ABSTRACT
Transverse (t)-tubule remodelling is a prominent feature of heart failure with reduced ejection fraction (HFrEF). In our previous research, we identified an increased amount of collagen within the t-tubules of HFrEF patients, suggesting fibrosis could contribute to the remodelling of t-tubules. In this research, we tested this hypothesis in a rodent model of myocardial infarction induced heart failure that was treated with the anti-fibrotic pirfenidone. Confocal microscopy demonstrated loss of t-tubules within the border zone region of the infarct. This was documented as a reduction in t-tubule frequency, area, length, and transverse elements. Eight weeks of pirfenidone treatment was able to significantly increase the area and length of the t-tubules within the border zone. Echocardiography showed no improvement with pirfenidone treatment. Surprisingly, pirfenidone significantly increased the thickness of the t-tubules in the remote left ventricle of heart failure animals. Dilation of t-tubules is a common feature in heart failure suggesting this may negatively impact function but there was no functional loss associated with pirfenidone treatment. However, due to the relatively short duration of treatment compared to that used clinically, the impact of long-term treatment on t-tubule structure should be investigated in future studies.
ABSTRACT
The disrupted organisation of the ryanodine receptors (RyR) and junctophilin (JPH) is thought to underpin the transverse tubule (t-tubule) remodelling in a failing heart. Here, we assessed the nanoscale organisation of these two key proteins in the failing human heart. Recently, an advanced feature of the t-tubule remodelling identified large flattened t-tubules called t-sheets, that were several microns wide. Previously, we reported that in the failing heart, the dilated t-tubules up to ~1 µm wide had increased collagen, and we hypothesised that the t-sheets would also be associated with collagen deposits. Direct stochastic optical reconstruction microscopy (dSTORM), confocal microscopy, and western blotting were used to evaluate the cellular distribution of excitation-contraction structures in the cardiac myocytes from patients with idiopathic dilated cardiomyopathy (IDCM) compared to myocytes from the non-failing (NF) human heart. The dSTORM imaging of RyR and JPH found no difference in the colocalisation between IDCM and NF myocytes, but there was a higher colocalisation at the t-tubule and sarcolemma compared to the corbular regions. Western blots revealed no change in the JPH expression but did identify a ~50% downregulation of RyR (p = 0.02). The dSTORM imaging revealed a trend for the smaller t-tubular RyR clusters (~24%) and reduced the t-tubular RyR cluster density (~35%) that resulted in a 50% reduction of t-tubular RyR tetramers in the IDCM myocytes (p < 0.01). Confocal microscopy identified the t-sheets in all the IDCM hearts examined and found that they are associated with the reticular collagen fibres within the lumen. However, the size and density of the RyR clusters were similar in the myocyte regions associated with t-sheets and t-tubules. T-tubule remodelling is associated with a reduced RyR expression that may contribute to the reduced excitation-contraction coupling in the failing human heart.
ABSTRACT
Hyaluronan is a linear glycosaminoglycan that forms the backbone of perineuronal nets around neurons in the cerebral cortex. However, it remains controversial whether neurons are capable of independent hyaluronan synthesis. Herein, we examined the expression of hyaluronan and hyaluronan synthases (HASs) throughout cortical neuron development in vitro. Enriched cultures of cortical neurons were established from E16 rats. Neurons were collected at days in vitro (DIV) 0 (4 h), 1, 3, 7, 14, and 21 for qPCR or immunocytochemistry. In the relative absence of glia, neurons exhibited HAS1-3 mRNA at all time-points. By immunocytochemistry, puncta of HAS2-3 protein and hyaluronan were located on neuronal cell bodies, neurites, and lamellipodia/growth cones from as early as 4 h in culture. As neurons matured, hyaluronan was also detected on dendrites, filopodia, and axons, and around synapses. Percentages of hyaluronan-positive neurons increased with culture time to ~93% by DIV21, while only half of neurons at DIV21 expressed the perineuronal net marker Wisteria floribunda agglutinin. These data clearly demonstrate that neurons in vitro can independently synthesise hyaluronan throughout all maturational stages, and that hyaluronan production is not limited to neurons expressing perineuronal nets. The specific structural localisation of hyaluronan suggests potential roles in neuronal development and function.
Subject(s)
Cerebral Cortex/metabolism , Hyaluronic Acid/biosynthesis , Neurons/metabolism , Animals , Cells, Cultured , Cerebral Cortex/cytology , Neurons/cytology , RatsABSTRACT
Parkinson's disease (PD) is not only associated with degeneration of dopaminergic (DAergic) neurons in the Substantia Nigra, but also with profound loss of noradrenergic neurons in the Locus Coeruleus (LC). Remarkably, LC degeneration may exceed, or even precede the loss of nigral DAergic neurons, suggesting that LC neurons may be more susceptible to damage by various insults. Using a combination of electrophysiology, fluorescence imaging and electrochemistry, we directly compared the responses of LC, nigral DAergic and nigral non-dopaminergic (non-DAergic) neurons in rat brain slices to acute application of rotenone, a mitochondrial toxin used to create animal and in vitro models of PD. Rotenone (0.01-5.0µM) dose-dependently inhibited the firing of all three groups of neurons, primarily by activating KATP channels. The toxin also depolarised mitochondrial potential (Ψm) and released reactive oxygen species (H2O2). When KATP channels were blocked, rotenone (1µM) increased the firing of LC neurons by activating an inward current associated with dose-dependent increase of cytosolic free Ca2+ ([Ca2+]i). This effect was attenuated by blocking oxidative stress-sensitive TRPM2 channels, and by pre-treatment of slices with anti-oxidants. These results demonstrate that rotenone inhibits the activity of LC neurons mainly by activating KATP channels, and increases [Ca2+]ivia TRPM2 channels. Since the responses of LC neurons were smaller than those of nigral DAergic neurons, our study shows that LC neurons are paradoxically less sensitive to acute effects of this parkinsonian toxin.
Subject(s)
Action Potentials/drug effects , Insecticides/pharmacology , Locus Coeruleus/cytology , Neurons/drug effects , Pars Compacta/cytology , Rotenone/pharmacology , Animals , Animals, Newborn , Antihypertensive Agents/pharmacology , Calcium/metabolism , Diazoxide/pharmacology , Hydrogen Peroxide/metabolism , Hypoglycemic Agents/pharmacology , In Vitro Techniques , Membrane Potential, Mitochondrial/drug effects , Neurons/classification , Patch-Clamp Techniques , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , TRPM Cation Channels/metabolism , Tolbutamide/pharmacologyABSTRACT
Clara cell protein (CC16) is an anti-inflammatory protein, which is expressed in the airway epithelium. It is involved in the development of airway inflammatory diseases, including chronic obstructive pulmonary disease and asthma. However, the exact molecular mechanism underlying its antiinflammatory action remains to be fully elucidated. The aim of the present study was to define the protein profiles of the antiinflammatory effect of CC16 in lipopolysaccharide (LPS)treated rat tracheal epithelial (RTE) cells using shotgun proteomics. Protein extracts were obtained from control RTE cells, RTE cells treated with LPS and RTE cells treated with LPS and recombinant CC16 (rCC16). Subsequent labelfree quantification and bioinformatics analyses identified 12 proteins that were differentially expressed in the three treatment groups as a cluster of five distinct groups according to their molecular functions. Five of the twelve proteins were revealed to be associated with the cytoskeleton: Matrix metalloproteinase9, myosin heavy chain 10, actinrelated protein3 homolog, elongation factor 1α1 (EF1α1), and acidic ribosomal phosphoprotein P0. Five of the twelve proteins were associated with cellular proliferation: DNAdependent protein kinase catalytic subunit, EF1α1, tyrosine 3monooxygenase, caspase recruitment domain (CARD) protein 12 and adenosylhomocysteinase (SAHH) 3. Three proteins were associated with gene regulation: EF1α1, SAHH 3 and acidic ribosomal phosphoprotein P0. Three proteins were associated with inflammation: Tyrosine 3monooxygenase, CARD protein 12 and statinrelated protein. ATPase (H+transporting, V1 subunit A, isoform 1) was revealed to be associated with energy metabolism, and uridine diphosphate glycosyltransferase 1 family polypeptide A8 with drug metabolism and detoxification. The identified proteins were further validated using reverse transcriptionquantitative polymerase chain reaction. These protein profiles, and their interacting protein network, may facilitate the elucidation of the molecular mechanisms underlying the antiinflammatory effects of CC16.
Subject(s)
Epithelial Cells/metabolism , Inflammation/genetics , Recombinant Proteins/genetics , Trachea/metabolism , Uteroglobin/genetics , Animals , Chromatography, Liquid , Epithelial Cells/pathology , Humans , Inflammation/chemically induced , Inflammation/metabolism , Inflammation/pathology , Lipopolysaccharides/toxicity , Protein Biosynthesis/genetics , Proteomics , Pulmonary Disease, Chronic Obstructive , Rats , Recombinant Proteins/administration & dosage , Recombinant Proteins/metabolism , Tandem Mass Spectrometry , Trachea/pathology , Uteroglobin/administration & dosage , Uteroglobin/metabolismABSTRACT
Toxoplasmosis is one of the most widespread zoonoses worldwide. It has a high incidence and can result in severe disease in humans and livestock. Effective vaccines are needed to limit and prevent infection with Toxoplasma gondii. In this study, we evaluated the immuno-protective efficacy of a recombinant Toxoplasma gondii phosphoglycerate mutase 2 (rTgPGAM 2) against T. gondii infection in BALB/c mice. We report that the mice nasally immunised with rTgPGAM 2 displayed significantly higher levels of special IgG antibodies against rTgPGAM 2 (including IgG1, IgG2a and IgAs) and cytokines (including IFN-γ, IL-2 and IL-4) in their blood sera and supernatant of cultured spleen cells compared to those of control animals. In addition, an increased number of spleen lymphocytes and enhanced lymphocyte proliferative responses were observed in the rTgPGAM 2-immunised mice. After chronic infection and lethal challenge with the highly virulent T. gondii RH strain by oral gavage, the survival time of the rTgPGAM 2-immunised mice was longer (P < 0.01) and the survival rate (70%) was higher compared with the control mice (P < 0.01). The reduction rate of brain and liver tachyzoites in rTgPGAM 2-vaccinated mice reached approximately 57% and 69% compared with those of the control mice (P < 0.01). These results suggest that rTgPGAM 2 can generate protective immunity against T. gondii infection in BALB/c mice and may be a promising antigen in the further development of an effective vaccine against T. gondii infection.
Subject(s)
Phosphoglycerate Mutase/immunology , Protozoan Proteins/immunology , Protozoan Vaccines/immunology , Toxoplasma/immunology , Toxoplasmosis, Animal/prevention & control , Administration, Intranasal , Animals , Brain/parasitology , Cells, Cultured , Cytokines/blood , Drug Evaluation , Female , Immunity, Mucosal , Immunoglobulin A, Secretory/analysis , Immunoglobulin G/blood , Liver/parasitology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Peyer's Patches/immunology , Phosphoglycerate Mutase/genetics , Protozoan Proteins/genetics , Protozoan Vaccines/administration & dosage , Recombinant Proteins/immunology , Spleen/immunology , Toxoplasma/enzymology , Toxoplasma/genetics , Toxoplasma/growth & development , Toxoplasmosis, Animal/blood , Toxoplasmosis, Animal/immunology , Toxoplasmosis, Animal/parasitology , Vaccination , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunologyABSTRACT
Toxoplasma gondii is an obligate intracellular apicomplexan parasite that affects humans and various vertebrate livestock and causes serious economic losses. To develop an effective vaccine against T. gondii infection, we constructed a DNA vaccine encoding the T. gondii rhoptry protein 17 (TgROP17) and evaluated its immune protective efficacy against acute T. gondii infection in mice. The DNA vaccine (p3×Flag-CMV-14-ROP17) was intramuscularly injected to BALB/c mice and the immune responses of the vaccinated mice were determined. Compared to control mice treated with empty vector or PBS, mice immunized with the ROP17 vaccine showed a relatively high level of specific anti-T. gondii antibodies, and a mixed IgG1/IgG2a response with predominance of IgG2a production. The immunized mice also displayed a specific lymphocyte proliferative response, a Th1-type cellular immune response with production of IFN-γ and interleukin-2, and increased number of CD8(+) T cells. Immunization with the ROP17 DNA significantly prolonged the survival time (15.6 ± 5.4 days, P < 0.05) of mice after challenge infection with the virulent T. gondii RH strain (Type I), compared with the control groups which died within 8 days. Therefore, our data suggest that DNA vaccination with TgROP17 triggers significant humoral and cellular responses and induces effective protection in mice against acute T. gondii infection, indicating that TgROP17 is a promising vaccine candidate against acute toxoplasmosis.
Subject(s)
DNA, Protozoan/genetics , Protozoan Proteins/immunology , Toxoplasma/immunology , Toxoplasmosis, Animal/prevention & control , Vaccines, DNA/immunology , Virulence Factors/immunology , Animals , Antibodies, Protozoan/blood , CD8-Positive T-Lymphocytes/immunology , Cytokines/metabolism , DNA, Protozoan/immunology , DNA, Recombinant/genetics , DNA, Recombinant/immunology , Female , Immunoglobulin G/blood , Injections, Intramuscular , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Protozoan Proteins/genetics , Th1 Cells/immunology , Toxoplasmosis, Animal/immunology , Vaccination , Vaccines, Synthetic/immunology , Virulence Factors/geneticsABSTRACT
Clara cell protein (CC16) is a well-known anti-inflammatory protein secreted by the epithelial Clara cells of the airways. It is involved in the development of airway inflammatory diseases such as chronic obstructive pulmonary disease and asthma. Previous studies suggest that CC16 gene transfer suppresses expression of interleukin (IL)-8 in bronchial epithelial cells. However, its role in the function of these cells during inflammation is not well understood. In this study, we evaluated the effect of CC16 on the expression of matrix metalloproteinase (MMP)-9 in lipopolysaccharide (LPS)-stimulated rat tracheal epithelial cells and its underlying molecular mechanisms. We generated recombinant rat CC16 protein (rCC16) which was bioactive in inhibiting the activity of phospholipase A2. rCC16 inhibited LPS-induced MMP-9 expression at both mRNA and protein levels in a concentration-dependent (0-2 µg/mL) manner, as demonstrated by real time RT-PCR, ELISA, and zymography assays. Gene transcription and DNA binding studies demonstrated that rCC16 suppressed LPS-induced NF-κB activation and its binding of gene promoters as identified by luciferase reporter and gel mobility shift assays, respectively. Western blotting and immunofluorescence staining analyses further revealed that rCC16 concentration dependently inhibited the effects of LPS on nuclear increase and cytosol reduction of NF-κB, on the phosphorylation and reduction of NF-κB inhibitory IκBα, and on p38 MAPK-dependent NF-κB activation by phosphorylation at Ser276 of its p65 subunit. These data indicate that inhibition of LPS-mediated NF-κB activation by rCC16 involves both translocation- and phosphorylation-dependent signaling pathways. When the tracheal epithelial cells were pretreated with chlorpromazine, an inhibitor of clathrin-mediated endocytosis, cellular uptake of rCC16 and its inhibition of LPS-induced NF-κB nuclear translocation and also MMP-9 production were significantly abolished. Taken together, our data suggest that clathrin-mediated uptake of rCC16 suppresses LPS-mediated inflammatory MMP-9 production through inactivation of NF-κB and p38 MAPK pathways in tracheal epithelial cells.
Subject(s)
Lipopolysaccharides/pharmacology , Matrix Metalloproteinase 9/biosynthesis , NF-kappa B/metabolism , Uteroglobin/pharmacology , Animals , Cell Line , Clathrin-Coated Vesicles/metabolism , Endocytosis , Epithelial Cells/metabolism , I-kappa B Proteins , NF-KappaB Inhibitor alpha , Phospholipases A2/metabolism , Phosphorylation , RNA, Messenger/metabolism , Rats , Recombinant Proteins , Signal Transduction , Trachea/cytology , Trachea/metabolism , Uteroglobin/genetics , Uteroglobin/physiology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
OBJECTIVE: Although the spectrum of white matter injury (WMI) in preterm infants is shifting from cystic necrotic lesions to milder forms, the factors that contribute to this changing spectrum are unclear. We hypothesized that recurrent hypoxia-ischemia (rHI) will exacerbate the spectrum of WMI defined by markers of inflammation and molecules related to the extracellular matrix (hyaluronan (HA) and the PH20 hyaluronidase) that regulate maturation of the oligodendrocyte (OL) lineage after WMI. METHODS: We employed a preterm fetal sheep model of in utero moderate hypoxemia and global severe but not complete cerebral ischemia that reproduces the spectrum of human WMI. The response to rHI was compared against corresponding early or later single episodes of HI. An ordinal rating scale of WMI was compared against an unbiased quantitative image analysis protocol that provided continuous histo-pathological outcome measures for astrogliosis and microglial activation. Late oligodendrocyte progenitors (preOLs) were quantified by stereology. Analysis of hyaluronan and the hyaluronidase PH20 defined the progressive response of the extracellular matrix to WMI. RESULTS: rHI resulted in a more severe spectrum of WMI with a greater burden of necrosis, but an expanded population of preOLs that displayed reduced susceptibility to cell death. WMI from single episodes of HI or rHI was accompanied by elevated HA levels and increased labeling for PH20. Expression of PH20 in fetal ovine WMI was confirmed by RT-PCR and RNA-sequencing. CONCLUSIONS: rHI is associated with an increased risk for more severe WMI with necrosis, but reduced risk for preOL degeneration compared to single episodes of HI. Expansion of the preOL pool may be linked to elevated hyaluronan and PH20.
Subject(s)
Hypoxia-Ischemia, Brain/pathology , White Matter/injuries , White Matter/pathology , Animals , Animals, Newborn , Cell Adhesion Molecules/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Female , Fetus/metabolism , Fetus/pathology , Hyaluronic Acid/metabolism , Hyaluronoglucosaminidase/metabolism , Hypoxia-Ischemia, Brain/metabolism , Necrosis/metabolism , Necrosis/pathology , Oligodendroglia/metabolism , Oligodendroglia/pathology , SheepABSTRACT
Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that infects a variety of mammals, including humans. An effective vaccine for this parasite is therefore needed. In this study, RH strain T. gondii rhoptry protein 17 was expressed in bacteria as a fusion with glutathione S-transferase (GST) and the recombinant proteins (rTgROP17) were purified via GST-affinity chromatography. BALB/c mice were nasally immunised with rTgROP17, and induction of immune responses and protection against chronic and lethal T. gondii infections were investigated. The results revealed that mice immunised with rTgROP17 produced high levels of specific anti-rTgROP17 IgGs and a mixed IgG1/IgG2a response of IgG2a predominance. The systemic immune response was associated with increased production of Th1 (IFN-γand IL-2) and Th2 (IL-4) cytokines, and enhanced lymphoproliferation (stimulation index, SI) in the mice immunised with rTgROP17. Strong mucosal immune responses with increased secretion of TgROP17-specific secretory IgA (SIgA) in nasal, vaginal and intestinal washes were also observed in these mice. The vaccinated mice displayed apparent protection against chronic RH strain infection as evidenced by their lower liver and brain parasite burdens (59.17% and 49.08%, respectively) than those of the controls. The vaccinated mice also exhibited significant protection against lethal infection of the virulent RH strain (survival increased by 50%) compared to the controls. Our data demonstrate that rTgROP17 can trigger strong systemic and mucosal immune responses against T. gondii and that ROP17 is a promising candidate vaccine for toxoplasmosis.
Subject(s)
Antigens, Protozoan/immunology , Protozoan Proteins/immunology , Recombinant Proteins/immunology , Toxoplasma/immunology , Toxoplasmosis, Animal/prevention & control , Administration, Intranasal , Animals , Antibodies, Protozoan/immunology , Antigens, Protozoan/administration & dosage , Cytokines/metabolism , Female , Immunization , Immunoglobulin A/immunology , Immunoglobulin G/immunology , Lymphocyte Activation/immunology , Lymphocytes/immunology , Lymphocytes/metabolism , Mice , Protozoan Proteins/administration & dosage , Recombinant Proteins/administration & dosage , Toxoplasmosis, Animal/parasitologyABSTRACT
Nasal vaccination is an effective therapeutic regimen for preventing certain infectious diseases. The mucosal immune response is important for resistance to Toxoplasma gondii infection. In this study, we evaluated the immune responses elicited in BALB/c mice by nasal immunisation with recombinant T. gondii receptor for activated C kinase 1 (rTgRACK1) and their protective efficacy against T. gondii RH strain during both chronic and lethal infections. Nasal vaccination with rTgRACK1 increased the level of secretory IgA in nasal, intestinal and vesical washes, and the level of IFN-γ and IL-2 in intestinal washes, indicating that rTgRACK1 vaccination promotes mucosal immune responses. The mice immunised with rTgRACK1 also displayed increased levels of rTgRACK1-specific IgA, total IgG, IgG1 and in particular IgG2a in their blood sera, increased production of IFN-γ, IL-2 and IL-4 but not IL-10 from their isolated spleen cells, and enhanced splenocyte proliferation in vitro. rTgRACK1-vaccinated mice were effectively protected against infection with T. gondii RH strain, showing over 50% reduction of tachyzoite burdens in their liver and brain tissues during a chronic infection, and also a 45% increase in their survivals during a lethal challenge. These results indicate that rTgRACK1 might represent an intriguing immunogen for developing a mucosal vaccine against toxoplasmosis.
Subject(s)
Antigens, Protozoan/immunology , Protozoan Vaccines/immunology , Receptors, Cell Surface/immunology , Toxoplasma/immunology , Toxoplasmosis/prevention & control , Administration, Intranasal , Animals , Antigens, Protozoan/genetics , Cell Proliferation , Cytokines/metabolism , Disease Models, Animal , Female , Immunity, Mucosal , Immunoglobulin A, Secretory/analysis , Immunoglobulin G/blood , Leukocytes, Mononuclear/immunology , Mice, Inbred BALB C , Protozoan Vaccines/administration & dosage , Protozoan Vaccines/genetics , Receptors for Activated C Kinase , Receptors, Cell Surface/genetics , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Spleen/immunology , Survival Analysis , Toxoplasmosis/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/genetics , Vaccines, Synthetic/immunologyABSTRACT
Previous studies suggested that amyloid ß (Aß)-induced disruption of astrocytic Ca(2+) signalling and oxidative stress play a major role in the progression towards neuronal and glial death in Alzheimer's disease. We have recently demonstrated that Ca(2+)-permeable TRPV4 channels are highly expressed in rat hippocampal astrocytes and are involved in oxidative stress-induced cell damage. The aim of this study was to test the hypothesis that TRPV4 channels also contribute to hippocampal damage evoked by Aß. Synthetic Aß40 evoked cell death in hippocampal slice cultures in a concentration (0-20µM) and time (12-48h) dependent manner, after cultures were preconditioned with sublethal concentration of buthionine sulfoximine (1.5µM) which enhanced endogenous ROS production. As demonstrated by propidium iodide fluorescence, damage was observed in the granule cell layer of the dentate gyrus and to a smaller degree in pyramidal neurons of the CA1-CA3 region, as well as in glia cells mainly at the edge of the slice. Immunocytochemistry revealed an altered pattern of TRPV4 and GFAP protein expression, and reactive astrogliosis surrounding pyramidal CA1-CA3 neurons. Neuronal and astrocytic damage was attenuated by the antioxidant Trolox, TRPV4 channel blockers Gd(3+) and ruthenium red (RR), and a specific inhibitor of the redox and Ca(2+)-sensitive phospholipase A2 enzyme (MAFP). In disassociated co-cultures of hippocampal neurons and astrocytes without BSO preconditioning, Aß40 evoked pronounced neuronal damage, enhanced the expression of TRPV4 and GFAP proteins (indicative of reactive astrogliosis), and increased intracellular free Ca(2+) concentration in astrocytes. The latter effect was attenuated by RR and in Ca(2+)-free media. These data show that Aß40 can activate astrocytic TRPV4 channels in the hippocampus, leading to neuronal and astrocytic damage in a Ca(2+) and oxidative stress-dependent manner.
Subject(s)
Amyloid beta-Peptides/pharmacology , Astrocytes/drug effects , Calcium Signaling/drug effects , Hippocampus/cytology , Neurons/drug effects , Peptide Fragments/pharmacology , TRPV Cation Channels/metabolism , Animals , Animals, Newborn , Antioxidants/pharmacology , Arachidonic Acids/pharmacology , Cadmium Chloride/pharmacology , Cell Death/drug effects , Cells, Cultured , Chromans/pharmacology , Coculture Techniques , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Organ Culture Techniques , Organophosphonates/pharmacology , Rats , Rats, Wistar , Time FactorsABSTRACT
The conventional view of the mode of action of bisphosphonates is that they are taken up by bone surfaces and then ingested by bone-resorbing osteoclasts, the activity of which they inhibit through their actions on the enzyme, farnesyl pyrophosphate (FPP) synthase. This model suggests that these compounds should only have effects on osteoclasts, and does not provide an explanation for their other actions, such as the epithelial abnormalities seen in osteonecrosis of the jaw, and their possible prolongation of disease-free survival in some malignancies. The present studies set out to determine whether cells other than osteoclasts are affected by bone-bound bisphosphonates. Bone slices were incubated overnight in PBS or in solutions of bisphosphonates (100 µM), washed, then transferred to 96-well plates (1 slice/well). Cells from 2 cell lines were seeded onto the bone slices: Caco-2 human colorectal adenocarcinoma epithelial cells and Chinese hamster ovary (CHO) cells. Cell proliferation (cell numbers and thymidine incorporation) was assessed at 4-72 h. Cell adhesion at 4 h was normal on bone slices pre-treated with bisphosphonates, but there were progressive reductions in cell numbers from 48 h and even greater reductions in thymidine incorporation from 24 h (>90% with zoledronate at 72 h). Growth inhibition was related to the clinical potency of the bisphosphonate used. There was no evidence of increased apoptosis in cells grown on bisphosphonate-coated bone, but levels of unprenylated Rap1A were increased, indicating inhibition of FPP synthase. Similar growth inhibition was observed in primary cultures of rat osteoblasts on bone, indicating that this was not specific to transformed cells. It is concluded that bisphosphonates bound to a bone surface can act on adjacent non-bone cells and inhibit their growth. This greatly widens the range of potential target cells for these drugs.
Subject(s)
Bone and Bones/drug effects , Bone and Bones/metabolism , Diphosphonates/pharmacology , Animals , Blotting, Western , CHO Cells , Caspase 3/metabolism , Cattle , Cell Adhesion/drug effects , Cell Count , Cell Line , Cell Proliferation/drug effects , Cricetinae , Cricetulus , Humans , In Vitro Techniques , Osteoblasts/cytology , Osteoblasts/drug effects , Prenylation/drug effects , Rats , Thymidine/metabolism , Time FactorsABSTRACT
BACKGROUND: Survivin is a member of the inhibitor-of-apoptosis (IAP) family which is widely expressed by many different cancers. Overexpression of survivin is associated with drug resistance in cancer cells, and reduced patient survival after chemotherapy and radiotherapy. Agents that antagonize the function of survivin hold promise for treating many forms of cancer. The purpose of this study was to investigate whether a cell-permeable dominant-negative survivin protein would demonstrate bioactivity against prostate and cervical cancer cells grown in three dimensional culture. RESULTS: A dominant-negative survivin (C84A) protein fused to the cell penetrating peptide poly-arginine (R9) was expressed in E. coli and purified by affinity chromatography. Western blot analysis revealed that dNSurR9-C84A penetrated into 3D-cultured HeLa and DU145 cancer cells, and a cell viability assay revealed it induced cancer cell death. It increased the activities of caspase-9 and caspase-3, and rendered DU145 cells sensitive to TNF-α via by a mechanism involving activation of caspase-8. CONCLUSIONS: The results demonstrate that antagonism of survivin function triggers the apoptosis of prostate and cervical cancer cells grown in 3D culture. It renders cancer cells sensitive to the proapoptotic affects of TNF-α, suggesting that survivin blocks the extrinsic pathway of apoptosis. Combination of the biologically active dNSurR9-C84A protein or other survivin antagonists with TNF-α therapy warrants consideration as an approach to cancer therapy.
ABSTRACT
A reliable model system of epileptiform insult would facilitate investigation into the underlying biological mechanisms. Epileptiform insult was induced in hippocampal slice cultures by lowering extracellular Mg(2+), (+)-bicuculline, or (-)-bicuculline methochloride, a stable salt form of bicuculline (both forms block GABA(A) receptors). Cell death was assessed by propidium iodide uptake. Low Mg(2+) or (+)-bicuculline did not produce cell death regardless of dose or incubation period. Exposure to 100 microM (-)-bicuculline methochloride for 48 hr resulted in prominent CA1 cell death. These findings demonstrate that not all pro-epileptic drugs/ion changes used routinely for electrophysiological recording of seizure activity lead to cell death in hippocampal slice cultures and that treatment with bicuculline methochloride can be used as a reliable model for epileptiform insult.
Subject(s)
Bicuculline/toxicity , Hippocampus/metabolism , Hippocampus/pathology , Magnesium/metabolism , Animals , Animals, Newborn , Cell Death/drug effects , Cell Death/physiology , Convulsants/toxicity , Hippocampus/drug effects , Organ Culture Techniques , Rats , Rats, WistarABSTRACT
TRPM2 and TPPV4 channels, two members of TRP channel family, are known to be widely expressed in the brain but their exact expression pattern and function are not well understood. Due to their high Ca(2+) permeability and gating by reactive oxygen species (TRPM2), or cell swelling, low pH and high temperature (TRPV4), they are likely to be involved in cell damage associated with various brain pathologies. The aim of this study was to investigate the expression of these channels and their potential role in oxidative stress-induced cell damage in organotypic hippocampal slice cultures, a model that retains the complex interaction between neurons and astrocytes. Channel expression was confirmed with RT-PCR and western blotting, while immunocytochemistry demonstrated TRPM2 in CA1-CA3 pyramidal neurons and TRPV4 in astrocytes. Oxidative stress induced by exogenous application of H(2)O(2) (600 microM) caused preferential damage of pyramidal neurons, while oxidative stress evoked with mercaptosuccinate (MCS; 400 microM) or buthionine sulfoximine (BSO; 4 microM) mainly damaged astrocytes, as identified by propidium iodide fluorescence. Antioxidants (Trolox 500 microM; MitoE 2 microM) reduced both neuronal and astrocytic cell death. Blockers of TRPV4 channels (Gd(3+) 500 microM; Ruthenium red 1 microM) increased the viability of astrocytes following MCS or BSO treatments, consistent with the expression pattern of these channels. Blockers of TRPM2 channels clotrimazole (20 microM), N-(p-amylcinnomoyl)anthranilic acid (ACA, 25 microM) or flufenamic acid (FFA, 200 microM) failed to protect pyramidal neurons from damage caused by exogenous H(2)O(2), and increased damage of these neurons caused by MCS and BSO. The differential expression of stress-sensitive TRPM2 and TRPV4 channels in hippocampal neurons and astrocytes that show distinct differences in vulnerability to different forms of oxidative stress suggests the specific involvement of these channels in oxidative stress-induced cell damage. However, the exact relationship between TRPM2 channel activation and cell death still remains to be determined due to the lack of protective effects of TRPM2 channel blockers.
Subject(s)
Cell Death/physiology , Hippocampus/metabolism , Oxidative Stress/physiology , TRPM Cation Channels/metabolism , TRPV Cation Channels/metabolism , Animals , Antioxidants/pharmacology , Astrocytes/metabolism , Cell Culture Techniques , Cell Death/drug effects , Hippocampus/drug effects , Oxidative Stress/drug effects , Pyramidal Cells/metabolism , Rats , TRPM Cation Channels/antagonists & inhibitors , TRPV Cation Channels/antagonists & inhibitorsABSTRACT
Amyloid deposition is a common feature of Alzheimer's disease and type 2 diabetes related to beta-amyloid peptides (betaA) and human amylin (hA), respectively. Both betaA and hA form aggregates and fibrils and kill cultured cells. To investigate whether betaA and hA display peptide-specific toxicity on cultured islet beta-cells, we examined the effects of (1-40)betaA and (25-35)betaA peptides on hA-mediated cell death and [(125)I-Tyr(37)]hA precipitation. Synthetic hA aggregated in solution and evoked both conformation- and sequence-dependent cell death. While neither (1-40)betaA nor (25-35)betaA was toxic to islet beta-cells, they suppressed hA-evoked cell death in a concentration-dependent and saturable manner. Only (1-40)betaA, but not (25-35)betaA, showed trophic effects on cultured islet beta-cells and inhibited the precipitation of [(125)I]hA caused by hA. These results suggest that (25-35)betaA does not interfere with hA-mediated fibril formation. Suppression of hA-evoked death of cultured pancreatic islet beta-cells by the betaA peptides is likely to occur through a competing interaction at these cells.
Subject(s)
Amyloid beta-Peptides/pharmacology , Amyloid/metabolism , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Peptide Fragments/pharmacology , Amyloid/pharmacology , Animals , Cell Death/drug effects , Cell Line , Cell Survival/drug effects , Humans , Islet Amyloid Polypeptide , Peptides/pharmacology , RatsABSTRACT
Expression of the canine 180-kDa ribosome receptor p180 in yeast induces the synthesis of RER, and increases the mRNAs of secretory pathway proteins, and protein secretion. To assess whether p180 is a master regulator of cell secretion in mammalian cells, we stably expressed red fluorescent forms of the human p180 variants p180DeltaR (no tandem repeats), p180R (26 repeats), and full-length p180FR (54 repeats) containing different lengths of the tandem repeat ribosome-binding domain in rat pancreatic RINm5F islet beta-cells. All three fluorescent p180 variants localized exclusively to the RER. Cells transfected with p180R were filled with ribosome-studded karmellae, whereas p180DeltaR and p180FR transfectants contained only increased amounts of mostly smooth ER. Unlike in yeast, over-expression of p180R failed to increase the secretory pathway proteins calnexin, SEC61beta, and calreticulin, or ribosome biogenesis. The data suggest that alternative splicing of the p180 tandem repeat domain is a means of regulating the ribosome-binding activity of p180, and potentially the secretory activity of the cell. However, p180 is not a master regulator of mammalian cell secretion as it does not concomitantly trigger the synthesis of protein machinery required to enhance protein synthesis and cell secretion.
Subject(s)
Alternative Splicing , Endoplasmic Reticulum, Rough/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Animals , Cell Line , Epithelial Cells/metabolism , Gene Expression , Green Fluorescent Proteins , Humans , Insulin-Secreting Cells/metabolism , Protein Transport/genetics , Rats , Ribosomes/metabolism , Secretory Vesicles/metabolism , Tandem Repeat SequencesABSTRACT
Kinectin (KNT) is a candidate membrane receptor for kinesin in the movement of intracellular organelles along microtubules. Isoforms of KNT exist containing different combinations of six small (residues 23-33) variable domains (vd) vd1-6 within the C-terminus. Here we investigate a role for KNT and its isoform KNTvd4(-) in the transport of amylin and insulin-containing secretory vesicles in the pancreatic islet beta-cell line RINm5F. KNTvd4(-) lacks vd4 that forms the kinesin-binding domain, and hence its role in the cell is an enigma. We report that amylin-containing vesicles also contained insulin, and exhibited microtubule, and small G-protein-dependent secretion. Knockdown of KNT by small interference RNA (siRNA) inhibited amylin expression and secretion. In contrast, recombinant KNTvd4(-) overexpressed in RINm5F cells associated with amylin-containing vesicles and inhibited amylin secretion, but had no discernible affect on amylin expression. The data suggests that both KNT and KNTvd4(-) participate in microtubule-dependent secretion of amylin in islet beta-cells.