Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein

The 2.1-Å resolution crystal structure of wild-type green fluorescent protein and comparison of it with the recently determined structure of the Ser-65 → Thr (S65T) mutant explains the dual wavelength absorption and photoisomerization properties of the wild-type protein. The two absorption maxima are caused by a change in the ionization state of the chromophore. The equilibrium between these states appears to be governed by a hydrogen bond network that permits proton transfer between the chromophore and neighboring side chains. The predominant neutral form of the fluorophore maximally absorbs at 395 nm. It is maintained by the carboxylate of Glu-222 through electrostatic repulsion and hydrogen bonding via a bound water molecule and Ser-205. The ionized form of the fluorophore, absorbing at 475 nm, is present in a minor fraction of the native protein. Glu-222 donates its charge to the fluorophore by proton abstraction through a hydrogen bond network, involving Ser-205 and bound water. Further stabilization of the ionized state of the fluorophore occurs through a rearrangement of the side chains of Thr-203 and His-148. UV irradiation shifts the ratio of the two absorption maxima by pumping a proton relay from the neutral chromophore’s excited state to Glu-222. Loss of the Ser-205–Glu-222 hydrogen bond and isomerization of neutral Glu-222 explains the slow return to the equilibrium dark-adapted state of the chromophore. In the S65T structure, steric hindrance by the extra methyl group stabilizes a hydrogen bonding network, which prevents ionization of Glu-222. Therefore the fluorophore is permanently ionized, causing only a 489-nm excitation peak. This new understanding of proton redistribution in green fluorescent protein should enable engineering of environmentally sensitive fluorescent indicators and UV-triggered fluorescent markers of protein diffusion and trafficking in living cells.     

Postobstructive pulmonary edema: a case for hydrostatic mechanisms

BACKGROUND: Postobstructive pulmonary edema is a well-recognized complication of upper airway obstruction. The mechanisms of edema formation are unclear and may be due to increased hydrostatic forces generated by high negative inspiratory pressure or by increased permeability of the alveolar capillary membrane. Measurement of the edema fluid/plasma protein ratio and the rate of net alveolar fluid clearance are two well-validated methods for classifying the underlying mechanism of edema formation. The goal of the current study was to investigate the mechanisms of pulmonary edema formation in patients with postobstructive pulmonary edema by serial sampling of undiluted pulmonary edema fluid. METHODS: A retrospective review of 341 patients who had pulmonary edema fluid collected prospectively after the acute onset of pulmonary edema. All patients had serial samples of edema fluid and plasma collected over the first 8 h after intubation. RESULTS: Ten of the 341 patients with acute pulmonary edema were identified as having postobstructive pulmonary edema. The mean (+/- SD) edema fluid/plasma protein ratio in these patients was 0.54 +/- 0.15. The mean rate of alveolar fluid clearance over 8 h was 14.0 +/- 17.4% per hour. Nine of the 10 patients survived the hospitalization. CONCLUSION: Measurement of the edema fluid/plasma protein ratio and the presence of net alveolar fluid clearance in 10 patients with postobstructive pulmonary edema supports a hydrostatic mechanism for edema fluid formation. The predominantly fast rates of alveolar fluid clearance may explain the rapid resolution of clinical postobstructive pulmonary edema that is typically described.     

Esophagopharyngeal acid regurgitation: dual pH monitoring criteria for its detection and insights into mechanisms.

BACKGROUND & AIMS: A valid technique for the detection of esophagopharyngeal acid regurgitation would be valuable to evaluate suspected reflux-related otolaryngologic and respiratory disorders. The aim of this study was to derive pH criteria that optimally define esophagopharyngeal acid regurgitation and to examine patterns of regurgitation. METHODS: In 19 healthy controls and 15 patients with suspected regurgitation, dual or quadruple pH sensors were used to monitor pharyngeal and esophageal pH. For each combination of the 2 variables, DeltapH and nadir pH, proportions of pH decreases that occurred during or independent of esophageal acidification were calculated to determine the likelihood that an individual pharyngeal pH decrease was a candidate regurgitation event or a definite artifact. RESULTS: Overall, 92% of pharyngeal pH decreases of 1-2 pH units and 66% of pH decreases of this magnitude reaching a nadir pH of <4 were artifactual. Optimal criteria defining a pharyngeal acid regurgitation event were a pH decrease that occurred during esophageal acidification, had a DeltapH of >2 units, and reached a nadir of <4 units in less than 30 seconds. Regurgitation occurred more frequently in subjects in an upright (32 of 35) than in a supine (3 of 35 events; P 相似文献   

The photoisomerization of retinal in bacteriorhodopsin: Experimental evidence for a three-state model

The primary events in the all-trans to 13-cis photoisomerization of retinal in bacteriorhodopsin have been investigated with femtosecond time-resolved absorbance spectroscopy. Spectra measured over a broad range extending from 7000 to 22,400 cm⁻¹ reveal features whose dynamics are inconsistent with a model proposed earlier to account for the highly efficient photoisomerization process. Emerging from this work is a new three-state model. Photoexcitation of retinal with visible light accesses a shallow well on the excited state potential energy surface. This well is bounded by a small barrier, arising from an avoided crossing that separates the Franck–Condon region from the nearby reactive region of the photoisomerization coordinate. At ambient temperatures, the reactive region is accessed with a time constant of ≈500 fs, whereupon the retinal rapidly twists and encounters a second avoided crossing region. The protein mediates the passage into the second avoided crossing region and thereby exerts control over the quantum yield for forming 13-cis retinal. The driving force for photoisomerization resides in the retinal, not in the surrounding protein. This view contrasts with an earlier model where photoexcitation was thought to access directly a reactive region of the excited-state potential and thereby drive the retinal to a twisted conformation within 100–200 fs.     

The primary process of vision and the structure of bathorhodopsin: a mechanism for photoisomerization of polyenes.

A model for the primary process of vision is proposed, which involves a novel concerted-twist motion. Application of such motions to rhodopsin and bathorhodopsin successfully accounts for the properties of bathorhodopsin and related intermediates, including specific assignment of molecular structures to bathorhodopsin, to lumirhodopsin, and, less specifically, to hypsorhodopsin.     

The mechanisms of sulfonylurea-induced immune hemolysis: case report and review of the literature

Chlorpropamide, a sulfonylurea antidiabetic drug, was found to be the etiologic agent in a patient with immune hemolytic anemia. Hemolysis was severe, and ceased promptly when the drug was discontinued. The direct antiglobulin test was positive for complement, and the indirect antiglobulin test was also positive when the drug, patient serum, and fresh serum complement were all present. In this patient, and in four other patients described in the literature, hemolysis was mediated by the immune complex mechanism. In reports of two other patients taking a sulfonylurea drug, a milder form of hemolysis was mediated by the hapten mechanism. Sulfonylurea drugs are widely used. Since immune complex-mediated hemolysis in these patients is dramatic, it is not likely to be missed. However, the slower, milder hemolysis mediated by the hapten mechanism may be more common than is realized. Such patients may have only mild anemia and a near-normal reticulocyte count, making the diagnosis difficult. It is thus important to consider drug-immune hemolysis in patients who become anemic while taking sulfonylurea drugs.     

Testing mechanisms of action for intensive case management

AIM: This study identified factors that predict, mediate or moderate the effects of intensive case management (ICM) on longer-term drug abstinence outcomes in women on welfare. DESIGN: In a parent study women were assigned randomly to usual care (UC) or intensive case management (ICM). Treatment was provided for 12 weeks and follow-up continued for 15 months after study intake. A set of hypothesized mediators was assessed at month 3 and a rigorous four-step mediational model was tested using outcomes in months 4-15. PARTICIPANTS: Participants were 302 drug-dependent women applying and eligible for federal welfare and not currently in drug abuse treatment. Interventions ICM provided intensive treatment engagement including voucher incentives for treatment attendance and case management services; UC provided primarily referral to community treatment programs. MEASUREMENT: Substance use outcomes were assessed using the time-line follow-back interview and confirmed using biological and collateral measures. FINDINGS: Participants in ICM had more case manager contacts, better treatment engagement and more self-help attendance than did those in UC. Each of these variables predicted, and was shown to be a mediator of outcome, but case management contact was an especially robust mediator. Further, ICM effects were strongest for those who attended treatment least. Contrary to prediction, greater psychopathology and environmental stressors did not predict worse outcomes. CONCLUSIONS: Findings suggest that case management is an active intervention that may both facilitate and substitute for formal drug abuse treatment.     

The role of intersection topography in bond selectivity of cis-trans photoisomerization

Ab initio methods are used to characterize the ground and first excited state of the chromophore in the rhodopsin family of proteins: retinal protonated Schiff base. Retinal protonated Schiff base has five double bonds capable of undergoing isomerization. Upon absorption of light, the chromophore isomerizes and the character of the photoproducts (e.g., 13-cis and 11-cis) depends on the environment, protein vs. solution. Our ab initio calculations show that, in the absence of any specific interactions with the environment (e.g., discrete ordered charges in a protein), energetic considerations cannot explain the observed bond selectivity. We instead attribute the origin of bond selectivity to the shape (topography) of the potential energy surfaces in the vicinity of points of true degeneracy (conical intersections) between the ground and first excited electronic states. This provides a molecular example where a competition between two distinct but nearly isoenergetic photochemical reaction pathways is resolved by a topographical difference between two conical intersections.     

Targeting mechanisms of hypertensive vascular disease with dual calcium channel and renin-angiotensin system blockade

Patients with hypertension, particularly those with diabetes mellitus, are at heightened risk for cardiovascular and renal disease. Accumulated evidence indicates that the majority of hypertensive patients at high risk will require more than one antihypertensive agent to reach their blood pressure (BP) target. A reasonable strategy is to use agents with complementary mechanisms of action to enhance BP-lowering efficacy and prevent target organ damage. In experimental models, the combination of a calcium channel blocker (CCB) with an agent that blocks the renin-angiotensin system (RAS), an angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker, improves measures of endothelial function, inflammation, ventricular remodelling and renal function to a greater degree than these classes given as monotherapy. In clinical trials, calcium channel/RAS blockade combination therapy has been shown to provide greater BP reductions and improve renal function in patients with diabetic and nondiabetic renal disease earlier and to a greater extent than monotherapy. In addition, dual calcium channel/RAS blockade increases arterial compliance, arterial distensibility and flow-mediated vasodilation. Expanding upon extensive research on the benefits of calcium channel blockade and RAS blockade for the prevention of vascular events and preclinical and clinical trial evidence suggests added effects of combination therapy by targeting the underlying mechanisms of hypertensive vascular disease.     

A case of intra-ventricular dissociation with dual ventricular tachycardia

A 63 year old woman with chronic heart failure was admitted to our hospital for palpitation attack on 26th Apr 1988. The patient was died by cardiogenic shock and recurrent ventricular fibrillation 12-hours after admission. The autopsy revealed diffuse myocardial fibrosis and disarray which was compatible with dilated cardiomyopathy. The electrocardiogram on admission showed a peculiar wide QRS tachycardia with atrioventricular dissociation. After intravenous injection of 400 mg of procainamide, the QRS was separated into two types. The one type was left bundle branch block (LBBB) type with right axis deviation (type A), which was similar as that documented on Jan 1985, and the other was LBBB with normal axis (type B). Each wide QRS tachycardias were sustained independently and simultaneously either with RR 440 msec. or with RR 600 msec as if they were dissociated intraventricularly. The different wide QRS tachycardia documented on Feb 1986 was suspected as the fusion beats with type A and the QRS resembling type B. Although ventricular tachycardia with beat-to-beat changes of QRS morphology was generally regarded as bidirectional tachycardia, double foci were considered as origins of the two types of wide QRS tachycardia simultaneously observed in this patient.     

Extracellular nucleotides block bone mineralization in vitro: evidence for dual inhibitory mechanisms involving both P2Y2 receptors and pyrophosphate

Extracellular nucleotides, signaling through P2 receptors, may act as local regulators of bone cell function. We investigated the effects of nucleotide agonists [ATP, ADP, uridine triphosphate (UTP), and uridine diphosphate] and pyrophosphate (PPi, a key physiological inhibitor of mineralization) on the deposition and mineralization of collagenous matrix by primary osteoblasts derived from rat calvariae. Our results show that extracellular ATP, UTP, and PPi strongly and selectively blocked the mineralization of matrix nodules; ADP and uridine diphosphate were without effect. Significant inhibition of mineralization occurred in the presence of relatively low concentrations of ATP, UTP, or PPi (1-10 microm), without affecting production of fibrillar or soluble collagen. In cultures treated with 10 microm ATP or UTP, the expression and activity of alkaline phosphatase, which promotes mineralization by hydrolyzing PPi, was inhibited. The potent inhibitory actions of ATP and UTP on bone mineralization are consistent pharmacologically with mediation by the P2Y(2) receptor, which is strongly expressed by mature osteoblasts. In support of this notion, we found 9-17% increases in bone mineral content of hindlimbs of P2Y(2)-deficient mice. We also found that osteoblasts express ectonucleotide phosphodiesterase/pyrophosphatase-1, an ectonucleotidase that hydrolyzes nucleotide triphosphates to yield PPi, and that addition of 10 microm ATP or UTP to osteoblast cultures generated 2 microm PPi within 10 min. Thus, a component of the profound inhibitory action of ATP and UTP on bone mineralization could be mediated directly by PPi, independently of P2 receptors.     

The retinal chromophore/chloride ion pair: structure of the photoisomerization path and interplay of charge transfer and covalent states

Ab initio multi-reference second-order perturbation theory computations are used to explore the photochemical behavior of two ion pairs constituted by a chloride counterion interacting with either a rhodopsin or bacteriorhodopsin chromophore model (i.e., the 4-cis-gamma-methylnona-2,4,6,8-tetraeniminium and all-trans-nona-2,4,6,8-tetraeniminium cations, respectively). Significant counterion effects on the structure of the photoisomerization paths are unveiled by comparison with the paths of the same chromophores in vacuo. Indeed, we demonstrate that the counterion (i) modulates the relative stability of the S0, S1, and S2 energy surfaces leading to an S1 isomerization energy profile where the S1 and S2 states are substantially degenerate; (ii) leads to the emergence of significant S1 energy barriers along all of the isomerization paths except the one mimicking the 11-cis --> all-trans isomerization of the rhodopsin chromophore model; and (iii) changes the nature of the S1 --> S0 decay funnel that becomes a stable excited state minimum when the isomerizing double bond is located at the center of the chromophore moiety. We show that these (apparently very different) counterion effects can be rationalized on the basis of a simple qualitative electrostatic model, which also provides a crude basis for understanding the behavior of retinal protonated Schiff bases in solution.     

Disruption of myofibrillar energy use: dual mechanisms that may contribute to postischemic dysfunction in stunned myocardium

The abnormalities in regional function produced by myocardial ischemia persist after the ischemic episode resolves. Since a close functional coupling exists between myofibrillar creatine kinase and myosin ATPase, a disruption of this coupling could adversely influence myocardial function and might provide a mechanism for the myocardial dysfunction observed. The purpose of the present study was to determine if an alteration in the activity of creatine kinase associated with the myofibril occurs in the postischemic period. Anesthetized open-chest dogs (n = 6) underwent coronary occlusion for 15 minutes, followed by reperfusion for 15 minutes. In reperfused myocardium, adenine nucleotide content was decreased (72 +/- 10% of nonischemic myocardium, p less than 0.05), documenting the presence of previous ischemia. The creatine phosphate content of reperfused myocardium returned to normal, indicating resumption of myocardial energy production. The creatine kinase activity of purified myofibrils isolated from reperfused myocardium was decreased by 17 +/- 7% compared to that of nonischemic myofibrils (p less than 0.03). In addition, the free adenosine diphosphate concentration in reperfused myocardium was calculated to be 96 microM and was less than the Km of adenosine diphosphate determined for myofibrillar creatine kinase (105 microM). The results suggest two putative mechanisms for disruption of energy use in postischemic myocardium: decreased creatine kinase activity associated with the myofibril, and limitation of substrate necessary for maximal creatine kinase activity.     

Infection and diabetes: The case for glucose control

This review summarizes data concerning the host resistance to infection in diabetes and the influence of an acute infection upon the endocrinologic-metabolic status of the diabetic patient. While it is well known that acute infections lead to difficulty in controlling blood sugar levels and that infection is the most frequently documented cause of ketoacidosis, controversy persists as to whether or not patients with diabetes mellitus are more susceptible to infection than age- and sex-matched nondiabetlc control subjects. Our data obtained from the charts of 241 diabetic patients who were being followed as outpatients show a striking direct correlation between the overall prevalence of infection (p < 0.001) and the mean plasma glucose levels (representing three or more fasting glucose determinations taken at times when no evidence of infection existed). There is a significant diminution in intracellular bactericidal activity of leukocytes with Staphylococcus aureus and Escherichia coli in subjects with poorly controlled diabetes in comparison with the control group. Serum opsonic activity for both Staph. aureus and E. coli were significantly lower than in the control subjects. Taken together, the results from published reports as well as our data suggest to us that good control of blood sugar in diabetic patients is a desirable goal in the prevention of certain infections (Candida vaginitis, for example) and to ensure maintenance of normal host defense mechanisms that determine resistance and response to infection.     

库欣病与特发性颅内高压:病例报告和潜在的病理生理机制的复习

特发性颅内高压(ⅡH),也就是良性颅内高压或假性脑瘤,主要见于育龄期肥胖女性,表现为头痛、恶心、呕吐、视乳头水肿和颅内压升高,而没有颅内占位性病变.已有报告一些与ⅡH发生有关的诱发因素,包括炎症/自身免疫性疾病、某些药物、肥胖和妊娠等.另外,神经内分泌系统在某些ⅡH的发生发展中似乎也起到一定的作用,有几项研究报告了一个或多个下丘脑-垂体轴的缺陷与ⅡH的关系.特别是,下丘脑-垂体-肾上腺(HPA)轴的功能异常与ⅡH的发生有关.     

The innovation of composite core dual coil coronary guide-wire technology: A didactic coronary chronic total occlusion revascularization case report

The treatment of coronary chronic total occlusions (CTO) continues to solicit technical innovations. As success primarily depends on crossing the lesion with a wire, all aspects regarding tip shape retention, torque precision, and penetration ability of the guide-wire have greatly influenced new techniques and strategies. The world of interventional cardiology has to look carefully at these developments, and to use them accordingly to improve the success rate in ordinary percutaneous coronary interventions. We present a didactical case report of a CTO revascularization treated with a new ‘dual core’ technology guide-wire.