[High expression of CAMSAP2 promotes invasion and metastasis of gastric cancer cells by upregulating TGF-ß signaling].

OBJECTIVE: To investigate the expression of calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) in gastric cancer and its effect on gastric cancer cell invasion and metastasis. METHODS: The association of CAMSAP2 expression levels with progression and prognosis of gastric cancer was analyzed using public cancer data and in 106 patients receiving radical gastrectomy in our hospital from October, 2013 to October, 2017. The biological functions of CAMSAP2 were predicted using bioinformatics analysis. Gastric cancer MGC803 cells with CAMSAP2 overexpression and knockdown were observed for epithelial-mesenchymal transition (EMT), migration and invasion. A nude mouse model bearing orthotopic gastric cancer cell xenografts was established for verifying the results and exploring the underlying molecular mechanism. RESULTS: Gastric cancer tissues expressed high levels of CAMSAP2, which were positively correlated with CEA and CA19-9 (P<0.001). Cox regression analysis showed that CAMSAP2 expression level was an independent risk factor affecting the 5-year survival rate of gastric cancer patients (HR=2.969, 95% CI: 1.031-8.548). Enrichment analysis suggested that CAMSAP2 was involved in epithelialmesenchymal transition (EMT) and TGF-ß signaling. In gastric cancer cells, CAMSAP2 overexpression significantly increased the expressions of vimentin and N-cadherin, inhibited the expression of E-cadherin, and enhanced cell migration and invasion (P<0.05); CAMSAP2 knockdown produced the opposite effects in the cells (P<0.05). In the tumor- bearing mice, xenografts overexpressing CAMSAP2 showed enhanced metastasis (P<0.05), increased vimentin and N-cadherin expressions and lowered E-cadherin expression (P<0.05), and the xenografts with CAMSAP2 knockdown showed the opposite changes (P<0.05). Both the in vivo and in vitro experiments showed that CAMSAP2 overexpression increased and CAMSAP2 knockdown lowered the levels of TGF-ß and p-Smad2/3 in the gastric cancer cells (P<0.05). CONCLUSION: The high expression of CAMSAP2 contributes to disease progression and poor prognosis of gastric cancer possibly by upregulating TGF-ß signaling to promote EMT.

Effects of embryonic cadmium exposure on erythrocyte indices and morphology in newly hatched Gallus gallus domesticus chicks.

The aim of the current study was to assess the influence of embryonic exposure to cadmium on basic and derived erythrocyte indices, the morphology and morphometric properties of erythrocytes, as well as erythrocyte spectrin distribution in newly hatched Gallus gallus domesticus chicks. The eggs were injected with cadmium (Cd) at a dose of 2 µg, 4 µg, 6 µg, or 8 µg per egg on the sixth day of incubation. Blood samples were collected on the first day after hatching. Exposure to cadmium resulted in higher levels of red blood cell count, hemoglobin concentration, and hematocrit value, while derived erythrocyte indices were lower (mean corpuscular volume) or higher (mean corpuscular hemoglobin concentration) in comparison to the control. These changes occurred in animals exposed to higher doses of this toxic agent. In cadmium-treated individuals (2 and 8 µg of Cd), the percentage of erythrocytes which exhibited changed shape increased. Increases in the length (6 and 8 µg) and width (2, 6, and 8 µg) of erythrocytes and the length and width of the nucleus (2-8 µg) of red blood cells were observed. Changes in spectrin distribution were also observed, which indicate alterations at structural and molecular levels.

Identification of novel anti-angiogenic factors by in silico functional gene screening method.

Angiogenesis, the formation of new blood vessels out of pre-existing capillaries, occurs in a variety of pathophysiological conditions, and is regulated by a balance of angiogenic activators and inhibitors. To identify novel angiogenic factors, we developed a gene screening method by combining the prediction analysis of transcription factor (TF) binding site and the chromosomal localization analysis. First, we analyzed the promoter sequences from known angiogenesis-related factors using the MATINSPECTOR program in TRANSFAC database. Interestingly, we found that the binding site of LMO2 complex is highly conserved in the promoter regions of these factors. Second, we analyzed chromosome loci based on the hypothesis that angiogenesis-related factors might be co-localized in a specific chromosomal band. We found that angiogenesis-related factors are localized in specific 14 chromosomal bands including 5q31 and 19q13 using AngioDB and LocusLink database mining. From these two approaches, we identified 32 novel candidates that have the LMO2 complex binding site in their promoter and are located on one of 14 chromosomal bands. Among them, human recombinant troponin T and spectrin markedly inhibited the neovascularization in vivo and in vitro. Collectively, we suggest that the combination of the prediction analysis of TF binding site and the chromosomal localization analysis might be a useful strategy for gene screening of angiogenesis.

Fodrin inhibits phospholipases A2, C, and D by decreasing polyphosphoinositide cell content.

Brain fodrin inhibited in a dose dependent manner the GTPgammaS-stimulated cytosolic PLA2 (cPLA2), PLC, and PLD activities in differentiated HL-60 cells permeabilized with streptolysin O. cPLA2 and PLD were inhibited by the same concentrations of fodrin (IC50=1.5-2 nM) but PLC was inhibited by lower concentrations (IC50=0.3 nM). Moreover, the rates of inhibition were different between the phospholipases. Spectrin, which shares 50% homology with fodrin, had similar effects on the three phospholipases. However, using cytosol-depleted cells or recombinant PLD1, we showed that fodrin was not a direct inhibitor. Studying the potential mechanisms of these inhibitions, we demonstrated that a major decrease in membrane phosphatidylinositol 4-monophosphate (PtdIns(4)P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) amounts was induced by fodrin. Exogenous PtdIns(4,5)P2 partly reversed fodrin inhibition of GTPgammaS-stimulated phospholipase C activity. Hence, inhibition of PLC, cPLA2, and PLD activities observed with fodrin could be related to the decrease of PtdIns(4,5)P2, substrate of PLC, a cofactor of PLD and an enhancer of cPLA2 activity.

In vitro motility of immunoadsorbed brain myosin-V using a Limulus acrosomal process and optical tweezer-based assay.

To facilitate functional studies of novel myosins, we have developed a strategy for characterizing the mechanochemical properties of motors isolated by immunoadsorption directly from small amounts of crude tissue extracts. In this initial study, silica beads coated with an antibody that specifically recognizes the tail of myosin-V were used to immunoadsorb this motor protein from brain extracts. The myosin-containing beads were then positioned with optical tweezers onto actin filaments nucleated from Limulus sperm acrosomal processes and observed for motility using high resolution video DIC microscopy. The addition of brush border spectrin to the motility chamber enabled the growth of stable actin filament tracks that were approximately 4-fold longer than filaments grown in the absence of this actin crosslinking protein. The velocity of myosin-V immunoadsorbed from brain extracts was similar to that observed for purified myosin-V that was antibody-linked to beads or assessed using the sliding actin filament assay. Motile beads containing myosin-V immunoadsorbed from brain extracts bound poorly to nucleated actin filaments and were incapable of linear migrations following the addition of a different antibody that specifically recognizes the motor-containing head domain of myosin-V. Myosin-V motility was most robust in the absence of Ca2+. Interestingly, skeletal muscle tropomyosin and brush border spectrin had no detectable effect on myosin-V mechanochemistry. Myosin-V containing beads were also occasionally observed migrating directly on acrosomal processes in the absence of exogenously added actin.(ABSTRACT TRUNCATED AT 250 WORDS)

Tropomyosin from human erythrocyte membrane polymerizes poorly but binds F-actin effectively in the presence and absence of spectrin.

Actin in the human erythrocyte forms short protofilaments which are only long enough to accommodate tropomyosin monomers (Shen, B.W., Josephs, R. and Steck, T.L. (1986) J. Cell Biol. 102, 997-1006). This interaction between actin and tropomyosin monomers is predicted to be weak, since tropomyosin polymerization parallels its affinity for F-actin. We examine the binding of human erythrocyte tropomyosin to actin in the presence and absence of spectrin and its ability to polymerize. The binding of human erythrocyte tropomyosin to F-actin is not affected appreciably by the present of spectrin. Saturating F-actin with erythrocyte tropomyosin, however, weakens the binding of spectrin dimers to actin. Although tropomyosin from human erythrocyte and rabbit cardiac muscle have similar affinity for F-actin, the polymerizability of erythrocyte tropomyosin as determined by viscosity measurements is much reduced relative to muscle tropomyosin. This unusual property of erythrocyte tropomyosin is likely due to differences in its primary structure from other known tropomyosin at the amino and carboxyl terminal regions which are responsible for its head-to-tail polymerization and cooperative binding to F-actin. Analysis of the distribution of tyrosine by 2-dimensional tryptic mapping of 125I-labelled erythrocyte tropomyosin shows that tyrosine at positions 162, 214, 221, 261 and 267 in rabbit cardiac tropomyosin are conserved in human erythrocyte tropomyosin but Tyr-60 is absent. This observation suggests that erythrocyte tropomyosin has a carboxyl terminal region similar to its muscle counterparts but its amino terminal region resembles that of platelet tropomyosin which also lacks Tyr-60.

Is Ca2+ effect on passive K+ transport mediated by spectrin--dependent ATPase?

The aim of this report was to find which part of the membrane is responsible for the Ca2+ dependence of membrane permeability to K+. We found that the enzyme activity of large contractile complex of membrane proteins, the so called spectrin-dependent ATPase (sp-ATPase) increases at certain Ca2+ concentrations when K+ permeability decreases and vice versa. Ca2+ apparent dissociation constant for sp-ATPase is 6 X 10(-7) M which is the value corresponding to findings of Porzig and Stoffel (1978) for Ca2+ binding to membrane with low K+ permeability. Moreover, 20 chemically quite different substances which inhibit sp-ATPase activity simultaneously increase in the same concentrations K+ permeability and vice versa. No exception was found. The results show that low membrane permeability to K+ occurs at such conformation of sp-ATPase at which its enzyme activity may fully be manifested whereas at other conformations permeability to K+ increases. The conformation of sp-ATPase seems to affect gating mechanism of a respective channel for passive K+ transport through membrane.

Proteolytic self-digestion of bovine erythrocyte membranes.

"Self-digestion" of bovine erythrocyte membrane proteins was studied in isolated membrane preparations during prolonged incubation at 37 C. Protease activities associated with the membrane result in progressive degradation of all main erythrocyte membrane proteins, in particular spectrin and Band 3, and formation of lower molecular weight products which have been tentatively assigned to parent molecules. Membrane protein "self-digestion" occurs in a broad pH range (2-11), is inhibited by increased ionic strength and by inhibitors of metalloproteases, cysteine and serine proteases, and activated by low concentrations of SDS. "Self-digestion" also takes place in NaOH-stripped erythrocyte membranes. The activity of a protease involved in the "self-digestion", of apparent molecular weight of about 35,000, was renatured after SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins.

Actin assembly and filament cross-linking in the presence of TW 260/240, the tissue-specific spectrin of the chicken intestinal brush border.

TW 260/240 is a tissue-specific spectrin found in the terminal web region of the chicken intestinal brush border. We have examined the effects of TW 260/240 on assembly rates and critical concentrations (Co's) for monomer addition at the barbed and pointed ends of the actin filament. For these studies, acrosomal processes (AP) from Limulus sperm were used as nuclei for actin assembly. Under conditions which favor the interaction of TW 260/240 for actin (20-75 mM KCl, 2 mM Mg++) no effect on either elongation rates or Co's at either end of the actin filament was observed in the presence of this spectrinlike protein. The Limulus AP nucleation assay also allowed visualization of the kinetics of filament binding and cross-linking by TW 260/240. Ultrastructural analysis of TW 260/240 binding to actin filaments at their growing ends indicates that TW 260/240 tetramers bind laterally to the filament. Finally, evidence is presented that indicates that filaments cross-linked by TW 260/240 are stabilized against shear-dependent breakage.

On the mechanism of ATP-induced shape changes in human erythrocyte membranes. I. The role of the spectrin complex.

Human erythrocyte ghosts have been shown, by scanning electron microscopy, to undergo ATP-dependent shape changes. Under appropriate conditions the ghosts prepared from normal disk-shaped intact cells adopt a highly crenated shape, which in the presence of Mg-ATP at 37 degrees C is slowly converted to the disk shape and eventually to the cup shape. These changes are not observed with other nucleotides or with 5'-adenylyl imidodiphosphate. Anti-spectrin antibodies, incorporated along with the Mg-ATP into the ghosts in amounts less than equivalent to the spectrin, markedly accelerate the shape changes observed with the Mg-ATP alone. The Fab fragments of these antibodies, however, have no effect. The conclusion is that the structural effect produced by the ATP is promoted by the cross-linking of spectrin by its antibodies, and may therefore itself be some kind of polymerization or network formation involving the spectrin complex on the cytoplasmic face of the membrane. The factors that contribute to the shape of the ghost and of the intact erythrocyte are discussed in the light of these findings.