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1.
BMC Cancer ; 20(1): 1175, 2020 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-33261560

RESUMO

BACKGROUND: Early diagnosis is important for the timely treatment of gallbladder carcinoma (GBC) patients and may lead to increased survival outcomes. Here, we have applied serological proteome analysis (SERPA), an immunoproteomics approach, for the detection of 'tumor-associated antigens (TAAs) that elicit humoral response' in early stage GBC patients. METHODS: Total protein from pooled tumor tissue of GBC patients (n = 7) was resolved by two-dimensional gel electrophoresis (2-DE) followed by immunoblotting using pooled blood plasma from healthy volunteers (n = 11) or gallstone disease (GSD) cases (n = 11) or early stage GBC (Stage I and II) (n = 5) or GBC stage IIIA (n = 9). 2-D gel and immunoblot images were acquired and analyzed using PDQuest software to identify immunoreactive spots in GBC cases in comparison to controls. Proteins from immunoreactive spots were identified by liquid chromatography- tandem mass spectrometric analysis (LC-MS/MS). Autoantibody levels for two of the functionally relevant proteins were investigated in individual plasma samples (52 cases and 89 controls) by dot blot assay using recombinant proteins. RESULTS: Image analysis using PDQuest software identified 25 protein spots with significantly high or specific immunoreactivity in GBC cases. Mass spectrometric analysis of 8 corresponding protein spots showing intense immunoreactivity (based on densitometric analysis) in early stage GBC or GBC stage IIIA cases led to the identification of 27 proteins. Some of the identified proteins include ANXA1, HSPD1, CA1, CA2, ALDOA and CTSD. Among the two proteins, namely ANXA1 and HSPD1 verified using a cohort of samples, significantly elevated autoantibody levels against ANXA1 were observed in early stage GBC cases in comparison to healthy volunteers or GSD cases (unpaired t-test, p < 0.05). Receiver operating characteristic (ROC) curve analysis for ANXA1 showed an Area under the Curve (AUC) of 0.69, with 41.7% sensitivity against a specificity of 89.9% for early stage GBC. IHC analysis for ANXA1 protein showed 'high' expression levels in 72% of GBC cases whereas all the controls showed 'low' expression levels. CONCLUSIONS: The study suggests that the ANXA1 autoantibody levels against ANXA1 may be potentially employed for early stage detection of GBC patients. Other proteins could also be explored and verified in a large cohort of clinical samples.

2.
Neuroreport ; 30(16): 1087-1094, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31503205

RESUMO

OBJECTIVE: Growing evidences suggest systemic pathogen-induced neuroimmune interaction is a major risk factor for several neurological disorders. Our goal was to investigate whether asymptomatic peripheral carriage of Staphylococcus aureus, a widespread opportunistic pathogen, could modulate selective molecular features in brain tissues. METHODS: To address this, a peripheral infection model was developed by challenging Wistar rats repeatedly with a clinical strain of S. aureus. Animals infected with S. aureus (10 CFU for three times in 10 days) showed significant changes in acetylation profile of selective lysine (K) residues K9 (H3K9), K14 (H3K14) and K27 (H3K27) of histone H3 in the hippocampus and prefrontal cortex (PFC). RESULTS: Although S. aureus was restricted peripherally, the infection induced hypoacetylation of H3K9, H3K14 and H3K27 in the hippocampus and H3K27 in the PFC. Histone H3 hypoacetylation in the hippocampus and PFC was also detected when rats were challenged with an engineered invasive strain of E. coli K12, SK3842. This confirmed that modulation of epigenetic landscape in distal brain tissues may not be specific to S. aureus. Moreover, the tyrosine hydroxylase protein, the rate limiting enzyme in dopamine synthesis pathway whose gene-expression is regulated by H3 acetylation at the promoter, was remarkably reduced in the brain tissues of the infected hosts. CONCLUSION: The results indicate that commensals like S. aureus, in spite of being largely restricted to the peripheral tissues, could modulate the homeostasis of molecular features in brain tissues whose maintenance is critical for preserving normal neurological functions.


Assuntos
Encéfalo/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Acetilação , Animais , Escherichia coli , Expressão Gênica/genética , Histona Desacetilases/genética , Masculino , Regiões Promotoras Genéticas/genética , Ratos Wistar , Staphylococcus aureus
3.
IEEE Trans Biomed Eng ; 65(3): 608-618, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28541892

RESUMO

OBJECTIVE: Diabetic retinopathy (DR) is characterized by the progressive deterioration of retina with the appearance of different types of lesions that include microaneurysms, hemorrhages, exudates, etc. Detection of these lesions plays a significant role for early diagnosis of DR. METHODS: To this aim, this paper proposes a novel and automated lesion detection scheme, which consists of the four main steps: vessel extraction and optic disc removal, preprocessing, candidate lesion detection, and postprocessing. The optic disc and the blood vessels are suppressed first to facilitate further processing. Curvelet-based edge enhancement is done to separate out the dark lesions from the poorly illuminated retinal background, while the contrast between the bright lesions and the background is enhanced through an optimally designed wideband bandpass filter. The mutual information of the maximum matched filter response and the maximum Laplacian of Gaussian response are then jointly maximized. Differential evolution algorithm is used to determine the optimal values for the parameters of the fuzzy functions that determine the thresholds of segmenting the candidate regions. Morphology-based postprocessing is finally applied to exclude the falsely detected candidate pixels. RESULTS AND CONCLUSIONS: Extensive simulations on different publicly available databases highlight an improved performance over the existing methods with an average accuracy of and robustness in detecting the various types of DR lesions irrespective of their intrinsic properties.


Assuntos
Retinopatia Diabética/diagnóstico por imagem , Técnicas de Diagnóstico Oftalmológico , Interpretação de Imagem Assistida por Computador/métodos , Retina/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Algoritmos , Humanos , Programas de Rastreamento , Pessoa de Meia-Idade
4.
PLoS One ; 12(7): e0180953, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28732061

RESUMO

Oxaliplatin (Oxa) treatment to SH-SY5Y human neuroblastoma cells has been shown by previous studies to induce oxidative stress, which in turn modulates intracellular signaling cascades resulting in cell death. While this phenomenon of Oxa-induced neurotoxicity is known, the underlying mechanisms involved in this cell death cascade must be clarified. Moreover, there is still little known regarding the roles of neuronal mitochondria and cytosolic compartments in mediating Oxa-induced neurotoxicity. With a better grasp of the mechanisms driving neurotoxicity in Oxa-treated SH-SY5Y cells, we can then identify certain pathways to target in protecting against neurotoxic cell damage. Therefore, the purpose of this study was to determine whether one such agent, melatonin (Mel), could confer protection against Oxa-induced neurotoxicity in SH-SY5Y cells. Results from the present study found Oxa to significantly reduce SH-SY5Y cell viability in a dose-dependent manner. Alternatively, we found Mel pre-treatment to SH-SY5Y cells to attenuate Oxa-induced toxicity, resulting in a markedly increased cell viability. Mel exerted its protective effects by regulating reactive oxygen species (ROS) production and reducing superoxide radicals inside Oxa-exposed. In addition, we observed pre-treatment with Mel to rescue Oxa-treated cells by protecting mitochondria. As Oxa-treatment alone decreases mitochondrial membrane potential (Δψm), resulting in an altered Bcl-2/Bax ratio and release of sequestered cytochrome c, so Mel was shown to inhibit these pathways. Mel was also found to inhibit proteolytic activation of caspase 3, inactivation of Poly (ADP Ribose) polymerase, and DNA damage, thereby allowing SH-SY5Y cells to resist apoptotic cell death. Collectively, our results suggest a role for melatonin in reducing Oxa induced neurotoxicity. Further studies exploring melatonin's protective effects may prove successful in eliciting pathways to further alter the neurotoxic pathways of platinum compounds in cancer treatment.


Assuntos
Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Melatonina/farmacologia , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Compostos Organoplatínicos/toxicidade , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citocromos c/metabolismo , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , Oxaliplatina , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Proteína X Associada a bcl-2/metabolismo
5.
Cell Death Dis ; 8(3): e2667, 2017 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-28300841

RESUMO

Commensal Escherichia coli has been identified as a major protagonist of microbe-induced colorectal oncogenesis. Its tumour-promoting attribute is linked to the expression of DNA-damaging genotoxins. Using a constitutively invasive variant of non-pathogenic E. coli, we demonstrate that chronic presence of internalized E. coli leads to enhanced oncogenicity in colon cancer cells. Instead of genomic damage, the tumorigenic effect is mediated through an expansion of the cancer stem cell (CSC) population, likely through dedifferentiation of lineage-committed intestinal epithelial cells. Stemness-linked intestinal tumorigenicity is directly correlated to absence of microbial virulence factor expression and is specific for intestinal cells. The enriched CSC fraction remains stable in the absence of the instigating bacteria and can foster stemness traits in unexposed cells through secreted factors. Mechanistically, aberrant host invasion leads to realignment of multiple host signal transduction cascades, notably mutually re-enforcing NF-κB and ß-catenin activation, through reciprocal modulation of microbe sensing pathways Nod1/Rip2 and TLR/MyD88. The expanded tumorigenic CSC population is marked by enhanced malignancy traits, long-term self-renewal capacity and robust tumorigenic ability, both in vitro and in vivo. Our study shows that microbe-induced oncogenicity is not a strict correlate of commensal virulence and can be invoked by even non-pathogenic E. coli by engendering tumorigenic stemness in host cells.


Assuntos
Carcinogênese/metabolismo , Escherichia coli/patogenicidade , Intestinos/microbiologia , Intestinos/patologia , Células-Tronco Neoplásicas/microbiologia , Células-Tronco Neoplásicas/patologia , Animais , Células CACO-2 , Carcinogênese/patologia , Linhagem Celular Tumoral , Colo/metabolismo , Colo/microbiologia , Colo/patologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Células HCT116 , Células HT29 , Células Hep G2 , Humanos , Mucosa Intestinal/metabolismo , Masculino , Camundongos , Camundongos Nus , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Células-Tronco Neoplásicas/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais/fisiologia , beta Catenina/metabolismo
6.
Mol Neurobiol ; 54(9): 6960-6969, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-27785753

RESUMO

Phytanic acid, a saturated branched chain fatty acid and a major constituent of human diet, is predominantly found in dairy products, meat, and fish. It is a degradation product from the phytol side chain of chlorophyll. Degradation of PA is known to occur mainly in peroxisomes via α-oxidation and in mitochondria via ß-oxidation. Due to its ß-methyl group present at the 3-position of the carbon atoms, PA cannot be ß-oxidized. Although alteration in the metabolism of PA may play an important role in neurodegeneration, the exact mechanism behind it remains to be evaluated. In this study, we have described the potential of PA to induce neurotoxicity as an in vitro model (neuronal cell line, SH-SY5Y cells). Cells were pretreated with melatonin (10 µM) for 1 h followed by with and without PA (100 µM) for 24 h. In the present study, our data has confirmed that PA markedly increased both intracellular reactive oxygen species and reactive nitrogen species levels. Our results have shown that PA treatment did not induce cell death by cleavage of caspase-3/PARP-1 mediated by mitochondria through intrinsic pathways; however, PA induced nitric oxide-dependent apoptosis in SH-SY5Y cells. Additionally, melatonin pretreatment reduced the cell death in SH-SY5Y cells. Melatonin also effectively exerted an antiapoptotic and anti-inflammatory action by regulating Bax, Bcl-2, p-NFκB, and iNOS expressions in SH-SY5Y cells. These results suggested that melatonin acted as an antioxidative and antiapoptotic agent by modulating ROS, apoptotic proteins, and inflammatory responses under BCFA-induced neurotoxic conditions. The protective effects of melatonin depend on direct scavenging activity of free radicals and indirect antioxidant effects. Further deciphering of the cellular and molecular mechanism associated with neuroprotection by melatonin is warranted in BCFA-induced neurotoxicity.


Assuntos
Apoptose/efeitos dos fármacos , Melatonina/farmacologia , Mitocôndrias/metabolismo , Neurotoxinas/toxicidade , Ácido Fitânico/toxicidade , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/efeitos dos fármacos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/metabolismo
7.
Comput Methods Programs Biomed ; 133: 111-132, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27393804

RESUMO

BACKGROUND AND OBJECTIVES: Extraction of blood vessels on retinal images plays a significant role for screening of different opthalmologic diseases. However, accurate extraction of the entire and individual type of vessel silhouette from the noisy images with poorly illuminated background is a complicated task. To this aim, an integrated system design platform is suggested in this work for vessel extraction using a sequential bandpass filter followed by fuzzy conditional entropy maximization on matched filter response. METHODS: At first noise is eliminated from the image under consideration through curvelet based denoising. To include the fine details and the relatively less thick vessel structures, the image is passed through a bank of sequential bandpass filter structure optimized for contrast enhancement. Fuzzy conditional entropy on matched filter response is then maximized to find the set of multiple optimal thresholds to extract the different types of vessel silhouettes from the background. Differential Evolution algorithm is used to determine the optimal gain in bandpass filter and the combination of the fuzzy parameters. Using the multiple thresholds, retinal image is classified as the thick, the medium and the thin vessels including neovascularization. RESULTS: Performance evaluated on different publicly available retinal image databases shows that the proposed method is very efficient in identifying the diverse types of vessels. Proposed method is also efficient in extracting the abnormal and the thin blood vessels in pathological retinal images. The average values of true positive rate, false positive rate and accuracy offered by the method is 76.32%, 1.99% and 96.28%, respectively for the DRIVE database and 72.82%, 2.6% and 96.16%, respectively for the STARE database. Simulation results demonstrate that the proposed method outperforms the existing methods in detecting the various types of vessels and the neovascularization structures. CONCLUSIONS: The combination of curvelet transform and tunable bandpass filter is found to be very much effective in edge enhancement whereas fuzzy conditional entropy efficiently distinguishes vessels of different widths.


Assuntos
Entropia , Lógica Fuzzy , Vasos Retinianos , Humanos
8.
Comput Biol Med ; 70: 174-189, 2016 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-26848729

RESUMO

This paper proposes an automatic blood vessel extraction method on retinal images using matched filtering in an integrated system design platform that involves curvelet transform and kernel based fuzzy c-means. Since curvelet transform represents the lines, the edges and the curvatures very well and in compact form (by less number of coefficients) compared to other multi-resolution techniques, this paper uses curvelet transform for enhancement of the retinal vasculature. Matched filtering is then used to intensify the blood vessels' response which is further employed by kernel based fuzzy c-means algorithm that extracts the vessel silhouette from the background through non-linear mapping. For pathological images, in addition to matched filtering, Laplacian of Gaussian filter is also employed to distinguish the step and the ramp like signal from that of vessel structure. To test the efficacy of the proposed method, the algorithm has also been applied to images in presence of additive white Gaussian noise where the curvelet transform has been used for image denoising. Performance is evaluated on publicly available DRIVE, STARE and DIARETDB1 databases and is compared with the large number of existing blood vessel extraction methodologies. Simulation results demonstrate that the proposed method is very much efficient in detecting the long and the thick as well as the short and the thin vessels with an average accuracy of 96.16% for the DRIVE and 97.35% for the STARE database wherein the existing methods fail to extract the tiny and the thin vessels.


Assuntos
Modelos Cardiovasculares , Disco Óptico/irrigação sanguínea , Disco Óptico/cirurgia , Procedimentos Cirúrgicos Vasculares/métodos , Feminino , Humanos , Masculino
9.
Soft Matter ; 11(7): 1345-51, 2015 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-25574757

RESUMO

In this study two positional isomeric dipeptides Boc-m-ABA-Aib-OMe () and Boc-Aib-m-ABA-OMe () synthesized by reversal of the positions of two rigid amino acids (m-ABA: m-aminobenzoic acid, Aib: α-aminoisobutyric acid) showed marked difference in morphology under the same environmental conditions. Investigation of single crystal structures reveals the difference in crystal packing and higher order self-assembly pattern for both the isomeric peptides, which might be the responsible factor for their different morphological patterns. Moreover, these isomeric dipeptides have produced different cellular viability effects towards normal bone cells. These two peptides would have utilities in the model study of isomeric peptides/proteins, where morphological difference under identical conditions brings changes in their individual bio-activities and where the reversal of sequence causes different cellular viability and generates health hazard.


Assuntos
Dipeptídeos/química , Animais , Linhagem Celular , Dipeptídeos/síntese química , Dipeptídeos/toxicidade , Isomerismo , Camundongos , Nanotubos/química , Osteoblastos/efeitos dos fármacos , Polimerização , Estrutura Secundária de Proteína
10.
Soft Matter ; 10(40): 8075-82, 2014 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-25170841

RESUMO

A single ω-amino acid based molecule "Boc-ß-Ala-N,N'-dicyclohexylurea" can form diverse nanostructures such as nano-vesicles, nano-tubes, nano-rods and nano-fibrils by self-assembly, in response to various environmental conditions. Interestingly, the nano-vesicular structures generated from this molecule can encapsulate the highly potent anticancer drug methotrexate, which can be released by salt triggered disruption of these vesicles. This phenomenon indicates the probability of its use in targeted delivery of drugs or any bio-active molecule, utilizing this encapsulation efficiency. Moreover, a surface-induced morphological transformation of these nano-vesicles to nano-fibers can be realized while they interact with hydrocarbon-functionalized surfaces. This phenomenon indicates the probability of their utilization in model study of peptide interaction behavior at liquid-solid interfaces and can be used in advanced study of various biological phenomena and bio-technological applications.


Assuntos
Alanina/química , Portadores de Fármacos/química , Metotrexato/química , Nanoestruturas/química , Ureia/análogos & derivados , Ureia/química
11.
Chem Commun (Camb) ; 50(20): 2638-41, 2014 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-24469335

RESUMO

A small peptide mimetic molecule can form diverse nanostructures such as nano-vesicles, nano-tubes and nano-ribbons/fibrils by self-assembly, in response to various physical and chemical stimulations.


Assuntos
Biomimética , Nanoestruturas/química , Peptídeos/química , Cristalografia por Raios X , Microscopia Eletrônica de Varredura
12.
Bioeng Bugs ; 3(2): 133-7, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22539026

RESUMO

The status of E. coli K-12 as an exclusively non-invasive, non-pathogenic bacterium has almost been incontrovertible. Our recent finding that a mutation in one of its main architectural protein, HU, converts E. coli K-12 to an actively invasive form suggests that gaining host cell entry might be an expedient survival tactic for traditional commensals during certain altered host conditions. The mutant E. coli (SK3842) exhibits properties usually associated with pathogenic bacteria: host cell invasion, phagosomal disruption and intracellular replication. However, unlike the situation with some pathogens, internalized SK3842 imparts anti-apoptotic and cyto-protective effects rather than lethality on the host cell, both in vitro and in vivo. Here, we show that SK3842 also provides colonization resistance against other invasive pathogens--a trait not shared by the parental commensal strain. Thus, the altered lifestyle of SK3842 encompasses characteristics both from traditional pathogens as well as beneficial probiotic strains.


Assuntos
Escherichia coli K12/classificação , Escherichia coli K12/crescimento & desenvolvimento , Apoptose/efeitos dos fármacos , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Escherichia coli K12/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Probióticos/metabolismo
13.
mBio ; 2(5)2011.
Artigo em Inglês | MEDLINE | ID: mdl-21896677

RESUMO

UNLABELLED: The HUα(E38K, V42L) mutant of the bacterial histone-like protein HU causes a major change in the transcription profile of the commensal organism Escherichia coli K-12 (Kar S, Edgar R, Adhya S, Proc. Natl. Acad. Sci. U. S. A. 102:16397-16402, 2005). Among the upregulated genes are several related to pathogenic interactions with mammalian cells, as evidenced by the expression of curli fibers, Ivy, and hemolysin E. When E. coli K-12/ HUα(E38K, V42L) was added to Int-407 cells, there was host cell invasion, phagosomal disruption, and intracellular replication. The invasive trait was also retained in a murine ileal loop model and intestinal explant assays. In addition to invasion, the internalized bacteria caused a novel subversion of host cell apoptosis through modification and regulation of the BH3-only proteins Bim(EL) and Puma. Changes in the transcription profile were attributed to positive supercoiling of DNA leading to the altered availability of relevant promoters. Using the E. coli K-12/HUα(E38K, V42L) variant as a model, we propose that traditional commensal E. coli can adopt an invasive lifestyle through reprogramming its cellular transcription, without gross genetic changes. IMPORTANCE: Escherichia coli K-12 is well established as a benign laboratory strain and a human intestinal commensal. Recent evidences, however, indicate that the typical noninvasive nature of resident E. coli can be reversed under specific circumstances even in the absence of any major genomic flux. We previously engineered an E. coli strain with a mutant histone-like protein, HU, which exhibited significant changes in nucleoid organization and global transcription. Here we showed that the changes induced by the mutant HU have critical functional consequences: from a strict extracellular existence, the mutant E. coli adopts an almost obligate intracellular lifestyle. The internalized E. coli exhibits many of the prototypical characteristics of traditional intracellular bacteria, like phagosomal escape, intracellular replication, and subversion of host cell apoptosis. We suggest that E. coli K-12 can switch between widely divergent lifestyles in relation to mammalian host cells by reprogramming its cellular transcription program and without gross changes in its genomic content.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Escherichia coli K12/genética , Escherichia coli K12/patogenicidade , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação de Sentido Incorreto , Animais , Apoptose , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Feminino , Expressão Gênica , Humanos , Íleo/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Virulência
14.
Protein Pept Lett ; 18(9): 886-97, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21443496

RESUMO

In the biomimetic design two hydrophobic pentapetides Boc-Ile-Aib-Leu-Phe-Ala-OMe (I) and Boc-Gly-Ile-Aib-Leu-Phe-OMe (II) (Aib: α-aminoisobutyric acid) containing one Aib each are found to undergo solvent assisted self-assembly in methanol/water to form vesicular structures, which can be disrupted by simple addition of acid. The nanovesicles are found to encapsulate dye molecules that can be released by the addition of acid as confirmed by fluorescence microscopy and UV studies. The influence of solvent polarity on the morphology of the materials generated from the peptides has been examined systematically, and shows that fibrillar structures are formed in less polar chloroform/petroleum ether mixture and vesicular structures are formed in more polar methanol/water. Single crystal X-ray diffraction studies reveal that while ß-sheet mediated self-assembly leads to the formation of fibrillar structures, the solvated ß-sheet structure leads to the formation of vesicular structures. The results demonstrate that even hydrophobic peptides can generate vesicular structures from polar solvent which may be employed in model studies of complex biological phenomena.


Assuntos
Ácidos Aminoisobutíricos/química , Materiais Biomiméticos/química , Corantes/administração & dosagem , Oligopeptídeos/química , Sequência de Aminoácidos , Cristalografia por Raios X , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Estrutura Secundária de Proteína , Solventes
15.
Protein Pept Lett ; 16(9): 1063-73, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19508219

RESUMO

Single crystal X-ray diffraction studies and solvent dependent (1)H NMR titrations reveal that a set of four tetrapeptides with general formula Boc-Xx(1)-Aib(2)-Yy(3)-Zz(4)-OMe, where Xx, Yy and Zz are coded L-amino acids, adopt equivalent conformations that can be described as overlapping double turn conformations stabilized by two 4-->1 intramolecular hydrogen bonds between Yy(3)-NH and Boc C=O and Zz(4)-NH and Xx(1)C=O. In the crystalline state, the double turn structures are packed in head-to-tail fashion through intermolecular hydrogen bonds to create supramolecular helical structures. Field emission scanning electron microscopic (FE-SEM) images of the tetrapeptides in the solid state reveal that they can form flat tape-like structures. The results establish that synthetic Aib containing supramolecular helices can form highly ordered self-aggregated amyloid plaque like human amylin.


Assuntos
Ácidos Aminoisobutíricos/química , Oligopeptídeos/química , Amiloide/química , Cristalização , Cristalografia por Raios X , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Microscopia Eletrônica de Varredura , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Estrutura Secundária de Proteína
16.
J Biol Chem ; 281(52): 40144-53, 2006 Dec 29.
Artigo em Inglês | MEDLINE | ID: mdl-17062578

RESUMO

In bacteria, the contribution of global nucleoid organization in determining cellular transcription programs is unclear. Using a mutant form of the most abundant nucleoid-associated protein HU, HUalpha(E38K,V42L), we previously showed that nucleoid remodeling by the mutant protein re-organizes the global transcription pattern. Here, we demonstrate that, unlike the dimeric wild-type HU, HUalpha(E38K,V42L) is an octamer and wraps DNA around its surface. The formation of wrapped nucleoprotein complexes by HUalpha(E38K,V42L) leads to a high degree of DNA condensation. The DNA wrapping is right-handed, which restrains positive supercoils. In vivo, HUalpha(E38K,V42L) shows altered association and distribution patterns with the genetic loci whose transcription are differentially affected in the mutant strain.


Assuntos
Proteínas de Transporte/química , DNA Super-Helicoidal/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Histonas/química , Fatores de Transcrição/química , Proteínas de Transporte/genética , Proteínas de Transporte/ultraestrutura , Cromatina/química , Cromatina/genética , DNA Super-Helicoidal/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Dimerização , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestrutura , Marcadores Genéticos , Ácido Glutâmico/genética , Histonas/genética , Leucina/genética , Lisina/genética , Microscopia de Força Atômica , Mutação , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/ultraestrutura , Fatores de Transcrição/genética , Fatores de Transcrição/ultraestrutura , Valina/genética
17.
Proc Natl Acad Sci U S A ; 102(45): 16397-402, 2005 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-16258062

RESUMO

Bacterial nucleoid organization is believed to have minimal influence on the global transcription program. Using an altered bacterial histone-like protein, HUalpha, we show that reorganization of the nucleoid configuration can dynamically modulate the cellular transcription pattern. The mutant protein transformed the loosely packed nucleoid into a densely condensed structure. The nucleoid compaction, coupled with increased global DNA supercoiling, generated radical changes in the morphology, physiology, and metabolism of wild-type K-12 Escherichia coli. Many constitutive housekeeping genes involved in nutrient utilization were repressed, whereas many quiescent genes associated with virulence were activated in the mutant. We propose that, as in eukaryotes, the nucleoid architecture dictates the global transcription profile and, consequently, the behavior pattern in bacteria.


Assuntos
Proteínas de Bactérias/fisiologia , Cromossomos Bacterianos , Proteínas de Ligação a DNA/fisiologia , Transcrição Genética , Evolução Biológica , DNA Super-Helicoidal , Temperatura , Virulência
18.
Proc Natl Acad Sci U S A ; 102(34): 11993-8, 2005 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-16040799

RESUMO

Most HIV transmission occurs on the mucosal surfaces of the gastrointestinal and cervicovaginal tracts, both of which are normally coated by a biofilm of nonpathogenic commensal bacteria. We propose to genetically engineer such naturally occurring bacteria to protect against HIV infection by secreting antiviral peptides. Here we describe the development and characterization of Nissle 1917, a highly colonizing probiotic strain of Escherichia coli, secreting HIV-gp41-hemolysin A hybrid peptides that block HIV fusion and entry into target cells. By using an appropriate combination of cis- and transacting secretory and regulatory signals, micromolar secretion levels of the anti-HIV peptides were achieved. The genetically engineered Nissle 1917 were capable of colonizing mice for periods of weeks to months, predominantly in the colon and cecum, with lower concentrations of bacteria present in the rectum, vagina, and small intestine. Histological and immunocytochemical examination of the colon revealed bacterial growth and peptide secretion throughout the luminal mucosa and in association with epithelial surfaces. The use of genetically engineered live microbes as anti-HIV microbicides has important potential advantages in economy, efficacy, and durability.


Assuntos
Fármacos Anti-HIV/farmacologia , Escherichia coli/metabolismo , Engenharia Genética/métodos , Proteína gp41 do Envelope de HIV/metabolismo , Infecções por HIV/prevenção & controle , HIV/efeitos dos fármacos , Proteínas Hemolisinas/metabolismo , Peptídeos/farmacologia , Animais , Fármacos Anti-HIV/metabolismo , Ceco/microbiologia , Colo/microbiologia , Escherichia coli/genética , Feminino , HIV/metabolismo , Humanos , Mucosa Intestinal/microbiologia , Camundongos , Modelos Biológicos , Peptídeos/metabolismo , Vagina/microbiologia
19.
Biochemistry ; 44(14): 5373-80, 2005 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-15807530

RESUMO

DNA transaction reactions require formation of nucleoprotein complexes that involve multifaceted DNA-protein and protein-protein interactions. Genetic and biochemical studies suggested that the higher order Gal repressosome structure, which governs the transcription of two tandem galpromoters in Escherichia coli, involves sequence-specific binding of GalR repressor dimers to two operators, O(E) and O(I), located 113 bp apart, binding of GalR to the sequence-nonspecific DNA binding protein HU, interaction of HU with an architecturally critical DNA site between the two operators, and interaction between two DNA-bound GalR dimers generating a loop of the intervening DNA segment. In this paper, we demonstrate and determine the thermodynamic parameters of several of these interactions, GalR dimer-O(E), GalR tetramerization, HU-GalR, and HU-GalR-O(E) interactions, by analytical ultracentrifugation, fluorescence anisotropy, and fluorescence resonance energy transfer. The physiological significance of several of these interactions was confirmed by the finding that a mutant HU, which is unable to form the repressosome in vivo and in vitro, failed to show the HU-GalR interaction. The results help to construct a pathway of Gal repressosome assembly.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Sequência de Bases , Oligodesoxirribonucleotídeos , Espectrometria de Fluorescência , Ultracentrifugação
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