Alessandro Cestelli

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.

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.

Characterization and developmental changes of UDP-galactose-ceramide galactosyl transferase in a rat CNS axolemma-enriched fraction. Differences and similarities of the enzyme associated with the microsomal and myelin fractions.

Abstract— The properties of rat CNS UDP‐galactose‐ceramidc galactosyltransferase in an axolemma‐enriched fraction (AXL), microsomes, and myelin simultaneously isolated with the AXL was characterized using a newly developed assay system. The microsomal enzyme utilized either magnesium or manganese equally well as the divalent cation at 3.3 mm, while both the myelin and AXL enzyme preferred manganese over magnesium at this concentration. The microsomal enzyme was more stable to heat inactivation than the myelin or AXL enzyme. The AXL galactosyltransferase had the highest specific activity at 15 days (8‐fold higher than that of the microsomes) and dramatically decreased in specific activity with development. The developmental profile of the myelin enzyme paralleled that of the AXL although the absolute specific activity was lower than that of AXL. In contrast, the specific activity of microsomal enzyme was quite low at the earliest age then sharply increased to 25 days and gradually decreased with further development. The specific activity of the enzyme in AXL isolated from Quaking mouse was dramatically decreased (about 5% of control levels) whereas both whole homogenate and microsomal specific activity were decreased to 35% of control levels. These data indicate that AXL and myelin contain a galactosyltransferase with properties which are unique relative to those of the microsomal fraction. The possible functional significance of these findings with respect to myelination is discussed.

PIPPin Is a Brain-specific Protein That Contains a Cold-shock Domain and Binds Specifically to H1° and H3.3 mRNAs

During maturation of mammalian brain, variants of both linker (i.e. H1°) and core (i.e. H3.3) histone proteins accumulate in nerve cells. As the concentration of the corresponding transcripts decreases, in postmitotic cells, even if the genes are actively transcribed, it is likely that regulation of variant histone expression has relevant post-transcriptional components and that cellular factors affect histone mRNA stability and/or translation. Here we report that PIPPin, a protein that is highly enriched in the rat brain and contains a cold-shock domain, binds with high specificity to the transcripts that encode H1° and H3.3 histone variants. Both mRNAs are bound through the very end of their 3′-untranslated region that encompasses the polyadenylation signal. Although PIPPin is present both in the cytoplasm and the nucleus of nerve cells, PIPPin-RNA complexes can be obtained only from nuclear extracts. The results of two-dimensional electrophoretic analysis suggest that a relevant proportion of nuclear PIPPin is more acidic than expected, thus suggesting that its RNA binding activity might be modulated by post-translational modifications, such as phosphorylation.

H1° and H3.3B mRNA levels in developing rat brain

Two overlapping rat cDNAs, covering a continuous region of 1107 base pairs, have been isolated and sequenced. The clones contain identical open reading frames, encoding a 136 amino acid long polypeptide which exhibits 100% identity to other mammalian H3.3 histone variants. We show that the inserts derive, in particular, from the H3.3B gene. We used these inserts and an insert from an H1° encoding clone, previously described (6), as probes to study the accumulation of mRNAs encoding the corresponding histone replacement variants (namely, H1° and H3.3) during rat brain development. We found that the concentration of both H1° and H3.3B mRNAs decreases from the embryonal day 18 (E18) to the postnatal day 10 (P10), with inverse correlation to protein accumulation.

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.

Posttranscriptional regulation of H1° and H3.3B histone genes in differentiating rat cortical neurons

Accumulation of mRNAs encoding H1° and H3.3, two histone replacement variants, was studied in differentiating cortical neurons, cultured in a serum-free medium, with or without triiodothyronine (T3) supplementation. We found that the levels of both H1° and H3.3B mRNAs decrease inisolated neurons between the 2nd and 5th day of culture to the same extent as in vivo. At the same time, an active synthesis of the corresponding proteins was evidenced. The effects of transcription inhibition by actinomycin D and the results of nuclear run-on experiments suggest that H1° and H3.3 expression is regulated mainly at the posttranscriptional level. Concerning T3, only marginal effects were noticed, apart from up-regulation of both histone mRNAs at 2 days in culture. We propose one model for posttranscriptional regulation of the analyzed genes and discuss potential relationships to remodelling of chromatin.

Expression of synapsin I gene in primary cultures of differentiating rat cortical neurons

Synapsin I is a neuron-specific protein which is present in two isoforms, Ia and Ib. In the last few years this protein has been demonstrated to play a central role in the regulation of neurotransmitter release and synaptic plasticity. In this paper the developmental expression of this protein has been investigated in primary neuronal cultures from fetal rat brain cortices. The presence of thyroid hormone in the culture medium stimulates an early expression of the protein without exerting any effect at the level of mRNA transcription and accumulation. These observations implicate a T3-dependent regulation of this neuron-specific gene at the level of mRNA translation.

Triiodothyronine-induced shortening of chromatin repeat length in neurons cultured in a chemically defined medium.

Abstract: At the time of terminal differentiation, mammalian cortical neurons undergo a dramatic change in the structural organization of their chromatin: the nucleosomal repeat length shortens from ∼200 base pairs in fetuses to a value of 165 base pairs after birth. These events occur several days after the end of neuronal proliferation. Previously, we reported that rat cortical neurons cultured in a very selective synthetic medium were not yet programmed to these events at the end of mitotic cycles. Herein, we report that addition of triiodothyronine to neuronal cultures induces a shortening of the chromatin repeat length comparable to the natural one.

H10 RNA-binding Proteins Specifically Expressed in the Rat Brain

During brain maturation, histone H10 accumulates in both nerve and glial cells. The expression of this “linker” histone, the role of which still remains unclear, is a complex process, having both transcriptional and post-transcriptional regulatory components. In particular, the expression of H10 in rat cortical neurons is regulated mainly at the post-transcriptional level, and unknown cellular proteins are likely to affect H10 mRNA stability and/or translation. In looking for such factors, we tested the ability of rat brain extracts to protect H10 RNA probe from degradation by T1 RNase. The results reported here demonstrate that rat brain contains at least one major (p40) and two minor (p110 and p70) binding factors, specific for H10 RNA, all of which are much more or exclusively expressed in adult rat brain, when compared with other tissues. The binding of the factors is confined to a portion of the 3′-untranslated region (3′-UTR), which is highly conserved among murine and human H10 mRNAs. These findings suggest that the proteins identified play a critical role in regulating the expression of H10 histone in the brain of mammals.