RESUMO
The structural, optoelectronics, and transport properties of TlTaO3 compounds were determined utilizing the full potential augmented plane wave approach using first-principle method. We have considered the generalized gradient approximation for structural optimization and modified Becke-Johnson for electronic properties. The electronic properties reveal that the studied TlTaO3 possesses direct bandgap of magnitude 1.52 eV. Between 0 and 12 eV, optical spectra calculations are made, taking into account the real and imaginary parts of the dielectric function, refractive index, and loss function. The transport properties are estimated considering Boltzmann transport theory. The Seebeck coefficient, electrical conductivity, thermal conductivity, and power factor are all assessed using the Boltzmann transport theory. The optimized thermoelectric response of the examined TlTaO3 is produced by the improved carrier mobility, which also improves the thermoelectric efficiency of the TlTaO3. The obtained results will act as a theoretical road map for upcoming experimental and commercial TlTaO3 applications.
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AIMS: ß-lactamase inhibitor resistance (BLIR) among the uropathogenic Escherichia coli (UPEC) minimizes treatment options. This study aimed to identify inhibitor-resistant TEM (IRT) ß-lactamase that impart BLIR phenotype and explore non-ß-lactams as alternative therapeutics. METHODS AND RESULTS: Thirty BLIR UPEC isolates were detected by Kirby-Bauer disc diffusion technique using ß-lactam-ß-lactamase inhibitor combination. Conjugal transfer of BLIR was successful from 17 isolates. PCR and sequencing of the TEM ß-lactamases from the transconjugants indicated 14 TEM-84 (IRT) and three novel IRT variants (pUE184TEM, pUE203TEM, pUE210TEM). Three-dimensional models of the latter were predicted and validated. Molecular docking of selected non-ß-lactams (morin, catechin, naringenin triacetate) with the variants using AutoDock 4.2 showed comparable docking scores with significant hydrogen bond and hydrophobic interactions. Molecular dynamics simulation study confirmed stability of the non-ß-lactams inside the catalytic pocket of the enzymes. Moreover, all three non-ß-lactams were found to inhibit the purified TEM ß-lactamase variants in vitro. Microbroth dilution method indicated naringenin triacetate 64 µg ml-1 in combination with ceftazidime (CAZ) 30 µg ml-1 to be most effective against the BLIR transconjugants. CONCLUSIONS: BLIR phenotypes were primarily attributed to the production of IRT ß-lactamases. Administration of the non-ß-lactams with CAZ demonstrated an alternative therapeutic strategy against the IRT ß-lactamase producers. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates high risk of transmission of IRT ß-lactamases and suggests ß-lactam-non-ß-lactam combination therapy to combat BLIR.
Assuntos
Antibacterianos/farmacologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Inibidores de beta-Lactamases/farmacologia , Ceftazidima/farmacologia , Cefalosporinas/farmacologia , Análise por Conglomerados , Flavanonas/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Fenótipo , Reação em Cadeia da Polimerase , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , beta-Lactamas/químicaRESUMO
Tyro3, Axl, MERTK (TAM) receptor tyrosine kinases are implicated in the regulation of the innate immune response through clearance of apoptotic cellular debris and control of cytokine signaling cascades. As a result they are pivotal in regulating the inflammatory response to tissue injury. Within the liver, immune regulatory signaling is employed to prevent the overactivation of innate immunity in response to continual antigenic challenge from the gastrointestinal tract. In this review we appraise current understanding of the role of TAM receptor function in the regulation of both innate and adaptive immunity, with a focus on its impact upon hepatic inflammatory pathology.
Assuntos
Hepatopatias/imunologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Citocinas/genética , Citocinas/metabolismo , Humanos , Imunidade Inata , Hepatopatias/patologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais , c-Mer Tirosina Quinase , Receptor Tirosina Quinase AxlRESUMO
The structural, magnetic, electronic, elastic, vibrational, optical, thermodynamic as well as thermoelectric properties of newly predicted quaternary LiZrCoX (X = Ge, Sn) Heusler compounds are evaluated intricately with the aid of ab initio techniques developed under the framework of density functional theory. The computed structural properties are found to be in tandem with the existing analogous theoretical and experimental facts. Structural optimization has been carried out in three different structural arrangements, i.e., Type-1, Type-2, and Type-3. Further analysis of the optimization curves reveals that the Type-3 phase, which has the least amount of energy, is the most stable structure for the compounds under consideration. The tabulated cohesive energy and formation energy of these compounds depict their chemical as well as thermodynamic stability. The absence of negative phonon frequencies in the phonon band spectrum of the studied compounds depicts their dynamic stability. Similarly, the tabulated second-order elastic constants (Cij) and the linked elastic moduli show their stability in the cubic phase. The calculated value of Pugh's ratio and Cauchy pressure reveal that LiZrCoGe is brittle whereas LiZrCoSn is ductile. Additionally, the optical characteristics of the compounds are studied in terms of the dielectric function, refractive index, extinction coefficient, absorption coefficient, reflectivity, energy loss function, and optical conductivity. The obtained high value of power factor and figure of merit of the studied lithium-based quaternary compounds predict good thermoelectric behavior in these compounds. Thus, LiZrCoX (X = Ge, Sn) compounds can therefore be used to create innovative and intriguing thermoelectric materials as well as optoelectronic and energy-harvesting equipment.
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Old World, monopartite begomoviruses associated with satellite DNA ß were observed in papaya showing symptoms of leaf curl disease sampled randomly over five years from within a radius of 250 km in north-central India. Three groups of DNA A sequences were evident. One group resembled chili leaf curl virus infecting tomatoes (ChiLCuV). Another group resembled tomato leaf curl New Delhi virus (ToLCuNDV). The third group was novel (tentatively named papaya leaf crumple virus, PaLCrV), with less than 89% identity to known begomovirus sequences in the GenBank database. At least seven DNA A sequences were putative recombinants. The AC4-encoding regions exhibited highest numbers of non-synonymous substitutions. Most DNA ß sequences resembled tomato leaf curl virus-associated DNA ßs. A few DNA ß sequences were similar to that of croton yellow vein mosaic virus-associated DNA ß (CroYVMVß). One DNA ß sequence was novel and showed <65% similarity to its counterparts. Mixed infections and sequence diversity among 25 cloned av1 genes indicated that papayas grown in plantations, kitchen gardens and feral patches in the region are vulnerable to disease outbreak. No geographic or temporal patterns were discernable in the distribution of these viruses.
Assuntos
Begomovirus/classificação , Begomovirus/genética , Carica/virologia , Variação Genética , Doenças das Plantas/virologia , Sequência de Aminoácidos , DNA Viral/genética , Regulação Viral da Expressão Gênica/fisiologia , Genoma Viral , Anotação de Sequência Molecular , Dados de Sequência Molecular , Filogenia , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Yellow mosaic disease causes severe yield loss in grain legumes in Indian subcontinent and south east Asia. The disease is caused by two virus species, Mungbean yellow mosaic India virus (MYMIV) and Mungbean yellow mosaic virus (MYMV). They have genome organization typical of Old World begomoviruses, the unique feature being the presence of an open reading frame (ORF) AV2 upstream of coat protein gene. In order to elucidate its function, ORF AV2 of blackgram isolate, Mungbean yellow mosaic India virus-[India:New Delhi:Blackgram 3:1991] MYMIV-[IN:ND:Bg3:91] and cowpea isolate, Mungbean yellow mosaic India virus-[India:New Delhi:Cowpea7:1998] MYMIV-[IN:ND:Cp7:98], respectively, were over expressed in Escherichia coli in fusion with maltose binding protein (MBP). The recombinant protein did not show efficient binding to DNA. However, both MBP-BgAV2 and MBP-CpAV2 proteins modulated nicking and ATPase activity of replication initiation protein (Rep). Even low concentration, 20 ng of MBP-BgAV2 and MBP-CpAV2 could bring 20 folds increase in nicking activity of Rep. Similarly in the presence of AV2 protein, two to three fold increase in ATPase activity was observed. It is hypothesized that AV2 protein may play a role of accessory protein modulating Rep activities.
Assuntos
Adenosina Trifosfatases/metabolismo , Begomovirus/enzimologia , DNA Helicases/metabolismo , DNA Viral/metabolismo , Desoxirribonuclease I/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Clonagem Molecular , Desoxirribonuclease I/genética , Escherichia coli/genética , Proteínas Virais/genéticaRESUMO
OBJECTIVE: Cystic fibrosis associated liver disease (CFLD) is the third largest cause of mortality in CF. Our aim was to define the burden of CFLD in the UK using national registry data and identify risk factors for progressive disease. METHODS: A longitudinal population-based cohort study was conducted. Cases were defined as all patients with CFLD identified from the UK CF Registry, 2008-2013 (n = 3417). Denominator data were derived from the entire UK CF Registry. The burden of CFLD was characterised. Regression analysis was undertaken to identify risk factors for cirrhosis and progression. RESULTS: Prevalence of CFLD increased from 203.4 to 228.3 per 1000 patients during 2008-2013. Mortality in CF patients with CFLD was more than double those without; cirrhotic patients had higher all-cause mortality (HR 1.54, 95% CI 1.09 to 2.18, p = 0.015). Median recorded age of cirrhosis diagnosis was 19 (range 5-53) years. Male sex, Pseudomonas airway infection and CF related diabetes were independent risk factors for cirrhosis. Ursodeoxycholic acid use was associated with prolonged survival in patients without cirrhosis. CONCLUSIONS: This study highlights an important changing disease burden of CFLD. The prevalence is slowly increasing and, importantly, the disease is not just being diagnosed in childhood. Although the role of ursodeoxycholic acid remains controversial, this study identified a positive association with survival.
Assuntos
Fibrose Cística/epidemiologia , Cistos/epidemiologia , Doenças do Sistema Digestório/epidemiologia , Cirrose Hepática/epidemiologia , Hepatopatias/epidemiologia , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Fibrose Cística/complicações , Fibrose Cística/patologia , Cistos/complicações , Cistos/patologia , Doenças do Sistema Digestório/complicações , Doenças do Sistema Digestório/patologia , Feminino , Humanos , Cirrose Hepática/complicações , Cirrose Hepática/patologia , Hepatopatias/complicações , Hepatopatias/patologia , Masculino , Fatores de Risco , Índice de Gravidade de Doença , Reino Unido/epidemiologia , Adulto JovemRESUMO
The cDNA encoding p43, a DNA binding protein from pea chloroplasts (ct) that binds to cognate DNA polymerase and stimulates the polymerase activity, has been cloned and characterised. The characteristic sequence motifs of hydroxyproline-rich glyco-proteins (HRGP) are present in the cDNA corres-ponding to the N-terminal domain of the mature p43. The protein was found to be highly O-arabinosylated. Chemically deglycosylated p43 (i.e. p29) retains its binding to both DNA and pea ct-DNA polymerase but fails to stimulate the DNA polymerase activity. The mature p43 is synthesised as a pre-p43 protein containing a 59 amino acid long transit peptide which undergoes stromal cleavage as evidenced from the post-translational in vitro import of the precursor protein into the isolated intact pea chloroplasts. Surprisingly, p43 is found only in pea chloroplasts. The unique features present in the cloned cDNA indicate that p43 is a novel member of the HRGP family of proteins. Besides p43, no other DNA-polymerase accessory protein with O-glycosylation has been reported yet.
Assuntos
Cloroplastos/enzimologia , DNA Polimerase Dirigida por DNA/metabolismo , Glicoproteínas/metabolismo , Pisum sativum/genética , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Arabinose/metabolismo , Sequência de Bases , Transporte Biológico , Cloroplastos/metabolismo , Clonagem Molecular , DNA Complementar/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Especificidade de Órgãos , Pisum sativum/citologia , Pisum sativum/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas , RNA Mensageiro/análise , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Pyridine nucleotides are critical during oxidative stress due to their roles in reductive reactions and energetics. The aim of the present study was to examine pyridine nucleotide changes in six brain regions of mice after an intracerebroventricular injection of the oxidative stress inducing agent, t-butyl hydroperoxide (t-BuOOH). A secondary aim was to investigate the correlation between NAD+ levels and DNA fragmentation. Here, we demonstrate that t-BuOOH induced a rapid oxidation of NADPH and a slow depletion of NAD+ in most brain regions. A slight increase in NADH also occurred in five brain regions. NAD+ depletion was associated with increased DNA fragmentation. This suggests the initiation of a death cascade involving poly(ADP-ribose) polymerase (PARP), NAD+, ATP depletion and consequent cell death in brain tissue. PARP activity was accelerated in some brain regions after 20 min of oxidative stress. To counteract oxidative stress induced toxicity, NAD+ levels were increased in the brain using an intraperitoneal injection of nicotinamide. A surplus of brain NAD+ prevented DNA fragmentation in some brain regions. Nicotinamide administration also resulted in higher brain NADH, NADP+ and NADPH levels in some regions. Their synthesis was further upregulated during oxidative stress. Nicotinamide as a precursor for NAD+ may provide a useful therapeutic strategy in the treatment of neurodegeneration.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Fármacos Neuroprotetores/farmacologia , Niacinamida/farmacologia , Nucleotídeos de Purina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Fragmentação do DNA , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , NADP/metabolismo , Oxirredução , Estresse Oxidativo , Poli(ADP-Ribose) Polimerases/metabolismo , terc-Butil Hidroperóxido/toxicidadeRESUMO
Oral administration of 250 mg hydrochlorothiazide/kg, with previous i.v. injection of 4 i.u. soluble insulin or s.c. injection of protamine-zinc insulin, has been found to produce stable hyperglycaemia in rabbits irrespective of sex. It is thought that insulin helps the selective action of hydrochlorothiazide on the beta cells by removing the protective glucose barrier. Although stimulation of glucagon or some extra-pancreatic mechanism at the initial stage could not be altogether ruled out, the central action of the drug is supported by the finding that the hydrochlorothiazide-induced hyperglycaemia was lowered by the sulphonylurea derivative, chlorpropamide.
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Diabetes Mellitus/induzido quimicamente , Hidroclorotiazida/farmacologia , Administração Oral , Animais , Clorpropamida/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Hidroclorotiazida/administração & dosagem , Hidroclorotiazida/antagonistas & inibidores , Insulina/administração & dosagem , Insulina de Ação Prolongada/administração & dosagem , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , CoelhosRESUMO
One group of rats received, an oral dose of 25 mg/kg body weight of Cyclosporin A daily for 21 consecutive days in olive oil, whereas the control group received an equal amount of the vehicle for the same period. On the 22nd day animals selected at random from both the groups were subjected to glucose tolerance (1.5 g/kg body weight/oral). The blood sugar values indicated glucose intolerance in experimental rats compared to vehicle-treated rats. The remaining animals were killed, and the pancreases were separated for islet isolation. After incubation of islets for 3 hr at 37 degrees with different glucose challenges, a highly significant (P less than 0.001) lowering of (pro)insulin biosynthesis, significant decrease in IRI release as well as significant inhibition in activities of acid phosphatase and cathepsin B was seen.
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Ciclosporinas/farmacologia , Insulina/biossíntese , Ilhotas Pancreáticas/metabolismo , Fosfatase Ácida/análise , Animais , Glicemia/análise , Ciclosporinas/administração & dosagem , Técnicas In Vitro , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Masculino , Proinsulina/biossíntese , RatosRESUMO
Earlier studies have demonstrated the inhibitory nature of epinephrine and norepinephrine on pancreatic insulin release. The present study reports their effect on beta-cell IRI release in an isolated islet system. Results show that epinephrine and norepinephrine inhibit islet-IRI release at the 100-10 microM level. The alpha adrenergic blocker phentolamine (100 microM) but not the beta adrenergic blocker propranolol (100 microM) can reverse this catecholamine induced inhibition of islet-IRI release. This clearly suggests that epinephrine and norepinephrine inhibit insulin release via alpha-adrenergic pathway.
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Epinefrina/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Norepinefrina/farmacologia , Animais , Técnicas In Vitro , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Fentolamina/farmacologia , Propranolol/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Nine districts in West Bengal, India, and 42 districts in Bangladesh have arsenic levels in groundwater above the World Health Organization maximum permissible limit of 50 microg/L. The area and population of the 42 districts in Bangladesh and the 9 districts in West Bengal are 92,106 km(2) and 79.9 million and 38,865 km(2) and 42.7 million, respectively. In our preliminary study, we have identified 985 arsenic-affected villages in 69 police stations/blocks of nine arsenic-affected districts in West Bengal. In Bangladesh, we have identified 492 affected villages in 141 police stations/blocks of 42 affected districts. To date, we have collected 10,991 water samples from 42 arsenic-affected districts in Bangladesh for analysis, 58,166 water samples from nine arsenic-affected districts in West Bengal. Of the water samples that we analyzed, 59 and 34%, respectively, contained arsenic levels above 50 microg/L. Thousands of hair, nail, and urine samples from people living in arsenic-affected villages have been analyzed to date; Bangladesh and West Bengal, 93 and 77% samples, on an average, contained arsenic above the normal/toxic level. We surveyed 27 of 42 districts in Bangladesh for arsenic patients; we identified patients with arsenical skin lesions in 25 districts. In West Bengal, we identified patients with lesions in seven of nine districts. We examined people from the affected villages at random for arsenical dermatologic features (11,180 and 29,035 from Bangladesh and West Bengal, respectively); 24.47 and 15.02% of those examined, respectively, had skin lesions. After 10 years of study in West Bengal and 5 in Bangladesh, we feel that we have seen only the tip of iceberg.
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Arsênio/análise , Poluentes Químicos da Água/análise , Arsênio/toxicidade , Arsênio/urina , Bangladesh , Saúde Ambiental , Cabelo/química , Humanos , Índia , Concentração Máxima Permitida , Unhas/química , Segurança , Pele/química , Dermatopatias/induzido quimicamente , Poluentes Químicos da Água/toxicidade , Poluentes Químicos da Água/urinaRESUMO
DNA is a primary site of damage during oxidative stress in the brain. DNA fragmentation occurs within minutes of induction of oxidative stress. This DNA fragmentation probably results from the attack of free radicals on DNA and from the activation of endonucleases. Oxidative stress was induced by intracerebroventricular injection of t-butylhydroperoxide. This results in a very rapid flux of t-butylhydroperoxide, which is cleared from the brain within minutes. This flux of t-butylhydroperoxide results in the formation of hydroxyl radical in the brain and probably in the nuclei of brain cells. Necrosis results from extensive DNA fragmentation caused by massive oxidative stress. Cresyl violet stained brain sections demonstrated necrosis in many brain regions. In addition, previous electron microscopy studies showed degradation of cellular nuclei caused by tBuOOH toxicity. Low doses of t-butylhydroperoxide can induce apoptosis, which is a delayed form of cell death. Apoptosis was found in brains stained to visualize apoptotic DNA fragments. Experiments performed in mice aged 2, 8 or 24 months will be discussed. We have also found that apoptosis and DNA fragmentation can be prevented by pretreating mice with the vitamin micotinamide. Nicotinamide is a precursor for NAD. DNA repair requires high levels of NAD in the nucleus for the activity of poly(ADP-ribose) polymerase. Oxidative stress in the brain produces both necrosis and apoptosis, probably as the result of DNA fragmentation. Senescence is associated with an increase in the production of DNA fragments during brain oxidative stress, which probably leads to more necrosis and apoptosis than in younger mice.
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Envelhecimento/metabolismo , Apoptose , Encéfalo/metabolismo , Estresse Oxidativo , Animais , Córtex Cerebral/metabolismo , DNA/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Tálamo/metabolismoRESUMO
Apoptosis is a characteristic form of cell death which has been implicated in neurodegeneration. In this study we document the induction of apoptosis and DNA fragmentation in vivo by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin. MPTP selectively damages dopaminergic neurons in the substantia nigra of the midbrain. It is a potent inducer of oxygen radicals. Nicotinamide, a precursor of NAD, is able to block the apoptosis induced by MPTP. Nicotinamide also quenches some of the radicals formed by xanthine oxidase. Nicotinamide may be of interest in the treatment of neurodegeneration.
Assuntos
Apoptose/efeitos dos fármacos , Encéfalo/enzimologia , NAD/fisiologia , Neurônios/efeitos dos fármacos , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/farmacologia , Animais , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , NAD/metabolismo , Niacinamida/farmacologia , Substância Negra/citologia , Substância Negra/enzimologiaRESUMO
The vitamin nicotinamide can protect against oxidative stress-induced apoptosis in the brain when used as a precursor for nicotinamide adenine dinucleotide (NAD+). The intracerebroventricular administration of tertiary-butylhydroperoxide (t-buOOH) to mice was used to simulate physiologic oxidative stress and apoptosis which may occur in some neurodegenerative conditions. t-buOOH produced characteristic apoptotic nuclear degeneration in neurons with extensive fragmentation of DNA. In this report we show that the elevation of NAD+ by nicotinamide prevents DNA fragmentation during apoptosis or necrosis in the brain as stimulated by t-buOOH administration. NAD+ levels can be increased by 50% in the brain. This may prevent the critical depletion of NAD+ by poly(ADP-ribose) polymerase (PARP) and provide additional substrate during the repair of DNA. Nicotinamide may be of particular interest in the treatment of neurodegeneration.
Assuntos
Apoptose/efeitos dos fármacos , Encéfalo/citologia , NAD/metabolismo , Niacinamida/farmacologia , Peróxidos/antagonistas & inibidores , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , DNA/metabolismo , Reparo do DNA , Imuno-Histoquímica , Injeções Intraventriculares , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/efeitos dos fármacos , Niacinamida/metabolismo , Estresse Oxidativo , Peróxidos/administração & dosagem , Peróxidos/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , terc-Butil HidroperóxidoRESUMO
In this study, the effect of intracerebroventricular administration of the free radical generator, tertiary butylhydroperoxide, on DNA, was quantitated. Previous studies had established DNA as a very important site of free radical attack. The purpose of the study was to detect whether DNA was one of the primary targets of the toxin as well as to detect any apoptosis that may have been induced by the toxin. The DNA fragmentation assay clearly showed DNA damage within 20 min of administration of 109.7 mg/kg t-BuOOH almost in all brain regions in both 2-month and 8-month-old C57BL/6 mice. In Situ Apoptosis Detection assay, where brain sections were stained with Apoptag, demonstrated that t-BuOOH induces apoptosis in many brain regions. Electron microscopy was done to show nuclear damage and DNA fragments appearing in the cytoplasm. Cresyl violet staining was done to show that while low dose (21.9 mg/kg) t-BuOOH induces apoptosis, it may also induce necrosis in other cells of the same brain region. Thus, from this study we can conclude that DNA may be one of the primary target sites of free radical attack in the brain, and results in both necrosis and apoptosis. This can have a profound effect on neurodegeneration.
Assuntos
Apoptose , Encéfalo/efeitos dos fármacos , DNA/efeitos dos fármacos , Peróxidos/farmacologia , Animais , Radicais Livres/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Necrose/induzido quimicamente , Degeneração Neural/fisiologia , Putamen/efeitos dos fármacos , Putamen/fisiopatologia , terc-Butil HidroperóxidoRESUMO
Combinations of nucleoside analog drugs, such as F-araA and ara-C, combined with Topoisomerase II inhibitors, such as anthracyclines, are synergistic against human leukemic T-cells and induce apoptotic cell death. Similarly, nucleoside analog drugs followed by mitotic inhibitors also have a synergistic effect. Sequence specific combinations of F-araA followed by ara-C and Taxotere (docetaxel) in CEM/0 cells showed a 2- to 3-fold synergism over the two drug (F-araA + ara-C) combinations and 2- to 4-fold synergism over Taxotere alone. This synergism was evident due to enhanced cellular apoptosis. In the CEM/ara-C/7A cell line, which is partially resistant to ara-C, the synergy observed with the triple drug combination was 9-fold greater than the F-araA plus araC combination, and 3-fold greater than Taxotere alone, making this three-drug regimen collaterally sensitive to ara-C. This study describes the mechanisms of the synergistic effect in regards to apoptosis achieved by three-drug regimens comprised of two nucleoside analog drugs and a mitotic inhibitor in comparison with the combination of two nucleotide analog drugs. The study also demonstrates that the possible biochemical mechanism of cellular toxicity and drug synergism is attributed to induction of apoptosis following drug treatment and the onset of the apoptotic cascade is primarily regulated by p21/WAF-I, which is transcriptionally activated by p53 following DNA damage. The anti-apoptotic protein, bcl-2, seemed to have no effect in inhibiting apoptosis following treatment with the two or three drug regimens in this in vitro leukemia model. The three-drug combination induced greater cellular apoptosis than the two-drug combination or Taxotere monotherapy. We conclude that the greater drug synergism observed in human leukemic cells, sensitive or resistant to ara-C, by Fludarabine + ara-C + Taxotere can be explained by the greater oligonucleosomal DNA fragmentation indicative of increased cellular apoptosis. The mechanism of this increased cytotoxic action is due to the upregulation of p53 and p21/WAF-1 with a down regulation of bcl-2. These studies are encouraging, and testing this three drug regimen in a clinical setting may result in improved antileukemic therapies.
Assuntos
Apoptose/fisiologia , Ciclinas/biossíntese , Citarabina/toxicidade , Paclitaxel/análogos & derivados , Taxoides , Proteína Supressora de Tumor p53/biossíntese , Vidarabina/análogos & derivados , Apoptose/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21 , Docetaxel , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Inibidores Enzimáticos , Humanos , Cinética , Leucemia de Células T , Matemática , Mitose/efeitos dos fármacos , Modelos Biológicos , Paclitaxel/toxicidade , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Transcrição Gênica , Células Tumorais Cultivadas , Vidarabina/toxicidadeRESUMO
The major limitation of treatment with antimetabolite drugs is that they produce resistant clones both in vitro and in patients who either do not respond to treatment or relapse soon after response has been documented. To better understand the phenomenon of cross-resistance, we developed seven CEM/ara-C-resistant leukemic clones from the CEM/0 (wt) cell line. These clones ranged from 4- to 3.5 x 10(8)-fold more resistant to ara-C than the wt CEM/0 cell line. Using this model, we determined IC50 concentrations to several chemotherapeutic agents and gamma radiation, and we also studied pro- (p53) and anti-apoptotic (bcl-2) proteins, as well as P-glycoprotein (P-gp) and multidrug resistance related protein (MRP). The cell viability assays showed that these clones were cross-resistant to 6-thioguanine (6-TG) or 6-mercaptoguanosine (6-TGuo) from 1.1- to 8.8-fold with ara-C; cross-resistance to vincristine (VCR) was from 200- to 1 x 10(4)-fold with ara-C. Taxotere (TXR) showed cross-resistance with ara-C from 1.39- to 3.03 x 10(3)-fold; dexamethasone (DEX) also showed a significant degree of cross-resistance from 27.4- to 3.87 x 10(7)-fold. Gamma radiation treatments from 0.77 Gy to 12.3 Gy showed a radiation dose-dependent cross-resistance with ara-C from 1.43- to 2.93-fold. Idarubicin was collaterally sensitive with ara-C from 4.6- to 1 x 10(9)-fold in these cell lines. The CEM/ara-C/G resistant cell line was 3-fold more sensitive to 6-TG or VCR than CEM/0 (wt), and 5-fold more sensitive to 6-TGuo. This cell clone expressed p53 and did not overexpress bcl-2 protein. All of the cell lines studied, CEM/0 (wt) and the ara-C resistant clones, showed functional p53 protein. The cell treatment with 0.1, 1 and 10 microM ara-C for 48 hours showed increased p53 protein expression in most of these lines. No increase in bcl-2 protein expression was seen in the wt cell line after ara-C treatment for 48 hours. Three cell lines resistant to ara-C (CEM/ara-C/B, CEM/ara-C/D and CEM/ara-C/I) showed an important increased expression of bcl-2 protein after treatment with 1 microM ara-C, but not after 10 microM. This alteration may lead to resistance to apoptosis and enhanced cell survival. The ratio of bcl-2 to p53 was increased significantly in these three clones, thus favoring an anti-apoptotic drive. All of the cell lines examined were negative for MRP expression and only two, CEM/ara-C/B and CEM/ara-C/J, were positive for MRP functional activity. However, three ara-C resistant cell clones, CEM/ara-C/7A, CEM/ara-C/B and CEM/ara-C/G, were positive for P-gp expression and functional activity. It is apparent that selection for ara-C resistance confers cross-resistance to many other classes of drugs and gamma radiation, probably due to bcl-2 protein overexpression or P-gp and MRP expression, as independent mechanisms.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Citarabina/farmacologia , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Guanosina/análogos & derivados , Leucemia/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Taxoides , Tioguanina/farmacologia , Tionucleosídeos/farmacologia , Vincristina/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/análise , Transportadores de Cassetes de Ligação de ATP/análise , Dexametasona/farmacologia , Docetaxel , Raios gama , Guanosina/farmacologia , Humanos , Leucemia/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Proteínas de Neoplasias/análise , Células-Tronco Neoplásicas/química , Células-Tronco Neoplásicas/efeitos da radiação , Paclitaxel/análogos & derivados , Paclitaxel/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/análise , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/efeitos da radiação , Proteína Supressora de Tumor p53/análiseRESUMO
AIMS AND OBJECTIVES: To ascertain whether therapeutic endoscopic retrograde cholangiopancreatography (ERCP) for benign biliary disease in frail elderly patients with comorbid conditions can be safely undertaken in a district general hospital, and whether the procedure is facilitated by the use of short-acting general anaesthesia. SETTING: District general hospital in South East England. DESIGN OF STUDY: Clinical study of 25 consecutive patients with benign biliary disease. METHODS: Describes the process of bile duct clearance by therapeutic ERCP under short-acting general anaesthesia in 25 patients with co-morbidity aged > or = 80 years and gives details of the general anaesthesia and monitoring. RESULTS: Twenty-two patients had their bile ducts successfully cleared locally and one patient was stented for a benign biliary stricture. The ampullae of two other patients were lying within diverticula, which hindered cannulation and only pancreatograms were obtained; one of the patients had a successful bile duct clearance at a tertiary centre, the other refused further intervention. Complications (melaena, bronchopneumonia and a Clostridium difficile infection) occurred in two patients (8%). There was no morbidity associated with the anaesthesia, and no mortality occurred within 30 days of the procedure. CONCLUSIONS: Bile duct clearance by therapeutic ERCP can be safely carried out in frail elderly patients in a district general hospital and the process is facilitated by the use of short-acting general anaesthesia. The importance of optimizing the patient's condition before ERCP, and not overfilling the pancreatic duct, is highlighted.