A key angiogenic role of monocyte chemoattractant protein-1 in hemangioendothelioma proliferation

Angiomatous lesions are common in infants and children. Hemangioendotheliomas (HE) represent one type of these lesions. Endothelial cell proliferation and the development of vascular/blood cell-filled spaces are inherent in the growth of HE. Therefore, understanding mechanisms that regulate the proliferation of these lesions should provide key insight into mechanisms regulating angiogenesis. A murine model was used to test the significance of monocyte chemoattractant protein (MCP)-1 in HE proliferation. EOMA cells, a cell line derived from a spontaneously arising murine HE, generate these lesions with 100% efficiency when injected subcutaneously into syngeneic mice. MCP-1 produced by EOMA cells recruit macrophages, which were shown to induce angiogenic behavior in EOMA cells by stimulating transwell migration and inducing sprout formation on type I collagen gels. When EOMA cells were injected into MCP-1^–/– mice, only 50% of the mice developed tumors, presumably because the low levels of MCP-1 expressed by the injected EOMA cells were enough to overcome any host deficits of this chemokine. When EOMA cells were coinjected with a neutralizing antibody to MCP-1, tumors failed to develop in any of the treated mice, including syngeneic 129P3, C57Bl/6 (wild type), and MCP-1^–/–. These results present the first evidence that MCP-1 is required for HE proliferation and may promote the growth of these lesions by stimulating angiogenic behavior of endothelial cells. This study has produced the first in vivo evidence of a complete response for any neoplasm, specifically a vascular proliferative lesion, to anti-MCP-1 therapy in animals with intact immune systems. endothelium; vascular; macrophage; redox; angiogenesis     

Use of a herbicide or lysine plus threonine for non-antibiotic selection of transgenic chickpea

A desensitized aspartate kinase (AK) gene has been developed as a non-antibiotic selection marker for use in the production of transgenic chickpea (Cicer arietinum L.). Transgenic shoots regenerated from embryo explants bombarded with the desensitized AK gene were selected on media containing two amino acids, lysine and threonine (LT). Approximately 15% of the putative transgenic shoots of vars. P-362 and P-1042 survived after 4 weeks of growth on MSB5 medium (MS mineral salts and B5 vitamins) containing 2 µM thidiazuron (TDZ) and 2 mM lysine and 2 mM threonine. These shoots were subsequently grown on MSB5 medium supplemented with 2 µM TDZ and 5 mM lysine and 5 mM threonine, and nearly 1% continued to grow after 16 weeks of selection. A phosphinothricin (PPT) selection system for Agrobacterium-mediated chickpea transformation was also developed. Three varieties of chickpea, P-362, P-1042 and P-1043, were successfully used for Agrobacterium transformation. Following Agrobacterium infection, 3-8% of the regenerated shoots remained green and continued to grow on MSB5 medium supplemented with 2.5 mg l^-1 PPT. Increasing the concentrations of PPT to 15 mg l^-1 reduced transgenic shoot production in P-362, P-1042 and P-1043 to 0.7%, 1.2% and 1.1%, respectively. Selected putatively transformed shoots of all three varieties were rooted and grown to maturity. Southern hybridization analysis revealed single as well as multiple integration of genes in selected transgenic lines. The level of AK activity detected in LT-selected plants was higher than that detected in the non-transformed control.Abbreviations AK: Aspartate kinase - CP: Chlorophenol red - GUS: -Glucuronidase - IBA: Indole-3-butyric acid - Kn: Kinetin (6-furfuryl aminopurine) - LT: Lysine plus threonine - MSB5: Murashige and Skoog salts with B5 vitamins - nptII: Neomycin phosphotransferase II - pat: Phosphinothricin-acetyltransferase - PPT: Phosphinothricin - TDZ: Thidiazuron [1-phenyl-3-(1,2,3-thiadiazol-5-YL) urea]Communicated by P. LakshmananJ. Sen and N. Tewari-Singh have contributed equally to this article.     

Theoretical studies on competitive binding of caldesmon and myosin S1 to actin: prediction of apparent cooperativity in equilibrium and slow-down in kinetics of S1 binding by caldesmon

Several laboratories have reported cooperative binding of S1 to actin in the presence of caldesmon. This cooperative binding has been interpreted with a model similar to that proposed for the binding of S1 to regulated actins in which the binding affinity of S1 is controlled by the position of the tropomyosin filaments. In a recent paper [Sen, A., Chen, Y., Yan, B., and Chalovich, J. M. (2001) Biochemistry 40, 5757-64], we showed qualitatively that S1 binding resulted in rapid dissociation of caldesmon from actin or actin-tropomyosin. This suggests that the cooperativity observed in the case of caldesmon is not due to a conformational change in actin-caldesmon but to the displacement of caldesmon. We show in this paper that the pure competitive binding model, in which both S1 and caldesmon are competing for the same binding sites on actin, can simulate quantitatively the effect of caldesmon on both the equilibrium and the kinetics of S1 binding to actin. This model successfully predicts an apparent cooperativity for the binding of S1 to actin-caldesmon without the need to assume multiple actin-caldesmon structures and produces a decreased rate of S1 binding to actin in the presence of caldesmon. This suggests that the inhibitory action of caldesmon on the actin-activated ATPase activity of myosin in solution and on the generation of active force in a contracting muscle may be simply due to the blocking of myosin binding sites on actin by caldesmon.     

Characterization of perceived hyperoxia in isolated primary cardiac fibroblasts and in the reoxygenated heart

Under normoxic conditions, pO2 ranges from 90 to <3 torr in mammalian organs with the heart at approximately 35 torr (5%) and arterial blood at approximately 100 torr. Thus, "normoxia" for cells is an adjustable variable. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of pO2 results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the pO2 to which cells are adjusted, results in the activation of specific O2-sensitive signal transduction pathways that alter cellular phenotype and function. Thus, reperfusion causes damage to the tissue at the focus of ischemia while triggering remodeling in the peri-infarct region by means of perceived hyperoxia. We reported first evidence demonstrating that perceived hyperoxia triggers the differentiation of cardiac fibroblasts (CF) to myofibroblasts by a p21-dependent mechanism (Roy, S., Khanna, S., Bickerstaff, A. A., Subramanian, S. V., Atalay, M., Bierl, M., Pendyala, S., Levy, D., Sharma, N., Venojarvi, M., Strauch, A., Orosz, C. G., and Sen, C. K. (2003) Circ. Res. 92, 264-271). Here, we sought to characterize the genomic response to perceived hyperoxia in CF using GeneChips trade mark. Candidate genes were identified, confirmed and clustered. Cell cycle- and differentiation-associated genes represented a key target of perceived hyperoxia. Bioinformatics-assisted pathway reconstruction revealed the specific signaling processes that were sensitive to perceived hyperoxia. To test the significance of our in vitro findings, a survival model of rat heart focal ischemia-reperfusion (I-R) was investigated. A significant induction in p21 mRNA expression was observed in I-R tissue. The current results provide a comprehensive molecular definition of perceived hyperoxia in cultured CF. Furthermore, the first evidence demonstrating activation of perceived hyperoxia sensitive genes in the cardiac I-R tissue is presented.     

Expression and purification of the Bacillus anthracis protective antigen domain 4

The protective antigen (PA) of Bacillus anthracis plays a crucial role in the pathogenesis of the anthrax disease. The fourth domain of PA (PA-D4) is responsible for initial binding of the anthrax toxin to the cellular receptor, and thus, is an attractive target for structure-based drug therapies. A synthetic gene for PA-D4 has been prepared by recursive PCR. PA-D4 has been expressed as a fusion protein in Escherichia coli. PA-D4 has been purified to near homogeneity and its identity has been verified by mass spectrometry. The recombinant PA-D4 exhibits CD and NMR spectra that suggest that it is folded and amenable for biophysical studies. Moreover, recombinant PA-D4 binds to HeLa cells, which suggests that recombinant PA-D4 is functional to bind to its cellular receptor.     

The 14-3-3 proteins Rad24 and Rad25 negatively regulate Byr2 by affecting its localization in Schizosaccharomyces pombe

In Schizosaccharomyces pombe, rad24 and rad25 have been identified to be homologous to mammalian 14-3-3 genes and found to be involved in many cellular events, including checkpoint and meiosis. In the present study, we present evidences that Rad24 and Rad25 act as negative regulators of Byr2 (mitogen-activated protein kinase [MAPK] kinase kinase). Overexpression of rad24 or rad25 reduced mating and sporulation in homothallic wild-type cells. In contrast, the mating and sporulation efficiency of rad24- or rad25-null cells was higher than that of wild-type cells. Deletion of rad24 or rad25 increased sporulation efficiency in ras1-null diploid cells but not in byr2-, ste4-, byr1-, and spk1-null cells. Rad24 and Rad25 had no effect on the activity of constitutively active Byr1(S214DT218D). Rad24 and Rad25 bound to both the N-terminal and the C-terminal domains of Byr2 when these bacterially expressed proteins were examined. The formation of complexes in vivo between Byr2 and either Rad24 or Rad25 was also confirmed by immunocoprecipitation. Furthermore, we showed negative regulation of Byr2 by Rad25, by monitoring the mRNA level of mam2, which is regulated by both the Ras1/MAPK pathway and ste11, in various combinations of mutants. In addition, the cellular localization of Byr2 in living cells was observed by using fusion to green fluorescent protein. Byr2 was mainly localized in the cytoplasm during vegetative growth and then concentrated at the plasma membrane in response to nitrogen starvation. Deletion of rad24 or rad25 fastened the timing of Byr2 translocation. Our results are consistent with the hypothesis that one of the roles of 14-3-3 is to keep Byr2 in the cytoplasm and to affect the timing of Byr2 translocation in response to sexual developmental signal.     

Novel identification of the different types of cones in the retina of the chicken

1. Although single cones and double cones in the chicken retina had been documented for more than 30 years, the exact morphology of these cells had never been studied by the scanning electronmicroscopy. In this brief report, we present the evidence for the first time the existence of two types of single cones and three types of double cones (termed as types A, B, and C), each with distinct morphology.2. The proportion of type A:type B:type C double cones, as estimated from the midperipheral and central regions of our scanning specimens, was about 30%:50%:20%.3. Based on the literature data that red oil droplets reside in single cones and yellow oil droplets in chief cones of the double cones, the proportion of single to double cones was deciphered and the relative proportion was estimated to be 1:0.91 in the central region, 1:0.92 in the midperiphery, and 1:0.84 in the periphery.     

Spike-timing-dependent plasticity: common themes and divergent vistas

Recent experimental observations of spike-timing-dependent synaptic plasticity (STDP) have revitalized the study of synaptic learning rules. The most surprising aspect of these experiments lies in the observation that synapses activated shortly after the occurrence of a postsynaptic spike are weakened. Thus, synaptic plasticity is sensitive to the temporal ordering of pre- and postsynaptic activation. This temporal asymmetry has been suggested to underlie a range of learning tasks. In the first part of this review we highlight some of the common themes from a range of findings in the framework of predictive coding. As an example of how this principle can be used in a learning task, we discuss a recent model of cortical map formation. In the second part of the review, we point out some of the differences in STDP models and their functional consequences. We discuss how differences in the weight-dependence, the time-constants and the non-linear properties of learning rules give rise to distinct computational functions. In light of these computational issues raised, we review current experimental findings and suggest further experiments to resolve some controversies.