Structural destabilization of synaptosomal particles by lysis and sequential chemical treatments

Synaptosomes from rat brains were subjected to a sequence of treatments: osmotic lysis, buffered saline wash, nonionic detergent, EGTA and EDTA. After each treatment, particulate samples were fixed in 2% glutaraldehyde-1% formaldehyde and centrifuged to form pellets which were then processed for and examined by electron microscopy. Five morphological classes of synaptic particle were defined in terms of character and presence of synaptic vesicles, flocculent and stranded material, designated as intervesicular scaffolding (IVS), and presynaptic membrane. During osmotic lysis, the presynaptic compartment was altered by loss of most, but not all, small synaptic vesicles, by increase in proportion of large vesicles, and by disappearance of the presynaptic densities. The retention of vesicles was interpreted in terms of IVS struts interconnecting anchorage sites on synaptic vesicles and the presynaptic junctional membrane. Treatment of lysed synaptosomes with nonionic detergent or EGTA resulted in loss of vesicles and IVS from the junctional region in most particles. The apposition of pre-and postsynaptic junctional membranes along the synaptic cleft was disrupted more by EGTA than by detergent. The final result of the sequential treatments was a sediment containing a high proportion of synaptic particles, about half of which had lost their presynaptic junctional membranes.     

Extracellular adenosine 5'-triphosphate (ATP) in the endolymphatic compartment influences cochlear function

There is strong evidence for the presence of P2 purinoceptors on cochlear tissues, but the role of extracellular ATP in cochlear function is still unclear. Our previous studies have determined the presence of ATP in the cochlear fluids and indicated that the purinoceptors are substantially localized to the tissues lining the endolymphatic compartment. This implies that extracellular ATP may have an humoral role confined to the endolymphatic space. In order to study the influence of extracellular ATP in the endolymphatic space, a series of studies were undertaken in which ATP (10 microM to 10 mM) in artificial endolymph (EL) (test solution: 2-12.5 nl) was injected into the scala media and the effect on the cochlear microphonic (CM) and endocochlear potential (EP) evaluated. A double-barrelled pipette, with one barrel containing the test solution and the other artificial EL (control solution) was inserted into scala media of the third turn of the guinea-pig cochlea. A known volume (2-12.5 nl) of test or control solution was then pressure-injected into the space. ATP had a significant dose-dependent suppressive effect on both EP and CM with a threshold of approximately 2 x 10(-14) mol; the response was readily reversible, also in a dose-dependent fashion. Artificial EL of the same volume had no effect on EP and CM. The ATP effect on EP was blocked by the P2 purinoceptor antagonists suramin and reactive blue 2 (RB2). Neither adenosine (2 x 10(-13) to 2 x 10(-11) mol) nor suramin or RB2 on their own had any effect on EP and CM. This study provides the first evidence for an effect of extracellular ATP in the endolymphatic compartment on cochlear function which is mediated via P2 purinoceptors. This provides supporting evidence for an humoral role for extracellular ATP in the modulation of cochlear function.     

Temporal reduction in acute rejection after heart transplantation is not associated with a reduction in cell-mediated responses to donor-specific vascular endothelium

BACKGROUND: Over the first 6 months after clinical transplantation, the incidence of rejection falls despite typically substantial decreases in maintenance immunosuppression. Despite this, chronic vascular rejection, manifested by an accelerated form of coronary artery disease is usually evident by the first annual angiogram and continues to progress over subsequent years. METHODS: To investigate this phenomenon further, peripheral blood mononuclear cells were prepared from blood samples obtained from 42 cardiac allograft recipients at 1 week, 3 months, and 6 months after transplantation and co-cultured with endothelial cells isolated and cultured from the aortas of their specific cardiac allograft donors. Donor-specific alloreactivity was assessed by (1) peripheral blood mononuclear cell proliferation (3H-thymidine incorporation) and (2) up-regulation of endothelial cell major histocompatibility complex class I and class II antigens and ICAM-1 expression (flow cytometry) at all three time points. RESULTS: Over this 6-month period, rejection incidence fell from 0.68 rejections/patient to 0.12 rejection/patient. Cyclosporine dose was reduced from 5.6 +/- 0.3 mg/kg (mean +/- standard error of the mean) to 4.5 +/- 0.2 mg/kg, prednisone dose was reduced from 0.58 +/- 0.08 mg/kg to 0.17 +/- 0.02 mg/kg, and azathioprine remained constant at approximately 2 mg/kg over the 6-month period. Despite this reduction in rejection and immunosuppression, no measure of in vitro donor-specific cell-mediated response to endothelial cells decreased over the 6-month time period. Peripheral blood mononuclear cell proliferation in response to donor-specific endothelial cells was unchanged between 1 week (916 +/- 139 counts/min [cpm]) and 3 months (896 +/- 135 cpm) and increased at 6 months (1738 +/- 243 cpm, p < 0.01). The increase in endothelial cell major histocompatibility complex class II expression in response to recipient peripheral blood mononuclear cells likewise was unchanged between 1 week (42.5 +/- 7.8 mean channel shift [mcs]) and 3 months (34.7 +/- 6.6 mcs) and increased substantially at 6 months (95.4 +/- 17.2 mcs, p < 0.02). The magnitude of the increase in endothelial cell major histocompatibility complex class I antigen and ICAM-1 expression in response to co-culture with recipient peripheral blood mononuclear cells did not change over the 6-month period. CONCLUSIONS: These data suggest an important dichotomy between cell-mediated responses to allograft parenchyma versus those to allograft vasculature and may provide an explanation for progressive vascular disease despite the absence of acute rejection.     

Use of multiple difference-of-Gaussian filters to verify geometric models

Experiments are reported with a program which recognizes London buses in busy street scenes. The program uses a 3D model of a bus. together with knowledge of camera and scene parameters, to compute the visual appearance of possible target events. In earlier work this took the form of 2D templates, which identify locations within the image where abrupt discontinuities of grey-value are expected. Each template corresponds to an hypothesis which is verified by measuring the agreement between the predicted features and the response of a Sobel filter to the image data. This paper reports an extension to the program which offers improved selectivity, and a more general-purpose method of linking predicted features to detected imageattributes. Multiple difference-of-Gaussian filters perform an initial object-independent analysis of the image to provide an intermediate domain in which object-dependent features, predicted by hypothesized instances of the model, are tested.     

Thymic carcinoma. Five distinctive histological variants

Five histologically distinct variants of thymic carcinoma are described: mixed small cell undifferentiated squamous cell carcinoma (three cases), basaloid carcinoma (two cases), mucoepidermoid carcinoma (one case), clear cell carcinoma (one case), and sarcomatoid carcinoma (one case). While forming a heterogeneous group, these tumors bear the common features of an anterior mediastinal location and lack of evidence of a primary tumor elsewhere, marking them as primary thymic neoplasms. All except the sarcomatoid variant are morphologically related to similar malignant neoplasms of other organs. These tumors should be recognized as morphological variants of primary thymic carcinoma and demonstrate the ability of thymic epithelium to differentiate toward a variety of different cell types.     

A decrease of reelin expression as a putative vulnerability factor in schizophrenia

Postmortem prefrontal cortices (PFC) (Brodmann's areas 10 and 46), temporal cortices (Brodmann's area 22), hippocampi, caudate nuclei, and cerebella of schizophrenia patients and their matched nonpsychiatric subjects were compared for reelin (RELN) mRNA and reelin (RELN) protein content. In all of the brain areas studied, RELN and its mRNA were significantly reduced (approximately 50%) in patients with schizophrenia; this decrease was similar in patients affected by undifferentiated or paranoid schizophrenia. To exclude possible artifacts caused by postmortem mRNA degradation, we measured the mRNAs in the same PFC extracts from gamma-aminobutyric acid (GABA)A receptors alpha1 and alpha5 and nicotinic acetylcholine receptor alpha7 subunits. Whereas the expression of the alpha7 nicotinic acetylcholine receptor subunit was normal, that of the alpha1 and alpha5 receptor subunits of GABAA was increased when schizophrenia was present. RELN mRNA was preferentially expressed in GABAergic interneurons of PFC, temporal cortex, hippocampus, and glutamatergic granule cells of cerebellum. A protein putatively functioning as an intracellular target for the signal-transduction cascade triggered by RELN protein released into the extracellular matrix is termed mouse disabled-1 (DAB1) and is expressed at comparable levels in the neuroplasm of the PFC and hippocampal pyramidal neurons, cerebellar Purkinje neurons of schizophrenia patients, and nonpsychiatric subjects; these three types of neurons do not express RELN protein. In the same samples of temporal cortex, we found a decrease in RELN protein of approximately 50% but no changes in DAB1 protein expression. We also observed a large (up to 70%) decrease of GAD67 but only a small decrease of GAD65 protein content. These findings are interpreted within a neurodevelopmental/vulnerability "two-hit" model for the etiology of schizophrenia.