Computed Tomography-Guided Microcoil Localization of Pulmonary Nodules: Effects of Multiple Punctures.

BACKGROUND: The aim of the study is to analyze the effect of multiple punctures in computed tomography (CT)-guided microcoil localization of pulmonary nodules with other risk factors for common complications. METHODS: Consecutive patients who underwent CT-guided microcoil localization and subsequent video-assisted thoracoscopic surgery (VATS) between January 2020 and February 2021 were enrolled. Nodules successfully located after only one puncture were defined as the single puncture group, and nodules requiring two or more punctures were defined as the multiple puncture group. Binary logistic regression analysis was performed to assess the relationship between the number of punctures and pneumothorax and intrapulmonary hemorrhage. RESULTS: A total of 121 patients were included. There were 98 (68.1%) pulmonary nodules in the single puncture group compared with 46 (31.9%) nodules in the multiple puncture group. The frequencies of pneumothorax and intrapulmonary hemorrhage were higher in the multiple puncture group than in the single puncture group (p = 0.019 and <0.001, respectively). Binary logistic regression demonstrated that independent risk factors for developing pneumothorax included lateral positioning of the patient (p < .001) and prone positioning (p = 0.014), as well as multiple punctures (p = 0.013). Independent risk factors for intrapulmonary hemorrhage included the distance between the distal end of the coil and the surface of the pleura (p = 0.033), multiple punctures (p = 0.003), and passage through the pulmonary vasculature (p < 0.001). CONCLUSION: Multiple punctures resulted in an increased incidence of pneumothorax and intrapulmonary hemorrhage compared with single puncture during CT-guided microcoil localization of pulmonary nodules and were independently associated with both pneumothorax and intrapulmonary hemorrhage.

Outcomes of CT-Guided Deeper Localization Technique for Superficial Pulmonary Nodules.

BACKGROUND: The possibility of coil dislocation in computed tomography (CT)-guided microcoil localization of superficial pulmonary nodules is relatively high. The aim of the study is to investigate the outcomes of deeper localization technique during CT-guided microcoil localization of superficial pulmonary nodules before video-assisted thoracoscopic surgery (VATS). METHODS: Fifty-seven identified superficial pulmonary nodules (nodule-pleural distance ≤ 1 cm on CT image) from 51 consecutive patients underwent CT-guided microcoil localization, and subsequent VATSs were included. The rate of technical success, complications, and excised lung volume were compared between deeper localization technique group and conventional localization technique group. RESULTS: The technical success rate of the localization procedure was 100% (25/25) in the deeper localization group and 81.3% (26/32) in the conventional localization group (p = 0.030). Excluding one case of lobectomy, the excised lung volume in the deeper localization group and the conventional localization group was 39.3 ± 23.5 and 37.2 ± 16.2 cm3, respectively (p = 0.684). The incidence of pneumothorax was similar between the deeper localization group and the conventional localization group (24.0 vs. 21.9%, respectively, p = 0.850). The incidence of intrapulmonary hemorrhage in the deeper localization group was higher (16.0%) than that in the conventional localization group (6.3%), but the difference was not statistically significant (p = 0.388). CONCLUSION: CT-guided microcoil localization of superficial pulmonary nodules prior to VATS using a deeper localization technique is feasible. Deeper localization technique reduced the occurrence of dislocation but did not increase excised lung volume.

Percutaneous transluminal forceps biopsy with an adjustable curved sheath for obstructive jaundice.

PURPOSE: To evaluate the feasibility and safety of percutaneous transluminal forceps biopsy (PTFB) with an adjustable curved sheath in patients with obstructive jaundice. MATERIAL AND METHODS: Forty-two patients who underwent PTFB with an adjustable curved sheath were analyzed retrospectively. Sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV) and accuracy were calculated for all populations and in different situations. Technical success and safety were evaluated. RESULTS: The technical success rate was 100%. Thirty-five of 42 cases were diagnosed malignant diseases, the sensitivity of PTFB with an adjustable curved sheath was 74.29% (26/35), the specificity was 100%, the positive predictive value was 100%, the negative predictive value was 43.75% (7/16), and the accuracy rate was 78.57% (33/42). There was a better sensitivity for bile duct malignancies when compared with non-bile duct malignancies (p = 0.012). No statistical difference was found in the sensitivity of the upper part of the biliary tree and the lower part of the biliary tree, and none in the sensitivity of different approaches (left vs. right). The complication rate was 11.90%, and no serious complications were observed. CONCLUSIONS: PTFB with an adjustable curved sheath is an effective and safe technique, without being limited by approaches and obstruction sites.

Ligand binding to a remote site thermodynamically corrects the F508del mutation in the human cystic fibrosis transmembrane conductance regulator.

Many disease-causing mutations impair protein stability. Here, we explore a thermodynamic strategy to correct the disease-causing F508del mutation in the human cystic fibrosis transmembrane conductance regulator (hCFTR). F508del destabilizes nucleotide-binding domain 1 (hNBD1) in hCFTR relative to an aggregation-prone intermediate. We developed a fluorescence self-quenching assay for compounds that prevent aggregation of hNBD1 by stabilizing its native conformation. Unexpectedly, we found that dTTP and nucleotide analogs with exocyclic methyl groups bind to hNBD1 more strongly than ATP and preserve electrophysiological function of full-length F508del-hCFTR channels at temperatures up to 37 °C. Furthermore, nucleotides that increase open-channel probability, which reflects stabilization of an interdomain interface to hNBD1, thermally protect full-length F508del-hCFTR even when they do not stabilize isolated hNBD1. Therefore, stabilization of hNBD1 itself or of one of its interdomain interfaces by a small molecule indirectly offsets the destabilizing effect of the F508del mutation on full-length hCFTR. These results indicate that high-affinity binding of a small molecule to a remote site can correct a disease-causing mutation. We propose that the strategies described here should be applicable to identifying small molecules to help manage other human diseases caused by mutations that destabilize native protein conformation.

Structural stability of purified human CFTR is systematically improved by mutations in nucleotide binding domain 1.

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is an ABC transporter containing two transmembrane domains forming a chloride ion channel, and two nucleotide binding domains (NBD1 and NBD2). CFTR has presented a formidable challenge to obtain monodisperse, biophysically stable protein. Here we report a comprehensive study comparing effects of single and multiple NBD1 mutations on stability of both the NBD1 domain alone and on purified full length human CFTR. Single mutations S492P, A534P, I539T acted additively, and when combined with M470V, S495P, and R555K cumulatively yielded an NBD1 with highly improved structural stability. Strategic combinations of these mutations strongly stabilized the domain to attain a calorimetric Tm > 70 °C. Replica exchange molecular dynamics simulations on the most stable 6SS-NBD1 variant implicated fluctuations, electrostatic interactions and side chain packing as potential contributors to improved stability. Progressive stabilization of NBD1 directly correlated with enhanced structural stability of full-length CFTR protein. Thermal unfolding of the stabilized CFTR mutants, monitored by changes in intrinsic fluorescence, demonstrated that Tm could be shifted as high as 67.4 °C in 6SS-CFTR, more than 20 °C higher than wild-type. H1402S, an NBD2 mutation, conferred CFTR with additional thermal stability, possibly by stabilizing an NBD-dimerized conformation. CFTR variants with NBD1-stabilizing mutations were expressed at the cell surface in mammalian cells, exhibited ATPase and channel activity, and retained these functions to higher temperatures. The capability to produce enzymatically active CFTR with improved structural stability amenable to biophysical and structural studies will advance mechanistic investigations and future cystic fibrosis drug development.

Assessing risk factors for complications in computer tomography-guided lung biopsy: quantitative analysis for predicting pneumothorax.

BACKGROUND: Computer tomography (CT)-guided lung biopsy carries the risk of pneumothorax. A variety of other risk factors affect the occurrence of pneumothorax. OBJECTIVE: Assess the incidence and risk factors associated with pneumothorax complications in CT-guided lung biopsy, and to conduct a quantitative analysis of the variables among the significant risk factors to identify more effective indicators for predicting pneumothorax complications. DESIGN: Retrospective logistic. SETTING: Single center in China. PATIENTS AND METHODS: From June 2017 to May 2021, consecutive patients who underwent CT-guided lung biopsy were identified from the medical record system. Binary logistic regression analysis was used to identify potential risk factors for pneumothorax. Receiver operating characteristic (ROC) curves were constructed for continuous variables to determine cutoff values that optimized sensitivity and specificity. MAIN OUTCOME MEASURES: The incidence and risk factors of pneumothorax in CT-guided lung biopsy. SAMPLE SIZE: 132 patients. RESULTS: The incidence of pneumothorax was 28.9% (38/132), with 6.8% (9/132) of patients requiring chest tube insertion. Results indicated that smaller lesion size (OR 0.724; 95% CI 0.619-0.848; P=.0001), longer needle tract length (OR 1.320; 95% CI 1.145-1.521; P=.001), multiple passes through the pleura (OR 4.618; 95% CI 1.378-15.467; P=.013), and needle tract length/lesion diameter (L/D) ratio (OR 0.028; 95% CI 0.002-0.732; P=.007) were independent risk factors for pneumothorax. ROC curve analysis determined a cut-off value of 0.81 for the L/D ratio (sensitivity=89.5%, specificity=71.3%). The area under the ROC curve (AUC) values of maximum diameter, needle tract length, and L/D ratio for pneumothorax were 0.749, 0.812, and 0.850, respectively. CONCLUSIONS: The L/D ratio, multiple passes through the pleura, longer needle tract length, and smaller lesions were independent risk factors for pneumothorax. A L/D ratio of less than 0.81 may indicate a pneumothorax. It may be necessary to use the proper sealing procedure for this patient group. LIMITATIONS: Due to its retrospective nature, there may be inherent selection bias.

Application of the 5F JL4 Catheter in Bronchial Artery Embolization With the Opening in the Inferior Wall of the Aortic Arch.

BACKGROUND: We investigated the efficacy and safety of bronchial artery embolization (BAE) using a 5F JL4 catheter in patients with hemoptysis and a bronchial artery opening in the inferior wall of the aortic arch. METHODS: This was a single-center retrospective study. Seventeen patients underwent BAE using 5F JL4. We then evaluated technical success (TS), clinical success (CS), incidence of complications, and hemoptysis recurrence rate (RR). RESULTS: The TS rate of microcatheter superselective catheterization and CS rate after surgery were 100%, and the incidence of severe complications and postoperative RR were 17.6%. CONCLUSIONS: Bronchial artery embolization for hemoptysis with a BA opening in the inferior wall of the aortic arch using the 5F JL4 catheter could be a safe method. The short- and medium-term results were excellent.

FEASIBILITY STUDY OF LOW TUBE VOLTAGE COMPUTER TOMOGRAPHY ANGIOGRAPHY (CTA) FOR BRONCHIAL ARTERY IMAGING IN PATIENTS WITH HEMOPTYSIS.

OBJECTIVE: To investigate the application of low tube voltage computer tomography angiography (CTA) in bronchial artery (BA) imaging in hemoptysis patients. METHODS: Between January 2017 and December 2021, 119 patients were studied, including 31 in the 80-kV group, 39 in the 100-kV group and 49 in the control group (120 kV). The CT dose index-volume (CTDIvol) (mGy) and effective dose (ED) (mSv) of each group were comparatively analysed. Image quality evaluation included the signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR) and subjective 5-scores. RESULTS: Statistically significant differences were noted in CTDIvol, ED, SNR, CNR and image quality scores of the groups (P < 0.05). Comparative analysis showed no statistical difference in CTDIvol, ED and image quality scores between the 80- and 100-kV groups. CONCLUSION: Low tube voltage CTA is useful in BA imaging for hemoptysis patients. Tube voltages of 100 kV have better image quality and lower radiation dose.

CT-guided microcoil localization for scapula-blocked pulmonary nodules using penetrating lung puncture before video-assisted thoracic surgery.

PURPOSE: To retrospectively analyze the effectiveness and safety of computed tomography (CT)-guided microcoil localization for scapula-blocked pulmonary nodules using penetrating lung puncture prior to video-assisted thoracic surgery (VATS). METHODS: One hundred thirty-eight patients with 138 pulmonary nodules were included in this single-center retrospective study. Among them, 110 patients who underwent CT-guided microcoil localization using the routine puncture technique formed the routine group; the other 28 patients who underwent the CT-guided microcoil localization using the penetrating lung puncture technique formed the penetrating lung group. The main outcomes were the success rate and complication rate of the two groups. RESULTS: The localization success rate was 95.5% (105/110) in the routine group and 89.3% (25/28) in the penetrating lung group (P = 0.205). There was no statistical difference in any of the complications (pneumothorax, intrapulmonary hemorrhage, or moderate and severe chest pain) in both groups (P = 0.178, P = 0.204, P = 0.709, respectively). Localization procedure time was significantly increased in the penetrating lung group compared with the routine group (31.0 ± 3.0 min vs. 21.2 ± 2.8 min, P < 0.001). CONCLUSION: CT-guided microcoil localization for scapula-blocked pulmonary nodules using penetrating lung puncture prior to VATS resection is effective and safe. However, the deployment of the microcoil using penetrating lung puncture required more time than the routine puncture method.

Comparative Study on Nosocomial Biliary Tract Infection Rate Between Biliary Stent Loaded with Radioactive 125 I Seeds and Conventional Biliary Stent in the Treatment of Distal Malignant Biliary Obstruction.

BACKGROUND: The purpose of this prospective randomized study was to compare the nosocomial biliary tract infection rate of biliary stent implantation with a biliary stent loaded with radioactive 125 I seeds (radioactive biliary stent, RBS) and conventional biliary stent (CBS); additionally, to preliminary discuss the causes of postoperative cholangitis. Moreover, the results will provide clinical evidence for the prevention of postoperative biliary tract infection. MATERIALS AND METHODS: We prospectively analyzed the nosocomial infection rate of the distal malignant biliary obstruction (MBO) treatment by stent implantation with RBS and CBS. All MBO patients who initially visited our tertiary hospital between July 2015 and December 2019 (n= 196) were evaluated, enrolled, and randomly divided into 2 groups, RBS (n=97) and CBS (n=99) group. χ 2 test was used to evaluate the categorical data, and t test was used to evaluate the numerical data. RESULTS: Our analysis of the study showed the incidence of postoperative infections of a biliary tract of the RBS group (23.7%) was significantly higher than the CBS group (11.1%). The difference was statistically significant (χ 2 =5.425, P =0.020). Our study also showed the most common pathogenic bacteria after surgery was Escherichia coli (26.5%). CONCLUSION: Treatment for distal MBO with biliary stent loaded with radioactive 125 I seeds had a higher nosocomial infection rate, and the most common pathogenic bacteria was E coli. , Supplemental Digital Content 1, http://links.lww.com/sle/A350.

CT-guided placement of microcoil end in the pleural cavity for video-assisted thoracic surgical resection of ground-glass opacity: a retrospective study.

BACKGROUND: The aim of the study was to investigate and summarize the effectiveness and safety of CT-guided microcoil localization before video-assisted thoracic surgery (VATS) for the removal of ground-glass opacity (GGO). METHODS: A total of 147 patients with GGO who were treated at our hospital between January 2019 and February 2021 were retrospectively analyzed. They were divided into two groups according to the final position at the end of the microcoil: intracavity (n = 78) and extracavity (n = 69), which were compared based on puncture complications and influence of the coil end position on VATS. RESULTS: The proportions of supine and prone positions in the intracavity group were significantly higher than those in the extracavity group (82.1% vs. 66.7%, P < 0.05). The incidence of intrapulmonary hemorrhage, chest pain, and coil displacement in the intracavity group was significantly lower than that in the extracavity group (28.2% vs. 46.4%, 19.2% vs. 39.1%, 1.3% vs. 11.6%, P < 0.05, respectively); however, the incidence of pneumothorax was not significantly different (P > 0.05). The time of VATS and the rate of conversion to thoracotomy in the intracavity group were significantly lower than those in the extracavity group (103.4 ± 21.0 min vs. 112.2 ± 17.3 min, 0% vs. 5.8%, P < 0.05, respectively). CONCLUSION: CT-guided placement of the microcoil is a practical, simple, and convenient localization method before VATS, with a high success rate and few complications. Furthermore, it is a better alternative method to place the end of the coil in the pleural cavity because of the lower complication rate, shorter VATS time, and lower rate of thoracotomy conversion.

[Acute superior mesenteric ischemia: a contrast study on short-and mid-term result between stent implantation and pharmaceutical thrombolysis].

OBJECTIVE: To compare the short-term and medium-term result of stent implantation with pharmaceutical thrombolysis in patients with acute superior mesenteric artery occlusion. METHODS: From January, 2004 to December, 2008, thirty-five patients diagnosed acute superior mesenteric ischemia, 12 patients treated with stent implantation (interventional therapy group) and 23 patients with pharmaceutical thrombolysis (thrombolytic therapy group). Interventional therapy group treated with balloon dilatation and stent implantation assisted with anticoagulation, antiplatelet and vascular dilation agents. Thrombolytic therapy group used urokinase combined with anticoagulation, antiplatelet and vascular dilation agents. All patients had taken clopidogrel and aspirin orally after discharged and followed up. The clinical effects of both groups were evaluated separately and the Fisher exact test was used to analysis the significant differences. RESULTS: In the 23 cases of thrombolytic therapy group, 7 cases was effective, 16 cases was ineffective (7 cases aggravated or died, 9 cases turn to surgical operation). In the 12 cases of interventional therapy group, 10 cases treated with stent implantation (1 case died of acute cardiac infarction 3 days after interventional operation), 2 cases failed in recanalizing. All patients were followed up after discharged (range 1 - 48 months, mean 15 ± 12 months), 1 case in thrombolytic therapy group was stable, 6 cases died of the recurrence of acute superior mesenteric artery occlusion; 7 cases in interventional therapy group was stable, 1 case died of acute cardiac infarction 20 months after interventional operation (intestinal ischemia not appeared), 1 case had intestinal ischemia reoccurred and recovered by superior mesenteric artery thrombolysis. CONCLUSIONS: In the treatment of acute superior mesenteric ischemia, stent implantation was obviously superior to pharmaceutical thrombolysis in improving intestinal ischemia and survival, therefore it could provided a reliable choice for the patients who had not appeared intestinal necrosis.

Retrospective analysis of T-lymphocyte subsets and cytokines in malignant obstructive jaundice before and after external and internal biliary drainage.

OBJECTIVE: To study changes in T lymphocyte subsets, cytokines, and liver enzymes in patients with malignant obstructive jaundice (MOJ) before and after external biliary drainage (percutaneous transhepatic cholangiography drainage, PTCD) and internal biliary drainage (percutaneous transhepatic insertion of biliary stents, PTIBS). METHODS: MOJ patients undergoing PTCD (n = 44) and PTIBS (n = 38) at our hospital were enrolled in the study from January 2017 until December 2019. Peripheral blood total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), CD3+%, CD4+%, CD4+/CD8+ ratio, interleukin (IL)-2, IL-6, and tumor necrosis factor (TNF)-α were measured before and 1 week after biliary drainage. RESULTS: There was no significant difference in any parameter between the two groups before biliary drainage. TBIL, DBIL, AST and ALT following PTCD were significantly lower than before PTCD. By contrast, CD3+%, CD4+%, CD4+/CD8+ ratio, IL-2, IL-6 and TNF-α showed no significant difference before and 1 week after PTCD. TBIL, DBIL, AST, ALT, IL-6 and TNF-α were significantly lower following PTIBS than before PTIBS. CD3+%, CD4+%, CD4+/CD8+ ratio and IL-2 were significantly higher following PTIBS than before PTIBS. CONCLUSION: Both PTCD and PTIBS were effective for treatment of MOJ, but PTIBS was more beneficial for recovery of immune function.

Restoration of NBD1 thermal stability is necessary and sufficient to correct ∆F508 CFTR folding and assembly.

Cystic fibrosis transmembrane conductance regulator (CFTR) (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis. The most common cystic-fibrosis-causing mutation, the deletion of F508 (ΔF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The ΔF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question, we varied the extent of stabilization of NBD1 using different second-site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on ∆F508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the ΔF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function.

Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains.

Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification.

Structural kinetics of cardiac troponin C mutants linked to familial hypertrophic and dilated cardiomyopathy in troponin complexes.

The key events in regulating cardiac muscle contraction involve Ca(2+) binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca(2+) movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca(2+) binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca(2+)-induced opening and closing of the N-domain by Förster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca(2+). L29Q had no effect on the closing rate of the N-domain triggered by release of Ca(2+), but reduced the Ca(2+)-induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca(2+) dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC.

Effects of PKA phosphorylation of cardiac troponin I and strong crossbridge on conformational transitions of the N-domain of cardiac troponin C in regulated thin filaments.

Regulation of cardiac muscle function is initiated by binding of Ca2+ to troponin C (cTnC) which induces a series of structural changes in cTnC and other thin filament proteins. These structural changes are further modulated by crossbridge formation and fine-tuned by phosphorylation of cTnI. The objective of the present study is to use a new Förster resonance energy transfer-based structural marker to distinguish structural and kinetic effects of Ca2+ binding, crossbridge interaction, and protein kinase A phosphorylation of cTnI on the conformational changes of the cTnC N-domain. The FRET-based structural marker was generated by attaching AEDANS to one cysteine of a double-cysteine mutant cTnC(13C/51C) as a FRET donor and attaching DDPM to the other cysteine as the acceptor. The doubly labeled cTnC mutant was reconstituted into the thin filament by adding cTnI, cTnT, tropomyosin, and actin. Changes in the distance between Cys13 and Cys51 induced by Ca2+ binding/dissociation were determined by FRET-sensed Ca2+ titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca2+ and strong binding of myosin head to actin was required to achieve a fully open structure of the cTnC N-domain in regulated thin filaments. Equilibrium and stopped-flow studies suggested that strongly bound myosin head significantly increased the Ca2+ sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca2+ sensitivity and kinetics of the structural transition of the cTnC N-domain but showed no global structural effect on cTnC opening. These results provide an insight into the modulation mechanism of strong crossbridge and cTnI phosphorylation in cardiac thin filament activation/relaxation processes.

Structural transition of the inhibitory region of troponin I within the regulated cardiac thin filament.

Contraction and relaxation of cardiac muscle are regulated by the inhibitory and regulatory regions of troponin I (cTnI). Our previous FRET studies showed that the inhibitory region of cTnI in isolated troponin experiences a structural transition from a beta-turn/coil motif to an extended conformation upon Ca(2+) activation. During the relaxation process, the kinetics of the reversal of this conformation is coupled to the closing of the Ca(2+)-induced open conformation of the N-domain of troponin C (cTnC) and an interaction between cTnC and cTnI in their interface. We have since extended the structural kinetic study of the inhibitory region to fully regulated thin filament. Single-tryptophan and single-cysteine mutant cTnI(L129W/S151C) was labeled with 1,5-IAEDANS at Cys151, and the tryptophan-AEDANS pair served as a donor-acceptor pair. Labeled cTnI mutant was used to prepare regulated thin filaments. Ca(2+)-induced conformational changes in the segment of Trp129-Cys151 of cTnI were monitored by FRET sensitized acceptor (AEDANS) emission in Ca(2+) titration and stopped-flow measurements. Control experiments suggested energy transfer from endogenous tryptophan residues of actin and myosin S1 to AEDANS attached to Cys151 of cTnI was very small and Ca(2+) independent. The present results show that the rate of Ca(2+)-induced structural transition and Ca(2+) sensitivity of the inhibitory region of cTnI were modified by (1) thin filament formation, (2) the presence of strongly bound S1, and (3) PKA phosphorylation of the N-terminus of cTnI. Ca(2+) sensitivity was not significantly changed by the presence of cTm and actin. However, the cTn-cTm interaction decreased the cooperativity and kinetics of the structural transition within cTnI, while actin filaments elicited opposite effects. The strongly bound S1 significantly increased the Ca(2+) sensitivity and slowed down the kinetics of structural transition. In contrast, PKA phosphorylation of cTnI decreased the Ca(2+) sensitivity and accelerated the structural transition rate of the inhibitory region of cTnI on thin filaments. These results support the idea of a feedback mechanism by strong cross-bridge interaction with actin and provide insights on the molecular basis for the fine tuning of cardiac function by beta-adrenergic stimulation.

Protein preparation, crystallization and preliminary X-ray crystallographic studies of a thermostable hypoxanthine-guanine phosphoribosyltransferase from Thermoanaerobacter tengcongensis.

Native and His-tagged mutant (L160I) hypoxanthine-guanine phosphoribosyltransferase (HGPRT) from Thermoanaerobacter tengcongensis were cloned, expressed in Escherichia coli and purified. Both proteins were crystallized with polyethylene glycol as the main precipitant at 293 K using the hanging-drop vapour-diffusion method. The crystal of native HGPRT belongs to space group C222(1), with unit-cell parameters a = 65.77, b = 137.73, c = 95.27 A, and diffracted to 2.2 A resolution on an in-house X-ray generator. The crystal of the His-tagged mutant (L160I) HGPRT belongs to the space group I222, with unit-cell parameters a = 52.21, b = 88.36, c = 93.03 A, and diffracted to 1.7 A resolution in-house.

Protein preparation, crystallization and preliminary X-ray analysis of human adrenal gland protein AD-004.

The adrenal gland protein AD-004 was identified in the human adrenal gland. Full-length AD-004 contains 172 amino acids, with a predicted molecular weight of about 20 kDa. In attempts to crystallize human AD-004, the gene was subcloned into a modified pET vector, pET21-DEST, with an N-terminal His(5) tag using the Gateway cloning system, followed by protein expression in Escherichia coli strain BL21(DE3). The protein was purified in two steps to near-homogeneity and was crystallized. The crystals belong to space group P6(1) or P6(5), with unit-cell parameters a = b = 99.56, c = 57.19 A. A complete 2.0 A data set has been collected at a rotating-anode X-ray source and structure determination is under way.