BpV (phen) induces apoptosis of RINm5F cells by modulation of MAPKs and MKP-1

We investigated the mechanism of toxicity of peroxovanadium complex bpV (phen) in RINm5F cells. Treatment with bpV (phen) provoked cell death, predominantly by apoptosis. This compound induced strong and sustained JNK and p38 MAPK activation. However, ERK phosphorylation was not affected. The level of expression of MAPK phosphatase MKP-1 was suppressed after bpV (phen) treatment. In addition, this compound did not stimulate proteolytic processing of procaspase-3, suggesting that caspase-3 is not activated during the course of bpV (phen)-induced apoptosis. A correlative inhibition of JNK activation by immunosuppressive drug FK 506 induced ERK activation and MKP-1 expression, and suppressed RINm5F cell death. A specific p38 inhibitor SB 203580 also stimulated ERK activation and cell survival. Furthermore, simultaneous pretreatment with both FK 506 and SB 203580 almost completely abolished cell death. Thus, our results suggest that stress kinases and MKP-1 have a role in bpV (phen)-induced apoptosis of RINm5F cells.     

A novel streptomycete lipase: cloning,sequencing and high-level expression of the Streptomyces rimosus GDS(L)-lipase gene

An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.     

Inhibition of high affinity choline uptake in the rat brain by neurotoxins: effect of monosialoganglioside GM1.

Mustard derivatives of ethyl-choline and hemicholinium-3 have been suggested as possible specific cholinergic neurotoxins. In this study a structural analog of hemicholinium-3, a,a'-bis[di(2-chloroethyl)amino]-4,4'-2-biacetophenone (toxin 7), was added to synaptosomes prepared from the cortex, striatum or hippocampus of rat brain. Synaptosomal high affinity choline uptake (HACU) was significantly decreased in a dose-dependent manner by addition of toxin 7, while synaptosomal uptake of GABA or dopamine was not changed. Incubation of cortical synaptosomes with the monosialoganglioside GM1 prevented the decrease in HACU seen following administration of toxin 7. This preventative effect of GM1 was greater if GM1 was added prior to or concomitant with toxin 7, than if GM1 was added following toxin 7. Two newly synthesized hemicholinium-3 analogs, 4-[3'-di(2-chloroethyl)aminopropionyl]biphenyl (toxin 5) and 4-[3'-di(2-bromoethyl)aminopropionyl]biphenyl (toxin 6) caused a large decrease in HACU when added to cortical synaptosomes, this decrease was significantly greater than that seen with the same dose of toxin 7 or ethyl-choline aziridinium (AF64A). Ultrastructural changes in the synaptosomal membrane following incubation with toxin 7 or toxin 7 with GM1 were examined by electron microscopy. Development of a compound which is both a potent neurotoxin, and is specific for cholinergic neurons will allow new insights into the normal function of the cholinergic system in the CNS and provide animal models of disease states in which cholinergic degeneration is an important element.     

Type 2 diabetes is associated with suppression of autophagy and lipid accumulation in β‐cells

Both type 2 diabetes (T2D) and obesity are characterized by excessive hyperlipidaemia and subsequent lipid droplet (LD) accumulation in adipose tissue. To investigate whether LDs also accumulate in β‐cells of T2D patients, we assessed the expression of PLIN2, a LD‐associated protein, in non‐diabetic (ND) and T2D pancreata. We observed an up‐regulation of PLIN2 mRNA and protein in β‐cells of T2D patients, along with significant changes in the expression of lipid metabolism, apoptosis and oxidative stress genes. The increased LD buildup in T2D β‐cells was accompanied by inhibition of nuclear translocation of TFEB, a master regulator of autophagy and by down‐regulation of lysosomal biomarker LAMP2. To investigate whether LD accumulation and autophagy were influenced by diabetic conditions, we used rat INS‐1 cells to model the effects of hyperglycaemia and hyperlipidaemia on autophagy and metabolic gene expression. Consistent with human tissue, both LD formation and PLIN2 expression were enhanced in INS‐1 cells under hyperglycaemia, whereas TFEB activation and autophagy gene expression were significantly reduced. Collectively, these results suggest that lipid clearance and overall homeostasis is markedly disrupted in β‐cells under hyperglycaemic conditions and interventions ameliorating lipid clearance could be beneficial in reducing functional impairments in islets caused by glucolipotoxicity.     

Dynamics and regulation of lipid droplet formation in lipopolysaccharide (LPS)-stimulated microglia

Lipid droplets (LDs) are neutral lipid-rich organelles involved in many cellular processes. A well-known example is their accumulation in leukocytes upon activation by pro-inflammatory stimuli such as lipopolysaccharides (LPS) derived from gram-negative bacteria. A role of LDs and LD-associated proteins during inflammation in the brain is unknown, however. We have now studied their dynamics and regulation in microglia, the resident immune cells in the brain. We find that LPS treatment of microglia leads to the accumulation in them of LDs, and enhancement of the size of LDs. This induction of LDs was abolished by triacsin C, an inhibitor of triglyceride biosynthesis. LPS strongly activated c-Jun N-terminal kinase (JNK) and p38 MAPK stress signaling pathways and increased the expression of LD-associated protein perilipin-2 (ADRP) in a time-dependent manner. Immunostaining showed that perilipin-2 in LPS-treated microglia predominantly colocalized with LDs. Inhibitors of p38 α/β (SB203580) and PI3K/Akt pathway (LY294002), but not that of JNK (SP600125), reduced LPS-induced LD accumulation and eliminated the activating effect of LPS on perilipin-2. In addition, cytosolic phospholipase A₂ (cPLA₂-α), a key enzyme for arachidonic acid release, colocalized with LPS-induced LDs. These observations suggest that LDs may play an important role in eicosanoid synthesis in activated microglia; they provide a novel insight into the regulation of LDs in inflammatory cells of the brain and point to a potential role of p38 α/β in LPS-induced LD accumulation. Collectively, our findings imply that LD formation and perilipin-2 induction could be microglial biomarkers of inflammation in the central nervous system.     

Amylin-induced cytotoxicity is associated with activation of caspase-3 and MAP kinases

Nanomolar concentrations of human amylin promote death of RINm5F cells in a time- and concentrationdependent manner. Morphological changes of chromatin integrity suggest that cells are predominantly undergoing apoptosis. Human amylin induces significant activation of caspase-3 and strong and sustained phosphorylation of stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, that precedes cell death. Extracellular signal-regulated kinase (ERK) activation was not concomitant with JNK and/or p38 activation. Activation of caspase-3 and mitogen-activated protein kinases (MAPKs) was detected by Western blot analysis. Addition of the MEK1 inhibitor PD 98059 had no effect on amylin-induced apoptosis, suggesting that ERK activation does not play a role in this apoptotic scenario. A correlative inhibition of JNK activation by the immunosuppressive drug FK506, as well as a selective inhibition of p38 MAPK activation by SB 203580, significantly suppressed procaspase-3 processing and the extent of amylin-induced cell death. Moreover, simultaneous pretreatment with both FK506 and SB 203580, or with the caspase-3 inhibitor Ac-DEVD-CHO alone, almost completely abolished procaspase-3 processing and cell death. Thus, our results suggest that amylin-induced apoptosis proceeds through sustained activation of JNK and p38 MAPK followed by caspase-3 activation.     

MKP-1 as a target for pharmacological manipulations in PC12 cell survival

Dual specificity mitogen activated protein kinase phosphatase-1 (MKP-1) inactivates extracellular signal-regulated kinase (ERK), p38 and/or c-jun N-terminal protein kinase (JNK) by dephosphorylation via a negative feed-back loop. The aim of the present study was to assess the role of expression of MKP-1 and phosphorylation status of mitogen-activated protein kinases (MAPKs) in promoting cell survival in PC12 cells. We used FK506 and three different monoperoxovanadium complexes (mpVs) as pharmacological tools for manipulation of MKP-1 expression. Peroxovanadium compounds, known to be insulinomimetic agents and protein tyrosine phosphatase inhibitors, are cytotoxic to the cells, they activate JNK and down-regulate MPK-1. On the other hand, FK 506 has transient effect on ERK activation. However, when the agents are used in combination, ERK phosphorylation is prolonged and intensified, MKP-1 expression is increased, and cell survival is enhanced. The concomitant alterations observed in intensities and duration of phospho-ERKs and phospho-JNKs signals suggest that monoperoxovanadium complexes in combination with FK 506 enhance survival of PC12 cells by an induction of MKP-1 expression.     

Islet-neogenesis-associated protein enhances neurite outgrowth from DRG neurons

Islet-neogenesis-associated protein, INGAP, is a 175-amino-acid pancreatic acinar protein that stimulates pancreatic duct cell proliferation in vitro and islet neogenesis in vivo. To date, the mitogenic activity of INGAP has been identified only in nonneural tissues. The aim of this study was to examine the effects of a pentadecapeptide of INGAP (INGAP peptide), the biologically active portion of the native protein, in cultured dorsal root ganglia (DRG) explants from C57BL/6 mice. The present study provides evidence that INGAP peptide acts as a mitogen in the peripheral nervous system (PNS), and that it enhances neurite outgrowth from DRGs in vitro in a time- and dose-dependent manner. The neuritogenic action of INGAP peptide correlates with an increase in [(3)H]thymidine incorporation (P < 0.0001) and mitochondrial activity (P < 0.001). Results from these studies suggest that INGAP peptide promotes Schwann cell proliferation in the DRG which releases trophic factors that promote neurite outgrowth.     

Cellular internalization of poly(ethylene oxide)-b-poly(epsilon-caprolactone) diblock copolymer micelles

Poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-b-PCL) block copolymers self-assemble into micelles in aqueous solution. We have examined whether these micelles can internalize into P19 cells in vitro. Fluorescently labeled PEO(45)-b-PCL(23) block copolymer was prepared by conjugating a tetramethylrhodamine molecule to the end of the hydrophobic PCL block. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) studies yielded 24 +/- 2 and 25 +/- 2 nm, respectively, for the diameters of the micelles. The studies also showed that chemical labeling did not effect the morphology or size. When the rhodamine-labeled PEO(45)-b-PCL(23) block copolymer micelles were tested in vitro, time-, concentration-, and pH-dependence of the internalization process suggested that internalization proceeded by endocytosis. The results from these studies provide the first direct evidence for the internalization of PEO(45)-b-PCL(23) micelles. Future studies will utilize multiple labeling of these micelles, allowing questions to be addressed related to the fate of internalized micelles as drug carriers, the destination of the incorporated drugs or fluorescent probes released from micelles, and the identification of the subcellular localization of the whole drug-carrier system within cells, both in vitro and in vivo.