The use of liposomes as acceptors for the assay of lipid glycosyltransferases from rat brain.

Preparation and characterization of sonicated vesicles of various lipid composition containing hydroxy and normal fatty acid ceramides are reported. Such vesicles have been successfully used for the first time as acceptors for the assays of lipid glycosyltransferases, UDP-galactose:ceramide galactosyltransferase and UDPglucose: ceramide glucosyltransferase. Stability of the vesicles and the optimal enzyme activities were the criteria used to select the final composition of the vesicles. The activities of the glycosyltransferases were dependent not only on the appropriate assay conditions but also on the type and source of the phospholipids used to form the liposomes. Ceramides containing normal fatty acids were incorporated into phosphatidylcholine vesicles in a molar ratio of 1 : 3.4 and used as the acceptor for the assay of UDPglucose:ceramide glucostyltransferase. For the assay UDP-galactose:ceramide galactosyltransferase, vesicles were prepared by sonication of bovine brain ethanolamine phospholipids, phosphatidylcholine and ceramide containing alpha-hydroxy fatty acids, in a molar ratio of 6 : 0.57 : 1. The size of the vesicles as determined by electron microscopic measurement ranged mostly between 200--500 A. The results obtained by selective labelling of the outer surface amino groups with the membrane-impermeable reagent, 2,4,6-trinitrobenzenesulfonic acid, indicated that the ethanolamine phospholipid-containing liposomes consisted of closed vesicles. After incubation with the appropriate cofactors and labelled sugar nucleotides, the radioactive reaction products were shown to cochromatograph with the authentic standards by thin-layer chromatography and autoradiography.

Synergistic effects of laminin and thyroid hormones on neuron polarity in culture.

This study is aimed to elucidate whether triiodothyronine (T3) and laminin have additive effects in regulating neural differentiation. We focused our attention on the expression of synapsin I, the 68 kDa component of the neurofilament triplet (NF-68), growth associated protein (GAP)-43 and microtubule associated protein (MAP)-2 as markers of synapses, cytoskeleton, axons and the somatodendritic domain, respectively. The addition of T3 to the medium of differentiating rat cortical neurons cultured on laminin did not have any effect on the concentration of these proteins, but was critical for their subcellular localization, suggesting a synergistic role of thyroid hormones and laminin in the establishment of neural polarity.

Specific neurons of brain cortex and cerebellum are PIPPin positive.

We recently cloned a cDNA encoding an RNA-binding protein, that we called PIPPin, which is highly enriched in the rat brain and contains two putative double stranded RNA-binding domains (PIP1 and PIP2) and a central cold shock domain (CSD). Here we report that PIPPin is specifically enriched in some pyramidal neurons of the cerebral cortex and in the Purkinje cells of the cerebellum. We also show that PIPPin inhibits translation of H1(o) and H3.3 mRNA in a cell-free system. The results reported suggest that PIPPin down-regulates histone variant expression in the developing rat brain.

Neurons and ECM regulate occludin localization in brain endothelial cells.

We report that extracellular matrix and neurons modulate the expression of occludin, one of the main components of tight junctions, by rat brain endothelial cells (RBE4.B). Of the three extracellular matrix proteins which we tested (collagen I, collagen IV, and laminin), collagen IV stimulated at the best the expression of occludin mRNA. The corresponding protein, however, was not synthesized. Significant amounts of occludin accumulated only when RBE4.B cells were cultured on collagen IV-coated inserts, in the presence of cortical neurons, plated on laminin-coated companion wells. Finally, occludin segregated at the cell periphery, only when endothelial cells were co-cultured with neurons for > or = 1 week.

Formulation of a novel synthetic medium for selectively culturing rat CNS neurons.

Dissociated cells from rat fetal cerebral hemispheres were grown in surface adhering culture using a novel synthetic medium (Maat medium) and compared with those grown either in the presence of serum or in the chemically defined medium described by Bottenstein and Sato. The addition of various compound combinations allowed us to lower insulin concentration to almost physiological levels. Maat medium improved the purity and longevity of neuronal cultures. The purity of neuronal cultures grown in different media was checked both by immunofluorescence and by the analysis of [3H]thymidine incorporation.

Functional feature of a novel model of blood brain barrier: studies on permeation of test compounds.

Drug delivery to the central nervous system (CNS) is subject to the permeability limitations imposed by the blood-brain barrier (BBB). Several systems in vitro have been described to reproduce the physical and biochemical behavior of intact BBB, most of which lack the feature of the in vivo barrier. We developed a fully formed monolayer of RBE4.B immortalized rat brain microvessel endothelial cells (ECs), grown on top of polycarbonate filter inserts with cortical neuronal cells grown on the outside. Neurons induce ECs to synthesize and sort occludin to the cell periphery. Occludin localization is regulated by both compositions of the substratum and soluble signals released by cortical co-cultured neurons. The observed effects do not require strict physical contact among cells and neurons. To assess the physiological function of the barrier we examined the transendothelial transfer of three test compounds: dopamine, L-tryptophan and L-DOPA. Polycarbonate filter inserts, where ECs were co-cultured with neurons, were assumed as open two compartments vertical dynamic models. Permeation studies demonstrated that the ECs/neurons co-cultures possess permeability characteristics approaching those of a functional BBB: the system behaved as a selective interface that excludes dopamine permeation, yet permits L-tryptophan and L-DOPA to cross. The movement of test compounds from the donor to the acceptor compartment was observed at a distinct time from the start of co-culture. Transfer was determined using standard kinetic equations. Different performance was observed after 5 and 7 days of co-culture. After 5 days dopamine, L-tryptophan and L-DOPA passively permeate through the membrane as indicated by fittings with a first-order kinetic process equation. After 7 days of co-culture, occludin localizes at ECs periphery, dopamine does not cross the barrier to any further extent, while the transfer of L-tryptophan and L-DOPA fits well with a saturable Michaelis-Menten kinetic process, thus indicating the involvement of a specific carrier-mediated transport mechanism. Permeation studies confirmed that culture of ECs in the presence of neurons induces the characteristic permeability limitations of a functional BBB.

RNA-protein interactions in the control of stability and localization of messenger RNA (review).

Growing evidence demonstrates the importance of regulating mRNA localization, stability and translation, in the control of gene expression, both in development and in differentiated cells. The signals responsible for specific regulation of mRNA metabolism reside in the RNA message itself: both 5' and 3' to the coding region, all transcripts contain variable lengths of untranslated sequences (5'-UTR and 3'-UTR) which contain the binding sites for a number of RNA-binding proteins (RBPs). Most RBPs assemble on the message at the moment of transcription, thus determining the future fate of the transcript from the very beginning. We discuss possible mechanisms through which mRNA, leaving from the nucleus as an RNA-protein complex, might reach its final intracellular destinations and how its access to the translational apparatus might be regulated in time and space. We also focus on a few known examples of aberrant RNA-protein interactions associated with human diseases, including cancer.

Expression of thyroid hormone receptor isoforms in the hypertrophic heart of spontaneously hypertensive rats.

Thyroid hormones (THs) enhance MHC alpha gene- and repress MHC beta gene-transcription in the heart, by interacting with specific nuclear receptors (TRs), that bind to regulatory sequences localized upstream of basal promoter of myosin heavy chain (MHC) genes. The overall effects of THs include an increase in V1- and a decrease in V3-myosin isozyme concentration in the heart. Myosin V1 contains two MHC alpha chains and has a higher ATPase activity than V3 isoform, which contains two beta chains. Previous studies on papillary muscles of spontaneously hypertensive rats (SHRs) showed that heart hypertrophy is accompanied by a shift from alpha to beta MHC accumulation. The present study was aimed at evaluating whether this event relates to differential expression of alpha1, alpha2, and beta1 isoforms of TRs. At the ages of 8 and 15 weeks, SHRs and Harlan Sprague-Dawley control rats were sacrificed under anesthesia and their hearts were dissected into left and right ventricles, free of atria and great vessels. The results of Western blot analyses showed that the levels of the three TR isoforms do not differ significantly between SHRs and control rats of the same age, either in the left or in the right ventricle. Thus, the expression of MHC beta in SHR hypertrophic heart does not seem to depend on changes in TR isoform concentrations.

Effect of aging and hypertension on beta-myosin heavy chain in heart of spontaneously hypertensive rats.

During aging rat myocardium undergoes structural changes characterized by a shift in the synthesis of myosin heavy chain (MHC) from V1 isoform, composed of two alpha-MHC, to V3 isoform, composed of two beta-MHC. In rat, besides ageing, cardiac hypertrophy as adaptive response to a superimposed pressure load (such as hypertension) is characterized by predominance of V3 myosin isoform. The aim of our study was to evaluate the expression of beta-MHC in right (RV) and left (LV) ventricles of spontaneously hypertensive rats (SHRs), a well defined animal model of hypertension, in relation to aging. We used very young (8-week old) and young (15-week old) SHRs and age-matched normotensive Harlan Sprague-Dawley control rats. By Western analysis, we found that beta-MHC is already present in both RV and LV of 8-week old SHRs, and is markedly predominant in RV and LV of 15-week old SHRs, when compared with age-matched control rats. Our study showed that the shift to V3 myosin isoform in SHRs is an early event, resembling accelerated senescence. We have also demonstrated that beta-MHC is actively synthesized also in young (15-week old) normal rats.

The relative proportion of H1(0) and A24 is reversed in oligodendrocytes during rat brain development.

1. The histone complement of oligodendrocyte chromatin at different stages of brain development was studied after acid extraction of nuclei. 2. HCl-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1(0) with differentiation. 4. The increase in H1(0) is accompanied by a concomitant decrease in the total amount of the ubiquitinated form of histone H2A (A24).

Neuronal cell cultures: a tool for investigations in developmental neurobiology.

The aim of this review is to describe environmental requirements for survival of neuronal cells in culture, and secondly to survey the complex interplay between hormones, neurotrophic factors, transport- and extracellular matrix- proteins, which characterize the developmental program of differentiating neurons. An overall reconsideration of the literature in this vast field is above the limits of the present paper; since progress and refinement in the techniques of neuronal cell cultures have paralleled the advancement in Developmental Neurobiology, we will run instead through the main steps which form the conceptual framework of neuronal cell cultures.

Cellular mechanism of action of thyroid hormones.

It has emerged in the last decade that the molecular mechanism of action of thyroid hormones resembles that of steroids; thyroid hormones indeed exert their effects mainly by directly regulating gene expression, on association with specific chromatin-bound receptors. Of the two thyroid hormones, thyroxine (T4) appears to be a sort of prohormone, whereas triiodothyronine (T3) seems to be the active form; in this respect, T4-deiodination, which occurs at the level of the target tissues, may be crucial in the local homeostasis of T3. Moreover, many cellular compartments, other than the nucleus, can bind thyroid hormone, and at least some of these further sites might play some role in modulating T3 supply to the nucleus. The binding of the T3-receptor complex to chromatin is likely to regulate the structural organization of specific genes and, in some instances, of the chromatin as a whole.

Developmental changes of neuron-specific enolase mRNA in primary cultures of rat neurons.

1. The level of mRNAs for neuron-specific enolase (NSE) and nonneuronal enolase (NNE) was studied in developing rat brain and in pure neuronal cultures of corresponding ages treated or not treated with triiodothyronine (T3). 2. In brain cortices both messages are already detectable at the earliest age (embryonal day 16; E16). During development the mRNA for NNE remains at a steady level, with a transient decline at postnatal day 5 (P5). 3. On the other hand, NSE mRNA follows a biphasic curve: the signal increases threefold from E-16 to P0 and threefold from P5 to P18, with a plateau between P0 and P5. 4. In neuronal cultures the NNE message is present at a constant level until day 10 and declines sharply thereafter, while in T3-treated cultures it reaches a minimum beforehand. 5. The NSE mRNA, on the other hand, increases continuously throughout the whole culture life span, and a slightly higher level is observed in T3-treated cells during the first ten days.

A low repeat length in oligodendrocyte chromatin.

The behavior of oligodendrocyte chromatin after micrococcal nuclease digestion of nuclei was assayed in brains of rats of four different ages. During oligodendrocyte differentiation, a decreasing sensitivity of the chromatin to enzymatic attack was observed. On the other hand, the nucleosomal repeat length showed a slight tendency to increase during development. It is worth noting that even the highest values reported here for "oligodendrocyte" chromatin repeat lengths are significantly lower than 200 base pairs, the value previously reported by others for "non-astrocytic glia."

Rat CNS cell culture. Enhancement of neuronal survival and delay of glial proliferation by serum from patients with multiple sclerosis. A morphological study.

The addition of serum from multiple sclerosis (MS) patients to the culture medium of dissociated cells from cerebral hemispheres of rat embryos caused a delay in glial proliferation and an enhancement of neuronal survival. Sera from normal individuals and patients with other neurological diseases failed to show this effect. These morphological observations are interpreted as the outcome of inhibition of in vitro gliogenesis.

Inhibition of glial proliferation in vitro by serum from patients with multiple sclerosis.

Primary cell cultures from fetal rat CNS have been employed to evaluate the effects caused by the addition of serum from patients affected by multiple sclerosis (MS). MS-serum supplemented media caused a decrease in [3H]-thymidine incorporation into the cultures, thus indicating an inhibitory effect on proliferating glial cells. Sera from patients in remission stage of the disease showed an inhibitory effect not significatively lower than those from patients in acute stage. These results suggest that glial cells may be a target of circulating factors present in MS.

Isolation of the plasma membrane from sea urchin embryos.

A method for isolation of sea urchin embryos plasma membranes is described. Purification of the obtained fraction was assayed by several enzymatic markers and electron microscopy. The isolated plasma membranes appear to be pure from contamination of other cell membranes (endoplasmic reticulum and mitochondria), and they can therefore be used for analytical studies on the composition and structure of plasma membrane.