Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.     

An Anti-β-Amyloid Vaccine for Treating Cognitive Deficits in a Mouse Model of Down Syndrome

In Down syndrome (DS) or trisomy of chromosome 21, the β-amyloid (Aβ) peptide product of the amyloid precursor protein (APP) is present in excess. Evidence points to increased APP gene dose and Aβ as playing a critical role in cognitive difficulties experienced by people with DS. Particularly, Aβ is linked to the late-life emergence of dementia as associated with neuropathological markers of Alzheimer’s disease (AD). At present, no treatment targets Aβ–related pathogenesis in people with DS. Herein we used a vaccine containing the Aβ 1–15 peptide embedded into liposomes together with the adjuvant monophosphoryl lipid A (MPLA). Ts65Dn mice, a model of DS, were immunized with the anti-Aβ vaccine at 5 months of age and were examined for cognitive measures at 8 months of age. The status of basal forebrain cholinergic neurons and brain levels of APP and its proteolytic products were measured. Immunization of Ts65Dn mice resulted in robust anti-Aβ IgG titers, demonstrating the ability of the vaccine to break self-tolerance. The vaccine-induced antibodies reacted with Aβ without detectable binding to either APP or its C-terminal fragments. Vaccination of Ts65Dn mice resulted in a modest, but non-significant reduction in brain Aβ levels relative to vehicle-treated Ts65Dn mice, resulting in similar levels of Aβ as diploid (2N) mice. Importantly, vaccinated Ts65Dn mice showed resolution of memory deficits in the novel object recognition and contextual fear conditioning tests, as well as reduction of cholinergic neuron atrophy. No treatment adverse effects were observed; vaccine did not result in inflammation, cellular infiltration, or hemorrhage. These data are the first to show that an anti-Aβ immunotherapeutic approach may act to target Aβ-related pathology in a mouse model of DS.     

Proliferation-associated expression of DNA methyltransferase in human embryonic lung cells

The cell cycle-dependent and proliferation-associated expression of the enzyme DNA methyltransferase has been evaluated immunocytochemically in synchronized L-132 human embryonic lung cells, using the anti-DNA methyltransferase monoclonal antibody M1F6D7/5C10. DNA methyltransferase-reactivity was firstly seen in mid-G1 cells. An intense and granular reaction in the cell nuclei with a sparing of the nucleoli was observed in addition to a homogenous and faint cytoplasmic staining. The staining intensity in the cell nuclei increased progressively up to mitosis. In early mitotic cells an intense perichromosomal staining was observed in addition to a homogenous staining of cyto- and karyoplasm after the resolving of the core membrane. In late mitosis the staining intensity decreased rapidly. Early G1 cells and density inhibited, resting G0 cells showed no DNA methyltransferase reactivity at all. Our results indicate that anti-DNA methyltransferase monoclonal antibodies could become valuable tools to detect proliferating cells in cell cultures and tissues.     

Stimulation of cellular autophagy by parathyroid hormone and cyclic adenosine 3′,5′: Monophosphate in isolated tubular fragments from the rat’s kidney cortex

Tubular fragments isolated from the cortex of the rat's kidney were qualitatively and quantitatively investigated with the electron microscope. The tubules frequently burst open and became "inverted" in such a way that the rarefied brush border now formed the outer circumference. By morphometry a decrease of the average cell volume in the proximal tubular fragments was ascertained. This was mostly caused by a loss of cytoplasmic ground substance, endoplasmic reticulum and ribosomes. Cytoplasmic herniations of the basal surface, filled with free ribosomes, suggested a partial shedding of the protein synthesizing apparatus. The number of autophagic vacuoles (AV) per unit area of cytoplasm was determined in proximal tubular fragments. After isolation alone, without further incubation, the number of AV was as low as the number found in an earlier study in proximal tubular cells in situ during the diurnal minimum. After control incubation the number of AV increased to about the mean value of the AV found in cells in situ during the whole diurnal cycle. By comparison with the control incubation the number of AV increased by a factor of 1.6 to 1.7, if cyclic adenosine 3',5': monophosphate (cyclic AMP) or parathyroid hormone (PTH) were added to the incubation-medium; it now reached about the number of AV found in situ during the diurnal maximum. The increase in the number of AV paralleled that of the production of ammonia and glucose from endogenous sources under the influence of cyclic AMP and PTH. This suggests that the breakdown of cytoplasmic components by cellular autophagy could be functionally related to gluconeogenesis. A quantitative comparison between the measured production of ammonia and glucose indicates, however, that in the system of isolated tubular fragments there may exist other mechanisms of degradation, and of the provision of substrates for gluconeogenesis, than cellular autophagy only.     

Influence of artificial air ionisation on the human electroencephalogram

Exposure of 20 subjects to negative ionisation was monitored by EEG. Negative ionisation was supplied by an Ionotron apparatus (Amcor-Amron, Herzlia-Israel) with an output of 3.5 × 10⁵ ions/(cm³ · sec) at 1 m distance. Objective findings in ten normal subjects showed reduction of the frequency of the alpha-waves from 10 or 11 down to 9 or 8 Hz, increase of the amplitude by up to 20%, advance of the alpha rhythm pattern from the occipital to the frontal area and general synchronisation of the EEG records of both hemispheres. These reactions were suppressed in 10 subjects by tranquillisers. Subjective findings included relaxation, alertness, improved working capacity and relief from the Serotonin Irritation Syndrome produced by the positive ionisation of hot, dry desert winds.This paper is devoted to the memory of Dr Igho H.Kornblueh, the pioneer of ion therapy, who passed away in 1973 in Philadelphia. Working on this project in 1957 he concluded "that further research on the effects of ionisation on the EEG is warranted".This report was prepared with the technical assistance of Mrs Suzi Alpern and B.Shalita.