Biomechanical basis of diazepam-induced neural tube defects in early chick embryos: a morphometric study

The biomechanical basis of diazepam (Valium/Roche)-induced neural tube defects in the chick was investigated using a combination of electron microscopy and morphometry. Embryos at stage 8 (four-somite stage) of development were explanted and grown for 6 hr in nutrient medium containing 400 micrograms/ml diazepam. Nearly 80% of these embryos exhibited neural tube defects that were most pronounced in the forming midbrain region and typified by a "relaxation" or "collapse" of neural folds. The hindbrain and spinal cord regions were less affected. Electron microscopy revealed that neuroepithelial cells in diazepam-treated embryos had smoother apical surfaces and broader apical widths than did controls. Morphometric measurements supported this observation and further showed that these effects were focused at sites within the wall of the forming neural tube that typically exhibit the greatest degree of bending and apical constriction (i.e., the floor and midlateral walls). Overall results indicate that neural tube defects associated with exposure to diazepam are due largely to a general inhibition of the contractile activity of apical microfilament bundles in neuroepithelial cells. These findings 1) emphasize the important contribution of microfilament-mediated apical constriction of neuroepithelial cells in providing the driving forces for bending of the neuroepithelium during neural tube formation and 2) suggest that agents or conditions that impair their contractile activity could play a role in the pathogenesis of certain types of neural tube defects.     

Value of apoptin’s 40-amino-acid C-terminal fragment for the differentiation between human tumor and non-tumor cells

Apoptin, a protein of the chicken anemia virus (CAV), consists of 121 amino acids (aa) and represents a novel, potentially tumor-specific therapeutic and diagnostic agent. The C-terminal part of Apoptin (aa 81–121) is believed to contain a bipartite nuclear localization signal (NLS) (NLS1: aa 82–88 and NLS2: aa 111–121), which is only active in tumor cells after phosphorylation of threonine¹⁰⁸ by tumor-specific cytoplasmic phosphokinases. Furthermore, a nuclear export signal (NES) (aa 97–105) seems to enable nuclear export of Apoptin only in healthy cells. The specificity for tumor cell nuclei also applies to the truncated C-terminal part of Apoptin (aa 81–121), which therefore represents a highly attractive peptide sequence for peptide synthesis. Here we describe for the first time the synthesis of fluorescein isothiocyanate (FITC)- and Dansyl-labelled conjugates containing this C-terminal part of Apoptin, with either phosphorylated or nonphosphorylated threonine¹⁰⁸. The phosphorylated conjugates were synthesized in an attempt to achieve nuclear accumulation in healthy cells, which lack cytoplasmic tumor-specific phosphokinases. Surprisingly, all the conjugates accumulated rapidly within the cell nuclei of both tumor and non-tumor cells from the bladder, brain and prostate and led to cell death. By coupling Apoptin^81–121 to FITC and DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) at either the C- or N-terminus we could exlude that the coupling site is decisive for tumor cell-specific nuclear localization. The labels FITC, DOTA and Dansyl were not responsible for cell death in healthy cells because cell death was not prevented by using an unlabelled Apoptin^81–121 peptide. Cellular and nuclear uptake of the FITC-labelled Apoptin^81–121 peptide was almost completely abolished after altering the NLS2 (replacement of five arginines with serines).     

Calcium-sensitive ROS-GC1 signaling outside of photoreceptors: A common theme

Until now, ROS-GC1 signal transduction system was thought to be exclusive to photoreceptors in the retina. Two recent reports, however, now show that this is not the case. In one, the ROS-GC1 signal transduction system has been identified and characterized in pinealocyte neurons. This signaling is modulated by norepinephrine. However, the response of the individual pinealocyte neuron to the norepinephrine signal depends on whether the GCAP1-linked (results in hyperpolarization) or S100-linked (results in depolarization) pathway is operational in the pinealocyte. The GCAP1-linked pathway results in hyperpolarization, while the S100-linked pathway, in depolarization. The two pathways are mutually exclusive. In the other report, the calcium-modulated ROS-GC1:GCAP1 signaling system has been discovered in mitral cells of the olfactory bulb. These findings raise the possibility that a common theme of calcium-modulated ROS-GC signaling may be utilized in a wide variety of neurosensory cells. This idea is also supported from evolutionary and functional perspectives.     

The Superoxide Dismutase-Mimic Copper-Putrescine-Pyridine Suppresses Lipid Peroxidation in CHO Cells. Implications for its Prooxidative and Antioxidative Mechanisms of Action

Copper-Putrescine-Pyridine (Cu-PuPy) is a membrane-permeable complex which efficiently dismutates superoxide. In excess of 0.1 mM it is highly cytotoxic and oxidizes cellular GSH with concomitant production of H₂O₂. Here we show that treatment of CHO cells with 0.2 mM Cu-PuPy (0-200 min) leads to an accumulation of H₂O₂. Organic hydroperoxides which are also formed at low levels in the presence of Cu-PuPy, increase significantly after removal of the copper complex. We conclude that Cu-PuPy acts as an oxidant until cellular GSH is depleted. However, by interfering with radical chain propagation reactions, it suppresses lipid peroxidation and thus substitutes for consumed physiological antioxidants in a later stage of treatment. This consistently explains our previous, seemingly paradox, finding that longer Cu-PuPy treatments may be significantly less toxic than shorter ones.     

Studies on the mechanisms of neurulation in the chick: morphometric analysis of the relationship between regional variations in cell shape and sites of motive force generation

Microfilaments, which are organized into bundles in the apical ends of neuroepithelial cells, are generally thought to play a major role in generating the driving forces for neural tube closure. Because of their proximity to the luminal surface, the contractile activity of these microfilament bundles results in conspicuous changes in the overall shape of neuroepithelial cells, most notably apical constriction and apical surface folding. In the present study, we have used morphometric methods and computer-assisted image analysis to reveal the distribution of microfilament-mediated forces in the developing midbrain during initial contact of apposing neural folds in chick embryos at Hamburger and Hamilton stage 8+ of development (Hamburger and Hamilton (1951) J. Morphol., 88:49-92). The degree of apical constriction, apical surface folding, and bending of the neuroepithelium was used as a barometer of local microfilament activity. Results indicate that cells forming the floor and midlateral walls of the developing midbrain consistently show a higher degree of apical constriction and surface folding than those at other locations. These same regions of the neuroepithelium also exhibit the greatest degree of bending. We conclude that the principal driving forces for closure of the neural tube, at the level of the midbrain, are concentrated in certain regions of the neuroepithelium (i.e., the floor and midlateral walls of the forming neural tube) rather than uniformly distributed.     

Plant lectin can target receptors containing sialic Acid, exemplified by podoplanin, to inhibit transformed cell growth and migration

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.     

Natural IgG Autoantibodies Are Abundant and Ubiquitous in Human Sera,and Their Number Is Influenced By Age,Gender, and Disease

The presence of self-reactive IgG autoantibodies in human sera is largely thought to represent a breakdown in central tolerance and is typically regarded as a harbinger of autoimmune pathology. In the present study, immune-response profiling of human serum from 166 individuals via human protein microarrays demonstrates that IgG autoantibodies are abundant in all human serum, usually numbering in the thousands. These IgG autoantibodies bind to human antigens from organs and tissues all over the body and their serum diversity is strongly influenced by age, gender, and the presence of specific diseases. We also found that serum IgG autoantibody profiles are unique to an individual and remarkably stable over time. Similar profiles exist in rat and swine, suggesting conservation of this immunological feature among mammals. The number, diversity, and apparent evolutionary conservation of autoantibody profiles suggest that IgG autoantibodies have some important, as yet unrecognized, physiological function. We propose that IgG autoantibodies have evolved as an adaptive mechanism for debris-clearance, a function consistent with their apparent utility as diagnostic indicators of disease as already established for Alzheimer’s and Parkinson’s diseases.     

Neural tube closure defects caused by papaverine in explanted early chick embryos.

Papaverine (50 micrograms/ml) preferentially inhibited uplifting of neural folds in explanted stage 8 chick embryos. Affected neuroepithelial cells often lost their wedge-shaped and elongated appearance. Also, luminal surfaces of most affected cells were smoother than usual as evidenced by the marked decrease in the number of cytoplasmic extensions, but the integrity of other structures (including cytoskeletal components) was not noticeably affected. The observed changes in cell surface topography were due, at least in part, to the imparied ability of apical microfilaments to contract and their eventual relaxation. The "relaxing" effect of papaverine on neural folds could be reversed by subsequent treatment with ionophore A23187. Since papaverine and ionophore A23187 are known to alter the normal distribution of intracellular Ca2+ and changes in cell surface topography are correlated with contractile activities of apical microfilaments, papaverine elicits neural tube closure defects by lowering intracellular free Ca2+ levels, thereby relaxing contracted apical microfilaments in neuroepithelial cells.     

Augmentation of Nitric Oxide,Superoxide, and Peroxynitrite Production During Cerebral Ischemia and Reperfusion in the Rat

The effect of ischemia produced by bilateral occlusion of the common carotid arteries (30 min) followed by 4 hours of reperfusion on total and inducible nitric oxide synthase (NOS) activity and the production of nitric oxide (NO), superoxide and peroxynitrite in the cerebral hemispheres was determined in the rat. Compared to sham-operated controls, cerebral ischemia-reperfusion resulted in a significant increase in total and inducible NOS activity and a significant increase in the production of NO and superoxide in the cerebral hemispheres. The level of NO in the plasma and the peripheral leukocyte count were also significantly increased. Immunohistochemical staining for nitrotyrosine (a marker of peroxynitrite production) showed that ischemia-reperfusion resulted in increased synthesis of cerebral peroxynitrite. Administration of the irreversible NOS inhibitor, N-nitro-L-arginine (L-NA), increased superoxide levels in the brain and significantly reduced plasma NO. Total and inducible NOS activity as well as NO and immunoreactive nitrotyrosine, in the cerebral hemispheres were reduced with L-NA administration. The number of leukocytes in the plasma was unaffected by administration of L-NA. These findings suggest that cerebral ischemia-reperfusion causes increased production of reactive oxygen species in the cerebral hemispheres and that the production of peroxynitrite, and not superoxide, may be dependent upon the availability of NO.     

Neural tube defects caused by local anesthetics in early chick embryos

The effects of local anesthetics (ketamine HCl, lidocaine HCl, procaine HCl, and tetracaine HCl) on stage 8 (four-somite) chick embryos were investigated. In general, embryos responded to drug treatment in a dose-related manner during the first 6 hr of incubation. Concentrations of 500 micrograms/ml (ca. 2 mM) or higher were embryolethal, whereas 100-200 micrograms/ml (0.1-0.8 mM) preferentially inhibited elevation of neural folds. The latter effect was detectable within 3 hr of treatment and was readily reversible. Tetracaine was the most potent among the four local anesthetics tested at any given dose. Compared to controls, cells in the defective neuroepithelium were less elongated and exhibited smoother apical (luminal) surfaces, thinner microfilament bundles, and less intense actin-specific fluorescence. Furthermore, the effects of local anesthetics (100-200 micrograms/ml) on stage 8 chick embryos were not identical to those of cytochalasin D (0.05 micrograms/ml), colchicine (1 microgram/ml), or ionophore A23187 (25 micrograms/ml), although all treatments produced neural tube defects. Overall results suggest that local anesthetics inhibit closure of the neural tube through their disruptive action on the organization and function of microfilaments in developing neuroepithelial cells.