Ultrastructural co-localization of calmodulin and B-50/growth-associated protein-43 at the plasma membrane of proximal unmyelinated axon shafts studied in the model of the regenerating rat sciatic nerve

Calmodulin and de-phosphorylated B-50/growth-associated protein-43 (GAP-43) have been shown to bind in vitro in a molecular complex, but evidence for an in situ association in the nervous system does not exist. Previously, we have reported that, in the model of the regenerating rat sciatic nerve, the B-50/GAP-43 immunoreactivity is increased and concentrated at the axolemma of unmyelinated axons located proximal to the site of injury and axon outgrowth. To explore a putative function of B-50/GAP-43, namely, the capacity of binding calmodulin to the plasma membrane, we examined the ultrastructural distribution of calmodulin in the proximal unmyelinated axon shafts of this model, using double immunolabelling and detection by fluorescent or gold probes conjugated to second antibodies. Immunofluorescence showed that seven days post-sciatic nerve crush the calmodulin immunoreactivity, similar to B-50/GAP-43 immunoreactivity, was intense in unmyelinated axon shafts located proximal to the site of injury of the regenerating nerve. Ultrastructurally, calmodulin was located at the axolemma of these regenerating unmyelinated axon shafts and inside the axoplasm, where it was associated with vesicles and microtubules. The plasma membrane labelling (approximately 69%) was significantly higher than the axoplasmic labelling. Over 60% of the plasma membrane-associated calmodulin co-localized with B-50/GAP-43 in a non-random distribution. Since normally calmodulin is largely present in the cytoplasm, these data suggest that calmodulin has been concentrated at the plasma membrane of unmyelinated axons, most probably by B-50/GAP-43. If the concentrating effect is due to B-50/GAP-43, then there is a possibility that these proteins may be present as a molecular complex in situ. The physiological significance could be that this association regulates the local availability of both B-50/GAP-43 and calmodulin for other interactions.     

Single-laser approach for fluorescence guidance of excimer laser angioplasty at 308 nm: evaluation in vitro and during coronary angioplasty

BACKGROUND AND OBJECTIVE: Spectroscopic guidance of laser angioplasty has been attempted using a diagnostic He-Cd laser in addition to the therapeutic laser system. This study evaluated a single-laser approach for simultaneous ablation and fluorescence excitation. STUDY DESIGN/MATERIALS AND METHODS: A spectroscopy system was coupled to a clinical XeCl excimer laser. Ablation of 162 human aortic samples in saline and blood with 45 mJ/mm2 per pulse yielded 676 fluorescence spectra validated histologically. The same equipment was used in 16 patients for angioplasty of 18 coronary stenoses applying 500 to 1,725 pulses with 45 to 60 mJ/mm2 under saline flushing. A total of 783 spectra were recorded and validated by intracoronary ultrasound (categories: atheroma, fibrous plaque, calcified lesion). RESULTS: In vitro, 5 types of spectra could be differentiated: (1) atheroma, (2) fibrous plaque, (3) calcified lesion in saline, (4) media, and (5) calcified lesion in blood. Discriminant analysis prospectively classified 576 validation spectra with the following sensitivity and specificity for each type: (1) 83.5 and 97.1%, (2) 85.7 and 96.8% (3) 100 and 98.5%, (4) 98.1 and 99.3%, (5) 98.9 and 100%, respectively. In vivo type 1, 2, 3, and 5 spectra were also observed, but not the media spectrum. The predominant sonographic category also prevailed in spectroscopy. Calcified lesions yielded type 3 and 5 as well as mixed spectra. CONCLUSIONS: Using an excimer laser for angioplasty allows combining ablation and fluorescence excitation without a diagnostic laser. Principal types of atherosclerotic lesions and the media can be differentiated spectroscopically with this approach.     

Evaluation of the uncertainty of molecular mass measurement by electrospray ionization mass spectrometry

Electrospray ionization (ES) is a novel method used in mass spectrometry (MS) for producing gas-phase ions from substances in solutions. Common practices for molecular mass estimation from ES spectra summarize the spectrum as a single peak giving no estimate of uncertainty or treat each peak as an independent molecular mass measurement. ES-MS data analysis showed that each peak in an ES spectrum does not always provide an independent measure of molecular mass. Underestimation of measurement uncertainty is a possible result. An elementary time series method, the Yule-Walker equations, was applied to molecular mass estimation from ES data.     

Evaluation of a new enzyme immunoassay for Clostridium difficile toxin A

AIM: To evaluate a new enzyme immunoassay (EIA) method for detection of Clostridium difficile toxin by comparing it to cytotoxicity assay. To investigate the nature of false negative and false positive EIA results by evaluating clinical and therapeutic parameters. METHODS: 737 consecutive diarrhoeal specimens collected from patients clinically suspected of having C difficile colitis were tested for the presence of C difficile toxin by EIA for toxin A and by cytotoxicity assay. Clinical data were evaluated in all cases positive by either method. RESULTS: With the cytotoxicity assay as a gold standard, the specificity of EIA for toxin detection was 99.3% and the sensitivity was 62.2%. No false negative EIA specimens were obtained from patients already being treated for C difficile colitis. Among patients with cytotoxicity positive specimens, those with EIA positive samples had no clinical features distinguishing them from patients with EIA negative samples. CONCLUSIONS: Although specific, the new EIA method directed against toxin A lacks sensitivity compared to cytotoxicity. False negative EIA tests are not associated with concurrent treatment for C difficile colitis nor with any specific clinical features examined in our study.     

Psychophysical evidence for a functional hierarchy of motion processing mechanisms

Current models of motion perception typically describe mechanisms that operate locally to extract direction and speed information. To deal with the movement of self or objects with respect to the environment, higher-level receptive fields are presumably assembled from the outputs of such local analyzers. We find that the apparent speed of gratings viewed through four spatial apertures depends on the interaction of motion directions among the apertures, even when the motion within each aperture is identical except for direction. Specifically, local motion consistent with a global pattern of radial motion appears 32% faster than that consistent with translational or rotational motion. The enhancement of speed is not reflected in detection thresholds and persists in spite of instructions to fixate a single local aperture and ignore the global configuration. We also find that a two-dimensional pattern of motion is necessary to elicit the effect and that motion contrast alone does not produce the enhancement. These results implicate at least two serial stages of motion-information processing: a mechanism to code the local direction and speed of motion, followed by a global mechanism that integrates such signals to represent meaningful patterns of movement, depending on the configuration of the local motions.     

A new non-random chromosomal translocation t(3;6)(q27;p21.3) associated with BCL6 rearrangement in two patients with non-Hodgkin's lymphoma

We describe two cases of non-Hodgkin's lymphoma associated with t(3;6)(q27;p21.3) and BCL6 rearrangement. The first case was in a 78-year old woman, whose performance status (PS) was 1, the serum lactate dehydrogenase (LDH) level was elevated, and the Ann Arbor stage was IIIA with no extra nodal lymphomatous site. The pathological diagnosis from a biopsy of the inguinal lymph node was 'malignant lymphoma (ML), follicular, small cleaved' according to the Working Formulation. Complete remission was achieved. Although she had relapse in 1992, remission was obtained again. The second case was in a 62-year old man, whose PS was 1, the serum LDH was normal, and Ann Arbor stage was IVA with the involvement of the small intestine. Histological diagnosis of the cervical lymph node was 'ML, diffuse, large cell'. Complete remission was obtained without relapse. The 3q27 translocations, found in 20-30% of non-Hodgkin's lymphoma, are unique in having multiple chromosomal translocation partners. Chromosome band 6p21.3 is one of these partner sites that may be the site of a novel gene. The two cases presented here show that this translocation is a non-random chromosomal change involving 3q27 and BCL6. Since t(3;6) was the sole karyotypic abnormality in one case, this translocation may play a role in lymphomagenesis.