Development and assessment of sensitive immuno‐PCR assays for the quantification of cerebrospinal fluid three‐ and four‐repeat tau isoforms in tauopathies

Characteristic tau isoform composition of the insoluble fibrillar tau inclusions define tauopathies, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP) and frontotemporal dementia with parkinsonism linked to chromosome 17/frontotemporal lobar degeneration‐tau (FTDP‐17/FTLD‐tau). Exon 10 splicing mutations in the tau gene, MAPT, in familial FTDP‐17 cause elevation of tau isoforms with four microtubule‐binding repeat domains (4R‐tau) compared to those with three repeats (3R‐tau). On the basis of two well‐characterised monoclonal antibodies against 3R‐ and 4R‐tau, we developed novel, sensitive immuno‐PCR assays for measuring the trace amounts of these isoforms in CSF. This was with the aim of assessing if CSF tau isoform changes reflect the pathological changes in tau isoform homeostasis in the degenerative brain and if these would be relevant for differential clinical diagnosis. Initial analysis of clinical CSF samples of PSP (n = 46), corticobasal syndrome (CBS; n = 22), AD (n = 11), Parkinson's disease with dementia (PDD; n = 16) and 35 controls revealed selective decreases of immunoreactive 4R‐tau in CSF of PSP and AD patients compared with controls, and lower 4R‐tau levels in AD compared with PDD. These decreases could be related to the disease‐specific conformational masking of the RD4‐binding epitope because of abnormal folding and/or aggregation of the 4R‐tau isoforms in tauopathies or increased sequestration of the 4R‐tau isoforms in brain tau pathology.     

Plant mitogen-activated protein kinase signaling cascades

Mitogen-activated protein kinase (MAPK) cascades have emerged as a universal signal transduction mechanism that connects diverse receptors/sensors to cellular and nuclear responses in eukaryotes. Recent studies in plants indicate that MAPK cascades are vital to fundamental physiological functions involved in hormonal responses, cell cycle regulation, abiotic stress signaling, and defense mechanisms. New findings have revealed the complexity and redundancy of the signaling components, the antagonistic nature of distinct pathways, and the use of both positive and negative regulatory mechanisms.     

Activation of mitogen-activated protein kinase by oxidized low-density lipoprotein in canine cultured vascular smooth muscle cells

Oxidized low-density lipoprotein (OX-LDL) contributes significantly to the development of atherosclerosis. However, the mechanisms of OX-LDL-induced vascular smooth muscle cell (VSMC) proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) by [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in canine cultured VSMCs. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenuated the OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating that these responses were mediated through a receptor coupled to a PTX-sensitive G protein. In cells pretreated with PMA for 24 h and with either the PKC inhibitor staurosporine or the tyrosine kinase inhibitor genistein for 1h, substantially reduced the [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to OX-LDL. Removal of Ca(2+) by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca(2+) for these responses. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK). Furthermore, we also showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G-protein-coupled receptor that involves the activation o Ras/Raf/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs.     

WNT5A selectively inhibits mouse ventral prostate development

The establishment of prostatic budding patterns occurs early in prostate development but mechanisms responsible for this event are poorly understood. We investigated the role of WNT5A in patterning prostatic buds as they emerge from the fetal mouse urogenital sinus (UGS). Wnt5a mRNA was expressed in UGS mesenchyme during budding and was focally up-regulated as buds emerged from the anterior, dorsolateral, and ventral UGS regions. We observed abnormal UGS morphology and prostatic bud patterns in Wnt5a null male fetuses, demonstrated that prostatic bud number was decreased by recombinant mouse WNT5A protein during wild type UGS morphogenesis in vitro, and showed that ventral prostate development was selectively impaired when these WNT5A-treated UGSs were grafted under under kidney capsules of immunodeficient mice and grown for 28 d. Moreover, a WNT5A inhibitory antibody, added to UGS organ culture media, rescued prostatic budding from inhibition by a ventral prostatic bud inhibitor, 2,3,8,7-tetrachlorodibenzo-p-dioxin, and restored ventral prostate morphogenesis when these tissues were grafted under immunodeficient mouse kidney capsules and grown for 28 d. These results suggest that WNT5A participates in prostatic bud patterning by restricting mouse ventral prostate development.     

Location and flexibility of the unique C-terminal tail of Aquifex aeolicus co-chaperonin protein 10 as derived by cryo-electron microscopy and biophysical techniques

Co-chaperonin protein 10 (cpn10, GroES in Escherichia coli) is a ring-shaped heptameric protein that facilitates substrate folding when in complex with cpn60 (GroEL in E. coli). The cpn10 from the hyperthermophilic, ancient bacterium Aquifex aeolicus (Aacpn10) has a 25-residue C-terminal extension in each monomer not found in any other cpn10 protein. Earlier in vitro work has shown that this tail is not needed for heptamer assembly or protein function. Without the tail, however, the heptamers (Aacpn10del-25) readily aggregate into fibrillar stacked rings. To explain this phenomenon, we performed binding experiments with a peptide construct of the tail to establish its specificity for Aacpn10del-25 and used cryo-electron microscopy to determine the three-dimensional (3D) structure of the GroEL-Aacpn10-ADP complex at an 8-Å resolution. We found that the GroEL-Aacpn10 structure is similar to the GroEL-GroES structure at this resolution, suggesting that Aacpn10 has molecular interactions with cpn60 similar to other cpn10s. The cryo-electron microscopy density map does not directly reveal the density of the Aacpn10 25-residue tail. However, the 3D statistical variance coefficient map computed from multiple 3D reconstructions with randomly selected particle images suggests that the tail is located at the Aacpn10 monomer-monomer interface and extends toward the cis-ring apical domain of GroEL. The tail at this location does not block the formation of a functional co-chaperonin/chaperonin complex but limits self-aggregation into linear fibrils at high temperatures. In addition, the 3D variance coefficient map identifies several regions inside the GroEL-Aacpn10 complex that have flexible conformations. This observation is in full agreement with the structural properties of an effective chaperonin.     

Schisandrin B stereoisomers protect against hypoxia/reoxygenation-induced apoptosis and inhibit associated changes in Ca2+-induced mitochondrial permeability transition and mitochondrial membrane potential in H9c2 cardiomyocytes

The effects of schisandrin B stereoisomers, (+/-)gamma-schisandrin [(+/-)gamma-Sch] and (-)schisandrin B [(-)Sch B], on hypoxia/reoxygenation-induced apoptosis were investigated in H9c2 cardiomyocytes. Changes in cellular reduced glutathione (GSH) levels, Ca(2+)-induced mitochondrial permeability transition (MPT), and mitochondrial membrane potential (Deltapsi(m)) values, were examined in (+/-)gamma-Sch-pretreated and (-)Sch B-pretreated cells, without or with hypoxia/reoxygenation challenge. The (+/-)gamma-Sch and (-)Sch B (2.5-5.0 microM) pretreatments protected against hypoxia/reoxygenation-induced apoptosis of H9c2 cells in a concentration-dependent manner, with (-)Sch B being more potent. The degrees of protection decreased, however, at the higher drug concentrations of 7.5 microM in both (+/-)gamma-Sch-pretreated and (-)Sch B-pretreated cells. The anti-apoptotic effects of the drugs were further evidenced by the suppression of hypoxia/reoxygenation-induced mitochondrial cytochrome c release and the subsequent cleavage of caspase 3 and poly-ADP-ribose polymerase after (-)Sch B pretreatment. Both (+/-)gamma-Sch and (-)Sch B pretreatments increased GSH levels in H9c2 cells, with (-)Sch B being more potent. Hypoxia/reoxygenation challenge caused a depletion in cellular GSH and the cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B was associated with enhancement of cellular GSH in H9c2 cells, as compared to the drug-unpretreated control. Whereas hypoxia/reoxygenation challenge increased the extent of Ca(2+)-induced MPT pore opening and decreased Deltapsi(m) in H9c2 cardiomyocytes, cytoprotection against hypoxia/reoxygenation-induced apoptosis afforded by (+/-)gamma-Sch/(-)Sch B pretreatments was associated with a decreased sensitivity to Ca(2+)-induced MPT and an increased Deltapsi(m) in both unchallenged and challenged cells, as compared to the respective drug-unpretreated controls. The degrees of protection against apoptosis correlated negatively with the extents of Ca(2+)-induced MPT (r=-0.615, P<0.01) and positively with the values of Deltapsi(m) (r=0.703, P<0.01) in (+/-)gamma-Sch/(-)Sch B-pretreated and hypoxia/reoxygenation challenged cells. The results indicate that (+/-)gamma-Sch/(-)Sch B pretreatment protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes and that the cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B may at least in part be mediated by a decrease in cellular sensitivity to Ca(2+)-induced MPT, which may in turn result from enhancement of cellular GSH levels by drug pretreatments.     

Vimentin supports mitochondrial morphology and organization

Vimentin is one of the intermediate filaments that functions in structural support, signal transduction and organelle positioning of a cell. In the present study, we report the contribution of vimentin in mitochondrial morphology and organization. Using subcellular fractionation, immunoprecipitation and fluorescence microscopy analyses, we found that vimentin was associated with mitochondria. Knockdown of vimentin resulted in mitochondrial fragmentation, swelling and disorganization. We further demonstrated that the vimentin cytoskeleton co-localized and interacted with mitochondria to a greater extent than other cytoskeletal components known to support mitochondria. Our results also suggest that vimentin could participate in the mitochondrial association of microtubules. As mitochondrial morphologies determine mitochondrial function, our findings revealed a potentially important relationship between the vimentin-based intermediate filaments and the regulation of mitochondria.     

Synergism of biochemical and mechanical stimuli in the differentiation of human placenta-derived multipotent cells into endothelial cells

There have been intensive studies on the differentiation of endothelial progenitor cells (EPCs) into endothelial cells. We investigated the endothelial differentiation of placenta-derived multipotent cells (PDMCs), a population of CD34(-)/CD133(-)/Flk-1(-) cells. PDMCs were cultured in basal media or media containing endothelial growth factors (EGM), including vascular endothelial growth factor (VEGF), for 3 days and then subjected to shear stress of 6 or 12dyn/cm(2) for 24h. Culture of PDMCs in EGM under static conditions resulted in significant increases in VEGF receptor-1 (Flt-1) and receptor-2 (Flk-1) expression. Application of shear stress at 12dyn/cm(2) to these cells led to significant increases in their expression of von Willebrand Factor and platelet-endothelial cell adhesion molecule-1 at both the gene and protein levels. Shear stress at 6dyn/cm(2) had lesser effects. Uptakes of acetylated low-density lipoproteins as well as formation of tube-like structures on Matrigel were significantly increased after subjecting to shear stress of 12dyn/cm(2) for 24h. Our findings suggest that the combined use of endothelial growth factors and high shear stress is synergistic for the endothelial differentiation of PDMCs.