RESUMO
There is a lack of large epidemiological studies focusing on the prevalence of lymphedema. Vital registration data from the United States (US) (1999-2020) and Veneto, Italy (2008-2021) were analyzed. Lymphedema-related deaths were identified using disease-specific ICD-10 codes and served to estimate the burden of disease in the general population. We studied (i) the lymphedema-specific proportionate mortality as a proxy of the disease-specific prevalence, (ii) the prevalence of lymphedema in key patient subgroups, and (iii) age and sex-specific mortality rates. The prevalence of lymphedema increased over the last two decades with marked sex-specific differences: in the US, the estimated prevalence of lymphedema was 2.7 per 10,000 deaths for women and 1.5 per 10,000 deaths for men. In Veneto, the prevalence was 3.0 per 10,000 deaths for women and 1.1 per 10,000 deaths for men. The prevalence of lymphedema was 2- to 20-times in specific subgroups of patients, including those with obesity, skin infections, hypertension, diabetes mellitus, breast/gynecological cancers, and venous thromboembolism. The estimated prevalence of lymphedema is 2- to 3-times higher than previously thought and has been increasing for the past two decades. These results will serve as a reference for future research in this field.
RESUMO
PURPOSE: Diabetes is a growing health problem. The aim of this study was to capture time trends in mortality associated with diabetes. METHODS: The mortality database of the Veneto region (Italy) includes both the underlying causes of death, and all the diseases mentioned in the death certificate. The annual percent change (APC) in age-standardized rates from 2008 to 2017 was computed by the Joinpoint Regression Program. RESULTS: Overall 453,972 deaths (56,074 with mention of diabetes) were observed among subjects aged ≥ 40 years. Mortality rates declined for diabetes as the underlying cause of death and from diabetes-related circulatory diseases. The latter declined especially in females - 4.4 (CI 95% - 5.3/- 3.4), while in males the APC was - 2.8 (CI 95% - 4.0/- 1.6). CONCLUSION: We observed a significant reduction in mortality during the period 2008-2017 in diabetes either as underlying cause of death or when all mentions of diabetes in the death certificate were considered.
Assuntos
Doenças Cardiovasculares , Complicações do Diabetes , Diabetes Mellitus/mortalidade , Mortalidade/tendências , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/mortalidade , Causas de Morte/tendências , Atestado de Óbito , Complicações do Diabetes/diagnóstico , Complicações do Diabetes/mortalidade , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/prevenção & controle , Feminino , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Fatores SexuaisRESUMO
OBJECTIVE: The aim of the study is to assess the impact of the COVID-19 pandemic on causes of mortality through multiple methodological approaches. MATERIALS AND METHODS: The causes of mortality in the Veneto region (Italy) during the first epidemic wave, March-April 2020, were compared with the corresponding months of the previous two years. Both the underlying cause of death (UCOD), and all diseases reported in the death certificate (multiple causes of death) were investigated; a further analysis was carried out through a simulation where the UCOD was selected after substituting ICD-10 codes for COVID with unspecified pneumonia. RESULTS: Overall 10,222 deaths were registered in March-April 2020, corresponding to a 24% increase compared to the previous two years. COVID-19 was mentioned in 1,444 certificates, and selected as the UCOD in 1,207 deaths. Based on the UCOD, the increases in mortality were observed for COVID and related respiratory conditions, diabetes mellitus, hypertensive heart diseases, cerebrovascular diseases, and ill-defined causes. Multiple causes of death and the simulation analysis demonstrated further increases in mortality related to dementia/Alzheimer and chronic lower respiratory diseases. CONCLUSIONS: This first report demonstrates an increase of several causes of death during the pandemic, underlying the need of a continuous surveillance of mortality records through different analytic strategies.
Assuntos
COVID-19/diagnóstico , COVID-19/mortalidade , Atestado de Óbito , COVID-19/complicações , Causas de Morte/tendências , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/mortalidade , Epidemias , Humanos , Itália/epidemiologia , Pneumonia/diagnóstico , Pneumonia/etiologia , Pneumonia/mortalidadeRESUMO
OBJECTIVE: To compare different methods assessing the burden of cardiovascular mortality in diabetes mellitus, which is usually underestimated by standard mortality statistics based on the underlying cause of death. PATIENTS AND METHODS: All residents in the Veneto Region (Italy) aged 30-89 years with co-payment exemption for diabetes in January 2010 (n=185,341) were identified and linked with mortality records (2010-2015). The underlying causes of death, as well as all the diseases mentioned in the death certificate (multiple causes), were extracted. The standardized mortality ratios (SMR) were computed with regional rates as a reference. RESULTS: After grouping diabetes and circulatory diseases as the underlying cause of death, the mortality rates were highly increased, especially among patients aged 30-54 years: SMR 4.24 (95% confidence interval 3.57-5.00) and 9.84 (7.47-12.72) in males and females, respectively. After re-assignment of the underlying cause in deaths from diabetes, the percentage of overall mortality caused by circulatory diseases increased from 33.8% to 41.7%. Based on multiple causes, the risk of death was increased for several cardiovascular diseases, including causes rarely emerging from standard mortality statistics such as atrial fibrillation/flutter. CONCLUSIONS: The re-assignment of the underlying cause and the analyses of the multiple causes of death allowed to estimate the whole burden of mortality associated with cardiovascular diseases.
Assuntos
Doenças Cardiovasculares/mortalidade , Causas de Morte , Efeitos Psicossociais da Doença , Complicações do Diabetes/mortalidade , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND AND AIMS: To investigate the risk of mortality from infections by comparing the underlying causes of death versus the multiple causes of death in known diabetic subjects living in the Veneto region of Northern Italy. METHODS AND RESULTS: A total of 185,341 subjects with diabetes aged 30-89 years were identified in the year 2010, and causes of death were assessed from 2010 to 2015. Standardized Mortality Ratios (SMRs) with 95% confidence intervals (CIs) were computed with regional mortality rates as reference. The underlying causes of death and all the diseases reported in the death certificates were scrutinized. At the end of the follow-up, 36,382 subjects had deceased. We observed an increased risk of death from infection-related causes in subjects with diabetes with a SMR of 1.83 (95% CI, 1.71-1.94). The SMR for death from septicemia was 1.91 (95% CI, 1.76-2.06) and from pneumonia was 1.47 (95% CI, 1.36-1.59). The use of the multiple causes of death approach emphasized the association of infectious diseases with mortality. CONCLUSION: The results of the present study demonstrate an excess mortality due to infection-related diseases in patients with diabetes; more interestingly, by routine mortality analyses, the results show a possible underestimation of the effect of these diseases on mortality.
Assuntos
Doenças Transmissíveis/mortalidade , Diabetes Mellitus/mortalidade , Pneumonia/mortalidade , Sepse/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Causas de Morte , Doenças Transmissíveis/diagnóstico , Diabetes Mellitus/diagnóstico , Feminino , Inquéritos Epidemiológicos , Humanos , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Pneumonia/diagnóstico , Prognóstico , Medição de Risco , Fatores de Risco , Sepse/diagnóstico , Fatores de TempoAssuntos
Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/mortalidade , Enterocolite/epidemiologia , Enterocolite/mortalidade , Hospitalização , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Infecções por Clostridium/microbiologia , Enterocolite/microbiologia , Feminino , Humanos , Lactente , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Análise de Sobrevida , Adulto JovemRESUMO
In this work, several aroma precursors present in Moscato Giallo grape juice were identified and characterized using LC-MS and NMR techniques. A preliminary separation of various fractions was obtained using adsorption on Amberlite(®) XAD resin and HPLC chromatography on a reverse phase column. Subsequently, U-HPLC with mass spectrometry allowed the identification of some compounds corresponding to mono- and disaccharides linked to terpenes. The MS-MS fragmentation step indicated which kind of glycosides, the moiety sequence and sometimes which kind of terpene were present. NMR enabled the correct identification of glycosides and terpene when the fraction analyzed was sufficiently concentrated and with few components. Twelve glycosidically bound terpenes were characterized: (E) and (Z)-furanosyl-linalooloxide-7-O-[α-D-apiofuranosyl-(1â6)-1-ß-D-glucopyranoside], (E)-furanosyl-linalooloxide-7-O-[1-ß-D-glucopyranoside], (Z)-8-hydroxylinalool-8-O-[1-ß-D-glucopyranoside], 1,2-dihydroxylinalool-1-O-[1-ß-D-glucopyranoside], linalool-3-O-[α-L-arabinofuranosyl-(1â6)-1-ß-D-glucopyranoside], linalool-3-O-[α-L-apiofuranosyl-(1â6)-1-ß-D-glucopyranoside], linalool-3-O-[α-L-rhamnopyranosyl-(1â6)-1-ß-D-glucopyranoside], nerol-1-O-α-D-apiofuranosyl-(1â6)-1-ß-D-glucopyranoside, geraniol-1-O-[α-L-arabinofuranosyl-(1â6)-1-ß-D-glucopyranoside], geraniol-1-O-[α-L-rhamnopyranosyl-(1â6)-1-ß-D-glucopyranoside], and a geranic acid disaccharide derivative. It is the first time that this kind of compounds are directly detected and identified in a mixture with these two techniques.
Assuntos
Bebidas/análise , Dissacarídeos/isolamento & purificação , Monossacarídeos/isolamento & purificação , Odorantes/análise , Terpenos/isolamento & purificação , Vitis/química , Vinho/análise , Cromatografia Líquida , Espectroscopia de Ressonância Magnética , Espectrometria de Massas em TandemRESUMO
A series of peptide-peptoid hybrids, containing N-substituted glycines, were synthesized based on the H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH(2) (Har = Homoarginine) as the parent parathyroid hormone (1-11) analog. The compounds were pharmacologically characterized in their agonistic activity at the parathyroid hormone 1 receptor.
Assuntos
Hormônio Paratireóideo/análogos & derivados , Fragmentos de Peptídeos/química , Peptoides/química , Células HEK293 , Humanos , Hormônio Paratireóideo/química , Hormônio Paratireóideo/farmacologia , Fragmentos de Peptídeos/farmacologia , Peptoides/farmacologia , Receptor Tipo 1 de Hormônio Paratireóideo/efeitos dos fármacosRESUMO
A rapid and quantitative (1)H nuclear magnetic resonance (NMR) method was developed to analyze histamine in cheeses. The procedure is simple because the acid extract is analyzed directly, without any need for further filtration, derivatization, or other manipulation. This NMR method was demonstrated to be specific by 2D total correlation spectroscopy (TOCSY) and heteronuclear multiple-quantum coherence (HMQC) experiments and reliable in terms of linearity, accuracy, recovery, repeatability, and limits of detection (LOD). Good precision, with relative standard deviation (RSD) <4%, recovery of 100%, and a range of 0.6-1 mg/kg for the LOD were obtained. The NMR method was successfully applied to different types of cheese, ranging from soft to hard. No interference from free amino acids, proteins, and other natural components was detected. The NMR method could be transferred to other kinds of food.
Assuntos
Queijo/análise , Histamina/análise , Espectroscopia de Ressonância Magnética/métodos , Controle de Qualidade , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: The study aimed to assess prevalence and risk factors for nosocomial infection (NI) in 21 hospitals of the Veneto Region (Italy). METHODS: In May 2003, a one-week-period prevalence study of NI was carried out in 21 hospitals, representing 63% of all hospital beds for acute patients of the Veneto Region. Intensive care units represented 84% of all intensive care beds of the Region. Long term care, neonatal intensive care, burn, psychiatric and dermatology units were excluded. RESULTS: Overall, 6,352 patients were surveyed. The prevalence of NI was 7.6% (range 2.6%-17.7%), while 6.9% of patients (range 2.6%-15.5%) were affected by at least one NI. The prevalence of patients with NI in medical, surgical and intensive care areas was 6.6%, 5.0% and 25.8%, respectively. The sites most frequently affected were the following: urinary tract (28.4%), surgical site (20.3%), blood stream (19.3%), pulmonary and lower respiratory tract (17.6%). At multivariate analysis risk factors independently associated to NI were: Charlson index score >1, severity of underlying disease, exposure to antibiotics, surgical intervention, trauma at admission, presence of central venous catheter >24 h, urinary catheter, intubation, tracheostomy, and duration since admission >15 days. CONCLUSION: The study provided baseline data of NI in the Veneto Region hospitals. It showed that NI are frequent, and display a wide inter-hospital variability of rates. The highest prevalence has been reported in intensive care units. The unusual high frequency of blood stream infections and the relatively lower prevalence rate of surgical site infections highlighted the limits of prevalence studies.
Assuntos
Infecção Hospitalar/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise de Variância , Antibacterianos/uso terapêutico , Criança , Pré-Escolar , Infecção Hospitalar/microbiologia , Enterococcus/isolamento & purificação , Feminino , Unidades Hospitalares , Humanos , Lactente , Itália/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Pseudomonas aeruginosa/isolamento & purificação , Fatores de Risco , Índice de Gravidade de Doença , Staphylococcus aureus/isolamento & purificaçãoRESUMO
Surveillance of surgical site infections (SSIs) with feedback to surgical personnel is pivotal in decisions regarding infection control. Prospective surveillance is time and resource consuming, so we aimed to evaluate a method based on data collected routinely during care delivery. The study was carried out at three acute hospitals in North-eastern Italy, from 1 January 2001 to 31 December 2001. Hospital discharge diagnoses (selected codes from the International Classification of Diseases, 9th Revision--Clinical Modification) and electronic microbiology reports (positive cultures from surgical wounds and drainages) were linked to identify suspected SSIs. A random sample of tracked events was submitted to total chart review in order to confirm the presence of SSIs retrospectively according to Centers for Disease Control and Prevention definitions. Of 865 suspected SSIs, 64.5% were identified from the microbiological database, 27.1% from discharge codes, and 8.4% from both. Four hundred and three admissions were sampled for review; the overall positive predictive value was 72% (95%CI=69-76%). Since inpatient individual antibiotic exposure is not registered in Italy, the combined use of discharge codes and microbiology reports represents the most feasible automated method for surveillance of SSIs developing during hospital stay.
Assuntos
Infecção Hospitalar/epidemiologia , Registro Médico Coordenado , Vigilância de Evento Sentinela , Infecção da Ferida Cirúrgica/epidemiologia , Infecção Hospitalar/microbiologia , Coleta de Dados , Registros Hospitalares , Humanos , Controle de Infecções , Classificação Internacional de Doenças , Itália/epidemiologia , Alta do Paciente , Projetos Piloto , Vigilância da População , Infecção da Ferida Cirúrgica/microbiologia , Ferimentos e Lesões/microbiologiaRESUMO
The N-terminal 1-34 segment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses in bone characteristic of the native intact PTH. Recent studies have demonstrated that N-terminal fragments presenting the principal activating domain such as PTH(1-11) and PTH(1-14) with helicity-enhancing substitutions yield potent analogues with PTH(1-34)-like activity. To further investigate the role of alpha-helicity on biological potency, we designed and synthesized by solid-phase methodology the following hPTH(1-11) analogues substituted at positions 1 and/or 3 by the sterically hindered and helix-promoting C(alpha)-tetrasubstituted alpha-amino acids alpha-amino isobutyric acid (Aib), 1-aminocyclopentane-1-carboxylic acid (Ac(5)c) and 1-aminocyclohexane-1-carboxylic acid (Ac(6)c): Ac(5)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (I); Aib-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (II); Ac(6)c-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (III); Aib-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (IV); Aib-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (V); S-V-Aib-E-I-Q-L-M-H-Q-R-NH(2) (VI), S-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (VII); Ac(5)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (VIII); Ac(6)c-V-S-E-I-Q-L-M-H-Q-R-NH(2) (IX); Ac(5)c-V-Ac(5)c-E-I-Q-L-M-H-Q-R-NH(2) (X); Ac(6)c-V-Ac(6)c-E-I-Q-L-M-H-Q-R-NH(2) (XI). All analogues were biologically evaluated and conformationally characterized in 2,2,2-trifluoroethanol (TFE) solution by circular dichroism (CD). Analogues I-V, which cover the full range of biological activity observed in the present study, were further conformationally characterized in detail by nuclear magnetic resonance (NMR) and computer simulations studies. The results of ligand-stimulated cAMP accumulation experiments indicated that analogues I and II are active, analogues III, VI and VII are very weakly active and analogues IV, V, VIII-XI are inactive. The most potent analogue, I exhibits biological activity 3500-fold higher than that of the native PTH(1-11) and only 15-fold weaker than that of the native sequence hPTH(1-34). Remarkably, the two most potent analogues, I and II, and the very weakly active analogues, VI and VII, exhibit similar helix contents. These results indicate that the presence of a stable N-terminal helical sequence is an important but not sufficient condition for biological activity.
Assuntos
Oligopeptídeos/química , Oligopeptídeos/farmacologia , Hormônio Paratireóideo/análogos & derivados , Hormônio Paratireóideo/química , Sequência de Aminoácidos , Linhagem Celular , Dicroísmo Circular , AMP Cíclico/metabolismo , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Secundária de Proteína , Receptor Tipo 1 de Hormônio Paratireóideo , Receptores de Hormônios Paratireóideos/genética , Receptores de Hormônios Paratireóideos/metabolismoRESUMO
Aib-rich side-chain lactam-bridged oligomers Ac-(Glu-Aib-Aib-Lys)n-Ala-OH with n = 1,2,3 were designed and synthesized as putative models of the 3(10)-helix. The lactam bridge between the side chains of L-Glu and L-Lys in (i)--(i + 3) positions was introduced in order to enhance the structural preference toward the right-handed 3(10)-helix. The conformational properties of the three peptides were studied in trifluoroethanol (TFE) solution by CD, NMR, and computer simulations. The structural information was derived mainly from the analysis of nuclear Overhauser effect spectroscopy spectra. The presence of alpha H(i)-HN(i + 2) and of alpha H(i)-HN(i + 3) connectivities and the absence of alpha H(i)-HN(i + 4) connectivities indicate that these peptides fold into a 3(10)-helix rather than into an alpha-helix. Based on these conformational features, stereospecific assignment of the Aib methyl groups was possible. The results of such experiments and of the subsequent distance geometry and restrained molecular dynamics simulations reveal a marked preference of these peptides for 3(10)-helix. The CD spectra of these peptides indicate that the helix content increases upon chain elongation. The CD spectrum of the trimer is characterized by a negative band at 200 nm and by a weak positive band around 220 nm. The CD spectrum in TFE is different from that observed in aqueous solution in the presence of SDS micelles, reported in our previous work, and from those reported by a different research group for 3(10)-helical peptides. A possible reason for these differences could rest in the presence of different equilibria of the conformer populations of the various peptides in different solvent systems.
Assuntos
Oligopeptídeos/química , Sequência de Aminoácidos , Hidrocarbonetos Aromáticos com Pontes/síntese química , Hidrocarbonetos Aromáticos com Pontes/química , Dicroísmo Circular , Lactamas/síntese química , Lactamas/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/síntese química , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
The N-terminal 1-34 fragment of parathyroid hormone (PTH) elicits the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(18) upon biological function, we synthesized and characterized the following human (h) PTH(1-34) analogues containing beta-amino acid residues: [analogues: see text]. Biological activity and binding affinity of analogue I are one order of magnitude lower than those of the parent compound. In analogue II, both binding affinity and biological activity are partially recovered. Analogues III and V have no binding affinity and very low biological activity. Both bioactivity and binding affinity are partially recovered in analogue IV. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, 2D-nuclear magnetic resonance and molecular dynamics calculations. The results confirmed the presence in all analogues of two helical segments located at the N-terminal and C-terminal sequences. The insertion of beta-amino acid residues around position 18 does not cause appreciable conformational differences in the five analogues. The differences in biological activity and binding affinity among the five analogues cannot be related to structural differences in the membrane mimetic environment reported in this study. Our results stress the importance of the side-chain functionalities in the sequence 17-19 for biological function.
Assuntos
Substituição de Aminoácidos , Hormônio Paratireóideo/química , Fragmentos de Peptídeos/química , Dicroísmo Circular , AMP Cíclico/metabolismo , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/metabolismo , Conformação ProteicaRESUMO
The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.
Assuntos
Ácidos Aminoisobutíricos/química , Hormônio Paratireóideo/química , Fragmentos de Peptídeos/síntese química , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Humanos , Fragmentos de Peptídeos/química , Conformação Proteica , Relação Estrutura-Atividade , Teriparatida/químicaRESUMO
The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1-34), each containing single or double substitutions with beta-amino acid residues around the putative hinge located at position 12: I. [Nle(8,18),beta-Ala(11,12),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); II. [Nle(8,18),beta-Ala(12,13),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); III. [Nle(8,18),beta-Ala(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); IV. [Nle(8,18),beta-hLeu(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); V. [Nle(8,18),beta-Ala(12), Nal(23),Tyr(34)]bPTH-(1-34)NH(2); VI. [Nle(8,18),beta-Ala(13), Nal(23),Tyr(34)]bPTH-(1-34)NH(2) (beta-hLeu = beta-homo-leucine; beta-Ala = beta-alanine; Nal = L-2-naphthyl-alanine; Nle = norleucine). Analogues I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue II, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analogue IV, and completely restored in analogues V and VI. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the alpha-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of beta-Ala dyads at positions 11,12 and 12,13 in analogues I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analogue IV, compared to the structurally similar analogue III, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues V and VI, the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single beta-amino acid residue in positions 11, 12, or 13 in analogues III-VI does not favor a disordered structure in this portion of the sequence.
Assuntos
Substituição de Aminoácidos , Leucina/análogos & derivados , Proteína Relacionada ao Hormônio Paratireóideo , Hormônio Paratireóideo/química , Hormônio Paratireóideo/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Dicroísmo Circular , AMP Cíclico/metabolismo , Humanos , Leucina/química , Leucina/metabolismo , Ligantes , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Hormônio Paratireóideo/síntese química , Fragmentos de Peptídeos/síntese química , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas/síntese química , Prótons , Receptores de Hormônios Paratireóideos/metabolismo , Relação Estrutura-Atividade , Termodinâmica , beta-Alanina/química , beta-Alanina/metabolismoRESUMO
Aib-rich side chain lactam-bridged oligomers with n =1, 2, 3, were designed and synthesized as putative models of the 3(10)-helix. These peptides were conformationally characterized in aqueous solution containing SDS micelles by CD, NMR, and computer simulations. The lactam bridge between the side chains of L-Glu and L-Lys in (i) and (i+3) positions was introduced in order to enhance the conformational preference toward the right-handed 3(10)-helix. The NMR results clearly indicate that there is an increase of 3(10)-helix formation upon chain elongation. In the dimer and trimer (n = 2 and n = 3, respectively, in the structure reported above) the observed NOE connectivities are compatible with the 3(10)-helical arrangement, confirmed by the temperature coefficients of the amide proton resonances which suggest the presence of a hydrogen-bonded structure. The phi and psi dihedral angles of the structures obtained by molecular dynamics calculations are also compatible with the 3(10)-helix. Identification of the hydrogen-bond pattern indicate that C=O(i)- - -HN(i+3) hydrogen bonds, typical of the 3(10)-helical conformation, are highly probable in all low-energy structures. The CD spectra of these Aib-rich lactam-bridged oligopeptides, obtained in the same solvent system used for NMR experiments, provide important insight into the spectroscopic characteristics of the 3(10)-helix.
Assuntos
Lactamas/química , Peptídeos/química , Estrutura Secundária de Proteína , Antibacterianos/química , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Peptídeos/síntese química , Soluções/químicaRESUMO
The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane-embedded G protein-coupled receptor (PTH1 Rc) present on the surface of cells in target tissues such as bone and kidney. This binding occurs in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34) PTH/PTHrP hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH in terms of bioactivity. The sites of incompatibility were identified at positions 5 in PTHrP and 19 in PTH. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two segmental hybrids: PTHrP(1-27)-[Tyr(34)]bPTH(28-34)-NH(2) (hybrid I) and PTHrP(1-18)-[Nal(23), Tyr(34)]bPTH(19-34)-NH(2) (hybrid II). Hybrid I is as active as PTH(1-34)NH(2) and more than two orders of magnitude more active than hybrid II. The conformational properties of the hybrids were studied in water/trifluoroethanol (TFE) mixtures and in aqueous solutions containing dodecylphosphocholine (DPC) micelles by CD, two-dimensional nmr and computer simulations. Upon addition of TFE to the aqueous solution, both hybrids undergo a coil-helix transition. The helix content in 1:1 water/TFE obtained by CD data is about 75% for both hybrids. In the presence of DPC, helix formation is observed at detergent concentrations above critical micellar concentration and the maximum helix content is of approximately 35 and approximately 30% for hybrid I and II, respectively. Combined nmr analysis, distance geometry, and molecular dynamics calculations suggest that, in both solvent systems, the biologically active hybrid I exhibits two flexible sites, centered at residues 12 and 19, connecting helical segments. The flexibility point at position 19 is not present in the poorly active hybrid II. Our findings support the hypothesis, proposed in our previous work, that in bioactive PTH analogues the presence and location of flexibility points between helical segments are essential for enabling them to fold into the bioactive conformation upon interaction with the PTH1 receptor.
Assuntos
Hormônio Paratireóideo/química , Peptídeos/química , Proteínas/química , Proteínas Recombinantes de Fusão/química , Dicroísmo Circular , AMP Cíclico/biossíntese , AMP Cíclico/metabolismo , Humanos , Modelos Moleculares , Proteínas de Neoplasias/química , Ressonância Magnética Nuclear Biomolecular , Proteína Relacionada ao Hormônio Paratireóideo , Peptídeos/síntese química , Estrutura Secundária de Proteína/efeitos dos fármacos , Receptores de Hormônios Paratireóideos/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Solventes/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1-34)PTH/PTHrP segmental hybrid peptides, the N-terminal 1-14 segment of PTHrP is incompatible with the C-terminal 15-34 region of PTH leading to substantial reduction in potency. The sites of incompatibility were identified as positions 5 in PTH and 19 in PTHrP. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two point-mutated PTH/PTHrP 1-34 hybrids in which the arginine residues at positions 19 and 21 of the native sequence of PTHrP have been replaced by valine (hybrid V(21)) and glutamic acid (hybrid E(19)), respectively, taken from the PTH sequence. Hybrid V(21) exhibits both high receptor affinity and biological potency, while hybrid E(19) binds weakly and is poorly active. The conformational properties of the two hybrids were studied in aqueous solution containing dodecylphosphocholine (DPC) micelles and in water/2,2, 2-trifluoroethanol (TFE) mixtures. Upon addition of TFE or DPC micelles to the aqueous solution, both hybrids undergo a coil-helix transition. The maximum helix content in 1 : 1 water/TFE, obtained by CD data for both hybrids, is approximately 80%. In the presence of DPC micelles, the maximum helix content is approximately 40%. The conformational properties of the two hybrids in the micellar system were further investigated by combined 2D-nmr, distance geometry (DG), and molecular dynamics (MD) calculations. The common structural motif, consisting of two helical segments located at N- and C-termini, was observed in both hybrids. However, the biologically potent hybrid V(21) exhibits two flexible sites, centered at residues 12 and 19 and connecting helical segments, while the flexibility sites in the weakly active hybrid E(19) are located at position 11 and in the sequence 20-26. Our findings support the hypothesis that the presence and location of flexibility points between helical segments are essential for enabling the active analogs to fold into the bioactive conformation upon interaction with the receptor.