Molecular Genetic and Crystal Structural Analysis of 1-(4-Hydroxyphenyl)-Ethanol Dehydrogenase from 'Aromatoleum aromaticum' EbN1.

The dehydrogenation of 1-(4-hydroxyphenyl)-ethanol to 4-hydroxyacetophenone represents the second reaction step during anaerobic degradation of p-ethylphenol in the denitrifying bacterium 'Aromatoleum aromaticum' EbN1. Previous proteogenomic studies identified two different proteins (ChnA and EbA309) as possible candidates for catalyzing this reaction [Wöhlbrand et al: J Bacteriol 2008;190:5699-5709]. Physiological-molecular characterization of newly generated unmarked in-frame deletion and complementation mutants allowed defining ChnA (renamed here as Hped) as the enzyme responsible for 1-(4-hydroxyphenyl)-ethanol oxidation. Hped [1-(4-hydroxyphenyl)-ethanol dehydrogenase] belongs to the 'classical' family within the short-chain alcohol dehydrogenase/reductase (SDR) superfamily. Hped was overproduced in Escherichia coli, purified and crystallized. The X-ray structures of the apo- and NAD(+)-soaked form were resolved at 1.5 and 1.1 Å, respectively, and revealed Hped as a typical homotetrameric SDR. Modeling of the substrate 4-hydroxyacetophenone (reductive direction of Hped) into the active site revealed the structural determinants of the strict (R)-specificity of Hped (Phe(187)), contrasting the (S)-specificity of previously reported 1-phenylethanol dehydrogenase (Ped; Tyr(93)) from strain EbN1 [Höffken et al: Biochemistry 2006;45:82-93].

Pseudilins: halogenated, allosteric inhibitors of the non-mevalonate pathway enzyme IspD.

The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis have been identified as attractive targets with novel modes of action for the development of herbicides for crop protection and agents against infectious diseases. This pathway is present in many pathogenic organisms and plants, but absent in mammals. By using high-throughput screening, we identified highly halogenated marine natural products, the pseudilins, to be inhibitors of the third enzyme, IspD, in the pathway. Their activity against the IspD enzymes from Arabidopsis thaliana and Plasmodium vivax was determined in photometric and NMR-based assays. Cocrystal structures revealed that pseudilins bind to an allosteric pocket by using both divalent metal ion coordination and halogen bonding. The allosteric mode of action for preventing cosubstrate (CTP) binding at the active site was elucidated. Pseudilins show herbicidal activity in plant assays and antiplasmodial activity in cell-based assays.

Intact versus fragmented 99mTc-monoclonal antibody imaging of infection in patients with septically loosened total knee arthroplasty.

OBJECTIVE: This prospective study compared the diagnostic accuracy of imaging using an intact murine antigranulocyte antibody 99mTc-besilesomab, and a murine antibody Fab fragment 99mTc-sulesomab, in patients with suspected septically loosened total knee arthroplasty. METHODS: Images from 20 patients who underwent threephase bone scintigraphy followed by imaging using 99mTc-besilesomab (n=10) or 99mTc-sulesomab (n=10) were evaluated and compared. Final diagnosis was determined by microbiological evaluation of aspirated synovial fluid, intraoperative samples through the clinical course, or by long-term follow-up. RESULTS: Prosthesis infection was shown in 18 patients. At 4 and 24 h after intravenous injection, absolute uptake of 99mTc-besilesomab was significantly higher than 99mTc-sulesomab in infected knee joints. Infected-to-healthy knee activity ratios were similar at 4 and 24 h for 99mTc-besilesomab and 99mTc-sulesomab. CONCLUSIONS: Both 99mTc-besilesomab and 99mTc-sulesomab had similar diagnostic accuracy for the detection of septic arthroplasty. If repeated use of immunoscintigraphy is needed for follow-up, 99mTc-sulesomab should be preferred over 99mTc-besilesomab since it is known to be well tolerated and without side effects or incompatibility reactions.

[Diagnostic imaging in patients with coronary artery disease: the nuclear medicine physicians' view]. / Diagnostische Bildgebung bei koronarer Herzkrankheit - die Sicht des Nuklearmediziners.

Actually myocardial perfusion scintigraphy (MIP) is best evaluated imaging modality in patients with coronary artery disease. It detects flow-limiting coronary artery disease with high sensitivity and specificity, enables recognition of the grade of severity and extensiveness of myocardial ischemia, and furthermore enables assessment of future cardiac events independently of clinical and diagnostic parameters. Due to rapid technical evolution in diagnostic tools there is need of comparison between MIP and other concurrent imaging modalities such as stress echocardiography, Cardio-CT and Cardio-MRI in patients with coronary artery disease.

Incidental non-secreting adrenal masses in cancer patients: intra-individual comparison of 18F-fluorodeoxyglucose positron emission tomography/computed tomography with computed tomography and shift magnetic resonance imaging.

The ability of integrated (18)F-fluorodeoxyglucose positron emission tomography and computed tomography (FDG PET/CT) to distinguish between benign and malignant incidental non-secreting adrenal masses was evaluated in cancer patients. Results were compared with those of CT and shift magnetic resonance imaging (MRI). A total of 1832 cancer patients who had undergone FDG PET/CT scans were retrospectively evaluated. Visual interpretation, tumour maximum standardized uptake value (SUV(max)), liver SUV(max) and tumour/liver SUV(max) ratios were correlated with the findings of CT, shift MRI and final diagnosis (based on biopsy or clinical/radiological follow-up). A total of 109 adrenal masses were found: 49 were malignant and 60 were benign on final diagnosis. A tumour/liver SUV(max) ratio threshold of 1.0 was more accurate in differentiating the tumour type than tumour SUV(max) or visual interpretation alone. Diagnostic accuracy of CT and shift MRI (92 - 97%) was similar to that for FDG PET/CT (94 - 97%). In conclusion, FDG PET/CT accurately characterizes adrenal tumours, with excellent sensitivity and specificity. Use of 1.0 as the threshold for the tumour/liver SUV(max) ratio seems to be promising for distinguishing benign from malignant adrenal masses in cancer patients.

(99m)Tc-Fab' fragments (sulesomab) for imaging septically loosened total knee arthroplasty.

The diagnostic accuracy of infection scintigraphy with (99m)Tc-labelled monoclonal antibody Fab' fragments (sulesomab) was studied in patients with suspected total knee arthroplasty (TKA) infection. Images from 26 patients were evaluated by two independent readers and compared with a quantitative interpretation of time-activity courses. Microbiological examinations and joint aspiration results were used as reference standards. Histologically, aseptic TKA loosening occurred in two patients and severe, moderate or mild septic loosening in four, nine and 11 patients, respectively. Diagnostic accuracy for severe infection was 100% for both readers, whereas for moderate infection accuracy decreased by 12% and 12% for readers one and two, respectively. For mild infection a further decrease of approximately 61% and 52% occurred for readers one and two, respectively. Quantitative evaluation gave significantly better results over visual interpretation with a diagnostic accuracy of 100% for severe infection and decreased by only 10% and 15% in patients with moderate and mild infection, respectively. Quantitative evaluation of (99m)Tc-Fab' fragments is highly sensitive and specific for diagnostic imaging of infection in patients with septically-loosened TKA.

[Nuclear medical imaging in case of painful knee arthroplasty]. / Nuklearmedizinische Diagnostik bei schmerzhafter Knieprothese.

RATIONALE: The aim of the present study was to calculate the overall diagnostic accuracy of nuclear medical imaging in patients with painful knee arthroplasty. MATERIAL AND METHODS: This retrospective study of all patients (n=87) where a (99m)Tc-triple phase bone scintigraphy (TPBS; n=120) and (99m)Tc-anti-granulocyte scintigraphy (BW 250/183; n=20) for a painful knee arthroplasty was performed between 2003 and 2007. RESULTS: A total of 87 patients with 94 knee arthroplasties were examined to detect septic and aseptic loosening and to differentiate between them. The sensitivity, specificity, the positive and negative predictive value and accuracy of TPBS for the detection of septic knee arthroplasty loosening was 100%, 85%, 55%, 100%, 73% and for BW 250/183 was 91%, 66%, 76%, 85%, 80% for sepsis, respectively. A significant increase in diagnostic accuracy with 94%, 88%, 89%, 95% und 89% (p <0.001) could be achieved when both methods were used in combination. CONCLUSION: Both methods alone have high negative predictive values, but the combination of both is complementary and significantly increases the diagnostic accuracy and positive predictive value for final diagnosis of knee arthroplasty loosening.

Quantitative [(123)I]FP-CIT pinhole SPECT imaging predicts striatal dopamine levels, but not number of nigral neurons in different mouse models of Parkinson's disease.

Single photon emission computed tomography (SPECT) using [(123)I]FP-CIT as radioligand for the dopamine transporter has become a widely used tool to monitor the integrity of the nigrostriatal dopaminergic projection in Parkinson's disease (PD). Previous studies with pinhole SPECT in small animals have demonstrated that the striatal [(123)I]FP-CIT binding indeed correlates with the striatal dopamine transporter protein level. It is unclear, however, if there is a stable relationship between the striatal [(123)I]FP-CIT binding and other functionally important parameters of the nigrostriatal system, such as the striatal dopamine levels and the number of dopaminergic neurons in the substantia nigra. To assess this question experimentally, we studied two different mouse models of PD, namely a mild 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intoxication paradigm, to model mild nigrostriatal damage and the intrastriatal 6-hydroxydopamine paradigm to model more advanced nigrostriatal damage. Our data demonstrate that the striatal [(123)I]FP-CIT binding measured by SPECT in vivo precisely predicts the striatal dopamine concentrations, but does not necessarily correlate with the nigral dopaminergic cell number. Thus, the present work underscores that FP-CIT SPECT does only allow judging the integrity of the striatal dopaminergic nerve terminals, but not the nigral dopaminergic cells in PD. This finding may have significant impact on the use of [(123)I]FP-CIT SPECT as a surrogate marker for clinical trials aimed at measuring neuroprotection.

Orally active thrombin inhibitors. Part 1: optimization of the P1-moiety.

The synthesis and SAR of novel nanomolar thrombin inhibitors with the common backbone HOOC-CH(2)-d-cyclohexylalanyl-3,4-dehydroprolyl-NH-CH(2)-aryl-C(=NH)NH(2) are described together with their ecarin clotting time (ECT) prolongation as measure for thrombin inhibition ex vivo. The aryl P1-moiety mimicking the arginine part of the d-Phe-Pro-Arg derived thrombin inhibitors turned out to be a key component for in vitro potency and in vivo activity. Optimization of this part led to compounds with improved antithrombin activity in rats and dogs after oral administration compared to the recently launched anticoagulant melagatran.

Orally active thrombin inhibitors. Part 2: optimization of the P2-moiety.

Synthesis and SAR of orally active thrombin inhibitors of the d-Phe-Pro-Arg type with focus on the P2-moiety are described. The unexpected increase in in vitro potency, oral bioavailability, and in vivo activity of inhibitors with dehydroproline as P2-isostere is discussed. Over a period of 24h the antithrombin activity of the most active inhibitors with IC(50)s in the nanomolar range was determined in dogs demonstrating high thrombin inhibitory activity in plasma and an appropriate duration of action after oral administration.

Crystal structure and enzyme kinetics of the (S)-specific 1-phenylethanol dehydrogenase of the denitrifying bacterium strain EbN1.

(S)-1-Phenylethanol dehydrogenase (PED) from the denitrifying bacterium strain EbN1 catalyzes the NAD+-dependent, stereospecific oxidation of (S)-1-phenylethanol to acetophenone and the biotechnologically interesting reverse reaction. This novel enzyme belongs to the short-chain alcohol dehydrogenase/aldehyde reductase family. The coding gene (ped) was heterologously expressed in Escherichia coli and the purified protein was crystallized. The X-ray structures of the apo-form and the NAD+-bound form were solved at a resolution of 2.1 and 2.4 A, respectively, revealing that the enzyme is a tetramer with two types of hydrophobic dimerization interfaces, similar to beta-oxoacyl-[acyl carrier protein] reductase (FabG) from E. coli. NAD+-binding is associated with a conformational shift of the substrate binding loop of PED from a crystallographically unordered "open" to a more ordered "closed" form. Modeling the substrate acetophenone into the active site revealed the structural prerequisites for the strong enantioselectivity of the enzyme and for the catalytic mechanism. Studies on the steady-state kinetics of PED indicated a highly positive cooperativity of both catalytic directions with respect to the substrates. This is contrasted by the behavior of FabG. Moreover, PED exhibits extensive regulation on the enzyme level, being inhibited by elevated concentrations of substrates and products, as well as the wrong enantiomer of 1-phenylethanol. These regulatory properties of PED are consistent with the presence of a putative "transmission module" between the subunits. This module consists of the C-terminal loops of all four subunits, which form a special interconnected structural domain and mediate close contact of the subunits, and of a phenylalanine residue in each subunit that reaches out between substrate-binding loop and C-terminal domain of an adjacent subunit. These elements may transmit the substrate-induced conformational change of the substrate binding loop from one subunit to the others in the tetrameric complex and thus mediate the cooperative behavior of PED.

Combination of 'idiopathic' REM sleep behaviour disorder and olfactory dysfunction as possible indicator for alpha-synucleinopathy demonstrated by dopamine transporter FP-CIT-SPECT.

REM sleep behaviour disorder (RBD) and olfactory dysfunction are common and very early features of alpha-synucleinopathies, in particular Parkinson's disease. To investigate the hypothesis that these two clinical features in combination are an indicator of evolving alpha-synucleinopathy, olfactory function was assessed in RBD. We studied 30 patients (18 male, 12 female; mean age 48 +/- 14 years, range 19-78 years) with clinical (idiopathic, n = 6; symptomatic, n = 13, mostly associated with narcolepsy) or subclinical (n = 11, associated with narcolepsy) RBD according to standard criteria and 30 age- and gender-matched healthy control subjects using standardized 'Sniffin' Sticks'. RBD patients had a significantly higher olfactory threshold (P = 0.0001), lower discrimination score (P = 0.003), and lower identification score (P = 0.001). Compared with normative data, 97% of the RBD patients had a pathologically increased olfactory threshold, 63% an impaired odour discrimination score, and 63% a decreased identification score. On neurological examination, signs of parkinsonism were newly found in five patients with clinical RBD (not associated with narcolepsy), who usually had a long history of 'idiopathic' RBD. Four of the five patients fulfilled the UK Brain Bank criteria for the clinical diagnosis of Parkinson's disease. The underlying nigrostriatal degeneration of clinical Parkinson's disease was confirmed by I-123-FP-CIT SPECT in one patient and early nigrostriatal degeneration was identified by SPECT in a further two patients with 'idiopathic' clinical RBD out of 11 RBD patients who agreed to undergo SPECT studies. Our study shows that RBD patients have a profound impairment of olfactory function. Five patients with clinical RBD not associated with narcolepsy had clinical or imaging signs of nigrostriatal degeneration. This new clinical finding correlates with the neuropathological staging of Parkinson's disease (stages 1-3) as proposed by Braak. In stage 1, the anterior olfactory nucleus or the olfactory bulb is affected (along with the dorsal motor nucleus of the glossopharyngeal and vagal nerves). In stage 2, additional lesions consistently remain confined to the medulla oblongata and pontine tegmentum, which are critical areas for RBD. Midbrain lesions are found only in stage 3, in particular degeneration of dopaminergic neurons in the substantia nigra pars compacta. Thus, 'idiopathic' RBD patients with olfactory impairment might present with stage 2 preclinical alpha-synucleinopathy. Since narcoleptic patients are not known to have an increased risk of developing parkinsonism, the pathophysiology and clinical relevance of hyposmia in RBD/narcolepsy patients requires further research.

Prospective study of presynaptic dopaminergic imaging in patients with mild parkinsonism and tremor disorders: part 1. Baseline and 3-month observations.

To record prospectively, from early presentation, the clinical features of parkinsonism and tremor disorders, in relation to evidence of dopaminergic deficit shown with [(123)I]-FP-CIT (DaTSCAN, Amersham Health) single photon emission computerised tomography (SPECT). Clinical signs were recorded in 62 patients, of whom 24 failed standard Parkinson's disease (PD) and essential tremor criteria, and 38 fulfilled UK Brain Bank step 1 PD criteria. Striatal radioligand uptake was graded visually as normal or abnormal, and specific:nonspecific ratios were calculated. Bradykinesia and rigidity showed significant overall association with abnormal scans (P < or = 0.003), but rest tremor did not (P = NS). In the 24 patients not fulfilling specific criteria (mean age 63 [SD 9] years, disease duration 3 [SD 4] years), 10 (42%) had abnormal visual SPECT assessment and 14 (58%) had normal scans. Of 38 patients with early PD by clinical criteria (mean age 60 [SD 9] years, disease duration 3 [SD 1.7] years), 33 (87%) were visually abnormal. Baseline clinical diagnosis corresponded with SPECT imaging results in 51 of 62 cases (82%), which increased to 56 of 62 cases (90%) with amendment of seven clinical diagnoses at 3 months (blind to SPECT results). Akinetic-rigid cardinal diagnostic features of parkinsonism associate well with dopaminergic deficit in patients with early and mild clinical features. When these clinical features are uncertain, or the patient fails clinical diagnostic criteria, testing for dopaminergic deficit with [(123)I]-FP-CIT SPECT may assist the diagnostic process.

D-Phe-Pro-Arg type thrombin inhibitors: unexpected selectivity by modification of the P1 moiety.

Synthesis of thrombin inhibitors and their binding mode to thrombin is described. Modification of the P1 moiety leads to an increased selectivity versus trypsin. The observed selectivity is discussed in view of their thrombin-inhibitor complex X-ray structures.

LeukoScan for imaging infection in different clinical settings: a retrospective evaluation and extended review of the literature.

PURPOSE: The aim of the current study was to determine the overall diagnostic accuracy of Tc-99m-labeled antigranulocyte monoclonal antibody Fab' fragments (LeukoScan) for the routine detection of bone and soft tissue infections in a retrospective evaluation. PATIENTS AND METHODS: 138 patients (63 men, 75 women; mean age, 58.29 +/- 25.38 years) with fever of unknown origin and possible endocarditis (n = 59), infection of arthroplastic joints (n = 20), arthritis (n = 16), peripheral (n = 15) and central bone infections (n = 14), soft tissue infection (n = 6), appendicitis (n = 4), pericarditis (n = 2), or vascular graft infection (n = 2) underwent imaging after injection of 555 to 925 MBq (15 to 25 mCi) Tc-99m-labeled antigranulocyte monoclonal antibody Fab' fragments (LeukoScan). RESULTS: True-positive results were found in 63 of 81 lesions. The overall sensitivity and specificity were 76% and 84%, respectively. In arthritis, seven of seven foci could be detected, whereas false-negative results were found in infections of the femoral bone in three of nine lesions and in periprosthetic infections of long bones in three of eight lesions. Good results were found in five of six soft-tissue infections, in four of six patients with endocarditis, in three of four atypical cases of appendicitis, in two of two infected vascular grafts, and in one of one patient with pericarditis. Subacute and chronic infections of the spine always showed photopenic areas in eight of eight patients. If photopenic lesions were included as diagnostic criteria, the sensitivity and specificity were 88% and 67%, respectively. CONCLUSIONS: Tc-99m-labeled antigranulocyte monoclonal antibody Fab' fragments can be used for imaging acute infections of peripheral bones and soft tissues. False-negative results are likely in patients with chronic infections. Sensitivity can be increased while decreasing specificity by including photopenic lesions in the spine as diagnostic criteria for localizing disease.

Follow-up of perfusion defects in pulmonary perfusion scanning after pulmonary embolism: are we too careless?

Persisting perfusion defects may still be found in pulmonary perfusion scintigraphy months or years after pulmonary embolism. The aim of this study was to investigate the rate of persisting perfusion defects and the pattern of scintigraphic follow-up of patients after pulmonary embolism. Only those patients were included into our study who received pulmonary perfusion scintigraphy between 1991 and 1999, and who had perfusion defects including at least one whole segment. These perfusion defects were considered as persisting perfusion defects if unchanged over at least 1 year. From 3640 patients examined, 451 (12.4%) had perfusion defects meeting the criteria of this study. Of those, 129 (28.6%) received a scintigraphic follow-up. In 62 patients (48.1%), a reperfusion of the defects was found. In 38 patients (29.5%), the defects persisted within a follow-up period of up to 12 weeks. However, no pulmonary perfusion scintigraphy was performed thereafter. Out of the 129 patients receiving a scintigraphic follow-up, only 29 (22.5%) had a follow-up over more than 1 year, 19 of those had persisting perfusion defects. It is concluded that our data show an inadequate scintigraphic follow-up of patients with pulmonary embolism which may lead to unnecessary anticoagulant treatment if persisting perfusion defects are misinterpreted as fresh pulmonary embolism. In many cases, there was no further follow-up even if reperfusion of the defects was lacking in early follow-up.

Tyrosine hydroxylase deficiency with severe clinical course: clinical and biochemical investigations and optimization of therapy.

Tyrosine hydroxylase deficiency was diagnosed after determination of cerebrospinal fluid neurotransmitters and DNA analysis in a child with severe axial hypotonia and hypokinesia associated with dystonic and ballistic movements. L-dopa therapy was unsuccessful, whereas a combination with selegiline, a selective monoamine oxidase-beta inhibitor, with low-dose L-dopa markedly improved the severe clinical picture.

[High dosage chemotherapy and peripheral blood stem cell transplantation in breast carcinoma]. / Hochdosis-Chemotherapie und periphere Blutstammzell-Transplantation beim Mammakarzinom.

Chemotherapeutic agents have been shown experimentally to have a dose-response relationship in drug-sensitive cancer. This suggests that high-dose chemotherapy (HDC) might improve the therapeutic outcome in breast cancer. The emergence of modern supportive measures like peripheral blood stem cell rescue has significantly decreased the toxicity and cost of HDC. HDC is being employed for patients with metastatic and high-risk breast cancer (> 10 involved axillary lymph nodes). Randomized phase III trials are urgently needed. At the moment patient with breast cancer should only be treated with HDC in context with a clinical trial.