Assessment of the stability of proanthocyanidins and other phenolic compounds in cranberry syrup after gamma-irradiation treatment and during storage.

Shelf life of commercial cranberry syrup irradiated with gamma radiation at a rate of 5 kGy and stored for 6 months at 25 °C and 60% relative humidity (RH) and under accelerated stability conditions was investigated. High-performance liquid chromatography coupled to electrospray ionisation quadrupole-time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS) was used to characterise cranberry syrup. Afterwards, these compounds were quantified by HPLC-ESI-QTOF-MS and 4-dimethylaminocinnamaldehyde (DMAC) assay. A significant increase in the content of procyanidin B isomer 1 (from 4.4 to 7.0 µg/ml) and procyanidin A2 (from 83 to 93 µg/ml) was observed after irradiation and compared with the non-irradiated syrup. Procyanidin B isomers and prodelphinidin were stable at 25 °C during the first month of storage, whereas quercetin and some derivatives remained constant for 3 months of storage at this temperature. In short, after gamma-irradiation in dose of 5 kGy, most compounds were highly stable for a month at 25 °C.

Phenylpropanoids and their metabolites are the major compounds responsible for blood-cell protection against oxidative stress after administration of Lippia citriodora in rats.

Lippia citriodora (lemon verbena) has been widely used in folk medicine for its pharmacological properties. Verbascoside, the most abundant compound in this plant, has protective effects associated mostly with its strong antioxidant activity. The purpose of this study was to test the effect of L. citriodora extract intake on the antioxidant response of blood cells and to correlate this response with the phenolic metabolites found in plasma. For this purpose, firstly the L. citriodora extract was characterized and its radical scavenging activity was measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. Then, catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GRed) activities were determined in lymphocytes, erythrocytes, and neutrophils isolated from rats after acute intake of L. citriodora. Phenolic metabolites were analyzed in the same plasma samples by HPLC-ESI-TOF-MS. Myeloperoxidase (MPO) activity in neutrophils, which has been proposed as a marker for inflammatory vascular damage, was also determined. After L. citriodora administration, the antioxidant enzymes activities significantly accelerated (p<0.05) while MPO activity subsided, indicating that the extract protects blood cells against oxidative damage and shows potential anti-inflammatory and antiatherogenic activities. The main compounds found in plasma were verbascoside and isoverbascoside at a concentration of 80±10 and 57±4 ng/ml, respectively. Five other metabolites derived from verbascoside and isoverbascoside were also found in plasma, namely hydroxytyrosol, caffeic acid, ferulic acid, ferulic acid glucuronide, and homoprotocatechuic acid, together with another eight phenolic compounds. Therefore, the phenylpropanoids verbascoside and isoverbascoside, as well as their metabolites, seem to be the responsible for the above-mentioned effects, although the post-transcriptional activation mechanism of blood-cell antioxidant enzymes by these compounds needs further investigation.

A metabolite-profiling approach allows the identification of new compounds from Pistacia lentiscus leaves.

Pistacia lentiscus L., commonly known as Mastic tree or lentisk, is a Mediterranean evergreen shrub widely used in traditional medicine to treat such diseases as eczema, diarrhoea, and throat infections. Furthermore, other properties are currently attributed to P. lentiscus, such as antioxidant capacity, hepatoprotective action, and anti-inflammatory effects. High-performance liquid chromatography with diode array coupled to electrospray ionization mass spectrometry (HPLC-ESI-QTOF-MS) was used for the comprehensive characterization of methanol extract from P. lentiscus leaves. After the optimisation of the HPLC-ESI-QTOF-MS method and the use of the negative ionization mode, 46 different compounds were identified, 20 of which were tentatively characterized for the first time in P. Lentiscus leaves. The majority of the compounds were quantified. Flavonoids, phenolic acids and their derivatives were the most abundant compounds, those with the highest concentrations being myricetin glycoside (6216.13 mg/kg of plant), catechin (3354.78 mg/kg of plant), ß-glucogallin (2214.461 mg/kg of plant), and quercitrin gallate (1160 mg/kg of plant). The importance of the knowledge of plants is increasing and our study may help in the future to formulate nutraceutical preparations and will provide the basis for new investigation into activities of the various compounds found in P. lentiscus.

A metabolite-profiling approach to assess the uptake and metabolism of phenolic compounds from olive leaves in SKBR3 cells by HPLC-ESI-QTOF-MS.

Olive leaves, an easily available natural low-cost material, constitute a source of extracts with significant antitumor activity that inhibits cell proliferation in several breast-cancer-cell models. In this work, a metabolite-profiling approach has been used to assess the uptake and metabolism of phenolic compounds from an olive-leaf extract in the breast-cancer-cell line SKBR3 to evaluate the compound or compounds responsible for the cytotoxic activity. For this, the extract was firstly characterized quantitatively by high-performance liquid chromatography coupled to electrospray ionization-quadrupole time-of-flight mass spectrometry (HPLC-ESI-QTOF-MS). Then, SKBR3 cells were incubated with 200 µg/mL of the olive-leaf extract at different times (15 min, 1, 2, 24, and 48 h). A metabolite-profiling approach based on HPLC-ESI-QTOF-MS was used to determine the intracellular phenolic compounds, enabling the identification of 16 intact phenolic compounds from the extract and four metabolites derived from these compounds in the cell cytoplasm. The major compounds found within the cells were oleuropein, luteolin-7-O-glucoside and its metabolites luteolin aglycone and methyl-luteolin glucoside, as well as apigenin, and verbascoside. Neither hydroxytyrosol nor any of its metabolites were found within the cells at any incubation time. It is proposed that the major compounds responsible for the cytotoxic activity of the olive-leaf extract in SKBR3 cells are oleuropein and the flavones luteolin and apigenin, since these compounds showed high uptake and their antitumor activity has been previously reported.

Evaluation of different extraction approaches for the determination of phenolic compounds and their metabolites in plasma by nanoLC-ESI-TOF-MS.

Sample preparation is an important step for the determination of phenolic compounds in biological samples. Different extraction methods have been tested to determine phenolic compounds and their metabolites in plasma by nano-liquid chromatography coupled to electrospray ionisation-time-of-flight mass spectrometry (nanoLC-ESI-TOF-MS). The sample treatment optimisation was performed using commercial foetal bovine serum spiked with representative phenolic standards, namely naringenin, luteolin, verbascoside, apigenin, rutin, syringic acid and catechin. Different protein-precipitation conditions were evaluated as well as enzymatic digestion with trypsin and solid-phase extraction using different phases such as C-18, ABN and ENV+, working at different pH values. The optimum extraction procedure consisted of a previous protein-precipitation step using HCl 200 mmol/L in methanol for 2.5 h at 50 °C followed by a solid-phase extraction using C-18 cartridges at pH 2.5. This procedure was finally applied to the plasma of rats overfed with a phenolic-rich Lippia citriodora extract. These samples were analysed by nanoLC-ESI-TOF-MS, enabling the identification of five compounds previously found in the administered L. citriodora extract and one metabolite.

Application of nanoLC-ESI-TOF-MS for the metabolomic analysis of phenolic compounds from extra-virgin olive oil in treated colon-cancer cells.

Crude phenolic extracts (PE) have been obtained from naturally bearing Spanish extra-virgin olive oil (EVOO) showing different polyphenol families such as secoiridoids, phenolic alcohols, lignans, and flavones. EVOO-derived complex phenols (especially from the Arbequina variety olive) have been shown to suppress cell growth of SW480 and HT29 human colon adenocarcinoma cell lines. Inhibition of proliferation by EVOO-PE Arbequina variety extract was accompanied by apoptosis in both colon-cancer-cell lines and a limited G2M cell-cycle arrest in the case of SW480 cells. The metabolized compounds from EVOO-PE in culture medium and cytoplasm of both cell lines were analyzed using nano-liquid chromatography (nanoLC) coupled with electrospray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS). The results showed many phenolic compounds and their metabolites both in the culture medium as well as in the cytoplasm. The main compounds identified from EVOO-PE were hydroxylated luteolin and decarboxymethyl oleuropein aglycone.

High-performance liquid chromatography with diode array detection coupled to electrospray time-of-flight and ion-trap tandem mass spectrometry to identify phenolic compounds from a lemon verbena extract.

High-performance liquid chromatography with diode array and electrospray ionization mass spectrometric detection was used to carry out the comprehensive characterization of a lemon verbena extract with demonstrated antioxidant and antiinflammatory activity. Two different MS techniques have been coupled to HPLC: on one hand, time-of-flight mass spectrometry, and on the other hand, tandem mass spectrometry on an ion-trap. The use of a small particle size C18 column (1.8 microm) provided a great resolution and made possible the separation of several isomers. The UV-visible spectrophotometry was used to delimit the class of phenolic compound and the accurate mass measurements on time-of-flight spectrometer enabled to identify the compounds present in the extract. Finally, the fragmentation pattern obtained in MS-MS experiments confirmed the proposed structures. This procedure was able to determine many well-known phenolic compounds present in lemon verbena such as verbascoside and its derivatives, diglucuronide derivatives of apigenin and luteolin, and eukovoside. Also gardoside, verbasoside, cistanoside F, theveside, campneoside I, chrysoeriol-7-diglucuronide, forsythoside A and acacetin-7-diglucuronide were found for the first time in lemon verbena.

From consent to choice in family planning: application of an international framework to the United States.

Building on the broad agenda set forth at the 1994 International Conference on Population and Development, this article articulates similarly broad expectations for informed choice in family planning service delivery. A framework developed by a Global Working Group in 1998 sets forth various issues into a single analysis of informed choice. In an effort to bring international perspectives to bear on the articulation of new objectives for the home front, the article applies this framework to the US research literature in family planning. Review of the literature indicates a heartening degree of attention to issues of access and choice, but it also uncovers challenges faced by clients and providers and gaps in what is known. Few studies look beyond access to how providers actually do or could better facilitate informed decision making within the family planning setting. Such studies could help improve innovative and ambitious service delivery approaches that go beyond meeting the reproductive health needs of clients to ensure rights, improve choice, and empower individuals.

Differentiation of monocytes to macrophages switches the Mycobacterium tuberculosis effect on HIV-1 replication from stimulation to inhibition: modulation of interferon response and CCAAT/enhancer binding protein beta expression.

HIV-1 replication is inhibited in uninflamed lung macrophages and is stimulated during tuberculosis. Attempts to recapitulate activation of HIV-1 replication in primary monocytes and macrophages ex vivo and in the untreated and PMA-treated THP-1 cell line model in vitro have produced opposite results depending on the state of differentiation of the cells. After infection with Mycobacterium tuberculosis, monocytes enhanced HIV-1 replication and produced a stimulatory 37-kDa CCAAT/enhancer binding protein beta (C/EBPbeta) transcription factor, whereas macrophages suppressed HIV-1 replication and produced an inhibitory 16-kDa C/EBPbeta transcription factor. IFN-beta induced inhibitory 16-kDa C/EBPbeta in macrophages, but had no effect on C/EBPbeta expression in monocytes. Macrophages, but not monocytes, were able to activate IFN-stimulated gene factor-3 (ISGF-3), a transcription factor composed of STAT-1, STAT-2, and IFN regulatory factor (IRF)-9, after infection with M. tuberculosis or stimulation with type I IFN. Macrophages expressed IRF-9 DNA-binding activity, but monocytes did not, and addition of the IRF-9 component reconstituted ISGF-3 in extracts of IFN-treated monocytes. Modulation of IFN responsiveness upon differentiation occurred at least in part through a post-transcriptionally regulated increase in IRF-9 expression. Both monocytes and macrophages maintained IFN responsiveness, activating STAT-1 homodimer formation and transcription of the STAT-1 gene after IFN stimulation. In addition, both monocytes and macrophages were able to activate NF-kappaB upon infection with M. tuberculosis. These results show that induction of ISGF-3, expression of the inhibitory 16-kDa C/EBPbeta, and suppression of HIV-1 replication via a transcriptional mechanism are macrophage-specific responses to infection with M. tuberculosis.

Opening a door to safe abortion: international perspectives on medical abortifacient use.

International experience compels us to revisit how we define and assess the safety and efficacy of medical abortifacients such as misoprostol. In some countries where safe abortion is neither accessible nor legal, even unsupervised, off-protocol use of misoprostol can provide women with a means to safely terminate pregnancy. This is due primarily to misoprostol-induced uterine contractions that cause bleeding, which in turn provides access to existing reasonable quality health services that would otherwise be unavailable. Several studies have suggested that an increase in the underground use of misoprostol in Brazil has already reduced serious complications from unsafe abortion. Thus, the availability of medical abortifacients combined with strengthened postabortion care services can legitimately be considered a public health success in countries in which safe abortion services do not exist and law reform is unlikely.

Activation of the p38 mitogen-activated protein kinase by type I interferons.

The p38 mitogen-activated protein (Map) kinase plays a critical role in the generation of signals in response to stress stimuli, but its role in interferon (IFN) signaling and its potential regulatory role in the activation of Jak-signal transducer and activator of transcription (Stat) pathway are not known. In the present study, we provide evidence that the p38 Map kinase is rapidly phosphorylated and activated during treatment of cells with Type I interferons (IFNalpha and IFNbeta). Furthermore, the Type I IFN-dependent activation of p38 regulates induction of the catalytic domains of MapKap kinase-2 and MapKap kinase-3, strongly suggesting the existence of an IFNalpha signaling cascade activated downstream of the p38 kinase. The engagement of this pathway in interferon signaling plays a critical role in interferon-dependent transcriptional regulation, as evidenced by the fact that inhibition of p38 activation results in abrogation of interferon-dependent gene transcription via interferon-stimulated response elements. Interestingly, inhibition of the kinase activity of the p38 blocks IFNalpha-induced gene transcription without inhibiting DNA binding or tyrosine phosphorylation of Stat proteins, suggesting that the p38 pathway acts in cooperation with the Stat pathway. Thus, the p38 kinase signaling cascade is activated by the Type I interferon receptor and plays a critical role in interferon signaling and interferon-dependent transcriptional regulation.

Fluoroquinolone action against clinical isolates of Mycobacterium tuberculosis: effects of a C-8 methoxyl group on survival in liquid media and in human macrophages.

When the lethal action of a C-8 methoxyl fluoroquinolone against clinical isolates of Mycobacterium tuberculosis in liquid medium was measured, the compound was found to be three to four times more effective (as determined by measuring the 90% lethal dose) than a C-8-H control fluoroquinolone or ciprofloxacin against cells having a wild-type gyrA (gyrase) gene. Against ciprofloxacin-resistant strains, the C-8 methoxyl group enhanced lethality when alanine was replaced by valine at position 90 of the GyrA protein or when aspartic acid 94 was replaced by glycine, histidine, or tyrosine. During infection of a human macrophage model by wild-type Mycobacterium bovis BCG, the C-8 methoxyl group lowered survival 20- to 100-fold compared with the same concentration of a C-8-H fluoroquinolone. The C-8 methoxyl fluoroquinolone was also more effective than ciprofloxacin against a gyrA Asn94 mutant of M. bovis BCG. In an M. tuberculosis-macrophage system the C-8 methoxyl group improved fluoroquinolone action against both quinolone-susceptible and quinolone-resistant clinical isolates. Thus, a C-8 methoxyl group enhances the bactericidal activity of quinolones with N1-cyclopropyl substitutions; these data encourage further refinement of fluoroquinolones as antituberculosis agents.

Type I interferon induces inhibitory 16-kD CCAAT/ enhancer binding protein (C/EBP)beta, repressing the HIV-1 long terminal repeat in macrophages: pulmonary tuberculosis alters C/EBP expression, enhancing HIV-1 replication.

We have previously observed that HIV-1 replication is suppressed in uninflamed lung and increased during tuberculosis. In vitro THP-1 cell-derived macrophages inhibited HIV-1 replication after infection with Mycobacterium tuberculosis. Suppression of HIV-1 replication was associated with inhibition of the HIV-1 long terminal repeat (LTR) and induction of ISGF-3, a type I interferon (IFN)-specific transcription factor. Repression of the HIV-1 LTR required intact CCAAT/enhancer binding protein (C/EBP) sites. THP-1 cell-derived macrophages infected with M. tuberculosis, lipopolysaccharide, or IFN-beta induced the 16-kD inhibitory C/EBPbeta isoform and coincidentally repressed HIV-1 LTR transcription. C/EBPbeta was the predominant C/EBP family member produced in THP-1 macrophages during HIV-1 LTR repression. In vivo, alveolar macrophages from uninflamed lung strongly expressed inhibitory 16-kD C/EBPbeta, but pulmonary tuberculosis abolished inhibitory C/EBPbeta expression and induced a novel C/EBP DNA binding protein. Therefore, in vitro, proinflammatory stimulation produces an IFN response inhibiting viral replication by induction of a C/EBPbeta transcriptional repressor. THP-1 cell-derived macrophages stimulated with type I IFN are similar to alveolar macrophages in the uninflamed lung in vivo. In contrast, the cellular immune response in active pulmonary tuberculosis disrupts this innate immunity, switching C/EBP expression and allowing high level viral replication.

Indications, outcomes, and provider volumes for carotid endarterectomy.

CONTEXT: While trials have demonstrated that carotid endarterectomy is superior to best medical therapy, most recently among asymptomatic patients, uses and outcomes of the procedure in more representative settings have not been established. OBJECTIVES: To profile the use and outcomes of carotid endarterectomy in a representative sample of Ohio's Medicare beneficiaries and to examine the relationships between provider-specific procedural volumes and patient outcomes. DESIGN: Retrospective cohort using Medicare Provider Analysis and Review files supplemented by detailed reviews of medical records on a random sample of patients. SETTING: Ohio hospitals performing carotid endarterectomy. PATIENTS: A random sample of 678 charts of the 4120 non-health maintenance organization Medicare beneficiaries who underwent carotid endarterectomy between July 1, 1993, and June 30, 1994. MAIN OUTCOME MEASURES: Nonfatal stroke or death within 30 days of surgery. RESULTS: The reviewed patients were similar to all eligible patients in sociodemographic characteristics and 30-day mortality rates. Among the 678 patients, indications for surgery were asymptomatic carotid stenosis in 167 (24.6%), transient ischemic attack in 294 (43.4%), completed stroke in 62 (9.1%), and nonspecific symptoms in 155 (22.9%). Thirty-two patients (4.7%) died or suffered nonfatal strokes by 30 days postoperatively. In univariate analyses, rates varied by hospital volume (P=.004) but not surgeons' volume (P=.47), although power to detect this difference was limited. Patients at higher- and lower-volume hospitals had similar indications and distributions of comorbidities. In analyses controlling for indications, comorbid conditions, and surgeon's volume, being operated on in a higher-volume hospital conferred a 71% reduction in risk for 30-day stroke or death (odds ratio, 0.29; 95% confidence interval, 0.12-0.69; P=.006). CONCLUSIONS: Almost half (47.5%) of the carotid endarterectomies among Ohio's Medicare population are performed on persons who are asymptomatic or who have nonspecific symptoms. These results highlight the importance of identifying patients and providers having the most favorable outcome profiles. The higher rate of adverse outcomes observed in lower-volume hospitals deserves further investigation, as it does not appear to be due to differences in patient selection.

Definition of the interferon-alpha receptor-binding domain on the TYK2 kinase.

Interferons and cytokines modulate gene expression via a simple, direct signaling pathway containing receptors, JAK tyrosine kinases, and STAT transcription factors. The interferon-alpha pathway is a model for these cascades. Two receptors, IFNaR1 and IFNaR2, associate exclusively in a constitutive manner with two JAK proteins, TYK2 and JAK1, respectively. Defining the molecular interface between JAK proteins and their receptors is critical to understanding the signaling pathway and may contribute to the development of novel therapeutics. This report defines the IFNaR1 interaction domain on TYK2. In vitro binding studies demonstrate that the amino-terminal half of TYK2, which is approximately 600 amino acids long and contains JAK homology (JH) domains 3-7, comprises the maximal binding domain for IFNaR1. A fragment containing amino acids 171-601 (JH3-6) also binds IFNaR1, but with reduced affinity. Glutathione S-transferase-TYK2 fusion proteins approximating either the JH6 or JH3 domain affinity-precipitate IFNaR1, suggesting that these are major sites of interaction within the larger binding domain. TYK2 amino acids 1-601 act in a dominant manner to inhibit the transcription of an interferon-alpha-dependent reporter gene, presumably by displacing endogenous TYK2 from the receptor. This same fragment inhibits interferon-alpha-dependent tyrosine phosphorylation of TYK2, STAT1, and STAT2.

Maintaining a focus on informed choice.

PIP: Informed choice refers to the process by which a person arrives at a decision about health care. It must be based upon access to, and full understanding of, all necessary information from the client's perspective. Much has been done over the past 20 years to build a strong global policy consensus for informed choice in family planning. That consensus rests upon a commitment to the health and rights of individuals, combined with the knowledge that family planning services do not meet clients' family planning needs when individual choice is not respected. However, despite this consensus, there remains a wide gap between policy objectives and the realities of informed choice at the service delivery level. There were widely published accounts in 1998 of people who have been forced to accept family planning, including sexual sterilization. Two particular reports referred to coercion in China and Peru. Informed choice remains a major concern. Interaction between clients and providers, providing information, informed consent protocols, targets and other evaluation measures, eligibility criteria for receiving services, and provider bias are discussed.^ieng

Convergence of TNFalpha and IFNgamma signalling pathways through synergistic induction of IRF-1/ISGF-2 is mediated by a composite GAS/kappaB promoter element.

The molecular basis for the well known synergistic biological effects of tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) is still poorly understood. This report demonstrates that expression of interferon-regulatory factor 1 (IRF-1), also known as interferon-stimulated-gene factor 2 (ISGF-2), is synergistically induced by these cytokines. The induction is a primary transcriptional response that occurs rapidly without a requirement for new protein synthesis. Synergism is mediated by a novel composite element in the IRF-1 promoter that includes an IFNgamma-activation site (GAS) overlapped by a non-consensus site for nuclear factor kappa B (NFkappaB). These sequences are bound strongly by signal transducer and activator of transcription 1 (STAT-1) and weakly by the p50/p65 heterodimer form of NFkappaB, respectively. However, the binding of STAT-1 and NFkappaB to the GAS/kappaB element in vitro seems to be mutually exclusive and independent. Synergistic induction of IRF-1 is likely to be an important early step in regulatory networks critical to the synergism of TNFalpha and IFNgamma. The GAS/kappaB element may mediate synergistic transcriptional induction of IRF-1 by other pairs of ligands that together activate NFkappaB and STAT family members. Other genes are likely to contain this motif and be regulated similarly.

Kinase-deficient forms of Jak1 and Tyk2 inhibit interferon alpha signaling in a dominant manner.

Signaling by interferon alpha (IFN alpha), an extracellular factor that mediates a number of anti-viral and growth-suppressive effects, requires two members of the Janus family of tyrosine kinases (JAK family): Jak1 and Tyk2. IFN alpha treatment of cells induces the rapid tyrosine phosphorylation of these two kinases, two subunits of the IFN alpha receptor, and two members of the signal transducer and activator of transcription (STAT) family of latent transcription factors. These proteins are believed to be direct substrates of one or both JAKs. Though the requirement for both Jak1 and Tyk2 in the IFN alpha-signaling cascade is well established, the order of activation and the relative contribution of the two kinases has not been elucidated completely. To address these questions, we have employed kinase-deficient mutants of both enzymes. Both mutant kinases suppress transcriptional activation as measured by an IFN alpha-dependent reporter-gene assay. Furthermore, in transient-transfection assays, the kinase-deficient versions of Tyk2 and Jak1 can act independently to inhibit STAT phosphorylation. Thus, kinase-deficient versions of JAK can act in a dominant-negative fashion to suppress IFN alpha signaling. The effects of the overexpressed mutant kinases on the phosphorylation of the kinases themselves, however, are unequal, suggesting that Jak1 functions upstream of Tyk2.