Detailed immunoautoradiographic mapping of enkephalinase (EC 3.4.24.11) in rat central nervous system: comparison with enkephalins and substance P

The metallopeptidase enkephalinase known to participate in the inactivation of endogenous enkephalins and, possibly, other neuropeptides such as tachykinins, was visualized by autoradiography using a [125I]iodinated monoclonal antibody. A detailed mapping of the enzyme in rat brain and spinal cord was established on 10-micron serial sections prepared in a frontal plane as well as a few sections in a sagittal plane. On adjacent sections, and for the purpose of comparison, substance P-like and enkephalin-like immunoreactivities were also visualized by autoradiography using a 125I-monoclonal antibody and a polyclonal antibody detected by a secondary 125I-anti-rabbit antibody respectively. Histological structures were identified on adjacent Nissl-stained sections. Using the highly sensitive 125I-probe, enkephalinase immunoreactivity was found to be distributed in a markedly heterogeneous manner in all areas of the central nervous system. Immunoreactivity was undetectable in white matter areas, for example the corpus callosum or fornix, and had a laminar pattern in, for example, the cerebral cortex or hippocampal formation. Hence, although immunodetection was not performed at the cellular level, a major neuronal localization of the peptidase is suggested. The latter is consistent with the detection of a strong immunoreactivity in a pathway linking the striatum to the globus pallidum, the entopeduncular nucleus and the substantia nigra, as well as with a series of biochemical and lesion data. The strong immunoreactivity also present in choroid plexuses and ependymal cells as well as in the intermediate lobe and in scattered cells of the anterior lobe of the pituitary suggests that populations of glial and endocrine cells also express the peptidase. The highest density of enkephalinase immunoreactivity was observed in basal ganglia and limbic areas (caudate putamen, globus pallidus, nucleus accumbens, olfactory tubercles) as well as in areas involved in pain control mechanisms (superficial layers of the spinal nucleus of the trigeminal nerve or of the dorsal horn of the spinal cord) which also display the highest immunoreactivities for both enkephalins and substance P (except in globus pallidus for the latter). These localizations account for the opioid-like analgesic and motor effects of enkephalinase inhibitors inasmuch as a selective or predominant participation of the peptidase in enkephalin inactivation is assumed. A number of other areas appear richly endowed in both enkephalinase and enkephalins whereas substance P is hardly detectable. This is particularly the case for the olfactory bulb, bed nucleus of the accessory olfactory tract, the cerebellum (where enkephalinase mainly occurs in the molecular layer) and the hippocampal formation (namely in the molecular layer of the dentate gyrus).(ABSTRACT TRUNCATED AT 400 WORDS)     

Beta 2-microglobulin amyloidosis (AB2M) in patients undergoing long-term hemodialysis. A new type of amyloid

Amyloidosis has been increasingly recognized in association with renal failure and chronic hemodialysis. This report describes three patients who had long-term hemodialysis (between 7-18 years), in whom deposits developed of a new type of amyloid of beta 2-microglobulin origin. Beta 2-microglobulin amyloid (AB2M) was found in multiple organs, i.e., bone, subendocardium, gastrointestinal blood vessels, tongue, and carpal tunnel connective tissue. AB2M displayed characteristic amyloid features on conventional light and polarized microscopic examination after congo red staining. However immunostaining with anti-amyloid A protein, kappa, and lambda antisera were negative. The studied material reacted positively with beta 2-microglobulin antisera, identifying AB2M in all three cases. Ultrastructural study revealed an unusual curvi-linear fibrillar configuration. AB2M appears to be a new subtype of systemic amyloidosis secondary to renal failure and long-term hemodialysis.     

Effect of compounds which disrupt proton gradients on secretion of neurosecretory proteins from PC12 pheochromocytoma cells

Treatment of PC12 cells with chroquine (10-50 microM) obliterated the low intragranular pH, as detected by Acridine Orange fluorescence, and depleted the cells of dopamine and norepinephrine. However, these concentrations of chloroquine did not prevent the release of the newly synthesized proteins which normally undergo stimulus-coupled secretion with the catecholamines. Higher concentrations of chloroquine (200 microM) and ammonium chloride (10 and 25 mM) inhibited the release of most of these proteins. This inhibition did not result from alterations in protein synthesis, since the profile of proteins synthesized was not substantially altered. Nor did the inhibition result from degradation of the neurosecretory proteins, since prelabeled proteins were capable of undergoing stimulated secretion from chloroquine-treated cells, as from normal cells. The findings indicated that the inhibition was at the step of packaging of the proteins into the neurosecretory granules. While release of the major secretory proteins, including chromogranin B, was inhibited with 200 microM chloroquine, chromogranin A was secreted upon stimulation of these cells. The results of this study indicate that an acidic intragranular pH is not a requirement for the packaging and secretion of neurosecretory proteins. Higher concentrations of chloroquine had a differential effect on the regulated secretion of different neurosecretory proteins.     

Baseline levels of chromosome instability in the human lymphoblastoid cell TK6

Induced genomic instability in the human B lymphoblastoid cell line TK6 manifests itself as increases in end-to-end chromosome fusions and non-reciprocal chromosome translocations. It is not associated with elevated frequencies of specific locus mutations or other cytogenetic alterations. Previous studies on a limited number of cells and end-points suggested that induced instability in TK6 mirrors spontaneous instability in terms of the types of alterations observed. In the present study we expanded on our previous analysis to include more cells and more end-points in order to derive a more precise measure of spontaneous instability in TK6 cells. The frequency of normal growth rate thymidine kinase mutants (TK(-/-)), measured in 44 independently isolated clones, was 2.73 +/- 0.78 x 10(-6)/cell, while that for slow growth mutants was 2.39 +/- 0.52 x 10(-6)/cell. These are similar to the frequencies observed for HPRT mutants in primary human cells. There was wide variation in chromatid break frequencies, but the average break frequency, at 0.04+/-0.01 breaks/cell, was only slightly higher than that reported for primary human cells. In contrast, the dicentric frequency of 0.006/cell was more than 10-fold higher for TK6 cells than that reported for normal primary human cells. Furthermore, the dicentrics in TK6 cells are unusual in that they are the result of end-to-end chromosome fusions. TK6 cells also show much higher levels of non-reciprocal chromosome translocations than are usually observed in primary human cells. The results suggest an inherent instability in TK6 cells that differs from what is observed in primary cells in that it affects the frequency of end-to-end chromosome fusions and non-reciprocal chromosome translocations, but not TK gene mutations or other cytogenetic alterations.     

Cross-reactivity distinguishes serum and nephritogenic anti-DNA antibodies in human lupus from their natural counterparts in normal serum

To distinguish the properties of anti-DNA antibodies in patients with lupus from those in normal individuals, we compared the ligand binding, idiotypic and charge properties of serum anti-DNA antibodies derived from: patients with active lupus; normal individuals; and among Ig eluted from the kidneys of two patients with active lupus nephritis (one with mesangial proliferation and the other with membranous nephropathy). The kidney eluate anti-DNA antibodies were the most cross-reactive; they cross-reacted with ssDNA, poly(GdC), poly(dT), poly(dG), poly(dC), ZDNA, SmRNP and the phospholipids cardiolipin and phosphatidyl serine. Lupus serum anti-DNA antibody cross-reacted with polynucleotides but not with phospholipids, whereas anti-DNA antibodies derived from normal serum reacted only with poly(dT). An anti-idiotype (anti-IdD; produced against serum anti-DNA antibodies from one patient) reacted with: anti-DNA antibodies in 8/9 lupus sera; antibodies in both kidney eluates; and anti-DNA antibodies from 5/7 normal sera. Anti-IdD did not react with Ig that did not bind to DNA. Isoelectric focusing of Ig showed that the charge of anti-DNA antibodies from lupus serum and normal serum were similar and unrestricted (pI 5.4-9.0); Ig in kidney eluates varied: membranous lupus pI 4.5-8.6; mesangial lupus pI 8.1-9.1. We conclude that idiotypically related anti-DNA antibodies in tissue lesions, lupus serum and normal serum from different individuals can be distinguished on the basis of their cross-reactive antigen-binding properties. Furthermore the cross-reactive properties of lupus auto-antibodies may influence their capacity to form glomerular immune deposits.