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.     

The functional metabolism of vitamin D in chicks fed low-calcium and low-phosphorus diets

Radioactively labelled cholecalciferol was administered continuously to chicks that were fed normal, low-calcium and low-phosphorus diets. It has been possible to show that under such steady state conditions with regard to cholecalciferol, and mineral restriction, the animal reacts by increased accumulation of 1,25-dihydroxycholecalciferol in the intestinal and the kidney cell, which was associated in the intestine with an increased calcium-binding activity. A similar accumulation of 1,25-dihydroxycholecalciferol in bone was not noticed.It is proposed that the intestine and the kidney, but not bone, are the main target organs for cholecalciferol in the maintenance of calcium homeostasis, and that both calcium and phosphorus play a role in the regulation of the formation and subsequent function of 1,25-dihydroxycholecalciferol.     

Isolation, Chemical Composition, and Ultrastructural Features of the Cell Membrane of the Mycoplasma-Like Organism Spiroplasma citri

Thin sections of Spiroplasma citri, a mycoplasma-like organism isolated from citrus infected with "Stubborn" disease, showed the organisms to be limited by a single trilaminar plasma membrane. An additional outer layer could, however, be frequently seen in freeze-etched preparations of unwashed cells. The organisms were found to be extremely sensitive to lysis by osmotic shock. The cell membrane of S. citri isolated in this way resembled that of mycoplasmas in ultrastructure and gross chemical composition. The isolated membranes showed the characteristic trilaminar shape in section and the typical particle-studded fracture faces in freeze-etched preparations. Protein and lipid formed over 80% of the total dry weight of the membrane, which had a density of ~1.180 g/cm(3). Cholesterol constituted over 20% of the total membrane lipid. Phosphatidyl-glycerol, synthesized by the organisms, was the major phospholipid. Significant amounts of hexosamine (15 to 35 mug/mg of membrane protein) could be found in the membrane preparations. Our results support the thesis that S. citri does not possess a cell wall, either of the gram-positive or the gram-negative type, though it may be coated by some other type of an envelope or by a slime layer, at least temporarily.     

Inhibition of human natural killer cell activity by a synthetic peptide homologous to a conserved region in the retroviral protein, p15E

It has been shown previously that the retroviral envelope protein p15E suppresses certain monocyte and lymphocyte functions. In this paper, we describe the effects on natural killer (NK) activity of a synthetic peptide (CKS-17) with homology to a region of p15E conserved among numerous retroviruses. Enriched human NK cells were assayed against K562 tumor target cells in a 51Cr-release cytotoxicity assay. Pretreatment of NK cells with CKS-17 at concentrations as low as 1.5 microM, but not with equivalent concentrations of control materials, markedly and reproducibly suppressed NK lytic activity. Prior exposure of NK cells to interferon-alpha (IFN-alpha) at 1000 U/ml did not alter their sensitivity to CKS-17-induced inhibition. Pretreating NK cells with CKS-17 almost entirely diminished their responsiveness to IFN-alpha and IFN-gamma, but not to interleukin 2 (IL 2). Kinetics experiments demonstrated that CKS-17-mediated suppression of both endogenous and activated NK cells was reversible after 18 hr at 37 degrees C. Experiments designed to examine the CKS-17 mechanism of action revealed that the peptide bound to all Leu-11+ lymphocytes, as shown by two-color flow cytometry. CKS-17 did not, however, inhibit effector cell/target cell conjugate formation. These data suggest a new mechanism for immune suppression mediated by retroviruses; inhibition of NK function. They moreover imply that the CKS-17 peptide interferes with the lytic phase of NK cytolysis.     

Acquisition of Sequences Homologous to Host DNA by Closed Circular Simian Virus 40 DNA III. Host Sequences

A preparation of serially passaged simian virus 40 (SV40) DNA, in which at least 66% of the molecules contain covalently linked cellular DNA sequences, was digested to completion with the Hemophilus influenzae restriction endonuclease. Polyacrylamide gel electrophoresis of the digest showed that the majority of the cleavage products migrated as nine classes of fragments, each class defined by a particular molecular weight. These classes of fragments differ in molecular weight from the fragments produced by the action of the same enzyme on plaque-purified virus DNA. Three classes of fragments were present in less than equimolar amounts relative to the original DNA. The remaining six classes of fragments each contain more than one fragment per original DNA molecule. DNA-DNA hybridization analysis (using the filter method) of the isolated cleavage products demonstrated the presence of highly reiterated cell DNA sequences in two of the nine classes of fragments. A third class of fragments hybridized with high efficiency only to serially passaged SV40 DNA; the level of hybridization to plaque-purified virus DNA was low and there was essentially no hybridization with cell DNA immobilized on filters. It is suggested that this class of fragments contains unique host sequences. It was estimated that at least 27% of the sequences in the substituted SV40 DNA molecules studied are host sequences. The majority of these are probably of the nonreiterated type.     

Human monoclonal antibodies derived from pleural effusion lymphocytes of a patient with breast carcinoma react with human breast cancer-associated antigens and mouse mammary tumor virus polypeptides

Four human hybridoma cell lines (PEB1-4) were established from a fusion of pleural effusion lymphocytes isolated from a breast cancer patient with metastatic disease, 6 years postmastectomy. The hybridomas secreted IgG-k (3 micrograms/ml/10(6) cells). These monoclonal antibodies (PEB1-4) reacted to different degrees with mouse mammary tumor virus (MMTV) and T47D particles (HuMTV). Immunological cross-reaction was also detected with antigens isolated from body fluids of breast cancer patients (BF-Ag). The binding capacity of the monoclonal antibodies (MAbs) PEB1-4 to the above-mentioned antigens was measured by RIA. The specificity of these antibodies was further demonstrated by radioimmunoprecipitation of MMTV, T47D (HuMTV) and BF-Ag. The binding of PEB1-4 to surface antigens of intact cells grown in culture was measured by RIA. Some of the MAbs were shown to bind more avidly to breast cancer cells than to nonbreast cancer cells or nonmalignant cells. The PEB1-4 human monoclonal antibodies may be found useful in analyzing the virus-breast cancer relationship.     

External electric field effects on photosynthetic membrane vesicles. Kinetic characterization of two electrophotoluminescence phases in hypotonically swollen chloroplasts

Strong externally applied electrical field pulses are known to stimulate delayed luminescence from preilluminated blebs (hypotonically swollen vesicles originating from thylakoid membranes of broken chloroplasts) by up to 3 orders of magnitude. This phenomenon is known as electrophotoluminescence. Previous analysis showed the kinetics of the electrophotoluminescence to be biphasic, displaying a rapid (R) phase which decays towards a slower one (S) (Ellenson, J.L. and Sauer, K. (1976) Photochem. Photobiol. 23, 113–123). We demonstrate that these two components represent different processes. At low pH, a good kinetic separation is obtained between the two phases, which become distinct, with the S phase manifesting also an initial rise period. Under these conditions, it is possible to estimate separately the approximate rise times of the two phases. It is shown that the R and S components have a different dependence on the pH and on the time between the actinic flash and onset of the field. The field dependence is also different, with the S phase requiring a lower threshold field than R. From these observations, it is concluded that the R and S luminescence components are formed by different precursors. The difference in behaviour of the two phases during formation of the bleb indicates that the precursors of the R and S phases belong to different parts of the bleb. We suggest that R precursors are located in the wall of the swollen thylakoid and S precursors in the membrane formations which are attached to this wall.     

Physiological adaptation to a newly observed low light intensity state in intact leaves,resulting in extreme imbalance in excitation energy distribution between the two photosystems

In intact leaves, a new physiological state is obtained reversibly at low light intensity (typically 1 W / m²), in which oxygen evolution yield, monitored by the photoacoustic method, approaches zero. In this ‘low-light’ state, irradiation with far-red (λ > 700 nm) background light immediately restores the normal oxygen yield, resulting in an unusually high Emerson enhancement ratio. Quantitative analysis of the enhancement ratio and the saturation curve of enhancement by far-red light shows that in the new state, short wavelength excitation does not reach PS I reaction centers, resulting in an extreme imbalance between the two photosystems. We suggest that adaptation to the low-light state occurs through loss of excitonic interaction between antennae of PS I and their reaction-centers. It appears also that the ‘far-red’ absorbing pigments do not participate in the disconnection and remain closely attached to the reaction centers of PS I. Their number is estimated to be less than 30 per reaction center. The disconnection of the antennae from the reaction center appears to be reversed by readaptation to ‘normal’ light levels, as well as by a brief preillumination with broad band (400–600 nm) light, acting as a trigger. In the last case, the transition to high oxygen yield state is transient. The quantum requirement of this recovery process is very small (approx. 10 hv / reaction center). The adaptation times after switching from higher to lower intensities and vice versa are in the range of minutes. The fluorescence yield remains virtually constant during adaptation to the low-light state in contrast to expectations, suggesting the possibility of cyclic electron flow around PS II in this state. In a chlorophyll-b-less barley mutant, which lacks the light-harvesting $\text{chlorophyll}\text{-}\text{a}\text{b}$ protein (LHC) (and possibly the newly discovered light-harvesting $\text{chlorophyll}\text{-}\text{a}\text{b}$ protein associated with PS I (LHC-I)), the ‘low-light’ state was absent. These results are consistent with the hypothesis that these antennae complexes participate directly in the adaptation to low light intensities.