A Simple Prognostic Scoring System for Hepatocellular Carcinoma Treated with Selective Internal Radiation Therapy.

INTRODUCTION: Selective internal radiation therapy (SIRT) is a local treatment option for patients with hepatocellular carcinoma (HCC). Its exact role next to other HCC therapies has yet to be defined. In order to identify patients most suitable for SIRT, a SIRT-specific prognostic score should be developed. METHODS: A cohort of 72 SIRT patients treated at the University Hospital of Munich was retrospectively analyzed. The prognostic performance of 12 HCC staging systems and prognostic scores was assessed. Cox-regression analysis was used to identify independent prognostic factors, which formed the basis of the Munich-SIRT score (M-SIRT). All scores were ranked by calculating the c-Index and Akaike information criterion (AIC). External validation was performed in a cohort of 128 SIRT patients treated at the University Hospital of Pamplona, Spain. RESULTS: median overall survival was 13 months (95% confidence interval 9.9-21.9). AFP (p = 0.005; hazard ratio [HR] 2.38), albumin (p < 0.001; HR 5.87), and alkaline phosphatase (p < 0.001; HR 8.38) were identified as independent prognostic factors. M-SIRT comprises 3 prognostic groups with a median survival of 38.9, 14.6, and 7.7 months, respectively (I vs. II: p = 0.003, II vs. III: p < 0.001). AIC (318) and concordance index (0.711) ranked M-SIRT superior to the established HCC staging systems, and the score successfully passed external validation in an independent SIRT cohort (I vs. II: p = 0.03; II vs. III: p = 0.007). CONCLUSION: Therapy-specific prognostic scores can facilitate treatment decisions and prognostication for HCC patients. Considering its performance in 200 SIRT patients, M-SIRT is a promising prognostic tool for HCC patients evaluated for SIRT.

Retrospective outcome analysis of rates and types of complications after 8654 minimally invasive radiological port implantations via the subclavian vein without ultrasound guidance.

PURPOSE: To evaluate the rate and types of complications after minimally invasive radiological central vein port implantation without ultrasound guidance. MATERIALS AND METHODS: We retrospectively evaluated 8654 patients who underwent port implantations in the subclavian vein without ultrasound guidance in our institution from 1998 to 2014 with regard to types and rates of peri-, early and late post-interventional complications according to the common classification for complications published by the Society of Interventional Radiology (SIR). Additionally, the impact of the training level of the operators on the rate of complications was analyzed. RESULTS: Successful port implantations were performed in 99.8% (8636/8654 procedures). From 1998 to 2014, a total of 565 (6.52%) complications were recorded. The overall percentage of the peri-, early and late post-interventional complications according to the SIR criteria was 1.69, 0.15 and 4.68, respectively. Significant differences due to the training level of the performing physician could be seen for the rates of pneumothorax, arterial puncture and hematoma. CONCLUSION: Minimally invasive radiological interventional port implantation is a safe treatment option with a low rate of complications even without ultrasound guidance.

Structured reporting of x-rays for atraumatic shoulder pain: advantages over free text?

BACKGROUND: To analyse structured and free text reports of shoulder X-ray examinations evaluating the quality of reports and potential contributions to clinical decision-making. METHODS: We acquired both standard free text and structured reports of 31 patients with a painful shoulder without history of previous trauma who received X-ray exams. A template was created for the structured report based on the template ID 0000154 (Shoulder X-ray) from radreport.org using online software with clickable decision trees with concomitant generation of structured semantic reports. All reports were evaluated regarding overall quality and key features: content, information extraction and clinical relevance. RESULTS: Two experienced orthopaedic surgeons reviewed and rated structured and free text reports of 31 patients independently. The structured reports achieved significantly higher median ratings in all key features evaluated (P < 0.001), including facilitation of information extraction (P < 0.001) and better contribution to subsequent clinical decision-making (P < 0.001). The overall quality of structured reports was significantly higher than in free text report (P < 0.001). CONCLUSIONS: A comprehensive structured template may be a useful tool to assist in clinical decision-making and is, thus, recommended for the reporting of degenerative changes regarding X-ray examinations of the shoulder.

Surgical Site Infections After Dermatologic Surgery in Immunocompromised Patients: A Single-Center Experience.

BACKGROUND: Immunosuppression is often considered as an indication for antibiotic prophylaxis to prevent surgical site infections (SSI) while performing skin surgery. However, the data on the risk of developing SSI after dermatologic surgery in immunosuppressed patients are limited. PATIENTS AND METHODS: All patients of the Department of Dermatology and Allergology at the University Hospital of RWTH Aachen in Aachen, Germany, who underwent hospitalization for a dermatologic surgery between June 2016 and January 2017 (6 months), were followed up after surgery until completion of the wound healing process. The follow-up addressed the occurrence of SSI and the need for systemic antibiotics after the operative procedure. Immunocompromised patients were compared with immunocompetent patients. The investigation was conducted as a retrospective analysis of patient records. RESULTS: The authors performed 284 dermatologic surgeries in 177 patients. Nineteen percent (54/284) of the skin surgery was performed on immunocompromised patients. The most common indications for surgical treatment were nonmelanoma skin cancer and malignant melanomas. Surgical site infections occurred in 6.7% (19/284) of the cases. In 95% (18/19), systemic antibiotic treatment was needed. Twenty-one percent of all SSI (4/19) were seen in immunosuppressed patients. CONCLUSION: According to the authors' data, immunosuppression does not represent a significant risk factor for SSI after dermatologic surgery. However, larger prospective studies are needed to make specific recommendations on the use of antibiotic prophylaxis while performing skin surgery in these patients.

Computed Tomography of the Spine : Systematic Review on Acquisition and Reconstruction Techniques to Reduce Radiation Dose.

The introduction of the first whole-body CT scanner in 1974 marked the beginning of cross-sectional spine imaging. In the last decades, the technological advancement, increasing availability and clinical success of CT led to a rapidly growing number of CT examinations, also of the spine. After initially being primarily used for trauma evaluation, new indications continued to emerge, such as assessment of vertebral fractures or degenerative spine disease, preoperative and postoperative evaluation, or CT-guided interventions at the spine; however, improvements in patient management and clinical outcomes come along with higher radiation exposure, which increases the risk for secondary malignancies. Therefore, technical developments in CT acquisition and reconstruction must always include efforts to reduce the radiation dose. But how exactly can the dose be reduced? What amount of dose reduction can be achieved without compromising the clinical value of spinal CT examinations and what can be expected from the rising stars in CT technology: artificial intelligence and photon counting CT? In this article, we try to answer these questions by systematically reviewing dose reduction techniques with respect to the major clinical indications of spinal CT. Furthermore, we take a concise look on the dose reduction potential of future developments in CT hardware and software.

Impact of radiation dose reduction and iterative image reconstruction on CT-guided spine biopsies.

This study aimed to systematically evaluate the impact of dose reduction on image quality and confidence for intervention planning and guidance regarding computed tomography (CT)-based intervertebral disc and vertebral body biopsies. We retrospectively analyzed 96 patients who underwent multi-detector CT (MDCT) acquired for the purpose of biopsies, which were either derived from scanning with standard dose (SD) or low dose (LD; using tube current reduction). The SD cases were matched to LD cases considering sex, age, level of biopsy, presence of spinal instrumentation, and body diameter. All images for planning (reconstruction: "IMR1") and periprocedural guidance (reconstruction: "iDose4") were evaluated by two readers (R1 and R2) using Likert scales. Image noise was measured using attenuation values of paraspinal muscle tissue. The dose length product (DLP) was statistically significantly lower for LD scans regarding the planning scans (SD: 13.8 ± 8.2 mGy*cm, LD: 8.1 ± 4.4 mGy*cm, p < 0.01) and the interventional guidance scans (SD: 43.0 ± 48.8 mGy*cm, LD: 18.4 ± 7.3 mGy*cm, p < 0.01). Image quality, contrast, determination of the target structure, and confidence for planning or intervention guidance were rated good to perfect for SD and LD scans, showing no statistically significant differences between SD and LD scans (p > 0.05). Image noise was similar between SD and LD scans performed for planning of the interventional procedures (SD: 14.62 ± 2.83 HU vs. LD: 15.45 ± 3.22 HU, p = 0.24). Use of a LD protocol for MDCT-guided biopsies along the spine is a practical alternative, maintaining overall image quality and confidence. Increasing availability of model-based iterative reconstruction in clinical routine may facilitate further radiation dose reductions.

Computed Tomography of the Head : A Systematic Review on Acquisition and Reconstruction Techniques to Reduce Radiation Dose.

In 1971, the first computed tomography (CT) scan was performed on a patient's brain. Clinical CT systems were introduced in 1974 and dedicated to head imaging only. New technological developments, broader availability, and the clinical success of CT led to a steady growth in examination numbers. Most frequent indications for non-contrast CT (NCCT) of the head include the assessment of ischemia and stroke, intracranial hemorrhage and trauma, while CT angiography (CTA) has become the standard for first-line cerebrovascular evaluation; however, resulting improvements in patient management and clinical outcomes come at the cost of radiation exposure, increasing the risk for secondary morbidity. Therefore, radiation dose optimization should always be part of technical advancements in CT imaging but how can the dose be optimized? What dose reduction can be achieved without compromising diagnostic value, and what is the potential of the upcoming technologies artificial intelligence and photon counting CT? In this article, we look for answers to these questions by reviewing dose reduction techniques with respect to the major clinical indications of NCCT and CTA of the head, including a brief perspective on what to expect from current and future developments in CT technology with respect to radiation dose optimization.

Low-dose multi-detector computed tomography for periradicular infiltrations at the cervical and lumbar spine.

Periradicular infiltrations are frequently performed in daily neuroradiological routine and are often guided by multi-detector computed tomography (MDCT), thus leading to radiation exposure. The purpose of this study was to evaluate MDCT with low dose (LD) and model-based iterative reconstruction for image-guided periradicular infiltrations at the cervical and lumbosacral spine. We retrospectively analyzed 204 MDCT scans acquired for the purpose of cervical or lumbosacral periradicular interventions, which were either derived from scanning with standard dose (SD; 40 mA and 120 kVp) or LD (20-30 mA and 120 kVp) using a 128-slice MDCT scanner. The SD cases were matched to the LD cases considering sex, age, level of infiltration, presence of spinal instrumentation, and body diameter. All images were reconstructed using model-based iterative image reconstruction and were evaluated by two readers (R1 and R2) using 5- or 3-point Likert scales (score of 1 reflects the best value per category). Furthermore, noise in imaging data was quantitatively measured by the standard deviation (StDev) of muscle tissue. The dose length product (DLP) was statistically significantly lower for LD scans (6.75 ± 6.43 mGy*cm vs. 10.16 ± 7.70 mGy*cm; p < 0.01; reduction of 33.5%). Image noise was comparable between LD and SD scans (13.13 ± 3.66 HU vs. 13.37 ± 4.08 HU; p = 0.85). Overall image quality was scored as good to very good with only minimal artifacts according to both readers, and determination of the nerve root was possible in almost all patients (LD vs. SD: p > 0.05 for all items). This resulted in high confidence for intervention planning as well as periprocedural intervention guidance for both SD and LD scans. The inter-reader agreement was at least substantial (weighted Cohen's κ ≥ 0.62), except for confidence in intervention planning for LD scans (κ = 0.49). In conclusion, considerable dose reduction for planning and performing periradicular infiltrations with MDCT using model-based iterative image reconstruction is feasible and can be performed without clinically relevant drawbacks regarding image quality or confidence for planning.

Outcome and Safety after 103 Radioembolizations with Yttrium-90 Resin Microspheres in 73 Patients with Unresectable Intrahepatic Cholangiocarcinoma-An Evaluation of Predictors.

Trans-arterial radioembolization (TARE) is increasingly evaluated for unresectable intrahepatic cholangiocarcinoma (ICC). Not all ICC patients benefit equally well from TARE. Therefore, we sought to evaluate variables predicting progression-free survival (PFS) and overall survival (OS). Patients with non-resectable ICC underwent TARE and were treated with 90Y resin microspheres. Baseline characteristics, biochemical/clinical toxicities, and response were examined for impact on PFS and OS. A total of 103 treatments were administered to 73 patients without major complications or toxicity. Mean OS was 18.9 months (95% confidence intervals (CI); 13.9-23.9 months). Mean and median PFS were 10.1 months (95% CI; 7.9-12.2) and 6.4 months (95% CI; 5.20-7.61), respectively. Median OS and PFS were significantly prolonged in patients with baseline cholinesterase (CHE) ≥ 4.62 kU/L (OS: 14.0 vs. 5.5 months; PFS: 6.9 vs. 3.2 months; p < 0.001). Patients with a tumor burden ≤ 25% had a significantly longer OS (15.2 vs. 6.6 months; p = 0.036). Median PFS was significantly longer for patients with multiple TARE cycles (24.4 vs. 5.8 months; p = 0.04). TARE is a considerable and safe option for unresectable ICC. CA-19-9, CHE, and tumor burden have predictive value for survival in patients treated with TARE. Multiple TARE treatments might further improve survival; this has to be confirmed by further studies.

Impact of dose reduction and iterative model reconstruction on multi-detector CT imaging of the brain in patients with suspected ischemic stroke.

Non-contrast cerebral computed tomography (CT) is frequently performed as a first-line diagnostic approach in patients with suspected ischemic stroke. The purpose of this study was to evaluate the performance of hybrid and model-based iterative image reconstruction for standard-dose (SD) and low-dose (LD) non-contrast cerebral imaging by multi-detector CT (MDCT). We retrospectively analyzed 131 patients with suspected ischemic stroke (mean age: 74.2 ± 14.3 years, 67 females) who underwent initial MDCT with a SD protocol (300 mAs) as well as follow-up MDCT after a maximum of 10 days with a LD protocol (200 mAs). Ischemic demarcation was detected in 26 patients for initial and in 64 patients for follow-up imaging, with diffusion-weighted magnetic resonance imaging (MRI) confirming ischemia in all of those patients. The non-contrast cerebral MDCT images were reconstructed using hybrid (Philips "iDose4") and model-based iterative (Philips "IMR3") reconstruction algorithms. Two readers assessed overall image quality, anatomic detail, differentiation of gray matter (GM)/white matter (WM), and conspicuity of ischemic demarcation, if any. Quantitative assessment included signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) calculations for WM, GM, and demarcated areas. Ischemic demarcation was detected in all MDCT images of affected patients by both readers, irrespective of the reconstruction method used. For LD imaging, anatomic detail and GM/WM differentiation was significantly better when using the model-based iterative compared to the hybrid reconstruction method. Furthermore, CNR of GM/WM as well as the SNR of WM and GM of healthy brain tissue were significantly higher for LD images with model-based iterative reconstruction when compared to SD or LD images reconstructed with the hybrid algorithm. For patients with ischemic demarcation, there was a significant difference between images using hybrid versus model-based iterative reconstruction for CNR of ischemic/contralateral unaffected areas (mean ± standard deviation: SD_IMR: 4.4 ± 3.1, SD_iDose: 3.5 ± 2.3, P < 0.0001; LD_IMR: 4.6 ± 2.9, LD_iDose: 3.2 ± 2.1, P < 0.0001).  In conclusion, model-based iterative reconstruction provides higher CNR and SNR without significant loss of image quality for non-enhanced cerebral MDCT.

Evaluation of Visualization Using a 50/50 (Contrast Media/Glucose 5% Solution) Technique for Radioembolization as an Alternative to a Standard Sandwich Technique.

BACKGROUND: Radioembolization (RE) with 90yttrium (90Y) resin microspheres generally employs a sandwich technique with separate sequential administration of contrast medium (CM), followed by vehicle (e.g., glucose 5% [G5] solution), then 90Y resin microspheres (in G5), then G5, and then CM again to avoid contact of CM and microspheres under fluoroscopic guidance. This study evaluates the visualization quality and safety of a modified sandwich technique with a 50/50-mixture of CM (Imeron 300) and G5 for administration of 90Y resin microspheres. MATERIALS AND METHODS: A retrospective analysis of 81 RE procedures in patients with primary or secondary liver tumors was performed. The quality of angiographic visualization of the hepatic vessels was assessed before the first injection and immediately before the whole dose has been injected. Visualization and flow rate were graded on a 5-point scale: 1 = very good to 5 = not visible/no antegrade flow. Univariate logistic regression models and multiple linear regression models were used to evaluate the prognostic variables associated with visualization and flow scores. RESULTS: Visualization quality was inversely related to flow rate, the lower the flow rate the better the grade of the visualization. Visualization quality was also inversely related to body-mass-index (BMI). Performing RE with the 50/50-CM/G5 mixture resulted in a mean injection time for 1 GBq of 15 min. No clinically significant adverse events, including radiation-induced liver disease were reported. CONCLUSION: RE with a 50/50-mixture of CM and G5 for administration of 90Y resin microspheres in a modified sandwich technique is a safe administration alternative and provides good visualization of hepatic vessels, which is inversely dependent on flow rate and BMI. Injection time was reduced compared with our experience with the standard sandwich technique.

Safety, Efficacy, and Prognostic Factors After Radioembolization of Hepatic Metastases from Breast Cancer: A Large Single-Center Experience in 81 Patients.

UNLABELLED: The present study evaluated safety, efficacy, and prognostic factors for (90)Y-yttrium microsphere radioembolization of unresectable liver metastases from breast cancer. METHODS: Eighty-one patients were treated with radioembolization. Acute toxicity was monitored through daily physical examination and serum tests until 3 d after radioembolization; late toxicity was evaluated until 12 wk after radioembolization. Overall survival and response according to (18)F-FDG PET (>30% decrease of tracer uptake) and CA15-3 serum level (any decline) were recorded. Pretherapeutic characteristics, including pretreatment history, liver function tests, and PET/CT parameters, were assessed by univariate and subsequent multivariate Cox regression for predicting patient survival. RESULTS: A toxicity grade of 3 or more based on clinical symptoms, bilirubin, ulcer, pancreatitis, ascites, or radioembolization-induced liver disease occurred in 10% or less of patients. Two patients eventually died from radioembolization-induced liver disease. Sequential lobar treatment and absence of prior angiosuppressive therapy were both associated with a lower rate of serious adverse events. On the basis of PET/CA15-3 criteria, 52/61% of patients responded to treatment. Median overall survival after radioembolization was 35 wk (interquartile range, 41 wk). Pretherapeutic tumor burden of the liver greater than 50% or more (P< 0.001; hazard ratio, 5.67; 95% confidence interval, 2.41-13.34) and a transaminase toxicity grade of 2 or more (P= 0.009; hazard ratio, 2.15; 95% confidence interval, 1.21-3.80) independently predicted short survival. CONCLUSION: Radioembolization for breast cancer liver metastases shows encouraging local response rates with low incidence of serious adverse events, especially in those patients with sequential lobar treatment or without prior angiosuppressive therapy. High hepatic tumor burden and liver transaminase levels at baseline indicate poor outcome.

In vitro study of physical properties of various embolization particles regarding morphology before, during and after catheter passage.

OBJECTIVE: To evaluate various embolization particles on their physical properties with special regard on morphological variability and elasticity. METHODS: 8 embolization particles (EmboCept®, Contour SE® Microspheres, Embosphere® Micorspheres 400 µm, 500 µm, 1300 µm, Embozene® Microspheres, DC Beads®, Embozene Tandem®) were evaluated and graduated from 1-6 microscopically due to morphologic changes in vitro before, during and after their catheter passage by 4 blinded reviewers. To facilitate comparison, microscopic images were provided with a scale. RESULTS: All tested particles showed a homogenous shape and morphology before passage through the simulation catheter. During the passage all particles were elastically deformable, where necessary. After the catheter passage no loss of basic shape was seen. Changes in size were found in 5/8 particles. Grading of morphologic changes varied between mean value of 1.0 and 3.0. No complete destruction or loss of function was seen. CONCLUSION: All tested embolization particles are, regarding their morphological properties in sense of homogenous shape and deformation after catheter passage, a safe treatment option. Tested in vitro no less of functionality regarding physical properties should be expected.

Comparative study of the corrosion behavior of peripheral stents in an accelerated corrosion model: experimental in vitro study of 28 metallic vascular endoprostheses.

PURPOSE: Clinical cases of stent-fractures show that corrosion behavior might play a role in these fractures. Implanted in vivo, especially in combination with other implanted foreign materials, these metallic products are exposed to special conditions, which can cause a process of corrosion. Here, we aimed to test the corrosion potential of stents made of different materials in an in vitro setting. METHODS: A total of 28 peripheral stents of different materials (nitinol, cobalt-chromium-nickel, tantalum, V4A) and surface treatments (electropolish, mechanical polish, no polish) were tested in vitro. Corrosion was accelerated by applying a constant voltage of 3.5 V and amperage of 1.16 mA in 0.9% NaCl. RESULTS: Nitinol stents showed the lowest susceptibility to corrosion and the longest period without damage. The Memotherm II® (BARD Angiomed®) was the only stent that showed neither macroscopic nor microscopic damages. The worst performing material was cobalt-chromium-nickel, which showed corrosion damages about ten times earlier compared to nitinol. Considering the reasons for termination of the test, nitinol stents primarily showed length deficits, while V4A and tantalum stents showed fractures. Cobalt-chromium-nickel stents had multiple fractures or a complete lysis in equal proportions. When placed in direct contact, nitinol stents showed best corrosion resistance, regardless of what material they were combined with. In terms of polishing treatments, electropolished stents performed the best, mechanical-polished stents and those without polishing treatment followed. CONCLUSION: The analysis of corrosion behavior may be useful to select the right stent fulfilling the individual needs of the patient within a large number of different stents.

Fluorescence studies on plasminogen activator inhibitor 1: reactive centre cysteine mutants remain active after fluorophore attachment.

To investigate structural-functional aspects of plasminogen activator inhibitor 1 (PAI-1) we have taken advantage of the lack of cysteines in the PAI-1 molecule and replaced Ser344 (P3) and Asn329 (P18) with cysteine residues, thereby creating unique attachment sites for extrinsic fluorescent probes. After expression in E. coli and purification to homogeneity, both of the mutant proteins were found to have similar biochemical characteristics as wild type PAI-1 (wtPAI-1). Following labelling with 4-chloro-7-nitrobenzofurazan (NBD) and 2-(4'-iodoacetamido-anilino)naphtalene-6-sulfonic acid (IAANS) the mutant inhibitors showed similar inhibitory activities and heat stability as wtPAI-1. The purified complex between uPA and NBD-labelled P3cys mutant was found to be extremely stable, suggesting that no slow cleavage or reversible reaction occurs in complexes that have been properly formed. The rate of labelling of both mutants was decreased when the mutants were in the latent form indicating that these cysteine residues may be less accessible in the latent configuration. The PAI-1 mutants labelled with both NBD and IAANS could convert from the active to the latent form, but P3cys labelled with the larger IAANS chromophore showed a two fold decrease in the rate of conversion to latency, suggesting that a large chromophore in the P3 position may interfere with the active to latent conversion. The fluorescence spectra of the two NBD labelled mutants were similar, but the intensity was three times higher for the P3cys mutant than for P18cys. No significant spectral changes could be seen when the P3cys mutant was transferred to latency. In contrast, the P18cys mutant showed a major change in the excitation spectra characteristic of migration of the NBD chromophore from a thiol to an amine. Complex formation with uPA had no effect on the fluorescence spectrum of P18cys-NBD while the spectrum of P3cys-NBD revealed changes consistent with a restriction of the mobility of NBD probe in the uPA-PAI-1 complex.

Conformational studies of plasminogen activator inhibitor type 1 by fluorescence spectroscopy. Analysis of the reactive centre of inhibitory and substrate forms, and of their respective reactive-centre cleaved forms.

The inhibitors that belong to the serpin family are suicide inhibitors that control the major proteolytic cascades in eucaryotes. Recent data suggest that serpin inhibition involves reactive centre cleavage followed by loop insertion, whereby the covalently linked protease is translocated away from the initial docking site. However under certain circumstances, serpins can also be cleaved like a substrate by target proteases. In this report we have studied the conformation of the reactive centre of plasminogen activator inhibitor type 1 (PAI-1) mutants with inhibitory and substrate properties. The polarized steady-state and time-resolved fluorescence anisotropies were determined for BODIPY(R) probes attached to the P1' and P3 positions of the substrate and active forms of PAI-1. The fluorescence data suggest an extended orientational freedom of the probe in the reactive centre of the substrate form as compared to the active form, revealing that the conformation of the reactive centres differ. The intramolecular distance between the P1' and P3 residues in reactive centre cleaved inhibitory and substrate mutants of PAI-1, were determined by using the donor-donor energy migration (DDEM) method. The distances found were 57+/-4 A and 63+/-3 A, respectively, which is comparable to the distance obtained between the same residues when PAI-1 is in complex with urokinase-type plasminogen activator (uPA). Following reactive centre cleavage, our data suggest that the core of the inhibitory and substrate forms possesses an inherited ability of fully inserting the reactive centre loop into beta-sheet A. In the inhibitory forms of PAI-1 forming serpin-protease complexes, this ability leads to a translocation of the cognate protease from one pole of the inhibitor to the opposite one.

Time-resolved polarized fluorescence spectroscopy studies of plasminogen activator inhibitor type 1: conformational changes of the reactive center upon interactions with target proteases, vitronectin and heparin.

Plasminogen activator inhibitor type 1 (PAI-1) is an important physiological inhibitor of the plasminogen activator system. To investigate the structure-functional aspects of this inhibitor, we have taken advantage of the lack of cysteine residues in the PAI-1 molecule and substituted Ser344 (P3) and Met347 (P1'), in the reactive center loop, with cysteines, thereby creating unique attachment sites for extrinsic fluorescent probe. Both cysteine mutants were purified and labeled with a sulfhydryl specific fluorophore, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacen yl-3-propionyl)-N- (iodoacetyl)ethylenediamine (BDYIA). The labeled mutants were found to reveal biochemical characteristics very similar to those of wild type PAI-1. Time-resolved fluorescence spectroscopy was used to examine orientational freedom of BDYIA in the reactive center loop of PAI-1. The orientational freedom of the probe was found to be greater in the latent form than in the active form of PAI-1, suggesting that the reactive center has a more relaxed conformation in the latent form than in the active form. Complex formation with target proteases, tissue type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA), caused decreased orientational freedom of BDYIA in the P3 position, while the orientational freedom of BDYIA in position P1' increased to a level similar to that of BDYIA in reactive center-cleaved PAI-1. In contrast, complex formation with modified anhydro-uPA, which is unable to cleave its substrate, largely restricted the orientational freedom of BDYIA probe in the P1' position.(ABSTRACT TRUNCATED AT 250 WORDS)

Polarized fluorescence and absorption spectroscopy of 1,32-dihydroxy-dotriacontane-bis-rhodamine 101 ester. A new and lipid bilayer-spanning probe.

We report on the properties of 1,32-dihydroxy-dotriacontane-bis-rhodamine 101 ester (Rh101C32Rh101) in lipid bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and in liquid solvents. The results are compared with those of rhodamine 101 octadecanyl ester (Rh101C18). Both molecules are solubilized in the lipid bilayer and the Rh101 moieties are anchored in the lipid-water interface, so that the electronic transition dipole moments (S 0 ↔S 1) are oriented preferentially in the plane of the bilayer. At low concentrations of the dyes in lipid bilayers of DOPC, the fluorescence relaxation is single exponential with a lifetime of τ=4.9±0.2 ns. The relative fluorescence quantum yield of ΦC32/ΦC18 ≈ 0.95 in DOPC vesicles. These results strongly suggest that only a small fraction of the Rh101C32Rh101 molecules are quenched, by, for example, intra- or intermolecular dimers in the ground state at mole fractions of less than 0.1% in the lipid bilayers. For Rh101C32Rh101 in lipid vesicles, the steady-state and time-resolved fluorescence anisotropies are compatible with efficient intramolecular electronic energy transfer. It is concluded that nearly every Rh101C32Rh101 molecule is spanning across the lipid bilayer of DOPC.

Chloride-sensitive fluorescent indicators.

Three fluorescent halide-sensitive quinolinium dyes have been produced by the reaction of the 6-methylquinoline heterocyclic nitrogen base with methyl bromide, methyl iodide, and 3-bromo-1-propanol. The quaternary salts, unlike the precursor molecule, are readily water soluble and the fluorescence intensity of these salts is reduced in the presence of aqueous chloride, bromide, and iodide ions, allowing halide solution concentrations to be determined using well-known Stern-Volmer kinetics. One of the dyes, dye 1, has a chloride Stern-Volmer constant of 255 mol(-1) dm(3) which is more than twice that of SPQ [6-methoxy-N-(3-sulfopropyl)quinolinium] used in recent physiological measurements to measure intracellular chloride levels. The dyes have been characterized using steady-state fluorescence spectroscopy and are compared to three similar dyes based on the 6-methoxyquinoline nucleus, reported earlier by the authors, and also to dyes reported by Krapf et al. (Anal. Biochem. 169, 142-150, 1988). The interference of aqueous anions and the potential for using these dyes in biological halide-sensing applications are discussed.

Donor-donor energy migration for determining intramolecular distances in proteins: I. Application of a model to the latent plasminogen activator inhibitor-1 (PAI-1).

A new fluorescence spectroscopic method is presented for determining intramolecular and intermolecular distances in proteins and protein complexes, respectively. The method circumvents the general problem of achieving specific labeling with two different chromophoric molecules, as needed for the conventional donor-acceptor transfer experiments. For this, mutant forms of proteins that contain one or two unique cysteine residues can be constructed for specific labeling with one or two identical fluorescent probes, so-called donors (d). Fluorescence depolarization experiments on double-labeled Cys mutant monitor both reorientational motions of the d molecules, as well as the rate of intramolecular energy migration. In this report a model that accounts for these contributions to the fluorescence anisotropy is presented and experimentally tested. Mutants of a protease inhibitor, plasminogen activator inhibitor type-1 (PAI-1), containing one or two cysteine residues, were labeled with sulfhydryl specific derivatives of 4,4-difluoro-4-borata-3a-azonia-4a-aza-s-indacence (BODIPY). From the rate of energy migration, the intramolecular distance between the d groups was calculated by using the Forster mechanism and by accounting for the influence of local anisotropic orientation of the d molecules. The calculated intramolecular distances were compared with those obtained from the crystal structure of PAI-1 in its latent form. To test the stability of parameters extracted from experiments, synthetic data were generated and reanalyzed.