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In the light of recent retrovirus pandemics, the issue of discovering new and diverse RNA-specific fluorochromes for research and diagnostics became of acute importance. The great majority of nucleic acid-specific probes either do not stain RNA or cannot distinguish between DNA and RNA. The versatility of polymethine dyes makes them suitable as stains for visualization, analysis, and detection of nucleic acids, proteins, and other biomolecules. We synthesized the asymmetric dicationic homodimeric monomethine cyanine dyes 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)pyridin-1-ium) bromide (Т1) and 1,1'-(1,3-phenylenebis(methylene))bis(4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium) bromide (M1) and tested their binding specificity, spectral characteristics, membrane penetration in living and fixed cells, cellular toxicity, and stability of fluorescent emission. Mesenchymal cells have diverse phenotypes and extensive proliferation and differentiation properties. We found dyes T1 and M1 to show high photochemical stability in living mesenchymal stem cells from apical papilla (SCAP) with a strong fluorescent signal when bound to nucleic acids. We found M1 to perform better than control fluorochrome (Hoechst 33342) for in vivo DNA visualization. T1, on the other hand, stains granular cellular structures resembling ribosomes in living cells and after permeabilization of the nuclear membrane stains the nucleoli and not the chromatin in the nucleus. This makes T1 suitable for the visualization of structures rich in RNA in living and fixed cells.
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BACKGROUND: Alzheimer's disease (AD) and Multiple sclerosis (MS) lead to neurodegenerative processes negatively affecting millions of people worldwide. Their treatment is still difficult and practically incomplete. One of the most commonly used drugs against these neurodegenerative diseases is 4-aminopyridine. However, its use is confined by the high toxicity. OBJECTIVES: The aim of this work is to obtain new peptide derivatives of 4-aminopyridine with decreased toxicity compared to 4-aminopyridine. METHODS: Synthesis was conducted in solution using a consecutive condensation approach. The new derivatives were characterized by melting points, NMR, and Mass spectra. Important ADME (absorption, distribution, metabolism, and excretion) properties have been studied in silico using ACD/Percepta v.2020.2.0 software. Acute toxicity was determined in mice according to a Standard protocol. All new derivatives were tested in vitro for cytotoxic activity in a panel of human (HEP-G2, BV-173) and murine (NEURO 2A) tumor cell lines via a standard MTT-based colorimetric method. ß-secretase inhibitory activity was determined by applying the fluorescent method. RESULTS: New derivatives of 4-aminopyridine containing analogues of the ß-secretase inhibitory peptide (Boc-Val-Asn-Leu-Ala-OH) were obtained. The in vivo toxicity of the tested compounds was found to be as high as 1500 mg/kg. Cell toxicity screening against tumor cell lines of different origins showed negligible growth-inhibitory effects of all investigated 4-aminopyridine analogues. CONCLUSION: Synthesis of new peptide derivatives of 4-aminopyridine is reported. Acute toxicity studies revealed a ca. 150 times lower toxicity of the new compounds as compared to 4-aminopyridine that may be ascribed to their peptide fragment.
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4-Aminopiridina , Enfermedad de Alzheimer , Ratones , Humanos , Animales , 4-Aminopiridina/toxicidad , 4-Aminopiridina/uso terapéutico , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos/farmacología , Línea Celular TumoralRESUMEN
Complex neurological disorders, including Alzheimer's disease, are one of the major therapeutic areas to which multitarget drug discovery strategies have been applied in the last twenty years. Due to the complex multifactorial etiopathogenesis of Alzheimer's disease, it has been proposed that to be successful the pharmaceutical agents should act on multiple targets in order to restore the complex disease network and to provide disease modifying effects. Here we report on the synthesis and the anticholinergic activity profiles of seven multitarget anti-Alzheimer compounds designed by combining galantamine, a well-known acetylcholinesterase inhibitor, with different peptide fragments endowed with inhibitory activity against BACE-1. A complementary approach based on molecular docking simulations of the galantamine-peptide derivatives in the active sites of acetylcholinesterase and of the related butyrylcholinesterase, as well as on inhibition kinetics, by global fitting of the reaction progress curves, allowed to gain insights into the enzyme-inhibitor mechanism of interaction. The resulting structure-activity relationships pave the way towards the design of more effective pharmacodynamic/pharmacokinetic multitarget inhibitors.
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Enfermedad de Alzheimer , Butirilcolinesterasa , Acetilcolinesterasa/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Butirilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/química , Galantamina/farmacología , Galantamina/uso terapéutico , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Fragmentos de Péptidos , Relación Estructura-ActividadRESUMEN
The Alzheimer's disease leads to neurodegenerative processes and affecting negatively million people worldwide. The treatment of the disease is still difficult and incomplete in practice. Galanthamine is one of the most commonly used drugs against the illness. The main aim of this work is design and synthesis of new derivatives of galanthamine comprising peptide moiety as well as study of their ß-secretase inhibitory activity and the anti-aggregating effect. All new derivatives of galanthamine containing analogues of Leu-Val-Phe-Phe (Aß17-Aß20) were synthesized in solution using fragment and consecutive condensation approaches. The new derivatives were characterized by melting points, NMR, and HPLC/MS. They were tested in vitro for ß-secretase inhibition activity by means of fluorescent method and were investigated in vitro for anti-aggregation activity on sheep platelet-rich plasma. Although the new compounds do not contain a structural element responsible for the ß-secretase inhibition, five of them show high or good ß-secretase inhibitory activity between 19.98 and 51.19% with IC50 between 1.95 and 5.26 nM. Four of the new molecules were able to inhibit platelet aggregation between 55.0 and 90.0% with IC50 between 0.69 and 1.36 µM. Four of the compounds were able to inhibit platelet aggregation and two of them have high anti-aggregating effects.
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Enfermedad de Alzheimer , Galantamina , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/prevención & control , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides , Animales , Galantamina/química , Galantamina/farmacología , Galantamina/uso terapéutico , Humanos , Péptidos/química , OvinosRESUMEN
BACKGROUND: Bile acid malabsorption is common in microscopic colitis, irritable bowel syndrome with diarrhea, and inflammatory bowel disease. We investigated the diagnostic accuracy of 7-alfa-hydroxy-4-cholesten-3-one and compared it with fibroblast growth factor-19 as biomarkers for bile acid malabsorption. METHODS: We enrolled consecutively 109 chronic diarrhea patients with standard laboratory tests, fecal calprotectin, and endoscopy separated into six groups: n = 30 with active inflammatory bowel disease, n = 21 with inflammatory bowel disease in remission reporting >3 bowel movements per day, n = 21 with inflammatory bowel disease after surgery, n = 23 with irritable bowel syndrome with diarrhea, n = 14 with microscopic colitis and 11 healthy subjects (controls). We defined bile acid malabsorption as >3 bowel movements and lower fibroblast growth factor-19 (<60 pg/ml). RESULTS: Median levels of 7-alfa-hydroxy-4-cholesten-3-one in inflammatory bowel disease active were 53.1 ng/ml, inflammatory bowel disease remission were 52.2 ng/ml, inflammatory bowel disease after surgery were 85.7 ng/ml, irritable bowel syndrome with diarrhea were 7.5 ng/ml, microscopic colitis were 69.3 ng/ml, and healthy controls were 3.7 ng/ml. We estimate a 7-alfa-hydroxy-4-cholesten-3-one cutoff of 48.9 ng/ml with 82.6% sensitivity and 84.3% specificity for detecting bile acid malabsorption. Both 7-alfa-hydroxy-4-cholesten-3-one >48.9 ng/ml and fibroblast growth factor-19 (<60 pg/ml) were found in 52% of the patients, compared with those 8% of patients below this 7-alfa-hydroxy-4-cholesten-3-one cutoff (P < 0.001). Serum 7-alfa-hydroxy-4-cholesten-3-one correlated with the number of bowel movements/day (r = -0.709; P < 0.001) and correlated inversely with fibroblast growth factor-19 (r = -0.741; P < 0.001). CONCLUSIONS: Serum 7-alfa-hydroxy-4-cholesten-3-one above 48.9 ng/ml and fibroblast growth factor-19 below 60 pg/ml identify patients with diarrhea likely attributable to bile acid malabsorption with high diagnostic accuracy and they can be used as screening biomarkers for bile acid malabsorption in microscopic colitis and inflammatory bowel disease.
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Ácidos y Sales Biliares/metabolismo , Colestenonas/sangre , Colitis Microscópica , Factores de Crecimiento de Fibroblastos/sangre , Enfermedades Inflamatorias del Intestino , Biomarcadores/sangre , Colitis Microscópica/complicaciones , Colitis Microscópica/diagnóstico , Diarrea/diagnóstico , Diarrea/etiología , Humanos , Enfermedades Inflamatorias del Intestino/complicaciones , Enfermedades Inflamatorias del Intestino/diagnósticoRESUMEN
Methanol-aqueous extracts from the aerial parts of Gypsophila glomerata (GGE), G. trichotoma (GTE) and G. perfoliata (GPE) were investigated for antioxidant potential using different in vitro models, as well as for phenolic and flavonoid contents. The possible anti-cholinesterase, anti-tyrosinase, anti-amylase and anti-glucosidase activities were also tested. The flavonoid variability was analyzed using ultra high-performance liquid chromatography (UHPLC) coupled with hybrid quadrupole-Orbitrap high resolution mass spectrometry (HRMS). Eleven C-glycosyl flavones and 4 O-glycosyl flavonoids, including 2"-O-pentosyl-6-C-hexosyl-apigenin/methylluteolin, as well as their mono(di)-acetyl derivatives were found in GGE. Both GGE and GTE shared 2"-pentosyl-6-C-hexosyl-luteolin together with the common saponarin, homoorientin, orientin, isovitexin and vitexin, while di C-glycosyl flavones were evidenced only in GPE. The highest radical scavenging in both ABTS and DPPH assays was noted in GPE, as well as ferric and cupric reducing abilities. However, GTE had the strongest metal chelating activity (17.44⯱â¯0.51â¯mg EDTAE/g extract). GPE and GGE were more potent as acetylcholinesterases inhibitors witnessed by 2.09⯱â¯0.02â¯mg GALAE/g extract and 1.59⯱â¯0.09 mgGALAE/g extract, respectively. All flavonoids were found in G. glomerata for the first time. Therefore, further isolation and structural elucidation of newly described acetylated flavonoids are needed in order to determine their relevance in the beneficial properties of the plant.
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Caryophyllaceae/química , Extractos Vegetales/química , Extractos Vegetales/farmacología , Amilasas/antagonistas & inhibidores , Antioxidantes/química , Antioxidantes/farmacología , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Cromatografía Líquida de Alta Presión/métodos , Flavonas/química , Flavonas/farmacología , Glucosidasas/antagonistas & inhibidores , Espectrometría de Masas/métodos , Monofenol Monooxigenasa/antagonistas & inhibidores , Fenoles/química , Fenoles/farmacologíaRESUMEN
INTRODUCTION: Geigeria alata is a traditional plant used in Sudanese folk medicine for treatment of diabetes, cough, epilepsy and intestinal complaints. OBJECTIVE: To analyze phenolic acids in Geigeria alata roots and leaves and to evaluate their antioxidant and antimicrobial activities. METHODOLOGY: Phenolic acids in the aqueous-methanol extracts were identified by LC-MS. Major compounds were isolated using low-pressure liquid chromatography. The quantitative analysis of phenolic acids was performed by a validated HPLC-UV method with limits of detection ranging from 0.04 to 0.57 µg/mL. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azinobis-(3-ethylbenzothiazine-6-sulphonic acid) (ABTS) and ferric reducing antioxidant power (FRAP) methods were used for antioxidant activity evaluation. In addition, the minimal inhibitory concentration and the minimal bactericidal concentration against a panel of pathogenic bacteria and fungi were determined by the broth microdilution test. RESULTS: For the first time protocatechuic, caffeic, p-coumaroylquinic, caffeoylsinapoylquinic, caffeoylferuloylquinic, three feruloylquinic, six caffeoylquinic acids, and a caffeic acid hexoside were detected in Geigeria alata roots by LC-MS. HPLC-UV analyses showed that 3,5-dicaffeoylquinic acid (25.96 ± 2.08 mg/g dry weight (DW)) was the most abundant phenolic acid in roots, while 4,5-dicaffeoylquinic acid (8.99 ± 0.56 mg/g DW) was the main compound present in leaves. 3,5-Dicaffeoylquinic acid demonstrated stronger radical scavenging activity and reducing power compared with the crude extracts and the positive control 5-caffeoylquinic acid. 3,4,5-Tricaffeoylquinic acid revealed the highest antibacterial potential against the penicillin sensitive and resistant Staphylococcus aureus strains, as well as methicillin-resistant S. aureus. CONCLUSION: The caffeoylquinic acids content of up to 6.22% in Geigeria alata roots establishes this species as a new source rich in these bioactive molecules. Copyright © 2016 John Wiley & Sons, Ltd.
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Antiinfecciosos/farmacología , Antioxidantes/farmacología , Cromatografía Líquida de Alta Presión/métodos , Geigeria/química , Antiinfecciosos/análisis , Antiinfecciosos/química , Antioxidantes/análisis , Antioxidantes/química , Ácidos Cafeicos/análisis , Ácido Clorogénico/análogos & derivados , Ácido Clorogénico/análisis , Cromatografía Liquida , Flavonoides/análisis , Espectrometría de Masas/métodos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Extractos Vegetales/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Raíces de Plantas/química , Plantas Medicinales/química , Polifenoles/análisis , Ácido Quínico/análogos & derivados , Ácido Quínico/análisisRESUMEN
Over the last 10 years, accumulated experimental and clinical evidence has supported the idea that AT1 receptor subtype is involved in epilepsy. Recently, we have shown that the selective AT1 receptor antagonist losartan attenuates epileptogenesis and exerts neuroprotection in the CA1 area of the hippocampus in epileptic Wistar rats. This study aimed to verify the efficacy of long-term treatment with losartan (10 mg/kg) after kainate-induced status epilepticus (SE) on seizure activity, behavioral and biochemical changes, and neuronal damage in a model of co-morbid hypertension and epilepsy. Spontaneous seizures were video- and EEG-monitored in spontaneously hypertensive rats (SHRs) for a 16-week period after SE. The behavior was analyzed by open field, elevated plus maze, sugar preference test, and forced swim test. The levels of serotonin in the hippocampus and neuronal loss were estimated by HPLC and hematoxylin and eosin staining, respectively. The AT1 receptor antagonism delayed the onset of seizures and alleviated their frequency and duration during and after discontinuation of treatment. Losartan showed neuroprotection mostly in the CA3 area of the hippocampus and the septo-temporal hilus of the dentate gyrus in SHRs. However, the AT1 receptor antagonist did not exert a substantial influence on concomitant with epilepsy behavioral changes and decreased 5-HT levels in the hippocampus. Our results suggest that the antihypertensive therapy with an AT1 receptor blocker might be effective against seizure activity and neuronal damage in a co-morbid hypertension and epilepsy.
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Conducta Animal/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipertensión/fisiopatología , Losartán/farmacología , Neuronas/efectos de los fármacos , Convulsiones/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Hipertensión/complicaciones , Losartán/administración & dosificación , Masculino , Ratas , Ratas Endogámicas SHR , Convulsiones/complicaciones , Convulsiones/fisiopatología , Estado Epiléptico/inducido químicamenteRESUMEN
Recently, we have shown that the blockade of AT1 receptor might be useful as an adjuvant treatment strategy for the prevention of oxidative stress and neurotoxicity caused by status epilepticus (SE) in rats. The purpose of the present study was to further assess the efficacy of long-term treatment with losartan (10mg/kg), the selective AT1 receptor antagonist, during kainate (KA)-induced epileptogenesis in Wistar rats. Losartan treatment started after onset of SE and continued for 4weeks. The rats were video- and EEG-recorded for 3months. Locomotor activity, anxiety and depressive-like behavior were evaluated 9weeks after SE, when all rats had developed chronic epileptic state. Neuronal damage in hippocampus was analyzed by hematoxylin while serotonin (5-HT) levels in hippocampus by HPLC. AT1 receptor antagonism increased the latent seizure-free period and decreased the frequency of spontaneous motor seizures. Losartan positively affected epilepsy-provoked behavioral changes, including impulsivity, low anxiety level and depression in a phase-dependent manner and restored the changes in diurnal fluctuation of motor activity. Losartan exerted neuroprotection selectively in the CA1 area of the hippocampus in the KA-treated rats and lowered the 5-HT levels both in normal and abnormal conditions. Our findings suggest that the AT1 receptor antagonist exerts disease-modifying effects during KA-induced epileptogenesis and neuronal damage in CA1 hippocampal area, attenuated some of the behavioral changes and restored diurnal variability in locomotor activity.
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Anticonvulsivantes/uso terapéutico , Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Ácido Kaínico/toxicidad , Losartán/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Bloqueadores del Receptor Tipo 1 de Angiotensina II/uso terapéutico , Animales , Masculino , Distribución Aleatoria , Ratas , Ratas Wistar , Resultado del TratamientoRESUMEN
Polyamines are essential polycations, playing important roles in mammalian physiology. Theoretically, the involvement of homocysteine in polyamine synthesis via S-adenosylmethionine is possible; however, to our knowledge, it has not been established experimentally. Here, we propose an original approach for investigation of homocysteine metabolites in an animal model. The method is based on the combination of isotope-labeled homocysteine supplementation and high-resolution accurate mass spectrometry analysis. Structural identity of the isotope-labeled metabolites was confirmed by accurate mass measurements of molecular and fragment ions and comparison of the retention times and tandem mass spectrometry fragmentation patterns. Isotope-labeled methionine, spermidine, and spermine were detected in all investigated plasma and tissue samples. The induction of moderate hyperhomocysteinemia leads to an alteration in polyamine levels in a different manner. The involvement of homocysteine in polyamine synthesis and modulation of polyamine levels could contribute to a better understanding of the mechanisms connected with homocysteine toxicity.
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Homocisteína/química , Homocisteína/metabolismo , Animales , Marcaje Isotópico , Estructura Molecular , Ratas , Ratas Wistar , Espermidina/biosíntesis , Espermina/biosíntesisRESUMEN
Melatonin is involved in the control of circadian and seasonal rhythmicity, possesses potent antioxidant activity, and exerts a neuroprotective and anticonvulsant effect. Spontaneously hypertensive rats (SHRs) are widely accepted as an experimental model of essential hypertension with hyperactivity, deficient sustained attention, and alterations in circadian autonomic profiles. The purpose of the present study was to determine whether melatonin treatment during epileptogenesis can prevent the deleterious consequences of status epilepticus (SE) in SHRs in the kainate (KA) model of temporal lobe of epilepsy (TLE). Spontaneous recurrent seizures (SRSs) were EEG- and video-recorded during and after the treatment protocol. Melatonin (10mg/kg diluted in drinking water, 8weeks) increased the seizure-latent period, decreased the frequency of SRSs, and attenuated the circadian rhythm of seizure activity in SHRs. However, melatonin was unable to affect the disturbed diurnal rhythms and behavioral changes associated with epilepsy, including the decreased anxiety level, depression, and impaired spatial memory. Melatonin reduced neuronal damage specifically in the CA1 area of the hippocampus and piriform cortex and decreased hippocampal serotonin (5-HT) levels both in control and epileptic SHRs. Although long-term melatonin treatment after SE shows a potential to attenuate seizure activity and neuronal loss, it is unable to restore epilepsy-associated behavioral abnormalities in SHRs.
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Antioxidantes/uso terapéutico , Conducta Animal/efectos de los fármacos , Encéfalo/patología , Epilepsia del Lóbulo Temporal/tratamiento farmacológico , Epilepsia del Lóbulo Temporal/fisiopatología , Melatonina/uso terapéutico , Animales , Antioxidantes/farmacología , Presión Sanguínea/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Ritmo Circadiano/efectos de los fármacos , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/inducido químicamente , Conducta Exploratoria/efectos de los fármacos , Preferencias Alimentarias/efectos de los fármacos , Ácido Kaínico/toxicidad , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Melatonina/farmacología , Ratas , Ratas Endogámicas SHR , Serotonina/metabolismo , Natación/psicología , Factores de TiempoRESUMEN
Melatonin is a potent antioxidant which showed anticonvulsant activities both in experimental and clinical studies. In the present study, we examined the effect of melatonin treatment (10mg/kg/day, diluted in drinking water, 8 weeks) during epileptogenesis on the consequences of a kainate (KA)-induced status epilepticus (SE) in rats. Melatonin increased the latency in the appearance of spontaneous recurrent seizures (SRSs) and decreased their frequency only during the treatment period. The behavioral alterations associated with hyperactivity, depression-like behavior during the light phase, and deficits in hippocampus-dependent working memory were positively affected by melatonin treatment in rats with epilepsy. Melatonin reduced the neuronal damage in the CA1 area of the hippocampus and piriform cortex and recovered the decrease of hippocampal serotonin (5-HT) level in rats with epilepsy. Taken together, long-term melatonin treatment after SE was unable to suppress the development of epileptogenesis. However, it showed a potential in reducing some of the deleterious alterations that develop during the chronic epileptic state in a diurnal phase-dependent mode.
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Depresores del Sistema Nervioso Central/uso terapéutico , Depresión/prevención & control , Hipercinesia/prevención & control , Melatonina/uso terapéutico , Estado Epiléptico/complicaciones , Análisis de Varianza , Animales , Cromatografía Líquida de Alta Presión , Depresión/etiología , Electroencefalografía , Agonistas de Aminoácidos Excitadores/toxicidad , Conducta Exploratoria/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Hipercinesia/etiología , Ácido Kaínico/toxicidad , Estimación de Kaplan-Meier , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Neuronas/patología , Ratas , Ratas Wistar , Serotonina/metabolismo , Estado Epiléptico/inducido químicamente , Estado Epiléptico/tratamiento farmacológico , Sacarosa/administración & dosificación , Natación , Factores de TiempoRESUMEN
INTRODUCTION: Polyamines (putrescine, spermidine, and spermine) are polycationic compounds that play a central role in keratinocytic proliferation, differentiation, and regulation. The objective was to elucidate the polyamine metabolic changes that occur in various benign and neoplastic skin proliferations. METHODS: The study included 58 patients: 31 with the plaque form of psoriasis vulgaris and 27 with non-melanoma skin tumors. The levels of putrescine, spermidine, and spermine were detected in lesional and non-lesional skin samples. RESULTS: Findings were representative (p < 0.05). Psoriatic lesions showed a twofold elevation of all polyamines in lesional skin compared to non-lesional skin. Spermine had the highest concentration, which suggested a leading position of propylamine synthesis in psoriatic pathogenesis. Results on the polyamine metabolism of basal cell carcinoma represented basic characteristics similar to those of psoriasis. Conversely, squamous-cell carcinoma lesions showed the highest concentration of putrescine, suggesting a crucial role of spermidine-spermine acetyltransferase in their pathogenesis. DISCUSSION: Our findings showed different polyamine metabolic changes in lesions from benign and neoplastic keratinocytic proliferations. Basal-cell carcinoma polyamine metabolism revealed a closer relationship to psoriasis than to squamous-cell carcinoma, which might explain its long-term benign course and non-metastatic nature.
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Carcinoma Basocelular/metabolismo , Carcinoma de Células Escamosas/metabolismo , Queratinocitos/fisiología , Poliaminas/metabolismo , Neoplasias Cutáneas/metabolismo , Adolescente , Adulto , Diferenciación Celular , Proliferación Celular , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
The purpose of this study was to explore whether the kainate (KA) model of temporal lobe epilepsy (TLE) can be used as a model of comorbid epilepsy and depression to study diurnal behavioral variations in rats. Development of chronic epilepsy was confirmed by the detection of spontaneous motor seizures (SMS) with video monitoring (24 hours/3-5 months after status epilepticus [SE]). KA-treated spontaneously hypertensive rats (SHRs) exhibited higher seizure frequency than Wistar rats during the light phase in the fourth and fifth months after SE. Although epileptic Wistar rats showed depression-like behavior and reduced anxiety mostly during the light phase, there were no diurnal variations in depression-like patterns in SHRs. Anxiety levels of control and epileptic SHRs were similar. Decreases in serotonin, tryptophan, and dopamine concentrations in the hippocampus were detected in epileptic Wistar rats compared with naive controls. However, monoamine levels of epileptic SHRs were close to those of their controls. Wistar rats and SHRs develop stable depression-like behavior during the chronic epileptic phase with strain-dependent diurnal differences.
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Trastornos Cronobiológicos/etiología , Ritmo Circadiano/fisiología , Depresión/etiología , Epilepsia del Lóbulo Temporal/complicaciones , Análisis de Varianza , Animales , Reacción de Prevención/efectos de los fármacos , Conducta Animal/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Catecolaminas/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Ritmo Circadiano/efectos de los fármacos , Modelos Animales de Enfermedad , Epilepsia del Lóbulo Temporal/inducido químicamente , Reacción de Fuga/efectos de los fármacos , Hipocampo/metabolismo , Ácido Kaínico/efectos adversos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Ratas Wistar , Estadísticas no Paramétricas , Sacarosa/metabolismo , Natación/psicología , Factores de TiempoRESUMEN
The specificity of 10 recombinant caspases was investigated using a set of competitive substrates. The caspase activity was determined by high-performance liquid chromatography using highly fluorescent peptides containing 2-aminoacridone (AMAC) as reporting group. The sequences of the used substrates were designed according to literature data for being specific for 10 of the caspases. The described approach allows the concentration changes of several substrates to be monitored simultaneously in a single sample. Because the substrates are in competitive conditions, the preferences of particular caspases to given peptide sequences are most clearly demonstrated. In the studied competitive assay conditions, all tested caspases except caspase 2 exhibit activity toward more than one substrate. None of the used peptide sequences was found to be highly specific for a defined caspase. The results obtained indicate that there is well-expressed group specificity among the caspases.
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Unión Competitiva , Caspasas/metabolismo , Péptidos/metabolismo , Proteínas Recombinantes/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Aminoacridinas/química , Caspasas/química , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Péptidos/química , Proteínas Recombinantes/química , Especificidad por SustratoRESUMEN
Synthesis and properties of a new fluorescent/fluorogenic substrate Ac-DEVD-AMAC for caspase-3 are reported. The substrate is obtained by conventional Fmoc-based solid phase peptide synthesis and its properties are investigated with regard to fluorescence, sensitivity, applicability and kinetic constants. A non-traditional approach to assay the proteases activity using 2-aminoacridone labeled peptides is proposed. This approach utilizes the decrease of fluorescence intensity of a sample as a measure for the enzyme activity.
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Acridonas/química , Aminoacridinas/química , Caspasa 3/análisis , Colorantes Fluorescentes/química , Oligopéptidos/química , Acridonas/síntesis química , Aminoacridinas/síntesis química , Caspasa 3/química , Línea Celular , Colorantes Fluorescentes/síntesis química , Humanos , Oligopéptidos/síntesis química , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/métodosRESUMEN
Design, synthesis and properties of new derivatization reagent N-(2-acridonyl)-maleimide (MIAC) for thiol groups is presented. The reaction of MIAC with aminothiols is specific, very fast and yield highly fluorescent products. The HPLC method for determination of homocysteine, cysteine and glutathione based on utilization of MIAC is developed. A baseline separation of derivatives is achieved by isocratic elution on reverse phase column within 6 min. The method is linear in the range of 0.5-25 microM for homocysteine and glutathione, and in the range of 0.5-200 microM for cysteine. The limits of detection for homocysteine, cysteine and glutathione are 1.2, 1.4 and 2.0 pmol, respectively, per 20 microl injection. Within and between-run precision expressed as relative standard deviations are in the range of 1.35-4.38% and 0.89-4.13%, respectively.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cisteína/química , Glutatión/química , Homocisteína/química , Maleimidas/química , Plasma/química , Cromatografía Líquida de Alta Presión/normas , Cisteína/sangre , Glutatión/sangre , Homocisteína/sangre , Humanos , Maleimidas/sangre , Estándares de Referencia , Sensibilidad y EspecificidadRESUMEN
A liquid chromatography method for simultaneous analysis of amino acids, polyamines, catecholeamines and metanephrines in human body fluids after derivatization with 9-fluorenylmethyloxycarbonyl chloride was developed. The chromatographic behavior of analytes at different pH of mobile phase was studied. Successful baseline resolution of all analyzed compounds was achieved using simultaneous gradient of pH and organic modifier in reverse phase mode of HPLC within 36 min. The repeatability of the proposed procedure in respect of retention time and peak area, expressed as RSD, ranges from 0.06 to 1.64% and 0.4 to 7.6%, respectively. The method linearity in the range of 1-200 microM for amino acids and in the range of 0.1-20 microM for polyamines, catecholeamines and metanephrines was found to be with correlation coefficients higher than 0.994. The limit of quantification (LOQ) was assessed to be in the range of 2.6-10 pmol for amino acids and 2-4 pmol for polyamines, catecholeamines and metanephrines.
Asunto(s)
Aminoácidos/sangre , Aminoácidos/orina , Aminas Biogénicas/sangre , Aminas Biogénicas/orina , Cromatografía Líquida de Alta Presión/métodos , Acetonitrilos , Fluorenos/química , Humanos , Concentración de Iones de HidrógenoRESUMEN
A high-performance liquid chromatography method for the simultaneous analysis of amino acids and biogenic polyamines, using a new procedure for pre-column derivatization of amino groups with N-(9-fluorenylmethoxycarbonyloxy)succinimide is described. The separation of 20 amino acids and 4 biogenic polyamines was achieved within 32 min on a sequence of three short (50 mm) reversed-phase C18, 5 microm columns by elution buffers based on dibutylamine phosphate. The method linearity, calculated for each amino acid and polyamine, has a correlation coefficient higher than 0.991, in concentrations ranging from 0.2 to 50 microM, except for spermine and methionine, where the correlation coefficients were r = 0.984 and r = 0.979, respectively. The stability of derivatives in acidified samples at 4 degrees C and room temperature was demonstrated. The limit of quantitation was estimated to be around 50 pM in 50 microl sample injection. The repeatability of the method, expressed as R.S.D., ranged from 1.1 to 6.7%. The presented method was applied for the quantitation of amino acid and polyamine contents in beer, wine, and cell culture samples, using 2-aminoheptanoic acid or 1,7-diaminoheptane as internal standard.