BackgroundThe skeletal elements of vertebrate embryonic limbs are prefigured by rod- and spot-like condensations of precartilage mesenchymal cells. The formation of these condensations depends on cell-matrix and cell-cell interactions, but how they are initiated and patterned is as yet unresolved.ResultsHere we provide evidence that galectins, β-galactoside-binding lectins with β-sandwich folding, play fundamental roles in these processes. We show that among the five chicken galectin (CG) genes, two, CG-1A, and CG-8, are markedly elevated in expression at prospective sites of condensation in vitro and in vivo, with their protein products appearing earlier in development than any previously described marker. The two molecules enhance one another's gene expression but have opposite effects on condensation formation and cartilage development in vivo and in vitro: CG-1A, a non-covalent homodimer, promotes this process, while the tandem-repeat-type CG-8 antagonizes it. Correspondingly, knockdown of CG-1A inhibits the formation of skeletal elements while knockdown of CG-8 enhances it. The apparent paradox of mutual activation at the gene expression level coupled with antagonistic roles in skeletogenesis is resolved by analysis of the direct effect of the proteins on precartilage cells. Specifically, CG-1A causes their aggregation, whereas CG-8, which has no adhesive function of its own, blocks this effect. The developmental appearance and regulation of the unknown cell surface moieties ("ligands") to which CG-1A and CG-8 bind were indicative of specific cognate- and cross-regulatory interactions.ConclusionOur findings indicate that CG-1A and CG-8 constitute a multiscale network that is a major mediator, earlier-acting than any previously described, of the formation and patterning of precartilage mesenchymal condensations in the developing limb. This network functions autonomously of limb bud signaling centers or other limb bud positional cues.

Long-term potentiation (LTP) of synaptic transmission in the CA1 region of the hippocampus depends on activation of N-methyl-D-aspartate receptors (NMDARs), which can be regulated by Ca2+-dependent release of D-serine from astrocytes. The detailed mechanism underlying astrocytic D-serine release is still unknown. In this study, we found that clamping astrocytic [Ca2+] at 100-150 nM or puffing artificial cerebrospinal fluid (ACSF) into the extracellular space (weak mechanical stimulation) enhanced synaptic activation of NMDARs. The enhancement was blocked by the NMDAR glycine site antagonist DCKA, glycine saturation, and infusion of astrocytes with D-Amino Acid Oxidase (DAAO) and the serine racemase inhibitor L-erythro-3-hydroxyaspartate (HoAsp), suggesting the involvement of astrocytic D-serine release. Intracellular 100-150 nM [Ca2+] or puffing ACSF stimulated astrocytes to generate D-serine-containing large vesicles (1-3 μm), exocytotic fusion of which released D-serine. The formation of astrocytic large vesicles involved intracellular fusion of small vesicles and/or other organelles. Spontaneous fusion of large vesicles occurred occasionally in astrocytes at rest, contributing to baseline D-serine levels, which increased the rising slope of NMDAR post-burst potentiation (PBP) without altering the PBP peak amplitude. Thus, under physiological conditions, astrocytic D-serine release by large vesicles facilitated weak theta-burst (TBS consisting of 5 bursts), but not strong TBS (TBS consisting of 10 bursts) stimulation-induced LTP.

Reduced bone morphogenetic protein (BMP) receptor (BMPR) expression and BMP signaling have been implicated in vascular cell proliferation and remodeling associated with pulmonary arterial hypertension (PAH). The low penetrance of the BMPR II disease gene in familial PAH suggests that additional genetic or environmental factors are involved in clinical manifestation of PAH. Smurf1 ubiquitin ligase, together with inhibitory SMAD 6/7, forms a negative feedback loop for the attenuation of BMP signals by downregulating BMPR and signaling molecules and, in addition, functions in the integration of MAPK/Ras mitogenic pathways. The present study found that Smurf1 was significantly elevated in pulmonary arteries of monocrotaline and hypoxia-induced PAH rats. In the pulmonary artery of hypoxia-exposed mice, elevation of Smurf1 and SMAD7 was correlated with reduced expression of BMPR II protein. Over-expression of Smurf1 in cultured cells induced ubiquitination and degradation of BMPR I and II whereas ligase-inactive Smurf1 reduced ubiquitination and elevated their protein levels, thus serving a dominant-negative function. Smurf1-induced receptor degradation was inhibited by both proteasomal and lysosomal inhibitors. Thus, Smurf1 reduces steady-state levels of BMPRs by ubiquitination and subsequent degradation involving proteasomes and lysosomes. Therefore, these results show that Smurf1 induction could be a key event for triggering downregulation of BMP signaling and causing vascular cell proliferation and remodeling in PAH and that abrogating Smurf1 function could be a strategy for PAH therapeutics.

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Recent studies indicate that astrocytes can play a much more active role in neuronal circuits than previously believed, by releasing neurotransmitters such as glutamate and ATP. Here we report that local application of glutamate or glutamine synthetase inhibitors induces astrocytic release of glutamate, which activates a slowly decaying transient inward current ( In addition to their nutritive and metabolic functions, astrocytes have been recently discovered to actively participate in and modify neuronal activity by releasing neurotransmitters, such as glutamate and ATP (1-6). Astrocytic Ca 2ϩ -dependent glutamate release modulates neuronal activity in hippocampal slices (7-9) and cell cultures (10, 11). However, there is still controversy about the mechanism by which astrocytes release glutamate. Several hypotheses have been proposed, including exocytosis of vesicles using a protein docking system similar to synaptic vesicles (12-17), swelling-induced anion channels (18), gap-junction hemichannels (19), P 2X receptor channels (20, 21), reverse transport (22), cystine/glutamate exchangers (23), and volume-sensitive nonselective channels (24). Recently, several groups have reported that a transient astrocytic glutamate release activates a slowly decaying transient inward current (SIC) 2 in hippocampal and thalamic neurons (7,8,25,26). Besides transient astrocytic glutamate release (ϳ1 s), a long lasting release of glutamate (several 10 s) from astrocytes has also been reported (7,21,24). Transient astrocytic glutamate release could play important roles in modulating neuronal activities under both physiological and pathological conditions (26,28,29). In a previous study, we reported that either bath application of 4-aminopyridine (4-AP) or infusion of inositol 1,4,5-trisphosphate (IP 3 ) together with high [glutamate] into astrocytes induced SICs, which were caused by fusion of a high [Ca 2ϩ ] large vesicle in astrocytes (26). In this study, we further demonstrate that local increases in [glutamate] o also induce spontaneous SICs and that SICs are activated by astrocytic release of glutamate through fusion of a large vesicle. EXPERIMENTAL PROCEDURESSlice Preparation-Brain slices were prepared as described previously (9). Briefly, 14 -20-day-old (P14 -P20) SpragueDawley rats of either sex were anesthetized with sodium pentobarbitone (55 mg/kg) and then decapitated. Transverse brain slices of 300 m thickness were cut with a vibratome (TPI, St. Louis, MO) in a cutting solution containing the following (in mM): 2.5 KCl, 1.25 NaH 2 PO 4 , 10 MgSO 4 , 0.5 CaCl 2 , 10 glucose,