Processing of the yeast pre-rRNA at sites A(2) and A(3) is linked.

Cleavage of the yeast pre-rRNA at site A(2) in internal transcribed spacer 1 (ITS1) requires multiple snoRNP species, whereas cleavage at site A(3),located 72 nt 3' in ITS1, requires Rnase MRP. Analyses of mutations in the pre- rRNA have revealed an unexpected link between processing at A(2) and A(3). Small substitution mutations in the 3' flanking sequence at A(2) inhibit processing at site A(3), whereas a small deletion at A(3) has been shown to delay processing at site A(2). Moreover, the combination of mutations in cis at both A(2) and A(3) leads to the synthesis of pre-rRNA species with 5' ends within the mature 18S rRNA sequence, at sites between + 482 and + 496. The simultaneous interference with an snoRNP processing complex at site A(2) and an Rnase MPRP complex at site A(3) may activate a pre-rRNA breakdown pathway. The same aberantpre-rRNA species are observed in strains with mutations in the RNA component of Rnase MRP, consistent with interactions between the processing complexes. Furthermore, genetic depletion of the snoRNA, snR30, has been shown to affect the coupling between cleavage by Rnase MRP and subsequent exonuclease digestion.We conclude that an sno-RNP-dependent processing complex that is required for A(2) cleavage and that recognizes the 3' flanking sequence at A(2), interacts with the RNase MRP complex bound to the pre-rRNA around site A(3).     

Cultured gill epithelia as models for the freshwater fish gill

We review recent progress in the development of models for the freshwater teleost gill based on reconstructed flat epithelia grown on permeable filter supports in primary culture. Methods are available for single-seeded insert (SSI) preparations consisting of pavement cells (PVCs) only from trout and tilapia, and double-seeded insert (DSI) preparations from trout, containing both PVCs (85%) and mitochondria-rich cells (MRCs, 15%), as in the intact gill. While there are some quantitative differences, both SSI and DSI epithelia manifest electrical and passive permeability characteristics typical of intact gills and representative of very tight epithelia. Both preparations withstand apical freshwater exposure, exhibiting large increases in transepithelial resistance (TER), negative transepithelial potential (TEP), and low rates of ion loss, but there is only a small active apical-to-basolateral "influx" of Cl(-) (and not of Na(+)). Responses to various hormonal treatments are described (thyroid hormone T3, prolactin, and cortisol). Cortisol has the most marked effects, stimulating Na(+),K(+)-ATPase activity and promoting active Na(+) and Cl(-) influxes in DSI preparations, and raising TER and reducing passive ion effluxes in both epithelia via reductions in paracellular permeability. Experiments using DSI epithelia lacking Na(+) uptake demonstrate that both NH(3) and NH(4)(+) diffusion occur, but are not large enough to account for normal rates of branchial ammonia excretion, suggesting that Na(+)-linked carrier-mediated processes are important for ammonia excretion in vivo. Future research goals are suggested.     

Targeting NADPH Oxidase and Phospholipases A2 in Alzheimer’s Disease

Alzheimer’s disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Aβ) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Aβ. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Aβ in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A₂ (PLA₂) and secretory PLA₂. In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Aβ NADPH oxidase and PLA₂ can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.     

Evaluation of the Qualitative and Automated Quantitative Microhemagglutination Assay for Antibodies to Treponema pallidum

Qualitative and quantitative microhemagglutination assays for antibodies to Treponema pallidum (MHA-TP) were performed on 314 syphilitic and 597 presumably nonsyphilitic sera, and the results were compared with those of the fluorescent treponemal antibody-absorbed (FTA-ABS), the Treponema pallidum immobilization (TPI), and the Veneral Disease Research Laboratory (VDRL) tests. MHA-TP sensitivity was similar to that of the other tests in all stages of syphilis except primary syphilis, in which MHA-TP reactivity was only 64% compared with 82% in the FTA-ABS test, 73% in the VDRL test, and 67% in the TPI test. MHA-TP specificity was satisfactory and comparable to that of the other treponemal tests. Quantitation of the MHA-TP test was automated by use of Autotiter II equipment. Titers tended to become elevated later in the course of syphilis and to remain elevated longer than did VDRL titers. Reproducibility of the quantitative MHA-TP test was satisfactory, with duplicate tests agreeing within one doubling dilution on 97.5% of 351 reactive sera. Poor reproducibility was obtained with sera giving minimal reactions in the qualitative test, and such sera should be routinely retested. The MHA-TP is less time-consuming and costly than the FTA-ABS test and could be used in conjunction with the VDRL or another reagin test for syphilis to eliminate a large number of the FTA-ABS tests now required.     

CGS 10746B: an atypical antipsychotic candidate that selectively decreases dopamine release at behaviorally effective doses

CGS 10746B, a benzothiadiazepine, has a behavioral profile in mice and monkeys similar to the atypical antipsychotic clozapine. Unlike clozapine, CGS 10746B suppresses dopamine neuron firing rates and, when administered at behaviorally effective doses by the oral or intraperitoneal route, decreases neostriatal dopamine release without changing dopamine metabolism or occupying D2 receptors. CGS 10746B is the first atypical antipsychotic candidate that selectively decreases dopamine release.     

Schistosoma mansoni: serotonin uptake and its drug inhibition

5-Hydroxytryptamine (serotonin) uptake by male and female Schistosoma mansoni was studied in vitro. Initial uptake was determined during the first 2 min of incubation with the indoleamine. There were two types of uptake: a saturable component that is apparent at low concentrations of serotonin, and a passive component that predominates at high concentrations. Female S. mansoni take up more 5-hydroxytryptamine per milligram protein than do the males. Studies on the distribution of 5-hydroxytryptamine taken up by the parasites show that a large proportion of the indoleamine is in the tegument. The female tegument had a greater proportion of the 5-hydroxytryptamine taken up than did the male tegument. The evidence indicated that 5-hydroxytryptamine associated with tegument represented the passive uptake while uptake in the main parasite body was both active and passive. Compounds that were shown to inhibit 5-hydroxytryptamine uptake include methylclonazepam, metergoline, imipramine, and ouabain. Maximal inhibition by these compounds ranged from 50 to 60%. Inhibition appeared to be confined to the saturable uptake component of the body fraction.     

Preparation of standards and determination of sizes of long-chain polyphosphates by gel electrophoresis

Procedures are presented for isolating fractions of long-chain polyphosphates which have a narrow range of sizes and for determining their chain lengths. The polyphosphates are isolated by elution from preparative polyacrylamide gels. Then, the lengths of these polymers are determined by a method of successive approximations of length from data obtained by electrophoresis on several different gels of varying polyacrylamide concentrations. Once sized, these isolated polyphosphates may be used as electrophoresis standards, making it possible to rapidly and accurately ascertain the size of other samples having unknown chain lengths. By comparison with two other procedures for sizing polyphosphates, it is shown that the method is definitely valid to a length of 450 and most likely to a length of at least 900. This electrophoresis procedure allows, for the first time, the determination of the range of sizes present and the average chain length with only 2-20 micrograms of polyphosphate.     

Pathogenicity of mouse hepatitis virus for preimplantation mouse embryos

Mouse embryos which were hatched from the zona pellucida in vitro in the presence of mouse hepatitis virus (MHV) or outgrown on coverslips and then exposed to MHV were shown by immunohistochemical staining to have virally infected trophoblast cells. Zona-intact embryos incubated with MHV for 48 h (2-cell embryos) or 1.5 h (blastocysts) were resistant to infection. Morulae and early blastocysts collected from donor mice experimentally infected with MHV were not infected, but the medium in which they were flushed from the uterine horns was contaminated with virus. No virus was detected after embryos were washed through three changes of uncontaminated medium. MHV was transmitted to foster mothers when embryos were transferred in medium contaminated with the virus. Fetal and decidual tissues were not infected. We suggest that embryo transfer is an effective and simple alternative to Caesarian rederivation of MHV-contaminated mice.     

LIPID AND PROTEIN CHANGES IN SCIATIC NERVE DURING WALLERIAN DEGENERATION

Lipid and protein changes have been measured and correlated during early Wallerian degeneration in the same rat sciatic nerve. The major structural glycoprotein disappears at a steady rate and little remains after 8 days. There is a concomitant appearance of a protein with the same molecular size as basic protein B₂ and which is probably formed from the glycoprotein. Basic protein (B₁) is slowly lost, but Wolfgram protein tends to increase possibly because of glial cell proliferation. Cholesterol ester has appeared 3 days after sectioning, while cholesterol and probably cerebroside are reduced. Triglyceride levels show considerable variation, but biphasic increases tended to occur at 3-4 and 10-14 days. Loss of phospholipids is a later event. The changes in lipid and protein are discussed in relation to the stability of the peripheral nerve myelin membrane.