Homeobox Genes, Retinoic Acid and the Development and Evolution of Dual Body Plans in the Ascidian Herdmania curvata

Ascidians, along with other urochordates, are the most evolutionarydistant group from vertebrates to display definitive chordate-specificcharacters, such as a notochord, dorsal hollow nerve cord, pharynxand endostyle. Most solitary ascidians have a biphasic lifehistory that has partitioned the development of these charactersbetween a planktonic microscopic tadpole larva (notochord anddorsal nerve cord) and a larger sessile adult (pharynx and endostyle).Very little is known of the molecular axial patterning processesoperating during ascidian postlarval development. Two axialpatterning homeobox genes Otx and Cdx are expressed in a spatiallyrestricted manner along the ascidian anteroposterior axis duringembryogenesis and postlarval development (i.e., metamorphosis).Comparisons of these patterns with those of homologous cephalochordateand vertebrate genes suggest that the novel ascidian biphasicbody plan was not accompanied by a deployment of these genesinto new pathways but by a heterochronic shift in tissue-specificexpression. Studies examining the role of all-trans retinoicacid (RA) in axial patterning in chordates also contribute toour understanding of the role of homeobox genes in the developmentof larval and adult ascidian body plans. Our studies demonstratethat RA does not regulate axial patterning in the developingascidian larval neuroaxis in a manner homologous to that foundin vertebrates. Although RA may regulate the expression of someascidian homeobox genes, ectopic application of RA does notappear to alter the morphology of the larval CNS. However, treatmentwith similar or lower concentrations of RA, have a profoundeffect on postlarval development and the juvenile body plan.These changes are correlated to a dramatic reduction of Otxexpression. Through these RA-induced effects we infer that whileRA may regulate the expression of some homeobox genes duringembryogenesis it has a far more dramatic impact on postlarvaldevelopment where regulative processes predominate.     

HLA-binding regions of HIV-1 proteins. I. Detection of seven HLA binding regions in the HIV-1 Nef protein.

The physical association of HLA class I or H-2 molecules with 36 HIV-1 Nef synthetic peptides was studied using a direct peptide binding assay (PBA) in solid phase. To assess the functional significance of the PBA results, the Nef peptides were also tested for their ability to inhibit the lytic activity of human or murine CTL. The PBA results showed that seven partly overlapping regions of the Nef protein contained MHC binding peptides (4-18, 46-67, 73-94, 100-128, 126-155, 182-198, and 192-206). Five of these seven regions included all the already described epitopes recognized by CD8+ human CTL. The two other regions, 4-18 and 46-67, are not yet described as antigenic for human CD8+ cells but they are located in the N-terminal part of Nef that was previously described as being stimulator for rat or chimpanzee CD4+ cells. Altogether, it can be concluded that 1) In virtually 100% of the cases, the PBA is capable to detect known antigenic peptides recognized by CTL. 2) The PBA and the functional inhibition assay provide similar results, supporting the functional significance of PBA results. 3) The PBA is easy to handle on a large scale, using multiple peptide and several MHC molecules, so that it can be used as a routine method for prevision of possibly epitopic sequences. 4) Systematic studies of peptides issued from the whole sequence of a given protein allow to map polyepitopic areas that are probably the most interesting parts of proteins for a vaccine purpose.     

Minimally-invasive Technique for Injection into Rat Optic Nerve

The rat optic nerve is a useful model for stem cell regeneration research. Direct injection into the rat optic nerve allows delivery into the central nervous system in a minimally-invasive surgery without bone removal. This technique describes an approach to visualization and direct injection of the optic nerve following minor fascial dissection from the orbital ridge, using a conjunctival traction suture to gently pull the eye down and out. Representative examples of an injected optic nerve show successful injection of dyed beads.     

Effect of inhibitors on conformational changes in hen lysozyme around thermal transition point in solution and solid state

Carbon-13 NMR spectroscopy has been used to further document the interaction, at low and high temperatures, of N-acetylglucosamine and its short polymers with hen egg-white lysozyme. The results have been compared with the corresponding X-ray crystallographic data. Two domains, the active site and the hydrophobic box, have been found by NMR to undergo conformational rearrangement while X-ray crystallography only detected changes located in the active site. The extent of the modifications induced by inhibitor binding was proportional to the inhibitor size. The two techniques concurred to show that even in the presence of monosaccharide (N-acetylglucosamine), more than one subsite of the enzyme was occupied at high temperature, the binding at the C-site being the best defined. The thermal transition of lysozyme still occurred in solution when inhibitors were bound. However, in the solid state, crystallographic data showed that the transition was hindered.     

Mechanisms of HIV-1 evasion to the antiviral activity of chemokine CXCL12 indicate potential links with pathogenesis

HIV-1 infects CD4 T lymphocytes (CD4TL) through binding the chemokine receptors CCR5 or CXCR4. CXCR4-using viruses are considered more pathogenic, linked to accelerated depletion of CD4TL and progression to AIDS. However, counterexamples to this paradigm are common, suggesting heterogeneity in the virulence of CXCR4-using viruses. Here, we investigated the role of the CXCR4 chemokine CXCL12 as a driving force behind virus virulence. In vitro, CXCL12 prevents HIV-1 from binding CXCR4 and entering CD4TL, but its role in HIV-1 transmission and propagation remains speculative. Through analysis of thirty envelope glycoproteins (Envs) from patients at different stages of infection, mostly treatment-naïve, we first interrogated whether sensitivity of viruses to inhibition by CXCL12 varies over time in infection. Results show that Envs resistant (RES) to CXCL12 are frequent in patients experiencing low CD4TL levels, most often late in infection, only rarely at the time of primary infection. Sensitivity assays to soluble CD4 or broadly neutralizing antibodies further showed that RES Envs adopt a more closed conformation with distinct antigenicity, compared to CXCL12-sensitive (SENS) Envs. At the level of the host cell, our results suggest that resistance is not due to improved fusion or binding to CD4, but owes to viruses using particular CXCR4 molecules weakly accessible to CXCL12. We finally asked whether the low CD4TL levels in patients are related to increased pathogenicity of RES viruses. Resistance actually provides viruses with an enhanced capacity to enter naive CD4TL when surrounded by CXCL12, which mirrors their situation in lymphoid organs, and to deplete bystander activated effector memory cells. Therefore, RES viruses seem more likely to deregulate CD4TL homeostasis. This work improves our understanding of the pathophysiology and the transmission of HIV-1 and suggests that RES viruses’ receptors could represent new therapeutic targets to help prevent CD4TL depletion in HIV+ patients on cART.     

The Use of Proton Chemical Shifts to Define the Solution Structure of a Dimeric Peptide

For flexible peptides, nuclear Overhauser Effects (NOE) experiments do not provide enough information to ensure a correct definition of their solution structure. The use of distance constraints, derived from the knowledge of proton chemical shifts, is developed to restrict the number of possible conformations. In the case of flexible molecules, randomization appears as an important factor of the correct estimation of the chemical shifts from the 3D structure. The refinement of the solution structure of the highly flexible AVP-like parallel dimer is described to illustrate this process.     

GLUT2 in pancreatic and extra-pancreatic gluco-detection

Detection of variations in blood glucose concentrations by pancreatic &#103 -cells and a subsequent appropriate secretion of insulin are key events in the control of glucose homeostasis. Because a decreased capability to sense glycemic changes is a hallmark of type 2 diabetes, the glucose signalling pathway leading to insulin secretion in pancreatic &#103 -cells has been extensively studied. This signalling mechanism depends on glucose metabolism and requires the presence of specific molecules such as GLUT2, glucokinase and the K ATP channel subunits Kir6.2 and SUR1. Other cells are also able to sense variations in glycemia or in local glucose concentrations and to modulate different physiological functions participating in the general control of glucose and energy homeostasis. These include cells forming the hepatoportal vein glucose sensor, which controls glucose storage in the liver, counterregulation, food intake and glucose utilization by peripheral tissues and neurons in the hypothalamus and brainstem whose firing rates are modulated by local variations in glucose concentrations or, when not protected by a blood-brain barrier, directly by changes in blood glucose levels. These glucose-sensing neurons are involved in the control of insulin and glucagon secretion, food intake and energy expenditure. Here, recent physiological studies performed with GLUT2 -/- mice will be described, which indicate that this transporter is ess ential for glucose sensing by pancreatic &#103 -cells, by the hepatoportal sensor and by sensors, probably located centrally, which control activity of the autonomic nervous system and stimulate glucagon secretion. These studies may pave the way to a fine dissection of the molecular and cellular components of extra-pancreatic glucose sensors involved in the control of glucose and energy homeostasis.     

Murine xenotropic type C viruses I. Distribution and further characterization of the virus in NZB mice.

The xenotropic mouse type C virus, originally detected in cultured embryo cells from New Zealand Black (NZB) mice, has been recovered from over 50 adult NZB animals and 15 NZB embryos. Its presence is best detected by measuring its ability to rescue a murine sarcoma virus (MSV) genome from a non-virus-producing MSV-transformed rat cell. The virus can serve as a helper for replication of MSV. It has a distinct type-specific coat and is a prototype for a third serotype of mouse type C viruses, NZB. The xenotropic virus may have an evolutionary role since it has a wide host range, including the ability to infect avian cells. It is produced spontaneously by all cells cultivated from NZB tissues and accounts for the high concentration of viral antigens associated with NZB tissues. The extent of virus production is similar in both male and female mice. All cell clones established from embryos also produce the virus. A variability in the intracellular regulation of virus replication is suggested since tissue cells from the same animal differ quantitatively in their ability to produce xenotropic viruses. Since enhanced spontaneous virus production is associated with cells from NZB mice, the virus may play a role in the autoimmune disease of this mouse strain.