Stabilization of the α2 isoform of Na,K-ATPase by mutations in a phospholipid binding pocket

The α2 isoform of Na,K-ATPase plays a crucial role in Ca²⁺ handling, muscle contraction, and inotropic effects of cardiac glycosides. Thus, structural, functional, and pharmacological comparisons of α1, α2, and α3 are of great interest. In Pichia pastoris membranes expressing human α1β1, α2β1, and α3β1 isoforms, or using the purified isoform proteins, α2 is most easily inactivated by heating and detergent (α2 ≫ α3 > α1). We have examined an hypothesis that instability of α2 is caused by weak interactions with phosphatidylserine, which stabilizes the protein. Three residues, unique to α2, in trans-membrane segments M8 (Ala-920), M9 (Leu-955), and M10 (Val-981) were replaced by equivalent residues in α1, singly or together. Judged by the sensitivity of the purified proteins to heat, detergent, “affinity” for phosphatidylserine, and stabilization by FXYD1, the triple mutant (A920V/L955F/V981P, called α2VFP) has stability properties close to α1, although single mutants have only modest or insignificant effects. Functional differences between α1 and α2 are unaffected in α2VFP. A compound, 6-pentyl-2-pyrone, isolated from the marine fungus Trichoderma gamsii is a novel probe of specific phospholipid-protein interactions. 6-Pentyl-2-pyrone inactivates the isoforms in the order α2 ≫ α3 > α1, and α2VFP and FXYD1 protect the isoforms. In native rat heart sarcolemma membranes, which contain α1, α2, and α3 isoforms, a component attributable to α2 is the least stable. The data provide clear evidence for a specific phosphatidylserine binding pocket between M8, M9, and M10 and confirm that the instability of α2 is due to suboptimal interactions with phosphatidylserine. In physiological conditions, the instability of α2 may be important for its cellular regulatory functions.     

Movement of protein and macromolecules between host plants and the parasitic weed Phelipanche aegyptiaca Pers.

Little is known about the translocation of proteins and other macromolecules from a host plant to the parasitic weed Phelipanche spp. Long-distance movement of proteins between host and parasite was explored using transgenic tomato plants expressing green fluorescent protein (GFP) in their companion cells. We further used fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite. Accumulation of GFP was observed in the central vascular bundle of leaves and in the root phloem of transgenic tomato plants expressing GFP under the regulation of AtSUC2 promoter. When transgenic tomato plants expressing GFP were parasitized with P. aegyptiaca, extensive GFP was translocated from the host phloem to the parasite phloem and accumulated in both Phelipanche tubercles and shoots. No movement of GFP to the parasite was observed when tobacco plants expressing GFP targeted to the ER were parasitized with P. aegyptiaca. Experiments using fluorescent probes of differing molecular weights to trace vascular continuity between the host plant and the parasite demonstrated that Phelipanche absorbs dextrans up to 70 kDa in size from the host and that this movement can be bi-directional. In the present study, we prove for the first time delivery of proteins from host to the parasitic weed P. aegyptiaca via phloem connections, providing information for developing parasite resistance strategies.     

Tricyclic quinoxalines as potent kinase inhibitors of PDGFR kinase,Flt3 and Kit

Here we report on novel quinoxalines as highly potent and selective inhibitors of the type III receptor tyrosine kinases PDGFR, FLT3, and KIT. These compounds, tricyclic quinoxalines, were generated in order to improve bioavailability over the highly hydrophobic bicyclic quinoxalines. Four of the highly active compounds were characterized in detail and are shown to inhibit PDGFR kinase activity of the isolated receptor as well as in intact cells in the sub-micromolar concentration range. We show that the most active inhibitor (compound 13, AGL 2043) is approximately 15-20 times more potent than its isomer (compound 14, AGL 2044). We therefore compared the three dimensional structures of the two compounds by X-ray crystallography. These compounds are also highly effective in blocking the kinase activity of FLT3, KIT, and the oncogenic protein Tel-PDGFR in intact cells. These compounds are potent inhibitors of the proliferation of pig heart smooth muscle cells. They fully arrest the growth of these cells and the effect is fully reversible. The chemical, biochemical and cellular properties of these compounds as well as the solubility properties make them suitable for development as anti-restenosis and anti-cancer agents.     

Type-II Myocardial Infarction – Patient Characteristics,Management and Outcomes

Background

Type-II MI is defined as myocardial infarction (MI) secondary to ischemia due to either increased oxygen demand or decreased supply. This categorization has been used for the last five years, yet, little is known about patient characteristics and clinical outcomes. In the current work we assessed the epidemiology, causes, management and outcomes of type II MI patients.

Methods

A comparative analysis was performed between patients with type-I and type-II MI who participated in two prospective national Acute Coronary Syndrome Israeli Surveys (ACSIS) performed in 2008 and 2010.

Results

The surveys included 2818 patients with acute MI of whom 127 (4.5%) had type-II MI. The main causes of type-II MI were anemia (31%), sepsis (24%), and arrhythmia (17%). Patients with type-II MI tended to be older (75.6±12 vs. 63.8±13, p<0.0001), female majority (43.3% vs. 22.3%, p<0.0001), had more frequently impaired functional level (45.7% vs. 17%, p<0.0001) and a higher GRACE risk score (150±32 vs. 110±35, p<0.0001). Patients with type-II MI were significantly less often referred for coronary interventions (36% vs. 89%, p<0.0001) and less frequently prescribed guideline-directed medical therapy. Mortality rates were substantially higher among patients with type-II MI both at thirty-day (13.6% vs. 4.9%, p<0.0001) and at one-year (23.9% vs. 8.6%, p<0.0001) follow-ups.

Conclusions

Patients with type-II compared to type-I MI have distinct demographics, increased prevalence of multiple comorbidities, a high-risk cardiovascular profile and an overall worse outcome. The complex medical condition of this cohort imposes a great therapeutic challenge and specific guidelines with recommended medical treatment and invasive strategies are warranted.     

Multiple Roles of the SO42?/Cl?/OH? Exchanger Protein Slc26a2 in Chondrocyte Functions

Mutations in the SO₄²⁻/Cl⁻/OH⁻ exchanger Slc26a2 cause the disease diastrophic dysplasia (DTD), resulting in aberrant bone development and, therefore, skeletal deformities. DTD is commonly attributed to a lack of chondrocyte SO₄²⁻ uptake and proteoglycan sulfation. However, the skeletal phenotype of patients with DTD is typified by reduction in cartilage and osteoporosis of the long bones. Chondrocytes of patients with DTD are irregular in size and have a reduced capacity for proliferation and terminal differentiation. This raises the possibility of additional roles for Slc26a2 in chondrocyte function. Here, we examined the roles of Slc26a2 in chondrocyte biology using two distinct systems: mouse progenitor mesenchymal cells differentiated to chondrocytes and freshly isolated mouse articular chondrocytes differentiated into hypertrophic chondrocytes. Slc26a2 expression was manipulated acutely by delivery of Slc26a2 or shSlc26a2 with lentiviral vectors. We demonstrate that slc26a2 is essential for chondrocyte proliferation and differentiation and for proteoglycan synthesis. Slc26a2 also regulates the terminal stage of chondrocyte cell size expansion. These findings reveal multiple roles for Slc26a2 in chondrocyte biology and emphasize the importance of Slc26a2-mediated protein sulfation in cell signaling, which may account for the complex phenotype of DTD.     

In Vitro Prenylation of the Small GTPase Rac13 of Cotton

Previous work (D.P. Delmer, J. Pear, A. Andrawis, D. Stalker [1995] Mol Gen Genet 248: 43-51) has identified a gene in cotton (Gossypium hirsutum), Rac13, that encodes a small, signal-transducing GTPase and shows high expression in the fiber at the time of transition from primary to secondary wall synthesis. Since Rac13 may be important in signal transduction pathway(s), regulating the onset of fiber secondary wall synthesis, we continue to characterize Rac13 by determining its ability to undergo posttranslational modification. In animals Rac proteins contain the C-terminal consensus sequence CaaL (where "a" can be any aliphatic residue), which is a site for geranylgeranylation (B.T. Kinsella, R.A. Erdman, W.A. Maltese [1994] J Biol Chem 266: 9786-9794). We have identified activities in developing cotton fibers that resemble in specificity the geranylgeranyl- and farnesyltransferases of animals and yeast. In addition, using prenyltransferases from rabbit reticulocytes, we show that Rac13, having a C-terminal sequence of CAFL, can serve as an in vitro substrate for geranylgeranylation but not farnesylation. However, the presence of the uncommon penultimate F residue appears to slow the rate of prenylation considerably compared with other acceptors.     

Pleiotropic effects of tobacco-mosaic-virus movement protein on carbon metabolism in transgenic tobacco plants

Transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants expressing wild-type or mutant forms of the 30-kDa movement protein of tobacco mosaic virus (TMV-MP) were employed to study the effects of the TMV-MP on carbon metabolism in source leaves. Fully expanded source leaves of transgenic plants expressing the TMV-MP were found to retain more newly fixed ¹⁴C compared with control plants. Analysis of ¹⁴C-export from young leaves of TMV-MP plants, where the MP is yet to influence plasmodesmal size exclusion limit, indicated a similar pattern, in that daytime ¹⁴C export was slower in TMV-MP plants as compared to equivalent-aged leaves on control plants. Pulse-chase experiments were used to monitor radioactivity present in the different carbohydrate fractions, at specified intervals following ¹⁴CO₂ labeling. These studies established that the-TMV-MP can cause a significant adjustment in short-term ¹⁴-C-photosynthate storage and export. That these effects of the TMV-MP on carbon metabolism and phloem function were not attributable to the effect of this protein on plasmodesmal size exclusion limits, per se, was established using transgenic tobacco plants expressing temperature-sensitive and C-terminal deletion mutant forms of the TMV-MP. Collectively, these studies establish the pleiotropic nature of the TMV-MP in transgenic tobacco, and the results are discussed in terms of potential sites of interaction between the TMV-MP and endogenous processes involved in regulating carbon metabolism and export.Abbreviations MP movement protein - SEL size exclusion limit - TMV tobacco mosaic virus - ts temperature sensitive This work was supported by United State-Israel Binational Agricultural Research Development Fund grant No. 90-00070 (S.W. and W.J.L). We thank Roger N. Beachy for generously providing some of the transgenic plant lines employed in this study. This paper is a contribution from the Uri Kinamon Laboratory. A.A.O. was supported by a scholarship from the Kinamon Foundation.     

Tyrosine Phosphorylation at a Site Highly Conserved in the L1 Family of Cell Adhesion Molecules Abolishes Ankyrin Binding and Increases Lateral Mobility of Neurofascin

This paper presents evidence that a member of the L1 family of ankyrin-binding cell adhesion molecules is a substrate for protein tyrosine kinase(s) and phosphatase(s), identifies the highly conserved FIGQY tyrosine in the cytoplasmic domain as the principal site of phosphorylation, and demonstrates that phosphorylation of the FIGQY tyrosine abolishes ankyrin-binding activity. Neurofascin expressed in neuroblastoma cells is subject to tyrosine phosphorylation after activation of tyrosine kinases by NGF or bFGF or inactivation of tyrosine phosphatases with vanadate or dephostatin. Furthermore, both neurofascin and the related molecule Nr-CAM are tyrosine phosphorylated in a developmentally regulated pattern in rat brain. The FIGQY sequence is present in the cytoplasmic domains of all members of the L1 family of neural cell adhesion molecules. Phosphorylation of the FIGQY tyrosine abolishes ankyrin binding, as determined by coimmunoprecipitation of endogenous ankyrin and in vitro ankyrin-binding assays. Measurements of fluorescence recovery after photobleaching demonstrate that phosphorylation of the FIGQY tyrosine also increases lateral mobility of neurofascin expressed in neuroblastoma cells to the same extent as removal of the cytoplasmic domain. Ankyrin binding, therefore, appears to regulate the dynamic behavior of neurofascin and is the target for regulation by tyrosine phosphorylation in response to external signals. These findings suggest that tyrosine phosphorylation at the FIGQY site represents a highly conserved mechanism, used by the entire class of L1-related cell adhesion molecules, for regulation of ankyrin-dependent connections to the spectrin skeleton.Vertebrate L1, neurofascin, neuroglial cell adhesion molecule (Ng-CAM),¹ Ng-CAM–related cell adhesion molecule (Nr-CAM), and Drosophila neuroglian are members of a family of nervous system cell adhesion molecules that possess variable extracellular domains comprised of Ig and fibronectin type III domains and a relatively conserved cytoplasmic domain (Grumet, 1991; Hortsch and Goodman, 1991; Rathgen and Jessel, 1991; Sonderegger and Rathgen, 1992; Hortsch, 1996). Members of this family, including a number of alternatively spliced forms, are abundant in the nervous system during early development as well as in adults. Neurofascin and Nr-CAM, for example, constitute ∼0.5% of the total membrane protein in adult brain (Davis et al., 1993; Davis and Bennett, 1994). Cellular functions attributed to the L1 family include axon fasciculation (Stallcup and Beasley, 1985; Landmesser et al., 1988; Brummendorf and Rathjen, 1993; Bastmeyer et al., 1995; Itoh et al., 1995; Magyar-Lehmann et al., 1995), axonal guidance (van den Pol and Kim, 1993; Liljelund et al., 1994; Brittis and Silver, 1995; Brittis et al., 1995; Lochter et al., 1995; Wong et al., 1996), neurite extension (Chang et al., 1987; Felsenfeld et al., 1994; Hankin and Lagenaur, 1994; Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995; Zhao and Siu, 1995), a role in long term potentiation (Luthl et al., 1994), synaptogenesis (Itoh et al., 1995), and myelination (Wood et al., 1990). The potential clinical importance of this group of proteins has been emphasized by the findings that mutations in the L1 gene on the X chromosome are responsible for developmental anomalies including hydrocephalus and mental retardation (Rosenthal et al., 1992; Jouet et al., 1994; Wong et al., 1995).The conserved cytoplasmic domains of L1 family members include a binding site for the membrane skeletal protein ankyrin. This interaction was first described for neurofascin (Davis et. al., 1993) and subsequently has been observed for L1, Nr-CAM (Davis and Bennett, 1994), and Drosophila neuroglian (Dubreuil et al., 1996). The membrane-binding domain of ankyrin contains two distinct sites for neurofascin and has the potential to promote lateral association of neurofascin and presumably other L1 family members (Michaely and Bennett, 1995). Nodes of Ranvier are physiologically relevant axonal sites where ankyrin and L1 family members collaborate, based on findings of colocalization of a specialized isoform of ankyrin with alternatively spliced forms of neurofascin and NrCAM in adults (Davis et al., 1996) as well as in early axonal developmental intermediates (Lambert, S., J. Davis, P. Michael, and V. Bennett. 1995. Mol. Biol. Cell. 6:98a).L1, after homophilic and/or heterophilic binding, participates in signal transduction pathways that ultimately are associated with neurite extension and outgrowth (Ignelzi et al., 1994; Williams et al., 1994a ,b,c,d; Doherty et al., 1995). L1 copurifies with a serine–threonine protein kinase (Sadoul et al., 1989) and is phosphorylated on a serine residue that is not conserved among other family members (Wong et al., 1996). L1 pathway(s) may also involve G proteins, calcium channels, and tyrosine phosphorylation (Williams et al., 1994a ,b,c,d; Doherty et al., 1995). After homophilic interactions, L1 directly activates a tyrosine signaling cascade after a lateral association of its ectodomain with the fibroblast growth factor receptor (Doherty et al., 1995). Antibodies against L1 have also been shown to activate protein tyrosine phosphatase activity in growth cones (Klinz et al., 1995). However, details of the downstream substrates of L1-promoted phosphorylation and dephosphorylation and possible roles of the cytoplasmic domain are not known.Tyrosine phosphorylation is well established to modulate cell–cell and cell–extracellular matrix interactions involving integrins and their associated proteins (Akiyama et al., 1994; Arroyo et al., 1994; Schlaepfer et al., 1994; Law et al., 1996) as well as the cadherins (Balsamo et al., 1996; Krypta et al., 1996; Brady-Kalnay et al., 1995; Shibamoto et al., 1995; Hoschuetzky et al., 1994; Matsuyoshi et al., 1992). For example, the adhesive functions of the calciumdependent cadherin cell adhesion molecule are mediated by a dynamic balance between tyrosine phosphorylation of β-catenin by TrkA and dephosphorylation via the LARtype protein tyrosine phosphatase (Krypta et al., 1996). In this example the regulation of binding among the structural proteins is the result of a coordination between classes of protein kinases and protein phosphatases.This study presents evidence that neurofascin, expressed in a rat neuroblastoma cell line, is a substrate for both tyrosine kinases and protein tyrosine phosphatases at a tyrosine residue conserved among all members of the L1 family. Site-specific tyrosine phosphorylation promoted by both tyrosine kinase activators (NGF and bFGF) and protein tyrosine phosphatase inhibitors (dephostatin and vanadate) is a strong negative regulator of the neurofascin– ankyrin binding interaction and modulates the membrane dynamic behavior of neurofascin. Furthermore, neurofascin and, to a lesser extent Nr-CAM, are also shown here to be tyrosine phosphorylated in developing rat brain, implying a physiological relevance to this phenomenon. These results indicate that neurofascin may be a target for the coordinate control over phosphorylation that is elicited by protein kinases and phosphatases during in vivo tyrosine phosphorylation cascades. The consequent decrease in ankyrin-binding capacity due to phosphorylation of neurofascin could represent a general mechanism among the L1 family members for regulation of membrane–cytoskeletal interactions in both developing and adult nervous systems.     

The crystal structure of the photosynthetic inhibitors DCMU and DTPU

X-ray diffraction studies have been carried out on a single crystal of the photosynthetic inhibitors N-(3,4-dichlorophenyl)-N′-dimethylurea (DCMU) and its newly synthesized spin-labeled analog N-(3,4-dichlorophenyl)-N′-(3,3,5,5-tetramethylpiperidine-4-oxyl)-urea (DTPU). The synthesis of DTPU as well as its crystallographic data are reported. The crystal system of both compounds is monoclinic with a space group P2₁/c. The cell constants of DCMU are a = 7.759(1), b = 14.737(3), c = 9.233(2) Å, β = 100.99(6)°; of DTPU they are a = 6.976(1), b = 11.998(2), c = 23.585(3) Å, β = 91.38(5)°. Comparison of conformational parameters of DCMU and DTPU reveal differences in the dihedral angle between the aromatic ring and the ureido plane. The measured volumes of DCMU and DTPU are 259.1 and 493.3 ų, respectively. These figures suggest the size of the binding site of the inhibitors in the photosynthetic membrane.