Neurotrophins,but not depolarization,regulate substance P expression in the developing optic tectum

Neurotransmitter expression can be regulated by both activity and neurotrophins in a number of in vitro systems. We examined whether either of these factors was likely to play a role in the in vivo optic nerve‐dependent regulation of a substance P‐like immunoreactive (SP‐ir) population of cells in the developing optic tectum of the frog. In contrast to our previous results with the adult system, blocking tectal cell responses to glutamate release by retinal ganglion cells with 6‐cyano‐7‐nitroquinoxaline‐2,3 dione (CNQX) did not affect the percent of SP‐ir cells in the developing tectum. Treatment with d‐(‐)‐2‐amino‐5‐phosphonovaleric acid (d‐AP‐5) was also ineffective in this regard, although both it and CNQX treatment disrupted visual map topography. Chronic treatment with brain‐derived neurotrophic factor (BDNF) and neurotrophin‐4/5 (NT‐4/5) produced increases in SP‐ir cells in the treated lobes of normal animals, which were significant in the case of NT‐4/5. Both substances also prevented the decrease of SP cells that would otherwise occur in the deafferented lobe of unilaterally optic nerve‐transected tadpoles. These changes in the percent of SP‐ir cells occurred without any detectable changes in the overall number of tectal cells. NGF had no effect on SP expression. Nor did it affect topographic map formation, which was disrupted by treatment with either BDNF or NT‐4/5. Our results demonstrate that different mechanisms regulate SP expression in the developing and adult tectum. They indicate that neurotrophin levels in the developing optic tectum may selectively regulate a specific neuropeptide‐expressing population of cells. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 131–149, 2001     

大鼠杏仁核5-HT_3受体参与免疫调制

实验通过大鼠侧脑室和杏仁核给予 5 HT3受体激动剂 1 phenylbiguanide (PBG) ,用 3H TdR掺入法测定脾细胞丝裂原 (concanavalinA ,ConA和lipopolysaccharide ,LPS)刺激增殖效应 ,用活化脾细胞增殖法测定IL 2生成 ,MTT法测定自然杀伤 (naturalkiller,NK)细胞活性和用放射免疫测定血浆皮质酮水平 ,以探讨大鼠杏仁核 5 HT3受体在免疫调控中的作用。结果表明 :5 HT3受体拮抗剂granisetron (GNT ,0 1～ 0 4mg/kgip)剂量依赖地增强ConA和LPS刺激的脾细胞增殖 ,作用在连续给药 5d最明显 ;双侧脑室给予PBG ( 5 μg/side)可增强ConA和LPS刺激的脾细胞增殖效应 ,作用在连续给药 3d最明显 ;双侧和单侧中央杏仁核给予PBG 0 5 μg均增强ConA刺激的脾细胞增殖和IL 2生成 ;底内侧杏仁核给予同剂量PBG仅增强LPS刺激的脾细胞增殖效应 ,不影响ConA刺激的脾细胞增殖和IL 2生成 ;中央杏仁核给予PBG升高血浆皮质酮的作用较底内侧杏仁核给予等量PBG引起的升高血浆皮质酮作用明显 (P <0 0 1)。侧脑室、中央杏仁核和底内杏仁核给予PBG对丝裂原刺激的脾细胞增殖效应影响不同 ,但均被同时同部位给予GNT所拮抗 ,提示杏仁核中央核和底内侧核的 5 HT3受体可能以不同方式参与ConA或LPS刺激的脾细胞增殖效应的调制     

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

The characteristics of Cl^– transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl^–-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K⁺ and valinomycin, increased the initial rate of Cl^– transport, demonstrating that Cl^– could be electrically driven into the vesicles. Chloride influx was unaffected by SO ₄ ^2- , but was competitively blocked by NO ₃ ^– , indicating that both Cl^– and NO ₃ ^– may be transported by the same porter. In some preparations, increases in free-Ca²⁺ concentration from 10^–8 to 10^–5 mol·dm^–3 caused a significant decrease in Cl^– influx, which may indicate that cytosolic Ca²⁺ concentration has a role in controlling Cl^– fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl^– transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.Abbreviations and Symbols BTP 1,3-bis[tris(hydroxymethyl)-methylamino]propane - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - membrane potential - pH pH gradient - SPQ 6-methoxy-1-(3-sulfonatopropyl)quinolinium - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine     

Production and characterisation of monoclonal antibodies to ovine interleukin-5

Monoclonal antibodies (MAbs) were developed against recombinant ovine interleukin-5 (IL-5) produced in the baculovirus expression vector system. One MAb, D11 (isotype IgG1), neutralised the activity of both recombinant and native sources of IL-5 in a biological assay (Baf cell assay) but was only weakly reactive in immunocytochemistry. Conversely, a second MAb, A8 (isotype IgA), successfully detected IL-5 in immunocytochemistry but did not display neutralising activity. The development of these MAbs will enable the assay of ovine IL-5 in vitro and permit studies into the role of hypersensitivity reactions in sheep by neutralisation of IL-5 in vivo.     

Synthesis of novel steroidal agonists,partial agonists,and antagonists for the glucocorticoid receptor

Adverse effects of glucocorticoids could be limited by developing new compounds that selectively modulate anti-inflammatory activity of the glucocorticoid receptor (GR). We have synthesized a novel series of steroidal GR ligands, including potent agonists, partial agonists and antagonists with a wide range of effects on inhibiting secretion of interleukin-6. Some of these new ligands were designed to directly impact conformational stability of helix-12, in the GR ligand-binding domain (LBD). These compounds modulated GR activity and glucocorticoid-induced gene expression in a manner that was inversely correlated to the degree of inflammatory response. In contrast, compounds designed to directly modulate LBD epitopes outside helix-12, led to dissociated levels of GR-mediated gene expression and inflammatory response. Therefore, these new series of compounds and their derivatives will be useful to dissect the ligand-dependent features of GR signaling specificity.     

Peptide modification by introduction ofα-trifluoromethyl substituted amino acids

Summary Methodology for the synthesis and incorporation of-trifluoromethyl substituted amino acids into N- and C-terminal position of peptides is described. The incorporation of-trifluoromethyl substituted amino acids into strategical positions of peptides enhances proteolytic stability and lipophilicity. Furthermore, it improves transport rates in vivo and permeability through certain body barriers.     

Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions

Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations.