Microtubules and cyclic amp in human leukocytes: on the order of things

We have shown previously that the β-adrenergic agonist isoproterenol (2μM) and the phosphodiesterase inhibitor isobutylmethylxanthine (1 mM) produce a much greater increase in cyclic AMP in human leukocytes that have been pretreated with colchicine (or with other agents that affect microtubule assembly) than in control leukocytes. The effects of colchicines were both time- and dose-dependant. These and other data suggested that the generation of cyclic AMP is normally restricted by an intact system of cytoplasmic microtubules. If so, then the same time and dose dependencies might apply to other colchicines-induced changes in leukocyte function. We have now assayed the distribution of concanavalin A (Con A)-receptor complexes on the leukocyte membrane, taking into account that leukocytes competent to assemble microtubules show a uniform distribution of surface- bound Con A whereas microtubule-deficient cells accumulate Con A in surface caps. We have found that the effect of colchicine on capping is also both time- and dose dependent, and that the dose-response relationships conform to those required to increase cyclic AMP levels. These findings provide further evidence that both colchicine-induced Con-A capping and colchicine- induced cyclic AMP generation depend upon the relaxation of constraints normally imposed by cytoplasmic microtubules upon the plasma membrane, which limit, respectively, lateral mobility of the lectin-receptor complexes, and expression of hormone-sensitive adenylate cyclase. Moreover, colchicine-induced Con-A cap formation is not affected even by very large changes in leukocyte cyclic AMP levels. Thus, elevated cyclic AMP levels do not appear to promote the dissolution of microtubules; rather, the dissolution of microtubules permits the generation of increased amounts of cyclic AMP.     

Cutting edge: activity of human adult microglia in response to CC chemokine ligand 21

The approximately 50 known chemokines are classified in distinct subfamilies: CXC, CC, CX3C, and C. Although the signaling of chemokines often is promiscuous, signaling events between members of these distinct chemokine classes are hardly observed. The only known exception so far is the murine CC chemokine ligand (CCL)21 (secondary lymphoid tissue chemokine, Exodus-2, 6Ckine), which binds and activates the murine CXC chemokine receptor CXCR3. However, this exception has not been found in humans. In this study, we provide evidence that human CCL21 is a functional ligand for endogenously expressed CXCR3 in human adult microglia. In absence of CCR7 expression, CCL21 induced chemotaxis of human microglia with efficiency similar to the CXCR3 ligands CXC chemokine ligand 9 (monokine induced by IFN-gamma) and CXC chemokine ligand 10 (IFN-gamma-inducible protein-10). Because human CCL21 did not show any effects in CXCR3-transfected HEK293 cells, it is indicated that CXCR3 signaling depends on the cellular background in which the CXCR3 is expressed.     

Cysteine Residues and the Structure of the Rat Renal Proximal Tubular Type II Sodium Phosphate Cotransporter (Rat NaPi IIa)

The rat renal Na/P _i cotransporter type IIa (rat NaP_i IIa) is a 637 amino acid protein containing 12 cysteine residues. We examined the effect of different cysteine modifying methanethiosulfonate (MTS)-reagents and the disulfide bond reducing agent tris(2-carboxyethyl)phosphine (TCEP) on the transport activity of wild-type and 12 single cysteine substitution mutants of rat NaPi IIa expressed in Xenopus laevis oocytes. The transport activity of the wild-type protein was resistant to three membrane impermeant MTS-reagents (MTSEA, MTSET and MTSES). In contrast, membrane permeant methyl methanethiosulfonate (MMTS) and TCEP inhibited the transport activity of both the wild-type, as well as all the single mutant proteins. This indicated the existence of more than one functionally important cysteine residue, not accessible extracellularly, and at least 2 disulfide bridges. To identify the disulfide bridges, three double mutants lacking 2 of the 3 cysteine residues predicted to be extracellular in different combinations were examined. This led to the identification of one disulfide bridge between C306 and C334; reconsideration of the topological model predictions suggested a second disulfide bridge between C225 and C520. Evaluation of a fourth double mutant indicated that at least one of two disulfide bridges (C306 and C334; C225 and C520) has to be formed to allow the surface expression of a functional cotransporter. A revised secondary structure is proposed which includes two partially repeated motifs that are connected by disulfide bridges formed between cysteine pairs C306-C334 and C225-C520. Received: 13 December 1999/Revised: 31 March 2000     

Positive inotropic and negative lusitropic effects of endothelin receptor agonism in vivo

The endothelin (ET) system is involved in the regulation of myocardial function in health as well as in several diseases, such as congestive heart failure, myocardial infarction, and septic myocardial depression. Conflicting results have been reported regarding the acute contractile properties of ET-1. We therefore investigated the effects of intracoronary infusions of ET-1 and of the selective ET(B) receptor-selective agonist sarafotoxin 6c with increasing doses in anesthetized pigs. Myocardial effects were measured through analysis of the left ventricular pressure-volume relationship. ET-1 elicited increases in the myocardial contractile status (end-systolic elastance value of 0.94 +/- 0.11 to 1.48 +/- 0.23 and preload recruitable stroke work value of 68.7 +/- 4.7 to 83.4 +/- 7.2) that appear to be mediated through ET(A) receptors, whereas impairment in left ventricular isovolumic relaxation (tau = 41.5 +/- 1.4 to 58.1 +/- 5.0 and t(1/2) = 23.0 +/- 0.7 to 30.9 +/- 2.6, where tau is the time constant for pressure decay and t(1/2) is the half-time for pressure decay) was ET(B) receptor dependent. In addition, intravenous administration of ET-1 impaired ventricular relaxation but had no effect on contractility. Intracoronary sarafotoxin 6c administration caused impairments in left ventricular relaxation (tau from 43.3 +/- 1.8 to 54.4 +/- 3.4) as well as coronary vasoconstriction. In conclusion, ET-1 elicits positive inotropic and negative lusitropic myocardial effects in a pig model, possibly resulting from ET(A) and ET(B) receptor activation, respectively.     

Cysteine mutagenesis reveals novel structure-function features within the predicted third extracellular loop of the type IIa Na(+)/P(i) cotransporter

The transport function of the rat type IIa Na(+)/P(i) cotransporter is inhibited after binding the cysteine modifying reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) to a cysteine residue substituted for a serine at position 460 (S460C) in the predicted third extracellular loop. This suggests that Ser-460 lies in a functionally important region of the protein. To establish a "structure-function" profile for the regions that flank Ser-460, the substituted cysteine accessibility method was employed. 18 mutants were constructed in which selected amino acids from Arg-437 through Leu-465 were substituted one by one for a cysteine. Mutants were expressed in Xenopus oocytes and transport function (cotransport and slippage) and kinetics were assayed by electrophysiology with or without prior treatment with cysteine modifying (methanethiosulfonate, MTS) reagents. Except for mutant I447C, mutants with cysteines at sites from Arg-437 through Thr-449, as well as Pro-461, were inactive. Cotransport function of mutants with Cys substitutions at sites Arg-462 through Leu-465 showed low sensitivity to MTS reagents. The preceding mutants (Cys substitution at Thr-451 to Ser-460) showed a periodic accessibility pattern that would be expected for an alpha-helix motif. Apart from loss of transport function, exposure of mutants A453C and A455C to MTSEA or 2-(triethylammonium)ethyl MTS bromide (MTSET) increased the uncoupled slippage current, which implicated the mutated sites in the leak pathway. Mutants from Ala-453 through Ala-459 showed less pH dependency, but generally stronger voltage dependency compared with the wild type, whereas those flanking this group were more sensitive to pH and showed weaker voltage dependence of cotransport mode kinetics. Our data indicate that parts of the third extracellular loop are involved in the translocation of the fully loaded carrier and show a membrane-associated alpha-helical structure.     

Interaction of the type IIa Na/Pi cotransporter with PDZ proteins

The type IIa Na(+)-dependent inorganic phosphate (Na/P(i)) cotransporter is localized in the apical membrane of proximal tubular cells and is regulated by an endocytotic pathway. Because molecular processes such as apical sorting, internalization, or subsequent degradation might be assisted by associated proteins, a yeast two-hybrid screen against the C-terminal, cytosolic tail of type IIa cotransporter was designed. Most of the potential proteins found belonged to proteins with multiple PDZ modules and were either identical/related to PDZK1 or identical to NHERF-1. Yeast trap truncation assays confined the peptide-protein association to the C-terminal amino acid residues TRL of type IIa cotransporter and to single PDZ domains of each identified protein, respectively. The specificity of these interactions were confirmed in yeast by testing other apical localized transmembraneous proteins. Moreover, the type IIa protein was recovered in vitro by glutathione S-transferase-fused PDZ proteins from isolated renal brush border membranes or from type IIa-expressing oocytes. Further, these PDZ proteins are immunohistochemically detected either in the microvilli or in the subapical compartment of proximal tubular cells. Our results suggest that the type IIa Na/P(i) cotransporter interacts with various PDZ proteins that might be responsible for the apical sorting, parathyroid hormone controlled endocytosis or the lysosomal sorting of internalized type IIa cotransporter.     

豆科黄华属植物种子表面特征的研究

在扫描电镜下观察了豆科黄华属Thermopsis 18种植物种子的表面纹饰,发现 T．alpina,T.bar- bata,T．inflata,T．lupinoides,T. licentiana,T.smithiana和T．turkestanica的种子表面为粗网状,T californica,T.divaricarpa,T. macrophylla,T．mollis的种子表面为细网状,T.gracilis,T.montana,T. fabacea的种子表面为相对平滑型纹饰,T．alterniflora的种子表面为不规则条形,T．chinensis的种子表 面为粘膜状,T.rhombifolia的种子表面为条形及 T．viuosa的种子表面为碎屑状纹饰。结果表明黄华属的种子表面特征对属下类群的划分有一定意义,对澄清某些混乱的种有一定价值。     

榆科榉属模式的考证

长期以来,Zelkova crenata Spach被认为是榉属(Zelkova)的模式。作者基于国际植物命名法规中的模式指定原则,并通过有关文献的考证,确认Zelkova carpinifolia(Pall.) K. Koch为榉属的合法模式,而并非Z. crenata Spach或Z. carpinifolia(Pall.) Dipp.。     

USP18 lack in microglia causes destructive interferonopathy of the mouse brain

Microglia are tissue macrophages of the central nervous system (CNS) that control tissue homeostasis. Microglia dysregulation is thought to be causal for a group of neuropsychiatric, neurodegenerative and neuroinflammatory diseases, called “microgliopathies”. However, how the intracellular stimulation machinery in microglia is controlled is poorly understood. Here, we identified the ubiquitin‐specific protease (Usp) 18 in white matter microglia that essentially contributes to microglial quiescence. We further found that microglial Usp18 negatively regulates the activation of Stat1 and concomitant induction of interferon‐induced genes, thereby terminating IFN signaling. The Usp18‐mediated control was independent from its catalytic activity but instead required the interaction with Ifnar2. Additionally, the absence of Ifnar1 restored microglial activation, indicating a tonic IFN signal which needs to be negatively controlled by Usp18 under non‐diseased conditions. These results identify Usp18 as a critical negative regulator of microglia activation and demonstrate a protective role of Usp18 for microglia function by regulating the Ifnar pathway. The findings establish Usp18 as a new molecule preventing destructive microgliopathy.