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Neurotrophin‐3 (NT‐3) has low‐affinity (Kd = 8 × 10(−10) M), as well as high‐affinity receptors (Kd = 1.8 × 10(−11) M) on embryonic chick sensory neurons, the latter in surprisingly high numbers. Like the structurally related proteins nerve growth factor (NGF) and brain‐derived neurotrophic factor (BDNF), NT‐3 also binds to the low‐affinity NGF receptor, a molecule that we suggest to designate low‐affinity neurotrophin receptor (LANR). NT‐3 dissociates from the LANR much more rapidly than BDNF, and more slowly than NGF. The binding of labelled NT‐3 to the LANR can be reduced by half using a concentration of BDNF corresponding to the Kd of BDNF to the LANR. In contrast, the binding of NT‐3 to its high‐affinity neuronal receptors can only be prevented by BDNF or NGF when used at concentrations several thousand‐fold higher than those corresponding to their Kd to their high‐affinity neuronal receptors. Thus, specific high‐affinity NT‐3 receptors exist on sensory neurons that can readily discriminate between three structurally related ligands. These findings, including the remarkable property of the LANR to bind three related ligands with similar affinity, but different rate constants, are discussed.

Notch and neurotrophins control neuronal shape, but it is not known whether their signaling pathways intersect. Here we report results from hippocampal neuronal cultures that are in support of this possibility. We found that low cell density or blockade of Notch signaling by a soluble Delta-Fc ligand decreased the mRNA levels of the nuclear targets of Notch, the homologues of enhancer-of-split 1 and 5 (Hes1/5). This effect was associated with enhanced sprouting of new dendrites or dendrite branches. In contrast, high cell density or exposure of low-density cultures to NGF increased the Hes1/5 mRNA, reduced the number of primary dendrites and promoted dendrite elongation. The NGF effects on both Hes1/5 expression and dendrite morphology were prevented by p75-antibody (a p75 NTR -blocking antibody) or transfection with enhancer-of-split 6 (Hes6), a condition known to suppress Hes activity. Nuclear translocation of NF-kappaB was identified as a link between p75 NTR and Hes1/5 because it was required for the up-regulation of these two genes. The convergence of the Notch and p75 NTR signaling pathways at the level of Hes1/5 illuminates an unexpected mechanism through which a diffusible factor (NGF) could regulate dendrite growth when cell-cell interaction via Notch is not in action.

We have previously shown that dendrite morphology of cultured hippocampal neurones is controlled by Notch receptor activation or binding of nerve growth factor (NGF) to its low affinity receptor p75 NTR , i.e. processes that up-regulate the expression of the Homologue of enhancer of split 1 and 5. Thus, the increased expression of these genes decreases the number of dendrites, whereas abrogation Neurotrophins have been shown to regulate dendrite morphology in a variety of experimental models (Mcallister et al. 1995(Mcallister et al. , 1997Baker et al. 1998;Jin et al. 2003). As neurotrophins are released in an activity-dependent manner, they may be fundamental in orchestrating the structural modifications that developing and mature neuronal circuits undergo (Whitford et al. 2002). However, the signalling pathways underlying the effects of neurotrophins on dendrite morphology are not fully understood. While most studies in this area have emphasized the importance of the Trk receptors, there is evidence that nerve growth factor (NGF) regulates dendrite morphology by binding to p75 NTR , the common neurotrophin receptor (Salama-Cohen et al. 2005) Address correspondence and reprint requests to Dr A. Rodríguez-Tébar, Instituto Cajal, CSIC, Avenue. Doctor Arce, 37, 28002 Madrid, Spain. E-mail: rodriguez@cajal.csic.esAbbreviations used: DIV, days in vitro; EGFP, enhanced green fluorescent protein; E/I, ratio, ratio of excitatory to inhibitory; GAD, glutamic acid decarboxylase; Hes, homologue of enhancer of split; Mash1: mouse achaete scute homologue 1; MAP, microtubule-associated protein; NGF, nerve growth factor; Ngn3, neurogenin 3; p75-Ab, p75 NTR blocking antibody; ROI, region of interest; Syp, synaptophysin; VgluT, vesicular glutamate transporter; VIAAT, vesicular inhibitory amino acid transporter.

The activity of penicillin-binding protein 3 of Escherchia coli has been studied both in vivo and in ether-permeabilized cells. The peptidoglycan transpeptidase activity of penicillin-binding protein 3 appears to use either nascent or exogenously added UDP-N-acetylmuramyl tripeptide-derived substrates as acceptors. By means of a defilamentation system which elicited the activity of penicillin-binding protein 3 in vivo, the structure of peptidoglycan made by this enzyme has been elucidated. This peptidoglycan, very probably of septal location, contained increased amounts of cross-linked peptidoglycan as well as a higher ratio of tripeptide-containing cross-linked subunits. Kraus et al. (13) shows that the structure of peptidoglycan synthesized by either ethertreated E. coli cells or isolated envelopes was different from that of peptidoglycan synthesized by the living cell.It is well known that PBP 3 from E. coli participates in cell division. Mutations affecting PBP 3 or selective inhibition of this PBP by 13-lactams impairs cell septation and produces filamentation of E. coli cells (29). Botta and Park, by selective inhibition of PBP 3 during defilamentation (a period of active septation), demonstrated that PBP 3 was involved in septal peptidoglycan synthesis (5). It is now believed that PBP 3 synthesizes, at least in part, the septal peptidoglycan. So far, little or nothing is known about either the activity of this PBP or the structure of septal peptidoglycan. A recent study by Olijhoek et al. (22) has shown that the degree of peptidoglycan cross-linkage was higher during septation. From these data, it was inferred that septal peptidoglycan is of a more cross-linked nature, with a higher ratio of both dimeric and trimeric subunits (12, 13 In this communication, we present biochemical data on the activity of PBP 3 both in vivo and in vitro, with regard to the substrates it uses and the products yielded, as well as an outline of the structure of the septal peptidoglycan of E. coli. A partial account of this study has appeared elsewhere (26). MATERIALS AND METHODSBacterial strains and culture conditions. E. coli PA3092 (Fthr-J leu-6 thi-J argHI thy his-i trp-J str-9 lacYJ malAl xyl-7 mtl-2 melffhuA2 supE44) and PAT84 (like PA3092, but dapA lysA sulB [27a]). Cells were grown aerobically in L broth (16) supplemented with D-glucose (4 mg/ml) and, for PAT84 strain, 2,6-meso-diaminopimelic acid (5 mg/liter). Other conditions varied from one experiment to another and are defined below.Detection of PBPs in cell extracts and cell envelopes. Usually, PBPs were detected in whole-cell extracts. Cells were collected from the cultures by rapid cooling and centrifugation at low speed. Pelleted cells were suspended in a small volume of ice-cold 20 mM sodium phosphate buffer, pH 7.0. Suspended cells (usually about 0.5 ml) were subjected to a 10-s burst of sonication in a 150-V sonic oscillator (MSE Instruments, Crawley, U.K.). Sonic treatment was carried out with a 1/8-in. (3 mm) probe at an amplitude of 6 ,um (peak to peak at...

During development, neuronal survival is regulated by the limited availability of neurotrophins, which are proteins of the nerve growth factor (NGF) family. Activation of specific trk tyrosine kinase receptors by the neurotrophins blocks programmed cell death. The trkA-specific ligand NGF has also been shown to activate the non-tyrosine kinase receptor p75, a member of the tumour necrosis factor (TNF) receptor and Fas (APO-1/CD95) family. Here we report that, early in development, endogenous NGF causes the death of retinal neurons that express p75 but not trkA. These results indicate that, as with cells of the immune system, the death of neurons in the central nervous system can also be induced by ligands, and that the effect of NGF on cell fate depends on the type of receptor expressed by developing neurons.

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