Haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice

AimsTo investigate whether haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice.Methods and ResultsExperiments were performed using irradiated LDL receptor-deficient (LDLR^−/−) mice with marrow from either TLR4-deficient (TLR4^−/−) or age-matched wild-type (WT) mice. After 12 weeks of being fed a high-cholesterol diet, TLR4^−/− → LDLR^−/− mice developed fewer atherosclerotic lesions in the aorta compared to WT → LDLR^−/− mice. This effect was associated with an increase in multilocular lipid droplets and mitochondria in perivascular adipose tissue (PVAT). Immunofluorescence analysis confirmed that there was an increase in capillary density and M2 macrophage infiltration, accompanied by a decrease in tumour necrosis factor (TNF)-α expression in the localized PVAT of TLR4^−/− → LDLR^−/− mice. In vitro studies indicated that bone marrow-derived macrophages (BMDMs) from WT mice demonstrated an M1-like phenotype and expression of inflammatory cytokines induced by palmitate. These effects were attenuated in BMDMs isolated from TLR4^−/− mice. Furthermore, brown adipocytes incubated with conditioned medium (CM) derived from palmitate-treated BMDMs, exhibited larger and more unilocular lipid droplets, and reduced expression of brown adipocyte-specific markers and perilipin-1 compared to those observed in brown adipocytes exposed to CM from palmitate-treated BMDMs of TLR4^−/− mice. This decreased potency was primarily due to TNF-α, as demonstrated by the capacity of the TNF-α neutralizing antibody to reverse these effects.ConclusionsThese results suggest that haematopoietic-specific deletion of TLR4 promotes PVAT homeostasis, which is involved in reducing macrophage-induced TNF-α secretion and increasing mitochondrial biogenesis in brown adipocytes.     

Toll-like receptor 3 plays a role in myocardial infarction and ischemia/reperfusion injury

Innate immune and inflammatory responses mediated by Toll like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. This study examined the role of TLR3 in myocardial injury induced by two models, namely, myocardial infarction (MI) and I/R. First, we examined the role of TLR3 in MI. TLR3 deficient (TLR3^−/−) and wild type (WT) mice were subjected to MI induced by permanent ligation of the left anterior descending (LAD) coronary artery for 21 days. Cardiac function was measured by echocardiography. Next, we examined whether TLR3 contributes to myocardial I/R injury. TLR3^−/− and WT mice were subjected to myocardial ischemia (45 min) followed by reperfusion for up to 3 days. Cardiac function and myocardial infarct size were examined. We also examined the effect of TLR3 deficiency on I/R-induced myocardial apoptosis and inflammatory cytokine production. TLR3^−/− mice showed significant attenuation of cardiac dysfunction after MI or I/R. Myocardial infarct size and myocardial apoptosis induced by I/R injury were significantly attenuated in TLR3^−/− mice. TLR3 deficiency increases B-cell lymphoma 2 (BCL2) levels and attenuates I/R-increased Fas, Fas ligand or CD95L (FasL), Fas-Associated protein with Death Domain (FADD), Bax and Bak levels in the myocardium. TLR3 deficiency also attenuates I/R-induced myocardial nuclear factor KappaB (NF-κB) binding activity, Tumor necrosis factor alpha (TNF-α) and Interleukin-1 beta (IL-1β) production as well as I/R-induced infiltration of neutrophils and macrophages into the myocardium. TLR3 plays an important role in myocardial injury induced by MI or I/R. The mechanisms involve activation of apoptotic signaling and NF-κB binding activity. Modulation of TLR3 may be an effective approach for ameliorating heart injury in heart attack patients.     

Cardioprotective effects of lipoic acid,quercetin and resveratrol on oxidative stress related to thyroid hormone alterations in long-term obesity

This study investigated possible mechanisms for cardioprotective effects of lipoic acid (LA), quercetin (Q) and resveratrol (R) on oxidative stress related to thyroid hormone alterations in long-term obesity. Female C57BL/6 mice were fed on high-fat diet (HFD), HFD + LA, HFD + R, HFD + Q and normal diet for 26 weeks. Body weight, blood pressure, thyroid hormones, oxidative stress markers, angiotensin converting enzyme (ACE), nitric oxide synthase (NOS) and ion pump activities were measured, and expression of cardiac genes was analyzed by real-time polymerase chain reaction. HFD induced marked increase (P < .05) in body weight, blood pressure and oxidative stress, while plasma triidothyronine levels reduced. ACE activity increased (P < .05) in HFD mice (0.69 ± 0.225 U/mg protein) compared with controls (0.28 ± 0.114 U/mg protein), HFD + LA (0.231 ± 0.02 U/mg protein) and HFD + Q (0.182 ± 0.096 U/mg protein) at 26 weeks. Moreover, Na⁺/K⁺-ATPase and Ca^2 +-ATPase activities increased in HFD mice whereas NOS reduced. A 1.5-fold increase in TRα1 and reduction in expression of the deiodinase iodothyronine DIO1, threonine protein kinase and NOS3 as well as up-regulation of AT1α, ACE, ATP1B1, GSK3β and Cja1 genes also occurred in HFD mice. Conversely, LA, Q and R inhibited weight gain; reduced TRα1 expression as well as increased DIO1; reduced ACE activity and AT1α, ATP1B1 and Cja1 gene expression as well as inhibited GSK3β; increased total antioxidant capacity, GSH and catalase activity; and reduced blood pressure. In conclusion, LA, resveratrol and quercetin supplementation reduces obesity thereby restoring plasma thyroid hormone levels and attenuating oxidative stress in the heart and thus may have therapeutic potential in heart diseases.     

Browning deficiency and low mobilization of fatty acids in gonadal white adipose tissue leads to decreased cold-tolerance of transglutaminase 2 knock-out mice

During cold-exposure ‘beige’ adipocytes with increased mitochondrial content are activated in white adipose tissue (WAT). These cells, similarly to brown adipose tissue (BAT), dissipate stored chemical energy in the form of heat with the help of uncoupling protein 1 (UCP1). We investigated the effect of tissue transglutaminase (TG2) ablation on the function of ATs in mice. Although TG2^+/+ and TG2^−/− mice had the same amount of WAT and BAT, we found that TG2^+/+ animals could tolerate acute cold exposure for 4 h, whereas TG2^−/− mice only for 3 h. Both TG2^−/− and TG2^+/+ animals used up half of the triacylglycerol content of subcutaneous WAT (SCAT) after 3 h treatment; however, TG2^−/− mice still possessed markedly whiter and higher amount of gonadal WAT (GONAT) as reflected in the larger size of adipocytes and lower free fatty acid levels in serum. Furthermore, lower expression of ‘beige’ marker genes such as UCP1, TBX1 and TNFRFS9 was observed after cold exposure in GONAT of TG2^−/− mice, paralleled with a lower level of UCP1 protein and a decreased mitochondrial content. The detected changes in gene expression of Resistin and Adiponectin did not provoke glucose intolerance in the investigated TG2^−/− mice, and TG2 deletion did not influence adrenaline, noradrenaline, glucagon and insulin production. Our data suggest that TG2 has a tissue-specific role in GONAT function and browning, which becomes apparent under acute cold exposure.     

Transcriptional responses of neonatal mouse lung to hyperoxia by Nrf2 status

Hyperoxia exposure can inhibit alveolar growth in the neonatal lung through induction of p21/p53 pathways and is a risk factor for the development of bronchopulmonary dysplasia (BPD) in preterm infants. We previously found that activation of nuclear factor erythroid 2 p45-related factor (Nrf2) improved survival in neonatal mice exposed to hyperoxia likely due to increased expression of anti-oxidant response genes. It is not known however, whether hyperoxic induced Nrf2 activation attenuates the growth impairment caused by hyperoxia in neonatal lung. To determine if Nrf2 activation modulates cell cycle regulatory pathway genes associated with growth arrest we examined the gene expression in the lungs of Nrf2^−/− and Nrf2^+/+ neonatal mice at one and 3 days of hyperoxia exposure.MethodsMicroarray analysis was performed in neonatal Nrf2^+/+ and Nrf2^−/− lungs exposed to one and 3 days of hyperoxia. Sulforaphane, an inducer of Nrf2 was given to timed pregnant mice to determine if in utero exposure attenuated p21 and IL-6 gene expression in wildtype neonatal mice exposed to hyperoxia.ResultsCell cycle regulatory genes were induced in Nrf2^−/− lung at 1 day of hyperoxia. At 3 days of hyperoxia, induction of cell cycle regulatory genes was similar in Nrf2^+/+ and Nrf2^−/− lungs, despite higher inflammatory gene expression in Nrf2^−/− lung.Conclusionp21/p53 pathways gene expression was not attenuated by Nrf2 activation in neonatal lung. In utero SUL did not attenuate p21 expression in wildtype neonatal lung exposed to hyperoxia. These findings suggest that although Nrf2 activation induces expression of anti-oxidant genes, it does not attenuate alveolar growth arrest caused by exposure to hyperoxia.     

New oleanane-type saponins: Leptocarposide B-D,from Ludwigia leptocarpa (Onagraceae)

Three new oleanane-type saponins, leptocarposide B-D (1–3), were isolated from the whole plant of Ludwigia leptocarpa (Nutt.) Hara, together with ten known compounds 4–13.The structures of these compounds were determined by interpretation of their spectral data, mainly HR-TOFESIMS, 1D-NMR (¹H, ¹³C) and 2D-NMR (¹H–¹H COSY, HSQC, HMBC, and NOESY), and by comparison with the literature data. The structures of the new compounds were established as 28-O-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-[α-l-arabinopyranosyl-(1 → 3)]-4-O-(3′-hydroxybutanoyloxy-3-hydroxybutanoyloxy)-β-d-fucopyranosyl zanhic acid (1); 3-O-β-d-glucopyranosyl-28-O-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-4-O-(3′-hydroxybutanoyloxy-3-hydroxybutanoyloxy)-β-d-fucopyranosyl medicagenic acid (2); 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranosyl-28-O-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-[α-l- arabinopyranosyl-(1 → 3)]-4-O-(3′-hydroxybutanoyloxy-3-hydroxybutanoyloxy)-β-d-fucopyranosyl zanhic acid (3).     

Dianthus erinaceus var. erinaceus: Extraction,isolation, characterization and antimicrobial activity investigation of novel saponins

A phytochemical analysis of Dianthus erinaceus Boiss. var. erinaceus (Caryophyllaceae) has led to the isolation of two novel triterpenoid saponins, containing an oleane type skeleton, named dianosides K and L (1, 2), along with six known triterpenoid saponins (3–8). On the basis of chemical and spectrometric data, the structures of the new compounds were elucidated as 3-O-[β-d-glucopyranosyl (1 → 3)]–[β-d-glucopyranosyl (1 → 6)]-β-d-glucopyranosyl-olean-12-ene-23α,28-β–dioic acid 28-O-β-d-glucopyranosyl ester (1) and 3-O-[β-d-glucopyranosyl (1 → 3)]–[β-d-glucopyranosyl(1 → 6)]-β-d-glucopyranosyl-olean-12-ene-23α,28-β-dioic acid 28-O-α-l-mannopyranosyl (1 → 6)-β-d-glucopyranosyl ester (2). All isolated natural compounds were structurally characterized by 1D- (¹H, ¹³C, DEPT); 2D- (COSY, HMQC, HMBC) NMR and HR-ESI/MS methods. The antimicrobial activity of compounds 1 and 2 were tested against four Gram-negative, three Gram-positive bacteria and the yeast Candida albicans by the MIC method.     

Remarkable induction of UV-signature mutations at the 3′-cytosine of dipyrimidine sites except at 5′-TCG-3′ in the UVB-exposed skin epidermis of xeroderma pigmentosum variant model mice

The human POLH gene is responsible for the variant form of xeroderma pigmentosum (XP-V), a genetic disease highly susceptible to cancer on sun-exposed skin areas, and encodes DNA polymerase η (polη), which is specialized for translesion DNA synthesis (TLS) of UV-induced DNA photolesions. We constructed polη-deficient mice transgenic with lacZ mutational reporter genes to study the effect of Polh null mutation (Polh^−/−) on mutagenesis in the skin after UVB irradiation. UVB induced lacZ mutations with remarkably higher frequency in the Polh^−/− epidermis and dermis than in the wild-type (Polh^+/+) and heterozygote. DNA sequences of a hundred lacZ mutants isolated from the epidermis of four UVB-exposed Polh^−/− mice were determined and compared with mutant sequences from irradiated Polh^+/+ mice. The spectra of the mutations in the two genotypes were both highly UV-specific and dominated by C → T transitions at dipyrimidines, namely UV-signature mutations. However, sequence preferences of the occurrence of UV-signature mutations were quite different between the two genotypes: the mutations occurred at a higher frequency preferentially at the 5′-TCG-3′ sequence context than at the other dipyrimidine contexts in the Polh^+/+ epidermis, whereas the mutations were induced remarkably and exclusively at the 3′-cytosine of almost all dipyrimidine contexts with no preference for 5′-TCG-3′ in the Polh^−/− epidermis. In addition, in Polh^−/− mice, a small but remarkable fraction of G → T transversions was also observed exclusively at the 3′-cytosine of dipyrimidine sites, strongly suggesting that these transversions resulted not from oxidative damage but from UV photolesions. These results would reflect the characteristics of the error-prone TLS functioning in the bypass of UV photolesions in the absence of polη, which would be mediated by mechanisms based on the two-step model of TLS. On the other hand, the deamination model would explain well the mutation spectrum in the Polh^+/+ genotype.     

Rheediinosides A and B,two antiproliferative and antioxidant triterpene saponins from Entada rheedii

Two triterpenoid saponins have been isolated from the seed kernels of Entada rheedii. Their structures have been established using 1D- and 2D-NMR and mass spectrometry as 3-O-β-d-xylopyranosyl-(1 → 3)-O-α-l-arabinopyranosyl-(1 → 6)-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside A, 1) and 3-O-β-d-glucopyranosyl-(1 → 3)-O-[β-d-xylopyranosyl-(1 → 3)-α-l-arabinopyranosyl-(1 → 6)]-2-acetylamino-2-deoxy-β-d-glucopyranosylentagenic acid 28-O-β-apiofuranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 2)-β-d-glucopyranoside (Rheediinoside B, 2). Compounds 1 and 2 were tested for their antiproliferative activity against T98G, A431, PC3 and B16-F1 cell lines, and further for their antioxidant properties. Moderate cytotoxic potency and antioxidant properties were found for these compounds whereas Rheediinoside B was in all assays more active than Rheediinoside A.     

Postoperative endometrial carcinoma treated with external beam irradiation plus vaginal-cuff brachytherapy. Is there a dose relationship with G2 vaginal complications?

AimTo analyse the possible relationship between the EQD2_(α/β=3Gy) at 2 cm³ of the vagina and late toxicity in vaginal-cuff-brachytherapy (VBT) after external-beam-irradiation (EBRT) for postoperative endometrial carcinoma (EC).Materials and methodsFrom 2014 to 2016, 62 postoperative EC patients were treated with EBRT + VBT. The median EBRT dose was 45 Gy (44 Gy–50.4 Gy). VBT involved a single 7 Gy dose. Toxicity was prospectively evaluated using the RTOG score for the rectum and bladder and the objective LENT-SOMA criteria for the vagina. EQD2_{(α/β = 3Gy)} at 2 cm³ of the most exposed part of the vagina was calculated by the sum of the EBRT + VBT dose. Statistics: Boxplot, Student’s t and Chi-square tests and ROC curves.ResultsMean follow-up: 39.2 months (15–68). Late toxicity: bladder:0 patient; rectum:2 patients-G1; Vagina: 26 patients-17G1, 9G2; median EQD2_(α/β=3Gy) at 2 cm³ in G0-G1 patients was 70.4 Gy(SD2.36), being 72.5 Gy(SD2.94) for G2p. The boxplot suggested a cut-point identifying the absence of G2: 100 % of G2p received >68 Gy, ROC curves showed an area under the curve of 0.72 (sensitivity of 1 and specificity of 0.15).ConclusionsDoses >68 Gy EQD2_(α/β=3Gy) at 2 cm³ to the most exposed area of the vagina were associated with late G2 vaginal toxicity in postoperative EC patients treated with EBRT + VBT suggesting a very good dose limit to eliminate the risk of G2 late toxicity. The specificity obtained indicates the need for prospective analyses.     

The effects of acute changes in temperature and oxygen availability on cardiac performance in winter flounder (Pseudopleuronectes americanus)

Studies on how flatfish cardiovascular function responds to environmental challenges are limited, and have largely relied upon indirect methodologies (i.e. Fick principle). Thus, we measured dorsal aortic blood pressure (P_DA) and cardiac function in 8 and 15 °C-acclimated flounder exposed to graded hypoxia, and in 8 °C-acclimated fish exposed to an acute temperature increase to their critical thermal maximum (CTM). The extent of bradycardia in 8 °C-acclimated fish (decrease in heart rate of 41%) was consistent with that observed for other teleosts, as was this species' CTM (25.8 ± 0.5 °C) and its cardiac response to increasing temperature. However, this study provides further examples of how cardiovascular function is controlled differently in the flounder as compared with other fishes. First, the onset of bradycardia in 8 °C-acclimated fish occurred earlier than expected for this inactive and hypoxia-tolerant species (60% water air saturation). Second, resting cardiac output was similar in flounder acclimated to 8 and 15 °C (～ 15 mL min^? 1 kg^? 1), and hypoxic bradycardia was surprisingly absent at 15 °C. Finally, systemic vascular resistance decreased when flounder were exposed to elevated temperature, and this resulted in a 26% fall in P_DA. These are novel findings, however, the extent to which the flounder's behaviour influenced some of the results is unclear.     

PAS-1, a protein from Ascaris suum,modulates allergic inflammation via IL-10 and IFN-γ, but not IL-12

Helminths and their products have a profound immunomodulatory effect upon the inductive and effector phases of inflammatory responses, including allergy. We have demonstrated that PAS-1, a protein isolated from Ascaris suum worms, has an inhibitory effect on lung allergic inflammation due to its ability to down-regulate eosinophilic inflammation, Th2 cytokine release and IgE antibody production. Here, we investigated the role of IL-12, IFN-γ and IL-10 in the PAS-1-induced inhibitory mechanism using a murine model of asthma. Wild type C57BL/6, IL-12^−/−, IFN-γ^−/− and IL-10^−/− mice were immunized with PAS-1 and/or OVA and challenged with the same antigens intranasally. The suppressive effect of PAS-1 was demonstrated on the cellular influx into airways, with reduction of eosinophil number and eosinophil peroxidase activity in OVA + PAS-1-immunized wild type mice. This effect well correlated with a significant reduction in the levels of IL-4, IL-5, IL-13 and eotaxin in BAL fluid. Levels of IgE and IgG1 antibodies were also impaired in serum from these mice. The inhibitory activity of PAS-1 was also observed in IL-12^−/− mice, but not in IFN-γ^−/− and IL-10^−/− animals. These data show that IFN-γ and IL-10, but not IL-12, play an important role in the PAS-1 modulatory effect.     

An unexpectedly lichenase-stable hexasaccharide from cereal,horsetail and lichen mixed-linkage β-glucans (MLGs): Implications for MLG subunit distribution

Mixed-linkage (1→3),(1→4)-β-d-glucan (MLG) is a biologically and technologically important hemicellulose, known to occur in three widely separated lineages: the Poales (including grasses and cereals), Equisetum (fern-allies), and some lichens e.g. Iceland moss (Cetraria islandica). Lichenase (E.C. 3.2.1.73) is widely assumed to hydrolyse all (1→4) bonds that immediately follow (1→3) bonds in MLG, generating predominantly the tetrasaccharide β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-d-Glc (G4G4G3G; MLG4), the corresponding trisaccharide (G4G3G; MLG3), and sometimes also laminaribiose (G3G; MLG2). The ratio of the oligosaccharides produced characterises each polysaccharide. We report here that digestion of MLG from barley (Hordeum vulgare), Equisetum arvense and C. islandica by Bacillus subtilis lichenase also yields the unexpectedly stable hexasaccharide, β-d-Glcp-(1→3)-β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-β-d-Glcp-(1→4)-β-d-Glcp-(1→3)-d-Glc (G3G4G4G4G3G, i.e. MLG2–MLG4), identified by thin-layer chromatography, gel-permeation chromatography, HPLC (HPAEC), β-glucosidase digestion, ¹H/¹³C-NMR spectroscopy and mass spectrometry. On HPLC, G3G4G4G4G3G is the major constituent of a peak previously ascribed solely to the nonasaccharide G4G4G4G4G4G4G4G3G. Because it was widely presumed that lichenase would cleave G3G4G4G4G3G to MLG2 + MLG4, our data both redefine the substrate specificity of Bacillus lichenase and show previous attempts to characterise MLGs by HPLC of lichenase-digests to be flawed. MLG2 subunits are particularly underestimated; often reported as negligible, they are here shown to be an appreciable constituent of MLGs from all three lineages. We also show that there is no appreciable yield of water-soluble lichenase products with DP > 9; potential identities of products previously labelled DP > 9 are suggested. Finally, this discovery also provides a opportunity to investigate the spatial distribution of subunits along the MLG chain. We show that MLG2 subunits in barley and Cetraria MLG are not randomly distributed, but predominantly found at the non-reducing end of MLG4 subunits.     

Hepatic stellate cells retain the capacity to synthesize retinyl esters and to store neutral lipids in small lipid droplets in the absence of LRAT

Hepatic stellate cells (HSCs) play an important role in liver physiology and under healthy conditions they have a quiescent and lipid-storing phenotype. Upon liver injury, HSCs are activated and rapidly lose their retinyl ester-containing lipid droplets. To investigate the role of lecithin:retinol acyltransferase (LRAT) and acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) in retinyl ester synthesis and lipid droplet dynamics, we modified LC–MS/MS procedures by including multiple reaction monitoring allowing unambiguous identification and quantification of all major retinyl ester species. Quiescent primary HSCs contain predominantly retinyl palmitate. Exogenous fatty acids are a major determinant in the retinyl ester species synthesized by activated HSCs and LX-2 cells, indicating that HSCs shift their retinyl ester synthesizing capacity from LRAT to DGAT1 during activation. Quiescent LRAT^−/− HSCs retain the capacity to synthesize retinyl esters and to store neutral lipids in lipid droplets ex vivo. The median lipid droplet size in LRAT^−/− HSCs (1080 nm) is significantly smaller than in wild type HSCs (1618 nm). This is a consequence of an altered lipid droplet size distribution with 50.5 ± 9.0% small (≤ 700 nm) lipid droplets in LRAT^−/− HSCs and 25.6 ± 1.4% large (1400–2100 nm) lipid droplets in wild type HSC cells. Upon prolonged (24 h) incubation, the amounts of small (≤ 700 nm) lipid droplets strongly increased both in wild type and in LRAT^−/− HSCs, indicating a dynamic behavior in both cell types. The absence of retinyl esters and reduced number of lipid droplets in LRAT-deficient HSCs in vivo will be discussed.     

Two new spirostanol saponins from the the roots and rhizomes of Tupistra chinensis

Two new spirostanol saponins (1) and (2), together with three known saponins (3–5), were isolated from the roots and rhizomes of Tupistra chinensis, and their structures were determined as (20S, 22R)-spirost-25(27)-en-1β, 3β, 4β, 5β-tetraol-5-O-β-d-glucopyranoside (1) and (20S, 22R)-spirost-25(27)-en-1β, 3β, 5β-triol-5-O-β-d-glucopyranoside (2), (20S, 22R)-spirost-25(27)-en-1β, 2β, 3β, 4β, 5β-pentaol-5-O-β-d-glucopyranoside (3), Δ²⁵⁽²⁷⁾-pentrogenin (4) and ranmogenin A (5) on the basis of physicochemical properties and spectral analysis. The isolated compounds were evaluated for their cytotoxic activities against A549 and H1299 tumor cell lines in vitro. Among them, compound 2 showed cytotoxicities against A549 cells (IC₅₀ 52.66 ± 3.12 μmol L⁻¹) and H1299 cells (IC₅₀ 57.29 ± 2.51 μmol L⁻¹), respectively.     

A diazen-1-ium-1,2-diolate analog of 7-azabenzobicyclo[2.2.1]heptane: Synthesis,nitric oxide and nitroxyl release,in vitro hemodynamic,and anti-hypertensive studies

1-(7-Azabenzobicyclo[2.2.1]heptane)diazen-1-ium-1,2-diolate (16) was designed with the expectation that it would act as a dual nitric oxide (NO) and nitroxyl (HNO) donor that is not carcinogenic or genotoxic. Compound 16, with a suitable half-life (17.8 min) in PBS at pH 7, released NO (19%) and HNO (22%) during a 2 h incubation in PBS at pH 7. In addition, compound 16 exhibited a significant in vitro positive inotropic effect, increased the rates of contraction and relaxation, and increased coronary flow rate, but did not induce a chronotropic effect. Furthermore, compound 16 (13.7 mg kg^?1, po dose) provided a significant reduction in the blood pressure of mice up to 3 h post-drug administration. All these data suggest that compound 16 constitutes an attractive ‘lead-compound’ that could have potential applications to treat cardiovascular disease(s) such as congestive heart failure.     

Novel dammarane saponins from Gynostemma pentaphyllum and their cytotoxic activities against HepG2 cells

Two new dammarane saponins, 2α,3β,12β-trihydroxydammar-20(22),24-diene-3-O-[β-d-glucopyranoxyl(1→2)-β-d-6″-O-acetylglucopyranoside (1, namely damulin C) and 2α,3β,12β-trihydroxydammar-20(21),24-diene-3-O-[β-d-glucopyranoxyl(1→2)-β-d-6″-O-acetylglucopyranoside (2, namely damulin D), were isolated from the ethanol extract of Gynostemma pentaphyllum, which had been heat processed by steaming at 125 °C. The NMR spectroscopic data of the novel saponins were completely assigned by using a combination of 2D NMR experiments including ¹H–¹H COSY, HSQC, and HMBC. Their cytotoxic activities of human liver adenocarcinoma HepG2 cells were evaluated in vitro. They showed cytotoxicities against HepG2 cell line with IC₅₀ of 40 ± 0.7 and 38 ± 0.5 μg/ml, respectively.     

Homocysteine decreases blood flow to the brain due to vascular resistance in carotid artery

An elevated level of Homocysteine (Hcy) is a risk factor for vascular dementia and stroke. Cysthathionine β Synthase (CBS) gene is involved in the clearance of Hcy. Homozygous individuals for (CBS−/−) die early, but heterozygous for (CBS−/+) survive with high levels of Hcy. The γ-Amino Butyric Acid (GABA) presents in the central nervous system (CNS) and functions as an inhibitory neurotransmitter. Hcy competes with GABA at the GABA_A receptor and affects the CNS function. We hypothesize that Hcy causes a decrease in blood flow to the brain due to increase in vascular resistance (VR) because of arterial remodeling in the carotid artery (CA). Blood pressure and blood flow in CA of wild type (WT), CBS−/+, CBS−/+ GABA_A−/− double knockout, and GABA_A−/− were measured. CA was stained with trichrome, and the brain permeability was measured. Matrix Metalloproteinases (MMP-2 and MMP-9), tissue inhibitor of metalloproteinase (TIMP-3, TIMP-4), elastin, and collagen-III expression were measured by real-time polymerase chain reaction (RT-PCR). Results showed an increase in VR in CBS−/+/GABA_A−/−double knockout > CBS−/+/ > GABA_A−/− compared to WT mice. Increased MMP-2, MMP-9, collagen-III and TIMP-3 mRNA levels were found in GABA_A−/−, CBS−/+, CBS−/+/GABA_A double knockout compared to WT. The levels of TIMP-4 and elastin were decreased, whereas the levels of MMP-2, MMP-9 and TIMP-3 increased, which indirectly reflected the arterial resistance. These results suggested that Hcy caused arterial remodeling in part, by increase in collagen/elastin ratio thereby increasing VR leading to the decrease in CA blood flow.     

CaV1.3 L-type channels,maxiK Ca2 +-dependent K+ channels and bestrophin-1 regulate rhythmic photoreceptor outer segment phagocytosis by retinal pigment epithelial cells

Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca^2 + regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Ca_v1.3 L-type Ca²⁺ channels (Ca1.3^−/−) and maxiK Ca²⁺-dependent K⁺ channels (BK^−/−). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca²⁺ homeostasis, affected phagocytosis in porcine RPE cultures. Blockage of voltage-gated L-type channels by (+)BayK8644 inhibitor reduced phagocytosis in vitro, in contrast L-type activation by (−)BayK8644 had no impact. The expression rate of Ca_v1.3, the predominant L-type Ca^2 + channel in RPE cells, varied at different times of day. Ca_V1.3^−/− RPE lacked peak phagocytic activity following morning photoreceptor shedding in wild-type RPE and retained a higher number of phagosomes at a later time of day. The BK-channel blocker paxilline lowered phagocytosis in RPE cultures in a concentration-dependent manner. BK^−/− RPE in vivo retained phagocytic capability but this activity, which is normally well synchronized with circadian photoreceptor shedding, shifted out of phase. Retinae of older BK^−/− mice showed shortened photoreceptor outer segments and diminished rhodopsin content. Store-operated Ca^2 + channels Orai-1 did not affect phagocytosis in cultured RPE. TRPV channel inhibition by ruthenium-red reduced phagocytosis, whereas activation at high concentrations of 2-APB increased phagocytosis. Our data demonstrate essential roles for bestrophin-1, BK, TRPV and L-type channels in regulating retinal phagocytosis. These data indicate further the importance of BK and Ca_V1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding.     

Highly glycosylated flavonols with an O-linked branched pentasaccharide from Iberis saxatilis (Brassicaceae)

Four flavonol glycosides isolated from non-flowering leafy shoots of Iberis saxatilis (Brassicaceae) were characterised by spectroscopic and chemical methods as saxatilisins A–D, the 3-O-β-d-glucopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)[β-d-glucopyranosyl-(1 → 2)-α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside, 3-O-β-d-glucopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside, 3-O-(6-O-E-sinapoyl)-β-d-glucopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)[β-d-glucopyranosyl-(1 → 2)-α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside, and 3-O-(6-O-E-feruloyl)-β-d-glucopyranosyl-(1 → 3)-α-l-rhamnopyranosyl-(1 → 2)[β-d-glucopyranosyl-(1 → 2)-α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside of isorhamnetin (3,5,7,4′-tetrahydroxy-3′-methoxyflavone), respectively. Analysis of ²J_HC correlations detected with the H2BC (heteronuclear two-bond correlation) pulse sequence aided the unambiguous assignment of glycosidic resonances in the ¹H and ¹³C NMR spectra of these compounds. Saxatilisins A, C, and D, are the first flavonol glycosides to be described with a pentasaccharide chain at a single glycosylation site. Several pentaglycosides of kaempferol and quercetin, tentatively assigned as saxatilisin analogues from LC–MS/MS analyses, were present as minor constituents of the extracts.