
Bertrand GarbayInstitut Polytechnique de Bordeaux · École Nationale Supérieure de Technologie des Biomolécules (ENSTBB)
Bertrand Garbay
Doctor of Philosophy
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Introduction
Publications
Publications (92)
Despite progress in bone tissue engineering, reconstruction of large bone defects remains an important clinical challenge. Here, a biomaterial designed to recruit bone cells, endothelial cells, and neuronal fibers within the same matrix is developed, enabling bone tissue regeneration. The bioactive matrix is based on modified elastin‐like polypepti...
Developing new biomaterials is an active research area owing to their applications in regenerative medicine, tissue engineering and drug delivery. Elastin-like polypeptides (ELPs) are good candidates for these applications because they are biosourced, biocompatible and biodegradable. With the aim of developing ELP-based micelles for drug delivery a...
Diblock copolymers based-on elastin-like polypeptide (ELP) have the potential to undergo specific phase transitions when thermally stimulated. This ability is especially suitable to form carriers, micellar structures for instance, for delivering active cargo molecules. Here, we report the design and study of an ELP diblock library based on ELP-[M1V...
Developing new biomaterials is an active research area owing to their applications in regenerative medicine, tissue engineering and drug delivery.
Hybrids of synthetic polymers and biopolymers are known to be macromolecular systems that merge the properties of each component and overcome some of their intrinsic limitations. Elastin-like polypeptides (ELPs) are a class of biopolymers known for their genetically-encoded synthesis, monodispersity, biocompatibility and absence of toxicity, which...
Selective lectin binding and sorting was achieved using thermosensitive glycoconjugates derived from recombinant elastin-like polypeptides (ELPs) in simple centrifugation-precipitation assays. A recombinant ELP, (VPGXG)40, containing periodically spaced methionine residues was used to enable chemoselective post-synthetic modification via thioether...
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,...
Despite significant progress in the field of biomaterials for bone repair, the lack of attention to the vascular and nervous networks within bone implants could be one of the main reasons for the delayed or impaired recovery of bone defects. The design of innovative biomaterials should improve the host capacity of healing to restore a functional ti...
Elastin-like polypeptides (ELPs) are biocompatible-engineered polypeptides, with promising interest in tissue engineering due to their intrinsic biological and physical properties, and their ease of production. The IKVAV (Ile-Lys-Val-Ala-Val) laminin-1 sequence has been shown to sustain neuron attachment and growth. In this study, the IKVAV adhesio...
Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing th...
We report the synthesis of methionine-containing recombinant elastin-like polypeptides (ELPs) of different lengths that contain periodically spaced methionine residues. These ELPs were chemoselectively alkylated at all methionine residues to give polycationic derivatives. Some of these samples were found to possess solubility transitions in water,...
We have designed and prepared a recombinant elastin-like polypeptide (ELP) containing precisely positioned methionine residues, and performed the selective and complete oxidation of its methionine thioether groups to both sulfoxide and sulfone derivatives. Since these oxidation reactions substantially increase methionine residue polarity, they were...
Besides its widely described function in the innate immune response, no other clear physiological function has been attributed so far to the Liver-Expressed-Antimicrobial-Peptide 2 (LEAP2). We used the Xenopus embryo model to investigate potentially new functions for this peptide. We identified the amphibian leap2 gene which is highly related to it...
Recombinant proteins are often produced in the periplasm of Escherichia coli because this facilitates the purification process, and the oxidizing environment favors the formation of disulfide bridges. We showed that the periplasmic expression of the human hepcidin 25 hormone (Hep25) fused to the maltose-binding protein (MBP) resulted in cell death....
Tuning the lower critical solution temperature (LCST) of temperature-responsive recombinant elastin-like polypeptides has usually been achieved by designing different protein sequences, in terms of amino acid composition and length, implying tedious molecular cloning steps. In the present work, we have explored the chemoselective alkylation of meth...
Acquisition of Pseudomonas aeruginosa is known as a negative prognostic factor in patients with cystic fibrosis. We started a pilot study to evaluate P. 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 P. ae...
An innovative process to purify mAb from CHO cell culture supernatant was developed. This three-step process involved two mixed mode resins and an anion exchange membrane. We used a human IgG mixture to determine the optimal conditions for each purification step. Thereafter, the whole process was evaluated and improved for the purification of a rec...
Background
Bacterial biofilms are predominant in natural ecosystems and constitute a public health threat because of their outstanding resistance to antibacterial treatments and especially to antibiotics. To date, several systems have been developed to grow bacterial biofilms in order to study their phenotypes and the physiology of sessile cells. A...
##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##
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 micro...
The prerequisite to monitor gene expression is the selection of reference genes for normalization of RT-qPCR results. Using 13 sputum samples collected from 9 CF patients, we demonstrated that PA2875 and PA3340 are better reference genes than the previously used clpX and oprL genes.
Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole-genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and prot...
Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long sought hormone that regulates iron homeostasis in mammals. The native peptide of 25 amino acids (Hepc25) contains four disulfide bridges that maintain a β-hairpin motif. The aim of the present study was to...
We evaluated mixed mode chromatography for the capture of recombinant antibodies from CHO cell culture supernatants. We studied PPA HyperCel, HEA HyperCel, MEP HyperCel and Capto adhere resins, which all contain hydrophobic and cationic groups. A microplate approach combined with DoE modeling allowed the exploration of the complex behaviors of thes...
Imatinib mesylate is a tyrosine kinase inhibitor used as first line treatment in chronic myeloid leukaemia. Despite a remarkable effectiveness, treatment failure cases have been reported in 20 percent of CML patients. The identification of biomarkers which can predict the response to imatinib is our point of interest.
Gene expression profiling micr...
Lack of compensatory or even reduced food intake is frequently observed in weight-losing cancer patients and contributes to increased morbidity and mortality. Our previous work has shown increased transcription factor expression in the hypothalamus and ventral striatum of anorectic rats bearing small tumors. mRNA expression of molecules known to be...
Objective : Imatinib mesylate is a tyrosine kinase inhibitor used as first line treatment in chronic myeloid leukaemia. Despite a remarkable effectiveness, treatment failure cases have been reported in 20 percent of CML patients. The identification of biomarkers which can predict the response to imatinib is our point of interest.
Methods : Gene exp...
Hepcidin was first identified as an antimicrobial peptide expressed in the liver. It was later demonstrated that hepcidin is in fact the long sought hormone that regulates iron homeostasis in mammals. Hepcidin is encoded as an 84 amino acid prepropeptide that is successively cleaved to yield prohepcidin and the mature 25 amino acid hepcidin. Both t...
Liver-expressed antimicrobial peptide 2 (LEAP-2) is a 40-residue cationic peptide originally purified from human blood ultrafiltrate. The native peptide contains two disulfide bonds and is unique regarding its primary structure. Its biological role is not known but a previous study showed that chemically synthesized LEAP-2 exhibited in vitro antimi...
Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the...
Antimicrobial peptides (AMPs) are cationic molecules that are good leads for new antiinfective drugs. To obtain sufficient amounts, recombinant AMPs are generally produced as fusion proteins in Escherichia coli. Fusion partners facilitate purification of recombinant proteins. Fusion proteins are then cleaved by specific proteases, and cationic pept...
Antimicrobial peptides are good leads to develop new antibiotics, but knowledge of their mode of action is a prerequisite. Destruction of the microbial membranes through a detergent-like mechanism is one of these modes of action. This is usually studied by using a fluorescent nucleic acid stain such as SYTOX Green, which is impermeable to living ce...
RNA from the aerial parts of 15-day-old eceriferum cer6-2 mutant and control plants were used in microarray experiments to screen for differentially expressed genes. Among those which were down-regulated in the mutant, several were known to be involved in various plant defense mechanisms. The most remarkable difference was measured for the transcri...
Plant epidermal wax forms a hydrophobic layer covering aerial plant organs which constitutes a barrier against uncontrolled water loss and biotic stresses. Wax biosynthesis requires the coordinated activity of a large number of enzymes for the formation of saturated very-long-chain fatty acids and their further transformation in several aliphatic c...
The expression of acetyl-CoA carboxylase (ACC) in mouse peripheral nervous system (PNS) was investigated. Both ACC 265 and ACC 280 isoforms were expressed in the sciatic nerve, although ACC 265 was predominant. ACC 265 transcripts originating from promoters P1 and P2 could be detected in the developing nerve, as well as the two splice products, whi...
The membrane-bound acyl-CoA elongases are responsible for the synthesis of Very Long Chain Fatty Acids (VLCFA) which are the primer of wax constituents and are the major fatty acid is some seeds. VLCFA result from elongation of oleoyl-CoA by malonyl-CoA through four successive reactions: condensation (CE), keto-acyl-CoA reduction (KCR), dehydratati...
The expression of fatty acid synthase (FAS) in rat and mouse sciatic nerves during postnatal development was investigated. FAS activity was not sensitive to the nutritional status of the animals. During development, the specific activity of FAS was low in rat and mouse nerves immediately after birth. Then, there was a steady increase in the activit...
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 expresse...
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 (d...
Abstract 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...
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 rapes...
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-ke...
On May 21, 2001 this sequence version replaced gi:9887343.
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 dist...
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 obta...
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 multifun...
Despite increasing insight into peroxisomal β-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 multifuncti...
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 fo...
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 C1...
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...
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...
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...
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...
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...
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 proliferati...
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 amo...
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 t...
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 imp...
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 fr...
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 anal...
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 st...
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...
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...
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, alt...
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 studi...
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 th...
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...
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 fro...
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 littermat...