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1.
J Inorg Biochem ; 203: 110886, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31707334

RESUMO

Gram-negative bacteria of the human gastrointestinal (GI) tract microbiome: (i) are capable of generating a broad-spectrum of highly neurotoxic, pro-inflammatory and potentially pathogenic molecules; and (ii) these include a highly immunogenic class of amphipathic surface glycolipids known as lipopolysaccharide (LPS). Bacteroides fragilis (B. fragilis), a commensal, Gram negative, non-motile, non-spore forming obligatory anaerobic bacillus, and one of the most abundant bacteria found in the human GI tract, produces a particularly pro-inflammatory and neurotoxic LPS (BF-LPS). BF-LPS: (i) is known to be secreted from the B. fragilis outer membrane into the external-medium; (ii) can damage biophysiological barriers via cleavage of zonula adherens cell-cell adhesion proteins, thereby disrupting both the GI-tract barrier and the blood-brain barrier (BBB); (iii) is able to transit GI-tract barriers into the systemic circulation and cross the BBB into the human CNS; and (iv) accumulates within CNS neurons in neurodegenerative disorders such as Alzheimer's disease (AD). This short communication provides evidence that the incubation of B. fragilis with aluminum sulfate [Al2(SO4)3] is a potent inducer of BF-LPS. The results suggest for the first time that the pro-inflammatory properties of aluminum may not only be propagated by aluminum itself, but by a stimulation in the production of microbiome-derived BF-LPS and other pro-inflammatory pathogenic microbial products normally secreted from human GI-tract-resident microorganisms.


Assuntos
Compostos de Alúmen/farmacologia , Bacteroides fragilis/efeitos dos fármacos , Lipopolissacarídeos/metabolismo , Bacteroides fragilis/metabolismo
2.
J Inorg Biochem ; 203: 110860, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31698325

RESUMO

The first successful attempt to obtain purified aluminum metal was accomplished by the Danish physicist and chemist Hans Christian Orsted in 1824, however it was not until about ~140 years later that aluminum's capacity for neurological disruption and neurotoxicity was convincingly established. The earliest evidence of the possible involvement of this biosphere-rich metallotoxin in Alzheimer's disease (AD) originated in the early-to-mid-1960's from animal and human research investigations that arose almost simultaneously from independent laboratories in the United States and Canada. This short communication pays tribute to the pioneering research work on aluminum in susceptible species, in AD animal models and in AD patients by the early investigators Drs. Robert D. Terry, Igor Klatzo and Henryk M. Wisniewski with special acknowledgement to the late Dr. Donald RC McLachlan, and their contemporary physician-scientist colleagues and collaborators. Together these researchers established the groundwork and foundation towards our understanding of the potential contribution of aluminum to progressive, age-related and lethal neurodegenerative diseases of the human central nervous system.


Assuntos
Alumínio/toxicidade , Neurociências/história , Síndromes Neurotóxicas/etiologia , Doença de Alzheimer/etiologia , Amiloide/efeitos dos fármacos , Animais , Encéfalo/patologia , História do Século XX , História do Século XXI , Humanos , Emaranhados Neurofibrilares/efeitos dos fármacos , Placa Amiloide/etiologia , Estados Unidos
3.
J Inorg Biochem ; 152: 206-9, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26213226

RESUMO

At least 57 murine transgenic models for Alzheimer's disease (Tg-AD) have been developed to overexpress the 42 amino acid amyloid-beta (Aß42) peptide in the central nervous system (CNS). These 'humanized murine Tg-AD models' have greatly expanded our understanding of the contribution of Aß42 peptide-mediated pro-inflammatory neuropathology to the AD process. A number of independent laboratories using different amyloid-overexpressing Tg-AD models have shown that supplementation of murine Tg-AD diets and/or drinking water with aluminum significantly enhances Aß42 peptide-mediated inflammatory pathology and AD-type cognitive change compared to animals receiving control diets. In humans AD-type pathology appears to originate in the limbic system and progressively spreads into primary processing and sensory regions such as the retina. In these studies, for the first time, we assess the propagation of Aß42 and inflammatory signals into the retina of 5xFAD Tg-AD amyloid-overexpressing mice whose diets were supplemented with aluminum. The two most interesting findings were (1) that similar to other Tg-AD models, there was a significantly accelerated development of Aß42 and inflammatory pathology in 5xFAD Tg-AD mice fed aluminum; and (2) in aluminum-supplemented animals, markers for inflammatory pathology appeared in both the brain and the retina as evidenced by an evolving presence of Aß42 peptides, and accompanied by inflammatory markers - cyclooxygenase-2 (COX-2) and C-reactive protein (CRP). The results indicate that in the 5xFAD Tg-AD model aluminum not only enhances an Aß42-mediated inflammatory degeneration of the brain but also appears to induce AD-type pathology in an anatomically-linked primary sensory area that involves vision.


Assuntos
Compostos de Alumínio/toxicidade , Doença de Alzheimer/patologia , Agregação Patológica de Proteínas/induzido quimicamente , Retina/efeitos dos fármacos , Compostos de Alumínio/efeitos adversos , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Proteína C-Reativa/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Fragmentos de Peptídeos/metabolismo , Retina/patologia
4.
Cell Death Differ ; 20(9): 1149-60, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23645208

RESUMO

Inflammasomes are protein complexes assembled upon recognition of infection or cell damage signals, and serve as platforms for clustering and activation of procaspase-1. Oligomerisation of initiating proteins such as AIM2 (absent in melanoma-2) and NLRP3 (NOD-like receptor family, pyrin domain-containing-3) recruits procaspase-1 via the inflammasome adapter molecule ASC (apoptosis-associated speck-like protein containing a CARD). Active caspase-1 is responsible for rapid lytic cell death termed pyroptosis. Here we show that AIM2 and NLRP3 inflammasomes activate caspase-8 and -1, leading to both apoptotic and pyroptotic cell death. The AIM2 inflammasome is activated by cytosolic DNA. The balance between pyroptosis and apoptosis depended upon the amount of DNA, with apoptosis seen at lower transfected DNA concentrations. Pyroptosis had a higher threshold for activation, and dominated at high DNA concentrations because it happens more rapidly. Gene knockdown showed caspase-8 to be the apical caspase in the AIM2- and NLRP3-dependent apoptotic pathways, with little or no requirement for caspase-9. Procaspase-8 localised to ASC inflammasome 'specks' in cells, and bound directly to the pyrin domain of ASC. Thus caspase-8 is an integral part of the inflammasome, and this extends the relevance of the inflammasome to cell types that do not express caspase-1.


Assuntos
Apoptose , Proteínas de Transporte/metabolismo , Caspase 8/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Nucleares/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Proteínas Adaptadoras de Sinalização CARD , Caspase 1/metabolismo , Caspase 8/genética , Caspase 9/genética , Proteínas de Ligação a DNA , Inflamassomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Interferência de RNA , RNA Interferente Pequeno , Receptor Toll-Like 9/genética
5.
Cell Death Differ ; 13(12): 2052-61, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16710361

RESUMO

Receptor-mediated programmed cell death proceeds through an activated receptor to which the death adaptor FADD and the initiator procaspases 8 and/or 10 are recruited following receptor stimulation. The adaptor FADD is responsible for both receptor binding and recruitment of the procaspases into the death-inducing signaling complex. Biochemical dissection of the FADD death effector domain and functional replacement with a coiled-coil motif demonstrates that there is an obligatory FADD self-association via the DED during assembly of the death-inducing signaling complex. Using engineered oligomerization motifs with defined stoichiometries, the requirement for FADD self-association through the DED can be separated from the caspase-recruitment function of the domain. Disruption of FADD self-association precludes formation of a competent signaling complex. On this basis, we propose an alternative architecture for the FADD signaling complex in which FADD acts as a molecular bridge to stitch together an array of activated death receptors.


Assuntos
Proteína de Domínio de Morte Associada a Fas/metabolismo , Receptores de Morte Celular/metabolismo , Transdução de Sinais/fisiologia , Caspase 10/genética , Caspase 10/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/metabolismo , Proteína de Domínio de Morte Associada a Fas/genética , Regulação da Expressão Gênica , Humanos , Células Jurkat , Substâncias Macromoleculares , Mutação , Ligação Proteica , Receptores de Morte Celular/genética
6.
Cytotherapy ; 8(2): 141-8, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16698687

RESUMO

BACKGROUND: PBMC can be expanded ex vivo into aggressive cytotoxic effector cells (CEC) comprising T, NK and NKT cells. We identified the phenotype, cytotoxicity and mechanisms of killing of these CEC. METHODS: CY- and G-CSF-mobilized PBMC from myeloma patients were placed in Aim-V serum-free medium, IL-2 (50 IU/mL) and OKT-3 (50 ng/mL). Cytotoxicity was evaluated by selectively blocking the TCR, MHC class I or NKG2D receptor. RESULTS: The CEC expanded three-fold by day 7 and aggressively lysed myeloma cells (41.9%) compared with day 0 (4%; P=0.012). CD8+ CD56+ NKT cells performed the majority of lysis. The CD8+ cells greatly increased NKG2D expression during culture (P=0.005). Cytotoxicity correlated with target NKG2D ligand expression (P=0.0002). Blocking the TCR or MHC class I did not affect cytotoxicity (P>0.22). CD8+ cell-mediated lysis dropped 48% when the NKG2D receptor was blocked. Day 7 CEC aggressively lysed myeloma cells in an MHC- and non-MHC-restricted fashion, through the NKG2D receptor. DISCUSSION: Because MHC expression is often down-regulated on tumor cells and the NKG2D ligands are generally specific to malignant cells, the adoptive transfer of CEC that kill through different pathways may circumvent tumor-resistant mechanisms and improve outcomes.


Assuntos
Linfócitos T CD8-Positivos/fisiologia , Mieloma Múltiplo/terapia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/fisiologia , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Citotoxicidade Imunológica , Humanos , Imunoterapia Adotiva , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Complexo Principal de Histocompatibilidade/imunologia , Mieloma Múltiplo/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Células Matadoras Naturais , Linfócitos T Citotóxicos/metabolismo
7.
J S Afr Vet Assoc ; 77(4): 210-4, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17458347

RESUMO

Two, sibling, male Golden retriever puppies, 13 weeks of age, were presented with congenital biliary cysts of the liver involving both hepatic and segmental bile ducts, as well as bilateral polycystic kidney disease. Ultrasonography of the livers of both pups demonstrated segmental cystic lesions that were contiguous with the bile ducts. Histopathology revealed cystic ectatic bile duct hyperplasia and dysplasia with variable portal fibrosis in the liver, while in the kidneys there were radially arranged, cylindrically dilated cysts of the collecting ducts, which extended through the medulla and cortex. This pathology was compatible with that of congenital dilatation of the large and segmental bile ducts (Caroli's disease) described in humans, dogs and rats. In humans Caroli's disease has an autosomal recessive inheritance pattern, while in rats activation of the MEK5/ERK cascade initiates the biliary dysgenesis of Caroli's disease in this species. However, the exact mode of inheritance and pathogenesis of Caroli's disease in dogs is as yet unknown. Previous reports on congenital hepatic cystic diseases of the dog have described Caroli's disease like lesions in various breeds, but these are believed to be the 1st reported cases in the Golden retriever breed.


Assuntos
Doenças dos Ductos Biliares/veterinária , Ductos Biliares Intra-Hepáticos/anormalidades , Doenças do Cão/congênito , Hepatopatias/veterinária , Animais , Doenças dos Ductos Biliares/congênito , Doenças dos Ductos Biliares/diagnóstico , Doenças dos Ductos Biliares/patologia , Cistos/congênito , Cistos/diagnóstico , Cistos/patologia , Cistos/veterinária , Doenças do Cão/diagnóstico , Doenças do Cão/patologia , Cães , Evolução Fatal , Hepatopatias/congênito , Hepatopatias/diagnóstico , Hepatopatias/patologia , Masculino
10.
Eur J Immunol ; 31(11): 3318-28, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11745349

RESUMO

Keratocytes express MHC class I molecules constitutively, and keratocytes stimulated with IFN-gamma express MHC class II molecules. Unstimulated keratocytes constitutively express B7-1 and ICAM-1, as well as low levels of CD40 and 4-1BBL. These findings indicate that keratocytes may deliver both antigen-specific and costimulatory signals to CD4(+) and CD8(+) T cells. To demonstrate that keratocytes expressing B7-1 provide a costimulatory signal to T cells, CD4(+) or CD8(+) mouse T cells were incubated with anti-CD3 mAb and irradiated keratocytes. Enhanced proliferation of both CD4(+) and CD8(+) T cells occurred, and could be inhibited by anti-B7-1 mAb, indicating T cell costimulatory activity by B7-1 on the keratocytes. To demonstrate that keratocytes can deliver an antigen-specific signal, CD4(+) and CD8(+) T cells from herpes-infected mice were incubated with HSV-1-infected, irradiated keratocytes. The resulting T cell proliferation and production of Th1 cytokines (IL-2, IFN-gamma) indicated T cell activation by antigens presented by the infected keratocytes. These results show that keratocytes in the corneal stroma of the mouse can function as antigen-presenting cells and, thus, may play a role in immune-mediated stromal inflammation such as herpetic stromal keratitis.


Assuntos
Células Apresentadoras de Antígenos/fisiologia , Córnea/citologia , Animais , Antígenos CD/análise , Antígeno B7-1/análise , Antígeno B7-2 , Antígenos CD40/análise , Células Cultivadas , Citocinas/biossíntese , Feminino , Antígenos de Histocompatibilidade Classe II/análise , Ceratite/imunologia , Ativação Linfocitária , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos BALB C , Células Estromais/fisiologia
11.
J Chromatogr B Biomed Sci Appl ; 760(2): 191-205, 2001 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-11530977

RESUMO

Soy isoflavones are becoming of increasing interest as nutritional agents which can be used to combat osteoporosis and hyperlipidemia, and are also being considered as potential cancer chemopreventive compounds. However, prior to their formulation and distribution as therapeutic agents, thorough pharmacokinetic and toxicological assessment needs to be completed in men and women in a variety of health conditions in order to ensure their therapeutic efficacy and safety. At this time, studies of purified soy isoflavones are possible, and are being designed to fully evaluate the pharmacological utility of these preparations. In support of these studies, quantitative analysis of soy isoflavones in biological fluids can be accomplished with a wide variety of methods and analytical instrumentation. However, the relatively ubiquitous presence of high-performance liquid chromatography with ultraviolet detection (HPLC-UV) in most analytical laboratories, the relative ease of its operation, and the lesser expense of this instrumentation as compared to more sophisticated techniques such as liquid chromatography-mass spectrometry, offers some distinct advantages for its use in pharmacokinetic studies. In this manuscript, the development and validation of an HPLC-UV method for the quantitation of the principal soy isoflavones, genistein, daidzein, and glycitein, and their primary metabolites, in human plasma and urine is described. This analytical approach allows for pharmacologically relevant concentrations of the analytes and their principle metabolites to be detected, and has been validated in close agreement with the US Food and Drug Administration's guidelines for the validation of methods to be used in support of pharmacokinetic studies.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Genisteína/farmacocinética , Isoflavonas/farmacocinética , Genisteína/sangue , Genisteína/urina , Humanos , Isoflavonas/sangue , Isoflavonas/urina , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrofotometria Ultravioleta
12.
Endocrinology ; 142(8): 3348-53, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11459777

RESUMO

IGF-I and the IGF-I receptor are necessary for normal embryonic growth. VIP is an important regulator of early postimplantation growth and acts indirectly through the release of other factors, including activity-dependent neurotrophic factor. The relationship of IGF-I growth regulation to VIP/activity-dependent neurotrophic factor-stimulated growth was examined with whole cultured embryonic d 9.5 mouse embryos. Somite numbers and DNA and protein contents were measured in embryos treated with IGF-I, anti-IGF-I, VIP, activity-dependent neurotrophic factor, and anti-activity-dependent neurotrophic factor-14 (antiserum to an activity-dependent neurotrophic factor agonist). IGF-I mRNA content was measured after incubation with and without VIP for 30 and 60 min using competitive RT-PCR. IGF-I induced a significant, dose-dependent increase in growth as measured by somite number, DNA levels, and protein content. Furthermore, anti-IGF-I inhibited embryonic growth and also prevented exogenous IGF-mediated growth. Both VIP- and activity-dependent neurotrophic factor-stimulated growth were blocked by anti-IGF-I, whereas anti-activity-dependent neurotrophic factor-14 had no detectable effect on IGF-I-induced growth. Treatment with VIP resulted in a 2-fold increase in embryonic IGF-I mRNA. These data suggest that IGF-I is a downstream mediator of VIP and activity-dependent neurotrophic factor in a regulatory pathway coordinating embryonic growth and that VIP may function as a regulator of IGF-I gene expression in the embryo.


Assuntos
Embrião de Mamíferos/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Fatores de Crescimento Neural/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Peptídeo Intestinal Vasoativo/fisiologia , Animais , Técnicas de Cultura , Desenvolvimento Embrionário e Fetal/efeitos dos fármacos , Desenvolvimento Embrionário e Fetal/fisiologia , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/farmacologia , Camundongos , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Neuropeptídeos , Oligopeptídeos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Peptídeo Intestinal Vasoativo/farmacologia
13.
J Bone Joint Surg Am ; 83(6): 877-83, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11407796

RESUMO

BACKGROUND: The marked loss of glenoid bone volume or alteration of glenoid version can affect glenoid component fixation in patients undergoing total shoulder arthroplasty. The purpose of this study was to evaluate the long-term results associated with the use of bone-grafting for restoration of glenoid volume and version at the time of total shoulder arthroplasty. METHODS: Twenty-one shoulders received an internally fixed, corticocancellous bone graft for the restoration of peripheral glenoid bone stock at the time of total shoulder arthroplasty between 1980 and 1989. Grafting was indicated when glenoid bone stock was insufficient to maintain adequate version or fixation of the prosthesis. Seventeen shoulders were available for follow-up; the average duration of follow-up for the thirteen shoulders that did not have prosthetic failure within the first two years was seventy months. Total shoulder arthroplasty was performed because of osteoarthritis in five shoulders, chronic anterior fracture-dislocation in five, capsulorrhaphy arthropathy in three, inflammatory arthritis in two, recurrent dislocation in one, and failure of a previous arthroplasty in one. All patients had some form of anterior or posterior instability preoperatively. There were five anterior and twelve posterior glenoid defects. Bone from the resected humeral head was used for grafting in fifteen shoulders, and bicortical iliac-crest bone was used in two. RESULTS: The average glenoid version after grafting was 4 degrees of retroversion, with an average correction of 33 degrees. The graft failed to maintain the original correction in three shoulders due to nonunion, dissolution, or shift. Five total shoulder replacements failed, necessitating glenoid revision at two to ninety-one months postoperatively. The failures were associated with recurrent massive cuff tears (one shoulder), persistent instability (two shoulders), improper component placement (one shoulder), and loss of graft fixation (one shoulder). There were no humeral component failures. According to the criteria of Neer et al., the functional result was rated as excellent in three shoulders, satisfactory in six, and unsatisfactory in eight. CONCLUSIONS: Despite the finding that eight shoulders had an unsatisfactory functional result at the time of longterm follow-up, corticocancellous grafting of the glenoid successfully restored glenoid version and volume in fourteen of the seventeen shoulders in the present study. Patients with glenoid deficiency often have associated glenohumeral instability, which may affect the results of total shoulder arthroplasty. Bone-grafting of the glenoid is a technically demanding procedure that can restore bone stock in patients with structural defects.


Assuntos
Artroplastia de Substituição , Transplante Ósseo , Escápula/cirurgia , Articulação do Ombro/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Artrite/cirurgia , Doença Crônica , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/cirurgia , Dor , Falha de Prótese , Radiografia , Reoperação , Escápula/patologia , Luxação do Ombro/cirurgia , Articulação do Ombro/diagnóstico por imagem , Articulação do Ombro/fisiopatologia
14.
Med Hypotheses ; 56(3): 348-56, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11359358

RESUMO

Stress has been shown to modulate an individual's immune system through the release of certain signal molecules such as catecholamines, cytokines and glucocorticoids. These signal molecules can significantly alter the host immune system and leave it susceptible to a primary or recurrent viral infection. Focusing on herpes simplex virus types-1 and -2 as examples, the authors explain how stress-associated immunomodulation can influence the recurrence of herpes simplex viral infections. Specific signal molecules such as epinephrine, interleukin-6, cyclic adenosine monophosphate, glucocorticoids and prostaglandins are upregulated during episodes of acute and chronic stress and have been implicated as effectors of herpes simplex viral reactivation and recurrent disease. The authors suggest that the release of immunomodulating signal molecules due to stress can compromise the host's cellular immune response and trigger herpes simplex viral reactivation.


Assuntos
Herpes Simples/imunologia , Herpes Simples/psicologia , Estresse Psicológico/imunologia , Animais , Humanos , Modelos Imunológicos , Modelos Psicológicos , Simplexvirus/crescimento & desenvolvimento , Ativação Viral
15.
J Pharmacol Exp Ther ; 297(2): 774-9, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11303069

RESUMO

Two peptides [NAPVSIPQ (NAP) and SALLRSIPA (ADNF-9)], that are associated with novel glial proteins regulated by vasoactive intestinal peptide, are shown now to provide protective intervention in a model of fetal alcohol syndrome. Fetal demise and growth restrictions were produced after intraperitoneal injection of ethanol to pregnant mice during midgestation (E8). Death and growth abnormalities elicited by alcohol treatment during development are believed to be associated, in part, with severe oxidative damage. NAP and ADNF-9 have been shown to exhibit antioxidative and antiapoptotic actions in vitro. Pretreatment with an equimolar combination of the peptides prevented the alcohol-induced fetal death and growth abnormalities. Pretreatment with NAP alone resulted in a significant decrease in alcohol-associated fetal death; whereas ADNF-9 alone had no detectable effect on fetal survival after alcohol exposure, indicating a pharmacological distinction between the peptides. Biochemical assessment of the fetuses indicated that the combination peptide treatment prevented the alcohol-induced decreases in reduced glutathione. Peptide efficacy was evident with either 30-min pretreatment or with 1-h post-alcohol administration. Bioavailability studies with [(3)H]NAPVSIPQ indicated that 39% of the total radioactivity comigrated with intact peptide in the fetus 60 min after administration. These studies demonstrate that fetal death and growth restriction associated with prenatal alcohol exposure were prevented by combinatorial peptide treatment and suggest that this therapeutic strategy be explored in other models/diseases associated with oxidative stress.


Assuntos
Transtornos do Espectro Alcoólico Fetal/patologia , Morte Fetal/prevenção & controle , Retardo do Crescimento Fetal/prevenção & controle , Oligopeptídeos/farmacologia , Oligopeptídeos/farmacocinética , Animais , Disponibilidade Biológica , Peso ao Nascer/efeitos dos fármacos , Etanol/sangue , Feminino , Morte Fetal/patologia , Retardo do Crescimento Fetal/patologia , Glutationa/metabolismo , Tamanho da Ninhada de Vivíparos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Gravidez
16.
Anal Biochem ; 290(2): 330-7, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11237336

RESUMO

Pseudomonas aeruginosa secretes multiple proteases that have been implicated as virulence factors and the detection of each specific enzyme can be difficult to determine. Unlike the three Pseudomonas enzymes that have been well characterized (elastase A, elastase B, and alkaline protease), the activity of protease IV in multiple assays has yet to be described. This study defines new assays for Pseudomonas proteases and compares protease IV activity to the activities of elastase A, elastase B, and alkaline protease. Six in vitro assays were studied: zymography, elastin congo red assay, staphylolytic assay, colorimetric peptide assay, solid-phase colorimetric peptide assay, and poly-l-lysine degradation. Casein zymography distinguished protease IV from elastase B and alkaline protease, and gelatin zymography differentiated all four proteases. The elastin congo red assay detected mainly elastase B while the staphylolytic assay was specific for elastase A. Protease IV activity was assayed specifically by the colorimetric assay and two new assays, the solid-phase colorimetric assay and degradation of poly-L-lysine in the presence of EDTA. Alkaline protease could be specifically assayed by poly-L-lysine degradation in the presence of N-alpha-p-tosyl-L-lysine chloromethyl ketone. The results identified three specific assays for protease IV, a new assay specific for alkaline protease, and showed that protease IV has a distinct enzymatic specificity relative to the three other Pseudomonas proteases.


Assuntos
Colorimetria/métodos , Peptídeo Hidrolases/análise , Pseudomonas aeruginosa/enzimologia , Vermelho Congo/química , Elastina/química , Elastase Pancreática/análise , Peptídeos/química , Polilisina/química , Pseudomonas aeruginosa/química , Serina Endopeptidases/análise
17.
Mol Cells ; 12(3): 304-12, 2001 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-11804328

RESUMO

4-1BB, a member of the tumor necrosis factor receptor (TNFR) superfamily, is induced on CD4+ and CD8+ T cells upon engagement of the T cell receptor (TCR)/ CD3 complex with the antigen bound to MHC. 4-1BB plays an important role in transmitting costimulatory signal during T cell activation. However, 4-1BB-mediatded signal transduction pathways have remained elusive. We conducted the yeast two-hybrid screening to identify intracellular signaling molecules that associate with 4-1BB. A novel leucine-rich repeat (LRR)-containing protein, herein named LRR-1, was found to specifically interact with the cytoplasmic domain of 4-1BB. Overexpression of LRR-1 suppressed the activation of NF-KB induced by 4-1BB or TNF receptor-associated factor (TRAF) 2. In addition, LRR-1 down-regulated JNK1 activity was induced by 4-1BB. These results indicate that LRR-1 negatively regulates the 4-1BB-mediated signaling cascades which result in the activation of NF-kappaB and JNK1.


Assuntos
Proteínas/genética , Receptores de Fator de Crescimento Neural/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Antígenos CD , Clonagem Molecular , Regulação para Baixo , Humanos , Proteína Quinase 8 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , NF-kappa B/metabolismo , Estrutura Terciária de Proteína , Proteínas/metabolismo , Sequências Repetitivas de Aminoácidos/genética , Proteínas Repressoras , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral , Técnicas do Sistema de Duplo-Híbrido
18.
Eur J Biochem ; 267(15): 4649-57, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10903497

RESUMO

The three-dimensional solution structure of conotoxin TVIIA, a 30-residue polypeptide from the venom of the piscivorous cone snail Conus tulipa, has been determined using 2D 1H NMR spectroscopy. TVIIA contains six cysteine residues which form a 'four-loop' structural framework common to many peptides from Conus venoms including the omega-, delta-, kappa-, and muO-conotoxins. However, TVIIA does not belong to these well-characterized pharmacological classes of conotoxins, but displays high sequence identity with conotoxin GS, a muscle sodium channel blocker from Conus geographus. Structure calculations were based on 562 interproton distance restraints inferred from NOE data, together with 18 backbone and nine side-chain torsion angle restraints derived from spin-spin coupling constants. The final family of 20 structures had mean pairwise rms differences over residues 2-27 of 0.18+/-0.05 A for the backbone atoms and 1.39+/-0.33 A for all heavy atoms. The structure consists of a triple-stranded, antiparallel beta sheet with +2x, -1 topology (residues 7-9, 16-20 and 23-27) and several beta turns. The core of the molecule is formed by three disulfide bonds which form a cystine knot motif common to many toxic and inhibitory polypeptides. The global fold, molecular shape and distribution of amino-acid sidechains in TVIIA is similar to that previously reported for conotoxin GS, and comparison with other four-loop conotoxin structures provides further indication that TVIIA and GS represent a new and distinct subgroup of this structural family. The structure of TVIIA determined in this study provides the basis for determining a structure-activity relationship for these molecules and their interaction with target receptors.


Assuntos
Venenos de Moluscos/química , Caramujos/química , Sequência de Aminoácidos , Aminoácidos/química , Animais , Cromatografia Líquida , Conotoxinas/química , Cisteína/química , Dissulfetos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Biossíntese Peptídica , Peptídeos/química , Conformação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
19.
Eur J Biochem ; 267(15): 4642-8, 2000 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10903496

RESUMO

A novel conotoxin belonging to the 'four-loop' structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical-directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino-acid residues and contains three disulfide bonds. The amino-acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4-transL-hydroxyproline. Two under-hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four-loop structural framework common to many peptides from Conus venoms including the omega-, delta- and kappa-conotoxins. However, TVIIA displays little sequence homology with these well-characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four-loop conotoxins.


Assuntos
Venenos de Moluscos/química , Venenos de Moluscos/toxicidade , Caramujos/química , Sequência de Aminoácidos , Aminoácidos/química , Animais , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Camundongos , Dados de Sequência Molecular , Venenos de Moluscos/síntese química , Venenos de Moluscos/isolamento & purificação , Biossíntese Peptídica , Peptídeos/química , Peptídeos/isolamento & purificação , Poecilia , Ligação Proteica , Ratos , Homologia de Sequência de Aminoácidos
20.
J Neuropathol Exp Neurol ; 59(12): 1051-62, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11138925

RESUMO

Intracerebral administration of ibotenate produces, through activation of N-methyl-D-aspartate (NMDA) receptors, neuronal heterotopias in the newborn hamster neocortex: high doses of ibotenate induce periventricular and subcortical neuronal heterotopias, while low doses of ibotenate produce intracortical heterotopias and molecular layer ectopias. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are closely related peptides with neurotrophic properties. They share common VPAC1 and VPAC2 receptors, which use cAMP as a second messenger. Previous studies have shown that VIP prevents excitotoxic neuronal death and exacerbates glutamate-induced c-fos neuronal expression. In order to gain new insight into the molecular control of neuronal migration, the present study examined the effects of VIP and PACAP on ibotenate-induced heterotopias in the newborn hamster. Co-treatment with VIP and a high dose of ibotenate produced a pattern of neuronal heterotopias similar to the one observed in animals treated with low doses of ibotenate alone. Pups co-injected with a low dose of ibotenate and a VIP antagonist displayed cortical dysgeneses similar to those observed in animals treated with high doses of ibotenate alone. The modulating effects of VIP on excitotoxin-induced heterotopias were mimicked by forskolin, PACAP, and by a specific VPAC2 receptor agonist but not by a VPAC1 agonist, and were blocked by a protein kinase A (PKA) inhibitor. Taken together, these data suggest that VIP and PACAP can attenuate ibotenate-induced heterotopias in newborn hamster and that this effect is mediated by the VPAC2 receptor utilizing the cAMP-PKA pathway.


Assuntos
Animais Recém-Nascidos/fisiologia , Encefalopatias/patologia , Coristoma/prevenção & controle , Neocórtex/patologia , Tecido Nervoso , Neuropeptídeos/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia , Animais , Sítios de Ligação , Coristoma/induzido quimicamente , Coristoma/patologia , Cricetinae , Sinergismo Farmacológico , Ácido Ibotênico , Mesocricetus , Neocórtex/efeitos dos fármacos , Neocórtex/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Peptídeo Intestinal Vasoativo/metabolismo
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