Orange carotenoid protein burrows into the phycobilisome to provide photoprotection

In cyanobacteria, photoprotection from overexcitation of photochemical centers can be obtained by excitation energy dissipation at the level of the phycobilisome (PBS), the cyanobacterial antenna, induced by the orange carotenoid protein (OCP). A single photoactivated OCP bound to the core of the PBS affords almost total energy dissipation. The precise mechanism of OCP energy dissipation is yet to be fully determined, and one question is how the carotenoid can approach any core phycocyanobilin chromophore at a distance that can promote efficient energy quenching. We have performed intersubunit cross-linking using glutaraldehyde of the OCP and PBS followed by liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS) to identify cross-linked residues. The only residues of the OCP that cross-link with the PBS are situated in the linker region, between the N- and C-terminal domains and a single C-terminal residue. These links have enabled us to construct a model of the site of OCP binding that differs from previous models. We suggest that the N-terminal domain of the OCP burrows tightly into the PBS while leaving the OCP C-terminal domain on the exterior of the complex. Further analysis shows that the position of the small core linker protein ApcC is shifted within the cylinder cavity, serving to stabilize the interaction between the OCP and the PBS. This is confirmed by a ΔApcC mutant. Penetration of the N-terminal domain can bring the OCP carotenoid to within 5–10 Å of core chromophores; however, alteration of the core structure may be the actual source of energy dissipation.Excess energy arriving at photochemical reaction centers (RCs) can be detrimental (1, 2), leading to loss of photosynthetic viability (photoinhibition; PI) (3, 4). Cyanobacteria have evolved several mechanisms for energy dissipation to deal with overexcitation (5, 6). The major light-harvesting complex (LHC) in cyanobacteria is the phycobilisome (PBS), a giant complex that can functionally transfer energy to two to four RCs (7–9). If overexcitation occurs rapidly, photoprotection can be achieved by decreasing the energy arriving at the RCs by increasing energy thermal dissipation (nonphotochemical quenching; NPQ) or by physical (or functional) disconnection of the PBS (10, 11). In most cyanobacterial species, NPQ is obtained by the presence a 35-kDa, water-soluble, orange carotenoid protein (OCP) (12, 13). OCP-dependent NPQ was shown to be induced (14, 15) by strong blue-green light. The OCP has two states—an inactive resting orange state (OCP^O) and an active red state (OCP^R). Upon illumination, the OCP undergoes carotenoid and protein conformational changes to yield the metastable OCP^R (16). The OCP^R binds to the PBS and significantly quenches excitation energy and PBS fluorescence (17). The OCP noncovalently binds a single keto-carotenoid chromophore, 3′-hydroxyechinenone (hECN). The high-resolution crystal structure of the OCP^O [from the cyanobacterium Synechocystis sp. PCC 6803; Syn; Protein Data Bank (PDB) ID code 3MG1] shows a two-domain structure connected by an unstructured loop, and revealed hECN to be concealed deep inside the protein scaffold and stabilized via hydrogen bonds from both domains (17). The N-terminal domain (residues 15–160; Nterm) is all α-helical, and the C-terminal domain (residues 196–317; Cterm) is composed of β-strands in a twisted sheet. The first 19 N-terminal amino acids (the “cap”) extend over and interact with a solvent-exposed face of the β-sheet of the Cterm. The keto group of hECN is involved in hydrogen bonding with Tyr201 and Trp288 in the Cterm (13, 16, 18). It was found that the Nterm containing the carotenoid is sufficient to induce quenching activity without the presence of the Cterm (19, 20). Very recently, the structure of the N-terminal domain of the activated state of the OCP^R (denoted RCP; red carotenoid protein) was determined to high resolution, revealing that upon release of the Cterm the carotenoid molecule translates 12 Å further into the Nterm (20). By this movement, hECN is protected from the solvent, and this movement is accompanied by conformational changes that result in the change in carotenoid absorption. It can be inferred from these results that upon light activation the Cterm and Nterm of the OCP disassemble, allowing the Nterm to tightly associate with the PBS and perform its functional role of energy transfer disruption. Circular dichroism (CD) spectra of the two isolated domains suggest that each set of secondary structures is not altered by their physical separation (19), as confirmed by the RCP crystal structure for the Nterm. The length that links the Nterm to the Cterm is at most 37 residues (160–196), and thus the two domains can reach a separation of 60–80 Å, potentially leaving the Cterm unattached to the PBS (20). In the OCP^o form the Cterm prevents the OCP from binding to the PBS, and in the OCP^R the Cterm interacts with the fluorescence recovery protein (FRP), an additional component of the NPQ system. Presence of the FRP greatly enhances the reversal of OCP^R to OCP^O and unbinding of the OCP from the PBS (21).The core of the PBS is made up of two to five cylinders (Fig. S1), each composed of four (αβ)₃ trimers of allophycocyanin (APC) (22, 23). Two basal core cylinders sit on top of the membrane, and contain the main form of APC (APC₆₆₀) and one copy each of three minor APC forms, ApcF (a β-like subunit) and ApcD and ApcE (α-like subunits; ApcE is also known as L_CM, and in addition to the chromophore-bearing domain also contains large linker domains of unknown position) (Fig. 1). ApcD and ApcE exhibit significant red-shifted fluorescence APC₆₈₀ that overlaps well with the absorption of chlorophyll a, thus enabling efficient energy transfer from the PBS to the RC. The core is surrounded by six to eight rods containing phycocyanin (PC), which is always proximal to the core, and in some cases other phycobiliproteins (PBPs) (7). Additional nonpigmented proteins called linker or tuner proteins (LPs) are found within the empty spaces formed by APC or PC trimerization. The core contains two LPs, a small 8.5-kDa protein (ApcC) that blocks the entrance to the basal cylinders (24) and a very long extension to the ApcE subunit that is critical for core assembly (25, 26). Several models of the OCP–PBS interaction have been suggested. Blankenship and coworkers have recently used chemical cross-linking of the OCP^R to the PBS of Syn (27). Using relatively long linkers (11.4 Å), they identified four cross-links between APC and the OCP by liquid chromatography coupled to tandem mass spectrometry (LC/MS-MS) analysis. Using the OCP^O and APC crystal structures, this study suggested that the Nterm is bound between one APC₆₆₀ and one APC₆₈₀ trimer, bringing the hECN to 26 Å from the closest phycocyanobilin (PCB) chromophore. This model also predicted that the closest chromophore would be of the bulk APC₆₆₀ type. However, Stadnichuk and coworkers (28, 29) have suggested that the OCP^R interacts directly with the ApcE terminal emitter in vitro, and from this it was proposed that, in vivo, proper energy propagation to the reaction center is interrupted at that site. Jallet et al. (30), using an ApcE mutant, demonstrated that the lack of the ApcE PCB also does not affect OCP-induced quenching. In addition, Jallet et al. (30) and Kuzminov et al. (31) demonstrated that single and double mutants lacking either the ApcD or ApcF subunits continue to exhibit the OCP-dependent quenching mechanism with the same kinetics. These findings have led to the conclusion that the OCP-related quenching most probably takes place at APC₆₆₀. van Amerongen and coworkers have shown that indeed the first site of quenching is at 660 nm and that, in cells, the total rate constant for quenching is 16 ± 4 ps⁻¹ (32). Because only one of the 66 APC₆₆₀ bilins found in the core is directly quenched by the hECN chromophore, the rate constant for molecular quenching of APC₆₆₀ has to be at most (240 ± 60 fs)⁻¹, which is extremely fast, thus promoting very efficient quenching (80%). The exact mechanism leading to such an efficient quenching is still controversial.Open in a separate windowFig. 1.Partial model of the Syn PBS. (A) The PBS is visualized from the direction of the thylakoid membrane. The model includes a single-basal core cylinder (including ApcA, ApcB, ApcC, ApcD, ApcE, and ApcF; colored in yellow, blue, light orange, gray, light green, and light blue, respectively) and a three-hexamer rod that includes the phycobiliproteins CpcA and CpcB (colored in pink and red, respectively) and the CpcC, CpcD, and CpcG linkers (in black). In all schematic models, linkers are shown as small objects because their position and structures are not known. Only the phycobiliprotein-type domain of ApcE is shown. (B) The same segment of the PBS is shown perpendicular to that in A (along the membrane). Colors are as in A.Open in a separate windowFig. S1.Model of the Syn PBS. (A) The Syn PBS is visualized from the side, above the thylakoid membrane, similar to Fig. 1A but complete. An additional core cylinder and four rods are not seen from this angle. (B) Same model, seen along the membrane as in Fig. 1B.In the study presented here, we have analyzed the interactions between the OCP^R and the PBS using the cross-linking mass spectrometry (MS) methodology mentioned above with the more general linker glutaraldehyde (GA). Our analysis is based on many more cross-links than previously obtained, and indicates that the OCP penetrates into the PBS core, thereby bringing hECN into close contact with multiple PCB chromophores and inducing changes in the position of the ApcC linker protein.     

Increasing cloning possibilities using artificial zinc finger nucleases

The ability to accurately digest and ligate DNA molecules of different origins is fundamental to modern recombinant DNA research. Only a handful of enzymes are capable of recognizing and cleaving novel and long DNA sequences, however. The slow evolution and engineering of new restriction enzymes calls for alternative strategies to design novel and unique restriction enzymes capable of binding and digesting specific long DNA sequences. Here we report on the use of zinc finger nucleases (ZFNs)—hybrid synthetic restriction enzymes that can be specifically designed to bind and cleave long DNA sequences—for the purpose of DNA recombination. We show that novel ZFNs can be designed for the digestion of specific sequences and can be expressed and used for cloning purposes. We also demonstrate the power of ZFNs in DNA cloning by custom-cloning a target DNA sequence and assembling dual-expression cassettes on a single target plasmid, a task that rarely can be achieved using type-II restriction enzymes. We demonstrate the flexibility of ZFN design and the ability to shuffle monomers of different ZFNs for the digestion of compatible recognition sites through ligation of compatible ends and their cleavage by heterodimer ZFNs. Of no less importance, we show that ZFNs can be designed to recognize and cleave existing DNA sequences for the custom-cloning of native target DNA molecules.     

EBUS-TBNA is Sufficient for Successful Diagnosis of Silicosis with Mediastinal Lymphadenopathy

Background

Silicosis is an occupational lung disease resulting from inhalation of respirable crystalline silica. Recently, an international silicosis epidemic has been noted among artificial stone workers.

Objective

Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is currently used for patients with unexplained lymphadenopathy. Since silicosis may present with prominent lymphadenopathy, the diagnostic yield of EBUS-TBNA in diagnosing silicosis was evaluated.

Methods

Twenty-eight patients with suspected silicosis referred for outpatient evaluation in three large tertiary hospitals were evaluated. Patients with mediastinal lymphadenopathy underwent EBUS-TBNA, while others underwent TBB and/or video-assisted thoracoscopic surgery (VATS).

Results

Eleven patients with mediastinal lymphadenopathy (39%) were evaluated using EBUS-TBNA. The diagnosis was accurate in all cases, demonstrating silica particles under polarized light, with no complications. Among the remaining patients, TBB was only 76% diagnostic, therefore requiring VATS.

Conclusions

EBUS-TBNA is a useful and sufficient tool to diagnose silicosis in patients with mediastinal lymphadenopathy along compatible exposure histories.

     

Interdependency of beta-adrenergic receptors and CFTR in regulation of alveolar active Na+ transport

Beta-adrenergic receptors (betaAR) regulate active Na+ transport in the alveolar epithelium and accelerate clearance of excess airspace fluid. Accumulating data indicates that the cystic fibrosis transmembrane conductance regulator (CFTR) is important for upregulation of the active ion transport that is needed to maintain alveolar fluid homeostasis during pulmonary edema. We hypothesized that betaAR regulation of alveolar active transport may be mediated via a CFTR dependent pathway. To test this hypothesis we used a recombinant adenovirus that expresses a human CFTR cDNA (adCFTR) to increase CFTR function in the alveolar epithelium of normal rats and mice. Alveolar fluid clearance (AFC), an index of alveolar active Na+ transport, was 92% greater in CFTR overexpressing lungs than controls. Addition of the Cl- channel blockers NPPB, glibenclamide, or bumetanide and experiments using Cl- free alveolar instillate solutions indicate that the accelerated AFC in this model is due to increased Cl- channel function. Conversely, CFTR overexpression in mice with no beta1- or beta2-adrenergic receptors had no effect on AFC. Overexpression of a human beta2AR in the alveolar epithelium significantly increased AFC in normal mice but had no effect in mice with a non-functional human CFTR gene (Deltaphi508 mutation). These studies indicate that upregulation of alveolar CFTR function speeds clearance of excess fluid from the airspace and that CFTRs effect on active Na+ transport requires the betaAR. These studies reveal a previously undetected interdependency between CFTR and betaAR that is essential for upregulation of active Na+ transport and fluid clearance in the alveolus.     

The Impact of Childhood and Adolescent Obesity on Cardiovascular Risk in Adulthood: a Systematic Review

Purpose of Review

Adult obesity and cardiovascular diseases are closely linked. Yet, the relationship of childhood and adolescent obesities with cardiovascular diseases in adulthood requires additional evidence. The goal of the review is to inspect the relationship between childhood- and adolescent-increased body mass index (BMI) and cardiovascular risk factors, fatal and non-fatal cardiovascular diseases in adulthood.

Recent Findings

Cardiovascular diseases in adulthood are linked by most of the studies to childhood and adolescent obesities.

Summary

Studies showed that childhood and adolescent obesities increased the incidence of cardiovascular disease risk factors and were linked to higher risk of cardiovascular morbidity and mortality in adulthood. Childhood and adolescent obesities were also associated, likely with a causal relationship, with an increased likelihood for various cardiovascular morbidities including ischemic heart disease, stroke, but also non-ischemic heart disease-related cardiac pathologies.

     

Comparative efficacy and safety of immediate-release and controlled-release hydralazine in black hypertensive patients

Twenty-nine black hypertensive patients were randomized to treatment with controlled-release hydralazine capsules administered BID or QD, or immediate-release hydralazine tablets administered TID, for at least four weeks in a double-blind, parallel study. Hydralazine was begun after a two-week to four-week period in which blood pressure was not adequately controlled with diuretics alone. Each patient initially received 75 mg/day of the assigned drug. The dosage could be increased to 150 or 300 mg/day at weekly intervals if sitting diastolic blood pressure was not adequately controlled (greater than 90 mmHg). A beta-blocker (80 mg/day of nadolol) was added only for patients who had beta-blocker-responsive adverse effects that could not be controlled otherwise. Nine patients were considered unevaluable because of protocol violations or withdrawal from the study before completion of four weeks of treatment, primarily because of adverse effects. Twenty patients were included in the efficacy evaluation. Controlled-release hydralazine BID produced statistically significant mean falls from baseline in sitting diastolic blood pressure and in standing systolic blood pressure and an almost significant drop in standing diastolic blood pressure. Although the other two treatment groups also had substantial falls in all blood pressure measurements, the changes from baseline were not statistically significant. No significant difference in response was noted between patients who received a beta-blocker and those who did not. There were no statistically significant differences among the three treatment groups in incidence and severity of adverse effects or electrocardiographic abnormalities. A statistically, but not clinically, significant fall in hemoglobin, hematocrit, and red blood cell count was observed in all three treatment groups.     

Retention of intratumor injections of cisplatinum in murine tumors and the impact on laser thermal therapy for cancer treatment

Recent studies using murine models of human squamous cell carcinoma (SCCA) have revealed a significant improvement in survival and cure rate of animals transplanted with human SCCA when treated with a combination of intratumor injections of chemotherapy and laser induced thermal therapy (LITT). These preliminary results suggest that this novel combination therapy may lead to improved clinical response compared to either treatment modality alone. Using a murine model of human SCCA we investigated two different modes of intratumor injection of cisplatin: a sustained-release cisplatin gel implant (CDDP/gel) versus cisplatin in solution (CDDP) at varying doses (range 1–3 mg/ml). In addition, we tested CDDP/gel combined with LITT. Results showed optimal drug concentration (30–300 nM) at tumor margins up to 4 h after injection of CDDP/gel implant compared to 3 nM at 5 min after injection with CDDP solution. Combined CDDP/gel and laser therapy significantly decreased tumor volume (P < 0.05), with recurrence in only 25% of animals tested, compared to 78% tumor regrowth after LITT alone. These results suggest that laser chemotherapy may be an effective treatment for head and neck SCCA. Presented at the 2005 Annual Meeting of the Academy of Otolaryngology, Head and Neck Surgery. September 26, 2005, Los Angeles, CA, USA.     

Effects of exposure to low concentrations of carbon monoxide on exercise performance and myocardial perfusion in young healthy men

OBJECTIVE: To assess the effects of exposure to low concentrations of carbon monoxide (CO), as commonly measured in atmospheric urban air pollution and certain occupational environments, on exercise performance and myocardial perfusion in young healthy men, and the possible need for tighter restrictions on ambient concentrations of CO. METHODS: 15 young, healthy non-smoking men, 18-35 years old, were exposed blindly and randomly to air or to a mixture of CO and air, followed by an exercise treadmill test with thallium heart scintigraphy. Blood was drawn for determination of carboxyhaemoglobin before and at the end of the exposure, and for lactic and pyruvic acid at the beginning and the end of the exercise test. The main outcome measures include the duration of the exercise test, the maximal effort expressed in metabolic equivalent units (METs), the mean plasma lactic to pyruvic acid ratio at the end of the ergometry, ECG changes in the exercise test, and perfusion deficits in thallium heart scintigraphy. RESULTS: At the end of exposure to CO, the mean (SD) blood carboxyhaemoglobin concentration rose from 0.59% (0.08%) to 5.12% (0.65%) (p < 0.0001). At the end of the exercise period, the mean (SD) plasma lactate/pyruvate ratio, which reflects the level of anaerobic metabolism (69.9 (5.9) after air and 75.9 (7.0) after CO), was not significantly different between the two experimental groups. Exercise induced electrocardiographic changes were noted in only one subject after exposure to CO. No arrhythmias were detected in any of the subjects. Significant differences were found in the mean duration of the exercise test (p = 0.0012) and the METs (p = 0.0001). The mean adjusted difference of exercise duration between exposure to air and CO was 1.52 minutes 95% confidence interval (95% CI) 0.73 to 2.32 minutes. The mean adjusted difference of METs between exposure to air and CO was 2.04 95% CI 1.33 to 2.76. The models for duration of exercise and METs showed no significant sequence and period effects. Thallium myocardial perfusion imaging disclosed normal perfusion in all regions of the heart, with no significant differences in perfusion between the two exercise tests (after air or CO). CONCLUSION: Acute exposure to a low concentration of CO which produces blood carboxyhaemoglobin concentrations of 4%-6% significantly decreases exercise performance in young healthy men. No ischaemic electrocardiographic changes or disturbances in myocardial perfusion were found by graded exercise with thallium scintigraphy. Our findings suggest that pollution of atmospheric air by CO at concentrations which are commonly found in urban and industrial environments may exert an adverse effect on skeletal muscles, manifesting as decreased exercise performance.