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Electrochemiluminescence (ECL) is a powerful transduction technique with a leading role in the biosensing field due to its high sensitivity and low background signal. Although the intrinsic analytical strength of ECL depends critically on the overall efficiency of the mechanisms of its generation, studies aimed at enhancing the ECL signal have mostly focused on the investigation of materials, either luminophores or coreactants, while fundamental mechanistic studies are relatively scarce. Here, we discover an unexpected but highly efficient mechanistic path for ECL generation close to the electrode surface (signal enhancement, 128%) using an innovative combination of ECL imaging techniques and electrochemical mapping of radical generation. Our findings, which are also supported by quantum chemical calculations and spin trapping methods, led to the identification of a family of alternative branched amine coreactants, which raises the analytical strength of ECL well beyond that of present state-of-the-art immunoassays, thus creating potential ECL applications in ultrasensitive bioanalysis.

Poly-alpha-2,8-sialic acid (polysialic acid) is a post-translational modification of the neural cell adhesion molecule (NCAM) and an important regulator of neuronal cell-cell interactions. The synthesis of polysialic acid depends on the two polysialyltransferases ST8SiaII and ST8SiaIV. Understanding the catalytic mechanisms of the polysialyltransferases is critical toward the aim of influencing physiological and pathophysiological functions mediated by polysialic acid. We recently demonstrated that polysialyltransferases are bifunctional enzymes exhibiting auto- and NCAM polysialylation activity. Autopolysialylation occurs on N-glycans of the enzymes, and glycosylation variants lacking sialic acid and galactose were found to be inactive for both auto- and NCAM polysialylation. In the present study, we have analyzed the number and functional importance of N-linked oligosaccharides present on polysialyltransferases. We demonstrate that autopolysialylation depends on specific N-glycans attached to Asn(74) in ST8SiaIV and Asn(89) and Asn(219) in ST8SiaII. Deletion of polysialic acid acceptor sites by site-directed mutagenesis rendered the polysialyltransferases inactive in vitro and in vivo. The inactivity of autopolysialylation-negative polysialyltransferases in vivo was not caused by the absence or default targeting of the enzymes. The data presented in this study clearly show that active polysialyltransferases are competent to perform autopolysialylation and provide strong evidence for a tight functional link between the two catalytic functions.

Polysialic acid (PSA) is a dynamically regulated posttranslational modification of the neural cell adhesion molecule (NCAM), which modulates NCAM binding functions. PSA biosynthesis is catalyzed by two polysialyltransferases, ST8SiaII and ST8SiaIV. The catalytic mechanisms of these enzymes are unknown. In Chinese hamster ovary cells, ST8SiaIV is responsible for PSA expression. In the complementation group 2A10, the ST8SiaIV gene is disrupted. Investigating the molecular defects in this complementation group, seven clones with missense mutations in ST8SiaIV were found. Mutations cause replacement of amino acids that are highly conserved in alpha2,8-sialyltransferases. To verify the physiological relevance of identified mutations, identical amino acid substitutions were introduced into epitope-tagged variants of hamster ST8SiaIV and murine ST8SiaII and recombinant proteins were tested in vivo and in vitro. None of these constructs reconstituted PSA synthesis in 2A10 cells, although the proteins were expressed and with the exception of the cysteine variants ST8SiaIV-C356F and ST8SiaII-C371F correctly targeted to the Golgi apparatus. Interestingly, two mutations (ST8SiaIV-R277G and -M333V and the corresponding mutants ST8SiaII-R292G and -M348V) could be partially rescued if tested in vitro. Although these mutants were negative for autopolysialylation, partial reconstitution of both auto- and NCAM polysialylation was achieved in the presence of NCAM. The data presented in this study suggest a functional link between auto- and NCAM polysialylation.

We investigated the radiological findings of five patients with primary leiomyosarcoma of the inferior vena cava (IVC) comparing cavography, computed tomography (CT), and magnetic resonance imaging (MRI). The radiographic presentation ranged from an intraluminal lesion with obstruction of the IVC to a tumor mass extending from the media musculature into the surrounding perivascular tissue with only slight protrusion into the lumen. The lesions were associated with late or few symptoms. Pathognomonic radiological findings could not be established. In cases with an extraluminal growth a lobulated, well-defined, encapsulated tumor with an inhomogeneous contrast uptake is characteristic in CT studies. Additional information was provided by MRI. Leiomyosarcomas with extraluminal involvement showed homogeneous intermediate signal intensity (SI) on T1-, and mixed intermediate/high SI on T2-weighted images. In cases with predominantly intraluminal growth, both CT and MRI demonstrated the IVC to be dilatated and allowed to differentiate between tumor extent and obliterating thrombosis. Modern imaging modalities allow an early and accurate pre-operative diagnosis resulting in a higher rate of surgical resection and improvement of survival.

A prospective study was conducted in 38 patients with nodular lesions on plain chest radiographs and the clinical suspicion of invasive pulmonary aspergillosis (IPA) to assess the diagnostic accuracy of magnetic resonance imaging (MRI) and computed tomography (CT). For early diagnosis of IPA (clinical signs and symptoms < 10 days), CT scans with demonstration of the halo sign had a high sensitivity (16/22) and specificity (8/8). Magnetic resonance imaging performed at the same time revealed a relatively higher sensitivity (22/22), but a very poor specificity (0/8). Gadolinium-diethylene-triamine-pentaacetic acid (Gd-DTPA) enhanced images did not improve specificity. In the later course of infection (clinical signs and symptoms > 10 days), MRIs showed typical nodular target-like lesions with Gd-DTPA enhancement of the rim area that was not seen in the early course of the disease or in patients with Pseudomonas or staphylococcal infection. In conclusion, MRI findings are not as characteristic as the CT halo sign in diagnosing IPA in the early course of the disease, but the MRI target sign with Gd-DTPA enhancement of the rim area and the "reverse target" on T2-weighted images are strongly suggestive of IPA at a later stage of the disease.

In a prospective study we examined the diagnostic ranking of CT and MR in 52 immunocompromised patients with nodular pulmonary lesions and clinical suspicion of invasive pulmonary aspergillosis (IPA). For early diagnosis of IPA (clinical symptoms having existed for less than 10 days) the CT halo sign proved highly sensitive and specific. MRT showed at this time a comparatively high sensitivity but only low specificity that could not be improved upon after Gd-DTPA. At a later stage of the aspergillosis infection (clinical symptoms manifested for more than 10 days) MR identified aspergillus-specific lesions with on-target characteristics (marked enhancement of margins after Gd-DTPA) or the so-called "reverse" target phenomenon (T2-weighted sequences). Such lesions were never seen in the early stage of the disease in patients with nodular pulmonary lesions of different aetiology (pseudomonal or staphylococcal pneumonia).

We report on the treatment of invasive aspergillosis with the new triazole antimycotic agent itraconazole. All 11 patients suffered from pulmonary invasive aspergillosis. Two patients also had cerebral aspergillosis; in one of these patients the paranasal sinuses were also invaded. Underlying diseases were acute lymphoblastic leukaemia (n = 3), acute myeloid leukaemia (n = 4); one patient underwent allogeneic bone marrow transplantation before he developed aspergillosis; another was transplanted after successful aspergillosis treatment, liver cirrhosis (n = 1), lung infarction after pulmonary embolism (n = 1), chronic bronchitis after pulmonary tuberculosis (n = 1) and AIDS (n = 1). In five cases initial diagnosis was established by means of mycological methods and clinical signs. In six patients invasive pulmonary aspergillosis was initially diagnosed due to the clinical criteria presented in this paper. Secondary mycological confirmation after onset of therapy was achieved in five out of these six patients. All of the patients initially responded to therapy. One female patient experienced a relapse of aspergillosis and died of cerebral involvement and relapsing leukaemia. Two further patients died due to underlying diseases (pulmonary embolism, relapsing leukaemia). Nine patients (82%) were cured of the mycosis, including the patient with cerebral involvement; two underwent surgical resection of residual pulmonary lesions. Itraconazole is a very effective drug for treatment of invasive aspergillosis. Therapeutic efficacy can be optimized by early diagnosis using clinical criteria and prompt start of treatment.

A case of a malignant triton tumor, a malignant schwannoma with a rhabdomyoblastic component is described. The tumor appeared in the right thyroid gland with no relationship to a peripheral nerve. Beneath pulmonary metastases the tumor produced bone metastases and showed responsiveness to radiotherapy and treatment with ifosfamide. These findings are uncommon in malignant triton tumors.

Escape of endotoxin from the intraintestinal site was investigated in experimental models of intestinal ischemia and during intraabdominal infection in rats. Following the instillation of Salmonella abortus equi endotoxin (S-form) into the proximal large bowel, we recorded the presence of the lipopolysaccharide molecule in the bowel wall, the intestinal lymph nodes, the peritoneal cavity, and in the liver sinusoids by immunohistochemical methods. At 3, 6, 12, 24, and 48 hr after the operative procedure, peritoneal fluid, blood, and tissue samples were taken. Survival rates were similar between the two test-groups (occlusion of the superior mesenteric artery [SMA] and cecal ligation and puncture [CLP]) and were not influenced by the amount of the injected endotoxin. There was no detectable morbidity in the sham-operated control animals with endotoxin doses up to 20 mg. Endotoxin could only be detected at 24 and 48 hr in the SMA group in the liver as well as in the peritoneal sediment and in intestinal lymph nodes. CLP and control samples remained negative throughout the observation period. Bacteria were found intraperitoneally within 12 to 24 hr in the SMA group and within 3 to 12 hr in the CLP group.