in inflammatory disease. We have investigated BP in the blood of patients with arthritis and septicaemia and have attempted to identify CRF and other BP-ligands in synovial fluid. The BP was found to be significantly elevated in the blood of patients with rheumatoid arthritis and septicaemia. There was less BP-ligand and CRF in synovial fluid from patients with rheumatoid arthritis that from those with osteo- or psoriatic arthritis. There was at least 10-fold more BP-ligand than CRF in the fluid of all three groups of patients. A small amount of immunoreactive human (h)CRF, eluting in the expected position of CRF-41, was detected after high-pressure liquid chromatography of arthritic synovial fluid; however, the bulk of material with BP-ligand binding activity eluted earlier, suggesting that synovial fluid contained novel peptides that interacted with the BP. These results would suggest that the BP and its ligands could play an endocrine immunomodulatory role in inflammatory disease.