Patient Characteristics and General Practitioners' Advice to Stop Statins in Oldest-Old Patients: a Survey Study Across 30 Countries.

BACKGROUND: Statins are widely used to prevent cardiovascular disease (CVD). With advancing age, the risks of statins might outweigh the potential benefits. It is unclear which factors influence general practitioners' (GPs) advice to stop statins in oldest-old patients. OBJECTIVE: To investigate the influence of a history of CVD, statin-related side effects, frailty and short life expectancy, on GPs' advice to stop statins in oldest-old patients. DESIGN: We invited GPs to participate in this case-based survey. GPs were presented with 8 case vignettes describing patients > 80 years using a statin, and asked whether they would advise stopping statin treatment. MAIN MEASURES: Cases varied in history of CVD, statin-related side effects and frailty, with and without shortened life expectancy (< 1 year) in the context of metastatic, non-curable cancer. Odds ratios adjusted for GP characteristics (ORadj) were calculated for GPs' advice to stop. KEY RESULTS: Two thousand two hundred fifty GPs from 30 countries participated (median response rate 36%). Overall, GPs advised stopping statin treatment in 46% (95%CI 45-47) of the case vignettes; with shortened life expectancy, this proportion increased to 90% (95CI% 89-90). Advice to stop was more frequent in case vignettes without CVD compared to those with CVD (ORadj 13.8, 95%CI 12.6-15.1), with side effects compared to without ORadj 1.62 (95%CI 1.5-1.7) and with frailty (ORadj 4.1, 95%CI 3.8-4.4) compared to without. Shortened life expectancy increased advice to stop (ORadj 50.7, 95%CI 45.5-56.4) and was the strongest predictor for GP advice to stop, ranging across countries from 30% (95%CI 19-42) to 98% (95% CI 96-99). CONCLUSIONS: The absence of CVD, the presence of statin-related side effects, and frailty were all independently associated with GPs' advice to stop statins in patients aged > 80 years. Overall, and within all countries, cancer-related short life expectancy was the strongest independent predictor of GPs' advice to stop statins.

Burden of cardiovascular disease across 29 countries and GPs' decision to treat hypertension in oldest-old.

OBJECTIVES: We previously found large variations in general practitioner (GP) hypertension treatment probability in oldest-old (>80 years) between countries. We wanted to explore whether differences in country-specific cardiovascular disease (CVD) burden and life expectancy could explain the differences. DESIGN: This is a survey study using case-vignettes of oldest-old patients with different comorbidities and blood pressure levels. An ecological multilevel model analysis was performed. SETTING: GP respondents from European General Practice Research Network (EGPRN) countries, Brazil and New Zeeland. SUBJECTS: This study included 2543 GPs from 29 countries. MAIN OUTCOME MEASURES: GP treatment probability to start or not start antihypertensive treatment based on responses to case-vignettes; either low (<50% started treatment) or high (≥50% started treatment). CVD burden is defined as ratio of disability-adjusted life years (DALYs) lost due to ischemic heart disease and/or stroke and total DALYs lost per country; life expectancy at age 60 and prevalence of oldest-old per country. RESULTS: Of 1947 GPs (76%) responding to all vignettes, 787 (40%) scored high treatment probability and 1160 (60%) scored low. GPs in high CVD burden countries had higher odds of treatment probability (OR 3.70; 95% confidence interval (CI) 3.00-4.57); in countries with low life expectancy at 60, CVD was associated with high treatment probability (OR 2.18, 95% CI 1.12-4.25); but not in countries with high life expectancy (OR 1.06, 95% CI 0.56-1.98). CONCLUSIONS: GPs' choice to treat/not treat hypertension in oldest-old was explained by differences in country-specific health characteristics. GPs in countries with high CVD burden and low life expectancy at age 60 were most likely to treat hypertension in oldest-old. Key Points • General practitioners (GPs) are in a clinical dilemma when deciding whether (or not) to treat hypertension in the oldest-old (>80 years of age). • In this study including 1947 GPs from 29 countries, we found that a high country-specific cardiovascular disease (CVD) burden (i.e. myocardial infarction and/or stroke) was associated with a higher GP treatment probability in patients aged >80 years. • However, the association was modified by country-specific life expectancy at age 60. While there was a positive association for GPs in countries with a low life expectancy at age 60, there was no association in countries with a high life expectancy at age 60. • These findings help explaining some of the large variation seen in the decision as to whether or not to treat hypertension in the oldest-old.

Bactericidal activity of cyclotides where phosphatidylethanolamine-lipid selectivity determines antimicrobial spectra.

Cyclotides are a family of plant peptides characterized by a cystine knot embedded in a macrocyclic backbone. They bind to and disrupt phospholipid membranes, which explain their lytic activity on cells. In this study, we expose the full antibacterial potency of cyclotides by avoiding its inhibition by rich growth media assay conditions. For that purpose a two-step microdilution assay protocol was developed, using non-growing conditions during initial peptide incubation. A diverse set of cyclotides was tested for antibacterial and antifungal activity, and the results show that most cyclotides are active under these conditions, especially against Gram-negative bacteria. Activity was observed at sub-micromolar concentrations for three of the cyclotides tested, surpassing that of the control peptides LL-37 and melittin. Noteworthy, two anionic cyclotides were active on Pseudomonas aeruginosa at low micromolar concentrations. Broad-spectrum activity was pronounced among cycloviolacin cyclotides, which included activity on Staphylococcus aureus and Candida albicans. The factors influencing their bactericidal spectrum were revealed by correlating antimicrobial activity with membrane permeabilization on various liposome systems and with the physiochemical properties of the cyclotides. Whereas general electrostatic and hydrophobic parameters are more important for broad-spectrum cyclotides; a phospholipid-specific mechanism of membrane permeabilization, through interaction with phosphatidylethanolamine-lipids, is essential for cyclotides active primarily on Gram-negative bacteria.

Variation in GP decisions on antihypertensive treatment in oldest-old and frail individuals across 29 countries.

BACKGROUND: In oldest-old patients (>80), few trials showed efficacy of treating hypertension and they included mostly the healthiest elderly. The resulting lack of knowledge has led to inconsistent guidelines, mainly based on systolic blood pressure (SBP), cardiovascular disease (CVD) but not on frailty despite the high prevalence in oldest-old. This may lead to variation how General Practitioners (GPs) treat hypertension. Our aim was to investigate treatment variation of GPs in oldest-olds across countries and to identify the role of frailty in that decision. METHODS: Using a survey, we compared treatment decisions in cases of oldest-old varying in SBP, CVD, and frailty. GPs were asked if they would start antihypertensive treatment in each case. In 2016, we invited GPs in Europe, Brazil, Israel, and New Zealand. We compared the percentage of cases that would be treated per countries. A logistic mixed-effects model was used to derive odds ratio (OR) for frailty with 95% confidence intervals (CI), adjusted for SBP, CVD, and GP characteristics (sex, location and prevalence of oldest-old per GP office, and years of experience). The mixed-effects model was used to account for the multiple assessments per GP. RESULTS: The 29 countries yielded 2543 participating GPs: 52% were female, 51% located in a city, 71% reported a high prevalence of oldest-old in their offices, 38% and had >20 years of experience. Across countries, considerable variation was found in the decision to start antihypertensive treatment in the oldest-old ranging from 34 to 88%. In 24/29 (83%) countries, frailty was associated with GPs' decision not to start treatment even after adjustment for SBP, CVD, and GP characteristics (OR 0.53, 95%CI 0.48-0.59; ORs per country 0.11-1.78). CONCLUSIONS: Across countries, we found considerable variation in starting antihypertensive medication in oldest-old. The frail oldest-old had an odds ratio of 0.53 of receiving antihypertensive treatment. Future hypertension trials should also include frail patients to acquire evidence on the efficacy of antihypertensive treatment in oldest-old patients with frailty, with the aim to get evidence-based data for clinical decision-making.

Liquid Chromatographic Method for the Determination of Caffeoylquinic Acid Derivates in Hieracium pilosella L.

INTRODUCTION: Hieracium pilosella L. is a medicinal plant encountered in Europe in traditional herbal medicinal products. Caffeoylquinic (ortho-dihydroxycinnamic) acid derivatives are characteristic constituents used as analytical markers in the quality control of the herbal material. Until now, the caffeoylquinic acid derivatives have been assayed using a colorimetric method according to the French Pharmacopoeia. OBJECTIVE: To develop an HPLC-method for quantification of caffeoylquinic acid derivatives in H. pilosella. METHODOLOGY: Samples were prepared by methanol extraction of H. pilosella, dried herb. An HPLC method suitable for analysis was developed and validated. The content of caffeoylquinic acid derivatives was determined and expressed as chlorogenic acid. Individual substances in the samples were identified by analyses of UV-MS/MS spectra and by comparisons with spectra and chromatographic retention times of authentic reference substances. RESULTS: Validation showed that the chromatographic method has good selectivity with no interfering peaks. Sensitivity, linearity, repeatability and accuracy were shown to be adequate. In analyses of several batches of H. pilosella, contents of caffeoylquinic acids ranging from 0.7 to 1.9% were found. Compared to the colorimetric method, this newly developed HPLC method is more specific and results in detection of nominally lower amounts of caffeoylquinic acid derivatives. A new acceptance limit of 1.0% instead of 2.5% caffeoylquinic acid, expressed as chlorogenic acid, for H. pilosella, is proposed when using this HPLC-method. CONCLUSION: A newly developed HPLC method is shown to be appropriate for quantitative determination of caffeoylquinic acid derivatives in H. pilosella. Copyright © 2017 John Wiley & Sons, Ltd.

Chemistry and biology of cyclotides: circular plant peptides outside the box.

Cyclotides stand out as the largest family of circular proteins of plant origin hitherto known, with more than 280 sequences isolated at peptide level and many more predicted from gene sequences. Their unusual stability resulting from the signature cyclic cystine knot (CCK) motif has triggered a broad interest in these molecules for potential therapeutic and agricultural applications. Since the time of the first cyclotide discovery, our laboratory in Uppsala has been engaged in cyclotide discovery as well as the development of protocols to isolate and characterize these seamless peptides. We have also developed methods to chemically synthesize cyclotides by Fmoc-SPPS, which are useful in protein grafting applications. In this review, experience in cyclotide research over two decades and the recent literature related to their structures, synthesis, and folding as well the recent proof-of-concept findings on their use as "epitope" stabilizing scaffolds are summarized.

Management of chest pain: a prospective study from Norwegian out-of-hours primary care.

BACKGROUND: Chest pain is a common diagnostic challenge in primary care and diagnostic measures are often aimed at confirming or ruling out acute ischaemic heart disease. The aim of this study was to investigate management of patients with chest pain out-of-hours, including the use of ECG and laboratory tests, assessment of severity of illness, and the physicians' decisions on treatment and admittance to hospital. METHODS: Data were registered prospectively from four Norwegian casualty clinics. Data from structured telephone interviews with 100 physicians shortly after a consultation with a patient presenting at the casualty clinic with "chest pain" were analysed. RESULTS: A total of 832 patients with chest pain were registered. The first 100 patients (corresponding doctor-patient pairs) were included in the study according to the predefined inclusion criteria. Median age of included patients was 46 years, men constituted 58%. An ECG was taken in 92 of the patients. Of the 24 patients categorised to acute level of response, 15 had a NACA-score indicating a potentially or definitely life-threatening medical situation. 50 of the patients were admitted to a hospital for further management, of which 43 were thought to have ischaemic heart disease. Musculoskeletal pain was the second most common cause of pain (n = 22). Otherwise the patients were thought to have a variety of conditions, most of them managed at a primary care level. CONCLUSIONS: Patients with chest pain presenting at out-of-hours services in Norway are investigated for acute heart disease, but less than half are admitted to hospital for probable acute coronary syndrome, and only a minority is given emergency treatment for acute coronary syndrome. A wide variety of other diagnoses are suggested by the doctors for patients presenting with chest pain. Deciding the appropriate level of response for such patients is a difficult task, and both over- and under-triage probably occur in out-of-hours primary care.

Chest pain out-of-hours - an interview study of primary care physicians' diagnostic approach, tolerance of risk and attitudes to hospital admission.

BACKGROUND: Acute chest pain constitutes a considerable diagnostic challenge outside hospitals. This will often lead to uncertainty in choosing the right management, and the physicians' approach may be influenced by their knowledge of diagnostic measures and their tolerance of risk. The aim of this study was to investigate primary care physicians' diagnostic approach, tolerance of risk and attitudes to hospital admission in patients with acute chest pain out-of-hours in Norwegian primary care. METHODS: Data were registered prospectively from four Norwegian casualty clinics. Data from structured telephone interviews with 100 physicians shortly after a consultation with a patient presenting at the casualty clinic with "chest pain" were analysed. Tolerance of risk was measured by the Pearson Risk Scale and the Tolerance of Risk Scale, the latter developed for this study. RESULTS: "Patient history and symptoms" was considered the most important, and "negative ECG" and "effect of sublingual nitroglycerine" the least important aspects in the diagnostic approach. There were no significant differences in length of experience or gender when testing "risk avoiders" against the rest. Almost all physicians felt that their risk assessment out-of-hours was reasonably good, and felt reasonably safe, but only 50% agreed with the statement "I don't worry about my decisions after I've made them". Concerning chest pain patients only, 51% of the physicians were worried about complaints being made about them, 75% agreed that admitting someone to hospital put patients in danger of being "over-tested", and 51% were more likely to admit the patient if the patient herself wanted to be admitted. CONCLUSIONS: Physicians working out-of-hours showed considerable differences in their diagnostic approach, and not all physicians diagnose patients with chest pain according to current guidelines and evidence. Continuous medical education must focus on the diagnostic approach in patients with chest pain in primary care and empowerment of physicians through training and emphasis on risk assessment and "tolerance of risk".

Circular proteins from plants and fungi.

Circular proteins, defined as head-to-tail cyclized polypeptides originating from ribosomal synthesis, represent a novel class of natural products attracting increasing interest. From a scientific point of view, these compounds raise questions of where and why they occur in nature and how they are formed. From a rational point of view, these proteins and their structural concept may be exploited for crop protection and novel pharmaceuticals. Here, we review the current knowledge of three protein families: cyclotides and circular sunflower trypsin inhibitors from the kingdom of plants and the Amanita toxins from fungi. A particular emphasis is placed on their biological origin, structure, and activity. In addition, the opportunity for discovery of novel circular proteins and recent insights into their mechanism of action are discussed.

Cyclotide-membrane interactions: defining factors of membrane binding, depletion and disruption.

The cyclotide family of plant-derived peptides is defined by a cyclic backbone and three disulfide bonds locked into a cyclic cystine knot. They display a diverse range of biological activities, many of which have been linked to an ability to target biological membranes. In the current work, we show that membrane binding and disrupting properties of prototypic cyclotides are dependent on lipid composition, using neutral (zwitterionic) membranes with or without cholesterol and/or anionic lipids. Cycloviolacin O2 (cyO2) caused potent membrane disruption, and showed selectivity towards anionic membranes, whereas kalata B1 and kalata B2 cyclotides were significantly less lytic towards all tested model membranes. To investigate the role of the charged amino acids of cyO2 in the membrane selectivity, these were neutralized using chemical modifications. In contrast to previous studies on the cytotoxic and antimicrobial effects of these derivatives, the Glu6 methyl ester of cyO2 was more potent than the native peptide. However, using membranes of Escherichia coli lipids gave the opposite result: the activity of the native peptide increased 50-fold. By using a combination of ellipsometry and LC-MS, we demonstrated that this unusual membrane specificity is due to native cyO2 extracting preferentially phosphatidylethanolamine-lipids from the membrane, i.e., PE-C16:0/cyC17:0 and PE-C16:0/C18:1.

Genotoxicity and cellular uptake of cyclotides: evidence for multiple modes of action.

Cyclotides are a family of ultra-stable, head-to-tail cyclic mini-proteins from plants, with each member comprising about 30 amino acid residues. Their stability derives from the unique structural topology where the cyclic backbone and two disulfide bonds make up an embedded ring, which is knotted by a third disulfide bond. The cyclotides find potential applications in the pharmaceutical industry as stable peptide-based scaffolds for unstable drugs, and also as medicinal agents, due to the wide range of their inherent pharmacological activities. However, there is a lack of fundamental toxicological studies on this type of compound. The current study determined the possible DNA-damaging effects of three cyclotides, i.e., cycloviolacin O2, vaby D, and kalata B1, in human lymphoma cells by use of the alkaline version of the comet assay. The three cyclotides induced massive DNA fragmentation at lethal concentrations. At a sub-lethal concentration, cycloviolacin O2 and vaby D gave a bell-shaped dose-response curve for their DNA-damaging effect. Kalata B1 caused no significant DNA damage at sub-cytotoxic concentrations. Single-cell micro-autoradiography was carried out on tritium-labeled cycloviolacin O2 in order to understand the mechanism behind the dose-response curve. The results revealed that the peptide is taken up into the cell, both at cytotoxic and at low concentrations. Most biological effects of the cyclotides have been taken to follow from the disruption of cell membranes, but even if the intracellular mechanisms and targets still remain unknown, the current study has unequivocally demonstrated that cyclotides also must have other dose-dependent modes of action.

Cytotoxic potency of small macrocyclic knot proteins: structure-activity and mechanistic studies of native and chemically modified cyclotides.

The cyclotides are a family of circular and knotted proteins of natural origin with extreme enzymatic and thermal stability. They have a wide range of biological activities that make them promising tools for pharmaceutical and crop-protection applications. The cyclotides are divided into two subfamilies depending on the presence (Möbius) or absence (bracelet) of a cis-Pro peptide bond. In the current work we report a series of experiments to give further insight into the structure-activity relationship of cyclotides in general, and the differences between subfamilies and the role of their hydrophobic surface in particular. Selective chemical modifications of Glu, Arg, Lys and Trp residues was tested for cytotoxic activity: derivatives in which the Trp residue was modified showed low effect, demonstrating the existence of a connection between hydrophobicity and activity. However, over the full set of cyclotides examined, there was no strong correlation between the cytotoxic activity and their hydrophobicity. Instead, it seems more like that the distribution of charged and hydrophobic residues determines the ultimate degree of potency. Furthermore, we found that while the Glu residue is very important in maintaining the activity of the bracelet cyclotide cycloviolacin O2, it is much less important in the Möbius cyclotides. Despite these differences between cyclotide subfamilies, a systematic test of mixtures of cyclotides revealed that they act in an additive way.

Cyclotides from an extreme habitat: characterization of cyclic peptides from Viola abyssinica of the Ethiopian highlands.

As part of ongoing explorations of the structural diversity of cyclotides, the cyclotide content of a native violet of the East African highlands, Viola abyssinica (which grows at altitudes up to 3400 m), was studied. Six new cyclotides, vaby A-E (1-5) and varv E (6), were isolated and characterized by employing HPLC and MS techniques and quantitative amino acid analysis. Cyclotides 1-5 were found to have new sequences, and 1-3 have a further novel feature in their sequences, an alanine moiety in loop 2. Two of the cyclotides (1 and 4) also exhibited cytotoxic properties in a flourometric microculture cytotoxicity assay. The findings corroborate the hypothesis that investigating the cyclotide contents of violets growing in diverse environments is a promising approach for extending our knowledge of both the structural and biological diversity of cyclotides.

Two brominated cyclic dipeptides released by the coldwater marine sponge Geodia barretti act in synergy as chemical defense.

The current work shows that two structurally similar cyclodipeptides, barettin (1) and 8,9-dihydrobarettin (2), produced by the coldwater marine sponge Geodia barretti Bowerbank act in synergy to deter larvae of surface settlers and may also be involved in defense against grazers. Previously, 1 and 2 were demonstrated to bind specifically to serotonergic 5-HT receptors. It may be suggested that chemical defense in G. barretti involves a synergistic action where one of the molecular targets is a 5-HT receptor. A mixture of 1 and 2 lowered the EC(50) of larval settlement as compared to the calculated theoretical additive effect of the two compounds. Moreover, an in situ sampling at 120 m depth using a remotely operated vehicle revealed that the sponge releases these two compounds to the ambient water. Thus, it is suggested that the synergistic action of 1 and 2 may benefit the sponge by reducing the expenditure of continuous production and release of its chemical defense substances. Furthermore, a synergistic action between structurally closely related compounds produced by the same bioenzymatic machinery ought to be the most energy effective for the organism and, thus, is more common than synergy between structurally indistinct compounds.

Antimicrobial and antineoplastic activities of agelasine analogs modified in the purine 2-position.

Agelasines are 7,9-dialkylpurinium salts found in marine sponges (Agelas sp.), which display a variety of antimicrobial and cytotoxic effects. We have synthesized simplified agelasine analogs modified in the purine 2-position and examined their antimicrobial and anticancer activities. The compounds were screened against Staphylococcus aureus, Escherichia coli, Mycobacterium tuberculosis, Candida krusei, and Candida albicans, protozoa causing tropical diseases (Plasmodium falciparum, Leishmania infantum, Trypanosoma cruzi, and Trypanosoma brucei), a panel of human cancer cell lines (U-937 GTB, RPMI 8226/s, CEM/s, and ACHN) as well as VERO and/or MRC-5 cells. The results indicate that the introduction of a methyl group in the purine 2-position is beneficial for antimycobacterial and antiprotozoal activity, and that amino groups may enhance activity against several cancer cell lines.

Acute chest pain - a prospective population based study of contacts to Norwegian emergency medical communication centres.

BACKGROUND: Acute chest pain is a frequently occurring symptom in patients with medical emergencies and imposes potentially life threatening situations outside hospitals. Little is known about the epidemiology of patients with acute chest pain in a primary care setting in Norway, and we aimed to obtain more representative data on such patients using data from emergency medical communication centres (EMCCs). METHODS: Data were collected prospectively during three months in 2007 from three EMCCs, covering 816 000 inhabitants. The EMCCs gathered information on every situation that was triaged as a red response (defined as an "acute" response, with the highest priority), according to the Norwegian Index of Medical Emergencies. Records from ambulances and primary care doctors were subsequently collected. International Classification of Primary Care - 2 symptom codes and The National Committee on Aeronautics (NACA) System scores were assigned retrospectively. Only chest pain patients were included in the study. RESULTS: 5 180 patients were involved in red response situations, of which 21% had chest pain. Estimated rate was 5.4 chest pain cases per 1000 inhabitants per year. NACA-scores indicated that 26% of the patients were in a life-threatening medical situation. Median prehospital response time was 13 minutes; an ambulance reached the patient in less than 10 minutes in 30% of the cases. Seventy-six per cent of the patients with chest pain were admitted to a hospital for further investigation, 14% received final treatment at a casualty clinic, while 10% had no further investigation by a doctor ("left at the scene"). CONCLUSIONS: The majority of patients with acute chest pain were admitted to a hospital for further investigation, but only a quarter of the patients were assessed prehospitally to have a severe illness. This sheds light on the challenges for the EMCCs in deciding the appropriate level of response in patients with acute chest pain. Overtriage is to some extent both expected and desirable to intercept all patients in need of immediate help, but it is also well known that overtriage is resource demanding. Further research is needed to elucidate the challenges in the diagnosis and management of chest pain outside hospitals.

The cyclotide cycloviolacin O2 from Viola odorata has potent bactericidal activity against Gram-negative bacteria.

OBJECTIVES: To determine the antibacterial activity of small cyclic plant proteins, i.e. cyclotides, and the importance of the surface exposed charged residues for activity. METHODS: Prototypic cyclotides, including the Möbius kalata B1 and the bracelet cycloviolacin O2 (cyO2), were isolated using reversed-phase HPLC. Initial activity screenings were conducted using radial diffusion assays (RDAs) and MIC assays with Salmonella enterica serovar Typhimurium LT2, Escherichia coli and Staphylococcus aureus as test strains. For the most active peptide, cyO2, time-kill kinetics was determined in sodium phosphate buffer (containing 0.03% trypticase soy broth) against several Gram-negative and Gram-positive bacterial species. Charged residues in cyO2 were chemically modified and activity was determined in time-kill assays. RESULTS: CyO2 was the most active cyclotide and efficiently inhibited the growth of S. enterica serovar Typhimurium LT2 and E. coli in RDAs and MIC assays, while the other peptides were less active. In time-kill assays, cyO2 also had bactericidal activity against the Gram-negative species Klebsiella pneumoniae and Pseudomonas aeruginosa. In contrast, none of the cyclotides had high activity against S. aureus. Chemical masking of the charged Glu and Lys residues in cyO2 caused a near total loss of activity against Salmonella, while masking Arg caused a less pronounced activity reduction. CONCLUSIONS: CyO2 is a cyclotide with potent activity against Gram-negative bacteria. The charged residues in cyO2 are all required for optimum antibacterial activity. In combination with its previously demonstrated cytotoxic activity against cancer cells and the general stability of cyclotides, cyO2 provides a promising scaffold for future drug design.

Isolation, characterization, and bioactivity of cyclotides from the Micronesian plant Psychotria leptothyrsa.

Cyclotides, the largest known family of head-to-tail cyclic peptides, have approximately 30 amino acid residues with a complex structure containing a circular peptide backbone and a cystine knot. They are found in plants from the Violaceae and Rubiaceae families and are speculated to function in plant protection. In addition to their insecticidal properties, cyclotides display cytotoxic, anti-HIV, antimicrobial, and inhibition of neurotensin binding activities. Although cyclotides are present in all violaceous species hitherto screened, their distribution and expression in Rubiaceae are not fully understood. In this study, we show that Psychotria leptothyrsa var. longicarpa (Rubiaceae) contains a suite of different cyclotides. The cyclotide fractions were isolated by RP-HPLC, and sequences of six new peptides, named psyles A-F, were determined by MS/MS sequencing. One of these, psyle C, is the first rubiaceous linear variant known. Psyles A, C, and E were analyzed in a fluorometric microculture assay to determine cytotoxicity toward the human lymphoma cell line U937-GTB. The IC(50) values of psyles A, C, and E were 26, 3.50, and 0.76 muM, respectively. This study expands the number of known rubiaceous cyclotides and shows that the linear cyclotide maintains cytotoxicity.