23 research outputs found

    Endogenous amyloidogenesis in long-term rat hippocampal cell cultures

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    <p>Abstract</p> <p>Background</p> <p>Long-term primary neuronal cultures are a useful tool for the investigation of biochemical processes associated with neuronal senescence. Improvements in available technology make it possible to observe maturation of neural cells isolated from different regions of the rodent brain over a prolonged period <it>in vitro</it>. Existing experimental evidence suggests that cellular aging occurs in mature, long-term, primary neuronal cell cultures. However, detailed studies of neuronal development <it>in vitro </it>are needed to demonstrate the validity of long-term cell culture-based models for investigation of the biochemical mechanisms of <it>in vitro </it>neuronal development and senescence.</p> <p>Results</p> <p>In the current study, neuron-enriched hippocampal cell cultures were used to analyze the differentiation and degeneration of hippocampal neurons over a two month time period. The expression of different neuronal and astroglial biomarkers was used to determine the cytochemical characteristics of hippocampal cells in long-term cultures of varying ages. It was observed that the expression of the intermediate filament nestin was absent from cultures older than 21 days in vitro (DIV), and the expression of neuronal or astrocytic markers appeared to replace nestin. Additionally, morphological evaluations of neuronal integrity and Hoescht staining were used to assess the cellular conditions in the process of hippocampal culture development and aging. It was found that there was an increase in endogenous production of Aβ<sub>1-42 </sub>and an increase in the accumulation of Congo Red-binding amyloidal aggregates associated with the aging of neurons in primary culture. <it>In vitro </it>changes in the morphology of co-existing astrocytes and cell culture age-dependent degeneration of neurodendritic network resemble features of <it>in vivo </it>brain aging at the cellular level.</p> <p>Conclusion</p> <p>In conclusion, this study suggests that long-term primary CNS culture is a viable model for the study of basic mechanisms and effective methods to decelerate the process of neuronal senescence.</p

    Genomic features of resistant <i>Klebsiella pneumonia</i>, isolated from the bloodstream and cerebrospinal fluid of pediatric hospital patients

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    Introduction. Carbapenemase-producing Klebsiella pneumoniae (CP-Kp), which are international high-risk clones, have become a problem of utmost importance. CP-Kps, adapting to the hospital environment, evolve into convergent pathotypes. Such variants combine traits of two genetic lineages: multidrug resistant (MDR) and hypervirulent. The pathotypes, along with MDR K. pneumoniae, pose an exceptional threat to young patients during systemic infection. The objective of this study is the detailed molecular genetic analysis of MDR isolates of K. pneumoniae detected during the monitoring of resistant Gram-negative bacteria at the National Medical Research Center for Children’s Health in 2014–2021. Materials and methods. Whole-genome sequencing with a subsequent bioinformatics analysis of eight MDR isolates from the bloodstream and cerebrospinal fluid. Results. MDR isolates belonged to 4 sublineages (SL): SL307, SL395, SL29 and SL1198. In the genomes of 6 pangrug-resistant (PDR) isolates, genes associated with resistance to all categories of antibiotics recommended for Enterobacteriaceae therapy were identified. Plasmids were present in all genomes. In 6 isolates, plasmids contained heavy metal ion resistance operons in addition to antibiotic resistance genes. Prophages within the plasmids were also involved in the transfer of resistance genes. The ST395 isolate from the cerebrospinal fluid belonged to the convergent pathotype in terms of resistance and virulence. Comparison of genomes within SLs revealed recombination events in the K- and O-locus regions and the Yersiniabactin operon. Conclusion. Thus, in a sample of resistant K. pneumoniae isolated from bloodstream and cerebrospinal fluid, 6 PDR isolates were detected, one of which belongs to the convergent pathotype ST395

    Soy Isoflavones Genistein and Daidzein Exert Anti-Apoptotic Actions via a Selective ER-mediated Mechanism in Neurons following HIV-1 Tat1–86 Exposure

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    HIV-1 viral protein Tat partially mediates the neural dysfunction and neuronal cell death associated with HIV-1 induced neurodegeneration and neurocognitive disorders. Soy isoflavones provide protection against various neurotoxic insults to maintain neuronal function and thus help preserve neurocognitive capacity.We demonstrate in primary cortical cell cultures that 17β-estradiol or isoflavones (genistein or daidzein) attenuate Tat(1-86)-induced expression of apoptotic proteins and subsequent cell death. Exposure of cultured neurons to the estrogen receptor antagonist ICI 182,780 abolished the anti-apoptotic actions of isoflavones. Use of ERα or ERβ specific antagonists determined the involvement of both ER isoforms in genistein and daidzein inhibition of caspase activity; ERβ selectively mediated downregulation of mitochondrial pro-apoptotic protein Bax. The findings suggest soy isoflavones effectively diminished HIV-1 Tat-induced apoptotic signaling.Collectively, our results suggest that soy isoflavones represent an adjunctive therapeutic option with combination anti-retroviral therapy (cART) to preserve neuronal functioning and sustain neurocognitive abilities of HIV-1 infected persons

    Protein oxidation and enzyme activity decline in old brown Norway rats are reduced by dietary restriction. Mech Ageing Dev

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    Abstract The effect of aging and diet restriction (DR) on the activity of creatine kinase (CK), glutamine synthetase (GS) and protein carbonyl formation in the cerebellum, hippocampus and cortex of male and female brown Norway (BN) rats has been investigated. It was demonstrated that CK activity in three different regions of the rat brain declines with age by 30%. Age-related decrease of GS activity was only 10 -13% and did not reach statistical significance. Consistent with previously published studies, age-related increase of protein carbonyl content in each brain area studied has been observed. Preventive effects of a caloric restricted diet on the age-associated protein oxidation and changes of the activity of CK and GS in the brain was observed for both aging male and female BN rats. DR delayed the accumulation of protein carbonyls. Age-related changes of CK activity in rat brain were abrogated by DR. The activity of GS in the brain of old rats subjected to the caloric restricted diet was higher than that in the brain of young animals fed ad libitum. The results are consistent with the notion that DR may relieve age-associated level of oxidative stress and lessen protein damage. © 1998 Elsevier Science Ireland Ltd. 100 (1998) 157-168 15

    Genistein and daidzein attenuate Tat-induced caspase activation in primary cortical cultures.

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    <p>Cortical cultures were treated with 1 µM GEN or DAI 24 hr prior to Tat exposure. Expression of activated apoptotic proteins <b>A</b>. Caspase 9 (4 hr of Tat exposure) and <b>B</b>. caspase 3 (4 hr Tat exposure) was assessed by ELISA experiments. Data represents mean values ± SEM, with experiments performed in triplicate, *<i>p≤</i>0.05 as compared to Tat-treated cultures.</p

    ER subtype specific effects against caspase activity and Bax expression.

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    <p>Similar to 17β-estradiol (<b>A</b>), GEN (<b>B</b>) and DAI (<b>C</b>) effects against Tat-induced caspase 3 activity were maintained in the presence of specific antagonists for ERα (MPP) and ERβ (PHTPP). ER subtype antagonists reveal that ERβ signaling was preferential for GEN (<b>E</b>) effects on Bax. DAI (<b>F</b>) effects on Bax were blocked in the presence of both ER subtype antagonists. Data represents mean values ± SEM, *<i>p≤</i>0.05 as compared to GEN/DAI+Tat treated cultures.</p

    Soy isoflavones genistein and daidzein protect primary cortical cultures from Tat neurotoxicity.

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    <p>Primary cortical neurons were exposed to estrogen (0.1, 2.0 and 10 nM), or isoflavones (0.05, 0.2 and 1 µM) 24 hr prior to the start of Tat<sub>1–86</sub> B (50 nM) treatment. Cell viability was assessed by Live/Dead assay. Live/Dead ratios were determined after 48 hr (<b>A-C</b>) or 5 days (<b>D-F</b>) of the continuous exposure to Tat or equal volume of vehicle in cell culture groups that were treated or not treated with estrogen, GEN, or DAI. Data represents mean values ± SEM, n of cultures analyzed  = 7–12 per each group. *- indicates significant (<i>p</i>≤0.05) protective effects of the selected compounds against Tat neurotoxicity (cell viability decrease) in cortical cell cultures. Repeated (2–3) trials using cell culture preparations from different litters were carried out to ensure the reproducibility of the results.</p
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