Postembedding Staining of Brain Tissue for Ultrastructural Visualization of Amyloid in Alzheimer's Disease

A postembedding staining method is presented for ultrastructural visualization of amyloid deposits in brain sections from patients with Alzheimer's disease. Methenamine silver stain is applied to thin sections of tissue embedded in the acrylic resin LR Gold. Senile plaques are easily labeled by silver granules and the ultrastructural detail is well preserved. When staining time is prolonged, silver precipitate also is deposited on neuronal paired helical filaments. This method overcomes the drawbacks of previously reported applications of the stain on Vibra-tome and Epon sections. Thin sections from the same tissue block can be immunostained with antibodies to various plaque components, thus allowing comparative studies at the electron microscope level.     

Increase of hybridoma productivity using an original dialysis culture system

Hybridoma cell growth and monoclonal antibody production were investigated with a laboratory-made system in which cells were grown in dialysis tubing (MW cut-off 25 kD). The dialysis system contained 10 ml of cell suspension and was immersed in 200 ml of culture medium which when replaced or was at 4-day intervals. With this system, monoclonal antibody concentrations similar to those observed in ascites (concentrations in the order of one gramme per liter) were obtained. With no medium replacement, the antibody production was 3.3 g/l and the cell productivity 3.2×10^–8 g of IgM produced per cell in one minute. With medium replacement the antibody production was higher, 4.4 g/l but the cell productivity was lower, 1.49×10^–8 g per cell in one minute. Cells cultivated in non-optimized conditions were better producers than cells growing in a good environment.Abbreviations FCS fetal calf serum - Ig immunoglobulin - MAb monoclonal antibody - MW molecular weight - MWCO molecular weight cut off - RM replaced medium - NRM non replaced medium     

Karyotype morphology in Hyperiella dilatata Stebbing 1888 (Amphipoda: Hyperiidae) from the Ross Sea (Antarctica)

Summary The chromosome complement and some karyological features were investigated in the pelagic amphipod Hyperiella dilatata Stebbing 1888 from the Ross Sea (Antarctica). The diploid karyotype consists of 48 metacentric and 10 submetacentric elements (2n = 58). The presence of secondary constrictions and supernumerary chromosomes is described. Available chromosome numbers of Hyperiidea exhibit a wide range of distribution, among which Hyperiella dilatata is the closest to the modal number of other amphipods.     

Soluble or Bound Laminin Elicit in Human Neuroblastoma Cells Short- or Long-Term Potentiation of a K+ Inwardly Rectifying Current: Relevance to Neuritogenesis

Changes in the resting potential (V_rest) and in the underlying ionic conductances were measured by the patch-clamp technique in SH-SY5Y human neuroblastoma cells exposed to substrate-bound or soluble Laminin (bLN: sLN), as compared to integrin-independent substrates (polylysine (PL); bovine serum albumin (BSA)). While PL and BSA were ineffective, both forms of LN caused an early (5-15 min) activation of a peculiar type of Inwardly Rectifying K^- current (I,_ir) characterised by a voltage-dependent inactivation in the range of membrane potentials around —50/0 mV. I_ir was blocked by Cs⁺ ions and by the antiarrhythmic drug E-4031, a specific inhibitor of the HERG-codified channels. In cells adherent to bLN, I,_ir potentiation (85%) persisted for 90-120 min and was accompanied by a similar, but transient, increase in the leakage conductance (G_l). Successively, the persistence of a high I_ir conductance and the decrease of G_l progressively bring V_rest from -12 to -30 mV in about 120 min. On the other hand, in cells adherent to PL, exposure to sLN produced a similar but not persistent activation of I_ir, without any increase in G_l: this caused a rapid, transient hyperpolarisation of V_rest The effects of bLN and sLN were mimicked by antibodies raised against the integrin β₁ subunit, suggesting a specific integrin-mediated mechanism. In fact, when bound to the culture dishes, these antibodies simultaneously activated the I_ir and G_l, whereas in soluble form they only activated I_ir. Cells adherent to bLN emitted neurites, a process impaired by the block of I_ir by E-4031 and Cs⁺. On the whole data suggest that the integrin-mediated activation of I_ir plays a crucial role in the commitment to neuritogenesis of neuroblastoma cells, independently on the effects of this activation on V_rest.     

Administration of 8-Hydroxy-2-(Di-n-Propylamino)tetralin in Raphe Nuclei Dorsalis and Medianus Reduces Serotonin Synthesis in the Rat Brain: Differences in Potency and Regional Sensitivity

Following administration of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.04-5.0 micrograms/0.5 microliter) in the raphe nucleus dorsalis (DR) or medianus (MR), the synthesis of serotonin (5-HT), as assessed by the accumulation of 5-hydroxytryptophan (5-HTP) after decarboxylase inhibition, was measured in various regions of the rat CNS. At all doses, 8-OH-DPAT in the DR significantly reduced 5-HTP accumulation in the striatum, nucleus accumbens, cortex, and prefrontal cortex, whereas even the highest dose had no effect in the hippocampus, hypothalamus, and spinal cord. One microgram of 8-OH-DPAT in the MR significantly reduced 5-HTP accumulation in the nucleus accumbens and prefrontal cortex, and 5 micrograms had an effect in all the areas except the striatum and spinal cord. One and 5 micrograms of 8-OH-DPAT, administered in either the DR or MR, did not significantly modify the accumulation of dihydroxyphenylalanine in the striatum and nucleus accumbens. The results confirm that DR and MR have different sensitivities to 5-HT1A receptor agonists, and that activation of 5-HT1A receptors in these nuclei produces different effects on 5-HT synthesis in different brain regions.     

Two Methods for Flat Embedding Sections in LR White Cut from Paraffin Blocks

Two simple methods are described for flat embedding of sections cut from paraffin blocks of brain tissue in the hydrophilic resin LR White. The ability of fresh resin to polymerize when added to already cured resin is exploited. In the first method, a tissue section with a drop of LR White is pressed to the bottom of a gelatin capsule using a blank block. In the second method, the section in a drop of resin is sandwiched between the sawed halves of a blank block. After curing, thin sections containing large areas of tissue can be collected and processed for immunocytochemistr y.     

Superoxide Reaction with Nitroxides

Stable, free radical nitroxides are commonly used ESR spectroscopy tools. However, it has recently been found that ESR observable signal from 5-membered ring spin-adducts or stable label nitroxides is lost or diminished by reaction with superoxide. A similar radical-radical annihilation was not found for six membered ring nitroxide radicals. To discern why six-membered ring nitroxides are not reduced under superoxide flux generated by hypoxanthine/xanthine oxidase, spectrophoprmetric (Cyt C) and chemilu-minescence (lucigenin) and ESR assays were used to follow the reactions. Spectrophotometry and chemi-luminescence clearly demonstrated that the six-membered piperidine-I-oxyl compounds (TEMPO, TEMPOL, and TEMPAMIN) rapidly react with superoxide: rate constants at pH 7.8 ranging from 7 × 10⁴ to 1.2 × 10^-5M^-1s^-l. The absence of detectable ESR signal loss results from facile re-oxidation of the corresponding hydroxylamine by superoxide. To fully corroborate the efficiency of the 6-membered nitroxide superoxide dismutase activity, they were shown to protect fully mammalian cells from oxidative damage resulting from exposure to the superoxide and hydrogen peroxide generating system hypoxanthine/ xanthine oxidase. Since six-membered cyclic nitroxides react with superoxide about 2 orders of magnitude faster than the corresponding 5-membered ring nitroxides. they may ultimately be more useful as superoxide oxide dismutase mimetic agents.     

Salinity stress increases cytoplasmic ca activity in maize root protoplasts

High concentrations of NaCl immediately elevated cytoplasmic Ca activity in maize (Zea Mays L. cv Pioneer 3377) root protoplasts, as measured with the fluorescent probe Indo-1. The effect of salinity was inhibited by Li pretreatment but restored by inositol, suggesting that phosphoinositides mediate the stress response.     

OVULE ABORTION IN ‘NONPAREIL’ ALMOND (PRUNUS DULCIS [MILL.] D. A. WEBB)

The earliest indication of ovule abortion in almond (Prunus dulcis [Mill.] D. A. Webb ‘Nonpareil‘) is the deposition of callose (as indicated by aniline blue fluorescence) 2 days after pollination which is 2 days before clear histological symptoms of ovule degeneration are evident and 6 days before fertilization of the viable ovule. Callose deposition begins in the chalazal region of the nucellus where the funicular trace enters the ovule and ramifies into the integuments. As ovule abortion progresses, callose deposition in the inner integument extends as a ring around the nucellus. Movement of the fluorescent dye disodium fluorescein (uranin) indicated that translocation from the vascular trace into abortive ovules becomes blocked at the chalazal position. The dye freely penetrates and diffuses into viable ovules but fails to penetrate abortive ovules. Lack of, or delayed and irregular, megagametophyte development was another characteristic of abortive ovules. Biochemical and histochemical analyses of abortive and viable ovules indicated that carbohydrate depletion parallels ovule abortion. These observations lead to the conclusion that ovule abortion is accompanied by blockage in metabolite supply although whether this blockage is the primary cause or a consequence of ovule abortion is uncertain.