Search

N-methyl-d-aspartate (NMDA)receptors (NMDARs) located on peripheral terminals of primary afferents are involved in the transduction of noxious mechanical stimuli. Exploiting the fact that both NMDARs and stretch-activated channels are retained in short-term culture and expressed on the soma of dorsal root ganglia (DRG) neurons, we examined the effect of NMDA on mechanically mediated changes in intracellular calcium concentration ([Ca2+]i). Our aims were to determine whether NMDARs modulate the mechanosensitivity of DRG neurons. Primary cultures of adult rat lumbosacral DRG cells were cultured for 1–3 days. [Ca2+]i responses were determined by Fura-2 ratio fluorescence. Somas were mechanically stimulated with fire-polished glass pipettes that depressed the cell membrane for 0.5 s. Voltage-activated inward Ca2+ currents were measured by the whole cell patch clamp. Stimulation of neurons with 100 μM NMDA in the presence, but not the absence, of co-agonist (10 μM d-serine) caused transient [Ca2+]i responses (101±9 nM) and potentiated [Ca2+]i peak responses to subsequent mechanical stimulation more than two-fold (P<0.001). NMDA-mediated potentiation of mechanically induced [Ca2+]i responses was inhibited by the selective protein kinase C (PKC) inhibitor GF109203X (GFX; 10 μM), which had no independent effects on NMDA- or mechanically induced responses. Short-term treatment with the PKC activator phorbol dibutyrate (1 μM PDBu for 1–2 min) also potentiated mechanically induced [Ca2+]i responses nearly two-fold (P<0.001), while longer exposure (>10 min) inhibited the [Ca2+]i transients by 44% (P<0.001). Both effects of PDBu were prevented by prior treatment with GFX. Inhibition of voltage-dependent Ca2+ channels with 25 μM La3+ had no effect on mechanically induced [Ca2+]i transients prior to NMDA, but prevented enhancement of the transients by NMDA and PDBu. NMDA pretreatment transiently enhanced nifedipine-sensitive, voltage-activated Ca2+ currents by a process that was sensitive to GFX. In conclusion, activation of NMDARs on cultured DRG neurons sensitize voltage-dependent L-type Ca2+ channels which contribute to mechanically induced [Ca2+]i transients through a PKC-mediated process. [Copyright &y& Elsevier]

In the presence of external Ca2+, pretreatment of neutrophils with 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1) inhibited the cyclopiazonic acid (CPA)-induced [Ca2+]i elevation in a concentration- but not a time-dependent manner, while YC-1 had no effect on the Ca2+ signals in a Ca2+-free medium. YC-1 failed to inhibit ATP- and interleukin-8 (IL-8)-induced [Ca2+]i changes. Addition of YC-1 after cell activation strongly inhibited the CPA-induced [Ca2+]i changes. In a classical Ca2+ readdition protocol, a similar extent inhibition of Ca2+ spike by YC-1 introduced either prior to or after CPA stimulation was obtained. In rat neutrophils, mRNA for endothelial differentiation gene (edg)1, edg5, edg6 and edg8, the putative targets for sphingosine 1-phosphate (S1P), could be detected. However, S1P was found to have little effect on Ca2+ signals. YC-1 did not inhibit but enhanced the sphingosine-induced [Ca2+]i changes. Inhibition by YC-1 of CPA-induced [Ca2+]i changes was not prevented by 7-nitroindazole and N-(3-aminomethyl)benzylacetamidine (1400W), two nitric oxide synthase (NOS) inhibitors, by aristolochic acid, a phospholipase A2 inhibitor, or by suspension in a Na+-deprived medium. YC-1 did not affect the mitochondrial membrane potential. Moreover, YC-1 did not alter [Ca2+]i changes in response to ionomycin after CPA and formyl-Met-Leu-Phe (fMLP) stimulation in a Ca2+-free medium. YC-1 had no effect on the basal [Ca2+]i level, the pharmacologically isolated plasma membrane Ca2+-ATPase activity, and Ba2+ entry into CPA-activated cells. YC-1 alone resulted in the accumulation of actin filaments in neutrophils, while significantly reduced the intensity of actin filament staining in the subsequent activation with CPA. These results indicate that YC-1 inhibited CPA-activated store-operated Ca2+ entry (SOCE) probably through the direct blockade of channel activation and/or the disruption of the integrity of the actin cytoskeleton necessary for supporting Ca2+ entry pathway in neutrophils. [Copyright &y& Elsevier]

Two experimental approaches were used to investigate the immunological responses of chickens to a commercial killed Salmonella enteritidis (SE) vaccine. In the first, the effects of host age on antigen-specific proliferative responses and cytokine production were examined. Compared with non-vaccinated controls, 4-wk-old vaccinated chickens showed higher proliferation to SE LPS and flagella. The lymphoproliferation responses to these antigens of 8-mo-old vaccinated chickens were not different compared to the non-vaccinated controls. Increased production of interferon-γ (IFN-γ) and interleukin-2 (IL-2) by antigen-stimulated splenocytes following vaccination were, in general, more often observed in 4-wk-old compared with 8-mo-old chickens, whereas serum levels of these cytokines were consistently higher in the vaccinated birds compared with controls regardless of age. The second set of experiments were designed to determine the effects of SE vaccination on mitogen- or antigen-induced splenocyte proliferation and serum nitric oxide (NO) and cytokine levels. Splenocytes from vaccinated chickens stimulated with SE flagella showed significantly increased numbers of TCRγδ+ cells at 7 days post-vaccination compared with non-vaccinated birds. In contrast, no differences were noted with CD4+, CD8+, or TCRαβ+ cells at any time points examined. Higher levels of NO production were observed following stimulation with SE flagella at 4, 7, 11, and 14 days after SE vaccination while serum levels of IFN-γ, IL-1, IL-6, and IL-8 were elevated only at day 7 post-vaccination. In conclusion, younger chickens mounted a more robust antigen-specific immune response to the SE vaccine compared with older birds and vaccination induced not only T-cell-mediated responses but also host innate and pro-inflammatory responses. [Copyright &y& Elsevier]

Promotion of inflammatory response is an important role of the complement system, but this kind of function is poorly documented for the lower vertebrates. Here we report chemotactic activity of purified anaphylactic fragments derived from the complement components C3, C4 and C5 of the common carp. The purified anaphylatoxins are two C5a-desArg peptides derived from the C5-I isotype, an intact form and a desArg form of C4a from C4-2 isotype, and an intact form and a desArg form of C3a from C3-H1 isoform. These were identified by N-terminal sequencing, mass spectrometry, and peptide mass fingerprinting. In the chemotaxis assay using carp kidney neutrophils, the two C5a-desArg fragments, which are probably allotypic variants, showed a potent chemotactic activity at 0.5–1 nM, whereas C3a or C4a showed no significant activity. The results suggest that C3a, C4a and C5a of bony fish have functionally diverged to the state similar to their mammalian homologs. [Copyright &y& Elsevier]

: Background/AimsWe previously described that the cold-induced apoptosis of cultured hepatocytes and liver endothelial cells is mediated by an increase in the cellular chelatable iron pool—in the absence of any increase in O2⋅−/H2O2 formation. As this is an unusual mechanism, we here set out to assess whether an increase in cellular chelatable iron per se is sufficient to trigger cell injury/apoptosis.: MethodsCultured rat hepatocytes were acutely loaded with iron using the membrane-permeable complex Fe(III)/8-hydroxyquinoline and incubated under otherwise ‘physiological’ conditions.: ResultsIncubation with Fe(III)/8-hydroxyquinoline (15 μM/30 μM) increased the cellular chelatable iron and induced strong hepatocellular injury with morphological features of apoptosis, but also of necrosis. The iron-induced cell injury was oxygen-dependent, and although it was not inhibitable by extracellular catalase, it was strongly inhibited by the novel membrane-permeable catalase mimic TAA-1/Fe. The experimentally induced increase in cellular chelatable iron triggered a mitochondrial permeability transition (MPT) as assessed using double-staining with calcein and tetramethylrhodamine methyl ester. The MPT inhibitor cyclosporine A partially and the well-known inhibitor combination trifluoperazine+fructose completely inhibited the iron-induced cell injury/apoptosis.: ConclusionsThese results show that iron per se can induce cell injury/apoptosis and that this injury is mediated via an MPT. [Copyright &y& Elsevier]

Farnesyl-O-acetylhydroquinone (IC50=2.5 μmol/l) suppressed the proliferation of murine B16F10 melanoma cells with a potency much greater than those of farnesol (IC50=45 μmol/l) and farnesyl anthranilate (IC50=46 μmol/l), its alcohol, and ester counterparts with proven anti-tumor activities in vivo. Geranyl-O-acetylhydroquinone (IC50=5.1 μmol/l) also had a much-improved activity compared to geraniol (IC50=160 μmol/l) and geranyl anthranilate (IC50=30 μmol/l). The suppression by farnesyl-O-acetylhydroquinone was concentration- and time-dependent and was accompanied by arrest of cell cycle at G1 and G2/M phases as shown by flow cytometry. Farnesyl-O-acetylhydroquinone and lovastatin had additive impact on B16 cell proliferation. Farnesyl-O-acetylhydroquinone also suppressed the proliferations of human cancer cells HL-60, DU145, PC-3, LNCaP, Caco-2, and A549. Our results suggested that farnesyl derivatives, suppressors of tumor 3-hydroxy-3-methylglutaryl coenzyme A reductase activities, have potential as chemopreventive or chemotherapeutic agents. [Copyright &y& Elsevier]

In ileal epithelial cells, apical sodium-dependent bile acid transporter (ASBT) is responsible for the uptake of bile acids from the lumen. Furthermore, ASBT is expressed in the apical plasma membrane of intrahepatic bile duct cells (BECs). Using cultured immortalized mouse intrahepatic BECs that form monolayers or cysts, vectorial transport of bile acids was studied. [3H]-taurocholic acid ([3H]-TCA) was transported through monolayers transcellularly almost exclusively from the apical to the basolateral side in a Na+- and a temperature-dependent manner. Transport of [3H]-TCA was inhibited by 59.3±18.6% in the presence of taurochenodeoxycholic acid. Uptake of lysyl fluorescein-conjugated bile acid, Cholyl-[N&epsiv;-NBD]-lysine, was seen in a Na+- and a temperature-dependent manner from the apical side of BECs that form monolayer or cysts. Reverse transcription-polymerase chain reaction for mRNAs in the cells showed presence of mRNAs for ASBT and farnesoid X receptor (FXR), a nuclear bile acid receptor. In conclusion, intrahepatic BECs transport bile acids mainly from the apical to the basolateral side in concert with ASBT and maybe FXR in the cells. [Copyright &y& Elsevier]

Cellular infiltrates consisting mainly of lymphocytes are commonly present in the arterial wall in abdominal aortic aneurysm (AAA). However, despite this, the precise nature of these populations has to date not been described in detail. The aim of this study was to perform an exhaustive phenotypic characterisation of the infiltrating lymphocytes in order to define the inflammatory process that develops in AAA. For this purpose, AAA-infiltrating lymphocytes from 25 fresh aneurysm wall samples and, as a control, peripheral blood (PB) lymphocytes from the same patients were purified. The expression of lineage specific markers, maturation molecules, activation antigens and adhesion molecules on T and B lymphocytes was examined by triple-colour immunofluorescence and flow cytometry analysis. The phenotype of AAA-infiltrating lymphocytes was compared with that of PB of the patients with AAA. The results reveal that AAA-infiltrating B and T lymphocytes consist of activated memory cells, with specific homing properties. In addition, they express molecules of B–T co-stimulation and, occasionally, exhibit phenotypical features indicative of the ectopic formation of lymphoid structures. These findings are discussed in the light of the similarities shared with the lymphoid infiltration occurring in other chronic autoimmune/inflammatory disorders. [Copyright &y& Elsevier]

We employed fluorescence correlation spectroscopy (FCS) to analyze the characteristics of biomolecules in living cells. Protein kinase C (PKC) changes its subcellular localization from cytosol to the plasma membrane by its ligand. Using FCS, we found PKCβI labeled with enhanced green fluorescent protein freely diffusing in cytosol. Upon 12-O-tetradecanoylphorbol-13-acetate activation, a large part of PKCβI is anchored in the plasma membrane but some PKCβI still moves freely near the plasma membrane. These results indicate that a diffusion-driven transport mechanism is appropriate for the molecular mechanism of the PKCβI localization change. [Copyright &y& Elsevier]

The present work describes an isozyme-related effect of collagenase perfusion on hepatocyte microsomal cytochrome (CYP)-dependent monooxygenase activities: CYP 1A1/2-, 2B1/2-, 3A1/2- and 2E1-dependent activities in microsomes from rat hepatocytes after isolation were about 60% of that of liver microsomes, and CYP 4A1-dependent activity was equivalent to liver microsomes. In contrast, the microsomal protein content of the various CYP isoforms was not affected by hepatocyte isolation. This is in accordance with the hypothesis of CYP inactivation during the process of hepatocyte isolation by collagenase digestion. l-NAME (1 mm) was found unable to protect from the decline of CYP-dependent monooxygenase activities following hepatocyte isolation. It is possible that the decrease in glutathione peroxidase activity observed in the presence of l-NAME, namely depression of defense against peroxynitrite, could counteract the beneficial effect of l-NAME on nitric oxide synthesis inhibition. The present work also shows that l-NAME could not avoid the progressive, isoform-specific, most probably turnover-related, decline of CYP proteins and related monooxygenase activities in cultured hepatocytes. Dysregulations in the mechanisms of CYP expression in rat hepatocyte cultures, presently unknown but nitric oxide independent, thus appear to occur in cultured rat hepatocytes. [Copyright &y& Elsevier]

We report a four-step procedure that optimizes the methodology for isolation of highly purified rat Kupffer cells (KC). We combined the previously reported techniques of enzymatic tissue treatment, density gradient centrifugation, centrifugal elutriation and selective adherence. ED-2 immunophenotyping and non-specific esterase histochemistry were used for cell identification. This combination resulted in a satisfactorily high yield of 80–100×106KCs per liver, over 95% positive for ED-2 and 98% viable cells. Cultures of isolated KCs were functionally intact and exhibited a concentration and time-dependent LPS-induced TNF-α and nitric oxide production. [Copyright &y& Elsevier]

Dissociated embryonic ventral mesencephalic tissue is a source of dopaminergic neurones in both cell culture and neural transplantation studies. Around 90% of grafted dopaminergic neurones die within 1 week after transplantation. Little is known about when the cell death is triggered and what forms of cell death predominate. Using electron microscopy, we characterised ultrastructural changes in dissected embryonic day 14 rat mesencephalic tissue before and after tissue dissociation. In addition, cell viability was evaluated using Trypan Blue and Hoechst/Ethidium Homodimer. Several cells exhibited leaky outer membranes (permitting entry of vital stains) and ultrastructural degeneration already immediately after the mesencephalon was dissected, and before it was mechanically disrupted. After 2 h at room temperature, 90% of the remaining cells had intact outer membranes. However, when estimating cells lost acutely in the tissue dissociation, in addition to cells exhibiting condensed chromatin and organellar changes, we suggest that only around 14% of the cells initially dissected in the mesencephalic tissue pieces remained healthy after 2 h. There was a peak in calpain activity (specific cleavage of fodrin) immediately following tissue dissociation, and it subsided during the next few hours. Caspase-3 activity was initially low, but increased almost 20-fold 4 h after tissue disruption. Interestingly, extensive degradation of caspase-3 occurred already directly after dissection and was at least partly calpain-dependent. Our data suggest that, in addition to cells undergoing primary necrosis, some cells undergo apoptotic or related changes soon after tissue harvesting, and eventually undergo a secondary necrosis. In summary, embryonic mesencephalic cells exhibit multiple degenerative changes very early on in the neural transplant/tissue culture preparation protocol. [Copyright &y& Elsevier]

Helicobacter pylori upregulates endothelial adhesion molecules but the pattern is unclear. Human umbilical vein endothelial cells (HUVEC) were exposed to control medium or H. pylori 60190. Binding of monoclonal antibodies against P-selectin, E-selectin, vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) was determined using enzyme-linked immunosorbent assay. Binding of polymorphonuclear leukocytes to HUVEC was determined on cells exposed as above. After 6 h exposure to H. pylori, there were 30%, 124%, 167% and 100% increases in P-selectin, E-selectin, VCAM-1 and ICAM-1 levels and a 400% increase in polymorphonuclear leukocyte adhesion in HUVEC exposed to H. pylori. Effects of incubation for other intervals between 0 and 18 h are also described. H. pylori exerts some of its effects on gastric mucosa via gastric vasculature. This study gives insight into the pattern of H. pylori-associated endothelial adhesion molecule upregulation. [Copyright &y& Elsevier]

We have examined the role of the indirect pathway of antigen recognition and T cells in neural xenografts rejection by using major histocompatibility complex (MHC) class II-deficient mice as xenograft recipients. Dissociated embryonic ventral mesencephalic tissue from Sprague–Dawley rats was stereotaxically injected as a cell suspension into the striatum of MHC class II-deficient adult mice as well as MHC class I-deficient and wild-type mice as controls. All of the MHC class II-deficient mice had surviving grafts in the striatum 4 weeks post-grafting. In contrast, only a few of the MHC class I-deficient mice exhibited very small grafts and none of the wild-type mice had any surviving grafts. The mean number of surviving transplanted dopamine neurons in the MHC class II-deficient group was significantly larger than that observed in the other two groups. Moderate levels of MHC class I antigen expression were seen in the transplantation sites of some animals in the MHC class II-deficient group. No helper or cytotoxic T cells were observed infiltrating into the graft sites of this group. However, there were markedly increased levels of expression of MHC class I and class II antigens, and a number of T cells infiltrating in the graft sites in both the MHC class I-deficient and wild-type groups. These results show that rat embryonic nigral tissue can survive transplantation in the brain of the MHC class II-deficient mice for at least 4 weeks without any overt signs of rejection, suggesting that the indirect pathway of foreign antigen recognition mediated by host MHC class II molecules and helper T cells plays an important role in the rejection responses to intracerebral xenografts. [Copyright &y& Elsevier]

A representative set of potent antibacterial 6-desfluoro-8-methylquinolones, in which the C-6 fluorine atom is replaced by –NH2 or –H, and their 6-fluoro counterparts, were investigated to evaluate their phototoxic potential and to explore the mechanism behind their phototoxicity. The capacity to photosensitize biological substrates (lipids, proteins, DNA) has been analyzed, as well as their photocytotoxicity on red blood cells and 3T3 murine fibroblasts. The results obtained show that the quinolones studied are able to photosensitize red blood cell lysis in an oxygen-dependent way and induce a high decrease in cell viability after UVA irradiation. A major correlation with phototoxicity lies in the structure of the individual antibacterials and their hydrophobicity; in particular, 6-amino derivatives are less phototoxic than corresponding unsubstituted and fluorinated compounds. Cellular phototoxicity was inhibited by the addition of free radical and hydroxyl radical scavengers (BHA, GSH and DMTU), suggesting the involvement of a radical mechanism in their cytotoxicity. A good correlation was observed between lipid peroxidation and phototoxicity, indicating that the test compounds exert their toxic effects mainly in the cellular membrane. Preliminary experiments on pBR322 DNA show that these derivatives do not photocleave DNA, differently from the two photogenotoxic fluoroquinolones, ciprofloxacin and lomefloxacin, used as reference compounds. [ABSTRACT FROM AUTHOR]

DMP 406 is a clozapine analogue developed by Dupont-Pharma for the treatment of schizophrenia. Unfortunately it caused agranulocytosis in dogs during preclinical studies. Clozapine also causes agranulocytosis and this is believed to be due to a reactive nitrenium ion metabolite produced by neutrophils. We studied the oxidation of DMP 406 by activated neutrophils and found that the major reactive species that is produced is not a nitrenium ion but rather an imine. This metabolite is similar to the reactive metabolite that has been proposed to be responsible for mianserin-induced agranulocytosis. Therefore we also studied the oxidation of mianserin by activated neutrophils and found that, although the major species is an iminium ion, it also bears a lactam moiety in the piperazine ring resulting from further oxidation. We usually find that HOCl is a good model system for the production of reactive metabolites of drugs that are formed by activated neutrophils, but in the case of both DMP 406 and mianserin, the products produced were significantly different than those formed by activated neutrophils. In contrast, the combination of horseradish peroxidase and hydrogen peroxide (HRP/H2O2) formed a very similar pattern of products, and this system was used to produce sufficient quantities of metabolites to allow for identification. The reactive metabolites of both DMP 406 and mianserin reacted with a range of nucleophiles, but in many cases the reaction was reversible. The best nucleophile for trapping these reactive metabolites was cyanide. It has been demonstrated that the products of clozapine oxidation by HRP/H2O2, presumably the nitrenium ion, induced apoptosis in neutrophils at therapeutic concentrations of clozapine. It has been suggested that this process is involved in the mechanism of clozapine-induced agranulocytosis. We tested DMP 406 and mianserin in this system to see if the ability of a reactive metabolite of a drug to cause apoptosis could predict the ability of that drug to cause agranulocytosis. We used clozapine as a positive control and we also tested olanzapine, a drug that forms a reactive metabolite similar to that of clozapine but is given at a lower dose and does not cause agranulocytosis. We found that DMP 406 did not increase apoptosis at concentrations below 50 μM, and although mianserin did increase apoptosis at 10 μM this is above the therapeutic concentration. Olanzapine caused an increase in apoptosis at the same concentration as clozapine (1 μM), but because its therapeutic concentration is lower, this concentration was above the pharmacological range. There was no increase in apoptosis with any drug in the absence of HRP/H2O2. These results indicate that this assay is unable to reliably predict the ability of different types of drugs to cause agranulocytosis. This is not a surprising result given that different drugs may induce agranulocytosis by different mechanisms. [Copyright &y& Elsevier]

BK channels are voltage- and calcium-dependent potassium channels whose activation tends to reduce cellular excitability. In hippocampal pyramidal cells, BK channels repolarize somatic action potentials, and recent immunogold and electrophysiological analyses have revealed a presynaptic pool of BK channels that can regulate glutamate release. Agents that modulate BK channel activity would therefore be expected to affect cell excitability and neurotransmitter release also under pathological conditions. We have investigated the role of BK potassium channels in a model of ischemia-induced nerve cell degeneration. Organotypical slice cultures of rat hippocampus were exposed to oxygen and glucose deprivation (OGD), and cell death was assessed by the fluorescent dye propidium iodide. OGD induced cell death in the CA1 region and to a lesser extent in CA3. Treatment with the BK channel blockers, paxilline and iberiotoxin, during and after OGD induced increased cell death in CA1 and CA3. Both BK channel blockers also sensitized the relatively resistant granule cells in fascia dentata to OGD. The effect of paxilline and iberiotoxin was evident from 3 h after OGD, indicating a role of BK channels early in the post-ischemic phase or during OGD itself. The BK channel opener, NS1619, turned out to be gliotoxic, and this effect was not counteracted by paxilline and iberiotoxin.Our data show that blockade of BK channels aggravates OGD-induced cell damage and suggest that BK channels act as a kind of ‘emergency brake’ during and/or after ischemia. Accordingly, the BK channel is a potential molecular target for neuroprotective therapy in stroke. [Copyright &y& Elsevier]

It has been recognized for some years that a prolonged Ca2+ elevation that is predictive of impending cell death develops in cultured neurons following excitotoxic insult. In addition, neurons exhibit enhanced sensitivity to excitotoxic insult with increasing age in culture. However, little is known about the processes that selectively regulate the post-insult Ca2+ elevation and therefore, it remains unclear whether it is associated specifically with age-dependent toxicity.Here, we tested the hypothesis that a group I metabotropic glutamate receptor antagonist selectively modulates the prolonged Ca2+ elevation in direct association with its protective effects against excitotoxicity. Rat hippocampal cultures of two ages (8–9 and 21–28 days in vitro) were exposed to a 5-min glutamate insult (400 μM in younger and 10 μM in older cultures) sufficient to kill >50% of the neurons, and were treated with vehicle or the specific group I metabotropic glutamate receptor antagonist 1-aminoindan-1,5-dicarboxylic acid (AIDA; 1 mM), throughout and following the insult. Neuronal survival was quantified 24 h after insult. In parallel studies, neurons of similar age in culture were imaged ratiometrically with a confocal microscope during and for 60 min after the glutamate insult. A large post-insult Ca2+ elevation was present in older but not most younger neurons. The N-methyl-D-aspartate receptor antagonist, MK-801, blocked the Ca2+ elevation both during and following the insult. In contrast, AIDA blocked only the post-insult prolonged Ca2+ elevation in older neurons. Moreover, AIDA was neuroprotective in older but not younger cultures.From these results we suggest that the post-insult Ca2+ elevation is regulated differently from the Ca2+ elevation during glutamate insult and is modulated by group I metabotropic glutamate receptors. Further, the prolonged Ca2+ elevation appears to be directly linked to an age-dependent component of vulnerability. [Copyright &y& Elsevier]

We examined the effects of the activation of metabotropic P2Y receptors on the intracellular Ca2+ concentration and the release of neuropeptide calcitonin gene-related peptide (CGRP) in isolated adult rat dorsal root ganglion neurons. In small-sized dorsal root ganglion neurons (soma diameter<30 μm) loaded with fura-2, a bath application of ATP (100 μM) evoked an increase in intracellular Ca2+ concentration, while the removal of extracellular Ca2+ partly depressed the response to ATP, thus suggesting that the ATP-induced increase in intracellular Ca2+ concentration is due to both the release of Ca2+ from intracellular stores and the influx of extracellular Ca2+. Bath application of uridine 5′-triphosphate (UTP; 100 μM) also caused an increase in intracellular Ca2+ concentration in small-sized dorsal root ganglion neurons and the P2 receptor antagonists suramin (100 μM) and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS; 10 μM) virtually abolished the response, indicating that the intracellular Ca2+ elevation in response to UTP is mediated through metabotropic P2Y receptors. This intracellular Ca2+ increase was abolished by pretreating the neurons with thapsigargin (100 nM), suggesting that the UTP-induced increase in intracellular Ca2+ is primarily due to the release of Ca2+ from endoplasmic reticulum Ca2+ stores. An enzyme-linked immunosorbent assay showed that an application of UTP (100 μM) significantly stimulated the release of CGRP and that suramin (100 μM) totally abolished the response, suggesting that P2Y receptor-mediated increase in intracellular Ca2+ is accompanied by CGRP release from dorsal root ganglion neurons.These results suggest that metabotropic P2Y receptors contribute to extracellular ATP-induced increase in intracellular Ca2+ concentration and subsequent release of neuropeptide CGRP in rat dorsal root ganglion neurons. [Copyright &y& Elsevier]

Dibenzoylmethane (DBM), a minor β-diketone constituent of licorice and sunscreens, has been shown to exhibit anti-neoplastic effects in chemically induced skin and mammary cancers in several animal models. To date, no mechanism for the growth inhibitory effects of DBM on prostate cancer cells has been proposed. In this study, we examined the effects of DBM on the growth and cell cycle kinetics of several human prostate carcinoma cell lines. Using an MTT cytotoxicity assay, IC50 values of 25–100 μM were observed following 72 h exposure to DBM. LNCaP, DU145, and PC-3 prostate carcinoma cell lines were particularly sensitive in comparison to the cells with the vehicle alone. Flow cytometric analyses showed deregulation of the cell cycle, which correlated with the observed cytostatic effects of DBM in prostate carcinoma cells. These data suggest a potential role for DBM in the prevention and treatment of prostate cancer. [Copyright &y& Elsevier]

Protein kinase C (PKC) is a key molecule for the expression of long-term depression at the parallel fiber–Purkinje cell synapse in the cerebellum, a well known model for synaptic plasticity. We have recently shown that activity of PKC also profoundly affects the dendritic morphology of Purkinje cells in rat cerebellar slice cultures suggesting that synaptic efficacy and dendritic development may be controlled by similar intracellular signalling pathways. Here we have analyzed the role of the γ-isoform of protein kinase C (PKCγ), which is strongly and specifically expressed in Purkinje cells, during dendritic development. After pharmacological treatment with PKC modulators, phosphorylation of PKCγ at serine 660 was altered in cerebellar slices suggesting that a change of PKCγ activity by these treatments was taking place within the Purkinje cells. In PKCγ-deficient mice, Purkinje cell dendritic trees were enlarged and had an increased number of branching points compared to wild-type mice indicating a role for the PKCγ isoform as a negative regulator of dendritic growth and branching. Furthermore, the branching-stimulating effects of the PKC inhibitors 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide and Go¨6976 found in wild-type cultures were abolished in the absence of PKCγ. In contrast, the strong inhibitory effect on dendritic growth by the PKC activator phorbol-12-myristate-13-acetate (PMA) did not require the presence of the PKCγ isoform since it was still present in the cultures of PKCγ-deficient mice.Our results clearly demonstrate an involvement of PKCγ in Purkinje cell dendritic differentiation in cerebellar slice cultures. [Copyright &y& Elsevier]

Axonal injury to CNS neurons results in apoptotic cell death. The processes by which axotomy signals apoptosis are diverse, and may include deprivation of target-derived factors, induction of injury factors, bursts of reactive oxygen species (ROS), and other mechanisms. Our previous studies demonstrated that death of a dissociated retinal ganglion cell, an identified CNS neuron, is ROS-dependent. To better define the mechanisms by which ROS induce retinal ganglion cell death after axotomy, we studied their effects in dissociated neonatal rat retinal cultures. Postnatal day 2–4 Long–Evans rat retinal ganglion cells were retrogradely labeled with the fluorescent tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI). Postnatal day 7–9 retinas were dissociated and cultured in the presence of specific ROS generating systems, scavengers, or redox modulators. Retinal ganglion cells were identified by DiI positivity and viability determined by metabolism of calcein-acetoxymethyl ester.We found that ROS scavengers protected against retinal ganglion cell death after acute dissociation, and the effects of ROS appeared to be due to shifts in the redox potential, as retinal ganglion cell survival was critically dependent on redox state, with greatest survival under mildly reducing conditions. Culture of retinal ganglion cell with the non-thiol-containing reducing agent tris(carboxyethyl)phosphine resulted in long-term survival equivalent to or better than with neurotrophic factors.Our data suggest that axotomy-associated neuronal death induced by acute dissociation may be partly dependent on ROS production, acting to shift the redox state and oxidize one or more key thiols. Understanding the mechanisms by which ROS signal neuronal death could result in strategies for increasing their long-term survival after axonal injury. [Copyright &y& Elsevier]

Fetal hippocampal CA3 cells show excellent survival when homotopically grafted into the kainic acid-lesioned CA3 region of the young adult hippocampus, a model of temporal lobe epilepsy. However, survival of these cells in the kainic acid-lesioned CA3 region of the aging hippocampus is unknown. We hypothesize that fetal CA3 grafts into the lesioned CA3 region of the middle-aged and aged hippocampus exhibit significantly diminished cell survival compared with similar grafts in the lesioned young adult hippocampus unless pre-treated and transplanted with factors that augment graft cell survival. We analyzed cell survival of 5′-bromodeoxyuridine-labeled embryonic day 19 CA3 grafts following their transplantation into the lesioned CA3 region of the middle-aged and aged rat hippocampus. Grafts were placed 4 days after an i.c.v. administration of kainic acid, and absolute cell survival of grafts was quantified 1 month after grafting using 5′-bromodeoxyuridine immunostaining of serial sections and the optical fractionator counting method. Grafts into both middle-aged and aged hippocampus exhibited analogous but significantly diminished cell survival (30% of injected cells) compared with similar grafts into the young adult hippocampus (72% cell survival). However, the extent of cell survival of CA3 grafts pre-treated and transplanted with a combination of neurotrophic factors brain-derived neurotrophic factor and neurotrophin-3 and the caspase inhibitor acetyl-tyrosinyl-valyl-alanyl-aspartyl-chloro-methylketone was significantly enhanced in both middle-aged and aged hippocampus (51–63% cell survival).These results underscore that aging impairs the conduciveness of the CA3 region for robust survival of homotopic fetal CA3 grafts after lesion. However, a combined neurotrophic supplementation and caspase inhibition significantly enhances survival of fetal CA3 cells in the lesioned aging hippocampus. Thus, pre-treatment and grafting of donor cells with a combination of factors that support growth of specific donor cells may considerably enhance survival and integration of fetal grafts into the lesioned aging CNS in clinical trials. [Copyright &y& Elsevier]

Calves lacking detectable serum antibodies against bovine respiratory syncytial virus (BRSV) were screened for virus-specific T-cell memory. Peripheral blood mononuclear cells were cultured in vitro with live BRSV and analyzed by dual-color flow cytometry for surface expression of CD25 on CD4+, CD8+, and γδ T-cells. Significant recall responses were detected in some of the seronegative calves. Modified live BRSV vaccine was administered to these and to a group of non-responding calves. Following vaccination, virus-specific IgG, virus neutralizing antibody, and T-cell recall responses were all elevated more rapidly in the group with BRSV-sensitive T-cells than in the T-cell-negative group, which suggested that calves in the first group were previously exposed to BRSV. This demonstrates that exposure to BRSV can induce T and B cell memory in young calves without causing seroconversion. The calves were presumably exposed to BRSV while they had maternal antibody, which inhibited the calves from developing an antibody response. [Copyright &y& Elsevier]

Introduction: Heart disease is a major cause of mortality worldwide, and the annual number of deaths due to heart disease has increased in recent years. Although heart failure is usually managed with medicines, the ultimate treatment for end-stage disease is heart transplantation or an artificial heart. However, the use of these surgical strategies is limited by issues such as thrombosis, rejection and donor shortages. Regenerative therapies, such as the transplantation of cultured cells and tissues constructed using tissue engineering techniques, are receiving great attention as possible alternative treatments for heart failure. Research is ongoing into the potential clinical use of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). However, the energy-producing capacity of cardiomyocytes maintained under previous culture conditions is lower than that of adult primary cardiomyocytes due to immaturity and a reliance on glucose metabolism. Therefore, the aims of this study were to compare the types of fatty acids metabolized between cardiomyocytes in culture and heart cells in vivo and investigate whether the addition of fatty acids to the culture medium affected energy production by cardiomyocytes., Methods: A fatty acid-containing medium was developed based on an analysis of fatty acid consumption by rat primary cardiomyocytes (rat-CMs), and the effects of this medium on adenosine triphosphate (ATP) production were investigated through bioluminescence imaging of luciferase-expressing rat-CMs. Next, the fatty acid content of the medium was further adjusted based on analyses of fatty acid utilization by porcine hearts and hiPSC-CMs. Oxygen consumption analyses were performed to explore whether the fatty acid-containing medium induced hiPSC-CMs to switch from anaerobic metabolism to aerobic metabolism. Furthermore, the effects of the medium on contractile force generated by hiPSC-CM-derived tissue were evaluated., Results: Rat serum, human serum and porcine plasma contained similar types of fatty acid (oleic acid, stearic acid, linoleic acid, palmitic acid and arachidonic acid). The types of fatty acid consumed were also similar between rat-CMs, hiPSC-CMs and porcine heart. The addition of fatty acids to the culture medium increased the bioluminescence of luciferase-expressing rat-CMs (an indirect measure of ATP level), oxygen consumption by hiPSC-CMs, and contractile force generated by cardiac tissues constructed from hiPSC-CMs., Conclusions: hiPSC-CMs metabolize similar types of fatty acid to those consumed by rat-CMs and porcine hearts. Furthermore, the addition of these fatty acids to the culture medium increased energy production by rat-CMs and hiPSC-CMs and enhanced the contractility of myocardial tissue generated from hiPSC-CMs. These findings suggest that the addition of fatty acids to the culture medium stimulates aerobic energy production by cardiomyocytes through β-oxidation. Since cardiomyocytes cultured in standard media rely primarily on anaerobic glucose metabolism and remain in an immature state, further research is merited to establish whether the addition of fatty acids to the culture medium would improve the energy-producing capacity and maturity of hiPSC-CMs and cardiac tissue constructed from these cells. It is possible that optimizing the metabolism of cultured cardiomyocytes, which require high energy production to sustain their contractile function, will improve the properties of hiPSC-CM-derived tissue, allowing it to be better utilized for disease modeling, drug screening and regenerative therapies for heart failure., Competing Interests: Tatsuya Shimizu is a shareholder of CellSeed Inc. Katsuhisa Matsuura and Tatsuya Shimizu are the inventors of bioreactor systems. Tokyo Women's Medical University and received research funding from CellSeed Inc. and Nihon Kohden Corporation. The other authors have no potential conflicts of interest., (© 2022 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

The centrosome lacks microtubule (MT)-nucleation activity in differentiated neurons. We have previously demonstrated that MTs were nucleated at the cytoplasm of mouse neurons. They are supposed to serve seeds for MTs required for dendrite growth. However, the factors that activate the cytoplasmic γ-tubulin ring complex (γTuRC) are unknown. Here we report an alternative splicing isoform of cyclin-dependent kinase 5 regulatory subunit-associated protein 2 (CKD5RAP2) as a candidate for the cytoplasmic γTuRC activator. This isoform lacked exon 17 and was expressed predominantly in the brain and testis. The expression was transient during the development of cortical neurons, which period coincided with the period we reported cytoplasmic MT nucleation. This isoform resulted in a frameshift and generated truncated protein without a centrosomal localization signal. When this isoform was expressed in cells, it localized diffusely in the cytoplasm. It was co-immunoprecipitated with γ-tubulin and MOZART2, suggesting that it can activate cytosolic γTuRCs. After cold-nocodazole depolymerization of MTs and subsequent washout, we observed numerous short MTs in the cytoplasm of cells transfected with the cDNA of this isoform. The isoform-overexpressing cells exhibited an increased amount of MTs and a decreased ratio of acetylated tubulin, suggesting that MT generation and turnover were enhanced by the isoform. Our data suggest the possibility that alternative splicing of CDK5RAP2 induces cytoplasmic nucleation of MTs in developing neurons., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article., (© 2022 The Authors.)

In most multispectral optical-resolution photoacoustic microscopy (OR-PAM), spatial scanning is repeated for each excitation wavelength, which decreases throughput and causes motion artifacts during spectral processing. This study proposes a new spectroscopic OR-PAM technique to acquire information on the photoacoustic signal intensity and excitation wavelength from single spatial scans. The technique involves irradiating an imaging target with two broadband optical pulses with and without wavelength-dependent time delays. The excitation wavelength of the sample is then calculated by measuring the time delay between the photoacoustic signals generated by the two optical pulses. This technique is validated by measuring the excitation wavelengths of dyes in tubes. Furthermore, we demonstrate the three-dimensional spectroscopic OR-PAM of cells stained with suitable dyes. Although the tradeoff between excitation efficiency and excitation bandwidth must be adjusted based on the application, combining the proposed technique with fast spatial scanning methods can significantly contribute to recent OR-PAM applications, such as monitoring quick biological events and microscale tracking of moving materials., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Introduction: Lung transplantation is the only effective treatment option for many patients with irreversible pulmonary injury, and the demand for lung transplantation is increasing worldwide and expected to continue to outstrip the number of available donors. Regenerative therapy with alveolar epithelial cells (AECs) holds promise as an alternative option to organ transplantation. AECs are usually co-cultured with mouse-derived 3T3 feeder cells, but the use of xenogeneic tissues for regenerative therapy raises safety concerns. Fabrication of AEC sheets under feeder-free conditions would avoid these safety issues. We describe a novel feeder-free method of fabricating AEC sheets that may be suitable for pulmonary regenerative therapy., Methods: Lung tissues excised from male outbred rats or transgenic rats expressing green fluorescent protein (GFP) were finely minced and dissociated with elastase. The isolated AECs were cultured under four different feeder-free conditions according to whether a rho kinase (ROCK) inhibitor was included in the low-calcium medium (LCM) and whether the tissue culture dish was coated with recombinant laminin-511 E8 fragment (rLN511E8). The expanded cells were cultured on temperature-responsive dishes and subsequently harvested as AEC sheets. Engraftment of GFP-AEC sheets after their transplantation onto a partially resected region of the left lung was assessed in athymic rats., Results: AECs proliferated and reached confluence when cultured in LCM containing a ROCK inhibitor on tissue culture dishes coated with rLN511E8. When both the ROCK inhibitor and rLN511E8-coated culture dish were used, the number of AECs obtained after 7 days of culture was significantly higher than that in the other three groups. Immunohistochemical analyses revealed that aquaporin-5, surfactant protein (SP)-A, SP-C, SP-D and Axin-2 were expressed by the cultured AECs. AEC sheets were harvested successfully from temperature-responsive culture dishes (by lowering the temperature) when the expanded AECs were cultured for 7 days in LCM + ROCK inhibitor and then for 3 days in LCM + ROCK inhibitor supplemented with 200 mg/L calcium chloride. The AEC sheets were firmly engrafted 7 days after transplantation onto the lung defect and expressed AEC marker proteins., Conclusions: AEC sheets fabricated under feeder-free conditions retained the features of AECs after transplantation onto the lung in vivo . Further improvement of this technique may allow the bioengineering of alveolar-like tissue for use in pulmonary regenerative therapy., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tatsuya Shimizu is a shareholder of CellSeed Inc. Tokyo Women's Medical University10.13039/501100010391Tokyo Women's Medical University received research funding from CellSeed Inc. The other authors have no competing interests to declare., (© 2022 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Sertoli cells (SeC) isolated from porcine testes have shown direct effects on muscle precursor cells sustaining C2C12 myoblasts proliferation and inhibiting oxidative stress and apoptosis in the early phase of the differentiation process, and stimulating myoblast fusion into myotubes and the expression of markers of myogenic differentiation in the late phase. This suggested that the cocktail of factors secreted by SeC stimulates proliferation in myoblasts without weakening their myogenic potential resulting in the formation of the critical myoblast amount necessary to rebuild the required muscle mass upon a damage. Here, we show that co-culturing C2C12 myoblasts with high doses of SeC microencapsulated in clinical grade alginate-based microcapsules (MC-SeC) for three days in differentiation medium (DM) translates into increased cell numbers and almost absence of myotube formation. However, after removal of MC-SeC, an intense fusion activity into myotubes was observed culminating in a fusion index similar to that of control after additional three days of culture in DM. These data definitely demonstrate that SeC-derived factors preserve the myogenic potential while sustaining cell proliferation in C2C12 myoblasts., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2021 The Authors. Published by Elsevier Inc.)

Polyethylene glycols (PEGs) in general use are polydisperse molecules with molecular weight (MW) distributed around an average value applied in their designation e.g. , PEG 4000. Previous research has shown that PEGs can act as P-glycoprotein (P-gp) inhibitors with the potential to affect the absorption and efflux of concomitantly administered drugs. However, questions related to the mechanism of cellular uptake of PEGs and the exact role played by P-gp has not been addressed. In this study, we examined the mechanism of uptake of PEGs by MDCK-mock cells, in particular, the effect of MW and interaction with P-gp by MDCK-hMDR1 and A549 cells. The results show that: (a) the uptake of PEGs by MDCK-hMDR1 cells is enhanced by P-gp inhibitors; (b) PEGs stimulate P-gp ATPase activity but to a much lesser extent than verapamil; and (c) uptake of PEGs of low MW (<2000 Da) occurs by passive diffusion whereas uptake of PEGs of high MW (>5000 Da) occurs by a combination of passive diffusion and caveolae-mediated endocytosis. These findings suggest that PEGs can engage in P-gp-based drug interactions which we believe should be taken into account when using PEGs as excipients and in PEGylated drugs and drug delivery systems., (© 2020 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Keratinocyte line cells HaCaT and FEPE1L-8 are used for skin model with type I collagen fibrils (gels). For this purpose, not only differentiation but also regulation of proliferation on type I collagen gels by exogenous calcium concentration is important. When exogenous calcium concentration is low, primary keratinocyte proliferation is repressed and eventually cells are induced to apoptosis on type I collagen gels. The apoptosis induced on type I collagen gels is suppressed by increasing calcium concentration in the medium. That is, higher exogenous calcium concentration is necessary for primary keratinocyte survival on type I collagen gels than for that on dish surface culture. Meanwhile much higher exogenous calcium causes cell differentiation and inhibition of proliferation. The optimal calcium concentrations for proliferation on type I collagen gels have not been clarified in keratinocyte line cells. HaCaT cells have a unique calcium sensitivity in comparison with primary keratinocytes, whereas FEPE1L-8 cells have a similar sensitivity to primary keratinocytes. In this study, we compared the effect of calcium concentrations on proliferation of HaCaT and FEPE1L-8 cells on type I collagen gels. On type I collagen gels, both line cells required higher calcium concentrations for proliferation than on dish surface. HaCaT cells proliferated better in a wider range of calcium concentrations than FEPE1L-8 cells.

Enrichment of tissues with ≥20-carbon n-3 PUFA like EPA is associated with positive cardiovascular outcomes. Stearidonic acid (SDA; 18 : 4n-3) and α-linolenic acid (ALA; 18 : 3n-3) are plant-derived dietary n-3 PUFA; however, direct comparisons of their impact on tissue n-3 PUFA content are lacking. Ahiflower(®) oil extracted from Buglossoides arvensis seeds is the richest known non-genetically modified source of dietary SDA. To investigate the safety and efficacy of dietary Ahiflower oil, a parallel-group, randomised, double-blind, comparator-controlled phase I clinical trial was performed. Diets of healthy subjects (n 40) were supplemented for 28 d with 9·1 g/d of Ahiflower (46 % ALA, 20 % SDA) or flax seed oil (59 % ALA). Blood and urine chemistries, blood lipid profiles, hepatic and renal function tests and haematology were measured as safety parameters. The fatty acid composition of fasting plasma, erythrocytes, polymorphonuclear cells and mononuclear cells were measured at baseline and after 14 and 28 d of supplementation. No clinically significant changes in safety parameters were measured in either group. Tissue ALA and EPA content increased in both groups compared with baseline, but EPA accrual in plasma and in all cell types was greater in the Ahiflower group (time × treatment interactions, P ≤ 0·01). Plasma and mononuclear cell eicosatetraenoic acid (20 : 4n-3) and docosapentaenoic acid (22 : 5n-3) content also increased significantly in the Ahiflower group compared with the flax group. In conclusion, the consumption of Ahiflower oil is safe and is more effective for the enrichment of tissues with 20- and 22-carbon n-3 PUFA than flax seed oil.