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Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants in the same venom. We showed that V. lebetina venom contains: (1) proteases that degrade fibrinogen, but not fibrin; (2) fibrinolytic enzyme (lebetase); (3) factor X activator (VLFXA); (4) factor V activator (VLFVA). Fibrinolytic enzyme and VLFXA are metalloproteases; the other studied enzymes are serine proteases. α-Fibrinogenase has no homolog among known serine proteases. β-Fibrinogenase is a typical thermostable arginine esterase that hydrolyzes esters and amides of arginine and attacks the β-chain of fibrinogen. Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins. We used the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique for the recovery and identification of peptides released by protease hydrolysis and for the detection of human factor X cleavage products after VLFXA hydrolysis. VLFXA cleaves the Arg52-Ile53 bond in the heavy chain of human factor X and the Arg226-Val227 bond in human factor IX precursor; VLFVA cleaves Arg1545-Ser1546 in factor V.

A penalized weighted least squares (PWLS) reconstruction algorithm is described that simultaneously estimates activity and attenuation distributions from emission sinogram data alone. This estimation technique is based on differential attenuation information and is applicable to any SPECT imaging isotope with emissions at two or more distinct energies, after compensating for Compton scatter. A rotation-based forward projector is used to efficiently model photon attenuation at multiple emission energies, as well as distance-dependent spatial resolution. The algorithm was tested using simulated scatter-free /sup 201/Tl projection data from a single-slice numerical cardiac phantom with a large cold myocardial defect. Poisson noise was added to the projection data to mimic clinically realistic count densities. The activity estimates resulting from the proposed method had fewer artifacts and were substantially more accurate than images reconstructed with filtered backprojection without compensation for attenuation. Several techniques were employed to reduce the time required for the iterative routine to converge and to improve the stability of the solution, including: (1) a pre-conditioning image variable transformation; (2) a coarse-to-fine grid initialization schedule; and (3) a convex hull image mask determined directly from the projection data. The combined effect of these techniques was a reduction in computation time by a factor of /spl sim/200.

The AIM of this study was to determine the clinical relevance of compensating SPECT data for patient specific attenuation by the use of CT data simultaneously acquired with SPECT/CT when analyzing the skeletal uptake of polyphosphonates (DPD). Furthermore, the influence of misregistration between SPECT and CT data on uptake ratios was investigated.Thirty-six data sets from bone SPECTs performed on a hybrid SPECT/CT system were retrospectively analyzed. Using regions of interest (ROIs), raw counts were determined in the fifth lumbar vertebral body, its facet joints, both anterior iliacal spinae, and of the whole transversal slice. ROI measurements were performed in uncorrected (NAC) and attenuation-corrected (AC) images. Furthermore, the ROI measurements were also performed in AC scans in which SPECT and CT images had been misaligned by 1 cm in one dimension beforehand (ACX, ACY, ACZ).After AC, DPD uptake ratios differed significantly from the NAC values in all regions studied ranging from 32% for the left facet joint to 39% for the vertebral body. AC using misaligned pairs of patient data sets led to a significant change of whole-slice uptake ratios whose differences ranged from 3,5 to 25%. For ACX, the average left-to-right ratio of the facet joints was by 8% and for the superior iliacal spines by 31% lower than the values determined for the matched images (p0.05).AC significantly affects DPD uptake ratios. Furthermore, misalignment between SPECT and CT may introduce significant errors in quantification, potentially also affecting left-to-right ratios. Therefore, at clinical evaluation of attenuation-corrected scans special attention should be given to possible misalignments between SPECT and CT.

Snake venoms contain four classes of metalloproteases that all have a typical zinc-chelating sequence (HEXXHGXXH). N-terminal sequences and internal sequences of different purified metalloproteases were determined using Edman sequencing and LC MS/MS technique. Oligonucleotides were designed and used as primers for cDNA cloning from Vipera lebetina venom gland cDNA library. We found that isoforms of fibrinolytic enzyme lebetase Le-4 and Le-3 are synthesized in different way: Le-4 is synthesized as P-I type metalloprotease, Le-3 is synthesized with disintegrin-like domain as P-II type protease and processed post-translationally. An endothelial cell apoptosis-inducing heterodimeric glycosylated metalloprotease, V. lebetina apoptosis-inducing protease (VLAIP), belongs to P-III type containing metalloprotease, disintegrin-like and cysteine-rich domains. All these enzymes hydrolyze the Aalpha-chain and more slowly the Bbeta-chain of fibrinogen. Treatment of HUVEC cells with VLAIP induces changes in the attachment of cells to the substrate and causes apoptosis. V. lebetina venom contains also P-IV type-specific coagulant factor X activator (VLFXA) that cleaves the Arg52-Ile53 bond in the heavy chain of human factor X. VLFXA is a glycoprotein composed of a heavy chain and two C-type lectin-like light chains linked by disulfide bonds. The heavy and light chains of VLFXA are synthesized from different genes.

The Pixon method, a statistically rigorous procedure for adaptive noise suppression that avoids the generation of spurious artifacts yet preserves all the statistically justifiable image features resident in the raw counts, is applied to nuclear studies. The present work focuses on adaptive postsmoothing and sharpening of OSEM-reconstructed data at various count levels, with the ultimate goals to (i) increase sensitivity for detection of lesions of small size and/or of small activity-to-background ratio, (ii) reduce data acquisition time, and (iii) reduce patient dose. We use simulated and measured data and human-observer studies, which are analyzed using quantitative measures. The detectability shows improvement, as does resolution, especially at low counts. Clinical trials would be required to assess this method of image postprocessing

A new transmission source-based attenuation correction system, c.clear, has been developed for the Siemens c.cam cardiac SPECT camera. The transmission sources are two arrays of 14 Gd-153 line sources placed in cassette wings affixed to the detectors. The source strengths decrease geometrically away from the array center. The absorbed radiation dose received from these sources is about 0.01% of that received from a conventional Tl-201 myocardial scan. The line sources are stationary relative to the detector during the SPECT scan. The emission and transmission radiation are acquired simultaneously and are inherently registered spatially and temporally, so avoiding misregistration artifacts. Transmission reconstruction is performed using an ordered subset maximum likelihood algorithm with gradient descent. It models the geometry of the sources and the radiation emanating from these sources as narrow fan beams. The parameters of this model are estimated from near and far blank calibration images. It includes correction for the Compton emission down-scatter into the transmission energy window that occurs during the simultaneous Tc-99m and Gd-153 imaging. The down-scatter is estimated from down-scatter measured for a short interval when the line sources are obstructed by shutters in the cassette wings. The down-scatter is estimated by linear filtering using the measured short-interval down-scatter and the emission data. The transverse FOV of the camera can truncate the transmission radiation, particularly for obese or improperly positioned patients. Compensation for such truncations is achieved by employing robust data extrapolation techniques and extending the model of the source geometry beyond the measured FOV. This attenuation correction method has been investigated using both phantom and clinical studies and has been shown to produce corrected emission reconstructions of excellent quality.

The Differential Attenuation Method (DAM) was developed to simultaneously estimate activity and attenuation distributions from multi-emission projection data alone. Previously, DAM was shown to improve the quality and quantitative accuracy compared with filtered-backprojection without attenuation compensation. Here, that work is extended by comparing the performance of DAM to an iterative penalized-weighted least-squares reconstruction with: (1) uniform attenuation, and (2) the true attenuation. A single-slice numerical torso phantom was used to simulate data from /sup 201/Tl cardiac studies. Noise-free projections and data with additive Poisson-distributed noise were simulated with and without a myocardial perfusion defect. Scatter was not simulated. Images reconstructed with DAM from data at realistic count densities were more accurate than those reconstructed with an assumed uniform attenuation distribution, and nearly as accurate as those obtained using the true attenuation distribution.

The Differential Attenuation Method (DAM) simultaneously estimates SPECT activity and attenuation distributions from emission data. In this work, we extend the investigation of DAM to measured data and compare the results obtained with FBP without compensating for attenuation, and Penalized Weighted Least Squares (PWLS) with uniform and no attenuation. An anthropomorphic torso phantom with a heart insert containing a myocardial defect filled with /sup 111/In is used to acquire data on a GE VG tomograph. An accurate scatter correction before reconstruction is crucial. Scatter is subtracted with an Energy Window Based Subtraction method (EWBS), with positions and widths of the energy windows selected using Monte Carlo simulated source distributions. The results show the potential benefits of DAM as the activity estimate has fewer artifacts and is more accurate than FBP without compensating for attenuation or PWLS with uniform attenuation. However, artifacts remain and additional constraints on the allowed attenuation values were required to produce acceptable images.

Our studies of the venom from the Levantine viper Vipera lebetina have demonstrated the existence of both coagulants and anticoagulants in the same venom. We showed that V. lebetina venom contains: (1) proteases that degrade fibrinogen, but not fibrin; (2) fibrinolytic enzyme (lebetase); (3) factor X activator (VLFXA); (4) factor V activator (VLFVA). Fibrinolytic enzyme and VLFXA are metalloproteases; the other studied enzymes are serine proteases. alpha-Fibrinogenase has no homolog among known serine proteases. Beta-fibrinogenase is a typical thermostable arginine esterase that hydrolyzes esters and amides of arginine and attacks the beta-chain of fibrinogen. Lebetase is a direct-acting fibrinolytic zinc metalloendopeptidase related in amino acid sequence to reprolysins. We used the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique for the recovery and identification of peptides released by protease hydrolysis and for the detection of human factor X cleavage products after VLFXA hydrolysis. VLFXA cleaves the Arg(52)-Ile(53 )bond in the heavy chain of human factor X and the Arg(226)-Val(227) bond in human factor IX precursor; VLFVA cleaves Arg(1545)-Ser(1546) in factor V.

The 1H, 13C and 15N NMR spectra of α−, β− and γ-amanitins and amaninamide were recorded at 11.7 T and analysed by various two-dimensional (2D) NMR spectroscopic techniques. All diastereotopic protons were assigned, and the side-chain conformations and the conformation of the hydroxyproline residue were determined. The observed 15N chemical shifts are caused by non-bonded interactions of the peptide nitrogens. 1H, 13C and 15N data are complementary to each other and show that the conformation of β-amanitin is slightly different from that of the other isomers, in accordance with the lower toxicity. The NOESY and carbonyl 13C1H chemical shift diagrams confirm the closeness of the conformations of amanitins in DMSO solution to that of β-amanitin in the crystalline state.