Effects of 20-hydroxyecdysone and serotonin on neurite growth and survival rate of antennal lobe neurons in pupal stage of the silk moth Bombyx mori in vitro

Effects of 20-hydroxyecdysone and serotonin on the morphological development and the survival of antennal lobe neurons from day-2 pupal brains of the silk moth Bombyx mori were investigated in vitro. Four morphologically distinct neuronal types could be identified in the cultured antennal lobe neurons: unipolar, bipolar, multi-polar and projection neurons. Antennal lobe neurons in culture with 20-hydroxyecdysone and serotonin showed different patterns of the morphological development from those described in Manduca sexta. Projection neurons extend their neurites remarkably by 20-hydroxyecdysone in B. mori, but there is no extension from antennal lobe neurons in M. sexta. Multi-polar neurons conspicuously increase only formation of new branches from their primary neurites by serotonin in B. mori, but there are both extension and branching of the neurites in M. sexta. On day-5, antennal lobe neurons in lower titers of 20-hydroxyecdysone had significantly higher survival rates than those in higher titers. Neurons cultured for 7 days at different levels of 20-hydroxyecdysone generally showed significantly lower survival rates than neurons cultured for 5 days under the same conditions.     

Lack of enhanced spinal regeneration in Nogo-deficient mice

The failure of regeneration of severed axons in the adult mammalian central nervous system is thought to be due partly to the presence of endogenous inhibitors of axon regeneration. The nogo gene encodes three proteins (Nogo-A, -B, and -C) that have been proposed to contribute to this inhibition. To determine whether deletion of nogo enhances regenerative ability, we generated two lines of mutant mice, one lacking Nogo-A and -B but not -C (Nogo-A/B mutant), and one deficient in all three isoforms (Nogo-A/B/C mutant). Although Nogo-A/B-deficient myelin has reduced inhibitory activity in a neurite outgrowth assay in vitro, tracing of corticospinal tract fibers after dorsal hemisection of the spinal cord did not reveal an obvious increase in regeneration or sprouting of these fibers in either mouse line, suggesting that elimination of Nogo alone is not sufficient to induce extensive axon regeneration.     

The zebrafish van gogh mutation disrupts tbx1, which is involved in the DiGeorge deletion syndrome in humans

The van gogh (vgo) mutant in zebrafish is characterized by defects in the ear, pharyngeal arches and associated structures such as the thymus. We show that vgo is caused by a mutation in tbx1, a member of the large family of T-box genes. tbx1 has been recently suggested to be a major contributor to the cardiovascular defects in DiGeorge deletion syndrome (DGS) in humans, a syndrome in which several neural crest derivatives are affected in the pharyngeal arches. Using cell transplantation studies, we demonstrate that vgo/tbx1 acts cell autonomously in the pharyngeal mesendoderm and influences the development of neural crest-derived cartilages secondarily. Furthermore, we provide evidence for regulatory interactions between vgo/tbx1 and edn1 and hand2, genes that are implicated in the control of pharyngeal arch development and in the etiology of DGS.     

Directional shape complementarity at the protein-DNA interface

Nature utilizes various styles of architecture for DNA-binding proteins to recognize diverse DNA sequences, a process facilitated by a complementary surface between protein and DNA. However, the extent and ways this 'shape complementarity' occurs at the protein-DNA interface have yet to be characterized. Here, by analyzing a set of diverse protein-DNA complexes of known three-dimensional structures, we investigated whether the normal vectors of a protein surface at the interface exhibited any relationship with DNA conformation. Generally, the normal vectors of a DNA-contacting protein surface distinctly preferred certain angles, enabling them to align with certain axes characterizing the conformation of DNA. Thus, a new geometric property of DNA-binding protein is demonstrated, i.e. the "shape complementarity" of protein-DNA recognition clearly bears the property of "directionality".     

Calreticulin inhibits the MEK1,2-ERK1,2 pathway in alpha 1-adrenergic receptor/Gh-stimulated hypertrophy of neonatal rat cardiomyocytes

In cardiac myocytes, stimulation of alpha(1)-adrenoceptor (AR) leads to a hypertrophic phenotype. The G(h) protein (transglutaminase II, TGII) is tissue type transglutaminase and transmits the alpha(1B)-adrenoceptor signal with GTPase activity. Recently, it has been shown that the calreticulin (CRT) down-regulates both GTP binding and transglutaminase activities of TGII. To elucidate whether G(h) mediates norepinephrine-stimulated intracellular signal transductions leading to activation of extracellular signal-regulated kinases (ERKs) and neonatal rat cardiomyocyte hypertrophy, we examined the effects of G(h) on the activation of ERKs and inhibitory effects of CRT on alpha(1)-adrenoceptor/G(h) signaling. In neonatal rat cardiomyocytes, norepinephrine-induced ERKs activation was inhibited by an alpha(1)-adrenoceptor blocker (prazosin), but not by an beta-adrenoceptor blocker (propranolol). Overexpression of the G(h) protein stimulated norepinephrine-induced ERKs activation, which was inhibited by alpha-adrenoceptor blocker (prazosin). Co-overexpression of G(h) and CRT abolished norepinephrine-induced ERKs activation. Taken together, norepinephrine induces hypertrophy in neonatal rat cardiomyocytes through alpha(1)-AR stimulation and G(h) is partly involved in norepinephrine-induced MEK1,2/ERKs activation. Activation of G(h)-mediated MEK1,2/ERKs was completely inhibited by CRT.     

Leucine-rich repeat-mediated intramolecular interactions in nematode recognition and cell death signaling by the tomato resistance protein Mi

The root-knot nematode resistance gene Mi from tomato encodes a nucleotide-binding/leucine-rich repeat (NB/LRR) protein with a novel amino-terminal domain compared to related disease-resistance genes. The closely linked paralog Mi-1.1, which does not confer nematode resistance, encodes a protein 91% identical to the functional copy, Mi-1.2. The chimeric construct Mi-DS3, which encodes the 161 amino-terminal residues from Mi-1.1 fused to the remainder of Mi-1.2, induces localized necrosis when transiently expressed in Nicotiana benthamiana leaves. We produced mutant constructs that exchanged sequences encoding each of the 40 amino acid differences from the Mi-1.1 LRR region into Mi-DS3 and into Mi-1.2. For 23 of the substitutions, necrosis was lost upon transient expression of the mutated Mi-DS3 in N. benthamiana, and nematode resistance was lost when the altered Mi-1.2 was expressed in the tomato roots. One substitution, R961D, failed to give Mi-DS3-induced necrosis, but produced a dominant lethal phenotype when introduced into Mi-1.2. This gain-of-function phenotype was suppressed by co-expression with the amino-terminal region of Mi-1.1, suggesting that residue 961 is critical for negative regulation by the corresponding N-terminal region. Substitutions of Mi-1.1 residues 984-986 retained the ability to cause necrosis in Mi-DS3, but resulted in loss-of-nematode resistance in Mi-1.2, suggesting that these residues are essential for nematode recognition. None of the loss-of-function mutations in Mi-1.2 had a dominant negative phenotype. These results indicate that the Mi-1.2 LRR is involved in regulation of the transmission of the resistance response as well as in recognition of the nematode.     

Phenylbutyrates as potent,orally bioavailable vitronectin receptor (integrin alphavbeta3) antagonists

In our continuing efforts to identify small molecule vitronectin receptor antagonists, we have discovered a series of phenylbutyrate derivatives, exemplified by 16, which have good potency and excellent oral bioavailability (approximately 100% in rats). This new series is derived conceptually from opening of the seven-membered ring of SB-265123.     

Docking studies of sulphamate inhibitors of estrone sulphatase in human carbonic anhydrase II

We describe the docking of selected steroidal and non-steroidal estrone sulphatase inhibitors, including the Phase I clinical trial candidate 667COUMATE (6), into the active site of human carbonic anhydrase II (hCA II). The docking scores are compared with the inhibition of hCA II and show good correlation with biological activity.