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1.
ACS Appl Mater Interfaces ; 11(20): 18681-18690, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31038908

RESUMO

In recent years, favorable enhanced wound-healing properties and excellent biocompatibility of keratin derived from human hair have attracted considerable attention. Recombinant keratin proteins can be produced by recombinant DNA technology and have higher purity than extracted keratin. However, the wound-healing properties of recombinant keratin proteins remain unclear. Herein, two recombinant trichocyte keratins including human type I hair keratin 37 and human type II hair keratin 81 were expressed using a bacterial expression system, and recombinant keratin nanoparticles (RKNPs) were prepared via an ultrasonic dispersion method. The molecular weight, purity, and physicochemical properties of the recombinant keratin proteins and nanoparticles were assessed using gel electrophoresis, circular dichroism, mass spectrometry, and scanning electron microscope analyses. The RKNPs significantly enhanced cell proliferation and migration in vitro, and the treatment of dermal wounds in vivo with RKNPs resulted in improved wound healing associated with improved epithelialization, vascularization, and collagen deposition and remodeling. In addition, the in vivo biocompatibility test revealed no systemic toxicity. Overall, this work demonstrates that RKNPs are a promising candidate for enhanced wound healing, and this study opens up new prospects for the development of keratin biomaterials.


Assuntos
Derme , Queratinas Específicas do Cabelo , Queratinas Tipo II , Queratinas Tipo I , Nanopartículas , Cicatrização/efeitos dos fármacos , Ferimentos e Lesões , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Derme/metabolismo , Derme/patologia , Humanos , Queratinas Específicas do Cabelo/química , Queratinas Específicas do Cabelo/farmacologia , Queratinas Tipo I/química , Queratinas Tipo I/farmacologia , Queratinas Tipo II/química , Queratinas Tipo II/farmacologia , Masculino , Nanopartículas/química , Nanopartículas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Ferimentos e Lesões/tratamento farmacológico , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologia
2.
J Struct Biol ; 204(3): 491-497, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30248462

RESUMO

The X-ray diffraction patterns of quill and hair, as well as other trichocyte keratin appendages, contain meridional reflections that can be indexed on an axial repeat of 470 Å. Unusually, however, many of the expected orders are not observed. A possible explanation, proposed by Fraser and MacRae (1983), was that the intermediate filaments (IF) that constitute the fibrillar component of the filament/matrix texture consist of 4-chain protofilaments arranged on a surface lattice subject to a helical dislocation. The radial projection of the resulting 8-protofilament ribbon was defined in terms of a two-dimensional unit cell characterized by vectors (a, b) with axial projections za ∼ 74 Šand zb ∼ 198 Å. This situation resembles that found in microtubules, where helical dislocations in subunit packing are also encountered, leading to a so-called "seam" along their length (Metoz and Wade, 1997). In keratin, however, the protofilaments are helical so the seam is inclined to the axis of the IF. Here we report details of the Patterson function that provides independent evidence for both the helical dislocation and the dimensions of the surface lattice. In addition, the observed meridional X-ray amplitudes have been compared with those predicted by various models of the axial distribution of electron density. A new model, adapted from one previously proposed, fits the data significantly better than has heretofore proved possible. An interpretation of the model in terms of either specific keratin-associated-protein (KAP) binding or the retention of IF symmetry by a portion of the head and/or tail domains is suggested.


Assuntos
Cabelo/química , Filamentos Intermediários/química , Queratinas Específicas do Cabelo/química , Porcos-Espinhos/metabolismo , Animais , Cristalografia por Raios X , Proteínas do Citoesqueleto/química , Modelos Químicos , Oxirredução , Difração de Raios X
3.
Adv Exp Med Biol ; 1054: 21-32, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29797265

RESUMO

Wool and hair fibres are primarily composed of proteins of which the keratins and keratin associated proteins (KAPs) are the major component. Considerable diversity is known to exist within these two groups of proteins. In the case of the keratins two major families are known, of which there are 11 members in the acidic Type I family and 7 members in the neutral-basic Type II family. The KAPs are even more diverse than the keratins, with 35 families being known to exist when the KAPs found in monotremes, marsupials and other mammalian species are taken into consideration. Human hair and wool are known to have 88 and 73 KAPs respectively, though this number rises for wool when polymorphism within KAP families is included.


Assuntos
Cabelo/química , Queratinas Específicas do Cabelo/química , Lã/química , Animais , Humanos
4.
Adv Exp Med Biol ; 1054: 47-56, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29797267

RESUMO

The major components of hair are keratins and keratin associated proteins (KRTAPs). KRTAPs form the interfilamentous matrix between intermediate filament bundles through extensive disulfide bond cross-linking with the numerous cysteine residues in hair keratins. A variable number of approximately100-180 genes compose the KRTAP gene family in mammals. KRTAP gene family members present a typical pattern of concerted evolution, and its evolutionary features are consistent with the evolution of mammalian hair. KRATP genes might be more important in determining the structure of cashmere fibers in domestic mammals like sheep and goats. KRTAP gene variants thus should provide information for improved wool by sheep and goat breeding.


Assuntos
Evolução Molecular , Queratinas Específicas do Cabelo/química , Lã/química , Animais , Cabras , Ovinos
5.
J Biomater Sci Polym Ed ; 29(7-9): 1081-1093, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29285991

RESUMO

The intrinsically high cysteine content in human hair keratins and keratin associated proteins confer hair its outstanding mechanical strength through the formation of strong intermolecular disulfide bonds. In addition, these proteins offer the potential to be exploited as potent antioxidants. This report presents our findings on the antioxidant effects of human hair protein extracts and their consequent protective role against oxidative stress in human dermal fibroblast (HDF) cultures. Protein extracts were obtained from human hair using sodium sulfide as the reducing agent, and characterized using SDS-PAGE, Western blotting, MALDI-ToF mass spectrometry and amino acid analysis. Cysteine was found to account for 11.2 mol % in the extracted fractions. By measuring 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging activity, the hair protein fractions were shown to possess significant antioxidant ability (IC50 = 16.22 µM). As a supplement in cell culture media, the extracts protected HDFs from H2O2 induced oxidative stress, which was demonstrated by the maintenance of cell viability and reduced reactive oxygen species production. Besides offering mechanical support as a scaffolding material, the unique antioxidizing ability of human hair protein extracts may also be exploited in biomedical applications.


Assuntos
Antioxidantes/farmacologia , Cabelo/química , Queratinas Específicas do Cabelo/farmacologia , Antioxidantes/química , Proliferação de Células/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Queratinas Específicas do Cabelo/química , Estresse Oxidativo/efeitos dos fármacos
6.
ACS Appl Mater Interfaces ; 9(49): 43004-43012, 2017 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-29160686

RESUMO

Biomaterials have been attracting attention as a useful building block for biocompatible and bioresorbable electronics due to their nontoxic property and solution processability. In this work, we report the integration of biocompatible keratin from human hair as dielectric layer for organic thin-film transistors (TFTs), with high performance, flexibility, and transient property. The keratin dielectric layer exhibited a high capacitance value of above 1.27 µF/cm2 at 20 Hz due to the formation of electrical double layer. Fully solution-processable TFTs based on p-channel poly[4-(4,4-dihexadecyl-4H-cyclopenta[1,2-b:5,4-b]dithiophen-2-yl)-alt[1,2,5]thiadiazolo[3,4-c]-pyridine] (PCDTPT) and keratin dielectric exhibited high electrical property with a saturation field-effect mobility of 0.35 cm2/(Vs) at a low gate bias of -2 V. We also successfully demonstrate flexible TFTs, which exhibited good mechanical flexibility and electrical stability under bending strain. An artificial electronic synaptic PCDTPT/keratin transistor was also realized and exhibited high-performance synaptic memory effects via simple operation of proton conduction in keratin. An added functionality of using keratin as a substrate was also presented, where similar PCDTPT TFTs with keratin dielectric were built on top of keratin substrate. Finally, we observed that our prepared devices can be degraded in ammonium hydroxide solution, establishing the feasibility of keratin layer as various components of transient electrical devices, including as a substrate and dielectric layer.


Assuntos
Queratinas Específicas do Cabelo/química , Capacitância Elétrica , Eletricidade , Humanos , Transistores Eletrônicos
7.
J Struct Biol ; 200(1): 45-53, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28890162

RESUMO

For the past 50years there has been considerable debate over the sub-structure of the fully differentiated (oxidised) trichocyte keratin intermediate filament. Depending on the staining and preparative procedures employed, IF observed in transverse section in the transmission electron microscope have varied in appearance between that of a "ring" and a "ring-core" structure, corresponding to the so-called (8+0) and (7+1) protofilament arrangements. In a new analysis of the fine structure of the 1nm equatorial region of the X-ray diffraction pattern of quill we show that the observed pattern is consistent with the (8+0) model and we are also able to assign values to the various parameters. In contrast, we show that the observed X-ray pattern is inconsistent with a (7+1) arrangement. Furthermore, in the (7+1) model steric hindrance would be encountered between the core protofilament and those constituting the ring. The appearance of a central "core" in transverse TEM sections, previously attributed to a central protofilament, is explained in terms of portions of the apolar, disulfide-bonded head and/or tail domains of the trichocyte keratin IF molecules, including the conserved H subdomains, lying along the axis of the IF, thereby decreasing the efficacy of the reducing agents used prior to staining. The H1 subdomain, previously shown to be important in the assembly of epidermal IF molecules at the two- to four-molecule level, is likely to have a similar role for the trichocyte keratins and may form part of a central scaffold on which the molecules assemble into fully functional IF.


Assuntos
Queratinas Específicas do Cabelo/ultraestrutura , Sequência de Aminoácidos , Animais , Sequência Conservada , Queratinas Específicas do Cabelo/química , Microscopia Eletrônica de Transmissão e Varredura , Oxirredução , Porcos-Espinhos , Estrutura Quaternária de Proteína
8.
Biopolymers ; 107(10)2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28741310

RESUMO

In the past two decades, keratin biomaterials have shown impressive results as scaffolds for tissue engineering, wound healing, and nerve regeneration. In addition to its intrinsic biocompatibility, keratin interacts with specific cell receptors eliciting beneficial biochemical cues. However, during extraction from natural sources, such as hair and wool fibers, natural keratins are subject to extensive processing conditions that lead to formation of unwanted by-products. Additionally, natural keratins suffer from limited sequence tunability. Recombinant keratin proteins can overcome these drawbacks while maintaining the desired chemical and physical characteristics of natural keratins. Herein, we present the bacterial expression, purification, and solution characterization of human hair keratins K31 and K81. The obligate heterodimerization of the K31/K81 pair that results in formation of intermediate filaments is maintained in the recombinant proteins. Surprisingly, we have for the first time observed new zero- and one-dimensional nanostructures from homooligomerization of K81 and K31, respectively. Further analysis of the self-assembly mechanism highlights the importance of disulfide crosslinking in keratin self-assembly.


Assuntos
Biopolímeros/química , Queratinas Específicas do Cabelo/química , Proteínas Recombinantes/química , Engenharia Tecidual , Biopolímeros/genética , Humanos , Queratinas Específicas do Cabelo/genética , Nanoestruturas/química , Multimerização Proteica , Proteínas Recombinantes/genética
9.
Int J Biol Macromol ; 101: 805-814, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28315768

RESUMO

We selected 1235 decapeptides from human hair proteins encoded by human genes of keratins and keratin associated proteins. The peptides were linked to glass arrays and screened for their affinity towards a solution of human hair extracted keratin fraction. Based on the physicochemical properties of the peptides, ten variables were studied: content of different types of amino acid side chains (cysteine, hydrophobic, polar, basic, acidic, aromatic rings, amide, alcohol side chains), isoelectric point, and net charge. We found differences statistically significant on the binding affinity of peptides based on their content of cysteine, hydrophobic and polar amino acids, mainly containing alcohols. These results point to the formation of hydrophobic interactions and disulfide bonds between small peptides and human hair keratins as the main driving forces for the interaction of possible cosmetic peptides, namely designed to strength human hair. As so, our results enlighten the nature of the interaction of keratin based materials with human hair, which are claimed to enhance hair fiber strength, and enable a more directed and sustained hair care peptide design.


Assuntos
Queratinas Específicas do Cabelo/metabolismo , Fragmentos de Peptídeos/metabolismo , Humanos , Queratinas Específicas do Cabelo/química , Análise Serial de Proteínas , Ligação Proteica
10.
Colloids Surf B Biointerfaces ; 154: 160-170, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28334693

RESUMO

In the present study, we aimed at fabricating an osteoinductive biocomposite scaffold using keratin obtained from human hair, jellyfish collagen and eggshell-derived nano-sized spherical hydroxyapatite (nHA) for bone tissue engineering applications. Keratin, collagen and nHA were characterized with the modified Lowry method, free-sulfhydryl groups and hydroxyproline content analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) and thermal gravimetric analysis (TGA) which confirmed the success of the extraction and/or isolation processes. Human adipose mesenchymal stem cells (hAMSCs) were isolated and the cell surface markers were characterized via flow cytometry analysis in addition to multilineage differentiation capacity. The undifferentiated hAMSCs were highly positive for CD29, CD44, CD73, CD90 and CD105, but were not seen to express hematopoietic cell surface markers such as CD14, CD34 and CD45. The cells were successfully directed towards osteogenic, chondrogenic and adipogenic lineages in vitro. The microarchitecture of the scaffolds and cell attachment were evaluated using scanning electron microscopy (SEM). The cell viability on the scaffolds was assessed by the MTT assay which revealed no evidence of cytotoxicity. The osteogenic differentiation of hAMSCs on the scaffolds was determined histologically using alizarin red S, osteopontin and osteonectin stainings. Early osteogenic differentiation markers of hAMSCs were significantly expressed on the collagen-keratin-nHA scaffolds. In conclusion, it is believed that collagen-keratin-nHA osteoinductive biocomposite scaffolds have the potential of being used in bone tissue engineering.


Assuntos
Substitutos Ósseos/química , Colágeno/química , Durapatita/química , Queratinas Específicas do Cabelo/química , Osteogênese , Engenharia Tecidual/métodos , Animais , Células Cultivadas , Casca de Ovo/química , Humanos , Teste de Materiais , Células-Tronco Mesenquimais/citologia , Nanocompostos/química , Nanocompostos/ultraestrutura , Medicina Regenerativa , Cifozoários/química , Tecidos Suporte/química
11.
Subcell Biochem ; 82: 131-149, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28101861

RESUMO

The intermediate filaments (IF) in trichocyte (hard α-) keratin are unique amongst the various classes of IF in having not one but two topologically-distinct structures. The first is formed at an early stage of hair development in a reducing environment within the cells in the lower part of the follicle. The second structure occurs at a later stage of hair development in the upper part of the follicle, where there is a transition to an oxidizing environment. Crosslinking studies reveal that molecular slippage occurs within the IF upon oxidation and that this results in many cysteine residues lying in near axial alignment, thereby facilitating disulphide bond formation. The disulphide bonds so formed stabilize the assembly of IF molecules and convert the keratin fibre into a tough, resilient and insoluble structure suitable for its function in vivo as a thermo-regulator and a protector of the animal against its external environment.


Assuntos
Queratinas Específicas do Cabelo/química , Queratinas Específicas do Cabelo/ultraestrutura , Animais , Folículo Piloso , Humanos
12.
Int J Biol Macromol ; 85: 476-86, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26756110

RESUMO

The current study describes the in vitro phosphorylation of a human hair keratin, using protein kinase for the first time. Phosphorylation of keratin was demonstrated by (31)P NMR (Nuclear Magnetic Resonance) and Diffuse Reflectance Infrared Fourier Transform (DRIFT) techniques. Phosphorylation induced a 2.5 fold increase of adsorption capacity in the first 10 min for cationic moiety like methylene blue (MB). Thorough description of MB adsorption process was performed by several isothermal models. Reconstructed fluorescent microscopy images depict distinct amounts of dye bound to the differently treated hair. The results of this work suggest that the enzymatic phosphorylation of keratins might have significant implications in hair shampooing and conditioning, where short application times of cationic components are of prime importance.


Assuntos
Cátions/química , Queratinas Específicas do Cabelo/química , Adsorção , Humanos , Concentração de Íons de Hidrogênio , Cinética , Azul de Metileno/química , Fosforilação , Termodinâmica
13.
Methods Enzymol ; 569: 139-54, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26778557

RESUMO

A growing body of evidence from several laboratories points at nonmechanical functions of keratin intermediate filaments (IF), such as control of apoptosis, modulation of signaling, or regulation of innate immunity, among others. While these functions are generally assigned to the ability of IF to scaffold other proteins, direct mechanistic causal relationships between filamentous keratins and the observed effects of keratin knockout or mutations are still missing. We have proposed that the scaffolding of chaperones such as Hsp70/40 may be key to understand some IF nonmechanical functions if unique features or specificity of the chaperoning activity in the IF scaffold can be demonstrated. The same criteria of uniqueness could be applied to other biochemical functions of the IF scaffold. Here, we describe a subcellular fractionation technique based on established methods of keratin purification. The resulting keratin-enriched fraction contains several proteins tightly associated with the IF scaffold, including Hsp70/40 chaperones. Being nondenaturing, this fractionation method enables direct testing of chaperoning and other enzymatic activities associated with IF, as well as supplementation experiments to determine the need for soluble (cytosolic) proteins. This method also permits to analyze inhibitory activity of cytosolic proteins at independently characterized physiological concentrations. When used as complementary approaches to knockout, knockdown, or site-directed mutagenesis, these techniques are expected to shed light on molecular mechanisms involved in the effects of IF loss of function.


Assuntos
Proteínas de Choque Térmico/química , Queratinas Específicas do Cabelo/química , Proteína Quinase C/química , Animais , Células CACO-2 , Fracionamento Celular , Humanos , Filamentos Intermediários/enzimologia , Mucosa Intestinal/citologia , Camundongos , Fosforilação , Dobramento de Proteína , Processamento de Proteína Pós-Traducional
14.
PLoS One ; 10(8): e0137233, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26317522

RESUMO

Hair-derived keratin biomaterials composed mostly of reduced keratin proteins (kerateines) have demonstrated their utility as carriers of biologics and drugs for tissue engineering. Electrostatic forces between negatively-charged keratins and biologic macromolecules allow for effective drug retention; attraction to positively-charged growth factors like bone morphogenetic protein 2 (BMP-2) has been used as a strategy for osteoinduction. In this study, the intermolecular surface and bulk interaction properties of kerateines were investigated. Thiol-rich kerateines were chemisorbed onto gold substrates to form an irreversible 2-nm rigid layer for surface plasmon resonance analysis. Kerateine-to-kerateine cohesion was observed in pH-neutral water with an equilibrium dissociation constant (KD) of 1.8 × 10(-4) M, indicating that non-coulombic attractive forces (i.e. hydrophobic and van der Waals) were at work. The association of BMP-2 to kerateine was found to be greater (KD = 1.1 × 10(-7) M), within the range of specific binding. Addition of salts (phosphate-buffered saline; PBS) shortened the Debye length or the electrostatic field influence which weakened the kerateine-BMP-2 binding (KD = 3.2 × 10(-5) M). BMP-2 in bulk kerateine gels provided a limited release in PBS (~ 10% dissociation in 4 weeks), suggesting that electrostatic intermolecular attraction was significant to retain BMP-2 within the keratin matrix. Complete dissociation between kerateine and BMP-2 occurred when the PBS pH was lowered (to 4.5), below the keratin isoelectric point of 5.3. This phenomenon can be attributed to the protonation of keratin at a lower pH, leading to positive-positive repulsion. Therefore, the dynamics of kerateine-BMP-2 binding is highly dependent on pH and salt concentration, as well as on BMP-2 solubility at different pH and molarity. The study findings may contribute to our understanding of the release kinetics of drugs from keratin biomaterials and allow for the development of better, more clinically relevant BMP-2-conjugated systems for bone repair and regeneration.


Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Ouro/metabolismo , Queratinas Específicas do Cabelo/metabolismo , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Proteína Morfogenética Óssea 2/química , Ouro/química , Cabelo/química , Humanos , Concentração de Íons de Hidrogênio , Queratinas Específicas do Cabelo/química , Ligação Proteica , Eletricidade Estática , Propriedades de Superfície
15.
Acta Biomater ; 23: 201-213, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25997587

RESUMO

Polymeric biomaterials that provide a matrix for cell attachment and proliferation while achieving delivery of therapeutic agents are an important component of tissue engineering and regenerative medicine strategies. Keratins are a class of proteins that have received attention for numerous tissue engineering applications because, like other natural polymers, they promote favorable cell interactions and have non-toxic degradation products. Keratins can be extracted from various sources including human hair, and they are characterized by a high percentage of cysteine residues. Thiol groups on reductively extracted keratin (kerateine) form disulfide bonds, providing a more stable cross-linked hydrogel network than oxidatively extracted keratin (keratose) that cannot form disulfide crosslinks. We hypothesized that an iodoacetamide alkylation (or "capping") of cysteine thiol groups on the kerateine form of keratin could be used as a simple method to modulate the levels of disulfide crosslinking in keratin hydrogels, providing tunable rates of gel erosion and therapeutic agent release. After alkylation, the alkylated kerateines still formed hydrogels and the alkylation led to changes in the mechanical and visco-elastic properties of the materials consistent with loss of disulfide crosslinking. The alkylated kerateines did not lead to toxicity in MC3T3-E1 pre-osteoblasts. These cells adhered to keratin at levels comparable to fibronectin and greater than collagen. Alkylated kerateine gels eroded more rapidly than non-alkylated kerateine and this control over erosion led to tunable rates of delivery of rhBMP-2, rhIGF-1, and ciprofloxacin. These results demonstrate that alkylation of kerateine cysteine residues provides a cell-compatible approach to tune rates of hydrogel erosion and therapeutic agent release within the context of a naturally-derived polymeric system.


Assuntos
Materiais Biocompatíveis/síntese química , Preparações de Ação Retardada/síntese química , Hidrogéis/química , Queratinas Específicas do Cabelo/química , Queratinas Específicas do Cabelo/farmacologia , Engenharia Tecidual/métodos , Células 3T3 , Alquilação , Animais , Adesão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos
16.
ACS Appl Mater Interfaces ; 7(9): 5187-98, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25690726

RESUMO

Human hair keratins are readily available, easy to extract, and eco-friendly materials with natural bioactivities. Keratin-based materials have been studied for applications such as cell culture substrates, internal hemostats for liver injury, and conduits for peripheral nerve repair. However, there are limited reports of using keratin-based 3D scaffolds for cell culture in vitro. Here, we describe the development of a 3D hair keratin hydrogel, which allows for living cell encapsulation under near physiological conditions. The convenience of making the hydrogels from keratin solutions in a simple and controllable manner is demonstrated, giving rise to constructs with tunable physical properties. This keratin hydrogel is comparable to collagen hydrogels in supporting the viability and proliferation of L929 murine fibroblasts. Notably, the keratin hydrogels contract less significantly as compared to the collagen hydrogels, over a 16-day culture period. In addition, preliminary in vivo studies in immunocompetent animals show mild acute host tissue response. These results collectively demonstrate the potential of cell-loaded keratin hydrogels as 3D cell culture systems, which may be developed for clinically relevant applications.


Assuntos
Fibroblastos/citologia , Hidrogéis/química , Queratinas Específicas do Cabelo/química , Animais , Varredura Diferencial de Calorimetria , Técnicas de Cultura de Células , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Feminino , Fibroblastos/transplante , Humanos , Concentração de Íons de Hidrogênio , Queratinas Específicas do Cabelo/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Reologia , Pele/patologia , Temperatura , Transplante Homólogo
17.
Protoplasma ; 252(1): 271-81, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25030517

RESUMO

Trichocyst-enriched fractions were isolated from the marine dinophyte Prorocentrum micans. Transmission electron microscopy revealed that most of the trichocysts were discharged and had elongated to long filaments. Some trichocysts were still condensed. Fragments of discharged trichocysts measured up to 20 µm in length and 260 nm in width, those still condensed measured up to 1 µm in width and 16 µm in length. A distinct banding pattern with a transversal periodicity of approximately 16-18 nm and a periodic longitudinal striation of 3-4 nm could be measured along the trichocyst filaments. At higher magnifications, a fragile, alveolated, net-like organisation became obvious which resembled the one shown for the trichocysts of ciliates. When trichocyst-enriched fractions were treated with sodium dodecyl sulfate and centrifuged subsequently, no trichocysts were registered any longer in the sodium dodecyl sulfate-insoluble fraction by electron microscopy. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of trichocyst-enriched fractions and of the SDS-soluble fractions revealed a protein banding pattern which was dominated by polypeptides of 50-30, 12.5, and approximately 8.5 kDa. The polypeptide banding pattern deviated significantly from those registered for ejectisomes of cryptophytes and of the prasinophyte Pyramimonas grossii, for the Reb polypeptides which constitute the R-bodies of Caedibacter taeniospiralis, and also from the banding pattern of trichocysts of Paramecium. An antiserum directed against trichocysts of Paramecium did not cross-react with the polypeptides present in the trichocyst-enriched fraction of Prorocentrum micans.


Assuntos
Dinoflagelados/química , Queratinas Específicas do Cabelo/química , Microscopia Eletrônica de Transmissão/métodos , Dinoflagelados/crescimento & desenvolvimento
18.
Proteins ; 83(2): 224-34, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25402195

RESUMO

The disulfide bond network within the cortex of mammalian hair has a critical influence on the physical and mechanical characteristics of the fiber. The location, pattern, and accessibility of free and crosslinked cysteines underpin the properties of this network, but have been very difficult to map and understand, because traditional protein extraction techniques require the disruption of these disulfide bonds. Cysteine accessibility in both trichocyte keratins and keratin associated proteins (KAPs) of wool was investigated using staged labeling, where reductants and chaotropic agents were used to expose cysteines in a stepwise fashion according to their accessibility. Cysteines thus exposed were labeled with distinguishable alkylation agents. Proteomic profiling was used to map peptide modifications and thereby explore the role of KAPs in crosslinking keratins. Labeled cysteines from KAPs were detected when wool was extracted with reductant only. Among them were sequences from the end domains of KAPs, indicating that those cysteines were easily accessible in the fiber and could be involved in forming interdisulfide linkages with keratins or with other KAPs. Some of the identified peptides were from the rod domains of Types I and II keratins, with their cysteines positioned on the exposed surface of the α-helix. Peptides were also identified from keratin head and tail domains, demonstrating that they are not buried within the filament structure and, hence, have a possible role in forming disulfide linkages. From this study, a deeper understanding of the accessibility and potential reactivity of cysteine residues in the wool fiber cortex was obtained.


Assuntos
Cistina/química , Queratinas Específicas do Cabelo/química , Lã/química , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Mapeamento de Peptídeos , Estrutura Secundária de Proteína , Carneiro Doméstico
19.
PLoS One ; 9(6): e98073, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24915193

RESUMO

The primary function of hair and fur covering mammalian skin is to provide mechanical and thermal protection for the body. The proteins that constitute hair are extremely resistant to degradation by environmental factors. However, even durable materials can be slowly broken down by mechanical stresses, biodegradation mediated by endogenous enzymes in the skin or host microbes. We hypothesised that the biodegradation products of hair may possess bioprotective properties, which supplement their physical protective properties. Although evolutionary processes have led to a reduction in the amount of hair on the human body, it is possible that the bioprotective properties of hair biodegradation products have persisted. The human skin is exposed to various environmental carcinogenic factors. Therefore, we hypothesised that the potential bioprotective mechanisms of hair degradation products affect melanoma growth. We used pepsin to partially digest hair enzymatically, and this process produced a water-soluble lysate containing a mixture of peptides, including fragments of keratin and keratin-associated proteins. We found out that the mixtures of soluble peptides obtained from human hair inhibited the proliferation of human melanoma cells in vitro. Moreover, the hair-derived peptide mixtures also inhibited the proliferation of B lymphoma cells and urinary bladder cancer cells. Normal human cells varied in their susceptibility to the effects of the lysate; the hair-derived peptide mixtures modulated the proliferation of normal human fibroblasts but did not inhibit the proliferation of human mesenchymal cells derived from umbilical cord stromal cells. These results suggest that hair-derived peptides may represent a new class of anti-proliferative factors derived from basically structural proteins. Identification of active regulatory compounds and recognition of the mechanism of their action might pave the way to elaboration of new anticancer drugs.


Assuntos
Antineoplásicos/farmacologia , Queratinas Específicas do Cabelo/química , Fragmentos de Peptídeos/farmacologia , Hidrolisados de Proteína/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Humanos , Queratinócitos/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos
20.
Ukr Biochem J ; 86(1): 131-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24834727

RESUMO

The keratin fibers contain small amount of the internal lipids which are in free state or bound with fiber proteins via tioester of 18-methyleicosanoic acid. Today the origin of these lipids, their composition and functional properties are still not found. Therefore, our objective was to examine the content and composition of internal lipids in sheep's wool with different defects. We observed that regardless of the type of fibers defect there are significant changes especially in the quality composition of the internal lipids, although the total content of free and covalently bound lipids in all cases is practically identical. Notably, both free and covalently bound lipids composition of felted and simultaneously felted and yellowed wool is characterized by changes in contents mainly of free fatty acids and ceramides whereas abnormal thinning of fibers is accompanied only by a decrease of sulfolipids.


Assuntos
Ceramidas/análise , Colesterol/análise , Lipídeos/análise , Lã/química , Animais , Cromatografia em Camada Delgada , Ácidos Eicosanoicos/química , Queratinas Específicas do Cabelo/química , Microscopia Eletrônica de Varredura , Ovinos , Lã/ultraestrutura
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