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3.
Mol Cell Biochem ; 436(1-2): 189-199, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28589370

RESUMO

The EDA+ fibronectin splicing variant is overexpressed in psoriatic non-lesional epidermis and sensitizes keratinocytes to mitogenic signals. However, regulation of its abundance is only partially understood. In our recent cDNA microarray experiment, we identified three SR-rich splicing factors-splicing factor, arginine/serine-rich 18 (SFRS18), peptidyl-prolyl cis-trans isomerase G (PPIG), and luc-7 like protein 3 (LUC7L3)-which might be implicated in the preactivated states of keratinocytes in psoriatic non-involved skin and could also contribute to the regulation of fibronectin mRNA maturation. In this study, we investigated the role of LUC7L3, PPIG, and SFRS18 in psoriasis and in the mRNA maturation process of fibronectin. Regarding tissue staining experiments, we were able to demonstrate a characteristic distribution of the splicing factors in healthy, psoriatic non-involved and involved epidermis. Moreover, the expression profiles of these SR-rich proteins were found to be very similar in synchronized keratinocytes. Contribution of splicing facwwtors to the EDA+ fibronectin formation was also confirmed: their siRNA silencing leads to altered fibronectin mRNA and protein expression patterns, suggesting the participation in the EDA domain inclusion. Our results indicate that LUC7L3, PPIG, and SFRS18 are not only implicated in EDA+ fibronectin formation, but also that they could possess multiple roles in psoriasis-associated molecular abnormalities.


Assuntos
Fibronectinas/biossíntese , Queratinócitos/metabolismo , Psoríase/metabolismo , Fatores de Processamento de RNA/biossíntese , Splicing de RNA , RNA Mensageiro/metabolismo , Adolescente , Adulto , Ciclofilinas/biossíntese , Feminino , Humanos , Queratinócitos/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares , Psoríase/patologia , Proteínas de Ligação a RNA/biossíntese
4.
Br J Dermatol ; 174(3): 533-41, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26471375

RESUMO

BACKGROUND: Data indicate that in psoriasis, abnormalities are already present in nonlesional skin. Transforming growth factor-ß and keratinocyte growth factor (KGF), together with fibronectin and α5ß1 integrin, were suggested to play a crucial role in the pathogenesis of psoriasis by influencing inflammation and keratinocyte hyperproliferation. OBJECTIVES: To investigate the expression of KGF, fibroblast growth factor receptor (FGFR)2, fibronectin (FN) and extra domain A (EDA)-positive FN in healthy and nonlesional psoriatic skin, and to study the effect of KGF on the regulation of FN and EDA(+) FN production by fibroblasts. METHODS: Healthy, nonlesional psoriatic skin and lesional psoriatic skin were immunostained for α5 integrin, KGF, FGFR2, EDA(+) FN and signal transducer and activator of transcription (STAT)1. KGF-treated cell cultures were analysed for FN and EDA(+) FN mRNA and protein by real-time reverse-transcriptase polymerase chain reaction and flow cytometry, respectively. The major downstream signalling of KGF was investigated by blocking experiments using inhibitors of mitogen-activated protein kinase (MAPK) kinase (MEK1), AKT1/2, STAT1 and STAT3. RESULTS: The expression of α5 integrin, EDA(+) FN, KGF and its receptor FGFR2 is elevated in psoriatic nonlesional skin compared with healthy skin. KGF mildly induced EDA(+) FN, but not FN expression in healthy fibroblasts through MAPK signalling. Fibroblasts express the FGFR2-IIIc splice variant. STAT1 negatively regulates both FN and EDA(+) FN expression in healthy fibroblasts, and this regulation is compromised in fibroblasts derived from nonlesional psoriatic dermis. We detected active STAT1 in healthy and lesional skin, similarly to a previous report. However, in the nonlesional skin STAT1 activation was absent in tissues far away from lesions. CONCLUSIONS: The production of FN and EDA(+) FN by fibroblasts and the signalling of STAT1 are abnormally regulated in psoriatic nonlesional skin.


Assuntos
Fator 7 de Crescimento de Fibroblastos/fisiologia , Fibroblastos/metabolismo , Fibronectinas/biossíntese , Psoríase/metabolismo , Adolescente , Adulto , Estudos de Casos e Controles , Células Cultivadas , Fibronectinas/metabolismo , Voluntários Saudáveis , Humanos , Queratinócitos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Melanócitos/metabolismo , Pessoa de Meia-Idade , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/biossíntese , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3 , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Adulto Jovem
5.
Med Hypotheses ; 83(5): 599-606, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25200646

RESUMO

Lipedema is a common, but often underdiagnosed masquerading disease of obesity, which almost exclusively affects females. There are many debates regarding the diagnosis as well as the treatment strategies of the disease. The clinical diagnosis is relatively simple, however, knowledge regarding the pathomechanism is less than limited and curative therapy does not exist at all demanding an urgent need for extensive research. According to our hypothesis, lipedema is an estrogen-regulated polygenetic disease, which manifests in parallel with feminine hormonal changes and leads to vasculo- and lymphangiopathy. Inflammation of the peripheral nerves and sympathetic innervation abnormalities of the subcutaneous adipose tissue also involving estrogen may be responsible for neuropathy. Adipocyte hyperproliferation is likely to be a secondary phenomenon maintaining a vicious cycle. Herein, the relevant articles are reviewed from 1913 until now and discussed in context of the most likely mechanisms leading to the disease, which could serve as a starting point for further research.


Assuntos
Adipócitos/citologia , Estrogênios/química , Lipedema/genética , Lipedema/fisiopatologia , Tecido Adiposo , Proliferação de Células , Feminino , Predisposição Genética para Doença , Hormônios/química , Humanos , Inflamação , Perna (Membro)/irrigação sanguínea , Lipídeos/sangue , Masculino , Modelos Cardiovasculares , Obesidade/complicações , Sobrepeso/complicações , Sistema Nervoso Periférico , Sistema Nervoso Simpático
6.
Proc Natl Acad Sci U S A ; 105(19): 6888-93, 2008 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-18456840

RESUMO

The kinetics of electrogenic events associated with the different steps of the light-induced proton pump of bacteriorhodopsin is well studied in a wide range of time scales by direct electric methods. However, the investigation of the fundamental primary charge translocation phenomena taking place in the functional energy conversion process of this protein, and in other biomolecular assemblies using light energy, has remained experimentally unfeasible because of the lack of proper detection technique operating in the 0.1- to 20-THz region. Here, we show that extending the concept of the familiar Hertzian dipole emission into the extreme spatial and temporal range of intramolecular polarization processes provides an alternative way to study ultrafast electrogenic events on naturally ordered biological systems. Applying a relatively simple experimental arrangement based on this idea, we were able to observe light-induced coherent terahertz radiation from bacteriorhodopsin with femtosecond time resolution. The detected terahertz signal was analyzed by numerical simulation in the framework of different models for the elementary polarization processes. It was found that the principal component of the terahertz emission can be well described by excited-state intramolecular electron transfer within the retinal chromophore. An additional slower process is attributed to the earliest phase of the proton pump, probably occurring by the redistribution of a H bond near the retinal. The correlated electron and proton translocation supports the concept, assigning a functional role to the light-induced sudden polarization in retinal proteins.


Assuntos
Bacteriorodopsinas/química , Elétrons , Halobacterium salinarum/química , Prótons , Radiação , Simulação por Computador , Transporte de Elétrons , Cinética , Óptica e Fotônica , Bombas de Próton , Fatores de Tempo
7.
Biophys J ; 81(6): 3432-41, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11721005

RESUMO

The photocycle of dried bacteriorhodopsin, pretreated in a 0.3 M HCl solution, was studied. Some properties of this dried sample resemble that of the acid purple suspension: the retinal conformation is mostly all-trans, 15-anti form, the spectrum of the sample is blue-shifted by 5 nm to 560 nm, and it has a truncated photocycle. After photoexcitation, a K-like red-shifted intermediate appears, which decays to the ground state through several intermediates with spectra between the K and the ground state. There are no other bacteriorhodopsin-like intermediates (L, M, N, O) present in the photocycle. The K to K' transition proceeds with an enthalpy decrease, whereas during all the following steps, the entropic energy of the system decreases. The electric response signal of the oriented sample has only negative components, which relaxes to zero. These suggest that the steps after intermediate K represent a relaxation process, during which the absorbed energy is dissipated and the protein returns to its original ground state. The initial charge separation on the retinal is followed by limited charge rearrangements in the protein, and later, all these relax. The decay times of the intermediates are strongly influenced by the humidity of the sample. Double-flash experiments proved that all the intermediates are directly driven back to the ground state. The study of the dried acid purple samples could help in understanding the fast primary processes of the protein function. It may also have importance in technical applications.


Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/fisiologia , Luz , Halobacterium/metabolismo , Cinética , Fotoquímica , Análise Espectral Raman , Termodinâmica , Fatores de Tempo
8.
Biophys J ; 79(5): 2705-13, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11053142

RESUMO

The photocycle of pharaonis halorhodopsin was investigated in the presence of 100 mM NaN(3) and 1 M Na(2)SO(4). Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR' state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide.


Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/efeitos da radiação , Fenômenos Biofísicos , Biofísica , Halorrodopsinas , Cinética , Natronobacterium/química , Fotoquímica , Prótons , Espectrofotometria , Termodinâmica
9.
Biochim Biophys Acta ; 1319(1): 59-68, 1997 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-9107316

RESUMO

A fitting analysis resolved the kinetics in the microsecond to second time range of the absorption changes in the bacteriorhodopsin photocycle at pH = 8.0-9.5 in 3 M KCl into seven exponential components. The time constants and/or amplitudes of all components are strongly pH-dependent. In the pH range studied, the logarithms of the pH-dependent time constants varied linearly with pH. The maximum absolute value of the corresponding slopes was 0.4, in contrast with the theoretically expected value of 1 for unidirectional reactions coupled directly to proton exchange with the bulk phase. This indicates that the extracted macroscopic rate constants are not identical to the microscopic rate constants for the elementary photocycle reaction steps. Unexpected differences were found in the kinetic parameters in CHES and borate buffers.


Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/fisiologia , Sistema Livre de Células , Halobacterium/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Concentração Osmolar , Fotoquímica , Análise Espectral
10.
Biochim Biophys Acta ; 1319(1): 69-85, 1997 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-9107317

RESUMO

Photoselection measurements with moderate excitation intensity on bacteriorhodopsin (bR) immobilized in a polyacrylamide gel soaked in 3 M KCl in the pH range 8.0-9.5 resulted in an unusual time-dependent anisotropy. In the microsecond region, the anisotropy exhibits a constant level that is considerably less than 2/5 theoretically expected for the vanishing excitation intensity, indicating partial saturation. In the millisecond region, it becomes time-dependent. Theoretical models for such a time-dependent anisotropy are presented. These models include a consideration of: (i) reorientation of the retinal chromophore during or after excitation, (ii) parallel reactions of differently saturated photoselected species of a heterogenous bR population preexisting in the ground state or photochemically induced, (iii) branching in a photochemical step, and (iv) cooperativity of molecules within a trimer. All of these models describe the anisotropy as a ratio of sums of exponentials, where the rate constants correspond to the kinetics of the photocycle. An analysis of the fitted amplitudes of the exponentials favors the models involving parallel processes rather than those invoking chromophore reorientation.


Assuntos
Bacteriorodopsinas/química , Resinas Acrílicas , Bacteriorodopsinas/metabolismo , Halobacterium/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Proteínas de Membrana/química , Modelos Biológicos , Movimento , Concentração Osmolar , Fotoquímica , Análise Espectral
11.
Biophys J ; 69(5): 2060-5, 1995 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8580349

RESUMO

Dried oriented purple membrane samples of Halobacterium salinarium were excited by 150 fs laser pulses of 620 nm with a 7 kHz repetition rate. An unusual complex picosecond electric response signal consisting of a positive and a negative peak was detected by a sampling oscilloscope. The ratio of the two peaks was changed by 1) reducing the repetition rate, 2) varying the intensity of the excitation beam, and 3) applying background illumination by light of 647 nm or 511 nm. All of these features can be explained by the simultaneous excitation of the bacteriorhodopsin ground form and the K intermediate. The latter was populated by the (quasi)continuous excitation attributable to its prolonged lifetime in a dehydrated state. Least-square analysis resulted in a 5 ps upper and 2.5 ps lower limit for the time constant of the charge displacement process, corresponding to the forward reaction. That is in good agreement with the formation time of K. The charge separation driven by the reverse phototransition was faster, having a time constant of a 3.5 ps upper limit. The difference in the rates indicates the existence of different routes for the forward and the reverse photoreactions.


Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/efeitos da radiação , Fenômenos Biofísicos , Biofísica , Eletroquímica , Halobacterium/química , Halobacterium/efeitos da radiação , Lasers , Fotoquímica
12.
Biophys J ; 65(6): 2478-83, 1993 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8312486

RESUMO

Absorption kinetic and electric measurements were performed on oriented purple membranes of D96N bacteriorhodopsin mutant embedded in polyacrylamide gel and the kinetic parameters of the photointermediates determined. The rate constants, obtained from fits to time-dependent concentrations, were used to calculate the relative electrogenicity of the intermediates. The signals were analyzed on the basis of different photocycle models. The preferred model is the sequential one with reversible reaction. To improve the quality of the fits the necessity of introducing a second L intermediate arose. We also attempted to interpret our data in the view of reversible reactions containing two parallel photocycles, but the pH dependencies of the rate constants and electrogenicities favored the model containing sequential reversible transitions. A fast equilibrium for the L2<==>M1 transition and a strong pH dependence of the M2 electrogenicity was found, indicating that the M1 to M2 transition involves complex charge motions, as is expected in a conformational change of the protein.


Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Halobacterium salinarum/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Luz , Matemática , Modelos Teóricos , Mutação Puntual , Fatores de Tempo
13.
Biosystems ; 27(4): 201-2, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1493206

RESUMO

This paper summarizes the results found in our laboratory investigating the ultrafast light-induced charge separation in bacteriorhodopsin. A special technique was elaborated for dried oriented samples of long term stability. An upper limit of 21 ps was found by a direct electric method for the early charge separation processes. A permanent electric field on the surface of illuminated samples was demonstrated. The potential application of such samples as ultrafast optoelectric signal transducers is discussed.


Assuntos
Bacteriorodopsinas/química , Transdução de Sinais , Eletroquímica
14.
Biophys J ; 50(2): 357-66, 1986 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19431682

RESUMO

On capturing a quantum of light, the bacteriorhodopsin of Halobacterium halobium undergoes a photocycle involving different intermediates. The exact scheme of the photocycle and especially the number of M intermediates are subjects of debate. For a quantitative analysis of many effects connected with the photocycle, e.g. the effect of the membrane potential on the kinetics of M decay (Groma et al., 1984. Biophys. J. 45:985-992), a knowledge of the exact photocycle is needed. In the present work sophisticated measurements were made on the decay kinetics of the M forms in cell envelope vesicles, purple membrane suspension and purple membrane fragments incorporated in polyacrylamide gel. The experimental data were analyzed by fitting one, two, and three discrete exponentials. Three different real components were found in the M decay of cell envelope vesicles in 4 M NaCl. All of them exhibited a temperature-dependence obeying the Arrhenius law. Two real components were found for the purple membrane in suspension and in gel in NaCl-free medium. The third phase appeared when the gel was soaked in 4 M NaCl. As an independent means of analysis, a continuous distribution of exponentials was also fitted to the M decay kinetics in cell envelope vesicles. This calculation also resulted in three processes with distinct rates or alternatively two processes with distributed rates.

15.
Biophys J ; 45(5): 985-92, 1984 May.
Artigo em Inglês | MEDLINE | ID: mdl-6329348

RESUMO

The cell membrane of Halobacterium halobium (H. halobium) contains the proton-pump bacteriorhodopsin, which generates a light-driven transmembrane protonmotive force. The interaction of the bacteriorhodopsin photocycle with the electric potential component of the protonmotive force has been investigated. H. halobium cell envelope vesicles have been prepared by sonication and further purified by ultracentrifugation on Ficoll/NaCl/CsCl density gradients. Under continuous illumination (550 +/- 50 nm) varied from 0 to 40 mW cm-2, the vesicles maintain a membrane potential of 0 to -100 mV. The membrane potential was measured by flow dialysis of 3H-TPMP+ uptake and could be abolished by the uncoupler carbonylcyanide-m-chlorophenylhydrazone. Time-resolved absorption spectroscopy was used to measure the decay kinetics of the M photocycle intermediate, which was initiated by a weak laser flash (588 nm), while the vesicles were continuously illuminated as above. The M decay kinetics were fitted with two exponential decays by a computer deconvolution program. The faster decaying form decreases in amplitude (70 to 10% of the total) and the slower decaying form increases in amplitude and lifetime (23 to 42 ms) as the background light intensity increases. Although any correlation between the membrane potential and the bacteriorhodopsin photocycle M-forms is complex, the present data will allow specific tests of the physical mechanism for this interaction to be designed and conducted.


Assuntos
Bacteriorodopsinas/metabolismo , Carotenoides/metabolismo , Halobacterium/metabolismo , Membrana Celular/metabolismo , Cinética , Potenciais da Membrana , Fotoquímica , Prótons
16.
Acta Biochim Biophys Acad Sci Hung ; 16(3-4): 211-7, 1981.
Artigo em Inglês | MEDLINE | ID: mdl-7347989

RESUMO

The absorption characteristics of bacteriorhodopsin chromophore cannot be understand on the basis of a simple protonated Schiff-base linkage. A possible hypothetical explanation may be an interaction of the aromatic amino acid residues also with retinal. Mixtures of retinal and tryptophan analogues were reacted in organic solvents. Many similarities were found in the absorption spectra of the different products of these reactions and in those of the main forms of bacteriorhodopsin photocycle. Such products are suggested to model the purple complex of bacteriorhodopsin as well as the chromophores of the photointermediates.


Assuntos
Bacteriorodopsinas , Carotenoides , Modelos Químicos , Clorofórmio , Indóis , Retinaldeído , Espectrofotometria
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