Sensory proteins must relay structural signals from the sensory site over large distances to regulatory output domains. Phytochromes are a major family of red-light sensing kinases that control diverse cellular functions in plants, bacteria, and fungi.1-9 Bacterial phytochromes consist of a photosensory core and a C-terminal regulatory domain.10,11 Structures of photosensory cores are reported in the resting state12-18 and conformational responses to light activation have been proposed in the vicinity of the chromophore.19-23 However, the structure of the signalling state and the mechanism of downstream signal relay through the photosensory core remain elusive. Here, we report crystal and solution structures of the resting and active states of the photosensory core of the bacteriophytochrome from Deinococcus radiodurans. The structures reveal an open and closed form of the dimeric protein for the signalling and resting state, respectively. This nanometre scale rearrangement is controlled by refolding of an evolutionarily conserved “tongue”, which is in contact with the chromophore. The findings reveal an unusual mechanism where atomic scale conformational changes around the chromophore are first amplified into an Ångström scale distance change in the tongue, and further grow into a nanometre scale conformational signal. The structural mechanism is a blueprint for understanding how the sensor proteins connect to the cellular signalling network.
Serial femtosecond crystallography using ultrashort pulses from X-ray Free Electron Lasers (XFELs) offers the possibility to study light-triggered dynamics of biomolecules. Using microcrystals of the blue light photoreceptor, photoactive yellow protein, as a model system, we present high resolution, time-resolved difference electron density maps of excellent quality with strong features, which allow the determination of structures of reaction intermediates to 1.6 Å resolution. These results open the way to the study of reversible and non-reversible biological reactions on time scales as short as femtoseconds under conditions which maximize the extent of reaction initiation throughout the crystal.
Abstract-Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4-and 70-kDa dextrans through the cytosol, and localization of 155-and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin-and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4-to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size. Key Words: ultrasound microbubble targeted delivery Ⅲ cell membrane pore Ⅲ endocytosis Ⅲ dextran Ⅲ endothelial cells C onventional delivery methods for drugs or genes, such as systemic administration via intravenous injection or oral administration, often do not suffice for therapeutic compounds such as peptides, silencing RNAs and genes. 1 A recent development in delivery systems for therapeutic compounds is the microbubble-ultrasound (US) interaction. 2,3 Before its use as a clinical modality, it is of utmost importance to obtain new physiological insights into the mechanisms of uptake by US and microbubble-exposed cells.Microbubbles were originally developed as US contrast agents and are administered intravenously to the systemic circulation to enhance scattering of blood in echocardiography. They consist of a gas core stabilized with an encapsulation, ranging from 1 to 10 m in diameter. 4 Nowadays, research focuses on the use of US and microbubbles for therapeutic applications. It has been demonstrated that USexposed microbubbles can achieve safe and efficient local delivery of a variety of drugs 5,6 and genes.
A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes following photon absorption. The initial step is often the photo-isomerization of a conjugated chromophore. Isomerization occurs on ultrafast timescales, and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans to cis isomerization of the chromophore in photoactive yellow protein. Femtosecond, hard X-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on PYP microcrystals over the time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.
Postoperative renal function deterioration is a serious complication after cardiac surgery with cardiopulmonary bypass and is associated with increased in-hospital mortality. However, the long-term prognosis of patients with postoperative renal deterioration is not fully determined yet. Therefore, both in-hospital mortality and long-term survival were studied in patients with postoperative renal function deterioration. Included were 843 patients who underwent cardiac surgery with cardiopulmonary bypass in 1991. Postoperative renal function deterioration (increase in serum creatinine in the first postoperative week of at least 25%) occurred in 145 (17.2%) patients. In these patients, in-hospital mortality was 14.5%, versus 1.1% in patients without renal function deterioration (P < 0.001). Multivariate analysis significantly associated in-hospital mortality with postoperative renal function deterioration, re-exploration, postoperative cerebral stroke, duration of operation, age, and diabetes. In patients who were discharged alive, during long-term follow-up (100 mo), mortality was significantly increased in the patients with renal function deterioration (n = 124) as compared with those without renal function deterioration (hazard ratio 1.83; 95% confidence interval 1.38 to 3.20). Also after adjustment for other independently associated factors, the risk for mortality in patients with postoperative renal function deterioration remained elevated (hazard ratio 1.63; 95% confidence interval 1.15 to 2.32). The elevated risk for long-term mortality was independent of whether renal function had recovered at discharge from hospital. It is concluded that postoperative renal function deterioration in cardiac surgical patients not only results in increased in-hospital mortality but also adversely affects long-term survival.
Background-The renin-angiotensin system (RAS) is a key player in the progression of heart failure. is thought to modulate the activity of the RAS. Furthermore, this peptide may play a part in the beneficial effects of angiotensin-converting enzyme inhibitors in cardiovascular disease. We assessed the effects of angiotensin-(1-7) on the progression of heart failure. Methods and Results-Male Sprague-Dawley rats underwent either coronary ligation or sham surgery. Two weeks after induction of myocardial infarction, intravenous infusion of angiotensin-(1-7) (24 g/kg per hour) or saline was started by minipump. After 8 weeks of treatment, hemodynamic parameters were measured, endothelial function was assessed in isolated aortic rings, and plasma angiotensin-(1-7) levels were determined. Myocardial infarction resulted in a significant deterioration of left ventricular systolic and diastolic pressure, dP/dt, and coronary flow. Raising plasma levels 40-fold, angiotensin-(1-7) infusion attenuated this impairment to a nonsignificant level, markedly illustrated by a 40% reduction in left ventricular end-diastolic pressure. Furthermore, angiotensin-(1-7) completely preserved aortic endothelial function, whereas endothelium-dependent relaxation in aortas of saline-treated infarcted rats was significantly decreased. Conclusions-Angiotensin-(1-7) preserved cardiac function, coronary perfusion, and aortic endothelial function in a rat model for heart failure.
All children were managed from admission onward according to a standardized protocol for head injury management. Children with raised intracranial pressure (ICP) were randomized to standardized management alone or standardized management plus cerebral decompression. A decompressive bitemporal craniectomy was performed at a median of 19.2 h (range 7.3-29.3 h) from the time of injury. ICP was recorded hourly via an intraventricular catheter. Compared with the ICP before randomization, the mean ICP was 3.69 mmHg lower in the 48 h after randomization in the control group, and 8.98 mmHg lower in the 48 hours after craniectomy in the decompression group (P=0.057). Outcome was assessed 6 months after injury using a modification of the Glasgow Outcome Score (GOS) and the Health State Utility Index (Mark 1). Two (14%) of the 14 children in the control group were normal or had a mild disability after 6 months, compared with 7 (54%) of the 13 children in the decompression group. Our conclusion was that when children with traumatic brain injury and sustained intracranial hypertension are treated with a combination of very early decompressive craniectomy and conventional medical management, it is more likely that ICP will be reduced, fewer episodes of intracranial hypertension will occur, and functional outcome and quality of life may be better than in children treated with medical management alone (P=0.046; owing to multiple significance testing P <0.0221 is required for statistical significance). This pilot study suggests that very early decompressive craniectomy may be indicated in the treatment of traumatic brain injury.
To understand how signaling proteins function, it is crucial to know the time-ordered sequence of events that lead to the signaling state. We recently developed on the BioCARS 14-IDB beamline at the Advanced Photon Source the infrastructure required to characterize structural changes in protein crystals with near-atomic spatial resolution and 150-ps time resolution, and have used this capability to track the reversible photocycle of photoactive yellow protein (PYP) following trans-to-cis photoisomerization of its p-coumaric acid (pCA) chromophore over 10 decades of time. The first of four major intermediates characterized in this study is highly contorted, with the pCA carbonyl rotated nearly 90°out of the plane of the phenolate. A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation. Density functional theory calculations confirm that this structure is chemically plausible and corresponds to a strained cis intermediate. This unique structure is short-lived (∼600 ps), has not been observed in prior cryocrystallography experiments, and is the progenitor of intermediates characterized in previous nanosecond time-resolved Laue crystallography studies. The structural transitions unveiled during the PYP photocycle include trans/cis isomerization, the breaking and making of hydrogen bonds, formation/ relaxation of strain, and gated water penetration into the interior of the protein. This mechanistically detailed, near-atomic resolution description of the complete PYP photocycle provides a framework for understanding signal transduction in proteins, and for assessing and validating theoretical/computational approaches in protein biophysics.time-resolved X-ray diffraction | photoreceptor | light sensor
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