RESUMEN
A highly sensitive method is proposed for obtaining the Raman spectra of low-concentration proteins and nucleic acids in an aqueous solution using liquid-liquid phase separation. This method uses water droplets formed by adding a large amount of polyethylene glycol into a biomolecular aqueous solution. Ordinary spontaneous Raman spectra are obtained with a high signal-to-noise ratio.
Asunto(s)
Polietilenglicoles , Proteínas , Soluciones , Espectrometría Raman , Agua , Agua/química , Polietilenglicoles/química , Proteínas/análisis , Proteínas/química , Relación Señal-Ruido , Separación de FasesRESUMEN
Liquid-liquid phase separation is a phenomenon that features the formation of liquid droplets containing concentrated solutes. The droplets of neurodegeneration-associated proteins are prone to generate aggregates and cause diseases. To uncover the aggregation process from the droplets, it is necessary to analyze the protein structure with keeping the droplet state in a label-free manner, but there was no suitable method. In this study, we observed the structural changes of ataxin-3, a protein associated with Machado-Joseph disease, inside the droplets, using autofluorescence lifetime microscopy. Each droplet showed autofluorescence due to tryptophan (Trp) residues, and its lifetime increased with time, reflecting structural changes toward aggregation. We used Trp mutants to reveal the structural changes around each Trp and showed that the structural change consists of several steps on different timescales. We demonstrated that the present method visualizes the protein dynamics inside a droplet in a label-free manner. Further investigations revealed that the aggregate structure formed in the droplets differs from that formed in dispersed solutions and that a polyglutamine repeat extension in ataxin-3 hardly modulates the aggregation dynamics in the droplets. These findings highlight that the droplet environment facilitates unique protein dynamics different from those in solutions.
Asunto(s)
Enfermedad de Machado-Joseph , Humanos , Ataxina-3/metabolismo , Enfermedad de Machado-Joseph/metabolismoRESUMEN
Fluorogenic probes for bioimaging have become essential tools for life science and medicine, and the key to their development is a precise understanding of the mechanisms available for fluorescence off/on control, such as photoinduced electron transfer (PeT) and Förster resonance energy transfer (FRET). Here we establish a new molecular design strategy to rationally develop activatable fluorescent probes, which exhibit a fluorescence off/on change in response to target biomolecules, by controlling the twisted intramolecular charge transfer (TICT) process. This approach was developed on the basis of a thorough investigation of the fluorescence quenching mechanism of N-phenyl rhodamine dyes (commercially available as the QSY series) by means of time-dependent density functional theory (TD-DFT) calculations and photophysical evaluation of their derivatives. To illustrate and validate this TICT-based design strategy, we employed it to develop practical fluorogenic probes for HaloTag and SNAP-tag. We further show that the TICT-controlled fluorescence off/on mechanism is generalizable by synthesizing a Si-rhodamine-based fluorogenic probe for HaloTag, thus providing a palette of chemical dyes that spans the visible and near-infrared range.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Colorantes Fluorescentes , Colorantes Fluorescentes/química , Rodaminas , IonóforosRESUMEN
Copper-zinc superoxide dismutase (SOD1) has been proposed as one of the causative proteins of amyotrophic lateral sclerosis (ALS). The accumulation of non-native conformers, oligomers, and aggregates of SOD1 in motor neurons is considered responsible for this disease. However, it remains unclear which specific feature of these species induces the onset of ALS. In this study, we showed that disulfide-linked oligomers of denatured SOD1 exhibit pro-oxidant activity. Substituting all the cysteine residues in the free thiol state with serine resulted in the loss of both the propensity to oligomerize and the increase in pro-oxidant activity after denaturation. In contrast, these cysteine mutants oligomerized and acquired the pro-oxidant activity after denaturation in the presence of a reductant that cleaves the intramolecular disulfide bond. These results indicate that one of the toxicities of SOD1 oligomers is the pro-oxidant activity induced by scrambling of the disulfide bonds. Small oligomers such as dimers and trimers exhibit stronger pro-oxidant activity than large oligomers and aggregates, consistent with the trend of the cytotoxicity of oligomers and aggregates reported in previous studies. We propose that the cleavage of the intramolecular disulfide bond accompanied by the oligomerization reduces the substrate specificity of SOD1, leading to the non-native enzymatic activity.
Asunto(s)
Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Cisteína/química , Disulfuros/química , Humanos , Mutación , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1/genéticaRESUMEN
Background The deep inferior epigastric perforator (DIEP) flap has been widely used in breast reconstruction. During surgery, many surgeons use closed suction drainage for both the donor site and the reconstructed breast. However, the criteria for drainage removal depend on the surgeon's preference and remain controversial. Moreover, it is well known that early postoperative showering is harmless to the surgical site and is recommended in many reports. However, it has not been discussed whether it is acceptable for patients with closed suction drainage to take a shower. Methodology We conducted a retrospective study of postoperative showering in 30 patients who underwent breast reconstruction with a DIEP flap. During the surgery, a total of three closed suction drains were connected to the patient's body (one was connected to the reconstructed breast, and the other two were connected to the abdominal donor site). After the surgery, patients were allowed to shower when the number of connected drainage tubes was ≤2. Results The patients were divided into three groups according to the number of remaining drainage tubes connected to their bodies when they started postoperative showering. Group A included patients with no drainage tubes. Group B included patients with one remaining drainage tube. Group C included patients with two drainage tubes. No significant differences in the incidence of postoperative individual complications were observed among the three groups. Conclusions Postoperative showering for patients with closed suction drainage is safe and does not increase the incidence of postoperative complications, including surgical site infection.
RESUMEN
Exciton delocalization in organic semiconducting polymers, affected by structures at a molecular level, plays a crucial role in modulating relaxation pathways, such as charge generation and singlet fission, which can boost device efficiency. However, the structural diversity of polymers and broad signals from typical electronic spectroscopy have their limits when it comes to revealing the interplay between local structures and exciton delocalization. To tackle these problems, we apply femtosecond stimulated Raman spectroscopy in archetypical conjugated oligothiophenes with different chain lengths. We observed Raman frequency dispersions of symmetric bond stretching modes and mode-specific kinetics in the S1 Raman spectra, which underpins the subtle and complex interplay between exciton delocalization and bond length alternation along the conjugation coordinate. Our results provide a more general picture of exciton delocalization in the context of molecular structures for conjugated materials.
RESUMEN
The association and dissociation of small ligands regulate the functions of proteins through structural changes in the protein. Such structural changes propagate long distances, and this allostery plays a key role in molecular functions. However, the mechanism by which structural changes are transmitted is poorly understood. Here we show that nonbonded atomic contacts play an essential role in driving the displacement of a helix in picosecond time scale primary structural changes following the dissociation of carbon monoxide from the heme group in myoglobin. The present time-resolved ultraviolet resonance Raman study revealed that the amplitude of this helix displacement was reduced upon substitution of Val68, which contacts the heme in wild-type myoglobin, with a less bulky side chain (Ala). Our findings provided the first direct evidence that structural changes are transmitted not only by covalent bonds, salt bridges and hydrogen bonds but also by nonbonded atomic contacts in the primary protein response upon ligand dissociation. Furthermore, the present results indicate the importance of dense atomic packing in a protein structure for responding to the association and dissociation of small molecules. The high compactness of protein structures makes possible the propagation of structural changes, providing useful clues to the design of molecular machines.
Asunto(s)
Hemo , Mioglobina , Monóxido de Carbono , Ligandos , Movimiento (Física)RESUMEN
Bacteriorhodopsin is a prototypical photoreceptor protein that functions as a light-driven proton pump. The retinal chromophore of bacteriorhodopsin undergoes C13âC14 trans-to-cis isomerization upon photoexcitation, and it has been believed to be the first event that triggers the cascaded structural changes in bacteriorhodopsin. We investigated the protein dynamics of bacteriorhodopsin using deep-ultraviolet resonance femtosecond stimulated Raman spectroscopy. It was found that the stimulated Raman signals of tryptophan and tyrosine residues exhibit significant changes within 0.2 ps after photoexcitation while they do not noticeably change during the isomerization process. This result implies that the protein environment changes first, and its change is small during isomerization. The obtained femtosecond stimulated Raman data indicate that ultrafast change is induced in the protein part by the sudden creation of the large dipole of the excited-state chromophore, providing an environment that realizes efficient and selective isomerization.
Asunto(s)
Bacteriorodopsinas/química , Colorantes/química , Procesos Fotoquímicos , Retina/química , Espectrometría Raman/métodos , Cinética , Conformación Proteica , Estereoisomerismo , Triptófano/química , Tirosina/química , Rayos UltravioletaRESUMEN
To facilitate the cytosolic delivery of larger molecules such as proteins, we developed a new cell-penetrating peptide sequence, named Pas2r12, consisting of a repeated Pas sequence (FFLIG-FFLIG) and d-dodeca-arginine (r12). This peptide significantly enhanced the cellular uptake and cytosolic release of enhanced green fluorescent protein and immunoglobulin G as cargos. We found that simply mixing Pas2r12 with cargos could generate cytosolic introducible forms. The cytosolic delivery of cargos by Pas2r12 was found to be an energy-requiring process, to rely on actin polymerization, and to be suppressed by caveolae-mediated endocytosis inhibitors (genistein and methyl-ß-cyclodextrin) and small interfering RNA against caveolin-1. These results suggest that Pas2r12 enhances membrane penetration of cargos without the need for cross-linking and that caveolae-mediated endocytosis may be the route by which cytosolic delivery is enhanced.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Portadores de Fármacos/metabolismo , Endocitosis , Arginina/análogos & derivados , Caveolas/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Péptidos de Penetración Celular/química , Citosol/metabolismo , Portadores de Fármacos/química , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Inmunoglobulina G/metabolismoRESUMEN
Heliorhodopsins (HeR) constitute a new rhodopsin family and show only <15% sequence identities with type-1 and type-2 rhodopsins. The large difference in amino acid sequence between HeRs and other rhodopsins raises a question whether their biological function is triggered by efficient and rapid photoisomerization of the retinal chromophore as in the case of other rhodopsins. We performed femtosecond time-resolved absorption measurements of two HeRs, HeR 48C12 and HeR from Thermoplasmatales archaeon SG8-52-1. Both HeRs exhibit excited-state absorption around 480 nm and stimulated emission in the >650 nm region, and these transient signals decay concomitantly with appearance of photoproduct absorption on a subpicosecond time scale. The observed spectral change indicates that ultrafast retinal photoisomerization proceeds in the femtosecond time region. The transient spectra and dynamics of HeRs are surprisingly similar to those of type-1 rhodopsins, despite remarkable differences in amino acid arrangement in the hydrophobic region of the retinal binding site.
Asunto(s)
Proteínas Arqueales/química , Rodopsinas Microbianas/química , Thermoplasmales/química , Isomerismo , Cinética , EspectrofotometríaRESUMEN
KR2 is the first light-driven Na+-pumping rhodopsin discovered. It was reported that the photoexcitation of KR2 generates multiple S1 states, i.e., "reactive" and "nonreactive" S1 states at physiological pH, but their origin remained unclear. In this study, we examined the S1 state dynamics of KR2 using femtosecond time-resolved absorption spectroscopy at different pH's in the range from 4 to 11. It was found that the reactive S1 state is predominantly formed at pH >9, but its population drastically decreases with decreasing pH while the population of the nonreactive S1 state(s) increases. The pH dependence of the relative population of the reactive S1 state correlates very well with the pH titration curve of Asp116, which is the counterion of the protonated retinal Schiff base (PRSB) in KR2. This strongly indicates that the deprotonation/protonation of Asp116 is directly related to the generation of the multiple S1 states in KR2. The quantitative analysis of the time-resolved absorption data led us to conclude that the reactive and nonreactive S1 states of KR2 originate from KR2 proteins having a hydrogen bond between Asp116 and PRSB or not, respectively. In other words, it is the ground-state inhomogeneity that is the origin of the coexistence of the reactive and nonreactive S1 states in KR2. So far, the generation of multiple S1 states having a different photoreactivity of rhodopsins has been mainly explained with the branching of the relaxation pathway in the Franck-Condon region in the S1 state. The present study shows that the structural inhomogeneity in the ground state, in particular that of the hydrogen-bond network, is the more plausible origin of the reactive and nonreactive S1 states which have been widely observed for various rhodopsins.
RESUMEN
Parvularcula oceani xenorhodopsin is the first light-driven inward proton pump. To understand the mechanism of inward proton transport, comprehensive transient absorption spectroscopy was conducted. Ultrafast pump-probe spectroscopy revealed that the isomerization time of retinal is 1.2 ps, which is considerably slower than those of other microbial rhodopsins (180-770 fs). Following the production of J, the K intermediate was formed at 4 ps. Proton transfer occurred on a slower timescale. Proton release and uptake were observed on the L/M-to-M and M decay, respectively, by monitoring transient absorption changes of pH-indicating dye, pyranine. Although a proton was released from Asp216 into the cytoplasmic medium, no proton-donating residue was identified on the extracellular side in mutation experiments. We revealed that a branched retinal isomerization (from 13-cis-15-anti to 13-cis-15-syn and all-trans-15-anti) occurred simultaneously with proton uptake. Furthermore, although the proton release showed a large kinetic isotope effect (KIE), the KIE of proton uptake was negligible. These results suggest that retinal isomerization is the rate-limiting process in proton uptake and that the regulation of p Ka of the retinal Schiff base by thermal isomerization enables the uptake from extracellular medium. This proton uptake mechanism differs from that of the outward proton pump with an internal proton donor and is important for understanding how the direction of ion transport by membrane proteins is determined.
Asunto(s)
Alphaproteobacteria/metabolismo , Rodopsina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cromatografía Líquida de Alta Presión , Deuterio/química , Isomerismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Mutagénesis Sitio-Dirigida , Protones , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Retinaldehído/química , Rodopsina/química , Rodopsina/genética , Espectrofotometría , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
In organisms, ion transporters play essential roles in the generation and dissipation of ion gradients across cell membranes. Microbial rhodopsins selectively transport cognate ions using solar energy, in which the substrate ions identified to date have been confined to monovalent ions such as H+, Na+, and Cl-. Here we report a novel rhodopsin from the cyanobacterium Synechocystis sp. PCC 7509, which inwardly transports a polyatomic divalent sulfate ion, SO42-, with changes of its spectroscopic properties in both unphotolyzed and photolyzed states. Upon illumination, cells expressing the novel rhodopsin, named Synechocystis halorhodopsin (SyHR), showed alkalization of the medium only in the presence of Cl- or SO42-. That alkalization signal was enhanced by addition of a protonophore, indicating an inward transport of Cl- and SO42- with a subsequent secondary inward H+ movement across the membrane. The anion binding to SyHR was suggested by absorption spectral shifts from 542 to 536 nm for Cl- and from 542 to 556 nm for SO42-, and the affinities of Cl- and SO42- were estimated as 0.112 and 5.81 mM, respectively. We then performed time-resolved spectroscopic measurements ranging from femtosecond to millisecond time domains to elucidate the structure and structural changes of SyHR during the photoreaction. Based on the results, we propose a photocycle model for SyHR in the absence or presence of substrate ions with the timing of their uptake and release. Thus, we demonstrate SyHR as the first light-driven polyatomic divalent anion (SO42-) transporter and report its spectroscopic characteristics.
Asunto(s)
Luz , Rodopsinas Microbianas/metabolismo , Sulfatos/metabolismo , Synechocystis/química , Aniones/química , Aniones/metabolismo , Rodopsinas Microbianas/química , Espectrofotometría Ultravioleta , Sulfatos/química , Synechocystis/metabolismoRESUMEN
Symmetrical and unsymmetrical pentacenes carrying two perfluoroalkyl (Rf) chains, at the 6 and 13 positions, were synthesized from easily available pentacene-6,13-quinone via facile three or four step reactions. After extensive evaluation, it was clearly found that the control of both the electron density of the aromatic rings on the pentacene core and molecular alignment in the crystalline state nicely affected their physical properties. Thus, we successfully prove in this article that (1) their anti-oxidation ability was significantly enhanced due to a decrease in the HOMO and LUMO energy and (2) a distinct difference in charge-transporting properties was observed between the symmetrical and unsymmetrical pentacenes.
RESUMEN
We report the first femtosecond time-resolved absorption study on ultrafast photoreaction dynamics of a recently discovered retinal protein, KR2, which functions as a light-driven sodium-ion pump. The obtained data show that the excited-state absorption around 460 nm and the stimulated emission around 720 nm decay concomitantly with a time constant of 180 fs. This demonstrates that the deactivation of the S1 state of KR2, which involves isomerization of the retinal chromophore, takes place three times faster than that of bacteriorhodopsin. In accordance with this rapid electronic relaxation, the photoproduct band assignable to the J intermediate grows up at â¼620 nm, indicating that the J intermediate is directly formed with the S1 â S0 internal conversion. The photoproduct band subsequently exhibits a â¼30 nm blue shift with a 500 fs time constant, corresponding to the conversion to the K intermediate. On the basis of the femtosecond absorption data obtained, we discuss the mechanism for the rapid photoreaction of KR2 and its relevance to the unique function of the sodium-ion pump.
RESUMEN
Background. Postresective mandibular reconstruction is common in cases of oral and mandibular tumors. However, complications such as infection, plate exposure, or plate fracture can occur. We identified several significant risk factors of complications after reconstructive surgery and compared the effectiveness of different surgical techniques for reducing the incidence of complications. Methods. This study is a retrospective analysis of 28 oromandibular cancer cases that required reconstructive surgery between January 1999 and December 2011 at Kobe University Graduate School of Medicine in Japan. All cases were classified using Hashikawa's CAT and Eichner's classification methods. Then, we determined whether these classifications and different treatment or surgical methods were significantly related to complications. Results. Complications after mandibular reconstruction occurred in 10/28 patients (36%). Specifically, five patients had plate fractures, four had plate exposures, and one had an infection. Radiation therapy and closure without any flaps were significantly related to infection or plate exposure. The wrap-around technique of securing reconstruction plates was used in 14 cases, whereas the run-through technique was used in two cases. Conclusions. The success of mandibular reconstruction depends on both mechanical and biological factors, such as the location of defects, presence of occlusions, and the amount of vascularization of the flap.
RESUMEN
BACKGROUND: The clinical diagnosis of osteomyelitis is difficult because of neuropathy, vascular disease, and immunodeficiency; also, with no established consensus on the diagnosis of foot osteomyelitis, the reported efficacy of magnetic resonance imaging (MRI) in detecting osteomyelitis and distinguishing it from reactive bone marrow edema is unclear. Herein, we describe a retrospective study on the efficacy of MRI for decision-making accuracy in diagnosing osteomyelitis in diabetic foot ulcers. METHODS: Twelve diabetic patients with infected foot ulcers underwent preoperative MRI between January 1, 2008, and December 31, 2011. The findings were compared with the histopathologic features of 67 parts of 45 resected bones, the cut ends of which were also histopathologically evaluated. RESULTS: Osteomyelitis was disclosed by MRI and histopathologically confirmed in 30 parts. In contrast, bone marrow edema diagnosed by MRI in 29 parts was confirmed in 23; the other six parts displayed osteomyelitis. Among 17 resected bones, 13 cut ends displayed bone marrow edema and four were normal. All of the wounds healed uneventfully. CONCLUSIONS: In the diagnosis of diabetic foot ulcers, osteomyelitis is often reliably distinguished from reactive bone marrow edema, except in special cases.
Asunto(s)
Pie Diabético/patología , Imagen por Resonancia Magnética , Osteomielitis/patología , Adulto , Anciano , Anciano de 80 o más Años , Enfermedades de la Médula Ósea/diagnóstico , Enfermedades de la Médula Ósea/etiología , Enfermedades de la Médula Ósea/cirugía , Toma de Decisiones Clínicas , Pie Diabético/complicaciones , Pie Diabético/cirugía , Edema/diagnóstico , Edema/etiología , Edema/cirugía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteomielitis/etiología , Osteomielitis/cirugía , Reproducibilidad de los Resultados , Estudios RetrospectivosRESUMEN
Internal vascular shunts for haemodialysis can cause different complications. One of the most serious complications is steal syndrome, which can result in disturbed peripheral circulation causing finger necrosis and lead to amputation. Thus, prevention of these complications is important. Measurement of skin perfusion pressure (SPP) has been used in various clinical settings, including wound-healing management, and its usefulness has been increasingly unveiled. The present study was undertaken to evaluate changes in haemodynamics after internal shunt creation by measuring SPP of the thumb and the little finger before and after surgery in five patients undergoing shunt surgery using the radial artery and the cephalic vein. The study revealed average changes of -22.8 mmHg in thumb SPP. The change in the thumb was statistically significant (p < 0.05). If the effect of surgery and the threshold for wound healing are taken into account, the present results indicate the necessity to pay extra attention to fingers with extremely low preoperative SPP values. For the prevention of serious disturbances of peripheral circulation (e.g. steal syndrome), routine preoperative SPP measurement seems effective for screening of high-risk patients.
Asunto(s)
Dedos/irrigación sanguínea , Isquemia/diagnóstico , Diálisis Renal/efectos adversos , Dispositivos de Acceso Vascular/efectos adversos , Anciano , Femenino , Estudios de Seguimiento , Humanos , Isquemia/etiología , Masculino , Persona de Mediana Edad , Perfusión/métodos , Cuidados Preoperatorios/métodos , Presión , Flujo Sanguíneo Regional , Diálisis Renal/métodos , Medición de Riesgo , Muestreo , Piel/irrigación sanguíneaRESUMEN
Umbilical reconstruction after total excision of the umbilicus represents a challenging problem for reconstructive surgeons. We describe herein a new method for one-stage umbilical reconstruction after resection of a urachal cyst. This case series included 6 patients, with laparoscopic urachal cyst removal in 5 and conventional transcutaneous surgery in 1. One-stage umbilical reconstruction was performed in all cases. When a conventional transcutaneous approach is indicated, umbilical reconstruction can be undertaken through the same skin incision used for total resection of the urachal cyst. Two triangular flaps were designed just below the umbilical defect. Flaps were rotated 180 degrees and sutured together to form one big triangular flap. This flap was then folded to create the new umbilicus. A deep umbilicus with good shape was constructed in all cases, and all patients were satisfied with the outcome. This method is simple, easy, and produces a natural-looking umbilicus.
Asunto(s)
Procedimientos de Cirugía Plástica/métodos , Colgajos Quirúrgicos , Ombligo/cirugía , Quiste del Uraco/cirugía , Adulto , Humanos , Masculino , Persona de Mediana Edad , Anclas para Sutura , Adulto JovenRESUMEN
Anabaena sensory rhodopsin (ASR) is well-known as the only retinal protein that achieves the photochromic reaction between the all-trans form (AT-ASR) and the 13-cis form (C-ASR). Although it is known that the structure of the hydrogen-bonding network of ASR is pH-dependent, it is so far unclear how pH affects the photoreaction of ASR. We investigated the pH dependence of the photoreaction of AT-ASR by means of time-resolved absorption spectroscopy and found it to be extremely dependent on pH. At pH 7 and 9, not only the L intermediate but also the K intermediate consisted of two decay components. The formation ratios of the two distinct L intermediates L(fast):L(slow) at pH 7 and 9 were different from each other, although the K(fast):K(slow) ratio was pH-independent. The photoreaction at pH 5 was entirely different from that at pH 7 and 9. Two K intermediates existed, but their formation ratio and lifetimes were different at pH 7 and 9. Moreover, only one L intermediate exists, with a longer lifetime relative to pH 7 and 9. The final product of the photoreaction of AT-ASR was C-ASR at all pH values. Finally, we successfully determined the pH-related photoreaction pathway of AT-ASR.