Nucleic acids complexation with cationic elastin-like polypeptides: Stoichiometry and stability of nano-assemblies.

Positively charged elastin-like polypeptides (ELPs) were synthesized for the compaction of genetic material. A recombinant ELP (VPGXG)40 with X = V,M (3:1) was post-modified in two steps to introduce chemoselectively either primary or secondary amine pendant groups at each methionine residue. Positively charged ELPs were characterized by SDS-PAGE, size exclusion chromatography, 1H NMR, potentiometric titrations and dynamic light scattering to assess their purity and determine their degree of functionalization, molecular weight, isoelectric point and thermo-responsive behaviour. Electrostatic complexation between the different ELP derivatives and nucleic acids was studied to determine the stoichiometry of ELPS/nucleic acids complex formation, and to find optimal conditions leading to stable nanoparticles with controlled size and surface potential. The stability of these complexes was investigated in the presence of salts at physiological concentrations and in the presence of surfactant. This study revealed that two regimes of stable nanoparticles in terms of size and charge can be obtained from the electrostatic complexation between the primary amine containing ELP derivative, ELP(-NH2), and plasmid DNA. Resulting complexes were found to be stable to dissociation for charge ratios up to 2.5 under physiological salt concentrations (154 mM NaCl), showing that plasmid DNA was completely condensed by the polycationic ELP and protected against electrolyte-mediated dissociation.

Exploring the expression of Pseudomonas aeruginosa genes directly from sputa of cystic fibrosis patients.

UNLABELLED: Acquisition of Pseudomonas aeruginosa is known as a negative prognostic factor in patients with cystic fibrosis. We started a pilot study to evaluate Ps. aeruginosa gene expression directly from the sputum of infected patients. Total RNA was purified from 15 sputum samples collected from 10 patients, and the expression levels of five genes from Ps. aeruginosa were measured by RT-qPCR. Expression of algD, algR, antB, lasB and pqsA genes was determined in sputa that contained Ps. aeruginosa cells. The resultant data provided an overview of the expression of these genes in CF patients. Except for the correlation between algD expression and the mucoid phenotype, the gene expression profile could not be associated with the clinical status of patients. However, beyond the heterogeneity of the Ps. aeruginosa phenotype in sputum, we observed a correlation between the expression of antB and pqsA and a low level of lasB transcripts. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas aeruginosa infection leads to high morbidity and mortality in cystic fibrosis patients. The identification of Ps. aeruginosa-assigned factors is important to eradicate the colonization. We started a pilot study to evaluate the gene expression of Ps. aeruginosa directly from the sputum of infected patients. Preliminary results suggest that beyond the heterogeneity of the Ps. aeruginosa phenotype in sputum, we observe a correlation between the expression of antB and pqsA and a low level of lasB transcripts. This approach could shed some light on the behaviour of Ps. aeruginosa during pulmonary infection and may reveal some important elements for optimizing therapy.

Cation exchange versus multimodal cation exchange resins for antibody capture from CHO supernatants: identification of contaminating host cell proteins by mass spectrometry.

We compared classical and multimodal cation exchange resins for the capture of recombinant antibodies from Chinese hamster ovary (CHO) cell culture supernatant. Both Capto S and Capto MMC resins present anionic groups while the multimodal Capto MMC also features a hydrophobic moiety. First we screened optimal binding and elution conditions in microplates with a pure antibody. We validated the results on the lab-scale with columns with a pure antibody and a CHO cell culture supernatant. Both resins achieved good yield and purity for the capture step of an antibody. However, the multimodal resin appeared more efficient and selective. Then we identified proteins in the antibody fraction by mass spectrometry in order to highlight the behavior of host cell proteins (HCPs).

The homeotic protein dlk is expressed during peripheral nerve development.

To investigate the molecular events controlling myelination of the peripheral nervous system, we compared gene expression of normal mouse sciatic nerves to that of the trembler mouse, whose Schwann cells are blocked in a pre-myelinating phenotype. Using cDNA array, we assessed expression levels of 1176 genes, and we found that delta-like protein (dlk), an epidermal growth factor-like homeotic protein, was expressed in the normal developing nerves, but at a low level in the dysmyelinating mutant trembler. Moreover, dlk expression was down-regulated when myelin protein expression was up-regulated, and no expression was observed in the developing brain. These results suggest that dlk expression is required for Schwann cell acquisition of the myelinating phenotype.

Acyl-CoA elongase expression during seed development in Brassica napus.

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.

Zinc finger proteins in the peripheral nerve of adult normal and Trembler mice.

By using monoclonal antibodies directed against the conserved zinc binding site of zinc finger proteins, we detected 2 prominent zinc finger proteins in rat peripheral nervous system (PNS) during development, and in adult normal and Trembler mice sciatic nerves. The protein of 55 kDa is abundant in adult normal mice and rats, but is weakly expressed in adult Trembler mice. The 29 kDa protein is expressed in neonatal rats and in the Trembler mouse, but is absent in adult rats and mice. These results suggest that the Schwann cell proliferation stage may be associated with the 29 kDa protein expression, and the 55 kDa protein may be implicated in the PNS myelination process.

Peroxisomal beta-oxidation enzyme gene expression in the developing mouse brain.

Using the northern blot technique, the steady-state levels for the mRNAs encoding acyl-CoA oxidase, pristanoyl-CoA oxidase, trans2, 3enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional enzyme type 2, 3-ketoacyl-CoA thiolase and sterol-carrier-protein x during postnatal brain development were measured. The developmental patterns obtained for each mRNA species studied were similar, with an increase in the mRNA level between birth and postnatal day 5, followed by a gradual decrease to 34-55% of the maximal value at postnatal day 30. These results are in agreement with a coordinately controlled expression of the genes involved in VLCFA beta-oxidation during brain development. Moreover, comparison of these developmental profiles with that obtained for ceramide galactosyltransferase showed that the set-up of the very-long-chain fatty acids beta-oxidation system is independent of the myelinating signal in the central nervous system.

Myelin synthesis in the peripheral nervous system.

By imposing saltatory conduction on the nervous impulse, the principal role of the myelin sheath is to allow the faster propagation of action potentials along the axons which it surrounds. Peripheral nervous system (PNS) myelin is formed by the differentiation of the plasma membrane of Schwann cells. One of the biochemical characteristics that distinguishes myelin from other biological membranes is its high lipid-to-protein ratio. All the major lipid classes are represented in the myelin membrane, while several myelin-specific proteins have been identified. During development, the presence of axons is required for the initiation of myelination, but the nature of the axonal signal is still unknown. The only certainties are that this signal is synthesized by axons whose diameter is greater than 0.7 microm, and that the signal(s) include(s) a diffusible molecule. Morphological studies have provided us with information concerning the timing of myelination, the mechanism by which immature Schwann cells differentiate into a myelinating phenotype and lay down the myelin sheath around the axon, and the accumulation and the structure of the myelin membrane. The last 20 years have seen the identification and the cDNA and gene cloning of the major PNS myelin proteins, which signalled the beginning of the knock-out decade: transgenic null-mutant mice have been created for almost every protein gene. The study of these animals shows that the formation of myelin is considerably less sensitive to molecular alterations than the maintenance of myelin. During the same period, important data has been gathered concerning the synthesis and function of lipids in PNS myelin, although this field has received relatively little attention compared with that of their protein counterparts.

Enzymic activities and gene expression of enzymes of the acyl-CoA elongase during rapeseed development.

Enzymic activities and gene expression of oleoyl-CoA elongase were studied during seed development using two different rapeseed cultivars, high-erucic-acid rapeseed (HEAR) and low-erucic-acid rapeseed (LEAR). The overall elongase activities were maximal in HEAR between the fourth and eighth weeks after pollination (WAP) and absent in LEAR. The 3-ketoacyl-CoA synthase (condensing enzyme, CE) mRNA levels and the developmental profiles in the two cultivars were different since maximal expression levels were detected in HEAR and LEAR at WAP 4 and WAP 6, respectively. Anti-CE antibodies revealed two proteins of 60 and 67 kDa in both cultivars and an additional reacting protein of 57 kDa in HEAR.

Trembler as a mouse model of CMT1A?

The Trembler mouse suffers from a dominantly inherited autosomal mutation that results in an abnormal myelination of the peripheral nervous system. Biochemical studies have shown that dysmyelination is the primary event, demyelination being a late-occurring process. The expression of myelin protein genes has been studied. The steady-state levels for PMP22 mRNA represent 10 and 5% of normal values in the nerves of heterozygous and homozygous Trembler, respectively. This is due to a reduced expression of the specific transcript driven by the promoter 1 of the PMP22 gene. Collective results indicate that Trembler dysmyelination is not necessarily the consequence of a large accumulation of the mutated PMP22 protein. Moreover, it appears that the situation in the Trembler is different from that encountered in most CMT1A patients, where an increased PMP22 gene dosage is responsible for the disease. Therefore, the Trembler mutant is perhaps not an ideal model for this human neuropathy.

High metabolism and subsequent elongation of 3-hydroxyeicosanoyl-CoA in very-long-chain fatty acid deficient PNS of Trembler mice.

The developmental patterns of the overall fatty acid elongation and of the last two partial activities of microsomal elongase (dehydration and reduction of 3-hydroxyacyl-CoA) were investigated in the PNS of normal and Trembler mice. Unexpectedly, Trembler microsomes synthesized normal C22-CoA amounts from 3-hydroxyeicosanoyl-CoA (3-OHC20-CoA), a C18-CoA elongation intermediate. Hydroxy- acyl-CoA dehydrase and enoyl-CoA reductase activities were found to be higher in the mutant than in the control, whatever the stage of development. Moreover, C20-CoA elongation led to normal C22-CoA and C24-CoA formation in the mutant whereas C20-CoA formation from C18-CoA was always far lower in Trembler than in control. C18-CoA condensing enzyme emerges as the only elongation step involved in the VLCFA deficit evidenced in Trembler PNS.

Expression of myelin proteins in the adult heterozygous Trembler mouse.

The Trembler mouse (Tr) suffers from a dominantly inherited autosomal mutation (glycine to aspartic acid. G150D) affecting the PMP22 gene, which results in an abnormal myelination of the peripheral nervous system. The Tr mouse represents an animal model for the human hereditary neuropathy, Charcot-Marie-Tooth disease. We compared the expression of PMP22, P0, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) in nerve biopsies from a 1-year-old heterozygous Tr mouse (Tr/+) with that of a control mouse of the same age. Quantitative and qualitative ultrastructural immunocytochemical analysis showed a significant decrease in PMP22, P0 and MBP and an abnormal location of the MAG in the sciatic nerve of the Tr/+ mouse. We also noted a marked reduction in number of myelinated fibers associated with the presence of two types of myelinated axons: a population of abnormally thin myelin sheaths with lower PMP22 labeling than that observed in myelinated fibers from the control mouse, and some others fibers with normal sheaths for axons of comparable diameter to those of normal mouse with no difference in the PMP22 immunolabeling. This pointed to an involvement of PMP22 in the structure of myelin sheaths.

[The involvement of myelin proteins in hereditary neuropathies]. / Implications des protéines myéliniques dans les neuropathies héréditaires.

Hereditary sensoro-motor neuropathies such as Charcot-Marie-Tooth disease (CMT) form a heterogeneous group including some genetic conditions whose clinical manifestations differ in severity within a group or even within a sub-group. Diagnosis is based on the clinical, electrophysiological and pathological findings along with a genetic analysis. The current classification of CMT encompasses the clinical signs, mode of transmission, genomic localization and identification of the proteins actually involved. Several authors have identified the mutations on genes coding for proteins of peripheral myelin in CMT patients. Recent advances in molecular genetics have thrown more light on the differences between phenotypes within a sub-group, and have established genotype-phenotype relationships. It has been shown recently that the severity of the clinical signs depends on the nature and site of various mutations affecting the genes coding for certain myelin proteins. These mutations give rise to "dominant negative effects" or "mutations with loss of function of the allele". These observations may be extended to other proteins as many of them belong to the super family of immunoglobulins and have similar structures. In this study, we present a review of the literature focussing on the principal myelin proteins and the genomic modifications observed in patients with CMT.

Hydroxyacyl-CoA dehydrase and trans-2,3-enoyl-CoA reductase activities are consistent with long-chain fatty acid accumulation during rat brain development.

The developmental changes of microsomal 3-hydroxyacyl-CoA dehydrase and trans-2,3-enoyl-CoA reductase activities were analyzed and compared to very-long-chain fatty acid content and biosynthesis in rat brain. Contrary to the elongation rate of eicosanoyl-CoA and 3-hydroxyeicosanoyl-CoA, which paralleled myelination during brain maturation, the two partial activities of fatty acid elongation were already present at the earliest stages of development. One day after birth, 3-hydroxyacyl-CoA dehydrase and trans-2,3-enoyl-CoA reductase specific activities already represented 54.8% and 49.6% of the adult values, respectively. As a contribution to the quantitative estimation of the brain's ability to form its own VLCFA, it is shown that dehydrase and reductase activities are sufficient to allow the biosynthesis of all rat brain VLCFA at any age.

Gene expression of peroxisomal beta-oxidation enzymes in rat brain.

Despite increasing insight into peroxisomal beta-oxidation, it is still not clear which enzymes catalyze very-long-chain fatty acid degradation. Using the northern blot and RT-PCR techniques, a brain-specific expression is demonstrated for acyl-CoA oxidase 3II mRNA, thiolase-A and trans2,3enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional enzyme type 2.

Regulation of oleoyl-CoA synthesis in the peripheral nervous system: demonstration of a link with myelin synthesis.

We studied the regulation of oleic acid synthesis in the PNS. During mouse postnatal development, the proportion of 18:1 rises in the sciatic nerve from 17% at 5 days of age to 33% at 25 days. However, this rise does not occur in the dysmyelinating mutant mouse trembler. In normal mouse development, the total stearoyl-CoA desaturase (SCD) activity measured in sciatic nerve homogenates is high during the first 3 weeks. Yet in trembler nerves, this SCD activity represents only 15% of normal values. Using the RT-PCR technique, we demonstrate that the SCD2 isoform is predominantly expressed in the PNS. Northern blot analysis showed that the mRNA levels for SCD2 parallel those of other specific myelin proteins in both normal mouse and trembler mutant development. Similar experiments in a rat demyelination-remyelination model confirmed that SCD2 mRNA levels are regulated in the PNS in a similar manner to myelin-specific proteins.

Expression of the amino acid dimorphism in the small myelin-associated glycoprotein cytoplasmic domain in rat peripheral nerves during postnatal development.

The myelin-associated glycoprotein (MAG) is one of the proteins expressed during the period of myelin formation and is believed to play a major role in the initiation of myelination. It exists as two differentially expressed isoforms, L- and S-MAG, that are generated by alternative mRNA splicing. A nucleotide dimorphism at the mRNA level resulting in an Arg/Pro dimorphism in the cytoplasmic tail of the S-MAG protein has previously been detected in the rat brain. In this study, we show that this dimorphism is detectable in the rat peripheral nervous system. We propose an allelic origin for the dimorphism and demonstrate the differential expression of the S-MAG alleles in the sciatic nerves of heterozygous rats during the period of active myelination. We also present data on the properties of the two S-MAG cytoplasmic domains produced as GST fusion proteins. The importance of this differentially expressed amino acid dimorphism is discussed, taking into account both its probable effect on the S-MAG cytoplasmic domain function and its significance in functional and structural studies concerning the S-MAG protein.

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.

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.