Electrochromic shift supports the membrane destabilization model of Tat-mediated transport and shows ion leakage during Sec transport

The mechanism and pore architecture of the Tat complex during transport of folded substrates remain a mystery, partly due to rapid dissociation after translocation. In contrast, the proteinaceous SecY pore is a persistent structure that needs only to undergo conformational shifts between “closed” and “opened” states when translocating unfolded substrate chains. Where the proteinaceous pore model describes the SecY pore well, the toroidal pore model better accounts for the high-energy barrier that must be overcome when transporting a folded substrate through the hydrophobic bilayer in Tat transport. Membrane conductance behavior can, in principle, be used to distinguish between toroidal and proteinaceous pores, as illustrated in the examination of many antimicrobial peptides as well as mitochondrial Bax and Bid. Here, we measure the electrochromic shift (ECS) decay as a proxy for conductance in isolated thylakoids, both during protein transport and with constitutively assembled translocons. We find that membranes with the constitutively assembled Tat complex and those undergoing Tat transport display conductance characteristics similar to those of resting membranes. Membranes undergoing Sec transport and those with the substrate-engaged SecY pore result in significantly more rapid electric field decay. The responsiveness of the ECS signal in membranes with active SecY recalls the steep relationship between applied voltage and conductance in a proteinaceous pore, while the nonaccelerated electric field decay with both Tat transport and the constitutive Tat complex under the same electric field is consistent with the behavior of a toroidal pore.

The twin arginine translocation pathway is uniquely able to transport fully folded substrates in an ATP-independent manner, relying instead on an electrochemical gradient (i.e., the proton motive force, or pmf) across the transporting membrane. It is crucial to the transport of substrates requiring various cofactors and hetero-oligomeric complexes in prokaryotes and of substrates vital to photosynthetic machinery in thylakoids (1). In plant mitochondria, the Rieske Fe/S protein required for the biogenesis of complex III is transported by the Tat pathway (2–6). It is implicated in both the virulence and antibiotic resistance of various infectious bacteria (7–12) and has been studied for its potential in biotechnology applications (13–15). The uniqueness of Tat functionality and its appearance across the kingdoms of life make it a valuable research target for crop modification, biotechnology, and pathogenesis. Unfortunately, much of the knowledge about its mechanism has been hard won, and the pore structure remains a mystery, likely due to the transient nature of the active complex.The active Tat complex in thylakoids consists of three core subunits, Tha4, Hcf106, and cpTatC, which are homologous to the bacterial TatA, TatB, and TatC, respectively (1, 16). An N-terminal signal peptide with a twin-arginine motif inserts into the cis-side leaflet at the TatBC receptor complex (17–19). Subsequent oligomerization of TatA subunits (20–22) at the TatBC receptor complex results in rings of varying sizes (22, 23), but it is unclear whether these structures represent transport pores. Of particular note is the short TatA transmembrane helix (TMH). Composed of only 16 residues, the solid-state NMR solution suggests that the TMH must tilt and draw a portion of the cis-side amphipathic helix (APH) into the membrane in order to cross the bilayer (24), establishing a resting state of hydrophobic mismatch. During transport, a conformational shift increasing the angle between the TMH and APH results in exacerbated hydrophobic mismatch, as the APH is moved radially away from the center and the TMH is pulled up toward the cis-side in the active state (25, 26). For both native and foreign substrates, the Tat-targeted signal peptide and the pmf are sufficient to cause assembly of the active translocon and achieve transport (27–31). After the translocation event, the complex dissociates into TatA and TatBC components (1, 15, 16) with the exception of some residual TatA bound to the receptor complex in a nonactive state and a spectrum of smaller TatA oligomers (32).Within the thylakoid membrane, it is useful to compare the Tat complex with the general secretory translocon (Sec) because they both function in the same membrane environment (1, 33). Sec translocation first requires recruitment of the substrate to the soluble SecA ATPase to form the substrate–SecA complex, which is then recruited to the SecY pore (1). In the inactive state, the proteinaceous SecY pore prevents ion leakage through a combination of a trans-side plug domain and an internal array of hydrophobic residues (34). Following substrate–SecA docking, a conformational shift in SecY allows substrate movement through the open pore in an ATP-dependent process driven by SecA (35). In the mammalian homolog Sec61, leakage of NAD⁺ ions is recorded during ribosome-bound nascent chain transport in a fluorescence quenching study, suggesting the pore can reach 4 to 6 nm in diameter (36). However, X-ray structures of substrate-fused SecA complexed with SecY (35), conductance studies in ribosome-bound substrates engaged with SecY (37), and SecY plug deletion mutants (38) in Escherichia coli have estimated the open SecY pore diameter to range from 7.3 to 8.8 Å, almost 10-fold lower. This small diameter likely contributes to the restriction of ion movement during Sec transport (39).While the Sec machinery only transports unfolded substrates (40), the Tat pore accommodates substrates ranging from a single unfolded chain in an engineered system (13) to a folded substrate with an average diameter of 70 Å (41). This extended size range raises an interesting question about the pore architecture. In the Sec translocon, X-ray crystallography of the SecY channel in Methanocaldococcus jannaschii (42) and Thermotoga maritima (43) reveals that the SecY pore channel excludes lipids in both the resting state and when engaged with its SecA partner. Further structural work on the E. coli substrate-engaged SecA–SecY complex shows that the SecY channel excludes lipids during transport as well (35). No such structural information about the Tat pore exists, but functional data suggest that TatA plays an important role in the pore (20, 44, 45) and cryogenic electron microscopy structures of TatA oligomers reveal rings of an internal diameter ranging from 30 to 70 Å (23). During transport of the 17-kDa subunit of the oxygen-evolving complex (OE23), the Tat pathway exhibits very low ion leakage (46), estimated to be less than 1 pS. This is despite the exchange of 80,000 protons per substrate (47). Extensive mapping of subunit–subunit and subunit–substrate contacts has revealed no putative plug domain (20, 48–51) that could be compared to that in the SecY protein.Pore architecture can be characterized by membrane conductance behavior. Conductance measured through proteinaceous pores representing the barrel-stave model has a very steep dependence on the voltage applied, whereas conductance in toroidal pores requires a larger voltage to be detected and increases more slowly in response to increasing voltage (52–54). Performing similar experiments on the Tat and Sec translocons would require functional reconstitution of both complexes into an in vitro setting. However, decay of the electrochromic shift (ECS) signal can be used as a measure of ion conductance (46). A transient absorption peak at ∼515 nm arising from carotenoid pigments in response to the native electric field generated by charge separation in the photosynthetic reaction centers (55) can be measured by delivering a single-saturating flash. The decay rate of such a signal is a direct measurement of how quickly the electric field is dissipated by ion movement across the membrane.In the experiments reported herein, ECS signal decay rates revealing the conductance states of resting isolated membranes and those engaged in ongoing transport and in the presence of a constitutively assembled and/or substrate-engaged translocon are used to probe the pore architecture in the Tat and Sec complexes. Increased conductance across the thylakoid membrane is indicated by a more rapidly decaying ECS signal. We find that conductance in thylakoid membranes during Sec-mediated transport and substrate-engaged SecY is consistently higher than that during Tat-mediated transport and with the constitutively assembled Tat complex, respectively, despite the much larger Tat pore required to transport a fully folded substrate. This points to a difference not only in mechanism but in pore architecture between the two. Conductance behavior of membranes undergoing Sec-mediated transport is consistent with that of a proteinaceous pore, while the resistance demonstrated by membranes undergoing Tat-mediated transport is more reminiscent of toroidal pores.     

Predictors of acquired lipodystrophy in juvenile-onset dermatomyositis and a gradient of severity

We describe the clinical features of 28 patients with juvenile dermatomyositis (JDM) and 1 patient with adult-onset dermatomyositis (DM), all of whom developed lipodystrophy (LD) that could be categorized into 1 of 3 phenotypes, generalized, partial, or focal, based on the pattern of fat loss distribution. LD onset was often delayed, beginning a median of 4.6 years after diagnosis of DM. Calcinosis, muscle atrophy, joint contractures, and facial rash were DM disease features found to be associated with LD. Panniculitis was associated with focal lipoatrophy while the anti-p155 autoantibody, a newly described myositis-associated autoantibody, was more associated with generalized LD. Specific LD features such as acanthosis nigricans, hirsutism, fat redistribution, and steatosis/nonalcoholic steatohepatitis were frequent in patients with LD, in a gradient of frequency and severity among the 3 sub-phenotypes. Metabolic studies frequently revealed insulin resistance and hypertriglyceridemia in patients with generalized and partial LD. Regional fat loss from the thighs, with relative sparing of fat loss from the medial thighs, was more frequent in generalized than in partial LD and absent from DM patients without LD. Cytokine polymorphisms, the C3 nephritic factor, insulin receptor antibodies, and lamin mutations did not appear to play a pathogenic role in the development of LD in our patients. LD is an under-recognized sequela of JDM, and certain DM patients with a severe, prolonged clinical course and a high frequency of calcinosis appear to be at greater risk for the development of this complication. High-risk JDM patients should be screened for metabolic abnormalities, which are common in generalized and partial LD and result in much of the LD-associated morbidity. Further study is warranted to investigate the pathogenesis of acquired LD in patients with DM.     

Techniques of biliary drainage for acute cholecystitis: Tokyo Guidelines

The principal management of acute cholecystitis is early cholecystectomy. However, percutaneous transhepatic gallbladder drainage (PTGBD) may be preferable for patients with moderate (grade II) or severe (grade III) acute cholecystitis. For patients with moderate (grade II) disease, PTGBD should be applied only when they do not respond to conservative treatment. For patients with severe (grade III) disease, PTGBD is recommended with intensive care. Percutaneous transhepatic gallbladder aspiration (PTGBA) is a simple alternative drainage method with fewer complications; however, its clinical usefulness has been shown only by case-series studies. To clarify the clinical value of these drainage methods, proper randomized trials should be done. This article describes techniques of drainage for acute cholecystitis.     

Remodeling of sinus node function after catheter ablation of right atrial flutter

INTRODUCTION: The purpose of this study was to investigate the effect of ablation of right atrial flutter upon sinus node function in humans. METHODS AND RESULTS: This study enrolled 35 patients. Twenty-four patients (16 men and 8 women; age 68 +/- 11 years) were referred for ablation of persistent atrial flutter (duration 8 +/- 11 months). After ablation, there was abnormal sinus node function defined as a corrected sinus node recovery time (CSNRT) > or = 550 msec. The control group consisted of 11 patients who were undergoing pacemaker implantation for sinus node disease but did not have a history of atrial dysrhythmias or ablation. Within 24 hours of ablation or pacemaker implantation, baseline maximal CSNRT was measured through a permanent pacemaker by AAI pacing at six cycle lengths: 600, 550, 500, 450, 400, and 350 msec. CSNRT then was measured in the same manner at 48 hours, 14 days, and 3 months after ablation/pacemaker implantation. P wave amplitude and duration, and percent atrial sensing also were assessed at the same intervals. For patients undergoing atrial flutter ablation, there was progressive temporal recovery of CSNRT (1,204 +/- 671 msec at baseline vs 834 +/- 380 msec at 3 months; P < 0.001) and a significant increase in the percent atrial sensing and P wave amplitude at 3 months compared with baseline (P < 0.001). In control subjects, there was no change in the CSNRT, percent atrial pacing, or P wave amplitude. CONCLUSION: After ablation of persistent atrial flutter, there is temporal recovery of CSNRT and increase in spontaneous atrial activity. These findings suggest that atrial flutter induces reversible changes in sinus node function.     

Metabolically controlled reperfusion in acute myocardial infarction: should the polarizing solution be given subselectively?

BACKGROUND: In working rat hearts, metabolic support of injured tissue enhances recovery after acute myocardial infarction. Clinical experience with a systemic "polarizing solution" supports this claim. OBJECTIVES: In a dog model of ischemia/reperfusion, we tested the feasibility of subselectively supplying adapted metabolic substrates before instituting blood reperfusion. METHODS: Thirty-five dogs underwent ligation of the proximal left anterior descending artery and collaterals for 90 minutes. The animals were randomly assigned to receive direct blood reperfusion (Group I), intracoronary glucose, insulin, and potassium (Group II), or intracoronary glucose, insulin, and potassium plus propionyl-L-carnitine (PLC) (Group III). After 30 minutes of artificial reperfusion, prograde blood flow was resumed in groups II and III. A routine necropsy was performed 3 to 5 days later. Primary endpoints were severe arrhythmias, death, markers of infarct size, and specific histologic features. RESULTS: We excluded 4 dogs for technical reasons and 2 others for preexisting cardiomyopathy. In the remaining 29 animals, large apical infarctions were documented ventriculographically during arterial ligation. One dog died of irreversible ventricular fibrillation during the initial ischemic period, and 9/28 dogs (32.1%) died during early reperfusion. Ventricular fibrillation was more common with 10% (versus 5%) dextrose concentrations and was eliminated by PLC. Irreversibly injured (versus jeopardized) areas of myocardium were more common in Group III (85.9 19.3%) than in Groups I and II (16.9 10.8%). CONCLUSION: Subselective infusion of metabolically supportive solutions during acute myocardial infarction is technically feasible. To prevent osmotic endothelial damage, the perfusate must have a low (< 5%) dextrose content.     

Evaluation of antihypertensive therapy with the combination of olmesartan medoxomil and hydrochlorothiazide

BACKGROUND: Most patients with hypertension require more than one agent to control blood pressure (BP). The purpose of this study was to assess the efficacy and safety of the angiotensin II receptor blocker olmesartan medoxomil in combination with hydrochlorothiazide (HCTZ). METHODS: This was a randomized, double-blind, factorial design study. After a placebo run-in period, eligible patients (n = 502) with a baseline mean seated diastolic blood pressure (SeDBP) of 100 to 115 mm Hg were randomized to one of 12 groups: placebo, olmesartan medoxomil monotherapy (10, 20, or 40 mg/day, HCTZ monotherapy (12.5 or 25 mg/day), or one of six groups of olmesartan medoxomil/HCTZ combination therapy. The primary endpoint was the change in mean trough SeDBP from baseline at week 8. Statistical analyses were conducted to determine whether at least one combination produced a larger reduction in SeDBP at week 8 than the individual corresponding component doses, but did not compare BP reductions with different combination doses. RESULTS: Olmesartan medoxomil plus HCTZ produced greater reductions in both SeDBP and seated systolic blood pressure (SeSBP) at week 8 than did monotherapy with either component. All olmesartan medoxomil/HCTZ combinations significantly reduced SeDBP and SeSBP compared with placebo in a dose-dependent manner. Reductions from baseline in mean trough SeSBP/SeDBP were 3.3/8.2 mm Hg, 20.1/16.4 mm Hg, and 26.8/21.9 mm Hg with placebo, olmesartan medoxomil/HCTZ 20/12.5 mg, and olmesartan medoxomil/HCTZ 40/25 mg, respectively. All treatments were well tolerated. CONCLUSIONS: Olmesartan medoxomil/HCTZ combination therapy produced BP reductions of up to 26.8/21.9 mm Hg and was well tolerated.