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1.
AAPS J ; 22(2): 36, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31997031

RESUMO

Biologics can potentially induce unwanted immune responses, leading to formation of antidrug antibodies (ADA) of various affinity, isotypes, and subclasses. Among them, antigen and drug-specific immunoglobulin E (IgE) antibodies have been reported to have potential correlation with hypersensitivity and anaphylaxis in particular. Recent regulatory guidance on immunogenicity testing has recommended the measurement of antigen-specific IgE antibodies for biologics with a reported high risk of anaphylaxis using assays with sensitivities in the high pg/mL to low ng/mL range. Nevertheless, IgE ADA remains challenging to detect due to their being the least abundant isotype in blood serum samples and the potential for interference in the bioanalytical methods due to high levels of endogenous immunoglobulin G (IgG) and immunoglobulin M (IgM) ADA, not to mention the nonspecific total serum IgE antibodies. Another challenge in developing IgE ADA assays is the need to create a surrogate drug-specific IgE antibody positive control to monitor the performance of the assay for the intended use. In this case study, utilizing a human IgE antidrug antibody positive control and a human IgE receptor as capture, an enzyme-linked immunosorbent assay (ELISA) method was developed for the measurement of IgE ADA, meeting the regulatory expectations, with excellent assay sensitivity, selectivity, specificity, and tolerance towards potential interference in serum samples. This assay format could be readily adapted and implemented to assess drug-specific IgE antibodies in the event of drug-related anaphylaxis in clinical and in nonclinical development programs.

2.
ACS Omega ; 3(5): 4776-4785, 2018 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-30023902

RESUMO

Gold nanoparticles (Au NPs) have been thoroughly investigated for anti-cancer therapy. However, their undesired high gold content remains a problem when injected into the body for drug delivery applications. In this report, we made an effort to conjugate the curcumin molecules on the surface of gold quantum clusters (Au QCs) by a novel in situ synthesis method which provides an alternative route to not only reduce the metallic content but also increase the water solubility of curcumin and the loading efficiency. Here, curcumin itself acts as a reducing and capping agent for the synthesis of Au QCs. The UV-vis absorption, fluorescence, transmission electron microscopy, and electrospray ionization mass spectrometry results confirmed the synthesis of fluorescent Au QCs. Curcumin-conjugated Au NPs (C-Au NPs) and glutathione (GSH)-conjugated Au QCs (GSH-Au QCs) were also synthesized to visualize the effect of particle size and the capping agent, respectively, on the cytotoxicity to normal and cancer cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that the curcumin-conjugated Au QCs (C-Au QCs) were less cytotoxic to normal cells while almost the same cytotoxic to cancer cells in comparison to curcumin itself, which indicates that curcumin preserves its anticancer property even after binding to the Au QCs. However, C-Au NPs and GSH-Au QCs did not show any cytotoxicity against the normal and cancer cells at the concentration used. The western blot assay indicated that C-Au QCs promote apoptosis in cancer cells. Further, the in vivo study on severe combined immunodeficiency mice showed that C-Au QCs also inhibited the tumor growth efficiently without showing significant toxicity to internal organs.

3.
Am J Physiol Cell Physiol ; 314(3): C349-C365, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29167152

RESUMO

Umbrella cells, which must maintain a tight barrier, modulate their apical surface area during bladder filling by exocytosis of an abundant, subapical pool of discoidal- and/or fusiform-shaped vesicles (DFVs). Despite the importance of this trafficking event for bladder function, the pathways that promote DFV exocytosis remain to be identified. We previously showed that DFV exocytosis depends in part on a RAB11A-RAB8A-MYO5B network, but RAB27B is also reported to be associated with DFVs, and knockout mice lacking RAB27B have fewer DFVs. However, the RAB27B requirements for DFV exocytosis and the relationship between RAB27B and the other umbrella cell-expressed RABs remains unclear. Using a whole bladder preparation, we observed that filling-induced exocytosis of human growth hormone-loaded DFVs was significantly inhibited when RAB27B expression was downregulated using shRNA. RAB27A was also expressed in rat urothelium; however, RAB27A-specific shRNAs did not inhibit exocytosis, and the combination of RAB27A and RAB27B shRNAs did not significantly affect DFV exocytosis more than treatment with RAB27B shRNA alone. RAB27B and RAB11A showed a small degree of overlap when quantified using Squassh segmentation software, and expression of dominant-active or dominant-negative mutants of RAB11A or RAB8A, or expression of a RAB11A-specific shRNA, had no significant effect on the size, number, or intensity of RAB27B-positive DFVs. Likewise, treatment with RAB27B-specific shRNA had no effect on RAB11A-positive DFV parameters. We conclude that RAB27B, but not RAB27A, regulates DFV exocytosis in bladder umbrella cells in a manner that may be parallel to the previously described RAB11A-RAB8A-MYO5B pathway.


Assuntos
Células Epiteliais/enzimologia , Exocitose , Mecanorreceptores/metabolismo , Mecanotransdução Celular , Vesículas Transportadoras/enzimologia , Bexiga Urinária/enzimologia , Urotélio/enzimologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Ratos Sprague-Dawley , Bexiga Urinária/citologia , Urotélio/citologia , Proteínas rab de Ligação ao GTP/genética
4.
Methods Mol Biol ; 1523: 21-31, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27975242

RESUMO

Alzheimer's disease (AD) is one of the neurodegenerative disease characterized by progressive neuronal loss in the brain. Its two major hallmarks are extracellular senile plaques and intracellular neurofibrillary tangles (NFTs), formed by aggregation of amyloid ß-42 (Aß-42) and Tau protein respectively. Aß-42 is a transmembrane protein, which is produced after the sequential action of ß- and γ-secretases, thus obtained peptide is released extracellularly and gets deposited on the neuron forming senile plaques. NFTs are composed of microtubule-associated protein-Tau (MAPT). Tau protein's major function is to stabilize the microtubule that provides a track on which the cargo proteins are shuttled and the stabilized microtubule also maintains shape and integrity of the neuronal cell. Tau protein is subjected to various modifications such as phosphorylation, ubiquitination, glycation, acetylation, truncation, glycosylation, deamination, and oxidation; these modifications ultimately lead to its aggregation. Phosphorylation is the major modification and is extensively studied with respect to Tau protein. Tau protein, however, undergoes certain level of phosphorylation and dephosphorylation, which regulates its affinity for microtubule and ultimately leading to microtubule assembly and disassembly. Our main aim was to study the native state of longest isoform of Tau (hTau40WT-4R2N) and its shortest isoform, (hTau23WT-3R0N), at various temperatures such as 10, 25, and 37 °C. Raman spectroscopic results suggested that the proportion of random coils or unordered structure depends on the temperature of the protein environment. Upon increase in the temperature from 10 to 37 °C, the proportion of random coils or unordered structures increased in the case of hTau40WT. However, we did not find a significant effect of temperature on the structure of hTau23WT. This current approach enables one to analyze the global conformation of soluble Tau in solution.


Assuntos
Análise Espectral Raman/métodos , Proteínas tau/química , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Emaranhados Neurofibrilares/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Conformação Proteica
5.
BMC Microbiol ; 16(1): 165, 2016 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-27464881

RESUMO

BACKGROUND: Chlamydia trachomatis is a human pathogen which causes a number of pathologies, including genital tract infections in women that can result in tubal infertility. Prevention of infection and disease control might be achieved through vaccination; however, a safe, efficacious and cost-effective vaccine against C. trachomatis infection remains an unmet medical need. C. trachomatis major outer membrane protein (MOMP), a ß-barrel integral outer membrane protein, is the most abundant antigen in the outer membrane of the bacterium and has been evaluated as a subunit vaccine candidate. Recombinant MOMP (rMOMP) expressed in E. coli cytoplasm forms inclusion bodies and rMOMP extracted from inclusion bodies results in a reduced level of protection compared to the native MOMP in a mouse challenge model. RESULTS: We sought to target the recombinant expression of MOMP to the E. coli outer membrane (OM). Successful surface expression was achieved with codon harmonization, utilization of low copy number vectors and promoters with moderate strength, suitable leader sequences and optimization of cell culture conditions. rMOMP was extracted from E. coli outer membrane, purified, and characterized biophysically. The OM expressed and purified rMOMP is immunogenic in mice and elicits antibodies that react to the native antigen, Chlamydia elementary body (EB). CONCLUSIONS: C. trachomatis MOMP was functionally expressed on the surface of E. coli outer membrane. The OM expressed and purified rMOMP elicits antibodies that react to the native antigen, Chlamydia EB, in a mouse immunogenicity model. Surface expression of MOMP could provide useful reagents for vaccine research, and the methodology could serve as a platform to produce other outer membrane proteins recombinantly.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Chlamydia trachomatis/genética , Escherichia coli/genética , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/biossíntese , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/química , Células Cultivadas , Infecções por Chlamydia/prevenção & controle , Clonagem Molecular , DNA Bacteriano/genética , Escherichia coli/metabolismo , Feminino , Imunogenicidade da Vacina , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
6.
Analyst ; 141(7): 2250-8, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26934683

RESUMO

The multivalent display of carbohydrates on the cell surface provides cooperative binding to improve the specific biological events. In addition to multivalency, the spatial arrangement and orientation of sugars with respect to external stimuli also trigger carbohydrate-protein interactions. Herein, we report a non-covalent host-guest strategy to immobilize heptavalent glyco-ß-cyclodextrin on gold-coated glass slides to study multivalent carbohydrate-protein interactions. We have found that the localization of sugar entities on surfaces using ß-cyclodextrin (ß-CD) chemistry increased the avidity of carbohydrate-protein and carbohydrate-macrophage interactions compared to monovalent-ß-CD sugar coated surfaces. This platform is expected to be a promising tool to amplify the avidity of sugar-mediated interactions on surfaces and contribute to the development of next generation bio-medical products.


Assuntos
Concanavalina A/análise , Ouro/química , Macrófagos/citologia , beta-Ciclodextrinas/química , Adesão Celular , Linhagem Celular , Humanos , Propriedades de Superfície
7.
FEBS Lett ; 589(24 Pt B): 4033-8, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-26554815

RESUMO

Amyloid aggregates display striking features of detergent stability and self-seeding. Human serum albumin (HSA), a preferred drug-carrier molecule, can also aggregate in vitro. So far, key amyloid properties of stability against ionic detergents and self-seeding, are unclear for HSA aggregates. Precautions against amyloid contamination would be required if HSA aggregates were self-seeding. Here, we show that HSA aggregates display detergent sarkosyl stability and have self-seeding potential. HSA dimer is preferable for clinical applications due to its longer retention in circulation and lesser oedema owing to its larger molecular size. Here, HSA was homodimerized via free cysteine-34, without any potentially immunogenic cross-linkers that are usually pre-requisite for homodimerization. Alike the monomer, HSA dimers also aggregated as amyloid, necessitating precautions while using for therapeutics.


Assuntos
Proteínas Amiloidogênicas/química , Substitutos do Plasma/química , Albumina Sérica/química , Proteínas Amiloidogênicas/efeitos adversos , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/ultraestrutura , Cromatografia em Gel , Cisteína/química , Detergentes/química , Dimerização , Portadores de Fármacos , Humanos , Peróxido de Hidrogênio/química , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Peso Molecular , Oxidantes/química , Oxirredução , Substitutos do Plasma/efeitos adversos , Agregação Patológica de Proteínas/etiologia , Estabilidade Proteica , Proteínas Recombinantes , Sarcosina/análogos & derivados , Sarcosina/química , Albumina Sérica/efeitos adversos , Albumina Sérica/genética , Albumina Sérica/ultraestrutura , Albumina Sérica Humana
8.
Nanoscale ; 7(47): 19985-20002, 2015 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-26564987

RESUMO

Herein, we report a detailed experimental study supported by DFT calculations to understand the mechanism behind the synthesis of cefradine (CFD--an antibiotic) labeled gold nanoparticles (Au NPs) by employing CFD as both a mild reducing and capping agent. The analysis of the effect of growth conditions reveals that a higher concentration of HAuCl4 results in the formation of an increasing fraction of anisotropic structures, higher temperature leads to the formation of quasi-spherical particles instead of anisotropic ones, and larger pH leads to the formation of much smaller particles. The cyclic voltammetry (CV) results show that when the pH of the reaction medium increases from 4 to 6, the reduction potential of CFD increases which leads to the synthesis of nanoparticles (in a pH 4 reaction) to quantum clusters (in a pH 6 reaction). The MALDI-TOF mass spectrometry results of supernatant of the pH 6 reaction indicate the formation of [Au8(CFD)2S6] QCs which show fluorescence at ca. 432 nm with a Stokes shift of ca. 95 nm. The blue luminescence from Au8 QCs was applied for sensing of Hg(2+) ions on the basis of an aggregation-induced fluorescence quenching mechanism and offers good selectivity and a high sensitivity with a limit of detection ca. 2 nM which is lower than the detection requirement of 10 nM by the U.S. EPA and 30 nM by WHO for drinking water. We have also applied the sensing probe to detect Hg(2+) ions in bacterial samples. Further, we have investigated the antibacterial property of as-synthesized Au NPs using MIC, growth curve and cell survival assay. The results show that Au NPs could reduce the cell survival very efficiently rather than the cell growth in comparison to the antibiotic itself. The scanning electron microscopy study shows the degradation and blebbing of the bacterial cell wall upon exposure with Au NPs which was further supported by fluorescence microscopy results. These Au NPs did not show reactive oxygen species generation. We believe that the bacterial cytotoxicity is due to the direct contact of the Au NPs with bacterial cells.


Assuntos
Antibacterianos/química , Ouro/química , Mercúrio/química , Nanopartículas Metálicas/química , Acetatos/química , Anisotropia , Proliferação de Células , Sobrevivência Celular , Cefradina/química , Concentração de Íons de Hidrogênio , Íons , Luminescência , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Nanotecnologia/métodos , Estresse Oxidativo , Pontos Quânticos , Espécies Reativas de Oxigênio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Luz Próxima ao Infravermelho , Eletricidade Estática
9.
J Biol Chem ; 289(25): 17497-514, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24798335

RESUMO

The AP-2 clathrin adaptor complex oversees endocytic cargo selection in two parallel but independent manners. First, by physically engaging peptide-based endocytic sorting signals, a subset of clathrin-dependent transmembrane cargo is directly collected into assembling buds. Synchronously, by interacting with an assortment of clathrin-associated sorting proteins (CLASPs) that independently select different integral membrane cargo for inclusion within the incipient bud, AP-2 handles additional cargo capture indirectly. The distal platform subdomain of the AP-2 ß2 subunit appendage is a privileged CLASP-binding surface that recognizes a cognate, short α-helical interaction motif. This signal, found in the CLASPs ß-arrestin and the autosomal recessive hypercholesterolemia (ARH) protein, docks into an elongated groove on the ß2 appendage platform. Tyr-888 is a critical constituent of this spatially confined ß2 appendage contact interface and is phosphorylated in numerous high-throughput proteomic studies. We find that a phosphomimetic Y888E substitution does not interfere with incorporation of expressed ß2-YFP subunit into AP-2 or alter AP-2 deposition at surface clathrin-coated structures. The Y888E mutation does not affect interactions involving the sandwich subdomain of the ß2 appendage, indicating that the mutated appendage is folded and operational. However, the Y888E, but not Y888F, switch selectively uncouples interactions with ARH and ß-arrestin. Phyogenetic conservation of Tyr-888 suggests that this residue can reversibly control occupancy of the ß2 platform-binding site and, hence, cargo sorting.


Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Fibroblastos/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Arrestinas/genética , Arrestinas/metabolismo , Linhagem Celular Transformada , Vesículas Revestidas por Clatrina/genética , Fibroblastos/citologia , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Fosforilação/fisiologia , Fosfotirosina/genética , Fosfotirosina/metabolismo
10.
Mol Biol Cell ; 24(7): 1007-19, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23389633

RESUMO

Multiple Rabs are associated with secretory granules/vesicles, but how these GTPases are coordinated to promote regulated exocytosis is not well understood. In bladder umbrella cells a subapical pool of discoidal/fusiform-shaped vesicles (DFVs) undergoes Rab11a-dependent regulated exocytosis in response to bladder filling. We show that Rab11a-associated vesicles are enmeshed in an apical cytokeratin meshwork and that Rab11a likely acts upstream of Rab8a to promote exocytosis. Surprisingly, expression of Rabin8, a previously described Rab11a effector and guanine nucleotide exchange factor for Rab8, stimulates stretch-induced exocytosis in a manner that is independent of its catalytic activity. Additional studies demonstrate that the unconventional motor protein myosin5B motor (Myo5B) works in association with the Rab8a-Rab11a module to promote exocytosis, possibly by ensuring transit of DFVs through a subapical, cortical actin cytoskeleton before fusion. Our results indicate that Rab11a, Rab8a, and Myo5B function as part of a network to promote stretch-induced exocytosis, and we predict that similarly organized Rab networks will be common to other regulated secretory pathways.


Assuntos
Exocitose , Miosinas/metabolismo , Bexiga Urinária/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/metabolismo , Humanos , Microscopia Confocal , Microscopia Eletrônica , Miosinas/genética , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Estresse Mecânico , Bexiga Urinária/citologia , Bexiga Urinária/ultraestrutura , Proteínas rab de Ligação ao GTP/genética
11.
Nanoscale ; 5(5): 1882-93, 2013 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-23348618

RESUMO

Curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione) is an active component of turmeric; it is responsible for its characteristic yellow color and therapeutic potential, but its poor bioavailability remains a major challenge. In order to improve the bioavailability of curcumin, various approaches have been used. One of the possible approaches to increase the bioavailability of curcumin is its conjugation on the surface of metal nanoparticles. Therefore, in the present study, we report the binding of curcumin on the surface of gold nanoparticles (AuNPs). The AuNPs were synthesized by the direct reduction of HAuCl(4) using curcumin in the aqueous phase, without the use of any other reducing agents. We found that curcumin acts both as a reducing and capping agent, stabilizing the gold sol for many months. Moreover, these curcumin-capped AuNPs also show good antioxidant activity which was confirmed by the DPPH (2,2-diphenyl-l-picrylhydrazyl) radical test. Thus, the surface functionalization of AuNPs with curcumin may pave a new way of using the curcuminoids towards possible drug delivery and therapeutics. Apart from the experimental study, a detailed quantum chemical calculation using density functional theory (DFT) has been performed, in order to investigate the formation of a complex of curcumin with Au(3+) ions in different possible conformational isomeric forms. Our theoretical calculations indicate the evidence of electron transfer from curcumin into the Au center and essentially indicate that as a consequence of complexation, Au(3+) ions are reduced to Au(0). Our theoretical results also propose that it is the breakage of intramolecular H-bonding that probably leads to the increased availability of curcumin in the presence of gold ions and water molecules.


Assuntos
Antioxidantes/química , Curcumina/química , Ouro/química , Nanopartículas Metálicas/química , Portadores de Fármacos/química , Ligações de Hidrogênio , Espectroscopia Fotoeletrônica , Teoria Quântica , Propriedades de Superfície , Termodinâmica , Água/química
12.
EMBO J ; 29(12): 1961-75, 2010 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-20461056

RESUMO

Compensatory endocytosis (CE) ensures recycling of membrane components and maintenance of plasma membrane size; however, the mechanisms, regulation, and physiological functions of clathrin-independent modes of CE are poorly understood. CE was studied in umbrella cells, which undergo regulated exocytosis of subapical discoidal/fusiform vesicles (DFV) during bladder filling, and may then replenish the pool of DFV by internalizing apical membrane during voiding. We found that voiding-stimulated CE, which depended on beta(1) integrin-associated signalling pathways, occurred by a dynamin-, actin-, and RhoA-regulated mechanism and was independent of caveolins, clathrin, and flotillin. Internalized apical membrane and fluid were initially found in ZO-1-positive vesicles, which were distinct from DFV, classical early endosomes, or the Golgi, and subsequently in lysosomes. We conclude that clathrin-independent CE in umbrella cells functions to recover membrane during voiding, is integrin regulated, occurs by a RhoA- and dynamin-dependent pathway, and terminates in degradation and not recapture of membrane in DFV.


Assuntos
Dinaminas/metabolismo , Endocitose , Integrina beta1/metabolismo , Bexiga Urinária/fisiologia , Urotélio/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Caveolinas/metabolismo , Clatrina/metabolismo , Proteínas de Membrana/metabolismo , Coelhos , Vesículas Transportadoras/fisiologia
13.
Am J Physiol Renal Physiol ; 297(6): F1477-501, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19587142

RESUMO

The uroepithelium sits at the interface between the urinary space and underlying tissues, where it forms a high-resistance barrier to ion, solute, and water flux, as well as pathogens. However, the uroepithelium is not simply a passive barrier; it can modulate the composition of the urine, and it functions as an integral part of a sensory web in which it receives, amplifies, and transmits information about its external milieu to the underlying nervous and muscular systems. This review examines our understanding of uroepithelial regeneration and how specializations of the outermost umbrella cell layer, including tight junctions, surface uroplakins, and dynamic apical membrane exocytosis/endocytosis, contribute to barrier function and how they are co-opted by uropathogenic bacteria to infect the uroepithelium. Furthermore, we discuss the presence and possible functions of aquaporins, urea transporters, and multiple ion channels in the uroepithelium. Finally, we describe potential mechanisms by which the uroepithelium can transmit information about the urinary space to the other tissues in the bladder proper.


Assuntos
Urotélio/citologia , Urotélio/fisiologia , Animais , Aquaporinas/metabolismo , Transporte Biológico , Membrana Celular/fisiologia , Endocitose , Exocitose , Humanos , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Regeneração , Células-Tronco/fisiologia , Junções Íntimas , Ureia/metabolismo , Bexiga Urinária/fisiologia , Água/metabolismo
14.
Mol Biol Cell ; 20(1): 282-95, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18987341

RESUMO

Epithelial cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are initiated and coordinated and the mechanotransduction pathways involved are not well understood. We observed that in response to a dynamic mechanical environment, increased apical membrane tension, but not pressure, stimulated apical membrane exocytosis and ion transport in bladder umbrella cells. The exocytic response was independent of temperature but required the cytoskeleton and the activity of a nonselective cation channel and the epithelial sodium channel. The subsequent increase in basolateral membrane tension had the opposite effect and triggered the compensatory endocytosis of added apical membrane, which was modulated by opening of basolateral K(+) channels. Our results indicate that during the dynamic processes of bladder filling and voiding apical membrane dynamics depend on sequential and coordinated mechanotransduction events at both membrane domains of the umbrella cell.


Assuntos
Membrana Celular/metabolismo , Células Epiteliais , Mecanotransdução Celular/fisiologia , Bexiga Urinária/citologia , Urotélio/citologia , Animais , Membrana Celular/química , Citoesqueleto/metabolismo , Eletrofisiologia , Endocitose/fisiologia , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Pressão Hidrostática , Ativação do Canal Iônico/fisiologia , Canais Iônicos/metabolismo , Modelos Biológicos , Ratos , Estresse Mecânico , Bexiga Urinária/metabolismo
15.
Proc Natl Acad Sci U S A ; 105(41): 15773-8, 2008 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-18843107

RESUMO

The discoidal/fusiform vesicles (DFV) of bladder umbrella cells undergo regulated exocytosis in response to stretch, but little is known about their biogenesis or the molecular machinery that modulates this process. We observed that Rab11a was expressed in umbrella cells (but not Rab11b or Rab25) and was associated with DFV. Using adenovirus-mediated delivery we transduced umbrella cells in situ with either dominant active (DA) or dominant negative (DN) mutants of Rab11a. DA-Rab11a stimulated an increase in apical surface area in the absence of stretch, whereas DN-Rab11a inhibited stretch-induced changes. Endocytosed fluid and membrane markers had little access to Rab11a-positive DFV, but virally expressed human growth hormone (hGH), a secretory protein, was packaged into DFV. Whereas expression of DA-Rab11a stimulated release of hGH into the bladder lumen, expression of DN-Rab11a had the opposite effect. Our results indicate that DFV may be biosynthetic in nature and that their exocytosis depends on the activity of the Rab11a GTPase.


Assuntos
Exocitose , Bexiga Urinária/citologia , Proteínas rab de Ligação ao GTP/fisiologia , Forma Celular , Vesículas Citoplasmáticas , Exocitose/efeitos dos fármacos , Hormônio do Crescimento Humano/metabolismo , Humanos , Proteínas Mutantes/administração & dosagem , Proteínas Mutantes/farmacologia , Transdução Genética , Bexiga Urinária/ultraestrutura , Proteínas rab de Ligação ao GTP/genética
16.
Environ Microbiol ; 10(5): 1285-95, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18279345

RESUMO

Xenorhabdus nematophila produces type 1 fimbriae on the surface of Phase I cells. Fimbriae mediate recognition and adhesion of the bacteria to its target cell. To investigate the role of fimbriae in the biology of X. nematophila, we have produced a fimbrial mutant strain by insertional inactivation of the mrxA gene, encoding the structural subunit of type 1 fimbriae. Phenotypic characterization of the mutant revealed loss of fimbriae on the cell surface. Cell surface characteristics like dye absorption, biofilm formation, red blood cell agglutination remained unaltered. The mrxA mutant was defective in swarming on soft agar, although swimming motility was not affected. Flagellar expression was suppressed in the mrxA strain under swarming conditions, but not swimming conditions. Agglutination and cytotoxicity of the mutant to larval haemocytes was also reduced. When the mutant cells were injected in the haemocoel of the fourth instar larvae of Helicoverpa armigera, an increase in the LT(50) of 9-12 h was observed relative to the wild-type strain. The nematode growth was slow on the lawn of the fimbrial mutant. The mrxA negative strain was unable to colonize the nematode gut efficiently. This study demonstrates importance of type 1 fimbriae in establishment of bacteria-nematode symbiosis, a key to successful pest management program.


Assuntos
Fímbrias Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Rabditídios/microbiologia , Simbiose , Xenorhabdus/crescimento & desenvolvimento , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mariposas/crescimento & desenvolvimento , Mariposas/microbiologia , Movimento , Mutação , Controle Biológico de Vetores , Xenorhabdus/genética , Xenorhabdus/metabolismo
17.
Biochem Biophys Res Commun ; 336(1): 346-56, 2005 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-16150307

RESUMO

Nitric oxide synthase (NOS) is amongst a family of evolutionarily conserved enzymes, involved in a multi-turnover process that results in NO as a product. The significant role of NO in various pathological and physiological processes has created an interest in this enzyme from several perspectives. This study describes for the first time, cloning and expression of a NOS-like protein, baNOS, from Bacillus anthracis, a pathogenic bacterium responsible for causing anthrax. baNOS was expressed in Escherichia coli as a soluble and catalytically active enzyme. Homology models generated for baNOS indicated that the key structural features that are involved in the substrate and active site interaction have been highly conserved. Further, the behavior of baNOS in terms of heme-substrate interactions and heme-transitions was studied in detail. The optical perturbation spectra of the heme domain demonstrated that the ligands perturb the heme site in a ligand specific manner. baNOS forms a five-coordinate, high-spin complex with l-arginine analogs and a six-coordinate low-spin complex with inhibitor imidazole. Studies indicated that the binding of l-arginine, N(omega)-hydroxy-l-arginine, and imidazole produces various spectroscopic species that closely correspond to the equivalent complexes of mammalian NOS. The values of spectral binding constants further corroborated these results. The overall conservation of the key structural features and the correlation of heme-substrate interactions in baNOS and mammalian NOS, thus, point towards an interesting phenomenon of convergent evolution. Importantly, the NO generated by NOS of mammalian macrophages plays a potent role in antimicrobicidal activity. Because of the existence of high structural and behavioral similarity between mammalian NOS and baNOS, we propose that NO produced by B. anthracis may also have a pivotal pathophysiological role in anthrax infection. Therefore, this first report of characterization of a NOS-like protein from a pathogenic bacterium opens up avenues for further studies in understanding the importance of this protein in pathogenicity.


Assuntos
Bacillus anthracis/enzimologia , Óxido Nítrico Sintase/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Dados de Sequência Molecular , Óxido Nítrico Sintase/química , Óxido Nítrico Sintase/isolamento & purificação , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de Aminoácidos , Análise Espectral
18.
J Bacteriol ; 186(19): 6465-76, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15375127

RESUMO

Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth- or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.


Assuntos
Proteínas de Fímbrias/isolamento & purificação , Inseticidas/isolamento & purificação , Aglutinação , Sequência de Aminoácidos , Animais , Sequência de Bases , Dicroísmo Circular , Clonagem Molecular , Proteínas de Fímbrias/química , Proteínas de Fímbrias/farmacologia , Hemócitos/metabolismo , Larva/efeitos dos fármacos , Dados de Sequência Molecular , Subunidades Proteicas , Proteínas Recombinantes/isolamento & purificação , Xenorhabdus
19.
Biochem Biophys Res Commun ; 316(2): 559-64, 2004 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-15020254

RESUMO

Protective antigen (PA) is the main immunogenic constituent of all vaccines against anthrax. It is known to lose its biological activity even at 37 degrees C. Its thermolabile nature has, thus, remained a cause of concern as even transient exposure of the vaccine to higher temperature could compromise its efficacy. Various types of cosolvent excipients have been used to stabilize a number of proteins with variable success. However, no comprehensive and systematic study to stabilize anthrax PA molecule using this approach has ever been undertaken. We have carried out a systematic study on the effect of osmoprotectants like glycine and its methyl derivatives, sarcosine, dimethylglycine, and betaine, on the thermostability of PA. The thermal stability of PA was found to be highly sensitive to pH with maxima at pH 7.9. All the cosolvent additives used were able to enhance the thermal stability of PA as inferred from an increase in T(1/2) values, the temperature at which 50% activity was retained during short-term incubation. Glycine was found to be the best stabilizer, while the ability of its methyl derivatives to stabilize PA decreased with an increase in the number of substituted methyl groups suggesting perturbation of hydrophobic interactions. On extended incubation at 40 degrees C the half-life of PA thermal inactivation increased more than four times in the presence of glycine. Thus, glycine could be used as an effective stabilizer to enhance the shelf life of recombinant vaccine against anthrax.


Assuntos
Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Glicina/farmacologia , Temperatura Ambiente , Animais , Vacinas contra Antraz , Linhagem Celular , Glicina/análogos & derivados , Meia-Vida , Concentração de Íons de Hidrogênio , Metilação , Camundongos , Osmose
20.
Biochem Biophys Res Commun ; 314(4): 943-9, 2004 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-14751223

RESUMO

Xenorhabdus nematophila is an insect pathogenic bacterium, known to produce protein toxins that kill the larval host. We have described a cytotoxic pilin subunit of X. nematophila, which is expressed on the cell surface and also secreted in the extracellular medium associated with outer membrane vesicles. A 17kDa pilin subunit was isolated and purified from X. nematophila cell surface. The protein showed cytotoxicity to larval hemocytes of Helicoverpa armigera in an in vitro assay, causing agglutination of the cells, and releasing cytoplasmic enzyme lactate dehydrogenase in the medium. The pilin protein was able to bind to the surface of larval hemocytes. The binding and cytotoxicity of the purified 17kDa protein to hemocytes was inhibited by antiserum raised against the pilin protein. The study demonstrates for the first time a cytotoxic structural subunit of pilin from an entomopathogenic bacterium X. nematophila that is excreted in the extracellular medium with outer membrane vesicles.


Assuntos
Proteínas de Fímbrias/isolamento & purificação , Xenorhabdus/química , Testes de Aglutinação , Animais , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Proteínas de Fímbrias/toxicidade , L-Lactato Desidrogenase/metabolismo , Mariposas/microbiologia , Ligação Proteica
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