Perdeuteration: improved visualization of solvent structure in neutron macromolecular crystallography.

The 1.8 Šresolution neutron structure of deuterated type III antifreeze protein in which the methyl groups of leucine and valine residues are selectively protonated is presented. Comparison between this and the 1.85 Šresolution neutron structure of perdeuterated type III antifreeze protein indicates that perdeuteration improves the visibility of solvent molecules located in close vicinity to hydrophobic residues, as cancellation effects between H atoms of the methyl groups and nearby heavy-water molecules (D2O) are avoided.

[Multislice CT of the pelvis: dose reduction with regard to image quality]. / Mehrzeilen-Spiral CT (MSCT) des Beckenskelettes: Dosisoptimierung unter Berücksichtigung der Bildqualität.

PURPOSE: To optimize the examination protocols of multislice CT (MSCT) of the pelvis for dose reduction with regard to image quality. MATERIALS AND METHODS: MSCT of the pelvis was performed on 5 cadaver specimens with stepwise reduction of the tube current at 140 kV (250, 200, 150, 100, 75, 50, 35, 25 mAs) and 120 kV (50, 27 mAs). The remaining scan parameters like collimation (4 x 1 mm) and table speed of 4 mm per rotation remained unchanged. Axial sections and coronal reconstructions were used to evaluate cortex, trabecular structures, subjective image quality, image noise and detail detectability (pelvis and SI joint), with evaluation performed independently by four blinded experienced radiologists on a 5-point scale. Kappa coefficient, accuracy of the observers to sort the films with regard to dose reduction and mean scores of image evaluation were determined for statistical analysis. RESULTS: The deterioration of the image quality was most pronounced for all criteria between 75 mAs and 50 mAs at 140 kV. Good results with adequate image quality were obtained for detail detectability at 50 mAs and 140 kV (effective dose [E]: 3.3 mSv) and for the remaining four criteria at 75 mAs (E: 4.9 mSv). There was a moderate agreement between the four observers (kappa coefficient: 0.27). All observers were excellent in arranging the images according to the increasing dose reduction. CONCLUSIONS: Image quality of MSCT of the pelvis appears to be acceptable at 75 mAs and 140 kV with the dose reduced to 46% of the average value of the nation-wide survey of the German Roentgen Society in 1999 for this type of examination.

[Marfan syndrome: pathogenesis, phenotypes and diagnostic value, of various imaging techniques]. / Das Marfan-Syndrom: Pathogenese, Phänotypien und Stellenwert bildgebender Verfahren.

Marfan syndrome is a genetic disorder with autosomal dominant inheritance. It is caused by mutations in the fibrillin-1 gene and leads to different disease manifestations. Seventy-five percent of the affected individuals develop an aneurysm of the ascending aorta, 41 % suffer from aortic dissections, and 93 % die of cardiovascular diseases. Skeletal changes occur in two-thirds of the patients, and lens dislocation is observed in 60 to 80 %. Without treatment, the life expectancy is 32 +/- 16 years. However, Marfan patients can live up to 60 years if they receive optimal therapy. Early diagnosis of the disease and it 's life-threatening sequelae is the prerequisite for early therapy. Radiologic diagnostic techniques are of pivotal importance in this context as they allow the identification of major and minor disease manifestations and the detection of severe dilatations and aortic dissections at an early stage. This overview describes the radiologically detectable multiple changes seen in Marfan syndrome and explains the diagnostic value of various imaging techniques in the diagnosis and therapy of Marfan syndrome.

[Detection of liver lesions with gadolinium-enhanced VIBE sequence in comparison with SPIO-enhanced MRI]. / Detektion von Leberläsionen mit der Gadolinium-verstärkten VIBE-Sequenz im Vergleich zur SPIO-verstärkten MRT.

PURPOSE: Detection of malignant liver lesions with gadolinium-enhanced volumetric interpolated breath-hold examination (VIBE) in comparison with SPIO-enhanced MRT (four different SPIO-enhanced T2w sequences) and histological and imaging follow-up in non-cirrhotic livers at 1.5 T. PATIENTS AND METHODS: Twenty-two patients with suspected focal liver lesions prospectively underwent a gadolinium-enhanced 3D VIBE. Four T2w sequences (HASTE sequence, fast spin-echo-sequence with and without fat-saturation, FLASH 2D gradient echo-sequence) after administration of superparamagnetic iron oxide (SPIO) served as gold standard combined with histological work-up in 17 patients and histological and imaging follow-up in five patients. The image quality was evaluated and the detectability of intrahepatic lesions was rated by the alternative free-response receiver operating characteristic (AFROC) analysis. In addition, the contrast-to-noise ratio was compared. RESULTS: Altogether 49 malignant and 35 benign liver lesions were found. Concerning the image quality, VIBE turned out to be of slightly poorer image quality than the SPIO-enhanced examination with HASTE sequence (4.95 vs. 5.0). The fast spin-echo-sequence without fat-saturation demonstrated the highest contrast-to-noise ratio. All sequences showed a comparable certainty in detecting lesion (area under the curve 0.68-0.73) and identifying malignant liver lesions. CONCLUSION: Despite the small number of patients, VIBE seems to be a comparable, inexpensive and fast method in diagnosing malignant liver lesions.

[The role of angiography in the diagnosis and therapy of gastrointestinal hemorrhage]. / Stellenwert der Angiographie in der Diagnose und Therapie gastrointestinaler Blutungen.

PURPOSE: Evaluation of angiography in the diagnosis and treatment of acute and chronic gastrointestinal bleeding. MATERIALS AND METHODS: Thirty-seven consecutive patients with clinically suspected gastrointestinal bleeding underwent selective angiography of the visceral arteries. If technically possible, patients with radiologically proven hemorrhage underwent selective embolization with microcoils. Not all angiographically detected bleeding vessels were embolized. Clinical outcome was determined from a review of the medical records. RESULTS: 37 patients underwent 43 angiographies, which demonstrated 25 bleedings considered acute and 18 considered chronic. Seventeen patients had previous intestinal surgery. In 9 of the 37 patients, 12 of the 43 (sensitivity: 28 %) hemorrhages were detected. A postoperative bleeding was found in 6 patients. With one exception, all angiographically positive cases were clinically considered to be acute. Transcatheter embolization of the bleeding vessels with microcoils was performed in 6 patients, with 3 patients having the bleeding stopped permanently and 3 patients requiring further surgical treatment. In 3 patients, embolization was not possible for technical reasons. No complications attributable to angiography were seen. CONCLUSION: Angiography should be performed in patients with spontaneous and postoperative gastrointestinal bleeding. Transcatheter embolization is an effective and safe therapeutical option in both clinical settings.

Characterization of the EYE2 gene required for eyespot assembly in Chlamydomonas reinhardtii.

The unicellular biflagellate green alga Chlamydomonas reinhardtii can perceive light and respond by altering its swimming behavior. The eyespot is a specialized structure for sensing light, which is assembled de novo at every cell division from components located in two different cellular compartments. Photoreceptors and associated signal transduction components are localized in a discrete patch of the plasma membrane. This patch is tightly packed against an underlying sandwich of chloroplast membranes and carotenoid-filled lipid granules, which aids the cell in distinguishing light direction. In a prior screen for mutant strains with eyespot defects, the EYE2 locus was defined by the single eye2-1 allele. The mutant strain has no eyespot by light microscopy and has no organized carotenoid granule layers as judged by electron microscopy. Here we demonstrate that the eye2-1 mutant is capable of responding to light, although the strain is far less sensitive than wild type to low light intensities and orients imprecisely. Therefore, pigment granule layer assembly in the chloroplast is not required for photoreceptor localization in the plasma membrane. A plasmid-insertion mutagenesis screen yielded the eye2-2 allele, which allowed the isolation and characterization of the EYE2 gene. The EYE2 protein is a member of the thioredoxin superfamily. Site-directed mutagenesis of the active site cysteines demonstrated that EYE2 function in eyespot assembly is redox independent, similar to the auxiliary functions of other thioredoxin family members in protein folding and complex assembly.

Structures of l-fuculose-1-phosphate aldolase mutants outlining motions during catalysis.

The crystal structures of l-fuculose-1-phosphate aldolase (FucA) with and without a ligated analogue of dihydroxyacetone phosphate (DHAP) and of a number of active center mutants have resulted in a model of the catalytic mechanism. This model has now been confirmed by structural analyses of further mutations at the zinc coordination sphere and at the phosphate site. In addition, these mutants have revealed new aspects of the catalysis: the hydroxyl group of Tyr113' (from a neighboring subunit), which sits just outside the zinc coordination sphere, steers DHAP towards a productive binding mode at the zinc ion; Glu73 contacts zinc in between the two ligand positions intended for the DHAP oxygen atoms and thus avoids blocking of these positions by a tetrahedrally coordinated hydroxy ion; the FucA polypeptide does not assume its minimum energy state but oscillates between two states of elevated energy as demonstrated by a mutant in a minimum energy state. The back and forth motion involves a mobile loop connecting the phosphate site with intersubunit motions and thus with the Brownian motion of the solvent. The phosphate group is bound strongly at a given distance to the zinc ion, which prevents the formation of too tight a DHAP:zinc complex. This observation explains our failure to find mutants that accept phosphate-free substitutes for DHAP. The FucA zinc coordination sphere is compared with that of carbonic anhydrase.

Eyespot-assembly mutants in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a single-celled green alga that phototaxes toward light by means of a light-sensitive organelle, the eyespot. The eyespot is composed of photoreceptor and Ca(++)-channel signal transduction components in the plasma membrane of the cell and reflective carotenoid pigment layers in an underlying region of the large chloroplast. To identify components important for the positioning and assembly of a functional eyespot, a large collection of nonphototactic mutants was screened for those with aberrant pigment spots. Four loci were identified. eye2 and eye3 mutants have no pigmented eyespots. min1 mutants have smaller than wild-type eyespots. mlt1(ptx4) mutants have multiple eyespots. The MIN1, MLT1(PTX4), and EYE2 loci are closely linked to each other; EYE3 is unlinked to the other three loci. The eye2 and eye3 mutants are epistatic to min1 and mlt1 mutations; all double mutants are eyeless. min1 mlt1 double mutants have a synthetic phenotype; they are eyeless or have very small, misplaced eyespots. Ultrastructural studies revealed that the min1 mutants are defective in the physical connection between the plasma membrane and the chloroplast envelope membranes in the region of the pigment granules. Characterization of these four loci will provide a beginning for the understanding of eyespot assembly and localization in the cell.

Nonsurgical reconstruction of thoracic aortic dissection by stent-graft placement.

BACKGROUND: The treatment of thoracic aortic dissection is guided by prognostic and anatomical information. Proximal dissection requires surgery, but the appropriate treatment of distal thoracic aortic dissection has not been determined, because surgery has failed to improve the prognosis. METHODS: We prospectively evaluated the safety and efficacy of elective transluminal endovascular stent-graft insertion in 12 consecutive patients with descending (type B) aortic dissection and compared the results with surgery in 12 matched controls. In all 24 patients, aortic dissection was diagnosed by magnetic resonance angiography. In each group, the dissection involved the aortic arch in 3 patients and the descending thoracic aorta in all 12 patients. With the patient under general anesthesia, either surgical resection was undertaken or a custom-designed endovascular stent-graft was placed by unilateral arteriotomy. RESULTS: Stent-graft placement resulted in no morbidity or mortality, whereas surgery for type B dissection was associated with four deaths (33 percent, P=0.09) and five serious adverse events (42 percent, P=0.04) within 12 months. Transluminal placement of the stent-graft prosthesis was successful in all patients, with no leakage; full expansion of the stents was ensured by balloon inflation at 2 to 3 atm. Sealing of the entry tear was monitored during the procedure by transesophageal ultrasonography and angiography, and thrombosis of the false lumen was confirmed in all 12 patients after a mean of three months by magnetic resonance imaging. There were no deaths or instances of paraplegia, stroke, embolization, side-branch occlusion, or infection in the stent-graft group; nine patients had postimplantation syndrome, with transient elevation of C-reactive protein levels and body temperature plus mild leukocytosis. All the patients who received stent-grafts recovered, as did seven patients who underwent surgery for type B dissection (58 percent) (P=0.04). CONCLUSIONS: These preliminary observations suggest that elective, nonsurgical insertion of an endovascular stent-graft is safe and efficacious in selected patients who have thoracic aortic dissection and for whom surgery is indicated. Endoluminal repair may be useful for interventional reconstruction of thoracic aortic dissection.

Suppressor analysis of mutations in the 5'-untranslated region of COB mRNA identifies components of general pathways for mitochondrial mRNA processing and decay in Saccharomyces cerevisiae.

The cytochrome b gene in Saccharomyces cerevisiae, COB, is encoded by the mitochondrial genome. Nuclear-encoded Cbp1 protein is required specifically for COB mRNA stabilization. Cbp1 interacts with a CCG element in a 64-nucleotide sequence in the 5'-untranslated region of COB mRNA. Mutation of any nucleotide in the CCG causes the same phenotype as cbp1 mutations, i.e., destabilization of both COB precursor and mature message. In this study, eleven nuclear suppressors of single-nucleotide mutations in CCG were isolated and characterized. One dominant suppressor is in CBP1, while the other 10 semidominant suppressors define five distinct linkage groups. One group of four mutations is in PET127, which is required for 5' end processing of several mitochondrial mRNAs. Another mutation is linked to DSS1, which is a subunit of mitochondrial 3' --> 5' exoribonuclease. A mutation linked to the SOC1 gene, previously defined by recessive mutations that suppress cbp1 ts alleles and stabilize many mitochondrial mRNAs, was also isolated. We hypothesize that the products of the two uncharacterized genes also affect mitochondrial RNA turnover.

[Intramural hemorrhage of the thoracic aorta]. / Intramurale Hämorrhagie der thorakalen Aorta.

PURPOSE: Intramural hemorrhage (IMH) of the aorta is considered to be a possible preliminary stage to dissection. We report on our experience with MRI, CT, and TEE in the diagnosis as well as in the correct evaluation of the extent of an IMH. METHOD: Between 1983 and 1997 a total of 26 patients were examined using MRI (n = 13) and/or contrast enhanced CT (n = 18) or TEE. The validation was effected in 17 cases through intraoperative or postmortem inspection and in the remaining cases either through continuous clinical or radiological monitoring (n = 6) or through the depiction of the results in 2 different imaging procedures (n = 3). RESULTS: The correct diagnosis of an IMH was made on all examined patients with all imaging modalities. In all cases, MRI enabled the correct identification of the affected aortic segments. Using CT the affected descending aorta was overlooked in one out of 15 cases; TEE failed in one out of two cases of intramural hemorrhage in the aortic arch. A false positive diagnosis was made with CT in one out of 9 cases in the ascending aorta, and using sonography, this occurred in two out of 3 cases in the area of the aortic arch. CONCLUSION: According to our experience, magnetic resonance imaging is the most accurate method for the diagnosis of an IMH.

Regulation of poly(A) site choice of several yeast mRNAs.

Several yeast genes produce multiple transcripts with different 3'-ends. Of these, four genes are known to produce truncated transcripts that end within the coding sequence of longer transcripts: CBP1 , AEP2 / ATP13 , RNA14 and SIR1 . It has been shown that the level of the truncated CBP1 transcript increases during the switch to respiratory growth while that of the full-length transcript decreases. To determine whether this phenomenon is unique to CBP1 , northern analysis was used to determine whether the levels of other truncated transcripts are regulated similarly by carbon source. The levels of the shortest transcripts of AEP2 / ATP13 and RNA14 increased during respiration while the shortest SIR1 transcript remained constant. However, two longer SIR1 transcripts were regulated reciprocally by carbon source. Mapping the 3'-ends of each transcript by sequencing partial cDNA clones revealed multiple 3'-ends for each transcript. Examination of the sequences surrounding the 3'-ends of the induced transcripts failed to identify a consensus sequence but did reveal weak putative 3'-end formation signals in all of the transcripts. Similarly, no consensus sequence was found when the sequences surrounding the 3'-ends of the longest transcripts were compared, but again weak putative 3'-end formation signals were identified. These data are suggestive of carbon source regulation of alternative poly(A) site choice in yeast.

[Marfan syndrome: diagnosis of cardiovascular manifestations]. / Das Marfan-Syndrom: Diagnostik der kardiovaskulären Manifestationen.

With the availability of modern imaging modalities such as magnetic resonance imaging (MRI), contrast-enhanced computed tomography (CT), and transesophageal echocardiography (TEE), diagnostic options in the evaluation of Marfan patients have been improved profoundly. The most recent diagnostic advances comprise noninvasive tissue characterization of the aortic wall, immunofluorescence studies or pulse chase analyses of skin and cultured dermal fibroblasts, and molecular analysis at the cDNA/DNA level of the fibrillin-1 gene. New diagnostic insights have eventually led to revised nosologic criteria for the diagnosis of Marfan syndrome. The diagnostic reliability as well as the advantage and limitation of these recent diagnostic strategies are discussed; moreover diagnostic concepts for patients with neonatal as well as classic Marfan syndrome are presented and discussed in the context of the clinical management during adolescence, gravidity as well as in the pre- and postoperative patient.

Genetic evidence for interaction between Cbp1 and specific nucleotides in the 5' untranslated region of mitochondrial cytochrome b mRNA in Saccharomyces cerevisiae.

The cytochrome b (COB) gene is encoded by the mitochondrial genome; however, its expression requires the participation of several nuclearly encoded protein factors. The yeast Cbp1 protein, which is encoded by the nuclear CBP1 gene, is required for the stabilization of COB mRNA. A previous deletion analysis identified an 11-nucleotide-long sequence within the 5' untranslated region of COB mRNA that is important for Cbp1-dependent COB mRNA stability. In the present study, site-directed mutagenesis experiments were carried out to define further the features of this cis element. The CCG sequence within this region was shown to be necessary for stability. A change in residue 533 of Cbp1 from aspartate to tyrosine suppresses the effects of a single-base change in the CCG element. This is strong genetic evidence that the nuclearly encoded Cbp1 protein recognizes and binds directly to the sequence containing CCG and thus protects COB mRNA from degradation.

Premature 3'-end formation of CBP1 mRNA results in the downregulation of cytochrome b mRNA during the induction of respiration in Saccharomyces cerevisiae.

The yeast mitochondrial genome encodes only seven major components of the respiratory chain and ATP synthase; more than 200 other mitochondrial proteins are encoded by nuclear genes. Thus, assembly of functional mitochondria requires coordinate expression of nuclear and mitochondrial genes. One example of coordinate regulation is the stabilization of mitochondrial COB (cytochrome b) mRNA by Cbp1, the product of the nuclear gene CBP1 (cytochrome b processing). CBP1 produces two types of transcripts with different 3' ends: full-length 2.2-kb transcripts and 1.2-kb transcripts truncated within the coding sequence of Cbp1. Upon induction of respiration, the steady-state level of the long transcripts decreases while that of the short transcripts increases reciprocally, an unexpected result since the product of the long transcripts is required for COB mRNA stability and thus for respiration. Here we have tested the hypothesis that the short transcripts, or proteins translated from the short transcripts, are also required for respiration. A protein translated from the short transcripts was not detected by Western analysis, although polysome gradient fractions were shown to contain both long and short CBP1 transcripts. A mutant strain in which production of the short transcripts was abolished showed wild-type growth properties, indicating that the short transcripts are not required for respiration. Due to mutation of the carbon source-responsive element, the long transcript level in the mutant strain did not decrease during induction of respiration. The mutant strain had increased levels of COB RNA, suggestive that production of short CBP1 transcripts is a mechanism for downregulation of the levels of long CBP1 transcripts, Cbp1, and COB mRNA during the induction of respiration.

[Computerized tomography of retroperitoneal liposarcoma]. / Computertomographie retroperitonealer Liposarkome.

Besides the malignant fibrous histiocytoma, liposarcoma is one of the most common soft-tissue sarcomas seen in adults. They are most often localized in the lower extremity, followed by sarcomas in the retroperitoneal space which differ from the former in clinical appearance, epidemiology, and prognosis. The prognosis is especially dependent on tumor histology which is mainly classified into four subtypes. The aim of the study was to investigate whether there are specific CT-features for each histologic subgroup, by analyzing the examination of 20 patients. The highest percentage in fat and the biggest tumors were seen with well-differentiated liposarcomas (n = 4), whereas myxoid ones (n = 6) showed the sharpest margins. Round-cell (n = 2) and pleomorphic (n = 5) and pleomorphic (n = 5) types showed mainly soft-tissue attenuation and could not be differentiated, either from each other or from other soft-tissue tumors. A clear identification of the histologic subgroup does not seem to be possible.

Analysis for sulfur as hydrogen sulfide incorporating zirconia pretreatment and preconcentration.

The method of analysis for sulfate by reduction of high oxidation state sulfur to hydrogen sulfide, followed by spectrophotometric analysis, has the advantages of allowing small quantities to be measured and some interfering species to be removed. However, it has been found that acid digested samples cannot be analysed by this method due to destruction of the reduction mixture. A column of zirconium(IV) oxide was successfully used to both, remove interfering ions (H(+), Cl(-) and NO(-)(3)) from a sediment digest, as well as perform preconcentration of sulfate. Recoveries from digests of standard sulfur samples were 101 +/- 1%, and from preconcentration solutions 98.8 +/- 1.2%. Comparison of results with independent analyses confirmed that not all sulfur species are detected with the same efficiency by the combined zirconia/reduction-spectrophotometric method.

Tyrosine phosphorylation of I kappa B-alpha activates NF-kappa B without proteolytic degradation of I kappa B-alpha.

The transcription factor NF-kappa B regulates genes participating in immune and inflammatory responses. In T lymphocytes, NF-kappa B is sequestered in the cytosol by the inhibitor I kappa B-alpha and released after serine phosphorylation of I kappa B-alpha that regulates its ubiquitin-dependent degradation. We report an alternative mechanism of NF-kappa B activation. Stimulation of Jurkat T cells with the protein tyrosine phosphatase inhibitor and T cell activator pervanadate led to NF-kappa B activation through tyrosine phosphorylation but not degradation of I kappa B-alpha. Pervanadate-induced I kappa B-alpha phosphorylation and NF-kappa B activation required expression of the T cell tyrosine kinase p56ick. Reoxygenation of hypoxic cells appeared as a physiological effector of I kappa B-alpha tyrosine phosphorylation. Tyrosine phosphorylation of I kappa B-alpha represents a proteolysis-independent mechanism of NF-kappa B activation that directly couples NF-kappa B to cellular tyrosine kinase.

[Secondary osteosarcoma in Paget disease]. / Sekundäres Osteosarkom bei Morbus Paget.

The incidence of Paget's disease in Germany is about 3%. Up to 10% of all affected individuals develop secondary osteosarcomas in the affected bone areas. Compared to primary osteosarcoma patients with Paget's sarcoma differ in regard to age, tumor localisation, therapy, prognosis and radiological symptoms. The following case report describes clinical features, therapy and the typical radiological findings.