Introduction: In normal B lymphocytes, B-cell receptor (BCR)-induced activation of PI3K-AKT kinas... more Introduction: In normal B lymphocytes, B-cell receptor (BCR)-induced activation of PI3K-AKT kinases and subsequent inactivation of FOXO1 is a critical pro-survival component of tonic BCR signaling. In murine models, conditional deletion of FOXO1 protected quiescent peripheral B cells from apoptosis mediated by inducible loss of the BCR, demonstrating that PI3K-AKT-FOXO1 axis plays a central role in B-cell homeostasis. Disruption of the BCR signaling by SYK inhibitor leads also to the apoptosis of BCR-dependent DLBCLs, at least in part via a mechanism involving decreased activity of PI3K/AKT axis. We investigated the role of FOXO1 in the toxicity of BCR pathway/SYK inhibition in human BCR-dependent lymphomas. Methods: BCR-dependent DLBCL cell lines were incubated with SYK inhibitor and AKT phosphorylation, FOXO1 activation and transcriptional activity were assessed by phospho-specific flow cytometry, Western Blot and qPCR. FOXO1 knock-down in DLBCL cell was achieved with shRNA. The e...
Biochimica et Biophysica Acta (BBA) - General Subjects
BACKGROUND Neurosecretion is the multistep process occurring in separate spatial and temporal cel... more BACKGROUND Neurosecretion is the multistep process occurring in separate spatial and temporal cellular boundaries which complicates its comprehensive analysis. Most of the research are focused on one distinct stage of synaptic vesicle recycling. Here, we describe approaches for complex analysis of synaptic vesicle (SV) endocytosis and separate steps of exocytosis at the level of presynaptic bouton and highly purified SVs. METHODS Proposed fluorescence-based strategies and analysis of neurotransmitter transport provided the advantages in studies of exocytosis steps. We evaluated SV docking/tethering, their Ca2+-dependent fusion and release of neurotransmitters gamma-aminobutyric acid (GABA) and glutamate in two animal models. RESULTS Approaches enabled us to study: 1) endocytosis/Ca2+-dependent release of fluorescent carbon nanodots (CNDs) during stimulation of nerve terminals; 2) the action of levetiracetam, modulator of SV glycoprotein SV2, on fusion competence of SVs and stimulated release of GABA and glutamate; 3) impairments of several steps of neurosecretion under vitamin D3 deficiency. CONCLUSIONS Our algorithm enabled us to verify the method validity for multidimensional analysis of SV turnover. By increasing SV docking and the size of readily releasable pool (RRP), levetiracetam is able to selectively enhance the stimulated GABA secretion in hippocampal neurons. Findings suggest that SV2 regulates RRP through impact on the number of docked/primed SVs. GENERAL SIGNIFICANCE Methodology can be widely applied to study the stimulated neurosecretion in presynapse, regulation of SV docking, their Ca2+-dependent fusion with target membranes, quantitative analysis of expression of neuron-specific proteins, as well as for testing the efficiency of pre-selected designed neuroactive substances.
Fluorescent pyrene-linker-nucleobase (nucleobase = thymine, adenine) conjugates with carbonyl and... more Fluorescent pyrene-linker-nucleobase (nucleobase = thymine, adenine) conjugates with carbonyl and hydroxy functionalities in the linker were synthesized and characterized. X-ray single-crystal structure analysis performed for the pyrene-C(O)CH2CH2-thymine (2) conjugate reveals dimers of molecules 2 stabilized by hydrogen bonds between the thymine moieties. The photochemical characterization showed structure-dependent fluorescence properties of the investigated compounds. The conjugates bearing a carbonyl function represent weak emitters as compared to compounds with a hydroxy function in the linker. The self-assembly properties of pyrene nucleobases were investigated in respect to their binding to single and double strand oligonucleotides in water and in buffer solution. In respect to the complementary oligothymidine T10 template in water, compounds 3 and 5 both show a self-assembling behavior according to canonical base-base pairing. However, in buffer solution, derivative 5 was mu...
Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process... more Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process known as therapy-induced senescence (TIS). Such cells may undergo atypical divisions, thus contributing to tumor re-growth. Accumulation of senescent cancer cells reduces survival of patients after chemotherapy. As senescence interplays with autophagy, a dynamic recycling process, we sought to study whether inhibition of autophagy interferes with divisions of TIS cells. We exposed human colon cancer HCT116 cells to repeated cycles of a chemotherapeutic agent - doxorubicin (doxo) and demonstrated induction of hallmarks of TIS (e.g. growth arrest, hypertrophy, poliploidization and secretory phenotype) and certain properties of cancer stem cells (increased NANOG expression, percentages of CD24+ cells and side population). Colonies of small and highly proliferative progeny appeared shortly after drug removal. Treatment with bafilomycin A1 (BAF A1), an autophagy inhibitor, postponed short te...
Abstract: A system and method of validating differences between measured values of fluorescence i... more Abstract: A system and method of validating differences between measured values of fluorescence intensities obtained from a fluorescence-based instrument, including: generating calibration histograms for calibration beads run through the instrument; calibrating a distance function configured to measure distances between the histograms by: constructing a metric using the distance function, which includes a bin-to-bin dissimilarity matrix; and populating the dissimilarity matrix to maximize the following conditions:(A) the ...
Redox activities associated with plasma membranes of nonphagocytic animal and plant cells have be... more Redox activities associated with plasma membranes of nonphagocytic animal and plant cells have been reported by several authors. However, the natural substrates, structure and biological role of these putative enzyme systems are not known. Data indicating extracellular reduction of a nitroxide free radical Cat1 (1-oxy-4-trimethylamine-2,2,6,6,tetramethyl-piperidine) by hepatocytes were thought to be artefactual. We report evidence in support of a notion that Cat1 as well as a tetrazolium salt, CTC (5-cyano-2,3-ditolyl tetrazolium chloride), are reduced extracellularly, probably at the cell surface, by human HepG2 hepatoma cells. These data provide evidence confirming the existence of a yet unidentified reducing activity associated with outer surface of plasma membranes of transformed human hepatocytes.
Latrepirdine/Dimebon is a small-molecule compound with attributed neurocognitive-enhancing activi... more Latrepirdine/Dimebon is a small-molecule compound with attributed neurocognitive-enhancing activities, which has recently been tested in clinical trials for the treatment of Alzheimer's and Huntington's disease. Latrepirdine has been suggested to be a neuroprotective agent that increases mitochondrial function, however the molecular mechanisms underlying these activities have remained elusive. We here demonstrate that latrepirdine, at (sub)nanomolar concentrations (0.1 nM), activates the energy sensor AMP-activated protein kinase (AMPK). Treatment of primary neurons with latrepirdine increased intracellular ATP levels and glucose transporter 3 translocation to the plasma membrane. Latrepirdine also increased mitochondrial uptake of the voltage-sensitive probe TMRM. Gene silencing of AMPKα or its upstream kinases, LKB1 and CaMKKβ, inhibited this effect. However, studies using the plasma membrane potential indicator DisBAC2(3) demonstrated that the effects of latrepirdine on T...
Introduction: In normal B lymphocytes, B-cell receptor (BCR)-induced activation of PI3K-AKT kinas... more Introduction: In normal B lymphocytes, B-cell receptor (BCR)-induced activation of PI3K-AKT kinases and subsequent inactivation of FOXO1 is a critical pro-survival component of tonic BCR signaling. In murine models, conditional deletion of FOXO1 protected quiescent peripheral B cells from apoptosis mediated by inducible loss of the BCR, demonstrating that PI3K-AKT-FOXO1 axis plays a central role in B-cell homeostasis. Disruption of the BCR signaling by SYK inhibitor leads also to the apoptosis of BCR-dependent DLBCLs, at least in part via a mechanism involving decreased activity of PI3K/AKT axis. We investigated the role of FOXO1 in the toxicity of BCR pathway/SYK inhibition in human BCR-dependent lymphomas. Methods: BCR-dependent DLBCL cell lines were incubated with SYK inhibitor and AKT phosphorylation, FOXO1 activation and transcriptional activity were assessed by phospho-specific flow cytometry, Western Blot and qPCR. FOXO1 knock-down in DLBCL cell was achieved with shRNA. The e...
Biochimica et Biophysica Acta (BBA) - General Subjects
BACKGROUND Neurosecretion is the multistep process occurring in separate spatial and temporal cel... more BACKGROUND Neurosecretion is the multistep process occurring in separate spatial and temporal cellular boundaries which complicates its comprehensive analysis. Most of the research are focused on one distinct stage of synaptic vesicle recycling. Here, we describe approaches for complex analysis of synaptic vesicle (SV) endocytosis and separate steps of exocytosis at the level of presynaptic bouton and highly purified SVs. METHODS Proposed fluorescence-based strategies and analysis of neurotransmitter transport provided the advantages in studies of exocytosis steps. We evaluated SV docking/tethering, their Ca2+-dependent fusion and release of neurotransmitters gamma-aminobutyric acid (GABA) and glutamate in two animal models. RESULTS Approaches enabled us to study: 1) endocytosis/Ca2+-dependent release of fluorescent carbon nanodots (CNDs) during stimulation of nerve terminals; 2) the action of levetiracetam, modulator of SV glycoprotein SV2, on fusion competence of SVs and stimulated release of GABA and glutamate; 3) impairments of several steps of neurosecretion under vitamin D3 deficiency. CONCLUSIONS Our algorithm enabled us to verify the method validity for multidimensional analysis of SV turnover. By increasing SV docking and the size of readily releasable pool (RRP), levetiracetam is able to selectively enhance the stimulated GABA secretion in hippocampal neurons. Findings suggest that SV2 regulates RRP through impact on the number of docked/primed SVs. GENERAL SIGNIFICANCE Methodology can be widely applied to study the stimulated neurosecretion in presynapse, regulation of SV docking, their Ca2+-dependent fusion with target membranes, quantitative analysis of expression of neuron-specific proteins, as well as for testing the efficiency of pre-selected designed neuroactive substances.
Fluorescent pyrene-linker-nucleobase (nucleobase = thymine, adenine) conjugates with carbonyl and... more Fluorescent pyrene-linker-nucleobase (nucleobase = thymine, adenine) conjugates with carbonyl and hydroxy functionalities in the linker were synthesized and characterized. X-ray single-crystal structure analysis performed for the pyrene-C(O)CH2CH2-thymine (2) conjugate reveals dimers of molecules 2 stabilized by hydrogen bonds between the thymine moieties. The photochemical characterization showed structure-dependent fluorescence properties of the investigated compounds. The conjugates bearing a carbonyl function represent weak emitters as compared to compounds with a hydroxy function in the linker. The self-assembly properties of pyrene nucleobases were investigated in respect to their binding to single and double strand oligonucleotides in water and in buffer solution. In respect to the complementary oligothymidine T10 template in water, compounds 3 and 5 both show a self-assembling behavior according to canonical base-base pairing. However, in buffer solution, derivative 5 was mu...
Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process... more Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process known as therapy-induced senescence (TIS). Such cells may undergo atypical divisions, thus contributing to tumor re-growth. Accumulation of senescent cancer cells reduces survival of patients after chemotherapy. As senescence interplays with autophagy, a dynamic recycling process, we sought to study whether inhibition of autophagy interferes with divisions of TIS cells. We exposed human colon cancer HCT116 cells to repeated cycles of a chemotherapeutic agent - doxorubicin (doxo) and demonstrated induction of hallmarks of TIS (e.g. growth arrest, hypertrophy, poliploidization and secretory phenotype) and certain properties of cancer stem cells (increased NANOG expression, percentages of CD24+ cells and side population). Colonies of small and highly proliferative progeny appeared shortly after drug removal. Treatment with bafilomycin A1 (BAF A1), an autophagy inhibitor, postponed short te...
Abstract: A system and method of validating differences between measured values of fluorescence i... more Abstract: A system and method of validating differences between measured values of fluorescence intensities obtained from a fluorescence-based instrument, including: generating calibration histograms for calibration beads run through the instrument; calibrating a distance function configured to measure distances between the histograms by: constructing a metric using the distance function, which includes a bin-to-bin dissimilarity matrix; and populating the dissimilarity matrix to maximize the following conditions:(A) the ...
Redox activities associated with plasma membranes of nonphagocytic animal and plant cells have be... more Redox activities associated with plasma membranes of nonphagocytic animal and plant cells have been reported by several authors. However, the natural substrates, structure and biological role of these putative enzyme systems are not known. Data indicating extracellular reduction of a nitroxide free radical Cat1 (1-oxy-4-trimethylamine-2,2,6,6,tetramethyl-piperidine) by hepatocytes were thought to be artefactual. We report evidence in support of a notion that Cat1 as well as a tetrazolium salt, CTC (5-cyano-2,3-ditolyl tetrazolium chloride), are reduced extracellularly, probably at the cell surface, by human HepG2 hepatoma cells. These data provide evidence confirming the existence of a yet unidentified reducing activity associated with outer surface of plasma membranes of transformed human hepatocytes.
Latrepirdine/Dimebon is a small-molecule compound with attributed neurocognitive-enhancing activi... more Latrepirdine/Dimebon is a small-molecule compound with attributed neurocognitive-enhancing activities, which has recently been tested in clinical trials for the treatment of Alzheimer's and Huntington's disease. Latrepirdine has been suggested to be a neuroprotective agent that increases mitochondrial function, however the molecular mechanisms underlying these activities have remained elusive. We here demonstrate that latrepirdine, at (sub)nanomolar concentrations (0.1 nM), activates the energy sensor AMP-activated protein kinase (AMPK). Treatment of primary neurons with latrepirdine increased intracellular ATP levels and glucose transporter 3 translocation to the plasma membrane. Latrepirdine also increased mitochondrial uptake of the voltage-sensitive probe TMRM. Gene silencing of AMPKα or its upstream kinases, LKB1 and CaMKKβ, inhibited this effect. However, studies using the plasma membrane potential indicator DisBAC2(3) demonstrated that the effects of latrepirdine on T...
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Papers by Tytus Bernas