Gilles Patey

Ontogenesis of enkephalinergic systems in rat brain: Post-natal changes in enkephalin levels, receptors and degrading enzyme activities

Abstract The post-natal changes in enkephalin (ENK) levels, ENK receptor density and ENK degrading enzyme activities have been established in cerebral cortex and striatum. Met- and Leu-ENK levels both increase by 7- to 11-fold, but in an independent manner compatible with their presence in distinct neuronal systems. 3 H-ENK binding sites increase only 4-fold in striatum, as reported for receptor sites labeled with 3 H-opiate antagonists. The development of striatal “enkephalinase” i.e. of the particulate enzyme activity cleaving the Gly-Phe bond of ENKs is more of less parallel in time-course to that of ENK levels and receptors, with a 6-fold increase from birth. In contrast total ENK hydrolysing activity shows little change. The developmental pattern of angiotensin-converting enzyme (ACE) is clearly distinct from that of “enkephalinase”, thus confirming that the two enzymes are different species.

Human NT2 neurons express a large variety of neurotransmission phenotypes in vitro.

The NT2 cell line, which was derived from a human teratocarcinoma, exhibits properties that are characteristic of a committed neuronal precursor at an early stage of development. NT2 cells can be induced by retinoic acid to differentiate in vitro into postmitotic central nervous system (CNS) neurons (NT2‐N cells). The commitment of NT2‐N cells to a stable neuronal phenotype is irreversible. Because it may be possible to transplant these human neurons to compensate for neuronal loss after traumatic injuries or neurodegenerative diseases of the CNS, knowledge of their phenotype is essential. This study aimed to characterize in detail the neurotransmission phenotype of NT2‐N cells by using immunocytochemical methods. Single peroxidase immunostaining demonstrated that NT2‐N cells expressed the γ‐aminobutyric acidergic (GABAergic), catecholaminergic, and cholinergic phenotypes to a large extent and expressed the serotonergic phenotype to a minor extent. NT2‐N cells also expressed different neuropeptides, such as neuropeptide Y, oxytocin, vasopressin, calcitonin gene‐related peptide, and Met‐ and Leu‐enkephalin. Double fluorescence immunostaining further indicated that a large number of NT2‐N cells could express GABA and another neurotransmitter or neuropeptide at the same time. Finally, electron microscopy demonstrated that these NT2 neurons elaborate classical synaptic contacts. The multipotentiality of these neurons, combined with their apparent functionality, suggests that they may represent useful material for a variety of therapeutic approaches aimed at replacing dead neurons after neurodegenerative diseases or lesions of the CNS. J. Comp. Neurol. 422:380–395, 2000.

Subcellular distribution of enkephalin-dipeptidyl carboxypeptidase (enkephalinase) in rat brain

The distribution of enkephalinase activity i.e. of the peptidase activity cleaving the Gly3-Phe4 bond of enkephalins, has been established in subcellular fractions of rat cerebral cortex and striatum and compared with that of opiate receptors labelled with [3H] dAla2-Met-enkephalinamide. The fractions were obtained by differential and density gradient centrifugations and their purity assessed by measuring levels of a series of biochemical markers. A major part of enkephalinase activity is associated with the P2 fraction containing isolated nerve-endings and the subfractions of lysed P2 containing synaptosomal membranes display the highest enrichment. Another important part of enkephalinase activity is associated with the microsomal P3 fraction, whereas the enzyme activity is hardly detectable in cytoplasm or synaptic vesicle fractions. The distribution of enkephalinase activity between the various fractions and subfractions strikingly parallels that of enkephalin receptors. These data support the hypothesis that enkephalinase is selectively located in neuronal membranes in the vicinity of enkephalin receptors, as suggested by previous regional distribution and lesion studies, and that it might be responsible for the inactivation of endogenous enkephalins.

Changes in dopamine receptors in mouse striatum following morphine treatments.

Abstract Presynaptic opiate receptors previously evidenced on striatal dopaminergic nerve-endings might mediate an inhibition of dopaminergic transmission by morphine. We have now assessed on behavioral and biochemical tests the development of disuse hypersensitivity to dopamine (DA) following morphine treatments. 1. 1. A long-lasting increase in behavioral responsiveness to apomorphine regarding the climbing behavior, a stereotyped motor behavior mediated by striatal DA receptors, is shown to develop after a single dose of morphine. However after chronic treatments the behavioral data are difficult to interpret. 2. 2. After an initial rise, striatal HVA levels are significantly depressed for several days. 3. 3. Binding of 3 H-domperidone to striatal DA receptors is slightly enhanced. All these changes, although less marked, are reminiscent of those observed during typical hypersensitivity developing following chronic blockade of DA receptors by neuroleptics. The primary action of morphine on DA transmission is discussed.

Primary structure of the rat gene encoding an inhibitor of the insulin receptor tyrosine kinase.

The gene (PP63) encoding the inhibitor (PP63) of the insulin receptor tyrosine kinase was isolated from a rat genomic library. The intron/exon organization was deduced from Southern-blot analysis and sequence data (i.e., the exons + the boundaries). The PP63 gene, which maps to chromosome 11, spans approx. 8 kb and contains seven exons separated by six introns of different sizes. All of the boundaries match the consensus GT/AG sequence for donor and acceptor splice sites. Primer extension and S1 mapping experiments were used to locate the transcription start point (tsp) 73 nt upstream from the translational initiator. Both in vitro transcription assays and transcription of a chimeric gene in intact hepatoma cells indicated that the sequence located immediately upstream from the tsp contained a promoter. Several putative cis-regulatory elements, including a TATA box and a C/EBP-binding site were found within the 250 bp preceding the tsp.

Identification of a New Virulence Factor, BvfA, in Brucella suis

ABSTRACT We report the identification of BvfA (for Brucella virulence factor A), a small periplasmic protein unique to the genus Brucella, which is essential for the virulence of Brucella suis. A BvfA knockout mutant was highly attenuated both in in vitro macrophage infection assays and in vivo in the murine model of brucellosis. Fluorescence-activated cell sorting analysis with green fluorescent protein fusions showed that the expression of bvfA is induced within macrophages by phagosome acidification and coregulated with the B. suis virB operon, suggesting that it too may play a role in the establishment of the intracellular replication niche.

Swapping of Periplasmic Domains between Brucella suis VirB8 and a pSB102 VirB8 Homologue Allows Heterologous Complementation

ABSTRACT A Brucella suis mutant with a nonpolar deletion in the virB8 gene was attenuated in a macrophage infection model. Complementation with the B. suis VirB8 protein expressed from the virB promoter restored virulence. Expression of TraJ, a VirB8 homologue from plasmid pSB102, did not restore virulence; however, virulence was partially restored by a chimeric protein containing the N terminus of the B. suis VirB8 protein and the C-terminal periplasmic domain of TraJ.

Interactions between Brucella suis VirB8 and Its Homolog TraJ from the Plasmid pSB102 Underline the Dynamic Nature of Type IV Secretion Systems

The proteinVirB8 plays a critical role in the assembly and function of the Agrobacterium tumefaciens virB type IV secretion system (T4SS). The structure of the periplasmic domain of both A. tumefaciens and Brucella suis VirB8 has been determined, and site-directed mutagenesis has revealed amino acids involved in the dimerization of VirB8 and interactions with VirB4 and VirB10. We have shown previously that TraJ, the VirB8 homologue from pSB102, and the chimeric protein TraJB8, encompassing the cytoplasmic and transmembrane (TM) domains of TraJ and the periplasmic domain of VirB8, were unable to complement a B. suis mutant containing an in-frame deletion of the virB8 gene. This suggested that the presence of the TraJ cytoplasmic and TM domains could block VirB8 dimerization or assembly in the inner membrane. By bacterial two-hybrid analysis, we found that VirB8, TraJ, and the chimeras can all interact to form both homo- and heterodimers. However, the presence of the TM domain of TraJ resulted in much stronger interactions in both the homo- and heterodimers. We expressed the wild-type and chimeric proteins in wild-type B. suis. The presence of proteins carrying the TM domain of TraJ had a dominant negative effect, leading to complete loss of virulence. This suggests that the T4SS is a dynamic structure and that strong interactions block the spatial flexibility required for correct assembly and function.

Differential expression of Bcl-2-related proteins in differentiating NT2 cells

&NA; Although the role of Bcl‐2‐related proteins as regulators of the apoptotic process has been well documented, recent studies suggest that they might also be implicated in neuronal differentiation. We have studied by immunocytochemistry, Western blotting and RT‐PCR the expression pattern of BclxL, Bcl‐2 and BAX in the in vitro model of neuronal differentiation constituted by retinoic acid (RA)‐treated NTera‐2/D1 (NT2/D1) cells. Whereas BAX level did not change significantly during the RA treatment, Bcl‐xL level increased markedly during the first week, before returning to basal level during the second week. Bcl–2 expression, undetectable in undifferentiated cells, increased progressively from the first week. From our results, we suggest that, at least in our model, Bcl‐2‐related proteins might be involved in neuronal differentiation.

A single amino acid change in the transmembrane domain of the VirB8 protein affects dimerization, interaction with VirB10 and Brucella suis virulence.

AtVirB10 physically interacts with AtVirB8 by two hybrid (View interaction)