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1.
Molecules ; 24(19)2019 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-31590314

RESUMO

Proteinaceous materials have numerous structures, many of which aid in the roles they perform. Some need to impart strength while others need elasticity or toughness. This study is the first to investigate the modification of both globular and fibrous protein, namely, zein, soy protein and gelatin, using deep eutectic solvents (DES) to form bioplastics, which may have application in drug delivery systems. The effects of DES content on the thermal and mechanical properties of the material were determined. Zein and soy are globular proteins, which both showed a significant change in the properties by the addition of DES. Both of these materials were, however, weaker and less ductile than the starch based materials previously reported in the literature. The material made from gelatin, a fibrous protein, showed variable properties depending on how long they were in contact with each other before pressing. Conductivity and NMR measurements indicate the existence of a continuous liquid phase, which are useful in the demonstrated application of transdermal drug delivery systems. It is shown that pharmaceutical DESs can be gelled with gelatin and this method is three times faster at delivering a pharmaceutical active ingredient across the skin barrier than from a corresponding solid formulation.


Assuntos
Preparações Farmacêuticas/síntese química , Escleroproteínas/química , Solventes/química , Sistemas de Liberação de Medicamentos , Gelatina/química , Preparações Farmacêuticas/química , Conformação Proteica , Solubilidade , Proteínas de Soja/química , Zeína/química
2.
Acta Neuropathol Commun ; 6(1): 54, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29961428

RESUMO

Parkinson disease (PD) is the second most common neurodegenerative disorder and the leading neurodegenerative cause of motor disability. Pathologic accumulation of aggregated alpha synuclein (AS) protein in brain, and imbalance in the nigrostriatal system due to the loss of dopaminergic neurons in the substantia nigra- pars compacta, are hallmark features in PD. AS aggregation and propagation are considered to trigger neurotoxic mechanisms in PD, including mitochondrial deficits and oxidative stress. The eukaryotic elongation factor-2 kinase (eEF2K) mediates critical regulation of dendritic mRNA translation and is a crucial molecule in diverse forms of synaptic plasticity. Here we show that eEF2K activity, assessed by immuonohistochemical detection of eEF2 phosphorylation on serine residue 56, is increased in postmortem PD midbrain and hippocampus. Induction of aggressive, AS-related motor phenotypes in a transgenic PD M83 mouse model also increased brain eEF2K expression and activity. In cultures of dopaminergic N2A cells, overexpression of wild-type human AS or the A53T mutant increased eEF2K activity. eEF2K inhibition prevented the cytotoxicity associated with AS overexpression in N2A cells by improving mitochondrial function and reduced oxidative stress. Furthermore, genetic deletion of the eEF2K ortholog efk-1 in C. elegans attenuated human A53T AS induced defects in behavioural assays reliant on dopaminergic neuron function. These data suggest a role for eEF2K activity in AS toxicity, and support eEF2K inhibition as a potential target in reducing AS-induced oxidative stress in PD.


Assuntos
Encéfalo/metabolismo , Quinase do Fator 2 de Elongação/metabolismo , Doença de Parkinson/patologia , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans , Linhagem Celular Tumoral , Modelos Animais de Doenças , Quinase do Fator 2 de Elongação/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação/genética , Neuroblastoma/patologia , Técnicas de Cultura de Órgãos , Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Escleroproteínas/toxicidade , alfa-Sinucleína/genética
3.
Mar Drugs ; 16(3)2018 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-29522478

RESUMO

The biosynthesis, chemistry, structural features and functionality of spongin as a halogenated scleroprotein of keratosan demosponges are still paradigms. This review has the principal goal of providing thorough and comprehensive coverage of spongin as a naturally prefabricated 3D biomaterial with multifaceted applications. The history of spongin's discovery and use in the form of commercial sponges, including their marine farming strategies, have been analyzed and are discussed here. Physicochemical and material properties of spongin-based scaffolds are also presented. The review also focuses on prospects and trends in applications of spongin for technology, materials science and biomedicine. Special attention is paid to applications in tissue engineering, adsorption of dyes and extreme biomimetics.


Assuntos
Materiais Biocompatíveis/química , Escleroproteínas/química , Animais , Biomimética/métodos , Humanos , Engenharia Tecidual/métodos , Tecidos Suporte
4.
Afr J Tradit Complement Altern Med ; 14(1): 110-122, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28480388

RESUMO

BACKGROUND: Biomaterials are non-drug substances used to treat, enhance or replace functions of body tissues or organs. Natural sources of biomaterials have recently become the focus of several research activities. Cowry shell constitutes one of the most promising natural sources of biomaterials because of its chemical stability, biodegradability and biocompatibility in the body. However, its applications may be limited due to immunogenic and toxic responses that may occur following implantation, hence this study. MATERIALS AND METHODS: Crude fibrous protein extracted with citrate buffer from pulverised cowry shells (Cypraea moneta (L)), was resolved into two components (CSP1 and CSP2) by gel filtration. Immunological studies were performed with antisera obtained from rabbits by double immunodiffusion and immunoelectrophoresis techniques. Mice treated with the proteins were observed for signs of toxicity and their liver, kidney, lungs and spleen were processed histologically. RESULTS: The native molecular weight of CSP1 and CSP2 determined by gel filtration were 91kDa and 33kDa respectively. CSP1 and CSP2 displayed single bands on SDS-PAGE with subunit molecular weight values of 19kDa and 19.5kDa respectively. Antisera obtained from rabbits immunised with the crude citrate buffer extracts precipitated the antigen in double immunodiffusion tests. Histopathological examinations revealed a dose-dependent damaging effect of the shell proteins on liver, kidney, lung and spleen tissues of the treated mice. CONCLUSION: This study showed that cowry shells contain fibrous proteins which are immunogenic and toxic in mice at relatively high concentrations, causing visible organ damage without concurrent physical manifestations.


Assuntos
Exoesqueleto/química , Fatores Imunológicos/química , Fatores Imunológicos/isolamento & purificação , Escleroproteínas/química , Escleroproteínas/isolamento & purificação , Caramujos/química , Animais , Fatores Imunológicos/efeitos adversos , Fatores Imunológicos/farmacologia , Rim/efeitos dos fármacos , Fígado/efeitos dos fármacos , Camundongos , Peso Molecular , Coelhos , Escleroproteínas/efeitos adversos , Escleroproteínas/farmacologia , Pele/efeitos dos fármacos , Baço/efeitos dos fármacos
5.
Int J Dermatol ; 56(5): 589-591, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28239832

RESUMO

BACKGROUND: The use of special silk textiles (Dermasilk) has shown positive effects on chronic inflammatory diseases like lichen sclerosus et atrophicus, atopic dermatitis, diabetic ulcerations, and vulvovaginal candidiasis. OBJECTIVE: Wearing T-shirts of this particular fabric could be useful in the management of patients with acne vulgaris on the back and trunk. MATERIAL AND METHODS: Dermasilk T-shirts were given to 14 patients with acne vulgaris papulopustulosa on the back. The patients wore these shirts every night for 6 weeks, and their acne lesions were monitored. Dermasilk represents a polymerisate of fibroin, a silk protein, and antimicrobial AEM5772/5, an unsoluble colorless, odorless ammonium with antifungal and antibacterial ability. RESULTS: Photographic documentation before and after 6 weeks showed a clinically significant reduction in acne lesions on the back without any concomitant treatment or change in lifestyle and living conditions. DISCUSSION: The use of Dermasilk textiles in other subacute-chronic inflammatory skin diseases has shown positive effects. This is the first report on their safe and effective use in the management of acne vulgaris papulopustulosa corporis.


Assuntos
Acne Vulgar/tratamento farmacológico , Compostos de Amônio/uso terapêutico , Anti-Infecciosos/uso terapêutico , Fibroínas/uso terapêutico , Escleroproteínas/uso terapêutico , Compostos de Amônio/administração & dosagem , Anti-Infecciosos/administração & dosagem , Dorso , Vestuário , Feminino , Humanos , Masculino , Projetos Piloto
6.
Subcell Biochem ; 82: 1-33, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28101857

RESUMO

During the 1930s and 1940s the technique of X-ray diffraction was applied widely by William Astbury and his colleagues to a number of naturally-occurring fibrous materials. On the basis of the diffraction patterns obtained, he observed that the structure of each of the fibres was dominated by one of a small number of different types of molecular conformation. One group of fibres, known as the k-m-e-f group of proteins (keratin - myosin - epidermin - fibrinogen), gave rise to diffraction characteristics that became known as the α-pattern. Others, such as those from a number of silks, gave rise to a different pattern - the ß-pattern, while connective tissues yielded a third unique set of diffraction characteristics. At the time of Astbury's work, the structures of these materials were unknown, though the spacings of the main X-ray reflections gave an idea of the axial repeats and the lateral packing distances. In a breakthrough in the early 1950s, the basic structures of all of these fibrous proteins were determined. It was found that the long protein chains, composed of strings of amino acids, could be folded up in a systematic manner to generate a limited number of structures that were consistent with the X-ray data. The most important of these were known as the α-helix, the ß-sheet, and the collagen triple helix. These studies provided information about the basic building blocks of all proteins, both fibrous and globular. They did not, however, provide detailed information about how these molecules packed together in three-dimensions to generate the fibres found in vivo. A number of possible packing arrangements were subsequently deduced from the X-ray diffraction and other data, but it is only in the last few years, through the continued improvements of electron microscopy, that the packing details within some fibrous proteins can now be seen directly. Here we outline briefly some of the milestones in fibrous protein structure determination, the role of the amino acid sequences and how new techniques, including electron microscopy, are helping to define fibrous protein structures in three-dimensions. We also introduce the idea that, from the known sequence characteristics of different fibrous proteins, new molecules can be designed and synthesized, thereby generating new biological materials with specific structural properties. Some of these, for example, are planned for use in drug delivery systems. Along the way we also introduce the various Chapters of the book, where individual fibrous proteins are discussed in detail.


Assuntos
Estrutura Secundária de Proteína , Escleroproteínas/química , Aminoácidos/química , Animais , Cristalografia por Raios X/história , Cristalografia por Raios X/métodos , História do Século XX , História do Século XXI , Humanos , Modelos Moleculares
7.
Int J Biol Macromol ; 96: 485-493, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28034822

RESUMO

The present study explores the preparation, characterization and the role of phenolic acid tethered fibrous protein in the management of induced oxidative stress studied under in vitro conditions. In brief, the biomaterial is prepared by engineering the fibrous protein with dihydroxy and trihydroxy phenolic acid moieties and subjected to characterization to ensure the tethering. The resultant biomaterial studied for its efficacy as a free radical scavenger using polymorphonuclear (PMN) cells with induced oxidative stress and also as an agent for cell migration using fibroblasts cells. Results revealed that induced oxidative stress in PMN cells after exposure to UVB radiation managed well with the prepared biomaterial by reducing the levels of superoxide anion, oxygen and hydroxyl radicals. Further, the protein and the phenolic acid interaction supports the cell migration as evidenced from the scratch assay. In conclusion, though phenolic acids are well known for their antimicrobial and antioxidant potential, indenting these acids directly to the wounds is not sensible, but tethering to protein explored the scavenging activity as expected. The present study infers that phenolic acid engineered protein has a significant role in managing the imbalance in the redox state prevailing in wounds and supports the healing at appreciable level.


Assuntos
Hidroxibenzoatos/química , Hidroxibenzoatos/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Escleroproteínas/química , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Compostos de Bifenilo/metabolismo , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Espaço Extracelular/efeitos da radiação , Radical Hidroxila/metabolismo , Metaloproteinases da Matriz/metabolismo , Neutrófilos/citologia , Neutrófilos/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Picratos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Suínos , Raios Ultravioleta
8.
Biochemistry ; 55(7): 1024-35, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26784838

RESUMO

The impacts of glycosylation on biomineralization protein function are largely unknown. This is certainly true for the mollusk shell, where glycosylated intracrystalline proteins such as AP24 (Haliotis rufescens) exist but their functions and the role of glycosylation remain elusive. To assess the effect of glycosylation on protein function, we expressed two recombinant variants of AP24: an unglycosylated bacteria-expressed version (rAP24N) and a glycosylated insect cell-expressed version (rAP24G). Our findings indicate that rAP24G is expressed as a single polypeptide containing variations in glycosylation that create microheterogeneity in rAP24G molecular masses. These post-translational modifications incorporate O- and N-glycans and anionic monosialylated and bisialylated, and monosulfated and bisulfated monosaccharides on the protein molecules. AFM and DLS experiments confirm that both rAP24N and rAP24G aggregate to form protein phases, with rAP24N exhibiting a higher degree of aggregation, compared to rAP24G. With regard to functionality, we observe that both recombinant proteins exhibit similar behavior within in vitro calcium carbonate mineralization assays and potentiometric titrations. However, rAP24G modifies crystal growth directions and is a stronger nucleation inhibitor, whereas rAP24N exhibits higher mineral phase stabilization and nanoparticle containment. We believe that the post-translational addition of anionic groups (via sialylation and sulfation), along with modifications to the protein surface topology, may explain the changes in glycosylated rAP24G aggregation and mineralization behavior, relative to rAP24N.


Assuntos
Gastrópodes/química , Glicoproteínas/química , Nácar/química , Processamento de Proteína Pós-Traducional , Escleroproteínas/química , Sequência de Aminoácidos , Animais , Calcificação Fisiológica , Biologia Computacional , Escherichia coli , Gastrópodes/ultraestrutura , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Peso Molecular , Polissacarídeos/química , Polissacarídeos/metabolismo , Agregados Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Escleroproteínas/genética , Escleroproteínas/metabolismo , Células Sf9 , Spodoptera
9.
Biotechnol Bioeng ; 113(5): 913-29, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26332660

RESUMO

Fibrous proteins, such as silk, elastin and collagen are finding broad impact in biomaterial systems for a range of biomedical and industrial applications. Some of the key advantages of biosynthetic fibrous proteins compared to synthetic polymers include the tailorability of sequence, protein size, degradation pattern, and mechanical properties. Recombinant DNA production and precise control over genetic sequence of these proteins allows expansion and fine tuning of material properties to meet the needs for specific applications. We review current approaches in the design, cloning, and expression of fibrous proteins, with a focus on strategies utilized to meet the challenges of repetitive fibrous protein production. We discuss recent advances in understanding the fundamental basis of structure-function relationships and the designs that foster fibrous protein self-assembly towards predictable architectures and properties for a range of applications. We highlight the potential of functionalization through genetic engineering to design fibrous protein systems for biotechnological and biomedical applications.


Assuntos
Biotecnologia/métodos , Clonagem Molecular/métodos , Engenharia de Proteínas/métodos , Escleroproteínas/genética , Seda/genética , Animais , Colágeno/química , Colágeno/genética , Colágeno/isolamento & purificação , Colágeno/metabolismo , Elastina/química , Elastina/genética , Elastina/isolamento & purificação , Elastina/metabolismo , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Escleroproteínas/química , Escleroproteínas/isolamento & purificação , Escleroproteínas/metabolismo , Seda/química , Seda/isolamento & purificação , Seda/metabolismo
11.
Biotech Histochem ; 89(5): 355-70, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24476562

RESUMO

Consistency in gold chloride staining is essential for anatomical analysis of sensory nerve endings. The gold chloride stain for this purpose has been modified by many investigators, but often yields inconsistent staining, which makes it difficult to differentiate structures and to determine nerve ending distribution in large tissue samples. We introduce additional steps and major changes to the modified Gairns' protocol. We controlled the temperature and mixing rate during tissue staining to achieve consistent staining and complete solution penetration. We subjected samples to sucrose dehydration to improve cutting efficiency. We then exposed samples to a solution containing lemon juice, formic acid and paraformaldehyde to produce optimal tissue transparency with minimal tissue deformity. We extended the time for gold chloride impregnation 1.5 fold. Gold chloride was reduced in the labrum using 25% formic acid in water for 18 h and in the capsule using 25% formic acid in citrate phosphate buffer for 2 h. Citrate binds gold nanoparticles, which minimizes aggregation in the tissue. We stored samples in fresh ultrapure water at 4° C to slow reduction and to maintain color contrast in the tissue. Tissue samples were embedded in Tissue Tek and sectioned at 80 and 100 µm instead of using glycerin and teasing the tissue apart as in Gairns' modified gold chloride method. We attached sections directly to gelatin subbed slides after sectioning with a cryostat. The slides then were processed and coverslipped with Permount. Staining consistency was demonstrated throughout the tissue sections and neural structures were clearly identifiable.


Assuntos
Compostos de Ouro/química , Células Receptoras Sensoriais/química , Coloração e Rotulagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Escleroproteínas/química , Células Receptoras Sensoriais/citologia , Ombro/anatomia & histologia
12.
J Struct Biol ; 186(3): 320-34, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24148884

RESUMO

As a result of X-ray fiber diffraction studies on fibrous proteins and crystallographic data on fragments derived from them, new experimental techniques across the biophysical and biochemical spectra, sophisticated computer modeling and refinement procedures, widespread use of bioinformatics and improved specimen preparative procedures the structures of many fibrous proteins have now been determined to at least low resolution. In so doing these structures have yielded insight into the relationship that exists between sequence and conformation and this, in turn, has led to improved methodologies for predicting structure from sequence data alone. In this personal retrospective a selection of progress made during the past 50years is discussed in terms of events to which the author has made some contribution.


Assuntos
Pesquisa Biomédica/história , Escleroproteínas/química , Biofísica/métodos , Cristalografia por Raios X , História do Século XX , Queratinas/química , Modelos Moleculares , Plaquinas/química , Conformação Proteica , Tropomiosina/química , Difração de Raios X
13.
Mol Nutr Food Res ; 57(10): 1695-707, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23765989

RESUMO

This paper gives an insight into key factors, which impair enzymatic protein digestion. By nature, some proteins in raw products are already poorly digestible because of structural peculiarities, or due to their occurrence in plant cytoplasmic organelles or in cell membranes. In plant-based protein, molecular and structural changes can be induced by genetic engineering, even if protein is not a target compound class of the genetic modification. Other proteins only become difficult to digest due to changes that occur during the processing of proteinaceous products, such as extruding, boiling, or acidic or alkaline treatment. The utilization of proteinaceous raw materials in industrial fermentations can also have negative impacts on protein digestibility, when reused as fermentation by-products for animal nutrition, such as brewers' grains. After consumption, protein digestion can be impeded in the intestine by the presence of antinutritional factors, which are ingested together with the food or feedstuff. It is concluded that the encircling matrix, but also molecular, chemical, and structural peculiarities or modifications to amino acids and proteins obstruct protein digestion by common proteolytic enzymes in humans and animals.


Assuntos
Proteínas na Dieta/metabolismo , Digestão/fisiologia , Animais , Proteínas na Dieta/química , Fermentação , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Escleroproteínas/química , Proteínas de Armazenamento de Sementes/química
14.
Biomacromolecules ; 13(12): 4264-72, 2012 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-23137042

RESUMO

Whereas there is growing interest in producing biomaterials containing coiled-coils, relatively few studies have made use of naturally occurring fibrous proteins. In this study, we have characterized fibrous proteins used by mother praying mantises to produce an extensive covering for their eggs called an ootheca and demonstrate the production of artificial ootheca using recombinantly produced proteins. Examination of natural oothecae by infrared spectroscopy and solid-state nuclear magnetic resonance revealed the material to consist of proteins organized predominately as coiled-coils. Two structural proteins, Mantis Fibroin 1 and Mantis Fibroin 2, were identified in ootheca from each of three species. Between species, the primary sequences of both proteins had diverged considerably, but other features were tightly conserved, including low molecular weight, high abundance of Ala, Glu, Lys, and Ser, and a triblock-like architecture with extensive central coiled-coil domain. Mantis fibroin hydrophobic cores had an unusual composition containing high levels of alanine and aromatic residues. Recombinantly produced mantis fibroins folded into coiled-coils in solution and could be fabricated into solid materials with high coiled-coil content. The structural features of mantis fibroins and their straightforward recombinant production make them promising templates for the production of coiled-coil biomimetics materials.


Assuntos
Materiais Biocompatíveis/química , Materiais Biomiméticos/síntese química , Fibroínas/química , Mantódeos/química , Óvulo , Alanina/química , Sequência de Aminoácidos , Animais , Dicroísmo Circular , Escherichia coli/genética , Feminino , Fibroínas/genética , Biblioteca Gênica , Ácido Glutâmico/química , Interações Hidrofóbicas e Hidrofílicas , Lisina/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Peso Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Escleroproteínas/química , Alinhamento de Sequência , Análise de Sequência de DNA , Serina/química
15.
Biochemistry (Mosc) ; 76(11): 1227-32, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22117549

RESUMO

A novel keratin-like fibrous protein K58 with molecular weight of about 58 kDa was discovered in bivalve Siliqua radiata ligament and identified by amino acid composition and MALDI-TOF-TOF analysis. We found that the protein is composed of cylindrical fibers (~160 nm in diameter) and contains high glycine (27.4%) and phenylalanine (10.5%) contents. Furthermore, it is homologous to keratin type II cytoskeletal 1, with repeat motifs of SGGG and SYGSGG. FTIR and secondary structure analysis indicate that K58 is composed of 46.2% ß-sheet, 33.4% ß-turn, 13.1% α-helix, and 4.7% disordered structure. This structure feature is closely related to the superior tensile strength, elasticity, and solvent resistance property of K58. These discoveries provide some evidence for evolution of keratin and fibrous proteins and prompt further studies of ligament fibrous proteins.


Assuntos
Bivalves/química , Escleroproteínas/química , Animais , Glicina/análise , Humanos , Queratinas/química , Ligamentos/química , Fenilalanina/análise , Estrutura Secundária de Proteína , Escleroproteínas/ultraestrutura , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
Mar Biotechnol (NY) ; 8(4): 415-24, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16670968

RESUMO

Two species of alcyonarian corals, Lobophytum crassum and Sinularia polydactyla, are closely related to each other. It is reported that the calcified organic substances in the skeletons of both contain a protein-polysaccharide complex playing a key role in the regulation of biocalcification. However, information on the matrix proteins of endoskeletal sclerite has been lacking. Hence we studied the proteinaceous organic matrices of sclerites for both species, to analyze the sequences and the functional properties of the proteins present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed four bands of proteins with apparent molecular masses of 102, 67, 48, and 37 kDa for L. crassum and seven bands of 109, 83, 70, 63, 41, 30, and 22 kDa for S. polydactyla. A major protein band of about 67 kDa in L. crassum and two bands of proteins of about 70 and 63 kDa in S. polydactyla yielded N-terminal amino acid sequences. Periodic acid-Schiff staining indicated that the 67-kDa protein in L. crassum, and 83- and 63-kDa proteins in S. polydactyla were glycosylated. For detection of calcium binding proteins, a Ca(2+) overlay analysis was conducted in the extract via (45)Ca autoradiography. The 102- and 67-kDa calcium binding proteins in L. crassum, and the 109- and 63-kDa Ca(2+) binding proteins in S. polydactyla were found to be radioactive. An assay for carbonic anhydrase (CA), which is thought to play an important role in the process of calcification, revealed specific activities. Newly derived protein sequences were subjected to standard sequence analysis involving identification of similarities to other proteins in databases. The significantly different protein expressions and compositional analysis of sequences between two species were demonstrated.


Assuntos
Antozoários/genética , Antozoários/fisiologia , Escleroproteínas/química , Acetazolamida/farmacologia , Sequência de Aminoácidos/genética , Animais , Antozoários/enzimologia , Antozoários/ultraestrutura , Isótopos de Cálcio/análise , Proteínas de Ligação ao Cálcio/isolamento & purificação , Inibidores da Anidrase Carbônica/farmacologia , Anidrases Carbônicas/análise , Anidrases Carbônicas/efeitos dos fármacos , Anidrases Carbônicas/metabolismo , Glicoproteínas/isolamento & purificação , Dados de Sequência Molecular , Escleroproteínas/isolamento & purificação , Alinhamento de Sequência , Especificidade da Espécie
17.
J Hum Genet ; 48(4): 177-82, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12730720

RESUMO

We have performed a comprehensive analysis of gene-expression profiles in human articular cartilage (hyaline cartilage) and meniscus (fibrocartilage) by means of a cDNA microarray consisting of 23,040 human genes. Comparing the profiles of the two types of cartilage with those of 29 other normal human tissues identified 24 genes that were specifically expressed in both cartilaginous tissues; these genes might be involved in maintaining phenotypes common to cartilage. We also compared the cartilage profiles with gene expression in human mesenchymal stem cells (hMSC), and detected 22 genes that were differentially expressed in cells representing the two cartilaginous lineages, 11 specific to each type, which could serve as markers for predicting the direction of chondrocyte differentiation. Our data should also provide useful information about regeneration of cartilage, especially in support of efforts to identify cartilage-specific molecules as potential agents for therapeutic approaches to joint repair.


Assuntos
Cartilagem Articular/metabolismo , Cartilagem/metabolismo , Perfilação da Expressão Gênica , Articulação do Joelho/metabolismo , Biomarcadores/análise , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Escleroproteínas/genética
18.
J Struct Biol ; 134(1): 25-34, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11469874

RESUMO

The juvenile shell of the brachiopod Discinisca consists of a mosaic of micrometer-sized siliceous tablets embedded in a chitinous substrate. The first-formed tablets are secreted on glycocalyx by a newly differentiated collective of outer epithelial cells. They are mainly rhombic but may also be ellipsoidal, discoidal, or deformed and sporadically overlap one another. On the surrounding juvenile shell, secreted by an incipient outer mantle lobe, the tablets are nearly all perfect rhombic plates in rhombic arrays. Their constant size, arrangement, and centripetal crystallization suggest intracellular assembly. The tablets, which are normally bilamellar, consist of discrete aggregates of crystalline spherules of silica in rhombic arrays within an organic matrix of fibrous protein and, presumably, a soluble polysaccharide(s). Mosaic secretion ceases at about the time when juveniles settle on the sea bed, which more or less coincides with the secretion of a ring of lamellae around the mosaic, induced by rapid advances and retractions of the outer mantle lobe prior to deposition of the organophosphatic mature shell. Energy dispersion X-ray analyses of pelagic and newly settled juveniles show that phosphatic secretion, even in the site of the first-formed outer epithelial collective, does not begin until all siliceous secretion has ceased.


Assuntos
Crustáceos/anatomia & histologia , Silício/metabolismo , Animais , Quitina/química , Quitina/metabolismo , Crustáceos/crescimento & desenvolvimento , Crustáceos/ultraestrutura , Cristalização , Cristalografia por Raios X , Células Epiteliais/metabolismo , Glicocálix/metabolismo , Microscopia Eletrônica de Varredura , Fosfatos/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Escleroproteínas/metabolismo , Silício/química
19.
Atherosclerosis ; 157(1): 251-4, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11427228

RESUMO

Plaques from the coronary arteries of explanted hearts showed massive calcification (15-fold increase) with a loss of scleroproteins (-36%), an increase in the collagen to elastin ratio (twofold) and activation (+15%) of matrix metalloproteinase-2 (MMP-2). Plaque-free portions of the coronary artery gave results similar to those obtained with the internal mammary artery. There was a significant correlation between plaque calcification and MMP-2 activation, suggesting that the two processes may be linked.


Assuntos
Doença das Coronárias/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Escleroproteínas/metabolismo , Adulto , Idoso , Calcinose/metabolismo , Doença das Coronárias/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Ativação Enzimática , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Pessoa de Meia-Idade
20.
J Vasc Res ; 38(3): 237-46, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11399896

RESUMO

Most previous studies of arterial wall elasticity and rheology have assumed that the properties of the wall are uniform across the thickness of the media and, therefore, that the relationship between stress and strain may be described by a constitutive equation based on a single strain energy function. The few studies where this assumption has been questioned, focussed on differences between the adventitia and the media rather than on differences within the media itself. Here, we report in vitro elasticity and residual strain measurements performed separately on the inner and outer half of the pig aortic media, together with a histomorphometric assessment of the radial distribution of elastin, collagen and smooth muscle cell numbers. Although we found that the pressure-diameter relationships of the two halves were dissimilar, when allowance was made for their different unloaded dimensions, their material properties agreed closely, a result in keeping with the observed uniform radial distribution of scleroprotein and vascular smooth muscle. We also found a difference in the opening angle (which is often taken as a measure of residual strain) between the inner and outer medial halves. However, strain analysis showed that the opening angle is an extremely sensitive measure of residual strain and that the difference in the actual magnitudes of residual strain between the two halves of the media was small. We conclude that the media of the porcine thoracic aorta has similar elastic properties throughout its thickness and that this uniformity is matched by a uniform distribution of matrix protein and vascular smooth muscle cells. Furthermore, the distribution of strain in the media can adequately be described by a single-layer model with uniform elastic properties throughout its thickness.


Assuntos
Aorta/química , Túnica Média/química , Anatomia Transversal , Animais , Aorta/anatomia & histologia , Colágeno/análise , Técnicas de Cultura , Elasticidade , Elastina/análise , Pressão , Escleroproteínas/análise , Estresse Mecânico , Suínos
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