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Background: Motor vehicle crash (MVC) is a major public health problem worldwide and contributes to a large burden of death, disability, and economic loss., Objective: To identify the predictors of hospital readmission in victims of MVC within 1 year after discharge., Methods: A prospective cohort study conducted with individuals who suffered MVC admitted to a regional hospital and who were followed up for 12 months after discharge. Predictors of hospital readmission were verified by means of Poisson regression models with robust variance, using a hierarchical conceptual model., Results: Of the 241 patients followed up, 200 were contacted and comprised the population of this study. Of these, 50 (25.0%) reported hospital readmission during the 12-month period after discharge. It was evidenced that being male (relative risk [RR] = 0.58; 95% CI [0.36, 0.95], p = .033) was a protective factor, whereas occurrences of greater severity (RR = 1.77; 95% CI [1.03, 3.02], p = .036), not receiving pre-hospital care (RR = 2.14; 95% CI [1.24, 3.69], p = .006), the occurrence of postdischarge infection (RR = 2.14; 95% CI [1.37, 3.36], p = .001), and having access to rehabilitation treatment (RR = 1.64; 95% CI [1.03, 2.62], p≤ .001) are configured as risk factors for hospital readmission in individuals who have suffered these events., Conclusion: It was found that gender, trauma severity, pre-hospital care, postdischarge infection, and rehabilitation treatment variables predict hospital readmission in MVC victims within 1 year after discharge., Competing Interests: The authors have no conflicts of interest or funding to declare., (Copyright © 2023 Society of Trauma Nurses.)

The study aimed to evaluate the work by the Expanded Family Health and Basic Healthcare Centers (NASF-AB), from the users' perspective, based on the attributes of comprehensive primary healthcare (PHC). This was a qualitative multiple case study conducted in six municipalities (counties) in the state of Bahia, Brazil. The theoretical and methodological frame of reference was an evaluative matrix based on the following attributes: access, longitudinality, and comprehensiveness. The matrix also considered the assumption of Inter-Consultation Support and the guidelines of the Family Health Strategy. Data and information were obtained from 44 interviews with users of the NASF-AB and observation of the centers' activities and routine work in the health units. The study showed that users have limited access to the NASF-ABs' activities. The availability of clinical care was insufficient, access was facilitated by home visits, and the acceptability was jeopardized by frustrations from unmet expectations. Longitudinal care has not been prioritized in the centers' work, with limited involvement by supporters in developing continuing care. Group activities showed the potential for forming bonds. In the attribute of comprehensiveness, the presence of NASF-ABs helped increase activities and case-resolution capacity, but the linkage with the healthcare network was negligible. In conclusion, the work by the NASF-AB displayed limitations for the development of comprehensive PHC. The results also suggest potentialities with the capacity to strengthen primary care that have not been fully explored.

Adipose stromal cells are promising tools for clinical applications in regeneration therapies, due to their ease of isolation from tissue and its high yield; however, their ability to transdifferentiate into neural phenotypes is still a matter of controversy. Here, we show that combined chemical and neurotrophin stimulation resulted in neuron-like morphology and regulated expression and activity of several genes involved in neurogenesis and neurotransmission as well as ion currents mediated by NMDA and GABA receptors. Among them, expression patterns of genes coding for kinin-B1 and B2, α7 nicotinic, M1, M3 and M4 muscarinic acetylcholine, glutamatergic (AMPA2 and mGlu2), purinergic P2Y1 and P2Y4 and GABAergic (GABA-A, β3-subunit) receptors and neuronal nitric oxide synthase were up-regulated compared to levels of undifferentiated cells. Simultaneously, expression levels of P2X1, P2X4, P2X7 and P2Y6 purinergic and M5 muscarinic acetylcholine receptors were down-regulated. Agonist-induced activity levels of the studied receptor classes also augmented during neuronal transdifferentiation. Transdifferentiated cells expressed high levels of neuronal β3-tubulin, NF-H, NeuN and MAP-2 proteins as well as increased ASCL1, MYT1 and POU3F2 gene expression known to drive neuronal fate determination. The presented work contributes to a better understanding of transdifferentiation induced by neurotrophins for a prospective broad spectrum of medical applications.

Background: Scorpion accidents have gained great visibility around the world because of the high frequency and severity with which they occur, and have become a global medical-sanitary problem., Objective: The aim of this study was to describe the sociodemographic, clinical and epidemiological profile of scorpionism in the municipality of Jequié, Bahia, Brazil, from 2007 to 2015., Design and Setting: Retrospective epidemiological study in the municipality of Jequié, Bahia, Brazil., Methods: This study was based on data collected from the epidemiological investigation notification forms of the injury information system., Results: There was an increase in the coefficient of incidence of scorpion accidents in Jequié from 23.4/100,000 in 2007 to 413.6/100,000 in 2015. There were 3565 cases: 54.9% were female, 58.8% were aged 20-59 years, 63.5% had brown skin color and 48.6% had incomplete primary education. Most accidents occurred in urban areas (93.1%). Homes were the main place of occurrence (84.5%) and upper limbs were the commonest sting sites (53.0%). Regarding clinical aspects, 66.4% of the cases received hospital assistance within one hour after the bite, 84.1% presented mild severity, 97.1% had local manifestations and 10.2% had systemic symptoms. Serum therapy was administered in 17.3% of the cases, and 99.9% evolved to cure., Conclusion: There was an increase in the incidence of scorpion accidents in the municipality, which demonstrates the need for investment in actions that reduce the morbidity and mortality caused by these accidents, such as educational campaigns and improvements in socioeconomic and health conditions.

Objective: The objectives of this study were to discover associations between anemia and functional performance indicators, and to identify the best functional performance test to screen for anemia in the elderly., Methods: A cross-sectional study analyzed data from 109 elderly residents in the community. For statistical analysis, the Logistic Regression and the Receiver Operating Characteristic (ROC) curve was used., Results: The prevalence of anemia in the elderly was 13.9%. There was an inverse association between handgrip strength (HGS) and anemia in women; the HGS test was an effective screening tool for anemia in the elderly., Conclusion: The HGS test can be used to screen for anemia in the elderly women., (© 2019 Wiley Periodicals, Inc.)

Background: Detection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells., Materials and Methods: The human adenocarcinoma cell line A549 was used for selection in seven cell SELEX cycles. We used human blood leukocytes for negative selection, and lung stem cell protein marker CD90 antibody binding A549 cells for positive selection., Results: The obtained oligonucleotide sequences after the seventh SELEX cycle were subjected to in silico selection analysis based on three independent types of bioinformatics approaches, selecting two closely related aptamer candidates in terms of consensus sequences, structural motifs, binding affinity (Kd) and stability (ΔG). We selected and identified the aptamer A155_18 with very good binding characteristics to A459 cells, selected for CD90 antibody binding. The calculated phylogenetic tree showed that aptamers A155_18 and the known A549 cell aptamer S6 have a close structural relationship. MEME sequence analysis showed that they share two unique motifs, not present in other sequences., Conclusions: The novel aptamer A155_18 has strong binding affinity for A549 lung carcinoma cell line subpopulation that is expressing stem cell marker CD90, indicating a possible stemness, characteristic for the A459 line, or a subpopulation present within this cell line. This aptamer can be applied as diagnostic tool, identifying NSLC circulating cells.

Studies have confirmed that bone marrow-derived mesenchymal stem cells (MSCs) can be used for treatment of several nervous system diseases. However, isolation of bone marrow-derived MSCs (BMSCs) is an invasive and painful process and the yield is very low. Therefore, there is a need to search for other alterative stem cell sources. Adipose-derived MSCs (ADSCs) have phenotypic and gene expression profiles similar to those of BMSCs. The production of ADSCs is greater than that of BMSCs, and ADSCs proliferate faster than BMSCs. To compare the effects of venous grafts containing BMSCs or ADSCs on sciatic nerve injury, in this study, rats were randomly divided into four groups: sham (only sciatic nerve exposed), Matrigel (MG; sciatic nerve injury + intravenous transplantation of MG vehicle), ADSCs (sciatic nerve injury + intravenous MG containing ADSCs), and BMSCs (sciatic nerve injury + intravenous MG containing BMSCs) groups. Sciatic functional index was calculated to evaluate the function of injured sciatic nerve. Morphologic characteristics of nerves distal to the lesion were observed by toluidine blue staining. Spinal motor neurons labeled with Fluoro-Gold were quantitatively assessed. Compared with sham-operated rats, sciatic functional index was lower, the density of small-diameter fibers was significantly increased, and the number of motor neurons significantly decreased in rats with sciatic nerve injury. Neither ADSCs nor BMSCs significantly improved the sciatic nerve function of rats with sciatic nerve injury, increased fiber density, fiber diameters, axonal diameters, myelin sheath thickness, and G ratios (axonal diameter/fiber diameter ratios) in the sciatic nerve distal to the lesion site. There was no significant difference in the number of spinal motor neurons among ADSCs, BMSCs and MG groups. These results suggest that neither BMSCs nor ADSCs provide satisfactory results for peripheral nerve repair when using MG as the conductor for engraftment., Competing Interests: None declared.

Aptamers compete with antibodies in many applications, in which high-affinity and specificity ligands are needed. In this regard, fluorescence-tagged aptamers have gained applications in flow and imaging cytometry for detecting cells expressing distinct antigens. Here we present prospective methods, as a starting point, for using these high-affinity ligands for cytometry applications.

Objective: to describe the characteristics of hospitalizations due to external causes involving contact with animals in a general hospital in the interior of Bahia State (BA), Brazil, from 2009 to 2011., Methods: this was a descriptive study using data on hospital admissions in Jequié-BA., Results: there were 246 admissions owing to accidents involving contact with animals, especially venomous ones (83.3% snake venom, and 6.5% scorpion venom); most involved male victims (66.7%), the 20-59 year age group (50.4%), and people resident in rural areas (91.2%); most hospitalizations occurred during the night shift (39.0%) and on weekdays (69.1%); most cases had 1 to 3 day inpatient stays (50.8%), and 97.6% were subsequently discharged., Conclusion: snakebites and scorpion stings were the most frequent accidents and predominantly affected young men living in the rural area.

The kinins bradykinin and des-arg(9) -bradykinin cleaved from kininogen precursors by kallikreins exert their biological actions by stimulating kinin-B2 and B1 receptors, respectively. In vitro models of neural differentiation such as P19 embryonal carcinoma cells and neural progenitor cells have suggested the involvement of B2 receptors in neural differentiation and phenotype determination; however, the involvement of B1 receptors in these processes has not been established. Here, we show that B1 and B2 receptors are differentially expressed in mouse embryonic E14Tg2A stem cells undergoing neural differentiation. Proliferation and differentiation assays, performed in the presence of receptor subtype-selective agonists and antagonists, revealed that B1 receptor activity is required for the proliferation of embryonic and differentiating cells as well as for neuronal maturation at later stages of differentiation, while the B2 receptor acts on neural phenotype choice, promoting neurogenesis over gliogenesis. Besides the elucidation of bradykinin functions in an in vitro model reflecting early embryogenesis and neurogenesis, this study contributes to the understanding of B1 receptor functions in this process., (© 2015 International Society for Advancement of Cytometry.)

This study aimed to describe the dynamics of intergenerational family relationships from the viewpoint of the elderly. It is a study of qualitative approach, developed with 32 elderly people registered in the coverage area of a Family Health Program in a city in the state of Bahia. Data were collected through semi-structured interviews during the first half of 2013, through home visits. We applied to thematic content analysis, and four categories emerged: harmonic intergenerational family relationships; conflicting intergenerational family relationships; family relationships permeated by intergenerational carefor the elderly; intergenerational relationships permeated by elderly caring for their grandchildren. The categories enabled an understanding of the dynamics of intergenerational family relationships from the viewpoint of elderly, unveiling family relationships guided in the care, support, family unity and even conflicts.

This study aimed to estimate the incidence of non-fatal work-related accidents in the informal commercial sector and analyze associated socio-demographic, occupational, workplace, and health factors, in a cross-sectional survey of 434 workers in the business district of Jequié, Bahia State, Brazil, in 2013. Logistic regression was used to assess factors associated with accidents. Incidence of accidents in the previous 12 months was estimated at 32.3%, and multivariate analysis showed higher odds of accidents in male sex workers (OR = 1.61), young individuals (OR = 4.62), meat or poultry workers (OR = 9.55), and workers performing heavy physical work (OR = 1.71). The results show the need for public policies to prevent accidents in the informal commercial sector.

Alzheimer's disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.

The objective of this study was to analyze the use of relationship technology in care provided to patients with arterial hypertension. This is a qualitative study, conducted in eight Family Health Units from Jequié-Bahia. The study subjects were policy makers, health professionals and patients, totaling sixteen informants. Semi-structured interviews, systematic observation and documental analysis were performed in 2012. The data were submitted to thematic content analysis. Results showed that the professionals, especially nurses and community health workers, use light technology in the search for comprehensive care. On the other hand, some professionals have demonstrated biology-based practice, with emphasis on rules and procedures. It is concluded that there is a need to change professional-patient interactions, considering the uniqueness and autonomy of the patient and family in the construction of new forms of care.

(-)-Carvone is an antinociceptive monoterpene found as the main active constituent of essential oils obtained from plants of the genus Mentha. Here, we have investigated the pharmacology of this monoterpene in dorsal root ganglia (DRG) neurons and TRPV1-expressing HEK293 cells. (-)-carvone at pharmacological active concentrations did not reveal significant cytotoxicity to the cells used in this study, as investigated by neutral red and propidium iodide flow cytometry assays. In calcium imaging experiments 1 mM (-)-carvone increased the cytosolic calcium levels in DRG neurons from 120.6 ± 5.0 nM (basal) to 310.7 ± 23.1 nM (P < 0.05). These effects were completely abolished when neurons were preincubated with calcium-free bath solution or ruthenium-red (5 µM) and capsazepine (10 µM), suggesting the possibility of TRPV1 channel-activation by (-)-carvone. Activity of (-)-carvone on TRPV1 channels was further investigated in HEK293 cells expressing recombinant human TRPV1 channels revealing dose-dependent calcium transients with an EC(50) of 1.3 ± 0.2 mM (Hill coefficient = 2.5). In conclusion, we show for the first time the ability of (-)-carvone to induce increases in cytosolic calcium concentration through TRPV1 activation., (Copyright © 2013 International Society for Advancement of Cytometry.)

Modern medicine will unequivocally include regenerative medicine as a major breakthrough in the re-establishment of damaged or lost tissues due to degenerative diseases or injury. In this scenario, millions of patients worldwide can have their quality of life improved by stem cell implantation coupled with endogenous secretion or administration of survival and differentiation promoting factors. Large efforts, relying mostly on flow cytometry and imaging techniques, have been put into cell isolation, immunophenotyping, and studies of differentiation properties of stem cells of diverse origins. Mesenchymal stem cells (MSCs) are particularly relevant for therapy due to their simplicity of isolation. A minimal phenotypic pattern for the identification of MSCs cells requires them to be immunopositive for CD73, CD90, and CD105 expression, while being negative for CD34, CD45, and HLA-DR and other surface markers. MSCs identified by their cell surface marker expression pattern can be readily purified from patient's bone marrow and adipose tissues. Following expansion and/or predifferentiation into a desired tissue type, stem cells can be reimplanted for tissue repair in the same patient, virtually eliminating rejection problems. Transplantation of MSCs is subject of almost 200 clinical trials to cure and treat a very broad range of conditions, including bone, heart, and neurodegenerative diseases. Immediate or medium term improvements of clinical symptoms have been reported as results of many clinical studies., (Copyright © 2012 International Society for Advancement of Cytometry.)

Conus venoms are rich sources of biologically active peptides that act specifically on ionic channels and metabotropic receptors present at the neuromuscular junction, efficiently paralyzing the prey. Each species of Conus may have 50 to 200 uncharacterized bioactive peptides with pharmacological interest. Conus regius is a vermivorous species that inhabits Northeastern Brazilian tropical waters. In this work, we characterized one peptide with activity on neuronal acetylcholine receptor (nAChR). Crude venom was purified by reverse-phase HPLC and selected fractions were screened and sequenced by mass spectrometry, MALDI-ToF, and ESI-Q-ToF, respectively. A new peptide was identified, bearing two disulfide bridges. The novel 2,701 Da peptide belongs to the cysteine framework I, corresponding to the cysteine pattern CC-C-C. The biological activity of the purified peptide was tested by intracranial injection in mice, and it was observed that high concentrations induced hyperactivity in the animals, whereas lower doses caused breathing difficulty. The activity of this peptide was assayed in patch-clamp experiments, on nAChR-rich cells, in whole-cell configuration. The peptide blocked slow rise-time neuronal receptors, probably α 3 β 4 and/or α 3 β 4 α 5 subtype. According to the nomenclature, the new peptide was designated as α -RgIB.

Nitrosamines are well known for their carcinogenic potential. Recently, it was found that some of them may also interact with human nicotinic acetylcholine receptor (nAChR) subtypes. This work studied the effects of N-nitrosonornicotine (NNN) on recombinant rat α3β4 nAChR in HEK cells as well as on nAChR endogenously expressed in PC12 pheochromocytoma cells and in BC3H1 muscle-type cells. Whole-cell recording in combination with the cell-flow technique for agonist and inhibitor application in the millisecond time region revealed that NNN inhibits the activity of neuronal nAChR expressed in HEK or PC12, whereas weak inhibitory effects on muscle-type nAChR were observed at NNN concentrations up to 3 mM. Pharmacological actions of NNN and the inhibition mechanism were studied in detail using recombinant α3β4 nAChR expressed in HEK cells as a model. NNN-induced inhibition of nicotine-evoked α3β4 nAChR activity was dose-dependent with an inhibitory constant (IC(50)) of 0.92 ± 0.05 mM. Analysis based on mathematical models indicated a noncompetitive inhibition mechanism of the rat α3β4 nAChR by NNN. NNN's mechanism of action involves acceleration of conversion of the receptor from active to desensitized forms. In summary, this work shows that NNN inhibits rat α3β4 nAChR in a noncompetitive way and interacts weakly with muscular nAChR.

Pericyte perivascular cells, believed to originate mesenchymal stem cells (MSC), are characterized by their capability to differentiate into various phenotypes and participate in tissue reconstruction of different organs, including the brain. We show that these cells can be induced to differentiation into neural-like phenotypes. For these studies, pericytes were obtained from aorta ex-plants of Sprague-Dawley rats and differentiated into neural cells following induction with trans retinoic acid (RA) in serum-free defined media or differentiation media containing nerve growth and brain-derived neuronal factor, B27, N2, and IBMX. When induced to differentiation with RA, cells express the pluripotency marker protein stage-specific embryonic antigen-1, neural-specific proteins β3-tubulin, neurofilament-200, and glial fibrillary acidic protein, suggesting that pericytes undergo differentiation, similar to that of neuroectodermal cells. Differentiated cells respond with intracellular calcium transients to membrane depolarization by KCl indicating the presence of voltage-gated ion channels and express functional N-methyl-D-aspartate receptors, characteristic for functional neurons. The study of neural differentiation of pericytes contributes to the understanding of induction of neuroectodermal differentiation as well as providing a new possible stem-cell source for cell regeneration therapy in the brain., (Copyright © 2011 International Society for Advancement of Cytometry.)

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds., (Copyright © 2011 International Society for Advancement of Cytometry.)

Energy metabolism in the adult brain consumes large quantities of glucose, but little is known to date regarding how glucose metabolism changes during neuronal differentiation, a process that is highly demanding energetically. We studied changes in glucose metabolism during neuronal differentiation of P19 mouse embryonal carcinoma cells, E14Tg2A embryonic stem cells as well as during brain development of BLC57 mice. In all these models, we find that neurogenesis is accompanied by a shift from oxidative to fermentative glucose metabolism. This shift is accompanied by both a decrease in mitochondrial enzymatic activities and mitochondrial uncoupling. In keeping with this finding, we also observe that differentiation does not require oxidative metabolism, as indicated by experiments demonstrating that the process is preserved in cells treated with the ATP synthase inhibitor oligomycin. Overall, we provide evidence that neuronal differentiation involves a shift from oxidative to fermentative metabolism, and that oxidative phosphorylation is not essential for this process.

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric alpha7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca2+ influx. However, the study of recombinant alpha7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for alpha3, alpha5, alpha7, beta2 and beta4 subunits was present during the course of differentiation, while mRNAs coding for alpha2, alpha4 and beta3 subunits were not expressed in PC12 cells. alpha7 subunit expression was highest following 1 day of induction to differentiation. Activity of alpha7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the alpha7 agonist choline. Increased alpha7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

RNA and DNA aptamers developed by systematic evolution of ligands by exponential enrichment (SELEX) have turned into important tools in diagnostics, research, and therapeutics. Unlike antibodies, high-affinity and specific aptamers identified through an in vitro selection process can be chemically modified to gain nuclease resistances in biological fluids and to extend their bioavailability in animals. Aptamers can be raised against virtually any target including those which are toxic or do not elicit any immune response in animals. They can be developed in automated processes against various protein targets and then easily modified by attaching fluorescence reporters, nanoparticles or biotin moieties, rival antibodies in high-throughput proteomics and cell separations. In this review, we will discuss the high competence of aptamers in recognizing biomarkers and molecular signatures of cell surfaces, and how these unique features can be exploited for the identification and isolation of cancer, stem cells and even detection of parasite-infected cells.

The first naturally occurring angiotensin-converting enzyme (ACE) inhibitors described are pyroglutamyl proline-rich oligopeptides, found in the venom of the viper Bothrops jararaca, and named as bradykinin-potentiating peptides (BPPs). Biochemical and pharmacological properties of these peptides were essential for the development of Captopril, the first active site-directed inhibitor of ACE, currently used for the treatment of human hypertension. However, a number of data have suggested that the pharmacological activity of BPPs could not only be explained by their inhibitory action on enzymatic activity of somatic ACE. In fact, we showed recently that the strong and long-lasting anti-hypertensive effect of BPP-10c [

The mechanism of eupalmerin acetate (EUAC) actions on the embryonic muscle nicotinic acetylcholine receptor (nAChR) in BC3H-1 cells was studied by using whole-cell and single-channel patch-clamp current measurements. With whole-cell currents, EUAC did not act as an agonist on this receptor. Coapplication of 30 microM EUAC with 50 microM, 100 microM, or 500 microM carbamoylcholine (CCh) reversibly inhibited the current amplitude, whereas, with 20 microM CCh, current was increased above control values in the presence of EUAC. EUAC concentration curves (0.01-40 microM) obtained with 100 microM and 500 microM CCh displayed slope coefficients, n(H), significantly smaller than one, suggesting that EUAC bound to several sites with widely differing affinities on the receptor molecule. The apparent rate of receptor desensitization in the presence of EUAC and CCh was either slower than or equal to that obtained with CCh alone. The major finding from single-channel studies was that EUAC did not affect single-channel conductance or the ability of CCh to interact with the receptor. Instead, EUAC acted by increasing the channel closing rate constant. The results are not consistent with the competitive model for EUAC inhibition, with the sequential open-channel block model, or with inhibition by increased desensitization. The data are best accounted for by a model in which EUAC acts by closed-channel block at low concentrations, by positive modulation at intermediate concentrations, and by negative allosteric modulation of the open channel at high concentrations.

Age-related macular degeneration (AMD) is the main cause of loss of sight in the world and is characterized by neovascularization of the macula. The factors producing choroidal vascularization involve various growth factors, including the vascular endothelial growth factor (VEGF(165)). In this context, the systematic evolution of ligands by exponential enrichment (SELEX) became a tool for developing new therapeutic agents for AMD treatment. The SELEX is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules (aptamers) are identified by their ability to bind their targets with high affinity and specificity. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands for a wide range of proteins of clinical importance. For instance, Pegaptanib sodium, a 28-nucleotide polyethylene glycol RNA aptamer that selectively binds to VEGF(165) and inhibits angiogenesis, was approved by the Food and Drug Administration for the treatment of wet AMD, thereby providing significant benefits to a great number of patients with minimal adverse effects.

The systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules are selected by their ability to bind their targets with high affinity and specificity, comparable to those of antibodies. Nucleic acids with high affinity for their targets have been selected against a wide variety of compounds, from small molecules, such as ATP, to membrane proteins and even whole organisms. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands, also known as aptamers, for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. The number of aptamers generated as inhibitors of various target proteins has increased following automatization of the SELEX process. Their diagnostic and therapeutic efficacy can be enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Several aptamers are currently being tested in preclinical and clinical trials, and aptamers are in the process of becoming a new class of therapeutic agents. Recently, the anti-VEGF aptamer pegaptanib received FDA approval for treatment of human ocular vascular disease.

P2X receptors play an important role in communication between cells in the nervous system. Therefore, understanding the mechanisms of inhibition of these receptors is important for the development of new tools for drug discovery. Our objective has been to determine the pharmacological activity of the antagonist suramin, the most important antagonist of purinergic receptor function, as well as to demonstrate its noncompetitive inhibition and confirm a competitive mechanism between ATP and TNP-ATP in 1321N1 glial cells stably transfected with the recombinant rat P2X(2) receptor. A radioligand binding assay was employed to determine whether suramin, TNP-ATP, and ATP compete for the same binding site on the receptor. TNP-ATP displaced [alpha-32P]ATP, whereas suramin did not interfere with [alpha-32P]ATP-receptor binding. To determine the inhibition mechanism relevant for channel opening, currents obtained in fast kinetic whole-cell recording experiments, following stimulation of cells by ATP in the presence of suramin, were compared to those obtained by ATP in the presence of TNP-ATP. Supported by a mathematical model for receptor kinetics [Breitinger, H. G., Geetha, N., and Hess, G. P. (2001) Biochemistry 40, 8419-8429], the inhibition factors were plotted as functions of inhibitor or agonist concentrations. Analysis of the data indicated a competitive inhibition mechanism for TNP-ATP and a noncompetitive inhibition for suramin. Taken together, both data support a noncompetitive inhibition mechanism of the rat recombinant P2X(2) receptor by suramin, confirm the competitive inhibition by TNP-ATP, and allow the prediction of a model for P2X(2) receptor inhibition.

Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid beta peptide (Abeta) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by Abeta has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking Abeta-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to Abeta. Using this approach, we identified a heptapeptide denoted IQ, which binds with nanomolar affinity to Abeta and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that Abeta inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by Abeta. These results indicate that the Abeta binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for Abeta neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble Abeta in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD.