María Lorenzo

Lovastatin inhibits the extracellular-signal-regulated kinase pathway in immortalized rat brain neuroblasts

We have shown previously that lovastatin, a 3-hydroxy-3-methyl- glutaryl coenzyme A reductase inhibitor, induces apoptosis in spontaneously immortalized rat brain neuroblasts. In the present study, we analysed the intracellular signal transduction pathways by which lovastatin induces neuroblast apoptosis. We showed that lovastatin efficiently inhibited Ras activation, which was associated with a significant decrease in ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Lovastatin also decreased CREB phosphorylation and CREB-mediated gene expression. The effects of lovastatin on the Ras/ERK1/2/CREB pathway were time- and concentration-dependent and fully prevented by mevalonate. In addition, we showed that two MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitors, PD98059 and PD184352, were poor inducers of apoptosis in serum-treated neuroblasts. However, these inhibitors significantly increased apoptosis induced by lovastatin treatment. Furthermore, we showed that pharmacological inhibition of both MEK and phosphoinositide 3-kinase activities was able to induce neuroblast apoptosis with similar efficacy as lovastatin. Our results suggest that lovastatin triggers neuroblast apoptosis by regulating several signalling pathways, including the Ras/ERK1/2 pathway. These findings might also contribute to elucidate the intracellular mechanisms involved in the central nervous system side effects associated with statin therapy.

Genetic determinants for cfxA expression in Bacteroides strains isolated from human infections

OBJECTIVESnTo identify genetic determinants that determine beta-lactamase expression in Bacteroides strains isolated from human infections.nnnMETHODSnBeta-lactam susceptibility and beta-lactamase enzyme expression were characterized in selected strains. Beta-lactamase genes and surrounding regions were analysed by PCR, inverse PCR and Southern hybridization.nnnRESULTSnHigh resistance to penicillins and cephalosporins was found among most isolated strains, in which all known beta-lactamase genes from Bacteroides are represented, but differences were found in their expression of enzyme activity. In contrast to the cepA gene, ubiquitously found but frequently inactive, or cfiA, which only confers carbapenem resistance in two strains, the detection of high beta-lactamase expression correlates closely with the presence of cfxA genes. This genetic determinant shares variability of upstream regulatory elements, including sequence tags from Tn4555, Tn4351 and IS614B, and polymorphisms of encoded amino acid sequences at positions G(57)C and Y(259)C, which might determine enzyme expression characteristics.nnnCONCLUSIONSnThe main determinant for beta-lactamase expression in Bacteroides strains is the cfxA gene, in which IS614B integration upstream of the coding sequence represents a molecular marker for higher levels of enzyme activity.

Implication of Akt, ERK1/2 and alternative p38MAPK signalling pathways in human colon cancer cell apoptosis induced by green tea EGCG.

We investigated apoptosis induced by the green tea component the epigallocatechin-3-gallate (EGCG) and the pathways underlying its activity in a colon cancer cell line. A complete understanding of the mechanism(s) and molecules targeted by green tea polyphenols could be useful in developing novel therapeutic approaches for cancer treatment. EGCG, which is the major polyphenol in green tea, has cytotoxic effects and induced cell death in HT-29 cell death. In this study, we evaluated the effect EGCG on mitogen-activated protein kinase (MAPK) and Akt pathways. EGCG treatment increased phospho-ERK1/2, -JNK1/2 and -p38α, -p38γ and -p38δ, as well as phospho-Akt levels. Using a combination of kinase inhibitors, we found that EGCG-induced cell death is partially blocked by inhibiting Akt, ERK1/2 or alternative p38MAPK activity. Our data suggest that these kinase pathways are involved in the anti-cancer effects of EGCG and indicate potential use of this compound as chemotherapeutic agent for colon cancer treatment.

Lovastatin inhibits the growth and survival pathway of phosphoinositide 3-kinase/protein kinase B in immortalized rat brain neuroblasts.

We previously showed that lovastatin, an HMG‐CoA reductase inhibitor, suppresses cell growth by inducing apoptosis in rat brain neuroblasts. Our aim was to study intracellular signalling induced by lovastatin in neuroblasts. Lovastatin significantly decreases the phosphoinositide 3‐kinase (PI3‐K) activity in a concentration‐dependent manner. Expression of p85 subunit and its association with phosphotyrosine‐containing proteins are unaffected by lovastatin. Lovastatin decreases protein kinase B (PKB)/Akt phosphorylation, and its downstream effectors, p70S6K and the eukaryotic initiation factor 4E (eIF4E) regulatory protein 1, 4E‐BP1, in a concentration‐dependent manner, and reduces p70S6K expression. Lovastatin effects are fully prevented with mevalonate. Only the highest dose of PI3‐K inhibitors that significantly reduce PI3‐K kinase activity induces apoptosis in neuroblasts but to a lower degree than lovastatin. In summary, this work shows that treatment of brain neuroblasts with lovastatin leads to an inhibition of the main pathway that controls cell growth and survival, PI3‐K/PKB and the subsequent blockade of downstream proteins implicated in the regulation of protein synthesis. This work suggests that inactivation of the antiapoptotic PI3‐K appears insufficient to induce the degree of neuroblasts apoptosis provoked by lovastatin, which must necessarily involve other intracellular pathways. These findings might contribute to elucidate the molecular mechanisms of some statins effects in the central nervous system.

Cleavage of focal adhesion proteins and PKCδ during lovastatin‐induced apoptosis in spontaneously immortalized rat brain neuroblasts

We have previously shown that lovastatin induces apoptosis in spontaneously immortalized rat brain neuroblasts. Focal adhesion proteins and protein kinase Cδ (PKCδ) have been implicated in the regulation of apoptosis. We found that lovastatin exposure induced focal adhesion kinase, Crk‐associated substrate (p130Cas), PKCδ cleavage and caspase‐3 activation in a concentration‐dependent manner. Lovastatin effects were fully prevented by mevalonate. The cleavage of p130Cas was almost completely inhibited by z‐DEVD‐fmk, a specific caspase‐3 inhibitor, and z‐VAD‐fmk, a broad spectrum caspase inhibitor, indicating that cleavage is mediated by caspase‐3. In contrast, the lovastatin‐induced cleavage of PKCδ was only blocked by z‐VAD‐fmk suggesting that PKCδ cleavage is caspase‐dependent but caspase‐3‐independent. Additionally, z‐VAD‐fmk partially prevented lovastatin‐induced neuroblast apoptosis. The present data show that lovastatin may induce neuroblast apoptosis by both caspase‐dependent and independent pathways. These findings may suggest that the caspase‐dependent component leading to the neuroblast cell death is likely to involve the cleavage of focal adhesion proteins and PKCδ, which may be partially responsible for some biochemical features of neuroblast apoptosis induced by lovastatin.

Gene Context and DNA Rearrangements in the Carbapenemase Locus of Division II Strains of Bacteroides fragilis

ABSTRACT The cfiA gene is clustered in a bicistronic operon encoding an N-acetyltransferase and an O-acetyltransferase related to resistance markers. This genetic context, exclusively found in strains of Bacteroides fragilis division II, has been highly rearranged by the successive integration of two new mobile sequences, a miniature element and ISBf9. Besides that, among the DNA polymorphisms detected in the cfiA locus, only the integration of IS942 at its promoter was a determinant for expression of carbapenemase activity.

CCK1 and 2 receptors are expressed in immortalized rat brain neuroblasts: Intracellular signals after cholecystokinin stimulation

Cholecystokinin (CCK) is one of the most abundant neuropeptides in the central nervous system (CNS) where it promotes important functions by activation of receptors CCK1 and CCK2. Our aim was to investigate CCK receptors expression and their downstream intracellular signaling in immortalized rat brain neuroblasts. Results show that CCK1 and CCK2 receptor mRNAs and CCK2 receptor protein are expressed in neuroblasts. CCK incubation of neuroblasts leads to stimulation in a time‐dependent manner of several signaling pathways, such as tyrosine phosphorylation of adaptor proteins paxillin and p130Cas, phosphorylation of p44/p42 ERKs as well as PKB (Ser473). Moreover, CCK‐8 stimulates the DNA‐binding activity of the transcription factor AP‐1. The CCK2 receptor agonist gastrin stimulates ERK1/2 phosphorylation in a comparable degree as CCK does. ERK1/2 phosphorylation activated by CCK‐8 was markedly inhibited by the CCK2 receptor antagonist CR2945. Incubation for 48 h with CCK‐8 increases neuroblasts viability in a similar degree as EGF. In summary, our data clearly identify CCK1 and CCK2 receptor mRNAs and CCK2 receptor protein in brain neuroblasts and show that incubation with CCK promotes cell proliferation and activates the phosphorylation of survival transduction pathways. Stimulation of ERK1/2 phosphorylation by CCK is mainly mediated by the CCK2 receptor. Moreover, this work might provide a novel model of proliferating neuronal cells to further study the biochemical mechanisms by which the neuropeptide CCK exerts its actions in the CNS. J. Cell. Biochem. 100: 851–864, 2007.

Antimicrobial resistance determinants among anaerobic bacteria isolated from footrot.

Antibiotic resistance has been evaluated among 36 Gram negative and anaerobic bacilli (10 Bacteroides, 11 Prevotella, 7 Porphyromonas and 8 Fusobacterium strains) isolated from clinical cases of caprine and ovine footrot (necrotic pododermatitis). The initial analysis on this bacterial consortium evaluates the relationships existing among antimicrobial resistance determinants, phenotype expression and mobilization potential. The Bacteroides strains were generally resistant to penicillins, first-generation cephalosporins, tetracycline and erythromycin, and expressed low level of β-lactamase activity. The main determinants found among the Bacteroides strains were cepA and tetQ genes, conferring resistance to β-lactams and tetracycline, respectively. A general susceptibility to β-lactams was shown for most Prevotella, Porphyromonas and Fusobacterium strains, where none of the β-lactamase genes described in Bacteroides was detected. Resistance to tetracycline and/or erythromycin was found among the three bacterial groups. Although tetQ genes were detected for several Prevotella and Porphyromonas strains, a unique ermF positive was revealed among Prevotella strains. The expression of resistance markers was not related with the polymorphism of their coding sequences. However, the finding of sequence signatures for conjugative transposons in the vicinities of tetQ and ermF suggests a mobilization potential that might have contributed to the spread of antimicrobial resistance genes.

Hyperosmotic Stress Induces Tau Proteolysis by Caspase-3 Activation in SH-SY5Y Cells

Tau is a microtubule‐associated protein implicated in the pathogenesis of Alzheimers disease and other related tauopathies. In this subset of neurodegenerative disorders, Tau auto‐assembles into insoluble fibrils that accumulate in neurons as paired helical filaments (PHFs), promoting cellular dysfunction and cytotoxic effects. Growing evidence suggests that abnormal post‐translational regulation, mainly hyperphosphorylation and aberrant cleavage, drives Tau to this pathological state. In this work we show that sorbitol‐induced hyperosmotic stress promotes Tau proteolysis in SH‐SY5Y neuroblastoma cells. The appearance of cleaved Tau was preceded by the activation of μ‐calpain, the proteasome system and caspase‐3. Tau proteolysis was completely prevented by caspase‐3 inhibition but unaffected by neither the proteasome system nor μ‐calpain activity blockade. Concomitantly, hyperosmotic stress induced apoptosis in SH‐SY5Y cells, which was efficiently avoided by the inhibition of caspase‐3 activity. Altogether, our results provide the first evidence that Tau protein is susceptible to caspase‐3 proteolysis under hyperosmotic stress and suggest a positive relationship between Tau proteolysis and apoptosis in SH‐SY5Y cells. J. Cell. Biochem. 117: 2781–2790, 2016.

JNK signaling pathway regulates sorbitol-induced Tau proteolysis and apoptosis in SH-SY5Y cells by targeting caspase-3

Growing evidence suggests that Diabetes Mellitus increases the risk of developing Alzheimers disease. It is well known that hyperglycemia, a key feature of Diabetes Mellitus, may induce plasma osmolarity disturbances. Both hyperglycemia and hyperosmolarity promote the altered post-translational regulation of microtubule-associated protein Tau. Interestingly, abnormal hyperphosphorylation and cleavage of Tau have been proven to lead to the genesis of filamentous structures referred to as neurofibrillary tangles, the main pathological hallmark of Alzheimers disease. We have previously described that hyperosmotic stress induced by sorbitol promotes Tau proteolysis and apoptosis in SH-SY5Y cells via caspase-3 activation. In order to gain insights into the regulatory mechanisms of such processes, in this work we explored the intracellular signaling pathways that regulate these events. We found that sorbitol treatment significantly enhanced the activation of conventional families of MAPK in SH-SY5Y cells. Tau proteolysis was completely prevented by JNK inhibition but not affected by either ERK1/2 or p38 MAPK blockade. Moreover, inhibition of JNK, but not ERK1/2 or p38 MAPK, efficiently prevented sorbitol-induced apoptosis and caspase-3 activation. In summary, we provide evidence that JNK signaling pathway is an upstream regulator of hyperosmotic stress-induced Tau cleavage and apoptosis in SH-SY5Y through the control of caspase-3 activation.