PubMed Health⌕ Search

Biomedical subjects

B Garbay

Publications and source records attributed to B Garbay.

At least 19 recordsLinked to original sources
PubMed1997

Acetyl-CoA carboxylase gene expression in the developing mouse brain. Comparison with other genes involved in lipid biosynthesis.

The present study documents the steady-state levels for the mRNAs encoding acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD2) and brain long-chain acyl-CoA synthetase (BLACS) during mouse brain development. It is shown that ACC and FAS mRNA levels are at a maximum 5 days after birth, a time when cell proliferation is intense in the mouse brain, and then decrease steadily to reach 20% of those maximal values at day 20. The ACC transcript isoforms, which were detected in the central nervous system (CNS), originated from promoter P2 of the ACC gene. They encode ACC enzymes which cannot be phosphorylated at the Ser-1200 locus, thus indicating that brain ACC is highly sensitive to citrate activation. The developmental pattern for the SCD2 mRNA level is different from that of true myelin genes, such as CGT. Indeed, the steady-state levels for SCD2 and CGT in 5-day-old brain represent 85% and 5% of their maximal values, respectively. BLACS expression rose during the developmental period studied, but a slow decrease in the mRNA levels was not observed after postnatal day 20, unlike in "myelin-specific' genes. Therefore, it appears that the expression of the genes involved in fatty acid biosynthesis is independent of the myelinating signal in the mouse CNS.

Acetyl-CoA Carboxylase↗
PubMed1996

Acylation of endogenous acyl acceptors by mouse sciatic nerve microsomes.

Phospholipid (chiefly phosphatidylcholine) labeling from radioactive acyl-CoAs by mouse sciatic nerve microsomes is observed in the absence of added acyl acceptors. The maximal acylation (ca 10% of administered) for 10 micrograms microsomal proteins is observed at relatively low amounts of oleoyl-CoA (0.2-0.3 nmol) and decreases as the acyl-CoA amount increases. Labeled lysophosphatidylcholine (almost exclusively esterified at position 2) is also observed, particularly when the [1-14C]oleoyl-CoA concentration is higher than 0.2-0.3 nmol/50 microliters. The labeled acyl group is mainly inserted in position 2] of the glycerophosphorylcholine. With 0.15 nmol labeled oleoyl-CoA, phosphatidylcholine acylation increases as a function of the protein amount and reaches 25% of the added label at 40 microgram proteins. It is evaluated that, in the presence of 10 microgram proteins, 2% of the microsomal phosphatidylcholine molecules are acylated from 0.1 nmol acyl-CoA. The acylation mechanism seems to involve an acyl exchange between acyl-CoA and phosphatidylcholine.

Acyl Coenzyme A↗
PubMed1995

Expression of the exon 1A-containing PMP22 transcript is altered in the trembler mouse.

The trembler mouse suffers from a dominantly inherited mutation of the peripheral myelin protein 22 (PMP22) gene which results in an abnormal myelination of its peripheral nervous system. The recent identification of two different PMP22 mRNA differing in their 5' non-translated region led us to monitor their respective levels of expression in the trembler peripheral nervous system (PNS) during the myelination period. We showed that the steady-state levels of the exon 1A-containing transcript, which is thought to be involved in the myelination process, were greatly reduced in heterozygous and homozygous trembler mice when compared to the normal animals. Such a difference was not observed for the exon 1B-containing transcript. Therefore, our results support the idea that the two alternatively used promoters of the PMP22 gene are under different regulation control, and that the up-regulation of the exon 1A-transcript is necessary for the normal myelination of the mouse PNS.

Animals↗
PubMed1995

Cerebroside formation in the peripheral nervous system of normal and Trembler mice.

The formation of cerebrosides by the galactosylation of ceramides was investigated in a microsomal fraction prepared from sciatic nerves of normal and Trembler mice. In the control, cerebroside synthesis is observed in the presence of uridine-diphosphate-galactose both from endogenously synthesized [1-14C]stearoyl-sphingosine (C18-ceramide), and from [1-14C]lignoceroyl-sphingosine (C24-ceramide). Cerebroside formation is also demonstrated by studying the galactosylation of exogenous ceramides with UDP[1-14C]-galactose. In the mutant, only trace amounts of labeled cerebrosides are formed from labeled stearoyl-sphingosine, whereas with lignoceroyl-sphingosine, no cerebroside synthesis is detected under conditions allowing their formation in the control. However, a higher rate of synthesis of short acyl chain-glucosyl ceramides is observed in the Trembler samples.

Animals↗
PubMed1995

Sphingolipid metabolic disorders in Trembler mouse peripheral nerves in vivo result from an abnormal substrate supply.

Sphingolipid metabolic pathways in the peripheral nerves of dysmyelinating Trembler mice were studied in vivo, using intraneurally injected [3H]palmitate as the exogenous substrate. The kinetic analysis of the experimental data obtained for the mutant revealed that, as in normal nerves, two metabolically and kinetically independent pathways are implicated in the biosynthesis of the major peripheral nerve sphingolipids: the ceramide pathway and another pathway in which there is no detectable labeled intermediate ("direct amidification"). The results also show that, in the Trembler mouse sciatic nerves: (a) The severely deficient sphingolipid biosynthesis results from the constitution of a qualitatively and quantitatively abnormal fatty acid substrate pool destined for metabolism via the ceramide pathway, which ensures the totality of the galactocerebroside labeling and two-thirds of that of sphingomyelin. The ceramide intermediates of this pathway are labeled only on their fatty acyl moiety, which contains only 16-carbon atom chains. (b) "Direct amidification" events implicated in sphingolipid labeling are decreased compared with normal and account for the remaining sphingomyelin formation.

Animals↗
PubMed1994

Expression of the ceramide galactosyltransferase gene during myelination of the mouse nervous system. Comparison with the genes encoding myelin basic proteins, choline kinase and CTP:phosphocholine cytidylyltransferase.

The present study documents the patterns of mRNA expression for the ceramide galactosyltransferase (CGT), the CTP:phosphocholine cytidylyltransferase (CT), and the choline kinase (CK) during the myelination period of the mouse central nervous system (CNS) and peripheral nervous system (PNS). Using the Northern blot technique with densitometric analyses, we show that the CK gene is not developmentally regulated during the period studied, whereas a peak of expression of the CT gene is observed around day 10. On the other hand, the expression of the CGT gene is similar to that of the MBP gene in the CNS and the PNS. Therefore, the synthesis of the galactosylceramides during the myelination period seems to be controlled at the level of the expression of the CGT gene. These results were compared to those of a neurological mutant, the trembler mouse, whose PNS myelination is deficient. Our results clearly indicate that the deficit in the accumulation of the galactosylceramides documented for this mutant is well correlated to a reduced CGT gene expression.

Animals↗
PubMed1994

Over-expression of MBP and PLP messenger RNA in 8-day-old trembler brain.

The trembler mouse suffers from a dominantly inherited mutation of the peripheral myelin protein 22 (PMP-22) gene which results in abnormal myelination of the peripheral nervous system. However, the clinical symptoms observed in the mutant suggest abnormalities in the central nervous system. Using the Northern blot technique, we investigated the steady-state levels of mRNA encoding myelin protein genes in 8-day-old normal and trembler brains. We measured a two- to four-fold increase for myelin basic protein (MBP) and proteolipid protein (PLP) mRNAS in the trembler samples. Whether a relationship exists between this observation and the onset of the clinical symptoms is still to be determined.

Animals↗
PubMed1993

Structure and developmental regulation of Golli-mbp, a 105-kilobase gene that encompasses the myelin basic protein gene and is expressed in cells in the oligodendrocyte lineage in the brain.

We have identified a novel transcription unit of 105 kilobases (called the Golli-mbp gene) that encompasses the mouse myelin basic protein (MBP) gene. Three unique exons within this gene are alternatively spliced into MBP exons and introns to produce a family of MBP gene-related mRNAs that are under individual developmental regulation. These mRNAs are temporally expressed within cells of the oligodendrocyte lineage at progressive stages of differentiation. Thus, the MBP gene is a part of a more complex gene structure, the products of which may play a role in oligodendrocyte differentiation prior to myelination. One Golli-mbp mRNA that encodes a protein antigenically related to MBP is also expressed in the spleen and other non-neural tissues.

Alternative Splicing↗
PubMed1992

DM20 mRNA splice product of the myelin proteolipid protein gene is expressed in the murine heart.

Northern blot analysis of poly A(+) RNA isolated from mouse heart revealed the expression of 3.3 and 2.4 kb mRNAs that hybridized with a cDNA for the mouse proteolipid protein (PLP). In order to examine the relationship of these RNAs to the myelin PLP/DM20 mRNAs, a mouse heart cDNA library was prepared and screened with a mouse PLP cDNA. A cDNA was isolated, sequenced, and found to encode the DM20 variant of PLP. Polymerase chain reaction (PCR) analysis of heart cDNA with three sets of primers confirmed the presence of DM20 mRNA in mouse heart and indicated that it is the major splice product of the PLP gene expressed in that tissue. In situ hybridization localized the expression of the DM20 mRNA to the myocardial cells. Northern blot analysis indicated that expression of the DM20 mRNA is developmentally regulated in the murine heart, increasing significantly in concentration after 12 days postpartum. Northern analysis also revealed the expression of the DM20 mRNA in the hearts of the jimpy and quaking mutants. These results indicate that the PLP gene is expressed in tissues other than brain and support the concept that products of the PLP gene may have some biological role other than as structural components of myelin.

Animals↗
PubMed1992

P0 protein in normal, trembler heterozygous/homozygous mice during active PNS myelination.

The quantification of P0 protein has been currently used to measure the myelination of the peripheral nervous system (PNS). Using immunological techniques, we show that, as early as postnatal day 8, the P0 content of mice homozygous and heterozygous for the Trembler mutation, represent respectively one tenth and half of the normal values. Thus, although the Trembler mutation is dominant, the myelination defect observed in heterozygous Trembler mice is more pronounced in the homozygous Trembler mice. This gene dosage effect should be taken into account when a molecular model of the Trembler mutation will be proposed.

Animals↗
PubMed1992

Expression of the PMP-22 gene in Trembler mutant mice: comparison with the other myelin protein genes.

The Trembler mouse suffers from a dominantly inherited autosomal mutation affecting the Schwann cell activities, which results in an abnormal myelination of the peripheral nervous system. Very recently, it has been shown that the mutation is in the PMP-22 gene. However, the level of expression of the mutated gene in trembler mice has not been studied. Therefore, we measured the steady-state levels of mRNA encoding PMP-22 in the sciatic nerve of normal and trembler mice, and we compared these results with the steady-state levels of mRNAs encoding the major peripheral nervous system myelin proteins. Our results show that all the myelin protein genes studied are affected by the trembler mutation but to a different extent, and that the PMP-22 gene is expressed at very low levels in the trembler nerve. This suggests that regulation of the expression of the PMP-22 gene is altered in the Trembler mutant.

Animals↗
PubMed1990

cDNA library construction from small amounts of unfractionated RNA: association of cDNA synthesis with polymerase chain reaction amplification.

We describe here a general and simple procedure for cDNA library construction making use of in vitro amplification of cDNA by polymerase chain reaction (PCR). The first-strand cDNA is synthesized from total RNA with a primer EcoRI-(dT)17 and oligo(dG) tailed. An oligonucleotide, EcoRI-BamHI-(dC)13, is used to prime the second-strand synthesis by the thermostable DNA polymerase of Thermus aquaticus. The double-stranded cDNA is then amplified directly by PCR. A study of the effect of the elongation time on the PCR products showed that a long extension time is necessary to overcome the size heterogeneity of the cDNA population. Starting from 1 microgram of total brain RNA, the products obtained ranged from 200 to more than 2000 bp. The presence of the myelin basic protein cDNA sequence was determined. A lambda gt10 library containing 2 x 10(6) clones was established with the amplified cDNA. No sequences originating from rRNA were detected by Southern blot analysis. The ability to produce representative cDNA libraries from minute amounts of total RNA by this protocol should have many applications to studies of gene expression in small amounts of tissues or cells.

Animals↗
PubMed1989

Developmental expression of the P0 glycoprotein and basic protein mRNAs in normal and trembler mutant mice.

Mice affected by the autosomal dominant Trembler mutation exhibit a severe hypomyelinization of the PNS. Previous biochemical studies have shown that the accumulation of the major PNS myelin proteins, P0 and myelin basic protein (MBP), is strongly diminished in Trembler sciatic nerves during postnatal development. We performed Northern blots which showed that the size of mRNA species for P0 and MBP in normal and mutant mice are indistinguishable. Densitometric analysis of Northern blots showed that, in normal mice, the proportion of P0 mRNA increases up to the 12th day, then decreases slowly. At day 40, the proportion is 60% of the maximal value. In the mutant, the proportion of P0 mRNA increases up to the 12th day and then decreases much faster than in the control. At days 12 and 40, the P0 mRNA proportion measured in Trembler sciatic nerves represents only 40% and 7%, respectively, of the proportion measured in control littermates. The MBP mRNA proportion in the normal mice increases up to the 16th day, and then decreases to attain 45% of the maximum level at day 40. In the Trembler mouse, there is a maximum level at day 12, representing 25% of the normal level, but the MBP mRNA is barely detectable at days 8 or 40. Thus, these data seem to indicate that in the Trembler sciatic nerves, the proportions of P0 and MBP mRNAs are too small to allow the synthesis of normal levels of the corresponding proteins.

Actins↗
PubMed1988

Po, MBP, histone, and DNA levels in sciatic nerve. Postnatal accumulation studies in normal and trembler mice.

We studied the quantitative changes in proteins (total, Po, MBP, and histones) and DNA from sciatic nerves of normal and Trembler mice during postnatal development. Polyacrylamide gel electrophoresis and immunoblotting procedures allowed an accurate characterization of Po, MBP, and histones, as well as the comparison of their respective amounts from d 2 to d 120 after birth. It was found that 1. The immunoblotting procedure ascertains the presence of Po in the sciatic nerve of Trembler. In the 2-d-old mice, Po is detected in essentially similar amounts in Trembler and normal PNS, whereas its level in adult mutant sciatic nerves is never greater than 20% of the control. The sharp increase in Po levels observed during the third week in the normal nerves is not observed in those of the mutant; 2. MBP species are at most 4% of the control in the 10- to 12-d-old Trembler mice, whereas they were not detectable in adult nerves. The distribution of the different MBP species is the same in both mutant and control mice; 3. In normal mice, Po and MBP accumulate at similar rates, but the 14 kDa MBP accumulates faster than the 18.5 kDa MBP; and 4. Histone and DNA contents decrease 3- to 5-fold in normal nerves, whereas they remain constant, or increase slightly, in the mutant.

Animals↗
PubMed1986

Correlation between the morphology and the lipid and protein compositions in the peripheral nervous system of individual 8-day-old normal and trembler mice.

The hereditary, hypertrophic interstitial neuropathy which afflicts the trembler mouse manifests itself about two weeks after birth. Consequently, the identification of these mutant mice was not possible before this age, except when double mutants were available. We show that the trembler mice can be easily distinguished from their normal littermates before the clinical symptoms appear by using an HPTLC/densitometry technique that allows the simple and rapid analysis of the polar lipids extracted from one sciatic nerve. The results presented in this paper demonstrate important differences between the polar lipid compositions of sciatic nerves from 8-day-old normal and trembler littermates, whose phenotypes were confirmed by the morphological analysis of the contralateral sciatic nerves. The small amount of material that is needed for this identification makes it possible to use the remaining nerve material for other studies. Furthermore, important differences between the sciatic nerve protein compositions of normal and trembler mice, identified according to their polar lipid composition, were also observed and these differences can, therefore, also be employed for the identification of the mutants before the manifestation of the clinical symptoms of the trembler neuropathy.

Animals↗