Profilin inhibits pollen tube growth through actin-binding,but not poly-<Emphasis Type="SmallCaps">l</Emphasis>-proline-binding

Previously, we have shown that excess profilin inhibits pollen tube growth at significantly lower concentrations than it blocks cytoplasmic streaming. To elucidate the mechanism by which profilin achieves this function, we have employed mutant profilins from Schizosaccharomyces pombe [J. Lu and T.D. Pollard (2001) Mol Biol Cell 12:1161–1175], which have defects in actin-binding, ability to inhibit polymerization, and poly-l-proline (PLP)-binding. Using Lilium longiflorum L. pollen and S. pombe profilins as wild-type (wt) standards, mutant profilins have been injected into pollen tubes of Lilium, and examined for their effects on growth rate and cell morphology. Our results show that mutant Y5D (68% actin-binding; 1.1% PLP-binding) is indistinguishable from wt-standard profilins. However mutant K81F (2.7% actin-binding; 77% PLP-binding) and especially mutant K67E (<1% actin-binding; 100% PLP-binding) are significantly less effective than wt-standard profilins in their ability to inhibit pollen tube growth. PLP also inhibits pollen tube growth. However, PLP is not different from K67E/PLP combined, which has no actin-binding, suggesting that PLP does not function by binding to profilin. In addition, there are differences in the morphology and F-actin organization in cells injected with PLP versus wt-profilin. Whereas wt-profilin causes a fragmentation and marked reduction in the amount of F-actin [L. Vidali et al. (2001) Mol Biol Cell 12:2534–2545], PLP generates an extensive disorganization without any apparent reduction in the amount of F-actin. We conclude that along with actin-binding activity of profilin, PLP-containing proteins also participate in the growth control process, and can do so independently of binding to profilin.Abbreviations 3D Three-dimensional - PLP Poly-l-proline - RMS Root mean square - wt Wild type     

Amyloid fibrils of mammalian prion protein induce axonal degeneration in NTERA2-derived terminally differentiated neurons

Defects in axonal transport and synaptic dysfunctions are associated with early stages of several neurodegenerative diseases including Alzheimer's, Huntington's, Parkinson's, and prion diseases. Here, we tested the effect of full-length mammalian prion protein (rPrP) converted into three conformationally different isoforms to induce pathological changes regarded as early subcellular hallmarks of prion disease. We employed human embryonal teratocarcinoma NTERA2 cells (NT2) that were terminally differentiated into neuronal and glial cells and co-cultured together. We found that rPrP fibrils but not alpha-rPrP or soluble beta-sheet rich oligomers caused degeneration of neuronal processes. Degeneration of processes was accompanied by a collapse of microtubules and aggregation of cytoskeletal proteins, formation of neuritic beads, and a dramatic change in localization of synaptophysin. Our studies demonstrated the utility of NT2 cells as valuable human model system for elucidating subcellular events of prion pathogenesis, and supported the emerging hypothesis that defects in neuronal transport and synaptic abnormalities are early pathological hallmarks associated with prion diseases.     

Steroids and triterpenoids from Eastern Nigeria mistletoe, Loranthus micranthus Linn. (Loranthaceae) parasitic on Kola acuminata with immunomodulatory potentials

In search of immunomodulatory constituents from the Eastern Nigeria mistletoe, Loranthus micranthus Linn, two new stigmastane steroids: stigmast-7,20 (21)-diene-3β-hydroxy-6-one (1) and 3β-hydroxy-stigmast-23-ene (2); three (two new and one known) lupeol-based triterpenoid esters: 7β,15α-dihydroxyl-lup-20(29)-ene-3β-palmitate (3), 7β,15α-dihydroxyl-lup-20(29)-ene-3β-stearate (4) and 7β,15α-dihydroxyl-lup-20(29)-ene-3β-eicosanoate (5) were isolated and characterized following bioactivity-guided fractionation. The new compounds, 1, 2, 4 and 5 at concentrations of 10, 25 and 100 μg/ml were subjected to cell proliferation and early activation marker (CD69) expression studies in C57Bl/6 mice splenocytes using flow cytometry techniques against Lipopolysaccharide (LPS; 10 μg/ml) and Concanavalin A (ConA; 2 μg/ml) standards. The stigmastane steroids (1 and 2) at the highest concentration of 100 μg/ml showed statistically significantly (p < 0.05) stimulatory activity on the C57B1/6 splenocytes compared to the controls with values of 46 ± 0.76% and 43 ± 0.46% compared to 7.69 ± 0.41% recorded for the negative control. The novel lupeol esters, 4 and 5 at same concentration of 100 μg/ml exhibited lower stimulations of 30 ± 0.41% and 29 ± 0.17% respectively compared to the controls above. The CD69 expression assay at the above doses showed that all the compounds have minimal stimulation. The present study supports the observed immunomodulatory property of the Eastern Nigeria mistletoe and thus confirms the efficacy of this plant in mitigating against wide array of disease conditions orchestrated by immunodeficiency.     

Effects of sequential acute stress exposure on stress-induced pituitary luteinizing hormone and prolactin secretion

The present study was carried out to determine the effects of repetitive acute stress exposure on pituitary secretion of both luteinizing hormone (LH) and prolactin (PRL). Adult male rats were exposed to sequential episodes of acute novel environment stress separated by intervals of either 60 or 120 minutes. Serial blood samples were obtained from animals before, during and after each stress episode via indwelling intra-cardiac cannulas. The imposition of 10 minute episodes of novel environment stress on an hourly basis eventually rendered the hypothalamic-hypophyseal LH axis refractory to the stimulatory effect of stress. If sequential stress was imposed at 120 minute intervals, LH release was significantly enhanced during each exposure. A different pattern of PRL release was observed during the same sequential stress schedule. After an initial increase in hormone release in response to the first hourly stress episode, PRL levels were unaltered during the second and third hourly stress exposures. Thereafter, plasma PRL levels showed a trend toward a progressive increase in release during each successive episode, and were significantly elevated above preceding baseline levels during the fourth and fifth hourly stress exposures. In rats exposed to stress every two hours, a significant increase in PRL levels occurred following the first, but not the second stress episode. Hormone release was again enhanced in response to the third exposure to novel environment. The present results demonstrate that the repetitive exposure to acute novel environment stress results in differential alterations in pituitary LH and PRL secretion over time, and that the timing of repeated episodes is an important determinant of continued responsiveness to stress, particularly with regard to LH release. These findings suggest that the LH and PRL hormonal responses to at least this specific stressor are mediated by independent neural mechanisms.     

Tocotrienol-induced caspase-8 activation is unrelated to death receptor apoptotic signaling in neoplastic mammary epithelial cells

Tocotrienols, a subclass in the vitamin E family of compounds, have been shown to induce apoptosis by activating caspase-8 and caspase-3 in neoplastic mammary epithelial cells. Since caspase-8 activation is associated with death receptor apoptotic signaling, studies were conducted to determine the exact death receptor/ligand involved in tocotrienol-induced apoptosis. Highly malignant +SA mouse mammary epithelial cells were grown in culture and maintained in serum-free media. Treatment with 20 microM gamma-tocotrienol decreased+SA cell viability by inducing apoptosis, as determined by positive terminal dUTP nick end labeling (TUNEL) immunocytochemical staining. Western blot analysis showed that gamma-tocotrienol treatment increased the levels of cleaved (active) caspase-8 and caspase-3. Combined treatment with caspase inhibitors completely blocked tocotrienol-induced apoptosis. Additional studies showed that treatment with 100 ng/ml tumor necrosis factor-alpha (TNF-alpha), 100 ng/ml FasL, 100 ng/ml TNF-related apoptosis-inducing ligand (TRAIL), or 1 microg/ml apoptosis-inducing Fas antibody failed to induce death in +SA cells, indicating that this mammary tumor cell line is resistant to death receptor-induced apoptosis. Furthermore, treatment with 20 microM gamma-tocotrienol had no effect on total, membrane, or cytosolic levels of Fas, Fas ligand (FasL), or Fas-associated via death domain (FADD) and did not induce translocation of Fas, FasL, or FADD from the cytosolic to the membrane fraction, providing additional evidence that tocotrienol-induced caspase-8 activation is not associated with death receptor apoptotic signaling. Other studies showed that treatment with 20 microM gamma-tocotrienol induced a large decrease in the relative intracellular levels of phospho-phosphatidylinositol 3-kinase (PI3K)-dependent kinase 1 (phospho-PDK-1 active), phospho-Akt (active), and phospho-glycogen synthase kinase3, as well as decreasing intracellular levels of FLICE-inhibitory protein (FLIP), an antiapoptotic protein that inhibits caspase-8 activation, in these cells. Since stimulation of the PI3K/PDK/Akt mitogenic pathway is associated with increased FLIP expression, enhanced cellular proliferation, and survival, these results indicate that tocotrienol-induced caspase-8 activation and apoptosis in malignant +SA mammary epithelial cells is associated with a suppression in PI3K/PDK-1/Akt mitogenic signaling and subsequent reduction in intracellular FLIP levels.     

Kinase-dependent activation of voltage-gated Ca2+ channels by ET-1 in pulmonary arterial myocytes during chronic hypoxia

Exposure to chronic hypoxia (CH) causes pulmonary hypertension. The vasoconstrictor endothelin-1 (ET-1) is thought to play a role in the development of hypoxic pulmonary hypertension. In pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic rats, ET-1 signaling is altered, with the ET-1-induced change in intracellular calcium concentration (Δ[Ca(2+)](i)) occurring through activation of voltage-dependent Ca(2+) channels (VDCC) even though ET-1-induced depolarization via inhibition of K(+) channels is lost. The mechanism underlying this response is unclear. We hypothesized that activation of VDCCs by ET-1 following CH might be mediated by protein kinase C (PKC) and/or Rho kinase, both of which have been shown to phosphorylate and activate VDCCs. To test this hypothesis, we examined the effects of PKC and Rho kinase inhibitors on the ET-1-induced Δ[Ca(2+)](i) in PASMCs from rats exposed to CH (10% O(2), 3 wk) using the Ca(2+)-sensitive dye fura 2-AM and fluorescent microscopy techniques. We found that staurosporine and GF109203X, inhibitors of PKC, and Y-27632 and HA 1077, Rho kinase inhibitors, reduced the ET-1-induced Δ[Ca(2+)](i) by >70%. Inhibition of tyrosine kinases (TKs) with genistein or tyrphostin A23, or combined inhibition of PKC, TKs, and Rho kinase, reduced the Δ[Ca(2+)](i) to a similar extent as inhibition of either PKC or Rho kinase alone. The ability of PKC or Rho kinase to activate VDCCs in our cells was verified using phorbol 12-myristate 13-acetate and GTP-γ-S. These results suggest that following CH, the ET-1-induced Δ[Ca(2+)](i) in PASMCs occurs via Ca(2+) influx through VDCCs mediated primarily by PKC, TKs, and Rho kinase.     

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and “pearls of wisdom”/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.     

Neurochemical evidence for two types of presynaptic alpha2-adrenoceptors

Neurochemical and pharmacological evidence has been obtained that noradrenergic varicosities (in mouse and rat vas deferens) and cholinergic varicosities (in the Auerbach's plexus) contain heterogenous alpha₂-adrenoceptors through which the release of [³H]noradrenaline and [³H]acetylcholine can be modulated. The quantitative data also support the hypothesis that different noradrenaline and xylazine sensitive alpha₂-adrenoceptors are present prejunctionally in the vas deferens and Auerbach's plexus preparations. Prazosin, although it has a presynaptic inhibitory effect on alpha₂-adrenoceptors of noradrenergic axon terminals, has no effect on cholinergic axon terminals. These data suggest that there are two different types of alpha₂-adrenoceptors at the presynaptic axon terminals.Special Issue Dedicated to Dr. Abel Lajtha