Isoforms of soluble alpha-tubulin in oocytes and brain of the frog (genus Rana): changes during oocyte maturation

Rana oocytes have previously been shown to contain much more soluble tubulin than does the brain, suggesting different assembly and disassembly dynamics of frog oocyte tubulin compared to that in brain. By using centrifugation, SDS-PAGE, two-dimensional gel electrophoresis and Western blots, probed with anti-α-tubulin monoclonal antibodies, polymorphic α-tubulins (isoforms) were compared in brains and follicle-enclosed oocytes of northern (Rana pipiens) and southern (R. berlandieri) frogs. Oocyte tubulin in both species had isoforms with greater ranges of isoelectric point (pI) than those of brain tubulins; in particular, the oocyte tubulin pIs ranged further into the acidic region of the isoelectric-focusing gels than corresponding brain tubulin. This difference may, in part, be responsible for the previously reported assembly differences between oocyte tubulin (undetectable assembly) and brain tubulin (high assembly). Isoforms of α-tubulin with relat ively acidic pI were more abundant in northern frog brain and oocyte soluble extracts than in analogous extracts from southern frogs. Furthermore, additional acidic α-tubulin isoforms were found in progesterone-treated oocytes (i.e., eggs), indicating increased heterogeneity of acidic a-tubulin isoforms during oocyte meiotic maturation. Among northern frog oocyte soluble components fractionated on Superose-6b columns, tubulin complexes with apparent molecular mass of about 1800 kDa were found to contain acidic α-tubulin isoforms while the putative oligomeric tubulins with an apparent molecular mass of about 250 kDa contained an additional relatively basic α-tubulin isoform. The acidic α-tubulin isoforms, therefore, are proposed to be associated with cold-adaptable cells of brain and oocytes, and may also be involved in stabilization of large soluble tubulin complexes in oocytes of the northern frog. Received 1 October 2002; accepted 9 October 2002 RID="*" ID="*"Corresponding author.     

The renal plumbing system: aquaporin water channels

Aquaporins are channels that facilitate movement of water across lipid bilayers. They are expressed in multiple tissues and are essential for regulation of body water homeostasis. The kidney is the main organ responsible for this regulation, and at least seven aquaporins are expressed at distinct sites in the kidney. Aquaporin expression correlates with observed water permeability of each nephron segment: proximal tubule and descending thin limb of Henle have constitutive high water permeability due to expression of AQP1, whereas collecting duct water permeability is tightly regulated by the antidiuretic hormone vasopressin via regulation of AQP2. This review aims at providing insight into renal aquaporins, with special focus on AQP2.Received 9 December 2004; received after revision 8 April 2005; accepted 11 April 2005     

Isoform specific phosphorylation of p53 by protein kinase CK1

The ability of three isoforms of protein kinase CK1 (α, γ₁, and δ) to phosphorylate the N-terminal region of p53 has been assessed using either recombinant p53 or a synthetic peptide reproducing its 1–28 sequence. Both substrates are readily phosphoylated by CK1δ and CK1α, but not by the γ isoform. Affinity of full size p53 for CK1 is 3 orders of magnitude higher than that of its N-terminal peptide (K _m 0.82 μM vs 1.51 mM). The preferred target is S20, whose phosphorylation critically relies on E17, while S6 is unaffected despite displaying the same consensus (E-x-x-S). Our data support the concept that non-primed phosphorylation of p53 by CK1 is an isoform-specific reaction preferentially affecting S20 by a mechanism which is grounded both on a local consensus and on a remote docking site mapped to the K²²¹RQK²²⁴ loop according to modeling and mutational analysis.     

CADM1 isoforms differentially regulate human mast cell survival and homotypic adhesion

Cell adhesion molecule 1 (CADM1), expressed by human lung mast cells (HLMCs), mediates their adhesion to airway smooth muscle (ASM), and contributes to ASM-dependent HLMC proliferation and survival. CADM1 is expressed in alternatively spliced isoforms, but those present in HLMCs and their function are not known. We cloned three functional and one cryptic non-functional isoform with alternative splicing between exons 7/11 and 1/2, respectively, from HLMCs and human MC lines (HMC-1 and LAD2). Differentiated HLMCs and LAD2 cells expressed the functional isoform SP4 containing exons 7/8/11 (~80% of clones), as well as SP1 (exons 7/8/9/11) and a novel SP6 (exons 7/8/9/10/11). In contrast, immature HMC-1 cells expressed only functional SP4. SP4 overexpression in HMC-1 cells and HLMCs augmented homotypic adhesion to a greater extent than SP1 in various conditions. In contrast, CADM1 downregulation abolished homotypic adhesion, indicating that CADM1 is the sole receptor mediating mast cell aggregation. CADM1-mediated adhesion was enhanced by the presence of cell survival factors. SP1 overexpression in HMC-1 cells compromised survival compared to SP4 overexpression or control. CADM1 downregulation resulted in reduced viability and decreased expression of the pro-survival protein Mcl-1(L), but not Blc-2 or Bcl-X(L), and increased caspase-3/7 activity in both HMC-1 cells and HLMCs. This coincided with decreased basal Kit levels in HLMCs. In summary, human MCs express multiple CADM1 isoforms which exhibit differential regulation of survival and homotypic adhesion. The most highly expressed SP4 isoform is likely to contribute to MC aggregation and longevity in mastocytosis, and augment the pathophysiology of allergic diseases.     

盐酸戊乙奎醚对大鼠感染性脑水肿AQP4表达的影响

目的：研究盐酸戊乙奎醚对感染性脑水肿保护作用的机制。方法：采用左颈内动脉注射脂多糖复制大鼠感染性脑水肿模型,将84只雄性SD大鼠随机分为对照组（C组）、水肿组（I组）、盐酸戊乙奎醚治疗组（P组）。检测6h、12h、24h、48h时各组大鼠脑组织含水量、病理形态;脑组织SOD活性、MDA含量;免疫组化法及RT—PCR法检测AQP4蛋白分布与AQP4mRNA含量。结果：（1）与C组相比,I组、P组脑含水量、MDA含量均升高,同时SOD活性、AQP4蛋白、AQP4mRNA表达均减少（P〈0．01）。与对应时间点P组相比,I组脑含水量、MDA含量均升高,同时SOD活性、AQP4蛋白、AQP4mRNA表达均减少,于24h达高峰,48h仍在较高水平（P〈0．01）。（2）脑组织光镜检查：I组大鼠脑组织损伤严重,P组损伤明显减轻。结论：盐酸戊乙奎醚能在一定程度上可减轻感染所致的脑组织损伤,上调AQP4表达,对脑水肿有一定治疗作用,其机制可能与抗氧化作用有关。     

Aquaporins with selectivity for unconventional permeants

The aquaporin protein family generally seems to be designed for the selective passage of water or glycerol. Charged molecules, metal ions and even protons are strictly excluded. Recently, particular aquaporin isoforms were reported to conduct unconventional permeants, i.e., the unpolar gases carbon dioxide and nitric oxide, the polar gas ammonia, the oxidative oxygen species hydrogen peroxide, and the metalloids antimonite, arsenite and silicic acid. Here, we summarize the available data on permeability properties and physiological settings of these aquaporins and we analyze which structural features might be connected to permeability for non-water, non-glycerol solutes. Received 31 March 2007; received after revision 3 May 2007; accepted 23 May 2007     

Structural features underlying the selectivity of the kinase inhibitors NBC and dNBC: role of a nitro group that discriminates between CK2 and DYRK1A

8-hydroxy-4-methyl-9-nitrobenzo(g)chromen-2-one (NBC) has been found to be a fairly potent ATP site-directed inhibitor of protein kinase CK2 (Ki = 0.22 μM). Here, we show that NBC also inhibits PIM kinases, especially PIM1 and PIM3, the latter as potently as CK2. Upon removal of the nitro group, to give 8-hydroxy-4-methyl-benzo(g)chromen-2-one (here referred to as “denitro NBC”, dNBC), the inhibitory power toward CK2 is almost entirely lost (IC₅₀ > 30 μM) whereas that toward PIM1 and PIM3 is maintained; in addition, dNBC is a potent inhibitor of a number of other kinases that are weakly inhibited or unaffected by NBC, with special reference to DYRK1A whose IC₅₀ values with NBC and dNBC are 15 and 0.60 μM, respectively. Therefore, the observation that NBC, unlike dNBC, is a potent inducer of apoptosis is consistent with the notion that this effect is mediated by inhibition of endogenous CK2. The structural features underlying NBC selectivity have been revealed by inspecting its 3D structure in complex with the catalytic subunit of Z. mays CK2. The crucial role of the nitro group is exerted both through a direct electrostatic interaction with the side chain of Lys68 and, indirectly, by enhancing the acidic dissociation constant of the adjacent hydroxyl group which interacts with a conserved water molecule in the deepest part of the cavity. By contrast, the very same nitro group is deleterious for the binding to the active site of DYRK1A, as disclosed by molecular docking. This provides the rationale for preferential inhibition of DYRK1A by dNBC.     

Novel diterpenoid diacylglycerols from marine molluscs: potent morphogens and protein kinase C activators

Five novel 1,2-sn-diacylglycerols with diterpenoid acyl moieties in thesn-1 position were isolated and characterized, together with the corresponding 1,3-sn-diacylglycerols, from three species of dorid nudibranchs molluscs. Their potent activity as morphogens in vivo in theHydra tentacle regeneration assay and their parallel activity as activators of rat brain protein kinase C (PKC) in vitro are reported here. Our findings promote the use of these compounds as useful molecular probes for both in vivo and in vitro studies on the participation of PKC in cell development.     

Alternative exon usage selectively determines both tissue distribution and subcellular localization of the acyl-CoA thioesterase 7 gene products

Acyl-CoA thioesterases (ACOTs) catalyze the hydrolysis of acyl-CoAs to free fatty acids and coenzyme A. Recent studies have demonstrated that one gene named Acot7, reported to be mainly expressed in brain and testis, is transcribed in several different isoforms by alternative usage of first exons. Strongly decreased levels of ACOT7 activity and protein in both mitochondria and cytosol was reported in patients diagnosed with fatty acid oxidation defects, linking ACOT7 function to regulation of fatty acid oxidation in other tissues. In this study, we have identified five possible first exons in mouse Acot7 (Acot7a–e) and show that all five first exons are transcribed in a tissue-specific manner. Taken together, these data show that the Acot7 gene is expressed as multiple isoforms in a tissue-specific manner, and that expression in tissues other than brain and testis is likely to play important roles in fatty acid metabolism. Received 5 February 2007: received after revision 3 April 2007; accepted 19 April 2007     

Polynomials and equations in arabic algebra

It is shown in this article that the two sides of an equation in the medieval Arabic algebra are aggregations of the algebraic “numbers” (powers) with no operations present. Unlike an expression such as our 3x + 4, the Arabic polynomial “three things and four dirhams” is merely a collection of seven objects of two different types. Ideally, the two sides of an equation were polynomials so the Arabic algebraists preferred to work out all operations of the enunciation to a problem before stating an equation. Some difficult problems which involve square roots and divisions cannot be handled nicely by this basic method, so we do find square roots of polynomials and expressions of the form “A divided by B” in some equations. But rather than initiate a reconsideration of the notion of equation, these developments were used only for particularly complex problems. Also, the algebraic notation practiced in the Maghreb in the later middle ages was developed with the “aggregations” interpretation in mind, so it had no noticeable impact on the concept of polynomial. Arabic algebraists continued to solve problems by working operations before setting up an equation to the end of the medieval period. I thank Mahdi Abdeljaouad, who provided comments on an earlier version of this paper, and Haitham Alkhateeb, for his help with some of the translations. Notes on references: When page numbers are separated by a “ / ”, the first number is to the Arabic text, and the second to the translation. Also, a semicolon separates page number from line number. Example: [Al-Khwārizmī, 1831, 31;6/43] refers to page 31 line 6 of the Arabic text, and page 43 of the translation.     

The diterpenoid alkaloid noroxoaconitine is a Mapkap kinase 5 (MK5/PRAK) inhibitor

The mitogen-activated protein kinase-activated protein kinase MK5 is ubiquitously expressed in vertebrates and is implicated in cell proliferation, cytoskeletal remodeling, and anxiety behavior. This makes MK5 an attractive drug target. We tested several diterpenoid alkaloids for their ability to suppress MK5 kinase activity. We identified noroxoaconitine as an ATP competitor that inhibited the catalytic activity of MK5 in vitro (IC₅₀ = 37.5 μM; K _i = 0.675 μM) and prevented PKA-induced nuclear export of MK5, a process that depends on kinase active MK5. MK5 is closely related to MK2 and MK3, and noroxoaconitine inhibited MK3- and MK5- but not MK2-mediated phosphorylation of the common substrate Hsp27. Molecular docking of noroxoaconitine into the ATP binding sites indicated that noroxoaconitine binds more strongly to MK5 than to MK3. Noroxoaconitine and derivatives may help in elucidating the precise biological functions of MK5 and may prove to have therapeutic values.     

Multimeric structures of HLA-G isoforms function through differential binding to LILRB receptors

The non-classical Human leukocyte antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and immuno-inhibitory functions. Most of the known functions of HLA-G concern the membrane-bound HLA-G1 and soluble HLA-G5 isoforms, which present the typical structure of classical HLA class I molecule: a heavy chain of three globular domains α₁–α₂–α₃ non-covalently bound to β-2-microglobulin (B2M) and a peptide. Very little is known of the structural features and functions of other HLA-G isoforms or structural conformations other than B2M-associated HLA-G1 and HLA-G5. In the present work, we studied the capability of all isoforms to form homomultimers, and investigated whether they could bind to, and function through, the known HLA-G receptors LILRB1 and LILRB2. We report that all HLA-G isoforms may form homodimers, demonstrating for the first time the existence of HLA-G4 dimers. We also report that the HLA-G α₁–α₃ structure, which constitutes the extracellular part of HLA-G2 and HLA-G6, binds the LILRB2 receptor but not LILRB1. This is the first report of a receptor for a truncated HLA-G isoform. Following up on this finding, we show that the α₁–α₃-Fc structure coated on agarose beads is tolerogenic and capable of prolonging the survival of skin allografts in B6-mice and in a LILRB2-transgenic mouse model. This study is the first proof of concept that truncated HLA-G isoforms could be used as therapeutic agents.     

Membrane permeability changes duringRana oocyte maturation

A transition from an open system to a closed one must occur during the complex process of meiotic maturation of the amphibian oocyte. Membrane permeability to urea inRana oocytes following progesterone stimulation was determined, and the largest decrease was found to coincide with germinal vesicle breakdown. These findings suggest that the timing of the disappearance of membrane permeability correlates with developmental events that prepare the oocyte for a hostile environment.     

Characterization of the 5-HT1A receptor of the honeybee (Apis mellifera) and involvement of serotonin in phototactic behavior

Serotonin plays a key role in modulating various physiological and behavioral processes in both protostomes and deuterostomes. The vast majority of serotonin receptors belong to the superfamily of G-protein-coupled receptors. We report the cloning of a cDNA from the honeybee (Am5-ht1A) sharing high similarity with members of the 5-HT₁ receptor class. Activation of Am5-HT_1A by serotonin inhibited the production of cAMP in a dose-dependent manner (EC₅₀ = 16.9 nM). Am5-HT_1A was highly expressed in brain regions known to be involved in visual information processing. Using in vivo pharmacology, we could demonstrate that Am5-HT_1A receptor ligands had a strong impact on the phototactic behavior of individual bees. The data presented here mark the first comprehensive study—from gene to behavior—of a 5-HT_1A receptor in the honeybee, paving the way for the eventual elucidation of additional roles of this receptor subtype in the physiology and behavior of this social insect.     

Metal complex formation by nicotianamine,a possible phytosiderophore

Summary The acid dissociation constants of nicotianamine (1) (pK₁=6.97, pK₂=9.13, pK₃=9.75; 0.1 M KClO₄, 25°C) and the stability constants for its 11 complexes with bivalent metal ions (log K_Cu=18.6, log K_Ni=16.1, log K_Co=14.8, log K_Zn=14.7, log K_Fe=12.1, log K_Mn=8.8, log K_Mg4.5; 0.1 M KClO₄, 25°C) were determined using potentiometric titrations in aqueous solution. Fe(III)-nicotianamine complexes were not detected under the same experimental conditions.Part 13 in the series On the normalizing factor for the tomato mutantchloronerva, for part 12 see Ripperger et al.⁴.     

The three-fingered protein domain of the human genome

Extracellular domains of some cellular receptors expressed in the organisms at different levels of development belong to three-fingered protein (TFP) fold. The Homo sapiens genome encodes at least 45 genes containing from one to three TFP domains (TFPDs), namely diverse paralogues of the Ly6 gene, CD59 and the receptors of activins, bone morphogenetic proteins, Mullerian inhibiting substance and transforming growth factor-β. C4.4a and urokinase/plasminogen activatory receptor contain two and three TFPD repeats, respectively. These diverse proteins have a low overall sequence similarity with each other and their hydrophobicity levels vary to a considerable degree. It is suggested that sequence differentiation within the TFPD led to distinct groups of proteins whose attributes were optimized to fit both the physicochemical properties specific to their functional microenvironment and selective targeting of their highly diversified extracellular cofactors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 7 August 2008; accepted 29 August 2008     

The phorbol ester TPA induces metamorphosis in Red Sea coral planulae (Cnidaria: Anthozoa)

Controlled experiments on the metamorphosis of marine invertebrate larvae require artificial inducers. These inducers can be used for studying the involvement of known signal transduction pathways in settlement and metamorphosis. The ability of the tumor-promoting phorbol ester TPA (12-O-tetradecanoylphorbol-13-acetate) to induce metamorphosis in planulae of the Red Sea soft coral speciesHeteroxenia fuscescens, Xenia umbellata, Dendronephthya hemprichii, Litophyton arboreum andParerythropodium fulvum fulvum, and the stony coralStylophora pistillata, was examined by using various concentrations of TPA. The chemical induced metamorphosis in all six species. The effect was unspecific and concentration-related. For all the corals except forX. umbellata the highest mean percentages of metamorphosis were obtained with 8.1×10^–7–10^–9 M TPA. ForX. umbellata, the percentage of metamorphosis was lower, and was obtained within a wider TPA concentration range. The present results, along with previous studies on Hydrozoa and Scyphozoa, demonstrate that TPA is the first common artificial inducer for these classes of Cnidaria. TPA is known to activate the enzyme protein kinase C (PKC) and therefore plays an important role in studying the phosphatidylinositol signal transduction system. Evidence for the involvement of this pathway in triggering metamorphosis has already been reported for Hydrozoa and Scyphozoa. Our results suggest that PKC is also involved in initiating metamorphosis in Anthozoa.     

A complex of Shc and Ran-GTPase localises to the cell nucleus

The three isoforms of the adaptor protein Shc play diverse roles in cell signalling. For example, the observation of p46 Shc in the nuclei of hepatocellular carcinoma cells suggests a function quite distinct from the better characterised cytoplasmic role. Ligands responsible for the transport of various Shc isoforms into organelles such as the nucleus have yet to be reported. To identify such ligands a far western approach was used to determine the p52 Shc interactome. The Ran-GTPase nuclear transport protein was identified and found to bind to p52 Shc in vitro with low micromolar affinity. Co-immunoprecipitation, pull down and fluorescence lifetime imaging microscopy experiments in stable cells confirmed cellular interaction and nuclear localisation. The nuclear transport factor protein NTF2, which functions in cohort with Ran, was shown to form a complex with both RAN and Shc, suggesting a mechanism for Shc entry into the nucleus as part of a tertiary complex. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 20 October 2008; received after revision 04 December 2008; accepted 15 December 2008     

NHE3 phosphorylation via PKCη marks the polarity and orientation of directionally migrating cells

Endogenous electric fields (EF) may provide an overriding cue for directional cell migration during wound closure. Perceiving a constant direction requires active sodium-hydrogen exchanger (pNHE3) at the leading edge of HEK 293 cells but its activation mechanism is not yet fully understood. Because protein kinase C (PKC) is required in electrotaxis, we asked whether NHE3 is activated by PKC during wound healing. Using pharmacological (pseudosubstrate and edelfosine) inhibition, we showed that inhibition of PKCη isoform impairs directional cell migration in HEK 293 cells in the presence of a persistent directional cue (0.25–0.3 V/mm of EF for 2 h). Further, we found that pNHE3 forms complexes with both PKCη and ?-tubulin, suggesting that these molecules may regulate the microtubule-organizing center. In addition, cellular pNHE3 content was reduced significantly when PKCη was inhibited during directional cell migration. Taken together, these data suggest that PKCη-dependent phosphorylation of NHE3 and the formation of pNHE3/PKCη/?-tubulin complexes at the leading edge of the cell are required for directional cell migration in an EF.