Phenotypic properties of catecholamine-positive cells that differentiate in avian neural crest cultures

We have investigated several phenotypic features of the catecholamine-positive (CA+) cell population that develops in quail neural crest cultures. The number, spatial distribution, and morphology of CA+ and tyrosine hydroxylase-positive (TH+) cells are similar at all ages examined, suggesting that these 2 cell classes are identical. Neither CA+ nor TH+ cell bodies or processes were stained using antisera that recognize the 70 or 160 kDa subunits of chicken neurofilament protein. Other cell bodies and fibers in the cultures (which were CA- and TH-) were stained with these neurofilament antisera. The uptake and storage of 3H-norepinephrine by neural crest cultures containing CA+ cells were inhibited in the presence of desmethylimipramine and by incubation at 0 degrees C, but were unaffected by normetanephrine. Overnight treatment with reserpine eliminated histochemically detectable CA fluorescence from the cultures. Chronic reserpine treatment from day 2 to 7 in vitro prevented the appearance of CA+ cells, while normal numbers of TH+ and somatostatin-like immunoreactive (SLI) cells developed. The number and light-microscopic morphology of the CA+ cells that developed in these cultures were not dramatically altered by either exogenous NGF or 6-hydroxydopamine. Using the method of Grillo et al. (1974), we have demonstrated that the CA+ cells observed in the light microscope corresponded to cells containing abundant cytoplasmic granular vesicles (GV) characteristic of catecholamine storage granules observed in other systems. The GV diameters were quite similar in cells examined after 5, 7, 14, and 21 d in vitro. Most GV were 50-200 nm in diameter and were distributed in a unimodal manner, with the observed modal values in the range of 85-115 nm at the ages examined. The number of GV/micron2 of cytoplasmic area remained quite constant at all ages examined. These data, taken together with other available information, suggest that the CA+ cells that differentiate in our neural crest cultures resemble, in many respects, the small, intensely fluorescent cells found in autonomic ganglia and extra-adrenal chromaffin tissue of many species. At present, we do not know if the CA+ cells that differentiate in our neural crest cultures are a stable endpoint of development or whether they are a developmental intermediate in adrenergic differentiation that is normally observed only transiently during the development of avian sympathetic ganglia in vivo, but that can persist under our tissue culture conditions.     

Solubility of neurofibrillary tangles and ultrastructure of paired helical filaments in sodium dodecylsulphate

Summary Temporal cortex from 14 cases of Alzheimer-type dementia and 6 cases of Down's syndrome, all selected for severe Alzheimer pathology, was homogenised in distilled water, NaOH, or sodium dodecylsulphate (SDS) containing 0.1% -mercaptoethanol. The homogenates were stained with Congo red, and the neurofibrillary tangles and plaque cores were counted under crossed-polarisation microscopy. The number of tangles and plaque cores in the water-treated extracts was not related to age, sex, postmortem interval or duration of dementia. The number of tangles after extraction in SDS or NaOH, as a percentage of tangles in water-treated extracts, was 57±25 (mean±SD) for 1% SDS, 43±17 for 5% SDS and 37±22 for 0.2 M NaOH. Plaque cores were essentially insoluble in all three agents. The percentage of tangles insoluble in 1% SDS did not correlated with age or post-mortem interval but decreased with increasing duration of dementia. Enhanced tangle solubility with increasing duration of dementia suggests that the nature of tangles changes with time; one possibility is that this reflects transformation of intracellular to extracellular tangles. Paired helical filament (PHF) length and the number of repeats per PHF were measured in electron micrographs of PHF prepared with and without treatment by 1% SDS. There was no significant multimodality of PHF length to suggest that PHF broke at regular intervals. The mean repeat length (PHF length/number of repeats) was greater for PHF isolated in the presence of 1% SDS than in its absence, showing that SDS affects ultrastructure by untwisting PHF. An untwisting process may also occur in vivo producing the straight filaments found, together with PHF, in tangles and neurites.Supported by Miss E. Buchan (to the MRC Brain Metabolism Unit) and the British Foundation for Age Research and the Wellcome Trust (to P. A. M. Eagles). S. Hussey was in receipt of an MRC Partnership Award.     

Nano-C60 cytotoxicity is due to lipid peroxidation

This study examines the biological effects of water-soluble fullerene aggregates in an effort to evaluate the fundamental mechanisms that contribute to the cytotoxicity of a classic engineered nanomaterial. For this work we used a water-soluble fullerene species, nano-C₆₀, a fullerene aggregate that readily forms when pristine C₆₀ is added to water. Nano-C₆₀ was cytotoxic to human dermal fibroblasts, human liver carcinoma cells (HepG2), and neuronal human astrocytes at doses 50 ppb (LC₅₀=2–50 ppb, depending on cell type) after 48 h exposure. This water-soluble nano-C₆₀ colloidal suspension disrupts normal cellular function through lipid peroxidation; reactive oxygen species are responsible for the membrane damage. Cellular viability was determined through live/dead staining and LDH release. DNA concentration and mitochondrial activity were not affected by the nano-C₆₀ inoculations to cells in culture. The integrity of cellular membrane was examined by monitoring the peroxy-radicals on the lipid bilayer. Subsequently, glutathione production was measured to assess the cell's reaction to membrane oxidation. The damage to cell membranes was observed both with chemical assays, and confirmed physically by visualizing membrane permeability with high molecular weight dyes. With the addition of an antioxidant, l-ascorbic acid, the oxidative damage and resultant toxicity of nano-C₆₀ was completely prevented.     

Splice variants of the relaxin and INSL3 receptors reveal unanticipated molecular complexity

LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.     

Serotonin mediates learning-induced potentiation of excitability

Sensitization potentiates excitability in an interneuron, the S-cell, that is critical for this form of learning in the whole-body shortening reflex of the medicinal leech. Serotonin (5-HT) also increases S-cell excitability, and serotonergic modulation is known to be critical for sensitization of whole-body shortening, suggesting that 5-HT mediates learning-induced enhancement of S-cell excitability. In this paper, the role of 5-HT in mediating sensitization-induced potentiation of S-cell excitability was examined. Potentiation of S-cell excitability by 5-HT was blocked by the 5-HT receptor antagonist methysergide and by intracellular injection of the G-protein inhibitor GDP-beta-S, indicating that a metabotropic 5-HT receptor was involved. Bath application of Rp-cAMP, an inhibitor of protein kinase A (PKA), blocked 5-HT-induced potentiation of excitability, whereas db-cAMP, a cAMP analogue that activates PKA, mimicked the potentiating effects of 5-HT on the S-cell. During sensitization of the shortening reflex in semi-intact preparations, methysergide and Rp-cAMP prevented learning-induced potentiation of S-cell excitability, as well as the increase in S-cell activity that normally occurs during sensitization. Furthermore, sensitization-induced increases in the shortening reflex did not occur in preparations treated with methysergide or Rp-cAMP. These results demonstrate that sensitization-induced enhancement of S-cell excitability is mediated by 5-HT and suggests that these changes may contribute to this form of learning.     

Disposition and immunogenicity of penicillin in the rabbit

The immunogenicity, disposition and irreversible protein binding of benzylpenicillin (BP) were studied in male New Zealand White rabbits. There was an increase in IgG anti-benzylpenicilloyl (BPO) antibody activity, as detected by enzyme-linked immunosorbent assay (ELISA) following daily intramuscular administration (for 4 consecutive days) of BP (2.7 and 1.6 mmol/kg) freshly dissolved in 0.15 M NaCl. Antibody activity reached a maximum approximately 14 days after the last injection. There was a smaller immune response when a dose of 270 mumol/kg was administered. The specificity of the IgG antibody response for the BPO determinant was confirmed by inhibition of binding by BPO-aminocaproate. [3H]BP, administered intravenously to rabbits at a dose of 2.7 mmol/kg was rapidly cleared from plasma, and unchanged BP was not detected at 1 h. After 3 h, irreversible binding accounted for less than 0.004% of the dose bound per milliliter of plasma, and this represented all the radioactivity present in plasma at this time. Covalent binding of BP to plasma proteins, in vitro, after 3 h was of the same magnitude for rabbit, rat and human plasma. Therefore, BP can induce a specific antibody response in the rabbit in contrast to the lack of immunogenicity observed previously in the rat.     

Neutralization test for BK virus: plaque reduction detected by immunoperoxidase staining

We developed an immunoperoxidase staining test to detect structural antigens of BK virus (BKV) in Vero cell cultures. This test was used to examine the neutralizing activity of human and immunized animal sera. It was shown that sera positive for BKV antibodies measured by hemagglutination inhibition test and enzyme-linked immunosorbent assay (ELISA) were able to prevent expression of BKV structural antigens in cell cultures. The correlation between titers in the hemagglutination inhibition test, levels of BKV IgG measured by ELISA, and the titers assayed by the immunoperoxidase neutralization test was high. We suggest that this type of test may be used instead of conventional neutralization tests for other viruses with slowly developing cytopathogenic effects.     

Localization of enkephalin-like immunoreactivity in the cat carotid and aortic body chemoreceptors

The purpose of this study was to determine if enkephalin-like immunoreactivity was present in the glomus cells of the carotid and aortic body peripheral arterial chemoreceptors. Cat carotid and aortic bodies were reacted with antisera to met- and leu-enkephalin using the indirect peroxidase-antiperoxidase immunocytochemical method of Sternberger (1979). Both the carotid and aortic bodies demonstrated clusters of immunoreactive cells for both met- and leu-enkephalin. Additionally, met-enkephalin-like immunoreactivity was observed in many of the dense-core vesicles of the glomus cells of the carotid body. The glomus cells of these chemoreceptors are known to contain catecholamines which may modulate chemoreceptor activity. The presence of opioid peptide-like substances co-existing with the glomus cell catecholamines, perhaps in the same vesicles, may have important implications for a trophic influence of these peptides on glomus cell chemoreceptor modulation.