Equipment for Simultaneous Processing of Groups of Slides for Radioautography

The equipment consists of commercially available 5-slide plastic grips (obtained from Lipshaw Manufacturing Co., 7446 Central Ave., Detroit, Mich. 48210) and specially designed lightproof exposure boxes to hold the grips. After deparaffinization and hydration, slides are inserted into the grips, dipped in liquid emulsion, drained, and sealed in the exposure box. After exposure, the 5-slide units are removed from the box (in darkness) and inserted into specially made Plexiglas racks that hold four 5-slide units (20 slides). Photographic developing, fixing, and washing are performed by immersing the 20-slide group in fluids contained in 30-slide glass staining dishes (Lipshaw No. 122 dishes). The equipment simplifies darkroom operations and provides means for standardizing the preparation of radioautographs.     

Serotonin Metabolism by Monoamine Oxidase in Rat Primary Astrocyte Cultures

The oxidative deamination of serotonin (5-HT) to 5-hydroxyindoleacetic acid (5-HIAA) by rat primary astrocyte cultures was investigated in intact cells using HPLC. All detectable 5-HIAA accumulated in the extracellular medium, and its rate of production was proportional to the 5-HT concentration over the tested range of 5 x 10(-7) to 10(-4) M. At 5 x 10(-7) M 5-HT, intracellular 5-HT was detectable only in astrocytes treated with monoamine oxidase (MAO) inhibitors. These findings are consistent with the idea that 5-HT taken up into astrocytes is not stored for re-release, but is rapidly metabolized to 5-HIAA, which is then extruded from the cell. At 5 x 10(-7) M 5-HT, 5-HIAA formation in intact cells was blocked 63% by the selective high-affinity 5-HT uptake inhibitor fluoxetine. 5-HT oxidation to 5-HIAA is carried out principally by MAO-A, because clorgyline was more effective at inhibiting the production of 5-HIAA than was pargyline. Radioenzymatic determinations of MAO activity in cell homogenates supported these findings, because under these conditions clorgyline was 1,000-fold more effective than pargyline at inhibiting MAO activity toward 14C-labelled 5-HT. However, the relatively selective MAO-B substrate beta-phenylethylamine (PEA) was also oxidized, showing that these cultures also contained MAO-B activity; the Km values for MAO-A oxidation of 5-HT and MAO-B oxidation of PEA were 135 and 45 microM, and Vmax values were 88 and 91 nmol/mg of total cell protein/h, respectively. Higher concentrations of PEA (greater than 20 microM) were oxidized by both MAO-A and MAO-B isozymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamines and Ca2+ Mediate Hyperosmolal Opening of the Blood-Brain Barrier: In Vitro Studies in Isolated Rat Cerebral Capillaries

We recently presented evidence that the reversible opening of the blood-brain barrier (BBB) by the infusion of 1.6 M mannitol into the rat internal carotid artery is mediated by a rapid stimulation of ornithine decarboxylase (ODC) activity and putrescine synthesis in cerebral capillaries. We have now investigated this hypothesis further, using isolated rat cerebral capillaries as an in vitro model of the BBB. The ODC activity of cerebral capillary preparations was enriched up to 15-fold over that of the cerebral homogenate. Hyperosmolal mannitol in physiological buffer evoked a rapid (less than 15 s), concentration- and time-dependent increase in capillary ODC activity and an accumulation of putrescine and spermidine which was blocked by the specific ODC inhibitor, alpha-difluoromethylornithine (DFMO, 10 mM). Mannitol (1 M), as well as 2 M urea, evoked a two- to fivefold increase in the temperature-sensitive influx of 45Ca2+ and uptake of horseradish peroxidase (HRP) and 2-deoxy-D-[1-3H]glucose (DG), but not alpha-[1-14C]aminoisobutyrate, during a 2-min incubation. DFMO (10 mM) abolished 1 M mannitol-mediated stimulation of 45Ca2+ influx and uptake of HRP and DG, whereas 1 mM putrescine replenished capillary polyamines and reversed the DFMO effects. Mannitol (1 M)-induced stimulation of ODC activity and membrane transport processes was Ca2+-dependent and verapamil- and nisoldipine-sensitive. Phorbol myristate acetate (PMA, 10 nM), a protein kinase C activator, also evoked a two- to threefold stimulation of 45Ca2+ transport and HRP and DG uptake. This PMA effect was abolished by DFMO, suggesting involvement of rapid, ODC-controlled polyamine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cell-specific fatty acylation of proteins in cultured cells of neuronal and glial origin

Distinct sets of cellular proteins were labeled with [³H]myristic and [³H]palmitic acids in primary (rat neurons and astroglia) and continuous (murine N1E-115 neuroblastoma and rat C6 glioma) cell cultures derived from the nervous system. Both soluble and membrane proteins were modified by myristate in a hydroxylamine-stable (amide) linkage, while palmitoylated proteins were esterlinked and almost exclusively membrane bound. Chain elongation of both labeled fatty acids prior to acylation was observed, but no protein amide-liked [³H]myristate originating from [³H]palmitate was detected. Fatty acylation profiles differed considerably among most of the cell lines, except for rat astroglial and glioma cells in which myristoylated proteins appeared to be almost identical based on SDS gel electrophoresis. An unidentified 47 kDa myristoylated protein was labeled to a significantly greater extent in astroglial than in glioma cells; the expression of this protein could be related to transformation or development in cells of glial origin.     

SSCP analysis of the tyrosine kinase domain of the insulin receptor gene: Polymorphisms detected in South African black and white subjects

The frequency of DNA polymorphisms in the tyrosine kinase domain (exons 17–21) of the insulin receptor gene was assessed in 30 black and 30 white South Africans, using single-stranded conformation polymorphism and direct sequencing analysis. A comparison of the frequencies of the normal versus the combined polymorphic alleles, found only in exon 17, showed a significant difference between black and white groups (P = 0.037). Received: 25 May 1995 / Revised: 1 September 1995     

Skeptical responding in rhesus monkeys (Macaca mulatta)

Signalers that misinform sufficiently open may become devalued as sources of information; however, “skepticism” and any comparison involved in testing reliability entail a cost that involves delays and energy expenditure. Skepticism may be less costly though, if, as a rule, animals are not equally skeptical of the signals of all conspecifics. Animals with the ability to recognize individual conspecifics and to recall past encounters with them may have the capacity to restrict skepticism to subsets of animals that are most likely to benefit from deception. We played tape-recorded alarm calls of high- and low-ranking rhesus monkeys(Macaca mulatta) to their groups in a feeding context once daily over 8 consecutive days at the Yerkes Primate Center Field Station. Over the sequence of playbacks, response was greater to the calls of high-ranking monkeys, adult response patterns were different from those of juveniles, and for adults especially, decline in responsiveness was punctuated by partial resurgences of response. These differences may be the consequence of the adults’ more extensive histories of interaction with group members that, though generally reliable, vary with respect to the potential benefits of deceptive signaling.     

A comparison of the predicted and X-ray structures of angiogenin. Implications for further studies of model building of homologous proteins

The three-dimensional structure of human angiogenin has been determined by X-ray crystallography and is compared here with an earlier model which predicted its structure, based on the homology of angiogenin with bovine pancreatic ribonuclease A. Comparison of the predicted model and crystal structure shows that the active-site histidine residues and the core of the angiogenin molecule, including most of the-strands and-helices, were predicted reasonably well. However, the structure of the surface loop regions and residues near the truncated C-terminus differs significantly. The C-terminal segment includes the active-site residues Asp-116, Gln-117, and Ser-118; Gln-117 in particular has been shown to be important in affecting the ribonucleolytic activity of angiogenin. Also, the orientation of one helix in the model differed from the orientation observed experimentally by about 20°, resulting in a large displacement of this chain segment. The difficulty encountered in predicting the surface loop regions has led to a new algorithm [Palmer and Scheraga (1991),J. Comput. Chem.,12, 505–526; (1992),J. Comput. Chem.,13, 329–350] for predicting the conformations of surface loops.