Wharton's jelly-derived mesenchymal stem cells in the treatment of four patients with alopecia areata.

BACKGROUND: Alopecia areata (AA) is the second most common cause of non-scarring alopecia. Little is known on the etiopathogenesis of AA. It is considered an autoimmune disease, with T lymphocytes and antibodies directed against hair follicle structures. Topical and systemic therapies are used for the treatment of AA, but none of the therapies used to date have a permanent therapeutic effect. OBJECTIVES: To evaluate the efficacy and safety of AA treatment through a single intradermal injection of a suspension of allogeneic MSCs extracted from Wharton's jelly (WJ-MSCs) into the alopecia foci. MATERIAL AND METHODS: The study involved 4 AA patients who underwent experimental therapy with a suspension of WJ-MSCs. The AA intensity was measured using the SALT score. This measure was performed 3 times during treatment: 1st measure (SALT0) prior to treatment; 2nd measure (SALT12) 12 weeks after the treatment; and 3rd measure (SALT24) 24 weeks after the treatment. Furthermore, during each follow-up visit (6, 12, 18, and 24 weeks after the administration of WJ-MSCs) the patient's general condition (physical examination) and local condition were assessed, their mood was evaluated, and a photo of the scalp was taken. RESULTS: Hair regrowth was observed in all patients by an average of 67% at the sites where the cell suspension was administered. In all cases, we observed greater dynamics of hair regrowth in the first 3 months after the treatment, with an average increase of 52.2%, compared to the following 3 months, with an average of 32%. CONCLUSIONS: The results of the applied intradermal injections of an allogeneic WJ-MSC suspension were positive with hair growth observed in all participants and the therapy was found to be safe, with no side effects.

Pack-year cigarette smoking affects the course of palmoplantar pustulosis.

BACKGROUND: Palmoplantar pustulosis (PPP) is a chronic inflammatory disease with poorly understood pathogenesis. The disease has a chronic course with improvements and exacerbations. Due to palmoplantar location, PPP has a severely negative impact on patients' quality of life. OBJECTIVES: To identify demographic and environmental factors, concomitant diseases, medications, and bacterial factors which may affect the course of PPP. MATERIAL AND METHODS: A total of 51 patients suffering from PPP took part in the study. They were classified according to the Palmoplantar Pustulosis Psoriasis Area and Severity Index (ppPASI) into 3 groups due to the severity of the disease. Pack-year of smoking score was established as a quotient of packets smoked every 24 h and the years of being addicted. Diagnosis of metabolic syndrome was based on the IDF criteria from 2009. Chlamydia trachomatis was detected using enzyme-linked immunosorbent assay (ELISA) technique, Staphylococcus aureus by the culture swabs. Contact hypersensitivity was examined with the T.R.U.E. test. RESULTS: Significantly high severity of PPP was observed in patients addicted to smoking with a high pack-year score (p = 0.03). Significantly lower intensity of PPP lesions was observed in patients treated with ibuprofen (p < 0.01). There was no correlation between severity of PPP skin lesions and comorbidities. CONCLUSIONS: Addiction to cigarette smoking and a high pack-year score aggravates the course of PPP. Treatment with ibuprofen can improve the course of the disease.

Infectious diseases of the skin in contact sports.

Although the benefits of practicing sports are unquestionable, it can contribute to the spread of skin diseases. Mechanical trauma, exposure to environmental and infectious agents, and contact with the skin of other athletes increase the chances of getting an infection. In contact sports, skin infections are responsible for up to 20% of lost training and competition time. In the USA, skin infections, with an incidence of 8.5-20.9%, are the 2nd cause (following upper respiratory infections) of all medical consultations among young wrestlers. The high morbidity of skin diseases poses a great challenge for the diagnosis and treatment of skin infections in athletes practicing contact sports, for whom recommendations may differ from those in the general population. In this review paper, we summarize and discuss the management of infectious diseases of the skin in contact sports. The review shows that the most frequent among athletes are bacterial infections, including folliculitis, erysipelas, furuncles and inflammation of the subcutaneous tissue; viral infections caused by herpes simplex virus, human papilloma virus and molluscum contagiosum virus; fungal infections such as tinea; and infestations, including pediculosis and scabies. Preventing the spread of the infection is the 2nd most important aspect of treatment, following pharmacotherapy. This includes avoiding contact with other athletes, protection or removal of lesions, disinfection of common sports equipment, not sharing towels or other personal equipment. We conclude that protecting against infection and transmission of pathogens in sports teams is crucial in avoiding unnecessary morbidity and minimizing disruption to the training and competition schedule.

The Cochrane Library and safety of systemic corticosteroids for acute respiratory conditions in children: an overview of reviews.

BACKGROUND: Acute respiratory conditions are a leading cause of childhood morbidity and mortality. Corticosteroids are effective and established treatments in some acute respiratory infections (e.g. croup) and asthma exacerbations; however, their role is controversial in other conditions owing to inconsistent effectiveness or safety concerns (e.g. bronchiolitis, acute wheeze). OBJECTIVES: To examine clinically relevant short-term safety outcomes related to acute single or recurrent systemic short-term (<2 weeks) corticosteroid use based on systematic reviews of acute respiratory conditions. METHODS: We searched the Cochrane Database of Systematic Reviews in February 2013 for systematic reviews comparing systemic corticosteroids with placebo for children (aged 0-18 years) with acute asthma, preschool wheezing, bronchiolitis, croup, pharyngitis/tonsillitis or pneumonia. We selected the following outcomes a priori: gastrointestinal (GI) bleeding and abdominal pain; behavioural effects (tremor or hyperactivity, jitteriness, irritability or emotional distress); hypertension; serious adverse events, including death, length of stay in hospital; and relapse leading to hospitalization. One reviewer extracted data and another reviewer independently verified data. Results were combined using Peto odds ratios and risk differences (RD) for dichotomous outcomes and mean differences for continuous outcomes. MAIN RESULTS: Seven reviews containing 44 relevant randomized controlled trials were included. Three reviews were on asthma and one each on bronchiolitis, croup, wheeze and pharyngitis/tonsillitis. Six trials (2114 patients) assessed GI bleeding and/or abdominal pain and showed no significant differences between corticosteroids and placebo (1.5% vs. 1.8%, respectively). Various behavioural effects and hypertension/blood pressure were measured in four trials each (838 and 1617 patients, respectively), with no significant differences reported. None of the trials reported deaths in any of the treatment groups. Based on 17 trials (2056 patients), there were significantly fewer admissions at day 1 with corticosteroids (risk differences = -0.11, 95% confidence interval -0.18 to -0.05; Peto odds ratios = 0.63, 95% confidence interval 0.52 to 0.78). Based on 16 trials (1502 patients) corticosteroids resulted in over 8 fewer hours in hospital compared with placebo (mean differences = -8.49 hours, 95% confidence interval -1.76 to -3.23). There were significantly fewer relapses leading to hospitalization (13 trials, 1099 patients) with corticosteroids (Peto odds ratios 0.42, 95% confidence interval 0.23 to 0.76). While differences favouring corticosteroids in hospital-related outcomes were restricted to asthma and/or croup, we did not find any increase in hospital admission at day 1, length of stay or re-hospitalization in the other acute respiratory conditions. AUTHORS' CONCLUSIONS: Practitioners may prescribe systemic corticosteroids in otherwise healthy children when indicated for the management of acute respiratory conditions (i.e. infections or asthma exacerbations) with minimal concern about short-term adverse effects.

Safety of regular formoterol or salmeterol in adults with asthma: an overview of Cochrane reviews.

BACKGROUND: For adults with asthma that is poorly controlled on inhaled corticosteroids (ICS), guidelines suggest adding a long-acting beta2-agonist (LABA). The LABA can be taken together with ICS in a single (combination) inhaler. Improved symptom control can be assessed in the individual; however, the long-term risk of hospital admission or death requires evidence from randomised controlled trials. Clinical trials record these safety outcomes as non-fatal and fatal serious adverse events (SAEs), respectively. OBJECTIVES: To assess the risk of serious adverse events in adults with asthma treated with regular maintenance formoterol or salmeterol compared with placebo, or when randomly assigned in combination with regular ICS, compared with the same dose of ICS. METHODS: We included Cochrane reviews on the safety of regular formoterol and salmeterol from a June 2013 search of the Cochrane Database of Systematic Reviews. We carried out a search for additional trials in September 2013 and incorporated the new data. All reviews were independently assessed for inclusion and for quality (using the AMSTAR tool). We extracted from each review data from trials recruiting adults (participants older than 12 or 18 years of age).We combined the results from reviews on formoterol and salmeterol to assess the safety of twice-daily regular LABA as a class effect, both as monotherapy versus placebo and as combination therapy versus the same dose of ICS.We did not combine the results of direct and indirect comparisons of formoterol and salmeterol, or carry out a network meta-analysis, because of concerns over transitivity assumptions that posed a threat to the validity of indirect comparisons. MAIN RESULTS: We identified six high-quality, up-to-date Cochrane reviews. Of these, four reviews (89 trials with 61,366 adults) related to the safety of regular formoterol or salmeterol as monotherapy or combination therapy. Two reviews assessed safety from trials in which adults were randomly assigned to formoterol versus salmeterol. These included three trials with 1116 participants given monotherapy (all prescribed background ICS) and 10 trials with 8498 adults receiving combination therapy. An additional search for trials in September 2013 identified five new included studies contributing data from 693 adults with asthma treated with combination formoterol/fluticasone in comparison with the same dose of inhaled fluticasone, as well as from 447 adults for whom formoterol monotherapy was compared with placebo.No trials reported separate results in adolescents. Overall, risks of bias for the primary outcomes were assessed as low. Death of any causeNone of the reviews found a significant increase in death of any cause from direct comparisons; however, none of the reviews could exclude the possibility of a two-fold increase in mortality on regular formoterol or salmeterol (as monotherapy vs placebo or as combination therapy versus ICS) in adults with asthma. Pooled mortality results from direct comparisons were as follows: formoterol monotherapy (odds ratio (OR) 4.49, 95% confidence interval (CI) 0.24 to 84.80, 13 trials, N = 4824), salmeterol monotherapy (OR 1.33, 95% CI 0.85 to 2.08, 10 trials, N = 29,128), formoterol combination (OR 3.56, 95% CI 0.79 to 16.03, 25 trials, N = 11,271) and salmeterol combination (OR 0.90, 95% CI 0.31 to 2.6, 35 trials, N = 13,447). In each case, we did not detect heterogeneity, and the quality of evidence was rated as moderate. Absolute differences in mortality were very small, translating into an increase of 7 per 10,000 over 26 weeks on any monotherapy (95% CI 2 less to 23 more) and 3 per 10,000 over 32 weeks on any combination therapy (95% CI 3 less to 17 more).Very few deaths were reported in the combination therapy trials, and combination therapy trial designs were different from those of monotherapy trials. Therefore we could not use indirect evidence to assess whether regular combination therapy was safer than regular monotherapy.Only one death occurred in the monotherapy trials comparing formoterol versus salmeterol, so evidence was insufficient to compare mortality. Non-fatal serious adverse events of any causeDirect evidence showed that non-fatal serious adverse events were increased in adults receiving salmeterol monotherapy (OR 1.14, 95% 1.01 to 1.28, I(2) = 0%,13 trials, N = 30,196) but were not significantly increased in any of the other reviews: formoterol monotherapy (OR 1.26, 95% CI 0.78 to 2.04, I(2) = 15%, 17 trials, N = 5758), formoterol combination (OR 0.99, 95% CI 0.77 to 1.27, I(2) = 0%, 25 trials, N = 11,271) and salmeterol combination (OR 1.15, 95% CI 0.91 to 1.44, I(2) = 0%, 35 trials, N = 13,447). This represents an absolute increase on any monotherapy of 43 per 10,000 over 26 weeks (95% CI 6 more to 85 more) and 16 per 10,000 over 32 weeks (95% CI 22 less to 60 more) on any combination therapy.Direct comparisons of formoterol and salmeterol detected no significant differences between risks of all non-fatal events in adults (as monotherapy or as combination therapy). AUTHORS' CONCLUSIONS: Available evidence from the reviews of randomised trials cannot definitively rule out an increased risk of fatal serious adverse events when regular formoterol or salmeterol was added to an inhaled corticosteroid (as background or as randomly assigned treatment) in adults or adolescents with asthma.An increase in non-fatal serious adverse events of any cause was found with salmeterol monotherapy, and the same increase cannot be ruled out when formoterol or salmeterol was used in combination with an inhaled corticosteroid, although possible increases are small in absolute terms.However, if the addition of formoterol or salmeterol to an inhaled corticosteroid is found to improve symptomatic control, it is safer to give formoterol or salmeterol in the form of a combination inhaler (as recommended by the US Food and Drug Administration (FDA)). This prevents the substitution of LABA for an inhaled corticosteroid if symptom control is improved on LABA.The results of three large ongoing trials in adults and adolescents are awaited; these will provide more information on the safety of combination therapy under less supervised conditions and will report separate results for the adolescents included.

Treatment of acute gastroenteritis in children: an overview of systematic reviews of interventions commonly used in developed countries.

BACKGROUND: Acute gastroenteritis (AGE) is an extremely common paediatric condition, which results in significant morbidity in children and is a financial burden to the society. OBJECTIVE: The purpose of this overview is to critically evaluate the evidence currently available in the Cochrane Database of Systematic Reviews (CDSR) regarding the efficacy and safety of commonly considered treatment options in children with AGE. METHODS: All Cochrane reviews evaluating the following treatments in children with AGE were eligible for inclusion: oral rehydration therapy, anti-emetics and probiotics. We excluded those focusing on the treatment of antibiotic associated or nosocomial diarrhoea, persistent (chronic) diarrhoea and the prevention of gastroenteritis. We focused on the following outcomes that were selected a priori as clinically important: rate of admission to the hospital; length of stay in hospital; rate of return visits; administration of intravenous (IV) therapy owing to failure of oral rehydration therapy; adverse events and dysnatremia. MAIN RESULTS: Children who received oral rehydration therapy had a shorter length of stay in hospital compared with children who received IV therapy [mean difference, MD = -1.20 days (-2.38, -0.02)]; however, the result was no longer significant when an outlying study was removed. Children who received IV therapy were at increased risk of developing phlebitis [risk difference, RD= - 0.02 (-0.04, -0.01)], while paralytic ileus was more common in children receiving ORT [RD = 0.03 (confidence interval, CI 0.01-0.05)]. Children who received oral ondansetron had lower hospital admission rates to the emergency department (ED) and lower rates of IV rehydration during their ED stay compared with children receiving placebo [risk ration, RR = 0.40 (CI 0.19-0.83) and RR = 0.41 (CI 0.29-0.59), respectively]. Children receiving IV ondansetron had lower hospital admission rates to the ED than patients receiving placebo [RR = 0.21 (0.05, 0.93)]. Probiotic use amongst children hospitalized following AGE reduced the mean duration of hospitalization by 1.12 days (CI -1.16, -0.38). CONCLUSIONS: Given that oral rehydration is less invasive than IV rehydration with no evidence of important clinical differences, it is the first choice for rehydration in children with AGE and mild-to-moderate dehydration. As the vast majority of children with AGE do not require IV rehydration, oral ondansetron administration to children with significant vomiting should be performed to reduce the use of IV rehydration and the need for hospital admission. In children deemed too unwell to receive oral rehydration therapy, IV ondansetron administration is an option, as its use is associated with lower hospital admission rates. Although probiotics appear to be an effective option for the treatment of AGE amongst hospitalized children, outpatient data is lacking and more studies are urgently needed to determine the optimal organism, dosing and duration of treatment.

Overview of reviews in child health: evidence synthesis and the knowledge base for a specific population.

BACKGROUND: Overviews of reviews are an evolving form of evidence synthesis. The Cochrane Child Health Field has been producing overviews since 2006, during which time the methods that have been used have changed, both due to the development of guidance within The Cochrane Collaboration and to the decisions made by individual author teams. This paper studies the first 29 overviews published in EBCH. OBJECTIVES: To describe some aspects of the approaches taken in EBCH overviews to producing evidence syntheses relevant to the healthcare needs of children; to highlight the contribution that overviews can make to the knowledge base for treatment for a particular population. METHODS: Data was extracted on: whether the overview included systematic review (SR) data only, or also data from individual trials not present in the included SRs; name(s) of the Cochrane Review Group (CRG) that prepared the included SRs; topics of the overviews as compared to the topics of the included reviews; age-subgroup analyses presented in the overviews. RESULTS: In 23 overviews, all published in 2012, the authors included trial data as well as SR data; two overviews addressed conditions not explicitly addressed by the included reviews; three overviews included pre-specified age-subgroup analyses. DISCUSSION: The aim of clinical relevance has been achieved by means such as: drawing from reviews produced by multiple CRGs; using SR evidence to explore clinically relevant topics that may not match exactly with the topics covered by the SRs; ensuring that the evidence in overviews is as up to date as possible by redoing searches and including trials not incorporated in the included SRs; and, where permitted by the data, using age-subgroup analyses to present the data in a way which matches the stages of childhood development. CONCLUSION: Overview authors are dependent on the nature of the data and methods reported in the included SRs. This suggests a need for further study about how SRs could be conducted in order to facilitate the conduct of overviews.

Safety of regular formoterol or salmeterol in children with asthma: an overview of Cochrane reviews.

BACKGROUND: Two large surveillance studies in adults with asthma have found an increased risk of asthma-related mortality in those who took regular salmeterol as monotherapy in comparison to placebo or regular salbutamol. No similar sized surveillance studies have been carried out in children with asthma, and we remain uncertain about the comparative safety of regular combination therapy with either formoterol or salmeterol in children with asthma. OBJECTIVES: We have used the paediatric trial results from Cochrane systematic reviews to assess the safety of regular formoterol or salmeterol, either as monotherapy or as combination therapy, in children with asthma. METHODS: We included Cochrane reviews relating to the safety of regular formoterol and salmeterol from a search of the Cochrane Database of Systematic Reviews conducted in May 2012, and ran updated searches for each of the reviews. These were independently assessed. All the reviews were assessed for quality using the AMSTAR tool. We extracted the data relating to children from each review and from new trials found in the updated searches (including risks of bias, study characteristics, serious adverse event outcomes, and control arm event rates).The safety of regular formoterol and salmeterol were assessed directly from the paediatric trials in the Cochrane reviews of monotherapy and combination therapy with each product. Then monotherapy was indirectly compared to combination therapy by looking at the differences between the pooled trial results for monotherapy and the pooled results for combination therapy. The comparative safety of formoterol and salmeterol was assessed using direct evidence from trials that randomised children to each treatment; this was combined with the result of an indirect comparison of the combination therapy trials, which represents the difference between the pooled results of each product when randomised against inhaled corticosteroids alone. MAIN RESULTS: We identified six high quality, up to date Cochrane reviews. Four of these related to the safety of regular formoterol or salmeterol (as monotherapy or combination therapy) and these included 19 studies in children. We added data from two recent studies on salmeterol combination therapy in 689 children which were published after the relevant Cochrane review had been completed, making a total of 21 trials on 7474 children (from four to 17 years of age). The two remaining reviews compared the safety of formoterol with salmeterol from trials randomising participants to one or other treatment, but the reviews only included a single trial in children in which there were 156 participants.Only one child died across all the trials, so impact on mortality could not be assessed.We found a statistically significant increase in the odds of suffering a non-fatal serious adverse event of any cause in children on formoterol monotherapy (Peto odds ratio (OR) 2.48; 95% confidence interval (CI) 1.27 to 4.83, I(2) = 0%, 5 trials, N = 1335, high quality) and smaller increases in odds which were not statistically significant for salmeterol monotherapy (Peto OR 1.30; 95% CI 0.82 to 2.05, I(2) = 17%, 5 trials, N = 1333, moderate quality), formoterol combination therapy (Peto OR 1.60; 95% CI 0.80 to 3.28, I(2) = 32%, 7 trials, N = 2788, moderate quality) and salmeterol combination therapy (Peto OR 1.20; 95% CI 0.37 to 2.91, I(2) = 0%, 5 trials, N = 1862, moderate quality).We compared the pooled results of the monotherapy and combination therapy trials. There was no significant difference between the pooled ORs of children with a serious adverse event (SAE) from long-acting beta(2)-agonist beta agonist (LABA) monotherapy (Peto OR 1.60; 95% CI 1.10 to 2.33, 10 trials, N = 2668) and combination trials (Peto OR 1.50; 95% CI 0.82 to 2.75, 12 trials, N = 4,650). However, there were fewer children with an SAE in the regular inhaled corticosteroid (ICS) control group (0.7%) than in the placebo control group (3.6%). As a result, there was an absolute increase of an additional 21 children (95% CI 4 to 45) suffering such an SAE of any cause for every 1000 children treated over six months with either regular formoterol or salmeterol monotherapy, whilst for combination therapy the increased risk was an additional three children (95% CI 1 fewer to 12 more) per 1000 over three months.We only found a single trial in 156 children comparing the safety of regular salmeterol to regular formoterol monotherapy, and even with the additional evidence from indirect comparisons between the combination formoterol and salmeterol trials, the CI around the effect on SAEs is too wide to tell whether there is a difference in the comparative safety of formoterol and salmeterol (OR 1.26; 95% CI 0.37 to 4.32). AUTHORS' CONCLUSIONS: We do not know if regular combination therapy with formoterol or salmeterol in children alters the risk of dying from asthma.Regular combination therapy is likely to be less risky than monotherapy in children with asthma, but we cannot say that combination therapy is risk free. There are probably an additional three children per 1000 who suffer a non-fatal serious adverse event on combination therapy in comparison to ICS over three months. This is currently our best estimate of the risk of using LABA combination therapy in children and has to be balanced against the symptomatic benefit obtained for each child. We await the results of large on-going surveillance studies to further clarify the risks of combination therapy in children and adolescents with asthma.The relative safety of formoterol in comparison to salmeterol remains unclear, even when all currently available direct and indirect trial evidence is combined.

Ensemble fits of restrained peptides' conformational equilibria to NMR data. Dependence on force fields: AMBER/8 ff03 versus ECEPP/3.

Two variants of NMR-based conformational analyses of flexible peptides are compared using two examples meeting the formula Tyr-D-Daa-Phe-Daa-NH2 (Daa=diamino acid): 1 combining D-Dab² (α,γ-diaminobutyryl) with Lys4, and 2 -D-Dap² (α,ß-diaminopropionyl) with Orn4. The ω-amino groups of D-Daa² and Daa4 are coupled with C=O into the urea, restraining 1 and 2 with 16- and 14-membered rings and leading to potent and impotent µ/δ opioid peptides, respectively. To the current task, we took from an earlier work (Filip et al, J. Pept. Sci. 11 (2005) 347-352) the NMR NOE- and J-data in H2O/D2O; and the selection of the ensembles of 1 and 2, 822 and 788 conformational families, respectively, obtained by using the EDMC/ECEPP3 method. Here, we generated ensembles of 1 and 2 using AMBER molecular dynamics in explicit water to eventually selected 686 and 761 conformers for 1 and 2, respectively. We did numbers of fits for both types of the conformational ensembles of 1 and 2 to their NOE- and J-data using a common method i.e. maximum entropy approach (Groth et al, J. Biomol. NMR 15 (1999) 315-330). Both types of the well structurally diversified ensembles fit to quite different equilibria in regressions to common experimental NOE- and J-restraints using maximum entropy principle, which is a disappointing message. Intriguing is startlingly small standard deviation in J-couplings: σ(JNHαH) ≈ 0.01 Hz for LES-MD/AMBER ensemble, contrary to σ(JNHαH) = 0.8 - 1.1 Hz for the EDMC/ECEPP ensemble, over the wide range of entropy, i.e. relatively insensitive to it. A similar feature is not the case when comparing σ(NOE) in both methods. Hence, at minute entropy contributions, it follows that J does or does not transpose "overfitted" into the final σ(J) in the AMBER or ECEPP ensemble, respectively. Could this be an effect of softness of the AMBER flexible-valence force field compared to ECEPP rigid-geometry, and its effect on ensemble sampling? We do not know an answer.

Cyclic enkephalin-deltorphin hybrids containing a carbonyl bridge: structure and opioid activity.

Six hybrid N-ureidoethylamides of octapeptides in which an N-terminal cyclic structure related to enkephalin was elongated by a C-terminal fragment of deltorphin were synthesized on MBHA resin. The synthetic procedure involved deprotection of Boc groups with HCl/dioxane and cleavage of the peptide resin with 45 % TFA in DCM. d-Lys and d-Orn were incorporated in position 2, and Lys, Orn, Dab, or Dap in position 5. The side chains of the dibasic amino function were protected with the Fmoc group. This protection was removed by treatment with 55 % piperidine in DMF, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate. Using various combinations of dibasic amino acids, peptides containing a 17-, 18-, 19- or 20-membered ring structure were obtained. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid activities were observed, depending on the size of the ring. Extension of the enkephalin sequence at the C-terminus by a deltorphin fragment resulted in a change of receptor selectivity in favor of the δ receptor. The conformational propensities of selected peptides were determined using the EDMC method in conjunction with data derived from NMR experiments carried out in water. This approach allowed proper examination of the dynamical behavior of these small peptides. The results were compared with those obtained earlier with corresponding N-(ureidoethyl)pentapeptide amides.

Advances in biological NMR circa WWMR 2010 in Florence.

We summarize the advances and breakthroughs of 'biological' NMR that were presented at the Joint EUROMAR 2010 - 17th ISMAR Conference - aka 'World Wide Magnetic Resonance 2010' in Florence, Italy.

The carboxyl-terminal segment of apolipoprotein A-V undergoes a lipid-induced conformational change.

Apolipoprotein (apo) A-V is a 343-residue, multidomain protein that plays an important role in regulation of plasma triglyceride homeostasis. Primary sequence analysis revealed a unique tetraproline sequence (Pro293-Pro296) near the carboxyl terminus of the protein. A peptide corresponding to the 48-residue segment beyond the tetraproline motif was generated from a recombinant apoA-V precursor wherein Pro295 was replaced by Met. Cyanogen bromide cleavage of the precursor protein, followed by negative affinity chromatography, yielded a purified peptide. Nondenaturing polyacrylamide gel electrophoresis verified that apoA-V(296-343) solubilizes phospholipid vesicles, forming a relatively heterogeneous population of reconstituted high-density lipoprotein with Stokes' diameters >17 nm. At the same time, apoA-V(296-343) failed to bind a spherical lipoprotein substrate in vitro. Far-UV circular dichroism spectroscopy revealed the peptide is unstructured in buffer yet adopts significant alpha-helical secondary structure in the presence of the lipid mimetic solvent trifluoroethanol (TFE; 50% v/v). Heteronuclear multidemensional NMR spectroscopy experiments were conducted with uniformly (15)N- and (15)N/(13)C-labeled peptide in 50% TFE. Peptide backbone assignment and secondary structure prediction using TALOS+ reveal the peptide adopts alpha-helix secondary structure from residues 309 to 334. In TFE, apoA-V(296-343) adopts an extended amphipathic alpha-helix, consistent with a role in lipoprotein binding as a component of full-length apoA-V.

Solution structure of the regulatory domain of human cardiac troponin C in complex with the switch region of cardiac troponin I and W7: the basis of W7 as an inhibitor of cardiac muscle contraction.

The solution structure of Ca(2+)-bound regulatory domain of cardiac troponin C (cNTnC) in complex with the switch region of troponin I (cTnI(147-163)) and the calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfinamide (W7), has been determined by NMR spectroscopy. The structure reveals that the W7 naphthalene ring interacts with the terminal methyl groups of M47, M60, and M81 as well as aliphatic and aromatic side chains of several other residues in the hydrophobic pocket of cNTnC. The H3 ring proton of W7 also contacts the methyl groups of I148 and M153 of cTnI(147-163). The N-(6-aminohexyl) tail interacts primarily with the methyl groups of V64 and M81, which are located on the C- and D-helices of cNTnC. Compared to the structure of the cNTnC*Ca(2+)*W7 complex (Hoffman, R. M. B. and Sykes, B. D. (2009) Biochemistry 48, 5541-5552), the tail of W7 reorients slightly toward the surface of cNTnC while the ring remains in the hydrophobic pocket. The positively charged -NH(3)(+) group from the tail of W7 repels the positively charged R147 of cTnI(147-163). As a result, the N-terminus of the peptide moves away from cNTnC and the helical content of cTnI(147-163) is diminished, when compared to the structure of cNTnC*Ca(2+)*cTnI(147-163) (Li, M. X., Spyracopoulos, L., and Sykes B. D. (1999) Biochemistry 38, 8289-8298). Thus the ternary structure cNTnC*Ca(2+)*W7*cTnI(147-163) reported in this study offers an explanation for the approximately 13-fold affinity reduction of cTnI(147-163) for cNTnC*Ca(2+) in the presence of W7 and provides a structural basis for the inhibitory effect of W7 in cardiac muscle contraction. This generates molecular insight into structural features that are useful for the design of cTnC-specific Ca(2+)-desensitizing drugs.

N-(ureidoethyl)amides of cyclic enkephalin analogs.

Novel N-(ureidoethyl)amides of cyclic enkephalin analogs have been synthesized. The p-nitrophenyl carbamate of 1-Boc-1,2-diaminoethane was coupled with 4-methylbenzhydrylamine (MBHA) resin. The Boc group was removed by treatment with HCl/dioxane, and the peptide chain was assembled using Boc strategy. For deprotection of amino function, HCl/dioxane was used. D-Lys or D-Orn were incorporated in position 2, and the side chains of Lys, Orn, Dab, or Dap in position 5 were protected with Fmoc group. Side chain protection was removed by treatment with 55% piperidine in DMF, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate to form a urea bridge. The peptide was cleaved from the resin by treatment with 45% TFA in DCM. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Divers opioid activities were observed, depending on the size of the ring. In comparison with [Leu(5)]enkephalin, all peptides were more active in the GPI assay (between 125 and 12 times), and some of them were also more potent in the MVD assay. The conformational propensities of each peptide were determined using the EDMC method in conjunction with NMR experiments. This approach allows treating the dynamical behavior of small peptides properly. The results were compared with those obtained previously for corresponding nonsubstituted amides and are in agreement with the biologically active conformation proposed by us earlier.

Conformational studies of vasopressin and mesotocin using NMR spectroscopy and molecular modelling methods. Part II: Studies in the SDS micelle.

There is much evidence to support the hypothesis that lipids play a role in the interaction of peptide hormones with their membrane receptors. This interaction through change of peptide conformation can facilitate the entry of the hormone into the microenvironment of the receptor. In the present study we have examined the interaction of vasopressin and mesotocin with a lipid-sodium dodecylsulfate (SDS) micelle-using 2D nuclear magnetic resonance (NMR) and theoretical methods. Solution structures of two hormones in solution with SDS were established using the nuclear Overhauser effect (NOE) and the (3)J(NHHalpha) couplings. The amino acid sequences of these peptides are: c[C(1)-Y(2)-F(3)-Q(4)-N(5)-C(6)]-P(7)-R(8)-G(9)-NH(2) ([Arg(8)]vasopressin, AVP) and c[C(1)-Y(2)-I(3)-Q(4)-N(5)-C(6)]-P(7)-I(8)-G(9)-NH(2) (MT). Each of the peptides was found to occur as one stable conformation. AVP adopts the cis configuration on the Cys(1)-Tyr(2) peptide bond, a finding not reported so far. The three-dimensional structures of the two peptides studied were determined by a method that consisted of time-averaged molecular dynamics in an explicit SDS micelle with the parm99 force field in AMBER8.0 package. All calculated structures of the studied peptides form beta-turns in their cyclic parts. The C-terminal fragment of MT is bent, whereas that of AVP is extended.

Positioning of the Alzheimer Abeta(1-40) peptide in SDS micelles using NMR and paramagnetic probes.

NMR spectroscopy combined with paramagnetic relaxation agents was used to study the positioning of the 40-residue Alzheimer Amyloid beta-peptide Abeta(1-40) in SDS micelles. 5-Doxyl stearic acid incorporated into the micelle or Mn(2+) ions in the aqueous solvent were used to determine the position of the peptide relative to the micelle geometry. In SDS solvent, the two alpha-helices induced in Abeta(1-40), comprising residues 15-24, and 29-35, respectively, are surrounded by flexible unstructured regions. NMR signals from these unstructured regions are strongly attenuated in the presence of Mn(2+) showing that these regions are positioned mostly outside the micelle. The central helix (residues 15-24) is significantly affected by 5-doxyl stearic acid however somewhat less for residues 16, 20, 22 and 23. This alpha-helix therefore resides in the SDS headgroup region with the face with residues 16, 20, 22 and 23 directed away from the hydrophobic interior of the micelle. The C-terminal helix is protected both from 5-doxyl stearic acid and Mn(2+), and should be buried in the hydrophobic interior of the micelle. The SDS micelles were characterized by diffusion and (15)N-relaxation measurements. Comparison of experimentally determined translational diffusion coefficients for SDS and Abeta(1-40) show that the size of SDS micelle is not significantly changed by interaction with Abeta(1-40).

Ureido group containing cyclic dermorphin(1-7) analogues: synthesis, biology and conformation.

Six cyclic peptides related to dermorphin(1-7) have been synthesized. The synthesis of linear peptides containing diamino acid residues in positions 2 and 4 was carried out on a 4-methylbenzhydrylamine resin, and cyclization was achieved by treatment with bis-(4-nitrophenyl)carbonate to form a urea unit. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse opioid agonist activities were observed, depending on the size of the ring. The results were compared with those obtained earlier for 1-4 dermorphin analogues. The conformations of all six dermorphin analogues were studied. The conformational space of the peptides was examined using the electrostatically driven Monte Carlo method. On the basis of NMR data, an ensemble of conformations was obtained for each peptide. The opioid activity profiles of the compounds are discussed in the light of the structural data.

Conformational solution studies of the SDS micelle-bound 11-28 fragment of two Alzheimer's beta-amyloid variants (E22K and A21G) using CD, NMR, and MD techniques.

The beta-amyloid (Abeta) is the major peptide constituent of neuritic plaques in Alzheimer's disease (AD) and its aggregation is believed to play a central role in the pathogenesis of the disease. Naturally occurring mutations resulting in changes in the Abeta sequence (pos. 21-23) are associated with familial AD-like diseases with extensive cerebrovascular pathology. It was proved that the mutations alter the aggregation ability of Abeta and its neurotoxicity. Among five mutations at positions 21-23 there are two mutations with distinct clinical characteristics and potentially distinct pathogenic mechanism-the Italian (E22K) and the Flemish (A21G) mutations. In our studies we have examined the structures of the 11-28 fragment of the Italian and Flemish Abeta variants. The fragment was chosen because it has been shown to be the most important for amyloid fibril formation. The detailed structure of both variants Abeta(11-28) was determined using CD, 2D NMR, and molecular dynamics techniques under water-SDS micelle conditions. The NMR analysis revealed two distinct sets of proton resonances for the peptides. The studies of both peptides pointed out the existence of well-defined alpha-helical conformation in the Italian mutant, whereas the Flemish was found to be unstructured with the possibility of a bent structure in the central part of the peptide.

Cyclic enkephalin and dermorphin analogues containing a carbonyl bridge.

Four cyclic enkephalin analogues and four cyclic dermorphin analogues have been synthesized. Cyclization of linear peptides containing basic amino acid residues of various side chain length in position 2 and 5 (enkephalin analogues) or 2 and 4 (dermorphin analogues) was achieved by treatment with bis-(4-nitrophenyl) carbonate to form a urea unit. The peptides were tested in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays. Diverse activity was observed, depending on the size of the ring and the location of the urea unit. The conformation of two dermorphin analogues has been studied: one of high activity (IC(50) = 4.15 nM in the GPI assay) and a second of low activity (IC(50) = 6700 nM in the GPI assay). The conformational space of these peptides was examined using the EDMC method. Using data from the NMR spectra, each peptide was described as an ensemble of conformers. Biological activity was discussed in light of the structural data.