Widespread occurrence of AP in amyloidotic tissues. An immunohistochemical observation

Plasma (P)-component of amyloid (AP or SAP), while not an integral part of the amyloid fibril, has been considered to be intimately associated with virtually every different type of amyloid. In the present study, we evaluated the distribution of AP in the organs frequently involved in two forms of human systemic amyloidosis (AA and AF) and in mouse AA amyloidosis, by use of immunohistochemistry with anti-AP. Although the amyloid deposits generally showed moderate reactions with anti-AP, they were not always clearly distinguished from the surrounding non-amyloid tissue elements which often stained as well. The basement membrane often showed even stronger reaction to anti-AP than the adjacent amyloid deposits, and liver sections demonstrated such a high overall reaction to anti-AP that the anti-AP reaction on the amyloid deposits was often obscurred. The present results suggest that the binding between AP and the amyloid fibril may not be monospecific, that AP by this technique occurs rather widely throughout the body, and therefore that anti-AP may not be considered as specific a marker for amyloid deposits in immunohistochemical and perhaps other studies as well.     

Limiting dilution analysis of the frequency of antigen-reactive lymphocytes isolated from the central nervous system of Lewis rats with experimental allergic encephalomyelitis

Lymphocytes were isolated from the spinal cord and draining lymph nodes of Lewis rats with acute experimental allergic encephalomyelitis (EAE) 12 days after immunization with myelin basic protein (MBP) and tetanus toxoid (TT). An average of 8.0 +/- 2.0 X 10(6) cells was obtained from the spinal cord. Of these 71.1 +/- 8.6% expressed the helper-T-cell marker W3/25 and 14.8 +/- 6.2% expressed the killer/suppressor-T-cell marker OX8. By limiting dilution analysis of cells exhibiting an antigen-specific proliferative response, the average frequencies of cells reactive to MBP and TT were 3.36 +/- 2.4 and 7.60 +/- 4.1 per 10(4), respectively. In the draining lymph nodes, the frequencies of cells reactive to MBP and TT were 2.24 +/- 1.7 and 2.69 +/- 2.5 per 10(4). At a relatively early stage of clinical EAE, MBP-reactive T cells comprise only a small minority of the cells which can be isolated from the spinal cord; lymphocytes reactive to a protein antigen irrelevant to EAE pathogenesis are present in comparable numbers. This finding suggests that most of these cells accumulate as a result of mechanisms not specific for MBP-reactive lymphocytes.     

In vitro anti-HIV activity of phosphorothioate alpha-anomeric oligodeoxynucleotides

Oligonucleotide analogs consisting exclusively of alpha-anomeric deoxynucleoside units bridged with phosphorothioate linkages have been synthesized and tested in vitro as antiviral agents against human immunodeficiency virus (HIV) in human T cells. Two 28-mers, an homopolymer alpha-S-dC28 and an oligomer alpha-S-anti-rev complementary to the initiation site of the regulatory viral gene rev exhibited antiviral activities comparable to those reported for the corresponding beta-anomeric phosphorothioate analogs. In contrast, a nuclease-resistant homopolymer, alpha-dC28 was inactive. Their preliminary results would indicate that the origin of oligonucleotide phosphorothioate anti-HIV activity is not exclusively correlated with their higher nuclease resistance.     

Chemical modification of the Na+/H+ exchanger of thymic lymphocytes. Inhibition by N-ethylmaleimide

A Na+/H+ exchanger is involved in the regulation of cytoplasmic pH and cellular volume in a variety of cells. Little is known about the molecular nature of this exchanger. The purpose of this study was to survey a variety of group-specific covalent reagents as potential inhibitors of the exchanger. Na+/H+ countertransport activity was assayed as the amiloride-sensitive rate of Na+-induced alkalinization in acid-loaded lymphocytes, or as the rate of swelling in cells suspended in sodium propionate medium. Activity was not affected by proteinases or by carboxyl-group and amino-group specific reagents. A significant inhibition was produced by diethylpyrocarbonate, a histidine-specific reagent and by N-ethylmaleimide, a sulfhydryl group reagent. A similarly reactive but nonpermeating sulfhydryl agent, glutathione-maleimide, failed to inhibit Na+-H+ exchange. Moreover, the reaction with N-ethylmaleimide was sensitive to changes in the cytoplasmic pH. The data suggest that the chemically reactive groups of the Na+/H+ exchanger of lymphocytes have limited exposure to the extracellular medium but that an internally located sulfhydryl group is critical for the cation-exchange activity.     

Genomic Consequences of Ecological Speciation in Astyanax Cavefish

The cave environment is consistently radically different than the surface environment because it lacks light, and animals adapting to cave life are subject to strong selective forces much different than those experienced by their ancestors who evolved in the presence of light. As such, their divergence from surface ancestors and eventual speciation is likely to be driven by the shift in ecology. We report here that hybrids between cave and surface Astyanax mexicanus fishes produce offspring with allelic frequencies that differ significantly from Mendelian expectations both for transmission ratios and for independent assortment of unlinked markers. Comparison of allelic content of DNA from fin clips and sperm pools show that the transmission ratio distortion likely occurs during spermatogenesis. Departures from expectations of independent assortment are essentially epistatic phenomena generating linkage disequilibrium. A novel analysis of the epistatic interactions reveals an apparent network of interactions among genes known or suspected to be involved in cave adaptation, implying that the epistasis arose as a “by product” of the divergence due to cave adaptation.     

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.